Automated alkaline lysis for industrial scale cGMP production of pharmaceutical grade plasmid-DNA |
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Authors: | Urthaler Jochen Ascher Christine Wöhrer Helga Necina Roman |
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Institution: | Boehringer Ingelheim Austria GmbH, Dr. Boehringer-Gasse 5-11, A-1121 Vienna, Austria. jochen.urthaler@vie.boehringer-ingelheim.com |
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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. |
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