Specialized Workshop Th1 - Training Topic List

"Mass Spec Advanced Method Development - Peptides, Oligos, SM and Biomarkers: Solving Complex Challenges with Innovative Approaches on Sensitivity & Selectivity, Unstable/Sticky Compounds; Column Chemistry, Orthogonal Separations, Tissue Bioanalysis"

New level of Sensitivity & Selectivity (S&S); “Very” Unstable Metabolites/Pro-drugs how to catch them; Back to basic with adsorption of very “sticky” Compounds; Novel column chemistry and orthogonal separations. Novel strategies for tissue bioanalysis

  • Dr Scott Summerfield, Sr. Director and UK Head of Bioanalysis and Biomarkers,GlaxoSmithKline
  • Dr. Hongbin Yu, Director Bioanalytical Mass Spectrometry, Boehringer Ingelheim

Method Development & Metabolites Quantification Challenges

Biomarker Methods for new levels of Sensitivity & Selectivity (S&S)

Peptides & Oligos - Solving Challenges with Innovative Approaches


Discussion Topic DETAILS of Th1 – Mass Spec Advanced Method Development

  • Dr Scott Summerfield, Sr. Director and UK Head of Bioanalysis and Biomarkers,GlaxoSmithKline
  • Dr. Hongbin Yu, Director Bioanalytical Mass Spectrometry, Boehringer Ingelheim
Method Development & Metabolites Quantification Challenges
  • Lesson 1
    Advanced Method Development Strategies to Avoid Pitfalls in Bioanalysis: Building on WRIB recommendations, evaluating the chemical structures and evolving trends
    - Dr. Scott Summerfield, Sr. Director and UK Head of Bioanalysis and Biomarkers,GlaxoSmithKline
    • How to succeed in method development and method robustness were discussed in the White Paper in Bioanalysis since 2010 and new strategies added in the recommendations every year (2010-2019)
    • Most important strategies to consider succeeding in method development
      • What to consider to development of robust and selective method
      • Importance to evaluate the chemical structure of the analyte before going to the bench
      • Best criteria for the selection of cleanup methods
      • Prediction of potential interference
        • Metabolites
        • Isomerization
        • Degradation
        • Matrix suppression
    • Considerations to delivery high quality methods
      • Strengthening the method during the development stage
      • Avoiding weak validation packages or too many rejected runs
      • Avoiding surprises during sample analysis
    • What is this chemical structure telling you?
    • Case studies on best strategies in method development and decision tree

  • Lesson 2
    Importance of Step by Step Investigations and Troubleshooting of Unexpected Bioanalytical Result: Lesson learnt from multiple complex case studies in small molecule bioanalysis
    - Dr. Sune Sporring, Manager LCMS Group Development Bioanalysis, Novo Nordisk
    • Successful strategies in Bioanalytical investigations
    • Issues triggering a comprehensive investigation
      • Physiological issues
      • Method issues
      • Sampling
      • Stability issues
      • Ensuring sufficient training for personnel
      • Potential issues during IS addition
    • Importance of using Orthogonal analysis in complex investigations
      • Using the unique features of HRMS to troubleshoot the method
      • Detection of unknowns
      • Degradation profiles
      • Defining specific trends during the investigation
    • Issues in transferring preclinical methods to clinical
      • What to consider when a method progresses into clinical
    • Case studies on step by step Investigations and troubleshooting of unexpected bioanalytical result

  • Lesson 3
    State-of-the-Art Method Development Strategies in HRMS: Building on WRIB recommendations, challenges with extraction windows, effect of different processing software, balancing quality/quantity, evolving trends
    - Dr. Lieve Dillen, Lead Assay Development Bioanalytical Dept, Janssen
    • Advanced method development using HRMS
      • HRMS (QTOF and Orbitrap) is now a very common instrument in Bioanalysis
      • Numerous progresses have been achieved in Bioanalytical applications
    • Building on 2015-2019 White Paper in Bioanalysis recommendations
    • Importance to learn the challenges with Extraction Windows in HRMS
      • Practical case studies in quantification in clinical and preclinical studies
      • Evaluation of different data processing software packages
    • HRMS complexity
      • Exhaustive explanations on MD, optimization, software parameters to succeed in
      • Balancing selectivity with sensitivity and quality and quantity
      • Ability to identify unanticipated metabolites and interferences
    • Optimal HRMS settings in bioanalysis
      • How to minimize analysis time
    • Case studies on insights in optimizing the HRMS bioanalysis.

