Process for the scaleable purification of plasmid DNA
View Patent ↗The present invention relates to a nonchromatographic-based process for the isolation of clinical grade plasmid DNA from bacterial cells. The exemplified methods described herein outline a scaleable, economically favorable protocol for the purification of clinical grade plasmid DNA from E. coli which includes CTAB-based precipitation of DNA in combination with adsorption of impurities to calcium silicate.
1. A method of purifying supercoiled plasmid DNA from a cell lysate of a large scale microbial fermentation which comprises adding hydrated, crystallized calcium silicate to the cell lysate to adsorb residual impurities away from the supercoiled plasmid DNA.
2. A method of claim 1 wherein the cell lysate is clarified prior to addition of the hydrated, crystallized calcium silicate.
3. A method of claim 2 wherein the cell lysate is clarified by addition of diatomaceous earth.
4. A method of claim 3 wherein the supercoiled plasmid DNA is further concentrated by a process selected from the group consisting of alcohol precipitation and ultrafiltration.
5. A method of claim 4 wherein the supercoiled plasmid DNA is concentrated by ethanol precipitation.
6. A method of claim 1 wherein the supercoiled plasmid DNA is further concentrated by a process selected from the group consisting of alcohol precipitation and ultrafiltration.
7. A method of claim 6 wherein the supercoiled plasmid DNA is concentrated by ethanol precipitation.
8. A method of claim 1 wherein the hydrated, crystallized calcium silicate is added in a stepwise manner.
9. A method of claim 8 wherein the cell lysate is clarified by addition of diatomaceous earth.
10. A method of claim 9 wherein the supercoiled plasmid DNA is further concentrated by a process selected from the group consisting of alcohol precipitation and ultrafiltration.
11. A method of claim 10 wherein the supercoiled plasmid DNA is concentrated by ethanol precipitation.
12. A method of claim 1 wherein the hydrated, crystallized calcium silicate has a surface area of greater than about 75 m 2 /g.
13. A method of claim 12 wherein the hydrated, crystallized calcium silicate is in a composition comprising 25-35% calcium oxide by weight and 45-55% silicon dioxide by weight.