Training Course Th-1 - 8am to 5:30pm, Thursday April 12, 2018

"Crucial importance of correct Internal Standards (IS) Response
Troubleshooting & Interpretation in Effective
LCMS Method Development, Validation and Sample Analysis"

Learn how to interpret “dangerous” IS behaviors & take actions through an in-depth evaluation of case studies and be ready to answer Regulatory Agencies’ concerns on IS variation & data accuracy

Outline of Training Course Th-1

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

 

Course Co-Chair: Dr. Seongeun (Julia) Cho, Division Director, US FDA
Course Co-Chair: Dr. Olivier Le Blaye, Inspector, France ANSM - TBC

Part 1: Regulatory Perspectives
Part 2: Focus on IS Investigations & CAPA
Part 3: Focus on Impact of IS on Method Robustness

 

DETAILS of Training Course Th-1

Course Co-Chair: Dr. Seongeun (Julia) Cho, Division Director, US FDA
Course Co-Chair: Dr. Olivier Le Blaye, Inspector, France ANSM - TBC

Part 1: Regulatory Perspectives
  • Lesson 1
    US FDA Perspective on Current Industry Standards for Internal Standards (IS) Monitoring
    - Dr. Seongeun (Julia) Cho, Division Director, US FDA
    • Regulatory input on the importance of performing adequate monitoring of IS across runs/batches/studies
      • IS Variation
        • Data Interpretation
        • Identification of anomalies
        • Troubleshooting
    • Regulatory experience on how IS monitoring is performed in the Industry
      • Similarities among bioanalytical laboratories
      • Differences among bioanalytical laboratories
    • IS significant issues
      • Impact on method robustness
      • Impact on data accuracy
    • Overall Regulatory concerns on the extent of IS investigations/CAPA performed by the Industry
    • Case Studies: Recent Internal Standard findings from US FDA audits/investigations
  •  

  • Lesson 2
    EU EMA Perspective on Current Industry Standards for Internal Standards (IS) Monitoring
    - Dr. Olivier Le Blaye, Inspector, France ANSM - TBC
    • European regulatory experience on how IS monitoring is performed in the Industry
      • Similarities among bioanalytical laboratories
      • Differences among bioanalytical laboratories
    • IS significant issues
      • Impact on method robustness
      • Impact on data reliability
    • Case Studies: Internal Standard findings from EU EMA audits/investigations
  •  

  • Lesson 3
    Brazil ANVISA Perspective on Current Industry Standards for Internal Standards (IS) Monitoring
    - Mr. Gustavo Mendes Lima Santos, Head of Bioequivalence, Brazil ANVISA
    • ANVISA regulatory experience on how IS monitoring is performed in the Industry
      • Similarities among bioanalytical laboratories
      • Differences among bioanalytical laboratories
    • IS significant issues
      • Impact on method robustness
      • Impact on data reliability
    • Case Studies: Internal Standard findings from Brazil ANVISA audits/investigations
  •  

  • Lesson 4
    How to be Ready to Answer UK MHRA Concerns on Internal Standard Variation & Data Accuracy
    - Mr. Stephen Vinter, Operations Manager, GLPMA & Laboratories Group Inspections, UK MHRA
    • Why should Bioanalytical Labs be worried to observe IS variability?
    • How should IS variability be evaluated and handled?
      • Only during validation
      • Only during sample analysis
      • During both validation & samples analysis
    • Regulatory expectations & requirements
      • IS response differences between unknown samples and standards & QCs
      • SOP IS acceptance criteria
        • Fixed (50-150% or 80-120%, others)
        • Flexible based on IS variation seen during Validation
      • Calculations for mean IS response from
        • Whole run
        • Only from standards & QCs
      • Very low IS response and impact on samples close to LLOQ levels
        • BLOQ samples due to low IS response
        • Inaccurate integration and impact on accuracy
    • Case Studies: UK MHRA inputs on type of investigations and documentations expected for IS monitoring, variation and issues

 

Part 2: Focus on IS Investigations & CAPA
  • Lesson 5
    Inconsistent Internal Standard (IS) Response
    - Dr. Daniela Fraier, Head of BA Managers Small Molecules Bioanalytics, F. Hoffmann-La Roche
    • Appropriate actions to take when IS response becomes a serious issue during sample analysis
      • Evaluation the possible root causes
        • Impact on recovery differences due to matrix
        • Major ion suppression/enhancement
    • IS variability evaluation to identify impact
      • Gathering additional information when IS responses may influence study samples reliability
      • How major variability in the IS response throughout a run can be related to severe issues with the method
      • Incapability of the method to handle significant matrix changes
    • Case Studies: Inability of stable labeled IS to well-compensate for instrumental and sample preparation variability
  •  

