Watch the presentation of this webinar here: https://bit.ly/3RijkHC Detailed description: Imagine you’ve just completed a manufacturing run for your viral vector. Identity testing is performed to confirm the vector sequence. But when the results come back the data reveals unexpected sequence variants! With an appropriate risk mitigation testing strategy, this situation can be prevented. The situation described above is not hypothetical, and happens more that you think, costing valuable time and resources. Investigatory testing has shown that sequence variants present in starting materials (e.g. plasmids) are likely to make their way to the final product. Adequate identification of low-level variants with an appropriately sensitive method is critical in ensuring the quality of the final product. A risk-based testing strategy, in the context of identity, for viral vector manufacturing will be presented, focusing on key testing points. NGS assays for identity and variant detection will be highlighted due to their extremely sensitive nature compared to traditional approaches. In this webinar, we'll explore: • Regulatory requirements for identity testing • NGS applications for identity testing as compared to traditional methods • A case study on the impact of not establishing a proper risk-based testing strategy Presented by: Bradley Hasson, Director of Lab Operations for NGS Services

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Identity Testing by
NGS as a Means of
Risk Mitigation
Brad Hasson
Director, Lab Operations
Next Generation Sequencing Operations

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

3. Critical Quality
Attributes
Safety
Identity
Potency Purity

4. What is identity (ID) testing?
4
Regulators set high expectations
on drug manufacturers to
establish the sequence identity
and stability of viral and non-viral
vectors developed to deliver a
drug product
CGT & vaccine discoveries are
fueled by molecular methods
NGS is one such state-of-the-art
tool that establishes sequence
identity and integrity, providing
robust detection and
characterization of variants due
to point or indel mutations
Biosafety testing techniques
have evolved to keep pace
Fully validated GMP methods
specifically tailored for each type
of product to be evaluated
ensures reliable sequence
confirmation and variant
identification
Assays designed for molecule
types ensure success

5. *FDA – entire sequence if vector is <40kb, for vectors >40kb modified regions of the vector only
Regulatory guidance for CGT and vaccines require
sequencing for identity
5
Plasmid Sequence Identification
EMA and FDA Guidelines include full sequencing of plasmids used
to produce gene therapy vectors
Virus Vaccine Sequence Identification
FDA Vaccine Guidance includes the analysis of master seed virus
sequences and potential analysis of subpopulations
Virus Vectors for Gene Therapy
EMA and FDA guidelines recommend sequencing of
entire vector genome.*

6. Sanger NGS
What are your options when it comes to performing ID
assays?
6

7. Test Specification Disposition
Mycoplasma None Detected PASS
Sterility Sterility Confirmed PASS
Titer Above 1e12 VGC/mL PASS
Residual Host Cell
DNA
Below 1 ng/mL PASS
Vector Sequence Match to Reference FAIL
Unexpected Results
Case Study: Product Release of an AAV
Huh? Didn’t
expect that!
7

8. 8
Unexpected Results
Case Study: Product Release of an AAV
Problem:
- Vector sequence identity did not meet
specification.
Questions:
- What happened?
- How different is it from the reference
sequence?
- Number of bases different?
- Location of bases?
What test was performed to identify these
issues?
- Impact to product?
- Possible sources of the variability
in sequence?
- Biologically derived?
- From raw materials?

9. Identity Results via Next Generation Sequencing
Case Study: Product Release of an AAV
ASSAY NUMBER SPECIFICATION RESULT*
706510GMP.BSV Report Result
Reference Sequence Coverage= 99.99%.
Similarity to Covered Reference = 99.78%.
IDENTITY TESTING (ID) RESULTS
Total # of
Reads Used
for Mapping
Reference
Sequence Used
for Mapping
Accession
Length
(Bases)
Total # of
Mapped
Reads
% of
Population
Mapped
Reads in
Aligned
Pairs
Average
Depth of
Coverage1
Consensus
Length
Generated By
Mapping2
%
Reference
Coverage
%
Similarity
to
Reference
Total Number of
Unmapped or
Low-Quality
Positions3
22,463,940 EXM-2345 7,557 413,176 1.84 322,734 5,396.69 7,556 99.99 99.78 1
What do the results look like?
- Specification was 100% match to reference sequence. Results show 99.78% (~17 bp)
- Does it matter?
- Why is there a problem?
- What are the actual variant locations and nature?
- Why is this showing up in my final product?
9

