Learn about existing and emerging methods to accelerate biosafety testing of biologic therapies. Speed to market for biologic therapeutics is ever more critical. However, the critical safety tests for these molecules, for example screening for adventitious agents such as viral contaminants, can be time consuming as well as challenging and laborious. Join us for this webinar as we explore how rapid methodologies are being used to not only accelerate this process, but also enhance quality by reducing testing complexity. Existing technologies as well as emerging trends will be discussed, along with the implications these may have on the regulatory landscape. In this webinar you will learn: ● Which existing and emerging technologies are having now, and will have in the future, an impact on biosaftey testing. ● The benefits as well as risks of employing rapid methods for biosafety screening. ● How the regulatory agencies are reacting to rapid testing methods as alternatives to existing methods.

1. August 17, 2017
ONLY FOR U.S. AND CANADA AUDIENCE
Afshin Sohrabi, PhD
Principal Scientist, Development Services
Process Solution Services
Rapid Methodologies
for Biosafety Testing
of Biologic Therapies

2. Microbial Contamination & Production of Biologics
2
Manufacturing of Biologic Drugs
Final
Drug
Product
Research
Cell Line
Production Cell Line
(“Cell Bank”)
Cell engineering, selection
& integration site analysis
Media development,
Raw Materials selection
Chromatography
resin selection, yield
optimization, product
characterization
Formulation
development, stability
testing, fill & finish
“In process”
testing
Bioreactor
Purified Bulk
Drug Substance
Unprocessed
Bulk Product
Upstream processing Downstream processing
Material (Cell, Virus, Raw Material) Characterization Lot Release Testing
• Production of biopharmaceuticals (i.e.,
biologics) typically involves the use of
living cells
• As a consequence, the manufacturing
process and products are prone to
contamination by exogenous
(Adventitious) agents and endogenous
microbial agents (e.g., retroviruses for
rodent cell lines)

3. 3
Historical contamination events for biologics
Why does adventitious agent testing need to be
performed?
Human Products:
• Contaminated with blood borne viruses:
HIV, HBV, HCV, B19 parvovirus
• vCJD (whole blood)
Excipients:
 Yellow fever vaccine formulated with
human serum albumin that was
contaminated with HBV
 Recent PCV (porcine circovirus) DNA
contamination in rotavirus vaccines
Cell Lines:
• Rhesus Monkey cells used to produce
polio vaccines contaminated with
SV40
 SV40 now classified as a
human pathogen
• Contamination events in CHO
cells : Reovirus; Epizootic
Hemorrhagic Disease Virus
(EHDV); Minute Virus of Mice
(MMV, a parvovirus); Cache
Valley Virus (Bunyavirus);
Calicivirus

4. Manufacturing of Generic Biologics
Large Scale Process with multiple handling points prone to contamination
with Adventitious Agents - Testing Points to mitigate risks
4
Current Testing Methodology
In Vitro virus Detection: Cell Culture Based Assay

5. 5
Regulatory Agencies
US Food and Drug Administration (FDA)
 Center for Drugs Evaluation and Research (CDER)
 Center for Biologics Evaluation and Research (CBER)
China
• China Food and Drug Administration (CFDA)
Japan
 Pharmaceuticals and Medical Device Agency (PMDA)
EU
• European Medicines Evaluation Agency (EMA)
 MHRA
Korea
 Ministry of Food and Drug Safety (MFDS)
World Health Organization
Biological Safety Testing is Mandated by Regulatory Agencies Worldwide
5

6. 7
Value of Scalable NRTT

7. Testing tools are rapidly evolving to better
evaluate and mitigate risk....
Standard PCR  qPCR  Digital PCR
Sanger  Next Generation Sequencing (NGS)
Traditional  Rapid Chemical based Testing
8

8. A poll will now
occur

9. 10
Once we
accept our
limits,
we go beyond
them.
Albert Einstein

10. Regulatory Documents in Support of Molecular Based Testing
Excerpt for Page 28 of FDA Guidance
11

11. Ph. Eur. Chapter 5.2.14: Substitution of in vivo method(s) by in vitro methods for the quality of
vaccines (Pharmeuropa 28.2; April 2106)
“…Novel sensitive molecular techniques with broad detection capabilities are available, including deep
sequencing or high throughput sequencing methods, degenerate PCR for whole virus families or random
priming methods…”
Regulations are not Opposed to Alternative Methods
12
More Regulatory Documents in Support of Molecular Based Testing