  • Lesson 6
    Learning How to Interpret “dangerous” Internal Standard (IS) behaviors
    - Dr. Eric Woolf, Director, Merck
    • Internal standard (IS) variability has been already discussed between Industry and Regulators in the 2011, 2014, 2015 and 2017 White Papers
      • 2011 Recommendations: Consensus was on no need for IS response acceptance criteria if stable-isotope-labeled IS (SIL-IS) and the drug coelute
      • 2014 Recommendations: IS response evaluation is a very complex topic and a variety of approaches are needed
      • 2015 Recommendations: Trends should be investigated to identify their root causes
      • 2017 Recommendations: IS variability and possible impact on accuracy
    • Why are Regulatory Agencies still concerned about IS variability?
    • Importance to perform investigations which are
      • Science-driven
      • With clear rationale
      • Well-documented
    • What about current industry standards in demonstrating accuracy using pre-dose samples for subject-specific IS responses?
      • Advantages in using this approach to answer Regulatory concerns on IS variability
    • What’s next on IS troubleshooting?
      • Evolution of this important topic over the years and current industry standards
    • Case Studies: Practical Applications of the 2011-2017 White Paper in Bioanalysis Recommendations on IS variation
  •  

  • Lesson 7
    Finding the Root-Cause for Very Complex IS Variation Impacting Data Reliability & Accuracy
    - Dr. Nico van de Merbel, Senior Director of Bioanalytical Science, PRA Health Sciences
    • IS variation has been recognized as a very complex issue (2014 & 2015 White paper in Bioanalysis) with potential impact on accuracy
      • Need for investigations to find Root-Cause(s)
        • Evaluation of numerical boundaries based on the IS variation of known/unknow samples
        • Trend analysis
        • Statistical methods to identify outliers
        • Establishing method-specific criteria
      • What is IS variation telling us?
        • Is QC really representative of subject samples?
        • Any correlation between major IS variation and ISR/ISS?
    • How to investigate non-reproducible IS variations?
      • Designing a scientific-driven full investigation
      • Multiple possible causes
      • Final CAPA
      • Is this kind of IS variation real? Why isn’t reproducible?
    • Focus on performing a thorough inspection of the chromatograms
      • Looking for non-apparent anomalies
      • Is there any retention time drifting?
      • Are there unknown peaks at specific retention time or moving from run to run?
    • In depth investigation on unknown samples
      • Data interpretation of QC prepared using a pre-dose matrix
      • Data interpretation of diluted unknown sample
      • Step by step investigation to isolate multiple related or independent root-causes
    • Case Studies: Effects of very complex IS variation on the accuracy of the drug concentration results in matrix
  •  

  • Lesson 8
    Investigations Beyond SOP on Internal Standard (IS)
    - Dr. Michael Buonarati, Senior Director, Intertek
    • Risk of blindly applying IS variation SOP
      • Acceptance of unreliable data
      • Is the IS variation SOP really able to point out inaccurate results?
      • Correct interpretation of mean IS response within acceptance criteria
        • All data pass even if there is a difference of IS in the unknown samples
        • All data pass even if there is a difference between IS variation in validation vs sample analysis
    • Limitations of acceptance criteria for IS
      • Fix vs flexible/study specific criteria
      • Investigation on results outside acceptance criteria
      • CAPA on IS variation
    • Best approach to successfully monitoring IS response
      • Ensuring analytical problems are resolved
      • Avoiding rejection of good data due to wrong interpretation of SOP IS criteria
      • Data evaluation based on scientific judgement
        • Comparing IS variation in validation with sample analysis
        • Comparing run-by-run IS variation
        • Comparing whole study IS variation
    • Case Studies: SOP IS criteria alone may not detect key IS issues, systematic differences or complex trends

 

