Training Session T1 - Tuesday April 2, 2019, 7am to 6:30pm

"Current & Effective Strategies for Critical Reagent Characterization, Storage, LT Stability, Re-testing and Life Cycle Management for LBA & Flow Cytometry PK, ADA and Biomarkers Assays & Bioanalytical Troubleshooting of Critical Reagents Issues"

Recent Advancements and New Frontiers for the Bioanalytical Lab

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


Part 1: Critical Reagents Management: Challenges & Approaches
Part 2: Characterization & Storage
Part 3: LT Stability & Re-testing
Part 4: Life Cycle Management
Part 5: Bioanalytical Troubleshooting
2019 White Paper on Critical Reagent
  • Consensus and Conclusions on Critical Reagent for 2019 White Paper in Bioanalysis


DETAILS of Training Course T-1

Part 1: Critical Reagents Management: Challenges & Approaches
  • Lesson 1
    Evolution of WRIB Recommendations on Critical Reagents from 2015 to 2018: Where are we now and what next?
    • How the 2015-2018 White Paper in Bioanalysis Recommendations on Critical Reagents have impacted the current industry practice and regulations
      • Focus on Generation, Characterization and Maintenance
      • On-going needs for Critical Reagents discussion/training within the Bioanalytical Community and importance of learning from each other
      • Importance to use these recommendations to influence reagent suppliers for harmonization and better characterization of commercial products
    • Understanding where we started in 2015 and where we are now
      • Progresses based on Industry/Regulators’ experience at WRIB
      • Current Guidance on Critical Reagents: FDA, EMA, ICH
    • Crucial importance of Critical reagents to ensure assay performance during method validation and sample analysis

  • Lesson 2
    Type of Critical Reagents used in Flow Cytometry Instrumentation and Application in Drug Discovery Development: Similarities and differences between LBA and Flow Cytometry Critical Reagents
    • Flow Cytometry as powerful technology for any Bioanalytical Lab from early discovery to post-marketing clinical trials
      • Instrumentation Key Component
      • Main Applications in Drug Discovery & Development
        • Cellular characterization and functional analysis
        • Multi-parameter analysis & quantitation of different cellular components
        • Measurements on surface expressed targets
        • Analysis of intra-cellular signaling
        • RO - determination of drug-receptor occupancy
    • Critical Reagents for Flow Cytometry
      • What are the Critical Reagents for Flow Cytometry?
      • Similarities and differences between LBA and Flow Cytometry Critical Reagents

  • Lesson 3
    Defining Processes for Developing Continuous Improvement in Workflows, Overcoming Dogma and Bringing Value in Critical Reagents Management
    • Workflows for LBA Critical Reagents able to cover from early discovery to post-marketing
      • Fully Integration in Bioanalytical lab Workflows of Critical Reagents Life Cycle Management, Characterization, Quality evaluation and Resupply with LBA development
      • Definition of Interdisciplinary activities with in a bioanalytical laboratory for a successful Critical Reagent strategy
    • Developing incremental initiatives and innovations to achieve best practices and continuous improvement in Critical Regent Management
      • Implementation of plans able to overcome issues and bring values
    • Strategies for defining a robust process between bioanalytical group and critical reagents group


Part 2: Characterization & Storage
  • Lesson 4
    LBA Critical Reagent Characterization & Storage: Key tests to ensure characterization vs. traditional qualification by assay performance. Pros & Cons
    • Pros & Cons to qualify critical reagents by assay performance
      • Current Best Practices for critical reagent characterization based on in-depth characterization
      • How to maintain an appropriate characterization from early discovery to post-marketing
        • Risk of lack of characterization in discovery
    • Essential tests to ensure characterization
      • Novel applications of biophysical characterization
        • Improvement of critical reagent controls with new orthogonal techniques
        • Surface plasmon resonance and high-throughput kinetic information
        • HRMS
    • Focus on where Critical Reagents had a major impact on assays performance
      • Case studies and lesson learnt from numerous biotherapeutic programs
      • Assay failures based on lack of in-depth biophysical characterization
      • Strategic reagent screening and characterization to minimize assays failure

