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In-vitro Potency Assay for
SAM RNA Vaccines
Qiongman Kong, Nicholas Manzo, Janet Yu, Kate
Luisi, Marcelo Samsa, Chenxing Zheng, Meng
Zhang, Xianzhi Zhou, & Nicolas Moniotte
GSK Vaccines, Rockville, MD, USA
CASSS DC Discussion Group
07Jun2018
– Introduction to RNA vaccines
– GSK’s SAM (self-amplifying mRNA) vaccines
– SAM/CNE flow-based potency assay
– Alternative methods and future development
2 CASSS DC Discussion Group Meeting 07Jun2018
Presentation Outline
Modern Vaccine Development
– Objectives:
– Safe
– Protective long-term immune memory, both prophylactic and therapeutic
– Limitations of current vaccines:
– Live attenuated vaccines
– potent cellular and humoral immunity, but:
– adverse reactions, risk of reversion to virulence
– complicated cell-based production processes
– Subunit vaccines
– safer, more stable, more amenable to mass production, but:
– less potent, require adjuvants, lack of cellular immunity
– To improve current vaccines, GSK has selected the SAM platform for the
development of highly effective vaccines.
Objectives and Limitations of Current Vaccines
CASSS DC Discussion Group Meeting 07Jun2018 3
Nucleic Acid-Based Vaccines
– Advantages:
– Capacity to trigger both antibody-mediated and cell-mediated immunity
– Potential simplified production processes
– DNA vaccines
– RNA vaccines:
– Potential higher delivery efficiency
– Avoid potential integration into the host cell genome
– Avoid immune tolerance from T cell exhaustion induced by protein accumulation
– Scalable production in cell-free system
– Potentially enable fast response within weeks of pathogen detection
– Avoid significant anti-vector immunity
– Can be lyophilized for easy storage and transportation.
Advantages and Classes
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GSK’s SAM Vaccines: SAM + Non-Viral Delivery System
Mechanism and Advantages
5
1. Iavarone et al, (2017) Expert Rev Vaccines.16:9,871-881
– Mechanism1:
– A target antigen sequence
– A non-viral delivery system
– Encode an RDRP complex
– RDRP-dependent template
amplification
– Produce the antigen of interest
to elicit immune responses
– Higher levels of antigen expression; greater immune responses.
CASSS DC Discussion Group Meeting 07Jun2018
Self-Amplifying mRNA Conventional mRNA
SAM/CNE Vaccines ‘Bed-Side’ Mixed Vaccine Proposed for Phase 1 Approach
RNA-CNE (SAM vaccines) Vial 1 (RNA) Vial 2 (CNE) (Frozen at -80C) (2-8C)
6
– Cationic Nano Emulsion CNE serves as the non-viral delivery system for SAM,
and substantially increases the potency of the vaccine.
– CNE formulation is based upon the ingredients in a commercially–available
adjuvant MF59 plus DOTAP for the cationic component1.
– CNE uses ingredients that have been already used in humans2-4.
RNA
CNE
CASSS DC Discussion Group Meeting 07Jun2018
1. Brito et al, 2014 Mol Ther. 22(12): 2118–2129
2. Del Giudice et al, 2006 Clin Vaccine Immunol. 13(9):1010-1113
3. Porteous et al, 1997 Gene Ther. 4(3):210-218
4. Lu et al, 2012 PLoS One. 7(4):e34833
Analytical Method Development in ARD
An Iterative Process - Continually Advanced, Challenged, and Replaced
Our mission is to support Regulatory document submission, to develop analytical methods in line
with the Critical Quality Attributes (CQA), and to support drug substance and drug product development.
Safety
Potency
Purity
Size Structure
Excipients Impurities
Identity
Efficacy
Method Development
Define Procedures
Extensive Characterization
Robustness
Periodically Refine
ATP
Analytical Technology Screening
Analytical Target
Profile (ATP) is the
starting point in the
analytical cycle
Further Increase
Product Knowledge
RNA + CNE
CASSS DC Discussion Group Meeting 07Jun2018
– Potency is a CQA, and development of potency assay is an iterative process.
SAM In-vitro Potency Assays
– Questions to be addressed:
– Can GSK’s SAM vaccine be delivered into cells and express the target
antigen? How efficient is each type of delivery vehicles?
