Accelerating

Vaccine Production
Using a
Nonviral
Enabling Technology
for Cell
Engineering
By Victor Ayala, PhD
ABL, Inc.
Sponsored Report J une 2018 16(6)i BioProcess International 1
Accelerating Vaccine
Production Using a
Nonviral Enabling Technology
for Cell Engineering
Victor Ayala

At the recent World Vaccine Conference, Victor Ayala,

PhD, an early stage investigator with Advanced
BioScience Laboratories, Inc. (ABL), discussed how to
accelerate vaccine production using a nonviral enabling
technology for cell engineering. ABL is a contract
research/manufacturing organization (CRO/CMO)
providing manufacturing and laboratory research
services to advance leading vaccines and therapies from yielding. One major advantage to using this
clinical development to the commercial market. The approach over generating a cell line is that it
company has been conducting R&D research for over elminiates the need for generating a master cell
55 years and performing CMO manufacturing for bank (MCB) every time you want to manufacture a
over 25 years. With more than 200 employees drug molecule. Shortcomings of using transcient
worldwide, ABL has sites in Rockville, MD, as well transfection include a finite production of proteins,
as Strasbourg and Lyon, France. Services include inefficiencies with Chinese hamster ovary (CHO)
preclinical and clinical immunobiology services, cells (the cell line preferred by industry for
product development, and biomanufacturing. The antibodies and recombinant glycoproteins such as
following are highlights of Dr. Ayala’s presentation. HIV envelopes), and concerns about product
variability.

W
The MaxCyte flow electroporation technology
hen it comes to protein expression, has been a breakthrough technology for ABL in
manufacturers have a choice among a many areas of research. We have been working
range of technologies for with MaxCyte for over five years and currently
implementation. However, such systems use the STX and VLX systems in process
all fall into one of two basic categories: stable cell development (PD) and manufacturing. ABL
lines or transient transfection. Industry and recently purchased an additional STX unit for
regulatory agencies prefer stable cell lines because R&D work to bridge the gap between product
they provide product consistency. They are well- innovation and PD.
characterized systems, and the source of The transfection process of the MaxCyte Flow
production is indefinite. But using stable cell lines Electroporation system begins with expanding
can be expensive and time consuming. Researchers healthy cells to the 3–5 million/mL range.
want to fire off as many candidate biologics as Harvested cells then are concentrated down
possible, especially in early phases of discovery or 40-fold and mixed with MaxCyte’s proprietary
development. If that process is so burdened that it electroporation buffer and the biomolecule of
becomes inhibitory to execute, it slows the interest. The combined material is placed into a
development train and ultimately increases the process assembly unit, which is the cassette used
medical costs associated with preventable diseases. to electroporate the cells aseptically.
An alternative method is to use transient ABL currently uses three process assemblies to
transfection, which can be low-cost and high- produce proteins at different scales: OC-400 (2 ×
Sponsored Report J une 2018 16(6)i BioProcess International 3
Figure 1: Small-scale feasiblity of simian immunodeficiency Figure 2: Comparing large-scale production of SIV envelopes
virus (SIV) envelopes in CHO-S cells in CHO-S cells
100 SIVmac251 gp140
100
SIVsmE660 gp145
Content (mg/L)

Content (mg/L)
10 10

SIVmac251 gp140
SIVsmE660 gp145
1 1
0 1 2 3 4 5 6 7 8 9 10 11 12 Cell MaxCyte Cell MaxCyte
Days Posttransfection Line Line

