GlyCLICK Applications

The novel and unique conjugation technology GlyCLICK offers opportunities for site-specific conjugation of antibodies, using a two step enzymatic process and click chemistry. GlyCLICK applications include e.g. ADC development, fluorescent imaging and in vivo imaging. GlyCLICK is a modular technology applicable to IgG from several species and subclasses and offer robust and quantitative conjugation of 2 labels per antibody.

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For details on pricing and how to order, please visit the GlyCLICK Product Page

2017_ATDC_Site-specific and Quantitative Conjugation of Functional Groups to Antibody N-glycans using the Novel GlyCLICKTM TechnologyDownload GlyCLICK Poster

Site-specific and Quantitative Conjugation of Functional Groups to Antibody N-glycans using the Novel GlyCLICK™ Technology. Download the poster

Site-Specific Conjugation

GlyCLICK combines GlycINATOR’s specific and efficient trimming of the Fc glycans and the SiteClick™* technology for stable and precise attachment of a variety of labels and functional molecules on Fc. GlycINATOR removes all Fc N-glycans, including high-mannose, hybrid type and bisected glycans, leaving the innermost GlcNAc intact. The subsequent activation at this GlcNAc is followed by a click chemistry reaction for selective attachment of a DIBO-label. The conjugation of the DIBO-label of choice occurs at activated sites on the Fc region, ensuring specific and complete conjugation (Figure 1). A quantitative incorporation of two labels per antibody occurs, and the degree of labeling will always be two (DOL=2). The site-specific conjugation on the Fc domain preserves the affinity of the antigen binding sites.

 

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Figure 1. RP-HPLC analysis of Trastuzumab (Herceptin®) was performed on Agilent 1290 using Waters Acquity UPLC® BEH300 C4, 1.7 μm, 2.1x100 mm column in an acetonitrile/isopropanol gradient at 65 °C.
A. Digested with FabRICATOR® into F(ab’)2 and Fc/2 fragments.
B. Conjugated with Alexa Fluor®488 and digested with FabRICATOR into F(ab’)2 and Fc/2 fragments.
C. Fluorescence signal of B.

 

GlyCLICK LC/MS Analysis

To evaluate the site-specific GlyCLICK conjugation of a cytotoxic drug to the Fc portion of the antibody, trastuzumab conjugated with MMAE using GlyCLICK was analyzed by LC/MS. The native antibody (A), deglycosylated antibody (B), and GlyCLICK conjugated antibody (C) were all digested with FabRICATOR to obtain subunit fragments Fc/2, Fd and LC prior to LC/MS analysis. The results, presented in Figure 2, demonstrates the site-specific conjugation of a cytotoxic drug to the Fc/2 fragment while the Fd and the LC remain intact. The data also shows the homogeneity of the end-product with only 1 drug coupled per Fc/2 fragment, that results in an overall degree of labeling of 2. The mass difference between deglycosylated Fc/2 and the GlyCLICK conjugated sample is derived from the addition of a GalNAz residue of 245 Da and the DIBO linker with MMAE drug of 1781 Da, together a total mass shift of 2026 Da.

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Figure 2.  All samples were digested with FabRICATOR prior to LC/MS analysis. Antibody subunit samples were separated by reversed phase on a BEH300 C4 column from Waters and analyzed on an Agilent 1290 UPLC system coupled to a Bruker Impact II ESI Q-TOF. Mass spectrum was deconvoluted using the MaxEnt algorithm.

A. Intact trastuzumab, Fc/2, Fd and LC

B. Trastuzumab deglycosylated with Immobilized GlycINATOR

C. GlyCLICK conjugation at the Fc/2 fragment with azide activation (245 Da) and a DIBO linker with MMAE cytotoxic drug (1781 Da), together 2026 Da.

Fluorescent Imaging

Labeling of antibodies with fluorescent dyes is common in imaging applications (Figure 3). Traditional labeling methods rely on unspecific attachment of dye to the antibody and may interfere with antigen binding and result in a pool of heterogenous labeled antibodies. The GlyCLICK technology allows specific attachment of dye at the Fc domain of the antibody, allowing full antigen binding capacity of the antibody. The degree of label added to the antibody (DOL) equals two, with high reproducibility and low batch-to-batch variations. This feature allows correct quantitation of antibodies in sensitive imaging applications.