– What is the quality of each lot of SAM vaccines?
– Assay development strategy:
– Measure the target antigen expression in-vitro
– Understand the tolerable range of delivery vehicle in-vitro
– Define the suitable SAM vaccine treatment dose range
– Determine treatment protocol
– Develop target antigen detection method (flow cytometry, ELISA, HCA,
etc.)
8
Strategy for Assay Development
CASSS DC Discussion Group Meeting 07Jun2018
SAM In-vitro Potency Assays
Flow Cytometry Platform-Based Approach
Flow Cytometry Analysis
Indirect Antibody Labeling Single Cell Suspension BHK-21 Cells
18hr
Physical SAM Delivery 0-Xng/1x106 cells Electroporation
SAM in-vitro Potency Assay
1. One antigen specific antibody needs to identified
(primary antibody screen)
2. Preoptimized secondary antibodies can be used
to detect multiple antigen specific antibodies
3. Easily adaptable to detect different or multiple
expressed antigens in single cell
Platform Based Approach
Seed Cells BHK-21 Cells 1x106, 3-4hrs
Non-viral SAM Delivery 0-Xng/mL, 0.8mL, 2hr RNA-CNE Complex
RNA-CNE
18hr
SAM/CNE
in-vitro Potency Assay
(initial protocol)
9 CASSS DC Discussion Group Meeting 07Jun2018
SAM/CNE Potency Assay Development
SAM-GFP/CNE Time Course
10
4hr Expression 8hr Expression 24hr Expression 0.5
hr
Tra
nsfe
cti
on
1.0
hr
Tra
nsfe
cti
on
2
.0h
r
Tra
nsfe
cti
on
CASSS DC Discussion Group Meeting 07Jun2018
Determining Optimal Transfection Timing and Expression Kinetics
SAM/CNE Potency Assay Development
– The assay characterizes the potency of SAM vaccines
on:
1. Delivery efficiency of SAM by CNE, and
2. Subsequent ability of SAM on amplification and expression
of target antigen
– Procedures:
– Cell seeding
– Delivery of SAM by CNE
– Post-transfection antigen expression
– Antigen detection by flow cytometry
– Experimental design:
– Standard curve with ref. std.
– Ref. std. tested as the plate control
– Sample tested and interpolated against the standard curve.
CASSS DC Discussion Group Meeting 07Jun2018 11
3~4hr
Seed BHK cells RNA+CNE
18-21hr
Harvest for staining &
flow cytometry analysis
3~4hr
In Opti-MEM
Flow Cytometry Analysis
Target Specific
Antibody Labeling
Procedures and Design
– Seeding density and seeding time
– Type of cell culture plate
– Treatment medium volume
– Type of treatment medium
– SAM treatment doses
– Post-treatment medium
– Post-treatment incubation time for antigen expression
– Washing intensity
– Antibody incubation and dilution
– Blocking conditions
– Total antigen detection vs. surface antigen detection
– Linearity range
– etc.
CASSS DC Discussion Group Meeting 07Jun2018 12
SAM/CNE Potency Assay Development
Conditions Tested and/or Optimized
SAM/CNE Potency Assay Development
Full Dose Curve
13
% of Antigen+ Cells Detected by Flow
CASSS DC Discussion Group Meeting 07Jun2018
Higher Transfection Efficiency; Good R2 and CV%
SAM Dose
R2=0.998 R2=0.999
SAM Dose
Initial protocol:
2nd version of protocol: to pursue full dose
curve for relative potency (RP).
3rd version of protocol: Replace RP from full
dose curves with RP from back-calculation
SAM/CNE Potency Assay Development
Linearity Test
14
In vitro Potency
• ICH Guidelines (Q6B)
– “The results of biological assays should be expressed in units of activity calibrated
against an international or national reference standard, when available and
appropriate for the assay utilized. Where no such reference standard exists, a
characterized in-house reference material should be established and assay results
of production lots reported as in-house units.
• IVRP = [Interpolated Dose (ng) / Theoretical Dose (ng)]*100
CASSS DC Discussion Group Meeting 07Jun2018
Recoveries at Three Doses (40%, 100%, and 160% of sample test dose)
IVRP: in vitro relative potency.
