BIOMARKER DEVELOPMENT AND VALIDATION PRACTICES & EXPERIENCES Shawn Li, M.D., Ph.D. October 1, 2012 CORPORATE OVERVIEW PRESENTATION OUTLINE Introduction Frontage Capabilities and Approaches Case Studies Challenges and Solutions: Measurement of Analyte in Presence of Endogenous Protein Qualified Assays CORPORATE OVERVIEW 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 CORPORATE OVERVIEW Classification: Type 0 - Natural history markers Type 1 - Drug activity markers Type 2 - Surrogate markers 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 CORPORATE OVERVIEW BIOMARKER ASSAY CATEGORIES Continuous Analytical Response Definitive Quantitative Relative Quantitative Biomarker levels determined using a ‘reference standard’ (i.e., calibration curve) –Reference standard needs to be representative of analyte CORPORATE OVERVIEW Categorical Response QuasiQuantitative Qualitative 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 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. CORPORATE OVERVIEW BIOANALYTICAL VS. BIOMARKER ASSAYS CORPORATE OVERVIEW SUMMARY OF VALIDATION PARAMETERS APPLICABLE TO EACH CATEGORY OF BIOMARKER ASSAY Validation Parameters Definitive quantitative Relative Quasi-quantitative Qualitative quantitative Sample stability X X X X Reagent stability X X X X Assay range X X Parallelism X X Dilution linearity X X Accuracy X X Precision X X X Sensitivity X X X X Specificity X X X X Example Assays Mass ELISAs spectrometry CORPORATE OVERVIEW Immunogenicity immunoassays Immunohistochemistry 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 CORPORATE OVERVIEW 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 CORPORATE OVERVIEW CASE STUDY 1: GENOTYPING OF FCGRIIA Biomarker Category: Qualitative FcgRIIa can have either histidine (H131) or arginine (R131) at amino acid position 131 located in the IgG-binding site H131 H131/R131 R131 CRP Binding NO YES YES CRP Activation of Fibrocytes NO YES TBD SAP Binding YES YES YES SAP Inhibition of Fibrocytes YES YES YES CRP cross-inhibit SAP NO TBD TBD 1 H R H 2 3 Patient No. R H R FcgRIIA CORPORATE OVERVIEW Patients with different genotype response differently to anti-inflammatory drugs CASE STUDY 2: FISH DETECTION OF mRNA Biomarker category: Qualitative/semi-quantitative In situ hybridization of target biomarker mRNA in cancer tissue Drug treated CORPORATE OVERVIEW Vehicle injected CASE STUDY 3: WESTERN BLOT SEMI-QUANTIFICATION OF BIOMARKERS Biomarker category: Quasi-quantitative Target protein A CORPORATE OVERVIEW 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 CORPORATE OVERVIEW Drug x Dosage 3 Drug x Dosage 2 Drug x Dosage 1 Positive control BDNF RT-PCR Amplification Plot Vehicle ΒDNF CASE STUDY 5: MSD Multiplex Assay TH1/TH2 (10-Plex) CORPORATE OVERVIEW 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 CORPORATE OVERVIEW CASE STUDY 6: ANTI-DRUG ANTIBODY ASSAY Biomarker Category: Quasi-quantitative Key parameters for Points to consider for assay development validation Titer-based assay Screening cut point PCs, prefer pAbs Specificity/confirmation cut point Detect low and high affinity Sensitivity Sensitivity/w drug System suitability controls(QCs) acceptance • Preclinical: 500-1000 criteria ng/mL Selectivity/Interference • Clinical: 250-500 ng/mL • Matrix components Screening cut point, 5% FP • Drug Confirmation and titration Precision steps are needed Robustness Stability CORPORATE OVERVIEW CASE STUDY 6: ANTI-DRUG ANTIBODY ASSAY Method development and validation of anti-X123 antibody assay Evaluation Analytical Method Matrix Positive Control Drug Screening Assay Cut Point (S/N) Confirmation Cut-Point High Positive Control Low Positive Control Negative Control Titration Hook Effect Drug Tolerance Interference by Hemolysis and Lipemia Immuno-depleted Control (LPC) Immuno-depleted Control Method Relative Sensitivity Method Selectivity Bench Top Stability Refrigerated Stability Freeze Thaw Stability CORPORATE OVERVIEW Summary ELISA Monkey Serum anti-X123 X123 1.44 22.08% Intra-Assay %CV: ≤ 12.6% Inter-Assay %CV: 13.9% Intra-Assay %CV: ≤ 14.5% Inter-Assay %CV: 17.5% Intra-Assay %CV: ≤ 16.5% Inter-Assay %CV: 21.5% The average assay titer was 1:55 for TPC No Hook effect up to 100000 ng/mL 4.13µg/mL at 500ng/mL of ADA No Interference observed Average: 34.88% Average: 92.66% 167.75 ng/ 90% of the spiked and unspiked samples were within 75%125% of the respective controls. Stable up to 24 hrs Stable up to 3 days Stable up to 6 cycles 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 CORPORATE OVERVIEW 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, CORPORATE OVERVIEW time-consuming 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 CORPORATE OVERVIEW CHALLENGES IN ELISA BIOMARKER ASSAY – SELECTIVITY EVALUATION Subtract basal level Human Plasma Lot Blank Result # (pg/mL) BRH600992 208.892 BRH600993 BQL BRH600999 BQL BRH601000 BQL BRH601002 265.448 BRH601003 BQL BRH601004 BQL BRH601005 BQL BRH601006 BQL BRH591695 330.846 BRH591696 BQL BRH591706 236.192 BRH591709 362.267 BRH591714 556.763 BRH591716 368.536 BRH601001 859.823 Corrected Spiked Sample Concentratio Result (pg/mL) n (pg/mL) BQL NA 303.002 NA 315.437 NA 249.797 NA 430.442 164.994 347.240 NA 341.945 NA 235.449 NA 401.342 NA 496.075 165.229 394.732 NA 415.751 179.559 518.714 156.447 786.363 229.600 699.857 331.321 1099.457 239.634 RE% NA NA NA NA -17.5 NA NA NA NA -17.4 NA -10.2 -21.8 14.8 65.7 19.8 Pass/Fail Fail Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Pass Fail Pass 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) CORPORATE OVERVIEW 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 CORPORATE OVERVIEW 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 serumSpectramax Human Complement C3a-plasmaSpectramax Human Complement Bb-plasmaSpectramax Podocin in human urine -Spectramax Nephrin in human urine –Spectramax Creatinine in human UrineSpectramax NPY Human Plasma/serum spectramax CORPORATE OVERVIEW 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-serumSpectramax Mouse Complement C3-plasmaSpectramax Mouse Complement C5a-plasmaSpectramax 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 CORPORATE OVERVIEW