Virus Safety in Vaccine Production

Priyabrata Pattnaik, PhD
Head of Biologics Operations - APAC
DCVMN & Merck Advanced Workshop: Bioprocess Optimization
7th February 2018 | Singapore
Virus safety in
vaccine production

Agenda
History of Adventitious virus contamination
Process risk assessment and strategy
Prevent
Detect
Remove
Conclusion
Virus safety in vaccine production | Priyabrata Pattnaik | DCVMN Workshop | 07 Feb 20182
1
2
3
4
5
6

History of
Adventitious virus
contamination
3

4 Virus safety in vaccine production | Priyabrata Pattnaik | DCVMN Workshop | 07 Feb 2018
Biopharma Virus Contaminations
Rare But Devastating
Genzyme 2009
 Hundreds of millions in lost revenue,
decontamination expenses, and regulatory fines
 Drug shortages jeopardized patient care
 Months-long plant shutdowns
 FDA fast-tracking of competing drugs
 Ongoing patient lawsuits
Genentech 1993
GSK 2010
What are the Costs?What are the Costs?

Viral Contamination
What is the industry saying?
“[Contamination is] a
question of when, not if”
- Michael Wiebe Consortium on
Adventitious Agent Contamination
in Biomanufacturing (CAACB)
"Nobody, including the media
company, had considered human
viruses contaminating the media.”
- Eli Lilly
“It is not possible to prove a negative result
…. therefore, testing, while a critical part of an
effective solution for viral contamination
prevention, is not sufficient alone,”
- Ivar Kljavin, director of adventitious agent management
Genentech

The Reality of Upstream Contamination*
Many contamination events are not publically disclosedMany contamination events are not publically disclosed
1950s -
60s: SV40
in Polio
Vaccine
Contaminiation events in Upstream
Processing have broadly affected the plasma,
vaccine and recombinant protein industry.
2012: Leptospira
licerasiae
contaminated seed
train bioreactor
1980s:
HIV
contaminated
Factor VIII
1994:
Hepatitis C
contaminated IgG
1996:
Minute Virus of
Mice contaminated
bioreactors
2009:
Vesivirus 2117
contaminated
bioreactors
2010:
Porcine
Circovirus
in Rotavirus
Vaccine
2011:
Mycobacteria
contaminated
bioreactor
2012:
Bacillus
thuringiensis
contaminated
bioreactor
*This timeline highlights major contamination events, but is not
comprehensive.

$7MPer 10kL batch
Up to 7.5 M
1Viral particle per
liter is sufficient
to infect
biopharmaceutical
manufacturing
0.04%
samples tested positive
for viral contamination of
unprocessed bulk drug
substance during in vitro
screening assays
A exisiting risk
Adventitious virus contamination

Historical examples of viral contamination in vaccines
& potential source of contamination
Vaccine Contaminant Route/comment
Yellow Fever
vaccine
Avian Leukosis
virus
Infected eggs
Polio & Adenovirus
vaccines
SV-40 Primary monkey
kidney cell culture
Polio Wild type Polio
MMR & Polio Bacteriophage Bovine sera
Yellow Fever
Vaccine
HBV Contaminated
human serum
MMR (1995) Reverse
transcriptase
Chicken cells
Rotavirus vaccines
(2010)
Porcine circovirus
(PCV-1)
Detected in MCB,
WCB, MVSS &
WVSS
Virus Potential Source Material tested
MVM Medium/unknown CHO cells/bulk
Human rhinovirus unknown BHK bulk
Bovine viral
diarrhea virus
Bovine serum Various cells
Bovine
polyomavirus
Fetal bovine serum Raw material (FBS)
Epizootic
Haemorragic
disease virus
Bovine serum CHO bulk
Reovirus Bovine serum CHO & BHK
cell/bulk
Nodavirus Latent infection Insect cells
2117 calcivirus unknown Bulk

“But it doesn’t affect me since I have an AOF process”
Plant derived materials (peptones,
recombinant proteins)
Parvovirus are present among the
manure used as fertilizers and are the
most resistant viruses

