Participate in the interactive webinar now: http://bit.ly/CGTWebinar This webinar will introduce phase-appropriate validation and why it may be advantageous for cell and gene therapy development. We will also describe how validated platform assays can help you meet your critical development timelines. Explore our webinar library: www.merckmillipore.com/webinars

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Is Phase-
Appropriate
Validation the Right
Choice for your Cell
or Gene Therapy?
Dr. Krista Spreng
Scientist, R&D Services
Dr. Nikolay Korokhov
Principal Scientist, R&D Services

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

3. Agenda
Defining Validation & Regulatory Guidelines
Validation Strategies
Applying Phase-Appropriate Validation
to a Potency Assay
1
2
3

4. Defining Validation
& Regulatory
Guidelines

5. Defining validation
5
Assay Development /
Optimization
Transfer
Validation
Verification
Assay design and method optimization; evaluate performance
characteristics prior to execution of a full validation;
determine validity and system suitability criteria
Assay performance comparison to demonstrate that parameters
validated at originating site can be reproduced at the receiving site
Formal process to demonstrate that the performance of an assay
meets the requirements for which it is intended
Formal process that demonstrates the performance of an assay is
followed per compendial chapter (USP, EP, JP, and CFR) without any
deviations

6. ConsensusRegulatory
Agencies
 Responsible for licensing
medicinal products in their
respective regions.
− Clinical trial approval
− Inspection of manufacturing
and testing facilities
 Country-specific organizations
 Publish monographs for
standard assays
 USP – US
 EP – Europe (40 member
council)
 JP - Japan
 Promotes harmonization of the
interpretation of technical
documents
 Reduce duplication of testing
 Founding members from US,
EU and Japan
− Comprised of representatives
from regulatory agencies and
industry organizations from
several countries
Compendia
6
Who sets the guidelines?

7. • FDA Guidance for Industry: Guidance for human somatic cell therapy and gene
therapy. Mar 1998
• FDA Guidance for FDA reviewers and sponsors: Content and review of chemistry,
manufacturing and control (CMC) information for human gene therapy Investigational
New Drug Applications (INDs). July 2008
• 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
Clinical:
• FDA Guidance for Industry : Gene Therapy Clinical Trials – Observing Subjects for
Delayed Adverse Events. Nov 2006
• FDA Guidance for Industry : Design and Analysis of Shedding Studies for Virus or
Bacteria-Based Gene Therapy and Oncolytic Products. Aug 2015.
The FDA’s “Cellular and
Gene Therapy Guidance”
web page contains
updates on guidance
documents covering:
• Vectors
• Clinical Trial Design
7
Regulatory landscape – US guidance

8. Draft Guidance for Industry: Chemistry,
Manufacturing, and Control (CMC)
Information for Human Gene Therapy
Investigational New Drug Applications
(INDs), dated July 2018
• AAV: 23 mentions
• Lentiviral vector: 3 mentions
• Adenovirus: 6 mentions
Guidance for FDA Reviewers and Sponsors:
Content and Review of Chemistry,
Manufacturing, and Control (CMC)
Information for Human Gene Therapy
Investigational New Drug Applications
(INDs), dated April 2008
• AAV: 4 mentions (exclusively rcAAV)
• Lentiviral vector: 0 mentions
• Adenovirus: 3 mentions (RCA)
8
Gene therapy regulatory facts

9. Drug development phases
9
Discovery
Pre-Clinical
Phase I-III
GMP
Licensed Product
GMP
Perform validation work towards the front end of drug
development instead of at the end
GMP: Testing for Drug Quality.
Clinical and Commercial Material Validated Assays a
Regulatory Requirement.

10. Validation
Strategies

11. 11
• No “one size fits all” approach
• Platform approach (generic validation and verification of
client material)
• In accordance with regulatory requirements and client need
Perform a level of validation based on the drug
development phase
“Phase Appropriate Validation”
A customized approach to validation

12. Phase-Appropriate Validation
 Early phase (I/II) clinical product
− Validation as per ICH – under the GMP
quality system
− Minimum required/sufficient level of
evaluation of key method parameters.
− Simplified protocol and report
“Qualification” Reserved for Equipment, Raw Material/Reagents
Validation
 Late phase (III) clinical and commercial
products
− Validation as per ICH – under the GMP
quality system
− In-depth evaluation of all method
characteristics
− Ensure acceptance criteria is met
12
Validation path

13. Validation documentation - development
13
Development
Report
Risk
Assessment
Optimization
Development
Protocol
Assay SOP
QC approved
and released
GMP reagents

14. Validation documentation - validation
14
Validation Execution - draft SOPs
and Batch Records
Validation Report
Trending Spreadsheets
Operational Implementation
1
2
3
4

15. Platform method approach
 Identify as
platform method
(rationale)
 Identify aspects
that can be
varied/adjusted
 Changes to
establish a
robust method
OptimizationRisk Assessment
 Defined
conditions and
specifications
 Full validation of
the platform
method with
representative
material
 Develop/qualify/
validate client
material
Validation
Protocol
 Review of assay
performance in
line with
validation
Regular Cycle of
Risk Assessment
 May be required
due to major
changes in assay
performance,
critical reagent
change, etc.
Revalidation
15

