1、 Bioanalytical Method Validation 05/24/18 Bioanalytical Method Validation Guidance for Industry U.S. De partme nt of He alth and Human Se rvice s Food and Drug Adminis tration Ce nte r for Drug Evaluation and Re s earch (CDER) Cen ter f or V eter i n a r y Med i ci n e (CV M) May 2018 Biopharmaceuti
2、cs Bioanalytical Method Validation 05/24/18 Bioanalytical Method Validation Guidance for Industry Additional copies are available from: Office of Communications, Divis io n of Drug Inf orma ti o n Center f or Drug Eval ua t io n and Resea rc h Food and Drug Admi ni st ra ti on 10001 New Hampshire Av
3、e., Hillandale Bldg., 4 thFloor Silver Spri n g, MD 209 9 3-0002 Phone: 855-543-3 78 4 or 301-796-3400; Fax: 301-431-6353 Email: druginfofda.hhs.gov http:/www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm and/or Policy and Reg ul a ti on s St af f , HFV-6 Center for Vet
4、erinary Medicine Food and Drug Admi ni st ra ti on 7500 Standish Place, Roc kville, MD 20855 http:/www.fda.gov/AnimalVeterinary/GuidanceComplianceEnforcement/GuidanceforIndustry/default.htm U.S. De partme nt of He alth and Human Se rvice s Food and Drug Administration Ce nte r for Drug Evaluation an
5、d Re s earch (CDER) Cen ter f or V eter i n a r y Med i ci n e (CV M) May 2018 Biopharmaceutics Contains Nonbinding Recommendations Bioanalytical Method Validation 05/24/18 TABLE OF CONTENTS I. INTRODUCTION 1 II. BACKGROUND 2 III. BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION . 4 A. Guiding Princi
6、ples 4 B. Bioanalytical Parameters of CCs and LBAs 5 1. Reference Standards and Critical Reagents 5 2. Calibration Curve. 6 3. Quality Control Samples 7 4. Selectivity and Specificity. 7 5. Sensitivity 8 6. Accuracy, Precision, and Recovery . 8 7. Stability 9 8. Dilution Effects .10 9. Partial and C
7、ross Validations11 C. Validated Methods: Expectations of In-Study Analysis and Reporting 12 IV. INCURRED SAMPLE REANALYSIS 14 V. ADDITIONAL ISSUES 15 A. Endogenous Compounds 15 B. Biomarkers .15 C. Diagnostic Kits 16 D. Bridging Data From Multiple Bioanalytical Technologies .17 E. Dried Blood Spots
8、18 VI. DOCUMENTATION . 18 A. Summary Information18 B. Documentation for Method Validation and Bioanalytical Reports .19 VII. APPENDIX. 20 Table 1. Recommendations and Acceptance Criteria for Bioanalytical Method Validation and In-Study Conduct. .20 Table 2. Documentation and Reporting 28 Table 3.Exa
9、mple of an Overall Summary Table for a Method Validation Report* or a Clinical Study Report.33 Table 4. Example of Summary Analytical Runs for a Bioanalytical Study Report36 VIII. GLOSSARY . 37 Contains Nonbinding Recommendations Bioanalytical Method Validation 05/24/18 1 Bioanalytical Method Valida
10、tion Guidance for Industry 1This 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 o
11、f 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 This guidance helps sponsors of investigational new drug applications (INDs) or applicants of new drug applications (NDAs),
12、 abbreviated new drug applications (ANDAs), biologic license applications (BLAs), and supplements validate bioanalytical methods used in human clinical pharmacology, bioavailability (BA), and bioequivalence (BE) studies that require pharmacokinetic, toxicokinetic, or biomarker concentration evaluati
13、on. 2This guidance can also inform the development of bioanalytical methods used for nonclinical studies that require toxicokinetic or biomarker concentration data. For studies related to the veterinary drug approval process such as investigational new animal drug applications (INADs), new animal dr
14、ug applications (NADAs), and abbreviated new animal drug applications (ANADAs), this guidance may apply to blood and urine BA, BE, and pharmacokinetic studies. The information in this guidance applies to bioanalytical procedures such as chromatographic assays (CCs) and ligand binding assays (LBAs) t
15、hat quantitatively determine the levels of drugs, their metabolites, therapeutic proteins, and biomarkers in biological matrices such as blood, serum, plasma, urine, and tissue such as skin. This final guidance incorporates public comments to the revised draft published in 2013 and provides recommen
16、dations for the development, validation, and in-study use of bioanalytical methods. The recommendations can be modified with justification, depending on the specific type of bioanalytical method. This guidance reflects advances in science and technology related to validating bioanalytical methods. I
17、n general, FDAs guidance documents do not establish legally enforceable responsibilities. Instead, guidances describe the Agencys current thinking on a topic and should be viewed only 1Thi s gui dan ce ha s be en prep ared by th e Offi ce of C l i ni cal Pharm acol ogy i n the C ente r for Drug E va
