Effect of shear on plasmid DNA in solution

This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene t herapy and DNA vaccination. Controlled shear was generated using a capillar y rheometer and a rotating disk shear device. Plasmid DNA was tested in a c larified alkaline lysate solution. This chemical environment is characteris tic of the early stages of plasmid purification. Quantitative data is repor ted on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid m olecular weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear using the capillary rheometer. The shear sensitivity of the three plasmids was similar to that observed f or the same plasmids in the clarified alkaline lysate. Further experiments were carried out using the 20 kb plasmid and the rotating disk shear device . In contrast with the capillary rheometer data, ultrapure DNA redissolved in TE buffer was up to eight times more sensitive to shear compared to plas mid DNA in the clarified alkaline lysate. However, this enhanced sensitivit y decreased when the ionic strength of the solution was raised by the addit ion of NaCl to 150 mM. In addition, shear damage was found to be independen t of plasmid DNA concentration in the range from 0.2 mu g/ml to 20 mu g/ml. The combination of shear and air-liquid interfaces caused extensive degrad ation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These findings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of dam age was found to be critically dependent on plasmid size and the ionic stre ngth of the environment. The interaction of shear with air-liquid interface s shows the highest potential for damaging SC plasmid DNA during bioprocess es.