Purification process for plasmid DNA
View Patent ↗Methods of isolating clinical-grade plasmid DNA from manufacturing processes, including large-scale fermentation regimes, are disclosed which encompass alternatives to two core unit operations common to plasmid DNA purification processes. The novel upstream and downstream purification processes disclosed herein provide for reduced production costs and increase process robustness. Either or both of the purification processes disclosed herein may be used in combination with additional purification steps known in the art that are associated with DNA plasmid purification technology.
1. A method of purifying supercoiled plasmid DNA from a cell lysate of a large-scale microbial fermentation which comprises:
(a) lysing microbial host cells containing supercoiled plasmid DNA, forming a host cell lysate;
(b) clarifying said host cell lysate by flocculating host cell debris with a polymer flocculant wherein said flocculant is at a concentration which does not precipitate said supercoiled plasmid DNA; and,
(c) removing the flocculated, host cell debris, generating a clarified lysate containing the supercoiled plasmid DNA.
2. A method of claim 1 , wherein said polymer flocculant is polyethylene glycol (PEG).
3. A method of claim 2 , wherein the host cells in step (a) are lysed in a standard STET buffer containing lysozyme.
4. A method of purifying supercoiled plasmid DNA from a cell lysate of a large-scale microbial fermentation which comprises:
(a) lysing microbial host cells containing supercoiled plasmid DNA, forming a host cell lysate;
(b) clarifying said host cell lysate by flocculating host cell debris with PEG wherein said PEG is at a concentration which does not precipitate said supercoiled plasmid DNA; and,
(c) removing the flocculated, host cell debris, generating a clarified lysate containing the supercoiled plasmid DNA.
5. A method of claim 4 , wherein the host cells in step (a) are lysed in a standard STET buffer containing lysozyme.
6. A method of claim 5 , wherein the STET buffer contains PEG.
7. A method of claim 4 , wherein PEG is added to the host cell lysate after lysis.
8. A method of claim 7 , wherein the molecular weight of the PEG flocculant is between about 1450 Da and about 15,000 Da.
9. A method of claim 7 , wherein the host cell lysate is subjected to an alkaline pH shift and subsequent neutralization prior to addition of PEG.
10. A method of claim 9 , wherein the alkaline pH shift and subsequent neutralization comprises raising the pH of the host cell lysate to an alkaline value of between about pH 12 and about pH 13 and then lowering the pH of said cell lysate to a value of between about pH 7 and about pH 9.
11. A method of claim 7 , wherein the host cell lysate is subjected to an alkaline pH shift and subsequent neutralization after addition of PEG.
12. A method of claim 11 , wherein the alkaline pH shift and subsequent neutralization comprises raising the pH of the PEG-containing, host cell lysate to an alkaline value of between about pH 12 and about pH 13 and then lowering the pH of said lysate to a value of between about pH 7 and about pH 9.
13. A method of purifying supercoiled plasmid DNA from a cell lysate of a large-scale microbial fermentation which comprises:
(a) lysing microbial host cells containing supercoiled plasmid DNA in a physiological lysis buffer, forming a host cell lysate;
(b) subjecting the host cell lysate of step (a) to an alkaline pH shift by raising the pH of said lysate to between about pH 12 and about pH 13;
(c) neutralizing the alkaline-shifted cell lysate of step (b) to approximately the pH of the lysis buffer;
(d) clarifying the cell lysate of step (c) by flocculating host cell debris with PEG wherein said PEG is at a concentration which does not precipitate said supercoiled plasmid DNA; and,
(e) removing said flocculated host cell debris of step (d), obtaining a supernatant containing soluble supercoiled plasmid DNA.
14. A method of claim 13 , wherein the physiological lysis buffer of step (a) is a standard STET buffer.
15. A method of claim 13 , wherein a detergent-induced precipitation step between steps (d) and (e) is added to precipitate debris and non-supercoiled plasmid, further clarifying the cell lysate.
16. A method of claim 1 , wherein the detergent is hexadecyltrimethylammonium bromide (CTAB).
17. A method of purifying supercoiled plasmid DNA from a cell lysate of a large-scale microbial fermentation which comprises:
(a) lysing microbial host cells containing supercoiled plasmid DNA in a physiological lysis buffer, forming a host cell lysate;
(b) clarifying said host cell lysate by flocculating host cell debris with a PEG flocculant wherein said PEG flocculant is at a concentration which does not precipitate said supercoiled plasmid DNA;
(c) precipitating said supercoiled plasmid DNA with PEG; and,
(d) concentrating said precipitated supercoiled plasmid DNA by microfiltration under a tangential flow filtration mode.
18. A method of claim 17 , wherein said host cells in step (a) are lysed in a standard STET buffer.
19. A method of claim 18 , wherein the host cell lysate is subjected to an alkaline pH shift and subsequent neutralization prior to addition of the PEG flocculant.
20. A method of claim 19 , wherein the alkaline pH shift and subsequent neutralization comprises raising the pH of the host cell lysate to an alkaline value of between about pH 12 and about pH 13 and then lowering the pH of said lysate to approximately its pH prior to the alkaline shift.
21. A method for the purification of supercoiled plasmid DNA from a cell lysate of a large-scale microbial fermentation, which comprises:
(a) harvesting microbial host cells containing supercoiled plasmid DNA from a fermentation broth;
(b) lysing the host cells in a sufficient amount of a physiological lysis solution;
(c) subjecting the host cell lysate to an alkaline pH shift by raising the pH of said lysate to between about pH 12 and about pH 13;
(d) neutralizing the alkaline-shifted cell lysate to approximately the pH of the lysis buffer;
(e) clarifying the cell lysate of step (d) by flocculating host cell debris with the addition of PEG wherein said PEG is at a concentration which does not precipitate said supercoiled plasmid DNA;
(f) removing said flocculated host cell debris, generating a clarified lysate containing the supercoiled plasmid DNA;
(g) selectively precipitating the supercoiled plasmid DNA with a hexadecyltrimethylammonium bromide-induced precipitation;
(h) redissolving the supercoiled plasmid DNA and adsorbing impurities in a well defined buffer of optimized ionic strength and further containing hydrated, crystallized calcium silicate;
(i) adsorbing remaining impurities with a second hydrated, crystallized calcium silicate-induced adsorption;
(j) precipitating the supercoiled plasmid DNA with PEG;
(k) concentrating the precipitated, supercoiled plasmid DNA by microfiltration under a tangential flow filtration mode;
(l) partially dehydrating the precipitated, supercoiled plasmid DNA with addition of ethanol;
(m) concentrating the dehydrated supercoiled plasmid DNA a stirred-cell filter dryer; and,
(n) drying to remove ethanol.
22. A method of claim 21 , wherein said microbial cells are lysed in a standard STET buffer.
23. A method of claim 21 , wherein a low-cut hexadecyltrimethylammonium bromide-induced precipitation step between steps (e) and (f) is added to precipitate debris and non-supercoiled plasmid, further clarifying the cell lysate.