Case Studies of Microbial Contamination in Biologic Product Manufacturing
Suvarna, K., Lolas, A., Hughes, P., Friedman, R. Biotechnology Manufacturing Team, Division of Manufacturing and
Product Quality, Office of Compliance, Center for Drug Evaluation and Research, Food and Drug Administration
Facility
Equipment
Process
Materials
Utilities
Personnel
Each source
is a potential
entry point for
microbial
contamination
Acholeplasma laidlawii (<0.2um)
Leptospira species (>5 um)
Potential Entry point of contamination in Vaccine Manufacturing
Minute virus of mice (MVM) ~18-24nm

Vaccine Process
Sources of Adventitious viral Contamination

Virus/
Cells
mAb & r-
proteins
 Size-based filtration
 Chemical inactivation (low pH, detergents)
 Separation by chromatography
 Product is as large as potential contaminant
 Product sensitive to inactivation
 Some clearance possible using chromatography
(depending on model contaminant viruses tested)
Effective clearance may not be possible!
Virus Clearance in Viral vaccine processes
Clearance is a Challenge When Product is a Virus or Cell

What can we do to protect vaccines from viral contamination?

Process risk
assessment
and strategy

Virus Safety for Vaccines and Viral Vectors | Damon Asher | September 201715
Material Selection
Risk-Based Approach
Kiss, B., Practicing Safe Cell Culture: Applied Process Designs for Minimizing Virus Contamination Risk
Proceedings of the PDA/FDA Adventitious Viruses in Biologics: Detection and Mitigation Strategies
Workshop in Bethesda, MD, USA; December 1–3, 2010
Adventitious Agent Risk Level
Highest Medium Lowest
Material
Source
Animal-derived
Biological origin
(i.e.: Plant)
Microbial
fermentation,
synthetic, other
Raw Material
MFG Process
Crude material
Some
purification
steps
Inactivation/
Clearance
conditions
(heat)
Raw Material
Supply Chain
Limited
segregation,
non-validated
CIP/SIP, rodent
food source,
numerous
handlers
Some
segregation
CIP/SIP
Raw vs.
finished
segregation,
validated
CIP/SIP
Amount of Raw
Material Used in
Biotech
Process
Large Moderate Small
• Raw material media constituents
used to promote and sustain cell
growth and recombinant protein
productivity should be considered
high risk.
• Items outlined in red present the
greatest risk of introducing
adventitious agents to a cell culture
process.

Provantage offeringDefining the risk
Consider the situation & process
Process
Are specific
steps in the
process more
prone to
contamination?
Raw materials
Does my process
includes serum?
Do I use Porcine
trypsin?
History
What are the
historical
contamination of the
process?
Have they been
identified?
Cell bank
What are the probable
virus contamination in
my cells?
Testing
What’s my testing
regime?
Is it adapted to my
process, regulatory
requirements and
risk?
Existing
clearance
Do I have anything
in place to reduce
potential viral
loads?

Different types of vaccines
Different risks to be assessed
Live attenuated viruses
Inactivated viruses
Inactivated viral subunits
Recombinant VLPs or
vectors
Attenuated strains via non-host cell passages.
Yellow fever vaccine, Rotavirus vaccine
Ex: Mammalian cell culture or eggs
Includes a chemical inactivation step. Formaldehyde or Beta-propio-
lactone.
Ex: Polio vaccine, FMD vaccine
Mammalian cell culture or eggs
Includes a chemical inactivation and detergent treatment steps
(Fromadehyde/BLP, Triton X-100).
Ex: Influenza vaccines
Mammalian cell culture or eggs
Recombinant particles
Ex: Hepatitis B, Human Papilloma virus vaccines.
Mammalian, yeast or insect cells

18
Defining the risk
Testing & Clearance – Limitations
Note: Figures and numbers above are theoretical examples and are not prescriptive
High virus load is likely detected
Detection improves when greater
fractions of material are tested.
Low virus load is likely cleared
Clearance improves with higher LRV.
Potential for a critical virus load that is
too much to clear, but too little to be
detected

