Monitoring Liraglutide Oligomeric Interconversion and Fibril Dissociation through High-Pressure NMR Spectroscopy.

Aggregation and conformational dynamics are key attributes influencing the quality of peptide and protein therapeutics, yet the oligomerization pathways that underlie these behaviors remain difficult to characterize with conventional analytical tools. Here, we establish high-pressure nuclear magnetic resonance (HP-NMR) as the centerpiece of an integrated analytical strategy for probing the aggregation landscape of peptide therapeutics in aqueous formulations. Using liraglutide, a GLP-1 receptor agonist with well-documented but incompletely resolved self-assembly behavior, as a model system, we demonstrate that hydrostatic pressure reversibly modulates interconversion between its two distinct oligomeric states and dissociates fibrils, enabling direct, nondestructive delineation of oligomer exchange and fibrillation pathways under controlled conditions. Flow-NMR is also introduced as a complementary tool that provides independent control over fibrillation kinetics, further expanding the analytical toolkit. Together, these approaches yield thermodynamic and kinetic parameters, such as apparent free-energy differences and partial molar volume changes that are inaccessible by conventional biophysical methods, offering a rigorous and generalizable framework for characterizing aggregation-prone drug modalities.