ࡱ> 35,-./012%` bjbj"x"x f@@!mmmmmmm$m???P^?<@m{ZHZH(HHHII Iȓʓʓʓʓʓʓ$WhP-mMIIMMmmHH4$ZZZMmHmHȓZMȓZZw$mm ~HNH pBb?hQJ{,$?<{4{SdX ~m ~IJ|tZKdlK>IIIZ^III{MMMMmmmDNmmmmmmmmmmmm May 17th, 2010 Submission of comments on 'Guideline on Validation of Bioanalytical Methods' (EMA/CHMP/EWP/192217/2009) Comments from: EQAC Name of organisation or individualEuropean Quality Assurance Confederation Please note that these comments and the identity of the sender will be published unless a specific justified objection is received. When completed, this form should be sent to the European Medicines Agency electronically, in Word format (not PDF). General comments Stakeholder number (To be completed by the Agency)General comment (if any)Outcome (if applicable) (To be completed by the Agency)Comment: This guideline does not only address aspects concerning validation, but also of routine analytical run acceptance see section 5. The title of the document is therefore not totally adapted to the content. Proposed change (if any): Change to e.g. Guideline on validation and acceptance criteria for bio-analytical methods and the analysis of study samples Comment: Method validation is different between physico-chemical analysis and bio-assays. We therefore suggest to have either a specific guideline for bio-assays or a specific paragraph no sensitivity, carry-over, LOQ, calibration curves, accuracy, precision, dilution integrity, matrix effects and stability. Comment: The present scope of the Guideline is huge due to the fact that: - it covers both analytical method validation and sample analysis in biological matrices - the analyte could be anything from an organic synthetic molecule over a protein and a nucleic acid to a living microorganism - the application of the analyses mentioned spans over a wide range where toxicokinetcs, pharmacokinetics and bioequivalence are mentioned - both GLP and GCP are mentioned as legal authority referentials, and the reporting according to GLP routines is further detailed, although GLP compliance cannot be claimed. Comment: It is felt that the Guideline needs a thorough review and update either: a) to fully cover this complex field and more clearly describe what the EMA expects in the regulatory applications from industry in relation to the existing OECD GLP requirements b) or to introduce more clear limitations concerning the scope of the Guideline. Comment: While looking through the guideline it is also noticed that some terms are not consistently used throughout the document. To avoid confusion we recommend to harmonize within the document and to include, as necessary the definition of terms. This concerns, for example: protocol vs. plan (line 388 study plan; line 397/398 analytical study protocol vs. study plan, line 439 study protocol, line 491 protocol, line 496 analytical protocol) in compliance vs. in accordance (line 69/70, 489, 492) What do you mean with these terms? Please define it inside this guideline for clarification.  Specific comments on text Line number(s) of the relevant text (e.g. Lines 20-23)Stakeholder number (To be completed by the Agency)Comment and rationale; proposed changes (If changes to the wording are suggested, they should be highlighted using 'track changes')Outcome (To be completed by the Agency)Introduction/ ScopeComment: This guideline only applies to the Human Pharmaceutical Industry. Can you confirm that it is not applicable to the validation of analytical methods for residues detection. Will this guideline also be applicable to veterinary medicines? Comment: Some sections should be harmonized with FDA Guidance for Industry Bioanalytical Method Validation, May 2001 and Bioequivalence Guideline from EMEA, January 2009. Comment: This Guideline discusses no aspects of recovery. If recovery is omitted on purpose, clarify that it is EMEAs opinion that this is not needed even though it is a requirement in the FDA guidance.39-43Comments: Executive summary do not cover whole content. Proposed change: Adjust Executive Summary to content of Guideline e.g. the guideline is about bioanalysis and it is more appropriate to use bioanalytical instead of pharmacokinetic in line 41. Comments: Please delete sentence "The guideline focuses on.." since we at present do not have different validation guidelines for a method depending on the purpose of the study. 45-54Comments: A clearer definition of matrices in this section would be useful (serum, plasma, urine, saliva and other solid tissues) and would allow to show the difference with the draft guideline VICH topic GL49. 55-62 59-60Comments: Define more clearly what the scope is. Is it only covering active ingredients and drug products in biological matrices or something else? Here is mentioned only toxico-kinetics and clinical trials, which is limiting the scope. Comment: Radio-labelled analysis methods should be completely out of scope (therefore this sentence should be deleted from this section). Comments: It is stated that ...this guideline will describe when ... cross validation may represent an appropriate alternative ... [to full]... validation .... How should one interpret section 4.3 in regards to the statement above? Proposed change (if any): Clarify, either in this paragraph or in 4.3 when cross validation is an appropriate alternative to full validation. 64Comments: A reference (4) is referred to after general principles in this line however no references are cited in the document. If this guideline is supposed to also cover validations and bio-analyses in the veterinary medicine domain a cross reference to the EMEA VICH guidelines would be appropriate. Proposed change (if any): Add references. 