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Vanhoenacker G.,Research Institute for Chromatography and Metablys | Vandenheede I.,Research Institute for Chromatography and Metablys | David F.,Research Institute for Chromatography and Metablys | Sandra P.,Research Institute for Chromatography and Metablys | Sandra K.,Research Institute for Chromatography and Metablys
Analytical and Bioanalytical Chemistry

Comprehensive two-dimensional liquid chromatography (LC×LC) is here proposed as a novel tool for peptide mapping of therapeutic monoclonal antibodies in both R&D and routine (QA/QC) environments. This is illustrated by the analysis of the tryptic digest of trastuzumab (Herceptin) applying a commercially available two-dimensional 2D-LC system. Three different LC×LC combinations, i.e., strong cationexchange × reversed-phase (SCX×RP), reversed-phase × reversed-phase (RP×RP), and hydrophilic interaction × reversed-phase (HILIC×RP), are reported. Detection was carried out using both UV detection (DAD) and mass spectrometry (MS). Several challenges related to the application of LC×LC in peptide mapping and the hyphenation to MS are addressed. The applicability of LC×LC in the assessment of identity, purity, and comparability is demonstrated by the analysis of different Herceptin innovator production batches, a Herceptin biosimilar in development and of stressed samples. The described methodology was shown to be precise in terms of peak volume and 2D retention time opening interesting perspectives for use in QA/QC testing. © Springer-Verlag Berlin Heidelberg 2014. Source

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