Specialized Workshop F2 - Discussion Topic List

"Cell & Gene Therapy Bioanalysis: Recent Advancements and New Frontiers for Bioanalytical Labs on Flow Cytometry, qPCR, ELISPOT, Regulatory Requirements, CAR-T and CRISPR Assays Case Studies"

Catching up with “explosion” of recent developments Cell & Gene Therapy Bioanalysis with specific focus on Flow Cytometry, qPCR, ELISPOT and Regulatory Requirements. Ongoing research in CAR-T and CRISPR Bioanalytical, Biodistribution Biomarkers and Immunogenicity Case Studies. Strategies to be prepared to handle the increasing demand of drug discovery/development bioanalytical data in support of ongoing and future research

  • Dr. Heba Degheidy, Staff Scientist, Division of Cellular and Gene Therapies, CBER, US FDA
  • Dr. Nirjal Bhattarai, Principal Investigator and Product Reviewer Division of Cellular and Gene Therapies, CBER, US FDA

Recent Advancements in Gene & Cell Therapy and Building on 2019 WRIB Recommendations

qPCR, ddPCR and NGS

Flow Cytometry for CAR-T & Lesson Learnt from Vaccine

Immunogenicity, Safety Risk Assessment and NAb/CDx Assays

ELISPOT for Gene Therapy/Vaccine & Single Cell Western Blot Assay


  • Panel Discussion with All the Regulators:

    Regulatory Feedbacks on Submitted Studies and Regulations on Gene & Cell Therapy and Vaccine

    • Dr. Sara Gagneten (US FDA)
    • Dr. Nirjal Bhattarai (US FDA)
    • Dr. Heba Degheidy (US FDA)
    • Dr. Xiaofei Wang (US FDA)
    • Dr. Andrew Exley (UK MHRA)
    • Dr. Therese Solstad Saunders (Norway NoMA)
    • Dr. Richard Siggers (Health Canada)

White Paper on Cell & Gene Therapy Bioanalysis

  • White Paper

    Consensus, Recommendations, and Panel Discussions on Cell & Gene Therapy Bioanalysis
    - Dr. Heba Degheidy, Staff Scientist, Division of Cellular and Gene Therapies, CBER, US FDA
    - Dr. Nirjal Bhattarai, Principal Investigator and Product Reviewer Division of Cellular and Gene Therapies, CBER, US FDA
    - and Cell & Gene Therapy Bioanalysis White Paper Working Experts Group


Discussion Topic DETAILS of F2 – Cell & Gene Therapy Bioanalysis

  • Dr. Heba Degheidy, Staff Scientist, Division of Cellular and Gene Therapies, CBER, US FDA
  • Dr. Nirjal Bhattarai, Principal Investigator and Product Reviewer Division of Cellular and Gene Therapies, CBER, US FDA
Recent Advancements in Gene & Cell Therapy and Building on 2019 WRIB Recommendations
  • Topic 1
    Gene & Cell Therapy Bioanalysis: Building on 2019 WRIB Recommendations and Updates on Gene & Cell Therapy Advancements
    - Dr. Mark Milton, Executive Director Pharmacokinetic Sciences Ophthalmology and Department Lead Gene Therapies, Novartis
    • Building on the 2019 White Paper in Bioanalysis recommendations on Gene & Cell Therapy Bioanalysis with evolved industry experience and recent advancements
    • How Gene & Cell Therapy Bioanalysis is evolving and what is next step in both Science & Regulations
    • Current challenges addressed & unanswered concerns
      • Specificity & tissue targeting
      • Specific tissue/cell tropism
      • Selection of viral vectors
      • Immunogenicity issues
      • Understanding pharmacology & safety
      • Regulatory expectations
    • Case studies on the latest insights & regulatory developments on this fast-evolving field
      • Focus on what bioanalysis should measure for assessing drug exposure, responses and immunogenicity

