Method Development for Reversed-Phase Separations of Peptides: A Rational Screening Strategy for Column and Mobile Phase Combinations with Complementary Selectivity

Field, Jennifer K.; Bruce, James; Buckenmaier, Stephen; Cheung, Ming Yui; Euerby, Melvin R.; Haselmann, Kim F.; Lau, Jesper F.; Stoll, Dwight; Sylvester, Marie; Thogersen, Henning and Petersson, Patrik (2022). Method Development for Reversed-Phase Separations of Peptides: A Rational Screening Strategy for Column and Mobile Phase Combinations with Complementary Selectivity. LCGC Europe, 35(10) pp. 440–449.

DOI: https://doi.org/10.56530/lcgc.eu.qp3971p2

Abstract

This review article summarizes the results obtained from the combined efforts of a joint academic and industrial initiative to solve the real-life challenge of determining low levels of peptide-related impurities (typically 0.05–1% of the drug substance) in the presence of the related biologically active peptide at a high concentration. A rational screening strategy for pharmaceutically important peptides has been developed that uses combinations of reversed‑phase ultrahigh-pressure liquid chromatography (UHPLC) columns and mobile phases that exhibit complementary reversed-phase chromatographic selectivity using either UV- or mass spectrometry (MS)-compatible conditions. Numerous stationary and mobile phases were categorized using the chemometric tool of principal component analysis (PCA), employing a novel characterization protocol utilizing specifically designed peptide probes. This was successfully applied to the development of a strategy for the detection of impurities (especially isomers) in peptide drug substances using two-dimensional liquid chromatography coupled with MS detection (2D-LC–MS).

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