Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture.
Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.
Global Protein Science & Supply, DECS, AstraZeneca R&D, Södertälje, S-15185 Sweden. aleksei.rozkov@astrazeneca.com
Aleksei
A
Rozkov
Bert
B
Larsson
Stefan
S
Gillström
Robert
R
Björnestedt
Stefan R
SR
Schmidt
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000302:isolation & purification*; D004731:Endotoxins / Q000302:isolation & purification*; D004926:Escherichia coli / Q000502:physiology*; D006801:Humans; D007218:Industrial Microbiology / Q000379:methods; D008322:Mammals; D010865:Pilot Projects; D010957:Plasmids / Q000096:biosynthesis* / Q000302:isolation & purification*; D014162:Transfection / Q000379:methods*
D004269:DNA, Bacterial; D004731:Endotoxins