  • Topic 2
    Next Generation Approaches for Tracking and Evaluating Gene/Cell Therapy: Looking beyond qPCR and Flow Cytometry
    - Dr. Heather Hirsch, Sr. Director Translational Pharmacology and Head Clinical Biomarkers, CRISPR Therapeutics
    • Advances in Gene & Cell Therapy Bioanalysis have created the need to use in bioanalysis less traditional instrumentations beyond Mass Spectrometry and LBA applied for small molecules and biotherapeutics
      • Gene & Cell Therapy as diverse class of biologics
      • Need for Bioanalysis to adapt
        • Novel bioanalytical approaches for measuring exposure to Gene/Cell Therapy
    • Current bioanalytical focus is on
      • Flow Cytometry
      • qPCR
    • Flow Cytometry and qPCR methods
      • Developed to evaluate the cellular kinetics of CAR-T cells
      • Approaches for tracking and evaluating Gene/Cell Therapy
    • Beyond Flow Cytometry & qPCR
      • What's the next generation of approaches/instrumentation Gene/Cell Therapy?
      • Single cell techniques
      • Imaging platforms
      • Novel technique for evaluation of a product in the patient by whole body scan


qPCR, ddPCR and NGS
  • Topic 3
    qPCR Current Application in AAV Gene Therapy Bioanalysis & Clinical Biomarkers
    - Dr. Jean-Claude Marshall, Sr. Director Clinical Biomarker Group and Precision Medicine, Pfizer
    • Building on the 2019 White Paper in Bioanalysis recommendations on qPCR Validation with novel case studies and evolved industry experience
    • Increasing number Gene Therapy development
      • Adeno-associated virus (AAV) based delivery approaches
    • Challenges with the bioanalytical support for AAV GT
      • Shedding kinetics
      • Infectivity of viral particles
    • Need for the quantification of shed viral vector in
      • Whole blood
      • Plasma/PBMCs
      • Saliva
      • Urine
      • Semen
      • Stool
    • qPCR-based assay
      • Level of assays validation
      • Acceptance Criteria
      • Current status of Regulatory Guidance
    • Case studies on qPCR unique challenge in assay development and Validation

  • Topic 4
    Best Practice in ddPCR to Measure Vector/Transgene DNA Quantities and Structural Dynamics
    - Dr. Lynne Jesaitis, Director BioAnalytical Sciences, BioMarin
    • Evaluation of efficacy & safety for GT
      • Measurement of target nucleotide sequence levels
    • Characterization of
      • Biological distribution
      • Potential to release into the environment.
    • qPCR vs droplet digital PCR (ddPCR)
      • Advantages of newer technologies – ddPCR
      • Similarities between qPCR and ddPCR
      • Common use of
        • Target-specific primers
        • Fluorescent probes
      • Differences between qPCR and ddPCR
        • Unique process by which source amplicons are quantitated
        • No need for a standard curve
        • Increased tolerance to variable efficiency of amplification
        • Structural analysis of nucleic acids
    • Factors affecting qPCR & ddPCR
      • Matrix limitations
      • Dynamic range
      • Precision
    • Case studies on ddPCR bioanalytical assays, their development to support AAV GT and evaluation of changes in vector integrity and structure after administration

  • Topic 5
    NGS vs. qPCR Assays: A head to head comparison of applications in monitoring patients’ on- and off-target indels and gene integration events for an in vivo genome editing program in the clinic
    - Dr. Yanmei Lu, Sr. Director Biomarker and BioAnalytical Sciences, Sangamo Therapeutics
    • Next Generation Sequencing (NGS)
      • Technology of choice for the quantification of indels
        • High discovery power for heterogenous indel variants
        • Ability to multiplex samples and analytes
        • Requirement for small DNA input material
      • Strategies in NGS assay development and validation
        • Precision,
        • Accuracy
        • Sensitivity
        • Reference standards
        • Quality controls
      • Case studies on application of NGS to monitor on- and off-target indel events from patient liver biopsies
    • qPCR-based assay
      • Reverse transcription method
        • Sensitive
        • Cost effective
        • Simple
      • Method to quantify transcripts with known sequences
      • A case study of determining percentage of fusion mRNA representing transgene integration in patients’ liver
    • Case studies on successful implementation of quantitative and fully qualified NGS and qPCR-based assays to assess editing efficiency and transgene integration events in clinical trials.


Flow Cytometry for CAR-T & Lesson Learnt from Vaccine
  • Topic 6
    How to Develop and Control Vaccine Flow Cytometry Assays: Development of analytical control strategies and QC samples tracking. What CAR-T Flow Cytometry Assays can learn from Vaccine experience?
    - Dr. Sylvie Bertholet, Director Preclinical Evidence Generation,Vaccines GlaxoSmithKline
    • Lack for a Regulatory Guidance for the applications of Flow Cytometry Assays in Regulated Bioanalysis
    • Vaccine advanced experience in controlling of Flow Cytometry-based assays in a regulated environment
      • Global strategies from the Vaccine community to monitor and control Flow cytometry assays
      • Setting minimum standards for the development and qualification of in-process and release QC
    • Flow Cytometry QC recommendations in the 2019 White Paper in Bioanalysis