69- 70The meaning of in accordance with the principles of GLP is not clear; The sentence applies to both validation of methods and analysis of study samples. To be consistent with current industry and US FDA approaches, it is preferred that method validation studies are not required to be compliant with GLP. The US FDA has stated that the GLPs do not apply to validation trials to confirm the analytical methods. It is considered that the section on GLP (Section 3 Legal basis) raises more questions than it answers and appears self- contradictory. The guideline indicates that validation of methods should be conducted in accordance with the principles of GLP. There are two issues arising from this; firstly that there has been no specific requirement up to now for method validation studies to be conducted in compliance with GLP even for pre-clinical work and secondly as the following sentence states GLP would not be appropriate for clinical studies. In the UK any claim for GLP compliance for clinical samples would not be supported and could therefore considered to be false. The guideline indicates that the analysis of study samples should be performed in accordance with GLP principles. For pre-clinical studies it would, however, depend upon the intended purpose of the study as to whether a claim of compliance is to be made. Section 4: In this section the validation is described. The requirement for a validation report is missing. As written this report is mentioned in section 7 and it is confusing as previous sections describes the analytical runs. Consequently there can be no reference to compliance with GLP in this report. Proposed change: To modify sentence to clearly define that method validations should use as a basis the applicable principles of GLP, but compliance to GLP is not a requirement. L 85 / 4.1 Comment: ..validation should be performed using the same anticoagulant as for the study samples. why use the word should if the same anticoagulant has to be used? Does this mean that a new complete validation must be performed if the anticoagulant is changed, or is a partial validation sufficient? The same question arises for the change in matrix. Proposed change: Validation has to be performed using the same anticoagulant as for the study samples. Comments: Clarify the EMEA standpoint regarding K2-EDTA/K3-EDTA should be considered same or different anticoagulant. Proposed change (if any): Include an example in the sentence. 80Comment: Indicate that this section 4.1 is decdicated to physico-chemical assays and that there is a specific section or guideline for ligand-binding assays.86Comment: A full validation is required for a change of species. What is the scientific rationale justifying to validate all the parameters required for a full validation. What would be the utility to perform stability of the stock and working solutions? We believe that a full validation is not necessary foreach individual species: e.g.stability of extracted samples, influence of haemolysis, dilution effect, etc.: as far as it has been established in one species, there isvery reduced chance that it would differ between species. Even the matrix variability, which is critical for human species, has less chance to beimpacted in preclinical species as the animals come from same strains and have similar food. Proposed change: The compromise would be to perform a partial validation focused on the differences linked to the species as it is described in the report of the conference Bio-analytical Method Validation a revisit with a decade of progress Workshop held in Arlington, VA, 2000. The partial validation would include selectivity, long-term stability, within-run accuracy and precision. 88Comment: When will it be possible to use synthetic matrixes (such as synthetic urine)? What about matrix to use for endogenous compound? 91Comment: There is no more need to evaluate the recovery? 93-94Comment: Should the stability of stock and working solutions be included in each full validation or can this be information be taken from other validations using these same materials which would have no effect on these parameters? 95-98Comments: The use of examples can be improved with e.g. microorganisms used as active ingredients. Do analytes also include provoked responses like antibodies or other biomarkers? 101-102Comment: An internal standard is normally used. Keep the possibility to not use an Internal Standard. Do the standards also include e.g. microorganisms or nucleic acids? 108Comment: Suitability of the reference standard should be scientifically justified. Does that only concern the fact to have a Certificate of Analysis or is additional scientific rationale expected? It is said The use of certified standards is not needed for IS, as long as, but in line 114 stated that it is essential that the labelled standard is of the highest isotope purity The text should clarify if there is the necessity for a certificate of analysis for labelled standards used as IS or identify that the text is speaking about standards of reference only. 114-117Comments: A wide range of types analytes are used by industry from simple organic molecules over proteins and nucleic acids to microorganisms. Proposed change (if any): Due to the large number of different analytes the examples given should be reviewed and improved. 123Comment: What about the selectivity regarding endogenous compounds? Could this test not be done? The % of the response of interfering components does not seem to be established for IS. Given that there is an acceptability range for the analyteis it necessary to apply another for the internal standard? If yes, what should this be and on what basis should it be chosen? 124Comment: The metabolites investigation may present technical difficulties if the metabolites are not available in the shops (feasibility to have study samples before the validation). Same for the back-conversion. 