title: Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA.
abstract: Plasmid DNA for biopharmaceutical applications is mainly produced in E. coli cells. The first and most crucial step for recovering the plasmid is the cell lysis. Governed by the physico-chemical properties of the polynucleotide, alkaline lysis has been the lysis-method of choice. This chemical disintegration technique was initially developed for the lab scale and non-pharmaceutical applications. A continuous, fully automated and closed system combining alkaline lysis, neutralization and clarification in one gentle and generic operation was developed. This system consists of a three units. One unit controls mixing and contact time during the alkaline treatment, another one controls the neutralization and the concurrent formation of flocs and a third one the separation of flocs and pDNA containing lysate. Based on optimization experiments the selected process parameters resulted in yields up to 100% and homogeneities comparable to that obtained by gentle manual lysis. The process does not need enzymes and it is scalable and routinely used for cGMP-production of pharmaceutical grade plasmid DNA from 200 L fermentations.
authors.affiliation: Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria. jochen.urthaler@vie.boehringer-ingelheim.com
authors.forename: Jochen
authors.initials: J
authors.lastname: Urthaler
authors.forename: Christine
authors.initials: C
authors.lastname: Ascher
authors.forename: Helga
authors.initials: H
authors.lastname: Wöhrer
authors.forename: Roman
authors.initials: R
authors.lastname: Necina
mesh_terms: D001331:Automation; D001433:Bacteriolysis; D006152:Cyclic GMP / Q000096:biosynthesis*; D004247:DNA / Q000096:biosynthesis* / Q000302:isolation & purification; D004926:Escherichia coli; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000295:instrumentation* / Q000379:methods*; D010957:Plasmids / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
chemical_list: D004247:DNA; D006152:Cyclic GMP