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.