124-139Comment: A precision could be included to identify which metabolites are to be followed and how (e.g. principle metabolite, % circulating level, list of potentially unstable metabolites etc.). It may also be necessary to investigate: the extent of any interference caused by metabolites of the drug(s), interference from degradation products, etc. The text should explain the procedure to investigate the interferences. On the other hand, the text explains that the possibility of back-conversion of a metabolite into parent analyte should be evaluated but does not explain how and when (in pre-clinical and/or clinical part?) 143-144Comments: The text states If it appears that carry-over is unavoidable, specific measures should be considered. The text should define what would be these specific measures. In this section there are no acceptability levels. Is it not possible to attach these to other acceptance limits such as those for selectivity? 155Comments: "response of the instrument" is mentioned. however, analytes can be evaluated by counting distribution in situ in tissues. Proposed change (if any): Review wordings to include other possibilities of quantification. 154-181Comments: It should also be allowed to use 2 calibration curves (one before and one after the samples; it is not clear from the text if this is allowed). In addition, it should be allowed to use calibration curves that are not freshly prepared as long as stability over the period of use is demonstrated. 172-173 175-178 Comments: At least 3 calibration curves should be evaluated. In case a calibration standard does not comply with During the analysis of study samples it is accepted the truncation of calibration curve due to rejection of LLOQ or ULOQ calibration standard. If this idea is maintained, this possibility should be reflected also during the validation. Proposed change (if any): At least 3 calibration curves with LLOQ and ULOQ should be evaluated 175Comment: At least 75% of the calibration curves standards with a minimum of six, must fulfil this criterion. Does that mean that the curve is composed of 6 points and 4 are acceptable or do we need 6 acceptable points so at least 8 in the curve? 176Comment: At least four out of six non-zero standards should meet the above criteria, including the LLOQ and the calibration standard at the highest concentration. Excluding the standards should not change the model used (FDA 2001). 176Comment: It would be useful to ensure coherence with the FDA requirements. 179-181Comment: Why should we use freshly prepared calibration curve if stability is demonstratedin a frozen matrix?If stability in a frozen matrix has been established, the validation could thenbe conductedusing frozen calibration curves so this section should be modified in that sense. What does freshly prepared calibration curve mean? Is it necessary to prepare the calibration samples freshly each day? If calibration samples are prepared within one week before the validation study and then stored in single use aliquots: Is it necessary to compare these samples once with a freshly (not stored) set of calibration samples or QC samples even if stability has previously been demonstrated? 182Comment: In the section 4.1.5the terms true concentration or nominal value are used. It would be useful to harmonize these terms for the whole document and to put in a definition for nominal value in the definitions section (L545).187-190Comment: The levels of QCs during validation are defined 3 x LLOQ, 50% and 75% calibration range. Calibration curves with LC/MS methods are often populated asymmetrically, i.e. more calibration samples in the low range, less in the high concentration range. So the mid QC is often not placed at 50% calibration range, but around the middle calibration samples, i.e. if you use 8 calibration samples the mid QC is often between the 4th or 5th standard. When one rejects calibration points other than for analytical reasons, must these values be included in the cumulated statistical analysis of the standard calibration curve? If yes, how should one manage the out of specification results which could have brought about a variation in the statistical values thus calculated? 196Comment:It is recommended to demonstrate accuracy of QC samples over at least one of the runs with a size equivalent to a prospective analytical run. What is the usefulness? The bench-top stability and the auto-sampler stability can cover this point. 199- 221Comment: The document does not specify whether the QCs prepared for these parameters come from the same prepared batch or whether it is necessary to prepare different QC batches for the between run accuracy and precision. 209-211Comment: What kind of statistical tests should we use? If a value is statically determined outlier, could the value be excluded from the test and the test validated? Concerning these outliers in the between run accuracy, what should be done with the individual values considered as outliers and what values should be used in the statistical calculations? The explanations in this paragraph are not very clear. The text states Reported method validation data.should include all outliers; however, calculations ..excluding values .should additionally be reported Is it necessary to include both calculations? 215Proposed Change: Give examples of standard statistical methods for the calculation of precision. 224-227Comment: Sample shall be diluted with blank matrix, this implies that it is the same species as for the samples. In case of limited matrix (mice, cyno) it might be useful to use another appropriate matrix e.g. human for dilution, given that it is demonstrated that there is no interference. Proposed change (if any): dilution of this sample with an appropriate matrix .. Comment: Is it necessary to perform this verification during the validation of the method, or can it not be done as a supplementary phase to the validation if and when it is seen during the routine analyses? This would allow the verification of the dilution parameters to be better chosen with respect to the dilutions performed. 