Watch the webinar here: https://event.on24.com/wcc/r/2003970/F5AFA4FE6C60AD00635D4D15BADB5D8E?partnerref=slideshare As gene therapies and gene-modified cell therapies show increasing promise, the need for innovative and proficient viral vector manufacturing continues to grow. Concurrently, increased regulatory guidance governing the manufacturing and testing of viral vectors adds complexity and increases the timelines to successfully produce high-quality virus ready for clinical use. This webinar will address how the implementation of both manufacturing templates and platform characterization and safety assays can increase the likelihood of success in process validation and reduce risk in the timeline to commercialization for your gene therapy product. Using adeno-associated virus (AAV) as a case study, we will demonstrate how our validated, templated process for production can reduce the need for qualification inherent in niche manufacturing workflows and anticipate forthcoming needs for process performance qualification. This webinar will also highlight benefits from a new, platform assay offering for characterization and safety testing of AAV. Because these assays are pre-qualified, they reduce the variability inherent in assay validation and subsequently the time needed to establish readiness for regulatory compliance. While these developments increase the standardization across the manufacturing and testing workflows, they remain flexible to clients' needs and are created to be scalable and as future-proof as possible, allowing for adaptability as the regulatory landscape of gene therapies evolves. In this webinar, you will learn: ● The unit operations in AAV manufacturing that are ideal for templating ● How the manufacturing workflow can be targeted to reduce variability in testing and improve readiness for commercial production ● How platform assays can ease the burden of assay qualification and improve overall commercialization timelines

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Employing Innovative Platform
Manufacturing and Biosafety Testing
for your Gene Therapy Product to
Reduce Time to Market
Elie Hanania, Ph.D.
Head of Process Development
Marian McKee, Ph.D.
Senior Director, Head Global Testing
R&D Services

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

3. Agenda
1 Targeting the manufacturing workflow to
reduce variability in testing and improve
readiness for commercial production
2 Utilizing platform assays to ease the
burden of assay qualification and improve
overall commercialization timelines

4. Where is Gene Therapy Heading?
4 Gene Therapy: Managing Development Timelines | 13.06.2019
 Surge of Cell and Gene Therapy Products (Early Development)
 Gene therapy can treat what is deemed incurable diseases
 Scientific progress and improvements
 Increase in the number of INDs
 Growth in the number of approvals
 … by 2025, we predict that the FDA will be approving 10 to 20 cell and gene
therapy products a year*
* Statement from FDA Commissioner Scott Gottlieb, M.D. and Peter Marks, M.D., Ph.D., Director of the Center for Biologics
Evaluation and Research on new policies to advance development of safe and effective cell and gene therapies – January 15, 2019

5. Gene Therapy Trials and Commercial Products
5
1644
2914
634
485
151
I I/II II II/III Total
Trial Phase
Source: The Journal of Gene Medicine, December 2018
Approved Gene Therapy Products:
• Gendicine® recombinant adenovirus
• Oncorine® oncolytic adenovirus
• Glybera® adeno-associated virus therapy
• Imlygic® oncolytic viral therapy
• Zalmoxis® allogeneic T cells
• Strimvelis® autologous CD34+ enriched cell
fraction
• Luxturna® adeno-associated virus vector-
based gene therapy
• Kymriah® CAR-T cell therapy
• Yescarta® CAR-T cell therapy
Gene Therapy: Managing Development Timelines | 13.06.2019

6.  Raw Materials and
Reagents
 Equipment
 Consumable Sets
 Single Use Technology
6
Path to commercialization – critical factors
 Product Quality
 Product Yield
 Titer
 Purity
 Equipment
 Unit Operations
 Mode of Operation
 Stability
 Tolerance
 Feasible Range
 Process Time
1
4
2
3
cGMP Compatible
Reproducible
Scalable
Robust
Gene Therapy: Managing Development Timelines | 13.06.2019

7. What Do We Need for Late Phase and
Commercial Manufacturing?
7
Suitable manufacturing technologies (large and small)
Efficient manufacturing approaches
Stable and reproducible product
Gene Therapy: Managing Development Timelines | 13.06.2019

8. 8
Interdependency of Process and Product
PRODUCT
PROCESS
Gene Therapy: Managing Development Timelines | 13.06.2019

9.  Transient transfection mediated by polyethylenimine (PEI): co-transfection of
AAV production cells with 3 plasmids:
• Plasmid with AAV ITR and the gene of interest
• Plasmid with AAV rep/cap
• Plasmid providing the helper genes isolated from adenovirus
 Wild-type adenovirus infection into cell lines with AAV rep/cap genes and AAV
vector.
 Infection using two HSV viruses harboring the gene of interest and the rep/cap
genes to produce AAV.
 Infecting Sf9 cells with two baculoviruses harboring the gene of interest and the
rep/cap genes to produce AAV.
 Stable producer cell lines.
Five Major Production Modes for Recombinant AAV
9 Gene Therapy: Managing Development Timelines | 13.06.2019

