Azide Activation of Human IgG
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Azide Activation of Human IgG using TransGLYCIT
Site-specific Fc N-glycan conjugation is a powerful tool with applications ranging from biomedical research to the development of diagnostic methods and antibody-drug conjugates (ADCs). A reliable conjugation technology is crucial to obtain a homogenous and reproducible conjugate while preserving the stability and immunoreactivity of the final product. Using TransGLYCIT, human IgG is azide-activated by transglycosylation within a few hours. During that process, the antibody is rendered to carry azide-activated Fc N-glycans. The introduced azide handles can then be successfully used for site-specific conjugation, resulting in a homogenous degree of labeling (DOL) of 4.
A site-specific labeling with a high DOL is desirable for fluorescent-based assays since it allows high sensitivity, and in ADCs, the drug-antibody ratio (DAR) impacts the potency. The TransGLYCIT technology ensures quantitative conjugation and intact immunoreactivity, features that are often limited when using random conjugation technologies. The TransGLYCIT workflow provides a valuable tool to enable site-specific and quantitative conjugation of human IgG.
The Azide Activation Transglycosylation Workflow
- 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. - Transglycosylation
The engineered glycosynthase TransINATOR™ catalyzes the transglycosylation reaction between the oxazoline glycoform azide and the core GlcNAc. The degree of fucosylation will be the same as on the original IgG.
The transglycosylation of IgG is followed by and affinity purification step.
Site-specific Azide Activation of Trastuzumab
The conserved N-linked glycosylation site on the CH2 domain of each heavy chain of IgG can be used as a site for conjugation. Here, TransGLYCIT Azide Activation was used for site-specific azide activation of trastuzumab using the modified N-glycan oxazoline carrying two azide functionalities. The reaction generated an IgG with four sites for conjugation, two sites on each Fc/2 (Fig. 1).
Figure 1. Azide activation of trastuzumab using the TransGLYCIT workflow. Deconvoluted mass spectra of the Fc/2 fragment of native trastuzumab (top), after deglycosylation by GlycINATOR (middle) and after transglycosylation by TransINATOR with the oxazoline glycoform azide as the substrate (bottom). The mAb was digested with FabRICATOR and the subunits were analyzed by reversed-phase LC-MS on a Waters™ BioAccord™ system equipped with a Waters™ BioResolve™ RP mAb column (2.1×50mm).
Comparison of Site-specific and Random Conjugation Technologies
The azide-activated Fc N-glycans on antibodies modified using the TransGLYCIT Azide Activation workflow can be used for site-specific conjugation with a label of choice via click chemistry, resulting in an antibody homogeneously labeled with four labels per molecule (DOL=4). Using site-specific conjugation instead of random conjugation technologies is favorable for preserving affinity and generating conjugates where quantitative properties are desired. To illustrate the impact of different labeling methods on the DOL of the generated conjugates, we analyzed trastuzumab conjugated with a fluorophore using either TransGLYCIT, GlyCLICK or random labeling by NHS-chemistry. The TransGLYCIT and GlyCLICK workflows resulted in homogenously labeled samples with defined DOL 4 or DOL 2 respectively, whereas the random technique resulted in a heterogenously labeled sample with a DOL varying from 2 to 10 (Fig. 2).
Figure 2. Conjugation of trastuzumab using site-specific and random technologies. Deconvoluted mass spectra of intact trastuzumab modified using TransGLYCIT Azide Activation followed by site-specific conjugation by strain-promoted azide alkyne click chemistry using DBCO-AF647 (DOL=4; top), conjugated using GlyCLICK AlexaFluor®647 (DOL=2; middle), and randomly labeled using NHS-activated AlexaFluor 647 resulting in heterogenous labeling (DOL=2-10; bottom). The randomly labeled sample was analyzed after deglycosylation by GlycINATOR. The intact masses of the trastuzumab conjugates were analyzed using a Bruker Impact II ESI-QTOF MS.
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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.
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.
GlycINATOR is an IgG specific endoglycosidase that hydrolyzes complex, hybrid and high mannonse type glycans on the conserved Fc site on IgG.
No, TransGLYCIT is based on IgG specific enzymes and will only transglycosylate IgG.
The TransGLYCIT platform is developed for transglycosylation of human IgG.
