Thermal Methods of

Analysis of Pharmaceuticals

Abhay T Sangamwar, PhD

Department of Pharmaceutics

NIPER, SAS Nagar

 Proposition of the Usual Dose (Concentration)
“The dose makes the poison”

DOSE
PK/PD

VARIABILI
TY

Paracelsus
(1493-1541)
 Drug Discovery and Development and Cost to
Public Health
Preclinical studies Clinical studies
Discovery Early Clinical Development
CHEMISTRY/ IND* PHASE I PHASE II PHASE III NDA** PHASE IV
PHARMA-
COLOGY
Continued
Search for Regulatory Efficacy Clinical Comparative Regulatory comparative
active review studies on studies on a studies on a review studies
substances *Investigational healthy limited scale large number
New Drug volunteers of patients
Toxicology, Application for
KNOWLEDGE
efficacy permission to 50–150 100–200
LEVEL Registration,
studies on administer a new persons patients
drug to humans market
various 500–5,000
**New Drug introduction
types of KNOWLEDGE patients
Application
animals LEVEL Application for
permission to market
a new drug

TIME SPAN

2–4 yrs. 2–6 months 3–6 yrs. 1–3 yrs.

Approximately 10–15 years from idea to marketable drug

 Drug Discovery and Development and Cost to
Public Health
1 Drug Manufacturable
Molecule
Patentable

Non-mutagenic
Non-teratogenic
1 Drug Regulatory filing
Durable Launch
Competitive profile
Reversible
Cost-effective manufacturing
Non-inducing
Carcinogenicity studies
Metabolically stable
Permeable Long term safety
Efficacy
Soluble
Physically stable Side effect profile
Dosing range
Potent
10,000 Drug Patient recruitment
Selective
Candidates Trial sites and investigators
Targeted
Stability

Formulation

10 Drug Safe and active in lab and animal models

Molecules
All discovery criteria met
5
6
7
Methods of Analysis of Pharmaceuticals

• Titrimetric Techniques: 1835 Gay Lussac

Benefecial for establishment of reaction
rates
• Chromatographic Techniques
Thin layer chromatography, HPTLC, HPLC,
UPLC
• Spectroscopy
• NMR
8
 Outline
■ Brief history of thermal analysis

■ Theory of thermal analysis techniques

– Thermal Gravimetric Analysis (TGA)
– Differential Scanning Calorimetry (DSC)

■ Generating valid data

– Calibration
– Sample preparation

■ Interpreting data and Applications

– Real events
– Artefacts

■ Recent advances

EYP 2006
 Calorimetry
■ Calorimetry
– The study of heat transfer
during physical and chemical
processes

■ Calorimeter
– A device for measuring the
heat transferred

Lavoisier and Laplace (1782-1784):

■ oil was burned in a lamp (Fig 9)
held in a bucket (Fig. 8) held in a
wire mesh cage (f)
■ surrounded by ice in spaces b and
a of the double walled container a
foot in diameter
■ lid (F) was topped with ice, as was
a mesh lid (not shown) beneath it
that covered the inner volume b
Adapted from Clare Rawlinson EYP 2006
 Oil lamps to Guinea
■
Pigs…
Measured heat production of the
metabolic processes in the ice bath
calorimeter
■ Outer jacket prevented conduction of
heat from the external environment
which would have also melted the ice
■ From latent heat of fusion for ice (334
J/gram ice at 0 ºC) Lavoisier converted
the rate of water formation to heat
production
■ In 10 hours 370 grams of ice melted

Guinea pig produced 12,358 J per hour of

heat (12.4 kJ/hr)

Adapted from Clare Rawlinson EYP 2006

 Different Techniques
• Thermometric Titration (TT)
– Heat of mixing
• Thermal Mechanical Analysis (TMA)
– Thermal Expansion Coefficient
• Dynamic Mechanical Analysis (DMA)
– Viscoelastic Properties
• Differential Scanning Calorimetric (DSC)
– Heat flow during Transitions
• Thermal Gravimetric Analysis (TGA)
– Weight Loss due to decomposition
– Derivative Thermogravimetric Analysis (DTG)
• Differential Thermal Analysis (DTA)
– Heat of Transitions
• Temperature Programmed Desorption (TPD)
– Temperature at which gas is desorbed from (catalyst) surface
– Emission gas Thermoanalysis (EGT)
 Basic Principle
• Sample is heated at a constant heating
rate
• Sample’s Property Measured
– Wt TGA
– Size TMA
– Heat Flow DSC
– Temp DTA
– Gas evolved TPD
 Basic Principles of Thermal
Analysis
Modern instrumentation used for thermal analysis
usually consists of four parts:

 sample/sample holder

 sensors to detect/measure a property of the sample and

the temperature

 an enclosure within which the experimental parameters

may be controlled

 a computer to control data collection and processing

 TGA and DSC
■ Thermogravimetric Analysis (TGA)
– mass change of a substance measured as function of
temperature whilst the substance is subjected to a
controlled temperature programme1
– mass is lost if the substance contains a volatile fraction

■Differential Scanning Calorimetry (DSC)

– provides information about thermal changes that do not
involve a change in sample mass1
– more commonly used technique than TGA
– Two basic types of DSC instruments: heat-flux and power
compensation

1Haines, P. J. (2002) The Royal Society of Chemistry, Cambridge.

