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 ( rh A1AT, rh C1INH) decorated with humanized N-glycans are therefore of clinical and commercial interest. Here, we present engineered CHO cell lines producing rh A1AT or rh C1INH 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 rh A1AT and rh C1INH are homogeneous and similar to the structures obtained from plasma-derived A1AT and C1INH, marketed as Prolastin ® -C and Cinryze ® , respectively. rh A1AT and rh C1INH 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.