ICH M 10 Bioanalytical Method Validation and Study Sample Analysis Guidance for Industry PDF

M10 BIOANALYTICAL METHOD VALIDATION AND STUDY SAMPLE ANALYSIS Guidance for Industry U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER)...

M10 BIOANALYTICAL METHOD VALIDATION AND STUDY SAMPLE ANALYSIS Guidance for Industry U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) November 2022 ICH  M10 BIOANALYTICAL METHOD VALIDATION AND STUDY SAMPLE ANALYSIS Guidance for Industry Additional copies are available from: Office of Communications, Division of Drug Information Center for Drug Evaluation and Research Food and Drug Administration 10001 New Hampshire Ave., Hillandale Bldg., 4 th Floor Silver Spring, MD 20993-0002 Phone: 855-543-3784 or 301-796-3400; Fax: 301-431-6353 Email: [email protected] https://www.fda.gov/drugs/guidance-compliance-regulatory-information/guidances-drugs and/or Office of Communication, Outreach and Development Center for Biologics Evaluation and Research Food and Drug Administration 10903 New Hampshire Ave., Bldg. 71, Room 3128 Silver Spring, MD 20993-0002 Phone: 800-835-4709 or 240-402-8010 Email: [email protected] https://www.fda.gov/vaccines-blood-biologics/guidance-compliance-regulatory-information-biologics/biologics-guidances U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER) Center for Biologics Evaluation and Research (CBER) November 2022 ICH  Contains Nonbinding Recommendations FOREWORD The International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) has the mission of achieving greater regulatory harmonization worldwide to ensure that safe, effective, and high-quality medicines are developed, registered, and maintained in the most resource-efficient manner. By harmonizing the regulatory expectations in regions around the world, ICH guidelines have substantially reduced duplicative clinical studies, prevented unnecessary animal studies, standardized safety reporting and marketing application submissions, and contributed to many other improvements in the quality of global drug development and manufacturing and the products available to patients. ICH is a consensus-driven process that involves technical experts from regulatory authorities and industry parties in detailed technical and science-based harmonization work that results in the development of ICH guidelines. The commitment to consistent adoption of these consensus- based guidelines by regulators around the globe is critical to realizing the benefits of safe, effective, and high-quality medicines for patients as well as for industry. As a Founding Regulatory Member of ICH, the Food and Drug Administration (FDA) plays a major role in the development of each of the ICH guidelines, which FDA then adopts and issues as guidance to industry.  Contains Nonbinding Recommendations TABLE OF CONTENTS I. INTRODUCTION (1)................................................................................................. 1 A. Objective (1.1).............................................................................................................. 1 B. Background (1.2).......................................................................................................... 1 C. Scope (1.3).................................................................................................................... 2 II. GENERAL PRINCIPLES (2).................................................................................... 3 A. Method Development (2.1)............................................................................................. 3 B. Method Validation (2.2)................................................................................................. 3 1. Full Validation (2.2.1)..................................................................................................... 3 2. Partial Validation (2.2.2)................................................................................................. 4 3. Cross Validation (2.2.3).................................................................................................. 4 III. CHROMATOGRAPHY (3)....................................................................................... 4 A. Reference Standards (3.1).............................................................................................. 4 B. Validation (3.2)............................................................................................................. 5 1. Selectivity (3.2.1)............................................................................................................ 5 2. Specificity (3.2.2)............................................................................................................ 6 3. Matrix Effect (3.2.3)........................................................................................................ 7 4. Calibration Curve and Range (3.2.4)................................................................................. 7 5. Accuracy and Precision (3.2.5)......................................................................................... 8 6. Carryover (3.2.6)............................................................................................................ 9 7. Dilution Integrity (3.2.7).................................................................................................. 9 8. Stability (3.2.8).............................................................................................................10 9. Reinjection Reproducibility (3.2.9)...................................................................................12 C. Study Sample Analysis (3.3)..........................................................................................13 1. Analytical Run (3.3.1)....................................................................................................13 2. Acceptance Criteria for an Analytical Run (3.3.2)...............................................................14 3. Calibration Range (3.3.3)...............................................................................................15 4. Reanalysis of Study Samples (3.3.4)..................................................................................15 5. Reinjection of Study Samples (3.3.5).................................................................................16 6. Integration of Chromatograms (3.3.6)..............................................................................17 IV. LIGAND BINDING ASSAYS (4)............................................................................. 17 A. Key Reagents (4.1).......................................................................................................17 1. Reference Standard (4.1.1)..............................................................................................17 2. Critical Reagents (4.1.2).................................................................................................17 B. Validation (4.2)............................................................................................................18 1. Specificity (4.2.1)...........................................................................................................18 2. Selectivity (4.2.2)...........................................................................................................19 3. Calibration Curve and Range (4.2.3)................................................................................19 4. Accuracy and Precision (4.2.4)........................................................................................20 5. Carryover (4.2.5)...........................................................................................................21 6. Dilution Linearity and Hook Effect (4.2.6).........................................................................21 7. Stability (4.2.7).............................................................................................................22 i  Contains Nonbinding Recommendations C. Study Sample Analysis (4.3)..........................................................................................23 1. Analytical Run (4.3.1)....................................................................................................23 2. Acceptance Criteria for an Analytical Run (4.3.2)...............................................................23 3. Calibration Range (4.3.3)...............................................................................................24 4. Reanalysis of Study Samples (4.3.4)..................................................................................25 V. INCURRED SAMPLE REANALYSIS (5)............................................................... 26 VI. PARTIAL AND CROSS VALIDATION (6)............................................................ 27 A. Partial Validation (6.1).................................................................................................27 B. Cross Validation (6.2)...................................................................................................28 VII. ADDITIONAL CONSIDERATIONS (7)................................................................. 29 A. Methods for Analytes That Are Also Endogenous Molecules (7.1)....................................29 1. Quality Control Samples for Methods for Analytes That Are Also Endogenous Molecules (7.1.1)..31 2. Selectivity, Recovery, and Matrix Effects for Methods for Analytes That Are Also Endogenous Molecules (7.1.2)..............................................................................................................31 3. Parallelism for Methods for Analytes That Are Also Endogenous Molecules (7.1.3).................32 4. Accuracy and Precision for Methods for Analytes That Are Also Endogenous Molecules (7.1.4)32 5. Stability for Methods for Analytes That Are Also Endogenous Molecules (7.1.5).....................33 B. Parallelism (7.2)...........................................................................................................33 C. Recovery (7.3)..............................................................................................................33 D. Minimum Required Dilution (7.4).................................................................................34 E. Commercial and Diagnostic Kits (7.5)............................................................................34 F. New or Alternative Technologies (7.6)...........................................................................35 1. Dried Matrix Methods (7.6.1)..........................................................................................35 VIII. DOCUMENTATION (8).......................................................................................... 36 A. Summary Information (8.1)..........................................................................................36 B. Documentation for Validation and Bioanalytical Reports (8.2)........................................37 GLOSSARY........................................................................................................................ 