Adapted from Clare Rawlinson EYP 2006

 DSC
• To determine the purity of a sample
• To determine the number of polymorphs
and to determine the ratio of each
polymorph
• To determine heat of solvation
• To determine the thermal degradation of
a drug or excipient
• To determine the glass transition
temperature of a polymer
16
 Heat Flux DSC
Sample holder :
▪sample and reference are connected by a low-resistance heat
flow path
▪ Aluminium, stainless, platinum sample pans heating
coil

Sensors:
■temperature sensors sample reference
■usually thermocouples pan pan

Furnace:
▪one block for both sample
and reference cells inert gas
vacuum thermocouples

Temperature controller:
•temperature difference the sample and reference is
between measured
 Power Compensated
DSC
Sample holder :
▪Aluminium, platinum, stainless steel pans
individual
heaters
Sensors:
▪Pt resistance sample pan reference pan
thermocouples.
▪Separate sensors
and heaters for the
sample and reference inert gas
vacuum
inert gas
vacuum
thermocouple T = 0
Furnace:
▪separate blocks for sample and reference cells

Temperature controller:
▪differential thermal power is supplied to the heaters to maintain the
temperature of the sample and reference at the program value
 Outline
 Brief history of thermal analysis

 Theory of thermal analysis techniques

– Thermal Gravimetric Analysis (TGA)
– Differential Scanning Calorimetry (DSC)

■ Generating valid data

– Calibration
– Sample preparation

■ Interpreting data and Applications

– Real events
– Artefacts

■ Recent advances

EYP 2006
 DSC Calibration

Baseline
Calibration

▪ evaluation of the
thermal resistance of
the sample and
reference sensors

▪ measurements over
the temperature
range of
interest
 Indium (calibration standard, purity > 99.999 %)
Conditions
Measuring cell: DSC821e
Pan: Alumina 40 µl, with pierced lid
Sample preparation: Indium pellet, pressed flat , premelted
DSC measurement: Heating from 120 °C to 180 °C at 10 K/min
Atmosphere: Nitrogen, 50 cm3/min

Evaluation
The onset temperature and the heat of fusion of indium are
determined. The fully automated evaluation performs a
validation which compares the measured values with
literature values. If, as in this case, the values lie within the
allowed limits then the message ‘The DSC module is within
specifications’ is displayed.

Measu Ref. Tolera

red value nce

Meltin
+/- 0.3
g point 156.75 156.60
°C
(onset)

Heatr
+/- 0.6
of 28.42 28.45
J/g
fusion
21
 DSC Calibration
▪ Temperature
• match melting onset temperatures to the known melting points
of standards analyzed by DSC
• should be calibrated as close to desired temperature range as
possible
▪ Heat flow
• use calibration standards of known heat capacity, slow accurate
heating rates (0.5–2.0 °C/min), and similar sample and
reference pan weights metals
• Indium 156.6 °C; 28.45 J/g
calibrants • Zinc 419.47C, 108.17 J/g
• high purity inorganics
• accurately known enthalpies • KNO3 128.7 °C
• thermally stable • KClO4 299.4 °C
• light stable organics
• not hygroscopic • polystyrene 105 °C
• do not react (pan, atmosphere) • benzoic acid 122.3 °C; 147.3 J/g
EYP 2006
 Sample Preparation
▪ accurately-weighed samples (~3-20 mg, usually powders)
3-5 mg for simple

▪ small sample pans (0.1 mL) of inert or treated metals (Al, Pt, stainless)
▪ several pan configurations, e.g., open , pinhole, or hermetically-sealed pans
▪ same material and configuration should be used for the sample and the
reference
▪ material should completely cover the bottom of the pan to ensure good
thermal contact
▪ avoid overfilling the pan to minimize thermal lag
from the bulk of the material to the sensor

* small sample masses and low heating rates

increase resolution, but but

resolution, at
the expense of
expense of Pt alumina
Al Ni Cu quartz
sensitivity
 Purge Gases
■ Sample may react with air - oxidising or burning
■ Control moisture content of atmosphere
■ Use inert gas e.g. nitrogen or argon
■ Flowing purge gas
■ In some cases deliberately choose reactive gas, e.g.
– hydrogen to reduce an oxide to metal
– carbon dioxide which affects decomposition of metal carbonate

■ Removes waste products from sublimation or

decomposition
 Outline
 Brief history of thermal analysis

 Theory of thermal analysis techniques

– Thermal Gravimetric Analysis (TGA)
– Differential Scanning Calorimetry (DSC)

 Generating valid data

– Calibration
– Sample preparation

■ Interpreting data and Applications

– Real events
– Artefacts

■ Recent advances

EYP 2006
 Typical Features of a DSC Trace
(Polymorphic System)
• endothermic events
Exo • melting sublimation
• solid-solid
transitions desolvation
• chemical reactions

• exothermic events
• crystallization solid-
solid transitions
sulphapyridine • decomposition
chemical reactions

• baseline shifts
• glass transition

EYP 2006
 Melting Processes by
DSC
Pure substances Impure
substances
• linear melting curve
• Broad,
• melting point eutectic
melt asymmetric
defined by onset melting peak
temperature
• melting
characterized at
Melting with
peak maxima
decomposition
• eutectic impurities
• exothermic
may produce a
second peak
• endothermic

EYP 2006
 Definition of Transition Temperature
0.5

156.50°C
28.87J/g
0.0
Exo
extrapolated
-0.5
onset temperature
Heat Flow (W/g)

