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

By Esposito, Simone; Mele, Riccardo; Ingenito, Raffaele; Bianchi, Elisabetta; Bonelli, Fabio; Monteagudo, Edith; Orsatti, Laura
From Analytical and Bioanalytical Chemistry (2017), 409(10), 2685-2696. Language: English, Database: CAPLUS, DOI:10.1007/s00216-017-0213-1

In drug discovery, there is increasing interest in peptides as therapeutic agents due to several appealing characteristics that are typical of this class of compds., including high target affinity, excellent selectivity, and low toxicity. However, peptides usually present also some challenging ADME (absorption, distribution, metab., and excretion) issues such as limited metabolic stability, poor oral bioavailability, and short half-lives. In this context, early preclin. 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-quant. detn. of metabolic stability of peptides and qual. identification/structural elucidation of their metabolites in preclin. plasma metabolic stability studies using liq. chromatog.-high-resoln. Orbitrap mass spectrometry (LC-HRMS). Sample prepn. was based on protein pptn.: exptl. conditions were optimized after evaluating and comparing different org. solvents in order to obtain an adequate extn. of the parent peptides and their metabolites and to minimize matrix effect. Peptides and their metabolites were analyzed by reverse-phase liq. chromatog.: a template gradient (total run time, 6 min) was created to allow retention and good peak shape for peptides of different polarity and isoelec. points. Three LC columns were selected to be systematically evaluated for each series of peptides. Targeted and untargeted HRMS data were simultaneously acquired in pos. full scan + data-dependent MS/MS acquisition mode, and then processed to calc. 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).