O-glycan Site Occupancy Assessment of Etanercept using GalNAcEXO®

Etanercept was digested using FabRICATOR to isolate the TNFR from the Fc-domain, the N-glycans were removed using PNGase F and the O-glycans were partially deglycosylated using SialEXO and GalactEXO. This generated a TNFR-domain was without N-glycans and O-glycans that had been trimmed to the core GalNAc. The deconvoluted spectrum for this sample shows the TNFR containing a range of 7-11 attached GalNAc residues. This partial deglycosylation approach allows, in a simple and effective way, characterization of the O-glycan site occupancy based on the numbers of attached GalNAc residues, and the relative abundance of each species. This is particularly useful during process characterization and comparability studies by highlighting any variation is the relative occupancy of the O-glycans.

To confirm that the partially deglycosylated species contain GalNAc residues, and to fully deglycosylate the protein for further analysis, the sample was then incubated overnight with GalNAcEXO. The deconvoluted spectrum confirms the removal of GalNAc residues using GalNAcEXO to leave the fully deglycosylated protein. A high level of enzymatic efficiency is important when deglycosylating proteins, as with all of the glycans fully removed, any underlying protein modifications or variants can be much more confidently identified and characterized. If the glycan removal was incomplete, what glycans remain may make it difficult to observe and unambiguously identify such modifications.