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1 Qualification of an SPR kinetics (CD64) and steady state affinity ( 1 Qualification of an SPR kinetics (CD64) and steady state affinity (

1 Qualification of an SPR kinetics (CD64) and steady state affinity ( - PowerPoint Presentation

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1 Qualification of an SPR kinetics (CD64) and steady state affinity ( - PPT Presentation

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

binding 100 spr sample 100 binding sample spr assay fcrn precision capture affinity inactivated repeatability intermediate antibody study kit

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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