  • Lesson 5
    Impact of LBA Critical Reagents Characterization, Handling and Storage on ADA Assays
    • Case Studies on ADA Assays & investigations to find the root cause
      • Method Troubleshooting using biophysical characterization
      • Optimization of desalting cycles
      • Choices of buffer exchange into formulations
      • Use of cryoprotectant excipients
    • Impact of Conjugated Critical Reagents Aggregation & Degradation on ADA assays results
      • Increase in background
      • Increase in ADA positive responses
      • Increased signal intensity from negative control (NC)
    • Impact on Handling & Storage conditions on Conjugated Critical Reagents stability
      • Signal changes with freeze-thaw (FT) cycles
      • Considerations on cryo-frozen storage
      • How much can storage conditions impact quality and integrity of critical reagents?

  • Lesson 6
    Critical Reagents Characterization and Storage for Flow Cytometry: Preparation & Staining of Cells, Current Protocol for Fluorescence Labeling of Purified Antibodies
    • Characterization of Flow Cytometry Critical Reagents used for analyzing
      • Expression of cell surface
      • Definition of different cell types
      • Cell size and volume
      • Intracellular antigens
    • Characterization Tests for fluorescent-labeled antibodies or ligands that bind to specific cell-associated molecules
      • Protocols for fluorescence labeling of purified antibodies
      • Evaluation on how long the cells with the fluorochrome remain stable
      • Risk that antibodies are internalized overtime
    • Procedures for direct and indirect staining of single-cell suspensions for
      • Tissue
      • Peripheral blood
    • Protocols for intracellular cells staining
      • Impact of detergent and staining of nonviable cells
      • Approaches for identification of dead cells in fixed/permeabilized cell preparations


Part 3: LT Stability & Re-testing
  • Lesson 7
    Critical Reagents Long Term Stability & Re-testing: How extensive should we characterize the stability of critical reagents, especially labeled reagents, used for LBA Assays?
    • What test to perform to show Regulatory Agencies that assay reagents are stable over the course of use?
      • What is meaningful for Reagent stability studies?
      • Current/established recommendations for critical reagent long term stability (LTS) and re-testing
    • Approaches when stability of critical reagents is unknown
      • How to define the recommended expiration and re-testing dates
      • How this lack of knowledge can impact assay performance
    • Decision making process for critical reagent expiration date extension and re-testing dates
      • Considerations on
        • Assay performance deterioration and required binding properties
        • Biophysical characterization profile and reagent stability indicators
      • Understanding the relationships between biophysical changes and their effect on assays performance
    • Use of stability-enhancing additives, surfactants and conformational stabilizers on Critical Reagents stability
    • Issues with Critical Reagents transportation
      • Effects of CO2 exposure due to dry ice and pH changes
        • Changes in tertiary/quaternary structure
        • Change in enzyme activity
        • Changes in aggregation
      • Precaution to minimize impact of transportation on stability

  • Lesson 8
    Lot-to-lot Reproducibility/Stability and Life Cycle Management of Antibody Reagents for Flow Cytometry
    • Strategies to minimize issues of Lot-to-lot reagents variability in Flow Cytometry
      • Use of reagent QC to control for lot-to-lot variability
      • Variation in conjugation
      • Variation in staining intensities
    • Current protocols to evaluate critical reagent consistently
      • Reagent carryover and Lot-To-Lot reproducibility
      • Fluorochrome-to-antibody ratio and mean fluorescence intensity of antibody lots
      • Reproducible fluorescence intensities over time and Life Cycle Management
      • Evaluation and compensation of dye degradation trends
      • Issues produced by significant differences in Lot-to-lot fluorescence stability between manufacturers
    • Certificate of Analysis as crucial part of Critical Reagents Life Cycle Management
      • Identification of the source, characterization and quality of the reagent
      • Storage and overtime performance
      • Taking precautions in storing enough reagent of the same lot to complete both validation and study
    • Current status of Industry recommendations for production of Flow Cytometry fluorescence-labelled reagents


