pp 1-33 | Cite as

Glycoengineering of Mammalian Expression Systems on a Cellular Level

  • Kelley M. Heffner
  • Qiong Wang
  • Deniz Baycin Hizal
  • Özge Can
  • Michael J. Betenbaugh
Part of the Advances in Biochemical Engineering/Biotechnology book series


Mammalian expression systems such as Chinese hamster ovary (CHO), mouse myeloma (NS0), and human embryonic kidney (HEK) cells serve a critical role in the biotechnology industry as the production host of choice for recombinant protein therapeutics. Most of the recombinant biologics are glycoproteins that contain complex oligosaccharide or glycan attachments representing a principal component of product quality. Both N-glycans and O-glycans are present in these mammalian cells, but the engineering of N-linked glycosylation is of critical interest in industry and many efforts have been directed to improve this pathway. This is because altering the N-glycan composition can change the product quality of recombinant biotherapeutics in mammalian hosts. In addition, sialylation and fucosylation represent components of the glycosylation pathway that affect circulatory half-life and antibody-dependent cellular cytotoxicity, respectively. In this chapter, we first offer an overview of the glycosylation, sialylation, and fucosylation networks in mammalian cells, specifically CHO cells, which are extensively used in antibody production. Next, genetic engineering technologies used in CHO cells to modulate glycosylation pathways are described. We provide examples of their use in CHO cell engineering approaches to highlight these technologies further. Specifically, we describe efforts to overexpress glycosyltransferases and sialyltransfereases, and efforts to decrease sialidase cleavage and fucosylation. Finally, this chapter covers new strategies and future directions of CHO cell glycoengineering, such as the application of glycoproteomics, glycomics, and the integration of ‘omics’ approaches to identify, quantify, and characterize the glycosylated proteins in CHO cells.

Graphical Abstract


Chinese hamster ovary CHO CRISPR/Cas9 Fucosylation Glycoengineering Glycomics Glycoproteomics Mammalian expression systems N-linked glycosylation O-linked glycosylation Sialylation TALEN ZFN 



Antibody-dependent cellular cytotoxicity




Baby hamster kidney


Complement-dependent cytotoxicity


Chinese hamster ovary


cytidine 5′-monophosphate (CMP)-sialic acid transporter


Clustered regularly interspaced short palindromic repeats


Dolichol phosphate




Endoplasmic reticulum


Electrospray ionization mass spectrometry


Fragment crystallizable


Fc gamma receptor IIIa




GDP-4-keto-6-d-deoxymannose epimerase/GDP-4-keto-6-l-galactose reductase


GDP-fucose pyrophosphorylase


GDP-fucose transporter




GDP-fucose 4,6-dehydratase


Uridine diphosphate-N-acetyl glucosamine 2-epimerase/N-acetyl mannosamine kinase

GnT-1 or Mgat1

N-acetylglucosaminyltransferase I

GnT-II or Mgat2

Beta-1,2-N-acetylglucosaminyltransferase II

GnT-III or Mgat3

Beta-1,4-N-acetylglucosaminyltransferase III

GnT-IV or Mgat 4

Beta-1,2-N-acetylglucosaminyltransferase IV

GnT-V or Mgat 5

Beta-1,2-N-acetylglucosaminyltransferase V


Human embryonic kidney


Hepatocyte nuclear factor 1-alpha


High-performance liquid chromatography


Acetyl lactosamine


Monoclonal antibody


Matrix-assisted laser desorption/ionization time-of-flight


Alpha-mannosidase II


N-glycolylneuraminic acid


Natural killer




Ricinus communis agglutinin I




Short hairpin RNA


Small interfering RNA


Solid phase extraction of glycosylated peptides


Transcription activator-like effector nuclease




Tissue plasminogen activator


Zinc finger nuclease


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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Kelley M. Heffner
    • 1
  • Qiong Wang
    • 1
  • Deniz Baycin Hizal
    • 1
  • Özge Can
    • 2
  • Michael J. Betenbaugh
    • 1
  1. 1.Department of Chemical and Biomolecular EngineeringJohns Hopkins UniversityBaltimoreUSA
  2. 2.Department of Medical EngineeringAcibadem Mehmet Ali Aydinlar UniversityIstanbulTurkey

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