106 to 4× 107 cells), CL-2 (5 × 108 to 1 × 1010 cells) weeks 18, 38, and 58. It is important to note that
and VLX-D (1 × 1010 to 2 × 1011 cells). The only four animals were used in the original study.
OC-400 is used for very small scales (e.g., 16-mL Despite the small number of animals, study
size cultures). The CL-2 process assembly enables researchers made important observations.
you to scale up to 4 L. And the VLX system can Neutralizing antibodies titers against Tier II viruses
be used to perform 50-L runs. What is most were induced, and there was a delay in acquisition
helpful about this platform is that you can establish in two of the animals. Most exciting was the fact
transfection conditions using the OC-400 assembly, that one of the animals was protected after repeated
and carry them all the way through to intravaginal challenges, while all controls had
manufacturing using the exact same conditions. become infected after two challenges. These pilot
The results are surprisingly highly comparable — results were the rationale for acquiring additional
even between shake flasks and bioreactors funding to perform the subsequent study.
In my case study presentation, I discussed how As soon as funding is approved, the clock starts
the MaxCyte technology is being used to support ticking, and you have to be able to react quickly. A
preclinical vaccine studies in nonhuman primates number of costs are associated with running those
and how similar approaches are being types of studies. Once funding is provided, the
implemented to accelerate vaccine development for expectation is that proteins, DNA, and everything
phase 1 studies. In this study, ABL produced else required to carry out a study can be obtained
immunogens using MaxCyte technology for a within a very short time.
preclinical HIV vaccine study in nonhuman What complicates that work is that some
primates. The goal of the study is to evaluate proteins are not easy to produce because they are
vaccine efficacy against a challenge with the virus, highly glycosylated and can form multiple
SIVmac251. SIVmac251 is a distant relative virus oligomeric species. For the planned study, we
of HIV that causes an AIDS-like disease in rhesus needed to produce two different SIV envelopes:
macaques. This animal model is the best paradigm SIVmac251 gp140 and SIVsmE660 gp145, which
we have for AIDS vaccine research. The planned are the major targets on the surface of the virus.
study is a follow-up to a smaller pilot study Because both proteins were produced using cell
published in 2017 (1) on DNA prime-protein boost lines for the original study, we initially performed a
strategy formulated in Advax (a novel small-scale feasibility study using the MaxCyte
polysaccharide adjuvant). In this study the protein OC-400 process assemblies to evaluate expression
was produced from stable cell lines. To outline the in a transient transfection system. The experiment
initial pilot study further, the DNA primes was set up in duplicate using conditions provided by
consisted of 500 µg injected intramuscularly and MaxCyte. The MaxCyte feeding protocol worked
then electroporated using a BLX system. The better than our standard approach because the
protein boosts were delivered in three doses: one feeding was every four days instead of every two
intranasally with two subsequent intramuscular days. After electroporation, cultures were allowed
boosts. to recover at 37 °C and 3% CO2 with shaking.
DNA primes were delivered at week zero and After 24 hours, incubator temperature was set to 32
week four. Protein boosts were administered at °C with 2% CO2, and valproic acid (1mM) was
4 BioProcess International 16(6)i J une 2018 Sponsored Report
Figure 3: Characterization of SIV envelopes (antibodies Figure 4: Expression of antiviral monoclonal antibody in
provided courtesy of Mario Roederer, PhD) CHO-S cells

SIVmac251 gp140 SIVsmE660 gp145 500

SDS PAGE BN PAGE SDS PAGE BN PAGE
kDa kDa kDa kDa
250 1,236 1,236 400
250

Content (mg/L)
1,048 1,048
150 720 150 720 300
100
75 100
480 75 480
200
50 50
242 242
37 37 146
100
146
66 25 66
25
20 20 0
15 15 Day 4 Day 5 Day 6 Day 7 Day 8 Filtered
αCD4bs αCD4i αV3 αV2 αV1
(ITS01) (ITS51) (ITS52) (IT209.01) (ITS06.02) Days Postelectroporation
1 1 1 1 1
SIVmac251

MaxCyte
gp140

purity for both proteins was relatively high, as

0 360 0 360 0 360 0 360 0 360
Binding Response (nm)

Time (s)
1 1 1 1 1 evaluated with sodium dodecyl sulfate
Cell Line
0 360 0 360 0 360 0 360 0 360
polyacrylamide gel electrophoresis (SDS PAGE),
1 1 1
Time (s)
1 1 and the appropriate oligomeric form (720 kDa) was
MaxCyte isolated as seen in the blue native PAGE (Figure 3).
SIVsmE660