Part 4: Life Cycle Management
  • Lesson 9
    Importance of LBA Critical Reagents Life Cycle Management for Successful Biotherapeutics Discovery & Development: PK & Biomarkers Assays
    • Influence of Critical Reagents Life Cycle Management on PK Assays for successful biotherapeutics discovery & development
    • How to minimize the risk of interruptions/delays to/of the supply chain of Critical Reagents
      • Bad planning, forecasting and management
      • Unavailability of the right reagents at the right time
      • Delays for method development and validation
      • Impact on program decisions, time and resources
      • Unreliable data sets
    • Common failures in Critical Reagents Life Cycle Management
      • Inefficient and insufficient implementation of adequate characterization technologies
      • Production of low-quality reagents
      • Impact on Bioanalytical Lab deliverables
      • Need to re-develop, re-validate and cross validate assays

  • Lesson 10
    Rigorous & Robust LBA Critical Reagent Life Cycle Management for Consistent and Reliable Performance of ADA Assays
    • ADA Assays Conjugated Critical Reagents Life Cycle Management
      • Consistent and reliable performance
      • Importance of formulation conditions overtime
    • Fundamental activities for Life Cycle Management to ensure smooth performance of ADA Assays
      • Inter-disciplinary aspects of correct Positive Control generation & supply for screening and confirmatory assays
      • Conjugated Reagents performance evaluation in each phase of development, validation and use during actual clinical studies
      • Strategies to mitigate the risk of large variations in expression, purification and conjugation overtime
      • Approaches for successful qualification of a new lots Positive Controls


Part 5: Bioanalytical Troubleshooting
  • Lesson 11
    Critical Reagent Issues & Solutions for PK Assays: Bioanalytical Troubleshooting
    • Case studies on bioanalytical troubleshooting of PK assays impacted by the characteristic of the Critical Reagents
      • Results interpretation in the measuring of analyte concentrations for PK determinations
      • PK assays sensitivity due to high-affinity, stable and well-characterized Critical Reagents
      • Issues with specific antidrug and/or anti-target Critical Reagents
      • Impact of Critical Reagents on the assay design and format
      • Critical Reagents cross reactivity
    • Crucial effect of fragments, aggregates and partially unfolded reagents on PK assay signal, specificity and activity
      • Signal amplification
      • Avidity and cross-linking effects
      • Loss of binding properties

  • Lesson 12
    Critical Reagent Issues & Solutions for ADA Assays: Bioanalytical Troubleshooting
    • Case studies on bioanalytical troubleshooting of ADA assays impacted by capture and detector Critical Reagent for bridging assay format
    • Importance of Conjugation and suitable Positive Controls
      • Evaluation of the potential risk of having the conjugation masking drug–ADA interactions
      • Positive Control for assay performance and determination of assay sensitivity
      • Impact of different lots of surrogate Positive Control for ADA
      • Altered drug tolerance in ADA assays due to Critical Reagents issues
    • Understanding the effect of Degree of Labelling determination (D.O.L) on
      • ADA Assays Noise and Cut point Determination
      • False Positive
      • Assays Sensitivity and Drug Tolerance

  • Lesson 13
    Critical Reagent Issues & Solutions for Biomarkers Assays & Bioanalytical Troubleshooting: Biomarkers Calibrator Material Selection and Resulting Calculations
    • Case studies on bioanalytical troubleshooting of Biomarkers assays impacted by Critical Reagent for quantification of target expression, safety and efficacy end points
      • Use of highly specific and selective Critical Reagents for Biomarkers quantification in complex biological fluids and tissue
      • Impact Critical Reagents minor changes on binding, sensitivity and overall performance of Biomarkers assays
    • Problems with Commercially Critical Reagents
      • “The assay kit issue”

  • Lesson 14
    Reagent Issues for Flow Cytometry Assays: Bioanalytical Troubleshooting
    • Importance of Quality Reagents in Flow Cytometry Applications for Clinical Studies
    • Thorough troubleshooting of a Flow Cytometry biomarker assay
      • Unexpected behavior during Clinical trials
      • Root Cause and need for a redeveloped the assay
    • Investigation of the critical reagents in the antibody panel and ultimately needs of changing the panel design to improve the assay performance











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