Dr. Lee C. Smith BEBPA, Basel 26 Sept 2013 Topics Covered What is - - PowerPoint PPT Presentation

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Dr. Lee C. Smith BEBPA, Basel 26 Sept 2013 Topics Covered What is - - PowerPoint PPT Presentation

Nov 04, 2023 138 likes •576 views

The Experimental Approaches Applied to Optimise and Control a Cell-based Potency Assay used to Test a Live Attenuated Dengue Vaccine Dr. Lee C. Smith BEBPA, Basel 26 Sept 2013 Topics Covered What is Dengue and the need for a vaccine The

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The Experimental Approaches Applied to Optimise and Control a Cell-based Potency Assay used to Test a Live Attenuated Dengue Vaccine

  • Dr. Lee C. Smith

BEBPA, Basel 26 Sept 2013

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Topics Covered

  • What is Dengue and the need for a vaccine
  • The bioassay potency readout for DENVax
  • The approach taken in assay development
  • Selection & screening of assay parameters
  • Optimisation of assay set points
  • Assay precision analysis & assay transfer
  • Assay preparation for phase III validation
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3

Tropical/sub-tropical disease

Dengue Fever

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Dengue is “the most important mosquito-borne viral disease in the world” affecting populations across Asia, Latin America and Africa2

Estimated annual global burden of Dengue

  • 400 million people infected
  • 100 million develop clinical illness
  • 500 thousand hospitalized
  • 20 thousand deaths, mostly in children

Annual infections In 2010

Dengue Fever

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  • Over one half of world population

threatened

  • 70 – 500 million dengue infections per

year

  • 36 million cases of dengue fever per year
  • 2.1 million cases of dengue hemorrhagic

fever

  • > 20,000 deaths per year
  • Mosquito-borne infection
  • Four serotypes: DEN-1 through DEN-4
  • No therapeutic options
  • No vaccine currently available
  • Any vaccine candidate must protect

against all four dengue serotypes

Dengue Fever

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Zoonotic virus

  • 1. Dengue fever
  • 2. Dengue haemorrhagic

fever*

*Ab dependent enhancement (ADE)

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4 Serotypes; all cause disease Any vaccine candidate must protect with neutralizing Abs against all four dengue serotypes

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Measuring Vaccine Potency

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Plaque Based Assays

  • Plaque assays - standard for virus concentration
  • A confluent cell monolayer is infected with the

virus & covered with a semi-solid medium to prevent the virus infection from spreading indiscriminately

  • A viral plaque is formed when a virus infects a

cell within the fixed cell monolayer which will lyse and spread the infection to adjacent cells where the infection-to-lysis cycle is repeated

  • The infected cell area will create a plaque (an

area of infection surrounded by uninfected cells) which can be seen visually or with an

  • ptical microscope after 1 – 2 weeks

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Immuno Focus Assay (IFA)

  • Rapid variation of plaque assay
  • Uses immunostaining with antibodies specific

for the viral Ag

  • Measure host cell infection before an actual

plaque is formed.

  • Shorter incubation period (e.g., 1-5 days)
  • Stain with 2°Ab enzyme conjugates
  • Substrate leads to coloured foci
  • Results are expressed as Focus Forming Units
  • r FFU/mL

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Spearman-Karber & Reed-Muench

=>

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Dengue Immuno Focus Assay (IFA)

  • Relatively slow progress to distinct CPE
  • Stain virus plaques with anti-Dengue Abs after few days
  • Count Focus Forming Units (FFU/mL)
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A Perfect Spot?

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  • We’re counting spots so we wish them to be:

– Clean round spots – Unambiguous

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– Separated from others – The Right Size

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Imperfections

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Comets Satellites Too big Too small

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An inability to count the foci

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Inaccuracy & imprecision = “The method needs to produce foci that are the right size, not merged, have no comets or satellites regardless of who performs it, where it is performed and when it is performed”

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DoE brings understanding

– which parameters have an effect upon the desired responses – which parameters interact – which parameters need to be focused upon and optimised and those that can be fixed – The design space & how close to failure we operate the assay

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System

Controllable (X) Factors Uncontrollable (N) Factors Response Measures

System Variables

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Scoping - Assay Deconstruction

Titer Comets Satellites

Dilution series Volume added Mixing Time uncovered Incubation Time Temp CO2 conc Incubation Overlay type Time Temp CO2 conc PBS Wash VolPressure Repeats Methanol fix temp vol Time Temp Conc Agitation Repeats Vol Blocking Vol conc Time Temp 2o Ab AP Conc Inubation time Stop trigger with wash Optional storage point Vol Agitation Overlay conc

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Discuss & Agree what’s important

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Risk Assessment Control Strategy Heat Map

