Journal article · Ahead of Print article
Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures
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
Sialic acid, a terminal monosaccharide present in N-glycans, plays an important role in determining both the in vivo half-life and the therapeutic efficacy of recombinant glycoproteins. Low sialylation levels of recombinant human erythropoietin (rhEPO) in recombinant Chinese hamster ovary (rCHO) cell cultures are considered a major obstacle to the production of rhEPO in fed-batch mode.
This is mainly due to the accumulation of extracellular sialidases released from the cells. To overcome this hurdle, three sialidase genes (Neu1, 2, and 3) were initially knocked-out using the CRISPR/Cas9-mediated large deletion method in the rhEPO-producing rCHO cell line. Unlike wild type cells, sialidase knockout (KO) clones maintained the sialic acid content and proportion of tetra-sialylated rhEPO throughout fed-batch cultures without exhibiting a detrimental effect with respect to cell growth and rhEPO production.
Additional KO of two pro-apoptotic genes, BAK and BAX, in sialidase KO clones (5X KO clones) further improved rhEPO production without any detrimental effect on sialylation. On day 10 in fed-batch cultures, the 5X KO clones had 1.4-times higher rhEPO concentration and 3.0-times higher sialic acid content than wild type cells.
Furthermore, the proportion of tetra-sialylated rhEPO on day 10 in fed-batch cultures was 42.2–44.3% for 5X KO clones while it was only 2.2% for wild type cells. Taken together, KO of sialidase and pro-apoptotic genes in rCHO cells is a useful tool for producing heavily sialylated glycoproteins such as rhEPO in fed-batch mode.
Language: | English |
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Year: | 2020 |
Pages: | 182-192 |
ISSN: | 10967184 and 10967176 |
Types: | Journal article and Ahead of Print article |
DOI: | 10.1016/j.ymben.2019.11.008 |
ORCIDs: | Ha, Tae Kwang , Hansen, Anders Holmgaard and Kildegaard, Helene Faustrup |