Extraction of plasmid DNA using reactor scale alkaline lysis and selective precipitation for scalable transient transfection

• Published in: Cytotechnology (Dordrecht) Vol. 35; no. 3; pp. 165 - 173
• Main Authors: WRIGHT, Jason L, JORDAN, Martin, WURM, Florian M
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer 01-05-2001; Springer Nature B.V; Kluwer Academic Publishers
• Subjects: Acetic acid; Ammonium acetate; Biological and medical sciences; Bioreactors; Biotechnology; Deoxyribonucleic acid; DNA; Escherichia coli; Fermentation; Fundamental and applied biological sciences. Psychology; Gene therapy; Lysis; Transfection; Vaccination
• ISSN: 0920-9069; 1573-0778
• DOI: 10.1023/A:1013106032341

DNA extracted and purified for vaccination, gene therapy or transfection of cultured cells has to meet different criteria. We describe herein, a scalable process for the primary extraction of plasmid DNA suitable for transient expression of recombinant protein. We focus on the scale up of alkaline lysis for the extraction of plasmid DNA from Escherichia coli, and use a simple stirred tank reactor system to achieve this. By adding a series of three precipitations (including a selective precipitation step with ammonium acetate) we enrich very quickly the plasmid DNA content in the extract. The process has been thus far used to extract up to 100 mg of plasmid from 1.5 l of clarified lysate, corresponding to an E.coli bioreactor fermentation of 3 l.