Considerations for Successful Biomarker Bioanalysis in Regulated … · 2018. 6. 14. · Darshana...

1GLOBALPRODUCTDEVELOPMENTCLINICAL ASSAYGROUP

GLOBALPRODUCTDEVELOPMENTCLINICAL ASSAYGROUP

Considerations for Successful Biomarker Bioanalysis in Regulated Environment

Darshana Jani, [email protected]

10th European Bioanalysis ForumNovember 15, 2017


Disclaimer

The contents of this presentation reflect the personal opinion of the author and may not represent the official perspectives of the affiliated organization


Scope

§ Biomarker Basics§ Core Validation Challenges and Solutions

§ Case Studies§ Sample Analysis

§ Pre-analytical Factors§ Data Handling

§ Summary


"Almostanythingyoucanmeasure"

BiomarkersDefinitionsWorkingGroup(2001)Clin PharmTher 69(3):89RFrankandRHargreaves(2003)NatureRevDrugDisc2:566.

What is a Biomarker?

Type 0: Markers of natural history of disease and that

correlate longitudinally with known clinical

indices.

Type I: Markers that demonstrate mechanism of action of a drug.

Type II: Markers that predict a clinical benefit (surrogates).

DIFFERENT TYPES

A characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.


Translational Lab Development Lab Clinical Study Clinical UtilityResearch/

Discovery

Key

act

iviti

es a

nd d

eliv

erab

les

Identification of Biomarkers

Generate a reasonable proposalEvaluate appropriate technology platforms

• Relevance to human subjects and target disease population

• In-house assay performance?

• Commercial Reagents?• Method transfer from

preclinical matrix to human matrix

• Assay optimization-Single assay vs multiplex format?

• Ensure operational feasibility-sample type, volume, throughput

• Fit for Purpose Method validation

• Data Generation • Control/sample• Performance and

trending• Review the

challenges-Fit for purpose

• Successful data generation

• Confirm assay utility

• Assay maintenance

• Establish or revise clinical utility based on intended use of the data

Biomarker Assay Flow from Research to Clinical Study


What are the biggest challenges?

Scientific

Technical

Regulatory

What are the common accepted approaches??

Concept appears simple!


Key Questions for Successful Biomarker Assay Development

Crystal City Report Bioanalysis 2016(8)

What is the purpose of the study?

Are we measuring what we intended to measure?

What is the anticipated modulation?

How much variability is acceptable?

How does sample handling conditions impact analytical procedures and conditions?


Focus on Ligand Binding Assays, Principles Apply to Other Formats

Method Development and Validation - Fit for Purpose Approach

§ Matrix§ Reference Standards§ Sensitivity§ Reproducibility§ Specificity/Parallelism§ Robustness§ Stability

Sample Analysis

Assay Maintenance

Core Challenges in Biomarker Measurement


Matrix

PROBLEMS POSSIBLE SOLUTIONS

• Commercial kits supply diluents as a surrogate matrix - do not reflect the complex biological matrix

• Calibration matrix must be devoid of target analyte

• Must demonstrate analytical equivalency between surrogate and control matrices (e.g. during parallelism)

• Must account for influence of binding partners

• Requires additional QCs and stability assessment

• Use pooled plasma/serum from healthy volunteers

• Assess background levels of analyte

• Consider possible assay interference from biological metabolites

• Consider assay format based on relevant matrix


Case Study: Matrix is Important Consideration!

IL-17A Assay

§ Ultra sensitive measurements are required for circulating cytokine concentration

§ Cytokine profiles are altered in disease tissue§ Matched set of samples collected from Psoriatic

patients

§ Serum, Lesional tissue and Non-Lesional tissue


Serum vs Tissue

Donor

PASI Serum (pg/mL) Skin biopsy (pg/mL)

Score IL17A IL17A normal IL17A lesion

101 26.0 0.210 nd 179

102 21.6 0.114 nd 3.70

103 23.9 0.267 nd nd

104 24.3 0.266 nd 3.50

105 23.9 1.93 nd 59.7

Serum analysis requires ultrasensitive assay• IL17A LLOQ <0.05ng/mL

Skin biopsy could have been tested using one of the many commercial assay (MSD, R/D assay)nd=not detected

