Ligand Binding Assays in the 21st Century Laboratory: Recommendations for Characterization and Supply of Critical Reagents

Abstract

Critical reagents are essential components of ligand binding assays (LBAs) and are utilized throughout the process of drug discovery, development, and post-marketing monitoring. Successful lifecycle management of LBA critical reagents minimizes assay performance problems caused by declining reagent activity and can mitigate the risk of delays during preclinical and clinical studies. Proactive reagent management assures adequate supply. It also assures that the quality of critical reagents is appropriate and consistent for the intended LBA use throughout all stages of the drug development process. This manuscript summarizes the key considerations for the generation, production, characterization, qualification, documentation, and management of critical reagents in LBAs, with recommendations for antibodies (monoclonal and polyclonal), engineered proteins, peptides, and their conjugates. Recommendations are given for each reagent type on basic and optional characterization profiles, expiration dates and storage temperatures, and investment in a knowledge database system. These recommendations represent a consensus among the authors and should be used to assist bioanalytical laboratories in the implementation of a best practices program for critical reagent life cycle management.

Ligand Binding Assay Critical Reagents and Their Stability: Recommendations and Best Practices from the Global Bioanalysis Consortium Harmonization Team

“…Identifying a reagent as critical has been raised by both regulatory agencies and in a number of publications (1)(2)(3)(4)(5)(6). The authors recommend, within the scope of the GBC initiative, the following critical reagent definition: LBA reagents that are analyte specific are most often considered as critical reagents (antibodies, peptides, proteins, conjugates (label)), drug as reagent, and ADA reagents including positive and negative control.…”

“…Reagent characteristics can include but are not limited to the following: identity, source, purity, concentration (or titer), binding affinity, isotype (monoclonal/polyclonal antibody), molecular weight, specificity, incorporation ratio, and aggregation level. Several recent publications discuss in detail critical reagent characterization and testing methods (5,6,10).…”

“…It has been recognized that some reagents are critical, and their changes significantly alter or curtail the performance of the assay that relies on them (1)(2)(3)(4)(5)(6). Control of critical reagents is fundamental to the quality and long-term performance of LBAs.…”

Calibration Curves in Quantitative Ligand Binding Assays: Recommendations and Best Practices for Preparation, Design, and Editing of Calibration Curves

“…Performance characteristics of ligand binding assay (LBA) components which include but are not limited to the solid or immobilized surfaces such as microtiter plates and the capture and detection antibodies should be thoroughly evaluated in the method development phase, and appropriate plans should be put in place to monitor lot to lot reagent consistency. The general requirements for the design of calibration curves, the acceptance criteria for individual calibrators, and the guidelines for the selection of an appropriate regression model have been defined in regulatory guidance documents and lead publications by subject matter experts (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Compliance with these guidelines and adherence to the published requirements would enhance reproducibility of a calibration curve across runs and across studies.…”

Best Practices in Performing Flow Cytometry in A Regulated Environment: Feedback from Experience Within The European Bioanalysis Forum

“…Although every reagent is critical to generate valid results, the term 'critical reagent(s)' is defined as the essential components that are used in ligand-binding assays, whose particular characteristics influence assay performance [15]. The reagents that contribute the most to assay variability are assigned as critical.…”