Process for the scaleable purification of plasmid DNA
OwnerRussel Jackson Lander, Michael Albert Winters +1inventors
InventorsRussel Jackson Lander, Michael Albert Winters, Francis Jeremiah Meacle
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Abstract
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.
Claims63
A method of purifying supercoiled plasmid DNA from a cell lysate of a large scale microbial fermentation which comprises:
- (a) precipitating supercoiled plasmid DNA by a detergent-induced precipitation;
- and,
- (b) redissolving the precipitate in a salt solution.
A method of purifying supercoiled plasmid DNA from a cell lysate of a microbial fermentation which comprises adding hydrated, crystallized calcium silicate to the cell lysate to adsorb residual impurities away from the supercoiled plasmid DNA.
A method of purifying supercoiled plasmid DNA from a cell lysate of a microbial fermentation, which comprises:
- (a) precipitating the supercoiled plasmid DNA by a detergent-induced precipitation;
- (b) redissolving the precipitate in a salt solution;
- (c) adding hydrated, crystallized calcium silicate to the resuspended supercoiled plasmid to adsorb residual impurities away from the supercoiled plasmid DNA, resulting in a solution containing the supercoiled plasmid DNA;
- and,
- (d) concentrating the supercoiled plasmid DNA.
A method for the purification of supercoiled plasmid DNA from a microbial fermentation, which comprises:
- (a) harvesting microbial cells from a fermentation broth;
- (b) adding to the harvested microbial cells a sufficient amount of a lysis solution;
- (c) heating the microbial cells of step b) to a temperature between 70° C. and 100° C. in a flow-through heat exchanger to form a cell lysate;
- (d) cooling the cell lysate;
- (e) clarifying the cell lysate using filtration with diatomaceous earth;
- (f) precipitating residual cell debris and impurities with a first hexadecyltrimethylammonium-induced precipitation (g) selectively precipitating supercoiled plasmid DNA with a second hexadecyltrimethylammonium-induced precipitation;
- (h) redissolving the supercoiled plasmid DNA in a well defined buffer of optimized ionic strength and salt composition;
- (i) adsorbing residual impurities onto calcium silicate within the buffer of step (h);
- (j) precipitating supercoiled plasmid DNA with ethanol;
- (k) filtering to collect and wash the precipitate;
- (l) drying to remove ethanol;
- (m) redissolving purified supercoiled plasmid DNA in a physiologically acceptable formulation buffer;
- and,
- (n) sterilizing by filtration through a 0.22 μm filter.
A method for the purification of supercoiled plasmid DNA from a cell lysate of a large scale microbial fermentation, which comprises:
- (a) harvesting microbial cells from a fermentation broth;
- (b) adding to the harvested microbial cells a sufficient amount of a lysis solution;
- (c) heating the microbial cells of step b) to a temperature between 70° C. and 100° C. in a flow-through heat exchanger to form a cell lysate;
- (d) cooling the cell lysate;
- (e) clarifying the cell lysate using filtration with diatomaceous earth;
- (f) precipitating supercoiled plasmid DNA with hexadecyltrimethylammonium;
- (g) redissolving the supercoiled plasmid DNA in a well defined buffer of optimized ionic strength and salt composition;
- (h) adsorbing residual impurities onto calcium silicate within the buffer of step (g);
- (i) precipitating supercoiled plasmid DNA with ethanol;
- (j) filtering to collect and wash the precipitate;
- (k) drying to remove ethanol;
- (l) redissolving purified supercoiled plasmid DNA in a physiologically acceptable formulation buffer;
- and,
- (m) sterilizing by filtration through a 0.22 μm filter.
A method for the purification of supercoiled plasmid DNA from a cell lysate of a large scale microbial fermentation, which comprises:
- (a) harvesting microbial cells from a fermentation broth;
- (b) adding to the harvested microbial cells a sufficient amount of lysozyme/alkaline/KOAc to promote cell lysis, forming a cell lysate;
- (c) clarifying the cell lysate using filtration with diatomaceous earth;
- (d) precipitating residual cell debris and impurities with a first hexadecyltrimethylammonium-induced precipitation;
- (e) selectively precipitating supercoiled plasmid DNA with a second hexadecyltrimethylammonium-induced precipitation;
- (f) redissolving the supercoiled plasmid DNA in a well defined buffer of optimized ionic strength and salt composition;
- (g) adsorbing residual impurities onto calcium silicate with the buffer of step (f);
- (h) precipitating supercoiled plasmid DNA with ethanol;
- (i) filtering to collect and wash the precipitate;
- (j) drying to remove ethanol;
- (k) redissolving purified supercoiled plasmid DNA in a physiologically acceptable formulation buffer;
- and,
- (l) sterilizing by filtration through a 0.22 μm filter.
A method for the purification of supercoiled plasmid DNA from a cell lysate of a large scale microbial fermentation, which comprises:
- (a) harvesting microbial cells from a large scale fermentation;
- (b) adding to the harvested microbial cells a sufficient amount of lysozyme/alkaline/KOAc to promote cell lysis, forming a cell lysate;
- (c) clarifying the cell lysate using filtration with diatomaceous earth;
- (d) precipitating supercoiled plasmid DNA with hexadecyltrimethylammonium;
- (e) redissolving the supercoiled plasmid DNA in a well defined buffer of optimized ionic strength and salt composition;
- (f) adsorbing residual impurities onto hydrated, crystallized calcium silicate;
- (h) precipitating supercoiled plasmid DNA with ethanol;
- (i) filtering to collect and wash the precipitate;
- (j) drying to remove ethanol;
- (k) redissolving purified supercoiled plasmid DNA in a physiologically acceptable formulation buffer;
- and,
- (l) sterilizing by filtration through a 0.22 μm filter.
Dependencies are inferred from the claim text.