Journal article
Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles
CHO Cell Line Engineering and Design, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark1
Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark2
CHO in Silico Engineering of Glycosylation and Protein Quality (CiSe), Research Groups, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark3
CHO Core, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark4
Pre-Pilot Plant, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark5
iLoop, Translational Management, Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark6
Section for Synthetic Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark7
Network Engineering of Eukaryotic Cell factories, Section for Synthetic Biology, Department of Biotechnology and Biomedicine, Technical University of Denmark8
Department of Biotechnology and Biomedicine, Technical University of Denmark9
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.
Language: | English |
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Year: | 2019 |
Pages: | 143-152 |
ISSN: | 10967176 and 10967184 |
Types: | Journal article |
DOI: | 10.1016/j.ymben.2018.11.014 |
ORCIDs: | Arnsdorf, Johnny , Amann, Thomas , Hansen, Anders Holmgaard , Kol, Stefan , Hansen, Henning Gram , Voldborg, Bjørn , Andersen, Mikael Rørdam and Kildegaard, Helene Faustrup |