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GlycINATOR™ – Hydrolysis of All Types of Fc N-glycans
GlycINATOR (EndoS2) is an IgG-specific endoglycosidase that hydrolyzes all glycoforms present at the Fc N-glycosylation sites.
SmartEnzymes™
GlycINATOR acts on native IgG and leaves the core GlcNAc intact, with or without fucose. The enzyme is used to reduce the complexity of antibody-based therapeutics to study core afucosylation, and as an initial step in the GlyCLICK and TransGLYCIT platforms.
Why GlycINATOR™?
- Highly efficient IgG-specific endoglycosidase
- Fast – hydrolyzes all Fc N-glycoforms within 30 minutes, including high-mannose and hybrid-type glycoforms
- Enables characterization and quality control of antibody-based biotherapeutics
- Available immobilized in ready-to-use spin columns
Human IgG1-4, Fc-fusion proteins, IgG from mouse, rabbit, rat, monkey, sheep, goat, cow and horse
30 min reaction
Requires native IgG fold
Hydrolyzes the β1,4-linkage between the two innermost GlcNAc residues. Active on all Fc N-glycans
Product Formats
GlycINATOR™ Immobilized
Immobilized enzyme for hydrolysis of all types of Fc N-glycans in spin columns
Buy productGlycINATOR™ Low Endotoxin
Lyophilized enzyme with low levels of endotoxin for hydrolysis of all types of Fc N-glycans
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About GlycINATOR™
GlycINATOR (EndoS2) is an endoglycosidase from Streptococcus pyogenes that specifically hydrolyzes glycans at the Fc N-glycosylation sites of IgG.
The enzyme has been demonstrated to remove all glycoforms on IgG, including high-mannose, hybrid, complex, and bisecting type glycans (Sjögren et al. 2015). It is active on all human IgG subclasses and IgG from many different species, including mouse, rabbit, rat, monkey, sheep, goat, cow, and horse. It has been reported to have a limited activity on glycans from alpha-1-acid glycoprotein (Sjögren et al. 2013). The enzyme is expressed in E. coli, contains a His-tag, and the molecular weight is 92 kDa.
The enzyme is used to reduce the complexity of antibody-based therapeutics or to study core afucosylation and N-glycan site occupancy. The rapid and mild reaction conditions ensure a minimized risk for sample preparation-induced modifications (Sjögren et al. 2015).
Sjögren, J. et al., 2013. EndoS2 is a unique and conserved enzyme of serotype M49 group A Streptococcus that hydrolyses N-linked glycans on IgG and α1-acid glycoprotein. The Biochemical Journal, 455(1), pp.107–118.
Sjögren, J. et al., 2015. EndoS and EndoS2 hydrolyze Fc-glycans on therapeutic antibodies with different glycoform selectivity and can be used for rapid quantification of high-mannose glycans. Glycobiology, 25(10), pp.1053–1063.
Applications
Fast and reliable middle-level analysis of antibody oxidation using FabRICATOR and GlycINATOR, supporting robust QC of critical quality attributes.
Removal of Fc N-glycans abolishes ADCC activity, enabling direct study of glycan-mediated Fc receptor interactions and antibody function.
A simple and rapid estimation of IgG core fucosylation and glycan occupancy using LC-MS analysis.
Citations
Resources
AppNote SmartEnzymes™ for mAb Oxidation
Download Application Notes
FAQ and Support
What is the difference between GlycINATOR Immobilized and IgGZERO Immobilized?
In both products the enzyme is immobilized on agarose beads for IgG-specific hydrolysis of the Fc glycans to the innermost GlcNAc. IgGZERO Immobilized contains IgGZERO (EndoS) and GlycINATOR Immobilized contains GlycINATOR (EndoS2). The GlycINATOR enzyme has much higher enzymatic activity for high mannose and some bisected and hybrid glycans that can occur on mAbs.
Which should I choose, GlycINATOR or IgGZERO?
Typically, GlycINATOR is the first choice since it displays enzymatic activity on all glycoforms present on IgG, including complex type, high mannose, bisected and hybrid type glycans. IgGZERO does not hydrolyze high mannose glycans. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.
Can I achieve Fc-specific deglycosylation on IgG with N-linked glycans in the Fab region?
Yes, the GlycINATOR enzyme binds specifically to the Fc region and therefore will not deglycosylate N-linked glycans in the Fab region.
Can I re-use the GlycINATOR Immobilized column?
We can only guarantee optimal deglycosylation for one-time use. We do not have a cleaning or regeneration protocol to provide. However, depending on the antibody and the following application the column can be reused. We do not recommend using with different antibodies due to the risk for contamination of carry-over from previous sample. If column reuse is desired store the column in 10-20% ethanol at +4-8°C.
What is the difference between GlycINATOR and IgGZERO?
IgGZERO (EndoS) and GlycINATOR (EndoS2) are both IgG-specific endoglycosidases that hydrolyses Fc glycans to the innermost GlcNAc. The GlycINATOR enzyme has a broader substrate selectivity and therefore much higher enzymatic activity on high mannose and some bisected and hybrid glycans that can occur on mAbs. IgGZERO deglycosylates some species faster as compared to GlycINATOR, for example goat IgG.
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