Bioanalytical Workshop Research Articles

Bioactivity Determination of a Therapeutic Recombinant Human Keratinocyte Growth Factor by a Validated Cell-based Bioassay.

The therapeutic recombinant human keratinocyte growth factor 1 (rhKGF-1) was approved by the FDA for oral mucositis resulting from hematopoietic stem cell transplantation for hematological malignancies in 2004. However, no recommended bioassay for rhKGF-1 bioactivity has been recorded in the U.S. Pharmacopoeia. In this study, we developed an rhKGF-1-dependent bioassay for determining rhKGF-1 bioactivity based on HEK293 and HaCat cell lines that stably expressed the luciferase reporter driven by the serum response element (SRE) and human fibroblast growth factor receptor (FGFR2) IIIb. A good responsiveness to rhKGF-1 and rhKGF-2 shared by target HEK293/HaCat cell lines was demonstrated. Our stringent validation was completely focused on specificity, linearity, accuracy, precision, and robustness according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, AAPS/FDA Bioanalytical Workshop and the Chinese Pharmacopoeia. We confirmed the reliability of the method in determining rhKGF bioactivity. The validated method is highly timesaving, sensitive, and simple, and is especially valuable for providing information for quality control during the manufacture, research, and development of therapeutic rhKGF.

Statistical considerations for assessment of bioanalytical incurred sample reproducibility.

Bioanalytical method validation is generally conducted using standards and quality control (QC) samples which are prepared to be as similar as possible to the study samples (incurred samples) which are to be analyzed. However, there are a variety of circumstances in which the performance of a bioanalytical method when using standards and QCs may not adequately approximate that when using incurred samples. The objective of incurred sample reproducibility (ISR) testing is to demonstrate that a bioanalytical method will produce consistent results from study samples when re-analyzed on a separate occasion. The Third American Association of Pharmaceutical Scientists (AAPS)/Food and Drug Administration (FDA) Bioanalytical Workshop and subsequent workshops have led to widespread industry adoption of the so-called "4-6-20" rule for assessing incurred sample reproducibility (i.e. at least 66.7% of the re-analyzed incurred samples must agree within +/-20% of the original result), though the performance of this rule in the context of ISR testing has not yet been evaluated. This paper evaluates the performance of the 4-6-20 rule, provides general recommendations and guidance on appropriate experimental designs and sample sizes for ISR testing, discusses the impact of repeated ISR testing across multiple clinical studies, and proposes alternative acceptance criteria for ISR testing based on formal statistical methodology.

A risk-based analysis of the AAPS conference report on quantitative bioanalytical methods validation and implementation

The 3rd American Association of Pharmaceutical Scientists (AAPS)/Food and Drug Administration (FDA) Bioanalytical workshop in 2006 concluded with several new recommendations regarding the validation of bioanalytical methods in a report published in 2007. It was aimed to conciliate or adapt validation principles for small and large molecules and an opportunity to revisit some of the major decision rules related to acceptance criteria given the experience accumulated since 1990. The purpose here is to provide a “risk-based” reading of the recommendations of 3rd AAPS/FDA Bioanalytical Workshop. Five decision rules were compared using simulations: the proposed pre-study FDA and Total Error Rules, the rules based on the β-Expectation Tolerance and β- γ-Content Tolerance Interval and, finally, the 4-6-20 rule for in-study acceptance of runs. The simulation results demonstrated that the β-Expectation Tolerance Rule controls appropriately the risk. The β- γ-Content Tolerance Interval was found to be too conservative, depending on the objective, and to lead to a high rate of rejection of procedures that could be considered as acceptable. On the other side, the FDA and the AAPS/FDA workshop Total Error Rule, combined or not, did not achieve their intended objective. With these rules, the risk is high to deliver results in study that would not meet the targeted acceptance criteria. This can be explained because, first, there is confusion between the quality of a procedure and the fitness of purpose of the results it could produce and, second, between the initial performances of a procedure, for example evaluated during pre-study validation and the quality of the future results.

Workshop Report and Follow-Up—AAPS Workshop on Current Topics in GLP Bioanalysis: Assay Reproducibility for Incurred Samples—Implications of Crystal City Recommendations

The Conference Report of the 3rd AAPS/FDA Bioanalytical Workshop (Crystal City III) endorsed the concept that assay methods supporting bioanalytical data in submissions must demonstrate assay reproducibility by using incurred samples. The present Workshop was convened to provide a forum for discussion and consensus building about incurred sample assay reproducibility for both nonclinical and clinical studies. Information about current regulatory perspectives on incurred sample reanalysis (ISR) was presented, implications of ISR for both large and small molecules were discussed, and the steering committee put forth recommendations for performing ISR. These recommendations from the Workshop, along with the subsequent evolution of approaches leading to a robust ISR program, may be used by scientists performing bioanalytical assays for regulated studies to provide additional confirmation of assay reproducibility for incurred samples.

Confirmatory reanalysis of incurred bioanalytical samples

Bioanalytical methods used to support the drug development process are validated to ensure that they function in the manner in which they are intended. "Incurred" or study samples can vary in their composition when compared with the standards and quality control samples used to validate the method and analyze these samples. During the 3rd American Association of Pharmaceutical Scientists(AAPS)/Food and Drug Administration(FDA) Bioanalytical Workshop, it was suggested that the reproducibility in the analysis of incurred samples be evaluated in addition to the usual prestudy validation activities performed. This manuscript provides recommendations concerning the number and types of samples that should be analyzed in such an evaluation, as well as the manner in which the resultant data should be analyzed. Suggestions as to follow-up activities and data reporting are also discussed. This approach is at best a beginning and is offered as a platform for future discussion, comments, and revision.

