Establishment of an HPLC-based method to identify key proteases of proteins in vitro

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 detection in vitro substantiated 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.