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Large‐scale production of endotoxin‐free plasmids for transient expression in mammalian cell culture
Author(s) -
Rozkov Aleksei,
Larsson Bert,
Gillström Stefan,
Björnestedt Robert,
Schmidt Stefan R.
Publication year - 2007
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.21603
Subject(s) - industrial fermentation , plasmid , chromatography , fermentation , ultrafiltration (renal) , lysis , alkaline lysis , plasmid preparation , bioreactor , recombinant dna , biology , chemistry , downstream processing , fed batch culture , diafiltration , biochemistry , dna , dna vaccination , membrane , microfiltration , botany , pbr322 , gene
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. Biotechnol. Bioeng. 2008;99: 557–566. © 2007 Wiley Periodicals, Inc.