LC-MS

6th Workshop on Recent Issues in Bioanalysis (6th WRIB)

Where Regulators and the Industry Convene

Discussing, Sharing perspectives, and Providing Potential Solutions on the Most Recent Issues in Bioanalysis

March 28-29, 2012, San Antonio, TX, USA
Courses: March 26-27, 2012
Hotel Marriott San Antonio Riverwalk

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Registration Fees   |   Online Registration   |   Hotel Accomodation   |   White Papers from Pre. Editions Workshop  

 

Course 2a:
Bioanalysis of Large Molecule & Biomarkers:
Advanced Ligand Binding Assays (LBA) Method Development and Troubleshooting Course
8am - 5pm, March 27, 2012
Instructor: Dr. Binodh Desilva, Bristol-Myers Squibb

Course Description:

This course has been specifically designed for those who work in drug discovery and drug development bioanalytical laboratories desiring training in Ligand Binding Assays (LBA) and Biomarkers method development.

Original method development strategies and method development case studies are discussed.

Course Syllabus:

  • LBA Advanced Method Development & Troubleshooting
    • Development activities vs. validation
    • How do you develop an assay?
      • Discuss alternatives and possible pitfalls
      • Titration of Reagents
    • Serum or Plasma?
    • The Most Critical Component
    • Monoclonal vs. Polyclonal?
    • Tricks of the Trade
    • Antibody Production
    • Protein G: The Industry Standard
      • Pros and Cons for Rodent IgG Purification
    • Nonspecific Nonspecificity - Matrix Effect
      • Ways to assess specificity
      • Serum BPs cause LBA interferenceInterferences
      • Blocking and non-specific reactions
      • ELISA Blocker Evaluation
      • Types of plates
      • Irradiated polystyrene "high binding plates"
    • Factors affecting coating process
      • Coating of solid phases
      • Coating Time and Temperature
      • Coating Concentrations
    • Incubation and mixing
    • Blocking and non-specific reactions
    • Assay Optimization
      • Parameters to examine
      • Assessment of variability

  • Immunogenicity: The Challenges
    • Assessment
    • Effects of antibodies to TPs
    • Positive "Clearing" Antibody
    • "Sustaining" Antibody
    • Drug Induces Neutralizing Antibody to Drug and to Endogenous Protein
    • Strategy for Immunogenicity Testing
    • "High" and "Low" Affinity Antibodies
    • Acid Dissociation Reduces Interference
    • Neutralizing Antibodies
    • Importance of Biological Assays for Neutralizing Activity
    • Alternatives to Bioassays for Neutralizing Antibodies

  • LBA Validation
    • Pre-study vs. in-study validation
    • Documentation Requirements
    • Validation Parameters
    • Standard Curve Model Selection
      • Curve-Fitting Algorithm: Importance of Weighting
      • Four Parameter Logistic Function (4PL)
      • Misconception about the "Linear Portion" of a Calibration Curve
    • General recommendations for Standard calibrators
    • Standard Curve Model Acceptability
    • Accuracy and Precision (A&P)
    • Design Recommendations & Analysis Recommendations
    • Total Error Example
    • Specificity and Selectivity
    • Recommendation to evaluate sample matrix from patients
    • Dilution Linearity
    • Robustness and Ruggedness
    • Calibration Checks

  • LBA Stability Issues
    • Critical reagents and ADAs stability
    • Impact on activity or binding or activity
    • Impact on F/T, bench top and LT stability

  • LBA Free/Total
    • Impact of reversible binding: bound, non-bound or partially bound
    • Difficulties in measuring a specific form and LBA
    • LBA platform and impact on binding equilibrium

  • In-Study Validation Considerations
    • Standard Curve Acceptance Criteria
    • Parallelism
    • System Suitability

  • Critical Reagents Issues
    • Capture Reagent
    • Detection Reagent
    • Considerations for Reagent Development
    • Analyte heterogeneity
    • Immunogenicity of the analyte
    • Degree of sequence homology with endogenous versions of the analyte
    • Kinetics of reagent binding
    • Quantity of reagent needed
    • Specific and non-specific interfering matrix components

  • Types of Validations
    • Full, partial, cross

  • Biomarkers
    • Introduction to biomarkers
    • Nomenclature. How do you define a biomarker?
    • Qualification versus validation - Acceptance criteria for fit-for-purpose assays
    • General issues with biomarker analysis
    • Types and methods for biomarker development & validation
    • Assay development
      • Choice of reference standards
      • Target range
      • QC conditions
      • Stability
      • Matrix effect
      • Use of sample controls
      • Specificity, and relative selectivity
    • Sample collection, handling and storage
    • Biomarkers statistical considerations

  • Biomarkers: Commercial kits
    • Use of Commercial kits in the development and validation of Biomarkers
      • Pros and cons of using commercial kits
    • Similarity and differences from PK assays & diagnostic application
    • Systematic analytical validation of commercial kits for the determination of novel biomarkers
    • Adaptation of commercial test kits to generate biomarker data to support regulated studies
      • Challenges and limitations
      • Verification of commercial test kit performance
    • Factors to consider when identifying a kit manufacturer
    • Commercial test kits
      • Research-use only and Diagnostics kits
      • Availability from several different vendors
      • Differences in performance
      • Differences in internal development and validation
      • Validation challenges to the regulatory laboratory with respect to documentation reagents and reference standards
    • Logical and systematic approach for defining the extent of analytical validation needed for application of commercial kits based upon the intended use of the biomarker data
      • Intended purpose approach to kit validation
      • Extent of validation & intended use of the data
      • Levels of validation include: none, kit verification, exploratory and advanced

  • Case Studies
    • Ab1 - Before and After DOE
      • Method Development Challenges
      • Reagents Available
      • DOE Design - Varied parameters
      • Interference/Specificity
      • Reagent Stability Evaluation
    • Recombinant Protein
      • Available reagents
      • Analytical challenge
      • Total Assay
      • Validation Protocol
    • Fc-Fusion Protein
      • Correlation comparing data between repeat results
      • Event Resolution SOP
      • Scatter-plots
      • Recommended graphical and analysis
      • Representative PK profile from original and repeated assays

 

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