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Structural stability–chromatographic retention relationship on exenatide diastereomer separation

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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.

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.

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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

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Acknowledgments

The authors would like to thank ScinoPharm Taiwan, Ltd., for financially supporting this project. The authors also thank the National Center for High-Performance Computing of Taiwan and Vger Computer Cluster at National Center University of Taiwan for providing computer time and facilities.

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Authors and Affiliations

  1. Department of Chemical and Materials Engineering, National Central University, Jhong-Li, 32001, Taiwan

    Ching-Wei Tsai, Wei-Hung Kao, Ruoh-Chyu Ruaan & Wen-Yih Chen

  2. ScinoPharm Taiwan, Ltd, Shan-Hua, Tainan, 74144, Taiwan

    Li-Chiao Chang

Authors
  1. Ching-Wei Tsai
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  2. Wei-Hung Kao
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  3. Li-Chiao Chang
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  4. Ruoh-Chyu Ruaan
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  5. Wen-Yih Chen
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Correspondence to Wen-Yih Chen.

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Tsai, CW., Kao, WH., Chang, LC. et al. Structural stability–chromatographic retention relationship on exenatide diastereomer separation. Anal Bioanal Chem 404, 2437–2444 (2012). https://doi.org/10.1007/s00216-012-6352-5

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  • DOI: https://doi.org/10.1007/s00216-012-6352-5

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