Training Course M-1 - 8am to 5:30pm, Monday April 9, 2018

"Biomarkers Troubleshooting: Successes & Failures in Biomarkers Assays Development & Validation (BAV)"

Lesson learnt from practical biomarkers case studies depicting real-life situations in which problems need to be solved in LBA, Flow Cytometry, LCMS, HRMS, and Hybrid LBA/LCMS assays

Outline of Training Course M-1

(You can click on each blue topic title below to see details, or simply scroll down to see details)


Course Co-Chair: Dr. Susan Spitz, Senior Director Clinical Research, Translational Sciences, Incyte
Course Co-Chair: Dr. Yan Zhang, Director Bioanalytical Sciences, Discovery & Biomarkers, BMS

Part 1: LBA Biomarkers Method Development & Validation
Part 2: Flow Cytometry Biomarkers Method Development & Validation
Part 3: LCMS & Hybrid LBA/LCMS Biomarkers Method Development & Validation


DETAILS of Training Course M-1

Course Co-Chair: Dr. Susan Spitz, Senior Director Clinical Research, Translational Sciences, Incyte
Course Co-Chair: Dr. Yan Zhang, Director Bioanalytical Sciences, Discovery & Biomarkers, BMS

Part 1: LBA Biomarkers Method Development & Validation
  • Lesson 1
    Non-parallelism: Successes & Failures in Biomarkers Assays Development & Validation
    - Dr. Shobha Purushothama, Associate Director, Biogen
    • Bioanalytical strategies to address non-parallelism
      • Still need for broader conceptual understanding of parallelism across Industry
      • Perspective on relevant concepts & approaches
      • Dealing with parallelism failures
    • An in-depth understanding of the fundamental nature of parallelism in development and validation of biomarker assays
      • Demonstration that sample diluted response curve is parallel to the STD concentrations response curve
        • Confirmation that the standard material used is suitable
        • Determining MRD
        • Selectivity
        • LLOQ of the endogenous analyte
      • Understanding specificity of reagents
    • What if samples with adequate levels of endogenous analyte are not available?
      • Can other approaches be applied? What is their value vs parallelism?
        • Production of “endogenous” analyte to use for spiking experiments
        • Spike recovery of recombinant/purified material on top of endogenous analyte
    • Is the Industry still doing spike recovery studies with recombinant analytes for biomarkers rather than performing parallelism?
      • Importance to always perform parallelism assessments as soon as the samples are available
    • Case Studies: Using parallelism to define biomarker assay parameters. Troubleshooting non-parallelism issues

  • Lesson 2
    Commercial Biomarker Assays: Challenges and Solutions
    - Dr. Sally Fischer, Principal Scientist, Genentech
    • Overcoming major challenges with commercial kits commonly used as biomarker tools in drug discovery & development:
      • Changes of kit lot over time (Lot-to-Lot Variability)
        • Approach to generate a correction factor to bridge lots
        • Correction factor equation
      • Selection of appropriate kit for the intended use
      • What’s new from what recommended already in the 2013 & 2015 White Papers?
        • What is the Industry doing to avoid these issues? What’s next?
    • Current strategies to ensure commercial kits:
      • Long-term availability and consistent assay performance
      • Reagent and commercial kit characterization
      • End-users’ method adaptation
        • Additional calibrators
        • Curve fitting algorithm
        • Controls in surrogate/biological matrix
      • Standardization & harmonization
    • Qualification of the kits in specific matrix/disease state of interest
    • Evaluation of matrix Interference and Impact on Quantitation of Biomarkers
    • What to do when commercial kits are found to be unsuitable for supporting their intended use?
      • Multiplexed kits lack of sensitivity and poor performance
    • Case Studies: Failed commercial kit Performance. Practical approach for qualification of the kits in specific matrix/disease state

