Decoding thermal instability in peptide therapeutics links microheterogeneity to bioactivity loss

Area of Science:

• Analytical Chemistry
• Pharmaceutical Science
• Biochemistry

Background:

• Peptide therapeutics bridge small molecules and biologics.
• Topological isomers arise during peptide synthesis and processing, causing cryptic microheterogeneity.
• These variants can significantly affect drug properties like receptor binding, stability, and in vivo behavior.

Purpose of the Study:

• To introduce and evaluate cyclic ion mobility-mass spectrometry (cIM-MS) for characterizing peptide topological isomers.
• To investigate the conformational states and stability of specific peptide therapeutics (Stlassin and Balixafortide).
• To link microheterogeneity in peptide isomers to macroscopic bioactivity.

Main Methods:

• Development and application of cyclic ion mobility-mass spectrometry (cIM-MS).
• Analysis of lasso peptide Stlassin and bicyclic peptide Balixafortide.
• Accelerated thermal-stability studies and target-binding assays.

Main Results:

• cIM-MS successfully separated and characterized topological isomers in complex peptide mixtures.
• Stlassin exhibited a unique conformation, while Balixafortide showed multiple conformational states.
• Balixafortide demonstrated rapid topological rearrangement and degradation under thermal stress, compromising efficacy and stability.

Conclusions:

• Peptide therapeutics can undergo thermal inactivation via degradation or conformational reorganization.
• Microheterogeneity in peptide isomers directly impacts drug efficacy and stability.
• cIM-MS provides a robust framework for understanding and controlling peptide isomerism in drug development.