Training Session Th1 - Thursday April 4, 2019, 7am to 6pm

"Mass Spectrometric Novel & Advanced Method Development Strategies for Large Molecules Bioanalysis: Therapeutic Proteins, ADC, Biomarkers, Oligonucleotides, Peptides & New Modalities"

Expand your Experience in PK Assays, Biotransformation and Biomarkers Assays, and Learn LCMS Technology Most Recent Updates and Refinements

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


Part 1: Focus on Hybrid Technology Updates/Refinements
Part 2: PK Assays
Part 3: Biotransformation
Part 4: Complex Peptide/Protein Biomarkers Assays
2019 White Paper on Hybrid Technology
  • Consensus and Conclusions on Hybrid Technology for 2019 White Paper in Bioanalysis


DETAILS of Training Course Th-1

Part 1: Focus on Hybrid Technology Updates/Refinements
  • Lesson 1
    Recent Advancements in Immunoaffinity (IA) Approaches and Techniques for Hybrid LBA/LCMS to maximize Specificity & Sensitivity: Method Development Strategies for Best Reagent Selection and Design of Capture Format
    • Advanced approaches and innovation for IA used in hybrid LBA/LCMS methods
      • IA applications for bioanalysis of protein therapeutics and biomarkers
      • Latest development for improving the IA
        • Improved bioanalytical IA tools to support development programs
      • IA as a critical but extensive sample processing
        • Current strategies to reduce IA time
    • Lesson learnt based on these recommendations and evolving considerations for the best application of IA to gain the sensitivity needed
      • Thorough reagent selection
      • Reagent characterization based on LBA Critical Reagents experience
      • Choosing the most appropriate capture format
    • How the method development decisions on IA capabilities can significantly impact
      • Reliability & Robustness of the hybrid LBA/LCMS bioanalytical assays
      • Specificity & Sensitivity
    • Recent case studies where a development of a selective IA was key in the quantitation of low abundant biotherapeutics and biomarkers
    • Evolution of the 2014-2018 White Paper in Bioanalysis Part 2 Recommendations on Immunoaffinity (IA) approaches

  • Lesson 2
    Mitigating Bias in Sample Preparation for Intact Protein Analysis by Mass Spectrometry
    • Strategies for development of a successful sample preparation for intact protein analysis
      • Sensitivity needs & assay range
      • Sample volume limitations, extraction methods
      • Characterization & definition of what is biologically relevant
    • Case studies on potential bias in sample preparation for intact protein
      • Presence of multiple forms/species
        • Glycosylation
        • PTMs
        • Variable drug loading (ADCs)
        • Relative ratios of each form/species can be dynamic in vivo
      • Enzymatic or chemical PTMs
      • Deconjugation
      • Differential clearance
      • CDR modifications
    • Reagent specific bias potential
      • Anti-idiotype, immobilized antigen, and anti-drug capture
      • Oversaturation of affinity reagents
      • Binding capacity
      • Saturation in the presence of multiple species

  • Lesson 3
    Recent Advancements in Digestion & Multiplexing Techniques for Hybrid LBA/LCMS: Lesson Learnt in Method Development for Best Digestion Reproducibility and Reliability of Multiplexing
    • Evolution of the 2014-2018 White Paper in Bioanalysis Part 2 Recommendations on Digestion & Multiplexing Techniques
      • Regulators’ concerns in digestion efficiency
        • Ensuring digestion completion in Regulated Bioanalysis
    • Denaturation, Reduction and Alkylation protocols before Digestion
      • What’s the impact? Are they always needed?
      • Is there an impact on signal of signature peptide measured?
    • Improved efficiency in enzymatic digestion
      • Any reliable alternative to Trypsin?
        • Glutamyl endopeptidase (Glu-C)
      • How to reduce digestion time
        • Trypsin column vs traditional digestion
        • Techniques to monitor digestion efficiency
        • Maximizing peptide abundance
    • Advancements in the development of Multiplexing Hybrid LBA/LCMS methods
      • What’s the reliability of Multiplexing in Hybrid LBA/LCMS mainly in Biomarker assays?
      • How to handle data on more than 5 protein biomarkers in multiplexing
      • Strategies for Peptide selection and final reporting data
        • How to determine the right peptide in biomarkers multiplexing


