Recommendations on The Interpretation of The New European Medicines Agency Guideline on Bioanalytical Method Validation By Global Cro Council For Bioanalysis (Gcc)
2012; Future Science Ltd; Volume: 4; Issue: 6 Linguagem: Inglês
10.4155/bio.12.18
ISSN1757-6199
AutoresMark Boterman, Mira Doig, Massimo Breda, Steve Lowes, Jim Jersey, Ronald E. Shoup, Fabio Garofolo, Isabelle Dumont, Suzanne Martinez, Shane Needham, Maria Cruz Caturla, Philippe Couerbe, Joelle Guittard, John Maltas, Tim Lansing, Masood Bhatti, Christine Schiebl, Petra Struwe, Curtis Sheldon, Richard B. Hayes, Timothy Sangster, Colin Pattison, Johanne Bouchard, Lee Goodwin, Rafiq Islam, Rudi Segers, Zhongping Lin, Jim Hillier, Wei Garofolo, Dieter Zimmer, Lois Folguera, Thomas Zimmermann, Maria Pawula, Marc Moussallie, Leonardo de Souza Teixeira, Thaís Chaves Rosendo Rocha, Daniel Tang, Paula Jardieu, James Truog, Jenny Lin, Richard Lundberg, Chris D. Cox, Alan P. Breau, Chiara Bigogno, Dick Schoutsen, C Dilger, Mohammed Bouhajib, Ann Lévesque, Sofi Gagnon-Carignan, Robert I. Nicholson, Rand Jenkins, Ming Hung Lin, Shane Karnik, Theo de Boer, Richard Houghton, Rachel Green, William DeMaio, Romuald Sable, Kirk H. Smith, Christoph Siethoff, Laura Cojocaru, Mike Allen, Tammy Harter, Saadya Fatmi, Farhad Sayyarpour, Michele Malone, Stuart Best, Xinping Fang,
Tópico(s)Statistical Methods in Clinical Trials
ResumoBioanalysisVol. 4, No. 6 White PaperFree AccessRecommendations on the interpretation of the new European Medicines Agency Guideline on Bioanalytical Method Validation by Global CRO Council for Bioanalysis (GCC)Mark Boterman, Mira Doig, Massimo Breda, Steve Lowes, Jim Jersey, Ronald Shoup, Fabio Garofolo, Isabelle Dumont, Suzanne Martinez, Shane Needham, Maria Cruz Caturla, Philippe Couerbe, Joelle Guittard, John Maltas, Tim Lansing, Masood Bhatti, Christine Schiebl, Petra Struwe, Curtis Sheldon, Roger Hayes, Timothy Sangster, Colin Pattison, Johanne Bouchard, Lee Goodwin, Rafiq Islam, Rudi Segers, Zhongping (John) Lin, Jim Hillier, Wei Garofolo, Dieter Zimmer, Lois Folguera, Thomas Zimmermann, Maria Pawula, Marc Moussallie, Leonardo de Souza Teixeira, Thais Rocha, Daniel Tang, Paula Jardieu, James Truog, Jenny Lin, Richard Lundberg, Chris Cox, Alan Breau, Chiara Bigogno, Dick Schoutsen, Carmen Dilger, Mohammed Bouhajib, Ann Levesque, Sofi Gagnon-Carignan, Robert Nicholson, Rand Jenkins, Ming Hung Lin, Shane Karnik, Theo De Boer, Richard Houghton, Rachel Green, William DeMaio, Romuald Sable, Kirk Smith, Christoph Siethoff, Laura Cojocaru, Mike Allen, Tammy Harter, Saadya Fatmi, Farhad Sayyarpour, Michele Malone, Stuart Best & Xinping FangMark BotermanABL, Groningen, The Netherlands, Mira DoigABS Laboratories, Welwyn Garden City, UK, Massimo BredaAccelera, Nerviano, Italy, Steve LowesAdvion BioServices, Ithaca, NY, USA, Jim JerseyAgilux Laboratories, Worcester, MA, USA, Ronald ShoupAIT Bioscience, Indianapolis, IN, USA, Fabio GarofoloAlgorithme / Simbec, Laval, QC, Canada, Isabelle DumontAlgorithme / Simbec, Laval, QC, Canada, Suzanne MartinezAlgorithme / Simbec, Laval, QC, Canada, Shane NeedhamAlturas Analytics, Moscow, ID, USA, Maria Cruz CaturlaAnapharm Europe, Barcelona, Spain, Philippe CouerbeAtlanbio, Saint-Nazaire, France, Joelle GuittardAvogadro, Toulouse, France, John MaltasBASi, Kenilworth, Warwickshire, UK, Tim LansingBioAgilytix Labs, Durham, NC, USA, Masood BhattiBioPharma Services, Toronto, Ontario, Canada, Christine SchieblCelerion, Fehraltorf, Switzerland, Petra StruweCelerion, Fehraltorf, Switzerland, Curtis SheldonCelerion, Lincoln, NE, USA, Roger HayesCetero Research, Houston, TX, USA, Timothy SangsterCharles River, Tranent, UK, Colin PattisonCharles River, Tranent, UK, Johanne BouchardCiToxLAB, Evreux cedex, France, Lee GoodwinCovance Laboratories, Harrogate, UK, Rafiq IslamEMD Millipore, St Charles, MI, USA, Rudi SegersEurofins, Breda, The Netherlands, Zhongping (John) LinFrontage Laboratories, Malvern, PN, USA, Jim HillierGen-Probe Life Sciences, Livingston, UK, Wei Garofolo* Author for correspondenceGlobal CRO Council (GCC), 15 Sunview Dr, Toronto, Ontario, L4H 1Y3, Canada. ; www.global-cro-council.org, Dieter ZimmerHarlan Laboratories, Itingen, Switzerland, Lois FolgueraHarlan Laboratories, Santa Perpetua de Mogoda, Spain, Thomas ZimmermannHarlan Laboratories, Santa Perpetua de Mogoda, Spain, Maria PawulaHuntingdon Life Sciences, Huntingdon, UK, Marc MoussallieHuntingdon Life Sciences, Somerset, NJ, USA, Leonardo