  • Lesson 4
    Specificity Issue Caused by Metabolite Interference in Regulated Bioanalysis Using LC-MS/MS
    - Dr. Jinhui Zhang, Chemist Division of Product Quality Research, US FDA
    • Specific discussions regarding the Impact of Metabolites on Bioanalytical Assay results began in 2009 White Paper in Bioanalysis. Update recommendations 2009-2016
    • Impact of metabolites on bioanalytical methods
      • Potential interfering effect of metabolite(s) on the quantitation of parent drug
    • Limitation of Validation
      • Two methods generating different results but both fully validated according to EMA guideline
    • Isobaric metabolites
      • Identification and mitigation of an isobaric sulfate metabolite to a phosphate prodrug
      • Isobaric metabolites with almost identical MS/MS profile as parent drugs
      • New isobaric metabolite of Fluvastatin observed during ISR
    • How to minimize the risk of metabolite interference
      • Additional LC methods for in-study samples
      • Use of multiple MRM channels for data acquisition
      • Use of orthogonal HRMS
    • New case studies to show how metabolite(s) can affect the quantitation of parent drug

  • Lesson 5
    Rising of a Novel Paradigm in Metabolite Quantification: Building on WRIB recommendations, pharma experience on evolving trends on what metabolite to quantify at what stage
    - Dr. Jian Wang, Sr. Principal Scientist Bioanalytical Sciences, Bristol-Myers Squibb
    • Building on 2013-2016 White Paper in Bioanalysis recommendations on Metabolite Monitoring and Quantitation Strategy with new case studies and advances
    • Evolution of the metabolite monitoring and quantitation strategy
      • Drug safety evaluation
      • Biotransformation
      • Clinical pharmacology
      • Bioanalysis
    • Lesson learnt
      • Monitoring “important” animal metabolites to evaluate their contribution to the potential toxicity
      • Start evaluating metabolite exposure in IND enabling tox studies
    • 2018 FDA BMV Guidance and ICH M10 draft BMV Guideline
      • Stability testing with Multiple analytes present
    • Case studies on complexity in bioanalytical method development and validation when metabolites are involved

  • Lesson 6
    Free Drug Quantification: Building on WRIB recommendations, what’s new & new case studies, strategies on how to ensure no impact from very unstable metabolites
    - Dr. Nicki Hughes, Vice President Laboratory Operations BioPharma Services
    • Building on 2015-2016 White Paper in Bioanalysis recommendations on Free Drug Assays with new case studies and recent developments
    • Method development strategies to measure total, free and conjugated
    • Unstable parent drug
      • Specific stabilizers in whole blood and plasma
    • Extremely unstable sulfate conjugate
      • major form circulating in plasma in vivo
      • Unstable reference standard
      • Rapidly hydrolysis to parent drug in solution
    • Novel case studies on efforts to design in vitro experiment to demonstrate that parent drug levels measured in clinical samples are that of “free” or unconjugated drug and not due to the ex-vivo hydrolysis of the sulfate conjugate


Biomarker Methods for new levels of Sensitivity & Selectivity (S&S)
  • Lesson 7
    Novel Method Development Approaches for Tissue Bioanalysis of Biomarkers
    - Ms. Anita Lee, Principal Scientist Translational Biomarkers Group, Merck
    • Building on 2014-2016 White Paper in Bioanalysis recommendations on Tissue Bioanalysis of Biomarkers with new case studies and recent developments
    • “It is recommended to wash solid tissue samples with PBS during sample collection to remove possible blood contamination and flash freeze as soon as possible”
    • “When heterogeneity of the analyte distribution is a concern, use of pulverization and
    • homogenization techniques is recommended for the entire tissue sample to ensure a representative overall measurement”
    • Focus on Biomarkers Tissues analysis
      • Overcoming Sensitivity & Selectivity
      • development strategies
      • Tissue specific distribution
    • Adaptation of LCMS method for complex matrices
      • Increasing LCMS sensitivity for low level-analyte applications
      • Development of a novel extraction to isolate and enrich protein in tissues
      • Use of a selective, high throughput trapping LCMS method
    • Case studies on Novel developments and applications of a very sensitive LCMS assay for quantification of biomarkers in tissues