Part 2: PK Assays
  • Lesson 4
    Development of Multiple Hybrid LBA/LCMS Methods for Quantification Protein-Drug Conjugates (PDC): Ensuring method selectivity & sensitivity for a Novel Biotherapeutics
    • Method Development and Validation approaches for Protein-Drug Conjugates (PDC) PK Assays
      • Adapting LCMS and Hybrid LBA/LCMS methods to novel scaffold proteins used in targeted delivery
      • Bioanalytical similarities and differences between ADC and PDC
      • Lesson learnt from ADC Recommendations on stability of ADC in the 2014-2018 White Paper in Bioanalysis Part 2
        • ADC bioanalysis PK evaluations: total antibody, ADC, unconjugated drug
        • Regulators still expect to review all these results as part of drug submissions
    • PK and distribution of PDC in plasma and tissues
      • Total protein assay
      • Conjugate
      • Free payload
    • Challenges in the development of sensitive, specific, and high throughput methods
      • Issue with uncontrolled hydrolysis of PDC linker during sample collection and digestion
      • Need for identification and quantification of hydrolysis products

  • Lesson 5
    Biotherapeutics Quantification Using Intact Protein HRMS vs Signature/Surrogate Peptide(s): What are the Pros and Cons?
    • 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

  • Lesson 6
    Method Development & BMV of Trapping-Nano-Hybrid LBA/LCMS for Clinical Studies: Recent Application of Highly Sensitive Methods for Biotherapeutics in Regulated Bioanalysis
    • In recent years, there has been an increasing interest in reducing the column diameter further to increase sensitivity in innovative Hybrid LBA/LCMS approaches vs ultra-sensitive traditional LBA
      • From microLC to nano or capillary LC
      • Is the attempt to further reduce the column diameter really useful for Large Molecules Bioanalysis by Hybrid LBA/LCMS?
    • Advantages claimed for NanoLC
      • Reduced cost of operation
      • Higher speed
      • Gain in sensitivity
    • Issue and Drawback in Regulated Bioanalysis
      • Sensitivity & sample volume correlation
      • Extra demonstrations of equipment’s robustness for routine analysis
      • MS operating condition
      • Dead volume issues vs. Traditional HPLC/UHPLC
    • Actual Trapping-Nano-Hybrid LBA/LCMS applications in Clinical Studies
      • Are Robustness & Reliability of these methods good enough for Regulated Bioanalysis of Biotherapeutics?
      • What about US FDA’s concerns? Any submitted study and Regulatory Feedback?
      • Isn’t this technology for PK assays, mature enough for establishing a regulatory framework (Guidance/Guideline)?
    • Issues and Drawbacks in using Trapping-NanoLC in Large Molecules Bioanalysis
      • What we have learnt so far on this high sensitive approach for Biotherapeutics based on the 2014-2018 White Paper in Bioanalysis Part 2 Recommendations

  • Lesson 7
    Overcoming the Unique Challenges Associated with Biotherapeutics in Ocular Matrices: Development & Validation of Hybrid LBA/LCMS Ocular Bioanalytical Methods
    • Hybrid LBA/LCMS potentials to overcome Ocular Matrices challenges
      • Strategies on how to overcome the unique challenges associated with the characteristics of ocular matrices
      • Development and validation of ocular bioanalytical methods for Large Molecules
        • Dealing with ocular biotherapeutic administration far away from the site of action
    • Learning how ocular matrices directly impact Hybrid LBA/LCMS bioanalytical methods development design and fit for purpose validation
      • Case studies on strategies for monitoring drug concentrations in the various ocular tissues and fluids
      • Approaches and strategies for fit for purpose validating methods in Ocular matrices as close as possible to Bioanalytical Guidance/Guidelines
      • Oculars method evolution: From early Discovery Bioanalysis to fully validated methods for Regulated Bioanalysis
    • Different method development challenges with different ocular matrices:
      • Tears, Conjunctiva, Iris Aqueous humor, Vitreous humor, Retina, Choroid, Lens, Eye lid, Sclera, Lacrimal gland, Trabecular meshwork, Cornea, Iris ciliary body
      • Application of specific platforms for Ocular Tissues homogenization and extraction
      • Use of surrogate matrices due to unavailability of ocular control matrices


