Fast analysis of antibody-derived therapeutics by automated multidimensional liquid chromatography - mass spectrometry

Pot, S.; Gstottner, C.; Heinrich, K.; Hoelterhoff, S.; Grunert, I.; Leiss, M.; ... ; Dominguez Vega, E. (2021)

Article / Letter to editor

Characterization of post-translational modifications (PTMs) of therapeutic antibodies is commonly performed by bottom-up approaches, involving sample preparation and peptide analysis by liquid chromatography-mass spectrometry (LC-MS). Conventional sample preparation requires extensive hands-on time and can increase the risk of inducing artificial modifications as many off-line steps - denaturation, disulfide-reduction, alkylation and tryptic digestion - are performed. In this study, we developed an on-line multidimensional (mD)-LC-MS bottom-up approach for fast sample preparation and analysis of (formulated) monoclonal antibodies and antibody-derived therapeutics. This approach allows on-column reduction, tryptic digestion and subsequent peptide analysis by RP-MS. Optimization of the 1D -and 2D flow and temperature improved the trapping of small polar peptides during on-line peptide mapping analysis. These adaptations increased the sequence coverage (95-98% versus 86-94% for off-line approaches) and allowed identification of various PTMs (i.e. deamidation of asparagine, methionine oxidation and lysine glycation) within a single analysis. This workflow enables a fast (<2 h) characterization of antibody heterogeneities within a single run and a low amount of protein (10 mu g). Importantly, the new mD-LC-MS bottom-up method was able to detect the polar, fast-eluting peptides: Fc oxidation at Hc-Met-252 and the Fc N-glycosylation at Hc-Asn-297, which can be challenging using mD-LC-MS. Moreover, the method showed good comparability across the different measurements (RSD of retention time in the range of 0.2-1.8% for polar peptides). The LC system was controlled by only a standard commercial software package which makes implementation for fast characterization of quality attributes relatively easy. (C) 2021 The Author(s). Published by Elsevier B.V.

• Multidimensional LC
• Mass spectrometry
• Automation
• On-line bottom-up
• Therapeutic antibodies
• Post-translationalmodifications

• All authors: Pot, S.; Gstottner, C.; Heinrich, K.; Hoelterhoff, S.; Grunert, I.; Leiss, M.; Bathke, A.; Dominguez Vega, E.
• Date: 2021-11-01
• Journal: Analytica Chimica Acta
• Volume: 1184
• DOI: doi:10.1016/j.aca.2021.339015
• Link: https://www.sciencedirect.com/science/article/pii/S0003267021008412?via%...