Bioanalytical method validation - Global regulatory chalenges

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Detailed comparison of the bioanalytcial method validation guidelines of Europe, Japan, Brazil and the United States

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Bioanalytical method validation - Global regulatory chalenges

  1. 1. Evaluating EMA and FDA guidance on bioanalytical method validation Peter van Amsterdam (Abbott/EBF/GBC) 10th Bioavailability / Bioequivalence, Dissolution and Biowaivers 14 May 2014, Budapest
  2. 2. Evaluating EMA and FDA guidance on bioanalytical method validation Contents 1. Guidance 2. Method validation 3. Sample analysis 4. Quality systems --------------------- Further reading 14 May 2014 2
  3. 3. Guidance 14 May 2014 Evaluating EMA and FDA guidance on bioanalytical method validation 3
  4. 4. What to look for  ‘Global’ – OECD – ICH – WHO  ‘Regional’ – FDA – EMA – ASEAN  National – HPFB – MHLW – CFDA – etc. Evaluating EMA and FDA guidance on bioanalytical method validation 414 May 2014 Where to look …..  Bioanalytical method validation guidelines  BA/BE specific guidelines  PK / phase I specific guidelines  TK guidelines  Analytical chemistry guidance documents  GLP  GC(L)P  G-other-P
  5. 5. ‘BMVs’  International: ICH (2005) Q2(R1): Validation of Analytical Procedures: Text and Methodology  USA: FDA (2001) Guidance for Industry: Bioanalytical Method Validation  Europe: EMA (2011) Guideline on Bioanalytical Method Validation  Brazil: ANVISA (2012) RESOLUÇÃO - RDC Nº 27, DE 17 DE MAIO DE 2012 Dispõe sobre os requisitos mínimos para a validação de métodos bioanalíticos empregados em estudos com fins de registro e pós-registro de medicamentos.  Japan: MHLW (2013) Guideline on Bioanalytical Method Validation in Pharmaceutical Development  USA: FDA (2013) DRAFT Guidance for Industry: Bioanalytical Method Validation Evaluating EMA and FDA guidance on bioanalytical method validation 514 May 2014
  6. 6. ‘BABEs’  USA: FDA (2003) Bioavailability and Bioequivalence Studies for Orally Administered Drug Products - General Considerations  China: CFDA (2005) Technical guideline for human bioavailability and bioequivalence studies on chemical drug products  India (2005) Guidelines for Bioavailability & Bioequivalence Studies  Europe: EMA (2010) Guideline on the Investigation of Bioequivalence  China: CFDA (2011) Guidance on Management of Laboratory for Drug Clinical Trial Biological Sample Analysis (interim)  Canada: HPFB (2012) Conduct and Analysis of Comparative BA Studies … and many more Evaluating EMA and FDA guidance on bioanalytical method validation 614 May 2014
  7. 7. ‘GLPs’  USA: FDA (1978) 21CFR Part 58 Good Laboratory Practice for Nonclinical Laboratory Studies  International: OECD (current) Principles of Good Laboratory Practice and Compliance Monitoring  International: WHO (2009) Good Clinical Laboratory Practice (GCLP)  Europe: EMA (2012) Reflection paper for laboratories that perform the analysis or evaluation of clinical trial samples  USA: FDA (1997) 21CFR Part 11 Electronic Records; Electronic Signatures  Europe: Eudralex (2010) Good Manufacturing Practice - Medicinal Products for Human and Veterinary Use - Annex 11: Computerised Systems … and many more Evaluating EMA and FDA guidance on bioanalytical method validation 714 May 2014
  8. 8. 14 May 2014 Evaluating EMA and FDA guidance on bioanalytical method validation 8
  9. 9. … let us try to simplify the matter Evaluating EMA and FDA guidance on bioanalytical method validation 914 May 2014
  10. 10. … and concentrate on EMA BMV (2011) Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 10 Guideline on the validation of bioanalytical methods http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf  Well written with a clear structure  Clear distinction between method validation and sample analysis  First BMV guideline addressing ‘all’ the specifics for LBA/macromolecules  Defines applicable quality systems: GLP (pre-clinical) and GCP (clinical)  Good match with current thinking in BA community  Good fit with EMA Bioequivalence guideline  Fits with developing concepts on GCP for bioanalytical laboratories  Well accepted within the global BA community  Final & Current – FDA is from 2001 + a challenged 2013 draft (not taking ‘updates’ of CC-III & CC-IV into account) – ANVISA is specific for Brazil – MHLW is quite similar to EMA
  11. 11. Table of contents EMA BMV  1. Introduction  2. Scope  3. Legal basis  4. Method validation – 4.1. Full validation of an analytical method o 4.1.1. Selectivity o 4.1.2. Carry-over o 4.1.3. Lower limit of quantification o 4.1.4. Calibration curve o 4.1.5. Accuracy o 4.1.6. Precision o 4.1.7. Dilution integrity o 4.1.8. Matrix effect o 4.1.9. Stability – 4.2. Partial validation – 4.3. Cross validation Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 11
  12. 12. Table of contents (continued)  5. Analysis of study samples – 5.1. Analytical run – 5.2. Acceptance criteria of an analytical run – 5.3. Calibration range – 5.4. Reanalysis of study samples – 5.5. Integration  6. Incurred samples reanalysis  7. Ligand binding assays – 7.1. Method validation o 7.1.1. Full validation (multiple subchapters) – 7.2. Partial validation and cross-validation – 7.3. Analysis of study samples o 7.3.1. Analytical run o 7.3.2. Acceptance criteria for study sample analysis o 7.3.3. Incurred samples reanalysis  8. Reports – 8.1. Validation report – 8.2. Analytical report  Definitions Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 12
  13. 13. Method Validation Evaluating EMA and FDA guidance on bioanalytical method validation 1314 May 2014
