Watch the presentation of this webinar here: https://bit.ly/3MJ4u9V Testing for presence of replication competent adenovirus (RCA) is a key component to ensure patient safety and a requirement for all biologicals manufactured using adenoviral vectors. For many adenoviral-based products, the RCA assay is a rate-limiting assay for lot release. Join this webinar to learn about a rapid RCA detection assay currently in development, which combines a 7-day culture assay with a highly sensitive molecular endpoint specific for RCA. The method can detect presence of as little as 1 RCA in adenoviral vector material at an approximate concentration of 5x107 - 2x108 vector particles (VP)/mL, making it a suitable method to meet regulatory requirements while accelerating your lot release timelines. In this webinar, you will learn about: • Regulatory framework for adenoviral vector products • Considerations for lot release testing of adenoviral-based therapies • Advantages of a rapid method for RCA testing on production lot material Presented by: Axel Fun, Ph.D., Principal Scientist Alberto Santana, MBA, Product Manager, Biologics Biosafety Testing

1. The life science business of Merck KGaA, Darmstadt, Germany
operates as MilliporeSigma in the U.S. and Canada.
Alberto Santana, MBA
Axel Fun, Ph.D.
Rapid Detection of Replication-
Competent Adenovirus (RCA) in
Adenoviral Vector Material

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

3. Agenda
Risk mitigation & testing strategies for Adenoviral based
therapies
Testing for RCA
A rapid alternative for RCA detection
1
2
3

4. Viral Risk Mitigation Strategy for gene & cell therapy
Safe sourcing and
testing of raw
materials
Verify absence of viral
contaminants at
appropriate
stages
Verify capacity of manufacturing process to remove
or inactivate potential viral contaminants
Difficult for enveloped viral vectors – but possible for Adenovirus
A virus safety risk assessment should be conducted
(process indicated in European Pharmacopoeia 5.1.7)
4

5. Increase in regulations for cell and gene therapy observed in
recent years
FDA: Content and review of CMC
information for
 Human gene therapy INDs
 Human somatic cell therapy INDs
EMA: Guideline on human cell-based,
gene therapy medicinal products
EMA: GMP for ATMPs
EU Draft : Annex 1 revision for
sterile medicinal products
PIC/S Annex 2A Draft guidance for
the manufacture of ATMPs
NIFDC. China: Quality Control of
CAR-T Cell Therapy Products and
Consideration for Non-clinical
Research
FDA: Potency tests for cellular and
gene therapy products
FDA Draft guidance on CMC and Retroviral
testing guidance
EMA Guideline on quality, non-clinical and
clinical aspects of medicinal products
containing genetically modified cells
FDA: Finalized guidance
 Testing of retroviral vector-based
human gene therapy products for
replication competent retrovirus during
product manufacture and patient
follow-up.
 Chemistry, manufacturing and control
(CMC) for human gene therapy INDs
ChP: General Chapter of Gene Therapy
Products for Human Use (draft)
2008 2011 2017 2018 2019 2020
5

6. Building a testing strategy – what and when to test
MCB WCB /
CAL
Vector
Production
Unpurified Bulk
Harvest
Final drug
product
raw materials
Seed Virus
Master
Virus Seed
Stock
(MVSS)
Working
Virus Seed
Stock
(MVSS)
Purified Bulk
Harvest
Identity
Titer
Biosafety
Replication Competent Adenovirus
The Ph. Eur. 5.14 states “Provided that the tests for bovine serum albumin (when bovine serum is used to manufacture
the vector) and replication-competent adenoviruses have been carried out with satisfactory results on the final bulk,
they may be omitted on the final lot”. The FDA CMC for Human gene therapy IND applications (2020) “We
recommend that you qualify your MVB for RCA and test either the DS or DP of each production lot for RCA”

7. 7
Building a testing strategy
Identity
Microbial & other
Contaminants
Replication Competent
Adenovirus
QPCR, NGS
Adenovirus Fluorescent Focus
Unit (FFU), Genomic Titer PCR,
Quantitation of Adenovirus
Particles
Sterility, PCRs, ELISA,
RCA
Titer
Biosafety
Days 7 14 28 35 42 48
Microbials &
Others
Titer
Identity
Replication Competent Adenovirus
Longest turn around time of each category
Reducing the RCA assay turn around time will represent a
significant improvement to accelerate the release of each
batch
Turn around time per assay category

