Fully automated 3D LC-MS workflow for the characterization of antibody glycosylation

March 19, 2020 | References |

Genentech used FabRICATOR®-HPLC to develop a fast and automated 3D LC-MS workflow for the characterization of glycosylation on therapeutic antibodies.


The glycosylation profile is a critical quality attribute of biopharmaceuticals including antibodies. This heterogenous quality attribute can be tackled using a range of different analytical technologies including separation of released glycans , intact glycoprotein mass analysis, subunit mass analysis and many more. An antibody subunit approach using FabRICATOR (IdeS) combined with separation on hydrophilic interaction chromatography (HILIC) offers a promising strategy for characterization of IgG glycosylation. Typically, the FabRICATOR digestion is done manually, offline, prior to LC-MS. In a growing number of cases, like online monitoring of glycosylation, it is desirable to automate such a workflow.


Researchers at Genentech in collaboration with University of Geneva recently published an automated workflow using three dimensional separation coupled to mass spectrometry. To automate the workflow, they employed the FabRICATOR-HPLC column to deliver online antibody subunit generation. In their setup, this was followed by on column reduction and separation of the subunits using a reversed phase column, coupled to HILIC column to separate the Fc/2 glycoforms. In summary, the system incorporated three columns controlled by two valves: FabRICATOR-HPLC x Reduction/RPLC x HILIC/MS (Fig. 1 below).


Camperi et al. compared their automated workflow to the standard manual digestion by testing two of their candidate mAbs. The authors concluded that the 3D automated workflow, which took 95 minutes per sample, could deliver comparable data for all N-glycan variants for the Fc/2 subunit and therefore could be applied to the analysis of not only mAbs but also antibody drug conjugated (ADCs) and bispecific antibodies. Finally, the scientists emphasize that the key strength of the 3D approach was not just the automation aspect but also the fact that no tedious sample preparation was required, and that it is applicable to routine analysis tasks.

Fig 1. 3D-LC Workflow for automated middle-up analysis of mAbs (Camperi et al., 2020.)

Camperi et al., 2020. Development of a 3D–LC/MS workflow for fast, automated and effective characterization of glycosylation patterns of biotherapeutic products. Anal. Chem. 2020, 92, 6, 4357-4363. doi:10.1021/acs.analchem.9b05193