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Biomarker Development and Validation Practices & Experiences. Shawn Li, M.D., Ph.D. October 1, 2012. Presentation outline. Introduction Frontage Capabilities and Approaches Case Studies Challenges and Solutions: Measurement of Analyte in Presence of Endogenous Protein Qualified Assays.

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Presentation Transcript
presentation outline
Presentation outline
  • Introduction
  • Frontage Capabilities and Approaches
  • Case Studies
  • Challenges and Solutions:
    • Measurement of Analyte in Presence of Endogenous Protein
  • Qualified Assays
biomarker definition
Biomarker Definition
  • A characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention. [Clinical Pharmacology & Therapeutics (2001) 69, 89–95]
  • Disease-related Biomarkers
  • Drug-related biomarkers
    • Diagnostic Biomarker
    • Safety Biomarker
    • PD Biomarker
    • Efficacy Biomarker
    • Surrogate Biomarker
    • Prognostic Biomarker
    • Predictive Biomarker
  • Classification:
    • Type 0 - Natural history markers
    • Type 1 - Drug activity markers
    • Type 2 - Surrogate markers
role of biomarker in drug development
Role of Biomarker in drug development

Pharmacology markers for

•pharmacodynamic performance

•pharmacokinetic correlation

Disease biomarkers for

•diagnosis & prognosis

•predisposition assessment

•early detection of “toxicity”

Safety & efficacy biomarkers for

•clinical response monitoring

•surrogate endpoints

•response prediction

  • Compound selection
  • Dose selection
  • Dose monitoring
  • Therapeutical performance
  • Patient selection

Utility of biomarkers to support decision making in drug development is directly related to quality of data

biomarker assay categories
Biomarker assay categories

Continuous Analytical Response

Categorical Response

Definitive Quantitative

Relative Quantitative



Biomarker levels determined

using a ‘reference standard’

(i.e., calibration curve)

–Reference standard needs to be representative of analyte

Biomarker levels are determined without using a reference standard (i.e., calibrators)–Reference material is not available, or–Not representative of test samples

biomarker assay categories and purpose
Biomarker Assay Categories and Purpose

The fit-for-purpose approach to biomarker method validation tailors the burden of proof required to validate an assay to take account of both the nature of technology utilized and position of the biomarker in the spectrum between research tool and clinical end point. Ultimately, fit-for-purpose requires an assessment of the technical ability of the assay to deliver against the predefined purpose

IHC=immunohistochemistry; LBA=ligand binding assay; MS=mass spectrometry; PD=pharmacodynamic; POM=proof of mechanism; POC=proof of concept.

biomarker capabilities at frontage labs
Biomarker Capabilities at Frontage Labs
  • DNA
  • Genotyping, Mutation, Haplotype determination, SNP
  • Epigenetic, Methylation, Accetylation
  • RNA
  • Gene expression (real time PCR), In situ hybridization
  • Protein/Peptide
  • Single and multiplex Ligand binding assays
  • ELISA, ECL, SDS-PAGE, Western Blot
  • LC/MS/MS
  • Cytology / Histopathology
  • Tissue array
  • Immunohistochemistry / Immunocytochemistry
  • Flowcytometry
  • Immunogenicity
  • Screening/Confirmation/Titer determination
  • Cell based assay, Neutralization antibody assay
validation approaches at frontage labs
Validation Approaches at Frontage Labs

Biomarker validations

– Fit-for-purpose (FFP)

- Validation parameters differ depending on the purpose of study and method categories

- Follow bioanalytical method validation guideline as close as possible

– Biomarker validation SOP and validation protocol implementation

-Design of the experiments

-Standard and QC preparations

-Acceptance criteria

-Data reporting

case study 1 genotyping of fcgriia
Case Study 1: Genotyping of FCgRIIa

Biomarker Category: Qualitative

FcgRIIacan have either histidine (H131) or arginine (R131)at amino acid position 131 located in the IgG-binding site





Patient No.


Patients with different genotype response differently

to anti-inflammatory drugs

case study 2 fish detection of m rna
Case Study 2: FISH Detection of mRNA

Biomarker category: Qualitative/semi-quantitative

In situ hybridization of target biomarker mRNA in cancer tissue

Drug treated

Vehicle injected

case study 3 western blot semi quantification of biomarkers
Case Study 3: Western Blot Semi-Quantification of Biomarkers

Biomarker category: Quasi-quantitative

Target protein A

case study 4 rt pcr quantification
Case Study 4: RT-PCR Quantification

Biomarker category: Quasi-quantitative or Quantitative

Real-time RT-PCR quantification of BDNF mRNA in brain tissue






Drug x

Dosage 1

Drug x

Dosage 2

Drug x

Dosage 3

BDNF RT-PCR Amplification Plot

case study 5 msd multiplex assay
CASE STUDY 5: MSD Multiplex Assay

TH1/TH2 (10-Plex)

Biomarker category: Quantitative

LLOD≠ LLOQ, Matrix effect

1 curve fail ≠ all curve fail


Case Study 6: Anti-drug Antibody Assay

Immunogenicity: Unwanted immune response in the patient to biologic drugs--Development of Anti-Drug Antibodies(ADA)

