Specialized Workshop T1Tuesday September 27, 2022: 7am - 5:30pm - Topic List

"Mass Spectrometry Novel Technologies, Novel Modalities, and Novel Challenges"

New Tools to Reach Max Sensitivity & Selectivity; Advanced Applications in Column Chemistry and Separations Techniques; Problem Solving for Novel Modalities, Non-Liquid and Rare Matrices Method Development/Validation

(You can scroll down to see the details of each topic)

Part 1: Novel Technologies

  • Topic 1:

    Myths and Realities in the Use of HILIC to Replace Ion-Pairing and Hexafluoroisopranol (HFIP) for Oligonucleotides Bioanalysis: Does it really work? Is it possible to reproduce the published results? Is sensitivity still a limitation with HILIC approaches?
    Dr. Ming Huang, Scientist, Regeneron

  • Topic 2:

    Use of Aptamers as a Novel Reagent in Regulated Bioanalysis for Large Molecule Quantification by Hybrid Assays (IA-MS): Advantaged and Disadvantages in using Aptamers in Immunoaffinity enrichment
    Ms. Yunlin Fu, Senior Principal Scientist, Novartis

  • Topic 3:

    EAD vs CID Fragmentation for Small Molecule and their Metabolites: Is EAD Technology going to replace CID or do we still need both?
    Dr. Christopher Kochansky, Principal Scientist, Merck

Part 2: Novel Modalities & Novel Method Development/Validation Challenges

  • Topic 4, 5:

    Novel/Advanced Strategies and Recent Advancements in ADC Bioanalysis by Mass Spectrometry

    • Topic 4
      Selecting the right Internal Standard
      Ms. Hetal Sarvaiya, Associate Director, DMPK Bioanalysis, AbbVie
    • Topic 5
      Method Development approaches for Biotransformation
      Dr. Yue Huang, Associate Director, AstraZeneca
  • Topic 6, 7:

    Novel/Advanced Strategies and Recent Advancements in RNAi and ASO Therapeutics Bioanalysis by Mass Spectrometry

    • Topic 6
      HRMS Method Development approaches for Metabolism/Biotransformation
      Dr. Jing Li, Group Leader, Alnylam
    • Topic 7
      Advantages of Hybridization Mass Spec vs “gold standard” Hybridization LBA
      Dr. Long Yuan, Associate Scientific Director & Bioanalytical Group Leader, Biogen

Part 3: Problem Solving for Non-Liquid and Rare Matrices

  • Topic 8:

    Peripheral Blood Mononuclear Cell (PBMC) by Mass Spectrometry and Sample Preparation Strategies for Candidate Biomarkers Identification and Quantification
    Dr. Michael Lassman, Lab Director Translational Biomarkers, Merck

  • Topic 9:

    Bioanalysis of Nucleotide Analogue Drugs in PBMC - Advanced strategies for intracellular quantitation, sensitivity enhancement, PBMC isolation/handling, and stability control
    Ms. Kasie Fang, Investigator, GlaxoSmithkline

  • Topic 10:

    Pushing the Sensitivity Limits of Mass Spec for Discovery Biomarkers Quantification in Non-Liquid Matrices by Novel Sample Preparation, Chromatography and Instrumentations
    Mr. Jay Johnson, LCMS Senior Scientist Quantitative Biomarkers and Biomeasures, Pfizer

  • Topic 11:

    Mass Spectrometry Advanced Application for Ophthalmology Molecules and Challenges in Multiple Ocular Matrices
    Dr. Ola Saad, Senior Principal Scientist Bioanalytical Sciences, Genentech

Part 4: Novel Applications in Bioanalysis

  • Topic 12:

    Recent Developments in Quantitative Bioanalysis of Protein Panel by Mass Spectrometry: What are the Acceptance Criteria and Regulatory Requirements for Targeted Proteomics?
    Dr. Naiyu Zheng, Scientific Associate Director Translational Medicine, Bristol Myers Squibb

  • Topic 13:

