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Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles.
Metab Eng 2019; 52:143-152ME

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.

Authors+Show Affiliations

The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark. Electronic address: ahoha@biosustain.dtu.dk.The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark.The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark; Department of Biological Sciences, KAIST, Daejeon, Republic of Korea.Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark.The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs. Lyngby, Denmark. Electronic address: hef@biosustain.dtu.dk.

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

30513349

Citation

Amann, Thomas, et al. "Glyco-engineered CHO Cell Lines Producing Alpha-1-antitrypsin and C1 Esterase Inhibitor With Fully Humanized N-glycosylation Profiles." Metabolic Engineering, vol. 52, 2019, pp. 143-152.
Amann T, Hansen AH, Kol S, et al. Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles. Metab Eng. 2019;52:143-152.
Amann, T., Hansen, A. H., Kol, S., Hansen, H. G., Arnsdorf, J., Nallapareddy, S., ... Kildegaard, H. F. (2019). Glyco-engineered CHO cell lines producing alpha-1-antitrypsin and C1 esterase inhibitor with fully humanized N-glycosylation profiles. Metabolic Engineering, 52, pp. 143-152. doi:10.1016/j.ymben.2018.11.014.
Amann T, et al. Glyco-engineered CHO Cell Lines Producing Alpha-1-antitrypsin and C1 Esterase Inhibitor With Fully Humanized N-glycosylation Profiles. Metab Eng. 2019;52:143-152. PubMed PMID: 30513349.
* Article titles in AMA citation format should be in sentence-case
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 - Lee,Gyun Min, AU - Andersen,Mikael Rørdam, AU - Kildegaard,Helene Faustrup, Y1 - 2018/12/01/ PY - 2018/07/19/received PY - 2018/10/30/revised PY - 2018/11/30/accepted PY - 2018/12/5/pubmed PY - 2019/6/19/medline PY - 2018/12/5/entrez KW - Biotechnology KW - CRISPR/Cas9 KW - Chinese hamster ovary (CHO) cells KW - Glyco-engineering KW - Multiplexing KW - Plasma proteins SP - 143 EP - 152 JF - Metabolic engineering JO - Metab. Eng. VL - 52 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. SN - 1096-7184 UR - https://www.unboundmedicine.com/medline/citation/30513349/Glyco-engineered_CHO_cell_lines_producing_alpha-1-antitrypsin_and_C1_esterase_inhibitor_with_fully_humanized_N-glycosylation_profiles L2 - https://linkinghub.elsevier.com/retrieve/pii/S1096-7176(18)30289-1 DB - PRIME DP - Unbound Medicine ER -