Main Workshop DAY 1 - Tuesday April 10, 2018

"SM, SM Biomarkers, Peptides & Oligos by LCMS
Novel Solutions in Method Development & Regulatory Challenges"

Day 1 Discussion Topic List

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

Small Molecule Bioanalysis
Chair: Dr. Jan Welink, Senior Pharmacokinetic Assessor, EU EMA

Small Molecule Biomarkers
Chair: Dr. Eric Yang, VP, Head of Bioanalysis Immunogenicity and Biomarkers, GlaxoSmithKline

Peptides & Oligos
Chair: Dr. Eric Yang, VP, Head of Bioanalysis Immunogenicity and Biomarkers, GlaxoSmithKline

SM, SM Biomarkers, Peptides & Oligos by LCMS Panel Discussion & Consensus for 2018 White Paper in Bioanalysis
Chair: Dr. Jan Welink and Dr. Eric Yang

  • Panelists Industry: Dr. Nicki Hughes, Dr. Robert Dodge, Dr. Yan Zhang, Dr. Jaya Goyal, Dr. Adrien Musuku, Dr. Steve Piccoli, Dr. Daniela Fraier, Dr. Eric Woolf, Dr. Nico van de Merbel, Dr. Michael Buonarati, Dr. Rachel Green, Dr. Tom Verhaeghe, Dr. Tim Sangster, Dr. Yuanxin, Dr. Mark Ma, Dr. Daniel Norris, Dr. Uma Kavita, Dr. Alex Bulychev, Dr. Chris Stebbins, Dr. Kirk Brown, Dr. Neil Henderson, Dr. Tate Owen, Dr. Alexander Behling, Dr. Meena, Dr. Lieve Dillen, Dr. Ingo Röhl, Dr. Zamas Lam, Dr. Daksha Desai-Krieger, Dr. Jens Sydor, Dr. Scott Summerfield, Dr. Charles Hottenstein, Dr. Lina Luo, Dr. Andre Iffland, Dr. Barry Jones, Dr. Ragu Ramanathan, Dr. Allena Ji, Dr. Amanda Wilson, Dr. Dina Goykhman, Dr. Anton Rosenbaum

  • Panelists Regulators: Dr. Sean Kassim, Dr. Sam Haidar, Dr. Seongeun (Julia) Cho, Dr. Christopher Leptak, Dr. Shashi Amur, Dr. Olivier Le Blaye, Mr. Stephen Vinter, Ms. Emma Whales, Mr. Gustavo Mendes Lima Santos, Dr. Anna Edmison, Dr. Akiko Ishii, Dr. Yoshiro Saito



Discussion Topic DETAILS of Day 1

Small Molecule Bioanalysis
Chair: Dr. Jan Welink, Senior Pharmacokinetic Assessor, EU EMA

  • Topic 1
    Sophisticated Bioanalytical Methods Drive the Quality of PKPD and PBPK Modeling: What are they? Integration & interpretation of bioanalytical data and downstream analysis
    - Dr. Jens Sydor, Director DMPK & Bioanalysis, AbbVie
    • What is Physiologically based pharmacokinetic (PBPK) modeling and why are bioanalytical data important?
    • How the integration & interpretation of Bioanalytical data can impact PBPK mathematical modeling technique for predicting the absorption, distribution, metabolism and excretion (ADME)?
    • What bioanalysts should consider providing an efficient Bioanalytical Support to PBPK models?
    • How sophisticated should be the bioanalytical assays to support the building of these models?
    • Importance of involving bioanalytical scientists who develop the methods and generate the data in the data interpretation and further downstream analysis of PBPK models
      • Opportunities for bioanalysts to be involved in multidisciplinary teams
      • What you need to know and what it is expected from a bioanalytical expert
    • Case Studies: Integration of bioanalytical data in PBPK models, bioanalytical applications with ADC and building cellular models to better understand the disposition of ADC and the downstream effects in cancer cells