Developing a comprehensive viral clearance strategy
A multifaceted approach
Detect
Verify that raw materials are free
from contaminants
Ensure Safety of Raw
Materials and Processes
Verify absence of contaminants in process intermediates
Optimize Sampling and Test Methodologies
Develop capacity of manufacturing process with
orthogonal steps that remove or inactivate
contaminants
Implement Robust Clearance
Technologies
Comprehensive
Risk Mitigation
Strategy
19

Virus Safety
3 Complementary Pillars
Lot Release Testing
 Testing the product at
appropriate steps of
production
Testing
Removal/Inactivation
 Assess capacity of the
process to clear viral
contaminants
 Performed in-process before
final fill
 Filtration
 Chemical inactivation
 Chromatographic separation
Clearance
Clearance is the last line of defense against viruses that have gone undetected.
Selective Sourcing
 Low-risk raw materials (e.g.
avoid animal derived when
possible)
Characterization
 Cell line characterization
(CLC)
 Virus seed characterization
 Testing of source materials
(e.g. media components,
“raw materials”)
Material Control

Virus Clearance in mAb process
In-Process Clearance Targets
Clearance is quantified in units of Log Reduction Value (LRV)
one LRV = 10-fold reduction in virus, two LRV= 100-fold reduction, etc.
Regulatory documents provide guidance on LRV targets for
individual steps and totals for the complete production process.
Key Regulatory Documents
ICH
• Q5A Viral Safety Evaluation of Biotechnology
Products Derived from Cell lines of Human or
Animal Origin. CPMP/ICH/295/95.
FDA
• Points to Consider in the Characterization of Cell
Lines Used to Produce Biologicals (1993)
• Points to Consider in the Manufacture and Testing
of Monoclonal Antibody Products for Human Use
(1997)
EMEA
• Note for Guidance on Virus Validation Studies:
The Design, Contribution and Interpretation of
Studies Validating the Inactivation and Removal of
Viruses. CPMP/BWP/268/95, revised 1996.
• Guideline on Virus Safety Evaluation of
Biotechnological Investigational Medicinal
Products. EMEA/CHMP/BWP/398498/2005
(2008).

22
Virus Contamination Mitigation
Secondary
Clarification
Column
Protection
Column
Protection
Final FillingFinal Sterile
Filtration
Concentration
and
Formulation
Bulk Storage
and Transport
Viral
Inactivation
Column
Protection
Chromatography
Purification
Chromatography
Polishing
TFF
Bioreactor
Column
Protection
Chromatography
Purification
Primary
Clarification
MCB
Pretreated
Raw
Materials
FiltrationHTST
γ Radiation
UV-CRaw
Materials
Virus
Resistant
Cell Line Seed Train
WCB
= Routes of contamination
= Upstream Virus Mitigation
= Downstream Primary Viral Clearance
= Downstream Secondary Viral Clearance

Virus Safety for Vaccines and Viral Vectors | Damon Asher | September 2017
Vector/Cell
Master Cell Bank (MCB)
Working Cell Bank
(WCB)
Process Development
(Growth/Production/Modification)
Master/Working Virus Bank
(MVB/WVB)
Drug Substance Drug Product
Identity
Purity
Safety
Identity
Safety
QA/QC
In-process
testing
In-process
testing
Container Closure
Stability
Lot Release Testing
Shipping
Identity
Purity
Safety
Expression
In-process
testing
Virus and Cell Safety and Characterization
23

1. Prevent
Adventitious Agent Safety of Upstream Raw Materials
Consider virus resistant cell lines
• Centinel™ technology, first commercially
available gene editing tool to confer
Minute Virus of Mice (MVM) resistance to
CHO cell lines
Replace animal derived
components in media
• Recombinant proteins (r-Insulin,
r-Trypsin, etc.)
• Serum alternatives
Source lower risk animal derived
components
• Source from lower risk geographies (GBR I or II)
• Irradiation of raw material
• Viral testing (CFR9) of raw material by
manufacturer
Adopt of chemically defined animal
derived component-free media
• Most conservative approach
• Adaptation challenges
1
2
3
4

1. Prevent
MVM Resistance by Genetic Engineering
Centinel™ technology provides the first commercially available gene
editing tool to confer Minute Virus of Mice (MVM) resistance to CHO
bioproduction cell lines in order to support our customers in mitigating
viral risks.
 MVM enters the cells via sialic
acid
 Targeting SLC35A1 with Zinc
Finger Nuclease (ZFN)
technology creates a cell line
that lacks sialic acid on its
surface
 MVM infection is prevented
26

