Decoding thermal instability in peptide therapeutics links microheterogeneity to bioactivity loss

Peptide therapeutics occupy an intermediate position on the molecular continuum between small-molecule drugs and large biologics. A defining characteristic of these agents is their propensity to form topological isomers during synthesis and processing, which are often overlooked and give rise to cryptic microheterogeneity. Such topological variants can substantially alter receptor binding, structural stability and the in vivo disposition of the drug substance. Routine discrimination and quantification of these variants remain challenging with conventional analytical approaches. Here we introduce cyclic ion mobility-mass spectrometry (cIM-MS) as a high-resolution platform to deconvolute and precisely characterize topological isomers in complex peptide mixtures. Using the lasso peptide Stlassin and the potent CXCR4 antagonist bicyclic peptide Balixafortide as model systems, we systematically evaluated the capability of cIM-MS to separate peptide topological isomers. We found that Stlassin adopts a unique, well-defined conformation, whereas Balixafortide populates multiple conformational states. Accelerated thermal-stability studies showed that Balixafortide undergoes rapid topological rearrangement and degradation under stress conditions. Multiple target-binding assays further demonstrated that these processes substantially compromise drug efficacy and stability, directly connecting microscopic isomer distributions to macroscopic bioactivity. Together, these results reveal two pathways of thermal inactivation in peptide therapeutics, namely degradation and conformational reorganization, and establish a framework for understanding how microheterogeneity can scale into biologically meaningful effects.