Biomarker Development and Validation Practices & Experiences

1. Biomarker Development and ValidationPractices & Experiences Shawn Li, M.D., Ph.D. October 1, 2012

2. Presentation outline • Introduction • Frontage Capabilities and Approaches • Case Studies • Challenges and Solutions: • Measurement of Analyte in Presence of Endogenous Protein • Qualified Assays

3. 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

4. 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

5. Biomarker assay categories Continuous Analytical Response Categorical Response Definitive Quantitative Relative Quantitative Quasi-Quantitative Qualitative 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

6. 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.

7. Bioanalytical vs. Biomarker Assays

8. Summary of validation parameters applicable to each category of biomarker assay

9. 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

10. 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

11. 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 1 2 3 H R H R H R Patient No. FcgRIIA Patients with different genotype response differently to anti-inflammatory drugs

12. 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

13. Case Study 3: Western Blot Semi-Quantification of Biomarkers Biomarker category: Quasi-quantitative Target protein A

14. Case Study 4: RT-PCR Quantification Biomarker category: Quasi-quantitative or Quantitative Real-time RT-PCR quantification of BDNF mRNA in brain tissue β-Actin ΒDNF Positive control Vehicle Drug x Dosage 1 Drug x Dosage 2 Drug x Dosage 3 BDNF RT-PCR Amplification Plot

15. CASE STUDY 5: MSD Multiplex Assay TH1/TH2 (10-Plex) Biomarker category: Quantitative LLOD≠ LLOQ, Matrix effect 1 curve fail ≠ all curve fail

16. 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

17. 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

18. Case Study 6: Anti-drug Antibody Assay Method development and validation of anti-X123 antibody assay

19. 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

20. 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

21. 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

22. 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)

23. 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

24. 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

25. 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: shawnli@frontagelab.com • www.frontagelab.com