Detect
27Virus safety in vaccine production | Priyabrata Pattnaik | DCVMN Workshop | 07 Feb 2018

28
2. Detect
Secondary
Clarification
Chromatography
Protein A
Column
Protection
Column
Protection
Filtration
Concentration
and
Formulation
Bulk Storage
and Transport
Viral
Inactivation
Column
Protection
Chromatography
Purification
Chromatography
Polishing
Virus Filtration
Clearance
TFF
Bioreactor
Column
Protection
Chromatography
Purification
Primary
Clarification
MCB
Pretreated
Raw
Materials
FiltrationHTST
γ Radiation
UV-CRaw
Materials
Virus
Resistant
WCB
= Points for testing

2.Detect

2. Detect
Cell Bank Testing
Testing for virus contamination is part of cell bank characterization. Key Regulatory Documents
• US FDA Guidance for Industry: Characterization
and Qualification of Cell Substrates and Other
Biological Starting Materials used in Production of
Viral Vaccines for the Prevention and Treatment of
Infectious Diseases, 2010
• WHO Technical Report Series 978, Annex 3:
Requirements for the Use of Animal Cells as In
Vitro Substrates for the Production of Biologicals,
2010
• WHO Technical Report Series 927:
Recommendations for the production and control
of influenza vaccines (inactivated)
• EP 5.2.3 Cell Substrates for the Production of
Vaccines for Human Use
• EP 2.6.16 Extraneous agents in vaccines for
human use
• ICH Q5D: Derivation and characterization of cell
substrates used for production of biotechnological
/ biological products
Virus Tests Master Cell
Bank (MCB)
Working Cell
Bank (WCB)
Cells at Limit of
Production (CAL)
Broadly specific
in vitro assays
Broadly specific
in vivo assays
Species specific assays
(e.g. human, simian, rodent,
canine, bovine, porcine)
Retroviruses (e.g. PCR,
TEM, PERT, infectivity)
• Critical starting
material
• Full characterization,
one time testing
• Small number of
passages beyond
MCB
• Reduced package
of testing
• ‘Worst case’ for
amplification of
contaminants
• Full characterization,
one time testing

2. Detect
Master Virus Seed Assays
Virus Tests Master Virus
Seed Stock
Broadly specific
in vitro assays
Broadly specific
in vivo assays
Species specific assays
(e.g. human, simian, rodent,
canine, bovine, porcine)
Retroviruses (e.g. PCR,
TEM, PERT, infectivity)
• Neutralization pre-
studies for in vitro
and in vivo
infectivity studies
 MVSS should be screened fully for adventitious bacteria, fungi,
mycoplasma and viruses taking account of the origin and isolation
of virus stock
 Neutralizing antiserum is required for infectivity assays to
specifically inactivate the master virus
― Should be prepared from a stock that is different from stock
used for production and prepared using SPF animals
― Stock should not be grown in the same cell line as used for
production
― Not of human or simian origin
 Pre-studies are required to ensure neutralization of virus stocks
before testing
 Where neutralizing antisera of high enough titer cannot be prepared
a panel of PCR assays may be used
 Production control cells (not inoculated with virus) grown in same
medium and handled alongside production cells are tested for
adventitious mycoplasma and viruses
 Replication defective gene therapy vectors tested for presence of
replication competent viruses

2. Detect
Detection assays limitations
• Infectivity assays may take up to 4 weeks
• Viruses in raw materials may need to adapt to
growth in cell culture
• Components of raw materials (e.g. antibodies,
antibody complexes) may inhibit detection of
viruses
• Components of test material may be cytotoxic
to detector cells used for infectivity assays
• Different assay formats for virus detection and
quantitation of virus contaminants
• How to detect ‘unknown’ viruses?
“You only find what
you are looking for »

2. Detect
Next generation sequencing: an additional tool
“Is the biologic system what
is believed to be”?
“ Is the system contaminated
or impure? “
Genetic properties of the
MVSS
(identity/purity/stability)
Adventitious agent testing Raw material qualification
In process testing Lot release testing

