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An efficient liquid chromatography-high resolution mass spectrometry approach for the optimization of the metabolic stability of therapeutic peptides

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Abstract

In drug discovery, there is increasing interest in peptides as therapeutic agents due to several appealing characteristics that are typical of this class of compounds, including high target affinity, excellent selectivity, and low toxicity. However, peptides usually present also some challenging ADME (absorption, distribution, metabolism, and excretion) issues such as limited metabolic stability, poor oral bioavailability, and short half-lives. In this context, early preclinical in vitro studies such as plasma metabolic stability assays are crucial to improve developability of a peptidic drug. In order to speed up the optimization of peptide metabolic stability, a strategy was developed for the integrated semi-quantitative determination of metabolic stability of peptides and qualitative identification/structural elucidation of their metabolites in preclinical plasma metabolic stability studies using liquid chromatography-high-resolution Orbitrap™ mass spectrometry (LC-HRMS). Sample preparation was based on protein precipitation: experimental conditions were optimized after evaluating and comparing different organic solvents in order to obtain an adequate extraction of the parent peptides and their metabolites and to minimize matrix effect. Peptides and their metabolites were analyzed by reverse-phase liquid chromatography: a template gradient (total run time, 6 min) was created to allow retention and good peak shape for peptides of different polarity and isoelectric points. Three LC columns were selected to be systematically evaluated for each series of peptides. Targeted and untargeted HRMS data were simultaneously acquired in positive full scan + data-dependent MS/MS acquisition mode, and then processed to calculate plasma half-life and to identify the major cleavage sites, this latter by using the software Biopharma Finder™. Finally, as an example of the application of this workflow, a study that shows the plasma stability improvement of a series of antimicrobial peptides is described. This approach was developed for the evaluation of in vitro plasma metabolic stability studies of peptides, but it could also be applied to other in vitro metabolic stability models (e.g., whole blood, hepatocytes).

Left: trend plot for omiganan and major metabolites. Right: stability plot for five antimicrobial peptidesafter incubation with mouse plasma

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Acknowledgements

The authors would like to thank Dr. Maria Veneziano and Dr. Fulvia Caretti for the helpful discussions and suggestions regarding method development.

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Authors and Affiliations

  1. DMPK, IRBM Science Park, Via Pontina km 30,600, 00071, Pomezia, Roma, Italy

    Simone Esposito, Riccardo Mele, Fabio Bonelli, Edith Monteagudo & Laura Orsatti

  2. Peptide Therapeutics, IRBM Science Park, Via Pontina km 30,600, 00071, Pomezia, Roma, Italy

    Raffaele Ingenito & Elisabetta Bianchi

Authors
  1. Simone Esposito
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  2. Riccardo Mele
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  3. Raffaele Ingenito
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  4. Elisabetta Bianchi
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  5. Fabio Bonelli
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  6. Edith Monteagudo
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  7. Laura Orsatti
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Correspondence to Simone Esposito.

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Esposito, S., Mele, R., Ingenito, R. et al. An efficient liquid chromatography-high resolution mass spectrometry approach for the optimization of the metabolic stability of therapeutic peptides. Anal Bioanal Chem 409, 2685–2696 (2017). https://doi.org/10.1007/s00216-017-0213-1

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  • DOI: https://doi.org/10.1007/s00216-017-0213-1

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