GMP/GDP certified

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American Pharmaceutical

The Review of American Pharmaceutical Business & Technology

Volume 20 Issue 6 | September/October 2017

SHOW ISSUE:
AAPS Annual
Meeting and Exposition
Eastern Analytical
Symposium & Exposition

Choosing an
Antimicrobial Preservative
Adoption of FMEA for
Microbiological Contamination
Risk Assessment to Implement
USP Chapter <1115>
Batch, Continuous or
“Fake/False” Continuous Processes
Kinetic and Thermodynamic
Profiling in Drug Discovery
www.americanpharmaceuticalreview.com
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2 | | September/October 2017
 September/October 2017 | Volume 20, Issue 6

COVER FEATURES
24 MICROBIOLOGY
Adoption of FMEA for Microbiological Contamination Risk Assessment to Implement USP Chapter <1115>
N. T. Gilles,1 E. Marty,1 D. Roesti,1 A. Staerk,1 and M. Goverde2
1
Microbiological Quality Control Unit, Novartis Pharma Stein AG, Switzerland
2
MGP Consulting GmbH, Binningen, Switzerland

74 MICROBIOLOGY
Antimicrobial Preservatives Part Two: Choosing a Preservative
David P. Elder, PhD and Patrick J. Crowley, F.R.PS
Independent CMC Consultants

86 MANUFACTURING
Batch, Continuous or “Fake/False” Continuous Processes
Girish Malhotra, PE
EPCOT International

133 DRUG DEVELOPMENT

Kinetic and Thermodynamic Profiling in Drug Discovery: Promises, Challenges and Outlook
Ying Wang and Anil Vasudevan
AbbVie Inc., North Chicago, IL

www.americanpharmaceuticalreview.com | | 3
IN THIS ISSUE »
36 MICROBIOLOGY 138 BIOPHARM PROCESSING
Data Integrity Issues in Microbial Testing Trends and Growth in Single-Use System (SUS) Adoption
Cheryl Platco and Tony Cundell Eric S. Langer
Pharmaceutical Microbiologist (Retired) and Microbiological Consulting, LLC President and Managing Partner, BioPlan Associates, Inc.

44 FORMULATION AND DEVELOPMENT 142 CPhI REPORT

Selecting In Vitro Dissolution Tests for Bioavailability Enhancing The Congruence Between Small Molecule Generic Medicine and Biosimilar
Oral Formulations Medicine Business Models
Michael Grass, PhD Alan Sheppard
Senior Research Chemist Lonza Pharma & Biotech Principal, Global Generics, IMS Health

92 DRUG DISCOVERY 148 RESEARCH

The Evolution of Macrocycles in Drug Discovery: From Technologies Contract Research Organization Growth and Investment
to Drugs
Nigel Walker
Mark L. Peterson, PhD Managing Director, Nice Insight/That’s Nice LLC
Chief Operating Officer, Cyclenium Pharma Inc.
152 ROUNDTABLE
100 SEPARATION PURIFICATION
Drug Delivery
Case Study: Qualifying a Commercially Available ELISA for HCP
Quantification and Its Ability to Inform Purification Process Decisions
Samantha Kecman, Victor Dellisola, and Stephen Raso SPECIAL FEATURES
Genocea Biosciences
8 Social Media Connections
106 FORMULATION AND DEVELOPMENT
Evolution of Bench Scale Mixing Devices: Getting Reliable Scale-Up Data 9 CNPerspectives

Dr. Suzanne Kresta and Marcio B. Machado 10 Corporate Profiles

Department of Chemical and Materials Engineering, University of Alberta
49 Whitepaper Review
112 MANUFACTURING
Second Level Thinking in Cleanroom Decision Making 126 An Interview with Catalent
Sidney Backstrom, Executive Director, Business Management
146 An Interview with Amerisource Interivew
Peter Makowenskyj, Sales Engineer
Maik W. Jornitz, Chief Executive Officer
156 P.I.N. Points
G-CON Manufacturing, Inc.

122 FORMULATION AND DEVELOPMENT

Strategies to Predict the Developability of Biopharmaceuticals REGULAR FEATURES
Dr. Stefan R. Schmidt
Chief Scientific Officer 6 A Message from the Editor
Rentschler Biopharma SE
7 Editorial Advisory Board
130 SPECTROSCOPY
128 Equipment Focus
Polarization-Resolved Raman Spectroscopy for
Pharmaceutical Applications 136 Editor's Top Tech
Johannes Kiefer
Technische Thermodynamik, Universität Bremen, Bremen, Germany 160 Advertiser's Index

4 | | September/October 2017
OUR TEST, YOUR CURE...

ENSURING A HEALTHY WORLD

YOUR Endotoxin Experts!

www.acciusa.com
 » A Message from the Editor »
“The Future Ain’t What It Used To Be.”

The late, great Yogi Berra was credited with saying a lot of crazy things.

From “It ain’t over till it’s over.” to “Nobody goes there anymore. It’s too crowded.” Berra was adept at mangling the English language but
yet, you kind of, sort of, knew what he meant.

Today, I’m here to talk about his line, “You can observe a lot by watching.” and how it relates to the pharmaceutical industry.

I have no compunction about admitting that I watch television - perhaps too much television. I am also one of those people who tend
to also watch commercials – and not just during the Super Bowl. Most are boring, or predictable, but some are clever and humorous.
The “Jake from State Farm” commercial is particularly good – and always gives me a chuckle. I also find the Subaru commercials that
feature dogs amusing and effective.

Commercials for pharmaceuticals are fairly predictable. Thirty seconds of silly, contrived situations designed to emulate what companies
think/hope will get potential consumers to identify with – all the while making sure to include all the required legalese and side-effect
information – most of which normal people can’t stomach.

While the style of the commercials is usually the same – the types of products being advertised is what is interesting, and as Berra once
said, “You can observe a lot by watching.”

For example, just a few short years ago advertisements for ED treatments were on ALL THE TIME. Most noticeable during sporting
events, you were practically guaranteed to see at least one ED advertisement during a commercial break. And you have my sympathies
if you ever had to explain what ED was to a young child who happened to be watching the game with you.

More recently, commercials for low testosterone filled the commercial breaks. Remedies for Low-T as it was called seemed to replace
many of the spots for ED during sporting events. Then, just as quickly as they arrived, they disappeared.

Today, there appears to be two new hot indications: irritable bowel syndrome, and psoriasis. Both must be very tough for marketers to
make “sexy” as they did for ED and Low-T, but they sure are trying. And, just like the prior ads, spots for these indications are now taking
over prime-time and sports programming.

So, if you are looking to see what’s on the minds of pharmaceutical marketers, you need look no further than your TV screen because, as
Yogi said, “I never said most of the things I said.”

Mike Auerbach
Editor-In-Chief
[email protected]

6 | | September/October 2017
» Editorial Advisory Board »
Shaukat Ali, PhD John Finkbohner, PhD Shane R. Needham, PhD
Technical Support Manager Director, Regulatory Affairs Laboratory Director
BASF Corporation MedImmune Alturas Analytics, Inc.

Ghulam Shabir Arain, PhD, CSci, CChem, Daniel L. Norwood, MSPH, PhD
Adam S. Goldstein
FRSC, FCQI Distinguished Research Fellow
Senior Manager, Clinical Purification, Operations/Development
Managing Director Boehringer Ingelheim Pharmaceuticals, Inc.
DGS Pharma Consulting Ltd., UK Genentech

Davy Guillarme, PhD Mehul Patel

Katherine Bakeev, PhD Global Marketing Director
Director of Analytical Services and Support Senior Lecturer, School of Pharmaceutical Sciences
Endotoxin and Microbial Detection
B&W Tek, Inc. University of Geneva, University of Lausanne
Charles River
Douglas J. Ball, MS Chris Halling
David Radspinner
Diplomate, American Board of Toxicology; Research Fellow, Senior Manager, Global Communications Director of Marketing and Applications Support for BioProcess
Drug Safety Research & Development and European Marketing Production
Pfizer Global Research & Development Catalent Pharma Solutions Thermo Fisher Scientific
Suraj Baloda, PhD
Brian Lingfeng He Aniruddha M. Railkar, PhD
Founder and President
Research Investigator Director of CMC
SARMICON, LLC.
Bristol-Myers Squibb Tarsa Therapeutics
Rory Budihandojo
Director, Quality Systems Audit Ronald Iacocca, PhD Gary E. Ritchie
Boehringer Ingelheim Shanghai Pharmaceuticals Senior Research Advisor, Product and Process Performance President
Co., Ltd. Eli Lilly & Co. Council For Near Infrared Spectroscopy

Harsh Chauhan, PhD Maik W. Jornitz Rodolfo J. Romañach, PhD

Assistant Professor Vice President of Business Development Professor of Chemistry
School of Pharmacy & Health Professions G-Con Manufacturing, LLC University of Puerto Rico, Mayagüez Campus
Creighton University
Hemant N. Joshi, PhD, MBA Shouvik Roy, PhD
Robert V. Chimenti Principal Scientist, Organizational Unit Leader in
Principal
Sr. Strategic Applications Engineer Drug Product Engineering
Tara Innovations LLC
Innovative Photonic Solutions Amgen
Adjunct Professor Ian Lewis, PhD
Jim Rydzak
Rowan University Director of Marketing
Investigator, Strategic Technology Division
Kaiser Optical Systems, Inc. GlaxoSmithKline
Emil W. Ciurczak, PhD
Doramaxx Consulting Ralph Lipp, PhD Ronak Savla
President and CEO Fellow
Rick E. Cooley
Retired Lipp Life Sciences LLC Catalent Applied Drug Delivery Institute
Eli Lilly & Co., Inc. Jack Lysfjord Ken Seufert
Weiguo Dai, PhD Principal Consultant Managing Director, North America
Scientific Director, Janssen Fellow, Drug Product Development Lysfjord Consulting LLC MEGGLE USA Inc.
Johnson and Johnson
Steven R. Maple, PhD Jaleel Shujath
Nila Das, PhD Head of Pharmaceutical Technology Development Dept. Industry Strategist, Life Sciences
Senior Research Investigator Lilly Research Laboratories, Eli Lilly & Co., Inc. OpenText
Bristol-Myers Squibb
Jerold M. Martin Donald C. Singer
Vivek Dave, PhD Senior Vice President, Scientific Affairs GSK Senior Fellow, R&D
Assistant Professor, Pharmaceutical Sciences Pall Life Sciences GlaxoSmithKline
St. John Fisher College, Wegmans School of Pharmacy
John P. Mayer Onkar N. Singh, PhD, MBA
Michael Dong, PhD Director, Product Development
Senior Research Scientist
Consultant Insys Therapeutics, Inc.
Indiana University
MWD Consulting
Allen Templeton, PhD
Michael J. Miller, PhD
Dr. Thomas Dürig Associate Vice President
President Formulation Sciences Merck Research Laboratories
Sr. R&D Director, Pharmaceutical and Food Ingredients
Ashland Inc. Microbiology Consultants, LLC
Zhenyu Wang, PhD
Walter Dziki, PhD Ronald W. Miller, PhD, MBA Associate Principle Scientist and Group Leader
Associate Research Fellow President , Technology Consultant Respiratory Product Development
Abbott Laboratories Miller Pharmaceutical Merck & Co.

Stuart Farquharson, PhD Ganapathy Mohan, PhD Larry Wigman, PhD

President & CEO Head of Global CMC Regulatory Affairs Senior Scientific Manager
Real-Time Analyzers, Inc. Merck, Sharp and Dohme Corp. Genentech

www.americanpharmaceuticalreview.com | | 7
 AMERICAN PHARMACEUTICAL REVIEW

Social Media Connections

Janssen Global Microbes&Infection

@JanssenGlobal @MicrobesInfect
#JNJ #Ebola vaccine candidate moves forward On this day in 1928, Alexander Fleming accidentally
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U.S. FDA Amgen Foundation

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Research Matters: Engineered antibody protects
monkeys from HIV-like virus http://dlvr.it/Ps73nS
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new #VitalSigns. http://bit.ly/2xkDGJp

Connect with American Pharmaceutical Review at:

facebook.com/AmericanPharmaceuticalReview
twitter.com/ampharmrev
linkedin.com/groups/American-Pharmaceutical-
Review-3889659

8 | | September/October 2017
CNPerspectives
American Pharmaceutical Review is one of several outstanding publications available from CompareNetworks, Inc. Here is a look at the
insightful content our readers may enjoy from four of our sister publications: Pharmaceutical Outsourcing, Dentalcompare, Labcompare,
and Biocompare.

Synthetic Route Selection for APIs: It Pays

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Process chemists developing Active Pharmaceutical Ingredients (APIs) for clinical trials today face a challenging array of business and regulatory
hurdles. The pressures to reduce costs while meeting more complex regulatory mandates, create a difficult challenge: how to develop a
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Beyond the Limit: The World of Super-Resolution Microscopy

Up until the last 15 years, researchers and scientists have been largely constrained by a long-established optical resolution limit related to
biological microscopy systems. This limit was first stipulated by Ernst Abbe more than 140 years ago and established the maximum resolution
of an optical system to be around 200 nm in the XY direction. What this limit has always meant for microscope users was that many subcellular
structures were not large enough for detailed observation. The imaging of intracellular structures and activities has typically been restricted by
the optical resolution limit of available imaging systems.
http://bit.ly/2xaDe1G

Sorting Metabolic Signals

All of the small molecules involved in an organism’s metabolism make up its metabolome. When it comes to all of the ’omes—genomes, proteomes,
lipidomes, and so on—the metabolome might be the most talked about in recent years. Although the term, metabolome, was coined almost 20
years ago, the research is rolling today more than ever. This work often involves liquid chromatography (LC) and mass spectrometry (MS).
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www.americanpharmaceuticalreview.com | | 9
 Products & Solution Areas
www.acdlabs.com ACD/Labs’ unique expertise lies in a world-class knowledge management
platform for handling of vendor agnostic, multi-techniques spectroscopic,
ACD/Labs spectrometric, and chromatographic analytical data. The ACD/Spectrus
Platform enables spectroscopic data processing and interpretation for NMR,
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MS, LC/MS, IR, RAMAN, UV, other optical, and hyphenated instrumental
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techniques; chemical structure confirmation, verification, and elucidation;
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and separation science-based chromatographic method development and
Email: [email protected] optimization. Spectrus offers chemically intelligent databasing to support
collaborative research and efficient data-driven decision-making.
These expert data analysis tools have been further refined to align with
About ACD/Labs R&D workflows and provide a new generation of applications that assist in
comprehensive metabolite identification (MetaSense) and more recently to
Advanced Chemistry Development, Inc., (ACD/Labs) is an
help establish effective process and impurity control strategies in process
informatics company that develops and commercializes
development (Luminata).
solutions in support of R&D. Our chemical and analytical
knowledge management platform helps organizations The ACD/Percepta Platform, built on the foundation of our industry-leading
preserve information with chemical context so it can physicochemical property prediction algorithms, provides a platform for
be searched and re-applied with ease. Our professional property evaluation, compound screening, and lead optimization.

services team delivers software configuration and

customization to align the capabilities of our technologies
with scientific workflows. We also provide integration with
existing informatics systems and undertake custom projects
including enterprise-level automation.

Background
A privately-held company that has served the R&D
community for over two decades, ACD/Labs has a global
sales and support presence, with offices in N. America,
Europe, and Asia. Our employees number over 160 dedicated
individuals, including many PhD level scientists, with
expertise in informatics and various chemical disciplines.

Markets Served
Our customers span the variety of industries that undertake
research and development for innovative materials
and products, including pharmaceutical and chemical
companies—food and beverage, environmental, water,
agrochemical, flavors and fragrance, consumer packaged
goods, fine chemicals—academic organizations, and
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10 | | September/October 2017
 Company Description
www.ashland.com/pharmaceutical Ashland is a manufacturer and marketer of pharmaceutical grade excipients
supported by global cGMP manufacturing and R&D with expertise in polymer
Ashland science, formulation development, solid dispersions and bioavailability
8145 Blazer Drive enhancement. Our global manufacturing sites are held to strict cGMP
Wilmington, DE 19808 standards ensuring consistent production of high-quality products.
Tel: +1 877 546-2782 Ashland meets formulators’ needs by providing an unparalleled range of
excipients and technologies, as well as longstanding polymer expertise
and technical support from benchtop to commercialization for oral solids,
liquids, topical and parenteral dosage forms.
Company Background Research taking place at Ashland is the foundation of technical solutions
A long and winding road has lead to a sharp focus on that will address drug delivery challenges in the future.
specialty chemicals.
Products, Services & Capabilities
What began as a small oil refinery in eastern Kentucky
just over 90 years ago has grown into Ashland, one of Ashland’s leading position in drug solubilization is underscored by its broad
the world’s leading specialty chemical companies. From network of technical support and laboratories down to the regional level.
the beginning, the company’s founder, Paul Blazer, The company operates pharmaceutical centers of excellence in Wilmington,
instilled in Ashland employees a passion for hard work, Delaware (USA) and Hyderabad, India, and regional supporting laboratories
integrity and results - qualities that endure today. in Düsseldorf, Germany; Istanbul, Turkey; São Paulo, Brazil; Buenos Aires,
Argentina and Shanghai, China. Products include:
Markets Served • Aqualon™ and Blanose™ sodium • Benecel™ HPMC custom grades
• Pharmaceutical carboxymethylcellulose (CMC) • Cavamax*, Cavitron™ and Cavasol*
• Nutraceutical/Dietary Supplements • Aqualon™ ethylcellulose (EC) cyclodextrins
• Animal Health • AquaSolve™ hypromellose • Klucel™ hydroxypropylcellulose (HPC)
• Personal Care/Skin Care acetate succinate (HPMCAS) • Natrosol™ hydroxyethylcellulose (HEC)
• Aquarius™ film coating systems • Plasdone™ povidone and copovidone
• Benecel™ DC HPMC • Polyplasdone™ crospovidone
• Benecel™ methylcellulose and • Pharmasolve™ N-methyl-2-pyrrolidone
hypromellose (HPMC)

To help further solve for pharmaceutical challenges, the Ashland team has a state-of-the-art pharmaceutical center of excellence in
Wilmington, DE. The facility, which primarily focuses on drug development and and bioavailability enhancement, expands Ashland’s global
network of pharmaceutical research and development centers. The facility also includes formulation development and supports early stage
clinical trials spray drying and extrusion processes.

www.americanpharmaceuticalreview.com | | 11
 Specializing in chromogenic and turbidimetric reagent technologies,
Associates of Cape Cod, Inc. (ACC) has been a leader in endotoxin detection
www.acciusa.com products and services for more than 40 years. ACC pioneered LAL testing
methodology and was the first FDA licensed company to manufacture
Associates of Cape Cod, Inc.
LAL reagents; ACC has grown to be an internationally recognized leader in
124 Bernard E. Saint Jean Drive endotoxin detection.
E. Falmouth, MA 02536
ACC also operates a Contract Test Services (CTS) Laboratory which has
Tel: 888-395-2221
specialized in testing for endotoxin contamination for over 30 years. Our
Email: [email protected]
CTS laboratory is GMP compliant, ISO registered and DEA licensed and is
capable of handling all controlled drug substances except those included in
Schedule 1. All testing services can be performed to FDA, USP, EP and/or JP
regulatory guidelines. In addition to routine testing, our CTS Laboratory will
customize endotoxin testing, troubleshoot difficult samples, develop and/
or transfer LAL test methods, design and produce custom depyrogenation
controls for oven validation and perform Low Endotoxin Recovery (LER)
studies/protocols.

ACC also offers a clinical diagnostics product line and operates a CLIA-
certified laboratory specializing in (1→3)-ß-D-glucan analysis to support the
diagnosis of Invasive Fungal Disease (IFD).

Our worldwide headquarters are located in East Falmouth,

Massachusetts. With a dedication to quality, ACC is
certified to I.S. EN ISO 13485:2012 and ISO 13485:2003.
We are FDA inspected and operate DEA Licensed and
CLIA-certified laboratories. Our endotoxin detection
reagents, instruments and software are used within
the Pharmaceutical, Medical-Device, Biotechnology,
Compounding Pharmacy and Dialysis industries for quality
control, product release and research. Our reagents are
FDA licensed and can be used for testing in compliance
with USP, EP and JP bacterial endotoxin test chapters, and
our software is 21 CFR Part 11 Compliant.

12 | | September/October 2017
 BASF creates chemistry for a sustainable future by offering intelligent

www.pharma.basf.com solutions to the pharmaceutical industry. With expertise in polymer

chemistry, R&D-capabilities and commitment to developing excipients, BASF
creates solutions for Instant & Modified Release, Solubilization, Softgels, Skin
BASF Corporation Delivery and Biologic applications. We are also a leading supplier of selected
100 Park Avenue APIs such as ibuprofen and omega-3.

Florham Park, NJ 07932 Our team of experienced industry specialists are here to support you in
Tel: 800-469-7541 developing effective, reliable solutions to the formulation challenges you
Email: [email protected] face today and tomorrow. At BASF, we know that innovation, speed-to-
market, and cost-effectiveness are crucial to pharmaceutical companies.
With expertise across the entire pharmaceutical value chain, we deliver on
all three accounts, from lab to launch.

Our Instant & Modified release solutions offer an unprecedented range of

functionality that can help you formulate pharmaceuticals with the exact
release properties you desire.

Our Skin Delivery platform provides a portfolio of excipients spanning a

wide range of solubility parameters for use in semi-solids, gels, liquids
and transdermal patches to increase drug penetration through the skin.
What’s more, our excipients are proven to be mild and non-irritating in
clinical studies.

Our Softgel portfolio offers leading-edge functional excipients to help

achieve the best possible results for each element of a softgel – whether
coating, shell, or fill. Moreover, all ingredients have been tested according
to the highest quality standards to minimize the possibility of crosslinking.

We offer a comprehensive range of innovative Solubilization polymers,

and have an unparalleled understanding of the corresponding process
technologies. This unique combination means that we can ensure you
achieve effective solubilization across a range of dosage forms – particularly
in solid dispersions. And because we are a pioneer in the application of hot-
melt extrusion technology in pharmaceutical production, we can help you
combine effectiveness with cost efficiency.

With over 50 years of experience in EO/PO chemistry, the anchor of our

Biologics platform is Kolliphor® P 188 Bio, which is specifically designed to
meet the stringent requirements of biologics manufacturers for purity,
consistency and performance in protecting cells from shear stress in
mammalian cell culture systems.

Rely on us to help solve your drug development challenges.

www.americanpharmaceuticalreview.com | | 13
 Company Description
bioMérieux is the worldwide leader in Microbiology - providing a
www.biomerieux-usa.com full range of solutions for microbiological control dedicated to the
pharmaceutical and biotechnology industries. We help optimize your
bioMérieux Industry lab so you can operate with greater efficiency, accuracy and confidence
from Concept to Care.
595 Anglum Road
Hazelwood, MO 63042 USA Company Background
Tel: (800) 634-7656
With its 50th anniversary in 2013, bioMérieux has celebrated a major
Fax: (314) 731-8678 milestone. Our development during these years has been driven by
a pioneering spirit and unrelenting commitment to improve public
health. We are present in more than 150 countries through 41
subsidiaries and a large network of distributors, providing diagnostic
solutions that improve patient health and ensure consumer safety.
In 2011, bioMérieux acquired AES-Chemunex, extending its solution
portfolio, through the Chemunex® cytometry range and the Labguard®
lab monitoring solutions.

Markets Served
Through constant innovation, we have established a worldwide
reputation as leader in Pharmaceutical microbiology. We propose
solutions for the environmental control of manufacturing plants (air,
surface, water), product sterility testing, identification, as well as non-
sterile product testing. Blood banks, tissue banks and biotechnology
companies also use our microbiology solutions. bioMérieux follows
quality assurance rules that are similar to those applied by the
pharmaceutical industry. Our manufacturing methods are based on
cGMPs (current Good Manufacturing Practices) and standardized
according to normalized procedures. Our manufacturing plants are
certified ISO 9001.

Products, Services & Capabilities

The Pharma Microbiology Pathway: Optimizing Concept to Care™ goes
beyond the automation of outdated processes. We take a comprehensive
look at your people, process and technology, identifying where we
can help reduce inefficiencies, minimize wasted effort and optimize
workflows, enabling your organization to accurately and quickly make
decisions with confidence. From environmental monitoring to sterility
testing, through rapid microbial detection (ScanRDI®, BacT/ALERT®),
QC strains (BioBall®, LyfoCults®), innovative media (LockSure®, 3P™),
identification in minutes (VITEK® MS), workflow optimization, validation
and training (bioMérieux Performance Solutions), bioMérieux offers an
adapted array of integrated solutions to improve efficiency from the lab
to manufacturing, in full compliance.

14 | | September/October 2017
 Liquid filling and processing

www.boschpackaging.com Bosch manufactures machines for sterile filling of liquid and powder
pharmaceuticals. Aside from filling and closing machines for ampoules, vials,

Bosch Packaging Technology bottles, sterile drop, spray solutions, cartridges and syringes, the product
range also contains barrier and inspection technology.
8700 Wyoming Ave. N.
Brooklyn Park, MN 55445 Oral solid dose
Bosch manufactures processing, filling and compression solutions for solid
Tel: 763-424-4700
dose pharmaceuticals and dietary supplements.

Terminal sterilization systems

Bosch SBM offers customized solutions for different applications and
processes for the sterilization of medical products and equipment.
Company Description
Medical device assembly systems
Bosch Packaging Technology – Business Unit Pharma is
The Bosch Moeller & Devicon portfolio contains single machines and
one of the leading providers of process technology and
complete lines for device assembly.
packaging solutions for the pharmaceutical industry.
Inspection and secondary packaging
The portfolio includes single units, complete lines and
For filled products, Bosch offers an extensive inspection product line that
integrated systems for the manufacturing and processing
includes visual inspection machines and automated systems. In addition,
of liquid and solid pharmaceuticals. It also includes process
track and trace, cartoning, and other secondary packaging solutions are
technology, primary packaging, inspection technology available to complete the process.
for different application fields and packaging types.
Customized solutions
Secondary packaging with qualification and validation,
With our special solutions team, you have one single point of contact for
software solutions for track and trace and after-sales all your needs. Backed by thorough pharmaceutical process expertise and
service are also available. long-term engineering competence, Bosch will develop a seamless solution.
The following product brands are part of the Bosch
Service
portfolio for the pharmaceutical industry: Hüttlin, The Bosch Packaging Services Inc. portfolio ranges from standard services
Klenzaids, Moeller&Devicon, Pharmatec, SBM Schoeller- to complete service packages. With modernization, spare parts, and field
Bleckmann Medizintechnik, Sigpack and Valicare. service, we focus on flexible solutions that improve packaging flexibility,
machine efficiency, and productivity.
Products, Services & Capabilities

Processing equipment for clean utilities

Bosch Pharmatech provides total solutions for the
production, storage, and distribution of pharmaceutical
clean utilities.

www.americanpharmaceuticalreview.com | | 15
 Markets Served
www.criver.com We lead the market with products
and services that meet the diverse
Charles River needs of the pharmaceutical, home,
251 Ballardvale Street beauty, dairy, beverage and food
Wilmington, MA 01887 industries. Our unique combination
Phone: 1.877.CRIVER.1 of Endosafe® endotoxin testing,
Celsis® rapid microbial detection and
Fax: 978‐988‐9236
Accugenix® microbial identification
Email: [email protected]
and strain typing keeps your
manufacturing operations running
Company Background efficiently and smoothly, lowers your
cost to manufacture and protects
With 65 facilities in 15 countries, Charles River offers a
your reputation.
wide range of products and services that span the entire
drug discovery and development continuum and can be Products, Services & Capabilities
tailored to specific research conditions. Our portfolio covers
four distinct phases: basic research, drug discovery, safety With a purposefully-designed portfolio and proven innovation, we offer QC
assessment and manufacturing support. testing solutions and methods that provide actionable data for consistent
product quality every time. Our products and services adhere to global
regulations, including EP, USP and JP documents. Many methods comply
with multiple regulatory requirements.
• Endosafe® endotoxin testing systems
• FDA-licensed LAL cartridges, reagents and accessories
• Endotoxin testing instrumentation and software
• Endotoxin contract testing services
• Celsis® microbial detection systems
• Accugenix® microbial identification and strain typing
Company Description products and services
Charles River is a global provider of comprehensive, • Data management solutions for environmental monitoring
innovative microbial QC product and testing solutions.
We can help you meet the necessary global regulatory
requirements to get to market and get your products to the
patients and consumers that need them the most. We offer
full-service solutions, including rapid endotoxin testing,
microbial detection and identification, and strain typing to
ensure the safe manufacture and timely release of product.
We are committed to providing precision, confidence
and robustness in QC testing and methods to help
manufacturers meet their standards for product safety
and customer confidence. Our investment in new product
research and development is matched by our high-quality
standards and dedication to client-focused problem solving.

16 | | September/October 2017
 Company Background
www.dfepharma.com Our roots are in dairy producing companies in both the Netherlands
and New Zealand. Companies that have a history of more than 100 years
DFE Pharma Find worldwide
sales offices at: have joined forces to become the world’s leading excipient manufacturer.
Klever Strasse 187 We are headquartered in Germany and have production locations in the
47568 Goch, Germany Netherlands, Germany, New Zealand and India.
Tel: +49 2823 9288 770
Email: [email protected] Markets Served
We serve the global pharmaceutical market, with dedicated colleagues at
our worldwide sales offices. Our customers are small to large pharmaceutical
Company Description companies, that we support with insights for formulation development and
reliable supply of excipients with relevant and differentiating functionality.
DFE Pharma is the global leader in excipient solutions.
We develop, produce and market excipients for oral solid
Products, Services & Capabilities
dose and dry powder inhalation. Our portfolio consists
of filler/binders (Lactose, Microcrystalline Cellulose, We are recognised for our quality, expertise and support. At DFE Pharma,
Starches), superdisintegrants (Croscarmellose Sodium excipient excellence is what guides us on our way to developing
and Sodium Starch Glycolate) and carriers for inhalation the best possible excipient solutions for all our customers worldwide.
(Lactose). Please visit www.dfepharma.com Today, tomorrow, always.

www.americanpharmaceuticalreview.com | | 17
 www.EurofinsLancasterLabs.com
Eurofins Lancaster Laboratories, Inc.
2425 New Holland Pike
Lancaster, PA 17601
Tel: 717-656-2300
Email: [email protected] Markets Serviced
We provide complete CMC Testing Services to support virtual and large
With a proven track record of providing quality testing bio/pharmaceutical companies and CMOs, including testing of all starting
services for the largest pharmaceutical and bio- materials, process intermediates, drug substances, finished products
pharmaceutical companies in the world, Eurofins Lancaster and manufacturing support through our broad technical expertise in
Laboratories is a global leader in bio/pharmaceutical Biochemistry, Molecular & Cell Biology, Virology, Chemistry and Microbiology.

laboratory services providing comprehensive, innovative

Comprehensive Testing Services
and timely solutions to streamline all of your CMC
• Method establishment, including method development,
testing requirements.
feasibility, optimization, cGMP qualification and validation,
As a member of Eurofins BioPharma Product Testing as well as verification of compendial methods
Group—the largest network of harmonized bio/ • Comprehensive stability and release programs for clinical
pharmaceutical GMP product testing laboratories and marketed products
worldwide—Eurofins Lancaster Laboratories provides • Complete biochemical and chemical characterization and
comprehensive laboratory services to support all microbial identification
functional areas of bio/pharmaceutical production. • Raw materials and excipient testing (USP/NF, EP, JP)
• Production and non-production cell banking, including
Facilities full characterization

With a global capacity of more than 1,000,000 square • Lot release/unprocessed bulk testing

feet, our network of GMP laboratories operates under • Process/facilities validation, including viral clearance,
residual impurities testing, extractables & leachables,
the same strict quality procedures, LIMS, and centralized
water testing, environmental monitoring, disinfectant
billing system across 28 locations worldwide to make
efficacy and on-site sample collection
working with any of our global operations seamless.
• Consulting/protocol writing
In addition to these laboratory locations, we also have
teams of scientists placed at more than 70 client facilities Major Product Innovations
throughout the North America and Europe through our
We offer the flexibility to manage your testing programs more efficiently
Professional Scientific Services® (PSS) insourcing program. through your choice of three unique service models, including standard Fee
We also provide secure 24-hour data access from all of our for Service, as well as our award-winning Professional Scientific Services®
laboratories via LabAccess.com. (PSS) insourcing solutions and Full-Time-Equivalent (FTE) service models.

18 | | September/October 2017
 Products, Services & Capabilities
www.icdd.com PDF-4/Organics 2018 - Material Identification Database

PDF-4/Organics provides comprehensive coverage as the world’s largest

International Centre for Diffraction Data X-ray powder diffraction database for organics and organometallics. The
12 Campus Boulevard PDF-4/Organics database is a highly targeted collection, with special focus
Newtown Square, PA 19073 on materials used in commercial and regulatory fields. It is designed to
T: 610.325.9814 solve difficult problems that are analyzed by powder diffraction analysis
Toll-free: 866.378.9331 (USA & Canada only) for a multitude of applications in the pharmaceutical, regulatory, specialty
F: 610.325.9823 chemical, biomaterials, and forensic fields.
E-mail: [email protected]
The PDF-4/Organics provides the best of both worlds by including single
crystal and powder diffraction data together in a single, edited, and
standardized database. We not only extract from the public literature
Company Description
like other databases, we add unique content by extracting patent data,
ICDD is a non-profit scientific organization dedicated to combining single crystal and powder references, adding common inorganics
collecting, editing, publishing, and distributing powder and polymers, and continuously adding targeted materials through grants
diffraction data for the identification of crystalline materials. and research proposals.
Our mission is to continue to be the world center for
quality diffraction and related data to meet the needs of
the technical community. We promote the application
of materials characterization methods in science and
technology by providing forums for the exchange of ideas
and information. We sponsor the Pharmaceutical Powder
X-ray Diffraction Symposium (PPXRD), Denver X-ray
Conference; its proceedings, Advances in X-ray Analysis
and the journal, Powder Diffraction. ICDD and its members
conduct workshops and clinics on materials characterization
at our headquarters in Newtown Square, Pennsylvania and
at X-ray analysis conferences around the world.

Markets Served
ICDD’s PDF-4/Organics database is designed for a
multitude of applications in the pharmaceutical, regulatory,
specialty chemical, biomaterials, and forensic fields. Its
design allows for easy interface with diffractometers
and data analysis systems of leading software
developers and manufacturers of X-ray equipment. www.icdd.com/products
The database is useful for scientists working in
consumer products, catalysis, forensic science, analytical
labs, drug discovery, and production.

www.americanpharmaceuticalreview.com | | 19
 Markets Served
www.malvernpanalytical.com Malvern Panalytical products and solutions for Discovery, Research &
Development, Process Control, and Product Quality Assurance are sold primarily
Malvern Panalytical into four market sectors: Pharma/Food/Bio, Metals/Mining/Minerals, Chemicals/
Petrochemicals, and Advanced Materials sector which includes many academic
117 Flanders Road and government research facilities around the world.
Westborough, MA 01581
Company Background
Tel: +1 508-647-1100
Malvern Panalytical was formed by the merger of Malvern Instruments (Malvern,
UK) and PANalytical (Almelo, The Netherlands) on 1st January 2017, and employs
over 2,000 people worldwide. With R&D and manufacturing sites in North
America, Europe and China and a global sales and service presence, Malvern
Panalytical provides unrivalled levels of customer support. Malvern Panalytical is
a strong player and innovator in the materials characterization market, providing
expert solutions for superior actionable insight.

Products, Services, and Capabilities

• Dynamic Light Scattering (DLS) – particle size and zeta potential [Zetasizer]
• X-ray Powder Diffraction (XRPD) – crystal phase/structure [Empyrean, Aeris]
• Gel Permeation Chromatography (GPC)/ Size Exclusion Chromatography
(SEC) – molecular weight, size and structure [OMNISEC]
Company Description • Image Analysis – particle size, shape and chemical ID [Morphologi G3]
Malvern Panalytical technologies are used by scientists and • Isothermal Titration Calorimetry (ITC) – protein binding affinity [MicroCal ITC]
engineers in a wide range of industries and organizations to • Laser Diffraction – particle size distribution [Mastersizer 3000]
solve the challenges associated with maximizing productivity,
• X-ray Fluorescence (XRF) – elemental analysis, USP<232>
developing better quality products and getting them to market
[Zetium, Epsilon 3XLE]
faster. Our mission is to create superior, customer-focused
solutions and services to deliver tangible economic impact • Nanoparticle Tracking Analysis (NTA) – nanoparticle size/concentration
through chemical, physical and structural analysis of materials. [NanoSight]
• Resonant Mass Measurement (RMM) – particle size and concentration
Underpinned by extensive industry knowledge and technical
[Archimedes]
and applications expertise, Malvern Panalytical instruments
help users better understand a wide variety of materials, • Rheometry – rheological properties and viscosity [Kinexus]
from proteins and polymers to metals and building materials. • Taylor Dispersion Analysis (TDA) – molecular size and relative viscosity
Malvern Panalytical is part of Spectris plc, the precision [Viscosizer TD]
instrumentation and controls company.
Our technologies enable the measurement of parameters
such as particle size, shape and zeta potential, biomolecular
interactions and stability, rheological properties, molecular
weight, elemental concentrations and crystallographic
structure. Highly reliable and robust characterization of
these properties is fundamental to predicting how a product
will behave during use, to optimizing its performance and
achieving manufacturing excellence.

20 | | September/October 2017
 Company Description
www.meggle-pharma.com MEGGLE Excipients & Technology is a leading lactose manufacturer for the global
pharmaceutical industry. MEGGLE provides supply chain security with manufacturing
facilities in Europe and North America, and offers a broad product portfolio, including
MEGGLE Group milled, sieved, spray-dried, and agglomerated α-lactose monohydrate, β-anhydrous
BG Excipients & Technology lactose, and DPI lactose grades.

MEGGLE USA: MEGGLE Excipients & Technology is a pioneer in co-processing technologies allowing
simple, yet robust formulation development and manufacture. By co-processing
[email protected] lactose with other excipients, MEGGLE has developed high performance excipients
Phone +1 844 4MEGGLE having unique qualities with applications in directly compressible immediate and
(+1 844 463 453) sustained release pharmaceutical solid dosage forms.

MEGGLE also possesses extensive knowledge in the manufacture of other excipient

MEGGLE OFFICE GERMANY:
products and provides contract manufacturing services to several well-known global
[email protected] excipient companies wanting to enhance their excipient performance and product quality.
Phone +49 8071 73 476
Company Background
Founded in 1887 as a small dairy in Wasserburg near Munich, Germany, MEGGLE is a
privately held, third generation, family owned business. With an emphasis on quality
and innovation, the company is a global leader and premiere manufacturer of dairy
products. The company is represented by more than 2,700 employees worldwide.

Markets Served
MEGGLE Excipients & Technologies serves the pharmaceutical and biotechnology
markets with a global network of offices and authorized agents. As an innovator in
co-processed technologies, MEGGLE also provides contract manufacturing services to
several other global excipient companies.

MEGGLE’s broad product portfolio, multiple manufacturing locations, technical

centers in major markets, and innovative technologies make MEGGLE the
preferred supplier and valued partner by large and small pharmaceutical product
manufacturers.

Products, Services & Capabilities

MEGGLE Excipients MEGGLE Excipients
& Technologies Products: & Technologies Services
• Lactose monohydrate • Spray drying
• Anhydrous lactose • Co-Processing
• Co-processed excipients • Agglomeration
- MicroceLac® • Product Customization
- Cellactose®
- StarLac®
- RetaLac®
- CombiLac®
• Lactose for inhalation
• Lactose for lyophilization and
parenteral applications
• Customized lactose products

www.americanpharmaceuticalreview.com | | 21
 Company Description
www.netzsch-grinding.com/pharma NETZSCH specializes in machinery for grinding and dispersion; we have been
an innovative technology leader in batch and continuous process equipment
NETZSCH Premier Technologies, LLC for wet and dry grinding and dispersion; lab size to complete custom
engineered systems.
125 Pickering Way
Exton, PA 19341 Company Background
Tel: (484)-879-2020 NETZSCH Premier Technologies, LLC, is the North American business unit
Fax: (610) 280-1299 of NETZSCH Feinmahltechnik GmbH. In business for 140 Years, NETZSCH has
E-mail: [email protected] been producing the highest quality machinery for grinding and dispersing,
Contact: Steve Miranda, Dir. of Sales 610-280-1220 meeting our global customers’ demands for engineered solutions to produce
products to exacting standards in all areas of the pharmaceutical industry.

Markets Served
While NETZSCH concentrates in the pharmaceutical and biotech industries, we
also engineer machinery for the food, coatings, cosmetics, inks, agro/chem and
battery industries.

Products, Services & Capabilities

The NETZSCH DeltaVita® has been specially designed to efficiently increase
solubility of API’s, thereby enhancing their efficacy. The DeltaVita® is completely
scalable and reproducible.

The versatile DeltaVita® is constructed of stainless steel or ceramic materials with

surface finishes to RA = 0.4μm for all product contact components. This machine
is designed for easy cleaning and sterilization. Adhering to strict guidelines, the
DeltaVita® is built in accordance with cGMP, GAMP, GAMP5, ASME, BPE, UL or CE
and FDA Guidelines.

With this machine, users can achieve consistent particle size distributions
below 100 nanometers to increase surface area, solubility, and bioavailability.
Nanometer-scale particles provide many advantages. Gene and vaccine delivery
become more targeted. Nanoparticles can enhance the properties of time
release molecules. Nanoreceptors can be added to drug surfaces to release drugs
exactly where needed. In addition, benefits of increased bioavailability may be a
more cost-efficient product with less risks and side effects for the patient.

The DeltaVita® 15-300 is designed with growth and flexibility in mind with
several available and interchangable chamber sizes in stainless steel and ceramic
construction. The Laboratory Series sizes range from 15 to 300 ml chamber
volume. The Pilot Series is the DeltaVita® 600. In the Production Series NETZSCH
offers models from 2000 to 60000 ml.

DeltaVita® systems are available in portable and permanently mounted

through wall installations. Each machine is designed specifically for your
process requirements.

22 | | September/October 2017
 Company Background
www.ssi.shimadzu.com Headquartered in Columbia, Maryland, SSI was established in 1975 as a
distribution center providing analytical solutions to laboratories in the
Americas. Today, SSI’s focus is expanded greatly beyond distribution. Steady
Shimadzu Scientific Instruments
and controlled growth has seen the opening of nine regional offices across
7102 Riverwood Drive the United States; Shimadzu U.S.A. Manufacturing, which supplies UV-Vis,
Columbia, MD 21046 HPLC, GCMS, and LCMS products to the U.S. market; and, most recently,
state-of-the-art Solution Center and Innovation Center laboratories.
Tel: 410-381-1227
Markets Served
With a diverse portfolio of instruments, Shimadzu’s products are used
Company Description in a wide range of industries, academia, and government. These include
Shimadzu Scientific Instruments (SSI) is the North American pharmaceuticals, biopharma, life sciences, clinical, environmental, chemicals,
subsidiary of Shimadzu Corp., headquartered in Kyoto, forensics, electronics, automotive, food and beverages, and material sciences.
Japan, a leading provider of analytical measurement and
testing instrumentation for a broad range of applications.
Shimadzu’s extensive portfolio of innovative high-quality
system platforms provides customers with unparalleled
solutions-based offerings and we encourage results-driven
collaborations that meet growing customer demands. With
a vast installed base and preferred vendor status at many
institutions, SSI’s instruments are used for quality control
and research across the globe by customers who can count
on the long-term stability, experience, and support only
Shimadzu offers. Products, Services, and Capabilities
Shimadzu’s instruments are used throughout the pharmaceutical pipeline, from
life science research and drug discovery and development to manufacturing
and QA/QC. Instruments include chromatographs (HPLC, UHPLC, SFC, GC),
mass spectrometers (LC-MS/MS, GC-MS/MS, MALDI-TOF), spectrophotometers
(UV-Vis, UV-Vis-NIR, FTIR), spectrofluorometers (fluorescence, luminescence),
atomic spectrometers (AA, ICP), X-ray fluorescence (EDXRF) spectrometers,
thermal & particle size analyzers, Total Organic Carbon analyzers, data
management (Workstation, DB, CS) systems, balances, and materials testers.
In addition to a wide array of instruments, Shimadzu actively pursues
collaborative research projects with customers and partners. Its new solution
center and innovation center enable SSI to more quickly develop new
software applications and focus-based solutions to better serve growing
customer demands.
Shimadzu also offers a Global Preferred Accounts Program. This program is
designed to ensure that our people, technologies and global capabilities
enable customers to achieve breakthrough results and operational efficiency
in pharmaceutical research and manufacturing.

www.americanpharmaceuticalreview.com | | 23
» MICROBIOLOGY »

Adoption of FMEA Abstract

In December 2014 the new chapter <1115> of the U.S. Pharmacopeial

for Microbiological Convention came into effect. The new chapter deals with micro-
biological contamination risk control for nonsterile product manu-
facturing. To follow the recommended structured risk analyses for

Contamination Risk microbiological contamination control, a firm rating system for the
three FMEA parameters severity, occurrence and detectability was
developed and applied over several nonsterile drug production areas.

Assessment to Implement The rating system provides an objective risk evaluation for every process
step. In a first step, an Ishikawa diagram was prepared and process
charts were created for each individual process. The single process

USP Chapter <1115> steps were then assessed using the FMEA approach. The application
of the FMEA provides a quantitative risk assessment and therefore
the advantage of risk prioritization. Limits were defined rationally to
classify process steps in risk categories and to determine whether
risk-reducing measures should be implemented. Furthermore, the use
of an Improvement Index and a Cost-Benefit Ratio of pre-evaluated
measures provide a good aid for decision-making in terms of which
measure should be implemented when reducing contamination risk
N. T. Gilles,1,3 E. Marty,1 D. Roesti,1 A. Staerk,1 while taking cost factors into consideration. The template developed
and M. Goverde2* here was used to assess the microbiological contamination risk for the
1
production of a highly active drug product.
Microbiological Quality Control Unit, Novartis Pharma Stein AG, Switzerland
2
MGP Consulting GmbH, Binningen, Switzerland
3
Current address: Miltenyi Biotec GmbH, Friedrich-Ebert-Straße 68, D-51429 Bergisch
Gladbach, Germany Introduction
*Corresponding author: MGP Consulting GmbH, Melchtalstrasse 21, CH-4102 Binningen,
Switzerland. Telephone: +41 79 820 85 66; Email: [email protected] In December 2014 a new chapter of the U.S. Pharmacopeial Convention
came into effect. USP chapter <1115> covers microbiological
contamination risk control for nonsterile product manufacturing. Risk-
based microbiological contamination management is demanded in
order to implement adequate controls in nonsterile drug production.
The risk to patients based on product attributes, route of administration
and target patient population should be considered as well as the
manufacturing process itself and materials affected.
USP chapter <1115> provides a list of nonsterile pharmaceutical
product categories ordered according to their risk of microbiological
contamination. Inhalants and nasal sprays are named as the highest
risk product categories. Oral tablets and powder-filled capsules hold
the lowest risk of microbiological contamination.
The main sources of microbiological contamination in nonsterile drug
products are water (as an ingredient), the pharmaceutical ingredients,

24 | | September/October 2017
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» MICROBIOLOGY »

the process equipment, the manufacturing personnel and the

manufacturing environment.
For nonsterile drug product manufacturing, USP chapter <1115>
recommends good microbiological quality of pharmaceutical
ingredients and excipients and the corresponding identification of
suitable suppliers. Furthermore a microbiological risk assessment of
the manufacturing process and packaging system should result in the
implementation of a risk based monitoring and control system. Also
the case-to-case identification of contamination risk for final drug
product and harm to patient should be included.
In the current article, the application of failure mode and effect Figure 1. Ishikawa diagram to summarize all the possible
analysis (FMEA) is presented to evaluate the microbiological factors that can affect the microbiological contamination risk
contamination risk for nonsterile manufacturing according to of a drug product.
USP <1115>. The aim was to develop a system that can be used
for different manufacturing processes and in particular to define
clear criteria to rate the different parameters objectively. The result When manufacturing drug products a major aspect of the
of this approach is presented herein and its application when contamination risk is human-related. So the first process chart
manufacturing a highly active film coated tablet. “Personnel” gives personnel-related contamination risk factors (Figure
2, violet process chart). In manufacturing areas the topics listed
are of importance. Training of personnel and their hygiene-related

Implementation of Microbiological behavior must be evaluated. This applies to gowning, i.e. what kind
of gowning material is used and the gowning procedure itself. Finally
Contamination Risk Assessment the type of tasks that a person executes (i.e. this relates to movement
and the risk of microbial contamination that increases as a result)
To implement a risk-based microbiological contamination control
and the number of people present also have an influence on the
program in nonsterile drug product manufacturing, the failure
mode and effect analysis (FMEA) approach was applied. The
considerable advantages of FMEA include that it is easy to carry out
and that the quantitative risk assessment of detailed steps allows
precise prioritization and pre-evaluation of contamination risk
mitigation measures.1

Preliminary Work (Ishikawa Diagram,

Process Charts)
Before evaluating the risk factors on the process with an FMEA it is
necessary to list extensively all potential microbiological contamination
risks. To this end, an Ishikawa diagram, invented by Kaoru Ishikawa, was
generated with the outcome titled: “Microbiological contamination
risk of drug product” and all the influencing factors collected from
brainstorming and research.2 In the Ishikawa diagram, factors like
Water, Facility, Lock in Process were sorted by the main areas “Personnel”,
“Production Process”, “Environment”, “Material & Equipment”, “Cleaning
& Disinfection” and “Airlocks & Room Concept” (Figure 1).
This structured overview of all the influencing factors is helpful to break
down the processes and factors into detailed steps. Process charts for
each factor help to clearly represent the process factors and therefore
provide an easy transition to the next stages,3 which is the FMEA in this
Figure 2. Summary of the process charts derived from the Ishikawa
case. In Figure 2 the process charts developed are summarized.
of Figure 1. Abbreviations: pharm. = pharmaceutical, C&D =
In the following paragraph, three different factors are described in Cleaning & Disinfection, Interm. = Intermediate
more detail as an example of the process charts.

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microbiological contamination risk for the manufacturing process. evaluated next. As a third step, detectability is assessed based on the
It must be mentioned that there is no overall hierarchical order for current controls.
the factors listed; therefore a risk analysis gives more information on
In FMEA the risk components severity, occurrence and detectability
individual risk.
are considered to quantify the risk of a specific failure and its effect.
Another important factor is “Cleaning & Disinfection”. Due to the The risk priority number RPN functions as quantification of the risk
different levels of interaction with the drug product, this item was and it is calculated by multiplying the severity SEV, occurrence OCC
broken down into cleaning and disinfection of equipment and cleaning and detectability DET values. There are plenty of variants, but in this
and disinfection of rooms. Because of the higher microbiological approach, single values are used as in Zimmermann 2011.4 The Risk
contamination risk involved, only “Cleaning & Disinfection of Priority Number is calculated by multiplying each of the three risk
Equipment” is further discussed (Figure 2, orange process chart). The factors: RPN = SEV*OCC*DET without additional factors.
most important aspect of the cleaning procedures of equipment and
The assessment and quantitative rating of the components increases
facilities is cleaning validation, which should show the reproducible
with higher risk relevance. In the present approach, values between 1
appropriate cleaning of the equipment. In Figure 2 the cleaning
and 10 were used for rating. It is challenging to rate risk components
process is separated into single steps which can then be assessed.
objectively without a fixed pattern. Therefore firm assessment tables
Finally, the reassembling of the cleaned equipment and its storage
on the basis of the main influences on the risk components were
or hold time can have an important effect on the microbiological
created to enable an objective and overall consistent assessment of
contamination risk and must be taken into consideration.
the process steps.
For “Production and Packaging”, every process step from dispensing
ingredients, mixing and process-related steps such as compressing, to
packaging steps are important for microbiological product quality (see
Figure 2, red process chart). In the overall risk assessment, intermediate
Calculation of Severity
storage or holding times, in-process control and campaign duration To start the FMEA, the severity of a potential failure is assessed. In
must be considered as additional factors. the case presented this calculation was broken down into two steps.
Because the patient risk of microbiological contamination in nonsterile
drug products is more differentiated than in sterile drugs,5 product-
Preparation of FMEA dependent factors must be considered. First, the risk for the product and
patient was calculated (Product & Patient Risk, Figure 4) by examining the
The actual preparation of the microbiological FMEA was performed three main characteristics of the drug product: water activity, which has
with a table template using Microsoft Excel software (Figure 3). a strong influence on the microbiological proliferation in drug product,
This table consists of nine basic columns for process step, potential dosage form and route of administration, which both have an important
failure, effect (influence of failure), severity SEV, potential root cause, impact on the risk of infection for the patient. The sum of these three
occurrence OCC, current control, detectability DET and risk priority values is formed and could be in the range from 0 to 6. This number is
number RPN. For each risk component (severity, occurrence and then integrated in the following severity assessment (Figure 5 and 6).
detectability), adding a column with enough space for a description Second, to assess severity, the potential failure was split into two factors:
of the component to provide better traceability of the assessment is Process Step (Figure 5) and Ingredients (Figure 6). For both assessments
recommended, especially if the FMEA is to be reviewed after some the Product & Patient Risk from table 1 that was previously rated (Figure
years or presented at an audit or inspection. For a better overview 4) is included in the first row. The second block represents the basic
it could be started with an additional column for the area of the assessment. For Process Steps the product contact is considered, for
individual process steps. Ingredients the amount of drug product. Finally, to get higher values
To start the FMEA, all the process steps defined from the process for a higher microbiological risk, influencing factors such as humidity of
charts are added to the Excel table. The potential failures are product/ingredient respectively of the process step or environment and
described for each process step (column “Potential failure”). The next other growth promoting properties can be added to the severity rating
step is to describe the influence(s) of the listed failures. Based on this, in the third block. The sum of all the values represents the SEV value,
the severity is assessed. To assess the risk component occurrence, which is entered in the FMEA table mentioned above. For values under
the potential root cause and its likelihood of occurring must be 1, the rating is set to 1 (lowest risk), for values over 10, the rating is set to
10 (highest risk).

Calculation of Occurrence (OCC) and

Figure 3. FMEA for risk assessment showing the different Detectability (DET)
columns. Abbreviations: SEV = Severity, OCC = Occurrence, DET
= Detectability, RPN = Risk Priority Number After defining the potential root cause, its likelihood of occurring is
rated under occurrence (OCC) with the assessment shown in Figure

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Figure 4. Assessment for Product & Patient Risk to be added to

the severity. Figure 5. Assessment for Severity for Process Steps.

7. As for the severity assessment (SEV), a basic assessment must also

be done for occurrence in the first block (Figure 8) that reflects the
probability of its occurrence. Similarly, additional contributing factors
such as human or technical errors are added and the final OCC rating
is calculated.
In the next step, the detectability (DET), i.e. probability to detect a
failure, is assessed with the assessment Figure 8 based on the current
controls. Again a basic and an additional assessment are performed.
The rating increases with a decreasing probability of detecting the
failure. The basic assessment reviews the product testing frequency,
while the additional factors increase the probability of detection and
therefore reduce the detectability value by subtraction.
For both risk components OCC and DET, similar to SEV, the rating is the
sum of all the values and must be between 1 and 10.

Risk Ranking by Risk Priority

Number (RPN)
As mentioned above, all the values calculated for SEV, OCC and DET
are added to the FMEA Excel table and the Risk Priority Number RPN
is calculated. To classify the risk by the RPN value, limits must be set. Figure 6. Assessment for Severity for Ingredients.
The present approach distinguishes between critical control points,
moderate critical and non-critical.

