Specialized Workshop T1 - Tuesday Sep. 28, 2021: 8am - 5pm, ET - Lecture List

"Mass Spectrometry of Biotherapeutics, Protein Biomarkers and Transgene Proteins: Recent Issues from Discovery to Regulatory Submissions"

Opening Lecture

Part 1: Protein Therapeutics

Part 2: Protein Biomarkers

Part 3: Transgene Proteins & Anti-Drug Antibodies (ADA)


Lecture DETAILS of T1 – Mass Spectrometry of Biotherapeutics, Protein Biomarkers and Transgene Proteins

Opening Lecture
  • Lecture 1
    Recent Advancements in Mass Spectrometry of Large Molecules
    - Dr. John (Jack) Kellie, Scientific Leader & Fellow, GlaxoSmithKline
    • Current landscape and rationale for LCMS measurements for Biotherapeutics and Protein Biomarkers
      • Bottom-up characterization
        • Digested into enzymatically derived peptides
        • Advanced quantitation approaches for biotherapeutics and biomarkers
        • Antibody-free Assays Mass Spec
        • Post-Translational Modifications (PTM)
        • Hybrid Assay (IA-MS)
      • Top-down (Intact Protein)
        • Protein characterization in the Bioanalysis lab
        • Contaminants for intact protein MS bioanalytical assays
        • Advantage of using intact protein bioanalysis for biotherapeutic discovery and development
      • Toward a Universal platform for Native Antibody Bioanalysis
        • Recent development in Native Protein MS analysis
          • Applications for Serum Spiking for a mAb and its Target Ligand
        • Native MS Analysis potential for performing protein analysis
          • Technology major advantage of preserving the non-covalent protein-protein interactions
        • Native Mass Spectrometry very specific application of ESI-MS
          • Large molecules and complexes transferred from a three-dimensional functional existence in a condensed liquid phase to the gas phase
      • Case Studies: Recent data in Bottom-up, Top-down, Native Mass Spectrometry of Biotherapeutics and Protein Biomarkers
    Part 1: Protein Therapeutics
    • Lecture 2
      Advancement in Mass Spec to resolve Glycosylation Issues in Protein Therapeutics
      - Dr. Y-J Xue, Scientific Director Nonclinical Disposition and Bioanalysis, Bristol-Myers Squib
      • Top quality attributes monitored by Mass Spec: amino acid sequence, molecular mass, disulfide bonds, glycosylation, C-terminal sequence variants, N-terminal sequence variants, deamidation and oxidation
        • Need for higher resolution and sensitivity in Mass Spec used to measure critical quality attributes
        • Increase in use of Mass Spec for quality control and clinical applications
      • Considerations on Protein Glycosylation issues
        • Impact of glycosylation to PK/PD of biotherapeutics
        • Glycosylation role in proteinrecognition, signaling and adhesion
        • Importance of carbohydrate composition for protein therapeutics safety & efficacy
      • Importance of performing Glycan Bioanalysis
        • Technologies for biologic glycoform profiling
        • Identification, characterization and quantification approaches
        • Strategies and Development of bioanalytical methods
          • Intact Protein
          • Glycopeptides quantification
          • Cleaved Glycans quantification
      • Case Studies: Current practices within Pharma to perform glycan bioanalysis using Mass Spec and glycoform profiling of protein therapeutics

    • Lecture 3
      Advanced Applications of Hybrid Assays and challenges in overcoming ADA Interferences
      - Dr. Huiyu Zhou, Director BioAnalytical Research & Development, BioMarin
      • Hybrid Assays demonstrated Superior Accuracy over LBA
        • LCMS, ELISA and ECLA head-to-head data comparison
        • Use of spike recovery experiment to ensure reliable comparison of the data
      • Hybrid Assays better ADA-tolerance
        • Recent advancement on Hybrid Assays (IA-MS) as complementary techniques for Biotherapeutics PK Assays to troubleshoot LBA common issues
        • Use of acid dissociation step to reduce ADA interference in clinical sample analysis
        • Reliable assessment of the ADA impact on the PK datais crucial for better PK/PD data interpretation
      • Lesson learned in using clinical samples duringmethod development
        • Importance to evaluate the impact of ADA on both LCMS and LBA assay performance
        • Considerations on the effects of ADA on the LBA
          • Diverse and difficult to predict based on the ADA isotype or titer
          • Due to the complex and dynamic nature of the ADA response over the course of the study
        • Potential drug interference on the ADA measurement
      • Case Studies: Evaluation and implementation of Hybrid Assays (IA-MS) to support clinical PK studies of therapeutic proteins.

