GlyCLICK Characteristics

GlyCLICK is a site-specific conjugation technology for IgG using enzymatic remodeling of the Fc-glycan sites and click-chemistry. The key characteristics of GlyCLICK facilitate the generation of tailored and robust conjugates for reproducible and reliable results independent of the label or payload.GlyCLICK site-specific conjugation

Site-specific conjugation

GlyCLICK is a site-specific conjugation technology that utilizes glycan remodeling using the Fc-specific GlycINATOR®enzyme and combines it with click-chemistry for a stable and precise glycan-mediated antibody conjugation. The GlycINATOR enzyme digests all IgG glycoforms, including high-mannose, hybrid, complex, and bisecting type glycans from several subclasses and species of IgG, resulting in complete labeling with a constant degree of label (DOL=2.0). Trastuzumab was labeled with AlexaFluor®488 using the GlyCLICK kit (T-GlyCLICK-AlexaFluor®488) and digested with FabRICATOR®to evaluate the specificity of the conjugation process. The resulting F(ab’)2 and Fc/2 fragments were separated using RP-HPLC with the fluorescent signal detected for the Fc domain only (Fig. 1), indicating that site-specific conjugation was achieved with GlyCLICK.

 

GlyCLICK site-specific conjugation fig 1

Figure 1. RP-HPLC analysis of FabRICATOR®digested Trastuzumab (Herceptin®) performed on an Agilent 1290 using Waters Acquity UPLC® BEH C4, 1.7 μm, 2.1x100 mm column in an acetonitrile/isopropanol gradient at 65 °C.

 

The efficacy of the conjugation process was analyzed using GlyCLICK-generated antibody-drug conjugates (ADCs) of monomethyl auristatin E (MMAE) labeled trastuzumab. LC-MS analysis of the conjugates showed mass shifts of the Fc/2 fragments corresponding to complete deglycosylation by GlycINATOR and subsequent conjugation with one drug payload to each Fc/2, without interfering with the Fd or LC fragments (Fig. 2).

 

GlyCLICK site-specific conjugation fig 2

Figure 2. All samples were digested with FabRICATOR prior to LC/MS analysis. Antibody subunit samples were separated by reversed phase on a BEH C4 column from Waters and analyzed on an Agilent 1290 UPLC system coupled to a Bruker Impact II ESI Q-TOF. Mass spectra were 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 an sDIBO linker with MMAE cytotoxic drug (1781 Da), together 2026 Da.

Reliable performance

The GlyCLICK technology is easy to work with and has a reliable performance that ensures the generation of stable conjugates with a consistent DOL of 2.0. The reproducible mode of incorporation is independent of the label or payload, and such reliable conjugation and performance can be applied to a range of applications. The performance of GlyCLICK was evaluated using LC-MS analysis of FabRICATOR-digested GlyCLICK conjugates compared to randomly generated conjugates of trastuzumab (Fig. 3a). Trastuzumab labeled with a toxic payload Monomethyl aurostatine E (MMAE) using GlyCLICK was also analyzed with LC-MS after digestion with FabALACTICA and compared to the intact Fc (Fig. 3b).

GlyCLICK site-specific conjugation fig 3

Figure 3. LC-MS analysis of a) trastuzumab-DFO GlyCLICK and random conjugates and b) trastuzumab-Monomethyl aurostatine EMMAE GlyCLICK conjugates. All samples were separated by RP-HPLC on a BEH C4 column from Waters and analyzed on a Bruker Impact II ESI-Q-TOF. Mass spectra were deconvoluted using the MaxEnt algorithm.

Preserved affinity

Site-specific conjugation at the Fc-glycan sites ensures intact immunoreactivity by preserving the antigen-binding capability of the antibody. Surface Plasmon Resonance (SPR) was performed on trastuzumab with various GlyCLICK conjugates and compared to randomly generated conjugates carrying the DyLight®488 label. The results show overlapping curves for the GlyCLICK conjugates and trastuzumab (Fig. 4). Although the affinity is unchanged for randomly generated conjugated material, the level of binding is severely impared.

GlyCLICK site-specific conjugation fig 4

Figure 4. SPR affinity analysis of non-conjugated and conjugated trastuzumab. Anti-human IgG (Fc) was used to capture non-conjugated, randomly conjugated DyLight®488 (DAR=10) material and GlyCLICK conjugates; AlexaFluor®488, DFO, Biotin and MMAE (DAR=2). HER2 was injected in a range to ensure sufficient curvature. All data were fitted against a 1:1 mathematical model.

Versatile technology

GlyCLICK is a versatile and scalable tool for conjugation of IgGs with a selection of labels and functional groups such as dyes, affinity tags or chelators. For example, stable conjugation of chelating agents and toxins (Fig. 5a) or biotin and fluorophores (Fig. 5b) is possible. In addition, any custom label or payload with suitable click-chemistry can be conjugated using the azide activation kit. All labels or payloads will be incorporated in the same reproducible manner.

GlyCLICK site-specific conjugation fig 5

Figure 5. RP-HPLC analysis performed on an Agilent 1290 using Waters Acquity UPLC® BEH C4, 1.7 μm, 2.1x100 mm column in an acetonitrile/isopropanol gradient at 65 °C.
A. Mouse IgG2a unmodifiedand conjugated with Biotin or AlexaFluor®488 and digested with FabRICATOR Z into F(ab’)2 and Fc/2 fragments.
B. Panitumumab (Vectibix®) unmodifiedand conjugated with DFO chelator or MMAE toxin(Monomethyl aurostatine E) and digested with FabRICATOR into F(ab’)2 and Fc/2 fragments.

 

The GlyCLICK technology simplifies the generation of conjugates suitable for in vitro and in vivo imaging applications, immunoassays and ADC drug development (Table 1). A range of kits are available with labels such as AlexaFluor®488, Biotin and the Chelator Deferoxamine (DFO), or a custom label (payload) of choice can be conjugated using the azide activation kit.

Table 1. Characteristics and examples of applications for available labels

Label Kit format  Characteristics Examples of Applications
Fluorophore AlexaFluor®488 Ex 496 nm/Em 519 nm In vitro imaging, IHC, FCM
Affinity Biotin Immuno assays, ELISA, western blot
Chelator Deferoxamine (DFO) Suitable for 89Zr In vivo immuno imaging
 No Label Azide Activation Custom conjugation Various

product-logo-glyclick-center

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

GlyCLICK Brochure

GlyCLICK® is a site-specific conjugation technology for IgG using enzymatic remodeling and click-chemistry. Download GlyCLICK Brochure

Order