45 ii  Contains Nonbinding Recommendations M10 BIOANALYTICAL METHOD VALIDATION AND STUDY SAMPLE ANALYSIS Guidance for Industry 1 This guidance represents the current thinking of the Food and Drug Administration (FDA or Agency) on this topic. It does not establish any rights for any person and is not binding on FDA or the public. You can use an alternative approach if it satisfies the requirements of the applicable statutes and regulations. To discuss an alternative approach, contact the FDA office responsible for this guidance as listed on the title page. I. INTRODUCTION (1) 2 A. Objective (1.1) This guidance is intended to provide recommendations for the validation of bioanalytical methods for chemical and biological drug quantification and their application in the analysis of study samples. Adherence to the principles presented in this guidance will ensure the quality and consistency of the bioanalytical data in support of the development and market approval of both chemical and biological drugs. The objective of the validation of a bioanalytical method is to demonstrate that it is suitable for its intended purpose. You may use an alternative approach from the recommendations in this guidance if it satisfies the requirements of applicable statutes and regulations. Alternative approaches should be supported by appropriate scientific justification. Applicants are encouraged to consult the regulatory authority(ies) regarding significant changes in method validation approaches when an alternate approach is proposed or taken. This guidance replaces the ICH draft guidance for industry M10 Bioanalytical Method Validation, issued on June 27, 2019.3 B. Background (1.2) Concentration measurements of chemical and biological drug(s) and their metabolite(s) in biological matrices are an important aspect of drug development. The results of studies employing such methods contribute to regulatory decisions regarding the safety and efficacy of 1 This guidance was developed within an Expert Working Group of the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) and has been subject to consultation by the regulatory parties, in accordance with the ICH process. This document was endorsed by the Regulatory Members of the ICH Assembly at Step 4 of the ICH process on May 24, 2022. 2 The numbers in parentheses reflect the organizational breakdown of the document endorsed by the Regulatory Members of the ICH Assembly at Step 4 of the ICH process, May 24, 2022. 3 We update guidances periodically. For the most recent version of a guidance, check the FDA guidance web page at https://www.fda.gov/regulatory-information/search-fda-guidance-documents. 1  Contains Nonbinding Recommendations drug products. It is therefore critical that the bioanalytical methods used are well characterized, appropriately validated, and documented in order to ensure reliable data to support regulatory decisions. This guidance intends to facilitate development of drugs in accordance with the principles of the 3Rs (Reduce, Refine, Replace) for animal studies, where valid. C. Scope (1.3) This guidance describes the validation of bioanalytical methods and study sample analysis that are expected to support regulatory decisions. The guidance is applicable to the bioanalytical methods used to measure concentrations of chemical and biological drug(s) and their metabolite(s) in biological samples (e.g., blood, plasma, serum, other body fluids or tissues) obtained in nonclinical toxicokinetic (TK) studies conducted according to the principles of Good Laboratory Practice (GLP), nonclinical pharmacokinetic (PK) studies conducted as surrogates for clinical studies, and all phases of clinical trials, including comparative bioavailability/bioequivalence (BA/BE) studies, in regulatory submissions. Full method validation is recommended for the primary matrix intended to support regulatory submissions. Additional matrices should be validated as necessary. For studies that are not submitted for regulatory approval or not considered for regulatory decisions regarding safety, efficacy, or labeling (e.g., exploratory investigations), applicants may decide on the level of qualification that supports their own internal decision-making. The information in this guidance applies to the quantitative analysis by ligand binding assays (LBAs) and chromatographic methods such as liquid chromatography (LC) or gas chromatography (GC), which are typically used in combination with mass spectrometry (MS) detection. For studies that are subject to GLP or Good Clinical Practice (GCP) requirements, the bioanalysis of study samples must conform to those requirements. The bioanalysis of biomarkers and bioanalytical methods used for the assessment of immunogenicity are not within the scope of this guidance. In general, FDA’s guidance documents do not establish legally enforceable responsibilities. Instead, guidances describe the Agency’s current thinking on a topic and should be viewed only as recommendations, unless specific regulatory or statutory requirements are cited. The use of the word should in Agency guidances means that something is suggested or recommended, but not required. 2  Contains Nonbinding Recommendations II. GENERAL PRINCIPLES (2) A. Method Development (2.1) The purpose of bioanalytical method development is to define the design, operating conditions, limitations, and suitability of the method for its intended purpose and to ensure that the method is ready for validation. Before or during the development of a bioanalytical method, the applicant is encouraged to, if feasible, understand the analyte of interest (e.g., the physicochemical properties of the drug, in vitro and in vivo metabolism, preferential distribution between red blood cells and plasma, and protein binding) and consider aspects of any prior analytical methods that may be applicable. Method development involves identifying the procedures and conditions involved with quantifying the analyte. Method development can include the characterization of the following bioanalytical elements: reference standards, critical reagents, calibration curve, quality control samples (QCs), selectivity and specificity, sensitivity, accuracy, precision, recovery, stability of the analyte, and minimum required dilution (MRD). Bioanalytical method development does not require extensive record keeping or notation. Once the method has been developed, bioanalytical method validation proves that the method is suited to the analysis of the study samples. If a problem is encountered with the method during the analysis of nonclinical or clinical study samples that requires that the analysis be stopped, any changes to the method and the rationale should be documented. B. Method Validation (2.2) 1. Full Validation (2.2.1) Bioanalytical method validation is important to ensure the acceptability of assay performance and the reliability of analytical results. A bioanalytical method is defined as a set of procedures used for measuring analyte concentrations in biological samples. A full validation of a bioanalytical method should be performed when establishing a bioanalytical method for the quantification of an analyte in clinical and in applicable nonclinical studies. Full validation should also be performed when implementing an analytical method that is reported in the literature and when a commercial kit is repurposed for bioanalytical use in drug development. Usually, one analyte has to be determined, but on occasion it may be appropriate to measure more than one analyte. This may involve two different drugs, a parent drug with its metabolites or the enantiomers or isomers of a drug. In these cases, the principles of validation and analysis apply to all analytes of interest. For chromatographic methods, a full validation should include the following elements, unless otherwise justified: selectivity, specificity, matrix effect, calibration curve (response function), range (lower limit of quantification (LLOQ) to upper limit 3  Contains Nonbinding Recommendations of quantification (ULOQ)), accuracy, precision, carryover, dilution integrity, stability, and reinjection reproducibility. For LBAs, the following elements should be evaluated, unless otherwise justified: specificity, selectivity, calibration curve (response function), range (LLOQ to ULOQ), accuracy, precision, carryover, dilution linearity, and stability. If necessary, parallelism can be conducted when appropriate study samples are available. The assessments that are performed during validation should be relevant to the sample analysis workflow. The matrix used for bioanalytical method validation should be the same as the matrix of the study samples, including anticoagulants and additives. In cases in which it may be difficult to obtain an identical matrix to that of the study samples (e.g., rare matrices such as tissue, cerebrospinal fluid, bile or in cases where free drug is measured), surrogate matrices may be acceptable for analytical method validation. The choice of surrogate matrix should be scientifically justified. Matrix differences within species (e.g., age, ethnicity, gender) are generally not considered different when validating a method. A specific, detailed, written description of the bioanalytical method and validation procedure should be established a priori. This description may be in the form of a protocol, study plan, report, notebook, or Standard Operating Procedure (SOP). 2. Partial Validation (2.2.2) Modifications to a fully validated analytical method may be evaluated by partial validation. Partial validation can range from as little as one accuracy and precision determination to a nearly full validation (refer to section VI.A (6.1)). The items in a partial validation should be determined according to the extent and nature of the changes made to the method. 3. Cross Validation (2.2.3) Cross validation is required to demonstrate how the reported data are related when multiple bioanalytical methods and/or multiple bioanalytical laboratories are involved (refer to section VI.B (6.2)). III. CHROMATOGRAPHY (3) A. Reference Standards (3.1) During method validation and the analysis of study samples, a blank biological matrix is spiked with the analyte(s) of interest using solutions of reference standard(s) to prepare calibration standards and QCs. Calibration standards and QCs should be prepared from separate stock solutions. However, calibration standards and QCs may be prepared from the same stock 4  Contains Nonbinding Recommendations solution provided the accurate preparation and stability of the stock solution should have been verified. A suitable internal standard (IS) should be added to all calibration standards, QCs, and study samples during sample processing. The absence of an IS should be justified. It is important that the reference standard is well characterized and the quality (e.g., purity, identity) of the reference standard and the suitability of the IS is ensured, as the quality will affect the outcome of the analysis and, therefore, the study data. The reference standard used during validation and study sample analysis should be obtained from an authentic and traceable source. The reference standard should be identical to the analyte. If this is not possible, an established form (e.g., salt or hydrate) of known quality should be used. Suitable reference standards include compendial standards, commercially available standards or sufficiently characterized standards prepared in-house or by an external organization. A certificate of analysis (CoA) or an equivalent alternative is recommended to ensure quality and to provide information on the purity, storage conditions, retest/expiration