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The optimal reaction conditions for sialylation of a target protein can be different for each application. Therefore, the presented protocol can only serve as a suggested starting protocol. Optimization of reaction conditions (e.g., with different enzyme-IgG-ratios) is recommended to determine the optimal conditions for each application.

1   Preparation of the reaction buffer, containing 6.1 mM CMP-NANA, 200 mM MES, pH 6.5.
•Dissolve 40 mg CMP-NANA and 390.4 mg MES in 6 ml of water.
•Adjust pH to 6.5.
•Adjust to a final volume of 10 ml with water.
2   Setting up the sialylation reaction, containing 500 μg CMP-NANA, 100 mg ST3Gal-6, 1 mg IgG.
Pipet the following solutions into a reaction tube:
•125 μl reaction buffer
•100 μl IgG (10 mg/ml, pH 6)
•18 μl α-2,3-Sialyltransferase
•7 μl water
3   Incubate the reaction at +37°C.
4   Stop the reaction by freezing the tube at -15 to -25°C.
Analyze the samples

It is recommended to set up several identical reactions and stop them at different time points (e.g., 2 h, 8 h, 24 h) to plot the kinetics for each application.


Samples were analyzed by electrospray ionization mass spectrometry and the content of G2+0SA, G2+1SA, and G2+2SA N-glycans was determined.
Instrument: Synapt G2 HDMS device (Waters, UK)
Software: MassLynx V 4.1
 ", "Country": "XG", "Code": "Protocols", "Name": "Protocols" }, { "Language": "en", "Value": "Unit of measure is \"mg\".", "Country": "XG", "Code": "CB - Order Information", "Name": "CB - Order Information" }, { "Language": "en", "Value": "As an example, highly galactosylated humanized monoclonal antibodies IgG1 and IgG4 are used for the kinetics.

Kinetics of α-2,3-Sialytransferase with IgG1
Maximal biantennary sialylation of IgG1 is observed after 24 hours (G2 + 2SA).

Kinetics of α-2,3-Sialytransferase with IgG4
Maximal biantennary sialylation of IgG4 is obtained after 8 hours (G2 + 2SA).", "Country": "XG", "Code": "Results", "Name": "Results" }, { "Language": "en", "Value": "For in vitro sialylation of all Galß1-4GlcNAc units on glycoproteins and complex molecules such as human monoclonal antibodies (MAB), use α-2,3-Sialyltransferase (ST3Gal-6) and CMP-N-Acetylneuraminic Acid (CMP-NANA).", "Country": "XG", "Code": "Applications (IFU)", "Name": "Applications (IFU)" }, { "Language": "en", "Value": "
Vial / Bottle Label Function / Description Content
1 α-2,3-Sialyltransferase rec. Enzyme
Storage buffer contains
50 mM MES, 200 mM NaCl, pH 6.4 at +4°C
", "Country": "XG", "Code": "Content", "Name": "Content" }, { "Language": "en", "Value": "No animal-derived material is added in fermentation, purification and final formulation of this enzyme.", "Country": "XG", "Code": "Quality Control", "Name": "Quality Control" }, { "Language": "en", "Value": "Incubation of α-2,3-Sialyltansferase and CMP-NANA with a glycoprotein leads to increased sialylation of the glycoprotein.

As α-2,3-Sialyltansferase rec. adds sialic acid only to galactosylated glycostructures, a galactosylation of the glycoprotein in a previous step helps to achieve higher sialylation levels. Galactosylation can be achieved by incubation of the target protein with ß-1,4-Galactosyltransferase and UDP-Galactose.", "Country": "XG", "Code": "Applications", "Name": "Applications" }, { "Language": "en", "Value": "α-2,3-Sialyltransferase, rec. can be used for in vitro glycoengineering of glycoproteins, e.g. therapeutic proteins.", "Country": "XG", "Code": "Positioning", "Name": "Positioning" }, { "Language": "en", "Value": "Benefits of using in vitro glycoengineering to modify glycosylation of therapeutic proteins:
  • Time- and cost-saving generation of glycan variants to facilitate drug development.
  • Optimized glycosylation without compromising other CQAs or product yield.
  • Improved lot-to-lot consistency to reduce risk of product quality variation and resulting delays.
  • Generation of glycoprofiles which may not otherwise be generated.
  • Streamlined analytics in comparability studies.
", "Country": "XG", "Code": "Benefits", "Name": "Benefits" }, { "Language": "en", "Value": "α-2,3-Sialyltansferase, rec. is a highly active recombinant human glycosyltransferase, expressed in HEK-293F.", "Country": "XG", "Code": "Product Description", "Name": "Product Description" }, { "Language": "en", "Value": "Appearance: Clear, colorless to slightly colored solution.
Composition: 50 mM MES, 200 mM NaCl, pH 6.4 ± 0.1 at +4°C
Specific activity: ≥80 U/μg
Protein A280nm ( 1 mg/mL) = 0.877: 5.5 ± 0.5 mg/mL
Purity: ≥90%
Bioburden: ≤100 CFU/mL (in evaluation)
Endotoxin: ≤10 EU/mg (in evaluation)
Stability: At -15 to -25°C within specification range for 12 months.
", "Country": "XG", "Code": "Specification", "Name": "Specification" }, { "Language": "en", "Value": "Molar mass: 34.5 kDa by cDNA", "Country": "XG", "Code": "Properties", "Name": "Properties" } ] } } ] }

α-2,3-Sialyltransferase, rec.

human, expressed in HEK-293F, solution


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