48 II. BACKGROUND 59-71 Proposed change: Remove “and/or biopharmaceutics” from line 61 as it is out of context. The sentence would then read: “Selective, sensitive, and validated analytical methods for the quantitative evaluation of drugs and their metabolites (analytes) and biomarkers are critical for the successful conduct of nonclinical and/or clinical studies for small-molecule drugs and biopharmaceutics.” The guidance does not mention specificity as one of the fundamental parameters for validation of LBA assays. Proposed change: Add “Specificity in case of LBA” to the list of fundamental parameters. 73-78 The guidance suggests that for exploratory methods “less validation may be sufficient” (line 78). Historically, exploratory methods (screening methods and those applied to studies) have not been within the scope of regulated bioanalysis and therefore have not been subject to formal validation. Recommendation: Remove this sentence. Alternatively, further clarify how fit-for-purpose validation should be applied. 80-86 The guidance states: modifications should be validated…”, Recommendation: Rather than using this statement, reference the partial validation section 98-116, which provides further detail on appropriate levels of validation for method changes. 88 The details on full, partial and cross-validation should be moved from the Background section to the Validation section.
194-199 EMA and Anvisa guidances require the assessment of selectivity in hemolyzed and lipemic matrix sources when is the target matrix. Recommendation: In order to align the draft guidance with those of the other agencies we recommend that lines 196-197 be revised to state “Analyses of blank samples of the appropriate biological matrix (plasma, urine, or other matrix) should be obtained from at least six independent sources; in the case of plasma or serum, these six sources may include those that are hemolyzed and/or lipemic”. We suggest that the agency consider aligning the need for method selectivity assessment to the inherent specificity of the detection approach that the method employs. Specifically, retention of the following text from the 2001 guidance should be considered: “for hyphenated mass methods, however, testing six independent matrices for interference may not be important.” Finally, we request that agency expectations with respect to the evidence needed to that the substance quantified is the intended analyte be stated. Is a match of the retention time of analyte peaks in standards and post-dose samples sufficient evidence for SRM LC/MS methods, or is additional information required?
229-231 The requirements for validation of dilution are not clear. Please clarify: If it is sufficient to simply demonstrate the ability to dilute from a concentration at least as high as the highest analyte concentration found in samples down into the standard curve range, or is it necessary to verify the ability to perform at each dilution factor applied. In order to address these points, consideration should be given to aligning the recommendations with those in the EMEA guidance. Proposed change: “Dilution integrity should be demonstrated by spiking the matrix with analyte at a concentration above the ULOQ of the assay that is at least equal to the highest anticipated concentration in study samples and diluting this sample with blank matrix using a dilution factor at least equal to the greatest factor to be applied to study samples. Accuracy and precision, based on n 5 determinations, should be within ±15%. “
668-672 a. Freeze and Thaw Stability 674-677 b. Bench-Top Stability 679-682 c. Long-Term Stability Proposed change: This reads as though a 5 year clinical study would require 5 year LTS to be demonstrated. We recommend changing the wording to “The storage time in a long-term stability evaluation shall equal or exceed the duration from time of sample collection to analysis for any given sample”. 684-690 d. Stock Solution Stability 692-695 e. Processed Sample Stability 697 C. Validated Method: Use, Data Analysis, and Reporting Recommendation: It appears this section was cut/pasted from the chromatography section. Certain aspects of which do not apply to LBAs. Recommend deleting portions that don’t apply and expanding on LBA expectations.
943-947 Lines 944-947: table needs to include reasons for the new method or additional validation & dates of final reports. Comment: It is the interpretation that is this is a form of the CTD table. Recommendation: That clarification be made if filing practices are not within the scope of this guidance. 948-950 The items listed required in the summary table are related to filing and should not appear as part of study data. Proposed change: Remove the summary table. 952-954 C. Documentation for Method Validation 959-960 Lines 959 – 960: Clarification is needed as to whether this is a description of the preparation of standards and QCs OR a table of all prepared standards/QC batches and the dates of preparation. As this information is already present in the raw data and method it is Recommendation: Limit this requirement to a table of preparation dates. 961-964 961-964: Section is primarily chromatography focused. Recommendation: broaden the language to include LBA. Line 961: Only suitability of internal standard should be required. Proposed change: Removal of the requirement for evidence of purity and identity of IS Line 962: States that the chromatogram of the analyte should be interference-free. No chromatogram can be totally interference-free. The current validation criteria that interferences, such as carryover and non-selective signal are allowed to the degree of 20% of the LLOQ. Proposed change: reword this requirement as “The chromatogram of the analyte should show interference that is below that set forth in the validation criteria.”
1081-1082 There are several types of Reproducibility: between lab, re-injection, incurred sample, between instrument, etc. Recommendation: that a more generic definition be provided or concisely define each type of reproducibility. 1084-1086 Recommendation: define selectivity and specificity separately for LBA in line 1084. 1089-1090 Recommendation: change “chemical” to “biochemical” in line 1089. 1100-1103 IX. APPENDIX Priority Recommendation: Use of hyperlinks for eCTD submissions sounds good but is fraught with issues in practice. The process needs someone with a full overview of study/program to manage submission/report/data links.