SmartEnzymes GlycINATOR workflow

Hydrolysis of All Fc N-glycans

GlycINATOR (EndoS2) is an IgG-specific endoglycosidase that hydrolyzes all glycoforms present at the Fc N-glycosylation sites.

The enzyme 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.
 

  • 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
Hydrolyzes the β1,4-linkage between the two innermost GlcNAc residues. Active on all Fc N-glycans
30 min reaction
Requires native IgG fold

GlycINATOR® Lyophilized

Lyophilized enzyme for deglycosylation of IgG

GlycINATOR® Low Endotoxin

Lyophilized enzyme with low levels of endotoxin for deglycosylation of IgG

GlycINATOR® Immobilized

Immobilized enzyme for deglycosylation of IgG in spin columns

About GlycINATOR®

GlycINATOR workflow

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, 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.

GlycINATOR® Lyophilized

Lyophilized enzyme for deglycosylation of IgG.

GlycINATOR Lyophilized is available as a lyophilized powder in 2000 unit vials for deglycosylation of 2 mg IgG.
 

  • Flexible format for method development
  • Possible to combine enzymes for improved efficiency
  • Amenable to automated workflows
  • Ready to use – just add water!

Available Products

GlycINATOR Lyophilized 2000 unitsLyophilized enzyme for deglycosylation of 2 mg IgGA0-GL1-020620.00Buy / Request a Quote

For information on how to order, visit Place an Order or contact us directly at order@genovis.com.

Unit Definition

One unit deglycosylates ≥ 95% of 1 μg human IgG when incubated in 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 at 37°C for 30 min.

Content and Storage

GlycINATOR Lyophilized is reconstituted by addition of water. No preservatives added. After reconstitution, the enzyme is stable for 1 month at +4-8°C. The product is shipped at ambient temperature, and should be stored at -20°C upon arrival.

Instructions

Product Specification

Safety Data Sheet

Certificate of Analysis

GlycINATOR® Low Endotoxin

Lyophilized enzyme with low levels of endotoxin for deglycosylation of IgG.
GlycINATOR Low Endotoxin is available as a lyophilized powder in 2000 unit vials for deglycosylation of up to 2 mg IgG. This product formulation has low levels of endotoxins per vial (< 0.2 EU).
 

  • Efficient digestion with documented low levels of endotoxin
  • Suitable for use in sensitive in vivo or cell-based assays

Available Products

GlycINATOR Low Endotoxin 2000 unitsLyophilized enzyme with low levels of endotoxin for deglycosylation of 2 mg IgGA0-GL8-020666.00Buy / Request a Quote

For information on how to order, visit Place an Order or contact us directly at order@genovis.com.

Unit Definition

One unit deglycosylates ≥ 95% of 1 μg human IgG when incubated in 10 mM sodium phosphate, 150 mM NaCl, pH 7.4 at 37°C for 30 min.

Content and Storage

GlycINATOR Low Endotoxin is reconstituted by addition of water. No preservatives added. After reconstitution, the enzyme is stable for 1 month at +4-8°C. GlycINATOR Low Endotoxin is shipped at ambient temperature and should be stored at -20°C upon arrival.

Product Specification

Safety Data Sheet

Certificate of Analysis

GlycINATOR® Immobilized

Immobilized enzyme for deglycosylation of IgG in spin columns.

The GlycINATOR Immobilized spin columns contain the GlycINATOR enzyme covalently coupled to agarose beads, for deglycosylation of Fc N-glycans without contaminating the final preparation with enzyme.

The spin columns are available for deglycosylation of up to 0.5 mg (Microspin), 10 mg (Midispin) or 100 mg (Maxispin) IgG. The smallest columns are available in three different pack sizes, with 2, 5 or 10 columns per pack.

 

  • Easy-to-use spin columns
  • Increased enzyme-to-substrate ratio for improved digestion efficiency
  • No enzyme in the final sample

Available Products

GlycINATOR Immobilized Microspin 2 x 0.5 mgImmobilized enzyme for deglycosylation of 2 x 0.5 mg IgG in spin columnsA0-GL6-010456.00Buy / Request a Quote
GlycINATOR Immobilized Microspin 5 x 0.5 mgImmobilized enzyme for deglycosylation of 5 x 0.5 mg IgG in spin columnsA0-GL6-0251,028.00Buy / Request a Quote
GlycINATOR Immobilized Midispin 1-10 mgImmobilized enzyme for deglycosylation of 1-10 mg IgG in spin columnsA0-GL6-1001,367.00Buy / Request a Quote
GlycINATOR Immobilized Microspin 10 x 0.5 mgImmobilized enzyme for deglycosylation of 10 x 0.5 mg IgG in spin columnsA0-GL6-0501,695.00Buy / Request a Quote
GlycINATOR Immobilized Maxispin 10-100 mgImmobilized enzyme for deglycosylation of 10-100 mg IgG in spin columnsA0-GL6-10004,078.00Buy / Request a Quote

For information on how to order, visit Place an Order or contact us directly at order@genovis.com.

Unit Definition

One spin column contains sufficient GlycINATOR-coupled agarose beads to deglycosylate 0.5 mg (Microspin), 10 mg (Midispin), or 100 mg (Maxispin) IgG.

Content and Storage

GlycINATOR Immobilized is supplied in 20% ethanol, shipped cold, and should be stored at +4-8°C upon arrival. Do not freeze the product!

FAQ and Support

Popular FAQ

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.

Yes, the GlycINATOR enzyme binds specifically to the Fc region and therefore will not deglycosylate N-linked glycans in the Fab region.

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.

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.

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.

 

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