  • Topic 2
    What is ISR/ISS Telling the Global Bioanalytical Community about its Methods/Procedures?
    - Dr. Scott Summerfield, Head of Bioanalysis, GlaxoSmithKline
    - Dr. Jan Welink, Senior Pharmacokinetic Assessor, EU EMA
    • Understanding the ISR/ISS most important values in the last decade and how they helped the Bioanalytical Community to improve methods performance
      • Lesson learnt from Industry/Regulators’ feedback
      • ISR major failures: why did they happen? Are we able to avoid them in the future?
      • What have we learnt about our bioanalytical methods?
    • What are the ISR/ISS future opportunities?
      • Where the next decade should lead?
      • Is the use of ISS well understood by the industry?
        • Application of 2012 & 2015 White Paper recommendations on ISS
      • ISR unresolved issues and no consensus points: 7% (why?) or 10/5%?
        • US FDA 2013 draft Guidance states that ISR should be performed on 7% of incurred samples in the study regardless of size
        • EU EMA and Japan MHLW Guidelines approach is based on using 10% of the first 1000 samples and 5% of the number of samples exceeding the 1000 samples
    • Case Studies: Industry/Regulators’ practical experience from evaluating ISR during the last 10 years

  • Topic 3
    US FDA Regulatory Stability Research: Supporting Data from Stable and Unstable Compounds
    - Dr. Sam Haidar, Deputy Director, US FDA
    • Health Canada contacted FDA in 2015 regarding a practice they observed during an inspection for stability testing (excerpt from WRIB 2017)
      • Ongoing discussion Industry/Regulators in 2016 & 2017 White Papers
        • Preliminary data presented in 2017
      • US FDA started its own researching single tube vs. three tubes stability
        • Outline of a research project discussed with Industry (GCC)
        • LT, Bench top & F/T stability tested
        • Multiple variables considered
        • 5 model compounds considered
      • Looking into new data collected April 2017-2018
    • Case Studies: Evaluation using low stability model compounds & LT testing with longer storage duration

  • Topic 4
    Next Wave of Automation & Next Wave of Issues to Overcome
    - Dr. Charles Hottenstein, Scientific Leader, GlaxoSmithKline
    • Thorough evaluation of new types of automation and their impact on Regulated Bioanalysis
    • What are the pros/cons of emerging automation in a regulated environment?
    • Looking into the future of Bioanalysis and the regulatory challenges Boanalysis is going to face with the next wave of automation
    • Developing new strategies for having a fully compliant state-of-the-art automation
    • How is the next wave of automation different from the current one?
    • An unbias evaluation of:
      • Increased of reproducibility in routine work
      • Increased of throughput & flexibility
      • Increased of method performance & robustness
      • Increased of integrated system automation features
      • Better high-quality bioanalytical data
      • Minimized operator attendance
      • Better user friendliness
      • Less time data acquisition and more time on data interpretation and problem solving
    • Implementing novel automated systems from experimental design to reporting
    • Automation-assisted systems for multivariate experiments
    • Novel options for custom-built software in combination with commercial liquid handling automated systems
    • Case Studies: Latest advancement in automation

  • Topic 5
    Regulatory Feedback/Interaction on VAMS Application in Pediatric & Phase III Clinical Trials: Requirements for home-sampling, BMV and bridging studies
    - Dr. Daksha Desai-Krieger, Executive Director Bioanalysis, Allergan
    • New approaches in sample collection for Pediatric and Clinical trials and impact on Regulated Bioanalysis
      • What is the most appropriate technology? And why?
      • Is better VAMS or CMS? – Applications of the recommendation from 2017 White Paper Part 1
      • Why is “home sampling” required in some clinical trials?
      • Requirements for successfully setting up VAMS for Phase III Clinical Trials to have reliable samples
      • Issues related to patient self-sampling and practical obstacles for sample collection/processing at site
    • Clinical applications of VAMS
      • Studies outcome
      • Validation and Supporting In-Study Data
    • Regulatory Feedback/Interaction
      • Request for Bridging studies
      • Status of microsampling in a clinical setting
    • US FDA vs EU EMA perspective on microsampling
      • Recent significant increase in microsampling usage in EU
      • Sharing the positive feedback received by the Industry from EU-EMA on microsampling applications
      • Need in the US to work closely with the Agency in sharing successful cases of microsampling application in a regulated environment with a specific focus to VAMS & CMS
    • Case Studies: Industry experience from Pediatric & Clinical Trials using microsampling


