The life science business of Merck operatesas MilliporeSigma in the U.S. and Canada.

WCBP 2019

Afshin Sohrabi, PhD

January 29, 2019

Principal Scientist

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Rapid Alternative Platforms to Cell & Animal Based Tests

Culture/Animal

Sequencing

PCR

1+ Month

Weeks

Days

BroadDetection

Target Specific

Highly Sensitive

Not all viruses can be detected

Sensitivity may be an issue

Only detect what you are looking for

Rapid Platform

Multiple Target detection,High sensitivity & Specificity

Detects previously seen targets and new variants

Days

3

Key Features of “FAST” Assay Platform

Purification Amplification Analysis

1. The assay uses nested PCR

2. The assay uses degenerate PCR primers

3. The assay uses multiplex reaction

4. The assay integrates PCR + sequencing

5. The Platform integrates automation and Electronic Lab Notebook (ELN)

Results in higher throughput & reduced testing time e.g., from 28 Days

to ~ 3 Days

WCBP| 29Jan2019 Note: “FAST”= Fragment Analysis Sequencing Test

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Regulatory Publications Indicate that a Degenerate PCR Approach is Acceptable

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 2016)

“…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…”

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Viral Target Selection: Members of virus families

Degenerate PCR Primer Design Target Whole Family of Viruses

DNA Family VirusesRNA Family Viruses

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A Degenerate PCR Approach with the Breadth of Detection Based on Amplicon Size

1 primer set1 target

1 primer setmultiple targets

Single-Plex PCR Multiplex Degenerate PCR

bp bp

Combines detection and sequencingEnd point detection

Variant 1

Variant 2

Variant n

RFU

RFU

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Primer Design Scheme

Multiple Alignment of Variants

F1 F2

R2

R1

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Secondary structure very strong at priming site

priming site

Secondary structures may effect primer binding site

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Assay Robustness and Ruggedness at DL

Approximately 5µg CHO DNA/rxn used as background material

Example of Primer design with weakrobustness

Robust amplification efficiency

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Multiplexing Degenerate Primers Can Deliver Broad Detection of Adventitious Virus

MMV degenerateprimer pair

virus strains

Parvo virus panel (proof of concept):

• multiple degenerate primer pairs

• 45 targets including MMV

Automated Extraction(1-10ml input,Or Cell Pellet)

Multiplex Amplification

(degenerate primer target virus families)

Detection & Reporting

(sequencing confirmation for positives)

3 Step Process

F:

R:

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Single Target Detection Scheme

1 10 100 10000GC #

1e6 CHOCells

DNA Extraction

Output Result

Extraction, 260/280 OD ReadingDetermine genomic copies (GC) using ddPCR

MMVVirusStock

Test Sensitivity and Specificity of Methodology:1. Add MMV to CHO Cell in a single reaction2. Extract genomic material from MMV & CHO Mix3. Perform PCR4. 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 in vitro infectivity assay

Input-GC Culture-PFU1000 GC 3.091100 GC 0.30910 GC 0.031

Ratio (pfu/GC)FAST

PCR

MMV GC

Detected

0 No

1 No

10 Yes

100 Yes

Note: 1 TCID50~0.7pfu

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In-silico Case Study

Parvo Degenerate Primers Bind to Newly (2018) Discovered Mouse Kidney Parvo Virus (MKPV)

Multiple Alignment Showing Conservation of ParvoPrimer Binding Region & MKPV Sequence

MKPVParvo-1Parvo-2Parvo-3

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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 2l BSA toPCR 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 confirmationSanger sequencing confirms presence of BPV-3 DNA with 99% identity

BPV-3

ParvoV Panel AssayMultiplex primers: Generate Specific BPV-3 Signal

Target Detected

PPV-3 No

HPV-4 No

MMV Group(All reported 19 MMV

Strains)

No

PPV-1 No

HBoVNo

HPV-B19 No

PBoVNo

BHoKo No

ChiP No

PPV-456 No

PPV-23HOKONo

BPV-1 No

BPV-2 No

BPV-3 Yes

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Spike Recovery Study

Bioreactor Material

* PhiX DNA 178bp

** Cell Density ~25x106 cells/ml

** Extract Rapid Degenerate

PCR• Detection

Analysis

MMV10 GC

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*

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Spike Recovery Study

Cell Pellet

*PhiX Internal Control (IC) & Murid

Herpes Virus 4 (MHV4) DNA each spikes at 100GC in 1ml

N=3; n=9

Extract Rapid Degenerate

PCR• Detection

Analysis

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*DNA/RNA Spike

1e7 Cell Pellet

NTC

PhiX IC Spike

MHV4 Spike

MMV Infected CHO Cells in vitro coupled with “FAST” Parvo Panel AnalysisComparability Study

v

N=3n=3

N=3n=3

N=3n=3

N=3n=3

~3 Day Testing

Time

0.1 TCID50 1 TCID50

“FAST” 3 Days 2 Days

MMV qPCR >3 Days > 2 Days

NGS >3 Days >3 Days

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Assay Validation Pass/Fail Criteria

*ROCs analysis shows the Rapid Degenerate PCR assay highly accurate at detection limit

Nominal Logistic Fit of Counts by Response

Statistics Formula Value 95% CI

Sensitivity𝑇𝑃

𝑇𝑃+𝐹𝑁41.67%

15.17% -72.33%

Specificity𝑇𝑁

𝐹𝑃+𝑇𝑁100%

73.54%- 100%

Accuracy

𝑇𝑃+𝑇𝑁

𝑇𝑃+𝐹𝑁+𝐹𝑃+𝑇𝑁 70.83%48.91% -87.38%

Statistics Formula Value 95% CI

Sensitivity𝑇𝑃

𝑇𝑃+𝐹𝑁100% 63% - 100%

Specificity𝑇𝑁

𝐹𝑃+𝑇𝑁100%

63%100%

Accuracy

𝑇𝑃+𝑇𝑁

𝑇𝑃+𝐹𝑁+𝐹𝑃+𝑇𝑁 10079.41% -

100%

*Receivers Operator Curve (ROCs)

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A Proof of concept assay is available now for GMP level testing, with commercial solutions available from 2019

2018 2019 2020

Parvo Panel

Alpha test platform for future assay development and customer input

CHO Panel

Virus families representing current/emerging threats from

rodent cells and animal derived raw material

Mouse/Hamster Antibody Production Panel

Alternative to current in vivo model

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Replacing 28 day cell based assay

CHO FAST AssayMAP-HAP

FAST Assay

Replacing a 28 day animal study

ParvoV

FAST Assay

Replacing a rapid molecular assay

(MMV qPCR)

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Accelerating viral screening has multiple potential benefits

Acceleration oftesting

Speed to market

Faster revenue

recognition

Reduce warehouse

costs

Increasefacility

utilization

Continuousprocessing

Patients waiting

treatment

Ethical concerns

Limited alternative

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Afshin Sohrabi

Afshin.sohrabi@milliporesigma.com

Audrey Chang

Quanyi (Charlie) LiTeng Vang

Michael WintersMelisa Wilson

Lateefat YusufMelisa Wilson

Bradley Hasson

Martin WisherReginald Clayton

Nikolay KorokhovAdam Inche

Karin HeffnerColette Cote

Alison Armstrong

David Onions

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