Wendell C. Smith

Recommendations for the Bioanalytical Method Validation of Ligand-binding Assays to Support Pharmacokinetic Assessments of Macromolecules

AbstractPurpose.With this publication a subcommittee of the AAPS Ligand Binding Assay Bioanalytical Focus Group (LBABFG) makes recommendations for the development, validation, and implementation of ligand binding assays (LBAs) that are intended to support pharmacokinetic and toxicokinetic assessments of macromolecules. Methods. This subcommittee was comprised of 10 members representing Pharmaceutical, Biotechnology, and the contract research organization industries from the United States, Canada, and Europe. Each section of this consensus document addresses a specific analytical performance characteristic or aspect for validation of a LBA. Within each section the topics are organized by an assays life cycle, the development phase, pre-study validation, and in-study validation. Because unique issues often accompany bioanalytical assays for macromolecules, this document should be viewed as a guide for designing and conducting the validation of ligand binding assays. Results. Values of ±20% (25% at the lower limit of quantification [LLOQ]) are recommended as default acceptance criteria for accuracy (% relative error [RE], mean bias) and interbatch precision (%coefficient of variation [CV]). In addition, we propose as secondary criteria for method acceptance that the sum of the interbatch precision (%CV) and the absolute value of the mean bias (%RE) be 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 run acceptance criteria of 4-6-30. Exceptions to the proposed process and acceptance criteria are appropriate when accompanied by a sound scientific rationale. Conclusions. In this consensus document, we attempt to make recommendations that are based on bioanalytical best practices and statistical thinking for development and validation of LBAs.

Validation of immunoassays for bioanalysis: a pharmaceutical industry perspective

Immunoassays are bioanalytical methods in which quantitation of the analyte depends on the reaction of an antigen (analyte) and an antibody. Although applicable to the analysis of both low molecular weight xenobiotic and macromolecular drugs, these procedures currently find most consistent application in the pharmaceutical industry to the quantitation of protein molecules. Immunoassays are also frequently applied in such important areas as the quantitation of biomarker molecules which indicate disease progression or regression, and antibodies elicited in response to treatment with macromolecular therapeutic drug candidates. Currently available guidance documents dealing with the validation of bioanalytical methods address immunoassays in only a limited way. This review highlights some of the differences between immunoassays and chromatographic assays, and presents some recommendations for specific aspects of immunoassay validation. Immunoassay calibration curves are inherently nonlinear, and require nonlinear curve fitting algorithms for best description of experimental data. Demonstration of specificity of the immunoassay for the analyte of interest is critical because most immunoassays are not preceded by extraction of the analyte from the matrix of interest. Since the core of the assay is an antigen-antibody reaction, immunoassays may be less precise than chromatographic assays; thus, criteria for accuracy (mean bias) and precision, both in pre-study validation experiments and in the analysis of in-study quality control samples, should be more lenient than for chromatographic assays. Application of the SFSTP (Societe Francaise Sciences et Techniques Pharmaceutiques) confidence interval approach for evaluating the total error (including both accuracy and precision) of results from validation samples is recommended in considering the acceptance/rejection of an immunoassay procedure resulting from validation experiments. These recommendations for immunoassay validation are presented in the hope that their consideration may result in the production of consistently higher quality data from the application of these methods.

Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products

Most biological drug products elicit some level of anti-drug antibody (ADA) response. This antibody response can, in some cases, lead to potentially serious side effects and/or loss of efficacy. In humans, ADA often causes no detectable clinical effects, but in the instances of some therapeutic proteins these antibodies have been shown to cause a variety of clinical consequences ranging from relatively mild to serious adverse events. In nonclinical (preclinical) studies, ADA can affect drug exposure, complicating the interpretation of the toxicity, pharmacokinetic (PK) and pharmacodynamic (PD) data. Therefore, the immunogenicity of therapeutic proteins is a concern for clinicians, manufacturers and regulatory agencies. In order to assess the immunogenic potential of biological drug molecules, and be able to correlate laboratory results with clinical events, it is important to develop reliable laboratory test methods that provide valid assessments of antibody responses in both nonclinical and clinical studies. For this, method validation is considered important, and is a necessary bioanalytical component of drug marketing authorization applications. Existing regulatory guidance documents dealing with the validation of methods address immunoassays in a limited manner, and in particular lack information on the validation of immunogenicity methods. Hence this article provides scientific recommendations for the validation of ADA immunoassays. Unique validation performance characteristics are addressed in addition to those provided in existing regulatory documents pertaining to bioanalyses. The authors recommend experimental and statistical approaches for the validation of immunoassay performance characteristics; these recommendations should be considered as examples of best practice and are intended to foster a more unified approach to antibody testing across the biopharmaceutical industry.

Workshop on bioanalytical methods validation for macromolecules: summary report.

1. To determine industry and regulatory standards established for bioanalytical method validation in support of the estimation and characterization of macromolecules in the preclinical and clinical stages of drug development. 2. To evaluate special validation considerations for quantitative, macromolecule-detecting technologies that have emerged since 1990 including immunoassays, cell-based assays, antibody titers, and automation in the laboratory. 3. To address the strengths/limitations and advantages/ disadvantages of assay-customized approaches to validation that focus on assay parameters specific to the intended use of the assay. 4. To develop a 2000 workshop report regarding appropriate bioanalytical validation criteria and standardization of terminology for the above to be used by regulatory agencies to draft new guidelines for the bioanalytical validation criteria for macromolecules quantitation.

