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.

The seemingly unrelated massive aggregation of free mRNA under certain solution conditions and the well-known autohydrolysis of mRNA are actually both closely linked through its secondary and possibly tertiary structure (s/t). This hypothesis posits that s/t partially stabilizes mRNA against both autohydrolysis and massive aggregation. Destabilization of s/t via denaturant guanidine-HCl (Gd), or temperature, has profound effects on both aggregation rates and final degree of autohydrolysis. These denaturant effects occurred for a variety of mRNA, ranging from 700 to 3000 nucleotides but showed very different quantitative behavior among themselves, suggesting some of the methods presented here might help characterize mRNA stability and robustness. Light scattering monitoring during dialysis of mRNA against Gd revealed an “aggregation window”, over 0.5–3 M Gd, whereas dialyzing against a nondenaturing electrolyte (NaCl) showed semireversible monotonic increase of aggregation up to 4 M. Massive aggregation of mRNA in solutions with monovalent ions and in denaturing solutions has not been previously reported. A phenomenological model involving intermolecular electrostatic repulsion and attractions due chiefly to π–π stacking helps interpret the various phenomena.