Part 3: Biotransformation
  • Lesson 8
    LCMS Challenges & Solutions in the Quantitation of ASOs and siRNAs in Plasma and Tissues
    • Up-to-dated strategies in Bioanalysis & Biotransformation of ASOs and siRNAs
      • New data and further considerations and understanding of the 2018 White Paper in Bioanalysis Part 1 Recommendation on Oligonucleotides
        • Recommended to increase sensitivity of LCMS methods by double liquid–liquid extractions using phenol and chloroform combination or SPE, IA enrichment, or selecting multiple target ions
        • Recommended a selection of an IS that matches chemistry and stereochemistry (if necessary) with the aim to obtain the same extraction efficiency as the analyte
        • BMV for binding-free LCMS assays based on chromatographic assay criteria
        • Challenges in tissue quantification and correlation of PK serum/plasma levels with tissues levels
    • Experience with HFIP and TEA additives in the mobile phase
      • Risk of LCMS contamination
      • Pros & cons and evaluation of suppressor chip to remove TEA from mobile phase
      • Issues in keeping the chromatographic performance without impacting MS sensitivity
    • Case studies on ASO and siRNAs quantification in tissues and plasma
      • Exposure comparison between LCMS and HPLC-fluorescence assays
      • How unexpected tissue levels triggered more evaluation
    • Current understanding how the modifications on the RNA/DNA ASOs and siRNAs influence the bioanalysis

  • Lesson 9
    Recent Applications of HRMS Oligonucleotides Bioanalysis: Method Development, Quantitation and Metabolic Profiling - Lesson Learned & Case Studies on Bioanalysis and Biotransformation
    • HRMS in the quantitation and metabolic profiling of siRNA
      • Scientific considerations on quantitative HRMS for specific applications where the obstacles were overcome using this tool that could not have been achieved otherwise
      • HRMS extra sensitivity & selectivity for siRNA compounds and metabolites in plasma and tissues
        • Is HRMS the method of choice in our bioanalytical toolbox to get the sensitivity & selectivity needed for siRNA & characterization of biotransformation?
    • What to consider when developing method in HRMS
      • Strategies to ensure that HRMS method id robust and high throughput
      • Optimizing sample extraction by solid phase extraction (SPE) and ion-pairing reagents in RP chromatography
      • HRMS parallel reaction monitoring (PRM) and full scan mode
    • Metabolic profiling data and biodistribution from liver and kidney tissues
      • Plasma/Tissues data correlation for PK, metabolite profiling and exposure
    • Future support to preclinical and clinical studies
      • Recommendation on expected use of HRMS for siRNA in a regulated environment
    • Application of 2018 White Paper in Bioanalysis Part 1 Recommendation on use of HRMS for Oligonucleotides quantification
      • Review of what discussed in 2018 and new solution to solve old challenges
        • “The principles of MIST should be assessed for oligonucleotides, when metabolism/catabolism may lead to potential efficacy and/or toxicity issues”

  • Lesson 10
    Hybrid LBA/LCMS Advanced Application in Understanding Complex Biotransformations of ADCs and their Impact
    • New applications of Hybrid LBA/LCMS to understand ADC stability and biotransformations
      • Challenges in assay development for ADC with non-cleavable linker
      • Evaluation of complex in vivo biotransformations
    • Case studies on ADC using DNA alkylating agents as payloads
      • Discovery of unexpected and complex biotransformations
      • Identification of the payload metabolic pathways
      • Impact of the biotransformations on
        • in vitro potency
        • in vivo efficacy
        • bioanalysis
    • Comparison of Hybrid LBA/LCMS vs LBA data for Total Antibody Assay
      • Considerations on potential bias due to biotransformations
      • Lesson learnt on application of Hybrid LBA/LCMS to understand unpredicted biotransformations their Impact on ADC PK Assays
    • Practical application of 2018 White Paper in Bioanalysis Part 2 Recommendations on novel ADCs and novel bioanalytical challenges with biodistribution

