Methyl Proton Spin Relaxation (<i>R</i>₂/<i>R</i>₁) Enables Sensitive Detection of pH-Dependent Oligomerization in GLP-1 Analogs.

Liquid buffer pH influences the peptide higher order structure (HOS), including oligomerization, with important implications for peptide drug formulation development and stability control. Although dynamic light scattering (DLS) and diffusion-ordered spectroscopy (DOSY) NMR are commonly used to assess particle size (e.g., radius ) through measurement of translational diffusion coefficients (), their sensitivity to subtle pH-dependent oligomerization changes can be limited. Here, using glucagon-like peptide-1 (GLP-1) analogs liraglutide and semaglutide as model peptides, we introduce the methyl proton spin relaxation rate ratio (/) as a sensitive NMR metric for detecting pH-dependent changes in peptide oligomerization. Diffusion coefficients measured using DLS and DOSY-NMR exhibited an overall increasing trend from pH 6.6 to 8.5, consistent with a shift toward smaller oligomers in basic solution; however, insignificant differences ( value > 0.05) were observed between pH 7.1 and 8.5 for liraglutide and between pH 6.6 and 8.2 for semaglutide. In contrast, the methyl proton / decreased significantly with increasing pH, even within the narrow pH range of 7.1-7.7 ( value < 0.05), demonstrating smaller oligomer formation and less exchange at basic pH. The improved sensitivity arises because / depends on both rotational diffusion ( ∝ ) and exchange kinetics, whereas DLS and DOSY depend on (∝ ). Consequently, the / metric offers enhanced discriminatory power for resolving subtle pH-dependent peptide oligomerization in solution and serves as a practical analytical approach for peptide formulation development and stability control.