Neutralizing antibodies: Prevent drug from binding to the target molecule either by binding directly to epitopes in active site or by steric hindrance: abolish effect of the drug

  • Hypersensitivity reactions
  • Neutralize the activity of an endogenous equivalent, resulting in a deficiency syndrome.
  • Efficacy
  • Altered drug PK profile due to change in clearance

Non-neutralizing antibodies: Bind to sites on the drug molecule without affecting target binding and efficacy


Case Study 6: Anti-drug Antibody Assay

Biomarker Category: Quasi-quantitative

  • Key parameters for validation
    • Screening cut point
    • Specificity/confirmation cut point
    • Sensitivity
    • System suitability controls(QCs) acceptance criteria
    • Selectivity/Interference
      • Matrix components
      • Drug
    • Precision
    • Robustness
    • Stability
  • Points to consider for assay development
    • Titer-based assay
    • PCs, prefer pAbs
    • Detect low and high affinity
    • Sensitivity/w drug
      • Preclinical: 500-1000 ng/mL
      • Clinical: 250-500 ng/mL
    • Screening cut point, 5% FP
    • Confirmation and titration steps are needed

Case Study 6: Anti-drug Antibody Assay

Method development and validation of anti-X123 antibody assay

challenges in elisa based biomarker assay
Challenges in ELISA-based Biomarker Assay
  • Measurement of analyte in presence of endogenous protein:
  • Endogenous compound can exist in multiple isoforms or clipped forms in matrix
  • Multiple configurations of LBA: options to measure different compound forms
  • Appropriate choice of binding reagent, incubation times, buffers, sample dilution etc
  • Analog contains specific epitopes (characterization required)
  • Specific reagent development may needed
  • Unexpected challenges encountered with most commercial assay kits
challenges in elisa biomarker assay
Challenges in ELISA Biomarker Assay
  • How to create STDs/QC when endogenous levels present in matrix
  • – Use of matrix with low endogenous levels
  • – Use of substituted matrix
  • – Prepared in buffer
  • Subtract basal level
  • – Analysis of blank sample (zero spike)
  • – Endogenous amount subtracted, nominal amount of added spike determined
  • Endogenous and therapeutic act in similar manner: correction factor applied
  • – If endogenous and therapeutic NOT linear, correction factor cannot be applied: total measured concentration reported
  • Matrix can be stripped (charcoal):
  • – Not typically recommended
  • – Incomplete removal?
  • – Expensive, time-consuming
challenges in elisa biomarker assay mrd determination
Challenges in ELISA Biomarker Assay – MRD Determination

MRD: The smallest dilution to which a sample must be diluted in buffer to optimize accuracy and precision in an assay run by reducing the signal to noise ratio

challenges in elisa biomarker assay selectivity evaluation
Challenges in ELISA Biomarker Assay – Selectivity Evaluation

Subtract basal level

Method LLOQ = 200 pg/mL

Acceptance Criteria: If the measured concentration in the blank is ≥ LLOQ, the endogenous level will be subtracted. If the measured concentration in the blank is < LLOQ, the spiked concentration should be between 150-450 pg/mL (blank range + LLOQ range)

lc ms ms method validation of endogenous compounds
LC/MS/MS Method Validation of Endogenous Compounds

Case Study 1

A Highly Sensitive and Selective Method for the Determination of Leukotriene B4 (LTB4) in Ex-vivo Stimulated Human Plasma by Ultra Fast Liquid Chromatography–Tandem Mass Spectrometry

Case Study 2

Determination of an Endogenous Biomarker - 4β-Hydroxycholesterol

in K2EDTA Human Plasma by LC-MS/MS

qualified assays complete biomarker list available
Qualified Assays (complete biomarker list available)
  • Human IFNg-plasma-MSD
  • Human IL6-plasma-MSD
  • Human IL1-b-plasma-MSD
  • Human TNF-α-MSD
  • Human PSA-serum-Spectramax
  • Human Testosterone serum-Spectramax
  • Human Complement C3a-plasma-Spectramax
  • Human Complement Bb-plasma-Spectramax
  • Podocin in human urine -Spectramax
  • Nephrin in human urine –Spectramax
  • Creatinine in human Urine-Spectramax
  • NPY Human Plasma/serum - spectramax
  • Fibronectin in rat urine-Spectramax
  • MCP-1 in rat urine-MSD
  • Collagen IV in rat urine-Spectramax
  • sGAG in rat urine-Spectramax
  • Mouse IFNg-plasma-MSD
  • Mouse IL6-plasma-MSD
  • Mouse IL1-b-plasma-MSD
  • Mouse TNF-a-plasma-MSD
  • Mouse PSA-serum-Spectramax
  • Mouse Testosterone-serum-Spectramax
  • Mouse Complement C3-plasma-Spectramax
  • Mouse Complement C5a-plasma-Spectramax
thank you
Thank You!
  • Shawn Li, M.D., Ph.D.
  • Director, Biologics Services
  • Frontage Laboratories, Inc.
  • 700 Pennsylvania Drive
  • Exton, PA 19341
  • Tel:  484-348-4860 /  Fax:  610.232.0101
  • Email: