Effect of shear on plasmid DNA in solution

Abstract

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 therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plasmid DNA was tested in a clari®ed alkaline lysate solution. This chemical environment is characteristic of the early stages of plasmid puri®cation. Quantitative data is reported on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid molecular 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 for the same plasmids in the clari®ed 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 plasmid DNA in the clari®ed alkaline lysate. However, this enhanced sensitivity decreased when the ionic strength of the solution was raised by the addition of NaCl to 150 mM. In addition, shear damage was found to be independent of plasmid DNA concentration in the range from 0.2 lg/ml to 20 lg/ml. The combination of shear and air-liquid interfaces caused extensive degradation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These ®ndings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of damage was found to be critically dependent on plasmid size and the ionic strength of the environment. The interaction of shear with air-liquid interfaces shows the highest potential for damaging SC plasmid DNA during bioprocesses.

Impact of engineering flow conditions on plasmid DNA yield and purity in chemical cell lysis operations

“…However, the prevailing fluid flow stresses normally associated with intense mixing may cause damage to the large macromolecules in the solution (Davison, 1959;Levy et al, 1999aLevy et al, , 1999bNguyen and Kausch, 1992). An important process engineering question that arises is what influence this kind of stress has on the yield and purity of the supercoiled plasmid DNA, and if the impact is detrimental, what mixing strategy may be adopted to minimize it.…”

“…An important process engineering question that arises is what influence this kind of stress has on the yield and purity of the supercoiled plasmid DNA, and if the impact is detrimental, what mixing strategy may be adopted to minimize it. It has been shown that plasmid DNA, typically below about 20 kb, does not suffer significantly from physical damage by fluid-induced stresses occurring in most items of process equipment, particularly in the presence of salt ions in solution (Levy et al, 1999b). Double-and singlestranded chDNA, however, are significantly bigger than plasmid DNA and may be damaged by fluid mechanical stresses, typical of those occurring in mechanically agitated lysis reactor.…”

Study of robustness of filamentous bacteriophages for industrial applications

“…High-energy turbulent conditions can occur throughout a bioprocess, such as at the fermentation, filtration, pumping, and continuous centrifugation stages (Boychyn et al, 2004;Levy et al, 1999;Zhang et al, 2007). From existing literature, the physical disruption of filamentous bacteriophages has been reported as a result of exposure to the extreme conditions of ultrasonication (Fareed and Valentine, 1968) and pressure homogenization (Grant et al, 1981).…”

“…1). Therefore, each virus is in effect a large protein rod 100-fold greater in mass than a full length IgG antibody and of contour length approaching the characteristic dimensions of a considerably shear-sensitive supercoiled 20 kb plasmid (Levy et al, 1999;Zhang et al, 2007). If M13 proves to be an extensively shear sensitive molecule then the choice of large-scale operations could be constrained.…”

“…In particular, a USD rotating disk shear device has been developed to predict the effects of turbulent fluid flow from large-scale pumps and continuous centrifuges (Boychyn et al, 2004;Hutchinson et al, 2006;Zhang et al, 2007). A solid base of small and large-scale verification experimentation exists, validating its use to determine patterns of shear-sensitive behavior for materials such as plasmid DNA, mammalian cell and antibody feeds (Biddlecombe et al, 2007;Hutchinson et al, 2006;Levy et al, 1999).…”