  14. 14. Full Validation Evaluating EMA and FDA guidance on bioanalytical method validation 1414 May 2014 EMA: A full method validation should be performed for any analytical method whether new or based upon literature. Generally a full validation should be performed for each species and matrix concerned Validation should be performed using the same anticoagulant as for the study samples If problematic for validation purposes to obtain an identical matrix compared to the matrix of the study samples, a suitable alternative matrix may be used. The main characteristics of a bioanalytical method that are essential to ensure the acceptability of the performance and the reliability of analytical results are: selectivity, LLOQ, the response function and calibration curve performance, accuracy, precision, matrix effects, stability of the analyte(s) in the biological matrix and stability of the analyte(s) and of the IS in the stock and working solutions and in extracts under the entire period of storage and processing conditions. The principles of validation and analysis apply to all analytes of interest. Draft FDA: Less defined and seems to be more lenient, leaving judgment to scientists ANVISA: Less defined, but follows same principles. Requires chromatographic method for ‘chromatographable analytes’ MHLW: Quite similar to EMA
  15. 15. Reference standards Evaluating EMA and FDA guidance on bioanalytical method validation 1514 May 2014 EMA: Suitable reference standards, include certified standards such as compendial standards (EPCRS, USP, WHO), commercially available standards, or sufficiently characterised standards prepared in-house or by an external non-commercial organisation. A certificate of analysis is required to ensure purity and provide information on storage conditions, expiration date and batch number. The use of certified standards is not needed for IS, as long as the suitability for use is demonstrated, e.g. lack of analytical interference is shown for the substance itself or any impurities thereof. A certificate of analysis is not required. Recommended to use stable isotope labeled IS for MS based assays However, it is essential that the labeled standard is of the highest isotope purity and that no isotope exchange reaction occurs. The presence of any unlabeled analyte should be checked and if relative amounts of unlabeled analyte are detected the potential influence has to be evaluated during method validation. Draft FDA: Similar to EMA, but requires expiration date and purity for IS. Stock solutions should not be used after ‘powder’ expires. SILIS not mentioned ANVISA: Prefers pharmacopeia reference standards, recommends SILIS and has detailed requirements on CoA information MHLW: Similar to EMA, but less explicit
  16. 16. Selectivity Evaluating EMA and FDA guidance on bioanalytical method validation 1614 May 2014 EMA: Selectivity should be proved using at least 6 individual sources of the appropriate blank matrix Normally, absence of interfering components is accepted where the response is less than 20% of the LLOQ for the analyte and 5% for the IS. It may also be necessary to investigate the extent of any interference caused by metabolites of the drug(s), degradation products and possible co-administered medications. Co-medications normally used in the subject population studied which may potentially interfere should be taken into account at the stage of method validation, or on a study specific and compound specific base. The possibility of back-conversion of a metabolite into parent analyte during the successive steps of the analysis should also be evaluated, when relevant (i.e. potentially unstable metabolites). Draft FDA: Similar, but a bit less explicit & no acceptance criteria given ANVISA: Similar, but no co-med or metabolites mentioned. Requires hemolyzed and lipemic MHLW: Similar, but no co-med or metabolites mentioned
  17. 17. Carry-Over Evaluating EMA and FDA guidance on bioanalytical method validation 1714 May 2014 EMA: Carry-over should be addressed and minimised during method development During validation carry-over should be assessed by injecting blank samples after a high concentration sample or calibration standard at the ULOQ. Carry over in the blank sample following the high concentration standard should not be greater than 20% of the LLOQ and 5% for the internal standard. If it appears that carry-over is unavoidable, study samples should not be randomised. Specific measures should be considered, tested during the validation and applied during the analysis of the study samples, so that it does not affect accuracy and precision Draft FDA: Described, but leaving judgment to scientists ANVISA: Similar, but also specifies how to conduct the experiment MHLW: Similar
  18. 18. Lower limit of quantification Evaluating EMA and FDA guidance on bioanalytical method validation 1814 May 2014 EMA: The LLOQ is the lowest concentration of analyte in a sample which can be quantified reliably, with an acceptable accuracy and precision... The LLOQ is considered being the lowest calibration standard. The analyte signal of the LLOQ sample should be at least 5 times the signal of a blank sample The LLOQ should be adapted to expected concentrations and to the aim of the study, e.g. for bioequivalence studies the LLOQ should be not higher than 5% of the Cmax Draft FDA: Similar plus 20% CV & 20% bias requirement, no words on adapting ANVISA: No separate section on LLOQ, but similar wording is in calibration section MHLW: Similar plus 20% CV & 20% bias requirement, no words on adapting
  19. 19. Calibration curve Evaluating EMA and FDA guidance on bioanalytical method validation 1914 May 2014 EMA: The calibration standards should be prepared in the same matrix as the intended study samples. A minimum of six calibration concentration levels should be used, in addition to the blank sample and a zero sample (processed matrix with IS). Ideally it should be known what concentration range is expected. This range should be covered by the calibration curve range, defined by the LLOQ and the ULOQ. The range should be established to allow adequate description of the PK of the analyte of interest. A relationship which can simply and adequately describe the response of the instrument with regard to the concentration of analyte should be applied. All the available (or acceptable) curves obtained during validation, with a minimum of 3 should be reported. The back calculated concentrations of the calibration standards should be within ±15% of the nominal value, except for the LLOQ for which it should be within ±20%. At least 75% of the standards, with a minimum of six calibration standard levels, must fulfill this criterion. The calibration curve should preferably be prepared using freshly spiked samples. Draft FDA: Similar. Instructions on exclusion of calibrators ANVISA: Quite similar plus requirement for weighting and a minimum of 8 calibration standards for non-linear models MHLW: Similar
