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
Chapter
Part of the Advances in Biochemical Engineering/Biotechnology book series

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.

Graphical Abstract

Keywords

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

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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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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