Plasmid DNA purification by selective calcium silicate adsorption of closely related impurities.

The selective adsorption of supercoiled plasmid, open-circular plasmid, and genomic DNA to gyrolite, a compound from the class of crystalline calcium silicate hydrates, is investigated and exploited for purification purposes. Genomic DNA and open-circular plasmid bind to gyrolite adsorbents with greater affinity than the more conformationally constrained supercoiled plasmid. As such, the gyrolite adsorbents are an economical and scaleable alternative to chromatographic purification for the removal of DNA impurities from solutions containing supercoiled plasmid. The advantage of gyrolite adsorbents is their lower unit price and ability to selectively adsorb DNA impurities without binding supercoiled plasmid under certain conditions. The effects of ionic strength, temperature, chelating agent, divalent cation, and lyotropic salts on adsorption of highly purified plasmid are studied to understand the forces that bind DNA to gyrolite, a structure with hydrophilic and hydrophobic characteristics. The results indicate that DNA binding is governed by hydrogen bonding, electrostatic bridging with divalent cations, shielding of electrostatic repulsion, hydrophobic adsorption, and disruption of integral surface water layer on gyrolite. On the basis of results from a range of Hofmeister series salts, strongly hydrated anions may enhance DNA adsorption by promoting hydrophobic interactions between DNA and gyrolite. Conversely, the very weakly hydrated chaotrope I(-) may enhance adsorption by strongly associating with hydrophobic siloxanes of gyrolite, thereby disrupting an integral water layer, which competes for hydrogen bonding sites.

Fractional precipitation of plasmid DNA from lysate by CTAB.

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.