  • Lesson 3
    Thorough Evaluation of QC Trends for Biomarkers LBA
    - Dr. Rafiqul Islam, Senior Director, Celerion
    • What we can learn from QC trend analysis
    • How to handle the acceptance criteria for QC
      • Consistent assay performance within run
      • Consistent assay performance between runs
      • Stability QC Test trend analysis
      • Assay robustness
      • Dilution and injection volume linearity
      • Eliminating nonspecific signal to enable generation of reliable results
    • Approaches to relative accuracy
      • Relative analyte concentrations based on the recombinant standards
      • Poor/no characterized standard
      • Crucial importance of longitudinal endogenous QC samples
        • Having Matrix QCs spanning the whole dynamic range
    • Importance of QC Trend for protein biomarker changes relative to their individual baseline
      • Lot changes QC trending
    • Case Studies: Biomarker assay characteristics improvement by QC trend analysis and Innovative strategies for biomarker assay development

  • Lesson 4
    Important Value of Biomarker Multiplex LBA
    - Dr. Paul Rhyne, Chief Scientific Officer, Biologic Development Services
    • Successful case studies of multiplex LBA
      • When to use multiplexing
      • Strategies to select the most suitable multiplex assay
    • Method development troubleshooting
      • Discussing main issues
      • Solutions to overcome issues
      • Interpretation of validation parameters
      • Overcoming interferences issues, sensitivity, cross-talk
    • Possibility of simplifying multiplexing with Emerging Technologies (ET)
    • Multiplex kit assays
    • Previous recommendations from 2016 White Paper Part 3
      • New data after the 2016 discussion
    • Case Studies: New applications of biomarker multiplex in LBA: method development and unique BAV


Part 2: Flow Cytometry Biomarkers Method Development & Validation
  • Lesson 5
    An in depth understanding of Flow Cytometry Strengths & Weakness in Biomarkers Assays development and comparison Flow Cytometry vs LCMS
    - Dr. Virginia Litwin, Vice President Immunology, Caprion
    - Dr. Lorella Di Donato, Senior VP Scientific Operations, Caprion
    • Flow Cytometry in the bioanalytical lab for Clinical Biomarker assays
      • Strength
        • Powerful technology for single-cell multiparameter analysis
        • Qualitative & Quantitative analysis
      • Weakness
        • Assay standardization/reproducibility
        • Sample accessibility & stability
    • Novel applications of Flow Cytometry in Clinical
      • Current best practices in
        • Validation
        • Standardization
    • Increasing performance in assay conditions
      • Staining quality
      • Subpopulations separation
    • Flow Cytometry vs LCMS
      • “Proof-of-concept” study create a decision tree in choosing which technology platform performs best in Biomarkers assays
      • Understanding on how different platforms compare and differ in terms of data interpretation
    • Case Studies: State-of-the-art strategies for successful implementation of robust clinical Flow Cytometry Biomarker assays in a bioanalytical lab and comparison with LCMS Biomaker assays

  • Lesson 6
    Flow Cytometry-based Immuno-Oncology Biomarkers: Successes & Failures in Biomarkers Assays Development & Validation
    - Dr. Catherine Aversa Fleener, Translational Oncology Lead, Pfizer
    • Strategies for development & validation of Immuno-oncology Biomarkers
      • Sensitivity and control on isotype and stained blood vs non-stained
      • Ensuring reliable quantification of immune cells
      • Ensuring enough sample stability
      • Importance of generating a good Positive Control (PC) material for BAV
    • Critical steps in the implementation of Flow Cytometry based Immuno-oncology Biomarkers biomarker assays
      • Phase I & Phase II studies
      • Development strategies for predictive & prognostic biomarkers in oncology
        • Patients’ stratification
        • Cancer immune responses
    • Flow Cytometry as the ideal technology to study the complex relationship between tumor and immune system
      • Confirming the importance of reproducible relationship between tumor microenvironment and peripheral cells for Flow Cytometry applications
      • Use of easily accessible human blood vs less accessible tumor tissues
        • Advanced applications/method development of peripheral and circulating cell populations quantification in blood
      • Unique advantages of real-time response of Flow Cytometry results to immunotherapy
    • Case Studies: Novel practical applications of Flow Cytometry in Oncology Clinical Trials

