FcRn assay based on the ICHQ2 R1 guidelines Michael Willcox Bioassay Department Eurofins BioPharma Product Testing Munich GmbH Overview Interpreting the FDA Guidance Document ID: 689121
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Slide1
1
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 GmbHSlide2
OverviewInterpreting the FDA Guidance Document for Kinetic AssaysCase Study 1: Kinetic Assay (CD64) QualificationCase Study 2: Steady State Affinity Assay (FcRn) Qualification
2Slide3
Interpreting the FDA Guidance Document for Kinetic AnalysisSpecificityLinearityAccuracyPrecision (Repeatability, Intermediate Precision)Range3Slide4
Interpreting the FDA Guidance Document for Kinetic AnalysisSpecificityLinearityAccuracyPrecision (Repeatability, Intermediate Precision)Range4Slide5
Interpreting the FDA Guidance Document for Kinetic AnalysisSpecificityLinearityAccuracyPrecision (Repeatability, Intermediate Precision)RangeAdditional Parameters to AssessBaseline StabilityCapture
Stability
Control Sample
Reproducibility
Immobilisation
Consistency
5Slide6
Biosimilar Panels6MoleculeAssaysSimponi (Golimumab)
Cell based assay, Fab binding to soluble TNFα (SPR), Fab binding to membranous TNFα
(FACS), ADCC (
Promega
kit), CDC, Fc
γ
RI binding (SPR), Fc
γ
RIIA131R binding (SPR), Fc
γ
RIIIA158V 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)
Cell based assay, ADCC (
Promega
kit), CDC
Avastin
(Bevacizumab)
Fab binding (ELISA)
Rituxan
(Rituximab)
ADCC (
Promega
kit), Binding to complement (C1q ELISA)
Several innovator molecules
Fc
γ
RI
binding (SPR), Fc
γ
RIIA131H binding (SPR), Fc
γ
RIIA131R binding (SPR), Fc
γ
RIIB/C binding (SPR), Fc
γ
RIIIA158F binding (SPR), Fc
γ
RIIIA158V binding (SPR), Fc
γ
RIIIB binding (SPR),
FcRn
binding (SPR), Binding to compliment (C1q SPR)Slide7
Case Study 1: Kinetic Assay Outline (CD64)
Step 1
Step 2
Step 3
Anti His
His
tagged
CD64
Golimumab
antibody
Binding
of
Golimumab
antibody
to
CD64
Concentration
range
30
nM
to
1920
nM
(1 in 2
dilution
)Slide8
Qualification Strategy8ExperimentAnalystChipInstrumentSample 1Sample 2
Sample 3
1
B
1
1
100%
100%
100%
2
A
1
2
100%
100%
100%
3
A
2
2
100%
100%
100%
4
B
2
1
100%
100%
100%
5
A
31100%
100%100%
6B
32100%100%100%7A1, 2 or 31 or 2
52 cycles 480 nM sampleNA
NA8
B1, 2 or 31 or 2non-specific proteinHeat inactivated sampleHeat inactivated sample + RS spikeSlide9
1:1 Langmuir Fit Model9Irving LangmuirGeneral Electric employee (1909 – 1950)Slide10
Intermediate Precision10Inter-assay PrecisionIntermediate PrecisionSlide11
Repeatability (Intra-assay Precision)11Slide12
Baseline Stability and Surface Performance12Slide13
Immobilisation Reproducibility13
Pre-concentration
NaOH
wash
EDC/NHS
Anti-His
antibody
EthanolamineSlide14
Specificity14Cimzia (Specificity sample)Heat Inactivated Sample
Heat
Inactivated
Sample +
Intact
SampleSlide15
System Suitability CriteriaAnti-His antibody immobilisation levels: 3450.6 RU to 5668.9 RUChip performance test (HBS-EP + 0.05% P20): Mean < 5 RU, St Dev < 3 RUChip performance test (50 ng/mL CD64): Mean 10.0 RU to 37.3 RU, St Dev < 10 RUChi Squared: <1.36 %CV (ka, kd, kD) between replicates <20%Control sample %CV <20%
15Slide16
16CD64 Validation Summary
Specificity
Precision (
Repeatability
, Intermediate Precision)
Additional Parameters
to
Assess
Baseline
Stability
Capture
Stability
Control Sample
Reproducibility
Immobilisation
ConsistencySlide17
Case Study 2: Steady State Affinity Assay Outline (FcRn)Binding of
Golimumab
antibody
to
FcRnSlide18
Case Study 2: Steady State Affinity Assay Outline (FcRn) using Biotin CAPture kitGolimumab Antibody
Biotinylated
FcRn
Sensor Chip CAP
Biotin
CAPture
Reagent
Concentration
range
7.81
nM
to
500
nM
(1 in 2
dilution
)Slide19
Qualification Strategy19ExperimentAnalystChipInstrumentSample 1Sample 2
Sample 3
1
B
1
1
100%
100%
100%
2
A
1
2
100%
100%
100%
3
A
2
2
100%
100%
100%
4
B
2
1
100%
100%
100%
5
A
31100%
100%100%
6B
32100%100%100%7B1, 2 or 31 or 2
non-specific proteinHeat inactivated sample
Heat inactivated sample + RS spikeSlide20
Case Study 2: Steady State Affinity Assay Outline (FcRn) using Biotin CAPture kit
Biacore
Steady
State
Affinity
Equation
Langmuir Adsorption IsothermSlide21
Intermediate Precision21Inter-assay PrecisionIntermediate PrecisionSlide22
Repeatability (Intra-assay Precision)22Slide23
FcRn capture levels23Consistent FcRn capture level within and between experiments
(52
cycles
per
run
)Slide24
Specificity24Cimzia (Specificity sample)Fab region onlyHeat Inactivated Sample
Heat
Inactivated
Sample +
Intact
SampleSlide25
System Suitability CriteriaChi Squared: <76.45%CV (kD) between replicates <20%Control sample %CV <20%25Slide26
26FcRn Validation Summary
Specificity
Precision (
Repeatability
, Intermediate Precision)
Additional Parameters
to
Assess
Capture
StabilitySlide27
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
27Slide28
Acknowledgements28EUROFINS BioPharma Product Testing MunichBIOASSAY departmentUlrike Graab Zdenka CicovaStefanie Löbens
Rebecca
Bomm
Alexander
Knorre
GE Healthcare
Anja
Drescher
Tim
Fagge