Opsonization from Industry Perspective

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Opsonization from Industry Perspective

• Branda T. Hu, Ph.D.
• Applied Immunology Microbiology
• Wyeth Vaccine Research
• June 5, 2005

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Vaccine potency data are collected across many
years and many trials Assays to measure
immunogenicity must be VALIDATED
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Major Issues in Measuring Vaccine Immunogenicity

• Consistency of Assay Performance
• Speed of Throughput

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Assay Consistency

• Four Major Components in PnOPA
• Bacteria --- S. pneumoniae
• Exogenous Complement --- human or baby rabbit
  complement
• Effector Cells (Phargocytic Cells) --- human
  PMNs or differentiated HL60 cells (or NB4 cells)
• Antibody Source --- human serum specimens

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Challenges to Validation of OPA

• Reliance on biologically active (labile)
  components
• Control of the critical components is important
  to minimize assay variability
• Demonstrate that OPA activity is Ab-mediated not
  non-specific

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Selection of Bacteria Strain

• Specific strains and isolates
• Degree of encapsulation
• Growth curve / condition
• Colony morphology
• Raised and shiny colonies are preferred
• Known antibiotic sensitivity

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Effector Cells (Phagocytic Cells) ---
Viability and Functionality

• PMNs
• Polymorphism in cell surface receptor expression
  present in human population
• Complement activation and Ab binding ? varying
  levels of OPA killing activity
• Solution
• Pool minimum of 6 donors to minimize the
  polymorphism impacting OPA outcome

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Effector Cells (Phagocytic Cells) ---
Viability and Functionality

• Differentiated HL60 cells
• Close monitoring
• Cell Viability Apoptotic/Necrotic cell
  population
• Cell surface receptor(s) expression CD35, CD71
• For both un-differentiated and differentiated
  cells

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Impact of ET Ratio on Assay Performance
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Exogenous Complement Source

• Human Complement
• Not Available for large scale testing
• Baby Rabbit Complement
• Potency
• Toxicity (non-specific killing)
• Stability through Storage

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In Vitro PnOPA Method
Transfer 10 l per well to the TSA blood agar
plate by tilt method
Serial 2X titration
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Control serum
C control
Antibiotic therapy control
Background control
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System Suitability Testing in PnOPA
Control Wells Bacteria Active C D C Effector D Serum Specimen
To Control Baseline Reference - - - -
C Control - - -
Tp Control Background and effector Control - -
Antibiotic Therapy Control - -
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System Suitability Testing in PnOPA

• 3-4 control sera with high, medium, and low
  levels of specific functional antibodies, are
  included in every run of PnOPA testing to monitor
  the consistency of the assay performance

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Qualification/Validation of an Assay

• Specificity
• Precision
• Linearity
• Accuracy
• Assay detection/quantitation range and limit
• International Conference of Harmonisation (1996)
  Guidance for IndustryQ2B-Validation of
  Analytical Procedures Methodology
• USDHHS, FDA, CDER CVM Guidance for Industry
  (2001) Bioanalytical Method Validation

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Assay Consistency can be achieved when
PnOPA

• Multiple biological components are carefully
  controlled
• System suitability is monitored
• Laboratory support equipment is routinely
  monitored and validated

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PnOPA Assay Consistency
Same assay performance consistency is seen in
other serotypes
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Acknowledgements

• Xinhong Yu
• Assay Development Clinical Serology teams
• Stephen Hildreth
• Phil Fernsten