In-vitro metabolite identification for MEDI7219, an oral GLP-1 analog, using LC-MS/MS with CID and EAD approaches

Oral peptide therapeutics typically have short half-lives due to rapid degradation by digestive enzymes. Systematic peptide engineering and formulation optimization led to the development of a clinical candidate MEDI7219, an orally bioavailable glucagon-like peptide 1 (GLP-1) peptide, with greater stability than wild-type GLP-1 or semaglutide:~60% of MEDI7219 remained intact after 2 h in vitro incubation with simulated intestinal fluid. This study further investigates proteolytic stability by elucidating biotransformation products of MEDI7219 using liquid chromatography-mass spectrometry (LC-MS) methods. Peptide metabolism was assessed using in vitro pancreatin assay followed by analysis utilizing liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID) and electron-activated dissociation (EAD) approaches. We have confidently identified 13 metabolites. Time course profiles of parent and metabolite peaks are consistent with sequential enzymatic cleavage pattern. The 13 metabolites mapped to 8 cleavage sites. Most of these cleavage sites can be explained by the specificity of digestive enzymes, <i>e.g.</i> trypsin, pepsin, and elastase. However, α-methyl-L-phenylalanine appeared to be well protected from chymotrypsin and pepsin digestion since no cleavage peptides ending with α-methyl-L-phenylalanine were observed. These study results provide further structural details explaining previously published stability data and provide new insights into potential GLP1 proteolytic liabilities for future engineering <b>What is this article about?</b> Oral peptide drugs are challenging to develop because oral drugs can be rapidly degraded by digestive enzymes in the gastrointestinal tract. To overcome this challenge, a GLP1 analog, MEDI7219, was developed as a clinical candidate. MEDI7219 is more stable than other GLP1 agonists such as semaglutide. A previous study showed that 60% of MEDI7219 remained stable after 2 h incubation with digestive enzymes. This study builds on the earlier findings and further characterizes the degradation products that occur after incubation of the peptide analog with digestive enzymes using advanced analytical instrumentation. <b>What were the results?</b> Our results identified a list of degradation products and time course patterns of this degradation. These results also revealed vulnerable spots in peptide sequence as well as protected regions. <b>What do the results of the study mean?</b> This information can help guide future oral peptide development to engineer more stable oral peptide products for chronic disease.