Small Molecule Biomarkers
Chair: Dr. Eric Yang, VP, Head of Bioanalysis Immunogenicity and Biomarkers, GlaxoSmithKline

  • Topic 6
    Recent Developments in the Quantification of Individual Bile Acids (IBA) as Biomarkers for Drug Induced Liver Injury: Method development approaches to confirm ethnic differences in serum concentrations
    - Dr. Lina Luo, Sr. Scientist, Pfizer
    • Method Development strategies to overcome the high variety of Bile Acids and optimize method selectivity & sensitivity (S&S)
      • Presence of numerous isobaric structural isomers
      • Wide range of in vivo baseline levels
      • Inefficient fragmentation properties for unconjugated Bile Acids
    • Available options beyond triple quadrupoles to achieve better selectivity & sensitivity with Bile Acids
      • HRMS
      • Ion mobility
      • Is accurate mass measurement a good approach to perform quantification and identification of Bile Acids simultaneously?
    • Issues with Surrogate Matrix approach for Bile Acids analysis
      • Matrix effect and recovery difference
      • Residue endogenous BAs in charcoal stripped matrices
      • Scientific justification for using a different species matrix as an alternative
        • Charcoal stripped dog plasma to measure human plasma samples
    • Issues with Surrogate Analyte approach for Bile Acids analysis
      • Lack of stable isotope labeled analogues for many Bile Acids
    • LCMS methodology for IBA: reference ranges, effects of age, gender, and ethnicity
      • Evaluation of IBA Ability to detect hepatic injury in patients with liver impairments
      • Conjugated bile acids vs non-conjugated bile acids evaluation for healthy subjects and hepatic impaired patients
    • Case Studies: Assessment of individual bile acids and their profiles as mechanistic biomarkers of liver injury for humans

  • Topic 7
    Very Long Chain Fatty Acids (VLCFA) Biomarker Qualification in Human CSF: Major Challenges in Solutions
    - Dr. Andre Iffland, Associate Director, Vertex Pharmaceuticals
    • LCMS method development approaches VLCFA biomarker qualification and BAV
    • Overcoming specific challenges with human Cerebrospinal Fluid (CSF)
      • What are the unique challenges with this matrix?
      • What is the biomarker community interest in measuring VLCFA in CSF?
    • Development of an improved extraction for LCMS quantification of VLCFA
      • How to reduce exogenous contamination by other fatty acids
      • Strategies to increase sensitivity of VLCFA
        • Getting more than 1000-fold in a single step
    • Fit for Purpose (FFP) Validation
      • Recommendations on FFP assay requirements for a full qualification and BAV in human CSF
    • Case Studies: Proof of concept measurement of basal levels of VLCFA in CSF of human healthy subjects

  • Topic 8
    BAV for Small Molecules Biomarkers by LCMS: Actual Case Studies in support of an alignment between the recommendations of Duke-Margolis/C-Path/US-FDA & 2016 & 2017 White Paper in Bioanalysis (Part 1)
    - Dr. Barry Jones, Associate Director, Q2 Solutions
    • The Duke-Margolis/C-Path/US-FDA White Paper is mainly focused on BAV for LBA Assays
    • Which parameters to include for SM Biomarker Assay Validation (BAV) by LCMS?
      • Small molecule biomarkers are very important as surrogates for disease diagnosis, progression, and prognosis and it is important to add specific guidelines in this document
      • 2016 & 2017 White Paper in Bioanalysis (Part 1) provide practical recommendations on SM BAV by LCMS
        • Can we include these recommendations in the Duke-Margolis/C-Path/US-FDA White Paper?
    • Other additions for SM BAV by LCMS vs LM BAV by LBA:
      • Surrogate Matrix & Surrogate Analyte experiments
      • Parallelism experiments for SM Biomarkers by LCMS are different if compared to LM Biomarkers by LBA
      • Matrix Effects/Factor and Ion suppression/enhancement
      • LCMS Multiplexing using multiple transitions
        • Simultaneous LCMS assays for multiple analytes with different ranges are routinely used
      • Criteria to define LLOQ and QCs levels in the calibration curve
      • Selectivity and specificity experiments
      • Should Total Allowable Error be considered also for LCMS?
      • For SM Biomarkers is possible to have reliable reference standards
      • Number of validation batches for LCMS (3) vs LBA (6)
      • Inclusion of chemical treatments like hydrolysis or derivativation
    • Case Studies: Parameters used in SM BAV by LCMS and why they were used