date and batch number of the reference standard. A CoA is not required for the IS as long as the suitability for use is demonstrated, e.g., a lack of analytical interference is shown for the substance itself or any impurities thereof. When MS detection is used, the use of the stable isotope-labeled analyte as the IS is recommended whenever possible. However, it is important that the labeled standard is of high isotope purity and that no isotope exchange reaction occurs. The presence of unlabeled analyte should be checked and if unlabeled analyte is detected, the potential influence should be evaluated during method validation. Stock and working solutions should only be prepared from reference standards that are within the stability period as documented in the CoA (either expiration date or the retest date). B. Validation (3.2) 1. Selectivity (3.2.1) Selectivity is the ability of an analytical method to differentiate and measure the analyte in the presence of potential interfering substances in the blank biological matrix. Selectivity should be evaluated using blank samples (matrix samples processed without addition of an analyte or IS) obtained from at least six individual sources/lots (non-hemolyzed and non- lipemic). Use of fewer sources may be acceptable in the case of rare matrices. Selectivity for the IS should also be evaluated. The evaluation of selectivity should demonstrate that no significant response attributable to interfering components is observed at the retention time(s) of the analyte or the IS in the blank samples. Responses attributable to interfering components should not be more than 20% of the 5  Contains Nonbinding Recommendations analyte response at the LLOQ and not more than 5% of the IS response in the LLOQ sample for each matrix. For the investigation of selectivity in lipemic matrices at least one source of matrix should be used. To be scientifically meaningful, the matrix used for these tests should be representative as much as possible of the expected study samples. A naturally lipemic matrix with abnormally high levels of triglycerides should be obtained from donors. Although it is recommended to use lipemic matrix from donors, if this is difficult to obtain, matrix can be spiked with triglycerides even though it may not be representative of study samples. However, if the drug impacts lipid metabolism or if the intended patient population is hyperlipidemic, the use of spiked samples is discouraged. This evaluation is not necessary for nonclinical studies unless the drug impacts lipid metabolism or is administered in a particular animal strain that is hyperlipidemic. For the investigation of selectivity in hemolyzed matrices at least one source of matrix should be used. Hemolyzed matrices should be obtained by spiking matrix with hemolyzed whole blood (at least 2% V/V) to generate a visibly detectable hemolyzed sample. 2. Specificity (3.2.2) Specificity is the ability of a bioanalytical method to detect and differentiate the analyte from other substances, including its related substances (e.g., substances that are structurally similar to the analyte, metabolites, isomers, impurities, degradation products formed during sample preparation, or concomitant medications that are expected to be used in the treatment of patients with the intended indication). If the presence of related substances is anticipated in the biological matrix of interest, the impact of such substances should be evaluated during method validation, or alternatively, in the pre- dose study samples. In the case of LC-MS based methods, to assess the impact of such substances, the evaluation may include comparing the molecular weight of a potential interfering related substance with the analyte and chromatographic separation of the related substance from the analyte. Responses detected and attributable to interfering components should not be more than 20% of the analyte response at the LLOQ and not more than 5% of the IS response in the LLOQ sample. The possibility of back-conversion of a metabolite into the parent analyte during the successive steps of the analysis (including extraction procedures or in the MS source) should also be evaluated when relevant (e.g., potentially unstable metabolites such as ester analytes to ester/acidic metabolites, unstable N-oxides or glucuronide metabolites, lactone-ring structures). It is acknowledged that this evaluation will not be possible in the early stages of drug development of a new chemical entity when the metabolism is not yet evaluated. However, this issue should be investigated, and partial validation performed, if needed. The extent of back- 6  Contains Nonbinding Recommendations conversion, if any, should be established and the impact on the study results should be discussed in the Bioanalytical Report. 3. Matrix Effect (3.2.3) A matrix effect is defined as an alteration of the analyte response due to interfering and often unidentified component(s) in the sample matrix. During method validation the matrix effect between different independent sources/lots should be evaluated. The matrix effect should be evaluated by analyzing at least three replicates of low and high QCs, each prepared using matrix from at least six different sources/lots. For each individual matrix sources/lots evaluated, the accuracy should be within ±15% of the nominal concentration and the precision (percent coefficient of variation (%CV)) should not be greater than 15%. Use of fewer sources/lots may be acceptable in the case of rare matrices. The matrix effect should also be evaluated in relevant patient populations or special populations (e.g., hepatically impaired or renally impaired) when available. An additional evaluation of the matrix effect is recommended using hemolyzed or lipemic matrix samples during method validation on a case-by-case basis, especially when these conditions are expected to occur within the study. 4. Calibration Curve and Range (3.2.4) The calibration curve demonstrates the relationship between the nominal analyte concentration and the response of the analytical platform to the analyte. Calibration standards, prepared by spiking matrix with a known quantity of analyte(s), span the calibration range and comprise the calibration curve. Calibration standards should be prepared in the same biological matrix as the study samples. The calibration range is defined by the LLOQ, which is the lowest calibration standard, and the ULOQ, which is the highest calibration standard. There should be one calibration curve for each analyte studied during method validation and for each analytical run. A calibration curve should be generated with a blank sample, a zero sample (blank sample spiked with IS), and at least six concentration levels of calibration standards, including the LLOQ and the ULOQ. A simple regression model that adequately describes the concentration-response relationship should be used. The selection of the regression model should be directed by written procedures. The regression model, weighting scheme, and transformation should be determined during the method validation. Blank and zero samples should not be included in the determination of the regression equation for the calibration curve. Each calibration standard may be analyzed in replicate, in which case data from all acceptable replicates should be used in the regression analysis. The calibration curve parameters should be reported (e.g., slope and intercept in the case of a linear model). The back-calculated concentrations of the calibration standards should be presented together with the calculated mean accuracy and precision values. All acceptable curves 7  Contains Nonbinding Recommendations obtained during validation, based on a minimum of three independent runs over several days, should be reported. The accuracy of the back-calculated concentrations of each calibration standard should be within ±20% of the nominal concentration at the LLOQ and within ±15% at all the other levels. At least 75% of the calibration standards with a minimum of six calibration standard levels should meet the above criteria. In the case that replicates are used, the criteria (within ±15% or ±20% for LLOQ) should also be fulfilled for at least 50% of the calibration standards tested per concentration level. In the case that a calibration standard does not comply with these criteria, this calibration standard sample should be rejected, and the calibration curve without this calibration standard should be reevaluated, including regression analysis. For accuracy and precision runs, if all replicates of the LLOQ or the ULOQ calibration standard in a run are rejected, then the run should be rejected, the possible source of the failure should be determined and the method revised, if necessary. If the next validation run also fails, then the method should be revised before restarting validation. The calibration curve should be prepared using freshly spiked calibration standards in at least one assessment. Subsequently, frozen calibration standards can be used within their defined period of stability. 5. Accuracy and Precision (3.2.5) a. Preparation of quality control samples (3.2.5.1) The QCs are intended to mimic study samples and should be prepared by spiking matrix with a known quantity of analyte, storing them under the conditions anticipated for study samples and analyzing them to assess the validity of the analytical method. Calibration standards and the QCs should be prepared from separate stock solutions in order to avoid biased estimations which are not related to the analytical performance of the method. If calibration standards and the QCs may be prepared from the same stock solution, the accuracy and stability of the stock solution should be verified. A single source of blank matrix may be used, which should be free of interference or matrix effects, as described in section III.B.3 (3.2.3). During method validation the QCs for accuracy and precision runs should be prepared at a minimum of 4 concentration levels within the calibration curve range: the LLOQ, within 3 times of the LLOQ (low QC), around 30% to 50% of the calibration curve range (medium QC) and at least 75% of the ULOQ (high QC). For non-accuracy and precision validation runs, low, medium, and high QCs may be analyzed in duplicate. These QCs, along with the calibration standards, will provide the basis for the acceptance or rejection of the run. b. Evaluation of accuracy and precision (3.2.5.2) 8  Contains Nonbinding Recommendations Accuracy and precision should be determined by analyzing the QCs within each run (within-run) and in different runs (between-run). Accuracy and precision should be evaluated using the same runs and data. Within-run accuracy and precision should be evaluated by analyzing at least five replicates at each QC concentration level in each analytical run. Between-run accuracy and precision should be evaluated by analyzing each QC concentration level in at least three analytical runs over at least two days. To enable the evaluation of any trends over time within one run, it is recommended to demonstrate accuracy and precision of the QCs over at least one of the runs in a size equivalent to a prospective analytical run of study samples. Reported method validation data and the determination of accuracy and precision should include all results obtained, including individual QCs outside of the acceptance criteria, except those cases where errors are obvious and documented. Within-run accuracy and precision data should be reported for each run. If the within-run accuracy or precision criteria are not met in all runs, an overall estimate of within-run accuracy and precision for each QC level should be calculated. Between-run (intermediate) precision and accuracy should be calculated by combining the data from all runs. The calibration curves for these assessments should be prepared using freshly spiked calibration standards in at least one run. If freshly spiked calibration standards are not used in the other runs, stability of the frozen calibration standards should be demonstrated. The accuracy at each concentration level should be within ±15% of the nominal concentration, except at the LLOQ, where it should be within ±20%. The precision (%CV) of the concentrations determined at each level should not exceed 15%, except at the LLOQ, where it should not exceed 20%. For non-accuracy and precision validation runs, at least 2/3 of the total QCs and at least 50% at each concentration level should be within ±15% of the nominal values. 