1. Opsonization from Industry Perspective Branda T. Hu, Ph.D. Applied Immunology & Microbiology Wyeth Vaccine Research June 5, 2005

2. “Vaccine potency data are collected across many years and many trials” Assays to measure immunogenicity must be VALIDATED

3. Major Issues in Measuring Vaccine Immunogenicity Consistency of Assay Performance Speed of Throughput

4. 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

5. 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

6. 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

7. 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

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

9. Impact of E:T Ratio on Assay Performance

10. Exogenous Complement Source Human Complement  Not Available for large scale testing Baby Rabbit Complement  Potency  Toxicity (non-specific killing)  Stability through Storage

11. In Vitro PnOPA Method Transfer 10  per well to the TSA blood agar plate by tilt method 634152798111210 Serial 2X titration Control serum Antibiotic therapy control C’ control Background control

12. System Suitability Testing in PnOPA Control WellsBacteriaActive C’  C’ Effector  Serum Specimen T o Control Baseline Reference + - - - - C’ Control + + - - - T p Control Background and effector Control + + - + - Antibiotic Therapy Control + - + - +

13. 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

14. Qualification/Validation of an Assay  Specificity  Precision  Linearity  Accuracy  Assay detection/quantitation range and limit - International Conference of Harmonisation (1996): Guidance for Industry:Q2B-Validation of Analytical Procedures: Methodology - USDHHS, FDA, CDER & CVM: Guidance for Industry (2001): Bioanalytical Method Validation

15. Assay Consistency can be achieved when Multiple biological components are carefully controlled System suitability is monitored Laboratory support equipment is routinely monitored and validated PnOPA

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

17. Acknowledgements Xinhong Yu Assay Development & Clinical Serology teams Stephen Hildreth Phil Fernsten