10. Production Flow Chart for rAAV Using Transient Transfection
10
Thaw and expand
HEK293 cells
Seed culture
vessels
PEI-mediated
transfection
Harvest
Benzonase® nuclease
treatment and
Clarification
Concentration and
Diafiltration
Affinity
Chromatography
2nd TFF
Concentration and
Final Formulation
Sterile Filtration
Fill and Finish
Ion Exchange
Chromatography
Gene Therapy: Managing Development Timelines | 13.06.2019

11. 11
Characterization: Process & Product
Early Stage Late Stage
• Mass Balance
• Process
appropriateness
• Material/supplies
appropriateness
• Scale assessment
ProductProcess
• Safety
• Reproducibility
• Trending
• Contaminants
• Residuals
• Potency
• Stability
• Identity
• Appearance
• Titer
• Infectious titer
• Purity
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12. 12
•Process Design
•Process Knowledge
(parameters and
attributes)
•Process Risk Assessment
•Design Space
•Control Space
•Validation Master Plan
Product
Design
•Equipment Qualification
•Assessment for
reproducibility
•Process
Characterization
Product
Reproducibility •Continued Process
Verification
•Continuous
improvement
Product
Performance
Process Validation
Quality, safety, and efficacy are designed into the product.
Each step of a manufacturing process must be controlled to assure that the finished
product meets all quality attributes
Gene Therapy: Managing Development Timelines | 13.06.2019

13. Value of Templated Process
13
Templated processes speed path clinical material and improve quality through standardization:
 Cell banking activities
 Tech transfer / most of process development
 Engineering run
 Custom Bill of Material / Batch Record creation
Largest benefit when leveraging production cells through downstream processing, avoiding:
 Minimize source of variation
 Defined product characterization
 Utilize defined production parameters
 Good basis for process validation
Gene Therapy: Managing Development Timelines | 13.06.2019

14. 14
Savings with Templated Process
1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
Limited
WCBMCB
Extensive
Process Development
Release to clinic
GMP1
Activity
Confirmation
Testing
BOM/BR Eng. Run
Clinical
Engineering
Limited
GMP1
Release to clinic
Cell Bank
Testing
Tech Transfer
Confirmation
Gap/Risk Analysis
Clinical
Process Development
TemplateCustom
Potential savings: 14-18 months
Gene Therapy: Managing Development Timelines | 13.06.2019

15.  Process Validation is designed to ensure the
delivery of Quality Product
 Operating Parameters and Output Parameters in
Manufacturing Viral Vectors has an impact on
Product Performance
 Templated processes can reduce time, cost, and
improve quality through standardization
Summary
15 Gene Therapy: Managing Development Timelines | 13.06.2019

16. Characterization
& Safety Testing
Title of Presentation | DD.MM.YYYY16

17. 17
Administration impacts testing
Viral Gene Therapy
In vivo (AAV)
Viral vector
is injected
DNA
(Gene)
Gene
encapsulated
in AAV
Gene Therapy
AAV
releases
gene into
cell
Gene
expresses
proteins
Target cell
Receptor
protein
Secreted
protein
Gene Therapy: Managing Development Timelines | 13.06.2019

18. 18
Safety and Characterization Testing
Plasmid or
Virus
Master Cell Bank (MCB)
Working Cell Bank
(WCB)
Process Development
(Growth/Production/Modification)
Plasmid Stock
Master/Working Virus Bank
(MVB/WVB)
Drug Substance
Drug Product
Cell
Identity
Safety
Stability
Lot Release
Testing
QA/QC
In-
Process
Testing
Identity
Purity
Safety
Identity
Purity
Safety
Gene Therapy: Managing Development Timelines | 13.06.2019

19. 19
Four Elements of Gene Therapy Testing
Identity Purity Potency Residuals
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20. 20
Identity
Confirm identity
of Viral Vector
Confirm identity
of Transgene
Confirm
Patient Cell ID
Identity Purity Potency Residuals
Genotypic
• Custom PCR
• Generic PCR
• Sanger
• NGS
• DNA fingerprinting
• Short tandem repeat analysis
• ELISA
• Mass spec
• HPLC
• Cell surface markers
(e.g. FACS)
Phenotypic
Gene Therapy: Managing Development Timelines | 13.06.2019

21. 21
NGS-AAT
Sterility
 Sterility
 BACT/ALERT® 3D
Microbial Identification
System
 Detects changes in
pH due to bacterial
growth
 Real time sample
monitoring
 Objective readout
 Adventitious Virus
 Detection and
identification of
adventitious agents
 Circumvents toxicity
and neutralization
issues
 Can be combined with
ID test
 Replication Competent
Virus
 Cellular assay
 Three or more rounds
of amplification
 CPE, qPCR or QPERT
endpoint
 Mycoplasma
 PCR
 Equivalent sensitivity
and specificity to
compendial method
 GMP and EP 2.6.7
compliant
 TAT and sample
requirements better
suited for cell
therapies
Mycoplasma rcVirus
http://www.med.upenn.edu/gtp/images/e20_
aav_med_full.jpg
Identity Purity Potency Residuals
Gene Therapy: Managing Development Timelines | 13.06.2019