    Strategies for the Quantification of Fecal Biomarkers for Inflammatory Bowel Disease (IBD) by Mass Spectrometry
    Dr. Ellen Casavant, Scientist, Genentech

  • Topic 14:

    Complex Mass Spec Development, Validation and Method Transfer of Total Lipid and Multiple Isoforms Biomarkers
    Dr. Allena Ji, Director, Global Diagnostics & Scientific Affairs Rare Diseases, Chiesi
    Dr. Lin Tao, Bioanalytical Scientist, Sanofi

Part 5: White Paper in Bioanalysis

  • 2022 White Paper on Biomarker Topics:

    Consensus & Conclusions on Mass Spectrometry Bioanalysis Topics for 2022 White Paper

 

Topic DETAILS of T1

Part 1: Novel Technologies
  • Topic 1

    Myths and Realities in the Use of HILIC to Replace Ion-Pairing and Hexafluoroisopranol (HFIP)
    for Oligonucleotides Bioanalysis: Does it really work? Is it possible to reproduce
    the published results? Is sensitivity still a limitation with HILIC approaches?

    Dr. Ming Huang, Scientist, Regeneron

    • Is it possible to overcome the major issues/drawbacks in the Mass Spec analysis of Oligos generated by the use of ion-pairing reagents?
      • Strong signal suppression
      • MS cleaning & downtime
      • Column cleaning and regeneration
    • What is the best chromatography for Oligos separation?
      • Does HILIC really work for Oligos?
      • HILIC to replace ion-pairing has been attempted by many unsuccessfully
      • Published methodologies seem not possible to reproduce
      • HILIC sensitivity seems to be a limitation when trying to explore the use of HILIC for Oligos
      • Overall consensus that using ion-pairing agents and HFIP is still the preferred approach
    • Novel strategies for the successful development of HILIC–MS/MS methods for Oligos
      • HILIC–MS/MS Analysis of DNA and RNA oligonucleotides
      • Separation of oligonucleotide (n-x) shortmer sequences and siRNAs
      • Automated MS/MS annotation achieved for unbiased sequence characterization.
      • Evaluation of absolute on-column sensitivity of a 20-mer phosphorothioate DNA oligonucleotide
    • HRMS analysis
      • Deconvoluted masses of oligonucleotides
      • Determination of siRNA single strands and duplexes
    • Case Studies: First successful approach in HILIC for Oligos the industry was able to use and reproduce
  •  

  • Topic 2

    Use of Aptamers as a Novel Reagent in Regulated Bioanalysis for Large Molecule Quantification by Hybrid Assays (IA-MS): Advantaged and Disadvantages in using Aptamers in Immunoaffinity enrichment
    Ms. Yunlin Fu, Senior Principal Scientist, Novartis

    • Advancements in Novel Capture Reagent for Hybrid Assay (IA-MS)
      • Impact of restrictions on Animal-Derived Antibodies on reagents
      • Animals are often used to generate capture antibodies for quantitative bioanalysis in support of drug development
    • EU Directive 2010/63/EU on the protection of animals used for scientific purposes
      • Animals should not be used in procedures
      • Where a non-animal alternative exists, which provide the same or higher level of information as obtained from animal procedures
    • Aptamers as Capture Reagent
      • Synthetic oligonucleotides that adopt a 3D structure and bind to target molecules
      • Major benefit of aptamers as synthetic reagents
      • Less lot-to-lot variability
      • Single-stranded DNA or RNA molecules
      • Binding to their target molecules with high affinity & specificity
    • Hybrid Assy development/validation using Aptamers
      • Support of preclinical bioanalysis of IG therapeutics
      • Use of Anti-human IgG aptamer Apt 8-2,
      • Extensive evaluation of Apt 8-2 as an alternative to animal-based antibody as affinity enrichment reagent
      • Workflow for testing different human IgGs (IgG1, IgG2 and IgG4) using the Apt 8-2 enrichment
      • BMV conventional acceptance criteria
    • Consideration on the overall use of Aptamer for general humanized IgG specificity with no cross reactivity in rat, mouse, rabbit, and dog.
      • Cost effective compared to the one with animal-based capture antibody.
    • Case Studies: Hybrid Assay for bioanalysis of IgG therapeutics using aptamer as affinity enrichment reagent
  •  