  20. 20. Accuracy Evaluating EMA and FDA guidance on bioanalytical method validation 2014 May 2014 EMA: To enable evaluation of any trends over time within one run, it is recommended to demonstrate accuracy and precision of QC samples over at least one of the runs in a size equivalent to a prospective analytical run of study samples. Within-run accuracy should be determined by analysing in a single run a minimum of 5 samples per level at a minimum of 4 concentrations which are covering the calibration curve range: the LLOQ, within three times the LLOQ (low QC), around 50% of the calibration curve range (medium QC), and at least at 75% of the upper calibration curve range (high QC). The mean concentration should be within 15% of the nominal values for the QC samples, except for the LLOQ which should be within 20% of the nominal value. For the validation of the between-run accuracy, LLOQ, low, medium and high QC samples from at least three runs analysed on at least two different days should be evaluated. The mean concentration should be within 15% of the nominal values for the QC samples, except for the LLOQ which should be within 20% of the nominal value. Note: between-run = ‘total’, interpret Me QC at 50% as on a geometric scale Draft FDA: Similar, however include outliers in calculation of accuracy and QC concentrations are to be related to sample concentrations rather than cal range. ANVISA: Similar, but 5 concentrations required: LLOQ, Lo, Me, Hi & ULOQ MHLW: Similar. Me QC at midpoint cal. curve
  21. 21. Precision Evaluating EMA and FDA guidance on bioanalytical method validation 2114 May 2014 EMA: Precision is expressed as the coefficient of variation (CV). Precision should be demonstrated for the LLOQ, low, medium and high QC samples, within a single run and between different runs, i.e. using the same runs and data as for the demonstration of accuracy. For the validation of the within-run precision, there should be a minimum of five samples per concentration level at LLOQ, low, medium and high QC samples in a single run. The within-run CV value should not exceed 15% for the QC samples, except for the LLOQ which should not exceed 20%. For the validation of the between-run precision, LLOQ, low, medium and high QC samples from at least three runs analysed on at least two different days should be evaluated. The between-run CV value should not exceed 15% for the QC samples, except for the LLOQ which should not exceed 20% Note: between-run = ‘total’, interpret Me QC at 50% as on a geometric scale Draft FDA: Similar, however include outliers in calculation of accuracy and QC concentrations are to be related to sample concentrations rather than cal range. ANVISA: Similar, but 5 concentrations required: LLOQ, Lo, Me, Hi & Dilution QC MHLW: Similar
  22. 22. Dilution integrity Evaluating EMA and FDA guidance on bioanalytical method validation 2214 May 2014 EMA: Dilution of samples should not affect the accuracy and precision. If applicable, dilution integrity should be demonstrated by spiking the matrix with an analyte concentration above the ULOQ and diluting this sample with blank matrix (at least five determinations per dilution factor). Accuracy and precision should be within the set criteria, i.e. within ±15%. Dilution integrity should cover the dilution applied to the study samples. Use of another matrix may be acceptable, as long as it has been demonstrated that this does not affect precision and accuracy Draft FDA: Described, but leaving judgment to scientists ANVISA: As part accuracy & precision MHLW: Similar to EMA
  23. 23. Matrix effect Evaluating EMA and FDA guidance on bioanalytical method validation 2314 May 2014 EMA: Matrix effects should be investigated when using mass spectrometric methods, using at least 6 lots of blank matrix from individual donors. Pooled matrix should not be used. For each analyte and the IS, the matrix factor (MF) and the IS normalised MF should be calculated for each lot of matrix. The CV of the IS-normalised MF should not be greater than 15 %. This determination should be done at a low and at a high level of concentration If the matrix is difficult to obtain, less than 6 different lots of matrix may be used, but this should be justified. However, matrix effects should still be investigated. If a formulation for injection to be administered to the subjects or animals contains excipients known to be responsible for matrix effects, matrix effects should be studied with matrix containing these excipients, in addition to blank matrix. In addition to the normal matrix it is recommended to investigate matrix effects on other samples e.g. haemolysed and hyperlipidaemic plasma samples. If applicable also samples from special populations (such as renally or hepatically impaired populations) Draft FDA: Described, but leaving judgment to scientists ANVISA: Similar, but: 8 samples (4 normal, 2 lipemic, 2 hemolyzed) and ‘the degree of hemolysis’ MHLW: Similar, but CV MF < 15%
  24. 24. Stability (1) Evaluating EMA and FDA guidance on bioanalytical method validation 2414 May 2014 EMA: Evaluation of stability should be carried out to ensure that every step taken … sample preparation, analysis and storage … do not affect the concentration of the analyte. Stability tests should be done similar to the conditions and materials used for the actual study samples. Reference to data published in the literature is not considered sufficient. Stability of the analyte in the studied matrix is evaluated using low and high QC samples. The QC samples are analysed against a fresh calibration curve and the mean concentration at each level should be within ±15% of the nominal concentration. The following stability tests should be evaluated: • stock solution and working solutions of the analyte and IS. SILIS not needed • freeze and thaw stability of the analyte in the matrix • short term stability of the analyte in matrix at room temperature or sample processing temperature • long term stability of the analyte in matrix stored in the freezer • stability of the processed sample at room temperature or under the storage conditions to be used during the study (dry extract or in the injection phase), • on-instrument/autosampler stability of the processed sample at injector or autosampler temperature.