  • Topic 9
    Endogenous SM Biomarkers for Renal and Hepatic Transporters: LCMS & HRMS advanced approaches, case studies and interaction with Regulators
    - Dr. Ragu Ramanathan, Associate Research Fellow, Pfizer
    • Transporters play a fundamental role in DMPK/ADME and they are presently studies using probe drugs
    • The use of endogenous biomarkers will be able to minimize the need to dose probe drugs in clinical trials
      • Identification, qualification and validation of endogenous biomarkers for renal and hepatic transporters by LCMS & HRMS
        • Importance to develop very sensitive bioanalytical method to quantify endogenous biomarkers at very low levels
      • Status of the identification of endogenous biomarkers able to facilitate subject phenotyping in clinical trial and drug–drug interaction (DDI) assessment
      • Current plans to expand the number of these endogenous biomarkers
      • Further characterization of existing biomarkers
    • New insights on “Transporter Endogenous Biomarkers” and interaction with regulators
      • A fast-evolving area and need for feedback/discussions between Industry & Regulators
      • Current issue: “unnecessary” clinical DDI studies based on in vitro projections
        • Target: Using endogenous biomarkers in FIH to minimize some of the targeted DDI studies
        • Need: Regulatory guidance in support of reliable endogenous biomarkers qualification to reduce the number clinical DDI trials using probe drugs
        • Need: Industry/Regulators’ recommendations on how to establish in vitro to in vivo translation of endogenous biomarkers
    • Case Studies: Method development challenges to identify, qualify and validate Endogenous SM Biomarkers for Transporters

  • Topic 10
    Boosting SM Biomarker Assay Sensitivity
    - Dr. Allena Ji, Director, Sanofi-Genzyme
    • Overcoming high endogenous level during biomarkers method development and validation
      • Difficulties to achieve sensitivity below endogenous level with standard addition method
        • Health volunteers vs patients plasma levels
        • Importance to consider patient population & disease states to develop a reliable biomarker assays
    • Performing the best proxy matrix evaluation
      • Options to consider mimicking regular plasma
      • Limitations of buffer matrix in Surrogate Matrix approach
        • Recommended tests to perform and decision-making process based on 2015-2017 White Papers Part 1 for endogenous analytes
      • Advantages of using delipidized plasma
        • Slope comparison of calibration curves: regular plasma vs delipidized plasma
    • Successful BAV for supporting pivotal clinical studies in patients
      • Development of a high sensitivity GL-1 assay
      • Validation for its intended use to measure GL-1 in human plasma
      • Applications to patient
      • Baseline evaluation and post-treatment measurement
      • Lesson learnt and future applications
    • Case Studies: Method development and BAV for surrogate biomarker for enzyme replacement and substrate reduction therapies


Peptides & Oligos
Chair: Dr. Eric Yang, VP, Head of Bioanalysis Immunogenicity and Biomarkers, GlaxoSmithKline

