Fc N-glycan Hydrolysis for Afucosylation Assessment using GlycINATOR™

Application

A simple and rapid estimation of IgG core fucosylation and glycan occupancy 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 N-glycan core, serve to decrease the ADCC mode-of-action. Methods to determine the overall fucosylation levels of the IgG N-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.

An approach combining GlycINATOR and FabRICATOR enables a simple and effective method for determination of core fucosylation on IgG. In the example here we use the human IgG1 trastuzumab. Following FabRICATOR digestion, to isolate the N-glycan containing scFc subunit, the trastuzumab was incubated with GlycINATOR. Digesting between the N-glycan core GlcNAc residues significantly reduces the glycan heterogeneity and the observed mass shift when analysing by LC-MS, confirms a high degree of digestion efficiency.

Highly efficient deglycosylation of Fc N-glycans

The nature of GlycINATOR digestion means that the remaining species are the scFc with and without core fucose. It is, therefore, simple to determine an estimation for the overall levels of core fucosylation much more quickly than using a released glycan approach, which could be beneficial for higher throughput analytics, or where speed is an important factor. This method also allows an assessment of 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.

Rapid assessment of antibody afucosylation and glycan site occupancy

Assessment of antibody afucosylation at the subunit level with GlycINATOR digestion. Deconvoluted mass spectra from LC-MS analysis of trastuzumab scFc subuit following FabRICATOR digestion (top) and following FabRICATOR and GlycINATOR digestion (bottom). The levels of core afucosylation can be determined based on the relatives abundances of the species with or without core fucose.

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