0 360 0 360 0 360 0 360 0 360

gp145

1 1 1
Time (s) 1
1 Antigenically, the proteins look similar to the
0 360 0 360 0 360 0 360 0 360
Cell Line proteins we generated in cell lines, as measured
Time (s) with biolayer interferometry (BLI). The analysis
was performed using broadly neutralizing
antibodies targeting specific epitopes on the
added to the culture. Valproic acid is a histone envelopes. A slight difference was observed in the
deacetylase (HDAC) inhibitor that enhances binding of the CD4-binding site antibodies
protein production in CHO cells. between the transient transfection and cell-
Figure 1 shows the expression levels of the two line‑derived SIVmac251 gp140. However, binding
SIV envelope proteins in culture from day 2 to day of the broadly neutralizing antibodies (bNAbs) to
10. The SIVsmE660 protein always has been a low the SIVsmE660 protein essentially were identical
producer in general because it is difficult to for both expression platforms. That indicated that
express. However, 10–12 mg/L was achieved for the proteins produced through transient
this culture, which plateaued on day 4. Production transfection are comparable with the immunogens
of the SIVmac251 protein was higher at about produced from stable cell lines (Figure 3).
60–70 mg/L, plateauing on day 7. Using the stable cell line development process
Ultimately, we decided to harvest the took ~10 months to generate about 4–5 mg. With
SIVsmE660 culture on day 5 and the SIVmac251 the MaxCyte system, process time was reduced by
culture on day 6. When it was time to scale up, six months, and almost four times as much of each
the CL-2 process assemblies were implemented protein was produced, thereby shortening
using the same feeding strategy, and production time by five to six months. This
electroporation conditions were established with approach already has led to big savings for our
the OC-400 assemblies. As expected, expression clients and collaborators.
was maintained at the larger scale (4 L). Figure 2 ABL also has started producing recombinant
shows the comparison between transient antibodies in the R&D space by transient
transfection and the historic values from cell lines transfection for use as reagents. Having this
used originally for the first pilot study. Using capability expands what we can do in the short
MaxCyte’s technologies, we achieved over a term for a lot of work, especially assay development
25-fold increase for the SIVmac251 protein and an and for ligands for immunoaffinity purification
eightfold increase for the SIVsmE660 protein. methods. Frequently, manufacturers may have
Within two months of starting the project, the permission to use a specific antibody from different
team was working on downstream purification of organizations, but there is a significant delay in
the envelopes. The proteins were purified by lectin acquiring the necessary quantities — especially for
affinity using a GNL column followed by size- more obscure antibodies. Producing them in house
exclusion chromatography to isolate trimers. The is a big advantage.
Sponsored Report J une 2018 16(6)i BioProcess International 5
Figure 5: ABL upstream process (GMP = good manufacturing practice, HDAC = histone deacetylase)
Maxcyte STX Maxcyte STX Maxcyte VLX
Research and
Research andDevelopment
Development Process Development
Process Development Manufacturing
Manufacturing

Gene�c
Genetic constructs
Constructs Media
Media Op�miza�on
Optimization Scale up
Scale-Up Full Scale Run
Full-Scale Run
16-mL Shake Flasks 16-mL Shake Flasks 2-L Bioreactors 50-L Scale

Antigen design, Base media, supplementation Agitation, pH control, Pilot engineering

promoter elements HDAC inhibitors dissolved O2 GMP

Figure 6: ABL downstream process

Maxcyte STX Maxcyte VLX

Research andDevelopment
Research and Development Process
ProcessDevelopment
Development Manufacturing
Manufacturing
Gene�c
Custom constructs
Affinity Ligands Media Op�miza�on
Feasibility Scale up
Scale-Up Full Scale Run
Full-Scale Run
Single
Antibodies Chain Receptor Biolayer Interferometry Affinity Column
Screen-binding partners Transient transfection
Binding conditions (50-L scale) and coupling
Elution conditions to resin (non GMP)

Development Development
Small-scale purification Scaled-up purification
Polishing step Polishing step