226Comment: Post extraction dilution remains feasible? 229Comment: Should we test in range dilution during validation to be used when insufficient sample volume is provided? If yes, what concentration levels should be tested? 226 - 229Comment: This paragraph does not identify at what concentration (one or several) these tests must be performed; mid range, LLOQ, ULOQ, Since you discuss the precision of dilution should these tests be performed in one single run or repeated several times. That is intra or inter run precision. 231 232Comment: What is the consequence if the CV is more than 15%? Does this render the method invalid? The matrix effect only applies to the mass spectrometric methods? It is not required for classic HPLC chromatography? It is unclear if we should use 6 lots of each of normal, haemolysed and hyperlipidaemic matrix. Could you indicate what degree of haemolysed and hyperlipidaemic matrix should be tested. How should this be documented? The use of 6 lots of matrix should only be used if possible. Should matrix effect be tested on hyperlipidaemic plasma for any validation or could it be related to some study only? In this case, how should we document this and what kind of study is concerned? Should this be tested in every validation study or is it sufficient to demonstrate it only if such samples are expected/observed? Could the test for special populations be implemented during the clinical/toxic study by spiking pre-dose study samples. Perhaps this requirement should be put in as an option. Proposed Change: Replace Matrix effects should be investigated when using mass spectrometric methods using at least 6 lots of normal, haemolysed and hyperlipidaemic matrices. Comment: The limitations concerning the renal and hepatic deficiency population could also apply to the cases of the different matrices (hyperlipaemic etc., In fact this restriction if necessary in place of if necessary could include the whole paragraphe and not just the end of the sentence.232Comment: We areof the opinion, that matrix effects in haemolysed and/or hyperlipidaemic matrix is problematical: What degree of haemolysis or hyperlipidaemic and how to document this? 233Comment: This is fairly easy for humans but it should be noted that it is not be required for other species. Proposed Change: This requirement should therefore be limited to clinical studies. 234-237Comment: There is now no discussion on recovery studies although this section comes close to it for MS. The difference however is that there is no extraction tested as in recovery testing. 231, 239, 250Comment: The term lots of matrix and batches of matrix could be harmonized which would help in preventing confusion in the interpretation of this guideline. 252Comment: Matrix effect with the excipient: as the excipient is known during the drug development, this will be performed as an additional part of the validation? 252-256Comment: This requirement should be limited to clinical studies. The type of excipient used during the pre-clinical phase is very variable and each change would require a complement to the validation. 257 257 258-269 269 257-299Comments: The stability of the blood after the collection and the separation of the plasma/serum should not be considered part of this method validation but part of a more global method development. Should a criteria of stability be taken into account for the analysis of the time zero with respect to the nominal value? Please could you add some precision. Concerning the deviation with time, should this be determined with respect to the initial value or with respect to the nominal value? Concerning the deviation for the stabilities, should this be determined with respect to each of the initial values or with respect to the mean value calculated at each level of concentration? Section 4.1.9 Stability; there are some contradictions in this section (initial versus nominal concentrations: line 260 versus line 269) and unclear. In addition, we dont understand why would we need to study the stability of the IS in the matrix. We propose that blood stability should be evaluated versus reference, instead of versus nominal. For assessment of stability of the analyte and IS in the studies matrix, are you expecting the results of QC samples to be compared to nominal concentrations, or should the results for the 'after storage' QC samples also be compared to the results for the freshly prepared QC samples? Stability cannot be proven by literature data. Agreed! Is it possible to refer to stability studies obtained by another method or obtained at another site by the same method (e.g. at a CRO). Proposed Change: Replace Any deviation from the initial concentration that does occur must be within acceptable limits by Any deviation from the nominal concentration that does occur must be within the nominal limits.269Comment: The stability is evaluated using 3 replicates. The text states The deviation should be within 15% Is the deviation of 15% applicable to the individual or mean value? The deviation should be evaluated in terms of precision and accuracy. The CV value should not exceed 15% for the QC stability. For accuracy if the value should be within 15% of the nominal value, any grade of non-stability is assumed for analytes. Proposed change (if any): The CV value should not exceed 15% for the QC samples (precision). The mean accuracy value should be within 20% the nominal values for the QC samples. 270Comment: What are the acceptance criteria for the stability of the stock and working solutions? 