An unexpected bioanalytical challenge caused by positive control antibodies in a clinical immunogenicity assay - A...

Anti-drug antibody (ADA) assays are an important element in the suite of bioanalytical methods required for assessment of the safety and efficacy of recombinant-protein therapeutics. As such, and following extensive optimization, there is an expectation that clinical ADA assays be fully validated for multiple performance parameters, including sensitivity, specificity, reagent stability, and robustness. Among critical reagents used in ADA assays, ADA positive controls (PCs) play a crucial role in multiple stages of assay development and validation, including selection of assay format, establishing assay cut-points, estimating assay relative sensitivity, assessing assay precision, as well as ensuring acceptable assay performance during sample testing. This manuscript highlights an unexpected and highly-impactful PC performance inconsistency attribute we encountered prior to validation of a clinical ADA assay. We also describe the investigation that identified the root cause of this problem: the immune complexation between the murine monoclonal antibody used as the surrogate PC and human anti-mouse antibody present in the serum used to prepare the PC. We conclude that murine monoclonal antibodies are not fully appropriate reagents for use as PCs in clinical ADA assays. Finally, potential approaches to circumvent or mitigate this specific problem in clinical ADA assays are discussed.