Establishment of an HPLC-based method to identify key proteases of proteins in vitro
Establishment of an HPLC-based method to identify key proteases of proteins in vitro
复制标题
建立基于 HPLC 的体外蛋白质关键蛋白酶鉴定方法
DOI：
10.1016/j.ab.2019.02.030
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发表时间：
2019
影响因子：
2.9
通讯作者：
Yao Wenbing
中科院分区：
文献类型：
--
作者：
Wei Qingqing;Tian Hong;Zhang Fan;Sai Wenbo;Ge Yang;Gao Xiangdong;Yao Wenbing
Given that the biological functions of proteins may decrease or even be lost due to degradation by proteases, it is of great significance to identify potential proteases that degrade protein drugs during systemic circulation. In this work, we describe a method based on high-performance liquid chromatography (HPLC) to identify key proteases that degrade therapeutic proteins in blood, including endopeptidases and exopeptidases. Here, the degradation of proteins was detected by competition with standard substrates of proteases and is shown as the relative residue rate. Four protein drugs were subjected to this method, and the results suggested that growth hormone was degraded by aminopeptidase N and kallikrein-related peptidase 5, pertuzumab was hardly degraded by the proteases, factor VII was degraded by carboxypeptidase B, neprilysin, dipeptidyl peptidase-4 and peptidyl dipeptidase A, and fibrinogen was degraded by carboxypeptidase B and kallikrein-related peptidase 5, findings consistent with the literature. The results were confirmed by microscale thermophoresis; additionally, activity detectionin vitrosubstantiated that the degradation of factor VII decreased its activity. We demonstrate that this method can be used to identify key proteases of proteins with high accuracy, precision and durability.
