DC Field | Value | Language |
---|---|---|
dc.contributor.author | Ha, Tae Kwang | ko |
dc.contributor.author | Hansen, Anders Holmgaard | ko |
dc.contributor.author | Kildegaard, Helene Faustrup | ko |
dc.contributor.author | Lee, Gyun Min | ko |
dc.date.accessioned | 2019-12-13T01:21:22Z | - |
dc.date.available | 2019-12-13T01:21:22Z | - |
dc.date.created | 2019-12-02 | - |
dc.date.created | 2019-12-02 | - |
dc.date.created | 2019-12-02 | - |
dc.date.issued | 2020-01 | - |
dc.identifier.citation | METABOLIC ENGINEERING, v.57, pp.182 - 192 | - |
dc.identifier.issn | 1096-7176 | - |
dc.identifier.uri | http://hdl.handle.net/10203/268754 | - |
dc.description.abstract | 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. | - |
dc.language | English | - |
dc.publisher | ACADEMIC PRESS INC ELSEVIER SCIENCE | - |
dc.title | Knockout of sialidase and pro-apoptotic genes in Chinese hamster ovary cells enables the production of recombinant human erythropoietin in fed-batch cultures | - |
dc.type | Article | - |
dc.identifier.wosid | 000506206200018 | - |
dc.identifier.scopusid | 2-s2.0-85075628992 | - |
dc.type.rims | ART | - |
dc.citation.volume | 57 | - |
dc.citation.beginningpage | 182 | - |
dc.citation.endingpage | 192 | - |
dc.citation.publicationname | METABOLIC ENGINEERING | - |
dc.identifier.doi | 10.1016/j.ymben.2019.11.008 | - |
dc.contributor.localauthor | Lee, Gyun Min | - |
dc.contributor.nonIdAuthor | Ha, Tae Kwang | - |
dc.contributor.nonIdAuthor | Hansen, Anders Holmgaard | - |
dc.contributor.nonIdAuthor | Kildegaard, Helene Faustrup | - |
dc.description.isOpenAccess | N | - |
dc.type.journalArticle | Article | - |
dc.subject.keywordAuthor | Chinese hamster ovary (CHO) cells | - |
dc.subject.keywordAuthor | CRISPR/Cas9 | - |
dc.subject.keywordAuthor | Fed-batch culture | - |
dc.subject.keywordAuthor | Glyco-engineering | - |
dc.subject.keywordAuthor | Sialidase | - |
dc.subject.keywordAuthor | Apoptosis | - |
dc.subject.keywordPlus | FC-FUSION PROTEIN | - |
dc.subject.keywordPlus | ENHANCED SIALYLATION | - |
dc.subject.keywordPlus | CHO-CELLS | - |
dc.subject.keywordPlus | GLYCOPROTEIN SIALYLATION | - |
dc.subject.keywordPlus | BCL-X(L) OVEREXPRESSION | - |
dc.subject.keywordPlus | GLYCOSIDASE ACTIVITIES | - |
dc.subject.keywordPlus | ACID | - |
dc.subject.keywordPlus | EXPRESSION | - |
dc.subject.keywordPlus | LINES | - |
dc.subject.keywordPlus | SUPPLEMENTATION | - |
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