de Souza TeixeiraICF, Goiania, Brasil, Thais RochaICF, Goiania, Brasil, Daniel TangICON APAC, Shanghai, China, Paula JardieuICON Development Solutions, Whitesboro, NY, USA, James TruogJCL BIoassay USA, Hoffman Estates, Illinois, USA, Jenny LinJCL BIoassay USA, Hoffman Estates, Illinois, USA, Richard LundbergMEDTOX Laboratories, St Paul, MN, USA, Chris CoxMillipore BioPharma Services, Abingdon, UK, Alan BreauMPI Research, Mattawan, MI, USA, Chiara BigognoNiKem Research, Baranzate, Italy, Dick SchoutsenNOTOX, Hertogenbosch, The Netherlands, Carmen DilgerNuvisan GmbH, Neu-Ulm, Germany, Mohammed BouhajibPharma Medica Research, Toronto, Ontario, Canada, Ann LevesquePharmaNet Canada, Quebec City, Quebec, Canada, Sofi Gagnon-CarignanPharmaNet Canada, Quebec City, Quebec, Canada, Robert NicholsonPPD, Virginia, USA, Rand JenkinsPPD, Virginia, USA, Ming Hung LinProtech Pharmaservices, Taipei City, Taiwan, Shane KarnikPyxant Labs, Colorado Springs, CO, USA, Theo De BoerQPS Netherlands BV, Groningen, The Netherlands, Richard HoughtonQuotient Bioresearch, Fordham, UK, Rachel GreenQuotient Bioresearch, Fordham, UK, William DeMaioRicerca Biosciences, Concord, OH, USA, Romuald SableSGS, Wavre, Belgium, Kirk SmithSmithers Pharma Services, Wareham, MA, USA, Christoph SiethoffSwiss BioQuant AG, Reinach, Switzerland, Laura CojocaruTandem Labs, West Trenton, NJ, USA, Mike AllenTandem Labs, Durham, NC, USA, Tammy HarterUnilabs York Bioanalytical Solutions, York, UK, Saadya FatmiWarnex Bioanalytical, Laval, Quebec, Canada, Farhad SayyarpourWIL Research Company, Ashland, OH, USA, Michele MaloneWorldwide Clinical Trials, Austin, TX, USA, Stuart BestXceleron, York, UK & Xinping FangXenoBiotic Laboratories, Plainsboro, NJ, USAPublished Online:28 Mar 2012https://doi.org/10.4155/bio.12.18AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit Keywords: European Medicines AgencyEMAFDAmatrix selectionreference standardsstabilitySenior representatives of GCC member companies have thoroughly evaluated and discussed the new European Medicines Agency (EMA; July 2011 [1]) Guideline on Bioanalytical Method Validation (BMV), during the 4th GCC (23 October 2011, Washington DC, USA) and 5th GCC (14 November 2011, Barcelona, Spain) Closed Forums. These North American and European events provided a unique opportunity for CRO leaders to openly share opinions and perspectives and to agree on unified bioanalytical recommendations specifically in relation with the new EMA guideline.The Global CRO Council for Bioanalysis (GCC) [101] is a global independent group of many Contract Research Organization (CRO) leaders. The GCC was formed in September 2010 to meet regularly and discuss bioanalytical issues and regulatory challenges, many of them unique to the outsourcing industry. The outcome of the discussions held as part of these GCC closed forum meetings are shared with the global bioanalytical community via pertinent publications [2–6] and appropriate conference presentations.Prior to the issue of this new guideline, the November 2009 Draft EMA Guideline on BMV defined the proposed guideline and criteria of the EMA on validation of bioanalytical methods and their application in the analysis of study samples from animal and human studies [7].The EMA Guideline states that the validation of the bioanalytical methods to be used on non-clinical pharmacotoxicological studies to be submitted in a marketing authorization application should be performed following the principles of Good Laboratory Practice (GLP) with any aspects of method validation not performed according to GLP clearly identified and their potential impact on the validation status of the method indicated. Additionally, the EMA Guideline states that the validation of bioanalytical methods and the analysis of study samples for clinical trials in humans should be performed following the principles of Good Clinical Practice (GCP). This is in contrast to the 2001 FDA Guidance [8] and will be discussed at a future GCC closed meeting.The GCC recommendations on the interpretation of the finalized EMA Guideline on BMV were grouped into the following topics: ▪ Reference Standards: certificates of analysis and internal standards (IS);▪ Calibration curve and accuracy;▪ IS stability, processed sample stability and matrix effect;▪ Analysis of study samples and incurred sample reanalysis (ISR);▪ Ligand binding assay (LBA)-specific issues.Some of the major differences between the finalized EMA