In this webinar, you will learn: - about the importance of characterising cell banks and virus seed stocks in order to meet worldwide regulatory requirements. - the difference between guidance documents from different organizations worldwide - new technologies for determining the identity of cell substrates and virus seed stocks - detecting adventitious agent contamination

1. Merck KGaA
Darmstadt, Germany
Dr Martin Wisher 5th October 2017
Vaccinecell bank and virus
seed characterization

2. 2
The life science business of
Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma
in the U.S. and Canada.

3. • Live virus vaccines
• Usually attenuated strains
• Inactivated virus vaccines
• Whole virus
• Subunit vaccines
• Recombinant virus vaccines
• Assembled virus particles
• Virus proteins
Types of virus vaccines
3

4. Developing a Comprehensive Risk Mitigation Strategy - Multifaceted Approach
Safety of Raw
Materials & Process
“Prevent”
Testing
“Detect”
Implementation of
Clearance Technologies
“Inactivate/Remove”
4
Ensure quality of raw materials
Treatment of media
• HTST
• Barrier filter
Testing of:
•MCB, WCB and ECB
•MVSS, WVSS
•Virus harvests
•Purified virus
Not possible for vaccines
with live enveloped viruses
Inactivated & recombinant
vaccines – validate
inactivation & removal steps

5. Regulatory Guidance for Vaccine Characterization
US FDA Guidance for Industry:
Characterisation 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
EP 5.2.3 Cell substrates for the production of vaccines for human use.
01/2018:50203
EP 2.6.16 Tests for extraneous agents in viral vaccines for human use
ICH Q5D:
Derivation and Characterisation of Cell Substrates Used for Production
of Biotechnological / Biological Products, 1997
5

6. Cell seed
•Restricted to identity testing and NGS for adventitious agents
MCB
 Starting material for the whole of the production process
 Full characterisation for microbial and viral contaminants
 One time testing
WCB
 Small number of passages beyond MCB
 Reduced package of testing on cells from 1st WCB
End of Production Cells (EOPC) or Extended Cell Bank (ECB)
 Cells at or beyond the maximum population doubling level used for production
 ‘Worst case’ for amplification of contaminants
 Full characterisation, one time testing at a scale of production
EP 5.1.7 Viral safety
 Amount of testing and where testing is performed should be based on a viral risk assessment
Rationale for cell bank testing
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7. Cell Bank Characterisation - MCB
Purity Genetic Stability Identity
Bacteria, fungi- sterility
Mycobacteria
Broad specificity - in vitro/in vivo assays
Species specific – human/simian/rodent
canine/bovine/porcine
Retroviruses – PERT/PCR/TEM/infectivity
Master Cell Bank (MCB)
Mycoplasma/Spiroplasma
Virus
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8. Isoenzyme analysis: only determines species of cell
Sole supplier of isoenzyme kit ceased trading
DNA Fingerprinting (Short Tandem Repeats):
can identify cells of the same species
Cytochrome oxidase 1 bar coding or species specific probes:
can determine species and sub-species
Karyology:
chromosome number and marker chromosomes identify cells of
The same or related species
Cell identity markers indicative of cell type, pluripotency,
lineage commitment or terminal differentiation
Identity testing of cells
8

9. • Microgenomic identification system
• Mitochondrial genome of animals is a better target for analysis than the nuclear genome
• Lack of introns
• Limited exposure to recombination
• Haploid mode of inheritance
• Cytochrome oxidase I (COI) gene
• Universal primers for this gene are very robust, enabling recovery of its 5’ end from all animal phyla
 COI possess a greater range of phylogenetic signal than any other mitochondrial gene
• Rate of molecular evolution (base substitutions at third-position nucleotides is 3x greater
than 12S or 16S rDNA
• Evolution of this gene is rapid enough to allow the discrimination of closely allied species
and phylogeographic groups within the same species
Cytochrome c Oxidase I (COI)
COI analysis now method of choice for taxonomic identity
and for cell line identity at cell culture collections.
9

10. 10
Region unique to speciesConserved
Primer Region
Conserved
Primer Region
E.g., Sequence
unique to each
species
BLAST sequence in Consortium for the Barcode of Life
Database (BOLD)
All species in BOLD database (~500,000)
Species Identification by Barcode Analysis
Confirmation of species against internally
verified sequences:
Human, mouse, Chinese hamster, Syrian hamster, African
Green Monkey, Insect
Mid H2-15 End H2-15

