Opsonization from Industry Perspective Branda T. Hu, Ph.D. Applied Immunology & Microbiology Wyeth...
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Transcript of Opsonization from Industry Perspective Branda T. Hu, Ph.D. Applied Immunology & Microbiology Wyeth...
![Page 1: Opsonization from Industry Perspective Branda T. Hu, Ph.D. Applied Immunology & Microbiology Wyeth Vaccine Research June 5, 2005.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d405503460f94a1a49c/html5/thumbnails/1.jpg)
OpsonizationOpsonization from Industry Perspectivefrom Industry Perspective
Branda T. Hu, Ph.D.Applied Immunology & MicrobiologyWyeth Vaccine ResearchJune 5, 2005
![Page 2: Opsonization from Industry Perspective Branda T. Hu, Ph.D. Applied Immunology & Microbiology Wyeth Vaccine Research June 5, 2005.](https://reader036.fdocuments.net/reader036/viewer/2022062516/56649d405503460f94a1a49c/html5/thumbnails/2.jpg)
“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 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
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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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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
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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
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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 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
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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
+ - + - +
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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
Industry:Q2B-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
Multiple biological components are carefully controlled
System suitability is monitored Laboratory support equipment is routinely
monitored and validated
PnOPA
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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
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Acknowledgements
Xinhong Yu Assay Development & Clinical
Serology teams Stephen Hildreth Phil Fernsten