Guideline, the 2001 FDA guidance and the Crystal City III White Paper [9] were also presented in a tabular format during the GCC Closed Forums (Table 1).Reference standards: certificates of analysis & ISThe new EMA BMV Guideline includes requirements regarding the reference standards used during method validation and analysis of study samples. Section 4.1 of the EMA Guideline states that "A certificate of analysis is required to ensure purity and provide information on storage conditions, expiration date and batch number of the reference standard."The content and quality of certificates of analysis varies depending on the supplier and, in some cases, certain information, for example expiry or re-test date, is missing. The supplier may sometimes be contacted to obtain the missing information and when this information is not provided some CROs have a general standard operating procedure (SOP) to assign expiry dates to reference standards.The GCC recommendation for certificates of analysis is shown in Box 1.Section 4.1 of the EMA Guideline also states that "The use of certified standards is not needed for the IS, as long as the suitability for use is demonstrated." The majority of CROs are currently performing tests to demonstrate the suitability of the IS. In general, the interference at the analyte retention time from the presence of the IS is tested in analytical solutions (via the interference check) and in matrix (via selectivity experiments). The suitability of the IS is demonstrated in method development and overall validation results. Additionally, the absence of interference from the IS is usually demonstrated in every analytical batch via blank samples containing only IS (zero sample).The GCC recommendation for IS is shown in Box 2.Calibration curve & accuracyRegarding the calibration curve requirements, section 4.1.4 of the EMA Guideline contains a statement that was not present in the draft version of the guideline: "In case all replicates of the LLOQ or the ULOQ calibration standard are rejected then the batch should be rejected from the validation, the possible source of the failure be determined and the method revised (if necessary). If the next validation batch also fails, then the method should be revised before restarting validation." It is a common approach to investigate the cause of the failures before continuing the validation. In general, failures with no assignable cause would prompt the method to go back to the development stage to be re-worked. Different approaches can be followed depending on whether or not there was an assignable cause for calibration standard failure at a limit(s) of quantitation, and dependent on the assay type. Remedial action for assignable causes and investigation of unassignable causes were preferred.The GCC recommendation for calibration curve is shown in Box 3.In the Accuracy section of the EMA Guideline (section 4.1.5), there is a requirement concerning quality control (QC) samples preparation that was also not present in the draft version of the guideline: "The QC samples should be spiked independently from the calibration standards, using separately prepared stock solutions, unless the nominal concentration(s) of the stock solutions have been established." The most common approach in the industry seems to be the use of two separate stocks, unless there is limited compound, and to not determine the nominal concentrations. However, the comparison (correlation) of stock solutions prepared from different compound weighings, with an acceptable percent difference of, for example, 5% between them, is still used.The GCC recommendation for accuracy is shown in Box 4.IS stability, processed sample stability & matrix effectSection 4.1.9 of the EMA Guideline mentions the following about stability of the IS: "It is not needed to study the stability of stable-isotope labeled internal standards if it is demonstrated that no isotope exchange reactions occur under the same conditions as the stability of the analyte was demonstrated." It is commonly demonstrated that no isotope exchange reaction occurs and the amount of unlabeled compound is evaluated either in method development, validation and/or with any sample analysis batch. The isotopic purity is not necessarily demonstrated but as previously mentioned, interference coming from the IS is generally evaluated by spiking blank matrix with the IS and evaluating any interfering peak at the analyte's retention time