-1.0

-1.5

peak melting
-2.0
temperature
157.81°C

-2.5
140 145 150 155 160 165 170 175
Exo Up Temperature (°C) Universal V3.3B TA Instruments

EYP 2006
 Enthalpy of Fusion
0.5

156.50°C
28.87J/g
0.0
Exo

-0.5
Heat Flow (W/g)

-1.0

-1.5

-2.0

157.81°C

-2.5
140 145 150 155 160 165 170 175
Exo Up Temperature (°C) Universal V3.3B TA Instruments

EYP 2006
 Enthalpy of Fusion by DSC
For a single (well-defined)
melting endotherm
▪area under peak
▪minimal
decomposition/sublimation
▪readily measured for high
melting polymorph
▪can be measured for low
melting polymorph Endo

More difficult where multiple thermal events leading to stable melt

▪ e.g. solid-solid transitions (A to B) before melt, or where melt /
recrystallisation before melt
▪ Estimate from sum all areas

EYP 2006
 Purity by
▪ 1-3 mg samples in hermetically- DSC
sealed pans are recommended
▪ Peak width a valuable measure • of purity: Exo
▪ impurities lower the
97%
melting point
▪ Less pure99%(non-perfect)
crystals melt first followed
by purer
benzoic acid larger
99.9% crystals

▪ polymorphism interferes with

purity determination, especially
when a transition occurs in the
middle of the melting peak
▪ Accurate measurement of Hf
Plato, C.; Glasgow, Jr., A.R. Anal. Chem., needs pure samples of polymorphs
1969, 41(2), 330-336. EYP 2006
Glass Transitions
(no heat absorbed or

Exo

EYP 2006
 Effect of Heating Rate
▪ many transitions (evaporation,
crystallization, decomposition, etc.) are
kinetic so shift to higher temp. when
heated at a higher rate
▪ increasing the scanning rate increases
sensitivity, while decreasing the
scanning rate increases resolution
▪ to obtain thermal event temperatures
close to the true thermodynamic value,
slow scanning rates (e.g., 1–5 K/min)
should be used
▪ Rapid scanning can obscure thermal
events
▪ Advantageous in fast
scan DSC,
e.g. 500K/min
EYP 2006
 Artifacts in DSC
• Artifacts are effects that are not caused by the
sample under investigation

34
 Recognizing Artefacts
sample mechanical
Sample Pan cool air entry
pan shock / knock
movement in moves in into cell
distortion bench
pan furnace

sensor
contamination

atmosphere Closing /
electrical effects, changes opening pan
power spikes, etc. hole, e.g.
burst of
pan lid sublimation
EYP 2006
• An abrupt change of the heat transfer between the sample and
pan
• Samples of irregular form can topple over in the pan
• Polymer films that have not been pressed against the base of the
pan first change shape on initial warming. Afterward, on melting,
they make good contact with the pan
• B1
• An abrupt change of the heat transfer between the pan and the
DSC sensor
• Distortion of a hermetically sealed Al pan due to vapor pressure
of the sample
• Slight shift of the Al pan during a dynamic temperature
• The measuring cell suffers a mechanical shock

36
• The entry of cool air into the measuring cell due to a poorly
adjusted measuring cell lid leads to temperature fluctuations
which cause a very noisy signal
• Discharhge of static electricity in a metallic part of the system
• A sudden change of room temperature
• The lid of the pan bursts as result of increasing vapor pressure of
the sample
• Intermittent closing of the hole in the lid of the pan due to
droplets that condense
• Contamination of the sensors caused by residues of a sample from
previous experiments

37
 Ensuring correct interpretation of DSC

■ You can’t
■ Can minimise misinterpretation
■ Essential to have valid data to interpret
– Calibration, reproducible data, appropriate sampling etc

■ Kinetics / thermodynamics at elevated temps

– High temp can speed kinetics – event would happen at
room temperature but slowly
– Effect activated by increased temp (overcome activation
energy)
- event would not happen at room temperature
■ DSC shows excipients interact at 120ºC
– Does not necessarily show interaction at room temp
EYP 2006
 Polymorph Screening and
––––– Form
––––– Form
Indentification
FormI I Form II
Form
FormII III
Form II Dihydrate
1.0

––––– FormIII
Variable
Acetic acidHydrate
solvate 0.5

––––– Dihydrate
––––– Acetic acid solvate
0.0

Heat Flow (W/g)

-0.5
■ thermal stability
– melting -1.0

– crystallization -1.5

– solid-state transformations
––––––– Form
Form I
––––––– Form
Form II
––––––– Form III Hydrate
Variable
––––––– Dihydrate
-2.0

– desolvation
––––––– Acetic acid solvate

– glass transition -2.5

50 100 150 200 250 300 350

Exo
0
ExExoo Temperature (°C)

– sublimation
UUpp

– decomposition ■ mixture analysis

■ heat flow – physical purity (crystal
forms, crystallinity)
– heat of fusion
– chemical purity
– heat of transition
– heat capacity ■ phase diagrams / interactions

EYP 2006
 Effect of Phase
Impurities
▪ lots A & B of polymorph (identical by XRD) are different by DSC:

2046742
Lot A - pure
FILE# 022511DSC.1

-1
Heat Flow (W/g)

-2

2046742
FILE# 022458 DSC.1 Form II ?
-3

Lot B - seeds
-4

-5
80 130 180 230 280
Exo Up Temperature (°C) Universal V3.3B TA Instruments

▪ Lot A: pure low melting polymorph – melting observed

▪ Lot B: seeds of high melting polymorph induce solid-state transition
below the melting temperature of the low melting polymorph

