Integrated Experimental and Molecular Modeling Techniques to Investigate the Buffer Effects on Glucagon Stability

Area of Science:

• Biochemistry and Pharmaceutical Sciences
• Chemical Engineering
• Molecular Biology

Background:

• Peptide drugs are crucial biologics, but buffer selection for their formulations is poorly understood.
• Limited knowledge exists on how buffers influence peptide stability and molecular interactions.

Purpose of the Study:

• To systematically evaluate buffer effects on glucagon stability.
• To elucidate the molecular mechanisms underlying buffer-peptide interactions.

Main Methods:

• Utilized glucagon as a model peptide.
• Integrated experimental methods (RP-HPLC, Thioflavin T assays, CD) with molecular dynamics (MD) simulations.
• Investigated buffer-peptide interactions using varying buffer systems and peptide concentrations.

Main Results:

• Citrate buffer significantly affected glucagon stability, preserving α-helical structure via strong binding.
• Citrate binding reduced ζ-potential and colloidal stability, leading to aggregation and precipitation.
• MD simulations revealed citrate anions forming dense solvation shells, driving glucagon oligomerization.

Conclusions:

• Citrate buffer exhibits a dual role: stabilizing secondary structure while destabilizing colloidal integrity.
• Uncovered molecular insights into peptide-excipient interactions.
• Provides guidance for optimizing peptide drug formulations.