Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA.
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.
Urthaler
Jochen
J
Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria. jochen.urthaler@vie.boehringer-ingelheim.com
Ascher
Christine
C
Wöhrer
Helga
H
Necina
Roman
R
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
D004247:DNA; D006152:Cyclic GMP