  • Topic 3:

    EAD vs CID Fragmentation for Small Molecule and their Metabolites: Is EAD Technology going to replace CID or do we still need both?
    Dr. Christopher Kochansky, Principal Scientist, Merck

    • Driver for Alternative Fragmentation and information collection in Mass Spectrometry
      • Definitive structure elucidation in real time (automated) to aid chemistry design
      • Feasibility and considerations of incorporating alternative-CID fragmentation
      • Use of Metabolite information acquisition into the current laboratory setting
    • Considerations with incorporating new technologies into existing data workflows
      • EAD
      • Ion mobility
      • Speed of the workflow (LC to data turnaround)
      • Use of software to collect/process/interpret data
    • Acquisition of EAD compared to CID
      • Monitoring of the number of cell divisions over a pre-determined period of time
      • Different Cell proliferation protocols
      • Influencing metabolite identification data
      • Progress & challenges
    • Case Studies: Head-to-head comparison of EAD and CID fragmentation and interpretation with respect to an existing workflow

 

Part 2: Novel Modalities & Novel Method Development/Validation Challenges
  • Topic 4 & 5:

    Novel/Advanced Strategies and Recent Advancements in ADC Bioanalysis by Mass Spectrometry

    • Topic 4:

      Selecting the right Internal Standard
      Ms. Hetal Sarvaiya, Associate Director, DMPK Bioanalysis, AbbVie

      • What’s the best IS for ADCs?
        • There are no specific guidelines on use of appropriate Internal Standards (IS) for ADC bioanalysis via LCMS
        • White Papers recommendations are more focused on mAb rather than ADC
        • How to determine the strategies for selecting IS for these complex molecules
      • Challenge in quantitative LCMS bioanalysis of ADCs
        • Importance for choosing on appropriate IS to successfully develop/validated ADC methods
        • IS should represent both the antibody and the small molecule part of the ADC
      • Current landscape on IS for ADC
        • Mixed opinion in the literature
        • Use of either protein or peptide based internal standards for biologics bioanalysis using LCMS
      • Comparison of different IS options for ADC bioanalysis using LCMS in terms of
        • Different sample preparation workflow
        • Assay Precision & Accuracy (P&A)
        • ADC in vivo stability
        • Findings for choosing most appropriate internal standards
      • Case Studies: Selection of the Internal Standard (IS) for LC-MS/MS Bioanalysis of ADC

    • Topic 5:

      Method Development approaches for Biotransformation
      Dr. Yue Huang, Associate Director, AstraZeneca

      • Current bioanalytical challenges and lesson learned due to the unique complexity and diversity of ADCs components
        • Protein backbone
        • Conjugation sites
        • Linkers chemistry
        • Warheads
      • Importance of comprehensive PK characterization for ADCs
        • Acquired experience from ADC programs
        • Biotransformation as a key component of ADC PK
      • Mass Spectrometry characterization of ADCs
        • Quantification of ADC and the major biotransformation species of ADC
        • Identifying the multiple ADC proteoforms from biotransformation
      • Overall considerations on the biotransformation of ADCs
        • Important part of the characterization of the pharmacological activities of ADC
        • Help with next generation ADC design
      • Case Studies: Assessments of ADC biotransformation by Mass Spectrometry

  • Topic 6 & 7:

    Novel/Advanced Strategies and Recent Advancements in RNAi and ASO Therapeutics Bioanalysis by Mass Spectrometry

    • Topic 6

      HRMS Method Development approaches for Metabolism/Biotransformation
      Dr. Jing Li, Group Leader, Alnylam