EYP 2006
•

THANKS

41
 Outline
■ Brief history of thermal analysis

■ Theory of thermal analysis techniques

– Thermal Gravimetric Analysis (TGA)
– Differential Scanning Calorimetry (DSC)

■ Generating valid data

– Calibration
– Sample preparation

■ Interpreting data and Applications

– Real events
– Artefacts

■ Recent advances

EYP 2006
 Microcalorimetry
■ High sensitivity DSC
■ Solutions
■ Scan range typically 0-
120 C
■ Scanning rate of 0-120
C/hr
■ Reverse scan rate 0-45
C/hr
(depending on efficiency of
cooling tank)
■ Useful for looking at low
energy modifications
■ e.g. protein relaxation and trehlose
refolding, polymer
characterisation
EYP 2006
 Modulated DSC
(MDSC)
▪ introduced in 1993; “heat flux” Modulated DSC Heating Profile
design
▪ sinusoidal (or square-wave or
sawtooth) modulation is
superimposed on the underlying heating ramp
▪total heat flow signal contains all of the thermal transitions of
standard DSC

▪Fourier Transformation analysis is used to separate the total heat

flow into its two components: reversing and non-reversing heat flow
▪increased sensitivity, resolution and the ability to separate multiple
thermal events

EYP 2006
 MDSC for Polymorph
Characterization
Heat capacity Kinetic
(reversing heat flow) (non-reversing heat flow)
glass transition crystallization
melting decomposition
evaporation

0.00 0.2
Reversing (heat flowcomponent) Non-reversing (heat flowcomponent)

-0.05 reversing heat flow non-reversing heat flow

-0.10 0.0

-0.15
Lot A Lot A

Nonrev Heat Flow (W/g)

Rev Heat Flow (W/g)

-0.20 -0.2

-0.25

Lot B Lot B
-0.30
-0.4

-0.35

-0.40
-0.6

-0.45 DSC010622b.1 483518 HCL (POLYMORPH 1) DSC010622b.1 483518 HCL (POLYMORPH1)

DSC010622d.1 483518 HCL DSC010622d.1 483518 HCL

-0.50
110 -0.8
120 130 140 150 160 170 180 120 130 140 150 160 170 180
110
Exo Up Universal V3.3B TA
Temperature (°C) Exo Up Temperature (°C) Universal V3.3B TA

EYP 2006
 ‘Hyper’ DSC
■ Fast scanning DSC
■ Only possible with power compensated
■ Normal equipment ≈ 100 ºC/min
■ Specialised up to 500 ºC/min
■ Increased sensitivity, loss of resolution
■ e.g. amorphous content in mainly crystalline sample
– change of specific heat at Tg is linear relationship to the
amorphous content
– Conventional DSC 10% amorphous limit of detection
– Hyper DSC <1% amorphous easily detected

Lappalainen, M., I. Pitkanen, et al. (2006). International Journal of

Pharmaceutics 307(2): 150-155.

EYP 2006
 Best Practices for Thermal
Analysis
▪ proper instrument calibration
▪ use purge gas (N2 or He) to remove corrosive off-gases
• temperature-sensing device
▪ small sample size
▪ proper sample encapsulation
▪ good thermal contact between the sample and the
▪ start temperature well below expected
transition temperature
▪ slow scanning speeds
• (Unless aiming to obscure thermal transitions, e.g fast scan DSC)

▪ avoid decomposition in the DSC

• (Run TGA first – its easier to clean up!)

EYP 2006
 Caution
■ It is a bulk tool …
– Analysing the gross average of events in a sample
– Conversely, small powder sample in DSC may not
represent packing of powder bulk in
decomposition studies

■ Instrument error in DSC typically ± 0.5 - 1ºC

■ In Scanning modes, thermal events may be

“smeared” by a thermal lag
– Sample temperature not keeping up with
instrument
– Bigger effect at higher heating rates
– Typically 1ºC at 10ºC/min

EYP 2006
 And more
caution!
■ Thermal analysis tells you what is happening at
the temperature it happens at!
– Care when extrapolating to room temperature
stability / interaction

■ Don’t over-interpret data

■ Care when using thermal analysis in isolation

▪ Artefacts / heating rate effects etc
▪ Couple with other analytical tools
– Heated X-ray, heated vibrational spectroscopy, hot
stage microscope

EYP 2006
 Acknowledgements

Professor Adrian Williams, University of Reading

Dr Ian Grimsey, University of Bradford
Dr Peter Timmins, Bristol Myers Squibb

Dr Wendy Hulse, University of Bradford Luciana

DeMatos, University of Sheffield

EYP 2006
Questions

EYP 2006
 Reversing and Non-
Reversing Contributions
to Total DSC Heat Flow
total heat flow dQ/dt = Cp . dT/dt + f(t,T)
resulting from
average heating
rate
reversing signal non-reversing
heat flow resulting from signal
sinusoidal temperature (kinetic
modulation component)
(heat capacity
component)

e.g. see Pharmaceutical Research: 17 (6): 696-700, June 2000 Craig, DQM et al.

EYP 2006
 Some Common Thermal Analysis Techniques
There are various techniques in which a physical property is measured as a
function of temperature, while the sample is subjected to a predefined
heating or cooling program.