16. TCID50
Current Platforms
Droplet
Digital PCR
QPCR Sequencing
16

17. Potency Assay
Case Study
Title of Presentation | DD.MM.YYYY17

18. An introduction to AAV
Belongs to the
dependoparvovirus
subfamily – requires
a helper virus to
replicate
Small non-
enveloped DNA
virus – 25nm Can be
manufactured on a
large scale
Does not cause any
known disease in
humans – high
percent of the
population is
seropositive
2
1
43
18
One of the most promising candidates for therapeutic gene transfer

19. Let’s look
at AAV
Production Systems
19
Mammalian Cells Insect Cells
Triple Transfection
Helper-virus system
Baculovirus System
Vector Construct
Serotype Plasmids
Packaging Plasmid
Helper Plasmid
Synthetic serotypes are
becoming more common
HEK293, HeLa, BHK-21;
adherent or suspension
Sf9, suspension
13 Serotypes
Transfer Plasmid
Rep/Cap
Adeno: E1A, E1b55k, E4orf6, E2A,
E4, VA, HSV
Transgene

20. 20
AAV characterization
Identity
PCR identity assay
AAV serotype
Sequencing of vector
Purity
Sterility
Mycoplasma
Adventitious viruses
Replication competent AAV
Size distribution by DLS
Empty/Full Capsid (2020)
Titer/Potency
TCID50 infectivity assay
Viral Particles (ELISA)
Genomic Particle Count
(ddPCR)
Potency Assay (Custom)
Residuals
Plasmid or helper virus
Host Cell Proteins
Host Cell DNA
Benzonase® nuclease
BSA
AAV Vector

21. AAV testing challenges
1
5
2
3
ID (Serotype)
ELISA kit availability
Residuals (Production
Process)
Various production
processes
Impurity - rcAAV
(Serotype)
Assay controls, cell
substrate
Infectivity (Serotype)
qPCR target, cell substrate,
assay controls
Virus particle quantification
Empty/full
4
21

22. 22
AAV infectivity: TCID50
1 ml
etc…
H
G
F
E
D
C
B
A
N
1
-0.7
2
-1.4
3
-2.1
4
-2.8
5
-3.5
6
etc.
789101112
1 ml 1 ml 1 ml 1 ml
4 ml 4 ml 4 ml 4 ml
Virus
Dilution
Proportion of
positive wells
1
1
0.75
0.5
0.375
0.125
0
0
0
0
0
0
Wells are scored as + or -
50% Infection
< 50% Infection
> 50% Infection
Infect replicate wells with serial dilutions of sample

23. 23
AAV infectivity: TCID50
 Content/Potency (ICH Q2(R1))
Parameters included in validation: accuracy, precision, specificity, linearity and range
Expected modifications
Reference
Material
rAAV2-RSM
Helper: Ad5
FDA encourages the use of the RSMs as benchmarking tools
for qualifying inhouse reference materials and controls*
Test System
HeLaRC32
licensed
Not optimal for all serotypes#
End-point assay CMV-qPCR Not a universal target (would AAV2 ITR be better?)
Assay essentials: Reference Material, Test System and Assay End-Point
* - Gavin DK. FDA statement regarding the use of adeno-associated virus reference standard materials. Hum Gene Ther Methods. 2015 Feb;26(1):3
# - rAAV2-RSM has 1:7.5 TCID50:vgc ratio
rAAV8-RSM 1:456
The generic assay is based on AAV Reference Standard Working Group (AAVRSWG) published protocols with few
modifications.

24. 24
AAV Infectivity: from generic to client-specific
Assay essentials: Reference Material, Test System and Assay End-Point
End-point
qPCR
Test system
(cells/helper)
Reference
(Control)
Virus
Client-specific assay Development
Abridged validation
End-point
qPCR
Assay platform based validation:
➢ Simplified Validation Protocol and Report
➢ Document templates
➢ Limited number of tests
Assay platform
Generic assay
End-point
CMV-qPCR
Test system
HeLaRC32
Ad5
Reference
Virus
rAAV2-RSM

25. • Product Specific Qualifications (PSQ)
• Demonstrate that product or product matrix does not interfere with validated specifications
• Early Phase Products (Pre-clinical to Phase II)
• Production Process not validated yet
• Perform spiking study to assess effect of matrix on validated method specifications
• Internal Controls in test methods can serve this purpose
• Late Phase/Commercial Product
• Required for regulatory submission for drug license
• Uses representative batches of a defined production process
Product specific qualification
25

26. Custom vs platform method development and validation
26
Assay
Development
(new method)
Assay
Transfer
Assay
Validation
Ready
Test Method
Phase
Appropriate
Validation
Full
Validation
Development
Transfer/
Feasibility
Abridged
Validation
Custom
Method
Platform
Method
GMP
Assay
GMP
Assay

27. Conclusion
1
2
3
Validation strategy is a key part of the development
considerations for cell & gene therapy
Regulatory, regulatory, regulatory
Don’t lose sight of the big picture

28. krista.spreng@milliporesigma.comnikolay.korokhov@milliporesigma.com
Dr. Krista SprengDr. Nikolay Korokhov
THANK YOU