22. 22
Product Related
Impurity
Nature of Lot Release Test Used
Empty Capsids
Chromatography (IEX), Ultracentrifugation;
EM
Nuclease resistant Host
cell DNA (encapsidated)
qPCR to target generic host cell sequences
or specific sequences of concern e.g. AdE1
Nuclease resistant helper
DNA (plasmid)
qPCR to target generic helper virus
sequence
Replication competent
Virus
Various depending on Vector system
Non-infectious particles Total viral particle (VP): infectious unit (IU)
Aggregated, oxidised,
degraded vector
Size exclusion chromatography,
electrophoresis, DLS and others.
Identity Purity Potency Residuals
Gene Therapy: Managing Development Timelines | 13.06.2019

23. 23
Potency
Key Elements:
• Sensitive enough to discriminate small differences in biological
activity and stability indicating
• Quantitative readout over a range of treatment concentrations
• Endpoint analysis is suited to consistently and accurately
measure the biological effect
– Easy to use & robust
• Relevant controls and appropriate data analysis methods
Identity Purity Potency Residuals
Gene Therapy: Managing Development Timelines | 13.06.2019

24. • Common attributes
• Cell infection (293, C1886, HeLa)
• Virus specific endpoint
• PCR
• Immunofluorescence
• Plaque/foci count
• Measure of amount of infectious titer present in
the virus stock – live virus
• Used to monitor production process (in process
testing)
• Used to guide dosing
• Patient
• Cell transduction
• Quantification methods vary depending on
virus.
• AAV – TCID50 (tissue culture infectious
units)
• Lentivirus -- TCID50
• Adenovirus – Focus forming units (FFU)
24
TCID50
Identity Purity Potency Residuals
Gene Therapy: Managing Development Timelines | 13.06.2019

25.  Droplet Digital PCR – ddPCR
 Divides the PCR reaction into 20,000 droplets
 Positive droplet – DNA or RNA present
 Negative droplet – DNA or RNA not present
 Result is based on an absolute count of positive
and negative droplets
 No standard curve is needed.
 Higher accuracy and precision of quantitation
than qPCR
 More robust to lower copy number levels
 Resolve smaller differences in copy number
 Higher number of technical replicates
(20,000)
 Measures the number of particle associated
vector genome copies.
 DNA copies for AAV
 Measures total virus content (infectious and non
infectious)
 Used to guide dosing
 Involves a PCR-based method
 CMV promoter
 Gene of Interest
25
Genomic Titer
Identity Purity Potency Residuals

26. 26
Residuals
Process Related Impurity Nature of Lot Release Test Used
Host cell protein
Immunoassay using HCP specific antibodies (ELISA,
chemiluminescent IA)
Host cell DNA
qPCR to target generic host cell sequences or
specific sequences of concern e.g. AdE1
Residual cell culture related
components e.g. BSA, HSA
Various approaches, however immunoassay
common for abundant proteins such as BSA, HSA
Residual process reagents
e.g., Benzonase®
nuclease,
chromatography ligands
Various approaches, however immunoassay
common
Residual plasmid DNA qPCR targeting plasmid sequence (non-vector)
Identity Purity Potency Residuals
Gene Therapy: Managing Development Timelines | 13.06.2019

27. • Identity: GOI
• Titer: TCID50 of viral vector
• Purity:
• Bioburden
• Mycoplasma
• Mycobacterium
• Adventitious viruses (in vitro
& in vivo)
• Replication competent AAV
(rcAAV)
Testing AAV Bulk and Final Lots
Unpurified
bulk
Purified
bulk
• Identity: GOI, ELISA, Vector genome
• Titer: TCID50 of viral vector, Genomic
titer
• Potency: Expressed protein, function
• Purity: Sterility, Endotoxin, rcAAV
• Residuals:
• Residual host cell DNA
• Residual DNA size distribution
• Host cell protein, Residual BSA
• Residual AAV Affinity Ligand
• Purity by SDS PAGE
• Empty: Full Capsid
• Identity: GOI
• Titer: TCID50 of viral vector,
Genomic titer
• Potency: Expressed protein,
function
• Purity: Sterility, Endotoxin
• Product characteristics:
• Vector aggregates
• Osmolality
• pH
• Extractable volume
• Appearance
• Particulates
Formulated
& vialed
final lots
27 Gene Therapy: Managing Development Timelines | 13.06.2019

28. SUMMARY
● Targeting the manufacturing workflow to
reduce variability in testing and improve
readiness for commercial production
● Utilizing platform assays to ease the
burden of assay qualification and improve
overall commercialization timelines

29. marian.mckee@milliporesigma.comelie.hanania@milliporesigma.com
Marian McKee, Ph.D.Elie Hanania, Ph.D.
THANK YOU