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GlyCLICK® – Site-specific Conjugation of IgG
GlyCLICK is a three-step conjugation technology for site-specific and quantitative conjugation of IgG from several species and subclasses.
With GlyCLICK, specific Fc N-glycan hydrolysis allows for site-specific conjugation at the core GlcNAcs using robust click-chemistry and results in a degree of label (DOL) or drug-antibody ratio (DAR) of 2. The reliable performance ensures quantitative conjugation and intact immunoreactivity for sensitive applications.
The technology is available in a range of kit formats, including conjugation with fluorophores, biotin, DFO, or toxins for ADC development. The format “Azide Activation” renders the antibody azide-activated, for conjugation with any alkyne-reactive label of choice.
Why GlyCLICK®?
- Site-specific conjugation of IgG
- Reliable performance ensuring quantitative conjugation and preserved immunoreactivity for sensitive applications
- Stable and homogenous conjugation generates a degree of labeling (DOL) of 2
Human IgG1-4, IgG from mouse, rabbit, rat, monkey, sheep, goat, cow and horse
2-3 day protocol
Fluorophore, Biotin, DFO, MMAE, PNU, Azide Activation
Conjugation occurs at the Fc N-glycan sites
Product Formats
GlyCLICK® Azide Activation
Site-specific conjugation of IgG with azide-alkyne click chemistry
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About GlyCLICK®
How GlyCLICK works
- Deglycosylation
The Fc specific endoglycosidase GlycINATOR® (EndoS2) hydrolyzes the Fc glycans to the inner most GlcNAc moiety on several subclasses and species of IgG. The enzyme removes all glycoforms, including; high-mannose, hybrid, complex, and bisecting type glycans. - Azide Activation
Azide-containing UDP-GalNAz is enzymatically attached to the exposed GlcNAc using the ß-1,4-Galactosyltransferase Y289L* (GalT) to generate an azide-activated antibody that is reactive with an alkyne-carrying label. - CLICK Reaction
The azide activated antibody is conjugated with a cyclooctyne-functionalized label of choice in a biorthogonal reaction through strain-promoted copper-free click chemistry (SPAAC) to form a stable triazole.
Applications
Site-specific ADC generation with defined DAR, preserving antigen binding and delivering potent, controlled cytotoxicity in target cells.
Improved PET/CT tumor targeting and biodistribution using site-specific antibody labeling with consistent DOL and preserved in vivo performance.
Supports high-quality immunofluorescence imaging with consistent signal intensity and reliable quantification through defined antibody labeling.
Higher-resolution flow cytometry with improved signal intensity and a 10-fold increase in separation index using site-specific antibody labeling.
Citations
Resources
GlyCLICK® for Generation of Dual-payload and Bispecific ADCs
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GlyCLICK® for Generation of Improved ADCs
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GlyCLICK® for Dual-payload ADCs
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GlyCLICK®: From Kit to Clinic
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GlyCLICK® for Generation and Analysis of ADCs
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GlyCLICK® for Generation of Site-specific Conjugates for Fluorescent Imaging
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FAQ and Support
How do I perform quality control of the conjugation process?
Characterization using LC-MS will give a complete and clear picture of mass shift associated with successful conjugation if the antibody is analyzed reduced or fragmented. LC or LC-MS analysis of HC from a reduced sample or scFc fragments generated by FabRICATOR digestion provides distinct elution peaks for conjugated scFc or mass shifts corresponding to the degree of labelling (DOL) following conjugation. On the application page you can see an example of such analysis. Analysis of fluorescently labeled antibodies can also be performed by absorbance measurements and calculate the DOL using the Extinction coefficient for the antibody and the label and the correction factor for the label.
Which isotopes can I use to radiolabel IgG after chelation using GlyCLICK?
The GlyCLICK technology can be used to conjugate IgG with Desferrioxamine (DFO) using the GlyCLICK DFO kit. The DFO is a chelating agent for radiolabeling with the radioisotope Zirconium-89 (89Zr) for PET-imaging. The Azide Activation kit can be used to conjugate a chelator of choice if it is alkyne-modified (carrying for example DIBO, DBCO or BCN) to be compatible with the click chemistry.
What is the conjugation efficacy using GlyCLICK?
All the steps (deglycosylation, azide activation and click reaction) in GlyCLICK are highly efficient. Normally, all molecules are conjugated when using GlyCLICK, resulting in DOL=2. On the application page we have shown by MS-analysis that all molecules have been conjugated and there is no remaining un-conjugated material.
What fluorescent labels can I use with the GlyCLICK technology?
GlyCLICK is available in kit formats with AlexaFluor®488, AlexaFluor®555, AlexaFluor®647 or in the Azide Activation kit format for conjugation of a label of choice. With the fluorescent label being alkyne-modified (carrying for example DIBO, DBCO or BCN) it is possible to combine it with the GlyCLICK azide activation kit.
Can I freeze antibodies after biotinylation using GlyCLICK?
Problems can arise from freezing of the conjugates. We cannot guarantee the quality after freezing and therefore recommend storage at +4°C.
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Genovis products are intended for research use only. They are not intended to be used for therapeutic or diagnostic purposes in humans or animals. All goods and services are sold subject to Genovis’ General Terms and Conditions of Sale.
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