Differential Thermal Analysis (DTA)

•the temperature difference between a sample and an inert reference material, T = TS -
TR, is measured as both are subjected to identical heat treatments

Differential Scanning Calorimetry (DSC)

•the sample and reference are maintained at the same temperature, even during a thermal
event (in the sample)

•the energy required to maintain zero temperature differential between the sample and the
reference, dq/dt, is measured

Isothermal titration calorimetry (ITC)

•The temperature of a “reaction” is kept constant whilst the energy change is measured

Thermogravimetric Analysis (TGA)

•the change in mass of a sample on heating is measured

EYP 2006
 Thermogravimetric Analysis (TGA)
• thermobalance to monitor
sample weight as a function
of temperature

• weight calibration using

known weights

• temperature calibration
b ased on ferromagnetic
120

transition of Curie point

standards (e.g., Ni)
100
12.15%

80 19.32%
Weight (%)

• larger sample masses, lower

60

temperature gradients, and 29.99%

h igher purge rates

40

minimize undesirable
20
0 20 40 60 80 100 120 140 160

effects buoyancy Time (min) Universal V3.7A TA Instruments

EYP 2006
 Differential Thermal Analysis
Sample holder: Sample and reference cells

Sensors: Thermocouples, one for the sample

and one for the reference

Furnace: Block containing sample and

reference cells

Temperature controller: Controls temperature

program

Advantages:
Disadvantages:
■instruments can be used at very high
temperatures ■uncertainty of heats of
■instruments are highly sensitive
■flexibility in sample volume/form
fusion and transition
■characteristic transition or reaction temperatures
temperatures can be determined

EYP 2006
 “Hyphenated”
Techniques
• thermal techniques alone are insufficient to prove the existence of polymorphs
and solvates
• other techniques should be used, e.g., microscopy, diffraction, and spectroscopy

• development of “hyphenated” techniques for simultaneous analysis

TG-DTA 120
4.2
15.55%
(0.9513mg)

TG-DSC 24.80°C
100.0%
3.2

80

Temperature Difference (µV/mg)

179.95°C
84.45%
2.2
TG-FTIR
Weight (%)

40 1.2

TG-MS
0.2

-0.8

-40 -1.8
20 70 120 170 220 270
Exo Up Temperature (°C) Universal V3.3B TA Instruments

TG-DTA trace of sodium tartrate

EYP 2006
 Overview
• Certain pharmaceutical products must be
sterile
– injections, ophthalmic preparations,
irrigations solutions, haemodialysis solutions

• Two categories of sterile products

– those that can be sterilized in final container
(terminally sterilized)
– those that cannot be terminally sterilized and
must be aseptically prepared
57
 Overview
Aseptic processing
• Objective is to maintain the sterility of a product,
assembled from sterile components
• Operating conditions so as to prevent microbial
contamination

58
 Overview
Objective
• To review specific issues relating to the manufacture of
aseptically prepared products:
– Manufacturing environment
• Clean areas
• Personnel
– Preparation and filtration of solutions
– Pre-filtration bioburden
– Filter integrity/validation
– Equipment/container preparation and sterilization
– Filling Process
– Validation of aseptic processes
59
– Specific issues relating to Isolators, BFS and Bulk
 Manufacturing Enviornment
Classification of Clean Areas
– Comparison of classifications

WHO GMP US 209E US Customary ISO/TC (209) EEC GMP

ISO 14644
Grade A M 3.5 Class 100 ISO 5 Grade A
Grade B M 3.5 Class 100 ISO 5 Grade B
Grade C M 5.5 Class 10 000 ISO 7 Grade C
Grade D M 6.5 Class 100 000 ISO 8 Grade D

60
 Manufacturing Enviornment
Classification of Clean Areas
– Classified in terms of airborne particles
Grade At rest In operation

maximum permitted number of particles/m3

0.5 - 5.0 µm > 5 µm 0.5 - 5.0 µm >5µ
A 3 500 0 3 500 0
B 3 500 0 350 000 2 000
C 350 000 2 000 3 500 000 20 000
D 3 500 000 20 000 not defined not defined

“At rest” - production equipment installed and operating

“In operation” - Installed equipment functioning in defined
operating mode and specified number of personnel present
61
Manufacturing Enviornment
Four grades of clean areas:
• Grade D (equivalent to Class 100,000, ISO 8):
– Clean area for carrying out less critical stages in
manufacture of aseptically prepared products eg. handling
of components after washing.
• Grade C (equivalent to Class 10,000, ISO 7):
– Clean area for carrying out less critical stages in
manufacture of aseptically prepared products eg.
preparation of solutions to be filtered.
• Grade B (equivalent to Class 100, ISO 5):
– Background environment for Grade A zone, eg. cleanroom
in which laminar flow workstation is housed.

