Opsonization from Industry Perspective
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Transcript of Opsonization from Industry Perspective
OpsonizationOpsonization from Industry Perspectivefrom Industry Perspective
Branda T. Hu, Ph.D.Applied Immunology & MicrobiologyWyeth Vaccine ResearchJune 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
Consistency of Assay Performance
Speed of Throughput
Assay Consistency
Four Major Components in PnOPABacteria --- 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
Differentiated HL60 cellsClose 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
Impact of E:T Ratio on Assay Performance
Killing Curve of A Non-immune Adult Serum in Pn9V OPA
0
50
100
150
200
250
300
Serum Dilution
CF
Us
PMN, 400:1
PMN, 50:1
HL60, 400:1
HL60, 50:1
64 128 256 512 1024 2048 4096 8192 16384 32768
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 per well to the TSA blood agar plate by tilt method63 41 52 7 98 11 1210
Serial 2X titration
Control serum
Antibiotic therapy control
C’ control
Background control
System Suitability Testing in PnOPA
Control Wells Bacteria Active C’ C’ Effector Serum Specimen
To Control
Baseline Reference
+
-
- - -
C’ Control
+
+ - - -
Tp Control
Background and effector Control
+ + - + -
Antibiotic Therapy 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
Multiple biological components are carefully controlled
System suitability is monitored Laboratory support equipment is routinely
monitored and validated
PnOPA
PnOPA Assay Consistency
Pn6B OPA Performance
y = 0.8526x + 0.5295R2 = 0.9274
0
2
4
6
8
10
12
14
16
0 2 4 6 8 10 12 14 16
Log2 base median OPA titers from 2003 Rochester site
Lo
g2
bas
e m
edia
n O
PA
tit
ers
fro
m
2004
Pea
rl R
iver
sit
e
r=0.963
Same assay performance consistency is seen in other serotypes
Acknowledgements
Xinhong Yu Assay Development & Clinical
Serology teams Stephen Hildreth Phil Fernsten