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Create Homogenous IgG Glycoforms

Transglycosylation of IgG

Glycoengineering of the IgG glycan profile is important for the development of next-generation therapeutic antibodies with enhanced or silenced effector functions. TransGLYCIT translgycosylated antibodies with defined Fc glycoforms to generate agalactosylated, galactosylated or sialylated glycan profiles. With the option to generate glycan profiles lacking the core fucose, antibodies that show increased binding to activating FcγIIIa receptors and thus an elevated ADCC response can also be obtained for direct comparison between fucosylated and afucosylated antibodies.

Using TransGLYCIT, IgG with the defined glycoforms G0, G1, G2 or G2S2 are obtained within three hours. With TransGLYCIT Afucosylated containing the FucosEXO™ 16 enzyme, it is possible to generate antibodies carrying afucosylated glycan profiles.
 

Transglycosylation Workflow

The IgG Transglycosylation Workflow

  1. Deglycosylation
    The Fc N-glycans are trimmed to the core GlcNAc using the IgG-specific Immobilized GlycINATOR® (EndoS2) enzyme that hydrolyses all Fc glycoforms, including high-mannose, hybrid, complex and bisecting glycans. To obtain afucosylated glycoforms, the optional Immobilized FucosEXO™ 16 enzyme hydrolyzes the α1-6 linked core fucose.
  2. Transglycosylation
    The engineered glycosynthase TransINATOR™ catalyzes the transglycosylation reaction between the oxazoline reactive G0, G1, G2 or G2S2 glycoform and the core GlcNAc.

 

Generating G0 Glycan Profiles

Agalactosylated IgG Fc glycans include both the fucosylated (G0F) and afucosylated (G0) glycoforms, where G0F accounts for a significant portion of the glycoforms observed in human serum and among several therapeutic antibodies. With TransGLYCIT, defined G0F and G0 glycan profiles can be generated for applications in both structural and functional analytical workflows for therapeutic antibodies.

 

Using the TransGLYCIT platform, the glycan profile of a therapeutic antibody was remodeled to carry G0 glycoforms within three hours and analyzed at the subunit level using LC-MS (Fig. 1). The resulting mass spectra show the heterogenous glycan profile of native trastuzumab (top) and the mass shifts after complete transglycosylation of the antibody to generate G0 glycoforms (middle) using the TransGLYCIT G0 kit. A homogenous afucosylated glycan profile was generated using the TransGLYCIT G0 Afucosylated kit (bottom).

 

Figure 1. Deconvoluted mass spectra of the Fc/2 fragment of native trastuzumab (top) and after transglycosylation with TransGLYCIT G0 (middle), or TransGLYCIT G0 Afucosylated (bottom). The mAb was digested with FabRICATOR and the subunits analyzed by reverse phase LC-MS on a Waters BioAccord system equipped with a Waters BioResolve RP mAb column (2.1 x 50 mm)

Galactosylated IgG Glycoforms

Terminal galactosylation of the IgG Fc glycans result in monogalactosylated (G1) or digalacosylated (G2) glycoforms, with additional fucosylated glycoform variants (G1F and G2F). By generating defined G1 and G2 glycan profiles, the differential impact of galactosylation on the structural and functional properties of the antibody can be analyzed and compared with other defined glycoforms, including both fucosylated and afucosylated glycan profiles.

 

 
Figure 2. Deconvoluted mass spectra of the Fc/2 fragment of native trastuzumab (top) and after transglycosylation with TransGLYCIT G1 (middle), or TransGLYCIT G1 Afucosylated (bottom). The mAb was digested with FabRICATOR and the subunits analyzed by reverse phase LC-MS on a Waters BioAccord system equipped with a Waters BioResolve RP mAb column (2.1 x 50 mm).

Galactosylated glycan profiles with and without core fucose were obtained by transglycosylation of trastuzumab using the TransGLYCIT and TransGLYCIT Afucosylated kits with G1 or G2 glycoforms. Analysis at subunit level using LC-MS (Fig. 2, 3) show mass spectra of the native glycan profile of trastuzumab (top) and the mass shifts after transglycosylation to generate G1 and G2 glycoforms (middle), or only afucosylated G1 or G2 glycoforms (bottom).

 

 
Figure 3. Deconvoluted mass spectra of the Fc/2 fragment of native trastuzumab (top) and after transglycosylation with TransGLYCIT G2 (middle), or TransGLYCIT G2 Afucosylated (bottom). The mAb was digested with FabRICATOR and the subunits analyzed by reverse phase LC-MS on a Waters BioAccord system equipped with a Waters BioResolve RP mAb column (2.1 x 50 mm).

Generating Sialylated Fc Glycoforms

Sialylated Fc glycans are rather rare on recombinantly expressed mAbs but are found in serum IgGs and have been reported to exert anti-inflammatory effects by regulating the antibody effector functions. Generating defined sialylated IgG glycoforms enables analysis of the impact on antibody structure and function, and the level of Fc glycan sialylation as a potential therapeutic strategy and diagnostic tool for autoimmune diseases.

 

Sialylated glycan profiles were generated using the TransGLYCIT platform by remodeling the glycan profile of a therapeutic antibody. The resulting mass spectra after subunit analysis using LC-MS (Fig. 4) show the glycan profile of trastuzumab (top) and mass shifts corresponding to transglycosylation of the antibody to generate G2S2 glycoforms (middle) or a homogenous afucosylated G2S2 glycan profile (bottom).

 

 
Figure 4. Deconvoluted mass spectra of the Fc/2 fragment of native trastuzumab (top) and after transglycosylation with TransGLYCIT G2S2 (middle), or TransGLYCIT G2S2 Afucosylated (bottom). The mAb was digested with FabRICATOR and the subunits analyzed by reverse phase LC-MS on a Waters BioAccord system equipped with a Waters BioResolve RP mAb column (2.1 x 50 mm).

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Popular FAQ

Yes, the transglycosylation reaction can be performed on all subclasses of human IgG. The reaction is somewhat slower on IgG2 and longer incubation times may be necessary to obtain over 95% transglycosylation.

Unfortunately no, the enzyme requires trimming of the Fc-glycan using GlycINATOR to enable access to the core fucose substrate.

GlycINATOR is an IgG specific endoglycosidase that hydrolyzes complex, hybrid and high mannonse type glycans on the conserved Fc site on IgG.

The TransGLYCIT platform is developed for transglycosylation of human IgG.

No, TransGLYCIT is based on IgG specific enzymes and will only transglycosylate IgG.

 

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