Polysorbate 20 (PS20) is widely used in biopharmaceutical formulations to stabilize therapeutic proteins. However, the degradation of PS20, particularly via enzymatic hydrolysis by host cell proteins (HCPs), can generate free fatty acids (FFAs) that could lead to undesirable particle formation. Although several potential HCPs responsible for PS20 degradation have been identified over the years, determining the primary cause of PS20 degradation remains a significant challenge in the biopharmaceutical industry. In this study, we investigated the cause of unexpected particle formation during the development of a monoclonal antibody (mAb-1) formulation. Using serine hydrolase inhibitors and activity-based protein profiling, we identified phospholipase A2 (PLA2) and phospholipase B-like 2 (PLBL2, encoded by Plbd2) as potential contributors to PS20 degradation. Targeted knockout of Pla2 in the production cell line used for mAb-1 production resulted in reduced PS20 degradation, thereby mitigating the formation of FFA particles in the mAb-1 drug product (DP). In contrast, the knockout of Plbd2 showed that PLBL2 was not a major contributor to PS20 degradation. This study identified PLA2 as the primary cause of PS20 degradation, and eliminating PLA2 in cells provides an effective solution to mitigate the challenge of HCP-induced PS20 degradation. Our findings presented a comprehensive verification strategy, combining biochemical and genetic methods, to identify the root cause of PS20 degradation and provided a scheme for controlling this risk in biopharmaceutical formulations.