Bioanalytical method validation for macromolecules in support of pharmacokinetic studies.

The development and validation of ligand binding assays used in the support of pharmacokinetic studies has been the focus of various workshops and publications in recent years, all in an effort to establish a guidance document for standardization of these bioanalytical methods. This summary report of the workshop from 2003 focuses on the issues discussed in presentations and notes points of discussion and areas of consensus among the participants.

Application of experimental design techniques to optimize a competitive ELISA

This paper describes the application of experimental design techniques to optimize a sensitive ELISA for a hapten molecule with a calibration range of 0-1000 pg/ml. Ten factors that were expected to affect the assay performance were initially screened, followed by factorial experiments to delineate the effects of the critical factors identified at the screening stage. Assay performance was evaluated using a unique rating system based on standard curve reproducibility, assay detection limits and the use of desirability functions. This rating system allowed multiple responses to be evaluated simultaneously. It was found that the substrate incubation time and enzyme label lot played an important role, while dilutions of the enzyme label and the anti-hapten antibody showed significant interaction. These observations were in good agreement with optimal assay conditions based on historical data collected over a period of two to three years. Application of experimental design techniques enabled us to confirm the significance of the factors affecting the assay within a three month period, with a minimum number of experiments. In addition, information on interaction between factors were determined.

Involvement of calcium channels in the sexual dimorphism of cadmium-induced hepatotoxicity

Cadmium toxicity has been evaluated in a number of in vivo and in vitro toxicological studies. In vivo Cd toxicity exhibits sexual dimorphism with females being more susceptible to Cd uptake, accumulation, and toxicity in the liver. Research to date does not explain why females are more sensitive to Cd-induced hepatotoxicity. Recent studies demonstrate that progesterone sensitizes female F(344) rats and TRL-1215 cells to Cd toxicity, however the mode of action is still unclear. Approximately one half of the Cd entering the cytoplasm does so through receptor operated Ca(2+) channels. Progesterone treatment of human spermatozoa and Xenopus laevis oocytes causes a rapid influx of Ca(2+) suggesting a possible mechanism. Since hepatocytes have progesterone receptors on their cellular membrane and Ca(2+) influx into the cytoplasm occurs following progesterone treatment we evaluated the hypothesis that progesterone facilitates the uptake and accumulation of Cd via Ca(2+) channels, leading to enhanced toxicity. Primary isolated rat hepatocytes were treated with Cd, progesterone, and/or verapamil for 4 h and cytolethality was measured. Pretreatment with the Ca(2+) channel blocker verapamil increased the Cd concentration producing 50% lethality (LC(50)) by 2-fold, thus decreasing Cd cytolethality. In contrast, pretreatment with progesterone decreased the Cd LC(50) by 2-fold resulting in enhanced Cd cytolethality. Verapamil treatment reversed the progesterone enhanced Cd cytolethality. Verapamil and/or progesterone in the absence of Cd did not affect hepatocyte viability. Overall, the results of this study demonstrate that inhibition of progesterone-induced Ca(2+) influx with the Ca(2+) channel blocker verapamil, decreases Cd cytolethality in primary isolated rat hepatocytes. These findings indicate that progesterone activation of receptor-mediated Ca(2+) channels is involved in the sexually dimorphic hepatotoxicity seen following acute Cd exposure.

Conceptual and statistical issues in the validation of analytic dilution assays for pharmaceutical applications

The discovery, research, and development of a pharmaceutical product relies on the availability of validated assays for assessing product characteristics and drug effects in vivo and in vitro. Development of a validated assay is a multifaceted activity that provides many interesting challenges for bioanalytical chemists and statisticians. In this paper, the similarity condition for fundamental validity of an analytic dilution assay is reviewed as a basic concept underlying the validation of assays for pharmaceutical applications. The distinction between the validity and the acceptability of an assay is considered in terms of the characteristics evaluated during four stages of validation. Recent guidelines on the validation of analytical procedures published by the U.S. Food and Drug Administration are appraised from a statistical perspective, and statistical issues in the validation process are discussed.

Expression of TRPM-2 during involution and regeneration of the rat liver

Increased message levels of testosterone-repressed prostate message-2 (TRPM-2) have been associated with programmed cell death in many tissues. To study its involvement in the apoptotic elimination of hepatocytes during liver involution and regeneration, levels of TRPM-2 message were evaluated in situ and by the ribonuclease protection assay. Although significant increases in apoptotic bodies were observed in rats 96 h following treatment with lead nitrate and ethylene dibromide, an increase in TRPM-2 message was not detected. Therefore, the expression of TRPM-2 mRNA may be a poor indicator of the extent to which apoptosis occurs during liver involution.

Error rate for testing a contrast after a significant f test

In statistical applications an experimenter often tests a particular contrast after a significant F test for the equality of means. This paper evaluates the overall error rate for this testing .procedure.