12. Continuing contamination events
Discovery of new non-culturable viruses
Development of new, rapid methods to detect contaminants
 How and when to use, interpreting results
 PCRs, rapid microbiology methods, Massively Parallel Sequencing
Use of new cell substrates
 New transformed cells for vaccine production, insect cell lines
New product types
 Advanced therapies: gene therapy, cell therapy including stem cells, tissue engineering
 Biosimilar products
New production processes
New endpoints for clinical trials
 Biomarkers, pharmacogenomic analysis
Why Seek Alternative Testing Methods
Challenges Facing Industry and Regulators
13
Scientific and Regulatory Drivers for Innovation in the Biological Safety Testing

13. 14
Challenges in cGMP Testing for Emerging Viruses
GMP Raw Materials: Viral Contaminants in Bovine Serum and Porcine Trypsin
1. The study by Dr. Carol Marcus-Sekura, James C. Richardson, Rebecca K. Harston, Nandini Sane
and Rebecca L. Sheets at BASI, ABSL & NIH (2011): Evaluation of the Human Range of Bovine
and Porcine Viruses that may Contaminate Bovine Serum and Porcine Trypsin Used in the
Manufacture of Biological Products, in Biologicals, 29 (6): 359-369
2. The authors pointed out that the current 9CFR (USDA) tests for bovine serum and porcine
trypsin were not sufficient, there is risk in missing viruses within virus families (such as new
viruses discovered using molecular methods).
3. Alternative methodologies should be evaluated. They recommended to incorporate virus-family
testing for raw materials:
1) Virus family-specific PCR assays
2) Better sensitivity
3) Direct detection of non-culturable viruses

14. A poll will now
occur

15. 16
Design Degenerate PCR Based Assay for Detection of Members of virus families
For Detection and Characterization of unknown virus in sample
Degenerate & Multiplex Primers
for families of DNA viruses
(Ref. Carol Marcus-Sekura et
al. 2011)
Degenerate & Multiplex Primers for families of RNA
viruses (Ref. Carol Marcus-Sekura et al. 2011)

16. Degenerate PCR Based Assay
3 Step Process: Sample Extraction, PCR Amplification, Detection
17
Automated Large
Volume (1-10ml)
magnetic-Bead
Based Extraction
Degenerate Primers target
virus families and
multiplexing approach to
increase throughput
The assay is an integration
of detection (PCR) and
identification (via amplicon
size/sequencing analysis)

17. 18
Single Target Detection Scheme
1 10 100 10000# of
Particles
1e6 CHO
Cells
DNA
Extraction
Output
Result
Extraction, 260/280 OD Reading
Determine genomic copies (GC) using ddPCR
MMV
Virus
Stock
Test Sensitivity and Specificity of Methodology:
1. Add MMV to CHO Cell in a single reaction
2. Extract genomic material from MMV & CHO Mix
3. Perform PCR
4. Detect and size the single Amplicon by Capillary End Point
Analysis
Mouse Minute Virus (MMV):
• Member of Parvovirus family
• Source of several bioreactor contamination events in industry
Detection of Mouse Minute Virus (MMV) in CHO Cell Matrix
Assay Sensitivity and Specificity equivalent to Compendial Method
MMV Particle
Spike in CHO
Cells
Detected
0 No
1 No
10 Yes
100 Yes
Input-GC Culture-PFU
1000 GC 3.091
100 GC 0.309
10 GC 0.031
Ratio (pfu/GC)
Polymer Capillary
End Point Analysis
Fluorescent Labeled
Based PCR

