1. Eurofins BioPharma Product Testing Munich GmbH
Qualification of an SPR kinetics (CD64) and steady state affinity (FcRn) assay based on the ICHQ2 (R1) guidelines
Michael Willcox
Bioassay Department
Eurofins BioPharma Product Testing Munich GmbH

2. Overview Interpreting the FDA Guidance Document for Kinetic Assays
Case Study 1: Kinetic Assay (CD64) Qualification
Case Study 2: Steady State Affinity Assay (FcRn) Qualification

3. Interpreting the FDA Guidance Document for Kinetic Analysis
Specificity
Linearity
Accuracy
Precision (Repeatability, Intermediate Precision)
Range

4. Interpreting the FDA Guidance Document for Kinetic Analysis
Specificity
Linearity
Accuracy
Precision (Repeatability, Intermediate Precision)
Range

5. Interpreting the FDA Guidance Document for Kinetic Analysis
Specificity
Linearity
Accuracy
Precision (Repeatability, Intermediate Precision)
Range
Additional Parameters to Assess
Baseline Stability
Capture Stability
Control Sample Reproducibility
Immobilisation Consistency

6. Biosimilar Panels Molecule Assays Simponi (Golimumab)
Cell based assay, Fab binding to soluble TNFα (SPR), Fab binding to membranous TNFα (FACS), ADCC (Promega kit), CDC, FcγRI binding (SPR), FcγRIIA131R binding (SPR), FcγRIIIA158V binding (SPR), FcRn binding (SPR), Binding to compliment (C1q ELISA / C1q SPR)
Enbrel (Etanercept)
Cell based assay, ADCC (Promega kit), CDC
Remicade (Infliximab)
Cell based assay
Humira (Adalimumab)
Avastin (Bevacizumab)
Fab binding (ELISA)
Rituxan (Rituximab)
ADCC (Promega kit), Binding to complement (C1q ELISA)
Several innovator molecules
FcγRI binding (SPR), FcγRIIA131H binding (SPR), FcγRIIA131R binding (SPR), FcγRIIB/C binding (SPR), FcγRIIIA158F binding (SPR), FcγRIIIA158V binding (SPR), FcγRIIIB binding (SPR), FcRn binding (SPR), Binding to compliment (C1q SPR)

7. Case Study 1: Kinetic Assay Outline (CD64)
Binding of Golimumab antibody to CD64
Step 3
Step 2
Concentration range 30 nM to 1920 nM (1 in 2 dilution)
Step 1
Anti His
His tagged CD64
Golimumab antibody

8. Qualification Strategy
Experiment
Analyst
Chip
Instrument
Sample 1
Sample 2
Sample 3 1 B
100% 2 A 3 4 5 6 7
1, 2 or 3
1 or 2
52 cycles 480 nM sample NA 8
non-specific protein
Heat inactivated sample
Heat inactivated sample + RS spike

9. General Electric employee
1:1 Langmuir Fit Model
Irving Langmuir
General Electric employee
(1909 – 1950)

10. Intermediate Precision
Inter-assay Precision
Intermediate Precision

11. Repeatability (Intra-assay Precision)

12. Baseline Stability and Surface Performance

13. Immobilisation Reproducibility
NaOH wash
Pre-concentration
EDC/NHS
Anti-His antibody
Ethanolamine

14. Heat Inactivated Sample + Intact Sample
Specificity
Cimzia (Specificity sample)
Heat Inactivated Sample
Heat Inactivated Sample + Intact Sample

15. System Suitability Criteria
Anti-His antibody immobilisation levels:
RU to RU
Chip performance test (HBS-EP % P20):
Mean < 5 RU, St Dev < 3 RU
Chip performance test (50 ng/mL CD64):
Mean 10.0 RU to 37.3 RU, St Dev < 10 RU
Chi Squared: <1.36
%CV (ka, kd, kD) between replicates <20%
Control sample %CV <20%

16. CD64 Validation Summary Specificity
Precision (Repeatability, Intermediate Precision)
Additional Parameters to Assess
Baseline Stability
Capture Stability
Control Sample Reproducibility
Immobilisation Consistency

17. Case Study 2: Steady State Affinity Assay Outline (FcRn)
Binding of Golimumab antibody to FcRn

18. Case Study 2: Steady State Affinity Assay Outline (FcRn) using Biotin CAPture kit
Golimumab Antibody
Biotinylated FcRn
Sensor Chip CAP
Biotin CAPture Reagent
Concentration range 7.81 nM to 500 nM (1 in 2 dilution)

19. Qualification Strategy
Experiment
Analyst
Chip
Instrument
Sample 1
Sample 2
Sample 3 1 B
100% 2 A 3 4 5 6 7
1, 2 or 3
1 or 2
non-specific protein
Heat inactivated sample
Heat inactivated sample + RS spike

20. Langmuir Adsorption Isotherm Biacore Steady State Affinity Equation
Case Study 2: Steady State Affinity Assay Outline (FcRn) using Biotin CAPture kit
Langmuir Adsorption Isotherm
Biacore Steady State Affinity Equation

21. Intermediate Precision
Inter-assay Precision
Intermediate Precision

22. Repeatability (Intra-assay Precision)

23. FcRn capture levels
Consistent FcRn capture level within and between experiments
(52 cycles per run)

24. Specificity Cimzia (Specificity sample) Fab region only
Heat Inactivated Sample
Heat Inactivated Sample + Intact Sample

25. System Suitability Criteria
Chi Squared: <76.45
%CV (kD) between replicates <20%
Control sample %CV <20%

26. FcRn Validation Summary
Specificity
Precision (Repeatability, Intermediate Precision)
Additional Parameters to Assess
Capture Stability

27. Assay Development Issues
anti-His antibody and R&D systems His tagged Human FcRn  no binding at pH 6.0
NTA chip  Variable Nickel capture and FcRn capture resulted in highly variable affinity data
Biotin CAPture kit and Immunitrack FcRn (with beta-2-microglobulin)  reproducible capture and affinity data

28. EUROFINS BioPharma Product Testing Munich
Acknowledgements
EUROFINS BioPharma Product Testing Munich
BIOASSAY department
GE Healthcare
Ulrike Graab
Zdenka Cicova
Stefanie Löbens
Rebecca Bomm
Alexander Knorre
Anja Drescher
Tim Fagge