Kovner Daniel, Yuk Inn H, Shen Amy, Li Hong, Graf Tobias, Gupta Sanjay, Liu Wenqiang, Tomlinson Anthony
Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, CA, USA.
Cell Culture and Bioprocess Operations, Genentech, 1 DNA Way, South San Francisco, CA, USA.
J Pharm Sci. 2023 May;112(5):1351-1363. doi: 10.1016/j.xphs.2023.01.003. Epub 2023 Jan 14.
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.
药物产品中聚山梨酯的酶促水解是生物制药行业面临的一项重大挑战。生物加工过程中共同纯化的宿主细胞蛋白（HCPs）导致的聚山梨酯水解会降低表面活性剂对活性药物成分的保护作用，并在长期储存过程中导致低溶解度降解产物的积累。由于在大多数生物药物的高效纯化过程后，这些HCPs的浓度极低，其身份难以捉摸。在这项工作中，通过正交方法重组表达、纯化并表征了20种因其已知或推定的水解活性以及降解聚山梨酯的潜力而被选择的酶。首先，在最近开发的高通量测定中，评估这些重组HCPs对荧光酯酶底物的水解活性。其次，在代表性的单克隆抗体制剂中筛选这些HCPs对聚山梨酯的水解活性。第三，对在前两个测定中表现出水解活性的HCPs针对聚山梨酯的酶动力学进行更详细的表征。最后，评估这些HCPs对不同聚山梨酯亚种的底物特异性。这项工作为靶向液相色谱-串联质谱（LC-MS/MS）方法鉴定相关聚山梨酯降解酶提供了关键的新见解，并支持改进去除此类HCPs的方法，包括在纯化过程中敲除或靶向去除。
