多肽药物关键水解酶体外高通量分析方法的建立

为了建立测定多肽药物体内代谢关键水解酶的高通量分析方法，本研究基于高效液相色谱技术，确定通用的体外酶解反应条件为:pH 7.8或9.0，缓冲体系为0.01 mol/L PBS或50 mmol/L Tris缓冲液，实现对多肽药物关键水解酶的统一高通量体外检测。利用该方法对多肽药物普兰林肽的关键水解酶进行分析，研究结果显示，基肽释放相关酶5和二肽基肽酶4对普兰林肽的水解作用最强，与微量热泳动分析结果一致。因此，本研究所建立的方法可用于分析多肽药物的关键水解酶，为优化多肽药物的酶稳定性提供方法参考与指导。

Establishment of a high-throughput analysis method for the identification of key proteases of peptide drugs in vitro

In order to establish an effective analytical method to detect the key proteases which affect the metabolism and the plasma half-life of peptides in vivo, a method to analyze the key proteases of peptide drugs based on high performance liquid chromatography in vitro was established. The general enzymatic reaction conditions in vitro were as follows: pH was 7. 8 or 9. 0 and the buffer system was 0. 01 mol/L PBS or 50 mmol/L Tris buffer. The results of pramlintide detected by this method showed that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 had the strongest hydrolysis on pramlintide. The result was consistent with that determined by microscale thermophoresis, which indicated that kallikrein-related peptidase 5 and dipeptidyl peptidase 4 were the key proteases of pramlintide. This analytical method provides the basis for high-throughput stability screening of peptides and can be used to analyze key proteases of peptide drugs. It can also provide guidance for optimizing the protease stability of peptide drugs.