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AGLink® Site-specific conjugation kit

The conjugation method chosen for antibody-based conjugated drugs plays a pivotal role in determining the Antibody-Drug Ratio (DAR) and the uniformity of the resulting product, directly impacting its efficacy, tolerability, and stability.

Conjugation methods are commonly categorized as random or site-specific. Site-specific conjugation is increasingly preferred for its ability to yield conjugated drugs with high uniformity and enhanced safety profiles.

Almost all monoclonal antibodies are glycosylated at (or around) Asn-297 of the Fc domain. Glycan -mediated conjugation, a well-established site-specific method, utilizes the N297 glycosylation sites of the antibody for conjugation.

Being distant from the antigen-binding region, conjugation at this site preserves the antibody's binding function, minimizing the risk of compromising its antigen-binding ability post-conjugation. This method eliminates the need for antibody engineering modifications, enhancing convenience, versatility, and significantly expediting early-stage research efficiency.

Through collaboration with Glyco-Therapy Biotechnology Co., Ltd., we have developed the AGLink® DAR2&4 site-specific conjugation kit. Based on Glyco-therapy's YTConju™ conjugation platform, the kit facilitates efficient conjugation of MMAE. Designed to empower early-stage research and biological experiments in the field of conjugated drugs.

Product Features

One-pot, One step method

DAR 2 or DAR 4 kits available with one kit

Achieve Uniform DAR values overnight to within 24 hours

Homogeneous and stable products after conjugation

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Product List

Cat. No.MoleculeProduct DescriptionOrder Now
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Mechanism of Conjugation

Almost all monoclonal antibodies are glycosylated at (or around) Asn-297 of the Fc domain. While the glycans have different isoforms, the types that typically dominate are G0F, G1F&G2F (＞90%)1. The AGLink® Antibody Conjugation Kit utilizes the YTConju™ platform, a glycan remodeling strategy developed by Glyco-therapy Biotechnology Co., Ltd. for the construction of site-specific antibody conjugates2.

For DAR4 Conjugation
Mechanism of Conjugation

For DAR2 Conjugation
Mechanism of Conjugation

AGLink® Site-specific conjugation kit

Application Scenarios

Conjugation with MMAE for antibody screening, preparation, and performance studies in the ADC field.

Assay data

High homogeneity conjugated antibodies
High homogeneity conjugated antibodies

HIC-HPLC analysis of MMAE ADCs (DAR 4 & DAR 2)

Preserved immunoreactivity after conjugation
Preserved immunoreactivity after conjugation

The analysis of the antigen-binding capacity of MMAE ADCs (DAR 4 & DAR 2), the results showing that binding to the HER2 antigen unaffected by the AGLink conjugation

Consistency in the site-specific labeling process ensures mitigate the aggregation
Consistency in the site-specific labeling process ensures mitigate the aggregation

Less than 5% of antibody aggregation by SEC-HPLC

The conjugates have high in-vitro plasma stability
The conjugates have high in-vitro plasma stability

MMAE ADCs (DAR 4 & DAR 2) are stable in human plasma in vitro

In-vitro cell-killing activity assay
In-vitro cell-killing activity assay

Validated in-vitro cell-killing activity of MMAE ADCs (DAR 4 & DAR 2)

Resources

FAQ

1. What is the minimum amount of antibody suitable for the AGLink® Conjugation Kit?

The minimum requirement is 200 μg, with an expected recovery rate exceeding 85%. To accommodate this quantity, scale down the reaction reagents proportionally according to the protocol provided.

2. Is it necessary to concentrate the antibody according to the protocol?

For optimal reaction efficiency, it is recommended to concentrate the antibody to the protocol’s suggested concentration. If concentration is not feasible, ensure that the antibody concentration remains above 1 mg/ml, and extend the reaction time to 48 hours to maintain conjugation efficiency.

3. Is this Conjugation Kit suitable for antibodies from all species?

This conjugation kit is highly effective for humanized antibodies conjugation. However, murine and rabbit antibodies, due to their complex structures and incomplete validation, may require an extended conjugation time of up to 48 hours to enhance conjugation efficiency.

4. Can the Linker in the AGLink® ADC Conjugation Kit be hydrolyzed, and if so, by what means?

The linker in the AGLink® ADC Conjugation Kit (MMAE, DAR 2 & 4, 1 mg & 5 mg) is vc-PAB, which can be cleaved by Cathepsin B. The cleavability of linkers in other kits depends on whether a cleavable linker is used during click chemistry conjugation.

5. How is the conjugation efficiency of the Conjugation Kit, and what methods are used to detect the Drug to Antibody Ratio (DAR) of conjugated product?

The conjugation efficiency of those kits can exceed 90%, and the DAR value can be analyzed using HIC-HPLC (MMAE) or LC-MS (MMAE/DBCO/Tetrazine/Biotin).

6. How to store the conjugated product and its stability under those conditions?

It is recommended to store the conjugated product in PBS buffer, where it can be preserved for over six months at -80°C. Avoid repeated freeze-thaw cycles.

7. What laboratory environment is required for this kit?

AGLink® conjugation kit can be used under normal laboratory conditions. Ensure you use personal protection equipment (lab coat, safety glasses, and chemical resistant nitrile gloves) while handling MMAE.