10. Questions:
1. What is the impact of the variants to my product?
2. Where are the variants located in the sequence?
1. Coding region?
2. ITR?
3. Promoter?
3. Where did the variants come from?
1. Is it Biologically derived?
2. Is it from my raw materials?
Next Generation Sequencing Results
Case Study: Product Release of an AAV
10
CHARACTERIZATION OF VARIANTS
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)
Max Number of
Reads
Supporting
INDEL (IDV)
Raw Read
Depth (DP)
Variant
Frequency
(%)
Variant Type
5,702 G GTACGGCC N/A N/A N/A N/A 1,047 1,298 80.66 Insertion
5,811 C T 5,401 286 2,267 714 N/A 9,349 34.39 Substitution
5,856 T C 7,306 517 3,798 1,997 N/A 18,848 42.55 Substitution
5,885 T A 4,672 905 1,917 995 N/A 13,726 34.3 Substitution
5,885 ATGACATC T N/A N/A N/A N/A 3,845 13,726 28.01 Deletion

11. Where does ID testing fit?
11
Following production, sequencing can be
used to establish vector identity and
production consistency
Identity testing is
established early in
the development
process, and
performed on
starting materials
such as plasmids or
virus seed stocks

12. Further sequencing of the raw material during investigation found the same sequence variations in the plasmid
used to produce the AAV vector.
- Importance of establishing the sequence early on in the process using the most sensitive methods
- QC testing using the SAME TECHNOLOGY on both the raw material and the final product
- RECOMMENDATION: Use of Next Generation Sequencing for QC of Raw Material and Final Product.
- Identification of Low-Level Variants that could have an impact on your final product.
- Low level variants are typically not seen via traditional methodology (e.g. Sanger Sequencing)
- Insurance Policy to mitigate unexpected results.
Importance of early testing for RISK MITIGATION
From Plasmid to Final Product
12
G
ATGACATC
T
C
T ATGACATC C T G T

13. ID Testing Workflow
13
Bioinformatics
- Read mapping against a client-provided reference
sequence (typically derived from Sanger consensus)
- Proprietary Variant Caller Algorithm is fully validated
- Detected variants:
- Insertions
- Deletions*
- Substitutions*
- Multi-nucleotide variants (MNV’s)
- Variant Detection included:
- Under 1%: No Variant Reported
- 1-10%: Low confidence variant reported for
informational purposes
- Above 10%: High confidence variant reported
- *Variant detection is limited to under ~20bp due to read
mapping algorithm used.
DNA Fragmentation
Library Preparation
DNA Extraction
DNA Sequencing
ATGCCAAGCAT
CGCTGTACGTA

14. Plasmid
ID
Vector
characterization,
yielding plasmid-only
results in the presence
of bacterial host DNA
Select the right NGS method based on your sample type
14
Release &
characterization testing
for viral vectors and
vaccines
Bacterial DNA
sequencing for whole
genome or gene-of-
interest analysis
RNA sequencing to confirm
identity of a vaccine or
therapeutic drug product
Customized
gene-of-interest
characterization to
evaluate rare, short
sequences
Targeted capture-
based sequencing to
evaluate long,
integrated sequences
Virus ID
Bacterial
ID
RNA ID
Amplicon
ID
Targeted
capture
ID

15. Select a service provider offering more than just raw data
15
 Dependable
bioinformatic
algorithms
 Dedicated
bioinformaticians
 Electronic data
delivery
 Secure data
management
 Expert-delivered
data analysis
 QA review &
approval
 Detailed reports
and raw data
available
 Industry-leading
scientific support
 Regulatory support
 Method
customization
when needed
 Global availability
 Facilities in the US
& UK
 Increasing capacity
to support growing
demand
 Validated
workflows, from
sample receipt to
final report (per
GMP guidelines)
 Assay validation
data is part of an
FDA Biologics
Master File (BMF)
Trusted GMP
services
Industry-leading
footprint
Secure data,
reliable results
Expert analysis
and QA review
Support when
you need it

16. 16
Lessons learned from the case study
1
3
2
Risk Mitigation using
appropriate testing,
even if not “required”
Use a reputable service provider
for continued support through
your regulatory process
QC check materials and
different parts in the process
using the same sensitive
technology
Avoid surprises

17. Bradley.Hasson@milliporesigma.com
Brad Hasson
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