  • Lesson 7
    Biomarkers Flow Cytometry for Clinical Studies: Successes & Failures in Biomarkers Assays Development & Validation
    - Dr. Yongliang Sun, Senior Research Investigator, Bristol-Myers Squibb
    • Novel application of Flow Cytometry in support of global clinical trials
      • Development of reliable quantitative assays according to the most current industry standards
      • Procedure for generation of a standardized assays
    • Lesson learnt from previous failures
      • What to do better
      • Importance of sample collection and thorough assessment of sample stability
      • Careful evaluation of multiparameter flow cytometric staining of blood samples
      • Comparison of permeabilization buffers
        • Ensuring a sufficient permeabilization in intracellular multicolor assays
        • Risk of insufficient exposure to the detection antibody
        • Need to preserve staining of the surface markers
    • Challenges with multiple surface and intracellular panels
      • Ensuring that share markers across the panels are comparable
        • Staining patterns
        • Intensities
    • Case Studies: Flow Cytometry Biomarker assay optimization, development and FFP validation to ensure excellent precision and sufficiently robust for clinical applications

  • Lesson 8
    Target Engagement (TE) Biomarker Assays by Flow Cytometry
    - Dr. Ian Catlett, Senior Research Investigator, Bristol-Myers Squibb
    • Why is Flow Cytometry the elective technique for Target Engagement (TE) assays?
      • TE assays and need for an accurate measuring technique
      • TE assays as unique type of biomarker assay to evaluate the the direct interaction of the drug molecule and target
    • TE assays challenges & solution in development, validation and implementation
      • Overcoming development obstacles
      • Strategies in performing a successful TE assay validation
        • FFP validation design
        • Sample stability evaluation
      • Designing the correct TE assay principle
    • Importance of TE biomarkers assays as PD markers
      • Impact on rational/optimal design of dosing regimen in clinical applications
        • Key parameter to define safe starting doses for FIH clinical trials
        • Estimation in rational dose selection
      • Proof-of-mechanism
      • In-vivo activity
      • Loss of efficacy
      • Exposure
    • Case Studies: Challenges in the development and validation and implementation of a Flow Cytometry Target Engagement Biomarker assay to support clinical trials


Part 3: LCMS & Hybrid LBA/LCMS Biomarkers Method Development & Validation
  • Lesson 9
    Hybrid LBA/LCMS Biomarker Assays Method Development Challenges
    - Dr. Joe Palandra, Principal Scientist, Pfizer
    • Reaching the Sensitivity & Selectivity (S&S) needed by Hybrid LBA/LCMS
      • Where we started and where we are now for S&S
        • Advanced application of immunoaffinity (IA)
        • Most sensitive Mass Spec instrumentations
    • LCMS approaches for relative quantification of protein biomarkers
    • Understanding of what is being measured
      • State-of-the-art approaches in using the signature peptide(s)
      • One or multiple peptides
    • Considerations on Reference Standards in Biomarkers with LCMS
      • Since in LCMS the exact same peptide on recombinant protein and endogenous protein is monitored, don’t we have a true reference standard?
        • Why can’t synthetized signature peptide be considered a “true reference standard”?
      • Are the problems with protein reference standards in LCMS the same as LBA where the binding is involved?
        • In Hybrid LBA/LCMS, the IA part is only for clean up
        • Does the recent AAPS White paper on Reference Standards apply to LCMS based on the previous considerations?
      • Answering all the questions by comparing same recombinant reference standards to both LCMS (signature peptide) and LBA (binding recombinant protein)?
    • Case Studies: Recent approaches to Hybrid LBA/LCMS Biomarkers assays with special considerations on “true signature peptide” reference standards