Pilot Engineering GMP

One example of the rapid antibody production STX technology to develop custom cellular targets
using MaxCyte is ABL’s production of an antiviral expressing a protein of interest. Thus, we will be
monoclonal antibody in CHO-S cells (Figure 4). able to produce both the antibody of interest and its
DNA was electroporated at a ratio of 1:1, heavy- to target for assay development, whether in soluble
light-chain plasmid DNA. By day 8 we had a titer form or cell bound. Two assays that ABL is
of ~375 mg/L. This experiment will be repeated to developing are the antibody-dependent cellular
determine whether the culture can maintain a high cytotoxicity (ADCC) and antibody-dependent
viability for 14 days. MaxCyte’s data demonstrate cellular phagocytosis (ADCP) assays. They are
that the system can achieve over one-gram intended to evaluate the functionality of antibodies
quantities for antibodies. Purification is a two-step by measuring binding to specific Fc receptors. That
process consisting of protein A and a polishing step. capability will be useful particularly for rapid
The antibody produced was evaluated using BLI to deployment of novel immunoassays needed for
assess potency. The binding affinity to its cognate biodefense and immuno-oncology studies.
target was in the nanomolar range, suggesting that
transient transfection systems can producce How to Accelerate Vaccine Development
functional proteins. A 2018 white paper discusses the many challenges
CMOs are facing for vaccine development (2).
Functional Antibody Assays These challenges include the need to reduce
Another example of how ABL is using MaxCyte’s timelines, shrink resources, increase productivity,
technology is the development of custom functional and simultaneously maintain adherence to
antibody assays. We intend to use the MaxCyte regulatory requirements. It is estimated that
6 BioProcess International 16(6)i J une 2018 Sponsored Report
producing one HIV envelope for phase 1 can cost for purifying HIV envelopes. Some experts think it
$5 million or more in production and can take from might not even be possible given the viral diversity.
three to five years. The goal at ABL is to support However, in light of the recent isolation of a
research by reducing costs and accelerating number of bNAbs, we are exploring just that. ABL
timelines. Because the phase 1 stage is still one of is just one of a few companies trying to assess
discovery, it does not make sense to spend a lot of whether a particular antibody specific for an
money on permanent processes that may or may not oligomeric species of HIV envelope can be used in
work. The ultimate goal is to test as many vaccine the capture step. The goal is to produce an envelope
candidates as possible. To achieve that, a drug for under $2 million with a delivery time of two
product has to be cost effective and produced in a years. This would entail a two-step purification
timely and efficient manner such that the product is process: capture and polishing. We believe the
still relevant to the field when it reaches capture ligand can be produced in the same manner
investigational new drug (IND) submission. The as the immunogen, using MaxCyte’s
MaxCyte transient transfection technology fits well electroporation using a “GMP light” process that is
strategically into both our upstream and robust and cost effective. The ligand can be
downstream processes. customized to target your protein of interest using
On the upstream side, we are saving costs by antibodies, single chains, aptomers, and so on. The
cutting out time-consuming steps and additional goal is to produce 30–40 g at the 50-L scale, which
fees for permanent cell line development. Other is sufficient for generating reagent-grade material.
technologies such as lentiviral or retroviral vectors Ultimately, specifications will be critical for
can have relatively higher costs because of your product, and those in turn depend on the
intellectual property, licensing fees, stable cell line quality of your analytics. The stronger the
development, MCB release, and additional residual analytics — especially on the characterization side
testing. With the MaxCyte platform, we can use — the better off you will be.
one MCB for all of our work, eliminating many
steps that can burden a process. Our goal is to be Acknowledgments
able to provide our downstream team with 4–5 g of This type of work requires a lot of collaborative teamwork
and execution, none of which would be possible if it were
target protein within a 50-L bulk harvest. To
not for the exceptional individuals at ABL. I give a special
achieve that, we are aggressively implementing thanks to Irene Kalisz, Stephen Whitney, Ranajit Pal, Maria
novel design of experiment (DoE) strategies on the Ferrari, Timothy Fouts, Beth Tebeau, Franck Lemiale, Rebecca
front end of the transfection development process Harston and Thomas VanCott for all of their support.
using OC-400 and CL-2 process assemblies. That
facilitates how we can optimize plasmid constructs, References
media formulation, feed strategies, and the addition 1 Menon V, et al. DNA Prime/Protein Boost
Vaccination Elicits Robust Humoral Response in Rhesus
of HDAC inhibitors to improve the quality and
Macaques Using Oligomeric Simian Immunodeficiency Virus
quantity of our target proteins. We are not saying Envelope and Advax Delta Inulin Adjuvant. J. General
that cell lines are irrelevant or unnecessary. On the Virology 98(8) 2017: 2143–2155.
contrary, at some stage in the vaccine process, cell 2 Challener C. Accelerating Vaccine Development and
lines are more practical and necessary for large-
November 2017. c
Manufacturing. Pharm. Technol. – BioPharm Int. eBook,
scale production. In fact, the beauty of the
MaxCyte platform is that it can be modified easily
to support stable cell line development if needed. Victor Ayala, PhD, is a microbiologist/immunologist with
The MaxCyte system has the flexibility to leverage ABL, Inc. (Rockville, MD). He has 17 years of experience in
all of those parameters on an upstream process in a the biomedical field. He serves as a key investigator on
streamlined manner. Implementing the MaxCyte several government contracts relating to the manufacture
strategy permits us to go from 16 mL to 50 L of vaccines in support of phase 1–2 clinical trials and as a
within months (Figure 5). We believe that if primary investigator for many preclinical studies for
transient transfection can bridge the gap cost- industry and academic clients. Dr. Ayala’s expertise spans
effectively between phase 1 and future clinical both the preclinical evaluation of vaccines and
trials, that is a good business niche to be in. therapeutics in animal models as well as the refinement of
On the downstream side, MaxCyte’s technology upstream and downstream manufacturing processes used
to accelerate timelines and reduce production costs.
can be used to accelerate vaccine development
(Figure 6). Currently, there is no universal platform
Sponsored Report J une 2018 16(6)i BioProcess International 7
Accelerating the Development and
Manufacturing of Your Next-Generation
Vaccines

Consistent, clinical-scale cell engineering

technology for high yield, cGMP-compliant
bioproduction.

• Rapid Response Vaccines

• Viral Vectors
• Subunit Vaccines
• VLP/VRPs
• Antibodies

Contact us today to discuss

your vaccine development
and production goals.

301-944-1700
[email protected]
www.maxcyte.com
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