62
 Manufacturing Enviornment
• Grade A (equivalent to Class 100 (US Federal Standard
209E), ISO 5 (ISO 14644-1):
– Local zone for high risk operations eg. product filling, stopper
bowls, open vials, handling sterile materials, aseptic
connections, transfer of partially stoppered containers to be
lyophilized.
– Conditions usually provided by laminar air flow workstation.
• Each grade of cleanroom has specifications for viable
and non-viable particles
– Non-viable particles are defined by the air classification (See
Table 2)

63
 Manufacturing Enviornment
• Limits for viable particles (microbiological
contamination)
Grade Air sample Settle plates (90mm Contact plates Glove print
(CFU/m3) diameter) (55mm (5 fingers)
(CFU/4hours) diameter) (CFU/glove)
(CFU/plate)
A <3 <3 <3 <3
B 10 5 5 5
C 100 50 25 -
D 200 100 50 -

– These are average values

– Individual settle plates may be exposed for less than 4 hours
• Values are for guidance only - not intended to represent specifications
• Levels (limits) of detection of microbiological contamination should be
established for alert and action purposes and for monitoring trends of air
quality in the facility
64
Manufacturing Enviornment
Environmental Monitoring
• Physical
– Particulate matter
– Differential pressures
– Air changes, airflow patterns
– Clean up time/recovery
– Temperature and relative humidity
– Airflow velocity

65
Manufacturing Enviornment
Environmental Monitoring - Physical
• Particulate matter
– Particles significant because they can contaminate and also
carry organisms
– Critical environment should be measured not more than 30cm
from worksite, within airflow and during filling/closing
operations
– Preferably a remote probe that monitors continuously
– Difficulties when process itself generates particles (e.g.
powder filling)
– Appropriate alert and action limits should be set and
corrective actions defined if limits exceeded

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Manufacturing Enviornment
Environmental Monitoring - Physical
• Differential pressures
– Positive pressure differential of 10-15 Pascals should be
maintained between adjacent rooms of different classification
(with door closed)
– Most critical area should have the highest pressure
– Pressures should be continuously monitored and frequently
recorded.
– Alarms should sound if pressures deviate
– Any deviations should be investigated and effect on
environmental quality determined

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 Manufacturing Enviornment
Environmental Monitoring - Physical
• Air Changes/Airflow patterns
– Air flow over critical areas should be uni-directional (laminar
flow) at a velocity sufficient to sweep particles away from
filling/closing area
– for B, C and D rooms at least 20 changes per hour are ususally
required
• Clean up time/recovery
– Particulate levels for the Grade A “at rest” state should be
achieved after a short “clean-up” period of 20 minutes after
completion of operations (guidance value)
– Particle counts for Grade A “in operation” state should be
maintained whenever product or open container is exposed
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Manufacturing Enviornment
Environmental Monitoring - Physical
• Temperature and Relative Humidity
– Ambient temperature and humidity should not be
uncomfortably high (could cause operators to generate
particles) (18°C)
• Airflow velocity
– Laminar airflow workstation air speed of approx 0.45m/s
± 20% at working position (guidance value)

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Manufacturing Enviornment
Personnel
• Minimum number of personnel in clean areas
– especially during aseptic processing
• Inspections and controls from outside
• Training to all including cleaning and maintenance
staff
– initial and regular
– manufacturing, hygiene, microbiology
– should be formally validated and authorized to enter
aseptic area
• Special cases
– supervision in case of outside staff
– decontamination procedures (e.g. staff who worked with 70
animal tissue materials)
Manufacturing Enviornment
Personnel (2)
• High standards of hygiene and cleanliness
– should not enter clean rooms if ill or with open wounds
• Periodic health checks
• No shedding of particles, movement slow and controlled
• No introduction of microbiological hazards
• No outdoor clothing brought into clean areas, should be
clad in factory clothing
• Changing and washing procedure
• No watches, jewellery and cosmetics
• Eye checks if involved in visual inspection
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Manufacturing Enviornment
Personnel (3)
• Clothing of appropriate quality:
– Grade D
• hair, beard, moustache covered
• protective clothing and shoes
– Grade C
• hair, beard, moustache covered
• single or 2-piece suit (covering wrists, high
neck), shoes/overshoes
• no fibres/particles to be shed
– Grade A and B
• headgear, beard and moustache covered,
masks, gloves
• not shedding fibres, and retain particles shed 72
Manufacturing Enviornment
Personnel (4)
• Outdoor clothing not in change rooms leading to Grade B
and C rooms
• Change at every working session, or once a day (if
supportive data)
• Change gloves and masks at every working session
• Frequent disinfection of gloves during operations
• Washing of garments – separate laundry facility
– No damage, and according to validated procedures
(washing and sterilization)
• Regular microbiological monitoring of operators

73
 Aseptic Processing
• In aseptic processing, each component is individually
sterilised, or several components are combined with the
resulting mixture sterilized.
– Most common is preparation of a solution which is filtered
through a sterilizing filter then filled into sterile containers (e.g
active and excipients dissolved in Water for Injection)
– May involve aseptic compounding of previously sterilized
components which is filled into sterile containers
– May involve filling of previously sterilized powder
• sterilized by dry heat/irradiation
• produced from a sterile filtered solution which is then aseptically
crystallized and precipitated
– requires more handling and manipulation with higher potential for
contamination during processing
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 Aseptic Processing
Preparation and Filtration of Solutions
• Solutions to be sterile filtered prepared in a Grade C
environment
• If not to be filtered, preparation should be prepared in a
Grade A environment with Grade B background (e.g.
ointments, creams, suspensions and emulsions)
• Prepared solutions filtered through a sterile 0.22μm (or
less) membrane filter into a previously sterilized container
– filters remove bacteria and moulds
– do not remove all viruses or mycoplasmas
• filtration should be carried out under positive pressure

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 Aseptic Processing
Preparation and Filtration of Solutions (2)
• consideration should be given to complementing filtration
process with some form of heat treatment
• Double filter or second filter at point of fill advisable
• Filters should not shed particles, asbestos containing filters
should not be used
• Same filter should not be used for more than one day unless
validated
• If bulk product is stored in sealed vessels, pressure release
outlets should have hydrophobic microbial retentive air
filters

