Pharmaceutical Development, Genentech Inc., 1 DNA Way, MS 562a, South San Francisco, CA, 94080, USA.
Department of Chemical and Biological Engineering, University of Colorado, Boulder, 3415 Colorado Ave, Boulder, CO, 80303, USA.
Pharm Res. 2021 Mar;38(3):397-413. doi: 10.1007/s11095-021-03011-1. Epub 2021 Mar 2.
Hydrolytic degradation of polysorbate during 2-8°C storage of monoclonal antibody drug products has been attributed to residual enzymes (e.g., esterases) from bioprocessing steps. Robust detection of esterase activity using sensitive, non-polysorbate surrogate substrates can provide an alternate method to assess polysorbate degradation risk, if the correlation between the esterase activity and polysorbate degradation is established.
A general esterase activity assay was developed as a monitoring and characterization tool during bioprocess development of monoclonal antibodies.
We report a fluorescence plate-based assay for quantifying esterase activity, utilizing 4-methylumbelliferyl caprylate (MU-C8) as the esterase substrate. The assay was first assessed for substrate, inhibitor and pH specificity using both model enzymes and purified protein samples. The assay was then extensively tested to understand sample matrix effects on activity rates.
The use of this high-throughput method will allow for rapid characterization of protein samples in under three hours. The esterase activity correlated directly with polysorbate degradation and can provide valuable information on polysorbate degradation risk throughout drug development.
在单克隆抗体药物产品 2-8°C 储存期间，聚山梨酯的水解降解归因于生物加工步骤中残留的酶（例如酯酶）。使用灵敏、非聚山梨酯替代底物对酯酶活性进行可靠检测，如果建立了酯酶活性与聚山梨酯降解之间的相关性，则可以提供评估聚山梨酯降解风险的替代方法。
开发了一种通用的酯酶活性测定法，作为单克隆抗体生物工艺开发过程中的监测和表征工具。
我们报告了一种荧光板基测定法，用于定量酯酶活性，使用 4-甲基伞形酮辛酸酯（MU-C8）作为酯酶底物。该测定法首先使用模型酶和纯化蛋白样品评估了底物、抑制剂和 pH 特异性。然后，对该测定法进行了广泛测试，以了解样品基质对活性率的影响。
使用这种高通量方法将允许在不到三个小时的时间内快速表征蛋白质样品。酯酶活性与聚山梨酯降解直接相关，并可在整个药物开发过程中提供有关聚山梨酯降解风险的有价值信息。