2. Remove
Secondary
Clarification
Column
Protection
Column
Protection
Filtration
Concentration
and
Formulation
Bulk Storage
and Transport
Viral
Inactivation
Column
Protection
Chromatography
Purification
Chromatography
Polishing
TFF
Bioreactor
Column
Protection
Chromatography
Purification
Primary
Clarification
MCB
Pretreated
Raw
Materials
FiltrationHTST
γ Radiation
UV-CRaw
Materials
Virus
Resistant
WCB

Process dependent, 2 robust steps implementation is a challenge
3. Remove
Inactivation Chromatography Nanofiltraton
(downstream)
Inactivated
Live
VLP
Vector
retrovirus filter
Only for virus size
<50nm (ex: AAV)
Provide some LRV
but not robust
Robust, but only
for killed viruses

3. Remove
What if « clearance » moves to upstream?
Raw materials Dowstream
Bioreactor

Technology Robust Clearance Media Compatibility Point of Use Scalability Cost Effective
HTST
(~102C
~10 sec)
Yes
Component
dependent
Yes
Challenging
for small to
mid-scale
Yes at
Large
Scale
UV-C
(254 nm)
Organism
dependent
Component
dependent
Yes
Challenging
at large
scale
Yes at
Small
Scale
γ
Radiation
Organism
dependent
Component
dependent
No
Small
batches
Yes
Downstream
Virus
Filters
If specifically
claimed.
Consistent
LRV
Yes but
designed for
downstream
fluids
Yes Yes
Not for
batch
processes*
Upstream
Virus Barrier
Filters
Yes by size
exclusion.
Consistent
LRV
Yes,
specifically
designed for
upstream
media
Yes Yes Yes
3. Remove
Protection of the bioreactor/CCM & feeds treatment
* Downstream viral clearance filters, are designed for very clean feed streams and would not be cost effective on upstream bioreactor media and feeds.

3. Remove
How to select the right method? Nature of feed, impact on
properties/growth
CCM, Feeds
CCM, feeds
CCM, feeds
Serum, consumables
High volumes, difficult solutions to filter,
Little sensitiveness to feed
Small volumes
New available nanofilters
Small to large volumes
Heat sensitive feeds

3.Remove
Schematic presentation of HTST setup
 High Temperature Short Time (HTST) also called flash pasteurization, especially used for high volume processing.
 Liquid is heated from ambient to 102 °C (F), hold at this temperature for a minimum of 10 seconds then cooled to 37°C before it
is sent to bioreactors

3.Remove
HTST treatment for Glucose 50% w/v - Cell Performance
0.0
20.0
40.0
60.0
80.0
100.0
0 4 6 7 10 12 14 15
%Viability
Days
Viability Average Glucose Testing
Treated #1 Untreated #1 Treated #2 Untreated #2
Treated #3 Untreated #3 No Glucose
0.0
100.0
200.0
300.0
400.0
500.0
600.0
700.0
800.0
7 10 12 14
ug/mL
Days
Productivity - Average (IgG) Glucose Testing
Treated #1 Untreated #1 Treated #2 Untreated #2
Treated #3 Untreated #3 No Glucose
 102°C up to 10 minutes
 Glucose used as challenging model
 Cell performance comparable across treated
and untreated
 Consistent LRV >4
41

11%
14% 14%
61%
28 in-scope media (chemically-defined, no hydrolysates)
were tested with the Viresolve® Barrier Filter – mix of
commercially-available and customer-proprietary
Membrane inlet
(upstream)
~0.5 - 10 µm
= 500 – 10,000 nm
Membrane outlet (downstream)~20 nm
Bacteria
~300 nm
Mycoplasma
~150 nm
Retrovirus (~MuLV,
enveloped)
~90 nm
Parvovirus (~MVM, non-
enveloped)
~23 nm
Membrane optimized for Cell Culture Media
3. Remove
42