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 Aseptic Processing
Preparation and Filtration of Solutions (3)
• Time limits should be established for each phase of
processing, e.g.
– maximum period between start of bulk product
compounding and sterilization (filtration)
– maximum permitted holding time of bulk if held after
filtration prior to filling
– product exposure on processing line
– storage of sterilized containers/components
– total time for product filtration to prevent organisms from
penetrating filter
– maximum time for upstream filters used for clarification or
particle removal (can support microbial attachment)
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 Aseptic Processing
Preparation and Filtration of Solutions (4)
• Filling of solution may be followed by lyophilization (freeze
drying)
– stoppers partially seated, product transferred to lyophilizer
(Grade A/B conditions)
– Release of air/nitrogen into lyophilizer chamber at completion
of process should be through sterilizing filter

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 Aseptic Processing
Prefiltration Bioburden (natural microbial load)
• Limits should be stated and testing should be carried out on
each batch
• Frequency may be reduced after satisfactory history is
established
– and biobuden testing performed on components
• Should include action and alert limits (usually differ by a
factor of 10) and action taken if limits are exceeded
• Limits should reasonably reflect bioburden routinely
achieved

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 Aseptic Processing
Prefiltation Bioburden (2)
• No defined “maximum” limit but the limit should not
exceed the validated retention capability of the filter
• Bioburden controls should also be included in “in-process”
controls
– particularly when product supports microbial growth and/or
manufacturing process involves use of culture media
• Excessive bioburden can have adverse effect on the quality
of the product and cause excessive levels of
endotoxins/pyrogens

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 Aseptic Processing
Filter integrity
• Filters of 0.22μm or less should be used for filtration of
liquids and gasses (if applicable)
– filters for gasses that may be used for purging or overlaying of
filled containers or to release vacuum in lyphilization chamber
• filter intergrity shoud be verified before filtration and
confirmed after filtration
– bubble point
– pressure hold
– forward flow
• methods are defined by filter manufacturers and limits
determined during filter validation
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 Aseptic Processing
Filter Validaton
• Filter must be validated to demonstrate ability to
remove bacteria
– most common method is to show that filter can retain a
microbiological challenge of 107 CFU of Brevundimonas
diminuta per cm2 of the filter surface
– a bioburden isolate may be more appropriate for filter
retention studies than Brevundimonas diminuta
– Challenge concentration is intended to provide a margin of
safety well beyond what would be expected in production
– preferably the microbial challenge is added to the fully
formulated product which is then passed through the filter

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 Aseptic Processing
Filter validation (2)
– if the product is bactericidal, product should be passed through
the filter first followed by modified product containing the
microbial challenge (after removing any bactericidal activity
remaining on the filter)
– filter validation should be carried out under worst case
conditions e.g. maximum allowed filtration time and maximum
pressure
– integrity testing specification for routine filtration should
correlate with that identified during filter validation

83
 Aseptic Processing
Equipment/container preparation and
sterilization
• All equipment (including lyophilizers) and product
containers/closures should be sterilized using validated
cycles
– same requirements apply for equipment sterilization that
apply to terminally sterilized product
– particular attention to stoppers - should not be tightly packed
as may clump together and affect air removal during vacuum
stage of sterilization process
– equipment wrapped and loaded to facilitate air removal
– particular attention to filters, housings and tubing
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 Aseptic Processing
Equipment/container preparation and
sterilization (2)
• CIP/SIP processes
– particular attention to deadlegs - different orientation
requirements for CIP and SIP
• heat tunnels often used for sterilization/depyrogenation
of glass vials/bottles
– usually high temperature for short period of time
– need to consider speed of conveyor
– validation of depyrogenation (3 logs endotoxin units)
• worst case locations
– tunnel supplied with HEPA filtered air
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 Aseptic Processing
Equipment/container preparation and
sterilization (2)
• equipment should be designed to be easily assembled and
disassembled, cleaned, sanitised and/or sterilized
– equipment should be appropriately cleaned - O-rings and gaskets
should be removed to prevent build up of dirt or residues
• rinse water should be WFI grade
• equipment should be left dry unless sterilized immediately after
cleaning (to prevent build up of pyrogens)
• washing of glass containers and rubber stoppers should be
validated for endotoxin removal
• should be defined storage period between sterilization and use
(period should be justified)
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 Aseptic Processing
Process Validation
• Not possible to define a sterility assurance level for
aseptic processing
• Process is validated by simulating the manufacturing
process using microbiological growth medium (media
fill)
– Process simulation includes formulation (compounding),
filtration and filling with suitable media using the same
processes involved in manufacture of the product
– modifications must be made for different dosage formats e.g.
lyophilized products, ointments, sterile bulks, eye drops filled
into semi-transparent/opaque containers, biological products

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 Aseptic Processing
Process Validation (2)
• Media fill program should include worst case
activities
– Factors associated with longest permitted run (e.g.
operator fatigue)
– Representative number, type, and complexity of
normal interventions, non-routine interventions and
events (e.g. maintenance, stoppages, etc)
– Lyophilisation
– Aseptic equipment assembly