  • Lesson 10
    Low-Level Therapeutic/Biomarker Protein Quantification in Tissues
    - Dr. Luca Ferrari, Laboratory Head, Small Molecules Bioanalytics, Roche
    • Focus on Biomarkers/Biotherapeutics Tissues analysis
      • Overcoming Sensitivity & Selectivity issue by Hybrid LBA/LCMS method development strategies
      • Tissue specific distribution
    • Adaptation of Hybrid LBA/LCMS method for complex matrices
      • Increasing LCMS sensitivity for low level-analyte applications
      • Development of a novel platform-based IA to isolate and enrich protein in tissues
      • Use of a selective, high-throughput trapping LCMS method
    • Extra significant sensitivity gain due to enrichment at peptide level to reach low levels in various tissues
    • Case Studies: Novel developments and applications of a very sensitive Hybrid LBA/LCMS assay for quantification of biotherapeutics/biomarkers in tissues

  • Lesson 11
    Protein Biomarker Quantification by Hybrid LBA/LCMS Assays: Successes & Failures with Enzymatic Digestion. What’s the lesson learnt?
    - Dr. Tim Sikorski, Exploratory Biomarkers, GlaxoSmithKline
    • Key importance of Enzymatic Digestion in Hybrid LBA/LCMS methods
      • Recent regulatory concerns on incomplete digestion
      • Current industry standards on Digestion Efficiency
        • It is often underestimated - how to ensure no impact?
        • How do we monitor it?
    • Decision making process for choosing the best present strategy
    • Novel approaches in protein biomarkers digestion
      • Digestion at elevated temperature with a novel trypsin reagent
      • Low grade trypsin – works? Or risk of protein side reactions?
      • Alternative to Trypsin
        • Glutamyl endopeptidase (Glu-C)
        • Others
        • How often are they used?
    • Case Studies: Issues with Enzymatic Digestion in the development and validation Protein Biomarker Hybrid LBA/LCMS Assays

  • Lesson 12
    State-of-the-art Method Development Approaches to Improve LCMS Biomarkers Assay Performance: Advanced applications of reagent-free LCMS methods
    - Dr. Jiang Wu, Director Global Bioanalytical and Biomarker, Shire
    • Reagent-free methods when applicable offer the best advantages vs Traditional LBA
      • No overall critical reagents issue
      • No binding issues due to interferences
      • No Lot-to-lot variability
    • Multiple clinical application of reagent-free methods
      • Development & validations strategies to achieve sensitivity & selectivity without IA cleanup/enrichment
      • Comparison with orthogonal LBA
    • Share experience and perspective on future directions for reagent-free LCMS vs Hybrid LBA/LCMS for Protein Biomarkers
    • Case Studies: Novel applications of reagent-free LCMS quantifications of Biotherapeutics/Biomarkers to support clinical trials

  • Lesson 13
    Fit for Purpose LCMS/HRMS Assay Development and Qualification for Novel Biomarkers
    - Dr. Naidong Weng, Scientific Director and Head of Bioanalytical and Pharmacokinetics US, Janssen
    • LCMS/HRMS Biomarker assays are now widely used and their application ranges from
      • Target and candidate selection and refinement in discovery
      • Safety and efficacy evaluation in drug development
      • Patient stratification and market differentiation at late phase development and post market
    • Improvement in mass spectrometry assays for novel biomarkers
      • At very low levels
      • Applied in early and late stages of drug development
    • How to successfully handle LCMS/HRMS BAV daunting task and its unique associated challenges
      • Selectivity & Sensitivity for endogenous analytes
      • Basal level fluctuation
      • Instability or pseudo-stability
    • FFP Validation and supported justification for Regulators
    • Case Studies: Importance of platform used: How the choice of LCMS vs LBA can also affect the outcome of the study






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