  25. 25. Stability (2) Evaluating EMA and FDA guidance on bioanalytical method validation 2514 May 2014 EMA: WRT LTS: For small molecules it is considered acceptable to apply a bracketing approach, i.e. in case stability has been proved for instance at -70°C and -20°C, it is not necessary to investigate the stability at temperatures in between. Study samples may be used in addition to QC samples, but the exclusive use of study samples is not considered sufficient. The results of long term stability should be available before the study report is issued. In case of a multi-analyte study and specific for bioequivalence studies, attention should be paid to stability of the analytes in the matrix containing all the analytes. Sufficient attention should be paid to the stability of the analyte in the sampled matrix directly after blood sampling of subjects and further preparation before storage, to ensure that the obtained concentrations by the analytical method reflect the concentrations of the analyte in the subject at the moment of sampling. A demonstration of this stability may be needed on a case-by-case basis, depending on the structure of the analyte. Draft FDA: Similar, but also IS stock stability and reinjection reproducibility ANVISA: Quite similar, but triplicate analysis MHLW: Similar, but triplicate analysis
  26. 26. Recovery Evaluating EMA and FDA guidance on bioanalytical method validation 2614 May 2014 EMA: Not addressed MHLW: Recovery is a measure of the efficiency at which an analytical method recovers the analyte through the sample-processing step. In order to elucidate the nature of analytical method, it is advisable to evaluate the recovery. The recovery is determined by comparing the analyte response in a biological sample that is spiked with the analyte and processed, with the response in a biological blank sample that is processed and then spiked with the analyte. It is important to demonstrate the reproducibility, rather than to show a higher recovery rate. ANVISA: Not addressed Draft FDA: Similar to MHLW, but 3 concentrations required
  27. 27. Endogenous analytes Evaluating EMA and FDA guidance on bioanalytical method validation 2714 May 2014 EMA: Not addressed Draft FDA: The biological matrix used to prepare calibration standards should be the same as the study samples and free of the endogenous analyte. To address the suitability of an analyte-free biological matrix, the matrix should be demonstrated to have (1) no measurable endogenous analyte and (2) no matrix effect or interference when compared to the biological matrix. The use of alternate matrices (e.g., buffers, dialyzed serum) for the preparation of calibration standards is generally not recommended … The QCs should be prepared by spiking known quantities of analyte(s) in the same biological matrix as the study samples. The endogenous concentrations of the analyte in the biological matrix should be evaluated prior to QC preparation (e.g., by replicate analysis). ANVISA: For endogenous compounds, the selectivity may be tested by a comparison of the gradients of, as a minimum, 6 standard addition curves in 6 samples from different sources of the biological matrix and the standard curve in solution or substitute matrix. The method is considered selective if the slopes of the curves are not significantly different. The validation tests of the calibration curve, accuracy, and carry-over may be carried out using calibration standards and QCs in solution or substitute matrix The validation tests of precision and stability tests must be carried out in the same biological matrix as the samples in the trial. MHLW: Not addressed
  28. 28. Partial validation Evaluating EMA and FDA guidance on bioanalytical method validation 2814 May 2014 EMA: In situations where minor changes are made to an analytical method that has already been validated, a full validation may not be necessary, depending on the nature of the applied changes. Changes for which a partial validation may be needed include transfer of the bioanalytical method to another laboratory, change in equipment, calibration concentration range, limited sample volume, another matrix or species, change in anticoagulant, sample processing procedure, storage conditions etc. All modifications should be reported and the scope of revalidation or partial validation justified. Note: Generally a full validation should be performed for each species (4.1 Full validation) Draft FDA: Similar, but OK with partial for matrix change within species & species change within matrix ANVISA: Mentioned MHLW: Similar
  29. 29. Cross validation Evaluating EMA and FDA guidance on bioanalytical method validation 2914 May 2014 EMA: Where data are obtained from different methods within and across studies or when data are obtained within a study from different laboratories, applying the same method, comparison of those data is needed and a cross validation of the applied analytical methods should be carried out. For the cross validation, the same set of QC samples or study samples should be analysed by both analytical methods For QC samples, the obtained mean accuracy by the different methods should be within 15% and may be wider, if justified. For study samples, the difference between the two values obtained should be within 20% of the mean for at least 67% of the repeats. FDA: Similar, but leaving judgment to scientists ANVISA: Not addressed MHLW: Similar, but 20% for QCs is acceptable
