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Recommendations for the Bioanalytical Method Validation Of Pharmaceutical Research, Vol. 20, No. 11, November 2003 (© 2003) Research Paper Recommendations for the studies was held more than a decade ago in Crystal City, VA (1,2). The proceedings and recommendations of that meeting Bioanalytical Method Validation of essentially became a de facto guideline for bioanalytical meth- Ligand-binding Assays to Support ods validation within the pharmaceutical industry. The con- ference addressed validation of bioanalytical methods in gen- Pharmacokinetic Assessments eral, but acknowledged differences between chromatographic of Macromolecules and nonchromatographic assays, including immunoassays and microbiological-based methods. After the original Crystal City conference, bioanalytical methods validation was ad- dressed subsequently several times at meetings and in publi- Binodh DeSilva,1 Wendell Smith,2 Russell Weiner,3 cations (3–6). Most emphasis to date has been on validation of Marian Kelley,4,11 JoMarie Smolec,5 Ben Lee,6 bioanalytical methods for conventional small molecular Masood Khan,7 Richard Tacey,8 Howard Hill,9 and weight drugs, principally due to the rapid growth during the Abbie Celniker10 1990s in the use of hyphenated mass-spectrometry as a rou- tine analytical tool. Because of the evolution in divergent analytical tech- Received July 2, 2003; accepted July 30, 2003 nologies for conventional small molecules and macromol- Purpose. With this publication a subcommittee of the AAPS Ligand ecules and the growth in the interest of macromolecular Binding Assay Bioanalytical Focus Group (LBABFG) makes recom- therapeutics, the topic of bioanalytical methods validation mendations for the development, validation, and implementation of was revisited in 2000 in 2 meetings, one focused on small ligand binding assays (LBAs) that are intended to support pharma- molecule analytes (7) and the other focused on macromol- cokinetic and toxicokinetic assessments of macromolecules. ecules (8). The macromolecule workshop addressed diverse Methods. This subcommittee was comprised of 10 members repre- topics and issues including quantitative immunoassays, bioas- senting Pharmaceutical, Biotechnology, and the contract research or- says, biomarkers, and assays for neutralizing antibodies. Ad- ganization industries from the United States, Canada, and Europe. ditionally, the workshop report provided some default criteria Each section of this consensus document addresses a specific analyti- for method and run acceptance of assays for macromolecules cal performance characteristic or aspect for validation of a LBA. across a broad range of analytical methodologies (8). Because Within each section the topics are organized by an assay’s life cycle, the development phase, pre-study validation, and in-study validation. this meeting was the first one focused solely on bioanalytical Because unique issues often accompany bioanalytical assays for mac- methods validation for assays of macromolecules and it cov- romolecules, this document should be viewed as a guide for designing ered numerous topics, it was clear at the meeting’s end that and conducting the validation of ligand binding assays. more in-depth discussions were needed to address the unique Results. Values of ±20% (25% at the lower limit of quantification analytical aspects of ligand-binding assays (LBAs) for mac- [LLOQ]) are recommended as default acceptance criteria for accu- romolecules and achieve a confluence of opinions for harmo- racy (% relative error [RE], mean bias) and interbatch precision nization of bioanalytical methods validation. Accordingly, a (%coefficient of variation [CV]). In addition, we propose as second- group of the meeting’s attendees formed the Ligand Binding ary criteria for method acceptance that the sum of the interbatch Assay Bioanalytical Focus Group (LBABFG) within the precision (%CV) and the absolute value of the mean bias (%RE) be American Association of Pharmaceutical Scientists (AAPS) less than or equal to 30%. This added criterion is recommended to help ensure that in-study runs of test samples will meet the proposed to create a forum to address issues and promote education for run acceptance criteria of 4-6-30. Exceptions to the proposed process bioanalysis of a broad range of analytes by LBAs. Because and acceptance criteria are appropriate when accompanied by a guidance is lacking for bioanalytical methods validation for sound scientific rationale. assays of macromolecules, a subcommittee of the LBABFG Conclusions. In this consensus document, we attempt to make rec- undertook development of this consensus report. This docu- ommendations that are based on bioanalytical best practices and sta- ment builds on recent publications for validation of immuno- tistical thinking