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The advances in gene therapy and genetic vaccination based on plasmid DNA result in an increasing demand of pharmaceutical grade plasmids produced under cGMP. A generic process consisting of a cell disintegration step followed by three different chromatography steps has been developed. Cell lysis is performed by an automated continuous reactor. The clarified lysate is further purified by hydrophobic interaction, anion exchange, size exclusion chromatography and by an ultra‐filtration step. The produced plasmid DNA was up to 98 % in the supercoiled form. The final genomic DNA content was lower than 10 μg/mg plasmid DNA, RNA was not detectable by agarose gel electrophoresis, the protein content was lower than 1 μg/mg plasmid DNA and the endotoxin content lower than 0.1 EU/mg plasmid DNA. An overall yield of 50 % and higher in the desired final buffer could be obtained. This process is scalable and does not require animal derived materials, detergents or organic solvents, meeting current standards for therapeutic applications.

A recombinant therapeutic protein expressed in Escherichia coliin form of inclusion bodies was used as a model system for comparative studies on the refolding efficiencies obtained by continuous matrix‐assisted refolding or batch‐dilution in a stirred tank reactor. Due to the strong aggregation tendency the refolding yield after dilution into an appropriate renaturation buffer was only 10 % at a protein concentration of 0.1 mg/mL. To improve the yield of active protein matrix‐assisted refolding using size‐exclusion chromatography was tested. A preparative continuous annular chromatography system (PCAC) equipped with a size‐exclusion resin was used for continuous processing of the solubilized unfolded protein. As the protein passed through the column, it was separated from the chaotropic agent and started to regain its native structure. Tightly bound aggregates produced during the refolding reaction were continuously removed from the rotating bed by 6 M guanidine‐hydrochloride. The aggregate fraction was recycled to the feed stream after concentrating by ultradiafiltration to increase the amount of refolded protein. The refolding yield of 27 % without recycling was increased to 45 % at a recycling rate of 0.51 at a protein concentration of 1 mg/mL. The stability of the reactor under continuous operation was shown for 15 hours without any significant loss of refolding efficiency or chromatographic resolution. Interdependencies of operational parameters such as protein concentration, loading factor and recycling rate on throughput of refolded protein are shown.