Main Workshop DAY 2A - Wednesday April 11, 2018

"Hybrid LBA/LCMS (Immunoaffinity LCMS) for Biotherapeutics, Biomarkers, ADA
New Developments and Achievements"

Day 2A Discussion Topic List

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

PK Assays by Hybrid LBA/LCMS
Chair: Dr. Timothy Olah, Group Director, Bristol-Myers Squibb

Biomarkers Assays by Hybrid LBA/LCMS
Chair: Dr. Hendrik Neubert, Research Fellow, Pfizer

Immunogenicity Assays by Hybrid LBA/LCMS
Chair: Dr. Hendrik Neubert, Research Fellow, Pfizer

Hybrid LBA/LCMS for Biotherapeutics, Biomarkers and ADA Panel Discussion & Consensus for 2018 White Paper in Bioanalysis
Chair: Dr. Timothy Olah and Dr. Hendrik Neubert

  • Panelists Industry: Dr. Susan Spitz, Dr. Omar Laterza, Dr. Lorella Di Donato, Dr. Joe Palandra, Dr. Luca Ferrari, Dr. Tim Sikorski, Dr. Jiang Wu, Dr. Naidong Weng, Dr. Steve Alley, Dr. Hongbin Yu, Dr. Anita Lee, Dr. Matthew Szapacs, Dr. Linzhi Chen, Dr. Gilles Miscoria, Dr. Ludovicus Staelens, Dr. Darshana Jani, Dr. Yan Zhang, Dr. Robert Dodge, Dr. Ola Saad, Dr. Eric Woolf, Dr. Steve piccoli

  • Panelists Regulators: Dr. Sean Kassim, Dr. Sam Haidar, Dr. Seongeun (Julia) Cho, Dr. Christopher Leptak, Dr. Shashi Amur, Dr. Joao Pedras-Vasconcelos, Dr. Brian Booth (TBC), Dr. Jan Welink, Dr. Olivier Le Blaye, Mr. Stephen Vinter, Ms. Emma Whales, Dr. Shirley Hopper, Mr. Gustavo Mendes Lima Santos, Dr. Anna Edmison, Dr. Akiko, Ishii, Dr. Yoshiro Saito



Discussion Topic DETAILS of Day 2A

PK Assays by Hybrid LBA/LCMS
Chair: Dr. Timothy Olah, Group Director, Bristol-Myers Squibb

  • Topic 1
    Quantitation of Intact Biotherapeutics by Hybrid LBA/LCMS: Current state-of-the-art workflow, successes & failures and future perspectives
    - Dr. Timothy Olah, Group Director, Bristol-Myers Squibb
    • Pros/Cons of using HRMS for quantitation of intact proteins and/or subunits
    • Why and How intact protein quantification should be performed
      • “Bottom-up” (signature peptide) or “Top-down” strategies in Bioanalysis
      • Limitation of signature peptide approach to provide sufficient information on the biotherapeutics measured
        • “Lost in digestion”
      • How to preserve the therapeutic protein for intact quantification
      • Identification and quantitation of intact biotherapeutics and their catabolites
        • Better understanding the various circulating biotherapeutic forms
        • Better of biotransformation
        • Glycoforms quantitation
        • Post-translational modifications
    • Overcoming sensitivity issues in intact protein quantification
      • Maximizing enrichment by IA
        • Best capturing antibody for improving mass spectrometry S/N
      • Deglycosylation
      • Subunits quantification
        • Digestion at subunits level - current approaches
      • Summing isotope signals
        • Charge state & isotope effects on S/N
        • Charge state coalescence with DMSO and gain in signal intensity
      • Optimizing extraction window (XIC) for quantitation
      • Declustering potential & accumulation time
      • Chromatographic options for intact proteins
        • Best stationary phases to use
      • Importance of stable isotope-labeled version of the protein IS
    • Considerations for applying Intact Biotherapeutics by Hybrid LBA/LCMS in a regulated environment
    • Case Studies: Optimization of Intact/subunits protein quantification by HRMS. Applications to different biotherapeutic classes, advantages/disadvantages of this approach and industry current standards

