Main Workshop DAY 2A - Discussion Topic List
June 19, 2020, 11am to 4pm ET

"Multiple Technologies to solve Complex Bioanalytical Issues - DAY 2A"

View Main Workshop DAY 2A Agenda

DAY 2A: Application of Multiple Technologies to solve Complex Bioanalytical Issues

Chair: Dr. Hendrik Neubert, Sr. Director Translational Biomeasures and Protein Biomarkers Group, Pfizer

 

Topic DETAILS of DAY 2A - Multiple Technologies

Day 2A: Application of Multiple Technologies to solve Complex Bioanalytical Issues

Chair: Dr. Hendrik Neubert, Sr. Director Translational Biomeasures and Protein Biomarkers Group, Pfizer

 

  • Topic 1
    Complementary qPCR & Hybrid LBA/LCMS for Transgene Product Quantification: Application of multiple technologies to solve complex bioanalytical issues in Gene Therapy
    - Dr. Hendrik Neubert, Sr. Director Translational Biomeasures and Protein Biomarkers Group, Pfizer
    • Gene Therapy is gaining major attention in addressing an unmet medical need
      • The aim is to achieve successful expression of the transgene in a targeted tissue
    • qPCR application for Integration Site Analysis
      • Determination of integration sites and all fusion sequences
      • Identification of viral vector genomic sequences
      • Quantitative assay used to analyze the samples for vector sequences
      • Ability of the assay to differentiate between the product and endogenous virus
      • If viral vector is detected in a tissue, the expression of the transgene is assessed by Hybrid LBA/LCMS
    • Hybrid LBA/LCMS
      • Innovative bioanalytical techniques for the measurements of the transgene product in in a targeted tissue
      • Overcoming technical challenges: efficient protein extraction, measurement specificity, sensitivity and accuracy
      • Peptide-base quantification of the transgene product
      • Workflows based on protein characteristics
    • Case studies where qPCR & Hybrid LBA/LCMS are used for viral vector detection and transgene quantification in a tissue
  •  

  • Topic 2
    Current Experience in ADA Isotype Analysis Using LBA (MSD), Hybrid LBA/LCMS and Genalyte Technologies: Application of multiple technologies to solve complex bioanalytical issues in Immunogenicity
    - Dr. Boris Gorovits, Sr. DirectorBioanalytical Lab, Pfizer
    • New developments in Hybrid assays for ADA Isotype Analysis and How sciences evolved from 2015-2017 White Paper in Bioanalysis Recommendations
    • Growing need to assess isotype distribution of ADA responses to biotherapeutics
      • Identifying isotype by various technologies
        • Hybrid LBA/LCMS
        • LBA
        • Genalyte
    • Ability of multiplex platforms to identify isotype and domain specificity of ADA
      • Screening and characterizing pre- and post-dose ADA reactivity
      • Technologies allowing for a multiplexed and streamlined evaluation
      • Evaluation of what can be accomplished with these technologies.
        • Sample pre-treatment
        • Technology specific read out step
        • Data interpretation
    • Hybrid LBA/LCMS potential to offer additional semi-quantitative comparison of signal observed for individual isotypes
    • Case studies on current experience in ADA isotype analysis using LBA (MSD), Hybrid LBA/LCMS and Genalyte technologies
  •  

  • Topic 3
    Successful Novel Cases of LBA & Hybrid LBA/LCMS Complementary & Orthogonal Application for PK Assays & ADA Assays: Solving multiple complex bioanalytical issues with multiple technologies
    - Dr. Mark Qian, Director Bioanalytical Sciences, Takeda
    • PK Assays
    • ADA Assays
      • Indirect detection of ADA by LBA
        • Traditional method for assessment of ADAs
        • Susceptibility to target and drug interferences
      • Direct measurement of ADA by Hybrid LBA/LCMS
        • Pros & Cos from previous recommendations
        • Recent improvements
          • This innovative topic was shared with the audience for the first time at the WRIB in 2015
          • The 2015-2017 White Paper recommendations produced the interest in Regulators to understand the advantages of Hybrid LBA/LCMS for immunogenicity testing vs traditional approaches
        • Ability to directly measure signature peptides originated from ADA
        • Specificity and opportunity of isotyping
    • New case studies on assessment of ADA by Hybrid LBA/LCMS
  •  

  • Topic 4
    Endogenous Biotin Interference for:
    • LBA - Potential bias and challenges with bridging ADA assays
    • Hybrid LBA/LCMS - Potential bias and challenges in quantitation of biotherapeutics & biomarkers
    - Dr. Kay-Gunnar Stubenrauch, Head Large Molecule Bioanalytical Sciences, Roche
    - Mr. William Mylott, Director Biologics by LC-MS/MS, PPD
    • Issues produced by biotin interference caused by dietary supplements
      • Excess of biotin in blood is getting more common due to supplement ingestion & over-ingestion
      • Endogenous biotin can interfere with both LBA and Hybrid LBA/LCMS
    • Impact of Endogenous biotin on Streptavidin based assays
      • Limitations of biotin-streptavidin system
      • Endogenous biotin in human plasma/serum as potential interfering factor
      • Competition between Endogenous biotin and biotinylated antibody to bind with streptavidin
    • How to minimize or eliminate this potential issue?
      • Extent of interference is assay specific
      • Assessment during development phase is highly recommended
      • Evaluation of the levels of biotin in plasma/serum which can impact the quantitative performance of the assay
        • MRD adjustment
        • Sample pre-treatment
    • Case studies on both LBA and Hybrid LBA/LCMS where endogenous biotin produced significant bias on bioanalytical data.
  •  

  • Topic 5
    Recent Advancements in Exosomes Bioanalysis as Liquid Biopsies: Solving complex exosomes bioanalytical issues with multiple technologies
    • Biological Challenges and Flow Cytometry
    • Hybrid LBA/LCMS
    • qPCR & Western Blots
    - Dr. Lindsay King, Assoc. Research Fellow BioMedicines Design, Pfizer
    - Dr. Hendrik Neubert, Sr. Director Translational Biomeasures and Protein Biomarkers Group, Pfizer
    - Dr. Jean-Claude Marshall, Sr. Director Clinical Biomarker Group and Precision Medicine, Pfizer
    • Bioanalysis fundamental role in Liquid Biopsies evaluation:
      • Current Facts, Challenges, and Future Perspectives
      • 2020 White Paper in Bioanalysis recommendations on Liquid Biopsies
    • Industry standards for Detection and Quantification in patient-derived liquid samples of
      • Circulating Tumor Cells (CTCs)
      • Circulating Tumor DNA (ctDNA)
      • Extracellular Vesicles
    • Exosomes are extracellular vesicles first described as such 30 years ago and since implicated in cell–cell communication and the transmission
      • Exosome-based Proteins from Liquid Biopsies
      • Exosomes special sample preparation and separation from soluble molecules
      • What types of protein biomarkers should be measured from plasma and other body fluids
    • Biologic properties of extracellular vesicles
      • Diagnostic value
      • Clinical implications of liquid biopsies
      • Advantages to provide real-time information on the dynamic changes in the cell populations that define the actual cancer stage
    • Using a combination of Flow Cytometry, Hybrid LBA/LCMS, qPCR and Western Blots to detect and characterize extracellular vesicles in blood with high sensitivity and selectivity
    • Case studies on the bioanalytical measurement of Biomarkers in Liquid Biopsies and method development strategies for this emerging sample type




Final Agenda Agenda at a Glance