  30. 30. Reporting Evaluating EMA and FDA guidance on bioanalytical method validation 3014 May 2014 EMA: The validation report should include at least the following information: • summary of the validation performances, • details of the applied analytical method • details of the assay procedure (analyte, IS, sample pre-treatment and analysis), • reference standards (origin, batch number, CoA, stability and storage conditions), • calibration standards and QC samples (matrix, anticoagulant, preparation, preparation dates, and storage conditions), • run acceptance criteria, • analysis: • table of all runs with dates, passed or failed and the reason for the failure • table of calibration results of all accepted analytical runs, • table of QC results of all accepted analytical runs (precision and accuracy); • stability data of stock solution, working solution, QC • data on selectivity, LLOQ, carry-over, matrix effect, dilution integrity; • unexpected results obtained during validation with full justification of the action taken, • deviations from method and/or SOPs Draft FDA: Similar, but with more detail and recommendation for a summary table ANVISA: Not addressed MHLW: Similar, but a bit less detail
  31. 31. One method, one validation, one report (1) Aspect Follow … and Full validation EMA, MHLW Reference standards EMA ANVISA: CoA information Selectivity EMA ANVISA: Lipemic & hemolyzed Carry-over EMA, MHLW ANVISA: experimental LLOQ Any Note: BE requirement EMA Calibration curve EMA ANVISA: weighting and non-linear models Accuracy & Precision EMA, MHLW ANVISA: add 5th QC = dilution QC Dilution integrity EMA, MHLW ANVISA: dilution is part of A & P Matrix effect EMA ANVISA: 2x lipemic & hemolyzed MHLW: CV_MF < 15% Stability EMA ANVISA, FDA, MHLW: triplicate Evaluating EMA and FDA guidance on bioanalytical method validation 3114 May 2014
  32. 32. One method, one validation, one report (2) Aspect Follow … and Recovery FDA, MHLW Endogenous analyte ANVISA, FDA Partial validation EMA, MHLW Note: species change within a matrix or matrix change within a species may require a full validation Cross validation EMA Reporting EMA, FDA Evaluating EMA and FDA guidance on bioanalytical method validation 3214 May 2014 Conclusions  You can use EMA BMV as the basis  CoA & selectivity: check ANVISA  Matrix effect: check ANVISA & MHLW  Stability: check ANVISA, FDA or MHLW  Recovery: use FDA or MHLW  Endogenous analyte: check ANVISA and/or FDA  Note: ANVISA requires dilution QC in all A & P validation batches
  33. 33. Sample Analysis Evaluating EMA and FDA guidance on bioanalytical method validation 3314 May 2014
  34. 34. Analytical run Evaluating EMA and FDA guidance on bioanalytical method validation 3414 May 2014 EMA: An analytical run consists of the blank sample (processed matrix sample without analyte and without IS) and a zero sample (processed matrix with IS), calibration standards at a minimum of 6 concentration levels, at least 3 levels of QC samples (low, medium and high) in duplicate (or at least 5 % of the number of study samples, whichever is higher), and study samples to be analysed. All samples (calibration standards, QCs, and study samples) should be processed and extracted as one single batch of samples in the order in which they intend to be submitted or analysed. A single batch is comprised of samples which are handled at the same time, i.e. subsequently processed without interruption in time and by the same analyst with the same reagents under homogeneous conditions Draft FDA: Similar, lot of detail ANVISA: Not specifically addressed MHLW: Not specifically addressed
  35. 35. Acceptance criteria of an analytical run Evaluating EMA and FDA guidance on bioanalytical method validation 3514 May 2014 EMA: The back calculated concentrations of the calibration standards should be within ±15% of the nominal value, except for the LLOQ for which it should be within ±20%. At least 75% of the calibration standards, with a minimum of six, must fulfil this criterion. If one of the calibration standards does not meet these criteria, this calibration standard should be rejected and the calibration curve without this calibration standard should be re-evaluated, and regression analysis performed. If the rejected calibration standard is the LLOQ, the LLOQ for this analytical run is the next lowest acceptable standard of the calibration curve. If the highest calibration standard is rejected, the ULOQ for this analytical run is the next acceptable lower standard of the calibration curve. The revised calibration range must cover all QC samples (low, medium and high). The accuracy values of the QC samples should be within ±15% of the nominal values. At least 67% of the QC samples and at least 50% at each concentration level should comply with this criterion.. Draft FDA: Similar ANVISA: Similar MHLW: Similar, but multi analyte case not addressed
  36. 36. Calibration range Evaluating EMA and FDA guidance on bioanalytical method validation 3614 May 2014 EMA: If a narrow range of analyte concentrations of the study samples is known or anticipated before the start of study sample analysis, it is recommended to either narrow the calibration curve range, adapt the concentrations of the QC samples, or add new QC samples to adequately reflect the concentrations of the study samples. If a narrow range of analysis values is unanticipated, but observed after the start of sample analysis, it is recommended that the analysis is stopped and either the calibration range narrowed, QC concentrations revised, or QC samples at additional concentrations are added. It is not necessary to reanalyse samples analysed before optimising the standard curve range or QC concentrations. The same applies if a large number of the study samples appear to be above the ULOQ. The calibration curve range should be extended, if possible, and QC samples added or their concentrations modified. At least 2 QC sample levels should fall within the range of concentrations measured in study samples. If the calibration curve range is changed, the bioanalytical method should be revalidated (partial validation) to verify the response function and to ensure accuracy and precision. Draft FDA: Similar, but less attention to inadequate calibration range ANVISA: Not addressed MHLW: Similar, but less attention to inadequate calibration range
