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

ZHANG Fan; TIAN Hong; YAO Wenbing

doi:10.11665/j.issn.1000-5048.20190312

Journal of China Pharmaceutical University > 2019 > 50(3): 352-356. > DOI: 10.11665/j.issn.1000-5048.20190312

ZHANG Fan, TIAN Hong, YAO Wenbing. Establishment of a high-throughput analysis method for the identification of key proteases of peptide drugs in vitro[J]. Journal of China Pharmaceutical University, 2019, 50(3): 352-356. DOI: 10.11665/j.issn.1000-5048.20190312

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

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Abstract

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.
- peptide,
- enzymatic catabolism,
- HPLC,
- proteases,
- high-throughput analysis,
- pramlintide

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