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Due to the multiplication of SEV, OCC and DET without additional

coefficients, the RPN range is between 1 and 1000. It was pointed
out by Shebl et al.6 that, especially for higher rates, not all values in
the ranking could be obtained. For example, the next lower value
to 1000 is 900. Thus there is no linear correlation of the values and
therefore an arithmetic mean cannot be used. A mean rating of all the
risk components (in the current case this would result in an RPN of 5
x 5 x 5 = 125) could be used but there are no regulations on the limit
definition.7 By increasing or decreasing one of the risk components by
1, the limits were set to 5 x 5 x 4 = 100 and 5 x 5 x 6 = 150 and an
appropriate classification was created (Figure 9).
According to the fragmentation described, all RPN are classified
as critical control point, moderate critical or non-critical. For critical
control points, where RPN is above or equal to 150, mitigation actions
will be undertaken and implemented. The aim is to reduce the risk of
all critical control points so that after implementation of actions the
RPNs are lower than 100, but maximum 149. RPN between 100 and
149 are classified as moderate critical. For all moderate critical steps
mitigation actions are recommended, but the prospectively analysis
of risk reduction and costs (see next section) is important to decide
for implementation or to accept risk without actions. In this case it
must be considered, that risk based monitoring activities are good
choices of mitigation actions concerning moderate critical steps by
decrease of detectability. RPN fewer than 100 are classified as non-
critical. In these cases no mitigation actions must be considered.
Figure 7. Assessment of Occurence. There is no need of risk reduction for non-critical steps. For some
non-critical steps it is even possible with prospective recalculation to
reduce monitoring activity.

Choice of Good Measures

As mentioned above, for critical control points risk-reducing measures
or actions must be initiated. To identify the potential of improvement,
a pre-evaluation of the planned measures by recalculating the RPN
after implementation is important. The documentation should be
entered directly into the FMEA table (Figure 10).

Figure 9. Summary of the three classes for evaluating the

criticality of the RPN (Risk Priority Number).

Figure 10. FMEA for the evaluation or risk reducing measures

in addition to the columns in Figure 3. Abbreviations: SEV =
Severity, OCC = Occurrence, DET = Detectability, RPN = Risk
Figure 8. Assessment of Detectability. Priority Number, Imp I = Improvement Index, CBR = Cost-
Benefit Ratio

32 | | September/October 2017
 « MICROBIOLOGY »

When choosing between measures, the Improvement Index (Imp I) is such as working time and costs. The value 1 would be a low cost, 2
helpful, which shows the improvement after implementation of the moderate and 3 high cost. It is possible to use a bigger range, but
planned measures.8 The following formula is used: Imp I = RPNbefore : in the approach presented here this easy range was effective when
RPNafter deciding between measures.

Based on the Improvement Index, the measure with the higher rate
should be preferred. In many cases it is not sufficient only to calculate
the Improvement Index for measures under consideration. By only Pilot Project for Implementation of
considering Improvement Index, the best measure would be nearly
aseptic production even for oral dosage forms. But it is neither in
FMEA in Nonsterile Drug Production
terms of patient nor of economic feasibility to initiate measures To implement the FMEA in nonsterile drug production, a pilot
where the ratio of the costs or investment is disproportionate to the project was chosen within a production area for a highly active film
improvement. Especially for moderate critical process steps or for coated tablet. Because of security priorities like negative pressure
measures under consideration that demand a substantial investment, of production rooms compared to the environment to keep the
the costs as opposed to the benefits must be considered. Therefore, in dust from active ingredients inside the area, there was conflict with
this application the Cost-Benefit Ratio (CBR) was calculated using the preventive measures for microbiological environment control such as
following formula: CBR = Imp I : Investment overpressure in the production area to the adjacent environment.
The Investment used was valued between 1 and 3 for an easy Using the FMEA approach described above, a total of 113 single
assessment and includes all the necessary investment factors, process steps were evaluated. On the basis of the quantitative

www.americanpharmaceuticalreview.com | | 33
» MICROBIOLOGY »

possible by classroom training for all production employees on

microbiological topics overall and especially about the disinfection of
surfaces in contact with the product. In addition a QA-Oversight was
implemented which increases the efficacy of the training and supports
continuous improvement. The Imp I, which at 2 was moderate, could
be compensated with very little effort with a value of 1. The resulting
CBR at 2 was at a good level.

Retain moderate critical process step without actions

or measures:
One of three moderate critical process steps, where the risk was
accepted and therefore no action or measure was initiated, was
weighing of the excipients. The excipients were sucked in with a
lance out of an open barrel. The microbiological contamination risk
was evaluated with a RPN of 100. The air in the production room was
already under microbiological monitoring; therefore a problem with
Figure 11. Overview of evaluated RPN (Risk Priority Number) for microbiological contamination of the air would lead to further product
the manufacturing of a high potent drug product. examination. Because this RPN is at the lower end of moderate critical
process steps, and because of the inappropriate high cost of risk
reducing measures (i.e. a high CBR was found), the risk was accepted.

evaluation of microbiological contamination risk, all the process steps

could be prioritized by their RPN. Overall 12 critical control points, 12
moderate critical and 89 non-critical process steps were identified Summary and Outlook
(Figure 11). After implementing appropriate risk-reducing measures,
In USP <1115> no method for microbiological contamination risk
all critical control points were reduced in risk and resulted in non-critical
analysis is dictated but the HACCP is suggested. Thanks to the
process steps. For 9 of the moderate critical process steps, risk-reducing
numerical ranking to support risk prioritization and the possibility
measures were implemented, for 3 moderate critical process steps no
to integrate the product and patient risk directly and easily into the
measures were initiated due to the inappropriately high investment
assessment, the FMEA approach was chosen instead of the suggested
for the potential improvement.
HACCP. As mentioned by Sandle7 and Shebl et al.,6 both methods are
To illustrate each of these three scenarios, one example is given: commonly used and have a lot of similarities (e.g. process charts, failure
analysis). In contrast to the HACCP, the FMEA has a clear numerical
Reduction of critical control point to non-critical ranking which supports the prioritization of failures.4

process step: For the present study, a highly objective and well-documented FMEA
approach was developed. After summarizing all the production steps,
No written procedure of disinfection was recorded for material
their severity, occurrence and detectability was calculated using
entering the production area. In line with the worst-case scenario that
clearly pre-defined assessment tables. With this – regardless of who is
incoming material is never disinfected, the RPN value was 320. The
performing the risk assessment or which production process is being
disinfection procedure was integrated in the SOP for the production
assessed – a very objective RPN number is calculated and prioritization
area and the personnel was trained accordingly. The implementation
of risk-reducing steps can be implemented.
of the measure resulted in a new RPN with a value of 64, simply by
reducing the possibility of the failure to occur and thereby reducing With the help of the FMEA presented here, the potential failures of
the OCC risk factor. With the Imp I value 5 and little effort (Value: 1), this process steps in a highly active production area could be tackled in a risk-
resulted in a CBR of 5. prioritized manner. Overall, a big improvement in the microbiological
contamination risk control in this pilot production area was achieved
and the sampling plans for microbiological environmental monitoring
Reduction of moderate critical to non-critical were adjusted based on the microbiological contamination risk values
process step: evaluated by the FMEA. By doing so, at least certain sampling points
A potential failure that was evaluated as moderate critical was could be reduced.
the inadequate disinfection of material in contact with the The resulting FMEA can be used to retrospectively analyze the
product, resulting in a risk of contamination through water-borne microbiological contamination risk in existing drug production areas or
microorganisms. This potential failure was assessed with a RPN value to evaluate the microbiological contamination risk of drug production
of 100. Thanks to a low effort measure, the RPN could be reduced areas in advance (e.g. design phase). This is also mentioned by Shebl
to 50 (non-critical), after a reduction in the OCC. This was made et al.,6 where the clear advantage of using FMEA in advance to reduce

34 | | September/October 2017
 « MICROBIOLOGY »

any later contamination risks can be seen. Furthermore, the system

developed here can easily be adapted for each nonsterile production Conclusion
process, especially since the process is highly objective, resulting in
the possibility of comparing different production processes or even Carrying out an FMEA is a very labor-intensive but also very objective
way to assess microbiological contamination risks. However, it must
different production sites.
be decided case by case which level of detail is to be addressed. In
Finally, applying a risk-based approach, forces the user to consider certain cases, e.g. where microbiological problems can be expected, a
the entire process at a high level of detail. This allows a much better higher degree of detail makes sense. Whereas in very easy processes
understanding of the process to be gained and further improvements (e.g. non-highly active, oral dosage forms with low water activity
can be implemented. In the present study, we found, among others, and antimicrobial process steps) a less detailed level can be chosen.
weak points in regard to personnel security that could be improved. The most important aspect for us was the objectivity of the rating.
Since an FMEA needs to be performed in a multidisciplinary team, the For this we developed assessment charts where the risk evaluation
common purpose brings with it many advantages such as better and for microbiological contaminations in nonsterile drug production
easier communication and better understanding of the problems and is objective for a consistent assessment of all the process steps over
point of view of the other field of activity. different production areas. By pre-evaluating risk-reducing measures,
the benefit can be calculated using the Imp I Improvement Index. The
In compliance with USP chapter <1115> after numeric evaluation Cost-Benefit Ratio CBR allows decision-making from an economic point
of microbiological contamination risk and prioritization, the FMEA of view. Finally the application of the FMEA shown here is very well
permits practicable management of risk-reducing actions or measures. documented and therefore the decisions taken can be followed even
The suggested actions or measures could be integrated in the after an interval of some years. Thus it provides complete traceability
assessment table and then by initiating the measures the reduction in and is especially suitable for inspection or audit presentations.
risk could be evaluated.

The developed firm assessment charts provide an objective evaluation

of the risk factors of potential failures and are applicable to all nonsterile Acknowledgments
drug production areas and processes. By defining RPN limits for critical
We thank Ylber Qusaj, Sascha Gasser and Alexander Schröder for their
control points, moderate critical and non-critical process steps, risk-
technical support, Ania Dardas for proofreading.
based conditions for measures were provided.

By evaluating the RPN with measures, before initiating the risk-

reducing measures and evaluating the costs involved, the Cost Benefit Conflict of Interest Declaration
Ratio (CBR) could be used for management of risk-reducing measures.
This proactive evaluation provides the possibility of the appropriate The authors declare that they have no competing interests.
choice of measures in the case of critical control points and furthermore
simplifies whether or not to initiate a measure in the case of moderate
critical process steps. In the example presented here (see example C References
above), the contamination risk during transfer of the excipient to the
1. ICH Q9, 2005. Harmonised Tripartite Guideline: Quality Risk Management. Available on
mixing process in the isolator could be reduced with new equipment. http://www.ich.org.
The equipment is expensive and qualification and validation require a
2. Wong, K. C., 2011. Using an Ishikawa diagram as a tool to assist memory and retrieval of
great deal of work. In this case, the financial investment outweighs the relevant medical cases from the medical literature. Journal of Medical Case Reports 5, 1-3.
microbial risk reduction, i.e. the CBR is low. 3. Carducci, A., Davini, G. and Ceccanti, S., 2013. The Application of Quality Risk Management
to the Bacterial Endotoxins Test: Use of Hazard Analysis and Critical Control Points. PDA
Although the FMEA approach for the evaluation of the microbiological
Journal of Pharmaceutical Science and Technology 67, 553-567.
risk of nonsterile production process presented here is very objective
4. Zimmermann, H. F., Hentschel, N., 2011. Proposal on How To Conduct a Biopharmaceutical
and detailed, it is also quite time consuming. The development of the Process Failure Mode and Effect Analysis (FMEA) as Risk Assessment Tool. PDA Journal of
different risk assessment charts (Figures 4 – 8) in particular needed a Pharmaceutical Science and Technology 65, 506-512.
lot of discussion. However, once the tables are developed they can 5. Guilfoyle, D.E., Friedman, R.L., Hughes, P.F. Hussong, D., Rosenberg, A.S., Brorson, K., 2013,
be used for different (maybe all) other processes. Furthermore, the Microbial Risk in Pharmaceutical Manufacturing and ICH Q9. PDA Journal of Pharmaceutical
Science and Technology 67, 79-80.
required level of detail should be defined before carrying out the
FMEA. For general risk evaluation of various production areas, it is 6. Shebl, N.A., F., Franklin, B.D., Barber, N., 2012. Failure mode and effects analysis outputs:
are they valid? BMC Health Services Research 12, 150.
recommended to combine similar process steps. For a retrospective
7. Sandle, T., 2006. Environmental Monitoring Risk Assessment. Journal of GXP Compliance
risk evaluation in a production area with microbiological problems, a
54-73.
higher level of detail could help to identify potential weak points and
8. Van Leeuwen, J.F., Nauta, M.J., de Kaste, D., Odekerken-Rombouts, Y.M.C.F., Oldenhof, M.T.,
improve the microbiological situation based on the individual risk of Vredenbregt, M.J., Barends, D.M., 2009. Risk analysis by FMEA as an element of analytical
the single process steps. validation. Journal of Pharmaceutical and Biomedical Analysis 50, 1085-1087.

www.americanpharmaceuticalreview.com | | 35
» MICROBIOLOGY »

Data Integrity Issues in Introduction

Microbial Testing
A lack of data integrity often is “just fraud,” says Howard Sklamberg,
FDA deputy commissioner for global regulatory operations and
policy. FDA relies on company information documenting adherence
to cGMPs, he explained at a July 2014 conference sponsored by the
Food and Drug Law Institute. Yet almost all recent warning letters
cite evidence of altered and falsified records. If data are “knowingly
incorrect, we take that very seriously,” Sklamberg stated, expressing
dismay that some manufacturers still fail to remedy record-keeping
problems despite repeated warnings from the agency. For Indian
Cheryl Platco1 and Tony Cundell2 pharmaceutical companies supplying generic drugs to the U. S. market
1
Pharmaceutical Microbiologist (Retired) this has meant import alerts excluding their drug products from the
2
Microbiological Consulting, LLC, Scarsdale, New York U.S. (Wechsler, 2014).

What is a Lack of Data Integrity

Many people in the pharmaceutical industry are confused by the
concept of data integrity. Data integrity is comprised of these following
broad actions to hide test failures and/or manufacturing deviations:

• Omission of data

• Errors in data recording

• Changing data

• Deleting data

• Destroying data

These actions may be both unintentional and intentional, representing

GMP violations that can have civil and criminal consequences to the
company and seriously damage the company’s business.

36 | | September/October 2017
 « MICROBIOLOGY »

Examples of a lack of data integrity in chemical testing include:

• Not documenting activities or failing to document activities WHO ARE THE
at the time performed (pre- or post-dating)
PEOPLE BEHIND THE SCIENCE?
• Testing and discarding failing data
• Testing and only reporting passing data.
• Fabricating data
• Using previously generated data
• Not following test procedures and sampling plans
• Missing, altered or raw data not captured on test report or
batch records
• Memorized or recorded data on loose pieces of paper
• Electronic records changed without an audit trial

Why Has Data Integrity Become a

Hot Topic?
Beginning in 2014 there was a marked increase in warning letters
to Indian drug manufacturers addressing data integrity as the FDA
uncovered problems during regulatory inspections (Unger, 2017). This
increase is illustrated in Table 1. They look like you. Like us.
Like bioMérieux employees and
customers. They ’re inquisitive
Is the Issue Limited to Indian and microbiologists who rely on their
Chinese Pharmaceutical Companies? experience and power ful tools to
find answers and solve problems.
In 2016, the FDA issued 39 warning letters citing a lack of data integrity
(Unger, 2017). The national distribution of warning letters was China They ’re people who are committed
13 (33%), India 9 (23%), U.S.A. 6 (5%), E.U. 6 (15%), Brazil 3(8%) and to keeping your lab running smoothly
Japan 2 (5%).
and ensuring that your products
The FDA completed a total of 5,615 GMP inspections of registered are safe.
drug and device establishments in FY 2015. Of these 4,055 (72%) were
domestic inspections and 1560 (28%) were foreign inspections. Based To learn more about the products
on the recent U.S. GAO-17-143 Report on the FDA’s Foreign Offices and our people are creating for you, visit
Drug Inspections in FY 2015 there were 842 foreign drug establishment thepeoplebehindthescience.com

Together, we are the people behind

Table 1. The Number of FDA Warning Letters Issued Addressing
Data Integrity the science.
Calendar Year Number of Warning Letters Most Frequently
addressing Data Integrity Cited Country
2008 3 None

2009 5 None

2010 5 China

2011 4 None

2012 6 None

2013 6 India

2014 10 India

2015 15 India

2016 39 China

www.americanpharmaceuticalreview.com | | 37
» MICROBIOLOGY »

inspections of which 527 (63%) were for GMP surveillance, 250 (30%) limitations. Microorganisms grow at different rates, different con-
for pre-approval and surveillance, 23 (3%) for preapproval only and ditions, evolve to different colony shapes, sizes, and colors that must
42 (5%) for cause. The report highlighted the high frequency that be accommodated. The addition of a high quality digital image of a
an establishment may have never been inspected. For example, in plate is helpful in terms of data integrity (verification of test results,
the FY 2017 catalog were 572 Indian establishments with 33% never data archival, data retrieval) and verification of the accuracy of results
inspected and 535 Chinese establishments with 45% never inspected. for the manual recording of colonies. Microbiologists may still have to
This suggests that Indian and Chinese drug manufacturing facilities override electronic results if spreading colonies, colonies embedded
will continue to be cited for data integrity issues into the foreseeable within other colonies in plate counts, or product precipitation or
future unless they aggressively address data integrity issues within flocculation in sterility tests makes interpretations by instrumentation
their companies. inaccurate or more challenging.

Data Integrity Directed Towards Risk Analysis

Microbiological Testing Many pharmaceutical companies are now evaluating data integrity
using standard risk assessment tools such as FMEA (Failure Mode and
Many of the cited data integrity issues are not associated with
Effects Analysis). In the opinion of the authors the tests which appear
microbiology but with chemical analyses, especially High-Performance
to be the most critical, subjective, and prone to data integrity issues
Liquid Chromatography (HPLC). The norm for microbiological testing is
are the sterility test, the gel clot Limulus Ambeocyte Lysate (LAL)
the visual inspection of media for the detection of microbial growth or
bacterial endotoxin assay and any microbial enumeration test which
the enumeration of microbial counts. Typically the results are recorded
involves counting microbial colonies which covers most of the other
on paper worksheets or more recently into electronic notebooks.
compendial tests. The sterility and LAL gel clot assays are subjective in
The data, but not always the original microbial cultures, are checked
their interpretations. The enumeration test accuracy depends on the
by a second person for completeness and the absence of recording
interpretation of discreet colony counts by the microbiologist reading
and arithmetical errors. The data are then entered into Laboratory
the plates.
Information Management Systems (LIMS) and approved by a laboratory
supervisor. These laboratory procedures place a high reliance on the Using risk assessment tools, the authors believe that data integrity
integrity, experience, and training of the individual analyst, the ability issues can be classified as high, medium and low risk processes.
of the peer reviewer or supervisory staff to detect poor data integrity, High Risk
and the culture of the company to set the highest standard. This type
• Analyst records incorrect data
of data could be susceptible to falsification. Microbial methods rarely
take advantage of modern analytical technologies. • No second opinion for subjective interpretation of test results

Contributing factors to reduced data integrity in the microbiology • Analyst performs wrong method or wrong sample preparation
laboratory are the subjectivity of scoring microbial growth in broth, • Missed critical data or incorrect information
analyst-to-analyst variability in counting colonies on plates, and the
• Falsification or lack of authentic data
inability to perform aseptic manipulations with the contempora-
neous recording of data. Often LIMS data fields for many microbial Medium Risk
measurements such as <10 CFU/g, Log 10 counts, and absence of • Real time data entry difficulties
specified microorganisms test results are lacking or inconsistent. For • Incomplete data entries
most microbiological tests, the absence of computer data collection,
• Chain of custody for samples issues
automatic calculations and generation of a finished report lack detail
and flexibility. • Delay in testing, times not documented

A broader discussion of the issue of data integrity in the area of Low Risk
microbiology could be useful in terms of setting industry and • Improper recording of non-critical data
regulatory expectations. Microbiological data has historically been
• Lack of real time entry of non-critical data (lot numbers,
evaluated and recorded manually by appropriately educated and
expiry dates, and recalibration dates.)
experienced microbiologists trained in the art of contamination
detection and colony counting. Microbiologists must use experience,
expertise and judgment for test interpretation, which may lead to
subjective and variable interpretation and documentation of test GMP Requirements with Respect to
results. While the use of rapid technology, digital image capture and Data Integrity
automated plate readers can remove most of the subjective data
interpretations and solve data integrity issues, the algorithms used Data integrity is addressed in 21 CFR 211.194 Laboratory Records where
to count colonies or detect contamination are subject to validation the GMP requirements are defined. Recently four guidance documents

38 | | September/October 2017
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The ATCC® Clinical Isolates Collection is an Features and Benefits

assembly of clinically relevant and extensively Each culture within the collection is provided with extensive
characterized antimicrobial-resistant strains characterization data, including minimum inhibitory concentration
with validated genotypic and phenotypic values and susceptibility profiles for targeted drugs, DNA sequence
activity against a variety of critical drug classes. information for antibiotic resistance genes and 16S rRNA genes, and
Browse the collection online at expanded levels of source metadata such as geography, collection
www.atcc.org/ClinicalIsolates. date, patient age and gender, and site of infection.

Beta-Lactam Resistance Combination Therapy Resistance

Enzymatic hydrolysis by beta lactamases The use of combination therapy
is a common method of resistance to offers an alternative method for the
beta-lactam antibiotics. Strains treatment of multidrug-resistant
within the ATCC® Clinical Isolates infections; here, two or more
Collection were evaluated for antibiotics, or an antibiotic and
beta-lactam resistance through an adjuvant, are combined in a
phenotypic testing against treatment regimen. We tested
various carbapenem, strains in the ATCC® Clinical
monobactam, and Isolates Collection against
cephalosporin antibiotics; three combination therapies:
here, strains demonstrated Amoxicillin-clavulanic acid,
varying levels of resistance or Piperacillin-tazobactam, and
susceptibility. Genotypic Trimethoprim-sulfamethoxazole.
characterization of the strains We found varying levels of
identified the presence of annotated antibiotic resistance and
genes predicted to encode Class A, B, C, susceptibility to
and D beta-lactamases, including, KPC, these combinations.
NDM, OXA, CTX-M, SHV, VIM, and ACT.

Aminoglycoside Resistance Colistin Resistance

Enzymatic modification is one of the most common mechanisms of Colistin is a decades-old drug that is commonly reserved
aminoglycoside resistance and can result in high-level resistance. Strains as a last-resort antibiotic for multidrug-resistant infections
within the collection were tested against the aminoglycoside Amikacin, caused by Gram-negative bacteria such as Pseudomonas
and were found to exhibit varying levels of susceptibility and resistance. aeruginosa and Acinetobacter baumannii. Representative
Genotypic analysis identified over 15 genes that were predicted to strains of these species within the ATCC® Clinical Isolates
function as aminoglycoside inactivation enzymes, including those Collection were evaluated against Colistin and were found
encoding various types of N-Acetyltransferase (AAC), O-Adenyltransferase to exhibit varying minimum inhibitory concentration levels
(ANT), and O-Phosphotransferase (APH) enzymes. conferring drug susceptibility.

www.atcc.org

www.americanpharmaceuticalreview.com | | 39
» MICROBIOLOGY »

addressing data integrity have been written by the U.K. Medicines & Critical deficiency (Impact to product with risk to patient health):
Healthcare Products Regulatory Agency (MHRA), Parenteral Drug
• Product failing to meet specification at release or within
Association (PDA), U.S. Federal Food and Drug Administration (FDA),
shelf life. Reporting of a “desired” result rather than an actual
and Pharmaceutical Inspection Co-operation Scheme (PIC/S). The
out of specification result when reporting of quality control
documents are:
(QC) tests, critical product or process parameters.
• 2015 MHRA GMP Data Integrity Definitions and Guidance
Major deficiency (Impact to product with no risk to patient health):
for Industry
• Data being misreported (e.g. original results “in specification,”
• 2016 PDA Elements of a Code of Conduct for Data Integrity
but altered to give a more favorable trend). Reporting of a
in the Pharmaceutical Industry
“desired” result rather than an actual out of specification
• 2016 Draft Guidance for Industry – Data Integrity and result when reporting of data, which does not relate to QC
Compliance with CGMP tests, critical product or process parameters. Failures arising
• 2016 PIC/S Guidance Good Practices for Data Management from poorly designed data capture systems (e.g. using Post-
and Integrity in Regulated GMP/GDP Environments its® to record information for later transcription) No impact
to product; evidence of widespread failure.
• Bad practices and poorly designed systems which may
Why These Documents result in opportunities for data integrity issues or loss of
traceability across a number of functional areas production,
Inadequately Deal with Data QA, QC, etc., though individually, each violation “has no
Integrity Issues in Microbiology direct impact to product quality”
Other (minor) deficiency (No impact to product with limited evidence
On April 14, 2016 the FDA published a Draft Guidance for Industry on
of failure):
data integrity that emphasized computer data management systems
but not specifically microbiological testing. The document defined • “Bad practice or poorly designed system, which results in
data integrity as to the completeness, consistency and accuracy of opportunities for data integrity issues or loss of traceability
data. They used the acronym ALCOA which stands for Attributable (A), in a discrete area. Limited failure in an otherwise
Legible (L), Contemporaneously recorded (C), Original or a true copy acceptable system”
(O) and Accurate (A).
On August 10, 2016, a draft PIC/S guidance document was published
to provide industry with a “consolidated, illustrative guidance on risk- Application of ALCOA
based control strategies”. The new guidance from PIC/S was applied Principles to Microbiological
on a 6-month trial basis by participating regulatory authorities. The
authors agree with the practical approach taken by PIC/S. Data Integrity Issues
The PIC/S guidance defines and deals with: Applying the ALCOA principles to microbiology testing may on the
• Data governance systems surface seem to be a common-sense approach to cGMP compliance.
However, after applying risk-based principles to the discreet tasks
• Organizational influences on successful data integrity
management related to microbiological testing, documentation can be difficult
and cumbersome. Approaches to applying the ALCOA principles can
• Specific data integrity principles and enablers
be minimal, moderate or extreme. Extreme approaches may coun-
• Data integrity considerations for paper-based and teract good documentation practices. A risk-based approach to the
computerized systems criticality and functionality of the task and its documentation should
• Integrity considerations for outsourced activities be used. Companies also need to decide if “data” includes ancillary
• Regulatory actions in response to data integrity findings items such as media, reagent lot numbers, and expiration dates, in-
strument numbers and their recalibration dates, and laminar flow
The guidance wisely states, “Management should aim to create a work hood numbers. Applying extreme approaches to ALCOA principles
environment (i.e., quality culture) that is transparent and open, one in for data integrity can result in a second person having to shadow
which personnel are encouraged to freely communicate failures and the microbiologist and witness every documented step. Microbial
mistakes, including potential data reliability issues, so that corrective tests also involve the use of aseptic techniques where real-time re-
and preventative actions can be taken.” cording of the tasks cannot be performed without jeopardizing the
The authors agree that the PIC/S draft guidance, unlike the other quality of the results. Manual documentation of hundreds of envi-
regulatory documents, details the various deficiencies linked to data ronmental monitoring, water, or enumerations plates are often batch
integrity failures that may have varying impacts on product quality read with “no growth detected” plates being separated from “growth
(i.e., a risk-based approach to a loss of data integrity): detected” plates. All documentation is batch recorded at the same

40 | | September/October 2017
 « MICROBIOLOGY »

time. Do the ALCOA principles require each plate read, documenta- plate readers and rapid technology instruments may come with
tion, and recording of a second person confirmation all have to oc- some logistical issues since one must assure that functions such as
cur at the same time? Do the principles require that a second person audit trails are turned on, and permissions for tasks appropriately
have to re-enumerate quantitative plates to confirm the accuracy of assigned in a manner that there is no conflict of interest. The ability
the colony forming units? The authors believe not. For microbiology to reproduce/retain the raw data in a readable form for archival and
testing, many tests are performed over days, even weeks. How do retrieval must also be achieved.
we document teamwork? Frequently multiple individuals work on
the same test, performing dilutions, culturing organisms, counting

Legible
plate colonies, and reading or interpreting results at different times
over a number of days? How are ALCOA principles applied? These
FDA 483 observations give an insight into the regulatory thinking
The second ALCOA principle is LEGIBLE. Companies have received
about data integrity as applied to microbiology. Microbiologists can
observations for obvious use of correction fluids, overwrites/
also evaluate how rapid technologies may be helpful in remediating
the problems. scribbles on handwritten data sheets, and not having appropriate
change controlling procedures in place. This includes access to and
use of signature stamps for electronic systems in lieu of handwritten

Attributable signatures. General data integrity principles have always mandated

that data must be readable throughout the data retention period
The first ALCOA principle is ATTRIBABLE. The data is linked to the and lifecycle, with permanent and identifiable changes using
person performing the action. Only that person may sign for the permanent ink, neatly, and following established date and time
work completed. An FDA 483 observation was given to a company formats. Electronic data has additional challenges. The rules apply
which read: “While multiple employees participate in a process to all raw data, meta data, and finished data reports which are stored
step that spans more than one shift, only the last person involved and backed up for archival and retrieval. Many computerized systems
is required to sign the batch record for completion of the activity.” store the raw data and use internal software to access, process,
Another observation read: “The inspection documented that all of and report the information. Computer systems and programs
your QC laboratory computerized instruments (redacted) were found
are constantly being updated. Therefore, the retired instrument
to be stand alone, and laboratory personnel demonstrated that they
programs may also need to be archived in a manner that raw data
can delete electronic raw data files from the local hard drive. Your
can be retrieved and reprocessed. Computer operating systems may
firm deleted multiple data files acquired in 2013 allegedly to clear
not be able to run the specialized software after many years. This
up hard drive space without creating backups. Your QC management
conundrum has yet to be resolved.
confirmed that there is no audit trail or other traceability in the
operating system to document the deletion activity. Furthermore,
your analysts do not have unique user names and passwords for the
computer and laboratory information systems; your QC analysts use a Contemporaneous
single shared user identifier and password to access and manipulate
multiple stand alone systems.” The third ALCOA principle is CONTEMPORANEOUS. For microbiologists,
this may pose the most challenging principle. These challenges include:
While these observations may not apply directly to microbiology
laboratories, there is a concern around the inability to attribute a • Signing/dating records at the time of activity completion
task or data set to a person or group of people. Often hundreds, even
• Back-dating or pre-dating data sheets
thousands of microbiology plates are read. There may be multiple
individuals all working together to complete the task spanning • Limiting access to change date/time of electronic data
more than one shift, or even more than one day. How granular and • Aseptic manipulations under laminar flow hoods or
frequent must the tasks be documented?
in isolators including the opportunity to document a
The requirements for data integrity were not intended to limit microbial test
laboratory flexibility or agility. Problems can arise when personnel
A recent 483 observation was …. “Specifically, an operator performed
must repeatedly log in and out of computers for each task completed
the in-process tablet (redacted) testing for the (redacted) mg tablet
to document individual contributions to the team. Shared electronic
batch #(redacted) without the batch record or a manufacturing form
notebooks can help, but care must be taken to disallow and/or
track overwrites and accidental omissions. In some ways, paper to document the results contemporaneously. …your operator stated
documentation is easier than electronic documentation. However, that he records the two weights with (redacted) significant figures
electronic documents can be templated, time stamped, and into the batch record (located in another room) from memory”. Do
changes tracked, which is a huge advantage. The advantage of tasks or data have to be recorded during microbial testing when
rapid technology automatic data capture readers is that they can activity occurs and how do we maintain aseptic technique while still
address many of the data integrity concerns. The use of automated complying with the requirements for data integrity?

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» MICROBIOLOGY »

the tests, your procedure needs to specify steps that enable objective
Original evaluation, such as assigning two operators for evaluation on the last
day.” This implies that the FDA expects a second microbiologist check
The fourth ALCOA principle is ORIGINAL. This can be difficult for all sterility test media at the end of the test time.
microbiologists. Many microbiologists write the plate counts on the
A third recent FDA 483 observation stated: “Not all laboratory data
plate. Many isolate zero count plates into one location to document
were recorded accordingly. On 5/25/2017 an FDA microbiologist
all negative or 0 CFUs later. The authors believe these are common
observed the following: a. “The firm’s microbiologist read the settling
practices, which allow the laboratory operations to remain productive
plate at location Vial Sealing Area Passive on 5/24/2017 and reported
and flexible.
“0” CFU. A FDA microbiologist observed 1 CFU for the same plate.”
Original data is data as the file or format in which is was generated,
The observation that the company reported no CFU and the FDA
preserving the integrity (accuracy, completeness, content and
investigator was finding 1 CFU is disconcerting and may be a serious
meaning) of the record
data integrity issue. The delay in examining the plates may have
• Retain all raw data and printouts been a factor - the plates were read 5/24/2017 and the inspection
• Original data must be recorded directly into GMP records conducted between 5/25 and 6/1/2017. Should one automatically
assume that there was falsification of the environmental monitoring
• Original data must be reviewed
results? The authors believe that only the CFUs reported within the
• Electronic raw data files must be retained recommended incubation time are valid. Since specifications are
A recent 483 observation stated: “Your firm repeatedly delayed, denied, for a defined incubation period, viewing colonies after the end of
limited an inspection or refused to permit the FDA inspection. An FDA the incubation may not be valid. It is possible that a slower growing
investigator identified the presence of torn raw data records in the colony was not visible at termination of the test. It is possible that one
waste area and asked one of your firm’s QA Officers to remove these opens the plates for a better view to read the plates. One should not
torn raw data records for the investigator’s review. This QA Officer automatically infer error or fraud for data which is acquired during a
presented the FDA investigator with approximately 20 paper records, set period of time and the analytes are known to be dynamic at the
none of which included raw data entries identified in the waste area end of a tests’ duration.
earlier during the inspection. The FDA investigator then revisited the
waste area and found that the raw data records had been removed and
placed in a different holding bag.” While this observation is an example Risk Mitigation
of a more egregious breach of data integrity, microbiologists struggle
with exactly what is original data. If plate counts are written on the How can the risk be mitigated with microbial test methods? What

plate and not recorded directly onto a report, are the plates raw data? should and should not industry do in response to data integrity
issues? The first step is to acknowledge the limitation of the classical
microbial test methods, (accuracy and precision), their dependency on

Accuracy the technical capabilities, honesty of the microbiologists conducting

the tests, and supervisors reviewing and approving the tests for
The fifth and last ALOCA principle is ACCURACY. This article addresses data integrity (Table 2). Ultimately the maintenance of data integrity
the Who, What, Where, When, Why and How data represents the is the joint responsibility of upper management, managerial and
complete set of factual data and meta data. supervisory staff, and bench-level microbiologists. The authors do not
advocate employing a second microbiologist to review all microbial
• Data must reflect the action/observation accurately
test results, but potential failures of critical tests should be reviewed
• All data must be controlled and retained for the lifecycle contemporaneously, if possible, by a second person. Test result reports
• Modifications to the data must be explained if not obvious should be reviewed by a superior for accuracy and completeness. Out-
of-specification results should be fully investigated. Unlike the opinion
• Meta data includes units of measure, method details, audit
of some FDA investigators, the authors believe that negative sterility
trail, date/time modifications
tests do not require a second person review.
Recent FDA 483 observations stated: “Processing each result file
individually using different processing parameters generates the
data for assay. The second person reviewer is not required to review Alternative Microbial Test Methods
each of the processing parameters used to generate results.” Another
observation is “Only one operator was assigned to evaluate sterility Are the risks different with alternative microbial test methods? Will
test on the last day of the incubation period. To ensure reliability of this difference drive the implementation of these newer methods?

42 | | September/October 2017
 « MICROBIOLOGY »

Table 2. Data Integrity Level of Classic Microbial Test Methods Alternative microbiological test methods usually depend on analytical
signals that are quantitative, more sensitive, and usually more reliable
Microbial Test Data Data Integrity Risk Mitigation
Generation Level than classical growth-based methods (Table 3). Many alternative
Microbial Colonies counted Moderate: Count Second person review; methods generate results in real time so can be described as rapid
Enumeration on a plate or subject to analyst use of automated plate
membrane; variability; readers with electronic
microbial methods (RMM). The signals generating results may not
turbidity in a Most susceptible to storage of the image be fully equivalent to colony-forming units counted in a traditional
Probable Number falsification;
(MPN) broth tube. impermanent record plate count. Analytical methods are typically objective, do not rely
Test for a Specified Characteristic Low to moderate: Second person review; the subjective scoring of colonies growing on a plate or turbidity of
Microorganism growth on Atypical growth electronic storage of
selective media pattern; subjective the image a broth, and can be captured electronic and archived which means
after enrichment interpretation; they have inherent data integrity. These characteristics of alternative
culture. presumptive
result that must microbial test methods should help promote their implementation
be confirmed
by microbial
in the pharmaceutical industry. However, many of the data integrity
identification. issues associated with chemical assays would likely be applicable to
Sterility Tests Presence of Low to moderate: Second person these newer microbial methods.
microbial growth Growth detected review; end point
in the liquid media as turbidity, determinations, e.g.,
surface pellicles, ATP determination,
floccular growth or gaseous exchange, etc.
precipitation
Concluding Statements on the Issue of
Microbial Probability of Moderate: Utilization Second person review
Identification
(Phenotypic)
identification
based on the
tests based in pH
and redox changes
Data Integrity
pattern of chemical
reaction The amount of recent regulatory activity in the area of data integrity
Antimicrobial Log reduction Moderate: Serial Second person review and the failure of four recently published guidance documents
Preservative of challenge dilution and plate
Effectiveness Test microorganism counts to address microbiological issues is a concern to pharmaceutical
at specified time microbiologists. The challenge to ensure data integrity in the
intervals
microbiology laboratory is unique. Computerized record keeping
Bacterial Gel Clot Visual Examination Moderate to Second person
Endotoxin Assay of gel formation high: subjective verification of results by is not as advanced as it is in chemistry laboratories. In most cases
interpretation of gel direct observation
formation contemporaneous data collection is not possible due to the necessity
Note: Second person review of test vessels (plates, canisters, tubes) should be limited to alert of using aseptic techniques or contamination control. Furthermore,
and action level results, out-of specification results or ambiguous results only.
the volume of plates to be read in support of environmental and water
monitoring makes second person review of all plates impractical.
The authors believe that the right response to this challenge is that
second person review be limited to alert and action level results, out-
Table 3. Data Integrity Level of Alternative Microbial Test Methods
of-specification results or ambiguous results only. Completed work
Microbial Test Technology Data Data Integrity
Generation Level sheets should be diligently reviewed by laboratory supervisors and
Microbial Automated Plate Colony Counting High laboratory test failures aggressively investigated. Ultimately, data
Enumeration Counter integrity can only be ensured by the maintenance of high ethical and
Test for a Specified RT-PCR DNA Extraction, PCR Moderate to High
professional standards throughout a pharmaceutical company.
Microorganism Amplification and
Detection

Sterility Tests ATP ATP level Moderate to High

Bioluminescence CO2 production and
Respiration O2 Consumption References
Detection of
Flow Cytometry fluorescent-labeled 1. FDA Draft Guidance for Industry – Data Integrity and Compliance with CGMP www.
Solid Phase cells
Cytometry fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/
UCM495891.pdf
Microbial MALDI-TOF Mass Riboprotein charge- High
Identification Spectrometry mass fingerprints 2. Wechsler, J. Data integrity key to GMP compliance. BioPharm International (September
(Phenotypic) 2014) www.biopharminternational.com/data-integrity-key-gmp-compliance
Microbial Vitamin Automated Zone Zones of inhibition High
and Antibiotic Readers on plates
3. Unger, B. 2017 An analysis of FDA warning letters on data governance and data integrity
Assays Pharmaceutical Online, Guest Column July 14, 2017 www.pharmaceuticalonline.com/doc/
Automated Turbidity in broth an-analysis-of-fda-warning-letters-on-data-governance-data-integrity
Turbidometers culture
4. U.S. Government Accountability Office GAO-17-143 Report on the FDA’s Foreign Offices and
Bacterial Endotoxin Kinetic Color change or High
Assay Turbidimetric or turbidity due to gel
Drug Inspections www.gao.gov/products/GAO-17-143
Chromogenic clotting 5. PIC/S Guidance Good Practices for Data Management and Integrity in Regulated GMP/GDP
Methods
Environments 2016 www.picscheme.org/en/publications

www.americanpharmaceuticalreview.com | | 43
» FORMULATION AND DEVELOPMENT »

Selecting In Vitro Dissolution Tests for Bioavailability

Enhancing Oral Formulations
Michael Grass, PhD
Senior Research Chemist
Lonza Pharma & Biotech

Bioavailability enhancing (BAE) technologies for oral drug delivery failure mechanisms of the formulations. This approach will maximize
may be designed to overcome many different hurdles to absorption. the likelihood of developing a successful formulation.
The critical aspect(s) limiting systemic exposure may include one or
The first step in designing a biorelevant dissolution test is to
multiple rate-limiting steps that formulation may address. These
determine the problem statement to be addressed. For BCS II and
may include (1) solubility, (2) dissolution rate; (3) precipitation of
IV drug products, the most common problem statements can be
drug in the gastrointestinal (GI) tract; (4) diffusion of drug through
categorized as relating to precipitation, dissolution rate, or solubility-
the aqueous boundary layer (ABL); (5) permeation across epithelial
permeability limited absorption. Within each of these categories,
cells; (6) efflux; (7) API chemical degradation in the GI tract; (8) and
there are several sub-classes. A simplified strategy for selecting a
drug-drug interactions. The specific rate limiting steps for absorption
dissolution test is shown in Figure 1. In reality, the design process is
depend on both the drug product and the individual drug substance.
more complex – each API and formulation is unique and a dissolution
Several classification systems have been proposed to aid in binning test should be designed to capture its individual critical performance
compounds for guiding both formulation and testing methodology. attributes. The goal of a formulator should be to select the simplest
The most commonly referenced system is the biopharmaceutics dissolution test that predicts relative in vivo performance of the
classification system (BCS), dividing compounds based on solubility formulations being assessed.
and permeability. The BCS II and IV classes have been further
Many BAE formulations are designed to lead to supersaturation in GI
divided into subclasses of acidic, basic, and neutral compounds to
fluids. While supersaturation can lead to improved absorption, it also
aid in the selection of in vitro methodology and parameters.1 Other
leads to the possibility of precipitation. A simple non-sink dissolution
classification systems are also useful for determining the rate limiting
test can often determine if there is a potential for an API to precipitate on
step(s) to absorption.
a biologically relevant timescale. If there is a potential for precipitation,
The fraction absorbed classification system (FaCS)2 distinguishes the mechanism and critical factors affecting precipitation inform the
cases where absorption is limited by dissolution rate, permeability, type and complexity of the dissolution test to use.
or the combination of solubility and permeability. Permeability
Depending on the API and excipients utilized, precipitation may
limitations are further categorized as being limited across the ABL
occur in either the stomach or the intestine. Additionally, intestinal
or the epithelial membrane. These classifications have been applied
precipitation may be dependent on exposure to gastric fluid with
to drug substances as well as drug product properties as a way to
sensitivity to the pH of the gastric fluid and the exposure time. In
predict food effects, identify enabling technologies, and determine
these cases, a non-sink transfer dissolution test should be utilized.
bioequivalence strategies.
The simplest transfer test involves first adding gastric media to
Formulators have many technologies to select from in order to solve the formulation and mixing for a set period of time. Subsequently,
BAE challenges. Selecting an effective technology requires matching a concentrated “intestinal buffer” is added to raise the pH of the
the problem statement for the active pharmaceutical ingredient (API) final mixture to a physiological intestinal pH (5 – 7) and the bile salt
with an appropriate formulation strategy. The problem statement is concentration to a desired level. These two-stage dissolution tests
described by the hurdles to absorption listed above together with are simple and can be performed in a USP dissolution apparatus, but
the required dosage form, chemical stability, cost, and other issues do not capture physiologically relevant transfer of material from the
defining the target product profile. Common BAE technologies stomach to the intestine.
include salt forms, micronization, amorphous formulations, cocrystals,
A more involved option for transfer tests is a multi-compartment
and lipid based formulations.
or controlled transfer dissolution test. The artificial stomach and
Once an appropriate strategy has been identified, it is important to duodenum (ASD),3 gastrointestinal simulator (GIS),4 and others involve
test formulations using in vitro methods that indicate performance first adding the formulation to a “gastric compartment” and then the
related to the in vivo rate limiting step(s) to absorption and/or likely apparatus transfers fluids and solids at a controlled feed rate from

44 | | September/October 2017
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impact of dissolution rate on absorption can be assessed by modelling

dissolution in conjunction with in vivo processes such as gastric
emptying, intestinal transit, GI hydrodynamics, and absorption rate.
When dissolution rate in the duodenum is critical and it is desirable to
compare dissolution rate relative to gastric emptying time, an ASD-like
test can be utilized. In such a test, it is clear when dissolution is slower
than gastric emptying and the impact on intestinal concentration
can be measured. Another option for comparing the dissolution rate
of formulations after gastric exposure, is to perform a two-stage test
where the second stage of the dissolution test is a sink condition.
An alternative method for assessing dissolution rate is the use of
a dissolution/permeation test. The key parameters for designing a
permeation test that is sensitive to dissolution rate have been discussed
in detail. One advantage of this approach is when dissolution rate
changes over time in intestinal media due to aggregation, Ostwald
Figure 1. Problem statement driven selection of in vitro tests for ripening, or use of controlled release formulations. With a dissolution/
BCS II and IV drug products permeation test, these changes can be monitored over physiological
time scales.9
the gastric compartment to a “duodenal compartment.” Many of For solubility-permeability limited absorption, dissolution tests with
these tests include gastric and bile secretion to mimic physiological a permeation step are ideally suited for formulation assessment.
conditions along the GI tract. In Figure 1, all of these tests are termed Depending on the API, the formulation, and the test setup, permeation
“ASD.” These tests typically provide more insight into precipitation may be limited by either ABL diffusion or diffusion across the
mechanisms than a two-stage transfer test. membrane or cell monolayer. This type of test is an excellent choice
for formulation assessment when designed to match the rate limiting
In both the two-stage and controlled transfer tests, the tendency to
step of the test with the rate limiting step known (or predicted) in
crystallize is a “worst case scenario” because API is not being removing
vivo. When membrane diffusion is limiting, drug activity determines
by permeation as it would in vitro. When permeation is slow these tests
the flux of drug across the membrane. When ABL diffusion is limiting,
can be discriminating for resistance to precipitation and sustainment
all rapidly diffusing drug species (e.g. neutral drug, charged drug,
of permeable API. When permeation is known to be rapid, however, it
micelle-bound drug, and nanoparticles) may impact flux.
may be appropriate to use a dissolution test with a permeation step.
By way of example, itraconazole (ITZ) is a very low solubility, highly
Dissolution tests with a permeation step include biphasic dissolution
permeable (BCS II) antifungal which is available commercially as an
tests where the simulated GI media is in contact with a water-
amorphous spray layered dispersion, Sporanox®. In a recent publication,
insoluble organic phase,5 membrane flux tests incorporating a
we explored SDDs of itraconazole that form varying concentrations
lipid-filled membrane (membrane is impermeable to micelles, of drug-rich colloids (ca. 150 nm) and compared their performance to
inclusion complexes, etc.),6 and dissolution/permeation systems Sporanox® both in vitro and in vivo.10 ITZ is solubility- ABL permeability
that incorporate Caco-2 or other cell monolayers to separate the limited (SL -U) according to the FaCS for both the crystalline and
dissolution and permeation compartments.7 By using a dissolution amorphous forms of the drug (Table 1). For SL-U compounds,
test with a permeation step, API is constantly removed from the formulations which increase the total solubilized drug (through
system and the degree of supersaturation may be lowered, reducing formation of micelles, colloids, nanoparticles, etc.) are predicted to
the tendency to crystallize. improve oral bioavailability by increasing diffusion across the ABL.
Lipid formulations contain digestible excipients (i.e. triglycerides Following the guidance of Figure 1, a dissolution/permeation assay
and diglycerides). Such formulations should therefore be evaluated was used to compare spray dried dispersions (SDDs) of ITZ. All three
using dissolution tests that include enzymatic digestion of the lipidic SDD formulations produced drug-rich colloids and the permeation
components to assess the effect of digestion on precipitation of rate in the in vitro assay increased with an increasing concentration
supersaturated drug. The digestion products of lipid formulations of drug-rich colloids. In order to predict the performance in an in vivo
often have a large impact on the degree of drug solubilization and rat study, a pH 6.5 buffer with a high concentration (27 mM) of bile
therefore precipitation and absorption.8 salt was selected as the donor media. The acceptor phase media (2%
When dissolution is the rate limiting step to absorption, the impact SLS) was selected to provide much higher solubility than the donor
of formulation and process parameters on dissolution rate is key media and the two compartments were separated by a lipid-filled
to an in vitro dissolution testing strategy. The simplest method for membrane. The apparent concentration of ITZ reached for the SDDs
assessing dissolution rate is a sink dissolution test. Sink dissolution was 2 – 20x higher than that of the control formulation. The resulting
tests are performed such that the rate of dissolution is insensitive to flux was 1.5 – 2.2x higher than for the control formulation (Figure 2).
the concentration of dissolved API in the media. Sink conditions are The measured in vitro flux increased with increasing concentration
often not biorelevant for BCS II or IV compounds, but the data are of drug-rich colloids. The in vitro flux was an excellent predictor of
valuable as input to mechanistic modeling software. For example, the relative absorption rate in rats (Figure 3).

46 | | September/October 2017
Automated UPLC
injection for dissolution
Efficient. Resolved. Traceable.
TM
Xtend on-line dissolution with the ACQUITY UPLC
H-Class from Waters.
The SOTAX SAM Sample Manager is more than the next generation of HPLC
injection for dissolution. In combination with the Waters ACQUITY UPLC H-Class,
it becomes more capable than anything that has come before. SAM acts as a plat-
form for off-line collection, on-line injection, and sample archival. On-line UPLC
is an ideal complement for both semi- and fully automated dissolution systems.

www.sotax.com/hplc
» FORMULATION AND DEVELOPMENT »

Table 1. Properties and classification of crystalline and amorphous This example demonstrates one case where careful selection of the in
itraconazole (200 mg dose). vitro test and the conditions of the test allows for in vivo prediction
Property Crystalline Amorphous beyond simple rank ordering of formulations. Utilizing relatively
Aqueous Solubility, pH 6.5 (µg/mL) 4 x 10-4 0.07
simple in vivo predictive dissolution tests early in development can aid
in selecting an optimal formulation and balancing the bioperformance
FaSSIF Solubility (µg/mL) 0.09 6.5
of formulations with other critical attributes of the drug product such
Dose Number, D0 9000 120
as stability and manufacturability.
Permeation Number, Pn 5.8

Dissolution Number, Dn 0.002 - 0.2 1 - 20 Selection of a proper in vitro test must be based on an understanding
FaCS Solubility – ABL Permeability Limited (SL-U)
of the hurdles to in vivo absorption. In fast-paced formulation
development labs it can be tempting to develop “one size fits all” tests
BCS BCS II
for formulations. Such an approach, however, can be misleading in an
environment where multiple compounds each with unique properties
are under development. By following the guidance outlined here, it is
possible to test formulations using in vivo predictive dissolution tests
in a streamlined fashion while individually addressing the variety of
problem statements presented by a diverse portfolio of compounds.

References
1. Tsume, Y., Mudie, D. M., Langguth, P., Amidon, G. E. & Amidon, G. L. The Biopharmaceutics
Classification System: Subclasses for in vivo predictive dissolution (IPD) methodology and
IVIVC. Eur. J. Pharm. Sci. 57, 152–163 (2014).
2. Sugano, K. & Terada, K. Rate- and Extent-Limiting Factors of Oral Drug Absorption: Theory
and Applications. J. Pharm. Sci. 104, 2777–2788 (2015).
3. Polster, C. S., Atassi, F., Wu, S. J. & Sperry, D. C. Use of artificial stomach-duodenum model
for investigation of dosing fluid effect on clinical trial variability. Mol. Pharm. 7, 1533–
1538 (2010).
4. Matsui, K., Tsume, Y., Takeuchi, S., Searls, A. & Amidon, G. L. Utilization of gastrointestinal
simulator, an in vivo predictive dissolution methodology, coupled with computational
approach to forecast oral absorption of dipyridamole. Mol. Pharm. 14, 1181–1189 (2017).
5. Shi, Y. et al. Assessing Supersaturation and Its Impact on In Vivo Bioavailability of a Low-
Solubility Compound ABT-072 With a Dual pH, Two-Phase Dissolution Method. J. Pharm.
Sci. 105, 2886–2895 (2016).
6. Stewart, A. M. et al. Development of a Biorelevant, Material-Sparing Membrane Flux Test
for Rapid Screening of Bioavailability-Enhancing Drug Product Formulations. Mol. Pharm.
14, 2032–2046 (2017).
Figure 2. Donor (top) and receiver (bottom) concentration
of itraconazole for 3 SDD formulations and the commercial 7. Kataoka, M. et al. Application of Dissolution/Permeation System for Evaluation of
spray layered dispersion, Sporanox® in a dissolution/ Formulation Effect on Oral Absorption of Poorly Water-Soluble Drugs in Drug Development.
permeation experiment. Pharm. Res. 29, 1485–1494 (2012).
8. Williams, H. D. et al. Toward the establishment of standardized in vitro tests for lipid-based
formulations, part 3: Understanding supersaturation versus precipitation potential during
the in vitro digestion of type I, II, IIIA, IIIB and IV lipid-based formulations. Pharm. Res. 30,
3059–3076 (2013).
9. Mudie, D. M. et al. Mechanistic analysis of solute transport in an in vitro physiological two-
phase dissolution apparatus. Biopharm. Drug Dispos. 32, 378–402 (2012).
10. Stewart, A. M. et al. Impact of Drug-Rich Colloids of Itraconazole and HPMCAS on Membrane
Flux in Vitro and Oral Bioavailability in Rats. Mol. Pharm. 14, 2437–2449 (2017).

Author Biography
Michael Grass is a Senior Research Chemist at Lonza Pharma
& Biotech where he develops methods, conducts testing,
and analyzes data for a wide range of pharmaceutical
Figure 3. Relative In vivo vs. in vitro absorption rate of three SDD
formulations relative to the commercial Sporanox® formulation formulations. His expertise includes formulation development
of challenging compounds, dissolution testing, and physical stability of
amorphous dispersions.

48 | | September/October 2017
Whitepaper Review
A collection of technological whitepapers
from American Pharmaceutical Review

www.americanpharmaceuticalreview.com | | 49
» SECTION TITLE
WHITEPAPER »

A Message from the Editor

Michael Auerbach
Editor in Chief

In this issue of American Pharmaceutical Review, we present our Whitepaper Review. This section is a collection of
whitepapers from a variety of pharmaceutical equipment and technology providers published to help you make the
best decisions regarding your future capital expenditures.
Pharmaceutical equipment and service vendors are at the forefront of developing and testing technology to make
drug manufacturing more efficient, less expensive and with the highest possible quality.
Pharmaceutical manufacturers always stress how important the knowledge, skill, and support provided by these
companies is to their overall success.
Please take a look at the following technology providers. I’m sure you will be able to find a trusted partner to further
your business goals.

Mke Auerbach
Editor-In-Chief
[email protected]

Table of Contents
51 Analysis of Two Active Pharmaceutical Ingredients (API) Products
Using UV Spectrophotometry with Multi-Component Analysis and
a Fiber Optic Dissolution Analyzer
64 Determination of Stress Conditions for Microorganisms for use in
Validation of Milliflex Rapid Detection System
Lamin Jallow, Microbiology Application Scientist, MilliporeSigma
A. Kielt,* I. Nir, J. Seely, and G. Inman, Distek, Inc. North Brunswick, New Jersey

54 USP <661> compliance for Total Organic Carbon (TOC) Analysis 66 Applicability of the LyoCapsule Mini Freeze Dryer for Pharmaceutical
Product Formulation and Process Development
GE Water and Process Technologies Emily Gong and William Kessler, Physical Sciences, Inc.

