Transcript Opsonization from Industry Perspective

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
Major Issues in Measuring Vaccine
Immunogenicity

Consistency of 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
Impact of E:T Ratio on Assay Performance
Killing Curve of A Non-immune Adult Serum in Pn9V OPA
300
250
CFUs
200
PMN, 400:1
PMN, 50:1
HL60, 400:1
150
HL60, 50:1
100
50
0
64
128
256
512 1024 2048 4096 8192 16384 32768
Serum Dilution
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
Serial 2X titration
1
2
3
4 5
6 7
8
Transfer 10 l per well to
the TSA blood agar plate
9 10 11 12 by tilt method
Control serum
C’ control
Antibiotic therapy control
Background control
System Suitability Testing in PnOPA
Control Wells
Bacteria
Active C’
D C’
Effector
D 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
PnOPA
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 Assay Consistency
Pn6B OPA Performance
Log2 base median OPA titers from
2004 Pearl River site
16
y = 0.8526x + 0.5295
R2 = 0.9274
14
r=0.963
12
10
8
6
4
2
0
0
2
4
6
8
10
12
14
Log2 base m edian OPA titers from 2003 Rochester site
Same assay performance consistency is seen in other serotypes
16
Acknowledgements

Xinhong Yu

Assay Development & Clinical
Serology teams

Stephen Hildreth

Phil Fernsten