6. Carryover (3.2.6) Carryover is an alteration of a measured concentration due to residual analyte from a preceding sample that remains in the analytical instrument. Carryover should be assessed and minimized during method development. During validation carryover should be assessed by analyzing blank samples after the calibration standard at the ULOQ. Carryover in the blank samples following the highest calibration standard should not be greater than 20% of the analyte response at the LLOQ and 5% of the response for the IS. If it appears that carryover is unavoidable, study samples should not be randomized. Specific measures should be considered, validated, and applied during the analysis of the study samples, so that carryover does not affect accuracy and precision. This could include the injection of blank sample(s) after samples with an expected high concentration, before the next study sample. 7. Dilution Integrity (3.2.7) Dilution integrity is the assessment of the sample dilution procedure, when required, to confirm that it does not impact the accuracy and precision of the measured concentration of the analyte. 9  Contains Nonbinding Recommendations The same matrix from the same species used for preparation of the QCs should be used for dilution. Dilution QCs should be prepared with analyte concentrations in matrix that are greater than the ULOQ and then diluted with blank matrix. At least five replicates per dilution factor should be tested in one run to determine if concentrations are accurately and precisely measured within the calibration range. The dilution factor(s) and concentrations applied during study sample analysis should be within the range of the dilution factors and concentrations evaluated during validation. The mean accuracy of the dilution QCs should be within ±15% of the nominal concentration and the precision (%CV) should not exceed 15%. In the cases of rare matrices, use of a surrogate matrix for dilution may be acceptable. It should be demonstrated that this does not affect precision and accuracy. 8. Stability (3.2.8) Stability evaluations should be carried out to ensure that every step taken during sample preparation, processing, and analysis as well as the storage conditions used do not affect the concentration of the analyte. The storage and analytical conditions applied to the stability tests, such as the sample storage times and temperatures, sample matrix, anticoagulant and container materials, should reflect those used for the study samples. Reference to data published in the literature is not considered sufficient. Validation of storage periods should be performed on QCs that have been stored for a time that is equal to or longer than the study sample storage periods. Stability of the analyte in the matrix is evaluated using low and high concentration QCs. Aliquots of the low and high QCs are analyzed at time zero and after the applied storage conditions that are to be evaluated. One bulk QC should be prepared at each concentration level. For each concentration tested, the bulk sample should be divided into a minimum of three aliquots that will be stored, stressed, and analyzed. The QCs should be analyzed against a calibration curve, obtained from freshly spiked calibration standards in a run with its corresponding freshly spiked QCs or QCs for which stability has been proven. The mean concentration at each QC level should be within ±15% of the nominal concentration. If the concentrations of the study samples are consistently higher than the ULOQ of the calibration range, the concentration of the high QC should be adjusted to reflect these higher concentrations. It is recognized that this may not be possible in nonclinical studies due to solubility limitations. For fixed dose combination products and specifically labeled drug regimens, the freeze-thaw, bench top, and long-term stability tests of an analyte in matrix should be conducted with the matrix spiked with all of the dosed compounds. 10  Contains Nonbinding Recommendations The following stability tests should be evaluated: (1) Stability of the Analyte in Matrix Freeze-Thaw Stability in Matrix To assess the impact of repeatedly removing samples from frozen storage, the stability of the analyte should be assessed after multiple cycles of freezing and thawing. Low and high QCs should be thawed and analyzed according to the same procedures as the study samples. QCs should be kept frozen for at least 12 hours between the thawing cycles. QCs for freeze-thaw stability should be assessed using freshly prepared calibration standards and QCs, or QCs for which stability has been proven. The number of freeze-thaw cycles validated should equal or exceed that of the freeze-thaw cycles undergone by the study samples, but a minimum of three cycles should be conducted. Bench Top (Short-Term) Stability in Matrix Bench top matrix stability experiments should be designed and conducted to cover the laboratory handling conditions for the study samples. Low and high QCs should be thawed in the same manner as the study samples and kept on the bench top at the same temperature and for at least the same duration as the study samples. The total time on the bench top should be concurrent; additive exposure to bench top conditions should not be used (i.e., time from each freeze-thaw evaluation should not be added up). Long-Term Stability in Matrix The long-term stability of the analyte in matrix stored in the freezer should be established. Low and high QCs should be stored in the freezer under the same storage conditions and at least for the same duration as the study samples. For chemical drugs, the stability at one temperature (e.g., -20°C) to can be extrapolated to lower temperatures (e.g., -70°/-80°C). For biological drugs, a bracketing approach can be applied, e.g., in the case that the stability has been demonstrated at -70°/-80°C and at -20°C, then it is not necessary to investigate the stability at temperatures in between those two points at which study samples will be stored. (2) Stability of the Analyte in Processed Samples The stability of processed samples, including the time until completion of analysis (in the autosampler/instrument), should be determined. For example: 11  Contains Nonbinding Recommendations Stability of the processed sample under the storage conditions to be used during the analysis of study samples (dry extract or in the injection phase) On-instrument/autosampler stability of the processed sample at injector or autosampler temperature The total time that a processed sample is stored should be concurrent (i.e., autosampler and other storage times should not be added together). (3) Stability of the Analyte and IS in Stock and Working Solutions The stability of the stock and working solutions of the analyte and IS should be determined under the storage conditions used during the analysis of study samples by using the lowest and the highest concentrations of these solutions. They should be assessed using the response of the detector. Stability of the stock and working solutions should be tested with an appropriate dilution, taking into consideration the linearity and measuring range of the detector. If the stability varies with concentration, then the stability of all concentrations of the stock and working solutions should be assessed. If no isotopic exchange occurs for the stable isotopically labeled IS under the same storage conditions as the analyte for which the stability is demonstrated, then no additional stability determinations for the IS are necessary. If the reference standard expires, or it is past the retest date, the stability of the stock solutions made previously with this lot of reference standard are defined by the expiration or retest date established for the stock solution. Stock and working solutions should not be made from reference standards solely for extending the expiration date for the use of the reference standard. In addition, the following test should be performed, if applicable: (4) Stability of the Analyte in Whole Blood Sufficient attention should be paid to the stability of the analyte in the sampled matrix (blood) directly after collection from subjects and prior to preparation for storage to ensure that the concentrations obtained by the analytical method reflect the concentrations of the analyte in the subject’s blood at the time of sample collection. If the matrix used is plasma, the stability of the analyte in blood should be evaluated during method development (e.g., using an exploratory method in blood) or during method validation. The results should be provided in the Validation Report. 9. Reinjection Reproducibility (3.2.9) Reproducibility of the method is assessed by replicate measurements of the QCs and is usually included in the assessment of precision and accuracy. However, if samples could be reinjected (e.g., in the case of instrument interruptions or other reasons such as equipment failure), reinjection reproducibility should be evaluated to establish the viability of the processed samples and to support their storage prior to reinjection. 12  Contains Nonbinding Recommendations Reinjection reproducibility is assessed by reinjecting a run that is comprised of calibration standards and a minimum of five replicates of the low, middle, and high QCs after storage. The precision and accuracy of the reinjected QCs establish the viability of the processed samples. The results should be included in the Validation Report or provided in the Bioanalytical Report of the study where it was conducted. C. Study Sample Analysis (3.3) The analysis of study samples can be carried out after validation has been completed, however, it is understood that some parameters may be completed at a later stage (e.g., long-term stability). By the time the data are submitted to a regulatory authority, the bioanalytical method validation should have been completed. The study samples, QCs, and calibration standards should be processed in accordance with the validated analytical method. If system suitability is assessed, a predefined specific study plan, protocol, or SOP should be used. System suitability, including apparatus conditioning and instrument performance, should be determined using samples that are independent of the calibration standards and QCs for the run. Subject samples should not be used for system suitability. The IS responses of the study samples should be monitored to determine whether there is systemic IS variability. Refer to Table 1 for recommendations regarding documentation. 