18、l uati on and Research and the Center for V eterinary Medicine at the Food and Drug Adm inistration. 2Thi s gui dan ce app l i es to bot h spo n so rs and appl i can t s. The use of the word sponsor applies to both s ponsors and applicants and hence, INDs , NDA s , BLA s , and A NDAs. Contains Nonbi
19、nding Recommendations Bioanalytical Method Validation 05/24/18 2 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. II. BACKGROUND The 2001 guidance for
20、 industry on Bioanalytical Method Validation was originally based on the deliberations of two workshops described in publications entitled: Analytical Methods Validation: Bioavailability, Bioequivalence, and Pharmacokinetic Studies 3 Bioanalytical Methods Validation: A Revisit With a Decade of Progr
21、ess 4Additional workshops, summarized in the following publications, have informed subsequent revisions (e.g., the 2013 draft guidance for industry entitled Bioanalytical Method Validation 5 ): Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and L
22、igand Binding Assays 6 The AAPS/FDA Workshop on Incurred Sample Reanalysis 7 The AAPS Work shop on Crystal City V Quantitative Bioanalytical Method Validation and Implementation: 2013 Revised FDA Guidance 83Shah, V P, KK Mi dha, S Di ghe, IJ McG i l veray, JP Sk el l y, A Y acobi , T L ayl off, CT V
23、is wan at h an, CE Co o k, RD McDowell, KA Pittm an, S Spector, 1992, A n aly tical M etho ds Valid atio n : Bio av ailability , Bio eq u iv alen ce, an d Pharm acokinetic Studies, Pharm Res , 9:588-592. 4Shah, V P, KK Mi dha, JW Fi ndl ay, HM Hi l l , JD Hul se, IJ McG i l veray, G McKay , KJ M ill
24、er, RN Patn aik, M L Po well, A To n elli, CT Vis wan ath an, A Yaco b i, 2000, Bioanalytical Methods V alidation: A Revisit With a Decade of Progress, Pharm Res , 17:1551-1557. 5When fi nal , thi s gui da n ce wi l l represent the FDAs curre nt thi nk i ng on th i s top i c. For the m ost rece nt v
25、ersi on of a gui dance , chec k the FDA Drugs gu i dan c e We b pag e at http:/www.fda.gov/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/default.htm 6V iswanathan, CT, B Surendra, B Booth, AJ DeStefano, MJ Rose , J Sailstad, V P Shah, JP Skelly, PG Swann, R Weiner, 2007, Quantitative Bioan
26、alytica l Methods V alidation and Im plem entation: Best Practice s for C hrom atograp h i c and L i gand B i ndi ng Ass ay s, Pharm R es, 2 4 : 1 9 6 2-1973. 7Fas t, DM, M Kelley, CT V iswanathan, J OShaughne ssy , SP Ki ng, A C haudhary , R Wei ner, AJ DeStefan o, D Tang, 2 0 0 9 , Work sho p R ep
27、ort an d Fol l ow-Up A APS W orks hop on Current Topics in GLP Bioanalys is : A ssay R eproduci b i l i ty for Incurred Sam pl es Im pl i cati ons of C rystal C i ty R ecom m endat i on s, AAPS J, 1 1 : 2 3 8-241. 8B ooth, B , ME Arnol d, B DeSi l va, L Am aravadi , S Dudal , E Fl uhl er, B G orovi
28、ts, SH Hai dar, J Kadavi l , S L owes, R Ni chol son, M R ock , M Sk el l y, L Stevenso n, S Subram ani am , R Wei ner, E Wool f, 2 0 1 5 , Work sho p R eport : Contains Nonbinding Recommendations Bioanalytical Method Validation 05/24/18 3 Validated analytical methods for the quantitative evaluation
29、 of analytes (i.e., drugs, including biologic products, and their metabolites) and biomarkers in a given biological matrix (e.g. blood, plasma, serum, or urine) are critical for the successful conduct of nonclinical, biopharmaceutics, and clinical pharmacology studies. These validated methods provid
30、e critical data to support the safety and effectiveness of drugs and biologic products. Validating the analytical method ensures that the data are reliable by addressing certain key questions, including: Does the method measure the intended analyte? For example, does anything interfere with the meas
31、urement, and is the method specific or selective for the analyte? What is the variability associated with these measurements? For example, what are the accuracy and precisionof the method? What is the range in measurements that provide reliable data? For example, what is the sensitivity of the metho
32、d (e.g., what is the lower limit of quantitation (LLOQ) of the method, and what is the upper limit of quantitation the method (ULOQ)?) How do sample collection, handling, and storage affect the reliability of the data from the bioanalytical method? For example, what steps need to be followed while c
33、ollecting samples? Do the samples need to be frozen during shipping? What temperatures are required to store the samples, and how long can the samples be stored? When changes are made to a validated method, the sponsor should conduct additional validation (i.e., partial or cross validation). The fit
34、-for-purpose (FFP) concept states that the level of validation should be appropriate for the intended purpose of the study. The key questions listed above should be evaluated relative to the stage of drug development. Pivotal studies submitted in an NDA, BLA, or ANDA that require regulatory decision