for development and validation of LBAs. assays (3,8,9) and makes specific recommendations for vali- KEY WORDS: best practices; bioanalytical assay; biological matri- dation of LBAs to support pharmacokinetics assessments of ces; consensus document; immunoassay. macromolecules, includes examples of prestudy validation data, summary tables, and description of calculations. For this HISTORICAL PERSPECTIVE document, LBA is synonymous with immunoassay and refers to any analytical method, in which quantification is based on The first conference on bioanalytic methods validation to macromolecular interactions. support bioavailability, bioequivalence, and pharmacokinetic 11 To whom correspondence should be addressed. (e-mail: 1 Amgen Inc, Thousand Oaks, California. [email protected]) 2 Lilly Research Laboratories, Greenfield, Indiana. ABBREVIATIONS: AAPS, American Association of Pharmaceuti- 3 Bristol-Myers Squibb, Princeton, New Jersey. cal Scientists; ANOVA, Analysis of variance; CSF, Cerebral spinal 4 Johnson and Johnson Pharmaceutical, Raritan, New Jersey. fluid; CV, Coefficient of variation; ELISA, enzyme-linked immuno- 5 Alta Analytical Laboratory, San Diego, California. sorbent assay; HQC, high quality control; LBABFG, Ligand-binding 6 Pfizer Global Research and Development, Ann Arbor, Michigan. Assay Bioanalytical Focus Group; LBA, ligand-binding assay; 7 Covance Laboratories, Chantilly, Virginia. LLOQ, lower limit of quantification; LQC, lower quality control; 8 PPD Development, Richmond, Virginia. MRD, minimum required dilution; MQC, mid-quality control; QC, 9 HLS, Cambridgeshire, UK. quality control; ULOQ, upper limit of quantification; RE, relative 10 Millennium Pharmaceuticals, Cambridge, Massachusetts. error; RIA, radioimmunoassay; SOP, standard operating procedure. 1885 0724-8741/03/1100-1885/0 © 2003 Plenum Publishing Corporation 1886 DeSilva et al. INTRODUCTION reagents, sample preparation, preliminary stability and a pre- liminary assessment of assay robustness. At the end of the An assay life cycle can be categorized into 3 general development portion of the assay life cycle, a draft method or phases: method development, prestudy validation, and in- an assay worksheet should be generated to be referred to study validation. During method development, an assay con- during the prestudy validation. cept is evaluated, which will be confirmed during the prestudy validation phase, and applied during the in-study validation Prestudy Validation phase. To ensure that an assay can be used in the quantification A validation plan should be written before the initiation of a macromolecule in support of pharmacokinetics assess- of the prestudy validation experiments. Alternatively, refer- ment and withstand regulatory scrutiny, specific assay com- ence to an appropriate standard operating procedure (SOP) ponents need to be evaluated, subsequently validated, and can be made to ensure that a documented outline exists for continually monitored. Therefore, validation is a continuing the experiments required for prestudy validation. This plan process as long as the assay is in use. can be a stand-alone document or can be contained in a labo- Validation is not a static process but a dynamic one. The ratory notebook or some comparable format. The documen- components to be evaluated and the recommendations for tation should include a description of the intended use of the each of the stages in the assay lifecycle are summarized in method under consideration and a summary of the perfor- Table I. This paper is divided into these same components. mance parameters to be validated that should include, but Under each of the components more complete details are may not be limited to standard curve, precision and accuracy, discussed for possible activities in each phase. range of quantification, specificity and selectivity, stability, dilutional linearity, robustness, batch size, and run acceptance DOCUMENTATION criteria. The plan should include a summary of the proposed experiments and the target acceptance criteria for each per- formance parameter studied. Method Development After completion of the validation experiments, a com- The information generated during the development of a prehensive report should be written. The format of the report method should be documented in a laboratory notebook or may be dictated by internal policies of the laboratory; how- other acceptable format. Information on the following assess- ever, such reports should summarize the assay performance ments should be generated during assay development: critical data and deviations from the method SOP or validation plan assay reagent selection and stability, assay format selection and any other relevant information related to the conditions (antibody[ies], diluents, plates, detection
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