Structural stability-chromatographic retention relationship on exenatide diastereomer separation

Area of Science:

• Analytical Chemistry
• Biochemistry
• Computational Chemistry

Background:

• Peptide diastereomers present separation challenges in chromatography.
• Exenatide, a peptide drug for type II diabetes, and its diastereomers were studied.
• Understanding structural stability in elution solvents is key for chromatographic separation.

Purpose of the Study:

• To investigate the relationship between structural stability and retention behavior of peptide diastereomers.
• To provide guidance for selecting elution solvents for improved peptide diastereomer separation.
• To explore the impact of sample loading solvent on chromatographic retention.

Main Methods:

• Reversed-phase chromatography (RPC) was employed to analyze retention behaviors.
• Implicit molecular dynamics (MD) simulations were used to assess solution structural stability.
• Exenatide and three D-serine mutated diastereomers were investigated.

Main Results:

• Solution structural stability order matched retention order in 36% acetonitrile/water.
• Sample loading solvent significantly influenced peptide diastereomer retention in RPC.
• MD simulations revealed a larger conformation energy difference for exenatide/D-Ser39 diastereomers in 32% tetrahydrofuran/water, enabling baseline separation.

Conclusions:

• The correlation between solution structural stability and chromatographic retention is a valuable tool for peptide purification.
• This approach is particularly effective for the separation of critical pairs of peptide diastereomers.
• Optimized solvent selection based on stability can enhance chromatographic purification efficiency.