3. Remove
Nanofiltration of CCM – Viresolve® Barrier filter
A filter designed
specifically for cell
culture media
outperforms filters
designed for the
downstream process
An example medium
is shown here, but
performance
improvement was
demonstrated in a
range of media
Throughput w/CD OptiCHO™ Medium
0
500
1000
1500
2000
2500
3000
0 100 200 300 400
Throughput(L/m2)
Time (min)
Merck Viresolve® Barrier
filter
Merck Viresolve® Pro
Device
Merck Viresolve® NFP
filter
Filter 1
Filter 2
Filter 3
Filter 4

Optimized Membrane = No Prefilter Needed

3. Remove
Viable Cell Density
No impact of virus filtration was observed on any cell growth attributes
CellVento® CHO-200 with MAb01 EX-CELL® Advanced™ CHO with MAb02
No changes in pH, osmolarity, glucose, glutamate, lactate, or NH3 levels were seen (as measured by BioProfile® FLEX).

3. Remove
Virus retention with Viresolve® Barrier filter

Heat Inactivation: Viral Vector / Gene Therapy
Adventitious or unwanted virus removal from Viral Vector
 Heat inactivation is based on the different
thermal stabilities of AAV and the helper
viruses (50-55 0C for 10 min)
 Small scale heat inactivation can be
optimized using Rocking Heated Mixer
(preferably covered to maintain better
temperature control)
 Scaled-up can be achieved using plate
heat exchangers.
 Care has to be taken to maintain proper
recirculation of content and eliminating
cool-spot and dead legs.
 In-line sensors can be introduced in the
loop and the complete heat inactivation
can be set-up in a fully integrated single
use process.
SOURCE: Hehir, KM; Armentano, D; Cardoaz, LM, et al. (1996) Molecular characterization of replication competent variants of adenovirus vectors and genome
modifications to prevent their occurrence”. J. Virol. 70:8459-8467

Heat Inactivation Set-up: Viral Vector / Gene Therapy
Adventitious or unwanted virus removal from Viral Vector
 In an AAV process, any active adenoviruses as
impurities and need to be inactivated and
removed during downstream purification.
 AAV particles are stable in a wide pH range (3
to 9) and can resist heating at 56 ⁰C for 1 hour.
 Human adenoviruses are extremely sensitive
to heat.
 To inactivate adenovirus, preparations can be
heat inactivated for 15 minutes at 56 C and
tested for the presence of replication
competent adenovirus by plaque assay or
cytopathic effect.
SOURCE: Barb Thorne (2016) Manufacturing a Viral Vector for Gene Therapy at the 2000L Scale. Cell Culture World Congress, 24 Feb 2016; Thorne Bio‐Consulting LLC

3. Removal
Layout of normal flow filtration used for removal of adventitious
virus from vaccine feed stream
 Retrovirus removal by NFF from VLP
vaccines.
 Different size of virus of VLP can be
managed by use of NFF

Prevent
Detect
Remove
• Genetic
modification
of host cell
• Pre-
treatment of
high risk
components
• Virus barrier filtration
• High Temperature
Short Time (HTST)
Treatment
• Testing
• Next Generation
Sequencing (NGS)
• PCR testing
• In vitro testing
• Next Generation
Sequencing (NGS)
• Large Virus
filtration
• Inactivation
• Chromatography
Minimizing virus contamination risks throughout the process of viral
vaccines follows a multilayered strategy
Conclusion

Acknowledgement
 Anissa Boumlic, Associate Director - Vaccine initiative, EMEA
 Damon Asher, Associate Director, Novel Therapy & Vaccine Segment Development
 Kate Smith, Principal Scientist, Development Services, BioReliance
 Martin Wisher, Regulatory Consultant, BioReliance

Thank You
Priyabrata Pattnaik, PhD
priyabrata.pattnaik@merckgroup.com
@pattnaik_p
https://sg.linkedin.com/in/priyabratapattnaik
https://plus.google.com/+PriyabrataPattnaik153

Virus Safety in Vaccine Production

Recommended

Recommended

More Related Content

What's hot

What's hot (20)

Similar to Virus Safety in Vaccine Production

Similar to Virus Safety in Vaccine Production (20)

More from Dr. Priyabrata Pattnaik

More from Dr. Priyabrata Pattnaik (13)

Recently uploaded

Recently uploaded (20)

Virus Safety in Vaccine Production