against the LLOQ (specificity) or by injecting replicates of an analytical solution of the IS compared to replicates of the LLOQ (interference check), or a mixture of both tests over the method development/validation and/or sample analysis. When using a new batch of IS, these tests are repeated, generally using the zero sample (i.e., analyte-free sample spiked with IS only) within every analytical batch.The GCC recommendation for IS stability is shown in Box 5.One of the subjects brought forth in the EMA Guideline is the stability of the analyte and the IS in extracts. The EMA Guideline, section 4.1, requires "…stability of the analyte(s) and of the internal standard … in extracts under the entire period of storage and processing conditions." Post-preparative sample stability following initial injection is normally evaluated, thus effectively assessing the stability of extracts containing both the analyte and the IS.Furthermore, section 4.1.9. of the EMA Guideline states: "…stability of processed sample … on-instrument/autosampler stability of the processed sample…" The re-injection reproducibility is performed by re-injecting a whole batch after a period of storage. The processed (post-preparative) sample stability is performed by re-injecting QC samples along with a freshly prepared calibration curve. However, the processed sample stability approach, as described above, arguably demonstrates both extraction stability and re-injection reproducibility.The GCC recommendation for processed sample stability is shown in Box 6.The matrix effect is also a hot topic since the EMA Guideline, section 4.1.8, states: "…the matrix factor (MF) should be calculated for each lot of matrix…" Presently, the MF is used by some in the industry to evaluate the matrix effect. For others, the matrix effect is evaluated by calculating the mean precision and accuracy of plasma concentration values in different matrix lots and by evaluating the suppression/enhancement by comparing responses in the presence and absence of matrix. The use of the matrix factor may not always be applicable, for example because of pure solutions adsorption/solubility issues.The GCC recommendation for matrix effect is shown in Box 7.In the same EMA Guideline section, the matrix effect is further discussed as follows: "If a formulation for injection to be administered to the subjects or animals contains excipients known to be responsible for matrix effects, for instance polyethylene glycol or polysorbate, the matrix effects should be studied with matrix containing these excipients … The matrix used for this evaluation should be obtained from subjects or animals administered the excipient, unless it has been demonstrated that the excipient is not metabolized or transformed in vivo." In general, this kind of investigation is not performed and/or it is considered unnecessary, as the impact, if any, is mostly due to the presence of the excipient, not its in vivo metabolism or transformation. Matrix spiked with the excipient is used to investigate potentially problematic excipients since the use of excipient-dosed subjects is difficult to obtain in practice, especially in the validation stage. Furthermore, as different formulations could be administered over several studies, it would increase the type of matrices to be assessed for matrix effect.The GCC recommendation when in presence of excipients known to affect matrix effect is shown in Box 8.The EMA guideline also recommends that when samples from special populations, for example renally or hepatically impaired populations, are to be analyzed then matrix effects using matrix from such populations should be investigated.The GCC recommendation for matrix obtained from special populations is shown in Box 9.Analysis of study samples & ISRThe analysis of study samples is described in detail in section 5 of the EMA Guideline. One of the requirements of this section that needs further interpretation was the following: "Before start of the analysis of the study samples the performance of the bioanalytical method should have been verified." It may not be clear how the analytical method should be verified; what period of time needs to elapse before verification is required; if system suitability is considered adequate; or if a "commissioning batch" should be required. Indeed, this section can be interpreted differently and can refer to system suitability; to a "test batch" containing calibration standards