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 is a platform technology that enables efficient and site-specific human IgG glycan remodeling. With TransGLYCIT, antibodies with defined and homogenous glycoforms are generated using fast and robust enzymatic workflows. 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 Remodeling, IgG with the defined glycoforms G0, G1, G2 or G2S2 are obtained. With TransGLYCIT Remodeling Afucosylated, containing the FucosEXO™ 16 enzyme, it is possible to generate antibodies carrying afucosylated glycan profiles, including G0, G1, G2, G2S2 and Man5. All within three hours!
The IgG Transglycosylation Workflow
- Deglycosylation
The Fc N-glycans are trimmed to the core GlcNAc using the IgG-specific GlycINATOR® Immobilized (EndoS2) enzyme that hydrolyses all Fc glycoforms, including high-mannose, hybrid, complex and bisecting glycans. To obtain afucosylated glycoforms, the optional FucosEXO™ 16 Immobilized enzyme hydrolyzes the α1-6 linked core fucose. - Transglycosylation
The engineered glycosynthase TransINATOR™ catalyzes the transglycosylation reaction between the oxazoline-reactive glycoform and the core GlcNAc.
Generate 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 TransGLYCIT Remodeling G0. A homogenous afucosylated glycan profile was generated using the TransGLYCIT Remodeling Afucosylated G0 (bottom).
Figure 1. Deconvoluted mass spectra of the scFc 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 scFc fragment of native trastuzumab (top) and after transglycosylation with TransGLYCIT Remodeling G1 (middle), or TransGLYCIT Remodeling Afucosylated G1 (bottom). The mAb was digested with FabRICATOR and the subunits analyzed by reversed-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 Remodeling and TransGLYCIT Remodeling 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 scFc fragment of native trastuzumab (top) and after transglycosylation with TransGLYCIT Remodeling G2 (middle), or TransGLYCIT Remodeling Afucosylated G2 (bottom). The mAb was digested with FabRICATOR and the subunits analyzed by reversed-phase LC-MS on a Waters™ BioAccord™ system equipped with a Waters™ BioResolve RP mAb column (2.1 x 50 mm).
Generate 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 scFc fragment of native trastuzumab (top) and after transglycosylation with TransGLYCIT Remodeling G2S2 (middle), or TransGLYCIT Remodeling Afucosylated G2S2 (bottom). The mAb was digested with FabRICATOR and the subunits analyzed by reversed-phase LC-MS on a Waters™ BioAccord™ system equipped with a Waters™ BioResolve RP mAb column (2.1 x 50 mm).
Generate Man5 Glycoforms
Of the high-mannose N-glycan structures, Man5 is the most common one present on therapeutic antibodies. Antibodies carrying Man5 have shown increased clearance rates compared to those carrying complex N-glycan structures. With TransGLYCIT, defined Man5 glycoforms of therapeutic antibodies can be generated to investigate the impact of this N-glycan structure on functional antibody properties such as serum half-life and clearance.
High-mannose N-glycan structures are biosynthetically generated without core fucose. Using the TransGLYCIT platform, trastuzumab was remodeled to carry the Man5 glycoform within three hours. The transglycosylated antibody was analyzed at the subunit level using LC-MS (Fig. 5). The mass spectra show the glycan profile of native trastuzumab (top) and the resulting homogeneity and mass shift after complete transglycosylation of the antibody using TransGLYCIT Remodeling Afucosylated Man5 (bottom).
Figure 5. Deconvoluted mass spectra of the scFc fragment of native trastuzumab (top) and after transglycosylation with TransGLYCIT Remodeling Afucosylated Man5 (bottom). The mAb was digested with FabRICATOR and the subunits analyzed by reversed-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
Unfortunately no, the enzyme requires trimming of the Fc-glycan using GlycINATOR to enable access to the core fucose substrate.
No, TransGLYCIT is based on IgG specific enzymes and will only transglycosylate IgG.
GlycINATOR is an IgG specific endoglycosidase that hydrolyzes complex, hybrid and high mannonse type glycans on the conserved Fc site on IgG.
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.
The TransGLYCIT platform is developed for transglycosylation of human IgG.