18. 19
Detection of 44 Individual Parvoviruses Targets represented by 13
Peaks
HPV –B19
HPV-4
MMV-Group
PPV-1
BPV-1
HBoV
BPV-3
PBoV
BHoko
ChiPV
PPV-456
PPV-23HOKO
BPV-2
MMV
Mouse Parvovirus 4a &4b
Hamster Parvovirus
Rat Parvovirus
Bat Parvovirus
Kilham rat virus
Feline panleukopenia virus
Tumor virus -X
Canine Parvovirus
Mink enteritis virus
Multiple Target Detection Scheme
44 Targets + Degenerate
Multiplex Primer Mix
Polymer Capillary
End Point Analysis
Assay Approach
1. Mix 44 Individual ParvoV Targets in one reaction
2. Perform PCR
3. Detect and size the Multiple Amplicons by Capillary End Point
Analysis
Multiplex Assay Panel Covers:
• Targets that have been previously identified
• Ability to detect targets that have not been formally identified – e.g., new variants
Target Detected
BPV-3 Yes
HPV-4 Yes
MMV Group
(All reported 19
MMV Strains)
Yes
PPV-1 Yes
HBoV
Yes
HPV-B19
Yes
PBoV
Yes
BHoKo
Yes
ChiP
Yes
PPV-456
Yes
PPV-23HOKO
Yes
BPV-1
Yes
BPV-2 YesFluorescent Labeled
Based PCR

19. 20
Testing Bovine Serum Albumin (BSA) using the Parvovirus Panel
Raw Material Case Study
Why Test BSA?
Used to Stabilize PCR
formulations
How was it tested?
Directly add 2l BSA to
PCR Mix
Outcome?
A peak/band in BSA
correlating to BPV-3
Detection and Identification of Viral Sequence (Bovine Parvovirus 3) in commercially available BSA
Peak confirmation
Sanger sequencing confirms
presence of BPV-3 DNA with
99% identity
BPV-3
ParvoV Panel Assay
Multiplex primers: Generate Specific BPV-3 Signal
Target Detected
BPV-3 No
HPV-4 No
MMV Group
(All reported 19 MMV
Strains)
No
PPV-1 No
HBoV
No
HPV-B19 No
PBoV
No
BHoKo No
ChiP No
PPV-456 No
PPV-23HOKO
No
BPV-1 No
BPV-2 No
BPV-3 Yes

20. 21
Raw Material Case Study
MMV Spiked Unprocessed Bulk Bioreactor Material Analyzed
Unprocessed Bulk
1ml of
Unprocessed
Bulk
1TCID50 MMV Spike
1ml of
Unprocessed
Bulk
Spike
1X PBS
Sample
Purification
Sample
Purification
Bioreactor
Target Detected
BPV-3 No
HPV-4 No
MMV Group
(All reported 19 MMV
Strains)
Yes
PPV-1 No
HBoV
No
HPV-B19 No
PBoV
No
BHoKo No
ChiP No
PPV-456 No
PPV-23HOKO
No
BPV-1 No
BPV-2 No
BPV-3 No
Target Detected
BPV-3 No
HPV-4 No
MMV Group
(All reported 19 MMV
Strains)
No
PPV-1 No
HBoV
No
HPV-B19 No
PBoV
No
BHoKo No
ChiP No
PPV-456 No
PPV-23HOKO
No
BPV-1 No
BPV-2 No
BPV-3 No
Results
Results

21. • High sensitivity and specificity for detection of Adventitious Agents
• Faster molecular test results would provide better control over the manufacturing process
• Broad spectrum helps further reduce risk
• Can detect both culturable and non-culturable viruses
• Earlier results = earlier release of product: months to days/hours: inventory, space, facility utilization
• Newer biologics have short shelf life and are not amenable to cryopreservation storage
Benefits of degenerate primer methodology
Advantages:
22
Disadvantages:
• Molecular methods are not able to distinguish between infectious virus vs. “free” DNA/RNA
 This risk can be mitigated by screening the starting material upfront. If a virus persists and
contaminates your product, then risk has been mitigated by early detection of the virus.

22. Team Acknowledgments
Quanyi (Charlie) Li, PhD
Teng Vang, MS
Audrey Chang, PhD
Reginald Clayton, PhD
Adam Inche, PhD
Colette Cote, PhD
Martin Wisher, PhD
Karin Heffner, PM
23

23. A poll will now
occur

24. Questions?
25
Dr Afshin Sohrabi
BioReliance,
Rockville, MD
afshin.sohrabi@sial.com