  37. 37. Reanalysis of study samples Evaluating EMA and FDA guidance on bioanalytical method validation 3714 May 2014 EMA: Possible reasons for reanalysis of study samples and criteria to select the value to be reported should be predefined in the protocol, study plan or SOP The following are examples of reasons for study sample reanalysis: • rejection of an analytical run • IS response significantly different from calibration standard and QC samples, • improper sample injection or malfunction of equipment, • the obtained concentration is above the ULOQ or below the run’s LLOQ (truncated) • identification of quantifiable analyte levels in pre-dose samples or placebo sample, • poor chromatography For bioequivalence studies, normally reanalysis of study samples because of a pharmacokinetic reason is not acceptable, The safety of trial subjects should take precedence over any other aspect of the trial. Consequently, there may be circumstances when it is necessary to reanalyse specific study samples, e.g. where an unexpected result is identified that may impact patient safety Draft FDA: Similar, but specifies number of replicates and no special description about safety concerns ANVISA: Similar, but no special description about safety concerns MHLW: Similar, but no special description about safety concerns
  38. 38. (Re)Integration Evaluating EMA and FDA guidance on bioanalytical method validation 3814 May 2014 EMA: Chromatogram integration and re-integration should be described in a SOP. Any deviation from this SOP should be discussed in the analytical report. Chromatogram integration parameters and in case of re-integration, initial and the final integration data should be documented at the laboratory and should be available upon request. Draft FDA: Similar, but requires reporting of both original and re-integration data ANVISA: Not addressed MHLW: Similar
  39. 39. Incurred samples reanalysis Evaluating EMA and FDA guidance on bioanalytical method validation 3914 May 2014 EMA: As a guide, 10% of the samples should be reanalysed in case the number of samples is less than 1000 samples and 5% of the number of samples exceeding 1000 samples. Furthermore, it is advised to obtain samples around Cmax and in the elimination phase. The concentration obtained for the initial analysis and the concentration obtained by reanalysis should be within 20% of their mean for at least 67% of the repeats. Large differences between results may indicate analytical issues and should be investigated. Incurred sample reanalysis should be done at least in the following situations: • toxicokinetic studies once per species • all pivotal bioequivalence trials • first clinical trial in subjects • first patient trial • first trial in patients with impaired hepatic and/or renal function Samples should not be pooled, as pooling may limit anomalous findings. NOTE: Since 2012, EMA does not accept BE studies anymore without ISR Draft FDA: Similar, but recommended ISR size is 7% ANVISA: Not addressed MHLW: Similar
  40. 40. ISR Considerations Evaluating EMA and FDA guidance on bioanalytical method validation 4014 May 2014  At least 67% within ± 20% of the their mean value < A few in every batch or all in one or more dedicated batches Overall result is OK, but a random X% shows extreme values Overall result is OK, but a subject/time/… specific X% shows extreme values Overall result is OK, but indicative of systematic difference between 1st and 2nd result Instability of the drug (analyte) Instability of metabolites
  41. 41. System suitability Evaluating EMA and FDA guidance on bioanalytical method validation 4114 May 2014 EMA: Not addressed FDA: SOP is needed. Do not use CALs, QCs or samples ANVISA: Not addressed MHLW: Analytical instruments used in bioanalysis should be well maintained and properly serviced. In order to ensure optimum performance of the instrument used for bioanalysis, it is advisable to confirm the system suitability prior to each run, in addition to periodical check. However, confirmation of the system suitability is not mandatory in bioanalysis, because the validity of analysis is routinely checked in each analytical run
  42. 42. Reporting Evaluating EMA and FDA guidance on bioanalytical method validation 4214 May 2014 EMA: The analytical report should include at least the following information: • reference standards (origin, batch, certificate of analysis, stability, storage conditions) • calibration standards and QC samples (storage conditions) • run acceptance criteria (short description, reference to specific protocol or SOP) • assay procedure (short description) • sample tracking (dates, sample conditions, storage location and conditions) • study sample analysis: • content of the analytical run, • table identifying all analytical runs and study samples, with run dates and results, • table of calibration results of all (passed) analytical runs, • table of QC results of all (passed) analytical runs; • failed analytical runs (identity, assay date, reason for failure), • deviations from method and/or SOPs • reassay, excluding reassay due to analytical reasons, such as failed run For bioequivalence studies, all chromatograms from the runs which include 20% of the subjects, including the corresponding QC samples and calibration standards. For other studies representative chromatograms should be appended to the report Draft FDA: Similar, lot of detail ANVISA: Not addressed MHLW: Similar