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Unit Operation Method Parameters Ranges or Set Point CNX Accuracy Precision Foci size, comets & satellites Priority or Risk Overlay Incubation (Time) 4 – 7 days X 9 9 9 9.00 Overlay Type CMC vs Avicel C 9 9 5 7.40 Plate & Sample Prep Confluency 80-100% C 7 9 5 6.80 Plate & Sample Prep Passage number Currently at 149 C 7 7 5 6.26 Plate & Sample Prep Mixing/pipetting Mixing time 5 - 30 secs X 9 9 3 6.24 Virus adsorption Time dry (from aspiration of 60 - 300 secs N 9 9 3 6.24 Virus adsorption Sample Addition (Vol) 100 – 500 μl X 9 9 3 6.24 Overlay Concentration 0.4 – 1.0% X 7 7 3 5.28 Signal Development Incubation Time 20-90 mins C 7 7 3 5.28 Virus adsorption Incubation (Time) 1.5 – 24 hours X 9 5 3 5.13 Plate & Sample Prep Plate manufacturer Currently Greiner, cat. No. 657160 C 5 5 5 5.00 Virus adsorption Incubation (Temp) 35-38oC C 5 5 3 4.22 Virus adsorption Agitation Rocking vs Manual X 5 5 3 4.22 Plate & Sample Prep Plate Age 1.5 - 2.5 days C 7 9 1 3.98 Signal Development Incubation Temp 22-27oC N 7 7 1 3.66 Plate Fixing Wash repeats 2 - 4 C 3 5 1 2.47 Plate Fixing Wash agitation 6-10 swirls N 3 5 1 2.47 Plate & Sample Prep Dilution Series Factor 2 to 5 C 9 1 1 2.08 Virus adsorption Incubation (CO2 Conc) 3.5-6.5% C 3 3 1 2.08 Overlay Incubation (Temp) 36-38oC C 3 3 1 2.08 Immuno-staining Wash repeats 1 to 3 C 3 3 1 2.08 Immuno-staining Wash volume 1-3 mL C 3 3 1 2.08 Immuno-staining Blocking temp 25-40oC C 3 3 1 2.08 Immuno-staining Blocking Time 15-60 mins C 3 3 1 2.08 Immuno-staining Blocking volume 0.5-2mL C 3 3 1 2.08 Immuno-staining Primary Ab Vol 0.8 - 1.2 mL C 3 3 1 2.08 Immuno-staining Primary Ab Conc 1:1000-1:3000 X 3 3 1 2.08 Immuno-staining Primary Ab incbtn time 1-6 hr X 3 3 1 2.08 Immuno-staining Secndry Ab Vol 0.4 - 0.6 mL C 3 3 1 2.08 Immuno-staining Secndry Ab Conc 1:500-1:2000 C 3 3 1 2.08 Immuno-staining Secndry Ab incbtn temp 35 - 39 ºC C 3 3 1 2.08 Immuno-staining Secndry Ab incbtn time 30-120 mins C 3 3 1 2.08 Signal Development AP Volume 0.3ml-0.5mL C 3 3 1 2.08 Plate Fixing Methanol fix time 20-60 mins C 1 1 3 1.44 Plate & Sample Prep pH 6.8 - 7.6 C 1 1 1 1.00 Overlay Incubation (CO2 Conc) 4-6% C 1 1 1 1.00 Overlay Volume 2-4mL C 1 1 1 1.00
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Adsorption time Inoculum volume

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Overlay Medium Overlay Concentration Incubation time Agitation Mixing method

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Screening Designs

Selected 2 level fractional factorial with CP for curvature

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Parameters were a mix

  • f Categorical (binary)
  • r Numerical
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Serotype 1 Screening data

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0.00 384791182.00 769582364.00 1154373546.00 1539164728.00 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf-N o rm a l % P ro b a b ility B-Adsorption time C-Overlay concentration D-Incubation time E-Mixing method BC DE

Titer

0.00 0.34 0.67 1.01 1.35 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf-N o rm a l % P ro b a b ility B-Adsorption time D-Incubation time F-Overlay medium BD

Foci Size Comets

0.00 4.20 8.39 12.59 16.78 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf-N o rm a l % P ro b a b ility A-Infection volume C-Overlay concentration

Satellites

0.00 4.10 8.20 12.31 16.41 20.51 24.61 10 20 30 50 70 80 90 95 99 |Standardized Effect| H a lf-N o rm a l % P ro b a b ility B-Adsorption time D-Incubation time E-Mixing method BD DE
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Serotypes 2, 3 & 4

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0.00 950.49 1900.98 2851.47 3801.96 4752.45 5702.94 6653.43 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o rm a l % P r o b a b ility A-Infection volume C-Overlay concentration D-Incubation time E-Mixing method F-Overlay medium AC CF DE 0.00 0.10 0.19 0.29 0.38 0.48 0.57 0.67 0.77 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o r m a l % P r o b a b ility A-Infection volume B-Adsorption time C-Overlay concentration D-Incubation time F-Overlay medium CF 0.00 5.64 11.28 16.92 22.56 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o rm a l % P r o b a b ility A-Infection volume B-Adsorption time C-Overlay concentration D-Incubation time AD BC CD

