Course 2b:
Bioanalytical Method Validation and Sample Analysis (BMV & SA) Course:
Challenges, Solutions, Applications, Global Industry Standards, and New Regulations
8am - 5pm, March 27, 2012
Instructor: Dr. Fabio Garofolo, Algorithme Pharma
Course Description:
The Bioanalytical Method Validation and Sample Analysis course has been specifically designed for scientists, QA auditors and bioanalytical experts who work in regulated bioanalytical laboratories to bring them up-to-speed with global industry standards, new regulations, and white papers consensus in bioanalysis.
This course is an advanced discussion of bioanalytical common problems and "What's new in Bioanalysis?"
Course Syllabus:
- Matrix Effect and Ion Suppression/Enhancement
- FDA Guidance
- Matrix Effect in Electrospray (ESI) LC-MS/MS
- Ion suppression evaluation
- Differential Suppression
- The 3 Different Scenarios of Matrix Effect
- Instrument Optimization to Reduce Matrix Effect
- Matrix effect with ESI, APCI and APPI and on different brands
- General criteria to fight Matrix Effect
- Carry-Over
- Definition
- Recent regulatory guidelines, recommendations and white papers
- Crystal City III; 2019-2011 White papers; FDA, EMA, GCC
- Industry standards
- Impact of Carry-Over in Bioanalysis
- Factors Contributing to Imprecision and/or Inaccuracy
- Molecular structure of Sticky Compounds
- Types of carryover
- Fighting Carry-Over
- Manual vs. automated extraction
- Accumulation effect
- Special autosampler aggressive wash
- Autosampler plumbing to avoid carryover
- Carryover vs. contamination
- Memory effect
- Evaluating Curve Models
- Quality Controls: Conventional and New Approaches
- Recent regulatory guidelines, recommendations and white papers
- Crystal City III; 2009-2011 White papers; FDA
- Acceptance of nonlinear calibration models
- Evaluating Curve Models: Graphic & Mathematical Comparison
- How much "quadratic" is acceptable?
- Extended Dynamic Range
- Linear With-Dilution versus Quadratic No-Dilution
- Case Studies: Losartan; Tramadol and Bicalutamide
- Evaluation of P & A and Dilution Reliability in Upper Portions of Quadratic Calibration
- Case Studies: Topiramate; Etodolac; Sidenafil
- Endogenous drugs: present challenges
- Common approaches for the reliable estimation of endogenous drugs
- Why is standard addition not widely used since it is an excellent way to measure endogenous compounds?
- Analyte-free matrix unavailable
- Reference Standard Material (RSM) and impact in Bioanalysis
- Quality of RSM and certificate of analysis (CoA)
- Metabolite RSM with significant levels of parent drug or isomers
- Stable labeled Internal Standard RSM with significant level of parent drug (D0)
- Contamination effect in stable labeled standard with high isotopic purity
- Case Studies: Oxycodone-d3 and Ursodiol-d5
- Incurred Sample Re-Analysis (ISR)
- Recent regulatory guidelines, recommendations and white papers
- Crystal City III; 2019-2011 White papers; FDA, EMA, GCC
- Reproducibility of QC vs. Incurred Samples
- Approach for ISR: global industry standard
- Deficiencies noted on ISR
- ISR failure: what to do?
- Investigations in Bioanalysis
- Root Cause Analysis during Out of Specification (OOS) investigations
- Anomalous results
- Regulation and guidances for investigation in bioanalysis
- Regulatory Agencies & OOS results
- Canada cGMP and USA FDA cGMP
- Importance of Trend Analysis in GLP Bioanalysis
- What to do when you investigate data but not assignable cause?
- How extensive should it be an investigation before it is determined that there is "no assigned cause"
- How to handle anomalous values and anomalous values "within acceptance criteria"
- Case studies: Investigations of matrix effect; calibrant acceptance; chromatography; extraction method
- Stability Issues in Bioanalysis
- Recent regulatory guidelines, recommendations and white papers
- Crystal City III; 2019-2011 White papers; FDA, EMA, GCC
- Autosampler Re-injection Reproducibility
- Method Evolution & Stability Measurements
- Internal Standard Stability
- Long term stability at -70C vs. - 20C
- Reference Standard Expiration Date vs. Stock Stability
- Definition of "fresh"
- Whole blood stability evaluation: industry standards
- Sample processing
- Blood Kinetic instead of Stability
- Difficult to perform true stability
- Qualified methods in whole blood (re: recovery, sensitivity issues)
- Validated methods in plasma
- Recent recommendations: pros & cons
- Stability at the blood collection stage & Freshly spiked vs. freshly extracted
- The co-administered/co-formulated stability issue
- Anticoagulants
- Anticoagulants salt forms: EDTA: K2, K3, Na and Heparin: Na, Li
- No guidelines are available. What evaluations are required?
- Recent articles and white papers
- Cross-Validation Between Different Anticoagulant Salt Forms
- Experimental Design to evaluate Potential Counterion Impact
- Case Studies: Granisetron; Fluconazole; Sirolimus
- Possible chelating effect
- Evaluation of short term, F/T stability and matrix effect
- Impact of plasma anticoagulant counterion on recovery and ionization effect
- Metabolites in Safety Testing (MIST) and Bioanalysis
- Determination of Metabolites During Drug Development
- Characterization of the pharmacokinetics of unique and/or major human metabolites (UMMs)
- Drug having potentially toxic metabolites
- Guidance for the Industry: Safety Testing of Drug Metabolites
- Disproportionate drug metabolites
- Challenges faced by the bioanalytical community in supporting these guidance policies
- Tiered approach to metabolite quantification: Bioanalytical strategy, appropriate level of validation and fit-for-purpose approach
- Definition of a major human metabolite; preclinical safety coverage for important human metabolites; validated method vs. qualified method when synthetic standards are not available
- Microdosing: Bioanalytical challenges
- "1/100th or less than the pharmacological dose" and the need for extremely sensitive bioanalytical methods to fully define the pharmacokinetic profile
- Methods commonly used: LC-MS/MS and Accelerator Mass Spectrometry (AMS)
- Microdosing: in drug discovery and in the drug development process
- Overviews of the concepts and examination on how & when microdosing can be most effectively applied to the DD and DD process: case studies
- Introduction to Therapeutic Bioactive Proteins
- Biopharmaceuticals and Biologics
- Issues with purification, formulation, pharmacokinetics, and immunogenicity
- Protein Structure
- Post-translational modifications
- Degradation/denaturation
- Aggregation
- Protein Biopharmaceuticals
- Immunoassays (Ligand Binding Assay LBA)
- 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
- Cell-based assays, others
- Multiplexing: pros and cons of multiplexing; advantages in performing multiple measurements
- Impact on Assay Formats
- Detector System/Conjugation
- LBA: Strengths and Limitations
- Cross-Reactivity Issues
- Binding Proteins/ Interfering Factors
- Testing Formats and Models
- LBA vs. LC-MS/MS bioanalytical method validation (BMV)
- Regulatory Guidances and White Paper available
- Types of LBA Protocols
- Reagents vs. Equipment
- Detector System
- Reference Standard
- Calibration Curve & Prozone
- Assay Validation Parameters
- Matrix interference