11. ICH Q5D
•‘In most cases isoenzyme analysis is sufficient to confirm species of origin of cell line’
•‘….other technologies may be substituted to confirm species of origin’
•‘Either confirmation of species of origin or presence of known unique cell line markers is
considered an adequate test of identity.’
US FDA Guidance, 2010
•Discusses isoenzyme analysis, karyology, DNA fingerprinting
•‘Tests such as …PCR.., expression of a gene of interest may be applied to distinguish an
engineered cell line from other cell lines.’
EP 5.2.3
•‘Nucleic acid fingerprinting and a relevant selection of the following are used to establish the
identity of the cells: isoenzyme analysis; immunological characteristics; cytogenetic markers;
Nucleic acid amplification techniques (NAT, PCR)’
Identity testing of cells - Regulatory guidance on identity assays
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12. General strategy
Regulatory guidance on adventitious agent testing
12
• US FDA and WHO suggest full characterisation of
MCB and maximum PDL used for production cells
(EOPC); minimal testing on WCB.
• EP 5.2.3 01/2009 suggested that all adventitious
virus testing should be performed on EOPC but
new revision (01/2018) indicates that the
appropriate testing stages should be selected
based on a viral risk assessment and accepts
alternative strategies that focus on more
extensive testing at the MCB or WCB level.
• EP 5.1.7 Viral safety, describes parameters that
should be considered in a viral risk assessment.

13. Assays to detect adventitious viruses
•All documents aligned in recommending using live cells or cell lysate (at 107
cells/ml in
conditioned medium)
•In Vivo assay
• FDA guidance recommends adult mice (20 observed for 21 days); suckling mice (20,
observed for total 28 days with passage at 14 days); embryonated eggs (allantoic and
yolk sac inoculation with passage); Guinea pigs (42 days observation to detect
Mycobacterium)
• WHO recommends adult mice (20 observed for 28 days); suckling mice (20 , observed for
28 days but no passage at 14 days); Guinea pigs but can be replaced by in vitro method
for Mycobacterium; embryonated eggs but only if testing avian cell lines or novel cell
substrates.
• EP 5.2.3 only recommends inoculation of suckling mice (> 10, observed for 28 days) if
risk assessment indicates it provides risk mitigation taking into account the overall testing
package. Embryonated eggs only required for avian cell substrates.
Regulatory guidance on adventitious agent testing (2)
13

14. Retrovirus testing
•Similar approach recommended by FDA, WHO and EP
•If the cell line is not known to produce retroviral particles:
• Test using a product-enhanced reverse transcriptase (PERT) assay and examine by
transmission electron microscopy (TEM)
• If PERT and TEM give a positive or equivocal result test using an infectivity assay
•If the cell line is known to produce retroviral particles (e.g. rodent and avian cells)
• Test by TEM and using an infectivity assay
• If both TEM and infectivity are negative test using a PERT assay.
Regulatory guidance on adventitious agent testing (3)
14

15. Tests for specific viruses
•The list of specific viruses to be tested should be defined based on a viral contamination risk
assessment (defined in EP 5.1.7 Viral safety).
•Use PCR in cases where viruses cannot readily be grown in culture.
Specific human viruses
•FDA recommendations:
• Hepatitis A, B & C; HIV 1 & 2; HTLV 1 & 2; B19 parvovirus; EBV, CMV, HHV 6,7 & 8
• Enteroviruses, Circoviruses, papillomaviruses, human polyoma viruses, human adenovirus
• Should also consider simian viruses that could infect humans.
Specific bovine and porcine viruses
•FDA and WHO recommend testing for 9 bovine viruses and porcine parvovirus (trypsin)
specified in 9CFR 113.47 & 113.53
•Additional assays should be considered for:
• Bovine: bovine polyoma virus, bovine circovirus, bunyaviruses (Cache valley virus), EHDV
• Porcine: porcine circovirus, porcine hepatitis E virus, anelloviruses, hokovirus, bocavirus
Regulatory guidance on adventitious agent testing (4)
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16. Cell bank Characterization - WCB
Purity Identity
Bacteria, fungi- sterility
Broad specificity - in vitro assay
Working Cell Bank (WCB)
Mycoplasma
Virus
16

