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Comprehensive two-dimensional liquid chromatography of therapeutic monoclonal antibody digests

Vanhoenacker, Gerd, Vandenheede, Isabel, David, Frank, Sandra, Pat, Sandra, Koen
Analytical and bioanalytical chemistry 2015 v.407 no.1 pp. 355-366
cation exchange, comprehensive two-dimensional liquid chromatography, hydrophilicity, mass spectrometry, monoclonal antibodies, peptide mapping
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 cation-exchange × 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 and²D retention time opening interesting perspectives for use in QA/QC testing.