Development and validation of a novel ELISA for accurate and precise pharmacokinetic analysis of bispecific antibody drug Faricimab

Faricimab, a bispecific monoclonal antibody drug, targets both vascular endothelial growth factor (VEGF) and angiopoietin 2 (Ang-2). It is indicated for the treatment of age-related macular degeneration and diabetic macular edema via stabilization of blood vessels in the retina. By blocking the action of these two growth factors, Faricimab decreases migration and replication of endothelial cells allowing for stabilization of vascular structures, thereby decreasing vascular leakage. It holds immense potential for therapeutic applications and additional treatments, however, precise pharmacokinetic analysis is crucial for understanding its behaviour in vivo. Characterizing the ability of bispecific antibody drugs to bind to both targets simultaneously is critical for development of biotherapeutics. Compared to SPR, which is complex to set up and is not widely used, and cell-based flow cytometry, that lacks precision in complex matrices, ELISA is readily available method, being widely and extensively applied in almost every laboratory, and can be easily optimized to offer accuracy and precision in complex matrices. In this study, we developed an immunoassay for accurate and sensitive quantification of Faricimab concentrations in biological samples. The immunoassay employs a combination of monoclonal antibodies, specifically designed for selective recognition of Faricimab's antigen-binding sites. We validated the assay's specificity, precision, accuracy, and linearity with extensive analytical characterization and comparison to standard methods. Our results demonstrate excellent performance, with an assay range of 0-10 ng/ml and low limits of detection (0.2 ng/ml) and quantification (0.3 ng/ml). The unique immunoassay presented in this study offers a valuable tool for PK analysis of Faricimab, facilitating clinical development and optimization, and enabling a deeper understanding of bispecific antibody drugs' pharmacokinetic properties and additional therapeutic possibilities.