FabRICATOR® in Correlative N-glycan Analysis of Cetuximab Produced in Different Expression Systems

A strategy for comparative characterization of cetuximab produced in a variety of expression cell lines is described in this paper from scientists at the National Institute for Bioprocessing Research and Training (NIBRT) in Dublin, Ireland. FabRICATOR (IdeS) is a critical tool in this study, allowing for site-specific glycan analysis to be performed following antibody digestion into F(ab’)2 and Fc/2 subunits.

Cetuximab is a chimeric antibody used for the treatment of metastatic colorectal, non-small cell lung cancer and head and neck cancers, however, in some cases, patients have experienced adverse reactions. Cetuximab contains a second occupied glycosylation site in its Fab region and is also expressed in murine myeloma cells which can impart non-human type glycosylation moieties, such as galactose-α1-3-galactose (‘α-gal’) and N-glycolylneuraminic acid (NGNA) sialylation, into the glycan profile of the final product. These characteristic non-human glycan features could drive an immunogenic response within patients through IgE interaction with the Fab region of mAb therapeutics which contain these moieties.
It is important to define and control the glycosylation profiles within therapeutic proteins to minimize potential safety risks and maximize efficacy and product quality. The defined glycosylation profile is highly dependent on the host cell-line used for recombinant protein expression and the advent of biosimilar and biobetter development encourages investigation into the use of different cell lines to most effectively meet the target criteria.
Here, the scientists at NIBRT have produced cetuximab in both human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cell lines and describe the differences between these molecules and the commercially available reference molecule which is produced in murine myeloma cells. They use a combination of orthogonal methods to characterise the cetuximab derived from the different cell lines, with a particular interest in the glycan populations.
Since glycosylation within the Fab region is of particular interest to this study, FabRICATOR was an ideal tool to support this work. Cetuximab samples were digested below the hinge, producing F(ab’)2 and Fc/2 subunits, the subunits separated by size-exclusion chromatography (SEC) and collected prior to glycan analysis. This allowed for site-specific assignment of the local glycan population at each of the glycosylation sites, within the Fc and Fab regions respectively, which could then be compared between the samples from different cell lines.

The authors observed that the HEK expressed sample showed similar levels of Fc-galactosylation, which is important for effector function, and total sialic content, important for maintenance of circulating half-life, when compared to the murine derived sample. Significantly, whilst the overall profiles were similar, the HEK-expressed samples contained neither α-gal nor NGNA-containing glycans, thus, removing moieties responsible for IgE-mediated immunogenic response. The CHO expressed cetuximab, however, whilst also absent in α-gal and NGNA-containing glycans, exhibited a different glycan profile to the other two samples. There was a lower level of overall Fc-galactosylation, a much lower overall sialic content, and increased abundance of high mannose type glycans which, as a combination, would have a detrimental impact the overall efficacy and lower the circulating half-life of the molecule.
The ability to characterise complex biotherapeutics at the subunit-level allows for a more in-depth understanding of these molecules. FabRICATOR has proved to be a valuable tool in this study and allowed the scientists at NIBRT to uncover site-specific glycosylation differences between molecules derived from different cell lines. The advent of biosimilars and biobetters, where the ability to produce functionally similar molecules whilst eliminating unwanted immunogenic responses is highly desired, highlights the important of this type of analysis.


Trappe et al., 2022. Correlative N-glycan and charge variant analysis of cetuximab expressed in murine, chinese hamster and human expression systems Journal of Chromatography. https://doi.org/10.1016/j.jchromb.2022.123186.









FabRICATOR® – Below hinge digestion of IgG