Watch the presentation of this webinar here: https://bit.ly/33WUiqE Ensuring the safety and quality of your lentiviral vector is of the utmost importance. Attend this webinar to learn about testing strategies to monitor replication competent lentivirus. You will also hear about recent changes in regulatory guidance with regards to sample types and volumes tested. The use of lentivirus vectors to produce groundbreaking gene therapies is on the rise. Ensuring the biosafety and quality of these vectors is achieved through a multi-tiered testing approach. For lentivirus-based therapies, generation of replication competent particles is a potential risk. While improvements in design and manufacturing have decreased the probability of producing replication competent viruses, regulatory agencies provide guidelines to test for their presence at multiple stages in production. This webinar reviews the strategies for monitoring replication competent lentiviruses. We describe current methods and address: Sample types, testing volumes, and expected results. In this webinar, you will learn about: • The latest FDA regulatory guidelines on replication competent lentivirus (RCL) testing • Methods used to monitor RCL • Considerations on sample type and volume requirements

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Biosafety in Gene
Therapy: Applying the
latest regulatory
guidance for RCL
testing
Leyla Diaz, PhD

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

3. Agenda
Review Recent Updates to the Replication Competent
Lentivirus (RCL) FDA Guidelines
Application of RCL Testing in the Manufacturing Process
Discussion on potential impact of updated guidelines
1
2
3

4. 44
Replication Competent Lentivirus Testing
Testing of Retroviral Vector-
Based Human Gene Therapy
Products for Replication
Competent Retrovirus During
Product Manufacture and
Patient Follow-up, January
2020
1
Provides recommendations on testing strategies
during Lentivirus vector production and cell
transduction.
2
Outlines general testing methods including types of
samples and amount of material.
3
Guidance recommendations are based on
accumulating experience with different vector
systems, detection assays and patient monitoring

5.  Efficient gene delivery
 Integration of desired gene into the host genome
 Use of pseudotype envelope increases cell
tropism
 Most commonly used vectors
 Gammaretroviruses – MuLV and FLV
 Lentiviruses – HIV, SIV
Lentiviruses in Gene Therapy
As gene delivery systems they have been used
for a variety of gene therapy applications
These vectors offer several advantages for
gene therapy applications
5
 Production strategies that minimize the possibility
of RCL generation
 Testing at multiple manufacturing points
 Patient Monitoring
 Recombinational events during manufacture may
lead to a replication competent, potentially
pathogenic virus
Safety Concern Risk Mitigation

6. • Divide virus genes into multiple
plasmids
• Reduce regions of complementarity
• Packaging signal restricted to vector
plasmid
• Self Inactivating Virus (third
generation)
• Deletion of U3 from LTR
• Results in vector that does not
produce full length RNA
Safety by
Design
6
Third Generation Packaging Vectors
Packaging Plasmid
Envelope Encoding Plasmid
REV Encoding Plasmid
Vector Plasmid
LTR gag
pol
env
vif
vpr
vpu
rev
tat
LTR gag
pol
RREPromoter
Promoter env
Promoter rev
ΔU3LTR ψRREPromoter Promoter transgene ΔU3LTR

7. RCL Testing Strategy
7
Master Cell
Bank (MCB)
Cell culture RCL
Cell Line CharacterizationStable Vector
Producing Cells
Or
Cell
Substrate
Packaging and
Transfer Vector
Plasmids
Transient
Transfection
Cell culture RCL
Vector Supernatant
EOP Cells
Transduction Infusion
Vector Transduced
Cells
Cell culture RCL
Product Release
Patient Monitoring
Serology
PCR

8. RCL Cell Culture Method
8
Culture on a permissive
cell line
RCL Detection
• Indicator cell line
• p24 detection
• Reverse transcriptase activity
• PCR amplification of viral
genes
RCL Amplification for at
least five cell passages

9. Use a permissive cell line appropriate for the
lentivirus most likely to be produced by the
cells
MCB
 Cells
 Supernatants from production of MCB
How much to test?
 1% of total cells or 1e8 cells (lesser amount)
9
Vector Producing Cell Testing
Master Cell
Bank (MCB)
Stable Vector
Producing Cells

10. Volume
What to test
Vector Supernatant Testing
 Supernatant containing vector
 End of production cells
 EOP cells – 1e8 cells
 Supernatant
 5% up to 300 mL OR
 Volume to demonstrate that preparation
contains <1 RCL/patient dose
 Based on Dose Equivalent
 Amount of virus needed to transduce
cells
 Amount of virus to be administered
directly to patient
 Used to calculate volume at which the
probability of detecting 1 RCL is 95%
10
10L Production Volume
Cells to be
Transduced
1x108 1x108 1x108
MOI 0.5 1 10
Vector Titer 1x107 1x107 1x107
Dose
Equivalent 5 mL 10 mL 100 mL
Volume
Tested 2020
Guidance
15 mL 30 mL 300 mL
Volume
Tested 2006
Guidance
300 mL 300 mL 300 mL

11. 11
Testing Considerations for Vector Supernatant
Positive Control
Negative Control
Spike Control
Test Sample
Number of flasks depends on:
• Volume Tested
• Volume in which a single RCL
can be detected
RCL Detection
Total Volume
30 mL for test
Spike control = 5 mL
Total volume for RCL = 35 mL
Number of Flasks
Detection of 1 RCL in 5 mL
6 flasks test sample flasks

12. Test all transduced cell products, regardless of
length of culture
• What to test
• Cells at 1% total or 1e8 (lesser amount)
• Culture supernatant – 5% up to 300 mL
Non-cryopreserved cells
• Must be administered before testing is complete
• Initiate RCL at time of administration
• Use alternative method to provide initial assessment
• Method should be developed in consultation with regulators
• PCR assays commonly used for this purpose
Reduce or Eliminate RCL Testing
• Provide information on vector safety features
• Provide information on testing according to regulations
• Accumulated manufacturing and clinical data
• Consistent RCL negative testing for ex-vivo cells
• Sample archive if RCL testing is not performed
ex vivo Transduced Cells
Title of Presentation | DD.MM.YYYY12

13. 13
Summary of Changes in 2020 Updated Guidance
Material to be Tested 2006 Guidance 2020 Guidance
MCB (producer cells) Yes Yes
WCB (producer cells) Yes No
Vector Supernatant* Yes Yes
EOP Cells Yes Yes
Transduced cells (>4
days culture)
Yes Yes
Transduced cells (<4
days culture)
No Yes

14. 14
Summary
Incorporate Updated 2020
Guidelines into Testing Plan
1
3
2Testing is required to
ensure the safety of
lentivirus based vectors
.
Early testing strategy
development
 To ensure material is available for
testing
 Gather sufficient, high quality data
for eventual reduction or removal of
RCL testing

15. Leyla.diaz@milliporesigma.com
Leyla Diaz, PhD
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