  • Topic 2
    Novel ADC & Novel Bioanalytical Challenges: How to cope with ADC’s increased complexity and diversity
    - Dr. Steve Alley, Sr Director Experimental Pharmacology, Seattle Genetics
    • Current 3rd Generation ADC vs Next Generation
    • What we have learnt on ADC Bioanalysis from 1st to 3rd Generation
      • Expected increasing in diversity and complexity in next generation ADC
      • New potent payload and specific MoA
        • Payload with multiple MoA
        • Drug combination
      • Stable linkers
        • Method Development using “peptide-linker-payload”
      • New conjugation site specific
      • Novel constructs
        • MDB, Bispecific, Probody, Humabody
    • Addressing novel ADC stability concerns
      • ADC conjugation site & antibody 3D-structure
        • Impact on new payload release
          • Minimized release in circulation
          • Maximized release in tumor
        • New considerations on catabolism
        • Bioanalytical challenges to understand in vivo stability
    • ADC for new/different medical needs
      • Unmet medical needs – anti-infectives and bacterial resistance
    • Case Studies: Importance of a fully integrated ADC bioanalytical strategy for next generation ADC

  • Topic 3
    Application of Hybrid LBA/LCMS Assays for Clinical Studies: Robustness & Reliability of Hybrid LBA/LCMS Assays in Regulated Bioanalysis for Biotherapeutics, Submitted Studies and Regulatory Feedback
    - Dr. Hongbin Yu, Director, Bioanalytical Mass Spectrometry, Boehringer Ingelheim
    • PK assays using Hybrid LBA/LCMS (IA-LC-MS/MS) for Biotherapeutics have been used by the industry for more than 7 years
      • This approach is now very well-refined & well-established for PK assays with numerous publications and white papers from discovery to clinical applications
      • After an initial incredible incremental growth, Hybrid LBA/LCMS methods seem to have reached maturity for PK assays but still evolving in Biomarker assays and innovative applications like ADA assays
      • Isn’t this technology for PK assays, mature enough for establishing a regulatory framework (Guidance/Guideline)?
    • Sharing recent experience of Hybrid LBA/LCMS supporting biotherapeutics in clinical studies
      • Knowledge gap (if any)
      • Robustness of Hybrid LBA/LCMS assays
        • High throughput analysis for large studies
        • Ability to perform well overtime
        • Pharma/CRO method transfer
        • Validation experiments to measure IA & Digestion efficiency and method selectivity/specificity based on signature peptide
    • Regulatory feedback/concerns using Hybrid LBA/LCMS assays
      • Agency Scientific Advice before using this technology
        • Justification for why using Hybrid LBA/LCMS or LCMS rather than LBA
        • Risk evaluation for over/underestimation vs LBA
        • Selectivity & Exclusivity of the signature peptide
        • Impact of completion of Enzymatic Digestion vs Recovery
        • Internal Standard tracking (IS) for Hybrid LBA/LCMS methods
    • Case Studies: Applications of Hybrid LBA/LCMS assays in Regulated Bioanalysis for Clinical Trials


Immunogenicity Assays by Hybrid LBA/LCMS
Chair: Dr. Hendrik Neubert, Research Fellow, Pfizer

  • Topic 4
    Expressed Protein Product Quantification by Hybrid LBA/LCMS for Gene Therapy
    - Dr. Hendrik Neubert, Research Fellow, Pfizer
    • Gene Therapy PD markers
    • Importance of the evaluation of expression of the protein product in Gene Therapy
      • Novel Method Development Strategies
      • Innovative Workflows Enabling New Measurements
      • Challenges in quantification of protein expression from transfected cells
      • Intracellular protein expression to target cells
    • Advancements in protein quantification by Hybrid LBA/LCMS
      • Tissue bioanalysis
      • Target biomeasure packagess
      • New extraction methods
    • Case Studies: New progresses in tissue analysis related to Gene Therapy PD markers

