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

• Published in: Metabolic engineering 2019-03, Vol.52, p.143-152
• Main Authors: Amann, Thomas, Hansen, Anders Holmgaard, Kol, Stefan, Hansen, Henning Gram, Arnsdorf, Johnny, Nallapareddy, Saranya, Voldborg, Bjørn, Lee, Gyun Min, Andersen, Mikael Rørdam, Kildegaard, Helene Faustrup
• Format: Article
• Language: English
• Subjects: Biotechnology; Chinese hamster ovary (CHO) cells; CRISPR/Cas9; Glyco-engineering; Multiplexing; Plasma proteins
• ISSN: 1096-7176; 1096-7184
• DOI: 10.1016/j.ymben.2018.11.014

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. •Humanization of N-glycans on two recombinant therapeutic proteins.•N-glycosylation similar to human in engineered Chinese hamster ovary cells.•Knock out of ten target genes and expression of two human genes.