270Comment: Regarding working solution stability test, what concentration levels should be tested? Should this stability test done if working solutions are prepared freshly everyday of use? 271Comment: Stability should be tested only if necessary since calibration curve, may be obtained from freshly prepared stock and working solutions. 278-279Comment: What is the difference between these two sentences? What does bench top really mean particularly with respect to duration? 288- 289 Comments: Freeze and thaw stability The samples should be completely thawed before they are refrozen Proposed change (if any): At each cycle, when completely thawed, samples should be frozen for at least 12 hours 293Comment: not acceptable to use study samples for evaluation of long term stability:We still believe this should be kept as an option when appropriate (e.g. known presence of a metabolite that can degrade back to the analytebut that is not available as reference to conduct stability studies); in that case, comparison would be based on initial concentration instead of nominal which is unknown. 294Comment: Evaluationof long term stab before study startwould be nice butwill beunrealistic in term of planning in most situations. What matters in the end is that the study results are covered with appropriate stability data and that the stability required to cover the period of the study is available before the finalization of the study report. The sentence lines 294-295 could be skipped or modified in this sense. 296-297Comment: Could you clarify Sufficient attention should be paid to the stability of the analyte in the sampled matrix directly after blood sampling of patients and further preparation before storage. Is it the evaluation of the stability in blood during sample preparation, like centrifugation? Regarding the stability between the sampling, the processing and the storage is there any proposal on ways of doing it? 295Comments: According to the draft guideline, long-term stability tests are recommended to be carried out BEFORE the start of the actual study? To avoid differences in approach, shouldn't it be defined in the guideline what periods of storage are considered to be classed as 'long-term' and should be performed before start of the study? Also we feel that this requirement be modified to identify that the long term stability be demonstrated for the period required and be available before the finalization of the report containing the results of the study. 300Comment: It would be useful if this section was more precise and identify the different types of validation for pre-clinical and clinical situations, for example: Typical bio-analytical method changes that fall into this category include, but are not limited to: Bio-analytical method transfers between laboratories or analysts Change in analytical methodology (e.g., change in detection systems) Change in anticoagulant in harvesting biological fluid Change in matrix within species (e.g., human plasma to human urine) Change in sample processing procedures Change in species within matrix (e.g., rat plasma to mouse plasma) Change in relevant concentration range Changes in instruments and/or software platforms Limited sample volume (e.g., pediatric study) Rare matrices Selectivity demonstration of an analyte in the presence of concomitant medicati Selectivity demonstration of an analyte in the presence of specific metabolites. 303Proposed Change: Complete the possibility to perform partial validations when there is a change of species, change of anti-coagulant, selectivity with metabolites or concomitant medication, change of extraction method ( as described in the report of the conference Bio-analytical Method Validation a revisit with a decade of progress Workshop held in Arlington, VA, 2000). 306Comment: How many Accuracyand Precision runs are required? 308-316 315 Comment: How many batches/ validation days are recommended for cross or partial validations? In which cases do we need to consider cross validation? Does this apply only to studies for which analyses are performed in two different laboratories or are there other cases? Cross validation: For cross validation we propose to add standards and unknown samples to QC samples and that a statistical analysis should be performed. Proposed Change: Modify to difference between two measurements should not exceed 40% .This is more in line with the other method validation criteria (i.e. accuracy and precision within 15%, if one method accuracy 115%, and the other 85%, then difference is 30%, not 15%). However, we think that the acceptance criteria appear too stringent and should be in line with the other method validation criteria. Why not apply the acceptance level of plus or minus 20% as in the re-analysis criteria? 309-316Comment: This requirement should be limited to clinical studies. 325-328Comment: We disagree with the sentence Therefore validation must cover issues such as batch to batch differences in glycolysation and metabolic differences in phosphorylation. We prefer to recommend that the standard to be used is the closest as possible as the one given to pre-clinical species or man. In case of change of batch, a comparison of batches will be performed. 331Comment: IDEM 4.1.8 This is difficult to apply to pre-clinical animal matrices. 