1. Analytical Run (3.3.1) An analytical run should consist of a blank sample (processed matrix sample without analyte and without IS), a zero sample (processed matrix with IS), calibration standards at a minimum of six concentration levels, at least three levels of QCs (low, medium, and high) in duplicate (or at least 5% of the number of study samples, whichever is higher) and the study samples to be analyzed. The QCs should be interspersed in the run in such a way that the accuracy and precision of the whole run is ensured. Study samples should always be bracketed by QCs. The calibration standards and QCs should be spiked independently using separately prepared stock solutions unless the accuracy and stability of the stock solutions have been verified. 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 are intended to be analyzed. Analyzing samples that were processed as several separate batches in a single analytical run is discouraged. If such an approach cannot be avoided, for instance due to bench top stability limitations, each batch of samples should include low, medium, and high QCs. For comparative BA/BE studies, it is advisable to analyze all samples of one subject together in one analytical run to reduce variability. The impact of any carryover that occurs during study sample analysis should be assessed and reported (refer to section III.B.6 (3.2.6)). If carryover is detected, its impact on the measured concentrations should be mitigated (e.g., non-randomization of study samples, injection of blank 13  Contains Nonbinding Recommendations samples after samples with an expected high concentration) or the validity of the reported concentrations should be justified in the Bioanalytical Report. 2. Acceptance Criteria for an Analytical Run (3.3.2) Criteria for the acceptance or rejection of an analytical run should be defined in the protocol, in the study plan, or in an SOP. In the case that a run contains multiple batches, acceptance criteria should be applied to the whole run and to the individual batches. It is possible for the run to meet acceptance criteria, even if a batch within that run is rejected for failing to meet the batch acceptance criteria. Calibration standards in a failed batch should not be used to support the acceptance of other batches within the analytical run. 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 standard concentrations, which should include a minimum of six concentration levels, should fulfill these criteria. If more than six calibration standard levels are used and one of the calibration standards does not meet the criteria, this calibration standard should be rejected and the calibration curve without this calibration standard should be reevaluated, and a new regression analysis performed. If the rejected calibration standard is the LLOQ, the new lower limit for this analytical run should be the next lowest acceptable calibration standard of the calibration curve. This new lower limit calibration standard should retain its original acceptance criteria (i.e., ±15%). If the highest calibration standard is rejected, the ULOQ for this analytical run should be the next acceptable highest calibration standard of the calibration curve. The revised calibration range should cover at least three QC concentration levels (low, medium, and high). Study samples outside of the revised range should be reanalyzed. If replicate calibration standards are used and only one of the LLOQ or ULOQ standards fails, the calibration range is unchanged. At least 2/3 of the total QCs and at least 50% at each concentration level should be within ±15% of the nominal values. If these criteria are not fulfilled the analytical run should be rejected. A new analytical batch should be prepared for all study samples within the failed analytical run for subsequent analysis. In the cases where the failure is due to an assignable technical cause, samples may be reinjected. Analytical runs containing samples that are diluted and reanalyzed should include dilution QCs to verify the accuracy and precision of the dilution method during study sample analysis. The concentration of the dilution QCs should exceed that of the study samples being diluted (or of the ULOQ) and they should be diluted using the same dilution factor. If multiple dilution factors are used in one analytical run, then dilution QCs need only be diluted by the highest and lowest dilution factors. The within-run acceptance criteria of the dilution QC(s) will only affect the acceptance of the diluted study samples and not the outcome of the analytical run. When several analytes are assayed simultaneously, there should be one calibration curve for each analyte studied. If an analytical run is acceptable for one analyte but has to be rejected for another analyte, the data for the accepted analyte should be used. The determination of the 14  Contains Nonbinding Recommendations rejected analyte requires reextraction and analysis only for the analyte that is reanalyzed. Only data for this reanalyzed analyte needs to be reported. The back-calculated concentrations of the calibration standards and QCs of passed and accepted runs should be reported. The overall (between-run) accuracy and precision of the QCs of all accepted runs should be calculated at each concentration level and reported in the analytical report (refer to section VIII. (8) Documentation and Table 1). If the overall mean accuracy and/or precision fails the 15% criterion, an investigation to determine the cause of the deviation should be conducted. In the case of comparative BA/BE studies, it may result in the rejection of the data. 3. Calibration Range (3.3.3) 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 QCs, or add new QCs at different concentration levels as appropriate, to adequately reflect the concentrations of the study samples. At the intended therapeutic dose(s), if an unanticipated clustering of study samples at one end of the calibration curve is encountered after the start of sample analysis, the analysis should be stopped and either the standard calibration range narrowed (i.e., partial validation), existing QC concentrations revised, or QCs at additional concentrations added to the original curve within the observed range before continuing with study sample analysis. It is not necessary to reanalyze samples analyzed before optimizing the calibration curve range or QC concentrations. The same applies if a large number of the analyte concentrations of the study samples are above the ULOQ. The calibration curve range should be changed, if possible, and QC(s) added, or their concentrations modified. If it is not possible to change the calibration curve range or the number of samples with a concentration above the ULOQ is not large, samples should be diluted according to the validated dilution method. At least two QC 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. 4. Reanalysis of Study Samples (3.3.4) Possible reasons for reanalysis of study samples, the number of replicates, and the decision criteria to select the value to be reported should be predefined in the protocol, study plan, or SOP before the actual start of the analysis of the study samples. For study samples in which multiple analytes are being analyzed, a valid result for one analyte should not be rejected if the other analyte fails the acceptance criteria. The number of samples (and percentage of total number of samples) that have been reanalyzed should be reported and discussed in the Bioanalytical Report. For comparative BA/BE studies, a separate table should report values from rejected runs. 15  Contains Nonbinding Recommendations Some examples of reasons for study sample reanalysis are: Rejection of an analytical run because the run failed the acceptance criteria with regard to accuracy of the calibration standards and/or the precision and accuracy of the QCs IS response significantly different from the response for the calibration standards and QCs (as predefined in an SOP) The concentration obtained is above the ULOQ The concentration observed is below the revised LLOQ in runs where the lowest calibration standard has been rejected from a calibration curve, resulting in a higher LLOQ compared with other runs Improper sample injection or malfunction of equipment The diluted study sample is below the LLOQ Identification of quantifiable analyte levels in pre-dose samples, control, or placebo samples Poor chromatography (as predefined in an SOP) For comparative BA/BE studies, reanalysis of study samples for a PK reason (e.g., a sample concentration does not fit with the expected profile) should not be done, as it may bias the study result. Any reanalyzed samples should be identified in the Bioanalytical Report and the initial value, the reason for reanalysis, the values obtained in the reanalyzes, the final accepted value and a justification for the acceptance should be provided. Further, a summary table of the total number of samples that have been reanalyzed for each reason should be provided. In cases where the first analysis yields a nonreportable result, a single reanalysis is considered sufficient (e.g., concentration above the ULOQ or equipment malfunction). In cases where the value needs to be confirmed (e.g., pre-dose sample with measurable concentrations) replicate determinations are required if sample volume allows. The safety of trial subjects should take precedence over any other aspect of the trial. Consequently, there may be other circumstances when it is necessary to reanalyze specific study samples for the purpose of a safety investigation. 5. Reinjection of Study Samples (3.3.5) Reinjection of processed samples can be made in the case of equipment failure if reinjection reproducibility has been demonstrated during validation or provided in the Bioanalytical Report where it was conducted. Reinjection of a full analytical run or of individual calibration standards 16  Contains Nonbinding Recommendations or QCs simply because the calibration standards or QCs failed, without any identified analytical cause, should not be done. 6. Integration of Chromatograms (3.3.6) Chromatogram integration and reintegration should be described in a study plan, protocol, or SOP. Any deviation from the procedures described a priori should be discussed in the Bioanalytical Report. The list of chromatograms that required reintegration, including any manual integrations, and the reasons for reintegration should be included in the Bioanalytical Report. Original and reintegrated chromatograms and initial and repeat integration results should be kept for future reference and submitted in the Bioanalytical Report for comparative BA/BE studies. IV. LIGAND BINDING ASSAYS (4) A. Key Reagents (4.1) 1. Reference Standard (4.1.1) The reference standard should be well characterized and documented (e.g., CoA and origin). A biological drug has a highly complex structure and its reactivity with binding reagents for bioanalysis may be influenced by a change in the manufacturing process of the drug substance. It is recommended that the manufacturing batch of the reference standard used for the preparation of calibration standards and QCs is derived from the same batch of drug substance as that used for dosing in the nonclinical and clinical studies whenever possible. If the reference standard batch used for bioanalysis is changed, bioanalytical evaluation should be carried out with QCs from the original material and the new material prior to use to ensure that the performance characteristics of the method are within the acceptance criteria. 