      • RNAi Therapeutics
        • Mass Spec bioanalytical strategies for biotransformation determination
        • Challenges with biotransformation of GalNAc-conjugated siRNA
      • Triple Quadrupole LC-MS/MS
        • Understanding Givosiran metabolism, the first approved GalNAc-conjugated RNAi therapeutics
        • Identification and quantitation of active metabolite of siRNA
        • in-depth examples of Mass Spec quantitation
      • HRMS
        • Introduction the deaminated metabolites
        • Approaches for the discovery of a novel deaminated metabolite on single-stranded oligonucleotide in vivo
        • Solving the complex characterize the deaminated metabolite of a double-stranded siRNA in vivo for Lumasiran, the second approved GalNAc-conjugated RNAi therapeutics
      • Case Studies: Understanding the importance RNAi Therapeutics biotransformation

    • Topic 7:

      Advantages of Hybridization Mass Spec vs “gold standard” Hybridization LBA
      Dr. Long Yuan, Associate Scientific Director & Bioanalytical Group Leader, Biogen

      • Hybridization LC-MS/MS methodology
        • Predominant use of ligand-binding assays (LBA) for Anti-sense oligonucleotide (ASO) bioanalysis
        • Novel approaches for ASO bioanalysis
      • Comparison Hybridization Mass Spec vs Hybridization LBA
        • High sensitivity and throughput
        • Poor metabolites selectivity
        • Low sensitivity for tissue samples
        • Cross reactivity
        • Demonstrated superior performance of Mass Spec vs LBA for ASO
      • Method development for Hybridization LC-MS/MS in different matrices
        • Quantitation of ASO (20-mer, MW 7kD)
        • Plasma/serum samples
        • CSF samples and overcoming CSF shortage
        • Tissue samples (brain, spinal cord, liver, and kidney)
        • in vitro ADME study samples
        • ion-pairing reversed-phase chromatography
      • Application of Hybridization LC-MS/MS methodology in Regulated Bioanalysis
        • Validated in GLP environment
        • Application for to pre-clinical toxicology studies
      • Case Studies: Application in Regulated Bioanalysis of a validated hybridization LC-MS/MS method for ASO bioanalysis

 

Part 3: Problem Solving for Non-Liquid and Rare Matrices
  • Topic 8:

    Peripheral Blood Mononuclear Cell (PBMC) by Mass Spectrometry and
    Sample Preparation Strategies for Candidate Biomarkers Identification and Quantification

    Dr. Michael Lassman, Lab Director Translational Biomarkers, Merck

    • Peripheral Blood Mononuclear Cell (PBMC) significance in Biomarker Discovery/Development
      • PBMC as rich biological matrix from which to probe biology and critical pathways associated with disease.
      • Many important Proteins Biomarker may be found in PBMC but not plasma or serum
      • At present the bulk of quantitative LC-MS methods have been developed in plasma/serum rather than PBMC
    • Measuring Proteins Biomarkers in PBMC by Mass Spectrometry
      • Current industry experience in this field
      • Demonstration of the importance of being able to measure proteins from PBMC by LC-MS
      • How these measurements can aid in understanding biology and drug development
    • Crucial importance of PBMC sample collection & handling
      • Strategies for collecting PBMC appropriately
    • Specific considerations regarding PBMC based LC-MS method development
      • Sample Extraction
      • Chromatographic and Mass Spectrometric optimization
      • Highlight the pros and cons of combining Ab based pulldowns and enrichment combined with both high and low flow LC-MS
    • Case Studies: Candidate Protein biomarkers quantification from PBMS by LC-MS/MS, sample collection, preparation and method development

  • Topic 9:

    Bioanalysis of Nucleotide Analogue Drugs in PBMC - Advanced strategies for intracellular quantitation, sensitivity enhancement, PBMC isolation/handling, and stability control
    Ms. Kasie Fang, Investigator, GlaxoSmithkline