56 Steps to Ensuring a Successful Audit: Effective Risk Assessment

Design 68 7 Key Factors When Considering a Benchtop X-Ray Diffractometer
Katherine Macchiarola, Malvern Panalytical
Gilberto Dalmaso, PhD, Particle Measuring Systems

58 Extractable and Leachables Studies: Designed and Performed to

Meet All Intended Needs
70 4 Factors Critical to the Financial Success of an Emerging
Pharmaceutical Company
Gilberto Dalmaso, PhD, Particle Measuring Systems
Thomas Lehman, PhD, Eurofins Lancaster Laboratories

60 Selecting Microspheres to Calibrate and Assess Online Water

Bioburden Analyzer System Performance
72 Expansion of Human Bone Marrow-Derived Mesenchymal Stem
Cells in BioBLU 0.3c Single-Use Bioreactors
Vincent Dufey, Aurélie Tacheny, Muriel Art, Ulrike Becken, Françoise De Longueville
Allison Scott, Ph.D., Azbil North America Research and Development, Inc. - BioVigilant Eppendorf AG Bioprocess Center, Juelich, Germany

50 | | September/October 2017
 WHITEPAPER »

Analysis of Two Active Pharmaceutical

Ingredients (API) Products Using
UV Spectrophotometry with
Multi-Component Analysis and a
Fiber Optic Dissolution Analyzer
A. Kielt,* I. Nir, J. Seely, and G. Inman
Distek, Inc. North Brunswick, New Jersey USA

example is given of accurately monitoring and quantifying the

Introduction dissolution profile of an actual commercial product containing two
APIs, demonstrating the elimination of the need to draw samples or to
UV Spectrophotometry has traditionally been a simpler and less perform HPLC analysis for many of these type of products.
time and labor consuming method for analyzing dissolution testing
In the case of dissolution, Classical Least Squares analysis involves the
samples. However, as soon as a product contained more than one
application of Multiple Linear Regression to the classical expression of
active pharmaceutical ingredient (API), analysis with UV was no longer
the Beer’s law. Since complete UV spectra are measured, Beer’s law can
considered an option. This is because both species often absorb over be expanded to incorporate absorbance of multiple components at
the same spectral region, causing deviations from Beer-Lambert Law. different wavelengths, λ:
This linear relation between absorbance and the absorbing species is
the basis for calculating concentration values based on the measured
absorbance at a specific wavelength. In these cases, separation (1)
techniques such as HPLC become the de facto analysis methods.
While it has been long shown that using Multicomponent Analysis Where:
(MCA) software and complete spectral and temporal profiles make it
possible to analyze such products using UV, the drawback has always A λ = Absorbance of the mixture of p components at wavelength λ
been the difficulty in acquiring all the required data. However, with a E λ j = Response sensitivity factor (molar absorptivity × probe path
modern fiber optic UV dissolution analyzer, these obstacles have been length) of component j at wavelength λ
removed. Analysis of the two spectrally overlapping components Cj = Concentration of component j in the mixture
is accomplished by applying the Classical Least Squares form of
Multiple Linear Regression to the complete spectral and temporal However, interactions between components including excipient
materials also need to adequately represented. This leads to the need
profiles obtained using these new analyzers. The algorithm uses a
to expand the simple equation above into a more complex matrix:
calibration matrix of extinction coefficients to calculate component
concentrations in an unknown mixture. These are derived from a
training set comprised of the spectra of multiple standard solutions. (2)
This white paper explains the theory behind the MCA algorithm
methodology. Then, used in tandem with in-situ fiber optics, the
Where:
accuracy of the technique is demonstrated by recovering the
concentration of two APIs in known mixed solutions. Finally, an A = Matrix of absorbance values for the calibration solutions

www.americanpharmaceuticalreview.com | | 51
» WHITEPAPER

K = Matrix of sensitivity factors determined from measured spectra of

mixtures with known component concentrations.
C = Matrix of known standard concentration values
K is calculated using the concentration matrix C, its transpose CT, and
the calibration set absorbance matrix Astd.

(3)

From K and its transpose KT, Kcal (referred to as the calibration or

regression matrix) can then be generated:

(4)

Figure 1. Absorbance spectra of Acetaminophen and

The least-squares solution to determining analyte concentrations in Caffeine standards.
an unknown mixture is then determined by the applying Kcal to the
measured absorbance values of the unknown mixture Aunk,

(5)

Cunk is the vector containing predicted concentration values (C1, C2, …,

Cn) for each analyte in the unknown mixture.
As an example of the ability of this technique to measure the
concentrations of components in a mixed solution, known mixtures of
two ingredients found in common OTC products, Acetaminophen and
Caffeine were measured.
The spectra of pure standards of Acetaminophen and Caffeine are
shown in Figure 1.
Figure 2. Comparison of measured versus actual results of
The technique was then used to analyze data collected using the standard mixtures.
Distek Opt-Diss 410 Fiber Optic Dissolution System from five mixtures
with varying amounts of Acetaminophen and Caffeine. The computed One can clearly see that the method accurately quantitates the
values produced by the Opt-Diss 410 MCA software are compared to amounts of Acetaminophen and Caffeine in mixtures with an error
the actual values in the Table 1 and represented graphically in Figure 2. well less than 2%.

Table 1. Measured versus actual percentage values of standard mixtures.

Acetaminophen Caffeine

Mixture % Actual % Measured % Error % Actual % Measured % Error

1 50 50.43 0.9% 30 29.60 1.3%

2 30 30.30 1.0% 50 50.08 0.2%

3 90 89.83 0.2% 70 70.40 0.6%

4 70 70.19 0.3% 90 90.16 0.2%

5 100 99.68 0.3% 100 100.96 1.0%

52 | | September/October 2017
 WHITEPAPER »

comprised the measured values of five

different mixtures of Aspirin and Caffeine plus
the 80% pure standards shown in Figure 3.

As the results in Figure 4 demonstrate,

the technique measures the simultaneous
dissolution rates of the two components,
readily resolving caffeine’s very fast release
rate as well as aspirin’s slower one.

Summary
UV spectrophotometry combined with MCA
has been demonstrated to yield accurate
analysis of the absolute concentrations
of each component in two component
mixtures. The technique has been also
successfully applied to measuring the
separate dissolution rates of two APIs in
a commercially available product. These
results demonstrate the method can
Figure 3. Absorbance spectra of Aspirin and Caffeine standards.
accurately quantify two components even
with highly overlapping spectra without the
need for a separation step. The key to this
process is using large data sets consisting of
large spectral regions instead of individual
wavelengths and complete temporal
profiles instead of a few points. This rich
data set collection is enabled by the use of
in-situ sampling utilizing fiber optics probes
which analyze the sample within the vessel.
This circumvents the limit of the speed of
moving the liquid from vessel to the analyzer
that encumber traditional methods such as
HPLC or conventional UV spectroscopy. An
additional benefit of the instantaneous data
collection of in-situ probes is that they allow
near real-time dissolution analysis.

As these measurements of commercial

products under real-world conditions
illustrate, the addition of MCA and fiber optic
Figure 4. Average dissolution profile of six Aspirin Caffeine tablets. in situ measurements allow formulation and
analytical chemists, as well as QC analysts to
realize the time and labor savings associate
To illustrate the applicability of the technique Complete spectra from all six vessels were with UV spectrophotometry even when
to real measurements, the dissolution of a collected every 10 seconds for 30 minutes, measuring products with two APIs.
tablet containing 400 mg Aspirin and 32 mg again using the Distek Opt-Diss 410 Fiber
Caffeine was analyzed. Absorbance spectra of Optic Dissolution System. The results were
pure standards of Aspirin and Caffeine at 80% then analyzed using the method described *Corresponding Author
are shown in Figure 3. above. In this case, the training set used [email protected]

www.americanpharmaceuticalreview.com | | 53
» WHITEPAPER

USP <661> compliance for

Total Organic Carbon (TOC) Analysis
GE Water and Process Technologies

Introduction and Challenge USP <661> Subsections

The Pharmaceutical Industry is heavily dependent on plastic There are two subsections to chapter <661>:
packaging material to bring products to market. This packaging <661.1> Plastic Materials of Construction. This sub-section is designed
comes in the form of bottles, disposable IV bags, pre-filled syringes to ensure that the individual materials are characterized for suitability
and others and may include many materials (myriad polymers and of use. This is dedicated to individual plastic materials.
additives). Ultimately, it must be demonstrated that these packaging
<661.2> Plastic Packaging Systems for Pharmaceutical Use. This
systems and their materials of construction do not interact with the
subsection is designed to ensure that the entire system, which consists
therapeutic products in such a way to alter their suitability for use.
of one or more materials, is characterized for suitability of use.
The USP <661> chapter was recently revised to become more
comprehensive in scope. This has allowed the regulation to
separately cover the materials used in packaging and the packaging
system as a whole.
Expected Evaluations of <USP> 661
Material Screening
• Evaluation of ingredients as probable extractables and
Total Organic Carbon (TOC) potential leachables

Regulations per USP Controlled Extraction Study

• Worst-case controlled extraction (simulation) study to
USP mandates testing of TOC in both Purified Water (PW) as well as
determine the extent to which extractables may become
Water for Injection (WFI). This is comprehensively documented in USP
probable leachables
Chapter <643>. The limit for TOC in both PW and WFI has been set for
0.5 ppm. Product Assessment

On May 1, 2016 USP made a major revision to General Chapter <661> • Actual-case measurement of confirmed leachables in the
and reintroduced the chapter as “PLASTIC PACKAGING SYSTEMS AND therapeutic product in the pharmaceutical packaging/
THEIR MATERIALS OF CONSTRUCTION”. In addition, two subsections delivery system intended for the commercial market
were written:
Table 1. TOC Limits per <USP> 661
• <661.1> Plastic Materials of Construction
USP Applies to TOC Specification*
• <661.2>Plastic Packaging Systems for Pharmaceutical Use <661.1> Individual plastic materials ≤ 5ppm

In addition to the delineation of materials and systems, a wider <661.2> Plastic packaging system ≤ 8ppm

range of testing methodologies and technologies were presented, * TOC Specification is a differential and requires blank correction
including TOC.
As noted above, this was done to understand the materials that
compose the packaging systems, as well as the packaging systems
themselves. This regulation has far reaching effects, as the revised Other Requirements for TOC to
chapter will now apply to: comply with USP <661>
• Manufacturers of finished dosage pharmaceuticals
TOC analysis performed:
• Manufacturers of plastic bags, vials, IV kits, etc. • Should have a limit of detection of 0.2 ppm
Primary responsibility for compliance to this regulation resides with • Should have a demonstrated linear dynamic range from
the owner of the regulatory approval for the packaged drug product. 0.2–20 ppm

54 | | September/October 2017
 WHITEPAPER »

A linearity protocol and spreadsheet for reference can be provided

Sievers® M9 TOC Analyzer and on request.
Compliance to USP <661> These standards, combined with the Sievers Investigative Failure

The Sievers Total Organic Carbon (TOC) M9 Analyzers offer time-tested Analysis Report (FAR), provide traceability and enable expedited Out
dependability with fast analysis. The Analyzers reduce the time to TOC of Specification (OOS) investigations.
results by 50%, enabling increased productivity.
The M9 Analyzer is offered in Laboratory and Portable versions for
Designed to facilitate compliance within a heavily regulated
ease of use. The Analyzer is capable of complying with USP <643>, USP
environment, Sievers TOC Analyzers exceed both regulatory and
analytical requirements. The broad linear range allows for excellent <645>, USP <661>, USP <1225>, 21CFR Part 11 and others, including
low-level sensitivity for ultrapure water samples and high-level globally equivalent standards.
capability for cleaning validation samples.
With its Linear Range of 0.03 ppb to 50 ppm, the M9 Analyzer easily
meets the USP <661> requirements for detection limit as well as
dynamic linearity range. All Sievers TOC Analyzers also comply with
References
USP <643> for PW and WFI. 1. USP General Chapter <661>
NIST traceable standards and standards Certified for ISO Guide 34 2. USP Subchapters <661.1> and <661.2>
and ISO/IEC 17025 are available to support the Analyzers and USP
3. USP 661 Briefing: http://www.usp.org/sites/de-fault/files/usp_pdf/EN/meetings.pdf
<661> compliance:
• Accuracy/Precision, 8 ppm (STD 77013)
• Accuracy/Precision Set, 5 ppm (STD 99011) For more information about TOC, USP <661> and standards and traceability
• USP <661> Linearity Set (STD 99012) visit: https://www.geinstruments.com/industries/traceability-standards

www.americanpharmaceuticalreview.com | | 55
» WHITEPAPER

Steps to Ensuring a Successful Audit:

Effective Risk Assessment Design
Gilberto Dalmaso, PhD
Particle Measuring Systems

as molecular compounds, such as chlorides or

Abstract amines. Both types of contamination originate Risk Assessment Tools
from multiple sources, such as personnel, sur-
When designing a process, product quality is faces and the surrounding air. FDA and European authorities have made
an important measure of success. To ensure Critical areas are often monitored for the purpose risk assessment mandatory, with supporting
product quality, it is imperative that the envi- of ensuring a specified level of product quality. document ICH Quality Risk Management
ronment is monitored for contamination. The Q9. Methods for performing a risk assess-
best way to locate sources of contamination When levels of product quality deviate out-
side the specified level, which are determined ment should fit the need, and several op-
is via risk assessment, best performed before
by risk assessment, the results are categorized tions are available.
a process has been implemented. There are
multiple tools to assist in completing a risk into three types of failures:
assessment, and once completed, it is impera- • Out-of-specification (OOS): the result FMEA
tive to continually update this body of knowl- falls outside of establish acceptance
Failure Mode Effect Analysis (FMEA) is the ideal
edge to guarantee a defensible monitoring criteria, determined by risk assess-
risk assessment tool to use for process manage-
program is developed and enacted. ment and regulatory bodies
ment. Failures likely to occur during manufac-
• Out-of-expectation (OOE): the result turing or during a filling operation for example,
is atypical within a series of results are categorized by severity, occurrence, and
Introduction obtained over a short period, but
detection ability. The categorization is used to
meets specifications
develop a score providing a numeric represen-
When related to Environmental Monitoring
• Out-of-trend (OOT): the result is tation of risk sources.
(EM), risk management often refers to a com-
outside the predictive model and
plete understanding of how often and how An example of FMEA score calculation is below.
may fail process control tests
severe a process can be affected by external
Similar to product quality, contamination can A score system is determined to be equal to
factors. The development of this knowledge, a
risk assessment, is the culmination of multiple deviate outside of acceptable levels and im- and between numbers 1 and 5, with 1 being
party’s efforts, including Quality Engineers, pact product results. The acceptable levels of ‘low’, and 5 being ‘high’.
Microbiologists, and Production staff. Actions contamination are determined in the same
Points of risk are identified and given a score.
involved in completing a risk assessment start way: with a risk assessment. With EM, sources
Risk point 1 and its final FMEA score calculation
at the very beginning of new cleanroom con- of contamination can be quickly identified,
leading to a speedier recovery of the process is determined:
struction with the identification of product im-
pact sources. The relative risk to product qual- with less downtime. EM also assists with the RP1: (2) severity, (5) occurrence, (5) detection
ity is given hierarchy using well-established resulting investigations that occur when a ability
OOS, OOE and OOT is found.
assessment tools.
2 x 5 x 5 = 50 = FMEA final score
In order to prepare for this process, it is impor-
tant to attain a strong base of EM knowledge.
HACCP
Hazard Analysis Critical Control Points (HACCP)
is the assessment tool often used for non-sterile
Monitoring Basics products, such as food. It is less statistical and
EM typically includes the tracking and enu- focused more on the entire process as a whole.
meration of both viable and nonviable par-
ticles. Viable particles include living things,
Figure 1. MiniCapt® Mobile Microbial
Others
such as bacteria, molds and yeasts. The group
of nonviable particles is comprised of every- Air Sampler and Lasair® III At least a dozen more tools exist, allowing for
Aerosol Particle Counter placement personalized risk assessment construction to
thing non-living, including but not limited to
adjacent to filling line
dust, hair, smoke and even particles as small fit the needs of multiple industries.

56 | | September/October 2017
 WHITEPAPER »

Table 1. Example of criticality factor and Table 2. Risk associated with range of
Criticality Factors monitoring frequency by location contamination rates
Criticality Monitoring Contamination Rate Risk
Locations in manufacturing environments as- Location
Factor Frequency < 0.03% Low
sociated with a rating system with adjusted Daily or each
1 ISO 5, filling points > 0.03 - 0.09% Medium
monitoring frequency are defined as criticality batch
≥ 0.1% High
factors. The range of value categories are deter- 2
ISO 7, surrounding of ISO 5
Weekly
ISO 7, gowning rooms
mined on an individual basis, aligning with the
needs of the particular process under evalua- ISO 5, depyrogenation
3 Weekly
tunnels
tion. For example, a location with a criticality
factor of 1 (set to be the highest risk level), is 4 ISO 8, preparation areas Monthly

determined from a risk assessment to have a ISO 9, sterilization/ Every four

5
monitoring frequency of daily or after each washing areas months Figure 3. BioCapt® Single Use
batch. From this location’s criticality factor, sur-
rounding areas are also evaluated (see Table 1). Contamination rate (%)= documentation of risk assessment conclusions

Table 1 is determined from a set of decision crite- Settle plate count x [(Product Area x Product are essential for successful audits and investiga-
ria, including but not limited to the following: exposure) / (Settle plate area x Settle plate ex- tions, because your practices have a firm back-
posure)] x 100 = 1 x [(1 x 1) / (64 x 240)] x 100 ing and have been tested and proven to be suf-
• cleanliness needs of the locations ficient for your needs. It is recommended that
= 0.0065 %
• personnel transit risk assessments be continually performed and
• incoming goods A result of 0.0065% would be placed in the ‘low’
rationales and procedures be constantly criti-
risk category (Table 2). However, settle plates
• component preparation cized and improved. Constant innovation and
have been proven to be unsuitable for ISO 5
• activity duration improvement must be part of company culture.
monitoring due to lack of validation. Active
• room temperature air and continuous monitoring techniques are
• wet/dry areas the preferred method for ISO 5 environments.
• open/closed process Specific examples, such as the MiniCapt Mobile Conclusion
• final formulation/filtration and BioCapt® Single Use, have been validated
• variations within rooms (active air, to ISO 14698 with data proving high biological From regulations required by the FDA and
surface samples, etc.) and physical efficiency. European authorities, risk assessments are a
mandatory part of process development and
• sampling criticality (ex. high sampling When determining which method to use, ask
understanding. Risk assessments performed
frequency creates more contamina- yourself how reliable you expect the results to
to ensure quality assist in identifying risk to
tion with operator intervention) be. If no variance to contamination rates are ob-
the process, management of that risk, and
served, how valuable is the test? Should other
controlling/monitoring scheme development.
methods be used? Do the alternate method
Assessments should be performed prior to
results conflict? These questions must be an-
Closer Look: Assessing swered to achieve a comprehensive and defen- process implementation, not after, allowing
Risks to Product Fills sible monitoring program. for proper installation of monitoring devices.
By taking the necessary steps to complete a
It is imperative that the appropriate method is
risk assessment, you can ensure a defensible
chosen to monitor contamination. Older meth-
ods, such as passive monitoring with settle
After a Risk Assessment monitoring program that helps facilitate au-
dits and investigations.
plates, once standard practice, are obsolete. The entire process of performing a risk assess-
Particle Measuring Systems offers a wide ar-
Let’s take a look at a typical example of post-fill ment can take weeks to months to complete,
ray of consultancy services. Contact us for
assessment using settle plates using Whyte’s with an average of one week per filling line. Once
more information.
method. In this method, contamination rate is the assessment has concluded, sampling ratio-
determined based on the total count and the nale that supports the locations and monitoring
frequency are worked into Standard Operating
ratio of product and settle plate area and ex-
posure. The result is categorized into low, me-
Procedures (SOPs) and used to determine vi- Author Biography
able and total particle counter installations. Final
dium or high risk. The example values given
Gilberto Dalmaso, PhD has over 25 years’ ex-
below are very commonly found.
perience in pharmaceutical microbiology and
• Settle plate count = 1 CFU
sterility assurance. His current work is focused
• Settle plate area (90 mm) = 64 cm2 on pharmaceutical microbiology and aseptic
• Settle plate exposure = 240 minutes processes, microbiological contamination con-
• Product area = 1 cm2 Figure 2. Passive air monitoring trol and rapid microbiological methods, Quality
• Product exposure = 1 minute (settle plates) by Design (QbD), and PAT in microbiology.

www.americanpharmaceuticalreview.com | | 57
» WHITEPAPER

Extractable and Leachables Studies:

Designed and Performed to Meet
All Intended Needs
Thomas Lehman, PhD
Director, Method Development and Validation and Extractables and Leachables
Eurofins Lancaster Laboratories

Since the FDA released their Container Closure Systems for Packaging evaluating single-use and multiple-use systems for extractables and
Human Drugs and Biologics guidance in 1999, evaluation of final leachables. There are also various workgroups that have published
packaging components for extractables and leachables has become guidance documents to assist the industry in designing and
the expectation within the industry. Additionally, the increase in performing these studies. The Product Quality Research Institute
the use of single-use systems in manufacturing has drawn scrutiny (PQRI), the Bio-Process Systems Alliance (BPSA), and the Bio-
as another potential source of extractables and leachables. These Phorum Operations Group (BPOG) have all released publications
single-use systems include bioprocess bags, filters, tubing, fittings, defining a best practices approach to performing the testing. The
connectors, bioreactors, etc. Many of these single-use systems are PQRI guidance document focuses primarily on performing studies
constructed from polymeric materials, increasing the concern of on Orally Inhaled and Nasal Drug Products. Both BPSA and BPOG
the introduction of leachable compounds to product. And with the focus on the evaluation of single-use technology. While the general
tremendous growth of the implantable device market and continuous approach to generating an extractables profile of a contact material
emergence of new medical device technologies, there is a greater is similar, the details in performing the studies differ between the
potential for extractables and leachables to negatively impact a guidances. In addition, ISO-10993 provides guidance specifically for
device’s biocompatibility. the chemical characterization of medical devices for extractables
Extractables are compounds that can be extracted from a product and leachables. Eurofins Lancaster Laboratories has experience in
contact material under exaggerated conditions such as elevated
temperatures, extended storage times, or exposure to harsh extraction
solutions. Leachables, which are typically a subset of extractables, are
compounds that leach from product contact materials into the drug
product under normal conditions of use. Sources of extractables
and leachables compounds include antioxidants, antiozonants,
UV stabilizers, plasticizers, processing aids, accelerants, coatings,
elastomers, inks and vulcanizing agents, to name a few. Both
extractables and leachables represent potential contaminants to a
final product and may affect the pH or color of the product, impact
the efficacy of the product, or alter the active ingredient. In addition,
extractables and leachables compounds may be toxic.
There are no definitive requirements on how to perform extractables
and leachables studies; however, the USP recently released two
chapters to provide guidance on performing E&L studies. Chapters
<1663> and <1664> discuss options and considerations in performing
both an extractables study and a leachables study. Additionally, USP
<665>, currently in the draft stage, presents recommendations for

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chemical ionization techniques are used

to generate the data. These ionization
techniques are considered soft ionization
and typically result in the mass spectrum
displaying the molecular mass. The resulting
mass spectra must then be evaluated to
determine identifications so the safety
assessment can be performed. However,
there are a limited number of choices of
commercially available databases that allow
for rapid identification of the observed peaks.
Therefore, Eurofins Lancaster Laboratories
has generated its own in-house, proprietary
database. The Eurofins Extractables Index (EEI)
contains over 1,500 non-volatile compounds
that are commonly used in the production
of plastics and polymers. This database
is integrated directly into the processing
software, allowing for automated searching
and comparison of spectra.

Gas chromatography-mass spectrometry

(both headspace and direct injection
sample introduction) is used to evaluate
the presence of both volatile and semi-
volatile compounds. Eurofins Lancaster
Laboratories utilizes the NIST and Wiley
databases to assist in the identification of
observed extractables compounds.

In addition, inductively coupled plasma with

either mass spectrometry or optical emission
spectroscopy detection is used to monitor
the presence of metals. Eurofins Lancaster
Laboratories evaluates metals listed in USP
<232> and ICH Q3D and has the ability to
evaluate additional metals.
performing studies following any of these then tested by a variety of different analytical
workgroup and/or industry guidances, as techniques to generate a broad extractables Eurofins Lancaster Laboratories performs

well as establishing custom study designs. profile that can then be used to perform a more than 250 controlled extraction studies
safety assessment. Typically, these analytical per year and designs extractables studies
Typically, an extractables study involves
techniques utilize mass spectrometry to to meet clients’ project objectives. Whether
exposure of components to solvents of
allow for identification of the observed the study will be performed on a container-
varying polarity at elevated temperatures.
extractable compounds. closure system, drug-delivery device, single-
The extraction may involve incubating
components at elevated temperatures, Liquid chromatography-mass spectrometry use system or a medical device, Eurofins
or using sonication, accelerated solvent is utilized to evaluate the presence of Lancaster Laboratories has the experience and
extraction (ACE), soxhlet or reflux to extract non-volatile organic compounds. Both expertise in designing and performing studies
the component. The resulting extracts are electrospray and atmospheric pressure to surpass expectations.

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Selecting Microspheres to Calibrate

and Assess Online Water Bioburden
Analyzer System Performance
Allison Scott, Ph.D.
Senior Applications Enginer
Azbil North America Research and Development, Inc. - BioVigilant

Online Water Bioburden Analyzers (OWBAs) are a type of rapid In general, for particles made of the same material that are in the Mie
microbiological method designed for online bioburden detection in scattering size range, the larger the particle traveling through the laser
pharmaceutical grade waters. With a target of detecting planktonic the greater the intensity of scattered light; with the scattered intensity
(single, free floating) microbes, the system must be capable of weak increasing at a rate that approaches the square of the particle size.1,2
Mie scatter and intrinsic fluorescence detection. For OWBA system This scattered light intensity, however, also greatly depends on the
calibration and assessment, microbe and interferent microsphere refractive index of the particle such that a larger relative particle size
surrogates that have a fluorescence and size similar to that of microbes will not guarantee a larger scatter intensity across materials. Refractive
index is a unit-less number that describes how light travels through
should be selected, and directly impact the overall sensitivity of the
a material, such that it can provide a measure of the bending of light
system. Factors like microsphere material, size, dye, and dye quantity
when the light passes from one material to another. If using a 0.5μm
are key parameters to consider when selecting a system assessment or
polystyrene (PS) sphere to calibrate a Mie scatter based system, all
calibration bead. In the case of OWBA systems, fluorescent reference
particles detected will be interrogated based on a comparison of
particles can be used to assess or confirm the system’s biologic
Mie scatter intensity to this reference bead. Due to the dependence
detection performance when a particle having fluorescence on the
of scatter intensity on size and refractive index, knowledge of these
order of magnitude of microbes is utilized. However, particles used to parameters for the target particles of interest can aid in the selection
set the particle detection threshold are also incredibly important to of a calibration particle that yields a Mie scatter intensity within the
overall system sensitivity as the system must first be able to detect the range of the target particles and ensure adequate sensitivity of the
particle before it can determine if a signal seen on the fluorescence OWBA system.
detector(s) is biologic or inert.
NIST-traceable PS particles are commonly utilized in the calibration
of particle detection instrumentation, such as liquid particle counters
and flow cytometers. Use of a 0.5μm PS sphere, for example, to set a
Particle Size Standard Selection – minimum particle detection threshold on OWBA systems, however,
may not provide adequate sensitivity and sufficient ability of the system
Calibrating the System’s Particle to detect similarly sized microorganisms. Because of the influence of
Sensitivity refractive index on scattered light intensity, the calibration particle
should have a refractive index as close as possible to microorganisms
if a particle similar in size to the target organisms is utilized. If a
Silica vs. Polystyrene: Material Matters calibration particle with a refractive index that is significantly different
Microsphere material and size are both critical parameters to consider from microorganisms is used to set the minimum particle detection
when selecting a particle size standard used to calibrate an OWBA threshold, the size of this bead must be considered in order to obtain
system’s minimum particle size sensitivity. OWBA systems currently a scatter intensity that is low enough to maintain microbial detection
on the market utilize Mie scatter to detect the presence of particles in sensitivity on the system.
sampled water. Laser light of 405nm interrogates the water sample as
it flows through these systems. If a particle is present in the sampled IMD-Wª System and Particle Size Standard Selection
water, the laser light is scattered by the particle onto the system’s In past generations of the IMD-W system, itself an OWBA, a 0.5μm
particle detector. The incident 405nm source and this scattered light NIST-traceable PS microsphere was utilized to set the minimum
have different properties, with the intensity of the scattered light particle detection threshold. Biochallenge results significantly below
being dependent upon the properties of the particle (i.e. particle size, the targeted IMD-W Bio Counts-to-CFU Ratio of one were obtained
shape and refractive index) and its surroundings. for a majority of the ten organisms tested on these systems, as

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shown in the generation eight (G8) data in Figure 1. This data and that of a 0.5μm SiO2 sphere or microbe. If a 0.5μm PS particle were
concerns over inadequate sensitivity led to an investigation and utilized to calibrate the IMD-W particle detection threshold, this
ultimate determination that a smaller PS bead, or similarly sized would theoretically equate to being able to see a large percentage
amorphous silica (SiO2) bead, was needed to set the IMD-W system’s of SiO2 particles 1μm and larger, and microorganisms greater than
particle detection threshold to that needed for adequate detection approximately 1μm in size, depending on their refractive index. Due
of microorganisms. to distribution tails and variability in microbe size, smaller particles
would still be observed, but with a much smaller efficiency.

Figure 2. IMD-W optical model theoretical data (lines) and IMD-W

empirical data (black dots) showing the normalized Mie scatter
intensity as a function of refractive index for eight different particle
diameters, with water as the suspension medium.

Because of this analysis, BioVigilant currently utilizes 0.5μm SiO2

particles and a liquid particle counter with a 0.2μm minimum size
channel, based on a calibration with PS beads, to set the particle
Figure 1. IMD-W Generation 8 versus Generation 9 threshold on the IMD-W system. The change was made from 0.5μm PS
Biologic counts/CFU ratio. to 0.5μm SiO2 in an effort to calibrate the system with sufficient particle
detection sensitivity to detect microorganisms. As gathered from the
As part of this investigation, an optical engineering software called data in Figure 2, a 0.2μm to 0.3μm PS microsphere could also be used
FRED was utilized to model Mie scatter intensity data as a function to calibrate the IMD-W system, as a PS bead within this size range
of refractive index for the IMD-W system. Results of this analysis are will yield approximately the same Mie scatter intensity as a 0.5μm
in Figure 2, which shows the theoretical Mie intensity of 405nm light SiO2 bead. Use of calibration beads within this size range requires a
scattered from various particle sizes and the variability in scattered reference liquid particle counter with a minimum particle size channel
light intensity as a function of the refractive index of the particle. The of 0.2μm or, ideally, 0.1μm due to the calibration of liquid particle
surrounding medium in the model is water, with a refractive index of counters with PS particles. BioVigilant has recently implemented a
1.34 at 405nm.3 liquid particle counter with a 0.1μm minimum particle size channel for
The black dots on the graph, with particle size and material listed, use in setting the IMD-W particle threshold.
represent empirical data obtained by running beads through the
IMD-W system. A high correlation is present between the FRED
model and empirical results obtained from the IMD-W system. SiO2 Fluorescent Particle Selection
has a refractive index of 1.47 at 405nm,4 which is quite close to the
approximate refractive index of microorganisms, listed in literature
as commonly being between 1.39 and 1.45.5,6 PS, however, has a
Particle Dye and Intensity
refractive index of 1.63 at 405nm,7 which is significantly higher than Commercial OWBA systems utilize autofluorescence to detect
that of microorganisms. It can be seen in the graph that the scattered microorganisms in sampled water.8,9 Fluorescent microspheres currently
light intensity for a 0.5μm PS sphere is significantly higher than available are predominantly intended for systems, like flow cytometers,

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that are designed to detect extrinsic fluorescence applied to a sample

using stains or reagents. As a result, many dyed microspheres are
orders of magnitude brighter than a non-stained planktonic microbe
and are not well suited to assess the sensitivity of intrinsic fluorescence
based OWBA systems. Selection of a dyed microsphere with excitation
at 405nm, and fluorescence emission similar to that of bacteria in
wavelength and intensity can thus be challenging.
Light Yellow and Yellow dyes from Spherotech, Inc. (Figure 3), utilized
in their SPHEROTM Fluorescent Particles, have excitation and emission
spectra ranges that make them potentially well suited for use with
OWBA systems using a 405nm excitation source.10 Figure 4 shows the
normalized emission spectra of a Spherotech Yellow Low Intensity
0.8um bead (STY, FL-0852-2); two waterborne microorganisms,
B. diminuta and P. putida; and three interferent materials, Teflon (PTFE),
Kalrez (FFKM), and silicone rubber. A Jasco spectrofluorometer was
utilized to obtain the fluorescence spectra. Figure 4. Comparison of microbial, interferent and STY fluorescence
within the IMD-W detection range. Data was obtained with a
Also shown on this plot are a blue and green band that represent the Jasco spectrofluorometer.
spectral regions for the IMD-W system’s two fluorescence detectors.
The center pink band represents the region where Raman (vibrational)
fluorescence is generated from the background water, with an Spherotech Light Yellow dye (STLY), on the other hand, shows a
emission peak centered at 469nm with 405nm excitation, and thus is a significant level of integrated fluorescence in both the PMT1 and
region that is excluded from the system’s detection.11 PMT2 detection ranges, which is similar to the fluorescence seen with
a number of interferents like Teflon, Kalrez and silicone rubber (Figure
With 405nm excitation, the emission range of the STY bead closely
4). Therefore, the fluorescence detection range(s) of the OWBA system,
resembles that of the two microbes, with a significantly larger
in addition to other system specific parameters like algorithm, may
integrated fluorescence in the PMT2 detection range as compared
result in STY or STLY being a more appropriate dye when targeting a
to the PMT1 detection range. A custom, 0.8μm bead made with
surrogate for microbial fluorescence.

Variability in Laser Wavelength

Variability in laser wavelength can have a significant impact on the
fluorescence emitted from a dyed microsphere. Ideally, a dye is chosen
such that variability in excitation source wavelength of ±5nm, which is
typical, will not have a significant impact on the fluorescence emitted
from the bead. However, given the weak fluorescence needed in a
targeted detection range, this may not be possible. As can be seen
in Figure 3, an excitation wavelength range of 400nm – 410nm falls
on the sharp leading edge of the STY excitation spectrum, which
may result in significant variability in the emitted fluorescence and
fluorescence detected by the OWBA instrument. Although this is an
element that can be taken into account and measures implemented
to control, this is yet another variable that should be considered when
selecting a fluorescent bead to characterize an OWBA system.

IMD-W System and Fluorescent Particle Selection

Two fluorescent microspheres are utilized in the calibration and
performance assessment of the IMD-W system. A STY bead (Spherotech
FL-0852-2), is utilized to assess the biologic detection performance of
the IMD-W system, while a custom STLY bead is utilized to assess the
interferent discrimination performance of the IMD-W system.
Figure 3. Excitation and emission spectra of the dyes
A number of commercially available Spherotech yellow and Spherotech
utilized by Spherotech in their fluorescent microspheres.
yellow low intensity beads were assessed on the IMD-W system,

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in the search for a bead with a low enough fluorescence intensity,

similar to that of microbes on the IMD-W system. Figure 5 shows Conclusions
the IMD-W system PMT2 signal histograms for thirteen organisms,
the STY bead, and STLY bead. The linear, vertical axis represents the • Both microsphere diameter and refractive index are
important when selecting a particle size standard to set the
normalized counts for each sample, while the horizontal axis shows
minimum particle detection threshold.
the signal intensity on a logarithmic scale to better distinguish sample
distributions. The graph in Figure 5 shows that the thirteen microbes » Choose a particle with a Mie scatter intensity similar to
have measured fluorescence on the same order of magnitude as that produced by target microorganisms.
the STY bead and STLY bead on the system’s second fluorescence • Fluorescent dye and intensity selection should take into
detector, PMT2. When running STY through the IMD-W system, a high account the OWBA system’s excitation wavelength, emission
percentage of total counts are classified as biologic by the algorithm. detection range, and desired application for the bead.
» If using the fluorescent particle to assess biologic
performance, the system’s algorithm may also need
consideration.
» Variability in laser wavelength can have an impact
on fluorescence emitted from particles containing
certain dyes.
• A reference liquid particle counter with a sensitivity down
to 0.1μm or 0.2μm, based on a PS calibration, is useful in
confirming the sensitivity of the OWBA system to detect
microorganisms.

References
1. P. E. Plantz, “Particle Size Measurement from 0.1 to 1000 um, Based on Light Scattering
and Diffraction,” in Modern Methods of Particle Size Analysis , New York, John Wiley & Sons,
Figure 5. IMD-W System PMT2 fluorescence histograms for the STLY 1984, pp. 173-208.
bead, STY FL-0852-2 bead, and 13 organisms.
2. K. Okuyama, Y. Kousaka and J. H. Seinfeld, “Aerosols,” in Encyclopedia of Environmental
Science and Engineering: A-L, CRC Press, 2006, pp. 15-28.
3. M. Daimon and A. Masumura, “Measurement of the refractive index of distilled water from
A number of Spherotech light yellow beads were also assessed on the the near-infrared region to the ultraviolet region,” Applied Optics, vol. 46, no. 18, pp. 3811-
IMD-W system in an effort to find a bead with a low level of integrated 3820, 2007.
fluorescence on both PMT1 and PMT2. All commercially available 4. I. H. Malitson, “Interspeciment comparison of the Refractive Index of Fused Silica,” Journal
Spherotech light yellow beads tested showed fluorescence that of the Optical Society of America, vol. 55, no. 10, pp. 1205-1209, 1965.
was significantly higher than the targeted low level of fluorescence, 5. Y. Liu, L. K. Chin, W. Ser, T. C. Ayi, W. M. Ho, P. H. Yap, Y. Leprince-Wang and T. Bourouina,
“A single living bacterium’s refractive index measurement by using optofluidic immersion
similar to the fluorescence signal of STY on PMT2. As a result, a refractometry,” in 17th International Conference on Miniaturized Systems for Chemistry
custom Spherotech light yellow bead, STLY, was commissioned. and Life Sciences, Freiburg, 2013.
The fluorescence targeted with this custom bead was 20% of the 6. A. E. Balaev, K. N. Dvoretski and V. A. Doubrovski, “Refractive index of Escherichi coli cells,”
fluorescence measured on the IMD-W system with the most weakly in Proceedings of SPIE, 2002.
fluorescing Spherotech light yellow bead found in Spherotech’s 7. Duke Scientific Corporation, “Technical Note 007B,” 1 December 1996. [Online]. Available:
catalog and research inventory. When the STLY bead is run through the https://www.thermofisher.com.

system, >90% of fluorescent particles are discriminated as interferents 8. Mettler-Toledo, “Continuous On-Line Microbial Monitoring for Pharmaceutical Waters,”
February 2016. [Online]. Available: http://www.mt.com/dam/non-indexed/po/pro/pdf/
by the system’s algorithm. This discrimination is based, amongst other ds/toc/DS_7000RMS_Microbial_Analyzer_en_58087053_Feb16.pdf.
metrics, on significant integrated fluorescence observed on both 9. Azbil North America, Inc. - BioVigilant Division, “Instantaneous Microbial Detection for
PMT1 and PMT2, which is typical of the fluorescence observed with Pharmaceutical Waters,” 2014. [Online]. Available: http://biovigilant.com/wp-content/
interferent materials (Figure 4). Due to the low level of fluorescence uploads/2014/10/IMD-W-Brochure.pdf.
and signal on both PMT1 and PMT2, STLY is utilized by BioVigilant 10. Spherotech, Inc., “SPHERO(TM) Fluorescent Particles,” [Online]. Available: http://www.
spherotech.com/2016%20Catalog%20Product%20Detail%20Pages/Spherotech%20
to confirm and detect changes in system performance. Due to the
Fluorescent%20Polystyrene%20Particles.pdf.
classification of STLY beads as interferents on the IMD-W system, STLY
11. Perkin Elmer, “An Introduction to Fluorescence Spectroscopy,” 2000.
is not ideally suited to assess the biologic detection performance of
the IMD-W system from a reported counts perspective as a very low
percentage of particles will be reported as biologic by the system. IMD-W™Azbil Corp.

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Determination of Stress Conditions for

Microorganisms for use in Validation of
Milliflex Rapid Detection System
Lamin Jallow
Microbiology Application Scientist, MilliporeSigma

Propionibacterium acnes (P. acnes)

Introduction Propionibacterium acnes was prepared per manufacturer’s instructions
and plated on CDC blood agar. A small portion was transferred into
In the pharmaceutical/biotechnology manufacturing industry, most of
a test tube with peptone saline after 7 days of incubation in 30-35°C
the microbial contaminants are believed to be stressed. The stressed
anaerobically, then serially diluted to 10^4. At time zero (T0) 10uL of
condition could arise from lack of nutrients, exposure to extreme
the final dilution was plated on CDC blood agar, in duplicate. The rest
temperature (heat or cold), UV light or even cleaning chemicals. During
of the dilution was immersed in a water bath pre-heated to 60 °C. At
Performance Qualification of microbiological testing equipment,
various time intervals (T1=1 minute, T2=2 minutes up to T5), 10uL of
it is recommended to stress organisms in order to prove that these the solution was plated in duplicate and incubated anaerobically at
stressed organisms will be detected by the equipment when they are 30-35°C for a minimum of 6 days. After incubation, the colonies were
present in the product. The objective of this study is to determine the counted and the average of the two plates were compared to the time
stress conditions that would result in ~50% recovery as determined by zero plates to determine recovery. Time points T1 through T4 did not
Gray et al, 2010, PDA J Pharm Sci Tech. The authors state that “artificially yield the approximate recovery rate of 50 percent. Only T5 yielded
stressed” microorganisms are recommended for the validation of the successful results as stated below in Table 1.
Rapid Sterility Detection system as most sterility testing samples do
not contain healthy microorganisms. Other rapid methods such as Aspergillus brasiliensis and Candidas albicans
rapid bioburden use natural water as a sample. Water samples do
A.brasiliensis, and C.albicans were purchased as ready to use and
contain naturally stressed organisms according to the authors. This
prepared following the manufacturer’s instructions. Two plates were
eliminates the need to “artificially stress” microorganisms.
inoculated with 10uL in duplicate on Sabouraud Dextrose Agar (SDA)
at (T0). The rest of the dilution was then immersed in a 60°C water bath
and 10uL samples were plated on SDA at various time points (T1=1
Materials and Methods min, T2=2 min, T3,T4 and T5) and incubated at 20-25°C for 5-7 days.
Post-incubation results were counted and compared to T0 plates for
Seven organisms were tested including all 6 of the USP <71> recovery. None of the time points generated satisfactory results (~50%
recommended microorganisms for sterility testing (Bacillus recovery). All the time points showed recovery greater than 75%. The
subtilis, Candida albicans, Aspergillus brasiliensis, Staphylococcus study was repeated with the water bath heated to 65°C. A. brasiliensis
aureus, Pseudomonas aeruginosa and Clostridium sporogenes) T5 plates yielded about 57% recovery. For C. albicans, T4 plates yielded
and Propionibacterium acnes. The organisms were prepared per the desired results of 53% recovery.
manufacturer’s instructions and plated on Tryptic Soy Agar (TSA).
The plates were incubated in their respective incubation conditions. Bacillus subtilis
After incubation, each organism was serially diluted to a range of 20-
Bacillus subtilis was prepared per manufacturer instructions tested
100 CFU (Colony Forming Units). If the organism was ready to use at using two conditions. A portion of the organism preparation was
10-100 CFU, it was used as intended from the manufacturer without stored in 2-8 °C refrigerator. Two Tryptic Soy Agar (TSA) plates were
dilution. Two parameters were used to stress the organisms in this inoculated with 10ul of the organism representing T0. The rest of the
study: 1) heating in a water bath AND/OR 2) starvation/stasis in 2-8 solution was immersed in a 60°C water bath for 1 min (T1) and 10uL
°C. Depending on the organism they were either exposed to heat was plated in duplicate up to T5 (5 minutes). The plates were then
or kept in 2-8°C for certain timeframe. Refer to Table 1 for successful incubated at 30-35°C aerobically for 3-5 days. The T4 plates yielded
parameters for each organism. ~51% recovery.

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After 6 days of starvation, two TSA plates were bath and 10uL of that solution was plated
inoculated with 10uL of the preparation that out on TSA after 1 minute (T1), and up to 5 Conclusion
was stored in 2-8°C refrigerator and incubated minutes (T5). The plates were then incubated
for 3-5 days at 30-35°C to represent T1. This at 30-35°C for 3-5 days and results recorded. Most organisms in production areas or clean
was repeated after day 7 (T2) and day 8 (T3). None of the time points (T1-T5) yielded the rooms are usually under a stressed state so
None of the plates yielded desired results. desired recovery. The study was repeated it is recommended to demonstrate that the
with the water bath heated to 65°C and the Milliflex Rapid System is capable of detecting
(T5) yielded~ 52% recovery. organism under these conditions.The above
Clostridium sporogenes
results shows that it is possible to “artificially
Clostridium sporogenes was prepared per
Pseudomonas aeruginosa stress” microorganisms for use in validation
manufacturer’s instructions. Two TSA plates
studies such as Performance Qualification
were inoculated with 10uL of the organism After preparation and plating of T0 plates on
(PQ) of the Milliflex Rapid detection System.
in duplicate (T0). The rest of the preparation TSA in duplicate, the solution was immersed
Despite the fact that some of the conditions
was stored at 2-8°C. Gray et al, 2010, PDA J in 60°C water bath and samples were plated as
with the above organisms. After incubation, could not be replicated, successful results
Pharm Sci Tech. successfully recovered about
T2 plates yielded about 53% recovery. were achieved by either increasing the heat,
50% after 15 days so this was chosen as T1,
or by extending the exposure time. Several
day 16 as T2 and day 17 as T3. On each of the
factors such as strain type, condition of
aforementioned days, 10uL of the organism
preparation was plated out on TSA at T1 Results microorganism could contribute to different
results. MilliporeSigma does not guarantee
through T3 and incubated anaerobically at
After repeating the experiment documented that these conditions will be successful each
30-35 °C for 3-5 days and the results were
in Gray et al, 2010, PDA J Pharm Sci Tech, time and recommend that each user perform
recorded. The results stated in Gray et al, were
only C. sporogenes and P. aeruginosa were their own study to determine the conditions
successfully replicated. Plates at T1 (Day 15)
recovered at or near the 50%. These results that would generate desired results for
yielded about 46% recovery. T2 and T3 had
very low recoveries. could be due to several factors including validation.
condition of microorganism as the authors
used environmental isolates whereas
Staphylococcus aureus
10uL of each prepared solution was plated
the BoMLab used commercially available
strains. The experiment was repeated for the
Remark
out in TSA in duplicate to represent T0. The remaining organisms to achieve the results in We provide information and advice to our
solution was then immersed in a 60°C water Table 1. customers on application technologies
and regulatory matters to the best of our
Table 1. Microorganism and stress conditions that were successful knowledge and ability, but without obligation
Gram Positive Gram Negative Gram Positive Gram Positive or liability. Existing laws and regulations are
Yeasts/mold
Sporulating Bacteria Bacteria Cocci Rods
to be observed in all cases by our customers.
A. brasiliensis 65°C , 5 min B.subtilis 60°C 4 min P.aeruginosa 60°C, 2 min S.aureus 65°C, 5 min P.acnes 60°C, 5 min
60°C, 3min 6d starvation (2-8°C) 60°C, 2min 60°C, 4min 60°C, 1min
This also applies in respect to any rights of
C.sporogenes
third parties. Our information and advice
C.albicans 65°C 4 min
60°C, 2m
15d starvation, 2-8°C do not relieve our customers of their own
15d starvation, 2-8°C
responsibility for checking the suitability of
*The conditions in blue are from Gray et al, 2010 for comparison. Conditions in black are from BioM Lab study
our products for the envisaged purpose.

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Applicability of the LyoCapsule Mini Freeze Dryer

for Pharmaceutical Product Formulation and
Process Development
Emily Gong and William Kessler
Physical Sciences, Inc. 20 New England Business Center, Andover, MA 01810

lab-scale dryer) and Rp to produce high qual-

Introduction ity pharmaceutical product. Methods
The LyoCapsule™ Freeze Dryer (seven 20cc
The development of biopharmaceuticals Experiments were performed in both the
vial) miniature product chamber freeze
often requires lyophilized formulations to LyoCapsule™ Freeze Dryer and the lab-scale
dryer was developed to bridge the gap
produce stable drug products.1 Development LyoStar 3 Freeze Dryer (both manufactured by
between laboratory and manufacturing-
of a drug formulation and lyophilization SP Scientific, Stone Ridge, NY). A 5% mannitol
scale lyophilizers. As shown in Figure 1, the
(freeze drying) process begins with (Sigma) formulation was used for all cycles.
LyoCapsule™ Freeze Dryer contains a small
laboratory experiments followed by scale- The LyoCapsule™ Freeze Dryer was loaded
freeze drying chamber with a cylindrical
up to larger lyophilizers. A challenge during with seven 20cc vials and the LyoStar 3 Freeze
inner chamber and utilizes wall temperature
formulation and process development is Dryer was loaded with 112 filled 20cc vials
control to emulate the radiation conditions
the limited supply of and cost of producing surrounded by a row of empty “dummy” vials
of edge or center vials. It has the ability
sufficient Active Pharmaceutical Ingredient and placed on the middle shelf. The fill volume
to freeze-dry a relatively small number
(API). Significant amounts of API may be of the 5% mannitol solution was 5mL in both
of vials under heat and mass transfer
required to properly screen formulations for dryers. For all cycles, product temperatures of
conditions typically encountered in larger
freeze drying feasibility, understand critical edge and center vials were measured using
product attributes, and define product and freeze dryers. It is outfitted with process
36-gauge thermocouple probes (SP Scientific)
process design space to create a robust analytical technology tools (thermocouples,
placed at the bottom center of the vial. For
freeze-drying process. Placing a few vials capacitance manometers, Pirani gauge, experiments in the LyoStar 3 Freeze Dryer,
within a larger freeze dryer does not provide Manometric Temperature Measurement the temperature of the metal band that forms
representative drying conditions due to (MTM) and Tunable Diode Laser Absorption the bottomless tray (band temperature) was
atypical radiation conditions created when Spectroscopy (TDLAS)) that enable measured with thermocouple probes (Omega)
vials are not situated in a close-packed the use of scientific and engineering placed on the inside wall of the band. During
formation or when vials do not fill the freeze principles for successful process scale up experiments in the LyoCapsule™ Freeze Dryer,
dryer shelf surface.2 Atypical radiation effects and economically viable drug product the wall temperature was controlled either
lead to differences in product temperature manufacturing. In addition, it may also be a to match the center vial temperature (CVT)
histories and drying times resulting in non- valuable tool to “scale down” a process cycle during the cycle (real-time feedback control)
representative cycles. from a large freeze dryer to a smaller freeze or to emulate the temperature of the metal
dryer to investigate process failures using a band in the LyoStar 3 Freeze Dryer using a pre-
Another restriction of current lab-scale ly-
limited amount of API. programmed recipe. TDLAS water vapor mass
ophilizers is their limited ability to emulate
larger lyophilizers due to differences in ra- flow data was collected for all cycles.
diative effects from warmer, non-tempera-
ture-controlled surfaces between lab and
production-scale lyophilizers.3 Also, differ- Experimental Results
ences in the product resistance to drying (Rp)
created due to differences in ice nucleation This study’s focus was to demonstrate
temperatures between laboratory and the the application of this new lyophilizer to
sterile, low particle conditions in manufac- emulate the product temperature history
turing-scale operations leads to differences of a larger laboratory dryer often used
in product temperatures and drying times.4 for process development. The aim was to
Adjustments must be made to shelf temper- compare the radiation environment of the
atures and drying times to compensate for Figure 1. Inner cylindrical chamber LyoStar 3 process development freeze dryer
differences in drying heterogeneity (due to of the LyoCapsule™ Freeze Dryer with to the LyoCapsule™ Freeze Dryer. Similar
temperature-controlled shelf and wall.
the larger ratio of edge to center vials in a experiments were reported by Obeidat et al.5

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However, a second generation LyoCapsule™ Freeze Dryer was used

in this study. Improvements include changes to the shelf geometry
from square to cylindrical to eliminate corner effects and the creation
of an inner chamber that has the ability to slide out from the main
chamber for ease of loading vials and placing thermocouples.
First, a freeze drying cycle was performed in the LyoStar 3 Freeze Dryer
to determine primary and secondary drying times, product temperature
profiles of edge and center vials, and the band temperature profile. Then,
Figure 3. TDLAS data for water vapor mass flow (g/s) and
the same cycle was performed in the LyoCapsule™ Freeze Dryer with the integrated water removed (g) for 5% mannitol in A) the LyoStar
wall temperature controlled either to follow the CVT or to emulate the 3 Freeze Dryer, B) the LyoCapsule™ Freeze Dryer with the wall
band temperature in the LyoStar 3 Freeze Dryer. The drying chamber temperature set to follow the CVT, and C) For the LyoCapsule™
pressure was maintained at 100mTorr for all cycles. Comparisons of Freeze Dryer with the wall temperature set to emulate the band
temperature in the LyoStar 3 Freeze Dryer.
product temperature profiles between the LyoStar 3 Freeze Dryer and
the LyoCapsule™ Freeze Dryer are shown in Figure 2.
When the wall temperature in the LyoCapsule™ Freeze Dryer was Conclusions
controlled to match the CVT, product temperatures and primary drying
times of both edge and center vials (Figure 2A) in the LyoCapsule™ Technology transfer of lyophilization cycles from laboratory to
Freeze Dryer resembled those of center vials observed in the LyoStar production-scale dryers typically involves preserving product
3 Freeze Dryer. When the wall temperature in the LyoCapsule™ Freeze temperature histories. This is usually accomplished by maintaining
Dryer was controlled to emulate the band temperature in the LyoStar the same drying chamber pressure setting and adjusting the shelf
3 Freeze Dryer, the product temperature and primary drying time of temperatures and drying times. This study focused on demonstrating
the edge vials (Figure 2B) in the LyoCapsule™ Freeze Dryer closely the application of the LyoCapsule™ Freeze Dryer for emulating product
resembled the edge vials in the LyoStar 3 Freeze Dryer. Additionally, temperature history between two laboratory dryers using adjustments
the center vial in the LyoCapsule™ Freeze Dryer more closely resembled to the radiation environment in the LyoCapsule™ Freeze Dryer to
the LyoStar 3 Freeze Dryer edge vials than the center vials. emulate center and edge vial drying conditions in the LyoStar 3 Freeze
Dryer. Vials in both dryers were exposed to similar particle-laden
TDLAS data was collected for all three freeze drying cycles. Figure 3
environments, likely resulting in similar product resistance to drying
shows the TDLAS-determined water vapor mass flow and integrated
values (although these have not yet been assessed). We have shown
water removed. The water vapor mass flow profiles for the two cycles
that product temperature histories and drying times can be conserved
performed in the LyoCapsule™ Freeze Dryer indicated a difference in
between the LyoCapsule™ Freeze Dryer and LyoStar 3 Freeze Dryer
primary drying time. The data show an increased primary drying time
through controlling the wall temperature.
for the cycle where the wall temperature was set to follow the CVT
(Figure 3B) compared to the cycle where the wall temperature was set The LyoCapsule™ Freeze Dryer can be a powerful tool for formulation and
to emulate the band temperature in the LyoStar 3 Freeze Dryer (Figure process development for lyophilized pharmaceuticals. The LyoCapsule™
3C). This further validates the product temperature data shown in Freeze Dryer can be used to ensure that critical formulation collapse
Figure 2. Further analysis will utilize TDLAS data to explore differences temperature is not exceeded during primary drying, define proper
in product resistance between the LyoCapsule™ Freeze Dryer and the shelf temperature and pressure selection for effective sublimation
LyoStar 3 Freeze Dryer. processes, plan process scale-up and perform “scale-down” experiments
to troubleshoot issues encountered in commercial freeze dryers while
utilizing minimal API.