Titer Size Comets

0.00 9170450.81 18340901.62 27511352.42 36681803.23 45852254.04 55022704.85 64193155.65 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o r m a l % P r o b a b ility A-Infection volume B-Adsorption time C-Overlay concentration D-Incubation time E-Mixing method F-Overlay medium AC BD EF 0.00 0.10 0.21 0.31 0.41 0.52 0.62 0.72 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o r m a l % P r o b a b ility C-Overlay concentration D-Incubation time F-Overlay medium CD 0.00 8.82 17.64 26.46 35.28 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o rm a l % P r o b a b ility C-Overlay concentration D-Incubation time CD

Titer Size Satellites

0.00 4994774.89 9989549.77 14984324.66 19979099.55 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o r m a l % P r o b a b ility A-Infection volume B-Adsorption time C-Overlay concentration D-Incubation time E-Mixing method F-Overlay medium AE BD 0.00 0.66 1.33 1.99 2.65 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o r m a l % P r o b a b ility B-Adsorption time C-Overlay concentration D-Incubation time E-Mixing method F-Overlay medium CF 0.00 3.70 7.39 11.09 14.79 18.48 10 20 30 50 70 80 90 95 99 Half-Normal Plot |Standardized Effect| H a lf- N o r m a l % P r o b a b ility A-Infection volume B-Adsorption time C-Overlay concentration D-Incubation time E-Mixing method F-Overlay medium BD CF

Titer Size Satellites

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IFA Main Effects & Interactions

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TITER PARAMETER RESULT Adsorption Time MAIN EFFECT Overlay Concentration MAIN EFFECT

  • Adsorp. tme/overly conc.

INTERACTION COMETS PARAMETER RESULT MIXING METHOD MAIN EFFECT ADSORPTION TIME MAIN EFFECT INCUBATION TIME MAIN EFFECT INCUBATION/MIXING INTERACTION ADSORPTN/INCUBATN INTERACTION FOCI SIZE PARAMETER RESULT ADSORPTION TIME MAIN EFFECT INCUBATION TIME MAIN EFFECT OVERLAY CONCENTRATION MAIN EFFECT ADSORPTION/INCUBATION INTERACTION SATELLITES PARAMETER RESULT INFECTION VOLUME MAIN EFFECT OVERLAY CONC MAIN EFFECT

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Response Surface Design

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Optimal design

  • Mix of numerical and

categorical

  • 1. Inoculum volume
  • 2. Adsorption time
  • 3. Incubation time
  • 4. Mixing by pipette
  • 5. Agitation

Fixed the:

  • Mixing method (pipette)
  • Chose Avicel for overlay

Results

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Optimisation - 3D Models

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Titer DENVax 1

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Numerical Optimisation within DX8

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Set desired Criteria Review Responses

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JMP Profiler

Optimal with a robust range

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Assay Control: control the parameters inside boundaries

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Final Set-points

All serotypes were reviewed and suitable set points were decided upon: i. Agitation = static

  • ii. Mixing by pipette = 9 cycles
  • iii. Inoculum volume = 500 μL
  • iv. Adsorption time = 1.5 h
  • v. Incubation time = 5 days
  • Range around set-points determined

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Recap - Data Driven Development

Scope

Explore mildest to most forcing conditions

Optimize

Estimate & utilize interactions to move towards optimum conditions

Verify

Rattle the cage to deliver a design space

QC/TT

Transfer to QC to validate on batches & bring into routine use Identify few potential key parameters Focus on vital few & narrow ranges

Screen

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Assay Transfer of Monovalent IFA

Nested Design to understand Variance sources, Site to Site differences & assay precision:

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There’s no point in replicating elements within an assay that contribute relatively little towards variance This information is extremely useful to decide where to power your replication

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Summary

Deconstructed Assay & Prioritised 7 parameters for evaluation Optimised the parameters that allowed set points to be selected for the 4 serotypes Performed a screening assay that selected 5 parameters for optimisation Successfully transferred assay between 3 international sites Nested analysis helped understand the variance sources of the assay for replication decisions

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Transfer learnings from monovalent assay into the Tetravalent assay for release of drug product Understand and optimise cell preparation for both assays

What Next?

Design space data will be used for robustness studies in assay validation Perform further Nested analysis to understand vial to vial variation relative to other sources Use data to decide replication regime and to set acceptance criteria for assay validation for imending phase III

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Acknowledgments

  • Dr. Paul Nelson – Prism TC Ltd
  • Mr. Stuart Wilson – Prism TC Ltd.

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