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

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

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

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

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

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)

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

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

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

Intermediate Precision Inter-assay Precision Intermediate Precision

Repeatability (Intra-assay Precision)

Baseline Stability and Surface Performance

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

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

System Suitability Criteria Anti-His antibody immobilisation levels: 3450.6 RU to 5668.9 RU Chip performance test (HBS-EP + 0.05% 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%

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

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

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)

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

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

Intermediate Precision Inter-assay Precision Intermediate Precision

Repeatability (Intra-assay Precision)

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

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

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

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

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

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