References
1. Wang, W. (2000). Lyophilization and development of solid protein pharmaceuticals.
International journal of pharmaceutics, 203(1), 1-60.
2. Patel, S. M., Jameel, F., & Pikal, M. J. (2010). The effect of dryer load on freeze drying
process design. Journal of pharmaceutical sciences, 99(10), 4363-4379.
3. Pikal, M. J., Bogner, R., Mudhivarthi, V., Sharma, P., & Sane, P. (2016). Freeze-drying
process development and scale-up: scale-up of edge vial versus center vial heat transfer
Figure 2. Product temperatures comparisons during primary and coefficients, Kv. Journal of pharmaceutical sciences, 105(11), 3333-3343.
secondary drying between the LyoCapsule™ Freeze Dryer and the 4. Rambhatla, S., Ramot, R., Bhugra, C., & Pikal, M. J. (2004). Heat and mass transfer scale-up
LyoStar 3 Freeze Dryer for 5% mannitol. A) The wall temperature of issues during freeze drying: II. Control and characterization of the degree of supercooling.
the LyoCapsule™ Freeze Dryer was set to follow the CVT. B) The wall AAPS PharmSciTech, 5(4), 54-62.
temperature of the LyoCapsule™ Freeze Dryer was set to emulate the
5. Obeidat, W. M., Sahni, E., Kessler, W., & Pikal, M. (2017). Development of a mini-freeze
band temperature in the LyoStar 3 Freeze Dryer. LC and LS denote
dryer for material-sparing laboratory processing with representative product temperature
LyoCapsule™ Freeze Dryer and LyoStar 3 Freeze Dryer respectively.
history. AAPS PharmSciTech, 1-11.

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7 Key Factors When Considering a

Benchtop X-Ray Diffractometer
Katherine Macchiarola
Malvern Panalytical

Having managed X-ray diffraction labs at peak position accuracy, 2Theta linearity should
previous jobs and in grad school, I know that be within +/-0.02 degrees 2Theta.
there are a number of factors to consider
Low angle performance also varies from
when it comes time to add or replace X-ray
system to system. If you need to analyze
diffraction (XRD) system to your lab. Benchtop
pharmaceuticals or other organic materials,
XRD systems may be a compact and cost
clays, or mesoporous materials, low angle
effective alternative to floor standing systems.
performance should be high on your
Deciding which factors are key requirements
criteria list. Low angle reflections will be
for your needs will help you select a Benchtop
key to correct phase identification and
system ideal for your situation.
Figure 2. a requirement for accurate standardless
quantitative analysis such as Rietveld analysis.
Low angle performance can be demonstrated
Determine What Level of with an appropriate low angle standard

Performance is Acceptable such as silver behenate. Excellent low angle

performance would show clearly discernible
for Your Application peaks with correct intensity ratio down to 1
degree 2Theta.
One of the key aspects of system performance
to consider when buying a Benchtop XRD
system is resolution. Compact XRD systems
typically have a smaller radius at which Match your Work Load
Figure 3.
the X-ray tube and detector sit from the
sample being measured, and this will impact
with the Throughput of
resolution. There is a range of resolution
looking for the presence of low amounts of
polymorphs or trace phases, resolution should
the System
performance available from the Benchtop
be an important factor in your evaluation A Benchtop XRD system can have varying
XRD market, so it is important to determine
process. The ability to distinguish small peaks capacity to measure samples within a day. If
what will meet your needs. If your work is
will be critical for indicating the presence of throughput time is an important factor for
routine, involves a low number of phases in
unexpected or unwanted phases, in particular you it is worth exploring the possible options
your samples, or you are looking at primarily
with patterns that have many peaks present. and select a benchtop diffractometer optimal
nanomaterials, resolution may not be critical
Resolution is reported as full width at half for your needs.
to obtaining data of sufficient quality to meet
maximum (FWHM) of an early reflection from
your analysis goals. The allowed power settings of the instrument
a suitable XRD standard (e.g. NIST SRM 660c -
But if you are looking at complex mixtures or have a significant impact on throughput –
lanthanum hexaboride). The lower the FWHM,
operating at 40KV instead of 30KV improves
the better the resolution. Excellent resolution
measurement speed and thus throughput
for a Benchtop XRD system is less than 0.04
by 25%. Another way to reduce the
degrees 2-theta FWHM.
measurement time is to use a high speed
Another measure of performance is the line detector instead of a point detector. With
linearity of the goniometer. A highly optimized instrument setup measurement
linear goniometer provides accurate peak times can be as low as 5 - 10 minutes. With
positions over the entire 2-theta range of the such quick measurements, you then may want
goniometer. This is important if you are making to consider a sample changer on the system
critical calculations from your XRD data, such to alleviate the human sample changer. At
as unit cell size of cracking catalyst. Linearity a certain work load, it may also make sense
is determined using a line position standard to be able to use automation such as a
Figure 1.
such as NIST 640e – silicon powder. For good robotic arm or a belt to feed samples into the

68 | | September/October 2017
 WHITEPAPER »

system, or join the XRD system with sample analysis and also reporting, and all of these
preparation or complimentary technologies factors should be evaluated. In the case of
such as an X-ray fluorescence (XRF) system. a site with a transient work force such as a
university, an easy to use Benchtop system
Automation may also refer to analysis
software, which can be tailored for your needs with good documentation will be desirable.
and execute data processing, quantification,
and reporting of results to printer, network,
or LIMS system. If automation is desired for Assess the Safety of
your lab, ensure it is a possibility with the XRD
system you are evaluating. the System
The obvious safety concern with an XRD
system is radiation safety. According to the
Determine the True Cost International Commission on Radiological
Protection (ICRP) the absorbed dose of
of Ownership radiation should not exceed 1 mSv/year for a
person. When choosing a benchtop XRD you
Of course an XRD system has an initial price Figure 5.
tag, but there may be additional one-time or should check if the instrument complies with
repeated costs that contribute to a true cost this requirement.
sample preparation was sufficient to produce
of ownership. Factors to consider are that XRD The system should also be safe for the operator,
fine particles (<50µms) which then produce
systems may require: no possibility of bodily harm from the moving
solid Debye rings (left image), or if the particle
• a water chiller to provide cooling components, electricity, etc. This is assured
size is too course, resulting in spotty Debye
of the system’s X-ray tube - this wil by the compliance with the Machine Directive
rings (right image).
be another piece of equipment to 2006/42/EC and EMC Directive 2004/108/EC,
mainain, it will consume resources CE mark, and UL/CSA 61010-1 and 61010-2- It may also become apparent if the crystallites
liek water and electricity, and also 091. It is also important that the instrument are oriented either by poor sample
could be a source of instrument itself is protected from harmful intrusion preparation, or perhaps by growth of crystals
downtime if it needs repair. from objects and even dust and debris. If preferentially in certain orientations during
• lab or bottled gases or compressed air dusty environment is an issue in your lab, pay heating. Preferred orientation would exhibit
to operate components in the system attention to an Intrusion Protection (IP) rating as variations in the intensity along the Debye
of a chosen diffractometer, the higher the IP rings (top blue image) versus a consistent
• an external computer to operate
number the better. Ensure that the selected intensity all along the ring, indicating
the systems.
bentop XRD system complies with the safety randomness of the crystallite orientations.
• a lab environment with a controlled requirements in your laboratory.
range of temperature and humidity.
Also XRD systems may have power
requirements requiring the installation Availability of Options Availability of
of a new electrical service. Additionally, a
for Your Benchtop Application Support
maintenance agreement would be a recurring
cost, but may be something to consider to
XRD System and Service Support
keep your system in top running condition, or
cover you for unexpected repairs. An XRD vendor should have available
A benchtop XRD is a tool optimized for simple
application scientists to give advice on how to
routine measurements and does not offer
properly analyze your materials and answer
the flexibility of a floor-standing instrument.
questions you may have, as well as educate
Evaluate Ease of Use However, depending on a brand you choose, a
their customers through courses, seminars,
number of additional options can be available.
Those options may include a sample changer, conference presentations and webinars. Just
With X-ray diffraction, there are users who can
collect data, and then there are diffractionists different X-ray tubes, stages for heating and as important is an XRD vendor with sufficient
who can translate desired material properties cooling your sample, linear and even area service engineers to maintain and repair
to be analyzed into appropriate measurement detectors. While an area detector may not be systems in the field. Local support is key to
programs and accurate analyses. Many a well known option on a benchtop XRD, its highest uptime and best use of your investment
labs will have people with a range of XRD benefits, particularly for research and teaching – ask how many service engineers are trained to
knowledge, and a diffraction system should labs, are clear. An area or 2-D detector allows service your Benchtop XRD system, where the
be able to accommodate all. Ease of use you to visualize the Debye rings diffracting nearest service engineer is located, and whether
applies to instrument care and operation, the from a sample at characteristic positions in the instrument is serviced on site or needs to be
software for data collection as well as data 2Theta. This will immediately illustrate if the shipped to manufacturer for depot service.

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4 Factors Critical to the Financial Success of an

Emerging Pharmaceutical Company
Company structure decision making, funding approaches, perfecting the investor
pitch and hitting key milestones – the exciting and challenging journey of a growing
pharmaceutical company

Fascinating developments within the biopharmaceutical sector have

been grabbing industry headlines for years now. The field is exciting Funding Strategy
and developing rapidly. However, the reality is that small, synthetic
molecules remain critically important to both drug development The early days of a pharmaceutical company are both exciting and fraught
pipelines and the market. with challenges and risk. For example, over-capitalizing a company too
early may cause it to fail because of premature scaling (a common risk).
With all of the discussion touting advances in biologics and
Likewise, underfunding is equally risky in that the lack of capital will starve
biosimilars, some have concluded that small- molecule drugs are
opportunity and prevent growth.
on their way out. This could not be further from the truth: some of
the newest, cutting-edge drugs are small-molecule drugs, such as There are a variety of options for funding emerging pharmaceutical
aripiprazole (Ambilify) and esomeprazole (Nexium). Novel approaches companies. Careful consideration should be given to the options, as each
to molecular modelling and the advent of antibody drug conjugates, has pros and cons.
which deploy a small molecule warhead, are resulting in more targeted Investment from a Collaborating Partner – To keep their drug development
alternatives in critical fields such as oncology. Increasingly, they are pipelines full, many large pharmaceutical companies are striking
being developed to address very specific patient populations based collaborative partnerships with emerging companies. These relationships
on better understanding of genetics. Small-molecule drugs continue can have many advantages for both sides. The large company gets access
to hold a critical place in the generic pharmaceutical landscape. to cutting-edge innovation and fresh thinking. The work of the emerging
It is no surprise, therefore, that companies of all sizes – including company can move forward due to the financial investment of their larger
small and emerging companies– have ample small-molecule projects collaborator. In addition, access to expert team members, equipment
in process. There are, however, special considerations for small or and other resources is generally a critical part of the relationship for the
emerging companies, such as company structure and capabilities, emerging company. However, the downside of the relationship for the
funding strategy, ongoing investor pitches and project management smaller partner can be that resource priorities often change within large
that ensures critical clinical milestones are achieved. companies, especially if there are signs of project delay or indications
that the project is not as promising as originally hoped. If not handled
effectively by the smaller player, the project can be starved of attention
and resources, potentially killing the opportunity.
Company Structure and Capabilities Incubators – Incubators provide start-up companies with resources,
A growing company must think about the talent it requires beyond various services and access to a strong network of experts within
immediate and short-term needs. Too often, growing companies the industry. The incubator often provides facility rental for very little
try to save money by delaying hiring for critical executive positions cost as well as access to equipment and support staff. Typically, an
within their teams. While the talents and experience of a high-caliber incubator will allow an emerging company to remain in the incubation
executive for a given area might not be fully utilized when he or she program for approximately three years or until the company achieves
is first brought on board, waiting to hire until key individuals are truly specific milestones.
needed is a mistake. It is not always easy to find the right people, and Angel Investors – Angel investors invest their money in companies
critical gaps within a leadership team during important phases (e.g. or projects. They are very active in the U.S. investment community. In
moving into clinical trials) can cause long and very costly delays. fact, there are groups of angel investors, knowledgeable about a given
Additionally, emerging or growing companies must also decide space, who pool their money and act like a venture-capital (VC) firm.
which capabilities they are going to build in-house and which they Compared to other investment options, angel investors typically expect
are going to outsource. Given the extremely capable community of a lower rate of return and typically agree to longer time periods for their
contract research organizations (CRO), analytical laboratories and return – usually five to 10 years. Angel investors nearly always claim a
contract development manufacturing organizations (CDMO), many stake in the business. Often, angel investors receive stock and require a
pharmaceutical companies are deciding to stay virtual or to virtualize position on the board as part of the deal.
many of their operations. The industry has clearly moved from State and Federal Funds – Federal funding is granted by the Small
using a “build it if you have the money” approach to asking a much Business Administration through Small Business Innovation Research
more strategic series of questions relating to the value of in-house (SBIR) and Small Business Technology Transfer (STTR) programs. The
capabilities versus selecting and managing skilled contract partners. Patient-Centered Outcomes Research Institute (PCORI), the National

70 | | September/October 2017
 WHITEPAPER »

Institute of Health (NIH) and other organizations also have grant What causes missed milestones? Within emerging companies, failure to
programs, as do many states. manage the project in a coordinated manner is often the cause. Given
Venture Capital – Venture capital firms generally consist of institutional that many emerging companies are fully or partially virtual, multiple
investors; their return typically comes two to three years from an capabilities need to be secured for project completion. When companies
IPO or the sale of the company. In most cases, VC firms only invest in contract with multiple analytical laboratories, CROs and CDMOs, they
companies with the potential to make $100 million in sales within five may not have the resources to coordinate the technical teams from
years and which can be sold for at least $400 million upon exit. The multiple organizations. In addition, a non-integrated project commonly
advantage of VC funding is that it can provide the needed investment suffers from critical gaps in expertise at strategic points.
to scale a business quickly, along with access to executive management In past decades, CROs and analytical laboratories were often seen as
expertise. In fact, several big pharma companies have launched venture a “pair of extra hands” to address a lack of capacity within the client’s
capital divisions. The companies have a mechanism to invest in pipeline organization. Today, full projects are commonly outsourced, resulting in
opportunities, and emerging companies gain access to the tremendous greater complexity.
guidance and expertise within larger organizations. The downside of VC
funding is that it creates a high-pressure situation that may result in a In many cases, products themselves have become more complex. For
company moving forward faster than it really should. these reasons, a partner that can integrate an entire project – avoiding
risky technology transfers, lack of technical team coordination and
communication challenges – is very attractive for many emerging
pharmaceutical companies.
A Strong Investor Pitch Comprehensive accountability is also important. Working strategically
Given that many pharmaceutical entrepreneurs are scientists, crafting with contract partners capable of integrating a project from end to end
an effective investor pitch for an audience of non-scientist investment avoids finger-pointing and each team blaming another. With integrated
professionals is often a very challenging task. Of critical importance is approaches to drug development, there is less risk, milestones are much
to avoid bogging down potential investors with technical details that more commonly met, more robust processes are developed and the
are not relevant to the decision of whether or not to invest. end products are of higher quality.

An investor pitch should start by stating the problem or opportunity

your business is designed to address. For example, “There are 6.8
million patients in America suffering from this disorder, and no viable Conclusion
drug treatment options are currently available.” The investor pitch
then needs to describe the new product that will solve the problem or Navigating the rocky waters that emerging pharmaceutical
leverage the opportunity. If appropriate, the pitch should discuss the companies must travel is no easy task. Critical to the success of the
competitive landscape, likelihood of market acceptance, size of the effort is solid decision making relating to company structure and
market and the opportunity within it, product-development status sources of funding. Pitching potential investors on the merits of the
and regulatory-approval outlook. It should also include a description drug development project in a compelling manner is also key. At the
of the business model and overviews of intellectual product assets, the forefront of the decision-making process for any life-sciences investor
leadership team and projected investor-exit timeframe, along with a are the strategies for minimizing risk and maximizing investor returns.
financial overview and risk-mitigation.
One of the most successful ways to minimize risk and hit the clinical
The length of time allotted for pitches will vary, so the pitch milestones essential to project funding is to select a contract partner,
presentation needs flexibility to be condensed as needed for short such as Alcami, that can integrate the entire development process
meetings. Novel scientific concepts important to your business will to avoid tech-transfer risks, damaging gaps in communication and
need to be simplified, while still conveying their importance. Most accountability issues.
importantly, the presentation needs to convey the excitement and the
strength of the business and investment opportunity.

References
Hitting Key Clinical Milestones 1. “Emerging Therapeutic Company Investment and Deal Trends,” David Thomas, CFA and Chad
Wessel, Biotechnology Innovation Organization, May 2016
Although there are several different approaches for funding an 2. “A Tale of Two Startup Worlds: Biotech And Tech VC Ecosystems,” Life Sci VC, May 9, 2016
emerging pharmaceutical company, one characteristic is common to 3. “The Venture Funding Boom In Biotech: A Few Things It’s Not,” Bruce Booth, Forbes, July 23, 2015
nearly all investment- or collaborative-partner agreements: funding is 4. “New Frontiers in Pharma R&D Investment,” Eric David, Amit Mehta, Troy Norris, Navjot Singh,
nearly always conditional on hitting drug product-development and and Tony Tramontin, McKinsey & Company, February 2010
clinical milestones. Failure to meet established milestones will put the 5. “What’s Behind Those Billion-Dollar Biotech Deals?,” Damian Garde, STAT, November 28, 2016
project in considerable jeopardy. In collaborative projects with large 6. “Raising Capital As A Private Biotech: Insights From Unum Therapeutics’ Series B Round,”
pharmaceutical companies, missing milestones can cause the next Christiana Stamoulis, Life Sci VC, April 6, 2016
injection of investment to be withheld and resources to be retracted, 7. “Small Molecules: The Silent Majority of Pharmaceutical Pipelines,” Yuval Cohen, Xconomy,
essentially leaving the project to die on the vine. November 23, 2015

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Expansion of Human Bone Marrow-Derived

Mesenchymal Stem Cells in BioBLU® 0.3c
Single-Use Bioreactors
Vincent Dufey, Aurélie Tacheny, Muriel Art, Ulrike Becken, Françoise De Longueville
Eppendorf AG Bioprocess Center, Juelich, Germany

To confirm that hMSCs-BM cultured in BioBLU® 0.3c Single-Use Vessels

Scalable Stem Cell Expansion Needed retained their differentiation capacity, we used them for in vitro
osteogenic and chondrogenic differentiation assays. The cells were
The use of adult stem cells holds great promise for new cell-based able to differentiate into osteocytes and chondrocytes, respectively,
therapies and drug discovery. For their routine application, large demonstrating that their expansion in stirred-tank bioreactors did not
numbers of cells have to be produced with consistently high quality. affect multipotency.
Two-dimensional cultivation systems, such as T-flasks, are widely
used, but they are limited in terms of control and scalability. Stem
cell expansion in rigid-wall, stirred-tank bioreactors, however, Safe and Controlled Stem Cell
facilitates the precise control of critical process parameters like pH
and dissolved oxygen, and allows a more straightforward scale-up to Expansion
larger process dimensions.
BioBLU® Single-Use Vessels with maximum working volumes of 250
mL and 3.75 L were previously used for the expansion of human
induced pluripotent stem cells as cell-only aggregates and of adipose-
Stem Cell Cultivation in BioBLU® c derived mesenchymal stem cells on microcarriers. These results, and
our current data, suggest that Eppendorf BioBLU® Single-Use Vessels
Single-Use Vessels are widely applicable for the expansion of different stem cell types at
various scales.
BioBLU® c Single-Use Vessels combine the benefits of single-use
bioreactor technology with the reliable performance of conventional
glass or stainless steel bioreactors. The rigid-wall stirred-tank vessels
have been specifically designed and optimized for cultivation of
mammalian cells.
We tested the suitability of Eppendorf BioBLU® 0.3c Single-Use
Vessels (working volume 250 mL) controlled by a DASbox® Mini
Bioreactor System for the expansion of human bone marrow-derived
mesenchymal stem cells (hMSC-BM). To do so, we cultured hMSCs-BM
in parallel on two microcarriers types, Cytodex type 1 and Cytodex
type 3. Cytodex type 1 microcarriers are based on a dextran matrix
covered with positively charged groups, while a layer of denatured
collagen is covalently bound on the Cytodex type 3 dextran surface.
We obtained the best proliferation rate on Cytodex type 1 microcarriers.
The cell number increased 17.5 fold to a maximum cell density of 1 x
108 cells/bioreactor at day 14, corresponding to 4 x 105 cells/mL. On
Cytodex type 3 microcarriers, 20 days of culture were needed to reach
a maximum cell number of 7 x 107 cells per bioreactor, which is 11.5-
fold higher than the initial seeded quantity and corresponds to 2.5 x DASbox® Mini Bioreactor System equipped with
BioBLU® 0.3c Single-Use Vessels.
105 cells/mL.

72 | | September/October 2017
 SPONSORED BY
Ingredient Expertise
American Pharmaceutical Review surveyed our readers to learn what factors are most
important to them when researching excipient suppliers. Below are the results of the
survey and provide insight into the needs and concerns of excipient buyers.

Do you produce oral solid dose drugs? Rank what are the most important to you when selecting an OSD excipient
(1= most important, 6= least important)
YES
ES
49% Cost Supplier 4.83
Familiarity/
history
with the
51% N
NO
4.36 Reputation excipient
3.25 2.88 2.81 2.87

¢¢¢ ¢¢¢ ¢¢¢ ¢¢¢ ¢¢¢ ¢¢¢ ¢¢¢

What factors are important to you when Excipient Compatibility Manufacturing

considering using co-processed excipients? Stability with API method intended
for the OSD

73.4% 78.7% 78.7%

What is your company's most often used manufacturing
method for the manufacture of OSD’s?

48%
31%

19%
3%
¢¢ ¢¢¢¢¢¢ ¢¢¢¢¢¢ ¢¢¢¢¢¢ ¢¢

Existence of a Cost in use Operational

monograph benefit benefit DDirect Wet Dry Other
compression granulation granulation

When considering film coatings, When considering the use of lactose in your OSD formulation,
rank the most important factors that you consider? please rank the key factors affecting the specific grade you select.
(1=most important, 5= least important) (1= most important, 6 = least important)

2.72 CCost 2.56 CCountry of origin

2.6 TTechnical Support

4.48 SStability of the lactose grade

5.28 CCompatibility with API

2.32 SSupplier Reputation

2.92 TTechnical support

3.77 UUnique benefits that the coating may provide
2.84 GGlobal availability

3.59 RRegulatory Compliance

2.92 P
Price
» MICROBIOLOGY »

Antimicrobial Preservatives Introduction

Part Two:
The second article in this series deals with the many constraints that
face the pharmaceutical scientist tasked with developing preservation
systems for multi-use oral, topical and parenteral medicinal products.

Choosing a Preservative
The key role that pH plays in antimicrobial efficacy, as well as general
stability considerations (both chemical and physical), will be covered.

Evaluating Performance
Compendial tests1-3 for antimicrobial efficacy set high performance
standards. Furthermore, these requirements are not harmonized and
European Pharmacopeia (Ph. Eur.) standards are significantly more
David P. Elder, PhD and Patrick J. Crowley, F.R.PS challenging than US (USP) or Japanese pharmacopoeias (JP) – see
Independent CMC Consultants Table 1.
It is a regulatory requirement to assess the antimicrobial efficacy of
the drug product (in its final container) at the end of the product’s
proposed shelf-life. Antimicrobial efficacy needs to be broad spectrum,
encompassing bacteria (Gram-positive and Gram-negative), yeasts,
fungi and molds; but not viruses. An effective preservative must
reduce a microbial population significantly and prevent subsequent
re-growth and these effects must be both microcidal and microstatic
in nature (see Table 1).
Combining preservatives may help meet performance standards.
Benzalkonium chloride (BKC) is ineffective against some strains of
Pseudomonas aeruginosa, Mycobacterium and Trichophyton4 but
combinations with EDTA, benzyl alcohol, 2-phenylethanol or
3-phenylpropanol enhances anti-Pseudomonad activity.5 Synergy is
also obtained in combination with cetrimide, 3-cresol, chlorhexidine
and organomercurials.6,7 The amino benzoic acid esters (parabens) are
more active against Gram-positive than Gram-negative bacteria, and
more active against yeasts and molds than bacteria. The antimicrobial
activity of these esters increases with increased alkyl chain length (bu-
tyl > propyl > ethyl > methyl) but aqueous solubility commensurately
decreases and consequently the parabens are often used in combi-
nation, e.g. methyl and propyl paraben to ensure adequate solubility

74 | | September/October 2017
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Table 1.
Product Oral Preparations Topical, Nasal Sprays and Metered Dose Inhalants (MDIs)
Preparations
Pharmacopeia Bacterial Requirements Fungal/Mold Requirements Bacterial Requirements Fungal/Mold Requirements

USP <51>1 1 log reduction from initial count, i.e. no increase from initial calculated count 2 log reductions from initial count, i.e. no increase from initial calculated count
106 after 14 days and no increase in at 14 and 28 days 106 after 14 days and no increase in at 14 and 28 days
count over the following 14 days count over the following 14 days

JP <19> Preservatives – 1 log reduction from initial count, i.e. no increase from initial calculated count 2 log reduction from initial count, i.e. no increase from initial calculated count
Effectiveness Tests 106 after 14 days and no increase in at 14 and 28 days 106 after 14 days and no increase in at 14 and 28 days
count over the following 14 days count over the following 14 days

Ph. Eur. 5.1.32 Efficacy of 3 log reduction from initial count, i.e. 1 log reduction from initial count, i.e. Criteria A; 2 log reduction from initial Criteria A: 2 log reduction from initial
Antimicrobial Preparation 106 after 14 days and no increase in 106 after 14 days and no increase in count, i.e. 106 after 2 days, 3 log count, i.e. 106 after 14 days and no
count over the following 14 days count over the following 14 days reduction after 7 days and no increase increase in count over the following
in count over the following 14 days 14 days

Criteria B: 3 log reduction from initial Criteria B: 1 log reduction from initial
count, i.e. 106 after 14 days and no count, i.e. 106 after 14 days and no
increase in count over the following increase in count over the following
14 days 14 days

1. USP. has additional requirements for antacids made from aqueous base of no increase from initial calculated count for all challenge organisms, i.e. bacteria, yeasts and molds
2. In Ph. Eur., the oral category includes oral, oro-mucosal and rectal preparations

in the vehicle. Parabens esters also show some synergy with EDTA,8 Table 2.
2-phenylethanol9 and imidurea.10
Preservative pH of optimum Reference
The complexity of multi-phase dermal products, formulated as creams, activity
lotions or ointments mean that adequate antimicrobiial efficacy may Aminobenzoate esters e.g. Parabens pH 4-8 [13]

not be readily attainable due to partitioning of preservative to the Quartemary ammonium compounds pH 4-10 [4,14]
(QACs) e.g. Benzalkonium Chloride (BKC),
lipophilic (oil) phase of the system. The best that might be achieved
Benzethonium chloride
is a microstatic effect. In practical terms such performance may be
Aryl acids e.g. Benzoic acid/salts <pH 4.5 [15]
acceptable. If the bioburden is low most preservative systems can
Aryl alcohols e.g. Benzyl alcohol <pH 5.0 [16]
adequately kill or attenuate growth of most organisms. Current
Quartemary ammonium compounds pH 7-9 [17]
GMP (good manufacturing practice) standards, encompassing
(QACs) e.g. Cetrimide
operating and sampling procedures, controls on input materials, use
Biguanides e.g. Chlorhexidine pH 5-7 [18]
of clean room and automated technologies in manufacturing and
Chlorocresol pH 4-9 [19]
packaging, when viewed holistically ensure that high standards of
Chloroxylenol Little pH effect [20]
microbial cleanliness can be routinely achieved in finished products.
Additionally, the state-of-the-art in packaging technology is now Formaldehyde donators e.g. Imidurea pH 3-9 [21]

such that contamination, prior to use is unlikely. Hence the risk of Formaldehyde donators e.g. Bronopol pH 5-8 [22]

contamination is probably greatest during patient use of these multi- Alkyl acids e.g. Propionic Acid pH 3.9 [23]
dose liquid products. Microstasis may be an acceptable performance Alkyl acids e.g. Sorbic acid/salts pH 4.5 [24]
standard for non-parenteral products at this stage, if the in-use period Phenolic compounds e.g. Phenol, m-cresol pH 4-9 [25,26]
is short (< 1-month).
Phenylmercuric salts e.g. acetate, borate, pH 5-8 [27,28,29]
nitrate

Thiomersal pH 5-8 [30]

Influence of Product pH
product attributes as well as the activity of the preservative system. pH
pH can affect the rate of growth of microbes, the interaction of the
effects also reflect the chemical structure of the preservative molecule.
preservative with cell wall components and the MIC (minimum
For instance, if activity is associated with the non-ionized moiety (i.e.
inhibitory concentration) of many preservatives.11,12 In general,
microbial growth is optimal between pH 6-8. Outside this range acids, alcohols and phenols) the effect is usually optimal at acidic pH
growth rate declines. Product pH may reflect the intrinsic pH of the but ultimately reflects the pKa of the individual preservative agent.
active pharmaceutical ingredient (API), or the product may require However, and almost inevitably, there are exceptions. For example,
pH modification to enhance solubility, stability, palatability or optimal phenol is most active in acidic solutions, despite its high pKa (10.0).
antimicrobial effectiveness (MICmax). Table 2 lists pH ranges for Substituted alcohols are also less reliant on pH. Bronopol (2-bromo-
optimum activity for common preservatives. 2-nitro-1, 3-propanediol) is not markedly influenced by pH in the
Excipients in a product may also influence product pH. Hence, pH range 5.0-8.0, perhaps reflecting that its main activity is via release of
adjustment to regions less favorable to microbial viability i.e. away from formaldehyde, whose microcidal activity is not significantly influenced
pH 6-8, may not be feasible or must take account of effects on other by pH.22

76 | | September/October 2017
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Phenolic preservatives tend to be active over a wider pH range than this is rarely the case; pragmatic decisions and compromise are often
alcohols or acids, but are less desirable for a number of reasons. required such as the selection of a formulation pH which is sub-
Chlorocresol19 is most effective in acidic solutions but can retain optimal for antimicrobial efficacy but is better aligned with product
activity at pH regions up to its pKa (9.2). Similarly, 3-cresol25 is effective performance. In those cases, higher concentrations of preservatives
at pH values below its pKa (9.6). Solution pH does not have a marked may be required (Table 3). Thus, sorbic acid has a pKa of 4.76 and the
effect on the anti-microbial efficacy of 4-chloroxylenol.31 recommended level of preservative is 0.2% at pH 4.5.24 At this pH,
64.5% of the preservative is unionized (the effective form), the effective
Esterification of acids can extend the pH span of antimicrobial activity.
concentration of unionized sorbic acid being 0.13%. Table 3 shows the
The parabens (benzoic acid esters) are active over the range pH 4-8.
effect of formulation pH on the required concentration of preservative.
Efficacy decreases at higher pH due to the formation of phenolate
If the optimal product pH was 6.0, from stability, solubility, palatability
ion (pKa ca. 8.4) and ester hydrolysis at approximately pH 9. Efficacy
perspectives etc the concentration of the preservative would need
increases with the longer alkyl chain, but conversely aqueous solubility
to be increased 11.9-fold for adequate preservation compared with
decreases as hydrophobicity increases.13
formulating the product at an optimal pH for preservation , i.e. pH 4.5.
In contrast to acid preservatives, the quaternary ammonium
compounds (QACs) such as benzalkonium chloride (BKC) and
benzethonium chloride demonstrate anti-microbial efficacy over a
wide pH range (pH 4-10), activity being associated with the ionized
Factors that Compromise
(cationic) moiety and optimal at high pH values.4,11,14 Efficacy is also Preservative Efficacy
linked with alkyl chain length (C18 > C16 > C14 > C12). Cetrimide17
has a slightly narrower effective pH range (7-9), probably caused by Preservatives possess reactive functional groups and may have
the presence of a methyl rather than a benzyl moiety (less effective pH-solubility profiles to be considered when formulating the drug
at stabilizing the charge). High pH causes the microbial cell wall to be product. The optimal pH for microbial efficacy may not be the same as
negatively charged, thereby favoring the binding of cationic species. that required for drug solubility and/or stability, requiring compromise
and pragmatic choices. Preservative efficacy can also be affected by
There are no reported pH constraints on the permeation enhancing
interactions with active ingredients, excipients, container/closures
capability of EDTA, probably a consequence of its multiple pKa values.
or other physicochemical behaviors. Reduction in microbial efficacy
However, its limited intrinsic anti-microbial efficacy means that it is
can occur during manufacture, throughout the product’s shelf life or
rarely used on its own, but in combination with other preservatives. 32,33
during the in-use period. These effects can be ascribed to:
pH-related effects can sometimes be more complex than those
• interactions with other components within the product
summarized in Table 1. The antifungal activity of benzoic acid is less
(drug, excipients, pack or delivery device).
susceptible to pH than are its antibacterial effects.15 The substituted
benzoic acid derivative thiomersal, which has a pKa of 3.1 is • chemical instability of the preservative.
bacteriostatic and fungistatic at neutral and even mildly alkaline pH’s. • physical losses or changes in preservative levels in solution
However, the antimicrobial activity of the organic mercury component
Possibilities for chemical degradation are manifold, but the risk
also needs to be taken into account.30 A similar effect is evident with
can be mitigated at the outset by a thorough risk assessment, an
propionic acid23 and sorbic acids,24 which have appreciable antifungal
understanding of all the product components and by appropriate
but little or no antibacterial activity at pH 6.0, but this effect may be
preformulation studies to determine interaction propensity. It is
concentration dependent.
important that such knowledge and awareness is generated at the
Biguanide anti-microbials are generally active over the pH range 3-9. product design stage utilizing QbD (Quality by Design) concepts and
However, chlorhexidine is effective over a narrower pH range (5-7) approaches. Pharmaceutical products generally have much longer
while chlorhexidine base may precipitate from aqueous solutions at shelf life requirements, i.e. 24-36 months at ambient temperatures
pH values greater than 8.18 Imidurea is effective over the pH range than food or beverage products; antimicrobial efficacy must be
(3-9), although optimum efficacy is seen at acidic pH.21 Organo- retained over these periods and during product use. The relatively
mercurial preservatives such as phenylmercuric salts, have broad
spectrum bactericidal and fungicidal activities, being more potent Table 3.
with increasing pH. Efficacy against Pseudomonad’s has also been pH of % unionized Recommended Increased level
demonstrated at pH 6 or below.27-29 These preservatives have been formulation Preservative levels of of preservative
utilized in several ophthalmic products having acidic pH values. Activity preservative (%)24 (multiple of
level at pH 4.5)
is enhanced at acidic pH in the presence of sodium metabisulphite,
4.5 64.5 0.20 N/A
which can enhance activity at low pH, but has the opposite effect at
5.0 36.5 0.35 1.75
alkaline pH.34,35 In topical products phenylmercurate salts have been
5.5 15.4 0.84 4.2
reported as being active at pH 5-8.36
6.0 5.4 2.39 11.9
In the ideal world the pH of optimal microbial efficacy is aligned with
6.5 1.8 7.17 35.8
pH of optimal product stability, solubility or palatability, etc. However,

78 | | September/October 2017
 « MICROBIOLOGY »

insensitive nature of preservative efficacy tests1-3 could mean that WE’RE THE PEOPLE
modest but inexorable deterioration of antimicrobial effectiveness CREATING DATA INTEGRITY
during shelf-life storage may take time to be manifested or significant.
A consequent reformulation and evaluation program can delay IN YOUR LAB.
product development timelines.
AND MINIMIZING HEADACHES.

Chemical Stability of Preservatives

Preservative levels during product shelf life need to remain within
limits that ensure acceptable antimicrobial efficacy. It should not
come as a surprise that most acidic preservatives e.g. benzoic, sorbic
and propionic acids are incompatible with strong bases.15,23,24 Strong
oxidizing agents degrade sorbic acid,24 2-phenylethanol,37 hexetidine,38
EDTA,32 thimerosal,30 propyl gallate39 and butylated hydroxyanisole
(BHA).40 This latter material is particularly unstable in the presence
of peroxides and permanganates and prolonged interaction may
even result in spontaneous combustion.40 Although, the presence of
such reactive materials in a dosage form might be unusual (although
benzoyl peroxide is formulated in topical lotions to treat acne),
excipients such as povidone, crospovidone, polyethylene glycol and
polysorbates may contain residual peroxides.41 These may be present
at low concentrations, but a high excipient-to-preservative ratio could
cause significant interaction and degradation.
The antimicrobial efficacy of several preservatives is also compromised
by surface-active agents that are common formulation additives,
particularly for poorly wetting active ingredients. Benzalkonium
chloride,4 benzethonium chloride14 and cetrimide,17 are cationic in
nature and incompatible with anionic surfactants or other excipients
Manually reading plates can be
such as the negatively charged sodium carboxymethyl cellulose.
synonymous with time, frustration, and
Benzyl alcohol,16 2-phenoxyethanol,42 4-chloroxylenol20 and 3-cresol25 potential for error. EviSight™ Compact
should not be formulated with non-ionic surfactants. Chlorobutanol43 — our smar t plate-reading incubator —
and 2-phenylethanol37 are also adversely affected by the presence of can detect colonies at 40 -250 microns;
non-ionic surfactants such as. Polysorbate 80.
providing high-resolution color images
Such interactions may not involve conventional chemical and enumeration in real time. Get alarms
transformations, but include more subtle binding phenomena e.g. for out-of-spec results earlier, reduce
hydrogen bonding, aggregation and complex formation. Although double reading of plates and ensure
the overall level of preservative in the product may not change (as
complete data integrit y.
measured by chemical analysis) the preservatives may be bound
to such excipients and not available in the “free” form; efficacy may To learn more about EviSight™

be reduced. Determination of preservative efficacy is therefore Compact and the other products
mandated.1-3 It is also important that analytical techniques to monitor our people are creating for you, visit
preservative content in stability testing programs are designed to thepeoplebehindthescience.com
determine levels in solution in liquid formulations or in the aqueous
Together, we are the people behind
phase in complex biphasic systems. Preservative needs to be in
the science.
aqueous solution to evince an antimicrobial effect.
Some preservatives are incompatible with other preservatives. EDTA
interacts with thimerosal, propyl gallate and phenylmercuric salts32.
Chlorhexidine can interact with benzoic acid18 whereas cetrimide is
incompatible with phenyl mercuric nitrate17. Such interactions can
limit the choice of preservative combinations.
Most available preservatives seem ostensibly to possess stable
chemical structures. This may explain why reports of intrinsic

www.americanpharmaceuticalreview.com | | 79
» MICROBIOLOGY »

chemical instability (i.e. that do not involve interaction with benzyl alcohol,16 2-phenoxyethanol,42 3-cresol,25 chlorocresol19 and
other product components) are not widespread. Paraben13,44,45 chlorbutanol43 are all volatile to greater or lesser extents. This renders
preservatives are susceptible to base-catalyzed ester hydrolysis at them susceptible to processing losses by sublimation or evaporation
high pH, degrading by classic pseudo-first order kinetics, shorter during product manufacture or throughout product life. 3-Cresol25
chain analog such as methylparaben being least stable.13 Stability and phenol26 are not suitable as preservatives for preparations that
in solution is not markedly affected by pH up to about pH 6.5, but need to be lyophilized due to their volatility. In addition, if any of the
degradation rates increase at pH 7.5 and above.46 As parabens container/closure components are permeable to gases, e.g. plastic
show antimicrobial efficacy over the pH range 4-8 caution may bottles or elastomeric closures, then this can result in the depletion
be advisable where product pH is likely to be higher than neutral. of these volatile preservatives during long term product storage.
Unfortunately, there are few if any alternative preservatives that are Polyvalent ions may cause precipitation of certain preservatives from
efficacious at neutral to slightly alkaline pH values. In the light of the solution, due to H-bonding,for example:
predictable and well characterized degradation kinetics of these
agents scientifically relevant accelerated (high temperature) stability • sorbic acid24 and chlorhexidine18 can be “salted out” by
Ca2+ ions.
studies at the formulation development stage may predict long term
stability in the final product. The formulation scientist may then • chlorobutanol44 and chlorhexidine18 interact with Mg2+ ions.
have the option to include overages to compensate for any losses.
• bronopol22 and phenylmercuric nitrate29 can be precipitated
The guiding principle with respect to inclusion levels is that these
by Al3+ ions.
be minimal but commensurate with adequate preservative efficacy
at the end of shelf-life.47 If the formulation pH is sub-optimal from an • Fe3+ ions can salt out BHA40 and butylated hydroxytoluene
antimicrobial effectiveness perspective higher concentrations than (BHT).51
typically accepted may be appropriate (Table 3). • EDTA32 is precipitated by most polyvalent cations.
Higher product pH values (>pH 8) are often inimical to microbial The overall level of the preservative in the product may remain
viability but effects on preservative stability also need consideration. unchanged but free concentration in solution is diminished due to
Despite its many advantages as a preservative sorbic acid is relatively precipitation or adsorption, reducing antimicrobial efficacy. Analytical
unstable in semi-solid and liquid preparations. The principal techniques to monitor preservative content need to reflect such
degradation pathway is via auto-oxidation resulting in acetaldehyde possibilities viz determine the level of preservative in solution, in the
and β-carboxyacreloin end-products as well as numerous other volatile aqueous phase in a complex liquid such as a cream or lotion.
aldehydes, e.g. malonaldehyde, acrolein, crotonaldehyde and related
Adsorptionby excipients, especially those with large surface areas or on
furans (2-methylfuran, 2-acetyl-5-methylfuran, 2,5-dimethylfuran).48
to container/closure systems can also bind and remove preservative(s)
Sorbic acid may be stabilized by phenolic anti-oxidants, for example
from solution. Table 4 lists examples.
0.02% w/w propyl gallate39 but presence of an anti-oxidant in the
formulation needs to be justified. Physical and chemical interactions can make the antimicrobial
preservation of antacid-containing formulations challenging. The pH of
Macromolecules can be adversely affected by preservatives. Benzyl
such products is typically neutral to slightly alkaline, i.e. pH 7-8, where
alcohol causes aggregation of recombinant human interferon
intrinsic preservative activity can be low. Additionally, the presence
(rhIFN), recombinant human granulocyte stimulating factor rhGCSF)
of polyvalent cations (e.g. Al 3+, Ca 2+, Mg 2+) in antacid products can
and, human interleukin-1 receptor antagonist (rhIL1ra).49 Several precipitate the preservative. Adsorpton on to the insoluble antacid is
biopharmaceutical products mandate that the diluent for constitution
must not contain preservative(s) because of potential adverse
Table 4. Preservatives Susceptible to Adsorption
interactions with the anti aggregant Polysorbate 80, present in
most recent biopharmaceuticals, particularly monoclonal antibody- Preservative Adsorbent/Substrate Reference
containing products. Benzalkonium chloride Hypromellose 52
Filter Membranes 53
Preservatives for multidose insulin preparations must be chosen Benzoic acid Kaolin 54
carefully. Insulin zinc suspensions cannot contain phenol as it destroys Benzyl alcohol Polyethylene, Natural Rubber 55
the crystallinity of the insulin; mixtures of parabens are used instead.
Cetrimide Bentonite 17
In contrast, neutral protamine insulin requires the presence of phenol
Chlorbutanol Polyethylene 56
or meta-phenol to form and preserve the crystal form that provides
Chlorhexidine Various polymeric excipients eg Na 57
the long-acting effect.50 carboxymethylcellulose

p-aminobenzoate esters Ion Exchange Resins, some plastics 58, 59

Phenoxy ethanol PVC, Cellulose-based excipients 60, 61

Physical Stability of Preservatives Phenylmercuric salts Various suspending agents 62-65

Sorbic acid/sorbates Polypropylene, PVC, Polyethylene 24

Preservative content in medicinal products can be depleted during
Thiomersal Polyethylene, other plastics, rubber 66, 67
manufacture, storage or use. The parabens.13,45,46 benzoic acid,15

80 | | September/October 2017
 « MICROBIOLOGY »

also possible. Such effects are additive and obvious synthons may act as coformers. Methylparaben has been reported as forming cocrystals
contribute to loss of preservative efficacy with the anti-malarial quinidine71 and with β-lactam antibiotics.72
making antacid suspensions notoriously
The chlorinated preservatives, chlorobutanol,43 chloroxylenol20 and chlorhexidine18 can
difficult to preserve.. This is reflected in lower
partition onto polymeric suspending agents by competitive displacement of water of
acceptance criteria for Antacids (category 4
solvation. Similarly, the antimicrobial efficacy of 2-phenoxyethano42 is reduced in the
products) in USP <51>1 viz ‘No increase (in
presence of cellulosic suspending agents such as methylcellulose, sodium carboxymethyl
bacteria, yeasts and molds) from the initial
cellulose and hydroxypropyl methylcellulose.61
calculated count at 14 and 28 days’ (footnote
to Table 1).
Multiphase semi-solid products such as
creams, ointments, foams and lotions, as well
as some parenteral and nasal/ophthalmic
products, can have aqueous and oily
phases stabilized by surface active agents.
Viscosity enhancers can also be included as
suspending agents. Such agents can remove
preservatives from the solution phase as
described earlier.
Preservatives can partition between oil

ACCELERATE YOUR
and aqueous phases and at the interface
containing the surface-active agent
(depending on partition coefficient) reducing
aqueous concentration and compromising
the antimicrobial effect. Such behaviors can
reduce efficacy of the parabens preservatives,
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Some preservatives can form ion-pairs (or in
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substances , e.g. timolol with sorbic acid. seamless scale-up and robust solutions for
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www.americanpharmaceuticalreview.com | | 81
» MICROBIOLOGY »

The possibilities for reduced anti-microbial efficacy in multi-phase 11. R.A. Fassihi, Preservation of Medicines against Microbial Contamination, in: S.A.Block (Ed.)
systems, has engendered efforts to devise in silico predictive Disinfection Sterilization and Preservation, 4th Edition, Lea and Febiger, 1991, pp. 871-886.
approaches to determine the impact of formulation parameters on 12. W.B. Hugo, A.D. Russell (Eds.), Pharmaceutical Microbiology, 6th Edition, Blackwell Science,
1998, pp. 201-262 and 365-373.
preservative activity. The influence of partition coefficients, binding
constants (surfactants and polymers), and oil-in-water ratios have all 13. R.Johnson, R.Steer, Methylparaben Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.),
Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 466-
been investigated but with limited success.12 To date the pragmatic
470.
approach, involving optimizing the preservation system and
14. A.H. Kibbe: Benzethonium Chloride Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.),
inclusion levels by conventional assessment techniques remains the Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 64-65.
desired approach.
15. P.J.Weller, Benzoic Acid Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 66-68.
16. E.Cahill, Benzyl Alcohol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
Conclusions Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 69-71.
17. S.C.Owen, Cetrimide Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
Preservatives, either singly or in combination remain necessary to Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 152-154.
prevent microbial contamination of multi-use aqueous liquid or semi- 18. S.C. Owen, Chlorhexidine Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook
solid medicinal products by opportunistic pathogens. Non-inclusion of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 163-167.
can result in serious patient health consequences. There are a limited 19. S.Nema, Chlorocresol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
number of approved preservatives that can be included in such multi- Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 171-173.
use oral or topical medicinal products and the number is constrained 20. L.M.E.McIndoe, Chloroxylenol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.),
Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 180-
even further in parenteral products. The optimal conditions for
181.
preservative efficacy (pH, physical and chemical stability) may not be
21. R.T.Guest, Imidurea Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
the same as for the drug product.Compromisesmay be necessary to
Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 359-361.
ensure an optimal product shelf-life.
22. S.P.Denyer, N.A.Hodges, Bronopol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.),
Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 76-78.
23. G.E.Amidon, Propionic Acid Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook
Acknowledgements of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 617-618.
24. W.Cook, Sorbic Acid Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
Dr. Paul Newby and Dr. Don Singer, GSK for their review and comments Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 710-712.
on this manuscript. 25. L.Y.Galichet, m-Cresol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 208-210.
26. R.T.Guest, Phenol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
References Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 514-516.
27. S.E.Hepburn, Phenylmercuric acetate Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.),
1. United States Pharmacopeia General Chapter <51> Antimicrobial Effectiveness Testing, USP Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 521-
34-NF29, US Pharmacopeia, Rockville, Maryland, USA, 2010. 523.
2. European Pharmacopoeia 5.1.3, Efficacy of Antimicrobial Preservation, EP 6.4, European 28. S.E.Hepburn, Phenylmercuric borate Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.),
Directorate for Quality of Medicines, Strasbourg, France, 2010. Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 524-
525.
3. Japanese Pharmacopeia, General Information: 19. Preservative Effectiveness Test, 15th
Edition, Society of Japanese Pharmacopeia, Tokyo, Japan. 29. S.E.Hepburn, Phenylmercuric nitrate Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.),
Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 526-
4. A.H. Kibbe: Benzalkonium Chloride Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), 529.
Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 61-63.
30. P.J.Weller, Thimerosal Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
5. R.M.E. Richards, R.J. McBride, Enhancement of Benzalkonium Chloride and Chlorhexidine Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 777-779.
Acetate Activity against Pseudomonas aeruginosa by Aromatic Alcohols, J. Pharm. Sci., 62:
31. J.Judis, Studies on the Mechanism of Action of Phenolic Disinfectants, J. Pharm. Sci., 51: 261-
2035-2037 (1973).
265 (1962).
6. P.J. Hugbo, Additivity and Synergism In Vitro as Displayed by Mixtures of Some Commonly 32. S.C. Owen, Edetic Acid Monograph, in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Handbook of
Employed Antibacterial Preservatives, Can. J. Pharm. Sci., 11: 17-20 (1976). Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, pp. 260-263.
7. T.J. McCarthy, J.A., Myburgh, N. Butler, Further Studies on the Influence of Formulation on 33. G.Whalley, Preservative Properties of EDTA, Manuf. Chem., 62: 22-23 (1991).
Preservative Activity, Cosmet. Toilet., 92: 33-36 (1977).
34. J.Buckles, M.W.Brown, G.S Porter, The Inactivation of Phenylmercuric Nitrate by Sodium
8. T.E. Haag, D.F. Loncrini, Esters of para-Hydroxybenzoic Acid, in: J.J. Kabara (Ed.), Cosmetic and Metabisulphite, J. Pharm. Pharmac., 23: 237S-238S (1971).
Drug Preservation, Marcel-Dekker, New York,1984, pp. 63-77.
35. R.M.E. Richards, A.F. Fell, J.M.E. Buchart, Interaction between Sodium Metabisulphite and
9. R.M.E. Richards, R.J. McBride, Phenylethanol Enhancement of Preservatives Used in PMN, J. Pharm. Pharmac, 24: 999-1000 (1972).
Ophthalmic Preparations, J. Pharm. Pharmac., 23: 141S-146S (1971).
36. M.S. Parker, The Preservation of Pharmaceuticals and Cosmetic Products, in Principles and
10. W.E. Rosen, P.A. Berke, T. Matzin, A.F. Peterson, Preservation of Cosmetic Lotions with Practices of Disinfection, Preservation and Sterilization, Editors: A.D.Russell, W.B. Hugo,
Imidizolidinyl Urea plus Parabens, J. Soc. Cosmet. Chem., 28: 83-87 (1977). W.B. G.A.J. Ayliffe, Oxford, Blackwell Scientific, (1982) pp. 287-305.