35、 making for approval, safety or labeling, such as BE or pharmacokinetic studies, should include bioanalytical methods that are fully validated. Exploratory methods that would not be used to support regulatory decision making (e.g., candidate selection) may not require such stringent validation. This
36、 FFP concept applies to drugs, their metabolites, and biomarkers. The analytical laboratory conducting toxicology studies for regulatory submissions should adhere to 21 CFR 58, Good Laboratory Practices (GLPs). 9The bioanalytical method for human BA, BE, and pharmacokinetic studies must meet the cri
37、teria specified in 21 CFR 320 Bioequivalence and Bioavailability Requirements (i.e., 21 CFR 320.29). Cry s t al Cit y V Quantitative Bioanalytical Method V alidation and Im plem entation: The 2013 Revised FDA G uidance, AAPS J, 17:277-288. 9For the Center for V eterinary Medicine, all BE studies are
38、 subject to G ood L aboratory Practice s. Contains Nonbinding Recommendations Bioanalytical Method Validation 05/24/18 4 The following sections discuss the development, validation, and in-study use of bioanalytica l methods and how best to document validation methods and results. Refer to the Glossa
39、ry for the definitions of assay parameters and analytical terms used in this guidance. III. BIOANALYTICAL METHOD DEVELOPMENT AND VALIDATION A. Guiding Principles The purpose of bioanalytical method development is to define the design, operating conditions, limitations, and suitability of the method
40、for its intended purpose and to ensure that the method is optimized for validation. Before the development of a bioanalytical method, the sponsor should understand the analyte of interest (e.g., determine the physicochemical properties of the drug, in vitro and in vivo metabolism, and protein bindin
41、g) and consider aspects of any prior analytical methods that may be applicable. The elements and acceptance cri teri a of method development and validation are summarized in Table 1. Table 2 describes how the sponsor should document the development and validation of the bioanalytical assay and where
42、 it should be stored or submitted. Method development involves optimizing the procedures and conditions involved with extracting and detecting the analyte. Method development includes the optimization of the following bioanalytical parameters (which are discussed in greater detail in section III.B)
43、to ensure that the method is suitable for validation: Reference standards Critical reagents Calibration curve Quality control samples (QCs) Selectivity and specificity Sensitivity Accuracy Precision Recovery Stability of the analyte in the matrix Bioanalytical method development does not require ext
44、ensive record keeping or notation. However, the sponsor should record the changes to procedures as wel l as any issues and their resolutions during development of the bioanalytical method to provide a rationale for any changes during the development of the method. Bioanalytical method validation pro
45、ves that the optimized method is suited to the analysis of the study samples. The sponsor should: Contains Nonbinding Recommendations Bioanalytical Method Validation 05/24/18 5 Conduct a full validation of any new bioanalytical method for the analysis of a new drug entity, its metabolite(s), or biom
46、arkers. Conduct a full validation for any revisions to an existing validated method that adds a metabolite or an additional analyte. Establish a detailed, written description (e.g., protocol, study plan, and/or standard operating procedure (SOP) for the bioanalytical method before initiating validat
47、ion. The description should identify procedures that control critical parameters in the method (e.g., environmental, matrix, procedural variables) from the time of collection of the samples to the time of analysis to minimize their effects on the measurement of the analyte in the matrix. Document an
48、d report (in the method validation report) all experiments used to make claims or draw conclusions about the validity of the method. Validate the measurement of each analyte in the biological matrix. The specific recommendations and acceptance cri teri a for each bi oanal yti cal parameter are l i s
49、ted i n Table 1. B. Bioanalytical Parame te rs of CCs and LBAs The bioanalytical parameters applicable to CCs and LBAs are discussed below. Issues unique to either CCs or LBAs are speci fi cal l y i denti fi ed. 1. Reference Standards and Critical Reagents The sponsor should appropriately characterize and document (e.g. determine the identity, purity, and stability) all reference standards and cri ti cal reagents, such as anti bodi es, l abel ed anal ytes, and matrices and store them under defined conditions. a. Reference standards