 

Available products

glyclick-icon-alexa-flour-488GlyCLICK AlexaFluor®488

Figure 3. Cells with high (b) or low (a) HER2 expression were incubated with Hoechst 33342 for staining of the nuclei and subsequently incubated with trastuzumab-AlexaFluor®488. The cells were analyzed by fluorescence microscopy using DAPI and FITC filters. High and specific signal was observed on HER2-positive cells (b) and no signal was observed on the HER2-negative cells (a).

Modular Technology

GlyCLICK is a versatile tool for conjugation of any IgG with a selection of labels and functional molecules, such as a dye, an affinity tag or a chelator. It is a unique technology where the same conjugation method is used to produce conjugates suitable for both imaging and various immunoassays in mAb characterization and ADC drug development (Table 1). All conjugates will have the same reproducible mode of attachment on the IgG, independent of the label. With the GlyCLICK technique, it is possible to conjugate a variety of molecules to the antibody, including toxic payloads in ADC development (Figure 4). GlyCLICK is also available for azide activation of the antibody without a DIBO label, allowing custom conjugation of any alkyne label.

Table 1. Characteristics and examples of applications for available labels

Label Name  Characteristics Examples of Applications
Fluorophore AlexaFluor® 488 Ex 496 nm/Em 519 nm FACS, IHC, in vitro cell imaging
Affinity Biotin   Immuno assays, ELISA, western blot
Chelator Deferoxamine (DFO) Suitable for 89Zr Immuno imaging in vivo
 No Label Azide Activation Custom conjugation of any alkyne label Various

Figure 4. RP-HPLC analysis of Panitumumab (Vectibix®) was performed on Agilent 1290 using Waters Acquity UPLC® BEH300 C4, 1.7 μm, 2.1x100 mm column in an acetonitrile/isopropanol gradient at 65 °C, for unmodified and conjugated with DFO chelator or MMAE toxin (Monomethyl aurostatine E) digested with FabRICATOR into F(ab’)2 and Fc/2 fragments.

Reliable Performance

GlyCLICK is designed as a robust method that can be used to conjugate labels or functional molecules on IgGs of many species and subclasses (Figure 4 and 5). GlycINATOR deglycosylates all human IgG subclasses and IgG from many other species such as mouse, rat, goat and rabbit. The GlyCLICK technology is easy to work with and the chemo-enzymatic specificity ensures generation of highly reproducible conjugations. The resulting DOL=2 is independent on the IgG used, since the attachment will be on the Fc.

 

Figure 5. RP-HPLC analysis of mouse IgG2a was performed on Agilent 1290 using Waters Acquity UPLC® BEH300 C4, 1.7 μm, 2.1x100 mm column in an acetonitrile/isopropanol gradient at 65 °C, for unmodified and conjugated with Biotin or AlexaFluor®488 digested with FabRICATOR Z into F(ab’)2 and Fc/2 fragments.

Intact Immunoreactivity

The GlyCLICK conjugation technology allows site-specific conjugation at the Fc domain of IgG of many species and subclasses. Site-specific conjugation at the Fc ensures that the antibody's ability to bind its antigen remains intact.  The binding of trastuzumab-AlexaFluor®488 to HER2 (AF488, green) was tested in a direct ELISA format and compared to a trastuzumab-Dylight488 random conjugate (DY488, purple). Although the dye per antibody varied substantially between AF488 and DY488 (right panel), the fluorescent signal of AF488 when bound to HER2 was in the same range as for the Dy488 (Figure 6).

Available Products

glyclick-icon-alexa-flour-488GlyCLICK AlexaFluor®488

Figure 6. Left: Comparison of binding of GlyCLICK trastuzumab-AF488 (AF488, DOL = 2) and Dylight trastuzumab-Dy488 (Dy488, DOL = 10) conjugates to HER2 on ELISA plate in dilutions from 0.5 mg/ml, 100 μl/well. Plotted as mean values. Right: Correlation between fluorescence and concentration between GlyCLICK trastuzumab-AF488 (AF488, DOL = 2) and Dylight trastuzumab-Dy488 (Dy488, DOL = 10).

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For details on pricing and how to order, please visit the GlyCLICK Product Page

2017_ATDC_Site-specific and Quantitative Conjugation of Functional Groups to Antibody N-glycans using the Novel GlyCLICKTM TechnologyDownload GlyCLICK Poster

Site-specific and Quantitative Conjugation of Functional Groups to Antibody N-glycans using the Novel GlyCLICK™ Technology. Download the poster