82 | | September/October 2017
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» MICROBIOLOGY »

37. S.C.Owen, 2-Phenylethanol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), 57. R.T. Yousef, M.A. El-Nakeeb, S. Salama, Effect of Some Pharmaceutical Materials on the
Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, Bactericidal Activities of Preservatives, Can. J. Pharm. Sci., 8: 54-56 (1973).
pp. 519-520. 58. D.P.Elder, A. Park, P. Patel, N. Marzolini, Development of a Palatable Liquid Formulation of
38. D.S.Jones, C.P.McCoy, Hexetidine Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller a Bitter Tasting Drug Using Ion-Exchange Resins for Taste Masking, Editor J.A. Greig, in Ion
(Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, Exchange at the Millenium Imperial College Press, pp. 306-313, (2002).
2006, pp. 323-324. 59. K.Kakemi, H. Sezaki, E. Arakawa, Interactions of Parabens and other Pharmaceutical
39. P.J.Weller, Propyl Gallate Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), Adjuvants with Plastic Containers, Chem. Pharm. Bull., 19: 2523-2529 (1971).
Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, 60. M.G. Lee, Phenoxyethanol Absorption by Polyvinyl Chloride, J. Clin. Hosp. Phar., 9: 353-355
pp. 619-621. (1984).
40. R.T.Guest, Butylated Hydroxy Anisole Monograph in: R.C. Rowe, P.J. Sheskey, P. J. 61. T.R.R. Kurup, L.S.C. Wan, L.W. Chan, Interaction of Preservatives with Macromolecules, Part
Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical II: Cellulose Derivatives, Pharm. Acta. Helv., 70: 187-193 (1995).
Press, 2006, pp. 79-80.
62. K. Eriksson, Loss of Organomercurial Preservatives from Medicaments in Different Types of
41. P.J. Crowley, L.G. Martini, Drug-Excipient Interactions, Pharm. Technology Europe, Containers, Acta. Pharm. Suec., 4: 261-264 (1967).
13(3): 26-34 (2001).
63. J.A. Aspinall, T.D. Duffy, M.B. Saunders, C.G. Taylor, The Effect of Low Density Polyethylene
42. S.C.Owen, 2-Phenoxyethanol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller Containers on Some Hospital - Manufactured Eye drop Formulations I: Sorption of
(Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, Phenylmercuric Acetate, J.Clin. Hosp. Pharm., 5: 21-29 (1980).
2006, pp. 517-518.
64. J.A. Aspinall, T.D. Duffy, C.G. Taylor, The Effect of Low Density Polyethylene Containers
43. R.A.Nash, Chlorobutanol Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller (Eds.), on Some Hospital - Manufactured Eye drop Formulations II: Inhibition of Sorption of
Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press, 2006, Phenylmercuric Acetate, J.Clin. Hosp. Pharm., 8: 223-240 (1983).
pp. 168-170.
65. T.J. McCarthy, Interactions between Aqueous Preservative Solutions and their Plastic
44. R.Johnson, R.Steer, Propylparaben Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller Containers, Pharm. Weekbl.,107: 1-7 (1972).
(Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press,
66. S. Wiener, The interference of Rubber with the Bacteriostatic Action of Thiomersalate, J.
2006, pp. 629-632.
Pharm. Pharmacol., 7: 118-125 (1955).
45. R.Johnson, R.Steer, Butylparaben Monograph in: R.C. Rowe, P.J. Sheskey, P. J. Weller
67. J. Birner, J.R. Garnet, Thimerosal as a Preservative in Biological Preparations. III: Factors
(Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical Press,
Affecting the Concentration of Thimerosal in Aqueous Solutions and in Vaccines Stored in
2006, pp. 83-85.
Rubber-Capped Bottles, J. Pharm. Sci., 53: 1424-1426 (1964).
46. S.M. Blaug, D.E. Grant, Kinetics of Degradation of the Parabens; J.Soc Cosmetic
68. M.Higashiyama, K.Indana, A.Ohtori, K.Kakehi, NMR Analysis of Ion Pair Formation between
Chemistry 25: 495-506 (1974).
Timolol and Sorbic Acid in Ophthalmic Preparations, J.Pharm. Biomed. Anal., 43: 1335-
47. Draft Note for Guidance on Excipients, Antioxidants and Antimicrobial Preservatives 1342 (2007).
in the Dossier for Application for Marketing Authorisation of a Medicinal Product,
69. S. Gangavaram, S. Raghavender, P. Sanphui, S.Pal, S. G. Manjunatha, S. Nambiar, A. Nangia.
Committee for Proprietary Medicinal Products (CPMP), The European Agency for the
Polymorphs and Cocrystals of Nalidixic Acid. Cryst. Grow. Design., 12(10), 4963-4971
Evaluation of Medicinal Products Evaluation of Medicines for Human Use, CPMP/
(2012).
QWP/419/03, London 20th February 2003.
70. S.L. Childs. Crystal engineering approach to forming co-crystals of amine hydrochlorides
48. S.Yarramaraju, V.Akurathi, K.Wolfs, A. Van Schepdael, J.Hoogmartens, E.Adams,
with organic acids. Molecular complexes of fluoxetine hydrochloride with benzoic, succinic,
Investigation of Sorbic Acid Volatile Degradation Products in Pharmaceutical
and fumaric acids. J. Am. Chem. Soc.,126, 13335–42 (2004).
Formulations using Static Headspace Gas Chromotography, J. Pharm. Biomed. Anal.,
44: 456-463 (2007). 71. M. Khan, V. Enkelmann, G. Brunklaus. Crystal engineering of pharmaceutical co-crystals:
Application of methy paraben as molecular hook. JACS, 132(14), 5254-5263 (2010).
49. B.K.Meyer, A.N.B Hiu, L Shi, Antimicrobial Preservative Use in Parenteral Products:
Past and Present, J.Pharm.Sci, 96 (12) 3155-3167 (2007) 72. J.G. Amos, J.M. Indelicato, C.E. Passini, S.M. reutzel. Bicyclic beta-lactam/parabens
complxes. US Patent 5,412,094, May 2, 1995.
50. J.Brange, Galenics of Insulin, Springer-Verlag (1987) Berlin.
51. R.T.Guest, Butylated Hydroxy Toluene Monograph in: R.C. Rowe, P.J. Sheskey, P. J.
Weller (Eds.), Handbook of Pharmaceutical Excipients, Fifth Edition, Pharmaceutical
Press, 2006, pp. 81-82. Author Biographies
52. R.M.E.Richards, Effect on Hypromellose on the Antibacterial Activity of Benzalkonium
Chloride, J.Pharm. Pharmacol., 28:264 (1976). David P. Elder has 40-years of experience in the pharmaceutical industry.
He was formerly a director in the pre-clinical group at GSK and is now an
53. T.Bin, A.K. Kulshreshtha, R.Al-Shakshir, S.L. Hem, Adsorption of Benzalkonium
Chloride by Filter Membranes: Mechanisms and Effect of of Formulation and independent CMC consultant. He has a PhD from Edinburgh University,
Processing Parameters, Pharm. Dev. Technol., 4: 151-165 (1999). UK. He is a member of the British Pharmacopoeia Commission and
54. C.D.Clarke, N.A. Armstrong, Influence of pH on the Adsorption of Benzoic Acid by an FRSC. He has written and lectured widely on the theme of product
Kaolin, Pharm. J., 209: 44-45 (1972). development and the challenges of preservation.
55. M.S.Roberts, A.E. Polack, G. Martin, H.D. Blackburn, The Storage of Selected
Substances in Aqueous Solution in Polyethylene Containers, Int. J. Pharm., 2: 295-
Patrick Crowley is a pharmacist by training (FRPhSGB). He worked in
306 (1979). the Pharmaceutical Industry for over 40 years and was a VP of product
56. N.E. Richardson, D.J.G. Davies, B.J. Meakin, D.A. Norton, The Interaction of development at GSK. He currently operates as a consultant and teaches
Preservatives with Polyhydroxymethylmethacrylate (polyHEMA), J. Pharm. Pharmaceutical Sciences at a number of Institutions. Has authored/
Pharmacol., 30: 469-475 (1978). presented on over 40 topics related to pharmaceutical sciences.

84 | | September/October 2017
The Pharma & Device Packaging & Labeling West Coast event will be returning for the 2th year this
November. For 2017 the program will once again cover every step needed to ensure great results in the
market for your product, led by pharma and medical device companies in the local industry.
This year the program brings you plenty of opportunity to get involved, with panel discussions and
roundtables. With representation from Allergan, Amgen, Genentech, Stryker, Nektar, NuVasive and
plenty of small to mid-sized pharma and medical devices, there is really something for everyone.
This year’s agenda will cover every step needed to ensure great results in the market for your
product, and will bring numerous opportunities for you to get involved in interactive panel
discussions and roundtables.

Key speakers revealed Feedback from last year

n John W. Smith, Director, Regulatory “I enjoyed the opportunity to meet other
Affairs, Allergan people in the industry and share learnings.
n Anthony Bantug, Principal Engineer, Thanks for your efforts to put this together” -
Amgen GlaxoSmithKline
n Robert S. Bottome, Executive Director, “This was the first Pharma Packaging
Global Supply Chain, BioMarin and Labeling conference I have attended.
n Lisa M. Kelsey, Head, Commercial Overall I felt it was a good conference.
Labeling, Genentech The presentations were informative and
n Santiago Beltran, Principal Packaging the vendor booths were varied in their
Engineer, NuVasive offerings. I would attend again” - Sunovion
n Catherine Segura, Senior Manager, Pharmaceuticals
Systems Engineering Operations -- BD “Great conference! Extremely informative and
Biosciences good learning opportunity for all” - Allergan
n Steve Kemmerrer, VP Engineering
“I thought the conference was excellent. It
Development, Inovio Pharmaceutical
was very informative and the presentations
were extremely insightful with how different
companies are dealing with the same issues.
Also the networking was a great experience
for meeting new customers”- Pfizer

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» MANUFACTURING »

Batch, Continuous
Use of “Fake and/or False” in our vocabulary has become very pervasive
since 2016. I thought it would be useful and helpful to re-visit the
definitions of batch and continuous production processes and try to

or “Fake/False”
see if either of them fit in the realm of False or Fake “B or C” process. My
observations are based on my education, experiences in chemistry and
chemical engineering and the prevailing practices established more

Continuous Processes
than 100 years ago. They are also based on actual process design and
development, scale up of developed processes and management of
manufacturing processes and operations in fine/specialty chemicals,
coatings, and resins that were produced using organic chemicals.
Many of the products were produced by reacting chemicals and others
were formulated by blending different chemicals.
In recent months certain pharmaceutical processes have been
labeled as a continuous process but no process information has been
shared. However, based on public information about the products
Girish Malhotra, PE their viability of being operated as a continuous process is extremely
EPCOT International doubtful. Additional details are shared later.
Established batch and continuous process definitions and examples
are reviewed after a brief overview of what chemical engineers are
taught. I do not want to go in details but the following are part of the
Ch.E. curriculum.

Fundamentals Taught in
Chemical Engineering
Chemical engineers are taught that every process should be safe,
sustainable, and economic and should produce quality product the
first time and all the time.
If a product has to be reworked or its stoichiometry has to be adjusted
during the process to produce a quality product, its cost goes up and
the profitability of the company is lowered.
It is well known and practiced in every manufacturing industry that the
market size demand influences the type of process used. Irrespective
of the process used, product quality is a must for the market
success of every product. These expectations are most stringent for
pharmaceuticals as they influence human life.
Chemical reactive processes are called Unit Processes1 that produce
a product that could be the final saleable product or could be

86 | | September/October 2017
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an intermediate for another reactive or becomes critical, especially for the APIs and available hours are used for the production
formulated product. Unit operations2 involve their formulations. Idle (down) time can of a single product defines the production
a physical change or chemical transformation be high. Down time in the pharmaceutical process. If less than 8,400 hours are used to
that are used in the chemical and allied industry is extremely high.7 produce a product such a process according
industries to facilitate unit processes to to the established norms5 will be a batch
For the pharmaceutical industry batch
produce the desired products. process. This holds true for API production
processes are the main stay in the
The combination of these makes a process manufacture of active ingredients (API) and and their formulations.
and depending on demand it could be a their formulations. This is due to two inherent Table 2 is an illustration of how many hours
batch or continuous process. reasons. Micrograms to milligrams of API are per year are needed to produce at @200,000
McGraw-Hill has published series3 of books needed in every dose. One kilogram of an tablets per hour at different doses. Production
that cover unit processes, unit operations active produces ONE million tablets of one equipment of higher tableting rates are
and the economic considerations that milligram each. Thus, not a large quantity of commercial and available. It is most likely
allow chemical engineers and chemists the API is needed to satisfy the need. Table 1 is most of the products would be formulated
to develop, design and commercialize an illustration of the needed API and potential using batch processes even when they could
processes that are economic and profitable production methods for different dosages be operated continuously.
to any individual or company. Besides these and number of patients. Additional analysis8 is
books there are many other books that available elsewhere.
have been written on the subject. Excellent Process economics, chemistry and Continuous Production
books have been written about process execution method will determine the type
control methods and strategies.4 of process used. Typical available hours for The definition of continuous9 production has
Most of these books have been written by production at any plant are about 8,400 long been established. Continuous usually
what I would call the “Hall of Famers” of the (24x7x50) hours per year. How many of these means operating 24 hours per day, seven
Chemical Engineering and are considered the
Bibles of Chemical Engineering profession. Table 1. Typical API need, Number of Patients and
API Production Process
None of these books are recommended or
sponsored. There is no affiliation or financial Dose, Patients API Kilograms needed/year @ API Production
mg (millions) one tablet per day per year
relationship with the author. Preferred Process Number of plants
Type
1 500 182,500 Batch One or More

Batch Production 200

10
0.1

100
7,300

365,000
Batch

Batch
One

One or More
5
Wikipedia’s definition for batch production: 100 50 1,825,000 Batch or Continuous Could be a single
continuous plant but most
Batch production is a technique used in likely batch
manufacturing, in which the object in question 500 20 2,650,000 Continuous Could be a single
is created stage by stage over a series of continuous plant but
generally batch due to
workstations (steps), and different batches of multiple sites
product/s are made. Batch production is most
common in bakeries and in the manufacture
Table 2. Tablets Needed, Tablet Run Time, Number of Plans and Patients Served
of sports shoes, specialty/fine chemicals, resins,
Dose, Patients Tablets Tablets, Run time Number of Preferred
pharmaceutical ingredients (APIs), purifying
mg (millions) Used Millions/ needed, hours plans operating Formulation
water, inks, paints and adhesives. There are per day year @200,000 7,140 hours Process
other formulated products and most prominent tablets/hr. per year
are finished pharmaceutical drug dosages. 1 50 1 18,250 91,250 13 Continuous but will
be multiple batch
Batch production processes generally re- plants

quire much lower investment and have an 10 20 2 14,600 73,000 10 Continuous but will
be multiple batch
advantage because several products can be plants
produced in the same equipment. However, if 100 15 3 16,425 82,125 12 Continuous but will
the same equipment is to be used for different be multiple batch
plants
products, productivity will be significantly
500 10 4 14,600 73,000 10 Continuous but will
lower6 compared to a process where the be multiple batch
equipment is dedicated to produce a single plants

product. If different products are to be 200 0.1 1 36.5 183 1 (Run time less than Batch only
200 hours)
produced in the same equipment, cleanliness

88 | | September/October 2017
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www.americanpharmaceuticalreview.com | | 89
» MANUFACTURING »

days per week with infrequent maintenance shutdowns, such as semi- batches per year would be needed. If each batch took 30 hours to
annual or annual. Time could be allocated for unexpected shutdowns. manufacture all of the API would be produced in about eight weeks. If
Any process that does not meet the defined and established definition they campaigned the whole production for first two months of every
and is operated for fewer hours than the established definition would year, this would not make their process a continuous process. If this
be difficult to be justified as a continuous process. Excellent examples campaign run was called a “continuous process” then it would be an
of continuous process are Earth’s rotation and our heartbeat. Can we incorrect characterization and I would label this process a “fake/false
imagine a “stop and go” movement of the Earth and the human heart? continuous process”.
Throughout the pharma landscape there are are ten or less APIs that
are being produced by continuous processes. There are additional APIs
that could be produced continuously10 but effort is needed to develop Formulation Example
and commercialize such processes. Different business models would
be needed. Alternate business models and consolidation can convert If the example of API above was to be formulated and tableted,
batch processes to continuous processes. There also a downside to the product can be produced at single plant in less than 200 hours
continuous processes as it will result in consolidation of operations at 200,000 tablets per hour. However, if the tableting were done at
especially among the contract API producers and formulators. 10,000 tablets per hour, it would take them six months to produce
the total demand. It still does not make the process continuous, as the
McNeil, a Johnson and Johnson subsidiary, due to the formulation
equipment would be sitting idle for six months every year. It would
volume of Tylenol could have built a continuous process but they
be a wasted investment earning no return if every reader of this post
did not, a missed opportunity. It seems that chemical engineers and
had to invest his or her own money. Again if the process is called a
chemists at some companies have forgotten FRUGAL SCIENCE and
continuous process, it again would be an incorrect characterization
FRUGAL ENGINEERING.
and I would label this process “fake/false continuous process”.
Vertex8 has claimed to have a continuous process for their cystic
fibrosis (CF) drug. With less than 80,000 patients worldwide, they do
Fake/False Continuous Processes not have a large enough global patient (~80,000) base for all of their
In the current pharmaceutical landscape, processes that are in CF drugs and cannot operate their equipment for 8,400 hours per
reality batch processes are being called continuous by the chemical year per drug. A similar situation exists for Janssen Pharmaceutical’s
engineers working in public and private sectors. We all need to drug Prezista.
understand and recognize that if multiple products can be processed To me, an incorrect characterization of established definitions seems
in the same calendar year in the same equipment by re-arranging the to have become a new fashion especially in the pharma landscape.
reactive chemistry or the formulations, their processes would not be Is it because the US FDA is mentioning continuous manufacturing in
called “continuous manufacturing”. Any process that has a wide spot their communications and the industry wants to look good by calling
in the manufacturing line [material is held for any time period], should a naturally batch process a “continuous process”? Thus, if we accept an
be referred to as a batch process. Processing steps before such a hold incorrect definition then we should be also ready to accept 2+2=6, The
can be called by any name but if it is different from the established Sun can rise from the west and a mammal can be half pregnant (my
definition, we are making fun of science and engineering. apologies to all readers). If that were the case the basic laws of science,
FDA11 does not have an established definition and I am told one is math and anatomy would be defied.
forthcoming. I wonder how different it will be from what are the
established and practiced methods. Even the press12 has chimed
in. Numerous articles have been written that include “continuous Quality Assurance in Batch and
manufacturing” but no one has put forth its definition or names of
the product or their production rates. I am sure many, me included,
Continuous Processes
want to see the proof. I am sure many of us remember the movie Meeting quality standards in batch as well as continuous processes
Jerry Maguire.13 have different rigors. Command of the processes is a must whether
it is a batch or a continuous process. If command and understanding
of the processing steps are lacking, invariably there could be a batch-
API Example to-batch and/or a lot-to-lot quality variation. Efforts to rework or
bring the material to quality can and often results in waste i.e. higher
Analyzing Table 1, we have an example of a drug that has 100,000 product costs.
patients.
In batch processes due to their stop and go nature quality can
Patients have to take 200 mg dose every day of the year. Total API be sometimes managed and the process adjusted to achieve the
needed is about 7,300 Kg. per year. This product would and should established quality benchmarks. Thus an absolute command of the
be produced using a batch process in the available equipment. If batch process is less stressful but still is necessary. Batch processes
each batch reaction produced about 250 kilos per batch about 30 are based on in-process quality checks and adjustments. This practice

90 | | September/October 2017
 « MANUFACTURING »

extends cycle time and adds to inventory [raw material, in-process, 3. McGraw Hill Chemical engineering series, https://www.librarything.com/tag/McGraw+Hi
work in process and finished goods] challenges. If the batch process ll+Chemical+engineering+series, Accessed July 6, 2017
cannot be adjusted to correct process deviation, significant waste can
4. Chemical process control, https://www.librarything.com/subject/Chemical+process+
result. In the simplest terms, batch processes have economic value for
control, Accessed July 6, 2017
products that do not require continuous production but their quality
testing can be an aggravation. Batch process = quality by aggravation 5. Batch Production Wikipedia, https://en.wikipedia.org/wiki/Batch_production Accessed
(QbA) unless the process repeatability is strictly controlled. July 6, 2017

Compared to batch processes, continuous processes have much higher 6. Malhotra, Girish: Square Plug In A Round Hole: Does This Scenario Exist in Pharmaceuticals?
process control demand. Since the process is running with minimal/no Profitability through Simplicity https://pharmachemicalscoatings.blogspot.com/2010/08/
stop time, it is extremely critical that process operating parameters do square-peg-in-round-hole-does-this.html August 17, 2010 Accessed July 11, 2017
not deviate outside the established process operating control limits. If
7. Benchmarking Shows Need to Improve Uptime, Capacity Utilization, Pharmaceutical
the process deviates outside the established control limits, significant
quantities of waste and financial loss can result. Continuous processes Manufacturing (http://www.pharmamanufacturing.com/articles/2007/144/), Sep 20,
demand that the quality be established through robust process design 2007 Accessed July 7, 2017
when the process is developed, designed and commercialized. This 8. Malhotra, Girish: A Blueprint for Improved Pharma Competitiveness, Contract Pharma,
would be a case of quality by desire (QbD). September 2014, pp. 46-49
I am sure the debate on what is a continuous or a fake/false continuous 9. Continuous Production, HYPERLINK “https://en.wikipedia.org/wiki/Continuous_
process will go on until the economic realities of investment that have
production” https://en.wikipedia.org/wiki/Continuous production, Accessed July 14, 2017
been well established are understood by most. Chemical engineers
and chemists are taught everything, values and virtues, of batch and 10. Malhotra, Girish: Strategies for Improving Batch or Creating Continuous Active
continuous processes there is to know about such processes but Pharmaceutical Ingredients Manufacturing Processes, Profitability through Simplicity
one thing is sure unless one has not justified, developed, designed (https://pharmachemicalscoatings.blogspot.com/2017/03/strategies-for-enhancing-
or commercialized a process, it is difficult to discern value of the active.html), March 20, 2017, Accessed July 17, 2017
developed process. I know no investment would ever be made unless
11. Drug Making Email exchange with Dr. Janet Woodcock, FDA July 13, 2016
it can be justified and meets established norms of the science,
economics and engineering. 12. Breaks Away From Its Old Ways, The Wall Street Journal (https://www.wsj.com/articles/
drug-making-breaks-away-from-its-old-ways-1423444049), February 8, 2015, Accessed
As I said earlier a batch process cannot be continuous and vise versa.
If we accept it otherwise then we have a case of “false/fake” science, July 17, 2017
engineering, economics and human intelligence. 13. Jerry Maguire (https://www.youtube.com/watch?v=mBS0OWGUidc), Accessed July 18,
2017

References
For more thoughts on improving manufacturing and processing
1. Shreve, R. Norris: Unit Process In Chemical Processing, Ind. Eng. Chem., 1954, 46 (4), pp.
672–672, http://pubs.acs.org/doi/abs/10.1021/ie50532a025?src=recsys efficiency and profitability visit Girish Malhotra’s blog: www.
2. Unit Operation, https://en.wikipedia.org/wiki/Unit_operation, Accessed July 11, 2017 pharmachemicalscoatings.blogspot.com

www.americanpharmaceuticalreview.com | | 91
» DRUG DISCOVERY »

The Evolution of Introduction

Macrocycles in
Even with an ever-increasing array of cutting edge technologies
at its disposal, the process of drug discovery continues to be a
difficult one. As has been well documented, increased investments

Drug Discovery:
in pharmaceutical research and development have not led to a
concomitant rise in the number of new drugs.1 This certainly has
not been for a lack of new targets, as seemingly every week results

From Technologies to Drugs

of genomic and proteomic studies report the association of one or
more genes or proteins to a disease state. These significant advances,
however, have not been followed by a concurrent increase in the
availability of new chemical matter against which to interrogate this
myriad of targets. In the 1990s, combinatorial chemistry was hailed as
a revolutionary approach that would permit a meteoric advance in the
identification of novel therapeutic molecules.2 Although successful to
some degree with associated techniques, such as parallel synthesis,
solid phase methodology, high-throughput compound profiling and
Mark L. Peterson, PhD purification, now entrenched into modern drug discovery efforts,3 the
perceived failure of this technology to deliver on its surrounding hype
Chief Operating Officer
Cyclenium Pharma Inc. and the subsequent unrealistic expectations built thereupon, resulted
in a hesitancy by many in the industry to rush into the adoption of new
chemical technologies.
Further, associated with the resurgence of novel targets, a dramatic shift
in drug discovery approaches to biological entities has contributed to
a greater number of antibody and protein drug products appearing
and advancing in the pipelines of pharmaceutical and biotech
companies. Considering the therapeutic success, faster approval,
lower clinical failure rate, and premium pricing being realized by
these biomolecules, the attraction is certainly alluring.4 Hence, while
advances such as CRISPR/Cas9 and CAR-T capture the headlines and
imagination of investors and scientists, chemical technologies have
often been forgotten. However, biologics do possess some significant
limitations. Importantly, they can only address cell surface targets,
since they are too large to cross cellular membranes. In addition, their
preparation is complex, expensive and difficult to scale, plus concerns
about soliciting an undesirable immune response are ever-present.5
Indeed, despite the growing impact of biologics, small molecule
chemical drugs still account for the lion’s share of global sales.

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Unfortunately, attempts to modulate intracellular pathways entered the field in 1999. Indeed, there was actually considerable
and targets that are inaccessible to biologics have also proven resistance to the acceptance of macrocycles as possible pharmaceutical
intractable to the traditional compounds that dominate historical agents since they did not fit the usual structural paradigm that had
corporate compound collections. The limitations of biological served the industry very well for decades – small molecular weight
molecules coupled with the difficulties encountered with small (MW) heterocyclic molecules with a limited number of functional
molecules has led to consideration of non-traditional structures for groups that, more often than not, conformed to what became known
modulation of these challenging targets, such as protein-protein as Lipinski’s rule of five8 or, more succinctly termed, “drug-likeness.”
interactions (PPI), protein-nucleic acid interactions and tran- Further, synthetic accessibility for macrocyclic structures has been
scription factors.6 a long-standing challenge, particularly in the more diverse library
format needed for the high throughput screening (HTS) efforts central
One particularly attractive chemical class that suits this purpose has
to the majority of modern drug discovery programs.
been macrocyclic compounds.7 Macrocycles are molecular structures
that contain one or more rings of at least 12 atoms with the unique As with many nascent fields, it fell to smaller entrepreneurial firms to
characteristic that they combine the benefits of large biomolecules, change this situation and alter the perception of macrocycles in the
such as high potency and exquisite selectivity, with those of small pharmaceutical industry. In 2000, only two companies were actively
molecules, including reasonable manufacturing costs, favorable pursuing macrocyclic structures as a core technology: Polyphor
pharmacokinetic properties, including oral bioavailability, ease of in Switzerland, and the author’s former company, NéoKimia (later
administration and lack of immunogenicity. Interest in these structures Tranzyme Pharma9) in Canada. Their proprietary technologies proved
originated in part from the many macrocyclic natural products successful in advancing macrocycles into the clinic: the antibiotic
that have proven utility as therapeutic agents (e.g. vancomycin, murepavadin (POL7080)10 and the gastrointestinal prokinetic agents
amphotericin B, romidepsin, sirolimus). Further, the pre-organized and ulimorelin11 and TZP-10212 (Figure 2).
semi-rigid character of these structures holds potential for excellent Roused by these successes, a realization gradually developed in the
activity, even against intractable targets. Indeed, these molecules industry that there actually were biologically interesting molecules in
possess dense functionality packed into a compact, accessible scaffold, this “beyond rule of five” (bRo5) space.6 Hence, the situation steadily
yet still maintain sufficient flexibility to enable effective binding at began to change from that humble beginning and, during the first
disparate sites. In addition, macrocycles possess defined topologies, decade of the twenty-first century, an array of innovative technologies
which position functional groups in specific regions of space for target were created that provided access to macrocyclic compounds of
interaction, thereby permitting high potency to be achieved, even varying size and composition. These approaches ranged from purely
early in a discovery program. In particular, they are capable of effective chemical in nature13 to hybrid strategies that combined biological
interactions at the large, shallow surfaces that characterize many of methods, such as phage or mRNA display, intein-mediated protein
the challenging target classes. splicing, engineered enzymes and directed in vitro translation systems,
with one or more chemical steps.14 The latter methods, in particular,
can generate very large numbers of compounds, but such macrocycles
Macrocycles: Technology also tend to be highly peptidic and in the higher MW range, bringing

Development
concerns regarding their ability to possess drug-like properties. In
contrast, technologies that rely solely on chemical transformations
generally produce compounds occupying the lower MW end of
Despite their favorable nature, however, not much excitement
surrounded the area of macrocyclic drug discovery when the author

Figure 1. Macrocycle Chemical Space7b Figure 2. Initial Macrocyclic Clinical Candidates

94 | | September/October 2017
 « DRUG DISCOVERY »

the macrocycle continuum with their However, initial concerns about the ability to character, which, in concert with their size,
concomitant improved potential for favorable scale-up macrocycles, which the author recalls create significant issues in attaining the nec-
physicochemical and pharmacokinetic (PK- being emphatically told at one point would essary pharmacokinetic profile required for
ADME) profiles. preclude such molecules from ever becoming development as therapeutic agents. Indeed,
Today, the landscape is dramatically different viable therapeutics, were able to be decisively it is likely that they fail to satisfy any of the
with approximately thirty companies surmounted by the results with the first iconic rule of five criteria.8 Even smaller MW
embracing macrocycle-based technologies clinical HCV macrocyclic protease inhibitor, macrocycles with high amide bond content
as a core asset within their drug discovery ciluprevir (BILN 2061), when a synthesis that can often encounter these same problems.
effort, and many others employing them was successful in preparing over 400 kg of the For that reason, alternative guidance (“rules”)
for selected projects (Table 1). Indeed, active ingredient was reported.15 specifically for macrocyclic molecules and
a sure sign this area has moved into the libraries has been defined employing some
“established” phase is that multiple vendors of the same measures that were used to for-
are now offering macrocyclic libraries for sale. Macrocycles: Challenges mulate rule of five criteria, as well as other pa-
The temptation then might be to consider rameters more appropriate for this structural
macrocycles as now becoming just another As mentioned earlier, a host of hybrid chem- class.16 Compared to small molecules, where
commodity, although that also would not be ico-biological technologies have resulted relevant data on large collections of diverse
appropriate, since a vast region of macrocycle in the production of very large numbers of compounds are available, such an undertak-
chemical space still remains unexplored generally high MW macrocyclic compounds. ing is limited by the number of macrocycles
with current compounds and certain key Such macrocycles have proven themselves for which sufficient information is available.
deficiencies in working with these structures very effective at modulating a range of Hence, this analysis is skewed by the sizeable
still exist. Indeed, molecular modeling and PK- pharmacologically relevant interactions and quantity of natural product structures includ-
ADME properties are two important aspects providing insight into the structural determi- ed, since they have, to a large extent, been
where significant challenges remain and nants thereof.14 However, these large macro- the most thoroughly studied compounds. As
current research efforts are being directed. cycles typically possess considerable peptidic an example, for a compilation of 125 orally

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www.americanpharmaceuticalreview.com | | 95
» DRUG DISCOVERY »

absorbed cyclic peptides, which violated the rule of five limits to dif-
ferent degrees, it was found that oral bioavailability was generally fa-
vored by a lower number of hydrogen bond donors and reduced flex-
ibility.17 Nonetheless, careful investigations of cyclic natural products
have provided insight into factors that are critical to obtaining favor-
able properties in macrocyclic compounds with an emphasis on pas-
sive cellular permeability as a surrogate marker for oral bioavailability.
In particular, cyclosporine is an 11-amino acid peptide with potent
immunosuppressive effects18 that is often presented as the poster
Figure 3. Chameleonic Behavior of Macrocycles19
child for macrocyclic peptide drugs. However, it is actually the only
one of the 30 approved macrocyclic peptide drugs that is dosed
orally. This has been explained by the chameleonic character of the
macrocycle in being able to adapt its structure to the local environ- reduced permeability was observed at MW > 1000 Da, suggesting
ment.19 Cyclization has been well-known to render peptidic mol- that this could represent an upper size limit for these compounds with
ecules more resistant to degradation by shielding vulnerable func- respect to oral availability.21
tionalities from metabolic enzymes. In an analogous fashion, the Although these elegant studies have advanced the understanding of
cyclosporine structure exposes its polar functionalities when in an the factors involved in imparting desired properties into a peptidic
aqueous medium, but adopts a folded, almost micellular, structure
structural framework, the applicability to non-peptidic macrocyclic
with the polar groups shielded on the interior through formation of
molecules was less clear. Recently, however, a detailed investigation
a network of intramolecular hydrogen bonds to enable traversal of
using 200 such macrocycles from a designed collection demonstrated
the lipid membrane (Figure 3).
that the key to their favorable drug-like properties also resided in their
That this ability is not unique to cyclosporine was shown by a study capacity to modify their conformation to match the polarity of the local
of 39 cyclic peptide natural products, where it was found that good environment.22 These studies revealed that stereo- and regiochemistry
passive cell permeability was exhibited by those that had low energy
also have a significant influence on some of these properties, including
conformations in which compounds could effectively hide polar
cellular permeability.
functionalities using tactics such as by N-methylation, bulky side
chains and intramolecular hydrogen bonds (IHB).20 Additionally, in Of course, as with traditional medicinal chemistry optimization,
an evaluation of cyclic peptide libraries containing only N-methyl modifications made specifically to benefit compound properties must
residues to remove the effects of IHB on permeability, considerably always be balanced with maintaining the desired level of target activity

Table 1. Selected Macrocycle Technology Companies and Library Vendors

Company Platform Type Web Site
Technology Companies
Aileron Therapeutics Stapled Peptides Chemical www.aileronrx.com

Bicycle Therapeutics Bicycles® Hybrid www.bicycletherapeutics.com

Circle Pharma Computational Chemical www.circlepharma.com

Cyclenium Pharma CMRT (“smart”) Chemical www.cyclenium.com

Encycle Therapeutics Nacellins Chemical www.encycletherapeutics.com

Ensemble Therapeutics DNA Programmed Chemistry (DPC) Chemical www.ensembletx.com

Inthera Bioscience Protein Domain Mimics Chemical www.investiere.ch/inthera-bioscience-ag

OncoDesign Nanocyclix® Chemical www.oncodesign.com

PeptiDream Peptide Discovery Platform System (PDPS) Hybrid www.peptidream.com

Polyphor Protein Epitope Mimic (PEM) Chemical www.polyphor.com

Protagonist Therapeutics Vectrix Hybrid www.protagonist-inc.com

Ra Pharma Extreme Diversity Hybrid www.rapharma.com

Ripptide Pharma Chemoenzymatic Hybrid www.ripptide-pharma.com

Library Vendors
AnalytiCon Discovery MacroEvoLution Chemical/Natural Products www.ac-discovery.com

Asinex na Chemical www.asinex.com

ChemBridge na Chemical www.chembridge.com

Princeton Biomolecular na Chemical www.princetonbio.com

96 | | September/October 2017
 « DRUG DISCOVERY »

and selectivity. In this respect, a recurring theme from these works is now contain modules specifically aimed at handling conformational
that the cyclic nature of these molecules is the proverbial double- searches of macrocyclic molecules.25 However, it likely will still be
edged sword, with their rigid nature and defined conformations some time before computer-assisted macrocycle design enters the
leading to high potency and selectivity. However, within a macrocycle, discovery lexicon.
small structural modifications can result in significant conformational
changes, so the balancing act between potency and other properties,
such as PK-ADME, can prove to be even more delicate than with
traditional small molecules. On the other hand, this characteristic
Macrocyclic Pharmaceuticals
also leads to optimization of desired properties with macrocycles Despite these continuing challenges, a growing number of synthetic
not requiring major structural additions, hence does not significantly macrocycles have progressed into the clinic with several already
increase MW, in contrast to the situation with traditional small being established in the marketplace. An excellent compendium of
molecules.23 From this discussion, it can be concluded that the factors macrocyclic compounds in clinical development and that have been
influencing the “drugability” of macrocyclic molecules are beginning approved for human treatment has been published.26 Importantly,
to be understood, but the worth of this knowledge in enabling drug their toxicological profiles have been found to more resemble those
discovery still remains to be proven. of traditional small molecules than biologics and, generally, have
Another aspect of macrocycle drug discovery that is consistent exhibited excellent safety. Although the impetus for the interest in
with small molecule efforts is that structural biology in conjunction macrocycles came from their potential for difficult targets, the many
with molecular modeling has proven to have significant utility. In advantages provided by this compound class have made them
particular, such studies are beneficial for determining if cyclization quite valuable for the modulation of the more easily druggable
of a structure might be an effective strategy for constraint into target classes as well. In particular, macrocycles have impacted
the bioactive conformation. Indeed, prior to the dedicated library therapy for hepatitis C, where macrocyclic HCV NS3-4A protease
efforts described herein, this was the primary manner in which inhibitors (Figure 4), grazoprevir (MK-5172), paritaprevir (ABT-450),
synthetic macrocycles found their way into pharmaceutical simeprevir (TMC-435350), and vaniprevir (MK-7009), have helped to
discovery. However, in contrast to small molecules, the use of revolutionize the treatment of this disease and, in combination with
other drugs, have led to this condition even being considered cured
computational methods for de novo design and virtual screening
in infected individuals.27
in the macrocyclic area are complicated by the limited ability of
current approaches to handle large ring systems, the accessibility of Advances in the construction of kinase inhibitors with unique
multiple low energy conformations, difficulties in pose prediction properties have also been at the forefront of progress in the macrocycle
and imperfect treatment of conformational dynamics.24 Despite area, with lorlatinib (Figure 5), an ALK/ROS1 tyrosine kinase inhibitor
this, improvements are being made and many commercial programs in Phase III clinical trials for metastatic non-small cell lung cancer,

www.americanpharmaceuticalreview.com | | 97
» DRUG DISCOVERY »

Figure 6. Examples of Stapled Peptides

relevant conformation, such as an α-helix, (Figure 6).32 An early

example of this type of compound, ATSP-7041, which reactivates
p53-mediated tumor suppression through dual inhibition of MDMX
and MDM2, demonstrated efficacy in multiple human xenograft
models.33 More recently, a later generation stapled peptide from this
class, ALRN-6924, has entered clinical investigation for a variety of
cancer indications.34 Applications for such molecules continue to be
reported with the majority of them being in the modulation of PPI
implicated in oncology.35

Figure 4. Marketed Macrocyclic Pharmaceuticals

Conclusion
From the discussion in this article, the impact of a new chemical class
on drug discovery, even in these days of excitement over the latest
biological breakthrough, cannot and should not be underestimated.
Although the fervor surrounding the macrocycle area has subsided,
careful, innovative and often elegant research from dedicated
scientists in industry and academia over the past twenty years has led
to the realization of the early promise of the macrocyclic compound
class. Only a sampling of the advances and continuing challenges in
Figure 5. Selected Clinical Macrocyclic Pharmaceuticals
this exciting area could be presented herein; the reader is directed
to the articles cited in the references for additional information
on the many inventive solutions that have been devised by the
even demonstrating the ability to cross the blood-brain-barrier.28 As growing research community interested in macrocycles. Despite
another example, pacritinib (Figure 5) is a dual JAK2 and FLT3 inhibitor being intimately involved in this field for much of the past twenty
that holds promise for the treatment of myelofibrosis and is also in years, the author is realistic enough to recognize that the progress
Phase III.29
in the macrocycle field falls short of being “revolutionary,” but its
Returning to one of the primary drivers for the pursuit of macrocycles impact is nonetheless highly significant. The evolution of this area
in drug discovery, the potential to address intractable targets such as has proceeded very nicely, albeit not always smoothly, from early
PPI, the unique and versatile nature of the macrocyclic framework considerations related strongly towards technology development
again has been demonstrated with increasing reports of successful to now becoming established as another important element in the
modulation of this target class with these molecules, although such armamentarium of medicinal chemistry.
applications are still too early stage to gauge their clinical impact.30
Interestingly, in a comprehensive analysis of how drug molecules
bind to their targets, it was found that macrocyclic drugs actually
possess shapes that appear to be particularly appropriate for binding
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Nat. Rev. Drug Discov. 2011, 10(6), 428-438.
Lastly, one novel macrocycle class that deserves specific mention in
the PPI area is “stapled” peptides. These molecules utilize side chain 2. Gallop, M.A.; Barrett, R.W.; Dower, W.J.; Fodor, S.P.; Gordon, E.M. Applications of
combinatorial technologies to drug discovery. 1. Background and peptide combinatorial
functional groups at particular residues that can react with each
libraries. J. Med. Chem. 1994, 37(9), 1233-1251; (b) Gordon, E.M. Barrett, R.W.; Dower,
other to introduce a structural constraint (e.g. double or triple bond, W.J.; Fodor, S.P.; Gallop, M.A. Applications of combinatorial technologies to drug discovery.
triazole ring) into the peptide anchored at those residues, thereby 2. Combinatorial organic synthesis, library screening strategies, and future directions. J.
stabilizing the secondary structure into a desired biologically Med. Chem. 1994, 37(10), 1385-1401.

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 « DRUG DISCOVERY »

3. Kennedy, P.; Williams, L.; Bridges, T.M.; Daniels, R.N.; Weaver, D.; Lindsley, C.W. Application 20. Ahlbach, C.L.; Lexa, K.W.; Bockus, A.T.; Chen, V.; Crews, P.; Jacobson, M.P.; Lokey, R.S.
of Combinatorial Chemistry Science on Modern Drug Discovery. J. Combi. Chem. 2008, Beyond cyclosporine A: conformation-dependent passive membrane permeabilities of
10(3), 345–354. cyclic peptide natural products. Fut. Med. Chem. 2015, 7(16), 2021-2030.
4. (a) Hay, M.; Thomas, D.W.; Craighead, J.L.; Economides, C.; Rosenthal, J. Clinical 21. Pye, C. R.; Hewitt, W. M.; Schwochert, J.; Haddad, T. D.; Townsend, C. E.; Etienne, L.; Lao,
development success rates for investigational drugs. Nat. Biotechnol. 2014, 32(1), 40–51; Y.; Limberakis, C.; Furukawa, A.; Mathiowetz, A. M.; Price, D. A.; Liras, S.; Lokey, R. S.
(b) Smietana, K.; Siatkowski, M.; Møller, M. Trends in clinical success rates. Nat. Rev. Drug Nonclassical size dependence of permeation defines bounds for passive absorption of large
Disc. 2016, 15(6), 379–380. drug molecules. J. Med. Chem. 2017, 60(5), 1665−1672.
5. Lybecker, K.M. The Biologics Revolution in the Production of Drugs, Fraser Institute, July 2016. 22. Over, B.; Matsson, P.; Tyrchan, C.; Artursson, P.; et al. Structural and conformational
6. Indeed, this has been termed the “beyond rule of five” (bRo5) space, to differentiate from determinants of macrocycle cell permeability. Nat. Chem. Biol. 2016, 12(12), 1065-1074.
the space defined by compounds that fit the reference 8 criteria: Doak, B.C.; Over, B.; 23. (a) Opera, T.I. Current trends in lead discovery: Are we looking for the appropriate
Giordanetto, F.; Kihlberg, J. Oral Druggable Space beyond the Rule of 5: Insights from Drugs
properties? J. Computer-Aided Mol. Design 2002, 16, 325-334; (b) Wenlock, M.C.; Austin,
and Clinical Candidates. Chem. Biol. 2014, 21(9), 1115-1142.
R.P.; Barton, P.; Davis, A.M.; Leeson, P.D. A Comparison of Physiochemical Property Profiles
7. (a) Diggers, E.M.; Hale, S.P.; Lee, J.; Terrett, N.K. The exploration of macrocycles for drug of Development and Marketed Oral Drugs. J. Med. Chem. 2003, 46(7), 1250-1256.
discovery-an underexploited structural class. Nat. Rev. Drug Disc. 2008, 7(7), 608-624; (b)
24. (a) Chen, I.J.; Foloppe, N. Tackling the conformational sampling of larger flexible
Terrett, N.K. Methods for the synthesis of macrocycle libraries for drug discovery. Drug Disc.
compounds and macrocycles in pharmacology and drug discovery. Bioorg. Med. Chem.
Today: Technologies 2010, 7(2), e97-e104; (c) Marsault, E.; Peterson, M.L. Macrocycles are
great cycles: applications, opportunities, and challenges of synthetic macrocycles in drug 2013, 21(24), 7898-7920; (b) Allen, S.E.; Dokholyan, N.V.; Bowers, A.A. Dynamic Docking
discovery. J. Med. Chem. 2011, 54(7), 1961-2004; (d) Mallinson, J.; Collins, I. Macrocycles in of Conformationally Constrained Macrocycles: Methods and Applications. ACS Chem. Biol.
new drug discovery. Fut. Med. Chem. 2012, 4(11), 1409-1438; (e) Yu, X.; Sun, D. Macrocyclic 2016, 11(1), 10-24.
drugs and synthetic methodologies toward macrocycles. Molecules 2013, 18(6), 6230- 25. Watts, K.S.; Dalai, P.; Tebben, A.J.; Cheney, D.L.; Shelley, J.C. Macrocycle Conformational
6268; (f) Yudin, A.K. Macrocycles: lessons from the distant past, recent developments, and Sampling with MacroModel. J. Chem. Info. Model. 2014, 54(10), 2680–2696.
future directions. Chem. Sci. 2015, 6(1), 30-49; (g) You, L.; An, R.; Liang, K.; Cui, B.; Wang, X.
26. Giordanetto, F.; Kihlberg, J. Macrocyclic drugs and clinical candidates: what can medicinal
Macrocyclic Compounds: Emerging Opportunities for Current Drug Discovery. Curr. Pharm.
chemists learn from their properties? J. Med. Chem. 2014, 57(2), 278-295.
Des. 2016, 22(26), 4086-4093.
8. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational 27. MCauley, J.A.; Rudd, M.T. Hepatitis C virus NS3/4a protease inhibitors. Curr. Opin.
approaches to estimate solubility and permeability in drug discovery and development Pharmacol. 2016, 30, 84–92; (b) Pillaiyar, T.; Namasivayam, V.; Manickam, M. Macrocyclic
settings. Adv. Drug Deliv. Rev. 1997, 23(1-3), 3-25. Hepatitis C Virus NS3/4A Protease Inhibitors: An Overview of Medicinal Chemistry. Curr.
Med. Chem. 2016, 23(29), 3404-3447.
9. Now Ocera Therapeutics, which divested the MATCH™ macrocycle technology of Tranzyme
to the Roche Group in December 2013. 28. (a) Johnson, T.W.; Richardson, P.F.; Bailey, S.; et al. Discovery of (10R)-7-amino-12-fluoro-
2,10,16-trimethyl-15-oxo-10,15,16,17-tetrahydro-2H-8,4-(metheno)pyrazolo[4,3-h]
10. Luther, A.; Moehle, K.; Chevalier, E.; Dale, G.; Obrecht, D. Protein epitope mimetic
[2,5,11]-benzoxadiazacyclotetradecine-3-carbonitrile (PF-06463922), a macrocyclic
macrocycles as biopharmaceuticals. Curr. Opin. Chem. Biol. 2017, 38, 45-51.
inhibitor of anaplastic lymphoma kinase (ALK) and c-ros oncogene 1 (ROS1) with
11. Hoveyda, H.R.; Marsault, E.; Gagnon, R.; et al. Optimization of the potency and preclinical brain exposure and broad-spectrum potency against ALK-resistant mutations.
pharmacokinetic properties of a macrocyclic ghrelin receptor agonist (Part I): Development J. Med. Chem. 2014, 57(11), 4720-4744; (b) Basit, S.; Ashraf, Z.; Lee, K.; Latif, M. First
of ulimorelin (TZP-101) from hit to clinic. J. Med. Chem. 2011, 54(24), 8305−8320. macrocyclic 3rd-generation ALK inhibitor for treatment of ALK/ROS1 cancer: Clinical and
12. Hoveyda, H.R.; Fraser, G.L.; Marsault, E.; et al. "Optimization of a macrocyclic ghrelin designing strategy update of lorlatinib. Eur. J. Med. Chem. 2017, 134, 348-356.
receptor agonist (Part II): Development of TZP-102." In E. Marsault, M.L. Peterson (Eds.)
29. (a) William, A.D.; Lee, A.C.; Goh, K.C.; et al. Discovery of kinase spectrum selective
Practical Medicinal Chemistry with Macrocycles: Design, Synthesis, and Case Studies,
macrocycle (16E)-14-methyl-20-oxa-5,7,14,26-tetraazatetracyclo[19.3.1.1(2,6).1(8,12)]
Wiley, 2017, pp 545-558.
heptacosa-1(25),2(26),3,5,8(27),9,11,16,21,23-decaene (SB1317/TG02), a potent
13. Peterson, M. L. “The Synthesis of Macrocycles for Drug Discovery.” In J. Levin (Ed.) inhibitor of cyclin dependent kinases (CDKs), Janus kinase 2 (JAK2), and fms-like tyrosine
Macrocycles in Drug Discovery, Royal Society of Chemistry, Cambridge, 2014, pp 398-486. kinase-3 (FLT3) for the treatment of cancer. J. Med. Chem. 2012, 55(1), 169-196; (b)
14. (a) Frost, J.R.; Smith, J.M.; Fasan, R. Design, synthesis, and diversification of ribosomally Verstovsek, S.; Komrokji, R.S. A comprehensive review of pacritinib in myelofibrosis. Future
derived peptide macrocycles. Curr. Opin. Struct. Biol. 2013, 23(4), 571-580; (b) Oncol. 2015, 11(20), 2819-2830.
Bashiruddin, N.K.; Suga, H. Construction and screening of vast libraries of natural product- 30. (a) Cardote, T.A. Ciulli, A. Cyclic and Macrocyclic Peptides as Chemical Tools to Recognize
like macrocyclic peptides using in vitro display technologies. Curr. Opin. Chem. Biol. 2015, Protein Surfaces and Probe Protein-Protein Interactions. ChemMedChem 2016, 11(8),
24, 131–138.
787-794; (b) Dougherty, P.G.; Qian, Z.; Pei, D.; Macrocycles as protein-protein interaction
15. (a) Nicola, T.; Brenner, M.; Donsbach, K.; Kreye, P. First Scale-Up to Production Scale of a inhibitors. Biochem. J. 2017, 474(7), 1109-1125.
Ring Closing Metathesis Reaction Forming a 15-Membered Macrocycle as a Precursor of
31. Doak, B.C.; Zheng, J.; Dobritzsch, D.; Kihlberg, J. How beyond rule of 5 drugs and clinical
an Active Pharmaceutical Ingredient. Org. Proc. Res. Develop. 2005, 9(4), 513-515; (b) Ye.
candidates bind to their targets. J. Med. Chem. 2016, 59(6), 2312–2327.
N.K.; Farina, V.; Houpis, I.N.; et al. Efficient large-scale synthesis of BILN 2061, a potent
HCV protease inhibitor, by a convergent approach based on ring-closing metathesis. J. Org. 32. (a) Walensky, L.D.; Bird, G.H. Hydrocarbon-stapled peptides: principles, practice, and
Chem. 2006, 71(19), 7133-7145. progress. J. Med. Chem. 2014, 57(15), 6275-6288; (b) Cromm, P.M.; Spiegel, J.; Grossmann,
16. Villar, E.A.; Beglov, D.; Chennamadhavuni, S.; Porco, J.A.,Jr.; Kozakov, D.; Vajda, S.; Whitty, A. T.N. Hydrocarbon stapled peptides as modulators of biological function. ACS Chem. Biol.
How proteins bind macrocycles. Nat. Chem. Biol. 2014, 10(9), 723-731. 2015, 10(6), 1362-1375.

