Glucagon-like peptide-1 (GLP-1) analogs (GLPA) exist primarily as micelle-like associations when free in aqueous solution. The results here indicate that anisotropic electrostatic interactions play a central role in the instability and aggregation, which appear to arise predominantly from multipole, orientation-dependent electrostatics: net dipole moment and charge in GLPA affect attraction and repulsion, features not captured by mean-field, spherically symmetric approaches. Whereas hydrophobicity drives the micelle formation, electrolyte-dependent aggregation appears to be governed by these electrostatic interactions. Increasing ionic strength screens the Coulomb repulsion between micelles, reducing the electrostatic stabilization barrier and allowing orientation-dependent multipole attractions to promote aggregation. This behavior contrasts with globular protein aggregation, typically dominated by the classical hydrophobic effect. Spectroscopically monitoring forward and reverse dialysis with a custom device, stability of liraglutide and semaglutide samples was mapped vs electrolyte (NaCl) and denaturant concentrations (guanidinium chloride, Gdn). Gdn+ cation binding to negatively charged amino acids reduces net charge and dramatically destabilizes GLPA. In contrast, simple cations, such as Na+, merely screen electrostatically, and no binding term is required to explain the data. Aggregation caused by both NaCl and Gdn+ was semi-irreversible. An electrostatic model, based on attractive, screened monopole-dipole, dipole-dipole, and repulsive monopole-monopole interactions was developed to interpret results. This model may be applicable to other peptides and biologics with asymmetric and patchy charge distributions, and dipole moments. The work establishes a stability-testing paradigm that may accelerate development of these biologics, as well as other therapeutic peptides.
