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Glycoengineering is increasingly recognized as an important strategy in the development of new biopharmaceuticals with optimized half-life, antigenicity, and efficacy. With years of experience and advanced technologies, Creative Biolabs has proposed different genetic strategies to circumvent issues like heterogeneity, nonhuman glycans in different glycoprotein production systems. Professional technical experts in Creative Biolabs are dedicated to providing the most comprehensive glycoengineering service and we offer our clients with high-quality services in your glycoprotein project to help you get milestone success.

Strategies of Genetic Glycoengineering

Glycosylation profile varies significantly across organisms and glycosylation strongly affects therapeutic activity and efficacy of glycoprotein biologics. In general, the process of glycosylation starts in the endoplasmic reticulum (ER). Many glycosylation steps are accomplished by Golgi-localized glycosyltransferases that subsequently generate mature glycan structures. Thereby, numerous genetic strategies have been proposed for manipulating glycosylation altering heterogeneity, sialylation, fucosylation, and branching structures and humanizing the glycosylation profile of expression hosts, such as genes knockdown, knockout and knockin, overexpression and alteration and so forth.

  • Knockdown
  • Many experimental cases have demonstrated the effectiveness of gene knockout in glycosylation engineering. For example, engineered Chinese hamster ovary (CHO) cells (with GnT-IV and -V overexpression and GalT4 knockdown) express a highly glycosylated human chorionic gonadotropin (hCG) hormone. An RNAi-mediated knockdown approach was used to reduce plant-specific glycosyltransferase activities. Besides, double siRNA knockdown of α1,6-fucosyltransferase (FUT8) and GDP-mannose 4,6-dehydratase (GMD) in CHO cells provide a new strategy for generating fully non-fucosylated therapeutic antibodies with enhanced ADCC.

  • Knockout and Knockin by Precision Genome Editing
  • In glycoengineering approaches towards optimizing activity, unwanted enzymes are typically inactivated to prevent the transfer of single monosaccharides to glycan structures. Knockout of core α1,6-fucosyltransferase (FUT8) in CHO, knockout of α1,6-mannosyltransferase (Och1p) in yeast, knockout of core α1,3-fucosyltransferase (FUT) and β-N-acetylglucosaminidase (β-hex) in insect cells, knockout of FUT and β1,2-xylosyltransferase (XylT) in plants have already been reported. The synthesis of undesired or host specific glycol-epitopes also could be eliminated by employing gene knockout strategies. Besides, knockin strategies using human-specific glycosyltransferases have emerged for the humanization of N-glycosylation in plants. Glycoengineering with the combined knockout/knockin approach of glycosylation-related enzyme genes has been recognized as an effective method for the introduction of human glycosylation machinery.

Schematic depiction of nuclease-based precision gene editing techniques Fig.1 Schematic depiction of nuclease-based precision gene editing techniques (Steentoft, 2014).

  • Overexpression
  • Besides knockout and knockin of sialyltransferase genes, increases in sialic acid content can be obtained by overexpressing specific genes, such as galactosyltransferase genes, anti-apoptotic genes, and genes involved in sialic acid biosynthesis and transport. Additionally, co-overexpressing several genes together enhanced sialylation more significantly than overexpressing single genes. The altered glycan ratios can be overcome by overexpressing ST6Gal 1 in CHO cells by using a plasmid expression vector. Enhancement of sialylation on humanized IgG-like bispecific antibody can be obtained by overexpression of α2,6-sialyltransferase derived from CHO cells. Co-overexpression of β1,4-N-Acetylglucoseaminyltransferase III (GnT III) and α-mannosidase II leads to further lower fucose content by introducing non-fucosylated hybrid-type glycans. Production of antibodies with increased antibody effector function was also achieved by overexpression of GnT-III in CHO cells. For sialylation, overexpression of α-2,3-sialyltransferase and β-1,4-galactosyltransferase elevates IgG sialylation and galactosylation.

Features of Genetic Glycoengineering Strategies

  • Optional genetic approaches for manipulating glycosylation
  • Different production system
  • Perfect after-sale service system
  • Cost-effective and time-saving

Manipulating glycosylation to increase desired glycans in glycoprotein biologics is of high scientific and commercial interest. Equipped with state-of-the-art research and manufacturing facilities, Creative Biolabs has gained significant knowledge and rich experience in genetic glycoengineering. We are more than happy to share our experience and help our customers in cell line glycoengineering development. Please contact us for more information and a detailed quote.


  1. Steentoft, C.; et al. Precision genome editing: a small revolution for glycobiology. Glycobiology. 2014, 24(8), 663-680.
For Research Use Only.

Related Services:

  1. Glycoprotein Production System

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