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.
Winters
Michael A
MA
Department of Vaccine Bioprocess Engineering, Merck Research Laboratories, Merck & Co., Inc., P.O. Box 4, West Point, Pennsylvania 19486-0004, USA.
Richter
Jesse D
JD
Sagar
Sangeetha L
SL
Lee
Ann L
AL
Lander
Russel J
RJ
D000327:Adsorption; D017610:Calcium Compounds / Q000737:chemistry*; D002614:Chelating Agents / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D004340:Drug Contamination / Q000517:prevention & control; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D007477:Ions; D046911:Macromolecular Substances; D010957:Plasmids / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D012680:Sensitivity and Specificity; D017640:Silicates / Q000737:chemistry*; D013696:Temperature
D017610:Calcium Compounds; D002614:Chelating Agents; D004269:DNA, Bacterial; D007477:Ions; D046911:Macromolecular Substances; D017640:Silicates; C031293:calcium silicate