Part 3: Focus on Impact of IS on Method Robustness
  • Lesson 9
    Putting in Place a Decision-Making Process during Method Development (MD) based on Internal Standard (IS) Response
    - Dr. Rachel Green, Head of Bioanalytical Sciences, LGC
    • What should be done during method development to avoid IS issues in validation and mainly during sample analysis?
    • How can matrix effect/matrix factor MD studies help minimizing the risk?
    • High variable IS
      • Is it an expected assay variability or intrinsic feature of unknown samples/specific matrix?
      • Was this variability present during method development? Is it well-tracked by the IS?
    • Performing experiments to limit IS variation during MD to ensure no problems in validation and during sample analysis
      • Complementary test to ensure assay robustness
        • Matrix effect lot-to-lot variability
        • Evaluation of IS correcting/tracking power
      • Is the stable-isotope-labeled IS (SIL-IS) able to compensate these variations and minimize the impact of matrix effect?
      • How to ensure during MD that IS is able to manage major variation of matrix effect in subject samples
        • Need to use matrix from patients/disease population during MD
    • Best choice for method specific IS concentration during method development
      • Is IS concentration around the geometric mean of the calibration curve the best choice?
        • Too high SIL-IS concentration may impact on LLOQ
        • Too low SIL-IS concentration may be impacted by QC High and ULOQ
    • Case Studies: Matrix effect/matrix factor studies involving highly variable IS responses
  •  

  • Lesson 10
    Internal Standard (IS) Variation and Impact on Method Robustness
    - Dr. Tom Verhaeghe, Director Development Bioanalysis, Janssen
    • How to ensure method robustness through IS tracking before it fails?
      • Samples from diseased subjects demonstrating deviating IS response
      • Triggered Investigations due to IS issues
      • Problem resolutions
    • Sample matrix impacting IS response
      • IS response differences between individual subjects
      • IS response differences between subject samples and standards and QCs
      • IS ability to correct for sample-to-sample variation and correlation with sample matrix
      • Investigation to examine if IS issues are impacting reliability and accuracy of analyte measurements
      • Measures taken to resolve the issue
    • Case Studies: Impact of disease state on IS response and impact of blank plasma source on IS response. Issue resolution
  •  

  • Lesson 11
    Maximizing Internal Standard (IS) Tracking in Microsampling
    - Dr. Tim Sangster, Head Bioanalysis and Immunology, Charles River
    • Strategies & Approaches to Develop & Validate Robust Bioanalytical Methods using
      • Capillary Microsampling (CMS)
      • Volumetric Absorbtive Microsampling (VAMS)
    • Microsampling by definition is a sampling technique that requires taking less than 50 µL of blood from subjects
      • Method development may be more complicated due to
        • Low sample volumes (CMS & VAMS)
        • Dried blood matrix (VAMS)
      • Ensuring method robustness by thorough IS tracking
    • Recommendation on potential issued with CMS & VAMS and current applications
      • 2014 White Paper Part 1 – CMS considerations for ISR and stability testing
      • 2016 White Paper Part 1 – VAMS improvements vs DBS on card and use of fresh blood only
      • 2017 White paper Part 1 - CMS & VAMS as the most used microsampling techniques
    • Case Studies: IS addition and tracking during VAMS & CMS Sample Preparation/Analysis
  •  

  • Lesson 12
    Selecting the Right Internal Standard (IS) to Develop & Validate Robust Bioanalytical Methods for Large Molecules
    - Dr. Ola Saad, Senior Scientist, Genentech
    • Current industry standards & issues on the use of internal standard for Large Molecules bioanalysis by Hybrid LBA/LCMS (Immunoaffinity-LCMS)
    • Pros & cons on the classes of internal standards presently used from SIL-IS peptides to SIL-IS proteins
      • 2016 White Paper Part 2 recommendations: “As long as validation tests demonstrate that the assay is reproducible, there was no preference on IS”
        • SIL whole proteins
        • Winged peptides
        • SIL exact peptides
    • At present most used: SIL-IS Winged Peptide
      • Pros: Compensate for chromatography, ionization, matrix effects and enzymatic digestion
      • Cons: Does not compensate for sample enrichment/cleanup (protein precipitation or immunoaffinity)
    • Advanced applications of SIL-IS Whole Protein
      • Compensate for variability during the entire procedure
        • Match native protein sequence
        • High & reliable incorporation of stable isotopes
        • Similar PTM to native protein (ie, glycosylation)
        • Ensure same digestion kinetics as native protein vs winged peptides
        • Similar enrichment/cleanup as native protein by Immunoaffinity techniques
    • Case Studies: Use of IS in biotherapeutics analysis by Hybrid LBA/LCMS to reduces variability associated with enrichment/cleanup and enzymatic digestion

 

 

©2016 WRIB. All Rights Reserved.