Degree of Afucosylation Assessment using IgGZERO™

Application

A simple and rapid estimation of IgG core fucosylation using LC-MS analysis.

There is an inverse relationship between core fucosylation level and antibody-dependent cell-mediated cytotoxicity (ADCC), where increased levels of fucose at the glycan core, serve to decrease the ADCC mode-of-action. Methods to determine the overall fucosylation levels of the IgG glycans often involves released glycan analysis. Although the speed and efficiency of these methods have improved in recent years, they can still be time-consuming and involve multi-step methods for glycan release, labelling and analysis. At the intact, or even subunit level, the heterogeneity of the overall glycan population can make it difficult to accurately determine the global core fucosylation level. 

By combining a subunit approach with IgGZERO digestion, we highlight a simple and effective method for quick determination of core fucosylation on IgG. Characterization of the antibody scFc domain is simplified following FabRICATOR digestion and the removal of Fc N-glycans using IgGZERO. IgGZERO removes the vast majority of the glycan heterogeneity, resulting in scFc with and without the core GlcNAc with fucose. Here, the main species contains core fucose, with the minor portion being afucosylated. It is, therefore, simple to estimate the overall levels of core fucosylation much more quickly than using a released glycan approach, and in this instance, it could be determined that the level of afucosylation is approximately 13%.

Rapid assessment of antibody afucosylation

An approach such as this is fast and simple which is very important for higher throughput analytical support, such as supporting process development activities or clone selection activities. This method also allows an assessment of N-glycan site occupancy to be carried out which can be determined from the relative intensity of any peak which relates to the scFc without a core GlcNAc present. In addition, because IgGZERO doesn’t remove high mannose glycans, this method can also be used to observe and monitor the relative presence of these high mannose N-glycans which could also be very beneficial in clone selection.

 
This text is summarized from the following publication:
Henninot, A. et al., 2015. Anal. Biochem.1-8.

Highly beneficial for process development or clone selection activities

Assessment of antibody afucosylation at the subunit level with IgGZERO digestion. Deconvoluted mass spectra from LC-MS analysis of tantibody scFc subunit following FabRICATOR and GlycINATOR digestion. The levels of core afucosylation can be determined based on the relatives abundances of the species with or without core fucose and is estimated to be around 13%.

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