    • Lecture 4
      Microflow and Ionization sources for Biotherapeutics Bioanalysis - Impact to LLOQ and Selectivity/Specificity
      - Dr. Shawna Hengel, Associate Director DMPK, Seattle Genetic
      • Comparison of LBA specificity/selectivity issues in human plasma vs LCMS results
        • Using Hybrid Assays to overcome severe interference in LBA
        • LCMS challenges due to the extremely low doses of administration
        • LBA interference from endogenous compounds
      • Validation and Sample Analysis challenges with nano scale chromatography
        • What to consider to ensure the development and validation of a robust Hybrid bioanalytical assay for Regulated Bioanalysis
        • How Microflow-LCMS techniques are able to provide sensitivity gains to enable new approaches or make formerly difficult approaches more routine
        • Method development challenges in using Microflow-LCMS for Biotherapeutics Bioanalysis
      • Latest progresses in Microflow-LCMS for Biotherapeutics applications
        • Is it robust enough for routine sample analysis?
        • Unbiased method development tips and drawbacks
          • Pitfalls in using microfluids devices in bioanalysis
        • Increase in S/N between with Microflow LC vs. conventional approaches
          • Increase in ionization efficiency, reduction of source contamination and solvent consumption
      • Case Studies: Strategies for the development of a very sensitive Hybrid Assay to reach sub ng/mL LLOQ for antibody therapeutics; overcoming specificity/selectivity issues in LBA by using Mass Spec

    • Lecture 5
      Novel Developments in the Next Level of Sensitivity for Bioanalysis of Biotherapeutics by Mass Spec used for Regulatory Submissions: Focus on Speed & Robustness in Hybrid Assays Regulated Bioanalysis
      - Dr. Robert Wheller, Associate Scientific Director LC-MS Bioanalysis, LGC
      • State-of-the-art applications of New Generation of Digestion Enzymes
        • Possibility of working at high temperature
        • Faster digestion time
        • How to avoiding alkylation and reduction
      • Improvement in the workflows for bottom-up quantitation approaches
        • Increasing robustness of assays used for regulatory submissions
        • Strategies for increasing speed of analysis
        • Reducing the multi-step approach in bottom-up analysis
      • Pushing the quantification limits of biotherapeutics by Hybris assay as lower as possible
        • Focus on HRMS selectivity to improve sensitivity
      • Working in a high regulated environment
        • Application of WRIB 2019 & 2020 Hybrids Assays Recommendations for ICH M10 BMV Guideline
      • Case Studies: Sharing the latest experience in the application of new generation of digestion enzymes and HRMS to increase S&S in Hybrid Assays in Regulated Bioanalysis


    Part 2: Protein Biomarkers
    • Lecture 6
      Novel applications of Hybrid Assays to quantify Protein Biomarkers
      - Dr. Wenying Jian, Group Leader, Bioanalytical and Pharmacokinetics, Janssen
      • Important considerations for tissue protein biomarker quantitation
        • Recovery and matrix effects
        • Liver protein biomarker by Hybrid Assay
        • Improved best practices and new case studies to ensure high sensitivity in tissue assay development
        • Homogenization and extraction
        • Blood contamination in the tissue
      • Utilization of Hybrid Assay using HRMS Intact Analysis
        • Novel and alternative approach to bottom-up assay for protein biomarker quantitation
      • Application of Different Configurations of Hybrid Assays for Protein Biomarkers
        • Target protein turnover rate determination
        • Quantitation of a Coagulation factor and measurement of its activation for target engagement measurement
      • Case Studies:Recent examples to demonstrate different aspects/approaches of Hybrid Assays (IA-MS) for protein biomarker quantitation

    • Lecture 7
      Recent Advancements in Hybrid Assays to measure Target Proteins & PD Biomarkers to support Target Engagement (TE) assessment
      - Dr. Lin-Zhi Chen, Senior Research Fellow, Boehringer Ingelheim
      • Considerations when Drug level is overwhelmingly higher than its Target
        • Development of Drug-bound and/or total target assays
        • Are there any benefits to measure free target?
      • Membrane-bound target proteins
        • Challenges in Monitoring shed, soluble target in biological fluid
        • Requirements for the reference standard
        • Is the use of recombinant target with domain only portion acceptable?
      • Major Differences in TE results between LBA vs Hybrid Assays
        • Impact of reagents, kits and assay format,
        • How to deal with more than 1000 times difference in reported target expression levels
        • What data to use?
      • Low level Target Expression
        • Demands for highly sensitive assays (pg/mL) which can be challenging for Hybrid Assays
        • What are the major hurdles for improving Hybrid assay sensitivity and how to address it?
      • Discrepancies of Results between TE Assay for Drug and PK Assay
        • How to handle discrepancies between the two sets of results?
      • Case Studies: Hybrid Assays for multiple TE applications, High drug levels, free target, membrane-bound target protein, low level target expression