    • Importance of quantitation of phosphorylated NA (nucleoside/nucleotide analogs) anabolites in PBMC
      • NAs must undergo intracellular phosphorylation to be active
      • Quantitation of phosphorylated NA anabolites in PBMC provide critical information of drug exposure and efficacy
    • Challenges and solutions in accurate quantitation of intracellular NA phosphorylated anabolites using LC-MS/MS
      • Challenges in Chromatography
      • Challenges in Mass Spec Selectivity & Sensitivity (S&S)
      • Derivatization strategy and how to mitigate potential pitfalls
    • Variability Control during PBMC collection, counting and processing
      • Strategies for accurate cell counting
      • Strategies for robust PBMC process
    • Analyte Instability control from sample collection till data acquisition
      • How to control chemical instability
      • How to minimize enzymatic conversion
    • Case Studies: Chemical derivatization as a novel strategy for selective and sensitive determination of intracellular di- and triphosphate anabolites in PBMC

  • Topic 10:

    Pushing the Sensitivity Limits of Mass Spec for Discovery Biomarkers Quantification in Non-Liquid Matrices by Novel Sample Preparation, Chromatography and Instrumentations
    Mr. Jay Johnson, LCMS Senior Scientist Quantitative Biomarkers and Biomeasures, Pfizer

    • Advanced Mass Spectrometry Strategies for Non-Liquid Matrices Bioanalysis
      • Increased demand for high quality tissue target measurements
    • Fit-for-purpose development for assays needed quickly to support many drug modalities
      • Protein degraders
      • Gene therapy
      • LNP-mRNA
      • Preclinical biodistribution
    • Improved Sensitivity by
      • Innovative utilization of SDS Lysing and Direct Digestion
      • Using Suspension Trapping (STrap) plates combined with online peptide IA-LC-MS/MS
      • Bypassing of protein level immunopurification
    • Establishment of Quantitative Bioanalysis of the STrap-IA-LC-MS/MS workflow
      • Transporters in liver lysates
      • Transporters in hepatocytes
      • Transporters in liver fine needle aspirates
      • Exosome markers
    • Utilization of SDS lysing and direct digestion using suspension trapping (STrap) plates combined with LC-MS/MS when peptide antibodies are not available
      • Intercalated disc protein gene therapy in mouse heart
    • Future Directions
      • Automation
      • Direct serum digest
    • Case Studies: Innovative approaches to increase sensitivity in non-liquid matrices by novel sample preparation and instrumentations

  • Topic 11:

    Mass Spectrometry Advanced Application for Ophthalmology Molecules and Challenges in Multiple Ocular Matrices
    Dr. Ola Saad, Senior Principal Scientist Bioanalytical Sciences, Genentech

    • Complexity and importance of Eye Compartments Bioanalysis
      • To determine exposure of the target organ to the drug
      • To help interpret local pharmacological or toxic effects
      • To evaluate systemic exposure to demonstrate sustained release of the drug from devices
    • Many ocular diseases still present unmet medical needs
      • Ongoing developments of New Ophthalmologic Drugs, new modalities, new modes of delivery
      • Pegylated molecules
      • Port Delivery System (PDS)
    • Development of challenging PK/PD assays in multiple matrices
      • Low Aqueous humor (AH) volumes
      • High sensitivity needed
      • Large dynamic range
      • Long wait times for sourcing control matrix (AH healthy/disease matrix)
    • Importance of Biotransformation & Drug Stability
      • Long residence times targeted in vivo
      • Immunogenicity assessment
      • Concerns around ocular inflammation
    • Hybrid Assay (IA-MS) application for PK and biotransformation across matrices
      • Fragment antigen-binding (Fab)
      • pegylated hexamer-Fab
    • Case Studies: Current approaches for Eye Compartments Bioanalysis by Mass Spec based on practical experience

 

Part 4: Novel Applications in Bioanalysis
  • Topic 12:

    Recent Developments in Quantitative Bioanalysis of Protein Panel by Mass Spectrometry: What are the Acceptance Criteria and Regulatory Requirements for Targeted Proteomics?
    Dr. Naiyu Zheng, Scientific Associate Director Translational Medicine, Bristol Myers Squibb

