Molecular Mechanisms of Liraglutide Aggregation Induced by Dual Air–Water and Silicone-Oil–Water Interfacial Stress

Prefilled syringes (PFSs) serve as primary containers for therapeutic peptides and proteins in combination drug products. Silicone oil, a lubricant in PFSs to facilitate plunger movement during injection, and the headspace from syringe fillings, have been reported to induce protein aggregation and particle formation as a result of the associated interfacial stress. However, their impact on the stability of peptide drugs is underexplored. In this study, we employed biophysical techniques, including size exclusion chromatography (SEC), circular dichroism (CD) spectroscopy, and thioflavin T fluorescence (ThT), to investigate aggregate formation, secondary structural changes, and fibrillation following physical stress on liraglutide in the presence of varying silicone oil concentrations, headspace, and agitation strength. In contrast to the minimal structural changes or formation of higher-molecular-weight aggregates observed under mild agitation or limited headspace, enhanced headspace and agitation promote fibrillation of liraglutide. Furthermore, we utilized advanced techniques to probe silicone-oil-liraglutide interactions and interfacial stress, including high-resolution label-free stimulated Raman scattering (SRS) for chemical imaging and nuclear magnetic resonance (NMR) spectroscopy for structural characterization. We observed, for the first time, the peptide adsorption on the silicone oil surface at submicrometer resolution by SRS. ¹H NMR showed line broadening and signal loss, consistent with peptide aggregation or surface adsorption, but no chemical shift changes, supporting the absence of strong, specific interactions. Therefore, our data suggest that the dual air-water and silicone-oil-water interfacial stress, rather than either alone, plays a significant role in liraglutide aggregation. These findings emphasize the interactive roles of several stress parameters, including silicone oil concentration, silicone oil interface, headspace, and agitation strength, on the stability of peptide combination drug products, and highlight the critical role of interfacial stress-induced biophysical instability.