Unified High-throughput Detection and Analysis in Vitro of Key Hydrolases of Peptide Drugs in Vivo
The role of peptide drugs in the treatment of human diseases is becoming more and more important, and the lengths of time they remain in the body greatly affects the dosage and therapeutic effect of the drugs. However, peptide drugs have poor stability, short half-life, and are susceptible to protease hydrolysis in vivo, which limits their biological activity. This has led to the inability of many biologically active polypeptide molecules to be used clinically as drugs. Searching for key hydrolases of peptide drugs helps to elucidate the catabolism of peptide drugs in vivo, which is of great significance for the rational design and modification of peptide drugs. However, due to the wide range of protease hydrolysis, the variety of proteases, and the unclear key constraints, the analysis of key hydrolase enzymes for peptide drugs has been lacking effective methods. Therefore, this paper chooses blood protease as the research object, based on the combination of liquid chromatography and mass spectrometry, taking the hydrolysis of key hydrolase of recombinant human interleukin-11 as a peptide drug as an example. The in vitro unified high-throughput detection and analysis method for the key hydrolase of peptide drug metabolism in vivo was developed. Through the modular optimization of the reaction conditions, this study established an in vitro analysis method for key hydrolyzing enzymes of peptide drugs, and verified the analytical methodology. The results showed that the detection method developed by this study had a detection limit (LOD) of less than 0.215μm/mL-1 for most peptides. The method of this study has the characteristics of high throughput, high sensitivity and wide coverage, and is suitable for high-throughput screening and detection of peptide drugs. It is hoped that the method established in this study can be used to analyze the key hydrolases of peptide drugs, and provide reference and guidance for optimizing the enzyme stability of peptide drugs.