8. Background on assays for the
detection of replication
competent adenovirus (RCA)
❖ Required for all biologicals manufactured using
adenoviral vectors
❖ Demonstrate ≤1 RCA in a specified quantity of
production material.
❖ 2020 FDA guidance recommends testing each lot
of drug substance or drug product and
demonstrate ≤1 RCA per 3 x1010 VP.
❖ Recommendations to include positive controls
spiked in the test article to assess whether there
are any inhibitory effects of the test article on
detection.
8

9. 9
Rapid Replication
Competent Adenovirus
(rRCA)
New Rapid RCA assay will provide a
faster solution for the detection of
RCA for cell therapy/vaccines
customers using an adenoviral
platform

10. Rapid Replication
Competent Adenovirus
(rRCA)

11. Gold standard assay for the detection of RCA
11
❖ Requires large vessels (e.g. roller bottles, T175 flasks) to be cultured for 28 days
❖ Turn-around-time (TAT) 42-56 days
Day 1 Day 8 Day 15 Day 22 Day 29
Feed of cultures
performed
Passage 1
performed
Passage 2
performed
Final day read
performed
RCA detected
No RCA
detected
Report
Further investigation
required

12. Novel assay for the rapid detection of RCA
12
Inoculation
Sample harvest
& PCR analysis
 Samples can be stored prior to endpoint
analysis
 DNA extracted using automated extraction
 PCR analysis targets E1A gene
 E1A PCR Detection Limit (DL) is 20
copies/reaction
 1 day cell preparation followed by 7
days culture plus 1-3 days for PCR
analysis
 Maintaining assay DL of 1 RCA (PFU)
 Expected TAT 14-21 days
Day 1 Day 8
Increased flexibility and capacity 50% reduction in turn-around-time

13. Cytotoxicity and interference pre-study design for the
Rapid RCA assay
13
 3 control flasks: negative, 5 PFU/flask and 1 PFU/flask Adenovirus type 5 (Ad5) positive control
 3 different Test Article (TA) concentrations tested in a quantile manner that corresponds to an exact
number of flasks required for the RCA assay
 If required, more than 3 TA concentrations can be tested
 7-day culture

14. Cytotoxicity and interference pre-study design for the
Rapid RCA assay
14
For testing
5x1010 VP
For testing
3x1010 VP
5x107
VP/ml
1.5x108
VP/ml
1x108
VP/ml
8 TA T175
flasks
12 TA T175
flasks
24 TA T175
flasks
8 TA T175
flasks
12 TA T175
flasks
20 TA T175
flasks
1x108
VP/ml
2.5x108
VP/ml
1.67x108
VP/ml

15. Example cytotoxicity and interference pre-study
15
Testing TA 1 at 1x108, 1.5x108 and 1x109 VP/ml
Sample Cq value
NTC
NEG
NEC -ve
NEC +ve
n/a
n/a
n/a
n/a
Negative control
Positive control 1 PFU
Positive control 5 PFU
n/a
29.9
18.3
TA 1@ 1e8 - 1 PFU - rep 1
TA 1@ 1e8 - 1 PFU - rep 2
TA 1@ 1e8 - 1 PFU - rep 3
26.0
27.8
32.2
TA 1@ 1e8 - 5 PFU 26.7
TA 1@ 1.5e8 - 1 PFU - rep 1
TA 1@ 1.5e8 - 1 PFU - rep 2
TA 1@ 1.5e8 - 1 PFU - rep 3
32.9
32.6
31.2
TA 1@ 1.5e8 - 5 PFU 22.8
TA 1@ 1e9 - 1 PFU - rep 1
TA 1@ 1e9 - 1 PFU - rep 2
TA 1@ 1e9 - 1 PFU - rep 3
31.5
30.9
33.5
TA 1@ 1e9 - 5 PFU 31.0
Std 2e1 (DL) 31.6
1x108 VP/ml
=
2.5x109 VP/flask
3x1010 VP
=
12 flasks

16. Example - Rapid RCA assay results – testing 3x1010 VP
16
Testing TA 1 at 1x108 VP/ml

17. 17
Example – Mock rRCA with ‘contaminated’ TA
3x1010 VP TA 1 in 300 mL spiked with 1 PFU, divided over 12 flasks

18. 18
18
Final takeaways
Days 7 14 28 35 42 48
Microbials &
Others
Titer
Identity
Rapid RCA
Longest turn around time of each category
Turn around time per assay category
New Rapid RCA assay will provide a fit-for-purpose,
faster solution for the detection of RCA for cell
therapy/vaccines customers using an adenoviral platform
Final Rapid RCA TAT to be confirmed ~14 to 21 days

19. Alberto.santana@milliporesigma.com
Axel.fun@merckgroup.com
Alberto Santana, MBA
Axel Fun, Ph.D.
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