Analytical and Bioanalytical Chemistry

, Volume 404, Issue 8, pp 2437–2444

Structural stability–chromatographic retention relationship on exenatide diastereomer separation

Authors

  • Ching-Wei Tsai
    • Department of Chemical and Materials EngineeringNational Central University
  • Wei-Hung Kao
    • Department of Chemical and Materials EngineeringNational Central University
  • Li-Chiao Chang
    • ScinoPharm Taiwan, Ltd
  • Ruoh-Chyu Ruaan
    • Department of Chemical and Materials EngineeringNational Central University
    • Department of Chemical and Materials EngineeringNational Central University
Original Paper

DOI: 10.1007/s00216-012-6352-5

Cite this article as:
Tsai, C., Kao, W., Chang, L. et al. Anal Bioanal Chem (2012) 404: 2437. doi:10.1007/s00216-012-6352-5

Abstract

In this study, the relationship of the structural stability of peptide diastereomers in elution solvents and their retention behaviors in reversed-phase chromatography (RPC) was examined to provide guidance on the solvent selection for a better separation of peptide diastereomers. We investigated the chromatographic retention behaviors of exenatide, a peptide drug for the treatment of type II diabetes mellitus and its three diastereomers using RPC and implicit molecular dynamics (MD) simulation analysis. Three diastereomers involved in the single serine residue mutation of d-form at the 11th, 32nd, and 39th residues were investigated in this study. Results show that the order of the solution structural stability of exenatide and its diastereomers is consistent with their retention order by 36 % acetonitrile/water elution. The sample loading solvent also affects the retention behaviors of exenatide peptide diastereomers in RPC column. Furthermore, a larger solution conformation energy difference of the critical pair of exenatide and its diastereomer (d-Ser39) at the elution solvent of 32 % tetrahydrofuran/water were obtained by MD simulation, and baseline separation was proved experimentally. In summary, we demonstrated that the solution structural stability–chromatographic retention relationship could be a powerful tool for elution solvent selection in peptide chromatographic purification, especially valuable for the separation of critical pair of diastereomers.

https://static-content.springer.com/image/art%3A10.1007%2Fs00216-012-6352-5/MediaObjects/216_2012_6352_Figa_HTML.gif
Figure

The structural stability and reversed-phase chromatography (RPC) retention relationship was investigated for a better chromatographic separation of peptides. Our results revealed that the rigid peptide with lower solution conformation energy exhibits a smaller retention factor in RPC column. Conversely, the flexible peptide with the higher solution conformation energy exhibits a larger retention factor. Based on this finding, we have examined that the baseline separation could be achieved by tuning the elution solvent composition to increase the structural stability difference between peptides. Consequently, the structural stability and RPC retention relationship could actually provide an important guidance on peptide separation.

Keywords

ExenatideDiastereomer separationReversed-phase chromatographyMolecular dynamics simulationStructural stability–chromatographic retention relationship

Abbreviations

ACN

Acetonitrile

CD

Circular dichroism

HPLC

High-performance liquid chromatography

MD

Molecular dynamics

MeOH

Methanol

RPC

Reversed-phase chromatography

TFA

Trifluoroacetic acid

THF

Tetrahydrofuran

Supplementary material

216_2012_6352_MOESM1_ESM.pdf (73 kb)
ESM 1(PDF 72.8 kb)

Copyright information

© Springer-Verlag 2012