2. Critical Reagents (4.1.2) Critical reagents, including binding reagents (e.g., binding proteins, aptamers, antibodies, or conjugated antibodies) and those containing enzymatic moieties, have direct impact on the results of the assay and, therefore, their quality should be assured. Critical reagents bind the analyte and, upon interaction, lead to an instrument signal corresponding to the analyte concentration. The critical reagents should be identified and defined in the assay method. Reliable procurement of critical reagents, whether manufactured in-house or purchased commercially, should be considered early in method development. The data sheet for the critical reagent should include at a minimum identity, source, batch/lot number, purity (if applicable), concentration (if applicable), and stability/retest date/storage conditions (refer to Table 1). Additional characteristics may be warranted. A critical reagent lifecycle management procedure is necessary to ensure consistency between the original and new batches of critical reagents. Reagent performance should be evaluated using 17  Contains Nonbinding Recommendations the bioanalytical method. Minor changes to critical reagents would not be expected to influence the method performance, whereas major changes may significantly impact the performance. If the change is minor (e.g., the source of one reagent is changed), a single comparative accuracy and precision assessment is sufficient for characterization. If the change is major, then additional validation experiments are recommended. Ideally, assessment of changes should compare the method with the new reagents to the method with the old reagents directly. Major changes include, but are not limited to, change in production method of antibodies, additional blood collection from animals for polyclonal antibodies, and new clones or new supplier for monoclonal antibody production. Retest dates and validation parameters should be documented in order to support the extension or replacement of the critical reagent. Stability testing of the reagents should be based upon the performance in the bioanalytical method and upon general guidance for reagent storage conditions. It can be extended beyond the expiration date from the supplier. The performance parameters should be documented in order to support the extension or replacement of the critical reagent. B. Validation (4.2) Most often, microtiter plates are used for LBAs and study samples can be analyzed using an assay format of one or more well(s) per sample. The assay format should be specified in the protocol, study plan or SOP. If method development and method validation are performed using one or more well(s) per sample, then study sample analysis should also be performed using one or more well(s) per sample, respectively. If multiple wells per sample are used, the reportable sample concentration should be determined either by calculating the mean of the responses from the replicate wells or by averaging the concentrations calculated from each response. Data evaluation should be performed on reportable concentrations. 1. Specificity (4.2.1) Specificity is related to the concept of cross-reactivity in LBA. It is important that the binding reagent specifically binds to the target analyte but does not cross-react with coexisting structurally related molecules (e.g., endogenous compounds, isoforms, or structurally related concomitant medication). Specificity is evaluated by spiking blank matrix samples with related molecules at the maximal concentration(s) of the structurally related molecule anticipated in study samples. The accuracy of the target analyte at the LLOQ and at the ULOQ should be investigated in the presence of related molecules at the maximal concentration(s) anticipated in study samples. The response of blank samples spiked with related molecules should be below the LLOQ. The accuracy of the target analyte in presence of related molecules should be within ±25% of the nominal values. In the event of nonspecificity, the impact on the method should be evaluated by spiking increasing concentrations of interfering molecules in blank matrix and measuring the accuracy of the target analyte at the LLOQ and ULOQ. It is important to determine the minimum 18  Contains Nonbinding Recommendations concentration of the related molecule where interference occurs. Appropriate mitigation during sample analysis should be employed, e.g., it may be necessary to adjust the LLOQ/ULOQ accordingly or consider a new method. During method development and early method validation, these “related molecules” are frequently not available. Additional evaluation of specificity may be conducted after the original validation is completed. 2. Selectivity (4.2.2) Selectivity is the ability of the method to detect and differentiate the analyte of interest in the presence of nonspecific matrix components. The matrix can contain nonspecific matrix component such as degrading enzymes, heterophilic antibodies or rheumatoid factor which may interfere with the analyte of interest. Selectivity should be evaluated at the low end of an assay where problems occur in most cases, but it is recommended that selectivity is also evaluated at higher analyte concentrations. Therefore, selectivity should be evaluated using blank samples obtained from at least 10 individual sources and by spiking the individual blank matrices at the LLOQ and at the high QC level. Use of fewer sources may be acceptable in the case of rare matrices. The response of the blank samples should be below the LLOQ in at least 80% of the individual sources. The accuracy should be within ±25% at the LLOQ and within ±20% at the high QC level of the nominal concentration in at least 80% of the individual sources evaluated. Selectivity should be evaluated in lipemic samples and hemolyzed samples (refer to section III.B.1 (3.2.1)). For lipemic and hemolyzed samples, tests can be evaluated once using a single source of matrix. Selectivity should be assessed in samples from relevant patient populations (e.g., renally or hepatically impaired patients, inflammatory or immune-oncology patients if applicable). In the case of relevant patient populations, there should be at least five individual patients. 3. Calibration Curve and Range (4.2.3) The calibration curve demonstrates the relationship between the nominal analyte concentration and the response of the analytical platform to the analyte. Calibration standards, prepared by spiking matrix with a known quantity of analyte, span the calibration range and comprise the calibration curve. Calibration standards should be prepared in the same biological matrix as the study samples. The calibration range is defined by the LLOQ, which is the lowest calibration standard, and the ULOQ, which is the highest calibration standard. There should be one calibration curve for each analyte studied during method validation and for each analytical run. If needed, the use of surrogate matrix should be scientifically justified. A calibration curve should be generated with at least six concentration levels of calibration standards, including LLOQ and ULOQ standards, plus a blank sample. The blank sample should not be included in the calculation of calibration curve parameters. Anchor point samples at 19  Contains Nonbinding Recommendations concentrations below the LLOQ and above the ULOQ of the calibration curve may also be used to improve curve fitting. The relationship between response and concentration for a calibration curve is most often fitted by a 4- or 5-parameter logistic model if there are data points near the lower and upper asymptotes. Oher models should be suitably justified. A minimum of six independent runs should be evaluated over several days considering the factors that may contribute to between-run variability. The accuracy and precision of back-calculated concentrations of each calibration standard should be within ±25% of the nominal concentration at the LLOQ and ULOQ, and within ±20% at all other levels. At least 75% of the calibration standards excluding anchor points, and a minimum of 6 concentration levels of calibration standards, including the LLOQ and ULOQ, should meet the above criteria. The anchor points do not require acceptance criteria since they are beyond the quantifiable range of the curve. The calibration curve should preferably be prepared using freshly spiked calibration standards. If freshly spiked calibration standards are not used, the frozen calibration standards can be used within their defined period of stability. 4. Accuracy and Precision (4.2.4) a. Preparation of quality control samples (4.2.4.1) The QCs are intended to mimic study samples and should be prepared by spiking matrix with a known quantity of analyte, stored under the conditions anticipated for study samples, and analyzed to assess the validity of the analytical method. The dilution series for the preparation of the QCs should be completely independent from the dilution series for the preparation of calibration standard samples. If they are prepared from the same stock solution (or working stock) the accurate preparation and stability should be verified. The QCs should be prepared at a minimum of five concentration levels within the calibration curve range: The analyte should be spiked at the LLOQ, within three times of the LLOQ (low QC), around the geometric mean of the calibration curve range (medium QC), and at least at 75% of the ULOQ (high QC) and at the ULOQ. For non-accuracy and precision validation runs, low, medium, and high QCs may be analyzed in duplicate. These QCs, along with the calibration standards, should provide the basis for the acceptance or rejection of the run. b. Evaluation of accuracy and precision (4.2.4.2) Accuracy and precision should be determined by analyzing the QCs within each run (within-run) and in different runs (between-run). Accuracy and precision should be evaluated using the same runs and data. 20  Contains Nonbinding Recommendations Accuracy and precision should be determined by analyzing at least three replicates per run at each QC concentration level (LLOQ, low, medium, high, ULOQ) in at least six runs over 2 or more days. Reported method validation data and the determination of accuracy and precision should include all results obtained, except those cases where errors are obvious and documented. Within-run accuracy and precision data should be reported for each run. If the within-run accuracy or precision criteria are not met in all runs, an overall estimate of within-run accuracy and precision for each QC level should be calculated. Between-run (intermediate) precision and accuracy should be calculated by combining the data from all runs. The overall within-run and between-run accuracy at each concentration level should be within ±20% of the nominal values, except for the LLOQ and ULOQ, which should be within ±25% of the nominal value. Within-run and between-run precision of the QC concentrations determined at each level should not exceed 20%, except at the LLOQ and ULOQ, where it should not exceed 25%. For non-accuracy and precision validation runs, at least 2/3 of the total QCs and at least 50% at each concentration level should be within ±20% of the nominal values. Furthermore, the total error (i.e., sum of absolute values of the errors in accuracy (%) and precision (%)) should be evaluated. The total error should not exceed 30% (40% at LLOQ and ULOQ). 