Consider Assay Format Based on the Matrix!PASI=Psoriasis activity and severity index


Reference Standards

PROBLEMS SOLUTIONS

• No consistency between commercial standards even when calibrated to WHO/NIBSC standards

• Variation in reagent production creating discrepancies in quantification between different kits

• Incomplete characterization• Available standards do not always

depict circulating form• Biology not well understood

• Use the same sourced standards • Perform batch-to-batch

comparisons to ensure consistency between own results


Biomarker Stability

• Clinical biomarker sample analysis doesn’t occur real time except for point of care devices

• Problem: Altered amount of analyte in matrix during sample collection, short term and long term storage

• Problem: Altered immunoreactivity in a matrix • Problem: Reference material spikes are often used for

biomarker stability, quite often not indicative of endogenous markers

• Solution: Incurred samples may provide better understanding of analyte stability


Biomarker Stability – Case Studies

TGF-β1 Stability

§ Spiked TGF- β1 into buffer and pooled urine samples§ Also fresh frozen diabetic urine samples

IL-13 Stability

§ Established using incurred samples from ongoing clinical studies


TGF- β1 Stability

Stabilitysamples Sampleconcentrationvalues

Sample1 Sample2 Sample3

Replicate1 Replicate2 Replicate1 Replicate2 Replicate1 Replicate2

PurifiedTGF-β1spikesinbuffer

Prefreeze–thaw 108 108 251 340 981 937

Postfreeze–thaw 119 94 257 296 966 1021

%recovery† 110 87 102 87 98 109

PurifiedTGF-β1spikesinurine

Prefreeze–thaw 133 119 124 1260 1280 1250

Postfreeze–thaw 93 78 76 921‡ 871‡ 892‡

%recovery 30 34 39 27 32 29

Diabeticurinesamples

Prefreeze–thaw 151 172 196 147 51 282§

Postfreeze–thaw 8 70 7 141 19 15

%recovery 5 41 4 96 37 5

Use Individual or Pooled Clinical Samples for Stability Testing


IL-13spikedsamplestability.QCconcentration

(pg/ml)%recoverymonth4

%recoverymonth5

20.3 112 571.1 94 379

% recovery: (concentration at time/initial concentration) × 100.

Unanticipated loss of stability at 5 months

IL-13 Stability (1/2)

IL-13 stability was established using serum spiked reference material


IL-13 Stability (2/2)

Sample Initial concentration (pg/ml)

Reassay concentration

(pg/ml)

% difference from original

conc.

Months in storage

1 7.5 0.3 183 9

2 0.3 0.3 -1.7 8

3 0.5 0.4 26.7 14

4 5.8 0.4 175 9

5 0.4 0.4 23 14

6 0.8 0.8 0.91 15

7 0.9 0.7 29 8

8 0.9 0.9 -2.02 15

6 of 8 met ± 30% criteria

Stability extendedto 15 months

Samples were stored beyond established stabilityIncurred samples were tested to confirm validated QC sample stability


Cross-reactivity

Cross-talk

MRD, Selectivity

Failed analyte(s)

VALIDATION

• Test with “missing man” technique

• Test with single analyte if possible

• Use highest MRD based on parallelism, reproducibility results• Confirm final conditions for all analytes• Test early on, prior to pre-validation/validation experiments

• Consider alternatives pre-validation• Rerun and evaluate failed analyte(s) data only

METHOD

Multiplex Method Validation


Samples that are most relevant to the intended clinical application based on biology

Adequate attention in specimen handling

Adequate attention for specimen integrity

§ Shared samples for various analyte § Shipping challenges§ Sample collection and processing§ Storage conditions

Range of samples/time points

Attention to Pre-analytical Factors


Data handling (work list preparation, data management, assay approval, data reporting)

All pass – if you must rerun one analyte, what do you do with data for other passing analytes from

1st run?

