Characterization of Recombinantly-Expressed Hydrolytic Enzymes from Chinese Hamster Ovary Cells: Identification of Host Cell Proteins that Degrade Polysorbate

Enzymatic hydrolysis of polysorbate in drug products is a major challenge for the biopharmaceutical industry. Polysorbate hydrolysis caused by host cell proteins (HCPs) co-purified during bioprocessing can reduce the protective effects of the surfactant for the active pharmaceutical ingredient and cause the accumulation of low-solubility degradation products over the long-term storage. The identities of such HCPs are elusive due to their extremely low concentrations after the efficient purification processes of most biopharmaceuticals. In this work, 20 enzymes-selected for their known or putative hydrolytic activity and potential to degrade polysorbate-were recombinantly expressed, purified, and characterized via orthogonal methods. First, these recombinant HCPs were assessed for hydrolytic activity against a fluorogenic esterase substrate in a recently-developed, high-throughput assay. Second, these HCPs were screened for hydrolytic activity against polysorbate in a representative mAb formulation. Third, HCPs that displayed hydrolytic activities in the first two assays were subjected to more detailed characterization of their enzyme kinetics against polysorbates. Finally, these HCPs were evaluated for substrate specificity towards different sub-species of polysorbates. This work provides critical new insights for targeted LC-MS/MS approaches for identification of relevant polysorbate-degrading enzymes and supports improvements to remove such HCPs, including knockouts or targeted removal during purification. & COPY; 2023 American Pharmacists Association. Published by Elsevier Inc. All rights reserved.

Automated summary

Enzymatic hydrolysis of polysorbate in drug products poses a major challenge for the biopharmaceutical industry. This study aimed to identify and characterize host cell proteins (HCPs) responsible for polysorbate hydrolysis and their substrate specificity. Twenty recombinant HCPs were assessed for their hydrolytic activity against a fluorogenic esterase substrate and polysorbate in a representative monoclonal antibody formulation. The results provide valuable insights for the development of LC-MS/MS approaches to identify polysorbate-degrading enzymes and improve purification processes to remove such HCPs.