17. Nielsen, D.S.; Shepherd, N.E.; Xu, W.; Lucke, A.J.; Stoermer, M.J.; Fairlie, D.P. Orally Absorbed 33. Chang, Y.S.; Graves, B.; Guerlavais, V.; et al. Stapled α-helical peptide drug development: a
Cyclic Peptides. Chem. Rev. 2017, 117(12), 8094-8128. potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy. Proc. Natl.
Acad. Sci. USA 2013, 110(36), E3445-E3454.
18. Wenger, R.M.; Payne, T.G.; Schreier, M.H. Cyclosporine: Chemistry, Structure-Activity
Relationships and Mode of Action. Prog. Clin. Biochem. Med. 1986, 3, 157-191. 34. Meric-Bernstam, F.; Saleh, M.N.; Infante, J.R.; et al. J. Clin. Oncol. 2017, 35(15 suppl.), 2505
(Oral presentation at 2017 ASCO Meeting, Chicago, IL, June 2017, Abstract 2505).
19. Whitty, A.; Zhong, M.; Viarengo, L.; Beglov, D.; Hall, D.R.; Vajda, S. Quantifying the
chameleonic properties of macrocycles and other high-molecular-weight drugs. Drug 35. Iyer VV. A Review of Stapled Peptides and Small Molecules to Inhibit Protein-Protein
Discov. Today 2016, 21(5), 712-717. Interactions in Cancer. Curr. Med. Chem. 2016, 23(27), 3025-3043.

www.americanpharmaceuticalreview.com | | 99
» SEPARATION PURIFICATION »

Host cell proteins (HCPs) are process-related impurities introduced into

Case Study: the manufacturing process by the host organism in which a biologic
is produced. These proteins have the potential to elicit unwanted
immunological responses in patients, which may affect the safety
Qualifying a Commercially of the final drug product. Furthermore, residual host cell proteins,
such as proteases, have the potential to degrade the biologic drug
substance (DS).1 As a result, regulatory authorities expect that HCPs
Available ELISA for in the DS will be evaluated and the most common method for HCP
measurement is a quantitative enzyme-linked immunosorbent assay

HCP Quantification (ELISA). In addition to quantitating HCPs in the final DS, the HCP ELISA
can also be used to quantitate HCPs in in-process samples to evaluate
process performance, and to inform decisions during purification

and Its Ability to process development. It is generally accepted that a commercially

available HCP ELISA can be used during preclinical and early stages
of clinical development before a more sensitive process-specific ELISA,

Inform Purification with greater antibody coverage for the antigen, is developed.1 In this
case study, a commercially available Sf9 HCP ELISA was evaluated
and optimized for use in quantitating HCPs in a recombinant protein

Process Decisions DS and in-process samples. Subsequently, information from the

commercially available ELISA was used to characterize and guide
downstream purification process improvement decisions.
HCP ELISAs are complex because they utilize a polyclonal anti-HCP
antibody reagent to detect multiple HCP analytes. Therefore, even a
commercially available HCP ELISA kit that has been qualified by the
manufacturer should undergo further qualification by the user to
demonstrate that the assay is capable of accurately detecting HCPs
Samantha Kecman, Victor Dellisola, and present in all of the sample types for which it is intended. In addition
Stephen Raso to the standard practice of determining the range, repeatability,
intermediate precision, limit of detection, and limit of quantitation
Genocea Biosciences
of the assay, there are particular qualification parameters which
require special attention when developing a multiple-analyte HCP
ELISA, including specificity, accuracy, and dilutional linearity. The
vendor-supplied antibody must be shown to be specific for HCPs and
not recognize (cross-react with) any DS proteins. To determine the
specificity of the anti-HCP antibody provided in the kit, cross-reactivity
of the antibody for DS is evaluated by 1D Western blot. A blot in which
DS proteins are probed with the kit-supplied antibody and a negative

100 | | September/October 2017

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» SEPARATION PURIFICATION »

result, or lack of recognition of the DS proteins by the kit-supplied

antibody, is desired. Qualification of a commercially available ELISA for
HCP detection should also include an assessment of the accuracy of
the assay in detecting HCPs in each of the sample types to be tested,
primarily to ensure a lack of sample matrix interference. To determine
accuracy, spike and recovery experiments are conducted, in which the
process-specific HCP standard is added to appropriate samples and a
spike recovery of ±30% is demonstrated over the range of the assay.1
In order to accurately quantitate the HCPs in a given sample, all of the
anti-HCP antibodies must be in excess of their respective antigens.
Determination of antibody excess is important because at high levels,
a particular analyte can saturate the available antibody, leading to an
underestimation of that analyte by the assay. To determine whether
each anti-HCP antibody is in excess of its antigen, the property of
dilutional linearity is examined. Dilutional linearity is demonstrated
when a sample is serially diluted two-fold for several points and the
calculated HCP concentration does not vary by more than ±20%,
so that the value for the HCP concentration in that sample remains Figure 1. Overview of downstream purification Processes A, B
and C. Elution from the MM chromatography column in Process
relatively constant over several dilutions.2 In contrast, if the antibody A is by phosphate gradient. Elution from the MM column in
is saturated by the analyte, the apparent HCP concentration in the Processes B and C is by pH change. Elution by pH is preceded by
sample will increase as the sample is diluted, leading to inaccurate a 1-step phosphate wash in Process B and a 2-step phosphate
wash in Process C.
quantitation of HCPs in that sample.
In addition to establishing dilutional linearity for each sample type
to ensure accurate quantitation of HCPs, dilutional linearity can also wash prior to elution by pH shift. The commercially available ELISA
be used to evaluate the clearance of HCPs by downstream process was used to evaluate HCP levels in the column loads and eluates in
steps. Dilutional linearity indicates that each antibody is in excess of each process, and ultimately guided the selection of the MM column
its antigen and therefore, a loss of dilutional linearity at any step in the elution conditions for optimal HCP clearance.
purification process indicates that one or more HCPs is being enriched
Dilutional linearity was examined to determine the effect of changing
by that step, so that one or more antibodies are no longer in excess of
the mode of elution from the MM column. Plotting the HCP to product
their antigen.
protein ratio (ng/mg) against the concentration of the product (mg/
To evaluate dilutional linearity, the ratio of HCP (ng/mL) to the product mL), shows that in Process A, dilutional linearity is maintained for each
protein concentration (mg/mL) is plotted against the concentration of process intermediate because the Sf9 protein ratio remains relatively
the product (mg/mL). Typically, in-process and DS samples are tested unchanged (measured value within 20% of previous dilution) at every
by HCP ELISA and dilutional linearity is established by demonstrating sample dilution (Figure 2). In contrast, dilutional linearity was not
that the HCP-to-product ratio remains relatively constant regardless maintained for Process B (Figure 3). In Process B, the first three in-
of the sample dilution. However, if dilutional linearity is lost during process samples demonstrate dilutional linearity, indicating that every
the purification process, one or more antigens are co-purifying with anti-HCP antibody is in excess of its antigen in that sample. However,
the product molecule and may even be enriched (assuming matrix for both the MM and anion exchange (AEX) eluate samples, dilutional
interference and antibody-product cross reactivity are not factors). linearity was not observed, indicating that not all of the anti-HCP
In this case study, the effect of varying the elution conditions of a antibodies are in excess of their antigens. Since the loss of dilutional
mixed mode (MM) chromatography column on HCP clearance was linearity occurs after changing the mode of elution from the MM
chromatography column, and because dilutional non-linearity is first
studied using a commercially available HCP ELISA. Several changes
evident in the MM eluate sample, the data suggests that the one-step
were made to the downstream purification process for a recombinant
phosphate wash prior to elution by pH shift is increasing the level of
protein, originally designated Process A, then Process B, and ultimately
HCPs co-purifying with the DS. This knowledge was used to inform the
Process C, as shown in Figure 1. The focus of this communication is
decision to include a second phosphate wash of the column prior to
on the MM chromatography step and the optimization that was
elution by pH shift, leading to Process C (Figure 4). Dilutional linearity
performed on this step to improve the clearance of HCPs by this
was achieved in Process C, demonstrating that inclusion of a second
column. Although an MM chromatography step is used in all three
phosphate wash prior to pH elution prevented enrichment of HCPs in
processes, the mode of elution from the column is varied. In Process
the MM eluent.
A, the elution from the MM column is performed using a phosphate
gradient. In Processes B and C, elution from the column employs a pH The ability to elute the drug substance from the MM chromatography
change. Process B uses a one-step phosphate wash prior to elution with pH instead of phosphate was an important change to the
by pH shift, whereas Process C incorporates a two-step phosphate downstream purification process that resulted in an overall

102 | | September/October 2017

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» SEPARATION PURIFICATION »

Figure 2. Dilutional linearity determination by Sf9 HCP ELISA Figure 4. Dilutional linearity by Sf9 HCP ELISA when eluting
of Process A purification intermediates. The Sf9 protein ratio from the MM chromatography with pH following a 2-step
(ng/mg) is calculated by comparing the Sf9 HCP result (ng/ phosphate wash of the column. The Sf9 protein ratio (ng/mg)
mL) to the product protein concentration (mg/mL) in a sample. is calculated by comparing the Sf9 HCP measurement (ng/mL)
Process pools from various purification steps were tested with to the product protein concentration (mg/mL) in a sample.
serial dilution and plotted for Sf9 protein ratio (ng/mg) and Process pools from various purification steps were tested with
product concentration (mg/mL). SIB: solubilized inclusion serial dilution and plotted for Sf9 protein ratio (ng/mg) and
body, AEX: anion exchange chromatography, MM: mixed-mode product concentration (mg/mL). SIB: solubilized inclusion
chromatography, HIC: hydrophobic interaction chromatography body, CEX: cation exchange chromatography, MM: mixed-
mode chromatography

Figure 5. Optimization of the MM chromatography wash step

Figure 3. Dilutional linearity by Sf9 HCP ELISA of Process B reduces overall HCP levels in the MM eluate and resulting
purification intermediates. The Sf9 protein ratio (ng/mg) is DS. In Process A (Panel A), there is less efficient clearance of
calculated by comparing the Sf9 HCP measurement (ng/mL) HCPs by the MM column using phosphate elution compared
to the product protein concentration (mg/mL) in a sample. to Process C (Panel B), resulting in higher HCP levels in
Process pools from various purification steps were tested with the DS. The Sf9 protein ratio (ng/mg) is calculated by
serial dilution and plotted for Sf9 protein ratio (ng/mg) and comparing the Sf9 HCP level (ng/mL) to the product protein
product concentration (mg/mL). SIB: solubilized inclusion concentration (mg/mL) in a sample. Process pools from
body, CEX: cation exchange chromatography, MM: mixed-mode various purification steps were tested and the Sf9 HCP-to-
chromatography, AEX: anion exchange chromatography product ratio (ng/mg) was plotted for each. SIB: solubilized
inclusion body, AEX: anion exchange chromatography, MM:
mixed-mode chromatography, HIC: hydrophobic interaction
chromatography, CEX: cation exchange chromatography, UF/
improvement in the removal of HCPs by the MM column and
DF: ultrafiltration/diafiltration
reduction of HCP in the DS. When eluting by phosphate, there was
not a significant reduction in HCP levels by the MM column in Process
A (Figure 5A). In contrast, the same MM purification step in Process complex nature of a multi-analyte ELISA, it is imperative to qualify
C (Figure 5B) where the mode of elution has been changed to pH a commercially available HCP assay for use in quantitating HCPs in
with a two-step phosphate wash prior to elution reduced HCP levels process-specific samples. It is especially important in the qualification
by two orders of magnitude. Therefore, the optimization of the MM of HCP ELISAs to determine specificity, accuracy, and dilutional linearity
chromatography step contributes greatly to the overall lower levels of to demonstrate that the commercial ELISA is capable of accurately
HCPs in the MM eluate and DS. quantitating HCPs in each type of downstream process sample. Once
Because residual host cell proteins in a biologic can have many the assay is qualified for testing process-specific samples, the results
unwanted effects, including a reduction in safety and efficacy of the can be used to evaluate downstream purification process changes
therapeutic, the level of host cell proteins should be quantitated and and optimize conditions to reduce overall HCP levels. In this case, the
minimized.1 The use of a commercially available Sf9 HCP ELISA is a data demonstrate how quantitation of HCPs by the commercial ELISA
powerful tool for quantitating HCPs in process-specific samples and was vital for characterizing downstream process changes. Even small
evaluating downstream purification process changes. Due to the alterations to the process, such as changing column wash conditions,

104 | | September/October 2017

 « SEPARATION PURIFICATION »

can have a significant impact on the overall clearance of HCPs by the Novartis in the biochemistry and biologics groups and is now a Senior
MM purification chromatography. In particular, examination of the Analytical Development Associate at Genocea, where she is leading
dilutional linearity is a powerful and specific method important for the project to develop process-specific HCP ELISAs. She received her
evaluating downstream purification changes on HCP clearance and
Bachelors in Biochemistry and Molecular Biology from Boston University
optimizing the MM chromatography column wash conditions to avoid
and her Masters in Biology from Harvard University.
enrichment of HCPs. In this study, the characterization of downstream
process changes not only led to improved clearance of HCPs by the
Victor Dellisola is a development associate at Genocea Biosciences.
MM chromatography, but also resulted in overall lower levels of HCPs
Victor is a major contributor to the HCP project and development of
in the purified DS.
a product specific HCP ELISA at Genocea. He previously specialized in
phenotypic screening and assay development at Corning and Horizon

AMA References: Discovery. He received his B.S. in Marine Sciences from the University of
Massachusetts, Dartmouth.
1. <1132> Residual Host Cell Protein Measurement in Biopharmaceuticals (USP 39-NF 34).
Vol 1. Rockville, MD: United States Pharmacopeia Convention; 2016: 1416-1436. Stephen Raso is the Director of Analytical Development at Genocea
2. Assay Qualification Template for Host Cell Protein ELISA. Cygnus Technologies. Available at: Biosciences. He has previously held positions at MIT, Pfizer and
https://cygnustechnologies.com/content/faq.html#1. Accessed August 28, 2017.
Biogen, working on biopharmaceutical development and CMC. As
an Adjunct Professor at the University of Massachusetts Lowell, Steve

Author Biographies taught graduate-level classes on the Isolation and Characterization of

Biomolecules. He received his B.S. in Chemistry from the University of
Samantha Kecman is an expert in host cell protein assay development Massachusetts Boston and his Ph.D. (Chemistry/Biophysics) from Texas
at Genocea Biosciences. She was formerly a Scientific Associate at A&M University.

www.americanpharmaceuticalreview.com | | 105
» FORMULATION AND DEVELOPMENT »

Evolution of Bench Scale

Mixing is a crucial step in many chemical processes. Poor mixing
can limit the efficiency of an industrial process or cause severe
complications in scale-up. Despite its importance, the replication

Mixing Devices: Getting

of mixing conditions at different scales still needs to be better
understood. There are several cases where scale-up procedures
are known to fail and the reasons are not always clear. When a

Reliable Scale-Up Data

process is being designed or replicated between scales, as many
processing conditions as possible should be held constant, but
this is unfortunately impossible for many cases involving reactions
or multi-phase flow. When this happens, the rate-limiting variables
should be prioritized. Identifying these variables is a decision that
needs to be supported by experiments and/or simulations.
The most classical scale-up/-down methodology is geometric
similarity where the relative dimensions of a mixing vessel (D/T, C/T…)
are kept identical and one of the dynamic properties is chosen to be
Dr. Marcio B. Machado and Dr. Suzanne M. Kresta replicated, usually the power per mass (P/ρV). Even when it is possible
Department of Chemical and Materials Engineering to keep geometric similarity between scales and dimensionless groups
University of Alberta are also held constant, this approach may not work if:
• The process involves competing rates that depend on local
concentration (formation of an undesired product from a
mixing sensitive competitive reaction).
• Large scale-up factors are involved. The processing of
mineral slurries is a good example. While bench scale tests
are run in vessels with a typical diameter of 24 cm, industrial
scale vessels may have a diameter larger than 15 m (50 times
larger). The particle size distribution rarely scales-down in a
straight forward way.
• It is impossible to keep the same geometry for different
scales. Biotechnology processes are usually first developed
in shake flasks while their industrial version may include
an agitated bioreactor with air spargers, different impeller
geometries and heat exchangers.
When geometric similarity is not possible, Machado and Kresta1
suggested that the mixing energy is a parameter that may be useful.
The mixing energy (J) takes into account the total energy provided
to the system and is defined as the product of the energy dissipation

106 | | September/October 2017

» FORMULATION AND DEVELOPMENT »

and the mixing time (J = ε*tmix). This concept is very useful for the the distribution of energy dissipation is more uniform than in single-
following cases: impeller stirred tanks, the maximum energy dissipation that can be
• When a process is shear sensitive but a high mixing energy achieved is very low: 0.002 W/kg for unbaffled flasks and 0.1 W/kg for
is needed, the energy dissipation (which is an instantaneous baffled flasks.6 The addition of baffles increases the maximum energy
value) may be lowered and the mixing energy increased by dissipation but also slightly increases the non-uniformity distribution
increasing the mixing time; of energy dissipation.8 These devices can also present very long
mixing times (over 100 s) which makes the measurement of local
• When geometric similarity is not feasible, a similar mixing
concentration effects more difficult.9
energy may be scaled between two different vessel
geometries. Jar tests (Figure 1b) are vessels operating with a single flat-blade
impeller and are commonly used for coagulation and flocculation
This approach has been successfully used to replicate industrial
experiments in water treatment.10-12 The flat blade impeller has a large
conditions at the bench scale for bitumen de-watering processing2
swept volume, but the local energy dissipation rate is 4-6 times higher
and for liquid draw-down.
in the impeller zone than the volume average dissipation rate.13, 14
Due to the number of options available, the choice of the bench
Stirred tanks are quite well characterized.3 They have a very
scale mixing device is a key part of this process. Figure 1 shows
inhomogeneous energy dissipation distribution, and can also drop
several mixing devices that can used for bench scale experiments.
from fully turbulent conditions at the mega-scale, where very large
The development of chemical processes relied for many years on Reynolds numbers can easily be reached, to transitional flow over
shake flasks, jar tests, and single-impeller stirred tanks. These vessels a large part of the vessel on the small scale.15 Even a stirred tank
are extremely useful and work well for many scenarios, but they also operating in fully developed turbulent flow frequently presents
present some problems that cannot be ignored. All of these test inactive zones (30% of the volume)16 and a non-homogeneous
geometries present a non-uniform turbulence distribution, regions turbulence distribution.17 There are significant differences in the local
where the flow is inactive or stagnant and parts of the volume which energy dissipation rate from the impeller region to the bulk, giving
operate in transitional flow. When any of these problems is present at εmaximum/εaverage ratios as high as 100. A stirred tank operating with a
the bench scale, the process design may be compromised (see Chapter multi-phase system (oil draw-down into water) is shown in Figure 1c.
2B in Kresta, et al.3).
In order to overcome the limitations of the shake flasks, jar tests and
Shake flasks, as shown in Figure 1a, are quite common in applications conventional stirred tanks, several mixing test geometries have been
as diverse as biotechnology, surfactant formulation or replication
proposed in recent years. The design of these mixing vessels tends
of ocean waves and are particularly useful when a large number
to either miniaturize the vessel18-21 and/or confine the flow.22, 23 Both
of experiments need to be carried out.4-6 On the other hand, many
approaches provide a more uniform distribution of turbulence, smaller
industrial processes cannot be replicated by these devices.7 Although
regions where the flow is inactive or in a transitional flow regime, and
better control of mixing.
Some of the most successful new mixing devices are summarized
below. Most of them were developed to operate in fully turbulent flow:
• The confined impinging jet reactor (CIJR),23 as shown in
Figure 2a, is a milliliter-scale reactor where two jets impinge
generating a region with very high local energy dissipation,
up to 1000 times higher than what can be achieved close to
an impeller. The CIJR is a continuous device suitable for very
intense mixing and short residence times. Figure 2b shows
the non-uniformity of turbulence in the vessel. It is used at
the bench scale when the process involves fast reactions and
is possibly most useful for precipitation reactions. This vessel
has been successfully operated with its two impinging jets
at flow rates up to 30% different.24 An extensive list of papers
describe its characterization23-27 and application.28-31

Figure 1. a) Shake flasks (adapted from Kresta, et al.3), b) jar tests • Liu, et al.32 designed a multi-inlet vortex mixer (MIVM),
(adapted from Kresta, et al.3), c) stirred tank operating with a which is a four-stream chamber used for flash nano-
multi-phase system, d) milliliter-scale bioreactor operating with precipitation. The equipment allows for well-controlled
a one-sided magnetic driven impeller (adapted from Hortsch, et
al.33), e) Kar Dynamic Mixer (KDM) showing blending experiments micromixing conditions at Re > 1 600. This design
and particle tracks from CFD simulation (adapted from Yu, et al.36), also allows for different volumetric flow rates. The
f) NETmix (adapted from Laranjeira, et al.37). supersaturation and the final solvent quality can be
controlled by varying the stream velocities.

108 | | September/October 2017

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» FORMULATION AND DEVELOPMENT »

Figure 3. Decolorization reaction in a confined impeller stirred

tank. The tank is filled with a solution of 75wt% of glucose
in water and is operating at Re = 150. The images show time
progressing from left to right. Due to the low Re, the vessel is
not able to sustain turbulent flow and several compartments
are formed.

Figure 2. a) The confined impinging jet reactor (CIJR) and b) the

local energy dissipation rate at a jet Reynolds number of 400
(adapted from Liu and Fox).26 • Laranjeira, et al.37 described the NETmix, which is a new
static mixer that consists of a network of spherical chambers
interconnected by cylindrical channels as shown in Figure 1f.
• Hortsch, et al.33presented a milliliter-scale stirred bioreactor This reactor allows for very well controlled mixing conditions
(V = 10 mL) which was able to ensure efficient gas-liquid because the network structures can be adjusted to ensure the
mass transfer in viscous media, distribute power uniformly desired yield and selectivity. NETmix has been successfully
and prevent wall growth. The vessel has a magnetically used to produce nanoparticles with high reproducibility of
driven one-sided paddle impeller with a very high swept the size distribution.38
impeller volume in an unbaffled tank, as shown in Figure 1d.
This article describes several new mixing devices that have been
This vessel was able to sustain fully turbulent flow at Re as
developed by the mixing research community. These new devices
low as 4 000.
can overcome the limitations of conventional devices (shake flasks, jar
• Bagheri, et al.34 presented a micro reactor (T = 12.5 mm) with tests and stirred tanks). Research and development professionals now
a magnetic stirrer to study the effects of mixing and catalyst have a range of well characterized mixing test cells to choose from but
choice and showed that mixing plays a role even at the the best choice still depends on a critical analysis of the process and on
millilitre-scale.
the correct assessment of the most critical process mixing constraint.
• Machado and Kresta22 characterized a stirred vessel called
a confined impeller stirred tank (CIST), which is a 1L tank
filled with 5-6 impellers, and showed that this geometry
provides a much more uniform turbulence distribution
References
when compared to standard bench scale stirred tanks. 1. Machado, M. B.; Kresta, S. M., When Mixing Matters: Choose Impellers Based on Process
The inactive zone is reduced from 30% to less than 5% of Requirements. Chemical Engineering Progress 2015, 111, (7), 27-33.
the total volume. Figure 3 shows the CIST operating with 2. Chong, J. Y.; Machado, M. B.; Arora, N.; Bhattacharya, S.; Ng, S.; Kresta, S. M., Demulsifier
a solution of 75wt% of glucose in water at a Re = 150. The Performance in Diluted Bitumen Dewatering: Effects of Mixing and Demulsifier Dosage.
Energy & Fuels 2016, 30, (11), 9962-9974.
mixing is observed through a decolorization reaction.35 The
CIST was not able to sustain turbulent flow at Re = 150 and 3. Kresta, S. M.; Etchells, A. W.; Dickey, D.; Atiemo-Obeng, V. A., Advances in Industrial Mixing:
A Companion to the Handbook of Industrial Mixing. Wiley: 2015.
several mixing compartments were observed in the vessel.
The authors recommend limiting its application to fully 4. Büchs, J., Introduction to advantages and problems of shaken cultures. Biochemical
Engineering Journal 2001, 7, (2), 91-98.
turbulent conditions.
5. Mancilla, E.; Palacios-Morales, C. A.; Córdova-Aguilar, M. S.; Trujillo-Roldán, M. A.; Ascanio,
• Yu, et al.36 developed the Kar Dynamic Mixer (KDM) impeller G.; Zenit, R., A hydrodynamic description of the flow behavior in shaken flasks. Biochemical
to be used on test tubes with high viscosity fluids. This Engineering Journal 2015, 99, 61-66.
impeller is composed of multiple twisted ribbon elements 6. Kaku, V. J.; Boufadel, M. C.; Venosa, A. D., Evaluation of mixing energy in laboratory flasks
attached to a rotating shaft. It can be used for fluids with used for dispersant effectiveness testing. Journal of Environmental Engineering-Asce 2006,
viscosities varying from 5,000 to 30,000 cP. The test tubes 132, (1), 93-101.
had diameters varying from 15 mm to 40 mm and the 7. Azizan, A.; Büchs, J., Three-dimensional (3D) evaluation of liquid distribution in shake flask
impeller/vessel diameter ratio was between 0.5 and 0.86. using an optical fluorescence technique. Journal of Biological Engineering 2017, 11, (1).
Figure 1e shows the operation of the vessel and also a 8. Peter, C. P.; Suzuki, Y.; Rachinskiy, K.; Lotter, S.; Buchs, J., Volumetric power consumption in
particle track from CFD simulations. baffled shake flasks. Chemical Engineering Science 2006, 61, (11), 3771-3779.

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 « FORMULATION AND DEVELOPMENT »

9. Rodriguez, G.; Anderlei, T.; Micheletti, M.; Yianneskis, M.; Ducci, A., On the measurement 25. Fonte, C. P.; Sultan, M. A.; Santos, R. J.; Dias, M. M.; Lopes, J. C. B., Flow imbalance and
and scaling of mixing time in orbitally shaken bioreactors. Biochemical Engineering Reynolds number impact on mixing in Confined Impinging Jets. Chemical Engineering
Journal 2014, 82, 10-21. Journal 2015, 260, 316-330.
10. Yu, W. Z.; Gregory, J.; Campos, L.; Li, G. B., The role of mixing conditions on floc growth, 26. Liu, Y.; Fox, R. O., CFD predictions for chemical processing in a confined impinging-jets
breakage and re-growth. Chemical Engineering Journal 2011, 171, (2), 425-430. reactor. AIChE Journal 2006, 52, (2), 731-744.
11. Kan, C. C.; Huang, C. P.; Pan, J. R. S., Time requirement for rapid-mixing in coagulation. 27. Li, W.-F.; Du, K.-J.; Yu, G.-S.; Liu, H.-F.; Wang, F.-C., Experimental study of flow regimes in
Colloids and Surfaces A-Physicochemical and Engineering Aspects 2002, 203, (1-3), 1-9. three-dimensional confined impinging jets reactor. AIChE Journal 2014, 60, (8), 3033-
3045.
12. Rossini, M.; Garrido, J. G.; Galluzzo, M., Optimization of the coagulation–flocculation
treatment: influence of rapid mix parameters. Water Research 1999, 33, (8), 1817-1826. 28. Gavi, E.; Marchisio, D. L.; Barresi, A. A., On the Importance of Mixing for the Production of
Nanoparticles. Journal of Dispersion Science and Technology 2008, 29, (4), 548-554.
13. Cheng, C.-Y.; Atkinson, J. F.; Bursik, M. I., Direct Measurement of Turbulence Structures in
Mixing Jar Using PIV. Journal of Environmental Engineering 1997, 123, (2), 115-125. 29. Chiou, H.; Chan, H.-K.; Heng, D.; Prud’homme, R. K.; Raper, J. A., A novel production method
for inhalable cyclosporine A powders by confined liquid impinging jet precipitation.
14. Stanley, S. J.; Smith, D. W., Measurement of Turbulent-Flow in Standard Jar Test Apparatus. Journal of Aerosol Science 2008, 39, (6), 500-509.
Journal of Environmental Engineering-Asce 1995, 121, (12), 902-910.
30. Ma, C. Y.; Liu, J. J.; Zhang, Y.; Wang, X. Z., Simulation for scale-up of a confined jet mixer
15. Machado, M. B.; Bittorf, K. J.; Roussinova, V. T.; Kresta, S. M., Transition from turbulent to for continuous hydrothermal flow synthesis of nanomaterials. The Journal of Supercritical
transitional flow in the top half of a stirred tank. Chemical Engineering Science 2013, 98, 218- Fluids 2015, 98, 211-221.
230.
31. Siddiqui, S. W.; Unwin, P. J.; Xu, Z. H.; Kresta, S. M., The effect of stabilizer addition and
16. Bittorf, K. J.; Kresta, S. M., Active volume of mean circulation for stirred tanks agitated with sonication on nanoparticle agglomeration in a confined impinging jet reactor. Colloids and
axial impellers. Chemical Engineering Science 2000, 55, (7), 1325-1335. Surfaces a-Physicochemical and Engineering Aspects 2009, 350, (1-3), 38-50.
17. Zhou, G. W.; Kresta, S. M., Impact of tank geometry on the maximum turbulence energy 32. Liu, Y.; Cheng, C.; Prud’homme, R. K.; Fox, R. O., Mixing in a multi-inlet vortex mixer (MIVM)
dissipation rate for impellers. AIChE Journal 1996, 42, (9), 2476-2490. for flash nano-precipitation. Chemical Engineering Science 2008, 63, (11), 2829-2842.
18. Brivio, M.; Verboom, W.; Reinhoudt, D. N., Miniaturized continuous flow reaction vessels: 33. Hortsch, R.; Stratmann, A.; Weuster-Botz, D., New milliliter-scale stirred tank bioreactors for
influence on chemical reactions. Lab Chip 2006, 6, (3), 329-44. the cultivation of mycelium forming microorganisms. Biotechnology and Bioengineering
2010, 106, (3), 443-51.
19. Fernandes, P.; Cabral, J. M. S., Microlitre/millilitre shaken bioreactors in fermentative and
biotransformation processes – a review. Biocatalysis and Biotransformation 2006, 24, (4), 34. Bagheri, S. R.; Gray, M. R.; Shaw, J. M.; McCaffrey, W. C., In Situ Observation of Mesophase
237-252. Formation and Coalescence in Catalytic Hydroconversion of Vacuum Residue Using a Stirred
Hot-Stage Reactor. Energy & Fuels 2012, 26, (6), 3167-3178.
20. Kumar, S.; Wittmann, C.; Heinzle, E., Minibioreactors. Biotechnology Letters 2004, 26, (1),
1-10. 35. Cabaret, F.; Bonnot, S.; Fradette, L.; Tanguy, P. A., Mixing Time Analysis Using Colorimetric
Methods and Image Processing. Ind. Eng. Chem. Res. 2007, 46, 5032-5042.
21. Lattermann, C.; Buchs, J., Microscale and miniscale fermentation and screening. Curr Opin
36. Yu, Z.; Cope, R. F.; Kar, K. K.; Even, R. C.; Guillaudeu, S. J., Mixing Performance of the Novel
Biotechnol 2015, 35, 1-6.
Kar Dynamic Mixer Impeller in Small Laboratory-Scale Systems. Industrial & Engineering
22. Machado, M. B.; Kresta, S. M., The confined impeller stirred tank (CIST): A bench scale Chemistry Research 2012, 51, (46), 15282-15292.
testing device for specification of local mixing conditions required in large scale vessels.
37. Laranjeira, P. E.; Martins, A. A.; Nunes, M. I.; Lopes, J. C. B.; Dias, M. M., NETmix®, a new
Chemical Engineering Research & Design 2013, 91, (11), 2209-2224.
type of static mixer: Experimental characterization and model validation. AIChE Journal
23. Johnson, B. K.; Prud’homme, R. K., Chemical processing and micromixing in confined 2011, 57, (4), 1020-1032.
impinging jets. AIChE Journal 2003, 49, (9), 2264-2282.
38. Silva, V. M. T. M.; Quadros, P. A.; Laranjeira, P. E. M. S. C.; Dias, M. M.; Lopes, J. C. B., A
24. Siddiqui, S. W.; Zhao, Y. A.; Kukukova, A.; Kresta, S. M., Characteristics of a Confined Novel Continuous Industrial Process for Producing Hydroxyapatite Nanoparticles. Journal
Impinging Jet Reactor: Energy Dissipation, Homogeneous and Heterogeneous Reaction of Dispersion Science and Technology 2008, 29, (4), 542-547.
Products, and Effect of Unequal Flow. Industrial & Engineering Chemistry Research 2009,
48, (17), 7945-7958. Editor's Note: All images are copyright of their original publishers.

www.americanpharmaceuticalreview.com | | 111
» MANUFACTURING »

Second Level Introduction

Articles like these generally have the same opening – a discussion

Thinking in Cleanroom of increasing expression rates in mammalian cell culture systems,

the advent of single use technology, advances in personalized and
orphan therapies, etc. and how they all either by themselves or in
Decision Making combination with each other have made smaller scale manufacturing
of pharmaceuticals and biopharmaceuticals a growing trend. This
trend has led to an increased interest in modular manufacturing. The
promise of modular manufacturing has been speed and flexibility.
Speed and flexibility in this context are relative, the question generally
being, is it faster than and more flexible than traditional brick and
mortar facilities? These two low barriers are easily overcome by most
modular options. When they are, the next question is, “What’s the
Sidney Backstrom cost per square foot?” This question won’t bother some, as they have
shaved costs to be the low cost leader. But for those who emphasize
Executive Director, Business Management
quality in their offerings, it hits like a punch in the gut. This paper
Peter Makowenskyj analyzes whether cost of a cleanroom should be the focal point in the
buying decision or whether other factors should be more prominently
Sales Engineer
considered above the cost per square foot data point. We don’t buy
filling machines or bioreactors because they are cheap. We don’t buy
Maik W. Jornitz single use bags and filters because they are cheap. And we certainly
Chief Executive Officer don’t buy Apple® products, Samsung appliances and even major
purchases like our homes because they are cheap. So the question
G-CON Manufacturing, Inc. is-why would we, after investing tens if not hundreds of millions of
dollars in research, development and process equipment, decide that
the environment where life-saving and life-improving therapies will be
made needs to be the cheapest available?
There are a number of key drivers to consider in deciding which
facility design option is the best for a particular need. While this paper
advances the notion that cost per square foot is not the appropriate
center point of that discussion, it won’t be disregarded as a factor.
Speed and flexibility will also be considered and not just in the
context of a comparison to brick and mortar. Other cost factors will
be considered as well. Only after considering all of these factors, can
an informed decision be made on the cleanroom option for a project.

Speed
There is a saying in sports that “Speed kills.” What that means is speed
makes up for a lot of other deficiencies on the playing field. An error

112 | | September/October 2017

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» MANUFACTURING »

on offense or defense in any of the major sports can be offset by

speed. Because of this, a high value is placed on speed in sports. In
therapeutics manufacturing, speed has the same level of importance
but it might be more appropriate to adjust the adage to “Speed adds.”
Why speed is important in this area is examined herein.
Speed in this context is really focused on time to market for the
manufacturer’s product. There are a number of reasons why
shrinking this timeline as much as possible is to the advantage of the
manufacturer. First, being able to produce fast may allow new drugs
for unmet indications to reach patients. An ancillary benefit of this is
putting the manufacturer in a first to market position. Being the first
product to be approved by regulatory agencies and insurers and also
be prescribed by care providers is a major advantage. Being second
to market requires agencies and insurers to approve an additional
therapy for a given condition and requires care givers to change
treatment regimens that have already been put in place. The old Figure 1. Time to first product run and manpower comparison for
adage “If it ain’t broke, don’t fix it” applies to the disadvantage of the facility options.
manufacturer who gets to market second.
Second, producing a product with speed increases the amount of time
that the intellectual property of the company can provide returns. building or refurbishment of the host facility. Such an approach is
While it may be distasteful to some to focus on the monetary aspect of not possible with the other methods, which must be constructed
drug development, there are two realities related to this topic: 1) drug after the host facility is substantially complete. An additional factor
developers are in business to generate shareholder value and 2) the that the study did not consider but that also provides time benefits,
cost of drug development is staggering.1 The Tufts Center for the Study is the fact that prefabricated cleanrooms allow for abbreviating the
of Drug Development estimated in 2014 that the cost of developing a typical design timelines. Instead of creating new designs from scratch,
drug was $2.6 billion.2 Processes are generally patented well before a standard designs can be utilized to reduce the design time and
product is approved by the Agency. And patent lives are also limited cost effort. To this extent, facility and infrastructure designs may be
in time. (In the U.S., the life of a patent is twenty years.) With drug available “off the shelf” using the prefabricated approach with minimal
development taking eight years on average, time is precious.3 So or no configuration.
being able to get to market in one year versus three years, for example,
This demonstrated speed of the prefabricated approach leads to
increases the finite amount of time the developer has to monetize
significant financial benefits. The study mentioned above considered
their significant investment.
the same and the results are set forth in Table 1 below. As expected,
Third, when a manufacturing facility can be built quickly, capital the simple cost per square foot analysis favored the traditional stick-
investment decisions and therefore the actual spending can be built (typically epoxy coated gypsum wall) approach. The higher
delayed.4 This delay not only allows for retaining funds as long as quality epoxy coated prefabricated aluminum structures are higher as
possible, but also provides the opportunity to gauge the success of expected. And the modular built came in between the two in terms of
the drug development, before investing in the capital side. If a decision cost alone. Notably though, the cost differences per square foot were
can be delayed until the end of Phase II, there will certainly be less not significant top to bottom with prefabricated at $574 versus the
execution risk in that decision than one made during Phase I.
stick built at $516.
Having established that speed is a critical factor to consider in the
When these costs are considered along with the financial benefits of
facility approach decision, we must now determine which facility
speed to market, the dollar per foot savings pale in comparison. The
method produces a faster result. A leading A&E firm considered
following example provides a framework for considering the financial
the amount of time to build a 2,000L mAb facility in a recent study.
benefits of a faster time to market.
That study considered three types of facility options: prefabricated
cleanrooms, stick-built, and a modular panel wall approach. Figure 1 • Batch Size: 2,000L (two production reactors)
presents their findings. • Titer: 2 g/L
As can be seen in the chart, the time differences between the three
• Process Yield: 65%
models are substantial, with prefabricated systems being much faster.
The prefabricated approach led to a facility being ready for operation • Revenue/Gram Mab: $10,000
sixteen (16) months after the start date compared with twenty-five • Overall Cycle Time/Production Reactor: 21 Days
(25) months for the paneled approach and twenty-seven (27) months
• Net Profit: 40%
for the stick built approach. The prefabricated system solution was
faster due in part to the cleanrooms being built in parallel with the • Facility Size: 45,455 ft2

114 | | September/October 2017

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» MANUFACTURING »

Table 1. Summary of Cost Buildup for a mAb Facility – Pre-fabricated, 1. Can more than one product
Modular and Stick-Built be produced, simultaneously
Prefabricated Stick-Built Modular-Built and concurrently?
DIRECT COST SUMMARY Total Direct $/sf Total Direct $/sf Total Direct $/sf 2. Can the amount of production
1 General Conditions $134,000 $3 $501,000 $11 $441,000 $10 change to meet demand
2 Building Costs $15,289,550 $315 $5,454,074 $120 $6,814,699 $150 without interruption in the
3 MEP Costs $938,162 $21 $4,727,618 $104 $4,727,618 $104 ongoing process?
4 Process Equipment $4,538,500 $109 $4,538,500 $109 $4,538,500 $109 3. Can the facility be moved to meet
5 Process Commodities Costs $2,131,840 $48 $2,131,840 $48 $2,131,840 $48 changing global or regional demand?
6 Misc Direct Costs $520,700 $11 $304,900 $7 $341,200 $8 4. Can additional process steps be added
TOTAL DIRECT COST $23,552,900 $507 $17,658,100 $399 $18,994,900 $428 without interrupting operations?
INDIRECT COST SUMMARY Total Indirect Total Indirect Total Indirect
5. Can the facility methodology be
7 Engr/Procurement/Constr Admin $904,500 $21 $1,765,900 $40 $1,899,500 $43
deployed in foreign jurisdictions?
8 Validation $545,900 $12 $623,900 $14 $623,900 $14
Considering the main options, stick built,
9 Construction Mgmt Services $663,100 $15 $1,412,700 $32 $1,519,600 $34
modular panels and prefabricated cleanrooms,
10 Commissioning $416,600 $10 $529,800 $12 $569,900 $13
it seems obvious that all are more flexible in
11 Construction Mgmt Fee $414,500 $10 $883,000 $20 $949,800 $21 regard to these areas than the legacy model,
TOTAL INDIRECT COST $2,944,600 $68 $5,215,300 $117 $5,562,700 $125 where brick and mortar facilities were built
TOTAL DIRECT & INDIRECT COST $26,497,500 $574 $22,873,400 $516 $24,557,600 $554 and dedicated to a single product.6 Such was
the approach of choice in the early pharma
Based on this hypothetical example and the the money saved by going with a stick built days when every product was assumed to be
known construction timeframes above, the approach is quickly lost via revenue lost from a future blockbuster drug. If the demand was
difference in overall value of each approach is the delayed entry into the market. never realized or fizzled to less than what was
significant. The revenue realized from getting forecasted, either because the patent expired
to market faster substantially outweighs the or a competitor or generic emerged, the facility
higher per foot cost of both the modular and Flexibility was typically shut down and mothballed.
There was no ability to re-purpose or move the
prefabricated approaches. See (Table 2).
Flexibility is another key driver in the facility facility. As such, an asset with a depreciable life
The prefabricated approach leads to an IRR approach decision. But what does flexibility of 35-40 years became obsolete well before
of 26% compared to and 11.7% for modular mean?5 The “flexible facility” description is that timeframe. With the increasing amount of
panels and a mere 8.5% for stick built. The often used to describe single-use processes mothballed “assets,” which really weren’t assets
net return on assets of the prefabriacted within bioprocesses. That however does not at all, flexibility of facilities and processes
approach is 10X higher than stick built and speak to the facility. For a facility to be flexible became a key criterion in facility decisions.
almost 4X higher than modular-built. Hence, some appropriate questions include: The failure of brick and mortar to provide
Table 2. Financial value of each facility approach. flexibility spawned the stick built, modular
panel and prefabricated cleanrooms. Each
Prefabricated Stick-Built Modular-Built
sought to provide more flexibility. But have
Construction/Commisioning Costs $26,497,500 $22,873,400 $24,557,600
they done so? The answers to the following
Cost/ft2 $583 $503 $540
questions shed light on that topic.
Time to Build 10 21 19

Batches/Week $26,000,000 $26,000,000 $26,000,000

Weeks/Month 4 4 4
Can the facility option support a
Revenue/Month $69,333,333 $69,333,333 $69,333,333 multi-product and multi-purpose
Value after 22 months $832,000,000 $69,333,333 $208,000,000 facility to meet a developer’s
Profit $332,800,000 $27,733,333 $83,200,000 changing pipeline?
Depreciable Life 5 to 7 8 to 10 10 to 15
Multi-product, multi-purpose facilities can be
Useful Life 20 to 30 8 to 10 10 to 15
provided using prefabricated autonomous
Return on Net Assets 12.56 1.21 3.39
cleanroom modules that are not interconnect-
Net Present Value $186,096,239 $27,210,530 $50,429,930
ed to other areas of the facility. The lack of com-
Internal Rate of Return 26.0% 8.5% 11.7%
mon HVAC between the units assures that sepa-
Payback Period 0 years 1.28 years 1.18 years
rate operations can occur within each module
Discounted Payback Period (@ 10%) 0 years 1.33 years 1.21 years
cluster. (Figure 2).7

116 | | September/October 2017

 « MANUFACTURING »

taking away capacity and rebalancing if rooms are added or taken

out of commission.

Can the facility be moved to meet changing global or

regional demand?
Up until recently, asking if a facility or facility infrastructure could be
relocated would at least get you an odd stare and at most get you
a trip to your friendly neighborhood behavioral health clinic. But
now moving cleanroom infrastructures is readily considered as an
advantage to certain approaches.5 Flexibility in this regard could be
moving capacity to meet demand in another country and even for
production of another product. With stick built infrastructures, this
flexibility simply is not present. Once built, the infrastructure cannot
be disassembled and moved or simply moved without disassembly.
For modular panels, while the panels themselves can be disassembled
Figure 2. Example of a multi-tenant or multi-product site and moved, the non-modular mezzanine structure housing the HVAC
equipment cannot be. So moving this type of facility is not possible
either. In the prefabricated approach, the prefabricated units are
With stick-built infrastructures, such an autonomous approach is not mobile. Some even employ air bearings for easy movement into,
likely given the expansive HVAC infrastructure typically required. And around and out of facilities. Their minimal connection to the facility
even if separate zones were planned, those zones would not be able and onboard HVAC equipment makes them fairly autonomous within
to accommodate changes once installed, thereby rendering them the larger facility as well. As such, these units can be relocated with
ultimately inflexible. ease. Disconnecting the units from each other and the host facility
takes a matter of days. Then the units can be individually moved on a
Modular wall panels generate a similar outcome. Generally, one HVAC
standard eighteen wheeler to the next site or port of choice.
system will serve the entire infrastructure, meaning that changes
to the process(es) cannot be accommodated without redesigning, With the mobility of prefabricated units, one can even imagine
rebuilding and retesting the entire system. a centralized storage location of the units, which would serve as
the supply center for cleanroom capacity when needed. When the
Can the amount of production change to meet cleanroom infrastructure is no longer needed at a particular location,
it can be shipped back to the central site for cleaning, sanitization,
demand without interruption in the ongoing process?
service, maintenance and re-calibration if needed. This centralized
Modular panel construction onsite, while initially flexible in terms of area could also be used if cleanroom assets from disparate regions
size and shape, is not very malleable once it is built. Wall panels, doors, require retrieval due to political unrest or unfavorable changes in tax
ceilings and the like are permanently placed in position. Ductwork and structure/economic incentives. With the increasing need to supply the
utilities are provided in intricate detail above the cleanroom structures. patient base on a local level, the ability to move cleanrooms may go
Adding to or reducing the footprint of the fixed structures requires from a “nice to have” to a “need to have” in very short order. As such,
onsite construction or demolition, which will interrupt ongoing cleanrooms may take on even more mobility in the future. (Figure 3)
production. Once construction and/or demolition are complete,
rebalancing of the HVAC system and revalidation of the cleanroom
space will be required.
Can additional process steps be added without
interrupting operations?
Prefabricated cleanrooms – While it may seem that changes in
capacity would not be possible with such discrete units, the contrary is With pre-fabricated units, if additional production capacity is needed,
true. New units can be added when needed and without interrupting an additional autonomous production suite can be added without
the existing process. They can be added in a linear fashion or added interrupting the existing unit operations. The same is true for filling
to the existing cluster via the use of a corridor or previously placed capacity. Due to the autonomous nature of each unit, an overall
“knock-out” panel. A reduction in capacity in this context does not HVAC rebalance is not required. On site construction is not required.
mean that previously placed units are shut down. Rather, they can be Shutdowns to accommodate the onsite construction are not required.
disconnected and moved out of the facility for deployment elsewhere. As such, adjusting the process to add or takeaway steps is not an issue.

Stick Built – Similar to modular construction onsite but is even less For the modular paneled approach and the stick built approach, a
flexible than the paneled approach. In the paneled approach, walls change in process would result in onsite construction, a shutdown to
can be moved more easily than a stick built wall which is most often accommodate that construction, additional or different HVAC design
gypsum board covered with epoxy. Moving a wall in this option and a rebalancing of the HVAC system upon completion. As such, the
means demolition. Centralized HVAC distribution requires adding or process cannot be readily changed with either approach.

www.americanpharmaceuticalreview.com | | 117
» MANUFACTURING »

A Real Life Example

The perspective of cell and gene therapy is a good example in
considering flexibility of the options being considered. In cell and gene
therapy production rigorous containment is required. Typically, a cell
therapy application requires a relatively small processing space, for a
patient by patient drug production. Patient samples must be handled
with the utmost care. The cleanroom structure must be sanitized
frequently, generally with vaporized or ionized hydrogen peroxide.
And cross contamination from other process spaces must be avoided.
Reviewing the options with this example in mind, it is clear that the
pre-fabricated autonomous, segregated cleanroom units stand well
above the others. Each unit having its own HVAC and providing
unidirectional flow achieves the results required. (Figure 4). The
autonomy of the HVAC units allows for cleaning runs to occur in each
unit when needed, e.g., after every patient sample is processed is
even possible. While the other systems can be designed to achieve
Figure 3. Truck or trailer based example of a unidirectional flow, neither of them will be able to provide autonomy
mobile processing system. of HVAC for each process space. As such, cleaning will have to occur
during facility wide shutdowns. Containment between process spaces
will not be feasible either given the shared HVAC design.
Can the facility methodology be deployed in foreign
jurisdictions?
Cost
The ability to produce internationally has become an area of more
interest for pharma and biotech recently. Multi-national production As noted at the beginning of this paper, cost is a factor but should
provides developers with numerous benefits: 1) avoiding import not be the sole factor in the facility decision making process. This is
duties and transportation costs; 2) being able to produce to actual especially true when, as shown above, costs for the three considered
approaches are fairly in line with each other.
demand instead of forecast; 3) not relying on a single site for
worldwide production; and 4) opening markets where importing
was not allowed. So the question of whether any of the three options
being considered can be reliably and rapidly deployed into numerous
foreign jurisdictions is very relevant today.

With prefabricated cleanrooms, the infrastructures are built at a

central site by an experienced workforce and shipped to the location.
A small crew from the manufacturer arrives with the structures and
installs and connects the modules to each other. This process can be
completed in a matter of days. The modules are then commissioned by
the manufacturer’s quality team over a two to three week period and
the process is complete.

With stick-built, the drug manufacturer has to identify a new

contractor and engineer for each project who will design and build
a custom facility each time. Costing, design, permitting and building
will be specific to the site for each project. There will be little to no
institutional knowledge in the design and build from one project to
the next.

In the use of a paneled approach, some efficiency is gained in the

use of standard materials, but the exercise of choosing engineers, Figure 4. Cell therapy example - prefabricated units can be scaled
contractors, designing, costing, etc. is largely the same and is subject without interrupting other units.
to the same issues and delays.

118 | | September/October 2017

 « MANUFACTURING »

There are other elements of cost that should also be considered. made with wall structures. If the walls are epoxy coated gypsum walls,
One such factor is project scope. Because of the similarity in pricing as are often provided in stick built structures, hairline cracks result
between the various options, many vendors battle each other in a providing a conduit for moisture into the permeable wall material.
race to the bottom, seeking to have the lowest cost per square foot Modular wall panels and prefabricated cleanroom systems do not
price. In the process, many vendors quote a scope that is only a portion
allow such moisture penetration. Moisture penetration has to be
of the overall project. As an example, some providers of wall panels
avoided at all costs, as it promotes mold growth. Resulting unplanned
simply quote the walls and ceiling and not the mechanical and utility
equipment and design and installation of the same, just to name a repair, interruption and shut-down costs will more than offset the
few. Unsuspecting customers may only realize later, when they have cheaper purchase price.
already paid for the limited scope, that more is needed to complete
the project. For this reason the legacy cost per square foot approach
should be viewed with a fair amount of skepticism. The end user Conclusion
should ensure that all costs are considered including electrical, HVAC,
fire suppression, flooring, commissioning costs, onsite insurance, Modern facility designs are changing due to the demand for more
etc. In addition, for onsite built projects, health and safety insurance, agile, smaller footprint and flexible facilities. The evolution of the
project oversight, permitting costs (and potential delays) and laydown offerings available requires new thinking when it comes to making
area requirements should be considered and included into the overall
a facility decision. To determine which facility design and cleanroom
cost calculation.
infrastructure is best for the specific application, cost, speed and
The cost of project changes should be considered too if a total cost flexibility of the options should be considered. As we have seen,
of ownership analysis is being performed. With respect to onsite
speed in this context generates significant value. Flexibility is also
construction methods, in this case stick built and modular panel, if a
important as a flexible option becomes a renewable asset within the
change is required, the project timeframe will be extended leading
to additional disruption of the existing processes or the need for buyer’s overall production capability, for one or multiple products,
additional oversight. With prefabricated structures, most changes domestically or otherwise. And cost must not only consider the
will occur off-site, before the systems are placed into the final facility. initial cost per square foot but the overall project cost, the quality,
Hence, additional direct and indirect costs are avoided. and the indirect cost ramifications of the chosen option. In analyzing
Qualification costs should also be considered. These costs are much the options of stick-built, modular wall panels and prefabricated
more predictable in the prefabricated system. Prefabricated modular cleanrooms in this comprehensive manner, we have seen that
cleanroom units undergo prequalification in an offsite factory prefabricated cleanrooms stand well above the other options and
acceptance test. Within this prequalification phase, all functions are should be given due consideration for facility projects.
tested and issues can be corrected off-site. In the other approaches,
qualification occurs on-site and if corrections are required additional
cost, time, contractor traffic and disruption to the entire site will occur.
With prefabricated, the owner can expect approximately six people
References
on site for 2-5 days to move the prefabricated units into place and 1. Herper, (2002) Solving the Drug Patent Problem, Forbes
connect them to each other. In the build on site approaches up to 100
2. Silverman (2014) “What Does It Cost to Develop a New Drug? Latest Study Says $2.6 Billion”
FTEs can be expected on-site for eight months and beyond to build a
Wall Street Journal
traditional cleanroom structures.
3. Jornitz, M., Backstrom, S. (2016) Evaluating the Benefits of Prefabricated Cleanroom
Another cost factor to be considered is depreciation. Traditional fixed Infrastructure Designs and Costs, Pharmaceutical Engineering, 77-81
installed infrastructures are depreciated over 30-40 years, as they are
4. Jornitz, M. (2013) Defining Flexible Facilities, Pharmaceutical Processing, 22-23
considered part of the facility for tax purposes. Prefabricated, modular
and mobile cleanroom units are depreciated as equipment over a 5. For additional thoughts on what constitutes flexibility in facilities, see Backstrom, (2017)
time period of 7-8 years.8 This accelerated depreciation provides real Analyzing the flexibility of Pharmaceutical and Biopharmaceutical Options, International
Journal of Pharmaceutical Science Invention.
economic advantages as it relates to taxes and cash flow. This benefit
is typically not considered by those only concerned with the lowest 6. Sutton, S. (2017) Podifying Cleanroom Processes, the Medicine Maker, 40-43
cost per square foot. 7. One can even envision a facility fit out with prefabricated modules being multi-stakeholder
Finally, cost considerations cannot be made in a vacuum. The quality where multiple entities operate on their own programs independent of one another.
Certain costs could be shared such as quality, shipping/receiving, maintenance, etc. In this
being supplied has to be considered.9 A quote often attributed to
way the sharing of administrative costs would lower the overhead for each stakeholder.
Benjamin Franklin brings the point home - “The bitterness of poor
quality remains long after the sweetness of low price is forgotten.” 8. Markarian, J. (2014) Continuous Solid-Dosage Manufacturing Platform Nears Prototype
With the advent of single-use process equipment, process equipment Installation, Pharmaceutical Technology, 52-54
has become more mobile, e.g., pallet tanks. When these systems are 9. Jornitz, M. (2013) Cleanroom Construction: Materials Matter, Blogs/Pharmaceutical and
moved through the cleanroom space, it is inevitable that contact is Biopharmaceutical Manufacturing, Pharma Evolution

www.americanpharmaceuticalreview.com | | 119
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How Polymer Science

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New developments in polymer science are broadening the role that for invasive procedures, thereby
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DRUG RELEASE WITH and allow complete dissolution in the having a resolution of diarrhea.
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dietary requirements make HPMC an showed that DRcaps acid resistance Hydroxypropyl methylcellulose
important capsule polymer. is not affected by the presence of up acetate succinate (HPMC-AS). While
to 40 percent alcohol (ethanol) in the the polymer blend differs from what
Capsugel’s introduction of an HPMC dissolution media, which may help the enTRinsic capsules use, Vcaps®
capsule manufactured through prevent alcohol dose dumping in Enteric offer a similar benefit: simpler
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dissolution. This gave the new option as an extended delayed-
HPMC capsules pH independent release oral dosage form. 3 These enteric capsules comply with
disintegration, and was shown relevant EP, JP and US
in a human biostudy to provide Another study – the results of which Pharmacopeia monographs and
bioequivalence compared to appeared in medical journals – have been evaluated in vitro across
a gelatin capsule.1 described how investigators at a number of compounds. The results
Massachusetts General Hospital show they protect the stomach from
used DRcaps for an unusual aggressive APIs and delay release to
ACID-RESISTANT CAPSULES
treatment of a serious medical provide maximum absorption.
Launched in 2011, DRcaps™ capsules problem. They used pre-screened
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capsules protect the ingredients cause of morbidity and mortality.
from fully releasing in the stomach, The capsules obviated the need
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Influence of gelling systems on HPMC capsules in In vitro dissolution of caffeine in Vcaps® Plus capsules
dissolution testing
100 100

90 90

80 pH 1.2 USP 80

% Caffeine dissolved
% Caffeine dissolved

pH 6.8 USP 70
70
pH 6.8 JP2
60 Simulated milk fluid
60 pH 1.2 USP
pH 1.2 – 2 g KCI/L 50
pH 6.8 USP
50
pH 1.2 – 9 g KCI/L pH 6.8 JP2
40 40
Simulated milk fluid
30 30 pH 1.2 – 2 g KCI/L
20 pH 1.2 – 9 g KCI/L
20

10 10

0 0
0 3 6 9 12 15 18 21 24 27 30 35 40 45 50 55 60 75 0 3 6 9 12 15 18 21 24 27 30 35 40 45 50 55 60 75

Time in minutes Time in minutes

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The enTRinsic™ drug delivery technology provides full
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90
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% dissolved

60

shown to rapidly release at pH 5.5, allowing optimal 50

Budesonide
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in Vcaps Enteric
Enteric Budesonide
enables formulators to accelerated product development 30 (Entocort)
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LOOKING FORWARD WITH FUNCTIONAL CAPSULES

improve drug delivery. From research to human dosing,
In-vivo tests have shown that soluble compounds are well HPMC capsules provide predictable delivery of simple,
absorbed from both HPMC-based Vcaps® Plus and gelatin immediate-release formulations and address the complex
capsules. In most cases, capsules of either material perform needs of targeted release, moisture protection, and enteric
similarly, but in some applications they don’t. delivery. The variety of HPMC capsules now available,
HPMC capsules, for example, can interact with poorly combined with a host of innovative strategies and
soluble APIs in a way that leads to a lower crystallization technologies for drug delivery, offer a means of addressing
rate in the GI tract. This can be important when there are the challenges of today’s APIs and provide a platform to
supersaturated APIs in the intestine, as can occur when develop patient centric formulations that incorporate the
dosing either a high-energy salt form or a weakly basic next generation of molecules in development.
API. In those cases, HPMC-based capsules can help
maintain super-saturation by inhibiting crystallization. REFERENCES
The degree to which crystallization inhibition affects 1. S. Stegemann, et. al. Comparative Human In-Vivo Study of an Immediate
Release Tablet Over-encapsulated by Gelatin and Hydroxyproply Methyl
in-vivo performance will depend on a particular application, Cellulose Capsules – Impact of Dissolution Rate on Bioequivalence.
but HPMC has the potential to play a role as a functional Amer Pharm Review, Nov./Dec., 2015. Vol. 18, Issue 7.
excipient which improves bioavailability.5 Capsugel’s 2. Amo R. DRcaps Capsules Achieve Delayed Release Properties
for Nutritional Ingredients in Human Clinical Study. A Capsugel-
Bend, OR, formulators predict approximately 40 percent of commissioned study conducted by Bio-Images Research in Glasgow,
molecules are weakly basic, having a basic pKa between Scotland, completed in 2013.
3. He XW, Groshens E, et al. Prolonged gastric acid resistance using a new
2 and 7, and almost all these compounds are poorly water double DRcaps approach. Capsugel R&D Hard Capsule Applications,
soluble. This indicates that there are many compounds that Colmar France.
could benefit from HPMC-based capsules.6 4. Youngster I, Russell GH, et al. Oral, Capsulized, Frozen Fecal Microbiota
Transplantation for Relapsing Clostridium difficile Infection. JAMA (2014).
Published online October 11, 2014.
SUMMARY 5. Richardson, M., Morgan, M. Next Generation HPMC Capsules Bioequivalence
and Functional Performance, Pharmaceutical Technology, April, 2016.
Today’s HPMC capsules are more than an alternative to 6. Capsugel data on file.
gelatin capsules. They offer an array of opportunities to
© 2017 Capsugel Belgium NV All rights reserved.
» FORMULATION AND DEVELOPMENT »

Strategies to Predict the Developability

of Biopharmaceuticals
Dr. Stefan R. Schmidt
Chief Scientific Officer
Rentschler Biopharma SE

costs. Secondly, speed to market must be taken into account. Any

Introduction frontloading of activities that result in an earlier access to the market
are worth millions as the patent lifetime is limited. Thirdly, improved
Over the last decades drug development expertise for biologicals quality will contribute to better patient satisfaction and therapeutic
has constantly increased. Nevertheless failures still happen and are efficacy. Therefore developability assessment is introduced to enable
mostly related to intolerable toxicity, low efficacy and undesired a ranking of several potential product candidates not only based on
physicochemical and biopharmaceutical attributes. A complete their biological activity. This article describes typical strategies to
overview can be found in Figure 1. Although overall the success rates predict the behavior of molecules in terms of aggregation, stability,
for biologicals are a little better in comparison to small molecules, potency, pharmacokinetics and -dynamics (PK/PD), solubility, viscosity,
just the attrition rate could be further reduced by a more systematic immunogenicity and safety, taking into account sample consumption,
approach, assessing the “developability” of new biological entities duration of the assays and amenability to high throughput. These
(NBE) at an early stage. This strategy positively impacts cost, time
methods have usually been established for monoclonal antibodies,
and quality. Firstly, it is favorable to know as much as possible about
however most of them are applicable to any other NBE as well. This
the molecule as early as possible to avoid any negative surprises at a
supplies scientists in drug development with an arsenal of tools
more costly time point. This includes also the consideration whether
which can be utilized beyond standardized platforms that work
the protein can be manufactured at production scale at competitive
only for certain molecule classes. Although most companies have
specific strategies to cover developability aspects,1 it can be observed
that the majority utilizes the tools at two distinct points in the drug
development pathway. An overview of the typical elements can be
seen in Figure 2.

