中国仓鼠卵巢细胞
重组DNA
载体(分子生物学)
质粒
生物
基因
生产(经济)
细胞培养
计算生物学
遗传学
宏观经济学
经济
作者
Adeline Poulain,Sylvie Perret,Félix Malenfant,Alaka Mullick,Bernard Massie,Yves Durocher
标识
DOI:10.1016/j.jbiotec.2017.06.009
摘要
To rapidly produce large amounts of recombinant proteins, the generation of stable Chinese Hamster Ovary (CHO) cell pools represents a useful alternative to large-scale transient gene expression (TGE). We have developed a cell line (CHOBRI/rcTA) allowing the inducible expression of recombinant proteins, based on the cumate gene switch. After the identification of optimal plasmid DNA topology (supercoiled vs linearized plasmid) for PEIpro™ mediated transfection and of optimal conditions for methionine sulfoximine (MSX) selection, we were able to generate CHOBRI/rcTA pools producing high levels of recombinant proteins. Volumetric productivities of up to 900 mg/L were reproducibly achieved for a Fc fusion protein and up to 350 mg/L for an antibody after 14 days post-induction in non-optimized fed-batch cultures. In addition, we show that CHO pool volumetric productivities are not affected by a freeze-thaw cycle or following maintenance in culture for over one month in the presence of MSX. Finally, we demonstrate that volumetric protein production with the CR5 cumate-inducible promoter is three- to four-fold higher than with the human CMV or hybrid EF1α-HTLV constitutive promoters. These results suggest that the cumate-inducible CHOBRI/rcTA stable pool platform is a powerful and robust system for the rapid production of gram amounts of recombinant proteins.
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