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Advances in Glycobiotechnology pp 37–69Cite as

Glycoengineering of Mammalian Expression Systems on a Cellular Level

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Part of the book series: Advances in Biochemical Engineering/Biotechnology ((ABE,volume 175))

Abstract

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.

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Abbreviations

ADCC:

Antibody-dependent cellular cytotoxicity

Asn:

Asparagine

BHK:

Baby hamster kidney

CDC:

Complement-dependent cytotoxicity

CHO:

Chinese hamster ovary

CMP-SAT:

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

CRISPR:

Clustered regularly interspaced short palindromic repeats

Dol-P:

Dolichol phosphate

EPO:

Erythropoietin

ER:

Endoplasmic reticulum

ESI-MS:

Electrospray ionization mass spectrometry

Fc:

Fragment crystallizable

FcγRIIIa:

Fc gamma receptor IIIa

FUT8:

α-1,6-fucosyltransferase

FX:

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

GFPP:

GDP-fucose pyrophosphorylase

GFT:

GDP-fucose transporter

GlcNAc:

N-acetylglucosamine

GMD:

GDP-fucose 4,6-dehydratase

GNE/MNK:

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

HEK:

Human embryonic kidney

HNF1-alpha:

Hepatocyte nuclear factor 1-alpha

HPLC:

High-performance liquid chromatography

LacNAc:

Acetyl lactosamine

mAb:

Monoclonal antibody

MALDI-TOF:

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

ManII:

Alpha-mannosidase II

Neu5Gc:

N-glycolylneuraminic acid

NK:

Natural killer

OST:

Oligosaccharyltransferase

RCA-I:

Ricinus communis agglutinin I

Ser:

Serine

shRNA:

Short hairpin RNA

siRNA:

Small interfering RNA

SPEG:

Solid phase extraction of glycosylated peptides

TALEN:

Transcription activator-like effector nuclease

Thr:

Threonine

tPA:

Tissue plasminogen activator

ZFN:

Zinc finger nuclease

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Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA

    Kelley M. Heffner, Qiong Wang, Deniz Baycin Hizal & Michael J. Betenbaugh

  2. Department of Medical Engineering, Acibadem Mehmet Ali Aydinlar University, Istanbul, Turkey

    Özge Can

Authors
  1. Kelley M. Heffner
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  2. Qiong Wang
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  3. Deniz Baycin Hizal
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  4. Özge Can
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  5. Michael J. Betenbaugh
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Corresponding author

Correspondence to Michael J. Betenbaugh .

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Editors and Affiliations

  1. Bioprocess Engineering, Max Planck Institute for Dynamics of Complex Technical Systems, Magdeburg, Germany

    Erdmann Rapp  & Udo Reichl  & 

  2. glyXera GmbH, Magdeburg, Germany

    Erdmann Rapp

  3. Otto-von-Guericke University Magdeburg, Magdeburg, Germany

    Udo Reichl

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Heffner, K.M., Wang, Q., Hizal, D.B., Can, Ö., Betenbaugh, M.J. (2018). Glycoengineering of Mammalian Expression Systems on a Cellular Level. In: Rapp, E., Reichl, U. (eds) Advances in Glycobiotechnology. Advances in Biochemical Engineering/Biotechnology, vol 175. Springer, Cham. https://doi.org/10.1007/10_2017_57

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