Selection and Optimization Phase

Usually the first entry point is during the initial candidate screening
and selection phase, when more than 10 molecules are available that
display sufficient biological activity in terms of affinity, selectivity and
reactivity. In case the molecules are derived from genetic libraries, an
Figure 1. Potential sources of failures during development (in red: in silico sequence analysis is performed. Here critical parameters such
parameters assessed for developability) as the isoelectric point (pI), charge distribution, aggregation-prone
regions (APR), positions for post translational modifications (PTM),

122 | | September/October 2017

 « FORMULATION AND DEVELOPMENT »
Optimize
API Stability

Figure 2. Developability assessments at the

discovery/development interface

potential recognition sites for proteases and the epitope based risk
for immunogenicity are evaluated. Information about the pI is useful

Super Refined Excipients

TM
for designing appropriate downstream processing (DSP) steps and
selecting suitable excipients for formulation. Charged amino acids
should be distributed across the molecule and not concentrated
into large patches that might lead to aggregation or adsorption on n Maintain drug integrity
biological tissues. PTM sites are quite relevant as in general unpaired n Maximize efficacy
cysteines should not be present to avoid disulfide scrambling or
n Optimize delivery
intermolecular crosslinking leading to aggregation. Recognition
sequences for N-linked glycosylation to asparagine residues being n Multi-compendial compliance
sources for heterogeneity or regions susceptible to proteolytic
n Multiple dosage applications
degradation should be omitted. On the other hand if these amino acids
are required for biological function such as proteolytic activation or to
To maximize API stability, it is crucial to optimize
enable a certain glycan pattern for molecular recognition, they should
be at the correct position and properly modified. APRs in therapeutic
the drug active’s environment. A high purity
proteins are identified by measuring the dynamic exposure of excipient can help by minimizing the occurrence
hydrophobic patches. If using homology modeling or working with a of impurities that could accelerate drug active
static structure, predictions become less accurate but can be obtained degradation.
much faster.2 The immunogenicity risk can be predicted by looking
for T- or B-cell epitopes that elicit either a cellular or humoral immune Offering the most highly-purified excipients
TM
response. Unfortunately it is almost impossible today to predict available, the Super Refined range of excipients
B-cell epitopes as they, in contrast to linear T-cell epitopes, have a from Croda is manufactured through a proprietary
conformational dependency. B-cell epitopes cause the generation of
process that provides maximized efficacy of
anti-drug antibodies (ADA) that can either neutralize the activity of
the drug, alter the pharmacokinetics or even worse, cross-react with the excipient. This process physically removes
autologous proteins.3 impurities without affecting the fundamental
In case the protein is not derived from a library, obvious sequence
structure of the excipient in any way for more
and structure liabilities should already be eliminated during stable APIs. Let Croda help you harness the
molecule design. This includes removal of free cysteines, or other power of purity with Super Refined excipients.
solvent exposed amino acids sensitive to oxidation as methionine
or tryptophan. Furthermore, asparagine residues are critical as Visit us at www.crodahealthcare.com
they are the anchor point for N-linked glycosylation and tend to
isomerize to aspartic acid or just like glutamine to deamidation.
Also the presence and position of lysine must be scrutinized,
Innovation you can build on TM

as it can be the target for glycation, the covalent conjugation of

reducing sugars to the free amine group. However, when dealing
with non-antibody proteins, homology modelling of structures
gets difficult as probably not enough information on structural
conformation is available in databases.

www.americanpharmaceuticalreview.com | | 123
» FORMULATION AND DEVELOPMENT »

For the next steps protein material is required. This is usually chromatography (SMAC). Retention times on this column type are
generated by transient expression in human embryonic kidney (HEK) inversely related to colloidal stability. As it is essentially a SEC analysis,
cells which can deliver the quantities for subsequent high throughput further important properties as monomer content or solubility can be
(HTP) assays. During the candidate selection phase information about assessed simultaneously, thus saving time and sampling.7
aggregation behavior, experimental hydrophobicity and stability is
desirable. The easiest assessment of aggregation can be achieved by
size exclusion chromatography (SEC) followed by multi angle laser
light scattering (MALLS). This powerful method can also identify low
Formulation
molecular weight species resulting from degradation or misassembled Today formulation development is increasingly important as many
fragments in the case of antibodies and their derivatives. The presence drugs are administered subcutaneously at high concentrations (>100 g
of exposed hydrophobic patches is determined by retention in L-1) and low volumes (<1.5 mL). This is related to enabling patient self-
standard hydrophobic interaction chromatography (HIC). Stability can administration and indications requiring high doses. The challenges
indirectly be evaluated through differential scanning fluorimetry (DSF) in manufacturing arise from too high viscosity and propensity to
that measures the fluorescence of a dye when bound to hydrophobic aggregate. The ideal formulation minimizes viscosity while maximizing
regions in a protein at different conditions.4 thermostability. A number of excipients (sugars, amino acids) have been
The information gained from in silico analysis and the experimental data established that positively influence both parameters.8
can now be used either to re-engineer and mutagenize the protein of High viscosity is often caused by protein self-interaction that depends
interest5 or to eliminate candidates with unfavorable properties. on non-uniform charge distribution with positive and negative
patches. This can be partly predicted by in silico models, but the
experimentally determined values from dynamic light scattering (DLS)
Characterization Phase with inert latex beads are more reliable for non-antibody molecules.
Solubility is indirectly deducted from experiments with HIC and
After the first round of molecule assessment during lead optimization aggregation studies but nowadays also HTP viscometer instruments
within discovery, the next step is beyond the interface within the are available.9
phase of preclinical development. Typically no more than four top
At the initial stage, a simple formulation might be sufficient to get
lead candidates remain that have been improved or chosen based
through early development phases. However, it is recommended to
on the information from the selection phase. The first step now is
complete a more thorough optimization as early as possible to have a
to generate stable cell lines to express the proteins of interest. The
final formulation that that is suitable up to commercialization.
selection of the best cell line is based on parameters such as doubling
time of approximately 24 h, a cell concentration of more than 1×107
mL-1, a protein output of more than 10 pg per cell and day, a cell
stability beyond 20 passages and an average viability above 95%. For Strategic Recommendation
all further experimental investigations not necessarily clonal cells are
required, sometimes pool material is sufficient. This is particularly true Developability assessments typically are installed at the interface
for cells generated by site directed integration with relatively uniform between discovery and development.10 They serve the purpose of
properties instead of random integration with hugely variable results. selecting the right candidate(s) to enter the development stage. At
this point the desired biological attributes are established, so other
Now material for the following more extensive studies must be
parameters can be selected to identify the ideal candidate. Most
supplied. The initial approach is a standard cultivation of cells in shake
companies follow a two-tier approach that facilitates the optimization
flasks delivering harvest for the initial capture step. In the case of
of a limited number of leads after the first round. This selection phase
antibodies this is relatively straight forward as affinity chromatography
combines in silico sequence analysis with several HTP assays supplied
with protein A resins exists. All other proteins must rely on specific
by small scale transient expression.
properties such as charge and pI to enable high capacity ion exchange
chromatography. The first eluate from the capture step should A handful of top candidates successfully passing that first filter will
already have a purity above 70% to be applicable for subsequent then be used to generate stable pools and ultimately stable cell lines.
more detailed stability studies simulating potential extreme and non- These cell lines are then the sources for material supply to support
physiological conditions during the process. This includes exposure to all further studies comprising extensive stability assessments under
elevated temperatures, freeze-thawing cycles, storage in a wide range non-physiological conditions and paving the way for proper up- and
of pH values, and mechanical stress such as pumping, filtration or downstream process development. Formulation is a special case as
mixing. Of course, oxidative stress and the influence of light that could it benefits from the information gathered in both phases, but might
degrade aromatic amino acids and the peptide backbone should not be fully established only after finalizing the manufacturing process
be neglected.6 The sensitivity towards proteases in body fluids can thus stretching into the clinical development. It is a thin line between
be assessed by incubation in human or primate serum. In general it performing a full blown formulation development too early and
is advised to distinguish between structural and colloidal stability. wasting money and effort on failing candidates or to invest too late,
One of the suitable methods is standup monolayer adsorption thus loosing valuable time.

124 | | September/October 2017

 « FORMULATION AND DEVELOPMENT »

As a lot of data is accumulated about many different candidates it The overall goal is to integrate discovery (lead selection and
is recommended to integrate the information into IT solutions that optimization activities) with process development by implementing
enable a systematic ranking as schematically displayed in Figure 3. methods that facilitate risk assessment with minimal material
requirements and maximal throughput. The focus is on balancing
efforts not to spend too much early, but sufficiently to move forward
with a suitable candidate. Interestingly, different strategies can be
observed in different company types. For instance small and young
companies being cash flow and time constrained might tend to
limit the efforts in order to reach only the next financial inflection
point for subsequent fund raising. Established companies on the
other hand are more willing to invest more early on in order to have
a solid understanding which candidate to pursue further. Contract
manufacturing organizations, often not involved in candidate
selection, still can benefit from this holistic approach to initiate process
development, as developability studies deliver a modular tool kit to
address typical reoccurring issues.

References
1. Jarasch, A. et al. J. Pharm. Sci. 104, 1885–1898 (2015).
2. Chennamsetty, N. et al. J. Phys. Chem. B 114, 6614–6624 (2010).
3. De Groot, A.S. et al. Curr. Opin. Pharmacol. 8, 620–626 (2008).
4. Senisterra, G.A. et al. Mol. BioSyst. 5, 217–223 (2009).
5. Seeliger, D. et al. MAbs 7, 505–515 (2015).
6. Hawe, A. et al. J Pharm Sci 101, 895–913 (2012).
7. Kohli, N. et al. MAbs 7, 752–758 (2015).
8. Yang, Y. et al. Biotechnol. Bioeng. 114, 2043–2056 (2017).
Figure 3. Ranking matrix for developability assessment
9. Deshmukh, S. et al. ACS Comb. Sci. 18, 405–414 (2016).
10. Yang, X. et al. MAbs 5, 787–794 (2013).

www.americanpharmaceuticalreview.com | | 125
An Interview With...

Elizabeth Hickman
Director of Sales & Marketing
Catalent Pharmatek

»
surfactants, to improve dissolution. If simple blend excipients do
Can you tell us what Bioavailability Enhancement not provide the necessary solubility, more conventional, yet proven
is – and why it has become such an important techniques, such as micronization and wet and hot-melt granulation
topic in the pharmaceutical industry? are explored. However, when such traditional technologies do not
work, developers must look to more advanced techniques, such as
Two of the most important pieces of information that are typically lipid-based delivery and amorphous dispersions, to increase solubility.
collected for each active pharmaceutical ingredient (API) are its ability The identification of a suitable formulation is not a trivial exercise, and
to dissolve when dosed to a patient, and its ability to permeate across choosing a more advanced type of formulation will, inevitably, extend
the gut wall into the blood. These data can then be used to classify timelines and increase the complexity of the development process.
the molecule according to both the Biopharmaceutics Classification This is why these options should be employed only when there
System (BCS) and Developability Classification System (DCS). It is are no other suitable alternatives. However, advanced formulation
estimated that only a small proportion — less than 10% — of the technologies can be very powerful tools to increase solubility and
molecules in the development pipeline have both adequate solubility bioavailability. And when formulation enhancement is necessary, to
(dose-solubility ratio > 500 ml) and permeability (> 1 x 10-4 cm/sec) solve suboptimal pharmacokinetics of lead compounds – where the
to achieve good (>90%) bioavailability.1 Solubility can be addressed main challenge is the solubility of molecules – investing early on in
during both API form selection and during drug delivery technology identifying an advanced formulation technology leads to cost and
selection. During API development, solubility and stability can be time savings by avoiding rework, program delays, or worse, shelving
improved by molecule engineering - optimizing the physical form the molecule’s development due to poor clinical performance at a
of a drug molecule’s salt, co-crystals, and polymorphs. In addition, later phase.
solubility can change when purity changes.
The DCS helps to determine the optimal drug delivery technology,2 Can you tell us about some of Catalent’s latest
and classifies a molecule based on its dose-solubility ratio and technologies for bioavailability enhancement?
effective permeability, and its position on the DCS plot will help guide Are they designed to work with specific types
the formulator on which drug delivery technologies are most likely to
of products? Can they be applied to both new
give a successful oral product.
products under development – or to enhance and
The position in the DCS grid will determine the optimal starting point extend the life of older products?
to choose formulation options. Those in Class I (good solubility and
permeability) have a relatively straightforward development path Each molecule is unique, and no one technology offers a one-
where a solution, suspension, tablet, capsule and even injectable will size fits all solution, and Catalent has a broad range of enabling
work; the choice will hinge on what is likely to be most acceptable to technologies, and the expertise to support developments. Our
patients, is cost effective, and whether there are any other factors that
would discount a particular technology (like incompatibility).

What are some traditional methods for enhancing

bioavailability? Have they fallen out of favor? Why
are so many pharma companies looking towards
newer technologies to optimize their products?
As the number of new molecular entities coming out of discovery
which exhibit poor solubility rises, developers are looking to
formulation technology to overcome this issue. Traditionally,
developers would look to solubility enhancing excipients, such as

126 | | September/October 2017

 specialized equipment, such a gas chromatography, and additional
method development and testing to ensure residual solvent levels are
safe. Additionally, given the inherent nature of amorphous material,
amorphous dispersion products must be tested for long-term stability
to confirm the material does not recrystallize over time. Catalent offers
comprehensive and deep capabilities for characterization, method
development, and product testing to ensure successful development
of enabled formulations.

Looking toward the future – will bioavailability

enhancement become a normal part of drug
development and optimization processes? Do you
see more pharmaceutical companies looking for
the expertise of a company like Catalent to broaden
the market appeal and enhance products?
As more new molecule entities coming out of research present
solubility limitations, the need for bioavailability enhancement will
increase and become a normal part of development. This creates a
primary need for technologies and expertise that can recognize and
address bioavailability issues. The ability of a partner to develop and
manufacture a stable and scalable finished dosage form goes beyond
advanced bioavailability enhancement technologies include salt form having access to these advanced technologies. Newer technologies
screening, Micron particle size engineering, Pharmatek® SD spray might prove successful in preclinical testing, but have limited scalability
drying technology, OptiMelt® Hot Melt Extrusion, and lipid-based beyond the bench. Technological expertise is needed to provide a
formulations with RP Scherer Softgel technologies. safe drug delivery platform that also provides some opportunity for
customization. Understanding how a formulation technology impacts
We have developed platform screening protocols to help quickly the finished dose becomes important as the molecule transitions
and inexpensively collect the data needed and, based on our deep from early to late phase development. The experience in taking a
expertise, determine which approaches are most suitable and which molecule from candidate selection to market, with comprehensive
molecules have inherent issues that need to be resolved. To enhance characterization, solid state chemistry and formulation science,
collaborative scientific development between Catalent, its customers process development and scale-up, manufacturing capabilities
and partners, we have created a new Science and Technology function is important for successful development and directly impact the
to accelerate the development of drug products through the use of desirable reproducibility and quality of final product. More than
advanced formulation and drug delivery technologies. This team ever before, drug companies are reaching out to contract research,
of dedicated, seasoned development scientists can help customers development and manufacturing partners for help.
identify challenges and opportunities for their candidates, and
Catalent has gained significant practical experience across many
understand what drug delivery technologies can and cannot do, and
products and with many different customers of all sizes, and is known
to help them identify the right solution faster.
for having supported the commercial launch of products utilizing
its bioavailability enhancement technologies. The company is also
What do companies need to know when they engaged in many more preclinical and clinical programs with smaller
are looking to enhance the bioavailability of companies, where the attrition rate is higher. Any company that has a
existing products? Are there regulatory concerns? bigger pipeline, will benefit from a “pipeline hindsight” – experience
Product testing concerns? How can Catalent help a from prior programs – and it may be in a better position to inform
those companies at the earlier stages of development as to what data
company efficiently test and market a product with
to collect, and how best to interpret any discrepancies that are seen.
enhanced bioavailability?
Most technologies used to enhance bioavailability require additional References
levels of characterization and product testing. For example, spray dried
1. Butler JM, Dressman JB. The developability classification system: application of
dispersions (SDD) are produced by spraying solutions of the active biopharmaceutics concepts to formulation development. J Pharm Sci. 2010; 99: 4940-54.
ingredient in solvents such as acetone or tetrahydofuran. Given the
2. Hauss, David “Oral Lipid-based Formulations: Addressing an Urgent Industrial Need”,
high volumes of solvent used in the process, the potential for residual New Jersey Centre for Biomaterials www.njbiomaterials.org/NJCB_Files/File/Skin%20
solvent species in the SDDs is not insignificant. Therefore, SDDs require Workshop/5.%20Hauss_Presentation.pdf

www.americanpharmaceuticalreview.com | | 127
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patented air jet and vacuum concept: circum-
hidden inside the electromagnetic response of
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» SPECTROSCOPY »

Polarization-resolved Raman spectroscopy has great potential as a

Polarization-Resolved means of process analytical technology in pharmaceutical applications.
Recent progress has even enabled enantioselective measurements,
which were previously believed to be impossible. The present article
Raman Spectroscopy compares experimental approaches to polarization-resolved Raman
spectroscopy and discusses them with regards to their pros and cons.

for Pharmaceutical Background

Applications Raman spectroscopy is an established analytical tool in the phar-
maceutical sector. Beyond the analysis of products in the lab,
Raman techniques find more and more applications as a means of
process analytical technology (PAT). However, the full potential is
currently, by far, not tapped. A key feature that is rarely exploited in
routine Raman analysis is the sensitivity of Raman scattering to the
polarization properties of the incident light. If the laser is linearly
polarized, the majority of the Raman signal will be polarized in the
Johannes Kiefer same way. A certain fraction of the scattered light, however, will be
Technische Thermodynamik polarized orthogonally with respect to the incident light. This frac-
Universität Bremen, 28359 Bremen, Germany tion is referred to as the depolarized signal. The ratio of the depolar-
ized and polarized signal components is the depolarization ratio ρ.
Its value ranges from zero for highly polarized signals to 0.75 for the
fully depolarized case.
The depolarization ratio allows deriving valuable information about
the symmetry of a vibrational mode. Symmetric vibrations are
usually highly polarized and anti-symmetric modes are depolarized.
Consequently, acquiring both the polarized and the depolarized
Raman spectrum permits an improved structural analysis and a
more accurate assignment of the observed peaks. This is particularly
true for overlapping vibrational bands. Another advantage of
polarization-resolved detection is the determination of the isotropic
and anisotropic Raman intensities. In the context of pharmaceutical
analysis, however, the recent development of enantioselective Raman
(esR) spectroscopy, which is based on polarization-resolved detection,
is a promising tool.1,2

130 | | September/October 2017

 « SPECTROSCOPY »

1b. In order to avoid polarization-dependent METROHM

Experimental
YOUNG
effects in the spectrograph, the polarization

Approaches direction of the laser is adjusted using a half-

There are several common experimental

wave plate. However, again, the two spectra
must be recorded in sequence. CHEMIST
approaches to acquire polarization-resolved
Raman spectra. Four general concepts are
For many process monitoring applications,
however, temporal resolution is crucial.
AWARD
illustrated in Figure 1. The main components, Therefore, recording the two spectra sequen-
i.e. a linearly polarized laser, a spectrograph tially is not an option as the sample may have
and a camera (or a spectrometer with an changed between the two measurements.
integrated detector), and lenses for focusing Acquiring both the polarized and depolarized
and collimating radiation, are similar in all signal components simultaneously is possible
four. The simplest setup (Figure 1a) employs using a polarizing beam splitter in the signal
a polarization filter, either a thin film polarizer path and two spectrometers. This is illustrated
or a polarizing prism, in the signal collection in Figure 1d. Unfortunately, this approach is
path. To obtain the vertical or the horizontal inherently expensive and requires significant
signal component, the polarizer is oriented alignment effort, and the detectors must be
accordingly. This approach is experimentally synchronized. A suitable alternative tech-
simple, but it has two disadvantages, i.e. the nique for acquiring both spectra at the same WIN
$10,000
two spectra must be recorded sequentially time has recently been demonstrated.3 The
and a polarization scrambler is required to schematic setup is displayed in Figure 1c.
compensate for the polarization-dependent The key components are the half-wave plate
efficiency of the optical components in the and the Wollaston prism. The half-wave plate
spectrograph. Such a scrambler may also is used to control the laser polarization such
reduce the signal intensity and hence calls that it is rotated by 45°. This means that half
for longer acquisition times. A different of the intensity of the beam is vertically and
experimental option is depicted in Figure the other half is horizontally polarized. The
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Figure 1. Schematic experimental setups for polarization-resolved Raman spectroscopy. P

= polarizer, L = lens, spec = spectrograph, Sc = polarization scrambler, λ/2 = half-wave plate,
WP = Wollaston prism, PP = polarizing beam splitter.

www.americanpharmaceuticalreview.com | | 131
» SPECTROSCOPY »

Wollaston prism splits the two polarization components into two in- Table 1. Pros and cons of the different setups.
dividual beams, see enlarged detail in Figure 1c. The lens focusses the
Setup 1a Setup 1b Setup 1c Setup 1d
two beams to two spatially separated spots in the sample. The result-
Initial alignment J J K L
ing Raman scattering from both spots is imaged onto the entrance slit
Adjustment during operation Yes yes no no
of an imaging spectrograph equipped with a two-dimensional detec-
Simultaneous measurement L L J J
tor, e.g. a sensitive charge-coupled device camera. The camera eventu-
ally records both spectra simultaneously as they appear on separated Spatial resolution J J K J

parts on the chip. Basically, this concept makes use of the approach in Costs J J K L

Figure 1b, but enables the recording of the polarized and depolarized
Raman signals at the same time. The disadvantage of the method is setup 1c places itself in between and thus represents a true alternative,
the spatial separation of the measurement volumes inside the sample. in particular when temporally resolved measurements are needed.
In other words, the sample must be homogenous on the length scale
In conclusion, polarization-resolved Raman spectroscopy is an
of this separation. This length scale can be less than a millimeter, de-
emerging analytical technique and has great potential in the field
pending on the optical alignment of the setup.
of pharmaceuticals. The enantioselective Raman (esR) method is
of particular interest in this context. The present article evaluated
the current experimental possibilities for acquiring the polarized
Comparison and Conclusion and depolarized Raman spectra. Four concepts were compared
All the experimental approaches described above have their pros and with respect to experimental simplicity, alignment effort, temporal
cons. As mentioned before, temporal resolution is a main criterion and spatial resolution as well as cost. The recent development of an
in process monitoring. Further important points are experimental approach3 that allows the recording of both spectra simultaneously
simplicity and robustness as well as cost. The setups in Figures 1a turns out to exhibit experimental as well as economic advantages.
and 1b are experimentally simple but they share the disadvantage Thus, its further development towards a robust analytical tool will
of requiring sequential acquisition of the two spectra. Moreover, one open up Raman spectroscopy for new applications in monitoring
component needs to be adjusted between the measurements. The
pharmaceutical processes.
rotation of polarizers and half-wave plates can be made automatic,
but it still means a source of error. The setups in Figures 1c and 1d are
more complicated, but they do not need to be touched and hence are
more robust. Acknowledgement
The costs of a setup for polarization-resolved Raman spectroscopy Part of this work was funded by the Deutsche Forschungsgemeinschaft
can vary significantly depending on the individual components.
(DFG) through grant KI1396/4-1.
Only a few commercial Raman instruments are capable of recording
both polarization components. Most devices on the market utilize
fiber-optics, e.g. in immersion probes for process monitoring. The
fibers usually do not maintain the polarization. Hence, custom-made References
solutions are needed. Considering simple and cheap options, the four
1. J. Kiefer, K. Noack, Universal enantiomeric discrimination by Raman spectroscopy, Analyst
setups can be compared with each other. They all contain a laser and a
140, 1787-1790 (2015).
minimum of three lenses. Setup 1a in addition exhibits two polarizers,
2. J. Kiefer, Enantioselective Raman Spectroscopy – A new tool for process monitoring in the
a scrambler and a spectrograph with detector. The cheapest option
pharmaceutical industry?, American Pharmaceutical Review 19, 52-54 (2016).
would be to replace the spectrograph, camera, and scrambler by
a fiber-coupled miniature spectrometer with an integrated back- 3. J. Kiefer, Simultaneous acquisition of the polarized and depolarized Raman signal with a
single detector, Analytical Chemistry 89, 5725-5728 (2017).
illuminated detector. The fiber will act as polarization scrambler. Setup
1b contains a half-wave plate and two polarizers. The spectrograph
and camera can again be replaced by a fiber-couple spectrometer. This
is not possible in setup 1c, as the concept is based on the functionality Author Biography
of an imaging spectrograph and a two-dimensional detector. In
addition, a Wollaston prism is necessary. Setup 1d, on the other hand, Prof. Dr. Johannes Kiefer is Chair Professor and Head of the division
requires two spectrographs and detectors. Both can be implemented Technische Thermodynamik at the University of Bremen, Germany. In
as fiber-coupled spectrometers. addition, he is an Honorary Professor at the University of Aberdeen,
The pros and cons of all four setups are compared in Table 1. The costs Scotland, and he holds a guest professorship of the Erlangen Graduate
underlying the ratings have been estimated on the basis of current School in Advanced Optical Technologies (SAOT) at the University
catalog prices. Setups 1a and 1b are almost identical in price, while the Erlangen-Nuremberg, Germany. His research interests are the
costs for setup 1d is almost the double amount as the spectrometers areas of developing and applying spectroscopic techniques for the
(incl. detector) represent the main cost drivers. The recently proposed characterization of advanced materials and processes.

132 | | September/October 2017

 « DRUG DEVELOPMENT »

Kinetic and Thermodynamic Profiling in Drug Discovery:

Promises, Challenges and Outlook
Ying Wang and Anil Vasudevan
AbbVie Inc., North Chicago, IL

Taken together, kinetic profiling may provide another layer of

Abstract differentiation for compounds that are otherwise indistinguishable
by binding affinity. Thus, it has been advocated that kinetic profiling
Over the last decade, there has been an increased awareness on of compounds should be incorporated at an early stage of the drug
kinetic and thermodynamic profiling of ligand-protein interactions discovery processes until a clear decision can be made on what
and its linkage to the clinical effectiveness of drug candidates. Herein constitutes a desirable kinetic profile for the target and the chemical
we provide an overview on where the pharmaceutical industry stands series of interest.
with kinetic and thermodynamic profiling in drug discovery. We also
discuss the challenges for current technologies as well as provide our Similarly, from a thermodynamic perspective, Equation 1 shows
views on future prospects to use kinetic and thermodynamic profiling the relationship between binding affinity and changes in enthalpy
for prospective drug design. (ΔH) and entropy (ΔS) associated with the complex formation. ΔH
represents the heat associated with the making and breaking of non-
covalent bonds in going from the free to the bound state. ΔS reports
on the overall change in the degree of freedom of a system. On
Introduction retrospective perusal of the drug thermodynamic data, it is apparent
Traditional drug discovery paradigm has focused primarily on that, despite similar ΔG values, the underlying changes in ΔH and ΔS
optimizing drug-target binding affinities and in vivo pharmacokinetics could be quite different for each interaction.
while developing lead compounds against a validated drug target. ΔG = ΔH-TΔS = RTLnKD Equation 1
However, in the last decade, it has been proposed that kinetic and
thermodynamic profiling of compounds may be better predictors of Freire et al had studied the thermodynamic profiling of HIV protease
compound selectivity and in vivo efficacy.1-4 drugs over the development time course of their development.4
The best in class HIV protease drug, Darunavir, had the highest
The relationship between a compound’s binding affinity to a drug
ΔH contribution compared to other drugs in its class. Similarly,
target and kinetic parameters, in many cases, could be defined as in
thermodynamic profiling of the binding of a series of statins to HMG-
Figure 1. Association rate constant kon and dissociation rate constant
CoA reductase also demonstrated that Rosuvastation, the best-in-class
koff are not intrinsically related to one another. Thus, they are correlated
drug, had significantly optimized enthalpic contribution compared to
with structural features of the ligand, target and the complex in
the first approved drug in its class, Fluvastatin. The view thus was put
different ways. It is believed that in general, slow koff offers advantages
on compound efficacy and safety profile while kon is controlled by the forward that ΔH contributions to binding provides a good parameter
diffusion limit. This came from retrospective analyses indicating many for compound selection as it maximizes the influence of forces other
best-in-class drugs possessed slowest koff in its class. One of the classical than hydrophobicity. Obviously, thermodynamic data offers another
examples to illustrate this point is the well-known muscarinic M3 level of understanding on the formation of protein-ligand interfaces in
receptor antagonists, Tiotropium, the first once-a day bronchodilator addition to kinetic profiling and binding affinity studies. Analogous to
that provided a significant advantage over other drugs in its class. Its koff in kinetic profiling, it is proposed that ΔH optimization is required to
long lasting effect was attributed to its exceptionally slow koff, which achieve high efficacy and selectivity. Needless to say, thermodynamic
translated into a half-life of 35 hr vs 2-30 mins for other drugs in this and kinetic profiling provides an approach for optimization of
class. It’s worth pointing out that the binding affinities of these drugs inhibitors guided by independent variables (kon, koff, ΔH and ΔS), rather
for M3 receptor are very similar.5 than by a compound variable (KD).

www.americanpharmaceuticalreview.com | | 133
» DRUG DEVELOPMENT »

For thermodynamic profiling, isothermal titration calorimetry (ITC) is

the gold standard to obtain thermodynamic parameters. ITC directly
measures the change in enthalpy at a constant temperature by titrating
the protein target and a ligand that form an equilibrium complex at
known concentrations. From an ITC experiment, binding affinity KD and
ΔH can be obtained directly and ΔS can consequently be calculated.
Figure 1. Dissociation equilibrium constant (KD) and kinetic
parameters –interaction between receptor ( R ) and ligand (L) to ITC allows the highly accurate determination of thermodynamic
form the complex RL parameters with no requirement for chemical modification, labelling
or immobilization.
With the increasing research on thermodynamic profiling, very
These studies suggested a more holistic approach including binding
recently, it has been reported that protein and compound purities
kinetic and thermodynamic data should be incorporated as early as
could significantly influence the ITC readout in addition to other
possible in the drug discovery process, to enable medicinal chemists
experimental parameters. We have also found chiral purity of a
to identify and prioritize hit and lead compounds with best-in-
compound, in addition to compound purity, could distort the ITC
class potentials. Despite the fact validation of some of the claims
data readout which resulted in false negative readout. These studies
remains to be established.6,7 The hope is that the proposed impact of
demonstrated that ITC experimental parameters must be rigorously
binding kinetics and thermodynamics will ultimately lead to clinical
monitored in the context of compound structure and purity to obtain
candidates. In this review, we will discuss the progresses, challenges
accurate and reliable thermodynamic data.
and outlook, as an industry, in understanding and utilizing kinetic and
thermodynamic profiling for prospective drug design. Fast instrument advancements have been seen in the last decade,
triggered partially by the increased interest and research in kinetic and
thermodynamic profiling. Currently, the throughput of SPR and ITC are
Discussion not comparable to traditional methods for obtaining binding affinity
data. In the review by Miller et al in 2012,8 they were able to extract
Thermodynamic and Kinetic Data Acquisition. Surface plasmon 398 compounds with both kon and koff values from the Pfizer biological
resonance (SPR) is one of the most prevalent methods used in drug data repository and literature sources. Overall, 1855 compounds had
discovery to obtain kinetic parameters. In SPR, the target protein either kon or koff values. This is evidently nowhere near the plethora of
is immobilized onto a sensor chip. Compounds of interest are then thermodynamic affinity data available in the literature. In one of the
flown over the sensor surface. As the compounds bind to the target kinetic and themodynamic profiling study we completed, while more
protein on the sensor chip, it induces a real time change in the than 800 TR-FRET binding affinities were obtained on one chemical
refractive index, which is directly proportional to the mass bound at series with fast turnaround times enabling rapid SAR iterations, less
the surface. SPR is a label free technology for obtaining kon and koff than 15% of these analogs were profiled in SPR and ITC, due to the
rates, from which binding affinities could be calculated. It is known longer testing cycles needed for data collection and processing,
that the SPR signal is highly sensitive to temperature variation and making the timely incorporation of this data in compound selection
changes in bulk solvent and measurements need to be performed much less feasible. This in turn, limited the practical use of kinetic
with a fully dissolved ligand. However, it has been reported that and thermodynamic information for prospective design, compared to
only 7% of the available published and in-house kinetic data at other parameters that are easily obtainable. Nevertheless, significant
Pfizer had the temperature for the determinations reported. The instrumentation improvements have been reported in recent years.
kinetic profile, kon and koff, of compounds within a chemical series, For example, Biacore 8K can potentially characterize 64 interactions in
may very likely span a broad range of values covering several orders 5 hours. New development in ITC technology allows the instrument to
of magnitudes. Generally, the upper limit for kon determination by be fully automated with capacity to run four 96-well plates unattended
SPR is 10-6 M-1 s-1, whereas the fastest koff that can be accurately with improved software for data analysis. With the ever increasing
measured is approximately 10-1 s-1. This inevitably causes technical interest and research in kinetic and thermodynamic profiling, we
challenges for kinetic profiling of compounds with fast kon and koff expect unprecedented instrumentation development to enable fast
rates that are beyond instrument detection limits. In our experience, kinetic and thermodynamic data acquisition that is comparable to the
it is not uncommon to have compounds with a fast kinetic profile, binding affinity data acquisition in the foreseeable future.
especially in the early stage of the drug discovery where the binding
SKR and STR Analysis. The kinetic and thermodynamic profile of
efficiency between the compound and the protein target is far from
structurally different compounds could reveal features and aspects
optimal. In one of the hit-to-lead programs we evaluated, more than
of compound-protein interactions and thus aid optimization. This
25% of the compounds profiled had fast kinetics thus making the
is defined as structure-kinetics relationships (SKR) and Structure-
selection of hits based on kinetic profile impractical in this case. It is
thermodynamic relationships (STR), analogous to the traditional
our view that in order to fully explore the potential utility of kinetic
structure-activity relationship (SAR) studies.
parameters in the early stages of a drug discovery program, further
advances in instrumentation technology for kinetic measurements The rational design of compounds with desirable or distinct kinetics
are warranted. signatures is still in its infancy primarily due to the lack of molecular

134 | | September/October 2017

 « DRUG DEVELOPMENT »

understanding of the factors that influence binding kinetics and to the same target binding side in STR studies. The rationale was that
the difficulty in characterizing transition states of the ligand-target because of the structural similarity of these compounds, the effects of
complexes. Despite these challenges, significant progress has many of the factors will largely cancel out.
been made by gaining knowledge from both the ligand and target Interplay of thermodynamic and kinetic data with other
perspectives, such as by ligand modifications with subtle changes parameters. To understand the impact of kinetic and thermodynamic
to probe relevant pharmacophores, mutation studies, and molecular profiling on the time-course of target occupancy and drug effect, one
dynamic studies to establish the corresponding “anchoring” sites on should realize that this profiling is one of the several parameters that
the receptor, to name a few. will influence drug dosing and effects. For example, many factors can
Many elegant and systematic SKR studies have been reported very influence the ultimate importance of kinetic profiling in the context
recently. The study of a series of tertiary amine muscarinic M3 receptor of in vivo efficacy such as the concentration profile of the free drug
antagonists by Glosshop et al9 nicely exemplified a systematic in plasma and at the target site, the concentration of the target,
combination of both SAR and SKR studies in the early phase of drug competition between drug and endogenous ligand binding, target
discovery process. It was found that the gem-dimethyl substitution turnover etc. It will also be interesting to see how kinetic profiling
had a profound (>38-fold) effect on the dissociation rate. Interestingly, interplays with thermodynamic profiling. Do compounds with better
this difference was not observed with the corresponding binding koff also possess more ΔH contribution? Do kinetic and thermodynatic
affinities. Prioritization of this structural moiety and further chemical profiling influence the efficacy and selectivity in similar ways? As more
modification yielded a clinical candidate maintaining the gem- and more SKR and STR studies come aboard, this will enable us to
dimethyl moiety which provided efficacious 24h bronchodilation from answer these questions and assess the interplay of these parameters
a single inhaled dose. in a global and systematic way.

It should also be pointed out that SKR studies are not limited to koff
only. Our internal data showed that kon could vary dramatically even
among close analogs. Recently literature examples also suggested Conclusion
in certain cases kon may play an important role in downstream
Despite all the challenges, significant progress has been made in the
processes.10,11 It is also feasible that for certain compound-target
study of kinetic and thermodynamic profiling in drug discovery. For
protein interactions, the kinetic profile cannot be simplified as kon/koff,
example, the Kinetics for Drug Discovery (K4DD) Consortium, charged
with more complex interaction pathways between the compound and
with the mission to investigate major open questions related to
the target protein. Systematic SKR studies should be able to help to
binding kinetics with public-private partnership, enables targeting
answer these questions.
drug-binding kinetics in a holistic way. The hope is that as progress
One of the challenges in utilizing STR in a predictive manner resulted continues in these areas, the new insights gained and methods
from the fact that multiple factors such as hydrophobic interactions, developed will translate to better decision making for selecting drug
solvation and local water structure may influence the thermodynamic candidate, ultimately placing kinetic and thermodynamic profiling at
readout. Good progress has been made recently in understanding the same or higher level as with binding affinity. In the long run, the
the effects of these factors and how to apply this knowledge in STR multipronged strategy will be applied to drug discovery to achieve a
studies.12 It is more fruitful to compare similar molecules which bind thorough understanding of the interplay between structure, kinetics

www.americanpharmaceuticalreview.com | | 135
 EDITOR’S

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Biopharmaceutical manufacturing directly supports the $240 billion

Trends and Growth in biologics industry, and small improvements in costs of manufacturing
can result in very significant savings. However, unlike other industries,
where adoption of new technologies are often quickly embraced,
Single-Use System (SUS) in bioprocessing, change evolves slowly. This is due to a few factors,
but one of the largest is the intense regulation of the industry.

Adoption Manufacturers and contract manufacturing organizations (CMOs)

must gain regulatory approval for any change in technology or process.
Therefore, change is slow to happen because once a manufacturing
process has been established and approved, the costs of change are
significant, and incentives to change are limited.
Despite the onerous regulatory and testing burdens facing
manufacturers who seek innovation and process improvements,
change does happen. New technologies are developed, and the
Eric S. Langer industry moves forward, albeit slowly. One such change over the
past 20 years has been the adoption of Single-Use System (SUS)
President and Managing Partner
and disposables. In recent years, we have seen fewer blockbuster
BioPlan Associates, Inc.
drugs, more biologics having higher potency that require smaller
production volumes, advances of biosimilars targeting smaller
markets, and ongoing incremental improvements in production yields
and efficiencies that create production operations at much smaller
scales. This has permitted the use of single-use devices, such as plastic
bioreactors, mixing systems, and containers that are now dominating
clinical production, and are moving toward commercial operations.
Having adaptable equipment and flexible facilities that can
manufacture multiple biopharmaceutical products at once, or in
tandem, rather than a single drug that will carry a company for many
quarters to come, is now a standard of the bioprocessing industry.
Single-use and disposable devices are being used for a range of
applications including upstream production, mixing, filtration,
purification, fill-finish, and storage, among others. These systems

138 | | September/October 2017

 « BIOPHARM PROCESSING »

provide faster change-overs and reduced times for production. BioPlan collecting these data, particularly the simpler and/or less-expensive
Associates has surveyed global biopharmaceutical manufacturers and products, such as “Sampling systems” and “Media bags, purchased dry”.
CMOs to gain insight into current and future trends in the industry. Many of these products achieved relatively high adoption in earlier
In BioPlan’s 14th Annual Report and Survey of Biopharmaceutical years. For example, disposable media bags were among the very first
Manufacturing Capacity and Production, we asked 227 bioprocessing single-use products, with single-use filters common even before this.
decision-makers where they are using SUS and disposables, and the
In contrast, some new(er) single use equipment, such as membrane
critical factors, trends and hurdles being seen in adoption.
adsorbers and perfusion/tangential flow filtration devices, simply are
newer and continue to have relatively low adoption rates. As we reach

How Common are

a market saturation point for single-use pre-commercial applications,
it will take greater regulatory acceptance (commercial product
Single-Use Devices? approvals) for plastics usage and/or more approvals of single-use-
manufactured commercial biologics to allow this market to capture
The most common single use devices are basic tubing, disposable more significant market shares and growth in sales.
filter cartridges, and connectors and clamps. Although we do note
In our study, we evaluated the growth (change) in disposables
that these devices must meet exceptionally high standards for quality,
applications over the past 11 years (See Figure 2), in terms of the
and performance, nearly 90% of respondents to our survey indicated
difference in percentage facilities actually implementing disposable
they are using these products at some scale. In fact, these devices are
reaching market saturation, at least at clinical scale. At the bottom of applications. This year, “Bioreactors” percentages continued to grow
the ‘adoption’ list are perfusion devices, membrane adsorbers, and rapidly, up from 21% in 2006 to 80.3% adoption; a 59.3% point
disposable chromatography devices. These are at the lower end of difference. “Mixing Systems” also saw a large point difference this
usage ranges because they tend to be newer; given the slow adoption year, 58.4%, from a point difference of 50.8% in 2016. “Perfusion
rates in this industry, they are still moving up on the growth curve. devices” reported 35.9% growth this year, up from 33.1% in 2016.
While many devices were tracking around a healthy 13% annual Other areas have grown at faster rates this year, than reported in
growth rate last year, the more saturated devices were showing only 2016, again reflecting either slower and/or earlier adoption and
single-digit growth. As more facilities use them, growth in adoption of associated higher baseline usage rates (e.g., exemplified by the areas
single-use devices necessarily slows as market saturation is reached. with the least growth – “Media bags (wet)”, “Media bags (dry)” both
For many, probably most of these product classes, slow usage growth long used in bioprocessing – and “Disposable chromatography” with
rates likely reflect their relatively widespread adoption prior to our a high initial baseline).

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» BIOPHARM PROCESSING »

more widely used in scale-up/clinical

production and process development than
commercial production. In scale-up/clinical
production-scale, adoption rates for nearly
every type of single-use product is over 70%,
with several areas over 80%. In contrast,
disposable chromatography, for example, is
used by only 20% at commercial scale. This
is not unexpected, since devices like larger
scale SUS chromatography are not (yet)
available, and membrane adsorbers have not
yet entered mainstream commercial markets.

Reasons for Adopting

Single-Use Technology
And Disposables
Figure 1. Usage of Disposables in Biopharmaceutical manufacturing, any Stage of R&D or Study participants cited reducing capital
Manufacture (Selected Data) investment in facilities and equipment as the
most critical reason for increasing disposable
use. This was cited by 27.7% of respondents,
an almost 50% increase over that response
in 2016. This is likely due to the fact that
manufacturers are continuing to focus on
productivity, efficiency, and short-term cost
savings and therefore see a decrease in facility
costs as a good way to accomplish these
goals. The next most critical reasons cited
for adopting SUS were to eliminate cleaning
requirements (15.2%), faster campaign
turnaround time (8.9%), decrease risk of
cross-contamination (8%), and flexibility of a
modular approach (7.1%).
We also asked the most critical reason for not
increasing disposable use. The number one
listed reason was the high cost of disposables,
Figure 2. Selected Devices-11-Year Percentage-Point Change in First-Usage of Disposables,
2006-2017 cited by 23.4% of respondents. The fact that
cost issues have risen to the top is indicative
of how SUS device manufacturers have
The average annual growth rate (CAGR) for required to allow these devices to reach generally begun to resolve concerns of the
past, including breakage, and leachables and
some of these devices between 2006 and market saturation.
extractables, both of which have taken the
2017 has been relatively high, around 13% for
top spots in prior years.
mixing systems, membrane adsorbers, and
bioreactors. Other more common devices Process Scales Where
that have seen less average growth due to
the fact that these were already in steady use SUS and Disposables are When Will Facilities
when BioPlan began collecting data in 2006. Being Used Be Using 100% Fully
In addition, some other single-use equipment
may be showing slower growth today, such as We looked at commercial production, scale-
Disposable Technology?
perfusion/tangential flow filtration devices, up/clinical production, process development, A majority of industry experts, 64.9%, either
because there is more regulatory approval and early R&D. By far, disposables are being said they ‘strongly agree’ or ‘agree’ that there

140 | | September/October 2017

 « BIOPHARM PROCESSING »

Denmark are chaining multiple 2000 liter bioreactors to create up to

12,000L batch sizes. Thus, further reducing the need for large stainless
steel tankage.

Complete single-use upstream processes can compete with larger

commercial-scale manufacturing in cost. And now, suppliers and
innovators are turning to downstream processing single-use systems
to find cost-effective and efficient solutions. Even facilities with
conventional steel facilities are creating hybrids with SUS to optimally
incorporate disposables for production.

As regulatory agencies become more comfortable with the

performance of SUS, the industry will see a wider adoption at
commercial scale. This will result in the market for SUS rapidly growing
far past its current size. The availability of current SUS has benefited
Figure 3. Single-use / Disposable Device Adoption Factors new biopharmaceutical start-ups in particular, allowing them to
spend much less capital and quickly advance the development of new
products. Single-use systems may therefore increase the competition
within the biopharmaceutical manufacturing industry as a whole,
will be a 100% fully disposable facility in operation in 5 years. This
allowing smaller and medium-sized companies to gain a quick
is up from 57.2% of respondents last year. It’s likely these facilities
would be new and using devices like single-use upstream bioreactors foothold, whereas in the past, they would have been prevented from
and downstream disposable chromatography and filtration systems. doing so by huge up-front facilities costs.
Nearly two-thirds of respondents (65.7%) said they anticipated
their own facility’s cGMP clinical/commercial operations would be
substantially using single-use devices in five years. This response References
keeps going up, from 51.8% in 2016 and 49.1% in 2015.
1. 14th Annual Report and Survey of Biopharmaceutical Manufacturing Capacity and
Production, BioPlan Associates, Inc. April 2017, www.bioplanassociates.com

Conclusions
Single-use systems, which are being used at clinical scale for well Author Biography
over 80% of bioprocessing operations, will continue to be adopted
by biomanufacturers and CMOs at larger commercial scale as pipeline Eric S. Langer is President and managing partner at BioPlan Associates,
products being produced in SUS are approved, and move into Inc., a biotechnology and life sciences marketing research and publishing
commercial production. Because most single-use disposable systems firm established in Rockville, MD in 1989. He is editor of numerous
are already being used in scale up/clinical production, much of the studies, including “Biopharmaceutical Technology in China,” “Advances
future growth will come from the growth of larger commercial scales, in Large-scale Biopharmaceutical Manufacturing”, and many other
increasing market growth of SUS since these are much costlier systems industry reports. [email protected] 301-921-5979. www.
to implement. As the industry matures, vendors are creating improved bioplanassociates.com
disposable technologies to differentiate themselves from competitors.
This bodes well for manufacturers and CMOs as it will drive down Survey Methodology: The 2017 Fourteenth Annual Report and Survey
prices and increase competition. of Biopharmaceutical Manufacturing Capacity and Production yields
Disposable processing equipment is now being considered a composite view and trend analysis from 227 responsible individuals
increasingly for more strategic reasons, such as reduction in overall at biopharmaceutical manufacturers and contract manufacturing
costs, and improved productivity. The ‘tactical’ reasons such as organizations (CMOs) in 25 countries. The methodology also included
reductions in cleaning and validation requirements and in cross- over 131 direct suppliers of materials, services and equipment to this
contamination events are still important decision factors, but are industry. This year’s study covers such issues as: new product needs,
being seen as relatively less critical. facility budget changes, current capacity, future capacity constraints,
As better upstream productivity in recent years has required lower expansions, use of disposables, trends and budgets in disposables, trends
and/or less frequent dosing, and production requirements can be in downstream purification, quality management and control, hiring
made at a tenth of the scale from a decade ago, more production issues, and employment. The quantitative trend analysis provides details
lines can be specified at single-use scales. At this scale, e.g., 2,000L and comparisons of production by biotherapeutic developers and CMOs.
or less, disposable bioreactors are viable and cost-effective. Further, It also evaluates trends over time, and assesses differences in the world’s
some facilities, such as CMC Biologics in Bothell, WA, and Copenhagen major markets in the U.S. and Europe.

www.americanpharmaceuticalreview.com | | 141
» CPhI REPORT »

The generic medicine industry has generated significant cost savings

The Congruence Between for payers over many years (over 100 billion euros in Europe alone
in 2014) and provided access to affordable medicines for millions of
patients. Competition has been generated through the availability
Small Molecule Generic of generic medicines from multiple sources delivering lower prices
and a choice of suppliers. One aspect of such competition is that in
order to maintain growth a generic portfolio has to be replenished or
Medicine and Biosimilar refreshed frequently. Many generic companies set targets of 20 new
products to be launched each year. Product development of new

Medicine Business Models generic molecules starts early in the life cycle of the originator product
so long term planning is essential and mainly based on product
patent expiries. Following the patent cliff in 2012 it became apparent
that the opportunities for developing a new range of ‘blockbuster’
generic medicines based on small molecules would be curtailed, such
molecules diminishing in numbers in the ensuing 5-10 years.
The opportunities for growth from the existing portfolio of legacy
products was also being constrained by the slow, or even declining,
growth of traditional therapy areas where generic medicines
Alan Sheppard
dominated. In Europe, analysis showed the challenge facing
Principal, Global Generics manufacturers of declining prices and low volume growth.
IMS Health

This article forms part of the CPhI 2017 Annual Report, which will be
released during the CPhI Worldwide event in Frankfurt (October 24-26,
2017) http://www.cphi.com/europe/cphi-annual-report

Figure 1.

142 | | September/October 2017

 « CPhI REPORT »

Figure 2. Figure 3.

Pipelines then turned to developing products that had devices as

part of their requirement such as the asthma inhalers and injectable
products. Alongside these came the range of added value generic
products including modified release, combinations and reformulations
to improve absorption, dosing or side-effect profiles. Analysis of
market trends illustrated the growing importance of medicines based
on biological technology showing that they had exhibited growth at
twice the rate of the small molecules.
Whereas historically the league table of top products featured mainly
small molecule medicines the current picture is very different. Biologics
now dominate the chart and future years will show a deepening of the
penetration by biologics.
Those companies that identified the growing importance of biologics
identified an opportunity to develop copies of these products. Thus
started the pursuit of biosimilar medicines.
Figure 4.
The evolution of the biosimilar medicine market has been an
interesting journey and one which has still to fully deliver on its

www.americanpharmaceuticalreview.com | | 143
» CPhI REPORT »

Figure 5. Figure 6.

promise. It is a classic example of how the players in the off-patent originator what is the difference? Having been used to bioequivalence
industry can tackle what initially was seen as a huge challenge (some with generic medicines this posed additional questions in the minds
even said an impossible one) and bring about regulatory change, of clinicians and payers. Thus a lack of understanding and the need for
adopt a clinical approach to development , gain market access in education was evident. Also speculation on discounts that they would
specialist fields and deliver complex molecules at affordable prices be in the range of 20-30% off of originator prices suggested that
for patients. Much of this ground breaking work was carried out with costs savings would not be as significant when compared to generic
the support of the Medicines for Europe team who have provided medicine discounts of 70-90%. Initial sales results were poor and sales
additional technical support where necessary and the lobbying of the forecasts varied widely from the absurdly positive to the lowest of
key decision makers within the European theatre. Europe is now seen negatives. Finally the concept of low volume/high price meant a shift
as the leader in biosimilar development with many countries around in the generic medicine doctrine of high volume/low price. This move
the world adopting the regulatory processes and positioning of the from volume to value was a major concern for many generic players.
biosimilar industry seen in Europe.
Moving from a business model based on supplying commodity
One common question from generic medicine manufacturers has generic medicines in high volumes to a wide spectrum of buyers at low
been ‘should I be in the biosimilar market?’ The answer to this question prices to the biosimilar model which was built on supplying specialist
at the advent of the introduction of the first biosimilar medicines may medicines into institutions usually on a contract or tendered business
well have been negative. The specialist requirements for developing scheme required different skills and capabilities. Mistakes were made
a biosimilar; the need for expensive comparative clinical trials; the at first but slowly a better understanding of what the clinicians wanted
failings in the available laboratory tests for characterisation and and how best to support the specialist needs of this market have
confirming amino acid sequencing and molecular structures; complex evolved. Over time the dynamics of the biosimilar market have moved
manufacturing plants and finding scientists with the requisite closer to the generic scenario. Thus we are seeing some congruence
knowledge were just a few of the issues put forward to suggest the especially in the following areas:
return on any biosimilar investment was fraught with danger. The
• Regulatory guidelines are now in place which should
initial experience with the first biosimilar molecules launched also
accelerate the access to the market for newer biosimilars.
tended to support the fact that this would be a risky venture. These
The need for comparative clinical studies is under review
first biosimilars had to be branded, would require promotion to
and it is felt that a more relaxed approach will be accepted.
clinicians and were in specialist areas of medicine, something which
A common dossier for use both in the USA and Europe is
most generic medicine companies had little experience of. Concerns
under discussion which will significantly reduce costs and
about the delivery system with the first biosimilar somatropin (HGH),
should speed up registrations.
clinician preference in selection of an erythropoietin (EPO) for dialysis
and a lack of understanding with regard to biosimilar filgrastim (GCSF) • We have an increased number of biosimilar players which
were just some of the reasons for a slow adoption of these biosimilars. has helped to increase awareness of biosimilars. Additionally
It should also be remembered that these first biosimilars represented some research based companies such as Pfizer, Novartis, and
just 8% of the total biologics market. Even explanation of the naming Boehringer Ingelheim are now biosimilar players and this
of this new innovation of off-patent medicines was difficult; if it also reinforces the positioning of biosimilars as a valuable
is called a biosimilar and this means that it is not the ‘same’ as the tool in controlling costs.

144 | | September/October 2017

 « CPhI REPORT »

• More players introduce more competition, especially in the

pricing arena. Thus discounts have increased with a resultant
improvement in savings and consequently increased interest
in payers in ensuring access to biosimilars.

• Several new biosimilar molecules have been launched across

a wider range of indications increasing awareness of the role
of biosimilars. The launch of the first biosimilar monoclonal
antibody into a primary care managed condition has
expanded the potential patient numbers adding a volume
dynamic. Approvals are across all the major markets making
the opportunity a global one.

• The resolution of guidelines on interchangeability and

substitution of an originator medicine by a biosimilar has
aligned biosimilars closer to the generic medicines scenario
albeit still under the control of a healthcare professional.
Figure 7.
As experience and familiarity grows with biosimilars then
we can expect an increase in the number of patients who
will have their therapy switched from the originator to the
biosimilar with cost becoming the driver. Uptake of the
latest biosimilars in Europe is extremely positive and is
starting to track the dynamics of small molecule products.

• Several generic manufacturers now have biosimilars

within their portfolios and this adds credence to the role
that generic companies can play across a wide spectrum
of therapies.

The congruence of the two business models is also further reinforced

by the investment in manufacturing facilities by some of the generic
players who now consider the two portfolios as an integral part of their
operations. Marketing tactics and sales force resources are harmonised
and utilised across the full range of products. As biosimilars move into
areas where patients have more of a role to play and pharmacists are
Figure 8. able to influence product selection then we can anticipate the two
markets aligning themselves even more closely. The use of biologics
in oncology may show to have different dynamics but the effect of
lowering of prices seen with existing biosimilar molecules in other
therapy areas would indicate that all other things being equal then
the biosimilar will win on price differentials.

Asking the same question on whether or not to be in the biosimilar

market would in today’s environment elicit differing responses. The
main answer would be ‘it depends’.

Some companies may feel that they have ‘missed the boat’ with respect
to biosimilars but all is not lost. As with small chemical molecules
there now exists biosimilar developers who are seeking partners for
marketing and distribution and contract manufacturing organisations
with biosimilar capabilities. If that is not an option then portfolio
growth from small chemical molecules will still offer opportunities.
The recent increase in launches of small chemical products forecasted
to be potential blockbuster molecules will offer significant generic
Figure 9.
opportunities in the years beyond 2025.

As the biosimilar pioneers found, it will pay to be patient.

www.americanpharmaceuticalreview.com | | 145
An Interview With...

