CRISPR/Cas9-multiplexed editing of Chinese hamster ovary B4Gal-T1, 2, 3 and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins

Thomas Amann, Anders Holmgaard Hansen*, Stefan Kol, Gyun Min Lee, Mikael Rørdam Andersen, Helene Faustrup Kildegaard

*Corresponding author for this work

Research output: Contribution to journalJournal articleResearchpeer-review

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Abstract

In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, the authors aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2, and -T3 with and without a disrupted B4Gal-T4 sequence resulted in only ≈1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. The authors revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ≈6% and ≈3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, the authors present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, the authors provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins.
Original languageEnglish
Article number1800111
JournalBiotechnology Journal
Volume13
Issue number10
Number of pages9
ISSN1860-6768
DOIs
Publication statusPublished - 2018

Keywords

  • Chinese hamster ovary cells
  • CRISP/Cas9
  • Erythropoietin
  • Glycoengineering
  • Multiplexing
  • N‐glycosylation
  • Rituximab

Cite this

@article{9615dda9824f44dc945a6dbf6dc22600,
title = "CRISPR/Cas9-multiplexed editing of Chinese hamster ovary B4Gal-T1, 2, 3 and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins",
abstract = "In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, the authors aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2, and -T3 with and without a disrupted B4Gal-T4 sequence resulted in only ≈1{\%} galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. The authors revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ≈6{\%} and ≈3{\%} galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, the authors present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, the authors provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins.",
keywords = "Chinese hamster ovary cells, CRISP/Cas9, Erythropoietin, Glycoengineering, Multiplexing, N‐glycosylation , Rituximab",
author = "Thomas Amann and Hansen, {Anders Holmgaard} and Stefan Kol and {Min Lee}, Gyun and Andersen, {Mikael R{\o}rdam} and Kildegaard, {Helene Faustrup}",
year = "2018",
doi = "10.1002/biot.201800111",
language = "English",
volume = "13",
journal = "Biotechnology Journal",
issn = "1860-6768",
publisher = "Wiley - V C H Verlag GmbH & Co. KGaA",
number = "10",

}

CRISPR/Cas9-multiplexed editing of Chinese hamster ovary B4Gal-T1, 2, 3 and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins. / Amann, Thomas; Hansen, Anders Holmgaard; Kol, Stefan; Min Lee, Gyun; Andersen, Mikael Rørdam; Kildegaard, Helene Faustrup.

In: Biotechnology Journal, Vol. 13, No. 10, 1800111 , 2018.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - CRISPR/Cas9-multiplexed editing of Chinese hamster ovary B4Gal-T1, 2, 3 and 4 Tailors N-Glycan Profiles of Therapeutics and Secreted Host Cell Proteins

AU - Amann, Thomas

AU - Hansen, Anders Holmgaard

AU - Kol, Stefan

AU - Min Lee, Gyun

AU - Andersen, Mikael Rørdam

AU - Kildegaard, Helene Faustrup

PY - 2018

Y1 - 2018

N2 - In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, the authors aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2, and -T3 with and without a disrupted B4Gal-T4 sequence resulted in only ≈1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. The authors revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ≈6% and ≈3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, the authors present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, the authors provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins.

AB - In production of recombinant proteins for biopharmaceuticals, N-glycosylation is often important for protein efficacy and patient safety. IgG with agalactosylated (G0)-N-glycans can improve the activation of the lectin-binding complement system and be advantageous in the therapy of lupus and virus diseases. In this study, the authors aimed to engineer CHO-S cells for the production of proteins with G0-N-glycans by targeting B4Gal-T isoform genes with CRISPR/Cas9. Indel mutations in genes encoding B4Gal-T1, -T2, and -T3 with and without a disrupted B4Gal-T4 sequence resulted in only ≈1% galactosylated N-glycans on total secreted proteins of 3-4 clones per genotype. The authors revealed that B4Gal-T4 is not active in N-glycan galactosylation in CHO-S cells. In the triple-KO clones, transiently expressed erythropoietin (EPO) and rituximab harbored only ≈6% and ≈3% galactosylated N-glycans, respectively. However, simultaneous disruption of B4Gal-T1 and -T3 may decrease cell growth. Altogether, the authors present the advantage of analyzing total secreted protein N-glycans after disrupting galactosyltransferases, followed by expressing recombinant proteins in selected clones with desired N-glycan profiles at a later stage. Furthermore, the authors provide a cell platform that prevalently glycosylates proteins with G0-N-glycans to further study the impact of agalactosylation on different in vitro and in vivo functions of recombinant proteins.

KW - Chinese hamster ovary cells

KW - CRISP/Cas9

KW - Erythropoietin

KW - Glycoengineering

KW - Multiplexing

KW - N‐glycosylation

KW - Rituximab

U2 - 10.1002/biot.201800111

DO - 10.1002/biot.201800111

M3 - Journal article

VL - 13

JO - Biotechnology Journal

JF - Biotechnology Journal

SN - 1860-6768

IS - 10

M1 - 1800111

ER -