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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.

Gene therapy and genetic vaccines promise to revolutionize the treatment of inherited and acquired diseases. Since viral vectors are generally associated with numerous disadvantages when applied to humans, the administration of naked DNA, or DNA packed into lipo- or polyplexes emerge as viable alternatives. To satisfy the increasing demand for pharmaceutical grade plasmids we developed a novel economic downstream process which overcomes the bottlenecks of common lab-scale techniques and meets all regulatory requirements. After cell lysis by an in-house developed gentle, automated continuous system the sequence of hydrophobic interaction, anion exchange and size exclusion chromatography guarantees the separation of impurities as well as undesired plasmid isoforms. After the consecutive chromatography steps, adjustment of concentration and final filtration are carried out. The final process was proven to be generally applicable and can be used from early clinical phases to market-supply. It is scaleable and free of animal-derived substances, detergents (except lysis) and organic solvents. The process delivers high-purity plasmid DNA of homogeneities up to 98% supercoiled form at a high yield in any desired final buffer.