Resolving conformational polymorphism in disulfide-rich peptide drugs

Abstract

Disulfide-rich peptide therapeutics often exhibit compact cystine knot-like architectures that endow them with high conformational rigidity, proteolytic stability, and target specificity. Yet their intrinsic polymorphism and structural heterogeneity are difficult to resolve with conventional analytical methods, which lack sufficient power to distinguish subtle structural variants within highly constrained peptide frameworks. Here, cyclic ion mobility-mass spectrometry (cIM-MS), combined with accelerated thermal stress testing, was applied to a manufacturing batch of ziconotide and linaclotide under native and thermally stressed conditions. Pronounced conformational heterogeneity was observed for both drugs even under native conditions. Upon thermal stress, notable degradation and structural reorganization occurred, revealing conformational changes that were not adequately captured by conventional methods. The high-resolution separation achieved by cIM-MS enabled detailed mapping of coexisting conformers and their stress-induced transitions. These results demonstrate that cIM-MS provides a spatially resolved analytical platform for interrogating higher-order structural heterogeneity in disulfide-rich peptide therapeutics, extending the capabilities of conventional mass spectrometry and offering a generalizable approach for structural characterization and quality assessment.