Identification of Acyl-Protein Thioesterase-1 as a Polysorbate-Degrading Host Cell Protein in a Monoclonal Antibody Formulation Using Activity-Based Protein Profiling

Polysorbate (PS) degradation in monoclonal antibody (mAb) formulations poses a significant challenge in the biopharmaceutical industry. PS maintains protein stability during drug product's shelf life but is vulnerable to breakdown by low-abundance residual host cell proteins (HCPs), particularly hydrolytic enzymes such as lipases and esterases. In this study, we used activity-based protein profiling (ABPP) coupled with mass spectrometry to identify acyl-protein thioesterase-1 (APT-1) as a polysorbate-degrading HCP in one case of mAb formulation with stability problems. We validated the role of APT1 by matching the polysorbate degradation fingerprint in the mAb formulation with that of a recombinant APT1 protein. Furthermore, we found an agreement between APT1 levels and PS degradation rates in the mAb formulation, and we successfully halted PS degradation using APT1-specific inhibitors ML348 and ML211. APT1 was found to co-purify with a specific mAb via hitchhiking mechanism. Our work provides a streamlined approach to identifying critical HCPs in PS degradation, supporting quality-by-design principles in pharmaceutical development. (c) 2024 The Authors. Published by Elsevier Inc. on behalf of American Pharmacists Association. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)