    • Lecture 8
      Validation of a Target Engagement (TE) Protein Biomarker Assay by Mass Spec
      - Ms. Anita Lee, Principal Scientist, Merck
      • Method Development and Optimization
        • Considerations for Assessing Drug-Target Engagement by Mass Spec
        • Advanced approaches to measuring Drug-Target Engagement in tissue
        • Strategy using Hybrid Assays to measure in single assay at the site of action
        • Total drug, Target concentration and Target bound to drug
        • Ensuring in vivo drug-target association/disassociation
      • Fit-for-purpose Biomarker Assay Validation (FFP BAV)
        • Challenges and solutions in designing the validation protocol
        • Recovery Test Evaluation
        • Ensuring Sensitivity& Selectivity (S&S)
      • Sample Analysis
        • Application in clinical studies for internal decision making
      • Case Studies: Fit-for-purpose Validationof LCMS-based target engagement PD protein biomarker assay

    • Lecture 9
      High Sensitivity Biomarker Analysis by Mass Spec: Drive for determination of low abundance Protein Biomarkers
      - Dr. Ludovicus Staelens, Director Translational Biomarkers and Bioanalysis, UCB
      • Improved best practicesto ensure high sensitivity Mass Spec methods for Biomarkers
        • Maximizing Immuno-Affinity (IA) to improve sensitivity
          • Advantages of using antipeptide IA enrichment
          • New data in IA enrichment to maximize LCMS sensitivity & selectivity
          • Development of a high affinity immunocapture method
            • Target affinity capture reagent
            • Antibody capture reagent
        • Optimization of digestion time efficiency and bead density
          • Use of additional sample cleanup using solid phase extraction (SPE) to increase sensitivity
            • Optimizing chromatography to improve sensitivity
              • Nanoflow/nano spray approaches for sensitivity improvements
            • Case Studies: Determination of low abundance Protein Biomarkers by high sensitive Mass Spec methods


          Part 3: Transgene Proteins & Anti-Drug Antibodies (ADA)
          • Lecture 10
            Recent Developments in the Quantification of ADAby Hybrid Assays (IA-MS)
            - Dr. Haibo Qiu, Associate Director, Regeneron
            • Evaluating immunogenicity of therapeutic protein products is critical for drug development
              • Continuous improvement of Hybrid Assays
              • Demonstrated potential of Hybrid Assays as orthogonal and/or complementary to LBA for immunogenicity (Anti-Drug Antibodies ADA) analysis
            • Key questions and challenges
              • Current application of Hybrid ADA Assays
              • ADA isotyping
              • Semi-quantification
            • Potential improvement, trends and perspectives related to immunogenicity analysis by Hybrid Assays
              • Indirect detection of ADA by LBA
                • Traditional method for assessment of ADAs
                • Susceptibility to target and drug interferences
              • Direct measurement of ADA by Mass Spec
                • Ability to directly measure signature peptides originated from ADA
                • Specificity and opportunity of isotyping
            • Case Studies: Recent Hybrid ADA Assays method development and applications in nonclinical studies for ADA isotyping and semi-quantification

          • Lecture 11
            Opportunities & Challenges of Mass Spec-based Transgene Protein analysis
            - Dr. Hiroshi Sugimoto, Group Lead and Principal Scientist, Takeda
            • Hybrid Assay for Transgene Protein detection/quatification
              • Differentiation of wild type and mutated transgene
              • Detection of post-translational modification (PTM)
            • Advantages of LCMS-based Transgene Protein assay
              • High selectivity with broader dynamic ranges
              • Fast and more streamlined method development
              • Multiplexing capability
              • Less stringency on reagent requirements
              • Minimization of endogenous interference
            • Immunocapture purification
              • Workflow for immunocapture-LCMS comprises
              • Risk of competition between the endogenous and transgene protein during the immunocapture purification
            • Case Studies: Applications of Hybrid Assays (IA-MS) to quantify endogenous (wild type) and transgene protein (mutated) in Gene Therapy

          • Lecture 12
            Transgene Protein Bioanalysis for Gene Therapy and Validation Consideration for Clinical Applications
            - Dr. Joe Palandra, Senior Principal Scientist, Pfizer
            • Fit-for-Purpose Validation for Transgene Protein by Mass Spec
              • Specific Assessments
                • Assessment of immunocapture efficiency
                • Digestion efficiency
                • Unique Assay novelties
              • Reference standard quality and testing
              • Acceptance criteria
                • Current BMV guidance for PK Assays not ideal
              • Stability assessments
                • Transgene protein
                • Tissue samples
              • Assay sensitivityin relation to normal basal levels
              • Normalization of data for BicinChoninicAcid (BCA), weight, housekeeping protein
            • Application in Gene Therapy of the Hybrid Assays as a technique increasingly employed for the quantification of protein targets
              • Advanced applications for quantification of expression of the protein product
              • Current workflow consisting of protein precipitation, pellet digestion and anti-peptide antibody enrichment prior to LCMS based quantification of a surrogate peptide
            • Case Studies: Fit-for-Purpose Validation for Transgene Protein by Mass Spec for Clinical Applications

          Agenda at a Glance Agenda at a Glance