and a varying number of replicates of QC samples (typically informal and not reported); or to a partial re-validation for the re-evaluation of precision and accuracy. Also the period of time could vary widely depending on the interpretation, for example in each batch for system suitability, and from 2 weeks to 2 years for partial re-validation.The GCC recommendation on analysis of study samples is shown in Box 10.In section 5.1 of the EMA Guideline it is mentioned that: "For bioequivalence studies … QC samples should be divided over the run in such a way that the accuracy and precision of the whole run is ensured." In general, half of the QC samples at the front and the other half at the end of the run with all study samples bracketed by the QC samples cannot be considered to be 'divided over the run'. Indeed, QC samples split front and back of an analytical run cannot be adequate to ensure the accuracy and precision of the whole run. Randomization throughout the analytical run can be considered optimal, though bracketing with QC samples may be also acceptable, for small batches or to minimize carryover.The GCC recommendation on placement of calibration standards and QC samples is shown in Box 11.Regarding the run acceptance criteria described in section 5.2 of the EMA Guideline, it is mentioned that "In case the overall mean accuracy and precision exceeds 15%, this should lead to additional investigations justifying this deviation. In the case of bioequivalence trials it may result in the rejection of the data." During discussions on this section it was pointed out that this is 'study' and not 'run' acceptance criteria and that run acceptance criteria should be adequate.Meeting these criteria for bioequivalence studies (BE) should be unproblematic as the method used should be robust and bioequivalence studies typically involve a large number of analytical runs. A variety of statistical tests to determine statistical outliers can be used, for example Grubbs or Dixons Q tests, and then present overall mean and accuracy, including and excluding these statistical outliers. The section 5.2 of the EMA Guideline could be also applied to ligand-binding assays.The GCC recommendation on acceptance criteria is shown in Box 12.It is stated in section 5.5 of the EMA Guideline that "Chromatogram integration and re-integration should be described in a SOP." Having a SOP on chromatogram integration is a general industry practice. However, the contents of the SOP can be very variable, specifically covering how to integrate, how to justify re-integration and how to ensure traceability of integration. An integration SOP should cover all three areas.The GCC recommendation on chromatogram integration is shown in Box 13.Section 6 of the EMA Guideline presents specific information and criteria on ISR. In that section, it is mentioned that "Large differences between results may indicate analytical issues and should be investigated." In evaluating this guideline, high outliers and trends should be taken into consideration, as well as when an investigation and/or reanalysis should be performed. For instance, there is no agreement in the industry on what to consider high outliers since values used may vary between bias greater than 30% and 100% from the average.It seems to be a common approach in the industry, if the ISR acceptance criteria are met, not to investigate any high outliers that did not form a trend. Trends were considered to be a series of results that show commonality, specifically in terms of runs, populations (including gender), subjects or animals, timepoints, dilutions, consistently positive or negative bias (even if ISR acceptance criteria are met), and these should be investigated.The GCC recommendation on ISR is shown in Box 14.LBA: reference standards, selectivity, matrix selection, parallelism & reagentsComparatively to the draft EMA Guideline, the requirements specific to LBA were considerably expanded in the final version of the EMA Guideline, where this topic appears in section 7 with several sub-sections.Regarding reference standards, section 7.1.1.1 of the guideline states "It is strongly recommended that the batch of the reference standard used for the preparation of the calibration standards and QC samples is the same as that used for dosing in the non-clinical and clinical studies." It seems this recommendation is already widely followed in the industry.This is also discussed in the EMA