  • Lesson 11
    Unique Characteristic of Hybrid LBA/LCMS to Study Catabolism of Biopharma Traditional and Novel Constructs: mAbs, Fusion proteins, Bi-specifics and MDBs
    • Understanding the impact of metabolism/catabolism of biopharma constructs
      • Bioanalytical support to Biotransformation of Biotherapeutics
      • Bioanalytical LCMS, HRMS and Hybrid LBA/LCMS expertise in protein analysis and top down and bottom up approaches
    • Specific case studies of catabolism of
      • mAbs
      • Fusion proteins
      • Bi-specifics
      • MDBs
    • Technical limitations of current LCMS platforms and opportunities
      • Mass range
      • Resolution
      • Sensitivity
    • Impact of Biotransformation and quantification in vivo
      • Asp isomerization
      • Asn deamidation profiling
    • Practical application of 2017 White Paper in Bioanalysis Part 2 Recommendations on Biotransformation
      • “New insights in understanding the biotransformation of biotherapeutics by hybrid LBA/LCMS: what are we actually measuring?”
      • “Hybrid LBA/LCMS is now the gold standard for studying the biotransformation of biotherapeutics”


Part 4: Complex Peptide/Protein Biomarkers Assays
  • Lesson 12
    The Complexity of Biomarkers Free Assays by Hybrid LBA/LCMS: A Serious Sensitivity & Selectivity Challenge
    • Understanding the complexity and the challenges of Free assays for Biomarkers
      • Need to maximize sensitivity but also selectivity for Biomarkers free assays
      • Combination of HRMS and multiplex peptide Hybrid LBA/LCMS
      • Difficulties in generating robust and reliable data
      • Ex vivo quantification vs actual in vivo amount of free form
      • Multiple sources of perturbation of the equilibrium during bioanalysis
      • Practical considerations for free vs total assessment
        • Lesson learnt and possible solution from actual case studies
    • Advanced strategies to overcome main free assays issues
      • Total/Free Equilibrium shifts due to dilution
      • Recovery across concentration range
      • Immunoaffinity (IA) reagents and assay formats designed to measure free form
      • Optimization of
        • Capturing step
        • Incubation times
        • Sample collection to avoid equilibrium shift
        • Sample process steps and assay conditions that dissociate the bound form

  • Lesson 13
    Complex Peptide Biomarkers Assays by Hybrid LBA/HRMS: QTOF Advantages in Method Development of Difficult to Fragment, PTM and/or Large Peptides
    • Specific applications of HRMS in large peptides quantification
      • HRMS advantages vs Triple Quad for targeted quantitation of peptides
        • Peptides difficult to fragment
        • High molecular weight peptides
        • Post-translationally modified peptides
    • Clinical case studies for peptide hormone
    • How to maximize sensitivity & selectivity by
      • Immunoaffinity (IA) enrichment
      • UHPLC-HRMS
        • Value of using full scan
        • Optimization of mass resolving power, extraction window size and target enhancement
        • Effect of summing multiple isotopes and charge states
      • NanoLC/nanospray source
    • Practical application of 2018 White Paper in Bioanalysis Part 1 Recommendations on HRMS applications for therapeutics peptides

  • Lesson 14
    Overcoming Method Development Complexity in the Accurate Measurements of Free & Total Target Engagement (TE) Biomarkers by Hybrid LBA/LCMS: Unravelling the IP-10 Case Study
    • Practical application of 2017 White Paper in Bioanalysis Part 2 Recommendations on Target Engagement (TE) Biomarkers by Hybrid LBA/LCMS
      • “Assessing drug–target engagement in tissue with new hybrid LBA/LCMS”
      • “Hybrid LBA/LCMS can directly measure drug exposure and target engagement, two critical pharmacological questions, with a single assay”
    • Challenges in TE evaluation due to the presence of high level of drug
      • Risk of drug/target dissociation due to addition of capture reagent
      • Risk of drug interference with the binding between the capture reagent/Target
      • Potential bias in estimation of drug/target complex vs free target
      • Use of dosed drug as the capture reagent
    • Details strategies for maximizing assay sensitivity
      • Selection of digestion routes
      • Optimization of IA to achieve full recovery and minimal matrix effect
      • Strategies for minimizing drug/ligand dissociation for free assay
    • Case studies on the development Hybrid LBA/LCMS assays for quantifying free and total IP-10
      • TE measurement
      • PK-PD correlation
      • Human dose projections



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