Immobilized GalactEXO for Fast Hydrolysis of Galactose

Powerful Galactosidase Activity In An Easy-to-use Format

Immobilized GalactEXO consists of the powerful GalactEXO immobilized on agarose beads and formatted in an easy-to-use microspin column format. GalactEXO is a mix of β-galactosidases for efficient removal of galactose residues on N- and O-glycosylated proteins. Derived from Akkermansia muciniphila and recombinantly expressed, the GalactEXO enzymes display effective hydrolysis of β1-3 and β1-4 linked galactose. The GalactEXO microspin columns are formatted to hydrolyze galactose from 0.5 mg glycoprotein in 30-60 minutes.


Schematic overview of the Immobilized GalactEXO 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

galactosidase from Etanercept GalactEXO

Hydrolyzing β1-3 and β1-4 Galactose from Etanercept

Immobilized GalactEXO displays enzymatic activity on both β1-3 and β1-4 linked galactose present on biopharmaceuticals decorated with both N- and O-linked glycosylations such as the Fc-fusion protein etanercept. Etanercept was incubated with immobilized GalactEXO for 60 min and the enzymatic activity was studied using LC-MS on the subunit level after FabRICATOR digestion (Fig 2.) The TNFR and Fc/2 fragments were analyzed separately as depticted in Fig 2. Incubation with Immobilized GalactEXO resulted in hydrolysis of galactose residues on both N-linked and O-linked glycans seen as a loss of signal in the galactosylated species. Analysis of released N-glycans analysis confirmed the LC-MS data (Fig 3).

 

Figure 1. Deconvoluted mass spectra of the TNFalphaR (top) and Fc/2 (bottom) fragments of etanercept treated with immobilized GalactEXO. The protein was digested with FabRICATOR, separated by reverse phase HPLC (Waters BioResolve RP mAb (2.1 x 50 mm) with an water/ACN gradient with 0.1% Ionhance DFA as mobile phase additive) and analyzed by ESI-Q-TOF mass spectrometry (Bruker Impact II). The UV chromatograms (middle panel) clearly show the absence of the GalactEXO enzymes (yellow shading) in the sample treated with immobilized GalactEXO.

Generating Antibodies with G0F Glycoforms galactosidase

G0F Antibodies in 30 minutes

The presence of galactose impacts the effector function elicited by therapeutic antibodies and to study the antibody mode of action, a clean removal of galactose residues may be necessary. The β1-4 galactosidase activity of Immobilized GalactEXO was demonstrated on trastuzumab using a 30 min incubation. The antibodies were digested into subunits using FabRICATOR® and analyzed using LC-MS (Fig. 1). The shift to G0F glycoforms can be seen using both GalactEXO and Immobilized GalactEXO, but the latter leaves no enzyme in the final preparation.

 

Figure 2. Deconvoluted mass spectra of the Fc/2  fragment of trastuzumab treated with immobilized GalactEXO (bottom panel). The mAb was digested with FabRICATOR, separated by reverse phase HPLC (Waters BioResolve RP mAb (2.1 x 50 mm) with an water/ACN gradient with 0.1% Ionhance DFA as mobile phase additive) and analyzed by ESI-Q-TOF mass spectrometry (Bruker Impact II). The UV chromatograms (middle panel) clearly show the absence of the GalactEXO enzymes (yellow shading) in the sample treated with immobilized GalactEXO.

galactosidase Array GalactEXO

Released Glycans from Etanercept and Trastuzumab

To confirm the LC-MS data on loss of galactose from both etanercept and trastuzumab, released N-glycan analyses were performed. The separation of the labeled N-glycans clearly demonstrates the loss of galactose on both etanercept and trastuzumab as indicated by a loss of signal from the galactosylated glycan species (Fig 3). The absence of any galactosylated structures indicates the complete hydrolysis of galactose residues and thus any remaining minor peaks in the LC-MS data could be annotated as glycation of the subunit.

 

Figure 3. HILIC-FLD UHPLC analysis of N-glycans released from trastuzumab (top) and etanercept (bottom) previously treated with Immobilized GalactEXO for 30 and 60 min respectively. N-glycans were released by PNGase F, fluorescently labeled with 2-AB and analyzed by HILIC-UHPLC (Waters ACQUITY UPLC Glycan BEH Amide column, 2.1 x 150 mm)

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