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| ID | Components | Size |
| ADC-P005-C01 | 20 x Reaction buffer | 55ul |
| ADC-P005-C02 | Cofactor A | 55ul |
| ADC-P005-C03 | Cofactor B | 55ul |
| ADC-P005-C04 | Substrate A | 30ul |
| ADC-P005-C05 | Substrate B | 30ul |
| ADC-P005-C06 | Enzyme A | 30ul |
| ADC-P005-C07 | Enzyme B | 30ul |
| ADC-P005-C08 | Enzyme C | 25ul |
| ID | Components | Size |
| ADC-P005-1-C01 | Binding buffer | 15ml |
| ADC-P005-1-C02 | Elution buffer | 1.5ml |
| ADC-P005-1-C03 | Neutralization buffer | 0.5ml |
| ADC-P005-1-C04 | 250 mM Tris-HCl buffer (pH7.5) | 1.5ml |
| ADC-P005-1-C05 | 10×PBS (pH7.2- 7.4) | 6ml |
| ADC-P005-1-C06 | Desalting column | 2per |
| ADC-P005-1-C07 | Concentrator tube | 2per |
| ADC-P005-1-C08 | ProteinA Resin | 100ul×2 |
AGLink® ADC Conjugation Kit (MMAE, DAR2&4, 1mg) is designed to conjugate 1mg mAb with toxic payload (MMAE) through site-specific enzymatic reaction.
It is for research use only.
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 A&Glink® 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 conjugates[2].
The A&Glink® DAR2&4 Antibody Conjugation Kit integrates the procedure for constructing DAR2 conjugates and DAR4 conjugates in one kit, providing flexibility in DAR design.
To construct the DAR4 conjugates, galactose is first added to the terminal GlcNAc catalyzed by β-1,4-galactosyltransferase (GT) to form LacNAc. Then, α-1,3-fucosyltransferase (FT) recognizes LacNAc and transfers the Fuc-Payload to the GlcNAc of the LacNAc to form a conjugate with a theoretical DAR of 4.
To construct the DAR2 conjugates, glycans are first removed from the core GlcNAc catalyzed by endoglycosidase. Galactose is then added to the GlcNAc catalyzed by β-1,4-galactosyltransferase (GT) to form core LacNAc. Finally, α-1,3-fucosyltransferase (FT) recognizes LacNAc and transfers the Fuc-Payload to the GlcNAc of the LacNAc to form a conjugate with a theoretical DAR of 2.
The DAR4 and DAR2 conjugation procedures share the same FT, GT and GDP-Fuc-Payload. The main difference between these two procedures is the addition of an endoglycosidase.
These procedures are performed in a “one-pot” manner with only one purification step, which is highly convenient and efficient. Antibody conjugates produced with this kit generally exhibit high homogeneity, high stability and high hydrophilicity.
ADC DAR value assessment by RP-HPLC
DAR (Drug Antibody Ratio) value of ADC product was analyzed by RP-HPLC (QC tested).
Column: TSK gel Butyl-NPR column (4.6 mm × 35 mm, 2.5μm; TOSOH)
Mobile phase A: 20 mM sodium phosphate, 1.5 M ammonium sulfate (pH 6.9)
Mobile phase B: 75% (v/v) 20 mM sodium phosphate, 25% (v/v) isopropanol (pH 6.9)
Flow rate: 0.4 mL/min
Column temperature: 30 °C Detection wavelength：280nm
Loading Amount:10 µg (5-10 µL)
DAR (Drug Antibody Ratio) value of ADC product was analyzed by RP-HPLC (QC tested).
DAR4 =(1*4.43+2*2.28+3*7.1+4*86.19)/100=3.7505
DAR2＝(1*0.52+2*99.48)/100=1.9948
ADC DAR value assessment by RP-HPLC
DAR (Drug Antibody Ratio) value of ADC product was analyzed by RP-HPLC (QC tested).
Column: TSK gel Butyl-NPR column (4.6 mm × 35 mm, 2.5μm; TOSOH)
Mobile phase A：20 mM sodium phosphate, 1.5 M ammonium sulfate (pH 6.9)
Mobile phase B：75% (v/v) 20 mM sodium phosphate, 25% (v/v) isopropanol (pH 6.9)
Flow rate: 0.4 mL/min
Column temperature: 30 °C Detection wavelength：280nm
Loading Amount:10 µg (5-10 µL)
DAR (Drug Antibody Ratio) value of ADC product was analyzed by RP-HPLC (QC tested).
DAR4 =(1*4.43+2*2.28+3*7.1+4*86.19)/100=3.7505
DAR2＝(1*0.52+2*99.48)/100=1.9948
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