Persistence and Fab-specific interaction of acyl-protein thioesterase-1 with monoclonal antibodies during downstream processing.

Difficult-to-remove host cell proteins (HCPs) can compromise monoclonal antibody (mAb) product quality and stability. This study investigated the persistence of acyl-protein thioesterase-1, a polysorbate-degrading HCP, during downstream processing and its interaction with a set of mAbs. Acyl-protein thioesterase-1 remained in the eluates after reprocessing a specific mAb with protein A affinity liquid chromatography, even when various wash buffer additives were used, indicating a strong product:HCP interaction that resists conventional purification strategies. Upon cleaving the mAb and purifying its Fab and Fc fragments, we used biolayer interferometry to show that acyl-protein thioesterase-1 selectively and tightly binds to the Fab region. Domain-specific sensor-immobilization approach suggested that the binding site was located on the antibody's CH1 domain, and the interaction was influenced by preincubation with the mAb's cognate antigen. These findings highlight the importance of characterizing HCP:mAb interactions as a basis for improving HCP reduction strategies.