When Assay Format Matters: a Case Study on the Evaluation of Three Assay Formats to Support a Clinical Pharmacokinetic Study

Data generated using various immunoassay methods are an integral part of the development of protein therapeutics. These assaysare used in clinical and preclinical studies to establish the pharmacokinetic (PK) and pharmacodynamic (PD) characteristics as well as to assess the immunogenicity properties of a therapeutic. PK assaysmeasure therapeutic levels post-administration which is essential for understanding the effective dose and dose regimen for a therapeutic. Anti-OX40L is a fully humanized monoclonal antibody designed for the potential treatment of an autoimmune disease. The anti-OX40L human PK assayis required to be sensitive, robust, and precise. To address challenges due to assay sensitivityand reproducibility, as well as assaytechnology limitations, during development of the anti-OX40L human PK assay, three different assays, including an MSD-based electrochemiluminescence assay(ECLA), a fluorometric enzyme-linked immunosorbent assay(ELISA), and a colorimetric ELISA, were evaluated. The MSD-based assaywas the most sensitive but posed risk of inter-well signal crosstalk. The fluorescence ELISA fell short on reproducibility. The colorimetric ELISA was ultimately chosen for supporting sample analysis. This paper presents characterization data obtained from each of these assayformats, challenges that were encountered in the development of the assay, and the rationale for selecting the ultimate assayformat.