Determination of carryover and contamination for mass spectrometry-based chromatographic assays

The Third American Association of Pharmaceutical Scientists/Food and Drug Administration Bioanalytical Workshop, held in 2006, reviewed and evaluated current practices and proposed that carryover and contamination be assessed not only during the validation of an assay but also during the application of the method in a study. In this article, the potential risks of carryover and contamination in each stage of a bioanalytical method are discussed, to explain to the industry why this recommendation is being made.

Specificity and selectivity evaluations of ligand binding assay of protein therapeutics against concomitant drugs and related endogenous proteins

Macromolecule drugs designed against specific target proteins/ receptors have been applied in combination therapies, especially for complex and related diseases such as cancer for synergistic efficacy and alleviation of side effects. Protein therapeutics are typically measured using ligand binding assays (LBA). Evaluating the specificity and selectivity of LBA against their target proteins or in instances where concomitantly administered drugs are given was brought up during a conversation at the 3rd American Association of Pharmaceutical Scientists/US Food and Drug Administration Bioanalytical Workshop but was not discussed at the meeting sessions. The purpose of this article is to discuss the challenges related to this issue and present a few approaches and experiences to elicit further discussions. Specificity and selectivity tests should be based on the anticipated levels of the individual therapeutics with reference to the dosing regimens defined in the clinical study protocol. When the concomitantly administered compound is available as a pure or well-defined material, various concentrations from zero to above the expected high levels are added to validation samples of the protein therapeutics to assess specificity. Recovery results from spiked samples of target patient populations on concomitant medications can also be compared with those from normal individuals for selectivity. If the drug has an endogenous counterpart, the baseline concentrations of each lot should be subtracted from the test samples in the selectivity assessment. This article illustrates a flexible approach to evaluating specificity and selectivity on samples from target patient populations receiving multiple medications.

Key elements of bioanalytical method validation for macromolecules

The Third American Association of Pharmaceutical Scientists/US Food and Drug Administration (FDA) Bioanalytical Workshop, which was held May 1 and 2, 2006, in Arlington, VA, addressed bioanalytical assays that are being used for the quantification of therapeutic candidates in support of pharmacokinetic evaluations. One of the main goals of this workshop was to discuss best practices used in bioanalysis regardless of the size of the therapeutic candidates. Since the last bioanalytical workshop, technological advancements in the field and in the statistical understanding of the validation issues have generated a variety of interpretations to clarify and understand the practicality of using the current FDA guidance for assaying macromolecular therapeutics. This article addresses some of the key elements that are essential to the validation of macromolecular therapeutics using ligand binding assays. Because of the nature of ligand binding assays, attempts have been made within the scientific community to use statistical approaches to interpret the acceptance criteria that are aligned with the prestudy validation and in-study validation (sample analysis) processes. We discuss, among other topics, using the total error criterion or confidence interval approaches for acceptance of assays and using anchor calibrators to fit the nonlinear regression models.

Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays

The Third AAPS/FDA Bioanalytical Workshop, entitled "Quantitative Bioanalytical Methods Validation and Implementation: Best Practices for Chromatographic and Ligand Binding Assays" was held on May 1-3, 2006 in Arlington, VA. The format of this workshop consisted of presentations on bioanalytical topics, followed by discussion sessions where these topics could be debated, with the goal of reaching consensus, or identifying subjects where addition input or clarification was required. The discussion also addressed bioanalytical validation requirements of regulatory agencies, with the purpose of clarifying expectations for regulatory submissions. The proceedings from each day were reviewed and summarized in the evening sessions among the speakers and moderators of the day. The consensus summary was presented back to the workshop on the last day and was further debated. This communication represents the distillate of the workshop proceedings and provides the summary of consensus reached and also contains the validation topics where no consensus was reached.

Workshop/conference report—Quantitative bioanalytical methods validation and implementation: Best practices for chromatographic and ligand binding assays

I NTRODUCTION Bioanalysis, employed for the quantitative determination of drugs and their metabolites in biological fl uids, plays a signifi cant role in the evaluation and interpretation of bioequivalence, pharmacokinetic (PK), and toxicokinetic studies. The quality of these studies, which are often used to support regulatory fi lings, is directly related to the quality of the underlying bioanalytical data. It is therefore important that guiding principles for the validation of these analytical methods be established and disseminated to the pharmaceutical community. The fi rst American Association of Pharmaceutical Scientists (AAPS)/Food and Drug Administration (FDA) Bioanalytical Workshop in 1990 focused on key issues relevant to bioanalytical methodology and provided a platform for scientifi c discussions and deliberations. The workshop and the report 1 raised awareness of the need for validated bioanalytical methods for the regulatory acceptance of bioequivalence and pharmacokinetic data. Although the workshop addressed bioanalysis in general, it acknowledged the differences between chromatographic and ligand binding (nonchromatographic based) methods. The workshop identifi ed the essential parameters for bioanalytical method validation, ie, accuracy, precision, selectivity, sensitivity, reproducibility, limit of detection, and stability. The outcome of the fi rst workshop and its report resulted in improved quality of data submissions to regulatory agencies. Following the fi rst workshop report 1 and the experience