317-369Comments: This section 4.4 should be detailed. Specific paragraphs on selectivity, carry-over, LOQ, calibration curve, accuracy, precision, dilution integrity, matrix effect and stability should be included. Proposed Change: To add: Selectivity Selectivity should be proven by using at lest 6 sources of appropriate blank matrix which are individually analysed and evaluated for interference. Absence of interfering compounds is accepted where the response is less than 25% of the LOQ for the analyte. Calibration Curve: A minimum of 6 calibration concentration levels should be used, excluding the blank sample (processed matrix sample without analyte and without IS). A relationship which can simply and adequately describe the response of the instrument with regard to the analyte should be applied. The blank and zero samples might be taken into account to calculate the calibration curve parameters. Stability: Stability of the analyte and IS in the studies matrix is evaluated using at least triplicate samples of low and high QC samples which are analysed immediately after preparation and after the applied storage conditions that are to be evaluated. The QC samples are analysed against a calibration curve obtained from freshly prepared calibration standards and the obtained concentrations are compared to the nominal concentrations. The deviation should be within plus or minus 20%. Ligand binding assays: QCs on micro-titre plate based assays. Samples are often analysed as two or three replicates on a plate. FDA requires two samples per QC level resulting in two times three wells. This draft guideline asks for three QC levels. Is it sufficient to include one sample per QC level, if each sample is analysed in two or three replicates on one plate? 334-335Comment: To be homogeneous with line 349, replace may exceed 20% of the LLOQ by should not exceed 25% of the LLOQ. 370-466Comments: Methods for Analysis of study samples may vary due to the large amount of analyte types. Proposed change (if any): Review the wordings so the text can accommodate the different possibilities. 371-372Comment: To define what is a complete validation of the analytical method. Proposed Change: To replace After complete validation of the analytical method by After the characterization of the main parameters such as within and between parameters, LLOQ, ULOQ, and matrix effect. 372Comment: When do we need to verify the performance of the analytical method before we run sample analysis? Could it be possible to clarify time period? 379-380Comment: What criteria should be used for blanks (double blank) and zero sample (single blank) during validation and study assays? How shall the high, and mid QC concentrations be handled during analysis of study samples? For ligand binding, a blank sample is considered as standard zero. Proposed Change: To replace An analytical run consists of the blank sample (processed matrix sample without analyte and without IS) and a zero sample (processed matrix with IS) by For physico-chemical assays, an analytical run consists of the blank sample (processed matrix sample without analyte and without IS) and a zero sample (processed matrix with IS). 382Comment: What is one single batch? How will be defined the acceptance criteria for each batch and for the full run? 384Comment: How should we interpret the differences between analytical runs and prepared runs. It is sometimes very difficult to prepare all samples for an analytical run at exactly the same time. 393-395Comment: The re-grouping of QCs at the beginning and end of runs appears acceptable in this document although this is not recommended by the FDA. Depending on the size of the run, it should specified that it is necessary to monitor the middle section. 400-401Comment: LLOQ values for ligand binding assays might be included. Proposed Changes: To replace 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%. By The back calculated concentrations of the calibration standards should be within 15% of the nominal value (or 20% for ligand binding assays) , except for the LLOQ for which it should be within 20% (or 25% for ligand binding assays). 401-402Comment: At least 75% of the calibration curves standards with a minimum of six, must fulfil this criterion. Does that mean that the curve is composed of 6 points and 4 are acceptable or do we need 6 acceptable points so at least 8 in the curve? Should it be clarified here that this relates to a minimum of 6 calibration samples at six different concentrations......? 404-405Comments: Proposed Changes: To replace Criteria for decision to exclude calibration standards or not, should be pre-defined in an SOP by Criteria for decision to exclude calibration standards or not should be pre-defined in an SOP or in the validation plan or in the study plan. 407-411Comments: This is not in agreement with the FDA recommendation and current practices. It is not allowed to change the range of quantification. If the LLOQ or ULOQ is not acceptable then the run should be rejected. Proposed Changes: To replace If the rejected calibration standard is LLOQ, it should be realised that the LLOQ for this analytical run is the next acceptable higher calibration standard concentration of the calibration curve. If the highest calibration standard, the ULOQ for this analytical run is the concentration of the next acceptable lower calibration standard concentration of the calibration curve. by If the rejected calibration standard is the LLOQ or the ULOQ, the run should be rejected. 412Comments: Values for ligand binding assays should be included. Proposed Change: To replace The accuracy values of the QC samples should be within 15% of the nominal values by The accuracy values of the QC samples should be within 15% of the nominal values, or 20% for ligand binding assays. 413Proposed Change: Change 50% to at least one of. 416Comment: The between run accuracy should already have been validated and therefore is not useful here. Is the within-run precision calculated with all QCs (including the failed ones)? The QC samples rejected should be excluded from the calculation of the between run (mean) accuracy. The acceptance criteria on overall statistics of study QCs are not acceptable as study results are based on individual run acceptance. 