    • Protein Biomarker panels in biological samples by Mass Spectrometry-based Targeted Proteomics
    • Key role of Protein Biomarker Panels in drug discovery & development
      • To confirm the biomarker hypothesis
      • To better understand the biology of diseases and related pharmacological applications during the clinical trials for immuno-oncology, fibrosis, or many other drug development programs
      • Challenges in quantification of 10-200 proteins/panel
    • Recent developments in Mass Spectrometry instrumentation and software related to major targeted proteomics platforms
      • MRM
      • PRM
      • DIA-based platforms
      • Current challenges in each platform
    • Feasibility of Calibration curve preparation
      • Selection of surrogate matrix
      • Preparation using recombinant proteins vs synthetic surrogate peptides
      • Calibration using multiple-point calibration curve or single point (e.g., SIL peptides)
    • Feasibility of Quality Control (QCs) sample preparation
      • Spiked QCs vs endogenous QCs
      • Total number proteins (or surrogate peptides) and concentration levels to be included in each QC concentration level
    • Potential quality issues in data processing and quantitation using automated software
      • Misidentification of peaks
      • Unacceptable S/N
    • Expectation and future improvements in acceptance criteria
      • Regulatory requirements related to calibration curves, QCs, and data processing for targeted proteomics
    • Case Studies: Quantitative bioanalysis of protein panel by Mass Spectrometry, acceptance criteria and Regulatory Requirements for Targeted Proteomics

  • Topic 13:

    Strategies for the Quantification of Fecal Biomarkers for Inflammatory Bowel Disease (IBD) by Mass Spectrometry
    Dr. Ellen Casavant, Scientist, Genentech

    • Recent Developments of Biomarkers for Inflammatory Bowel Disease (IBD)
      • Characterization of gastrointestinal (GI) inflammation
    • Applications of Fecal Proteomics by Mass Spec for IBD
      • Potential for frequent, non-invasive monitoring biomarkers in IBD
      • Revealing disease activity throughout the GI tract from a single sample
    • Disease proteins detected in stools
      • Data-independent acquisition (DIA) based LC-MS/MS approaches
      • Profiling the human fecal proteome of IBD patients vs. healthy controls
      • Assessing disease-related proteomic changes
    • Method Development challenges
      • Sample Extraction
      • Mass Spec Optimization
      • Value of creating stool specific spectral library from ulcerative colitis patients, Crohn’s disease patients and healthy control samples
    • Better understanding of IBD biology
      • Based on biomarkers of disease activity
      • Based on identification of pathways of GI dysregulation
    • Case Studies: Use of fecal proteomics for identification of non-invasive, pathway focused, biomarkers characterizing disease activity in the gut from IBD

  • Topic 14:

    Complex Mass Spec Development, Validation and Method Transfer of Total Lipid and Multiple Isoforms Biomarkers
    Dr. Allena Ji, Director, Global Diagnostics & Scientific Affairs Rare Diseases, Chiesi
    Dr. Lin Tao, Bioanalytical Scientist, Sanofi

    • Challenges of cross-lab Fit-for-purpose Biomarker Assays Validation (FFP BAV) of Sphingolipids with Multiple Isoforms by LC-MS/MS
    • Method Development/Validation
      • Understanding sphingolipid structure and isoform distribution for planning a successful method development
      • Effect of composition of short and long chain isoforms on total lipid concentration results
      • Effect of instrument parameters (Collision Energy) on isoform response
      • Actions to minimize Source of Variation for Total Sphingolipid Results
    • Method Transfer & Troubleshooting
      • Common problems in method transfer for sphingolipids with multiple isomers
      • Dried Blood Spots (DBS) Sphingomyelin (SPM)
      • Plasma Globotriaosylceramide (GL-3)
    • Case Studies: Method development/validation and method transfer for multiple Isoforms biomarkers




Agenda at a Glance Agenda at a Glance