  43. 43. One study, one method, one report Aspect Follow … and Analytical run EMA, FDA Acceptance criteria Any Calibration range EMA Reanalysis of study samples EMA, MHLW, ANVISA Note: EMA requirement with respect to patient safety Reintegration EMA, MHLW FDA: original & reintegration data Incurred sample reanalysis EMA, MHLW System suitability FDA, MHLW Reporting EMA, FDA Evaluating EMA and FDA guidance on bioanalytical method validation 4314 May 2014 Conclusions  You can use EMA BMV as the basis  System suitability: check FDA & MHLW  Note: EMA accepts/advocates reanalysis if an outlying result may indicate issues with patient safety
  44. 44. Quality systems Evaluating EMA and FDA guidance on bioanalytical method validation 4414 May 2014
  45. 45. GxP Evaluating EMA and FDA guidance on bioanalytical method validation 4514 May 2014 EMA: The validation of bioanalytical methods and the analysis of study samples for clinical trials in humans should be performed following the principles of GCP. Further can be found in the “Reflection Paper for Laboratories That Perform The Analysis Or Evaluation Of Clinical Trial Samples.” (EMA/INS/GCP/532137/2010). The validation of bioanalytical methods used in non-clinical pharmaco toxicological studies that are carried out in conformity with the provisions related to Good Laboratory Practice should be performed following the Principles of GLP. EMA BE: The bioanalytical part of bioequivalence trials should be performed in accordance with the principles of GLP. However, as human bioanalytical studies fall outside the scope of GLP, the sites conducting the studies are not required to be monitored as part of a national GLP compliance programme. Note: OECD GLP Draft FDA: Pre-clinical adhere to GLP (21 CFR 58), Clinical adhere to 21 CFR 320.29 ANVISA: Not addressed MHLW: Not clearly addressed
  46. 46. One study, one method, one quality system .. Aspect Follow … and Method validation EMA Note: discrepancy between EMA BMV (2011) & EMA BE (2010) Sample analysis EMA, FDA Evaluating EMA and FDA guidance on bioanalytical method validation 4614 May 2014 Conclusions  You can use EMA BMV as the basis  For method validation for pre-clinical and clinical use the GLP system as the standard quality system for the laboratory.  For sample analysis from GLP studies (pre-clinical): use GLP as the quality standard and claim compliance to GLP.  For clinical sample analysis: use the GLP system as the standard quality system for the laboratory  For clinical studies in Europe: adhere to the EMA ‘GCLP’ reflection paper
  47. 47. Acknowledgements  EBF members and Steering Committee for the learning experiences.  The GBC Founding & Steering Committee members and Harmonization Team leads for the learning experiences  Informa Life Sciences for giving me the opportunity to present at their meeting.  The regulators and inspectors for stimulating us to continuously improve our work  You, for your attention and prof. Ernő Rubik for the inventing the cube. Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 47
  48. 48. Further reading Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 48
  49. 49. Papers  Analytical methods validation: bioavailability, bioequivalence and pharmacokinetic studies. Shah VP , Midha KK , Dighe SV , et.al. Pharm Res. 9, 588 - 592 (1992)  Bioanalytical method validation - a revisit with a decade of progress. Vinod P. Shah, Kamal K. Midha, John W. A. Findlay, et.al. Pharmaceutical Research 17(12), 1551-1557 (2000)  Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective J.W.A. Findlay, W.C. Smith, J.W. Lee, et.al. J. Pharmaceutical and Biomedical Analysis 21, 1249-1273 (200)  Workshop on bioanalytical methods validation for macromolecules: summary report Krys J. Miller, Ronald R. Bowsher, Abbie Celniker et.al. Pharmaceutical Research 18(9), 1373-1383 (2001)  Recommendations for the bioanalytical method validation of ligand-binding assays to support pharmacokinetic assessments of macrmolecules Binodh DeSilva, Wendell Smith, Russell Weiner, et.al. Pharmaceutical Research 20(11), 1885-1900 (2003)  Workshop/Conference report - Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays C. T. Viswanathan, Surendra Bansal, Brian Booth, et.al. Pharmaceutical Research 24(10), 1962-1973 (2007)  European Bioanalysis Forum and the way forward towards harmonized regulations Berthold Lausecker, Peter van Amsterdam, Margarete Brudny-Kloeppel, et.al. Bioanalysis 1(5), 873-875 (2009) Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 49
  50. 50.  Workshop Report and Follow-Up-AAPS Workshop on Current Topics in GLP Bioanalysis: Assay Reproducibility for Incurred Samples-Implications of Crystal City Recommendations Douglas Fast, Marian Kelley, C. Viswanathan, et. al. The AAPS Journal 11(2), 238-241 (2009)  Incurred sample reproducibility: views and recommendations by the European Bioanalysis Forum Philip Timmerman, Silke Luedtke, Peter van Amsterdam, et.al. Bioanalysis 1(6), 1049-1056 (2009)  Request for global harmonization of the guidance for bioanalytical method validation and sample analysis Philip Timmerman, Steve Lowes, Douglas Fast et.al. Bioanalysis 2(4), 683 (2010)  International harmonization of bioanalytical guidance Surendra Bansal, Mark Arnold, Fabio Garofolo Bioanalysis 2(4), 685-687 (2010)  Towards harmonized regulations for bioanalysis: moving forward! Peter van Amsterdam, Berthold Lausecker, Silke Luedtke, et.al. Bioanalysis 2(4), 689-691 (2010)  Bioanalytical method validation: notable points in the 2009 draft EMA Guideline and differences with the 2001 FDA Guidance Greame Smith Bioanalysis 2(5), 929–935 (2010)  Regulated bioanalysis and the desire for harmonized regulations in China Daniel Tang, Dafang Zhong Bioanalysis 2(12), 1913-1919 (2010) Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 50