Flow-Based Potency Assay to Assess Multiple Endpoints
Standard curve
SAM RS + CNE RS
SAM DS/DP + CNE RS
SAM DS/DP + CNE DS/DP
SAM RS + CNE DS/DP
+
Evaluate SAM RNA quality
Evaluate SAM vaccine potency
Evaluate vaccine potency
& quality of RNA and CNE
Sample test
Evaluate CNE quality
– Platform assay: easily adapted for different SAM vaccine products:
– For a different antigen of interest:
– switch to another antibody that is specific to the new antigen
– For a different delivery system:
– optimize treatment condition fit for new delivery vehicle
CASSS DC Discussion Group Meeting 07Jun2018
Easily Adapted for Different SAM Vaccine Products
RS: reference standard; DS: drug substance; DP: drug product.
Alternative Methods
– Whole Cell ELISA: measure total antigen expression.
– with SAM-electroporated cells.
– Screened conditions: antibodies and dilutions, blocking, washing, detection reagent,
dose and dilution strategy for full curve, etc.
– with SAM/CNE-treated cells.
– Screened plate types, cell seeding densities, treatment dose/dilutions/time, antigen
expression time, blocking and washing for cell detachment concern, etc.
– Challenge is the cell detachment during washes after high-dose CNE treatment.
– HCA
– Sandwich ELISA
16 CASSS DC Discussion Group Meeting 07Jun2018
ELISA, HCA, etc.
Whole-Cell ELISA with SAM-Electroporated Cells
– Plot 1: SAM In-House REF
– Plot 2: SAM bad lot DS
Independent Fits Global Fit
– Estimated relative potency of the bad lot DS is 41%.
In-House REF vs. a Bad Lot Drug Substance (DS)
CASSS DC Discussion Group Meeting 07Jun2018
SAM Dose
Lum
inescenct S
ignal
R2=1.000
EC50=7.22
R2=0.999
EC50=16.98
R2=0.998
Rel. Pot.=41%
Whole-Cell ELISA with SAM/CNE-Treated Cells
18
Quick adaption of ELISA staining protocol from cells electroporated with
SAM to cells treated with SAM/CNE.
2hr Non-Viral Delivery of SAM + Overnight Antigen Expression
CASSS DC Discussion Group Meeting 07Jun2018
SAM Dose
R2=0.996
Alternative Methods
Dose-Dependent Expression of Antigen with SAM Vaccine by ICC and HCA
19
After delivery of SAM by another delivery system:
SAM Dose Response in Triplicates
CASSS DC Discussion Group Meeting 07Jun2018
High transfection efficiency of SAM; good R2 and CV%
Future Assay Development
– Continuous optimization of flow-based assay for new SAM products.
– Development of alternative methods:
– Whole Cell ELISA
– HCA
– Sandwich ELISA, etc.
– Test different types of GSK’s SAM vaccine (SAM + alternative delivery
systems).
– SAM potency assay with SAM delivered by commercial transfection kit
instead of electroporation.
20 CASSS DC Discussion Group Meeting 07Jun2018
ELISA, HCA, etc.
Acknowledgement
21
- GSK ARD - Nicolas Moniotte - Alan Ng - Mandy Xie - Tim Schofield
- GSK ARD Bioassay Group
- Xianzhi Zhou - Nicholas Manzo - Janet Yu - Meng Zhang - Cari Kessing - Li Ma - Matthew Brecher
- Ying Zhang - Judit W. Johnson
- GSK Preclinical - Kate Luisi - Marcelo Samsa - Chenxing Zheng - Dong Yu - Shanshan Xu - Asma Ashraf
- GSK DP
- Kelly Forney-Stevens
- GSK GDSNT - Jessica Cohen
- GSK CMC Statistical Sciences
- Ryan Yamagata - Minggang Cui
CASSS DC Discussion Group Meeting 07Jun2018
Transparency Section
– Funding/Sponsorship
– The work was sponsored by GlaxoSmithKline Biologicals SA.
– Declaration of Interest
– All authors are employees of the GSK group of companies.
– Author Contributions
– Experiments were performed by Kong Q/Manzo N/Yu J/Samsa M/Zheng C/Zhang M; Zhou X/Moniotte N/Luisi K provided technical guidance and support.
22 CASSS DC Discussion Group Meeting 07Jun2018
Thank you!