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CORPORATE OVERVIEW BIOMARKER DEVELOPMENT AND VALIDATION PRACTICES & EXPERIENCES Shawn Li, M.D., Ph.D. October 1, 2012.

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Presentation on theme: "CORPORATE OVERVIEW BIOMARKER DEVELOPMENT AND VALIDATION PRACTICES & EXPERIENCES Shawn Li, M.D., Ph.D. October 1, 2012."— Presentation transcript:

1 CORPORATE OVERVIEW BIOMARKER DEVELOPMENT AND VALIDATION PRACTICES & EXPERIENCES Shawn Li, M.D., Ph.D. October 1, 2012

2 CORPORATE OVERVIEW  Introduction  Frontage Capabilities and Approaches  Case Studies  Challenges and Solutions:  Measurement of Analyte in Presence of Endogenous Protein  Qualified Assays PRESENTATION OUTLINE

3 CORPORATE OVERVIEW 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 BIOMARKER DEFINITION  Classification:  Type 0 - Natural history markers  Type 1 - Drug activity markers  Type 2 - Surrogate markers

4 CORPORATE OVERVIEW 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 CORPORATE OVERVIEW BIOMARKER ASSAY CATEGORIES Biomarker levels determined using a ‘reference standard’ (i.e., calibration curve) –Reference standard needs to be representative of analyte 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 Definitive Quantitative Relative Quantitative Quasi- Quantitative Qualitative Continuous Analytical Response Categorical Response

6 CORPORATE OVERVIEW 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 CORPORATE OVERVIEW BIOANALYTICAL VS. BIOMARKER ASSAYS

8 CORPORATE OVERVIEW SUMMARY OF VALIDATION PARAMETERS APPLICABLE TO EACH CATEGORY OF BIOMARKER ASSAY Validation Parameters Definitive quantitative Relative quantitative Quasi-quantitativeQualitative Sample stabilityXXXX Reagent stabilityXXXX Assay rangeXX ParallelismXX Dilution linearityXX AccuracyXX PrecisionXXX SensitivityXXXX SpecificityXXXX Example Assays Mass spectrometry ELISAsImmunogenicity immunoassays Immunohisto- chemistry

9 CORPORATE OVERVIEW 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 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

11 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 H131H131/R131R131 CRP BindingNOYES CRP Activation of FibrocytesNOYESTBD SAP BindingYES SAP Inhibition of FibrocytesYES CRP cross-inhibit SAPNOTBD H R H R H R 123 Patient No. FcgRIIA Patients with different genotype response differently to anti-inflammatory drugs

12 CORPORATE OVERVIEW CASE STUDY 2: FISH DETECTION OF mRNA Biomarker category: Qualitative/semi-quantitative In situ hybridization of target biomarker mRNA in cancer tissue Drug treatedVehicle injected

13 CORPORATE OVERVIEW CASE STUDY 3: WESTERN BLOT SEMI-QUANTIFICATION OF BIOMARKERS Biomarker category: Quasi-quantitative Target protein A

14 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 ΒDNF Vehicle Positive control Drug x Dosage 1 Drug x Dosage 2 Drug x Dosage 3 BDNF RT-PCR Amplification Plot

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

16 CORPORATE OVERVIEW 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 CORPORATE OVERVIEW Points to consider for assay development Titer-based assay PCs, prefer pAbs Detect low and high affinity Sensitivity/w drug Preclinical: ng/mL Clinical: ng/mL Screening cut point, 5% FP Confirmation and titration steps are needed 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 Biomarker Category: Quasi-quantitative CASE STUDY 6: ANTI-DRUG ANTIBODY ASSAY

18 CORPORATE OVERVIEW Method development and validation of anti-X123 antibody assay CASE STUDY 6: ANTI-DRUG ANTIBODY ASSAY EvaluationSummary Analytical MethodELISA MatrixMonkey Serum Positive Controlanti-X123 DrugX123 Screening Assay Cut Point (S/N)1.44 Confirmation Cut-Point22.08% High Positive Control Intra-Assay %CV: ≤ 12.6% Inter-Assay %CV: 13.9% Low Positive Control Intra-Assay %CV: ≤ 14.5% Inter-Assay %CV: 17.5% Negative Control Intra-Assay %CV: ≤ 16.5% Inter-Assay %CV: 21.5% TitrationThe average assay titer was 1:55 for TPC Hook EffectNo Hook effect up to ng/mL Drug Tolerance4.13µg/mL at 500ng/mL of ADA Interference by Hemolysis and LipemiaNo Interference observed Immuno-depleted Control (LPC)Average: 34.88% Immuno-depleted ControlAverage: 92.66% Method Relative Sensitivity ng/ Method Selectivity 90% of the spiked and unspiked samples were within 75%125% of the respective controls. Bench Top Stability Stable up to 24 hrs Refrigerated Stability Stable up to 3 days Freeze Thaw Stability Stable up to 6 cycles

19 CORPORATE OVERVIEW 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-BASED BIOMARKER ASSAY

20 CORPORATE OVERVIEW 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

21 CORPORATE OVERVIEW 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 CORPORATE OVERVIEW CHALLENGES IN ELISA BIOMARKER ASSAY – SELECTIVITY EVALUATION Subtract basal level Human Plasma Lot # Blank Result (pg/mL) Spiked Sample Result (pg/mL) Corrected Concentratio n (pg/mL)RE%Pass/Fail BRH BQLNA Fail BRH600993BQL NA Pass BRH600999BQL NA Pass BRH601000BQL NA Pass BRH Pass BRH601003BQL NA Pass BRH601004BQL NA Pass BRH601005BQL NA Pass BRH601006BQL NA Pass BRH Pass BRH591696BQL NA Pass BRH Pass BRH Pass BRH Pass BRH Fail BRH 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 pg/mL (blank range + LLOQ range)

23 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

24 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 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 CORPORATE OVERVIEW Shawn Li, M.D., Ph.D. Director, Biologics Services Frontage Laboratories, Inc. 700 Pennsylvania Drive Exton, PA Tel: / Fax: THANK YOU!


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