8. Are there any patent restrictions on this product, and will they impact on CMC development and commercial production?

The intellectual property of our AGLink® conjugation kit is covered in part or wholly by a patent owned or controlled by Glyco-therapy Biotechnology Co., Ltd. For more information about commercial rights, please contact inquiry@acrobiosystems.com for any comments or concerns.

9. If the Fc N297 site is mutated, can site-specific conjugation still be performed?

The current conjugation kit is designed for site-specific modification at glycosylated sites, if the N297 Fc glycosylation site is removed, this kit cannot be used. However, site-specific conjugation can be achieved using alternative methods.

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References

[1] Van Geel R, Wiideven M A, Heesbeen R, Heesbeen R.et al. Chemoenzymatic coniugation of toxic payloads to theglobally conserved N-glycan of native mAbs provids homogeneous and highly efficacious antibody-drug conjugatesJ.Bioconjugate Chemistry, 2015,26(11):2233-2242.

[2] Yang y, Zhentao S, Tian T,et al. Reducing the Complexity of Fc Glycan Enables the construction ofAntibodvoniugates with Unexpected High Efficiency and Payload Capacity via Gycoengineering; bioRxiy preprint doi:https://doi.org/10.1101/2022.09.04.506510.

FAQ

Q

What is site-specific ADC conjugation?

Site-specific ADC conjugation is an approach that attaches cytotoxic payloads to defined locations on antibodies through controlled conjugation strategies. Compared with random conjugation methods, site-specific conjugation provides improved control over payload attachment and DAR distribution. This approach helps generate more homogeneous ADCs for downstream characterization and evaluation.
Q

How does glycan-mediated conjugation enable site-specific ADC preparation?

Glycan-mediated conjugation utilizes the conserved Fc glycan region of IgG antibodies, including the N297 glycosylation site, as a defined location for payload attachment. Through glycan remodeling, reactive groups can be introduced for site-specific conjugation without modifying the antibody sequence. This strategy enables controlled generation of antibody conjugates while maintaining the antibody structure required for antigen recognition.
Q

What DAR values can be achieved with the AGLink® site-specific conjugation kit?

The AGLink® site-specific conjugation kit is designed to generate MMAE antibody conjugates with controlled DAR 2 or DAR 4. Different DAR formats are available to support evaluation of ADC candidates with different payload loading levels. After conjugation, DAR distribution and product homogeneity can be characterized using analytical methods such as HIC-HPLC.
Q

Does glycan-mediated site-specific conjugation affect antibody binding activity?

Glycan-mediated conjugation occurs at the Fc glycan region, which is spatially separated from the antigen-binding Fab region. Therefore, antibody binding activity can be evaluated after conjugation to confirm preservation of antigen recognition. Binding assays such as BLI or SPR are commonly used to assess the immunoreactivity of resulting ADCs.
Q

What applications can AGLink® site-specific conjugation kits support?

AGLink® site-specific conjugation kits support the preparation of MMAE antibody conjugates for ADC-related research applications, including antibody screening, ADC candidate preparation, and functional performance evaluation. The controlled conjugation approach enables researchers to generate characterized ADC samples for downstream studies.
Q

How does ACROBiosystems support site-specific ADC conjugation and characterization?

ACROBiosystems provides the AGLink® DAR2&4 site-specific conjugation kit based on glycan-mediated conjugation technology, supporting controlled preparation of MMAE antibody conjugates. Complementary ADC research products, including target proteins, anti-payload antibodies, internalization detection reagents, and linker-cleaving proteases, support downstream characterization of ADC properties.
Q

What critical biophysical quality attributes make ACROBiosystems' anti-idiotypic antibodies highly reliable across multi-year clinical campaigns?

Multi-year clinical trials require reagents with unwavering batch consistency and minimal risk of assay drift. ACROBiosystems’ anti-idiotypic antibody portfolio features: Picomolar Affinity (KD): Characterized via Surface Plasmon Resonance (SPR), reaching picomolar binding strength (e.g., clone ADB-Y19 binds Adalimumab with an affinity constant of 1.36 pM). SEC-MALS Verification: Select clones undergo Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) to confirm purity and a defined monomeric molecular weight. High Thermal & Freeze-Thaw Stability: Validated through accelerated 37°C stress tests and repeated freeze-thaw cycles without activity loss.
Q

What critical biophysical quality attributes make ACROBiosystems' anti-idiotypic antibodies highly reliable across multi-year clinical campaigns?

Multi-year clinical trials require reagents with unwavering batch consistency and minimal risk of assay drift. ACROBiosystems’ anti-idiotypic antibody portfolio features: Picomolar Affinity (KD): Characterized via Surface Plasmon Resonance (SPR), reaching picomolar binding strength (e.g., clone ADB-Y19 binds Adalimumab with an affinity constant of 1.36 pM). SEC-MALS Verification: Select clones undergo Size-Exclusion Chromatography coupled with Multi-Angle Light Scattering (SEC-MALS) to confirm purity and a defined monomeric molecular weight. High Thermal & Freeze-Thaw Stability: Validated through accelerated 37°C stress tests and repeated freeze-thaw cycles without activity loss.
  • Background
  • Product Features
  • Product List
  • Mechanism of Conjugation
  • AGLink® Site-specific conjugation kit
  • Resources
  • FAQ