  • Topic 7
    Current Industry Standards on How to Reliably Assess the QC samples in CAR-T Flow Cytometry Assays
    - Dr. Naveen Dakappagari, Head Protein Biomarker Solutions, Navigate/Novartis
    - Dr. Shyam Sarikonda, Assoc. Director Flow Cytometry Immuno-Oncology Biomarker Development, Navigate/Novartis
    • Challenges in the development of Flow Cytometry bioanalytical methods to quantitatively measure CAR-T cell levels in adoptive cell therapy
      • Major issues in defining QC samples in CAR-T Flow Cytometry Assays
    • Working together for providing clarifications and mainly recommendations on how to reliably assess the QC samples in CAR-T Flow Cytometry Assays
      • PK assay for CAR-T
      • Current limitation to validate Flow Cytometry Assays
    • No Regulatory Guidance on for the measurement of CAR-T levels by Flow Cytometry
      • Regulators' concerns about what we are actually measuring
      • Crucial importance of actual QC values for Flow Cytometry PK assays
      • Building on what discussed in 2019 White Paper in Bioanalysis Link: https://www.future-science.com/doi/pdf/10.4155/bio-2019-0271
        • How and when to prepare QC for CAR-T PK assays
        • How to make QC for CAR-T PK assays reliable and consistent
        • What stability programs put in place
        • How to ensure that QC for CAR-T PK assays can identify assay
    • Thorough evaluation of Pros & cons of current approaches
      • Using CAR-T samples frozen as a QC
        • Spiking issues in whole blood
        • Problems to mimic the complexity of incurred samples
        • Stability issues and inconsistency
    • Regulators' concerns in using other approaches rather that actual QCs
      • Fluorescence Minus One (FMO) controls
        • Used as gating control to identify the gating boundary for the one antibody that is missing
        • Used to identify background staining due to fluorescence spillover
      • Isotype controls
        • Used for identifying nonspecific staining
      • Reagent stability
      • Instrument checks


Immunogenicity, Safety Risk Assessment and NAb/CDx Assays
  • Topic 8
    Gene & Cell Therapy Safety Risk Assessment and its Direct Influences on Bioanalytical Strategy
    - Dr. Swati Gupta, Director Immunology, Allergan
    • Importance of assessing the Safety Risk preclinically in Gene 8 Cell Therapy
      • Plan the bioanalytical strategy
      • Ongoing efforts to improve risk assessment tools
      • Finding ways to de-risk
    • Considerations regarding preclinical assays to assess the impact of the critical quality attributes
    • Considerations for non-traditional dosing routes
      • Evaluation of different preclinical approaches to strategize bioanalytical plan
    • Design clinical studies in a patient population to manage
      • Safety risk
      • Therapeutic efficacy loss.
    • Groundwork for developing regulatory requirements for
      • Program specific risk-based assessments
      • Mitigation strategies
    • 2019 FDA Immunogenicity Guidance
      • Regulatory Expectations for risk assessment
        • More comprehensive risk assessment being performed and provided prior to IND submission
    • This risk assessment
      • Immunogenicity & safety
      • Impact on the bioanalytical and preclinical/clinical strategy
      • Preclinical data (NHP)
        • how to monitor ADA in case the NHP react to human

  • Topic 9
    Application of Current Immunogenicity Guidance/Guideline (FDA/EMA) to Gene/Cell Therapy: Parallels and New Thoughts
    - Dr. Jim McNally, Chief Scientific Officer, BioAgilytix
    • Building on the 2019 White Paper in Bioanalysis recommendations on Current Immunogenicity approaches to Gene/Cell Therapy with novel case studies and evolved industry experience
    • Are the current Immunogenicity Guidance/Guideline fully applicable to Gene/Cell Therapy?
      • Focused discussion on the impact of immunogenicity to Gene/Cell Therapy
    • Impact of pre-existing immunogenicity
      • Inclusion/exclusion
      • Re-dosing
    • Cell based assay development for immunogenicity
    • Monitoring of vector and transgene specific immunity is crucial to drive Gene Therapy clinical development
    • Approaches for viral and transgene specific immune responses
      • Pre-treatment / Pre-existing Abs
      • Post-Dose
      • Cellular Immune Responses
      • Humoral Immune Responses
    • Understanding the relevance and need for pre/post treatment immunogenicity measurements
      • Specific considerations on over-expressed proteins from Gene Therapy
    • Immune responses to Transgene Protein
      • Potential risk factors
      • Patient Cross-Reactive Immunologic Material (CRIM) status
      • Host immune system status
      • T-cell responses to transgene
    • Immune responses to Vector
      • Delivery vector (AdV vs. AAV) dependent
      • Route and dose of administration

