Scaling E. coli disruption for a continuous mill design for plasmid recovery

The production of plasmid DNA (pDNA) is fundamental for creating DNA and RNA vaccines, which have proven to be a novel and effective alternative to traditional vaccines. The pDNA production process includes the stages of fermentation, primary recovery, intermediate recovery, and purification. Primary recovery involves cell harvest and rupture, which is a critical step in the bioprocess. In this research, a bead milling operation was employed as an alternative to the conventional alkaline lysis technique to enhance the efficiency of the primary recovery stage. Batch kinetic studies were performed with milling chambers with L/D of 0.51, 0.93 and 1.67. By fitting a kinetic model to the experimental data, the specific kinetic constants of 0.46, 0.44 and 0.21 min^-1, respectively, were determined. Electrophoretic analysis shows that more than 60% of the supercoiled plasmid was recovered in less than 5 min, with no RNA content. The results of the milling kinetics in batch mode were used to analyze the behavior of a continuous mill using the Perfectly Agitated Tanks in series model. The results indicate that the highest overall efficiency is achieved with a chamber having an L/D ratio of 0.51 in 4 stages.