Tn antigen characterization using GalNAcEXO

Tn antigen characterization on O-glycosylated biopharmaceuticals using GalNAcEXO

Immature truncated O-glycans contribute to the heterogeneity of complex biopharmaceuticals making their analysis yet more challenging. The core of an O-glycan is composed of an α-GalNAc residue linked to serine or threonine by a glycosidic bond and is referred to as the Tn antigenA suitable enzymatic strategy to remove these GalNAcs and simplify the characterization of a biopharmaceutical is now possible using GalNAcEXO.


The GalNAcEXO enzyme is an  exo-α-N-Acetylgalactosaminidase that quickly and efficiently hydrolyzes GalNAc residues from native O-glycoproteinsThe enzyme is derived from  Akkermansia muciniphila, expressed in  E. coli with a His-tag, and has a molecular weight of 52 kDaThe enzyme display activity on both Tn-antigen and α1-3 linked terminal GalNAcsHere we show how GalNAcEXO can be used to characterize complex biopharmaceuticals.

GalNAc removal workflow - GalNAcEXO
Schematic overview of the GalNAcEXO activity.

GalactEXO Key Characteristics

  • Galactosidase hydrolyzing galactose residues on N- and O-glycoproteins
  • β1-3 and β1-4 linked galactose
  • 2 h incubation
  • No co-factors required

Deglycosylation of theC1 Inhibitor

Figure 1. Total deglycosylation of recombinant C1 inhibitor Recombinant C1 inhibitor was analyzed by LC (Agilent 1290 with Waters ACQUITY UPLC Protein BEH C4 column, 1.7 µm, 2.1 x 100 mm) and ESI-Q-TOF mass spectrometry (Bruker Impact II). In its intact form (A) the protein is too complex and deconvolution of the mass spectra only yielded noise. After removal of N- and core 1 O- glycans the Tn antigens remain (B) and can efficiently be removed by incubation with GalNAcEXO (C, overnight at 37C)

During production of O-glycosylated biopharmaceuticals, Tn antigens may appear as a result of incomplete processing of the core GalNAc residue. This appears as a repeating mass shifts with a 203 Da HexNAc unit difference. To confirm the presence of the Tn antigen, the following workflow can be applied, here demonstrated on the heavily (26 sites) O-glycosylated C1 inhibitor (Fig 1A). First, the glycoprotein is trimmed of N-glycans using PNGaseF and core 1 O-glycans using OglyZOR and SialEXO. A pattern of repeating peaks can be observed in the mass spectra (Fig. 1B). Secondly, after incubation with the GalNAcEXO enzyme, the remaining GalNAc residues were completely removed leaving one single peak (Fig. 1C).  This workflow therefore confirms the presence of Tn antigens on O-glycosylated biopharmaceuticals and allows for quantification of the core GalNAc levels. An additional benefit of such a workflow is the reduction in remaining heterogeneity which facilitate the confirmation of the intact mass of the protein as well as revealing truncated fragments as shown in Fig 1C.

galactosidase Array GalactEXO

Performance testing on Etanercept 

Etanercept is another challenging model biopharmaceutical molecule carrying 13 O-glycan sites of which 9 to 10 are occupied on average. After removal of N-glycans by PNGaseF and truncation of the O-glycans by SialEXO and GalactEXO, several peaks related to GalNAcs were observed in the mass spectra (Fig. 2A). To remove the remaining GalNAc residues, the GalNAcEXO enzyme was applied (Fig. 2B). The single protein peak observed after treatment with GalNAcEXO shows the complete removal of the remaining GalNAcs.

Figure 2. Performance testing on Etanercept Deconvoluted mass spectra of etanercept digested with SialEXO, GalactEXO and PNGase F (A) and further treated with GalNAcEXO (B, O/N at 37˚C). Samples were digested with FabRICATOR, separated by reverse phase HPLC (Agilent 1290 with Waters BioResolve RP mAb, 2.7 µm, 2.1 x 100 mm) and analyzed by ESI-Q-TOF mass spectrometry (Bruker Impact II).

No cofactors or special buffers required 

GalNAcEXO is active in physiological buffers and neutral pH, making it compatible with most samples. Moreover, the activity of GalNAcEXO is not dependent on any cofactors, such as BSA, that might impair LC-MS analysis. Depending on the nature of the substrate, the GalNAcEXO reaction for glycoproteins is complete in 2 hours while more complex samples may require overnight incubation. GalNAcEXO is also available immobilized in spin columns for fast and convenient processing of samples without residual enzyme in the final preparation.

Table 1. Summary of key specifications for GalNAcEXO.
No cofactors or special buffers required table - GalNAc

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