  • Topic 5
    Parallelism Evaluation in Protein Biomarkers Assays by Hybrid LBA/LCMS and Reagent-free LCMS
    - Ms. Anita Lee, Associate Principal Scientist, Merck
    • Difference & similarities between Hybrid LBA&LCMS and LBA Biomarkers Assay in regard to 2017 Duke-Margolis/C-Path/US-FDA White Paper Recommendations
      • LBA parallelism
        • Conserving interaction between critical reagents with the standard material and the endogenous analyte in presence of competing interference in the matrix
      • Hybrid LBA/LCMS parallelism depends on
        • Internal standard tracking/compensation
        • IA cleanup & digestion
        • Extraction & chromatography
        • MS matrix effect and ion suppression/enhancement
    • Parallelism and LCMS matrix effect evaluation between the surrogate matrix and authentic biological matrix
      • Advantages of using linear calibration curves in LCMS
    • Defining Parallelism criteria
      • Different criteria to evaluate parallelism in Hybrid LBA/LCMS vs LBA
      • Considerations on lack of high quality reference standards
        • Use of recombinant proteins as standards
    • Key questions: After protein digestion, the signature peptide is the same in the endogenous biomarker protein and in the recombinant protein and it is possible to have actual reference standards for this peptide. Hence:
      • How can we use this advantage in LCMS reference material vs. LBA?
      • Are reagent-free LCMS protein biomarkers assays able to get accuracy in this case (actual reference material for signature peptide) in LCMS like for SM Biomarkers?
    • Are there cases in Hybrid LBA/LCMS biomarker assays where the concentration increases if samples are diluted like in LBA?
    • Should Hybrid LBA/LCMS method use the MRD approach as in LBA
    • Case Studies: Actual cases to support the addition of Parallelism for Hybrid LBA/LCMS Biomarker assays in the C-Path White Paper

  • Topic 6
    Recent Advancements on Discriminating Biomarkers Proteoforms by Hybrid LBA/LCMS Assays: Confirming the importance to quantify the correct proteoform for clinical significance
    - Dr. Matthew Szapacs, Manager, GlaxoSmithKline
    • Considering the unique & crucial advantages offered by LCMS in the protein biomarker space: “The measurement of specific proteoforms”
      • Proteoforms: isoforms, phosphoforms, glycoforms, point mutations and other PTMs
      • Why is LCMS the ideal technology for discriminating proteoforms?
    • Proteoforms analysis:
      • What are the right questions to ask and what are the challenges to overcome?
      • Protein biomarkers can be present in distinct multiple proteoforms
      • LBA results may be biased by these proteoforms variations in different subjects and between healthy and disease states (biological variability)
      • Hybrid LBA/LCMS assays can provide results for
        • Total protein biomarker
        • Isoform-specific biomarker
    • Increasing sensitivity by maximizing chromatographic separation to detect low abundant isoforms
      • Optimization of the isoform-specific tryptic peptides
      • Validation of an isoform-specific assay
        • Crucial experiment in plasma and surrogate matrix
        • Sensitivity & Selectivity
    • Comparison between Hybrid LBA/LCMS and LBA data
      • Where is the bias?
      • Under/overestimation?
    • Case Studies: Development of an Hybrid LBA/LCMS assay for simultaneous quantification of total and isoform-specific biomarker proteoforms


Immunogenicity Assays by Hybrid LBA/LCMS
Chair: Dr. Hendrik Neubert, Research Fellow, Pfizer

  • Topic 7
    The Most Recent Applications of Hybrid LBA/LCMS in Combination with Traditional LBA for ADA Assessment
    - Dr. Linzhi Chen, Sr. Research Fellow, Boehringer Ingelheim
    - Dr. Gilles Miscoria, Bioanalysis Laboratory Manager, Sanofi
    - Dr. Ludovicus Staelens, Head Regulatory Bioanalysis, UCB Pharma
    • Overcoming Drug Tolerance issues in ADA Assays
    • Data comparison & interpretation
    • A sensible proposal for the future of ADA measurement
      • LBA for screening/confirmation
      • Hybrid LBA/LCMS for quantification/isotyping
    • Advantages of simultaneous characterization of ADA using Hybrid LBA/LCMS
      • Applicability of ADA analysis by Hybrid LBA/LCMS
        • Demonstration that it is feasible and generates data which are comparable and in some case better to LBA data
    • Ongoing investigation to fully determine that Hybrid LBA/LCMS could be a more informative and beneficial platform over conventional LBA approaches
      • How should data be compared?
      • How can we interpret the concentration data against the titers?
      • Impact of Positive Control (PC) on both ADA assays (Hybrid vs LBA)
      • Impact of Drug Tolerance on ADA assays (Hybrid vs LBA)
    • Methodology advances in ADA assessment by Hybrid LBA/LCMS
      • Noise reduction approaches
        • Reducing non-specific binding to the capture phase
    • Cut point determination
    • ADA concentration vs Drug binding capacity
    • Selecting universal isotyping peptide panels
    • Measurement of pre-existing ADA
    • Case Studies: Overcoming severe drug interference in LBA ADA assay by using Hybrid LBA/LCMS. Data comparison between tradition LBA approach and novel Hybrid LBA/LCMS

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