Development of production methodologies for scFv-Fc conjugated critical reagents to support CAR-T clinical programs

Routine conjugation protocols are typically used by bioanalytical laboratories for production of their assay critical reagents. Novel molecules can pose unique challenges to the production of high-quality conjugated critical reagents required for clinical bioanalytical assays. Using routine conjugation protocols, we observed gross instability of conjugated-drug surrogate material for use in antidrug antibody (ADA) assays in clinical autologous Chimeric Antigen Receptor (CAR)-T cell programs, thus halting assay development. We highlight our approach in developing process conditions for CAR-T supporting conjugated critical reagents, where recombinant CAR scFv-hFc is the surrogate drug material to be conjugated. We show that when routine platform process approaches are not appropriate to produce conjugated novel molecule critical reagents, in-silico modeling can be used to determine conditions imparting repeatable generation of stable reagents. Implementing this modeling is advantageous as it decreases laborious efforts to develop stable novel critical reagent production methodologies. As a result of these studies herein, optimal stable conjugated critical reagents were produced which enabled successful development and validation of clinical CAR-T ADA assay. This work provides a framework that can be applied to the production of other bioanalytical assay critical reagents when platform conjugation approaches fail. Developing high-quality, stable critical reagents for novel biotherapeutic molecules like CAR-T cell therapies can be challenging with traditional laboratory methods. New research conducted in this study shows that using computer-based (<i>in silico</i>) modeling can streamline the process of creating stable scFv-hFc-based critical reagents for CAR-T therapy immunogenicity assays. Critical reagents stabilized using the approach developed in this article were successfully validated as reagents to detect anti-drug antibodies in CAR-T patient serum samples.