5. Carryover (4.2.5) Carryover is generally not an issue for LBA analyses. However, if the analytical platform is prone to carryover, the potential of carryover should be investigated by placing blank samples after the calibration standard at the ULOQ. The response of blank samples should be below the LLOQ. 6. Dilution Linearity and Hook Effect (4.2.6) Due to the narrow assay range in many LBAs, study samples may require dilution in order to achieve analyte concentrations within the range of the assay. Dilution linearity should be assessed to confirm: (i) that measured concentrations are not affected by dilution within the calibration range and (ii) that sample concentrations above the ULOQ of a calibration curve are not impacted by hook effect (i.e., a signal suppression caused by high concentrations of the analyte), whereby yielding an erroneous result. The same matrix as that of the study sample should be used for preparation of the QCs for dilution. Dilution linearity should be demonstrated by generating a dilution QC, i.e., spiking the matrix with an analyte concentration above the ULOQ, analyzed undiluted (for hook effect) and diluting this sample (to at least three different dilution factors) with blank matrix to a concentration within the calibration range. For each dilution factor tested, at least three independently prepared dilution series should be performed using the number of replicates that will be used in sample 21  Contains Nonbinding Recommendations analysis. The absence or presence of response reduction (hook effect) should be checked in the dilution QCs and, if observed and unable to be eliminated with reasonable measures, steps should be taken to mitigate this effect during the analysis of study samples. The calculated mean concentration for each dilution should be within ±20% of the nominal concentration after correction for dilution and the precision should not exceed 20%. The dilution factor(s) applied during study sample analysis should be within the range of dilution factors evaluated during validation. 7. Stability (4.2.7) Stability evaluations should be carried out to ensure that every step taken during sample preparation, processing, and analysis as well as the storage conditions used do not affect the concentration of the analyte. The storage and analytical conditions applied to the stability tests, such as the sample storage times and temperatures, sample matrix, anticoagulant, and container materials should reflect those used for the study samples. Reference to data published in the literature is not considered sufficient. Validation of storage periods should be performed on QCs that have been stored for a time that is equal to or longer than the study sample storage periods. Stability of the analyte in the studied matrix should be evaluated using low and high concentration QCs. Aliquots of the low and high QCs should be analyzed at time zero and after the applied storage conditions that are to be evaluated. One bulk QC should be prepared at each concentration level. For each concentration tested, the bulk sample should be divided into a minimum of three aliquots that will be stored, stressed, and analyzed. The QCs should be analyzed against a calibration curve, obtained from freshly spiked calibration standards in a run with its corresponding freshly spiked QCs or QCs for which stability has been proven. While the use of freshly prepared calibration standards and QCs is the preferred approach, it is recognized that in some cases, for macromolecules, it may be necessary to freeze them overnight. In such cases, valid justification should be provided, and freeze-thaw stability demonstrated. QCs should be kept frozen for at least 12 hours between the thawing cycles. The mean concentration at each QC level should be within ±20% of the nominal concentration. Since sample dilution may be required for many LBA methods due to a narrow calibration range, the concentrations of the study samples may be consistently higher than the ULOQ of the calibration curve. If this is the case, the concentration of the QCs should be adjusted, considering the applied sample dilution, to represent the actual sample concentration range. For fixed dose combination products and specifically labeled drug regimens, the freeze-thaw, bench top and long-term stability tests of an analyte in matrix should be conducted with the matrix spiked with all of the dosed compounds, on a case-by-case basis. 22  Contains Nonbinding Recommendations As mentioned in section III.B.8 (3.2.8), the investigation of stability should cover bench top (short-term) stability at room temperature or sample preparation temperature and freeze-thaw stability. In addition, long-term stability should be studied. For chemical drugs, the stability at one temperature (e.g., -20°C) can be extrapolated to lower temperatures (e.g., -70°/-80°C). For biological drugs, a bracketing approach can be applied, e.g., in the case that the stability has been demonstrated at -70°/-80°C and at -20°C, then it is not necessary to investigate the stability at temperatures in between those two points at which study samples will be stored. C. Study Sample Analysis (4.3) The analysis of study samples can be carried out after validation has been completed, however, it is understood that some parameters may be completed at a later stage (e.g., long-term stability). By the time the data are submitted to a regulatory authority, the bioanalytical method validation should have been completed. The study samples, QCs, and calibration standards should be processed in accordance with the validated analytical method. Refer to Table 1 for recommendations regarding documentation. 1. Analytical Run (4.3.1) An analytical run should consist of a blank sample, calibration standards at a minimum of six concentration levels, at least three levels of QCs (low, medium, and high) applied as two sets (or at least 5% of the number of study samples, whichever is higher) and the study samples to be analyzed. The blank sample should not be included in the calculation of calibration curve parameters. The QCs should be placed in the run in such a way that the accuracy and precision of the whole run is ensured taking into account that study samples should always be bracketed by QCs. Most often, microtiter plates are used for LBAs. An analytical run may comprise of one or more plate(s). Typically, each plate contains an individual set of calibration standards and QCs. If each plate contains its own calibration standards and QCs, then each plate should be assessed on its own. However, for some platforms the sample capacity may be limited. In this case, sets of calibration standards may be placed on the first and the last plate, but QCs should be placed on every single plate. QCs should be placed at least at the beginning (before) and at the end (after) of the study samples of each plate. The QCs on each plate and each calibration curve should fulfill the acceptance criteria for an analytical run (refer to section IV.C.2 (4.3.2)). For the calculation of concentrations, the calibration standards should be combined to conduct one regression analysis. If the combined calibration curve does not pass the acceptance criteria the whole run fails. 2. Acceptance Criteria for an Analytical Run (4.3.2) Criteria for the acceptance or rejection of an analytical run should be defined in the protocol, in the study plan, or in an SOP. In the case that a run contains multiple batches, acceptance criteria 23  Contains Nonbinding Recommendations should be applied to the whole run and to the individual batches. It is possible for the run to meet acceptance criteria, even if a batch within that run is rejected for failing to meet the batch acceptance criteria. Calibration standards in a failed batch should not be used to support the acceptance of other batches within the analytical run. The back-calculated concentrations of the calibration standards should be within ±20% of the nominal value at each concentration level, except for the LLOQ and the ULOQ, for which it should be within ±25%. At least 75% of the calibration standards, with a minimum of 6 concentration levels, should fulfill this criterion. This requirement does not apply to anchor calibration standards. If more than six calibration standards are used and 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 reevaluated, and a new regression analysis performed. If the rejected calibration standard is the LLOQ, the new lower limit for this analytical run should be the next lowest acceptable calibration standard of the calibration curve. If the highest calibration standard is rejected, the new upper limit for this analytical run should be the next acceptable highest calibration standard of the calibration curve. The new lower and upper limit calibration standard will retain their original acceptance criteria (i.e., ±20%). The revised calibration range should cover all QCs (low, medium, and high). The study samples outside of the revised assay range should be reanalyzed. Each run should contain at least three levels of QCs (low, medium, and high). During study sample analysis, the calibration standards and QCs should mimic the analysis of the study sample with regard to the number of wells used per study sample. At least 2/3 of the QCs and 50% at each concentration level should be within ±20% of the nominal value at each concentration level. Exceptions to these criteria should be justified and predefined in the SOP or protocol. The overall mean accuracy and precision of the QCs of all accepted runs should be calculated at each concentration level and reported in the analytical report. In the case that the overall mean accuracy and/or precision exceeds 20%, additional investigations should be conducted to determine the cause(s) of this deviation. In the case of comparative BA/BE studies, it may result in the rejection of the data. 3. Calibration Range (4.3.3) At least two QC sample levels should fall within the range of concentrations measured in study samples. At the intended therapeutic dose(s), if an unanticipated clustering of study samples at one end of the calibration curve is encountered after the start of sample analysis, the analysis should be stopped and either the standard calibration range narrowed (i.e., partial validation), existing QC concentrations revised, or QCs at additional concentrations added to the original curve within the observed range before continuing with study sample analysis. It is not necessary to reanalyze samples analyzed before optimizing the calibration curve range or QC concentrations. 