Upload
others
View
0
Download
0
Embed Size (px)
Citation preview
The life science business of Merck operatesas MilliporeSigma in the U.S. and Canada.
WCBP 2019
Afshin Sohrabi, PhD
January 29, 2019
Principal Scientist
WCBP| 29Jan20192
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
4
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…”
WCBP| 29Jan20195
Viral Target Selection: Members of virus families
Degenerate PCR Primer Design Target Whole Family of Viruses
DNA Family VirusesRNA Family Viruses
WCBP| 29Jan2019
6
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
7
Primer Design Scheme
Multiple Alignment of Variants
F1 F2
R2
R1
WCBP| 29Jan2019
8
Secondary structure very strong at priming site
priming site
Secondary structures may effect primer binding site
9
Assay Robustness and Ruggedness at DL
Approximately 5µg CHO DNA/rxn used as background material
Example of Primer design with weakrobustness
Robust amplification efficiency
WCBP| 29Jan2019
10
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:
11
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
WCBP| 29Jan201912
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
WCBP| 29Jan201913
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
14
Spike Recovery Study
Bioreactor Material
* PhiX DNA 178bp
** Cell Density ~25x106 cells/ml
** Extract Rapid Degenerate
PCR• Detection
Analysis
MMV10 GC
WCBP| 29Jan2019
*
15
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
WCBP| 29Jan2019
*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
WCBP| 29Jan201917
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)
WCBP| 29Jan201918
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
19
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)
WCBP| 29Jan201920
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
21
Afshin Sohrabi
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
WCBP| 29Jan2019
22