Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

Thomas Amann, Anders Holmgaard Hansen*, Stefan Kol, Henning Gram Hansen, Johnny Arnsdorf, Saranya Nallapareddy, Bjørn Voldborg, Gyun Min Lee, Mikael Rørdam Andersen, Helene Faustrup Kildegaard

*Corresponding author for this work

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Abstract

Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest.
Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.
Original languageEnglish
JournalMetabolic Engineering
Volume52
Pages (from-to)143-152
Number of pages10
ISSN1096-7176
DOIs
Publication statusPublished - 2019

Keywords

  • Chinese hamster ovary (CHO) cells
  • CRISPR/Cas9
  • Glyco-engineering
  • Biotechnology
  • Multiplexing
  • Plasma proteins

Cite this

@article{daf762f7774f40e4a472f8f6ca1fbf03,
title = "Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles",
abstract = "Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest.Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin{\circledR}-C and Cinryze{\circledR}, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.",
keywords = "Chinese hamster ovary (CHO) cells, CRISPR/Cas9, Glyco-engineering, Biotechnology, Multiplexing, Plasma proteins",
author = "Thomas Amann and Hansen, {Anders Holmgaard} and Stefan Kol and Hansen, {Henning Gram} and Johnny Arnsdorf and Saranya Nallapareddy and Bj{\o}rn Voldborg and {Min Lee}, Gyun and Andersen, {Mikael R{\o}rdam} and Kildegaard, {Helene Faustrup}",
year = "2019",
doi = "10.1016/j.ymben.2018.11.014",
language = "English",
volume = "52",
pages = "143--152",
journal = "Metabolic Engineering",
issn = "1096-7176",
publisher = "Academic Press",

}

TY - JOUR

T1 - Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles

AU - Amann, Thomas

AU - Hansen, Anders Holmgaard

AU - Kol, Stefan

AU - Hansen, Henning Gram

AU - Arnsdorf, Johnny

AU - Nallapareddy, Saranya

AU - Voldborg, Bjørn

AU - Min Lee, Gyun

AU - Andersen, Mikael Rørdam

AU - Kildegaard, Helene Faustrup

PY - 2019

Y1 - 2019

N2 - Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest.Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.

AB - Recombinant Chinese hamster ovary (CHO) cells are able to provide biopharmaceuticals that are essentially free of human viruses and have N-glycosylation profiles similar, but not identical, to humans. Due to differences in N-glycan moieties, two members of the serpin superfamily, alpha-1-antitrypsin (A1AT) and plasma protease C1 inhibitor (C1INH), are currently derived from human plasma for treating A1AT and C1INH deficiency. Deriving therapeutic proteins from human plasma is generally a cost-intensive process and also harbors a risk of transmitting infectious particles. Recombinantly produced A1AT and C1INH (rhA1AT, rhC1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest.Here, we present engineered CHO cell lines producing rhA1AT or rhC1INH with fully humanized N-glycosylation profiles. This was achieved by combining CRISPR/Cas9-mediated disruption of 10 gene targets with overexpression of human ST6GAL1. We were able to show that the N-linked glyco-structures of rhA1AT and rhC1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin®-C and Cinryze®, respectively. rhA1AT and rhC1INH produced in our glyco-engineered cell line showed no detectable differences to their plasma-purified counterparts on SDS-PAGE and had similar enzymatic in vitro activity. The work presented here shows the potential of expanding the glyco-engineering toolbox for CHO cells to produce a wider variety of glycoproteins with fully humanized N-glycan profiles. We envision replacing plasma-derived A1AT and C1INH with recombinant versions and thereby decreasing our dependence on human donor blood, a limited and possibly unsafe protein source for patients.

KW - Chinese hamster ovary (CHO) cells

KW - CRISPR/Cas9

KW - Glyco-engineering

KW - Biotechnology

KW - Multiplexing

KW - Plasma proteins

U2 - 10.1016/j.ymben.2018.11.014

DO - 10.1016/j.ymben.2018.11.014

M3 - Journal article

C2 - 30513349

VL - 52

SP - 143

EP - 152

JO - Metabolic Engineering

JF - Metabolic Engineering

SN - 1096-7176

ER -