Liu, K.; Huang, Y.; Wang, T.; Mu, R.; Rosenbaum, A. I.

Oral peptide therapeutics typically suffer from short half-lives as they are readily degraded by digestive enzymes. Systematic peptide engineering along with formulation optimization led to the development of a clinical candidate MEDI7219, an orally-bioavailable GLP-1 peptide, that is much more stable than wild type GLP-1 or semaglutide. In this study, we elucidated peptide biotransformation products using in vitro pancreatin assay that employed both collision-induced dissociation (CID) and electron-activated dissociation (EAD) LC-MS/MS methods. Using this approach, we have confidently identified a total of 13 metabolites. Relative quantification of these metabolites over time showed sequential cleavage pattern as peptides were further digested to smaller fragments. These 13 metabolites mapped to 8 cleavage sites on MEDI7219 structure. Most of these cleavage sites can be explained by the specificity of digestive enzymes, e.g. ,trypsin, pepsin and elastase. -methyl-L-phenylalanine appeared to be well protected from chymotrypsin and pepsin digestion since no cleavage peptides ending with -methyl-L-phenylalanine were observed. These study results expand upon previously published stability data and provide new insights on potential GLP1 proteolytic liabilities for future engineering. Furthermore, this study exemplifies the application of pancreatin in vitro system methodology as a valuable tool for understanding metabolism of oral peptide therapeutics in vitro. Additionally, orthogonal MS fragmentation modes offered improved confidence in identification for peptide unknown metabolites.