17. 17
Cells DNA Adventitious Agents
 Demonstrate removal of
intact cells
 Determine tumorigenic
potential (TPD50) of cells
 Demonstrate no capacity for
transformation
(oncogenicity)
 Demonstrate lack of
inherent agents
 Infectious
 Latent/occult
 Oncogenic
 Demonstrate removal
and/or inactivation of
potential agents
Special Considerations for Continuous Cell Lines
 Demonstrate lack of
oncogenicity
 Newborn mice, rats and
hamsters
 Demonstrate acceptable DNA
removal and/or inactivation
 < 10 ng/dose; <200-400 bp

18. ‘New sensitive molecular techniques with broad
detection capabilities are available, including
massively parallel sequencing (MPS) methods,
degenerate PCR for whole virus families or random-
priming methods (associated or not with sequencing),
hybridisation to oligonucleotide arrays and mass
spectrometry. These methods may be used either as
an alternative to in vivo or specific NAT tests or as a
supplement/alternative to in vitro culture tests, in
agreement with the competent authority.’
New virus detection methods
18
‘In agreement with the competent authority, broad molecular methods (e.g. High
Throughput Sequencing) may be used either as an alternative to in vivo tests and
specific NAT or as a supplement or alternative to in vitro culture tests based on
the risk assessment.’
EP 5.2.3 (01/2018)

19. 19
Sample Processing
Collect nucleic acids
RNA/DNA, it doesn’t matter; if you
can collect it, it can be sequenced.
You don’t need to know anything
about the sequence.
Construct the desired library
Convert extracted material to
dsDNA.
Sequencing
Perform the sequencing reaction
Generate the dataset(s)
Bioinformatics (BFX)
Perform the desired analysis in
powerful computing environments
(e.g. Google Cloud)
Compile/compress/qualify the data
“MP-Seq™”
Next Generation Sequencing
(High Throughput/Deep/Massively Parallel Sequencing)
High throughput, concurrent sequencing by synthesis with three basic steps:

20. • Extraction
 Extract total nucleic acids — purify all genomes (RNA/DNA/ss/ds/ linear/circular)
 Library must be dsDNA so convert all material using a cDNA synthesis kit (pre-processing)
• Library preparation
 Shear DNA to an appropriate size range
 Add adaptors to ends of fragments
 Quantify DNA
 Tagged DNA is then amplified to enrich for sequences containing the proper tags for sequencing
 Amplification means the assay cannot be quantitative
 There is no selection or targeting of virus sequences — all sequences are converted into the library
• Sequencing by synthesis
 Addition of fluorescently-labeled nucleotides to complementary DNA strand releases fluorescent probe
Sample Extraction and Library Preparation
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21. 21
Overview of AAT Algorithm
Quality control steps to eliminate
poor quality reads from the analysis
Multi-step BLAST-based analysis
Control = housekeeping genes to assess
breadth/depth of coverage
Ribosomal = eliminates ribosomal background
Filter = custom DB that allows us to subtract
defined Sequences (e.g. known VSS)
Query = viral/bacterial/fungal DB
NT = Complete NCBI nt + RefSeq
Preliminary qualification of
Results & reporting

22. Next Generation Sequencing (NGS)
Advanced technology enabling sequencing of millions to billions of DNA molecules rapidly & simultaneously.
NGS is sequence-agnostic: you do not need to know anything about the sequence in order to sequence it
and gain info….
22
Personalized
Medicine/
Clinical Applications
Biosafety Testing
Diagnostics
Whole Genome Sequencing
Identity Testing/Confirmation
Variant Detection
Contaminant Detection

23. Interest Group
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24.  MVSS should be screened fully for adventitious bacteria, fungi, mycoplasma,
mycobacterium and viruses taking account of the origin and isolation of virus stock
 Neutralising antiserum is required for infectivity assays
Should be prepared from a stock that is different from stock used for production and prepared using
SPF animals
Not of human or simian origin
 Pre-studies are required to ensure neutralisation of virus stocks before testing
 Where neutralising antisera of high enough titre 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
 EP 2.6.16 only recommends using suckling mice as the in vivo assay for adventitious
viruses.
 ‘Each virus seed lot is tested in suckling mice if the risk assessment indicates that this test provides a
risk mitigation taking into account the overall testing package.’
Master Virus Seed Characterisation
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25. 25
Purity Identity
Bacteria, fungi- sterility
Mycobacteria
Broad specificity - in vitro assay
Specific viruses - PCR
Mycoplasma
Virus
Virus Harvest testing