Guideline on the Clinical Investigation of the Pharmacokinetics of Therapeutic Proteins [10], "Contrary to conventional molecules, a pure reference material that can serve as a calibration standard is either difficult or sometimes impossible to obtain for this class of compounds. Therefore extreme care should be taken in order to ensure that the reference material used in the different analytical calibration processes is representative of the material used in clinical trials, including clinical pharmacokinetics."The GCC recommendation on reference standards is shown in Box 15.The EMA Guideline requires in section 7.1.1.3 that "Selectivity should be evaluated at the low end of an assay where problems occur in most cases. It may be prudent also to evaluate the selectivity at higher analyte concentrations. In cases where interference is concentration dependent, it is essential to determine the minimum concentrations where interference occurs." It seems that this is routinely assessed at both low and high concentrations or in some cases typically assessed at low concentrations, but additionally assessed at high concentrations when a client requests it. Furthermore, performing a hook effect (prozone effect) experiment would cover the higher concentration.The GCC recommendation on matrix effect is shown in Box 16.For matrix selection, the EMA Guideline mentions the possibility to use extracted or alternative matrices in some particular cases. Section 7.1.1.5 of the guideline states the following to that effect: "QC samples should be prepared in the actual sample matrix and the accuracy should be calculated to demonstrate the absence of matrix effect." It is a common approach to prepare QC samples in the actual matrix. This section may be interpreted in different ways, for example including typical matrix effect assessments as well as assessments for use of surrogate matrices.Moreover, discussions on the interpretation of this section may include when it is acceptable to use surrogate matrices and, as stripped matrices can be variable, whether or not a number of stripped matrices be investigated to ensure conformity against the QC samples prepared in actual sample matrix.The GCC recommendation on matrix selection is shown in Box 17.Section 7.1.1.10 of the EMA Guideline discusses parallelism evaluation in LBA: "If study samples are available, parallelism between the calibration standard curve and serially diluted study samples should be assessed (…)" This test is already commonly performed in the industry by assessing parallelism in incurred samples. However, in some cases parallelism assessments are performed only using a spiked sample due to the unavailability of appropriate incurred samples.Without any doubt, incurred samples are more appropriate for parallelism investigations due to the presence of metabolites, excipients and so on. since the levels of these compounds would vary depending on the timepoint selected. If parallelism in incurred samples is performed during a sample analysis study, rather than a validation study, the subsequent data should be reported in the corresponding bioanalytical report.The GCC recommendation on parallelism is shown in Box 18.For the evaluation of the stability of study samples at each temperature at which they will be stored, the EMA Guideline section 7.1.1.11 (part of the ligand binding sections) mentioned that "A bracketing approach may be considered". This approach is also mentioned in the stability section 4.1.9. However, in that same section, the following can be read: "For small molecules it is considered acceptable to apply a bracketing approach … For large molecules (such as peptides and proteins) stability should be studied at each temperature at which study samples will be stored." Although a bracketing approach is considered acceptable, presently it is common practice for LBA that the actual storage temperature is assessed: stability is performed at, or as close as possible, to the storage temperature of the incurred samples.A further consideration is that whereas for small molecules proven stability at 20°C would typically indicate acceptability for storage at -80°C, for large molecules this would not be the case as lower temperatures may cause stability issues, and vice versa.The GCC recommendation on stability is shown in Box 19.Section 7.1.1.12 of the EMA Guideline is related to reagents used in LBA: "…accordingly, when changing reagent batches during