421Comments: The between run precision should already have been validated and therefore is not useful here. However the text states The between run (mean) precision should not exceed 15% The QC samples rejected (or at least the outliers) should be excluded from the calculation of the between run (mean) precision Proposed Change: Complete with the between-run precision of the QCs samples should not exceed 15% as in line 416. 421Comment: The between run precision should already have been validated and therefore is not useful here. 416-421Comment: Acceptance criteria on overall statistics of study QCs is not acceptable as study results are based on individual runs; acceptance, or criteriafor overall statistics should be enlargedto be statistically compatible with the 15/4/6rule. 428Proposed Change: Change 50% to at least one of. 430Comment: Section 5.3 describes the possibility to extend the calibration range once having verified the precision and accuracy. It can also be necessary to verify the model of regression chosen. 431Comment: Large number of samples > ULOQ: not very clear:this would necessitate abetter definition of "large number". 447Comment: What are the criteria to be used regarding Internal Standard (IS) variability? What about stable labelled Internal Standards? 447-448Is this criteria of re-analysis considered to be critical point by point aspect of the validation? e.g. acceptance limits? 454Proposed Change: Propose to add Poor replicate analysis for ligand binding assays. 455An outlier /aberrant value can be observed on the pharmacokinetic profile and be the reflection of a handling error during the analytical phase. If an aberrant value is detected on a pharmacokinetic profile and if we do not reanalyse, it means considering that an aberrant value is acceptable. Isnt it critical? We could demonstrate a bioequivalence where there isnt and vice versa. It appears strange that it is not allowed to re-analyse samples that are considered by the responsible scientist to be out of the expected PK/TK profile? There may be situations when a sponsor of a contracted study would specifically request this in order to verify accuracy of the 1st result obtained. If such procedures for repeat analysis are documented in approved SOPs, is it really a problem? The SOP (and GLP) would require a pre-defined, standard approach, justified in the raw data and report. It is perhaps naive to indicate Normally the reanalysis of study samples because of a pharmacokinetic reason is not acceptable. There will always be questions coming from our TK/PK colleagues about specific sample data. The new EMEA Guidance should state that it is permissible for BA to re-run a sample for TK/PK reasons provided that there is a clear reason for it provided by the pharmacokinetic colleagues and that it is documented prior to start of the re-assay. If maintained, The text should clarify if the reanalysis might be included in the final results or definitively not. 464It is already necessary to have an SOP for integration of chromatograms. 465Comments: There may be more sources of bioanalytical data than chromatograms. It should also be possible to reflect the chromatogram integration method in the study report Proposed change (if any): Review the wordings so the text can accommodate the different possibilities. 467If we consider that the long-term stability in the storage conditions is validated, what is the scientific rationale to redo these analyses? What would be the percentage of samples to be reanalysed? 467-472Comment: How many samples should be reanalysed in percentage? It is important to specify this since in pre-clinical studies this could possibly impact the number of animals to be used. Anindication of number of study samples to be re-analyzed would be useful. 471-472Comments: It is unclear what you mean by over a certain time period. To avoid issues related to stability, it is recommended to evaluate incurred sample reproductiibility as closest as possible from the first analysis, in a separate batch and not over a certain time period. 479-481Comments: We recommend to adopt the same approach as the FDA and to do it in a subset of samples. Proposed Change: To replace For (human)v study samples evaluation of incurred samples should be carried out for every subject or patient population, unless otherwise justified. by For (human)v study samples evaluation of incurred samples should be carried out on a subset of samples. 487Comments: 7. Study Report Section is entitled Study Report, but contains references to final report, validation report, study report, and analytical report, which makes it difficult to interpret the Validation Report and sample analysis Study Report requirements. Proposed change: To modify text to clearly define both the common and separate requirements for the Validation Report and sample analysis Study Report; to modify Section title to be representative of both the Validation Report and sample analysis Study Report. 489Comment: Delete is. 488-490Comments: Bioanalysis in a GLP study: The Study Report is commonly referred to as the report which the Study Director is responsible for. In a multi-site study there is a Principal Investigator (PI) who is responsible for the bio-analytical part of the study. The PI provides the report for the bioanalytical part and this report is amended to the Study Report. Proposed change (if any): Study Reports are a central element in the OECD GLP therefore consider review and update of this part so it fits the roles and responsibilities concerning Test Facility/Site management, Study Director/Principal Investigator and QA Program. 