A human expression system based on HEK293 for the stable production of recombinant erythropoietin
| Autor: | Meng How Tan, Ali Gowher, Justin B. Goh, Harini Srinivasan, Andy Hee-Meng Tan, Hsueh Lee Lim, Christine Lin Chin, Raghuvaran Shanmugam, Terry Nguyen-Khuong, Matthew S F Choo, Wen Qin Tang, Kaiwen Ivy Liu, Say Kong Ng |
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| Přispěvatelé: | School of Chemical and Biomedical Engineering, Genome Institute of Singapore, A*STAR |
| Jazyk: | angličtina |
| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Glycosylation HEK293 Expression systems Genetic Vectors lcsh:Medicine Gene Expression Protein Engineering 01 natural sciences Models Biological Epitope Article law.invention 03 medical and health sciences Gene Knockout Techniques law Glutamate-Ammonia Ligase 010608 biotechnology Gene expression medicine Humans lcsh:Science Transcriptomics Erythropoietin chemistry.chemical_classification Multidisciplinary Expression vector Chemistry Chinese hamster ovary cell HEK 293 cells lcsh:R Chemical engineering [Engineering] Recombinant Proteins Cell biology 030104 developmental biology Chinese Hamster Ovary (CHO) HEK293 Cells Batch Cell Culture Techniques Genetic engineering Recombinant DNA lcsh:Q CRISPR-Cas Systems Glycoprotein medicine.drug |
| Zdroj: | Scientific Reports Scientific Reports, Vol 9, Iss 1, Pp 1-16 (2019) |
| ISSN: | 2045-2322 |
| Popis: | Mammalian host cell lines are the preferred expression systems for the manufacture of complex therapeutics and recombinant proteins. However, the most utilized mammalian host systems, namely Chinese hamster ovary (CHO), Sp2/0 and NS0 mouse myeloma cells, can produce glycoproteins with non-human glycans that may potentially illicit immunogenic responses. Hence, we developed a fully human expression system based on HEK293 cells for the stable and high titer production of recombinant proteins by first knocking out GLUL (encoding glutamine synthetase) using CRISPR-Cas9 system. Expression vectors using human GLUL as selection marker were then generated, with recombinant human erythropoietin (EPO) as our model protein. Selection was performed using methionine sulfoximine (MSX) to select for high EPO expression cells. EPO production of up to 92700 U/mL of EPO as analyzed by ELISA or 696 mg/L by densitometry was demonstrated in a 2 L stirred-tank fed batch bioreactor. Mass spectrometry analysis revealed that N-glycosylation of the produced EPO was similar to endogenous human proteins and non-human glycan epitopes were not detected. Collectively, our results highlight the use of a human cellular expression system for the high titer and xenogeneic-free production of EPO and possibly other complex recombinant proteins. NRF (Natl Research Foundation, S’pore) ASTAR (Agency for Sci., Tech. and Research, S’pore) Published version |
| Databáze: | OpenAIRE |
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