single-well analysis in ligand-binding assays

Single-Well Analysis in Ligand-Binding Assays: Retrospective Evaluation of Validation and Study Sample Data of PK and ADA Assays using ELISA

Enric Bertran, Herbert F. Birnboeck, Eginhard SchickClinical Pharmocology and Bioanalytical R&DpRED Pharmaceutical Sciences, Roche Innovation Center BaselEBF 10th Open Symposium, Barcelona 2017

Single-Well Analysis in Ligand-Binding AssaysOutline

2

Introduction

HistoricalPerspective

BACommunityandRegulator’sView

RetrospectiveEvaluation:Duplicatevs.Single-WellAnalysis

5 CaseStudies:PKandImmunogenicityAssays

Conclusion

E. Bertran, H. Birnboeck, E. Schick, 10th EBF Open Symposium, Barcelona 2017

Single-Well Analysis in Ligand-Binding AssaysSome important evolutions in Bioanalysis

3E. Bertran, H. Birnboeck, E. Schick, 10th EBF Open Symposium, Barcelona 2017

RIA1959 Yalow

First Microtiter Plates (MTP)1950’s, Takatsy

ELISA1960’s

GyrolabLate 1990’s

Standardization of MTPsLate 1990’s, SBS

First Autoanalyzer1957, Skeggs

Flow Injection Analyzer

1975

LC-MS1980’s

Single-Well Analysis in Ligand-Binding AssaysDifferent Technology Platforms and Assay Variability

4

Assay Variability

fully manual fully automated

RadioImmunoAssayAutoanalyzerMTP Assay Formats


Single-Well Analysis in Ligand-Binding AssaysEvolution of scientific and regulatory landscape

• Historically, duplicate or even triplicate analysis has been the standardapproach in LBA because of inherent variability in assay platforms andtechnical equipment

• Singlicate analysis has been the accepted standard approach in chromatographic assays in Small Molecule BA (HPLC-UV, GC-MS, LC-MS)

• Modern assay technologies and platforms with low variability can supportsinglicate analysis also for LBAs

• Regulators are open to singlicate analysis in LBA if appropriate performancecan be shown in assay validation


Single-Well Analysis in Ligand-Binding AssaysEvolution of scientific and regulatory landscape


EBF Workshop Brussels, June 2014

Conducting ELISAs: are duplicates always necessary or do singletssuffice?

QPS Presentation

Single-Well Analysis in Ligand-Binding Assays Case Studies - General Design

7

1• Select various assay formats using ELISA platform (PK and

ADA assays)

2• Evaluate validation and sample assay data sets retrospectively

using duplicate-well and single-well evaluation.

3• Compare data and outcome


Single-Well Analysis in Ligand-Binding Assays Case Study 1: PK Assay Validation

MTP based classical ELISA for quantification of a monoclonal Ab


Substrate

MTP with Capture Ab

Labeled Detection Ab

2nd Label AbAb Drug



• Outcome of validation parameters very similar, with minimal variation, regardless of evaluation method (duplicate vs. single well)

• Overall outcome of validation within acceptance range, regardless of evaluation method (duplicate vs. single well)

Single-Well Analysis in Ligand-Binding Assays Case Study 2: PK Assay Validation and Sample Analysis

MTP based classical ELISA for quantification of a monoclonal Ab


Substrate

2nd Label AbAb Drug Labeled Detection Ab

MTP with Target

Single-Well Analysis in Ligand-Binding Assays Case Study 2 (PK Assay Validation):


Single-Well Analysis in Ligand-Binding Assays Case Study 2 (Sample Analysis):

Correlation of duplicate well vs. single well results from individual samples


Single-Well Analysis in Ligand-Binding Assays Case Study 2: PK Assay Validation and Sample Analysis




• No systematic deviation observed between concentration data, regardless of evaluation method (duplicate vs. single well)