validation or sample analysis the analytical performance of the analytical method must be verified to ensure that it is not altered compared with the original or previous batch." It is necessary to clarify up front what are critical reagents and, for specific assays, which of the reagents are considered to be critical.The GCC recommendation on reagents is shown in Box 20.Future perspectiveThe GCC will continue to provide recommendations on hot topics in bioanalysis of global interest and expand its membership by coordinating its activities with the regional and international meetings held by the pharmaceutical industry. Some suggested future topics for discussion include how to perform matrix effect experiments with respect to hemolysis, hyperlipidemia and excipients, and the GLP conduct of validations. The next GCC Closed Forum is scheduled at the 6th Workshop on Recent Issues in Bioanalysis (6th WRIB) in San Antonio, Texas, USA in March 2012. Please contact the GCC for the exact date and time of the aforementioned meeting, and for all membership information.Table 1. Some major differences between European Medicines Agency and FDA guidance.SubjectEuropean Medicines Agency Guideline (2011)US FDA Guidance (2001)US FDA Crystal City III (2007)RFSCofA not required for ISDiscusses isotopic purity expectations of labeled RFS for ISNo reference to expectations regarding labeled RFSNo mention of labeled IS. CofA or purity of IS not always necessarySelectivity criteriaResponse of interference is <20% of the LLOQ for analyte and <5% for ISNo specific criteriaCarryover section; the analyte response at the LLOQ should be at least 5× blank responseSelectivity (co-medications)Discusses the investigation of interference from possible co-administered medicationsNot discussedNot discussedSelectivity (metabolites)Includes test for possible metabolic back-conversionNo specific metabolite tests recommendedCharacterization of metabolites should proceed using a flexible, 'tiered' approachLLOQ requirementNot higher than 5% of Cmax in BE studiesNo mention of CmaxNot discussedCalibration curveCalibrants stats of all acceptable runs reported; QCs stats of all runs reported; truncated curves not accepted in validationReporting of runs and truncated curves not discussedCalibrants and QCs stats of all runs reported; truncated curves not discussedRecoveryNot discussedRequiredNot discussedIncurred sample reanalysisRequired; number and criteria provided (10% of first 1000 samples, 5% of the rest)Not formally discussed in guidance, but enforced by the US FDARecommendedCarryoverCarryover in the blank following the high standard not greater than 20% of the LLOQ and 5% for the ISNot discussedLLOQ response at least 5× the response due to blank matrix. Randomization of samples to avoidMatrix effectIncludes specifics of evaluation and criteria (matrix factor)General statement that it should be investigatedIncludes specifics of evaluation and criteria (matrix factor)StabilityProvides criteria of 15% from nominalIn case of a multianalyte BE study, attention should be paid to stability of the analytes in the matrix containing all analytesNo specific criteria; no mention of multianalyte studiesNo specific criteria; no mention of multianalyte studiesPharmacokinetic outliersNot recommendedAllowedNot discussedDilution integrityIncludes specifics of evaluation and criteria (validated by dilution factors)Should be demonstrated by accuracy and precision parameters in the validationNot discussedBE: Bioequivalence studies; IS: Internal standard; RFS: Reference standards.Box 1. The GCC recommendation for certificates of analysis.▪ A standard format and content for certificates of analysis would be beneficial. However, it is recognized that establishing this standard will not happen prior to the effective date of the Guideline. The minimum information requested by the EMA Guideline reflects the minimum information that the industry would like to receive for reference standards. Therefore, the GCC strongly urges the suppliers of reference standards to provide all of the requested information and recommends that information not supplied is discussed in the bioanalytical report, including an assessment of the potential impact.Box 2. The GCC recommendation for IS.▪ It is recommended to continue performing these tests. Furthermore, the GCC recommends that during method development the threshold of intern
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