  51. 51.  SQA opinion paper on global harmonization of the bioanalytical method validation guidances Christopher Tudan, Stephen Rogenthien, Anthony Jones Bioanalysis 2(12), 1921-1925 (2010)  Building the Global Bioanalysis Consortium – working towards a functional globally acceptable and harmonized guideline on bioanalytical method validation Peter van Amsterdam, Mark Arnold, Surendra Bansal, et.al. Bioanalysis 2(11), 1801-1803 (2010)  Workshop/Conference Report on EMA Draft Guideline on Validation of Bioanalytical Methods Henning Blume, Erich Brendel, Margarete Brudny-Klöppel, et.al. European Journal of Pharmaceutical Sciences 42, 300–305 (2011)  Implication of differences in bioanalytical regulations between Canada, USA and south America Mark Arnold Bioanalysis 3(3), 253-258  Bioanalytical procedures and regulation: towards global harmonization Howard Hill Bioanalysis 3(4), 365-367 (2011)  Bioanalysis in Latin America: where are we and where are we going? Rafael Eliseo Barrientos-Astigarraga Bioanalysis 3(10), 1043-1045 (2011)  US FDA/EMA harmonization of their bioanalytical guidance/guideline and activities of the Global Bioanalytical Consortium Fabio Garofolo, Josée Michon, Virginie Leclaire, et.al. Bioanalysis (4)3, 231-236 (2012) Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 51
  52. 52.  2012 white paper on recent issues in bioanalysis and alignment of multiple guidelines Binodh DeSilva, Fabio Garofolo, Mario Rocci et.al. Bioanalysis 4(18), 2213-2226 (2012)  Managing scientific, technical and regulatory innovation in regulated bioanalysis: a discussion paper from the European Bioanalysis Forum Philip Timmerman, Neil Henderson, John Smeraglia et.al. Bioanalysis 5(2), 139-145 (2013)  The European Bioanalysis Forum community’s evaluation, interpretation and implementation of the European Medicines Agency guideline on Bioanalytical Method Validation Peter van Amsterdam, Arjen Companjen, Margarete Brudny-Kloeppel et.al. Bioanalysis 5(6), 645-659 (2013)  Regulated bioanalysis in Japan: where do we come from and where are we going? Noriko Katori Bioanalysis 5(11), 1321-1323 (2013)  Current regulations for bioanalytical method validations Mark E. Arnold, Rafael E. Barrientos-Astigarraga, Fabio Garofolo, et.al. in: Wenkui Li, Jie Zhang, Francis L.S. Tse (ed) Handbook of LC-MS Bioanalysis: Best practices, experimental protocols and regulations John Wiley & Sons, Inc. (2013)  A comparison of FDA, EMA, ANVISA and others on bioanalysis in support of bioequivalence/bioavailability studies Bradley Nash in: Wenkui Li, Jie Zhang, Francis L.S. Tse (ed) Handbook of LC-MS Bioanalysis: Best practices, experimental protocols and regulations John Wiley & Sons, Inc. (2013) Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 52
  53. 53. Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 53 Guidance  ANVISA: Resolution- RDC Nº 27, Requirements for the validation of bioanalytical methods. http://bvsms.saude.gov.br/bvs/saudelegis/anvisa/2012/rdc0027_17_05_2012.pdf  CDSCO: Guidelines for bioavailability and bioequivalence studies http://cdsco.nic.in/html/BE%20Guidelines%20Draft%20Ver10%20March%2016,%2005.pdf  CFDA: Technical guideline for human bioavailability an bioequivalence studies on chemical drug products http://www.cde.org.cn/attachmentout.do?mothed=list&id=167  EMA: Guideline on the investigation of Bioequivalence http://www.emea.europa.eu/docs/en_GB/document_library/Scientific_guideline/2010/01/WC500070039.pdf  EMA: Guideline on the validation of bioanalytical methods http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2011/08/WC500109686.pdf  EMA: Reflection Paper for Laboratories that perform the analysis or evaluation of clinical trial samples http://www.ema.europa.eu/docs/en_GB/document_library/Regulatory_and_procedural_guideline/2010/09/WC50 0096987.pdf  FDA: Guidance for Industry: Bioanalytical Method Validation http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/UCM070107.pdf  FDA: Guidance for Industry - Safety Testing of Drug Metabolites http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079266.pdf
  54. 54.  HPFB: Conduct and Analysis of Comparative BA Studies http://www.hc-sc.gc.ca/dhp-mps/alt_formats/pdf/prodpharma/applic-demande/guide- ld/bio/gd_cbs_ebc_ld-eng.pdf  ICH: E6(R!) Guideline for Good Clinical Practice http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Efficacy/E6_R1/Step4/E6_R1_ _Guideline.pdf  ICH: Q2(R1): Validation of Analytical Procedures: Text and Methodology http://www.ich.org/LOB/media/MEDIA417.pdf  ICH: M3(R2): Guidance on Non-Clinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals http://www.ich.org/MediaServer.jser?@_ID=5544&@_MODE=GLB  MHLW:Draft Guideline on Bioanalytical Method Validation in Pharmaceutical Development http://www.nihs.go.jp/drug/BMV/BMV_draft_130415_E.pdf  MHLW: Guideline on Bioanalytical Method Validation in Pharmaceutical Development http://bioanalysisforum.jp/images/T130918I0020.pdf  OECD Series on Principles of Good Laboratory Practice (GLP) and Compliance Monitoring http://www.oecd.org/chemicalsafety/testing/oecdseriesonprinciplesofgoodlaboratorypracticeglpandcom pliancemonitoring.htm Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 54
  55. 55. … and may our dreams come true Evaluating EMA and FDA guidance on bioanalytical method validation14 May 2014 55

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