Jiaqi Lu, Eric Pang, Kang Chen
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 r ) through measurement of translational diffusion coefficients ( D t ), 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 ( R 2 / R 1 ) 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 ( p 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 R 2 / R 1 decreased significantly with increasing pH, even within the narrow pH range of 7.1–7.7 ( p value < 0.05), demonstrating smaller oligomer formation and less exchange at basic pH. The improved sensitivity arises because R 2 / R 1 depends on both rotational diffusion ( D r ∝ r –3 ) and exchange kinetics, whereas DLS and DOSY depend on D t (∝ r –1 ). Consequently, the R 2 / R 1 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.