Rick Lozano
VP Biosimilars & Integrated Business Development
AmerisourceBergen

»
Biosimilars, unlike traditional generics, require a strong value these key stakeholders on the issues they find most important,
proposition and patient support programs to drive payer the full potential of biosimilars in the U.S. may not be realized.
coverage and provider uptake. Experience, resources and a keen Companies like AmerisourceBergen are able to harness expertise
understanding of today’s market dynamics are required to advance from many sides of the business to help innovator companies
a promising yet complex molecule into an accessible therapy for develop a successful, customized biosimilar model. For example,
patients. As a leader in the space, AmerisourceBergen helped biosimilar manufacturers, like their innovator competitors, should
manufacturers successfully bring the first wave of biosimilars to invest in and provide hub services or patient support programs.
market in the U.S. with commercialization programs that ensure the Patient access to education on proper administration, management,
support and broadest access possible for patients and providers. handling and storage can help maximize outcomes. Lash Group, a
In a conversation with American Pharmaceutical Review, Rick part of AmerisourceBergen, works with manufacturers to develop
Lozano, VP of Biosimilars & Integrated Business Development at and implement successful patient support programs.
AmerisourceBergen, shares insights for manufacturers on the
evolving biosimilars landscape. How do strategies to bring biosimilars into the
market differ from generics?
Where do biosimilars stand today in the health
care landscape? A big challenge for manufacturers is determining the life cycle of
the drug they are bringing to market. As biologics and biosimilars
Today, a unique category of drug is developing and the market are relatively new to the health care market, accurately predicting
is beginning to make room for biosimilars. Biosimilars occupy a their life cycle or the space they occupy can be a challenge. Unlike
new, hybrid space: they are intended to infuse cost-savings in traditional generics, manufacturing and patient support costs can
to the biologic drug market, similar to the savings we saw when affect the price of biosimilars. Therefore, manufacturers need to
generics first launched. The potential benefit to patients, providers strategize with partners who can help plan for future competition
and the overall industry is large. Their acceptance and broader and what the entire care continuum needs to do to adopt and
commercialization could mean greater options for patients for support the use of biosimilars.
many disease states and cost-savings. However, the nature of these
Once a second biosimilar enters the market, the manufacturer’s strategy
products requires more research and development, in addition to
will change from one of pricing to distribution. Full-line distribution
customized, end-to-end commercialization and patient support.
offers manufacturers the ability to grant the widest possible access for
Market factors, including regulatory challenges and litigation from their product. Today, the first biosimilar entrants for unique indications
innovators, are also affecting the pace of approvals for biosimilars in will drop the market price by 15 to 20 percent. However, experts predict
the U.S. Issues like the patent dance, reimbursement codes and the that percentage will increase in the future when there are two to three
big question of interchangeability are all leading to uncertainty from competitors for the same molecule, driving the costs down by as much
stakeholders in the U.S. and potentially causing new approvals to slow. as 50 percent. For an innovator, pivoting to a full-line distribution
model after the second entrant is in market will be difficult and could
There is no proven model for launching a biosimilar that already
cause disruption in the market from a pricing perspective, which will
exists in the marketplace, and all of these factors reinforce the
ultimately not benefit any stakeholders.
complexity and challenges a manufacturer faces. As such, it’s critical
manufacturers look at the launch from all perspectives – provider, Aside from distribution, patient and provider education is especially
payer, patient and regulator. If manufacturers are not educating crucial for biosimilars. In time, providers and patients will build

146 | | September/October 2017

confidence as biosimilars are used more widely and outcomes are
evaluated in broad patient populations under real-world conditions. What hurdles will manufacturers face in terms of
The unique space biosimilars occupy can raise questions from these products being adopted in the clinic?
patients and providers that manufacturers need to equip providers
In my previous role at AmerisourceBergen, I worked closely with our
to answer. Again, this is part of building a support strategy that
community oncology partners. Community oncologists consistently
accounts for the entire life cycle of the product. Companies like
provide high quality service at a lower cost than some larger health
AmerisourceBergen’s Xcenda, a strategic consultancy, offer market
systems. So, you would think that the promise of biosimilars, as a
insights and guidance to inform sound commercialization strategies.
lower cost treatment option for their patients would be met with
Research with payers, providers and patients can yield insights to
a lot of enthusiasm. But, regardless of generic, branded, innovator
help manufacturers shape their go-to-market strategies. or biosimilar, community oncologists are looking for specific things:
confidence in the product, a sustainable pricing strategy and patient
What do the next 5-10 years hold for biosimilars? support that can all be facilitated through a trusted partner.
In my experience, community oncologists and other providers are
Despite the slowing of approvals that we’ve recently seen over the
excited about the prospect of biosimilars, yet there is still a lot of
last six months there is general consensus in the industry that the
apprehension. Manufacturers must take the appropriate steps to
global market for biosimilars will grow exponentially in the coming
educate providers and establish a relationship as a trusted partner.
years, some projected that it will climb to $15 billion by 2020. In
This is a point we continue to reinforce to our manufacturer partners.
the United States, about $100 billion worth of biologics will lose
Community oncologists are savvy in buy and bill, and we’ve learned
patent exclusivity by 2020, creating an opportunity for more product
that price is just as important as clinical efficacy. Community
launches and fueling competition. oncologists, like all health care stakeholders today, are looking to
As the marketplace for biosimilars in the U.S. matures, issues such biosimilar products as one part of the solution for lowering the total
as reimbursement and patient support will evolve and questions cost of care while achieving the best patient outcomes.
concerning interchangeability will need to be addressed. In For its part, AmerisourceBergen is creating a program specifically
January 2017, the FDA took a step toward more clearly defining tailored to help drive our customers’ awareness and utilization
the regulation of interchangeability. However, manufacturers will of biosimilars. Our position in the market allows us to have
need to continue building confidence with providers to prescribe meaningful relationships with providers; we take that information to
biosimilars. Reimbursement rates will need to be clearly defined, manufacturers so they can learn from our experiences. Working with
and manufacturers can work with partners to affect legislative a partner who has already built trust in a key audience will benefit
change for coding. manufacturers as they bring biosimilars for oncology to market.

www.americanpharmaceuticalreview.com | | 147
» RESEARCH » SPONSORED CONTENT

CROs are consolidating to deliver key discovery and early stage

Contract Research efficiencies in the lab, as well as expert clinical stage trial services, to
accelerate drug development.

Organization Growth Much of what the industry has been looking for from its supply chain
over the past decade plus, is a way to better manage the risk and cost
associated with drug development, and support therapeutic efficacy

and Investment and commercial performance long after approval.

Rising demand for effective pharmaceuticals has fostered innovation

across the industry. With this, business models have evolved to better
manage the risk that drug development entails. Some of the world’s
leading drug developers are adopting strategies that are centered
on creating deeper relationships with contract services providers to
support successful drug development from the very beginning.
Nigel Walker The value of the global CRO market is expected to exceed $32 billion
Managing Director in 2017 and reach $45 billion by 2022, according to a report by Grand
Nice Insight/That’s Nice LLC View Research.1 Analysts identified patent expiries in an increasing
number of partnerships and biologics, as well as new compounds, as
the primary reasons behind this growth. Rising R&D costs, in general,
were also identified as one of the industry’s top outsourcing drivers.

Earlier this year, respondents to the 2017 Nice Insight Preclinical and
Clinical Contract Research Survey, indicated that while initially, many
companies outsource research, development and manufacturing
activities to reduce costs; cost containment goals are no longer a
top-line driver. Revealing outsourcing priorities, nearly one-third
responding to Nice Insight’s CRO Outsourcing survey (32%) listed
access to specialized technologies as the main reason they partner

148 | | September/October 2017

GLITTERING INSIGHT FROM INSIDE THE INDUSTRY...

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THE MARKET IN THIS PARTICLE ENGINEERING
TECHNOLOGY, MAKING THEM A PORT OF CALL FOR
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with CROs. They also seek to improve quality (12%), incorporate new drug approvals by September 2017,6 the downward indicator
outsourcing in their strategic plans (11%), look for operational and may likely be viewed as a calm period before growth in the demand for
subject matter expertise (9%) and leverage the regulatory expertise CRO services, leading to a sustained CAGR as projected by Grand View.
of CROs (7%).2

According to Grand View: “Even government organizations are

outsourcing their clinical trial activities to CROs so that they can carry Faster Development Pathways Ready
out the clinical trials with the required infrastructure, expertise, and to Sustain CRO Strategy Past 2018
minimize cost and timelines.” For many, speed-to-market concerns
further support and spur demand for strategic research and The FDA is changing the way it does business. The industry

development outsourcing partners. Commissioner of the FDA, Scott Gottlieb, is increasingly vocal in
articulating the agency’s strategy supporting drug development.6
Recent speeches reflect his and the agency’s desire to fix the economics
of drug R&D. Gottlieb and Center for Drug Evaluation and Research
Globalization Prompts CRO Growth (CDER) Director Janet Woodcock, want to streamline the approval
and Consolidation process and reduce the cost of bringing new drugs to market. With a
speedier, less costly path to approval, their efforts have the potential
Capstone Research notes the increasing complexity of clinical trials
to unleash a new wave of drug development. An investment increase
has boosted the demand for more sophisticated, globalized CRO
would sustain the demand for CRO services, especially in the dynamic
partners that can offer a broader range of advanced capabilities.3
biologics sector.7
Commenting to an industry publication in May, Mark Goldberg,
PAREXEL’s CEO remarked, “We continue to see an industry focus on Overall, the high level of M&A activity suggests that competition among
specialty indications such as oncology, autoimmune diseases, and rare CROs appears to be tightening, making it increasingly important for
diseases, which tend to require more complicated trials. We’ve seen outsourcing partners to grow and acquire the capabilities to meet
trial complexity increase significantly in terms of the amount of data the expectations of sponsors, drug owners and developers. Nice
collected per patient, the numbers of studies and tests performed, and Insight analysis finds that to be successful competitively, contract
the numbers of endpoints included.”4 service providers should differentiate themselves, deploying unique
technologies and structuring operations to increase flexibility,
Nice Insight data supports what many contract research service
efficiency and sustain quality. This includes fielding a culture that
providers have asserted; contract service organizations that serve
provides excellent customer service and the ability to rapidly acquire
as long-term partners deliver better value from the supply chain.5
new knowledge in response to customer needs.8
Comprehensive support, combined with access to novel and
proprietary technologies, remains key to accelerating this process, and
has become central to many drug development.
M&A Activity Reveals
Strategic Positioning
Is There a Temporary Pause Over recent months, CRO merger and acquisition activity in the CRO
in Spending? sector has lulled to a certain degree. Bloomberg reported in June that
M&A spending in the CRO industry in 2017 was $13 billion, down more
Nice Insight’s Preclinical and Clinical Contract Research Survey data
than half from the $24 billion in deals in 2016.9
revealed budgets might be somewhat slowing, noting that the
CROs are increasingly being acquired by private equity firms, perhaps
number of drug manufacturers that plan to spend more than $50
indicating that investors are expecting CROs to grow over the next
million/year on contract research services dropped from 56% in 2016
decade.10 As Business Insider reports, stocks of public CRO companies
to 47% in 2017. Similarly, in 2016, nearly 75% of respondents expected
are up by more than 25% over the past year, “signaling public-market
spending on contract research services to increase, while in 2017 that
investors are betting on the companies too, potentially in anticipation
number was just 40%, with 50% predicting a decline in spending over
of activity to come.” As it stands, in late 2017, eight of the top ten CROs
the next five years.5
were involved in a major merger or acquisition including:
The reduced optimism and slowed spending revealed by the survey
• Pamplona Capital Management’s acquisition of PAREXEL
paralleled a drop in the number of new drugs approved by the FDA in
International for $5 billion taking the CRO private in June.11
2016 – down from 41 in 2014 and 45 in 2015 to just 19 in 2016, as of
November 29, 2016. However, about half of the respondents (48%) to • Albany Molecular Research Inc. acquired and taken private
the 20176 survey expect the number of research services organizations by Carlyle Group and GTCR for $922 million.12
they partner with to increase. With the FDA back on track logging 32 • Chiltern’s purchase of Integrated Development Associates.13

150 | | September/October 2017

SPONSORED CONTENT « RESEARCH »

• Inc Research Holdings merger with InVentiv Health, a $4.6 6. https://www.fda.gov/drugs/developmentapprovalprocess/druginnovation/

billion deal.14 ucm537040.htm
7. https://endpts.com/scott-gottlieb-vows-to-shake-up-the-fda-backing-a-trend-toward-
• Charles River Laboratories accelerating its strategy, acquiring
faster-drug-development/
Agilux and Phoeninx.15
8. https://www.pharmasalmanac.com/articles/pharma-contract-services-consolidation-
• Quintiles Transnational’s 2016 merger with IMS Health in an wave-continues
$8.75 billion deal.16
9. https://www.bloomberg.com/news/articles/2017-06-21/biopharma-outsourcing-is-
• LabCorp’s $6.2 billion deal for Covance in 2015.17 going-through-deal-bonanza-in-u-s
10. http://www.businessinsider.com/mergers-and-acquisitions-in-the-contract-research-
As we near the end of 2017, the CRO industry’s potential to influence
organization-2017-7
the direction and prospects of the larger pharmaceutical research and
development effort has never been clearer. 11. https://www.parexel.com/company/news-events/press-releases/2017/parexel-
international-enters-definitive-agreement-be-acquired-pamplona-capital-
management-8810-share-cash

References
12. https://www.carlyle.com/media-room/news-release-archive/amri-signs-definitive-
agreement-be-acquired-carlyle-group-and-gtcr

1. http://www.grandviewresearch.com/press-release/global-healthcare-cro-market 13. https://www.chiltern.com/about-us/news/chiltern-acquires-japanese-clinical-research-

organization-integrated-development-associates-co-ltd/
2. 2017 Nice Insight CRO survey http://www.capstonellc.com/sites/default/files/Capstone%
20Pharmaceutical%20Outsourcing% 14. http://www.inventivhealth.com/our-ideas/press/2017/inventiv-health-and-inc-
research-to-merge
3. 20M%26A%20Report_Q1%202017_0.pdf
15. http://ir.criver.com/phoenix.zhtml?c=121668&p=irol-newsArticle&ID=2292131
4. http://www.contractpharma.com/issues/2017-05-01/view_features/cros-next-
gendrug-development/53328 16. http://www.criver.com/about-us/news-events/featured-stories/2016/charles-river-
laboratories-acquires-agilux-laborat
5. Changing Expectations in the Pharma Outsourcing Market Nice Insight – Interphex 2017
March 23, 2017- INTERPHEX-NY-2017-Technical-White-Paper-Thats-Nice-2 17. http://www.quintiles.com/news/2016/05/ims-health-and-quintiles-to-merge

GLITTERING INSIGHT FROM INSIDE THE INDUSTRY...

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www.americanpharmaceuticalreview.com | | 151
» ROUNDTABLE »

What are some current critical industry issues that

Drug Delivery have made drug delivery such a hot topic?
Yihong Qiu, Ph.D., Sr. Research Fellow, Drug Products, Operations

Roundtable Science and Technology, AbbVie: Poor water solubility continues

to be one of the major challenges facing the industry in terms of oral
drug delivery. Up to 90% of current candidates from the discovery small
molecule pipeline across many therapeutic areas, such as oncology,
infectious or rare diseases, are having solubility/dissolution problems.
Many of these compounds are extremely insoluble. Twenty to 30 years
ago, we thought of poorly soluble compounds in terms of 100 μg/mL,
Yihong Qiu, PhD
and today we are working on a few μg/mL or even ng/mL solubility. It
Sr. Research Fellow, Drug Products, Operations Science and Technology
causes tremendous difficulties in early preclinical and safety studies
AbbVie
that require high exposure in animal models. It also presents different
types of significant challenges when designing a stable and bioavailable
Dr. Donald Kelemen commercial product. The drugs we are developing and manufacturing in
Head of Corporate Business Development recent years are even less soluble than KALETRA® which was approved
ABITEC in 2000. Over the years, AbbVie has been conducting research at the
forefront to dig more deeply into the fundamental aspects of solubility/
dissolution and drug delivery technologies for increasing oral absorption.
Cornell Stamoran
Dr. Donald Kelemen, Head of Corporate Business Development,
Vice President of Corporate Strategy
ABITEC: The relevance and importance of drug delivery solutions across
Catalent Pharma Solutions
the pharmaceutical sector cannot be over emphasized. Novel drug delivery
systems offer impactful solutions for developing new or improving existing
therapeutics to address a broad range of clinical and patient objectives
David P. Elder, PhD
including bioavailability enhancement, improved safety, decreased toxicity
Independent CMC Consultant
and improved patient compliance.
Cornell Stamoran, Vice President of Corporate Strategy, Catalent
Pharma Solutions: The foremost driving issue is the increasing complexity
Chris Cassidy
of compounds in the pipeline. The average small molecule compound is less
Vice President Sales & Marketing
bioavailable, often combined with other technical or clinical formulation
SCHOTT North America
challenges, such as targeted complex release profiles for therapeutic
efficacy. Large molecule proteins pose other challenges, such as target site
Hibreniguss Terefe PhD administration/delivery, large volume requirements driving less desirable
Vice President Research & Development infusion delivery, and the frequent use of non-standard delivery devices.
ExxPharma Therapeutics LLC And new treatment modalities, such as cell and gene therapy, mRNA, and
RNAi will require further drug delivery innovation still.
Nick Grasman Compounding the increase in molecular complexity is the ever-increasing
Marketing Manager need for differentiated clinical outcomes, to gain market access and
Dow Food, Pharma & Medical reimbursement, written prescriptions and patient interest. There’s a long
history showing that drug delivery done right – the right molecule form,
Matthew Shaffer the right formulation, the right dose form and device – can enable products
Manager, Multiparticulate Product Developmen to deliver better outcomes for patients, and in the market.
Lonza Pharma & Biotech David Elder, Independent CMC Consultant: The adverse impact of high-
throughput and combinatorial chemistry has resulted in molecular weight
inflation of new chemical entities (NCEs), resulting in increased lipophilicity
and decreased aqueous solubility. Historically, small biopharma companies

152 | | September/October 2017

 « ROUNDTABLE »

have not adequately invested in formulation development/optimization for Nick Grasman, Marketing Manager, Dow Food, Pharma & Medical:
these type of molecules, believing that simple, cheap formulations that enable The number one issue that continues to drive research on drug delivery
fast development were the optimal approach. However, if the absorption is systems is the poor bioavailability of compounds emerging from discovery.
solubility-limited then without appropriate formulation intervention the Drug delivery issues are taking on increasing prominence in generic drugs
company will not be able to adequately explore the pharmacology in man. as well. Recently there have been a few prominent examples of generic
The development classification system (DCS) enables companies to rapidly formulations which deviated from the delivery mechanism of the reference
identify whether NCEs show dissolution rate limited or solubility rate limited listed drug and were found to perform differently in-vivo, resulting in
absorption and thereby define the optimal formulation approach.1 significant regulatory and market challenges. More than ever, formulators
and drug developers need to engage with excipient suppliers to address
Chris Cassidy - Vice President Sales & Marketing, SCHOTT North
these challenges.
America: Self-administration of injectable drugs is a trend that continues
to gain momentum. To facilitate patient safety and convenience of use,
pharmaceutical companies are increasingly giving consideration to drug
delivery pathways from the earliest stages of the drug development Can you tell us about some new drug delivery
process. The expanded use of self-administered injectable drugs to treat technologies and/or processes that are helping
chronic conditions such as diabetes and rheumatoid arthritis has led to
pharmaceutical companies bring new products
growth in the market for ready-to-use devices. Drug manufacturers are also
highly concerned with the quality and performance of primary packaging to market or are reviving older products? Is there
and components contained within delivery systems. The pharmaceutical a specific process or technology that you see as
industry invests a great deal of time and effort into the evaluation of most effective?
packaging, components, and materials for parenteral drugs. By partnering
with vendors during the early stages of the formulation process, Qiu: The enabling technologies AbbVie focuses on for low solubility/
pharmaceutical companies can ensure that they are selecting packaging bioavailability compounds include amorphous solid dispersions (ASDs)
and components that will perform appropriately in combination with drug using Hot Melt Extrusion (HME) or spray drying manufacturing technology.
products. This can help to avoid risk of interactions that could adulterate Conventional approaches to enhance dissolution and oral absorption rely
the drug product or impact efficacy. In addition, the risk of problems such on increasing either solubility or surface area. ASD works on both; it allows
as device failure, delamination, damage / breakage, and contamination dispersion of the amorphous drug at the molecular level in a stable polymeric
can be eliminated to avoid market recalls. system to increase apparent solubility while at the same time creating very
small or even nanometer-sized particles to increase surface area during
Hibreniguss Terefe Ph.D., Vice President Research & Development,
drug release. ASD formulation technologies, and associated manufacturing
ExxPharma Therapeutics LLC: A search for new molecular entities (NME)
processes, can provide the maximized benefit of rapid dissolution of poorly
using modern screening processes, such as combinatorial chemistry and
soluble drugs. Both HME and spray drying technologies can be used to
drug design, is resulting with lead compounds that are increasingly lipophilic,
produce ASD and both involve a continuous operation that is amenable
poorly water soluble and having higher molecular weight. Approximately
to continuous manufacturing. AbbVie has been working with HME for two
up to 90% of new drugs in the pipeline have bioavailability limitations, and,
decades. HME is preferred because it offers several advantages over spray
according to the Biopharmaceutical Classification System (BCS) fall into Class
drying: no solvents, smaller footprint, higher efficiency and lower operating
II (poorly soluble, highly permeable) and Class IV (poorly soluble, poorly
cost. Depending on API and formulation, it also offers the opportunity of
permeable).
direct shaping, necessitating fewer processing steps. However, HME has its
Patent expiration of brand drug products combined with shrinking R&D limitations depending on API properties. When it is not feasible for certain
pipelines and high development cost necessitated implementation of life API’s due to thermal instability or limited drug loading, spray drying can
cycle management strategies that prolong patent life by improving the be the alternative because it is applicable to a broader range of APIs and
efficacy, safety and compliance of existing medicines. The generic market polymers without excessive heat exposure during manufacturing.
is highly competitive and obtaining approval through paragraph IV filing
Kelemen: It is difficult to envision the development of novel drug delivery
has become extremely difficult, especially for the smaller companies.
technologies without the realization that nanotechnology-based approaches
Consequently, many generic companies often are shifting their focus
are showing tremendous promise both experimentally and clinically.
towards specialty generics.
Perhaps this statement is most relevant in regards to the development of
Therefore, drug delivery systems that improve efficacy, safety and next generation immunotherapeutics and diagnostics. Relying on a wide-
patient compliance are in great demand and are highly sought after by range of technical capabilities and scientific disciplines, nanoparticle-
pharmaceutical companies. Bioavailability enhancing drug delivery based therapeutics offer very appealing approaches, for example, in tumor
systems are instrumental in rendering poorly soluble new molecular targeting either by passive or receptor-mediated active tumor targeting.
entities viable medicines and in extending the life cycle of existing These bioengineered nanoparticles can deliver immunotherapeutic agents
medicines that are approaching patent expiration. Pharmaceutical that specifically kill cancer cells and, when combined with advanced drug
companies can utilize them to get products approved through the delivery technologies, can direct therapeutics into regions of the body
505(b)(2) regulatory pathway. Modified release, abuse deterrent and where classic formulations cannot effectively penetrate including across
fixed dose combination drug delivery systems also serve in making drug the blood-brain-barrier. The overarching benefits of nanoparticle-based
products more efficacious and safe. Moreover, they play an important drug delivery systems can be quite impactful on the therapeutic outcome
role in improving patience compliance by reducing dose frequency and for the patient. There is mounting evidence that nanoparticles can improve
pill burden, and making dosage forms easy to administer. Demography the therapeutics’ pharmacokinetics and pharmacodynamics profiles, reduce
tailored drug delivery systems, such geriatric and pediatric dosage forms, systemic or acute cellular toxicity and provide controlled and targeted
are also gaining importance. release of the therapeutic agent.

www.americanpharmaceuticalreview.com | | 153
» ROUNDTABLE »

Over the last several years there have been dedicated and significant Stamoran: There’s fascinating work going on at both ends of the
advances in the development of nanotechnology-based vaccines for innovation range – well established formulation and dose platforms
the invasive and non-invasive delivery of immunotherapeutic agents, are being adapted to solve new challenges, and novel new predictive
especially nucleic acids. While several strategies have been developed formulation optimization platforms are being developed to accelerate and
over the years for RNA vaccine delivery, lipid nanoparticle formulations are optimize formulation development.
being recognized as one of the more promising drug delivery systems for
I’d highlight two examples from Catalent’s own work. First, in 2016, for the
nucleic acids and show great promise towards achieving the goal of safe
first time ever - in 200 years of soft gelatin capsule history – a sustained
and efficient in vivo RNA delivery.
release softgel capsule received regulatory approval. Using a new shell
Matthew Shaffer, Manager, Multiparticulate Product Development, system, OptiShell® technology, enabled a new fill modality, which becomes
Lonza Pharma & Biotech: Multiparticulates (MPs) technologies are semi-solid after filling, in turn enabling a sustained rate of compound
increasingly being selected as they are a proven life cycle management release for patients.
tool and highly flexible for achieving modified release delivery profiles,
Secondly, I’d highlight that we have also created a predictive formulation
improved dosing regimens and fixed dose combinations. As an indication
platform of which I’m quite proud – the OptiForm® Solution Suite
of their increasing value, 22 new drug products with multiparticulates
formulation platform. Starting with the idea that there’s no “one size fits
were approved by the U.S. Food and Drug Administration between 2012
all” approach to addressing solubility and bioavailability challenges,
and 2016; 10 of the 22 approvals were for pediatric applications where
we combined a proven, solid-state chemistry high-throughput analysis
multiparticulate formats are accepted as having superior dosing flexibility
platform with each of the key formulation enhancement approaches
and ease of administration over monolithic formats.
practiced today. Combining that with our own delivery expertise, built on
Multiparticulates are frequently used for oral drug delivery when a over 80 years of formulation work, we’re able to quickly provide dose-form
compound is in need of microencapsulation, modified release, solubilization agnostic recommendations, saving customers the substantial amount
or combinations thereof. They are typically presented to the patient as of time and money it would take to do the same work experimentally,
capsules or sachets. The multi-unit particle approach presents advantages sequentially. We have now extended that into other areas, such as the
over other oral solids through reduced PK variability, flexibility in dosing and complexities of oral peptide delivery, and most recently with complex
versatility in dosage form presentations. release profile formulations.
The surge in utilizing multiparticulate technology is largely attributed to Elder: Dissolution rate limited absorption can be addressed by API size
the development of pediatric products – as pediatric product development reduction and the use of surfactants to facilitate rapid wetting. In contrast,
presents several unique challenges. Children are a vulnerable population at solubility rate limited absorption can be addressed using amorphous
the peak of changes in both anatomy and physiology. From the youngest stabilized formulations or alternatively using lipid formulations in softgel
to oldest subpopulations, a broad range of doses and flexible dosing capsules to pre-solubilize the drug substance. The DCS1 approach allows
strategies are needed to accommodate these differences. Moreover, the companies to rapidly invest in the most suitable formulation strategy
dosage form presentation and product palatability are important to this to optimize the potential for clinical success; thereby ensuring optimal
population and their caregivers. Specialized MP technologies such as lipid development efficiency, whilst at the same time minimizing costs.
multiparticulate technology provide inherent taste masking functionality
Cassidy: For drug products that must be lyophilized, double-chamber
through microencapsulation of the drug, and the flexibility to be delivered
syringes offer a benefit to patients who self-administer the treatment.
in multiple dosage forms. For example, in a sprinkle capsule presentation,
Freeze-dried medicines must be reconstituted with a diluent prior to
the capsule could be swallowed, or the easy-to-swallow contents could be
injection. Double-chamber syringes allow patients to easily mix and
emptied directly in the mouth, or combined with food or liquids.
administer medications through the use of a single, contained system. The
MPs have universal properties as well that can be adapted within a broad drug product and diluent are prepacked in the double-chamber syringe,
number of dosage forms for adults as well. The number of 505(b)2 approvals thereby avoiding the possibility of contamination and ensuring that the
in recent years is evidence of this fact. Utilizing the lipid multiparticulate patient self-administers the proper dosage.
technology with a melt-spray congeal process, there is an opportunity to
Terefe: Drug delivery is a broad topic but let us focus here on drug delivery
establish a platform-view on product development even for new compounds.
technologies applicable to solid oral dosage forms and, more specifically,
Using this technology, a single development pathway for all ages going right
drug delivery technologies that enhance bioavailability. To enhance the
into first-in-human trials is achievable. This approach could realize affordable
bioavailability of poorly soluble drugs, basically two distinct strategies have
medicines especially for niche indications, low margin markets, or even
been employed. The first strategy focuses at the API development stage
the geriatric population. Furthermore, it would enable speed to market
and involves salt formation, prodrugs and search for more soluble analogs.
by building product knowledge right out of the gate, thus minimizing the
The second strategy relies on drug delivery technologies that enhance the
number of bridging studies during the product development.
solubility of poorly soluble molecular entities, and hence drug absorption.
As multiparticulates continue to demonstrate acceptability by patients, Technologies that improve drug absorption in the gastro-intestinal tract
expect to see new, evolving presentations of the product and how it is include lipid-based formulations, micro-emulsions and self-emulsifying
administered – motivated by personalizing the delivery of medicine. drug delivery systems, nano-particles and amorphous solid dispersions.
Examples of personalization could include the development of reusable Thanks to the extensive research and development activities that are
devices for dispensing that maintain the dosing flexibility, or offer patient being carried out by many companies to address bioavailability issues,
choices to dosing aids that are more controlled than simply suggesting the knowledge-base in this area is expanding and drug delivery systems
foods and beverages on the label. Lastly, with solubilized drug forms and increasingly becoming sophisticated. Drug delivery technologies have
high potency compounds becoming more common place in the industry, already led to the successful approval of several new molecular entities and
control strategies for multiparticulate technologies will need to adapt to product enhancements of approved drug products. Several amorphous
see these compounds through to the market. solid dispersion-based drug products have been approved by the FDA and,

154 | | September/October 2017

 « ROUNDTABLE »

the trend is going to continue as the technology gains more traction. While should be produced from materials of the highest purity to reduce the risk
understanding the physicochemical properties of the target drug substance of extractables and leachables, and to prevent heavy metal contamination
and selecting appropriate formulation components is the first step, it is also or ion release. Cross-linked silicone reduces subvisible particulates. Certain
imperative that formulations are processed in the most effective way to sterilization methods have significant advantages over others when it comes
obtain physically and chemically stable amorphous solid dispersions that to the reduction of extractables and leachables. Material concerns play a
provide maximum bioavailability. Hot melt extrusion has been proven to be major role in medication safety.
a very efficient process to handle complex formulations when dealing with
Terefe: Functional excipients are critical components of drug delivery
solubility enhancement. It is environment friendly and amenable to process
systems. Because of strict regulatory requirements, the list of available
analytical technology. The technology is versatile and could also be used to
excipients that could be used for solubility enhancement is very limited.
manufacture other types of drug delivery systems, such as modified release, Recognizing this need, excipient manufacturers are coming up with
fixed dose combination and abuse deterrent formulations. improved and innovative solutions to address solubility issues. To mention
Grasman: Brand new drug delivery technologies are rare, but we are some, BASF’s Soluplus® is a new functional excipient that was developed
seeing resurgence and growth in some niche technologies that are playing to enhance solubility of poorly soluble drug substances during hot melt
an important role. For example, extended release dosing via transdermal extrusion. Dow chemicals has introduced Affinisol® systems, i.e. HPMC and
patches is an excellent means of improving compliance among HPMCAS grades that have improved thermal properties for successful HME
populations that struggle to adhere to a strict dosing regimen. Likewise, processing. Evonik is expanding the application of Eudragit® polymers for hot
multiparticulate dosing can help a formulator achieve multiple objectives melt extrusion applications. Gattefosse is offering a range of pharmaceutical
by offering flexibility in drug release profile in a form which is much easier excipients that could be used in the development of lipid based and self-
to swallow. Osmotic pump tablets have also demonstrated that they can emulsifying drug delivery systems.
be an effective way to mitigate variation in drug release as a result of fed/ Grasman: Without a doubt, active ingredients are becoming more targeted
fasted state. Finally, a number of innovative approaches are being explored and more demanding placing increasing pressure on excipients to step up
to help address intentional abuse of analgesic medications. to the challenge. Across the board, excipient providers have been elevating
their performance by improving batch to batch consistency and innovating
within compendial limits, but the challenges of the future will likely require
How have functional excipients/raw materials helped novel excipients. Creation of a pathway for novel excipients that allows for
with drug delivery? Are these excipients becoming excipients to demonstrate their safety and receive approval independently
from a drug application remains a key barrier to breakthrough development.
more important as solubility concerns arise?
Qiu: Functional excipients are definitely becoming more important as
solubility concerns arise; we cannot formulate drugs by ASD technologies As drug delivery issues become more commonplace
without them. The challenge is to find the appropriate polymers, – what is the role of suppliers to industry? How
surfactants or other raw materials that will work for the molecular structure
important is their product/formulation experience
and physicochemical properties of the compound, to achieve increased
dissolution and acceptable stability. You need a certain type of polymer to the success of any given product?
and its functional properties to enable formulation and function of ASD.
Qiu: The experience of a supplier or service provider is, of course, critical to
Elder: The DCS1 formulation strategies, particularly those applied for the success of drug delivery. For a CDMO, such as AbbVie, you need to be
solubility-limited NCEs can only be implemented using functional excipients. able to demonstrate your skills and experiences in delivering a drug product
In the case of softgel development these excipient facilitate solubility in a to market. Can you develop a formulation and handle the technology
lipidic vehicle that can rapidly form an emulsion (typically micro- or nano- transfer or scale it up for commercial manufacture? What happens when
sized) on contact with the gastro-intestinal environment. For amorphous you run into inevitable hiccups? Does the CDMO have processes in place
stabilized formulation approaches the functional excipients minimize and a culture of transparency, communication, and ownership to convey
molecular mobility by trapping the amorphous API in an extended H-bonded the problem and possible solutions? And the ability to work with the client
network. The amorphous API has intrinsically higher aqueous solubility than toward an agreed solution? In every respect, experience and knowledge
its crystalline counter-part, but suffer from reduced chemical and/or physical are highly valuable to pharmaceutical sponsors who already have invested
stability. Such approaches can produce products with realistic, commercially significantly in their compounds.
aligned shelf-lives, i.e. 24+ months by reducing chemical instability as well
Kelemen: As a cGMP manufacturer and supplier of functional lipid
as enhancing physical stability by retarding/preventing conversion of the
excipients for drug formulation and delivery applications, our level
amorphous API into the thermodynamically more stable crystalline entity.
of commitment towards providing the highest quality monographed
Cassidy: The rise of self-medication has led to the expectation that excipients to the pharmaceutical industry cannot be overstated. In support
parenteral devices should be easy to use. Silicone has been the primary of a diverse catalog of functional lipid products, we strongly believe that it
lubricant used in pre-filled syringes to reduce glide force and preserve is imperative to have first-hand knowledge and application experience in
patient comfort. However, the use of lubricants can result in the presence the use of our excipients. This knowledge base expands across a wide range
of visual particulates and may cause interactions with drug products. of formulation types and delivery challenges including the development of
Particulates and potential drug interactions are major areas of concern self-emulsifying drug delivery systems for BCS Class II-IV small molecules
that drug manufacturers consider when selecting drug delivery devices, into the development of complex lipid nanoparticle formulations for the
components, and packaging materials. Pharmaceutical companies must non-invasive delivery of biologic drugs. The author believes that it is the
partner with suppliers who effectively manage these risks throughout every obligation and responsibility of excipient suppliers to maintain a high-
step of the manufacturing process. Primary packaging and components degree of scientific knowledge in order to support the pharmaceutical

www.americanpharmaceuticalreview.com | | 155
» ROUNDTABLE »

industry in their quest for the successful development of safe and effective
next generation therapeutics. If a company is having drug delivery/solubility
Stamoran: There are a few different business models that suppliers of drug problems with a product in development is there
delivery services and technologies follow. Some purely supply an excipient or
a “checklist” of items/steps, based on the latest
component – a specific device, for example. Others can provide early-stage
formulation design, but lack capabilities to produce it at clinical or commercial technologies that they need to review before
scale. And then some providers – like Catalent – believe it’s important that shelving a product?
you formulate not just for outcomes but also for manufacturability, and
that it’s critical to be able to take your customer’s product from the first pre- Qiu: AbbVie goes through a comprehensive review before considering
clinical dose all the way through the millionth – or billionth – dose on the shelving a product. Essentially, we will re-examine everything that went into
market. And then follow through by making the investments to bring the development of the molecule, its formulation, and manufacturing to see if
“best in class” providers of those capabilities in house. anything can be modified or changed to improve the outcome. Of course
we try to course-adjust along the way, considering every angle as we go to
There’s substantial published research that suggests linkages between
achieve the best outcome. But if we find a problem we didn’t anticipate and
drug substance and drug product design decisions and key drivers of
need to go back, we do a rigorous review and bring in subject-matter experts
patient outcomes, such as patient adherence, side effect profiles, and
as well as new people, a fresh set of eyes to examine the problem. You can
discontinuation. Through the Catalent Applied Drug Delivery Institute,
see what we have in place is better than a simple check list.
we’re trying to formalize and extend this research, including via a research
collaboration on drug delivery for young pediatric patients that we’ve Stamoran: What I’ve often seen happen – a company’s R&D team will have
recently signed with the Rutgers University School of Pharmacy, and one or more people familiar with at least one formulation enhancement
ongoing work in oncology drug delivery too. platform, say particle-size reduction. They try to explore that approach
with an early target candidate, and then if it’s insufficient, they may
Elder: The key attributes that suppliers can provide is knowledge and
choose to move on to other target variants, or to sequentially try a second
expertise. Poor solubility is an issue that they are very familiar with and they
formulation approach. This consumes both time and money, both of which
will have extensive experience on the best strategies to address this problem.
are in short supply!
Cassidy: Suppliers have become partners and consultants to the
I have yet to see a checklist or off-the-shelf tool to do this – the intersection
pharmaceutical industry. It is vitally important that vendors of primary
between chemistry and biology is complex, and patients are diverse. I
packaging, components, and drug delivery systems are engaged by
believe using a platform such as the OptiForm® Solution Suite formulation
pharmaceutical manufacturers during the early stages of drug development.
platform, and partnering with an experienced early development
Very often, several vendors must work together for the benefit of their
provider (like Catalent), will deliver the best results. And the earlier in the
mutual pharma customer. Drug makers are increasingly asking packaging
development path, the better.
suppliers for testing related to toxicity and leachables in a product. Also, drug
products that have moderate to severe delamination risks must undergo Elder: The DCS1 approach is useful because in addition to providing a
additional compliance testing for shelf life and stability. The pharmaceutical framework for relevant formulation strategies designed to address poor
industry has particularly complex supply chains, and packaging is just one of aqueous solubility, this approach can identify the likelihood of success.
the many variables they must control. Pharmaceutical companies operating Based on relevant, cross-industry experience we know that DCS class
drug development processes that allow for integrated collaboration with 4 compounds (i.e. poor solubility and permeability) have significantly
suppliers reap significant benefits, and are more likely to avoid pitfalls in the increased likelihood of attrition and that it will take much longer to
late-stage clinical and post-launch phases. demonstrate this during clinical development. 2 Fridgeirsdottir et al.3 used
guidance maps and formulation decision trees to facilitate in the rapid
Terefe: The pharmaceutical industry is highly competitive, and risk-
identification of an appropriate -enabling formulation technology.
averse. Companies want to win the race by having the best possible
quality product, in the shortest possible time, with the minimum possible Cassidy: Solutions always exist. To find these solutions, drug manufacturers
expense and risk. For that to happen, a number of factors must be must work closely with their suppliers to examine the entire filling
satisfied. For successful product development, the supplier should have process to evaluate the intended use of each component utilized. A
not only knowledge and experience in solving drug delivery issues, but holistic approach is required. From the early stages of drug formulation,
also good understanding about the regulatory requirement throughout the drug manufacturers must identify materials that meet the particular
the product development phases. As every drug substance is unique, the requirements of the product in question. Careful consideration must
development process should be guided by good science. Cost-effective, be given to the many potential risks that exist. Does the drug require
phase-appropriate development plan that shortens development time is particularly inert packaging? Is there any flexibility on device design?
crucial, especially for small discovery companies that operate in funding Is there a need for tight tolerances? Do any of the components require
constrained environment. Such demanding service requires extensive superior break-force resistance? Packaging and component material
product and formulation development experience. candidates must be evaluated for compatibility with existing filling lines.
Patient safety and comfort must always remain in focus as top priorities.
Grasman: Suppliers represent a significant source of underutilized
Each material utilized with have strengths and weaknesses. It is up to
information to formulators and product developers. In my experience,
the drug manufacturers to obtain all of the relevant information to make
when formulators and excipient providers have truly and openly engaged
informed choices regarding components, packaging, and delivery devices.
with one another, significant challenges have been surmounted. All too
often, suppliers and developers have a relationship that extends only to Terefe: Understanding physicochemical and biopharmaceutical pro-
exchanging small bits of information which does not allow for the kind of perties of a drug substance is the first step to successful drug product
collaborative problem solving that today’s drug delivery challenges require. development. Having exhaustive pre-formulation studies and determining

156 | | September/October 2017

 « ROUNDTABLE »

BCS classifications help to plan and prioritize which technologies and Cassidy: In order to develop more effective cures and treatments, the
formulations to consider. For example, is the poor solubility crystal lattice pharmaceutical industry is fundamentally shifting from large batch, “one-
energy-limited or solvation-limited? For predominantly hydrophobic size-fits-all” drugs to targeted medications, therapeutic niche applications,
drugs that are solvation limited, lipid-based formulations and self- and high-value biologic drugs. These highly specialized therapies focus on
emulsifying drug delivery systems may be a place to start. For crystal smaller patient populations, and thus are produced in smaller volumes. In
lattice energy-limited poorly-soluble drugs, amorphous solid dispersion the past, pharmaceutical manufacturers have tended to build filling lines
may be the way to go. Relevant questions include: Does the drug exhibit that are geared toward high-quantity production batches. However, in the
high melting point and high hydrophobicity? What is the propensity of the
future, drug manufactures will need to fill small-quantity batches and will
drug to recrystallize once converted into amorphous form? Computational
utilize different types of containers on the same filling line. The pharma
methods that use molecular modeling and solubility parameters are helpful
production process will require the ability to make quick job changes and
to select appropriate carrier polymers for the development of amorphous
setups. Not only will a drug product change from batch-to-batch, but
solid dispersions. Depending on the physicochemical properties of the
the type of container being filled may change as well. During the course
drug substances, the most appropriate technologies with appropriate
of a week, the same filling line may be utilized to fill vials, syringes, and
formulations shall be rationally selected.
cartridges. Next generation filling lines will require such flexibility in order
Grasman: In today’s world, there are a wide variety of options available for
to reduce time-to-market and avoid the risk of drug shortages. One of
tackling a particular drug delivery challenge. Knowing where to start and
the best ways for pharmaceutical manufacturers to achieve flexibility
when to call it quits are key skills that anyone developing new formulations
in an era of smaller batch sizes is to rely on the traditional nest and tub
must acquire. There is a very real desire to try and create a sort of flow-sheet
configurations for ready-to-use containers, components and devices.
that can be used to help guide formulation development, but this idea is
These configurations work on a variety of machine types, and allow
based on the premise that one particular technology is more likely to work
pharmaceutical manufacturers to quickly and easily shift from vials to
than another in nearly every situation. More often, I find the challenge for
drug development is efficiently expanding options beyond the typical cartridges to pre-filled syringes all on the same line. By standardizing drug
checklist. CROs and suppliers can be excellent sources of information for manufacturing and filling, the time required for regulatory review of new
expanding the suite of options to make sure that a valuable discovery is processes, materials, and components can be reduced.
not discarded due to delivery mismatch. Terefe: Biopharmaceutical companies are discovering more and more
complex small and large molecules which pose difficult drug delivery
and manufacturing issues. The need for the development of drug delivery
What do you see as the major industry critical issues systems that accurately deliver drugs to targeted body sites must be
over the next five years in regards to drug delivery? addressed more than before. Another issue that is challenging is the
development of age-appropriate pediatric and geriatric patients-focused
Qiu: For solid oral products, and the APIs involved, we expect the trend of
versatile drug delivery systems.
poor solubility to continue. The compounds will only get more challenging
to formulate, but we’re confident we’ll meet those challenges. In the area Pediatric patients differ in their developmental status and dosing
of formulation technology, we’ll still be challenged with drug loading. Right requirements from adults. Geriatric patients have compromised
now the percentage of drug in a formulation is relatively low – often less than pharmacokinetics affected by co-morbidity, reduced organ function and
30% and more often about 10 - 20%. That can result in the need for multiple multiple drug use. Hence most conventional drug delivery systems are not
pills or single large pills that patients must swallow to get the therapeutic acceptable for these demographic groups.
dose. This is an area where we are constantly striving to improve through Grasman: With Biologics being the largest growth segment for the
product and process research. Finally, in the manufacturing process, product Pharmaceutical Industry, the biggest challenge will be patient’s
and manufacturing consistency is an issue regulators are paying increased compliance with the delivery of the API. Oral delivery is the largest delivery
attention to. Continuous manufacturing may be one of the solutions here, solution today and Biologics typical route is parental because of the API’s
and HME, which AbbVie uses, is amenable to continuous manufacturing. interaction with the gastrointestinal tract. I expect more innovation in the
Stamoran: Delivering to the target activity site while minimizing impact combination medical devices like the wearable insulin pump patch to
on other non-targeted areas will be increasingly important for biologics improve the parental delivery experience.
and other drugs, as will determining how best to deliver new modalities
like gene and cell therapy.

Finding ways to address the “adherence problem”: I believe designing drug References
products to improve patient outcomes will play a significant role, as will
1. J.M. Butler, J. Dressman. 2010. The Developability Classification System: Application of
identifying ways to deal with polypharmacy issue, including regimens that Biopharmaceutics Concepts to Formulation Development. J. Pharm. Sci., 99(12), 4940-4954.
are predominantly generic.
2. M.K. Bayliss, J.M. Butler, P.L. Feldman PL, et al. 2016. Quality guidelines for oral drug
Finally, product developers must be prepared to demonstrate proof of candidates:
incremental value versus currently available treatments, and if they’re using 3. dose, solubility and lipophilicity. Drug Discov. Today.;21(10), 1719-1727.
an enhancing drug delivery technology, proof that such actually has an 4. G.A. Fridgeirsdottir, R. Harris, P.M. Fischer, C.J. Roberts CJ. 2016. Support tools in
incremental impact; to justify formulary placement, market access or cost. formulation development for poorly soluble drugs. J Pharm. Sci. 105(8), 2260-2269.

www.americanpharmaceuticalreview.com | | 157
» »
SECTION TITLE

Pharmaceutical
P.I.N. Dendronized Polymers for Nucleic

Points
Patent Innovation News
Acid Delivery;
Z. Guan and H. Zeng; The Regents of the
University of California;
U.S. Patent # 9,745,421, August 29, 2017.
RNA interference (RNAi) is a biological process in which RNA
molecules inhibit gene expression or translation, by neutralizing
targeted mRNA molecules. The therapeutic treatment potential
The purpose of this column is to highlight

Article Title
of RNAi has been hampered due to its unsafe and inefficient
and summarize recent key patents in the intracellular delivery of siRNA (small interfering RNAs). The present
pharmaceutical arena issued by the US
Introduction
invention provides for an innovative biodegradable peptide-based
dendronized polymer (“denpol”) architecture, that can be used as a
Patent Office in July-August, 2017. Paragrapgh Text
carrier for the intracellular delivery of nucleic acids. The dendronized
polymers combine the multi-valency of dendrimers, with the
conformational flexibility of linear polymers for optimal binding of
Anvit Vasavada, M.S., Harshada Sant, M.S., nucleic acids (e.g., siRNA). The patent also claims a pharmaceutical
Amitkumar Lad, Ph.D. and composition comprising a dendronized polymer/siRNA polyplex
Hemant N. Joshi, Ph.D., MBA* and provides for a method of treating a disease or disorder.
Tara Innovations, LLC
www.tarainnovations.com
*[email protected]

Enhanced Delivery of Drug Compositions

to Treat Life Threatening Infections;
J.E. Hitt, T.L. Rogers, B.D. Scherzer,
Oral Name
Author Pharmaceutical I.B. Gillespie, P.C. Garcia, N.S. Beck, C.J. Tucker,
Composition
Author Title of Isotretinoin; T.J. Young, D.A. Hayes, R.O. Williams III,
Author Company
R. Rao, A.K. Fanda, S.K. Jain, and K.P. Johnston, J.T. McConville, J.I. Peters, R.
R.B. Singh; Sun Pharma. Ltd.; Talbert, and D.S. Burgess, Board of Regents,
U.S. Patent # 9,700,535; July 11, 2017. The University Texas System;
The oral bioavailability of a drug is affected by various U.S. Patent # 9,724,344; August 8, 2017.
factors, which include aqueous solubility, absorption of
The patent describes an enhanced delivery of antifungal agents into
drug through GI tract, first pass effect, or food effect. The
lungs and how the concentration stays at the therapeutic level for
food-drug interactions can either decrease or increase
at least one hour after being delivered to the lung. These drugs are
the extent of drug absorption. Isotretinoin is known
known to have poor bioavailability after pulmonary administration. The
to have a food effect, i.e., its absorption is dependent
method of preparation involves: mixing an effective ingredient with a
on the presence of food in the stomach. The present
solution agent; spraying the effective ingredient-solution agent mixture
invention provides an oral oil-free dispersion comprising
through an insulating nozzle located at or below the level of a cryogenic
isotretinoin, surfactants having HLB value of 10 or greater
liquid. The invention also developed other methods to produce porous
and co-solvents. The composition can also be filled into
respirable nanoparticles suitable for deep lung deposition. In one of
capsules. It exhibits reduced food effect in comparison to
the claims, the respirable nanoparticle aggregates were prepared by
the marketed Epuris™ capsules indicated by higher Cmax
a process comprising ultra-rapid freezing of an amorphous antifungal
and AUC in fasting state.
drug solubilized in a freezable solvent on a solid substrate to form the
respirable nanoparticle aggregates.

158 | | September/October
November/December2017
2011
 « SECTION TITLE »

Small Cationic Antimicrobial Pharmaceutical Composition for

Peptides; Transcolonic Absorption;
R.E.W. Hancock, K. Hilpert, A. T. Yokota, M. Murakami, and K. Nishina;
Cherkasov, and C. Fjell; The University National University Corporation, Tokyo
Of British Columbia; Medical and Dental University; U.S.
U.S. Patent # 9,707,282; July 18, 2017 Patent # 9,731,025; August 15, 2017.
The treatment of bacterial infections with antibiotics is one of the High molecular, water-soluble compounds such as hormones,
mainstays of human medicine. Unfortunately, the effectiveness cytokines, nucleic acids etc. have low epithelial permeability
of antibiotics has become limited due to an increase in bacterial and cell membrane permeability. The present invention aims
antibiotic resistance in the face of decreasing efforts and to provide a pharmaceutical composition for transcolonic
success in discovery of new classes of antibiotics. The present absorption capable of delivering a physiologically active
invention relates generally to peptides and more specifically to substance having an intracellular site of action into specific
antimicrobial and immunomodulatory host defense peptides. tissue cells with high specificity, noninvasively by a means of
The present invention claims an isolated immunomodulatory administration other than injection. The invention described
peptide comprising the amino acid sequence set forth in SEQ method of noninvasive administration of vitamin E-conjugated
ID NO: 1214, or a variant thereof comprising an Ile, Arg or siRNA (VE-siRNA). Endogenous chylomicrons are formed in the
Val at position 5, or an amino acid sequence having at least small intestine, the majority of which penetrate through the
90% identity thereto. The invention provides a bioinformatic mucosal epithelium of the small intestine into lymphatic vessels
method of predicting new peptides with good antimicrobial and ascend along the lymphatic vessels; after draining into
activity through the creation of a random library of peptides. the vein, they are metabolized into chylomicron remnants by
lipoprotein lipase (LPL), whereby the endogenous chylomicron
is delivered to the liver. The patent helps to deliver endogenous
chylomicrons comprising VE-siRNA into the liver cells by
absorption from the small intestine.

RNAi-Mediated Inhibition
of Histamine Receptor
H1-Related Conditions;
J.M. Yanni, J.E. Chatterton, D.A. Sustained Release Eye Drop
Gamache, and S.T. Miller; Arrowhead Formulations;
Research Corp.; U.S. Patent # 9,745,585; V.G. Wong and L.L. Wood; Ramscor, Inc.;
August 29, 2017. U.S. Patent # 9,737,606; August 22, 2017.
The present invention relates to the field of interfering RNA The present invention relates to biocompatible, biodegradable,
compositions for treatment of a histamine receptor H1 sustained-release formulations of active agents for topical
(HRH1)-related condition. Such conditions include allergic administration to the eye. Current topical eye drop formulations,
conjunctivitis, ocular inflammation, dermatitis, rhinitis, asthma, which often elicit some degree of irritation, provide only short-
or allergy, for example. The patent claims a composition term exposure of beneficial agents to the eye and thus, often
comprising an effective amount of a single-stranded siRNA require several administrations daily. The formulation consists
having a length of 19 to 49 nucleotides, or a double-stranded of 10% to 15% (w/w) dexamethasone or a derivative, analog,
siRNA wherein each strand has a length of 19 to 49 nucleotides. or a salt dispersed or suspended in 85% to 90% (w/w) of a
The invention also claims a composition which can be biocompatible, biodegradable excipient mixture. Their aim is to
administered via an aerosol, buccal, dermal, intradermal, deliver medications in a continuous and sustained manner. The
inhaling, intramuscular, intranasal, intraocular, intrapulmonary, formulations deliver therapeutic and non-toxic levels of active
intravenous, intraperitoneal, nasal, ocular, oral, otic, parenteral, agents over the desired extended time frame, often primarily at
patch, subcutaneous, sublingual, topical, or transdermal route. the site of administration.

www.americanpharmaceuticalreview.com | | 159
» ADVERTISER'S INDEX »

Company Page # Web Address

AAPS Annual Meeting and Exposition 129 www.aaps.org/annual meeting
ACD/Labs 10 www.acdlabs.com
Ashland 11 www.ashland.com/pharmaceutical
Associates of Cape Cod 5, 12 www.acciusa.com
ATCC 39, 77 www.atcc.org
Azbil Biovigilant 27 www.biovigilant.com
BASF Corporation 13 www.pharma.basf.com
BD 31 www.bd.com/WideMouth
bioMerieux, Inc. 14, 29, 37, 79 www.biomerieux-usa.com
Bosch Packaging Technology 15, 87 www.boschpackaging.com
Capsugel 89, 120-121 www.capsugel.com
Catalent CV4 www.catalent.com
Charles River 16, CV2 www.criver.com
CPhI Worldwide 83 www.gotocphi.com/register
Croda 123 www.crodahealthcare.com
DFE Pharma 17, 75 www.dfepharma.com
Distek, Inc. 45 www.distekinc.com
Dow 93 www.dowpharmasolutions.com
Eastern Analytical Symposium and Exposition 137 www.eas.org
Eurofins Lancaster Laboratories 18, 103 www.EurofinsLancasterLabs.com
Gattefosse 95 www.gattefosse.com
ICDD 19 www.icdd.com
IFPAC 115 www.IFPACglobal.org
KENX 1 www.kenx.org
Kerry Group 73 www.kerrygroup.com
Lonza 25 www.lonza.com/qcinsider
Malvern Panalytical 20 www.malvernpanalytical.com
MEGGLE Group Cover Tip, 21 www.meggle-pharma.com
Metrohm 131 www.metrohmusa.com
Micronizer 109 www.micronizer.com
NETZSCH Premier Technologies 22, 107 www.netzsch-grinding.com/pharma
Perfex 113 www.perfexonline.com
Pharma & Device Packaging and Labeling 85 www.arena-international/pharmapackagingwest
Servier CV3 www.servier-cdmo.com
Shimadzu Scientific Instruments 23, 101 www.ssi.shimadzu.com
Sotax 47 www.sotax.com/hplc
SP Scientific 81 www.spscientific.com
That's Nice 149, 151 www.thatsnice.com

The contact directory is for information purposes only. While every effort has been made to ensure it is accurate and complete, any errors or omissions are not the responsibility of the publisher.

160 | | September/October 2017

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