rv potency assay - cdn.ymaws.com...higher transfection efficiency; good r2 and cv% sam dose r2=0.998...

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


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


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


– 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


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)


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


Determining Optimal Transfection Timing and Expression Kinetics


– 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.


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.



Conditions Tested and/or Optimized


Full Dose Curve

13

% of Antigen+ Cells Detected by Flow


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


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


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


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


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)


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


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


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.


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


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.


Thank you!

rv potency assay - cdn.ymaws.com...higher transfection efficiency; good r2 and cv% sam dose r2=0.998...

Documents