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Updated: Oct 26, 2025

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System
Published on: May 1, 2019
Monoclonal antibody charge variant characterization by fully automated four-dimensional liquid chromatography-mass
Liesa Verscheure1, An Cerdobbel2, Pat Sandra1
1RIC group, President Kennedypark 26, Kortrijk 8500, Belgium; Separation Science Group, Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281-S4, Ghent B-9000, Belgium.
Abstract:
Fully automated characterization of monoclonal antibody (mAb) charge variants using four-dimensional liquid chromatography-mass spectrometry (4D-LC-MS) is reported and illustrated. Charge variants resolved by cation-exchange chromatography (CEX) using a salt- or pH-gradient are collected in loops installed on a multiple heart-cutting valve and consequently subjected to online desalting, denaturation, reduction and trypsin digestion prior to LC-MS based peptide mapping. This innovation which substantially reduces turnaround time, sample manipulation, loss and artefacts and increases information gathering, is described in great technical detail, and applied to characterize the charge heterogeneity associated with three therapeutic mAbs. Sequence coverages > 95% are obtained for major and minor charge variants (> 1.0%). Post-translational modifications (PTMs) and modification sites are readily revealed in a repeatable manner including unstable succinimide intermediates which are not maintained when performing classical in-solution overnight digestion of offline collected CEX peaks.
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