26. 26
• Alignment of millions of reads
against one or more defined
reference sequences
• Unlike Sanger (consensus)
sequencing, NGS enables
sequencing & reporting of millions
to billions of individual molecules
→ analysis on a per molecule basis
• Exquisite variant detection down to
a single molecule level
• Sub-1% variant detection is possible
→ can identify ultra-rare variants
Identity Testing/Variant Determination by NGS

27. 27
Total # of
Reads Used
for
Mapping
Reference
Sequence
Used for
Mapping
Reference
Length
(Bases)
Total #
of
Mapped
Reads
% of
Population
Mapped
Average
Depth of
Coverage
Across
Consensus
Consensus
Length
Generated
By Mapping
(High
Quality
Unique
Positions)
%
Reference
Coverage
%
Consensus
Similarity
(Identity)
to
Reference
Total
Number of
Unmapped
or Low
Quality
Positions
36,211,136 AF345290.1 15,384 27,793 0.08 470.82 15,302 99.47 99.41 82
Position
Reference
Base
Variant
Base
Number of
High-
Quality Ref
(fwd)
Number of
High-Quality
Ref (rev)
Number of
High-
Quality Var
(fwd)
Number of
High-Quality
Var (rev)
Variant
Frequency (%)
Variant Type
75 T C 0 0 13 5 100.00 Substitution
92 G C 0 0 17 7 100.00 Substitution
121 G C 0 0 29 14 100.00 Substitution
123 G C 0 0 29 14 100.00 Substitution
150 T C 0 0 37 26 100.00 Substitution
616 C T 1 0 183 158 99.71 Substitution
764 G A 211 171 93 63 29.00 Substitution
1126 G A 0 0 201 228 100.00 Substitution
1205 C A 0 0 232 232 100.00 Substitution
1827 C G 1 0 109 124 99.57 Substitution
1829 A C 0 0 111 127 100.00 Substitution
1833 T G 0 0 114 126 100.00 Substitution
1834 C T 0 0 113 124 100.00 Substitution
Example Output: VSS-ID
Mumps (ssRNA)
Low percentage of reads
mapped to reference,
indicative of unpurified
or low titer sample
- In this case -- able to
get >99% reference
coverage from the
reads.
Sequence length
generated by mapping
reads shorter than
consensus by 82 bases
-quality of reference
-hard to sequence
section of genome
Example of a mixed
population where only
29% of reads had a
specific variant base
compared to >99% for
all other variants.

28. Developing a Comprehensive Risk Mitigation Strategy - Multifaceted Approach
28
28
Ensure Safety of Raw
Materials and Processes
Implement Robust Clearance Technologies
Optimize Sampling and Test
Methodologies

29.  DSP steps validated for viral inactivation/removal
 Inactivation steps
 Formaldehyde
 Beta propriolactone (BPL)
 Detergents
 Removal steps
 Ion exchange chromatography
 Virus removing filters
Clearance Studies – Inactivated Vaccines
29

30.  ICH Topic Q5A. Note for Guidance on Quality of Biotechnological Products:
Viral safety evaluation of biotechnology products derived from cell lines of
human or animal origin (CPMP/ICH/295/95).
 EMA 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, 1996).
 EMA Guideline on Virus Safety Evaluation of Biotechnological Investigational
Medicinal Products (EMEA/CHMP/BWP/398498)
 WHO Technical Report Series 927:
Recommendations for the production and control of influenza vaccines
(inactivated)
Viral Clearance Guidelines
30

31.  Develop a multi-faceted virus contaminant risk mitigation strategy when developing
a viral vaccine
Prevent: Ensure quality of raw materials
Detect: Implement testing package for cell banks,
maximum PDL cells, virus seed stocks and virus harvests
Inactivate/remove: Where possible implement virus
contaminant clearance technologies
 Overall testing strategy for viral vaccines is shared by US FDA, WHO and EP
 EP is recommending reducing in vivo virus detection assays and supplementing classical
virus detection and identity assays with new technologies such as NGS analysis
Conclusions
31

32. BioReliance
Todd Campus
West of Scotland Science Park
Glasgow, G20 0XA, UK
www.bioreliance.com
Dr Martin Wisher
Senior Director,
Global Head of Regulatory Affairs
martin.wisher@sial.com
Contact details The vibrant M and BioReliance are trademarks of Merck KGaA, Darmstadt, Germany
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Detailed information on trademarks is available via publicly accessible resources.
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