  • Lesson 8
    Novel Multiplex HRMS Approaches in the Quantification of Clinical Biomarkers of Transporter Mediated in DDI: Building on WRIB recommendations, new case studies, evolving trends
    - Ms. Lina Luo, Team Lead Global Bioanalytical Laboratories, Pfizer
    • Building on 2018-2019 White Paper in Bioanalysis recommendations on Biomarkers of Transporter Mediated in DDI with new case studies and recent developments
    • Increased interest in utilizing endogenous probes for drug-drug interaction (DDI)
      • Limitation of current approaches
      • High false positive rates with
      • Organic cation transporters (OCT1 & OCT2)
      • Multidrug and toxin extrusion proteins (MATEs)
    • Focus on Clinical Small Molecule Biomarkers for FIH studies
      • HRMS Advanced Method development for
      • Isobutyryl-L-carnitine (IBC)
      • Carnitine
      • Thiamine
      • N1-methylnicotinamide (1-NMN)
      • Creatinine
      • Metformin
    • Chromatographic challenges
      • Developing a highly sensitive hydrophilic interaction chromatography (HILIC)
    • FFP Validation
      • Surrogate matrix approach
      • Avoiding interference from endogenous analytes
      • Parallelism to native plasma
    • Novel case studies on multiplexed assay utilized in clinical sample analysis

  • Lesson 9
    Method Development Challenges and Solutions in Biomarkers: Importance of thoroughly Optimized Chromatography and Column Chemistry
    - Dr. Jenny Zhang, Assoc. Director Precision Medicine Early Clinical Development, Pfizer
    • Building on 2017-2019 White Paper in Bioanalysis recommendations on Small Molecules Biomarkers Method Optimization & FFP Validation with new case studies and recent developments
    • Advantages of Surrogate Analyte approach
      • Stable isotope-labeled fructose and sorbitol
        • Surrogate standards
        • Internal standards
    • Fructose and Sorbitol in the clinical setting
      • Challenges to develop a reliable bioanalytical method
      • Multiple isomers of sugars present in human plasma
        • Interferences from glucose and mannitol
      • Low molecular weights of these monosaccharides
      • Challenging chromatographic characteristics.
        • No derivatization & Chromatographic separation on a hydrophilic interaction liquid chromatography
        • Importance to increase column temperature to increase the analytical throughput while maintaining acceptable separation efficiency
    • FFP Validation
    • Case studies on fructose as SM Biomarker for NASH (nonalcoholic steatohepatitis) and predictive biomarker for the total sugar intake

  • Lesson 10
    Challenges with Biomarkers Quantification in Rare Matrices: How to conduct method development for successful FFP BAV and sample analysis?
    - Dr. Allena Ji, Director Clinical Mass Spectrometry Laboratory Biomarkers and Clinical Bioanalyses, Sanofi
    • Issue in in developing biomarkers methods on Rare Matrices
      • Use of human cerebral spinal fluid (CSF)
        • High cost
        • Low availability of healthy subject CSF
    • Use as a surrogate matrix
      • Artificial CSF for method development
      • Experiments needed for comparison of authentic CSF with artificial CSF
      • Matrix to use to prepare QCs (authentic or artificial CSF).
    • Method development strategies for CSF
      • How to achieve pg/mL sensitivity
      • Assessment of parallelism
      • Matrix effect
      • Adsorption of analyte(s)
      • Acceptance criterion for slope of calibration curve
        • Surrogate matrix vs authentic biological matrix
    • Effect for the surrogate calibration curve was evaluated at three levels
    • Case studies to evaluate accuracy recovery after spiking analyte into endogenous analyte matrix and variation among authentic biological matrix lots


Peptides & Oligos - Solving Challenges with Innovative Approaches