24  Contains Nonbinding Recommendations 4. Reanalysis of Study Samples (4.3.4) Possible reasons for reanalysis of study samples, the number reanalyzed, and the decision criteria to select the value to be reported should be predefined in the protocol, study plan, or SOP, before the actual start of the analysis of the study samples. The number of samples (and percentage of total number of samples) that have been reanalyzed should be reported and discussed in the Bioanalytical Report. For comparative BA/BE studies, a separate table should report values from rejected runs. Some examples of reasons for study sample reanalysis are: Rejection of an analytical run because the run failed the acceptance criteria with regard to accuracy of the calibration standards and/or the precision and accuracy of the QCs The concentration obtained is above the ULOQ The concentration obtained is below the LLOQ in runs where the lowest calibration standard has been rejected from a calibration curve, resulting in a higher LLOQ compared with other runs Malfunction of equipment The diluted sample is below the LLOQ Identification of quantifiable analyte levels in pre-dose samples, control, or placebo samples When samples are analyzed in more than one well and nonreportable values are obtained due to one replicate failing the predefined acceptance criteria (e.g., excessive variability between wells, one replicate being above the ULOQ or below the LLOQ) For comparative BA/BE studies, reanalysis of study samples for a PK reason (e.g., a sample concentration does not fit with the expected profile) should not be done, as it may bias the study result. The reanalyzed samples should be identified in the Bioanalytical Report and the initial value, the reason for reanalysis, the values obtained in the reanalysis, the final accepted value, and a justification for the acceptance should be provided. Further, a summary table of the total number of samples that have been reanalyzed due to each reason should be provided. In cases where the first analysis yields a nonreportable result, a single reanalysis is considered sufficient (e.g., concentration above the ULOQ or excessive variability between wells). The analysis of the samples should be based on the same number of wells per study sample as in the initial analysis. In cases where the value needs to be confirmed, (e.g., pre-dose sample with measurable concentrations) multiple determinations are recommended where sample volume allows. 25  Contains Nonbinding Recommendations The safety of trial subjects should take precedence over any other aspect of the trial. Consequently, there may be other circumstances when it is necessary to reanalyze specific study samples for the purpose of an investigation. V. INCURRED SAMPLE REANALYSIS (5) The performance of study samples may differ from that of the calibration standards and QCs used during method validation, which are prepared by spiking blank matrix. Differences in protein binding, back-conversion of known and unknown metabolites, sample inhomogeneity, concomitant medications, or biological components unique to the study samples may affect measured concentrations of the analyte in study samples. Incurred sample reanalysis (ISR) is intended to verify the reliability of the reported sample analyte concentrations. ISR should be performed at least in the following situations: For nonclinical studies within the scope of this guidance, ISR should, in general, be performed at least once per species. All pivotal comparative BA/BE studies. First clinical trial in subjects. Pivotal early patient trial(s), once per patient population. First or pivotal trial in patients with impaired hepatic and/or renal function. ISR is conducted by repeating the analysis of a subset of samples from a given study in separate (i.e., different to the original) runs on different days using the same bioanalytical method. The extent of ISR depends upon the analyte and the study samples and should be based upon an in-depth understanding of the analytical method and analyte. However, as a minimum, if the total number of study samples is less than or equal to 1000, then 10% of the samples should be reanalyzed; if the total number of samples is greater than 1000, then 10% of the first 1000 samples (100) plus 5% of the number of samples that exceed 1000 samples should be assessed. Objective criteria for choosing the subset of study samples for ISR should be predefined in the protocol, study plan, or an SOP. While the subjects/animals should be picked as randomly as possible from the dosed study population, adequate coverage of the concentration profile is important. Therefore, it is recommended that the samples for ISR be chosen around the maximum concentration (Cmax) and some in the elimination phase. Additionally, the samples chosen should be representative of the whole study. Samples should not be pooled, as pooling may limit anomalous findings. ISR samples and QCs should be processed and analyzed in the same manner as in the original analysis. ISR should be performed within the stability window of the analyte, but not on the same day as the original analysis. 26  Contains Nonbinding Recommendations The percent difference between the initial concentration and the concentration measured during the repeat analysis should be calculated in relation to their mean value using the following equation: repeat value − initial value % difference = × 100 mean value For chromatographic methods, the percent difference should be within ±20% for at least 2/3 of the repeats. For LBAs, the percent difference should be within ± 30% for at least 2/3 of the repeats. If the overall ISR results fail the acceptance criteria, an investigation should be conducted and the causes remediated. There should be an SOP that directs how investigations are triggered and conducted. If an investigation does not identify the cause of the failure, the potential impact of an ISR failure on study validity should also be provided in the Bioanalytical Report. If ISR meets the acceptance criteria yet shows large or systemic differences between results for multiple samples, this may indicate analytical issues and it is advisable to investigate this further. Examples of trends that are of concern may include: All ISR samples from one subject fail All ISR samples from one run fail All aspects of ISR evaluations should be documented to allow reconstruction of the study and any investigations. Individual samples that are quite different from the original value (e.g., > 50%, “flyers”) should not trigger reanalysis of the original sample and do not need to be investigated. ISR sample data should not replace the original study sample data. VI. PARTIAL AND CROSS VALIDATION (6) A. Partial Validation (6.1) Partial validations evaluate modifications to already fully validated bioanalytical methods. Partial validation can range from as little as one within-run accuracy and precision determination to a nearly full validation. If stability is established at one facility it does not necessarily need to be repeated at another facility. For chromatographic methods, typical bioanalytical method modifications or changes that fall into this category include, but are not limited to, the following situations: Analytical site change using same method (i.e., bioanalytical method transfers between laboratories) A change in analytical method (e.g., change in detection systems, platform) 27  Contains Nonbinding Recommendations A change in sample processing procedures A change in sample volume (e.g., the smaller volume of pediatric samples) Changes to the calibration concentration range A change in anticoagulant (but not changes in the counter-ion) in biological fluids (e.g., heparin to ethylenediaminetetraacetic acid (EDTA)) Change from one matrix within a species to another (e.g., switching from human plasma to serum or cerebrospinal fluid) or changes to the species within the matrix (e.g., switching from rat plasma to mouse plasma) A change in storage conditions For LBAs, typical bioanalytical method modifications or changes that fall into this category include, but are not limited to, the following situations: Changes in LBA critical reagents (e.g., lot-to-lot changes) Changes in MRD A change in storage conditions Changes to the calibration concentration range A change in analytical method (e.g., change in detection systems, platform) Analytical site change using same method (i.e., bioanalytical method transfers between laboratories) A change in sample preparation A change in anticoagulant (but not changes in the counter-ion) in biological fluids (e.g., heparin to ethylenediaminetetraacetic acid (EDTA)) The parameters of the partial validations should meet the full validation criteria. If these criteria are not satisfied, additional investigation and validation is warranted. B. Cross Validation (6.2) Cross validation is required to demonstrate how the reported data are related when multiple bioanalytical methods and/or multiple bioanalytical laboratories are involved. Cross validation is required under the following situations: 28  Contains Nonbinding Recommendations Data are obtained from different fully validated methods within a study. Data are obtained within a study from different laboratories with the same bioanalytical method. Data are obtained from different fully validated methods across studies that are going to be combined or compared to support special dosing regimens, or regulatory decisions regarding safety, efficacy, and labeling. If data are obtained from different fully validated methods, and these data are not to be combined across studies, cross validation is not generally required. Cross validation should be performed in advance of study samples being analyzed, if possible. Cross validation should be assessed by measuring the same set of QCs (low, medium, and high) at least in triplicate and study samples (if available) that span the study sample concentration range (n≥30) with both methods, or in both laboratories. Bias can be assessed by Bland-Altman plots or Deming regression. Other methods appropriate for assessing agreement between two methods (e.g., concordance correlation coefficient) may be used too. Alternatively, the concentration vs. time curves for study samples could be plotted for samples analyzed by each method to assess bias. The use of multiple bioanalytical methods for the measurement of the same analyte in the conduct of one comparative BA/BE study is strongly discouraged. VII. ADDITIONAL CONSIDERATIONS (7) A. Methods for Analytes That Are Also Endogenous Molecules (7.1) For analytes that are also endogenous molecules (e.g., replacement therapies), the accuracy of the measurement of the analytes poses a challenge when the method cannot distinguish between the therapeutic agent and the endogenous molecule. Furthermore, the endogenous levels of the analyte may vary because of age, gender, race, diurnal variations, illness, or as side effect of drug treatment. This section describes some of the approaches that may be used to assess concentrations of analytes that are also endogenous molecules. As a reminder, biomarkers are outside of the scope of this guidance. If available, biological matrix to prepare calibration standards and QCs should be the same as the study samples (i.e., authentic biological matrix) and it should be free of matrix effect and interference, as described in sections III. (3) and IV. (4). The endogenous concentration in the biological matrix chosen should be low enough to obtain an adequate signal-to-noise ratio (e.g.,