Identical curve fitting

Lack of regulatory performance recommendations

VALIDATION

• Possible use of macros, built in templates• Contract IT resources• Work with instrument/software vendors to assist with solutions

• Report data for analytes that pass, repeat run for analytes that do not pass (2nd run should mask results for passing analytes). Analyte that does not meet sample analysis acceptance criteria (if repeat analysis fails) should not be included in final analysis.

• Use best fit approach• It is acceptable to use different curve fits/weighting for different

analytes, however, for a single analyte continue using same curve fitting after validation

• Utilize regulatory requirements that most closely meet the study requirements

• FDA Draft Guidance on Bioanalytical Method Validation includes a discussion on biomarkers

METHOD

Data Analysis in Regulated Environment - Multiplex

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development. The AAPS Journal (# 2015) DOI: 10.1208/s12248-015-9820-y


Assay Maintenance

Recommendations for Use and Fit-for-Purpose Validation of Biomarker Multiplex Ligand Binding Assays in Drug Development. The AAPS Journal (# 2015) DOI: 10.1208/s12248-015-9820-y

Variability in reagent lots

§ Implement a defined process to investigate lot-to-lot variability

§ Apply a correction factor

§ Screen multiple lots§ Acquire sufficient volumes of an original lot with expiration dating that

allows completion of the programAvailability of critical reagents

§ Use surrogate molecules, if needed§ Initiate an analyte/antibody production program in anticipation starting the

project§ Contact vendors to assist with sourcing

§ Review research literature for possible academic sources


• Study design and assay methodology determines bioanalytical strategy

• Diverse assays require multidisciplinary team execution

• Bioanalysis is only one piece of the puzzle – consider overall biology

• Biomarker assays are not PK assays

• Use scientific judgement as appropriate

Summary


ReferencesCritical Path Institute

• Points to consider topics

AAPS NBC 2014, 2015, 2016

• Biomarker themes, topics

Crystal City V 2013

• FDA draft guidance discussion

Crystal City VI 2015

– Biomarker assay discussion

– Lowes, Ackermann 2016

– 2nd Upcoming publication

WRIB 2014, 2015, 2016

– Biomarker assay discussions

Lee et al. 2005, 2006, 2009

• Biomarker assay validation

O’Hara et al. 2012

• Critical Reagents characterization

King et al. 2014

• GBC Harmonization white paper on critical reagents for LBAs

Bower et al. 2014

• Commentary paper on reference standards and reagents in BMV


Regulations Examples

Ø FDA BMV Guidance, 2001 (US) Ø EMA BMV Guideline, 2011 (EMA)Ø ANVISA BMV Guideline 2012 (Brazil)Ø MHLW BMV Guidelines 2013 + 2014 (Japan)Ø cFDA BMV Guidelines 2015 (China)Ø Coming up: ICH M10 (concept adopted June ‘16)


Acknowledgement

• Pfizer Colleagues• Many Scientists from organizations………


• Back-Up slides


Robustness/Reproducibility

PROBLEMS SOLUTIONS

• ‘Noisy’ background• Different laboratories often

perform with different reagents and equipment

• Assay format e.g. ELISA performance may depend on reagents

• Cross-comparison of different platforms within the same laboratory

• Run healthy volunteer controls • Evaluate extent of biological

variation• Batch-run longitudinal samples per

subject to Minimize effect of biological variation between individuals

• Run the same samples in parallel in different laboratories/different platforms to assess comparability

• Often, quantitative differences are found but longitudinal trends are comparable


Specificity

PROBLEMS SOLUTIONS

• Surrogate peptides not uniquely specific

• Methods are not always selective due to matrix complexity

• Antibody used be specific

• Test antibodies from various vendors and various lots

• Consider replacing format


• Intended application of biomarkers dictates the rigor of biomarker assay validation for clinical studies

• Limited validation in exploratory and proof-of-concept biomarkers• Extensive full validation for biomarkers which provide pivotal data for critical

decision making in the drug development process, as part of regulatory requirement

Validation of biomarkers for clinical studies - Fit for purpose approach

Rigor of Validation

Considerations for Successful Biomarker Bioanalysis in Regulated … · 2018. 6. 14. · Darshana...

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