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Opsonization from Industry Perspective. Branda T. Hu, Ph.D. Applied Immunology & Microbiology Wyeth Vaccine Research June 5, 2005. “Vaccine potency data are collected across many years and many trials” Assays to measure immunogenicity must be VALIDATED.
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Opsonizationfrom Industry Perspective Branda T. Hu, Ph.D. Applied Immunology & Microbiology Wyeth Vaccine Research June 5, 2005
“Vaccine potency data are collected across many years and many trials”Assays to measure immunogenicity must be VALIDATED
Major Issues in Measuring Vaccine Immunogenicity • Consistencyof Assay Performance • Speed of Throughput
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
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
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
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
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
Exogenous Complement Source • Human Complement • Not Available for large scale testing • Baby Rabbit Complement • Potency • Toxicity (non-specific killing) • Stability through Storage
In Vitro PnOPA Method Transfer 10 l per well to the TSA blood agar plate by tilt method Serial 2X titration 1 2 3 4 5 6 7 8 9 10 11 12 Control serum C’ control Antibiotic therapy control Background control
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
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
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
PnOPA Assay Consistency Same assay performance consistency is seen in other serotypes
Acknowledgements • Xinhong Yu • Assay Development & Clinical Serology teams • Stephen Hildreth • Phil Fernsten