Method Development Challenges, Regulatory Findings, and Innovations
in Bioanalysis
5th Workshop on Recent Issues in Bioanalysis (5th WRIB)
Short Course 1b:
Bioanalysis of Large Molecule: Ligand Binding Assays (LBA) method development and validation
8:30am - 5:00pm, April 11, 2011
Instructor: Dr. Binodh Desilva, Bristol-Myers Squibband Dr. Fabio Garofolo, Algorithme Pharma
Course Description:
This course on large molecules bioanalysis has been designed for scientists who are involved in LBA therapeutic protein analysis. The course focuses on large molecule analysis and state-of-the-art approaches in protein analysis by LBA.
Course Syllabus:
- Introduction to Therapeutic Bioactive Proteins
- Biopharmaceuticals and Biologics
- Issues with purification, formulation, pharmacokinetics, and immunogenicity
- Protein Analysis
- Protein Structure
- Post-translational modifications
- Degradation/denaturation
- Aggregation
- Protein Biopharmaceuticals
- Immunoassays
- Yalow & Berson
- Antibodies and Antigens
- Antibodies as Analytes
- Polyclonal antibodies
- Monoclonal antibodies
- Heterogeneity of a Monoclonal Antibody Product
- Law of Mass Action
- Valency and Avidity
- Types of Immunoassays
- Competitive Immunoassay
- Non-competitive Immunoassay
- Heterogeneous vs Homogenous Immunoassays
- Sandwich Assay Format
- Bridge Assay Format
- RIA
- EIA, FPIA, KIMS, Lateral Flow
- ELISA
- Microplate Reader-Based Technologies
- Electrochemiluminescence (ECL) - Meso Scale Discovery (MSD)
- Time Resolved Fluorescence - DELFIA
- Luminescent Oxygen Channeling Immunoassay (LOCI) - AlphaLISA
- Gyrolab by Gyros
- Impact on Assay Formats
- Detector System/Conjugation
- LBA: Strengths and Limitations
- Cross-Reactivity Issues
- Binding Proteins/ Interfering Factors
- Testing Formats and Models
- LC-MS/MS vs. LBA
- Types of LBA Protocols
- Reagents vs. Equipment
- Detector System
- Reference Standard
- Calibration Curve & Prozone
- Assay Validation Parameters
- Matrix interference
- Development and Validation
- Development activities vs. validation
- Pre-study vs. in-study validation
- Documentation Requirements
- Validation Parameters
- Standard Curve Model Selection
- 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
- Stability
- Robustness and Ruggedness
- 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
- Case Studies
- Validated Assay Format using ECL
- Method Development Challenges
- Reagents Available
- DOE Design - Varied parameters
- Ab1 - Before and After DOE
- Interference/Specificity
- Reagent Stability Evaluation
- Automation Utility
- Recombinant Protein
- Available reagents
- Analytical challenge
- Total Assay
- Validation Protocol
- Incurred Sample Reanalysis (ISR) - Fc-Fusion Protein
- Statistical Evaluation of Incurred Sample Reproducibility
- Correlation comparing data between repeat results
- Event Resolution SOP
- Scatter-plots
- Recommended graphical and analysis
- Representative PK profile from original and repeated assays