88
 Aseptic Processing
Process Validation (3)
• Worst case activities (cont)
– No of personnel and their activities, shift changes,
breaks, gown changes
– Representative number of aseptic additions (e.g.
charging containers, closures, sterile ingredients) or
transfers
– Aseptic equipment connections/disconnections
– Aseptic sample collections
– Line speed and configuration

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 Aseptic Processing
Process Validation (4)
• Worst case activities (cont)
– Weight checks
– Container closure systems
– Specific provisions in processing instructions
• Written batch record documenting conditions and
activities
• Should not be used to justify risky practices

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 Aseptic Processing
Process Validation (5)
Duration
– Depends on type of operation
– BFS, Isolator processes - sufficient time to include
manipulations and interventions
– For conventional operations should include the total filling
time
Size
– 5000 - 10000 generally acceptable or batch size if <5000
– For manually intensive processes larger numbers should
be filled
– Lower numbers can be filled for isolators
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 Aseptic Processing
Process Validation (6)
• Frequency and Number
– Three initial, consecutive per shift
– Subsequently semi-annual per shift and process
– All personnel should participate at least annually,
consistent with routine duties
– Changes should be assessed and revalidation carried out
as required
• Line Speed
– Speed depends on type of process

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 Aseptic Processing
Process Validation (7)
• Environmental conditions
– Representative of actual production conditions (no. of
personnel, activity levels etc) - no special precautions (not
including adjustment of HVAC)
– if nitrogen used for overlaying/purging need to substitute
with air
• Media
– Anaerobic media should be considered under certain
circumstances
– Should be tested for growth promoting properties (including
factory isolates)

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 Aseptic Processing
Process Validation (8)
• Incubation, Examination
– In the range 20-35ºC.
– If two temperatures are used, lower temperature first
– Inspection by qualified personnel.
– All integral units should be incubated. Should be
justification for any units not incubated.
– Units removed (and not incubated) should be consistent
with routine practices (although incubation would give
information regarding risk of intervention)
– Batch reconciliation
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 Aseptic Processing
Process Validation (9)
• Interpretation of Results
– When filling fewer than 5000 units:
• no contaminated units should be detected
• One (1) contaminated unit is considered cause for revalidation,
following an investigation
– When filling from 5000-10000 units
• One (1) contaminated unit should result in an investigation,
including consideration of a repeat media fill
• Two (2) contaminated units are considered cause for revalidation,
following investigation
– When filling more than 10000 units
• One (1) contaminated unit should result in an investigation
• Two (2) contaminated units are considered cause for revalidation,
95
following investigation
 Aseptic Processing
Process Validation (10)
• Interpretation of Results
– Media fills should be observed by QC and contaminated
units reconcilable with time and activity being simulated
(Video may help)
– Ideally - no contamination. Any contamination should be
investigated.
– Any organisms isolated should be identified to species
level (genotypic identification)
– Invalidation of a media fill run should be rare

96
 Aseptic Processing
Process Validation (11)
• Batch Record Review
– Process and environmental control activities should be
included in batch records and reviewed as part of batch
release
 In-process and laboratory control results
 Environmental and personnel monitoring data
 Output from support systems(HEPA/HVAC, WFI, steam generator)
 Equipment function (batch alarm reports, filter integrity)
 Interventions, Deviations, Stoppages - duration and associated
time
 Written instructions regarding need for line clearances
 Disruptions to power supply

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 Aseptic Processing
Additional issues specific to Isolator and BFS
Technologies
• Isolators
– Decontamination process requires a 4-6 log reduction of
appropriate Biological Indicator (BI)
– Minimum 6 log reduction of BI if surface is to be free of
viable organisms
– Significant focus on glove integrity - daily checks, second
pair of gloves inside isolator glove
– Traditional aseptic vigilance should be maintained

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 Aseptic Processing
• Blow-Fill-Seal (BFS)
– Located in a Grade D environment
– Critial zone should meet Grade A (microbiological)
requirements (particle count requirements may be
difficult to meet in operation)
– Operators meet Grade C garment requirements
– Validation of extrusion process should demonstrate
destruction of endotoxin and spore challenges in the
polymeric material
– Final inspection should be capable of detecting leakers

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 Aseptic Processing
• Issues relating to Aseptic Bulk Processing
• Applies to products which can not be filtered at point of fill
and require aseptic processing throughout entire
manufacturing process.
• Entire aseptic process should be subject to process simulation
studies under worst case conditions (maximum duration of
"open" operations, maximum no of operators)
• Process simulations should incorporate storage and transport
of bulk.
• Multiple uses of the same bulk with storage in between should
also be included in process simulations
• Assurance of bulk vessel integrity for specified holding times.

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 Aseptic Processing
• Bulk Processing (2)
• Process simulation for formulation stage should be
performed at least twice per year.
– Cellular therapies, cell derived products etc
• products released before results of sterility
tests known (also TPNs, radioactive preps,
cytotoxics)
• should be manufactured in a closed system
• Additional testing
– sterility testing of intermediates
– microscopic examination (e.g. gram stain)
– endotoxin testing
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 Useful Publications
• PIC/S Recommendation on the Validation of Aseptic Processes
• FDA Guidance for Industry- Sterile Drug Products Produced by
Aseptic Processing - Current Good Manufacturing Process
• ISO 13408 Aseptic Processing of Health Care Products
– Part 1: General Requirements
– Part 2: Filtration
– Part 3: Lyophilization
– Part 4: Clean-In-Place Technologies
– Part 5: Sterilization-In-Place
– Part 6: Isolator Systems

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Thank You