Single-Well Analysis in Ligand-Binding Assays Case Study 3: ADA Assay Validation

MTP based classical bridging ELISA for determination of ADA


Substrate

2nd Label AbADAMTP with Drug Labeled Drug





• Screening and confirmatory cut-points very similar

• Assay sensitivity at cut-point very similar

Single-Well Analysis in Ligand-Binding Assays Case Study 4: Evaluation of clinical data (quantification of drug in serum)


Specific drug assay

• Assay used for quantification of drug in human plasma

• Total drug: acid dissociation prior to assay• Active/free drug: short incubation times, no acid dissociation

Further Reading:Staack RF et al. Free analyte QC concept: a novel approach to prove correct quantification of free therapeutic protein drug/biomarker concentrations. Bioanalysis 6(4), 485-496 (2014).Schick E et al. Validation of a ligand binding assay for active protein drug quantification following the “free analyte QC concept”. Bioanalysis 9(1), 2537-2549, 2016



Active/free drugTotal drug

Relative Difference (%) Total drug Active/free drug

Mean 2.5 3.2

Min 0.0 0.0

Max 23.6 27.7

Relative Difference (%) = │well 1−well 2│average (well1/2)



Accuracy (%) Precision (%CV)

Total drug(well 1/2)

Active drug(well 1/2)



CalibrationStandards

98.5 to 101.697.7 to 103.6

98.4 to 102.498.4 to 101.6

0.5 to 2.91.0 to 3.9

0.9 to 7.00.6 to 5.7

QC Samples 94.7 to 106.391.3 to 108.1

101.0 to 104.0102.4 to 104.7

5.7 to 7.95.0 to 8.1

9.2 to 12.79.9 to 13.8

Precision and Accuracy

Incurred Sample Reanalysis



Relative Difference (well 1)Relative Difference (well 2)

-15.9 to 24.8-17.5 to 18.1

-31.3 to 41.3-36.9 to 40.9

% Pass rate (well 1)% Pass rate (well 2)

100100

93.293.2



• Excellent correlation between concentration data from well1/2 over the whole concentration range

• No systematic deviation observed between concentration data from well1/2

• Comparable precision and accuracy in calibration standards and QC samples from well1/2

• Similar ISR success rate and comparable Relative Difference data from well1/2

Single-Well Analysis in Ligand-Binding Assays Case Study 5: Evaluation of clinical data (immunogenicity testing)


ADA Bridging Assay

• Data evaluation performed for

• Screening assay (assay signal)• Confirmatory assay (% inhibition)• Titration assay (titer)



Screening Assay Confirmatory Assay Titration Assay



• Excellent correlation between results from well1/2 in screening and confirmatory assay

• False positive rate 3.4% for well 1 and well 2 data evaluation

• Good correlation between titration assay results from well1/2

• Identical titers in 89.7% of all cases

• Maximum observed deviation was one dilution step (factor 3)

Single-Well Analysis in Ligand-Binding Assays

Conclusion (1)

25

Ø Single-well evaluation of validation and study sample data delivers similar overall outcome compared to duplicate-well evaluation

Ø Key validation parameters are comparable within narrow ranges

Ø Back-calculated concentrations of study samples are comparable within narrow ranges

Ø Cut-points in immunogenicity assays are very similar


Single-Well Analysis in Ligand-Binding Assays

Conclusion (2)

26

Ø Processing of plates using single-well pipetting requires more focus when done manually (more samples per plate)

Ø Operational feasibility should be tested in realistic cases (studies)

Ø Two (or more) sets of singlicate QCs per plate may be adequate

Ø May be appropriate to increase the ISR rate in assays to build further confidence in single-well analysis approach


Acknowledgement

Lab of E Schick , RIC Basel

Caroline Kreuzer

Colleagues from Large Molecule Bioanalytical R&D:

RIC Munich & RIC Basel

pRED Pharmaceutical Sciences


Julia Heinrich

Lisa Benincosa

Thomas Singer

Doing now what patients need next

single-well analysis in ligand-binding assays

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