  • Topic 10
    Considerations on Development and Validation AAV Capsid Nab Assays (Transfection Inhibition): Should they be CDx?
    - Dr. John Kamerud, Director BioMedicine Design, Pfizer
    • Unique approaches used in the development of AAV Gene Therapy
      • NAb and Transfection Inhibition (TI) assays in AAV-GT
        • Importance to detect antibodies which may inhibit the ability of the viral capsid to transfect target cells
        • Presence of NAbs associated with poor therapeutic efficacy
    • CLIA application for assays used to make treatment decisions
      • LDTs (laboratory developed tests)
      • Use of NAb assay results as exclusion criteria for clinical trials
      • FDA-approved companion diagnostic (CDx)
    • In depth discussion and current understanding on the use of anti-capsid NAb or TI assays
      • Methodology to identify parameters which may impact performance
      • Regulatory requirements
        • Considerations on the need for developing CDx devices prior to marketing approval of Gene Therapeutic product


ELISPOT for Gene Therapy/Vaccine & Single Cell Western Blot Assay
  • Topic 11
    Characterization and Validation of a Single Cell Western Blot Assay to Measure Expression of Transgene Product
    - Mr. Jason DelCarpini, Sr. Manager Biomarkers and Clinical Assay Development, Bluebird Bio
    • Bioanalytical strategies and novel approaches to measuring exposure to GT
      • Expression of the transgene product as important PD marker
      • New techniques and platforms
        • Establishing meaningful method validation acceptance criteria
        • Limited Regulatory Guidance
    • Advanced applications of Multiplexed Western Blot assay to measure
      • Expression of a transgene product
      • Diseased state biomarker in single red blood cells
    • Use of Design of Experiment (DoE) to
      • Characterize assay variability
      • Establish meaningful validation acceptance criteria
      • Review of the validation results
        • Lessons learned
    • Case studies on a novel approach using Multiplexed Western Blot

  • Topic 12
    ELISPOT Method Development & Validation for Vaccine Assays: Building on WRIB Recommendations on ELISPOT, How to develop reproducible and sensitive Vaccine assays. What Gene & Cell Therapy ELISPOT can learn from Vaccine ELISPOT experience?
    - Dr. Lisa Kierstead, Executive Director Vaccine Sciences, PPD
    • Building on the 2019 White Paper in Bioanalysis recommendations on ELISpot Validation with novel case studies and evolved industry experience
    • Vaccine community advanced experience in the Enzyme-Linked ImmunoSpot (ELISpot) assay
      • ELISpot
        • Instrumentation
        • Scientific principles
        • Parameters for monitoring of cell-mediated immunity
      • Importance to correctly set up ELISpot for
        • High reproducibility
        • Highly sensitive cellular assay
    • High-throughput analysis of cell immune responses
      • Detection of a secreted cytokine at the single cell level
      • Sensitivity as low as 1 cell in 100,000
    • Wide application of EliSpot as assay standard in Vaccine development
      • Immunogenicity assessment in support of Vaccine
    • Case studies in developing, qualifying and validating ELISpot assays

  • Topic 13
    ELISPOT Method Development & Validation for Gene Therapy: Building on WRIB Recommendations on ELISPOT, practical case studies, lesson learnt and evolving trends.
    - Dr. Arno Kromminga, Chief Scientific Advisor, BioAgilytix
    • Novel case studies on ELISpot Assay expectations & performance
      • Cell recovery and viability
      • Critical steps in standardizing an ELISpot assay
      • Overcoming ELISpot assay Variability
      • State-of-the art tools to control assay performance over time or across laboratories
    • Specific applications and new data on Cellular Immune Responses for AAV-GT
      • Anti-capsid and transgene
      • Pre- and Post-dose possible and potentially impactful
    • Central role of Cellular Immune Responses in understanding of the host reaction to Gene Therapy
      • New progresses in monitoring of T cells
      • Development of sensitive and robust assays
      • Critical assays variable in cellular immune assessments and their impact on results
      • Evolution of cellular immune assessment from supporting early discovery decisions to late stage clinical development