Preparative-scale purification of plasmid DNA has been attempted by diverse methods, including precipitation with solvents, salts, and detergents and chromatography with ion-exchange, reversed-phase, and size-exclusion columns. Chromatographic methods such as hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), and size exclusion chromatography (SEC) are the only effective means of eliminating the closely related relaxed and denatured forms of plasmid as well as endotoxin to acceptable levels. However, the anticipated costs of manufacturing-scale chromatography are high due to (a) large projected volumes of the high-dosage therapeutic molecule and (b) restricted loading of the large plasmid molecule in the pores of expensive resins. As an alternative to chromatography, we show herein that precipitation with the cationic detergent, cetyltrimethylammonium bromide (CTAB), is effective for selective precipitation of plasmid DNA from proteins, RNA, and endotoxin. Moreover, CTAB affords novel selectivity by removal of host genomic DNA and even the more closely related relaxed and denatured forms of plasmid as earlier, separate fractions. Finally, plasmid that has been precipitated by CTAB can be purified by selectively dissolving under conditions of controlled salt concentration. The selectivity mechanism is most likely based upon conformational differences among the several forms of DNA. As such, CTAB precipitation provides an ideal nonchromatographic capture step for the manufacture of plasmid DNA.

Chemical lysis of bacterial cells using an alkaline solution containing a detergent may provide an efficient scalable means for selectively removing covalently closed circular plasmid DNA from high-molecular-weight contaminating cellular components including chromosomal DNA. In this article we assess the chemical lysis of E. coli cells by SDS in a NaOH solution and determine the impact of pH environment and shear on the supercoiled plasmid and chromosomal DNA obtained. Experiments using a range of plasmids from 6 kb to 113 kb determined that in an unfavorable alkaline environment, where the NaOH concentration during lysis is greater than 0.15 F 0.03 M (pH 12.9 F 0.2), irreversible denaturation of the supercoiled plasmid DNA occurs. The extent of denaturation is shown to increase with time of exposure and NaOH concentration. Experiments using stirred vessels show that, depending on NaOH concentration, moderate to high mixing rates are necessary to maximize plasmid yield. While NaOH concentration does not significantly affect chromosomal DNA contamination, a high NaOH concentration is necessary to ensure complete conversion of chromosomal DNA to single-stranded form. In a mechanically agitated lysis reactor the correct mixing strategy must balance the need for sufficient mixing to eliminate potential regions of high NaOH concentrations and the need to avoid excessive breakage of the shear sensitive chromosomal DNA. The effect of shear on chromosomal DNA is examined over a wide range of shear rates (10 1 À10 5 s À1) demonstrating that, while increasing shear leads to fragmentation of chromosomal DNA to smaller sizes, it does not lead to significantly increased chromosomal DNA contamination except at very high shear rates (about 10 4 À105 s À1). The consequences of these effects on the choice of lysis reactor and scale-up are discussed.

Supercoiled plasmid DNA is susceptible to fluid stress in large-scale manufacturing processes. A capillary device was used to generate controlled shear conditions and the effects of different stresses on plasmid DNA structure were investigated. Computational fluid dynamics (CFD) analysis was employed to characterize the flow environment in the capillary device and different analytical techniques were used to quantify the DNA breakage. It was found that the degradation of plasmid DNA occurred at the entrance of the capillary and that the shear stress within the capillary did not affect the DNA structure. The degradation rate of plasmids was well correlated with the average elongational strain rate or the pressure drop at the entrance region. The conclusion may also be drawn that laminar shear stress does not play a significant role in plasmid DNA degradation.