Dissecting Hidden Liraglutide Oligomerization Pathways via Direct Mass Technology, Electron-Capture Dissociation, and Molecular Dynamics.

Peptide therapeutics have revolutionized drug design strategies, yet the inherent structural flexibility and conjugated moieties of drug molecules present challenges in discovery, rational design, and manufacturing. Liraglutide, a GLP-1 receptor agonist conjugated with palmitic acid at its lysine residue, exemplifies these challenges by forming oligomers, which may compromise efficacy through the progressive formation of aggregates. Here, we incorporate native mass spectrometry platforms, including electron-capture dissociation (ECD), direct mass technology (DMT), and molecular dynamics (MD), to capture the early oligomerization process of liraglutide. Our findings reveal a restricted C-terminal region upon oligomer formation, as indicated by the reduced release of -ions in ECD analysis. Additionally, we identified the formation of higher-order oligomers ( = 25-62) by DMT, primarily stabilized by hydrophilic interactions involving preformed stable oligomers ( = 12-18). Together, these integrative mass spectrometry results delineate a dual-pathway oligomerization process for liraglutide, demonstrating the power of mass spectrometry to uncover hidden pathways of self-association. This approach underscores the potential of mass spectrometry as a key tool in the rational design and optimization of peptide-based therapeutics.