  • Topic 11
    The Bioanalytical Challenge of Oligonucleotides Biodistribution: Current industry approaches for biological fluids/tissues, unsolved issues & unanswered questions and future perspectives
    - Dr. Amanda Wilson, Head Pre-clinical Bioanalyis & Toxicokinetics, AstraZeneca
    • In preparation for this discussion and for fully benefit from it, it is strongly recommended to attend the specifically designed Training Course M2: “The Exciting World of Oligonucleotides: A multidisciplinary complex challenge for multitasking ingenious bioanalysts”
    • Oligonucleotides discovery & development are booming after the regulatory approval of Fomivirsen
      • However, the bioanalysis of Oligonucleotides is still a relatively new area where multi-technologies are needed: LCMS, HPLC, LBA and PCR
        • The technology used depends on target molecule, concentration expected and measured metabolites
      • The regulatory framework for Oligonucleotides in biological matrix is not well-established yet
        • What is the reality the bioanalytical labs are facing today with these molecules?
        • What BMV guidance/guidelines should we use for RT-qPCR methods?
        • Are there alternative ways to measure mRNA beyond PCR? What about LCMS?
        • Should we apply the same LBA BMV guidance/guideline for branched-DNA assay? Should we use “house keeping” genes?
        • How to validate LCMS for Oligonucleotides?
        • How has the Industry/Regulators received the recommendations of the 2017 White Paper Part 2? How are these recommendations evolving?
    • State-of-the-art Bioanalysis strategies for studying Oligonucleotides Biodistribution
      • Approaches to understanding Oligonucleotides breakdown products
      • Importance of having highly specialized teams to deal with the bioanalysis of Oligonucleotides interchangeably using many different technologies
      • Measurement in biodistribution studies (PK & PD) of
        • Oligonucleotide & Breakdown products
        • Expressed protein
        • Nanoparticle & LNP breakdown products (cationic lipids)
        • How is tissue analysis approached?
    • Oligonucleotides underdevelopment for several diseases, their diversity and their different bioanalytical requirements & challenges
    • Case Studies: Current bioanalytical approaches to assess the biodistribution of Oligonucleotide therapeutics. State-of-the-arts & limitations

  • Topic 12
    Robust LCMS and Hybrid LBA/LCMS Methods for Therapeutic Peptides: “What Works & What doesn’t”
    - Dr. Dina Goykhman, Director, Merck
    • Lesson learnt on the overall strategies to overcome method development issues for Therapeutic Peptides
      • Developing robust bioanalytical methods
    • Gaining ultra-sensitivity & selectivity based on
      • Peptide size
      • Overcoming
        • Peptides instability
        • Non-specific binding
        • Low recovery
        • Catabolism and more
    • Reagent-free LCMS vs Hybrid LBA/LCMS method for peptides
      • Immuno-affinity (IA) purification is mainly used for large molecules. Is IA with LCMS needed also for peptides? Isn’t a Reagent-free LCMS enough for peptides?
      • What is the current method of choice for peptides when extra sensitivity & selectivity is needed?
        • New data to support an evolution of the 2017 White Paper part 1 recommendation
    • Maximizing optimization:
      • Selection of the best IA capturing antibody & IA platform
      • Optimal MS precursor and fragment choice
      • Optimal LC conditions
    • Full Hybrid LBA/LCMS method evolution: from Drug discovery to Drug Development
      • Qualified method for a discovery exploratory study
      • Validated method in GLP preclinical study
      • Validate human plasma method for a clinical FIH study
    • Case Studies: Challenges with lipidated peptides in pre-clinical & clinical studies

  • Topic 13
    HRMS in Regulated Bioanalysis of Therapeutic Peptides: New Development
    - Dr. Anton Rosenbaum, Scientist, MedImmune
    • The most current industry standards on HRMS vs previous White paper recommendations
      • Mass selection window (XIC as critical parameter)
        • XIC impact on S/N
        • XIC definition during method development
        • XIC confirmation during BMV
      • Full scan: Novel challenge & opportunities
      • Recording all fragment ions generated
      • Validation and documentation of the HRMS processing methods
      • Verification of overall assay performance
    • Current perspective in using HRMS on Regulated Bioanalysis
      • How to ensure the development of robust & highly sensitive HRMS methods for Regulated studies
      • How to resolve potential issue with HRMS data re-elaboration after data acquisition
      • Limitation of full scan data mining in a regulated environment
        • Informed consent requirements for clinical studies using HRMS methods – what to write in the informed consent?
    • The most recent HRMS instrumentation advances
      • Unbias evaluations beyond vendor demos
      • Significant improvements in sensitivity
      • Extended dynamic range
      • “Bullet-proof” regulatory compliant HRMS software & its validation
    • Case Studies: Addressing HRMS strategies for overcoming peptides bioanalytical challenges in a regulated environment



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