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