492Comment: Should the validation be in compliance with GLP and ICH guidelines? The GLPs are designed to be applicable to non-clinical safety studies and validation has always been excluded since this occurs independently of the study. No other method validation is considered necessary to be performed under GLPS e.g. all medical laboratory analyses. Some authorities are specifically requiring that routine analysis of clinical samples must be performed according to GCP.493Comments: .. conducted audits/inspection should be included in the report. it is not clear if this reference is solely for sample analysis Study Reports or also for Validation Reports; If full GLP compliance is not required of method validations, then validation specific audit/inspections are not required. If QA statements are however required, OECD GLP prescribes who is responsible for the reports and how to perform and report audits/inspections in a QA Statement. Proposed change (if any): Consider review and update of this part so it fits the roles and responsibilities concerningthe QA Program. Modify the text to ..conducted audits/inspection should be included in the report for GLP compliant study sample analysis. 494Comments: SOPs for relevant analysis specific procedures should be appended to the study report - SOPs may not be available when validation studies start. Any SOPs available are already summarized in the content of the report. They are available in the documentary system of the company. If we start to append the SOPs in the report with this guideline, the drift could be to append the analytical SOPs in the study reports for other types of analyses, and eventually for all activities performed within a study. This requirement should be looked at again since it may be in conflict with the GLPs. Why should SOPs for relevant analysis specific procedures be appended to the study report when SOPs are written in the national language and not in English? It does not appear acceptable to attach SOPs in our reportssince we may have many documentsand from country to country, the SOPs, written in local languages, would necessitate translations into English. This does not seem realistic. Proposed Change: To replace SOPs for relevant analytical specific procedures should be appended to the study report by The report includes the exact description of assay used in the study. 495Comment: All individual data should be available in electronic format to be provided upon request. What would be the electronic format, and what would the support need to be: CD-Rom or access in the software for the chromatographic interpretation? In any case, This requirement should be deleted since there is no GLP requirement to have individual data in electronic format (i.e. for paper raw data). 498Comments: We suggest a summary of the validation performances instead of validation report in a table format. 501Comments: Summary of the assay procedure The purpose of validation study is to determine the procedures of measurement. Proposed change: details of the assay procedure. 507 Comments: We suggest sample preparation recommendations instead of sample tracking (conditions and duration of storage). 511Comments: For the calibration curve there should not be within-run precision? 517Comments: For the Matrix effect complete with if applicable as from this text it applies to the mass spectrometric analytical methods. 520-522Comments: All measurements with the individual calculated values have to be presented . Do we also have to present all rejected data and if yes, is it necessary to include them or not in the statistical calculations, knowing that this will obviously produce a bias in the results? Although the study sample report is separate from the validation report it is often not a stand-alone entity. It is usually a sub-report to an overall study report. For samples analyzed under GLPs, the report would be generated and the compliance statement signed by a Principal Investigator. It would include a QA statement and be forwarded to the Study Director for incorporation into the main report as an Appendix. Proposed Change: The situation for phase reports in pre-clinical studies should be addressed. 528Comments: Are the details needed in a report for sample tracking : dates of receipt and content, storage location? This information is necessary but should only need to be available for on site inspections since it will not be interpretable without information concerning the freezer calibrations etc. 531Comments: Can you clarify set-up of the analytical run. Is it the list of runs or the typical content of a run? 532Comments: Table of all analytical runs with analysis dates and results indicating which samples have been analysed in which analytical run. Is this level of details needed? 544Comments: Including 5 to 20% of the subjects represent a lot of chromatograms. We suggest that it be necessary to include only representative chromatograms e.g. 1 subject/animal by sex and by group. Is this really of benefit in report interpretation, particularly when this may represent only a small part (phase report) of the overall study report? 569Comments: Should this not be lower limit of quantification as in the previous line. 591-Comments: There is no Reference Section Proposed change (if any): Adda Reference Section.       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DM_Keywords" DM_Authors# DM_Status$ DM_version%DM_emea_doc_ref_id nulldate2005 nulldateEMA Comments nulldate293958emea_document02/02/2010 18:25:13Hearfield NeilHearfield Neil02/02/2010 18:25:13Schumacher DunjaSubmission of comments formComments-EMA/293958/2005$3.0, CURRENT, Ready for publicationEMA/293958/2005  !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMNOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#$%&'()*+,-./0123456789:;<=>?@ABCDEFGHIJKLMOPQRSTUVWXYZ[\]^_`abcdefghijklmnopqrstuvwxyz{|}~      !"#%&'()*+4Root Entry Fp5Eb6Data 1TableNgWordDocumentfSummaryInformation(DocumentSummaryInformation8$CompObjq  FMicrosoft Office Word Document MSWordDocWord.Document.89q