Claims
- 1. A process for isolating plasmid DNA comprising the steps of:
(a) lysing cells containing said plasmid DNA with a lysis agent, thereby forming a lysate; (b) treating said lysate with a high salt agent, thereby forming a treated solution; and (c) purifying said treated solution to provide isolated plasmid DNA.
- 2. The process of claim 1, wherein said plasmid DNA is a mixture of supercoiled plasmid DNA, nicked circle plasmid DNA, and linearized plasmid DNA.
- 3. The process of claim 1, wherein said process does not involve the use of RNase.
- 4. The process of claim 1, wherein said high salt agent is capable of precipitating a significant portion of any RNA molecules from said cells.
- 5. The process of claim 1, wherein said high salt agent comprises one or more salts at a pH above approximately 5.5.
- 6. The process of claim 5, wherein said high salt agent comprises a mixture of 1M potassium acetate and 7M ammonium acetate at a pH between 7.0 and 9.0.
- 7. The process of claim 6, wherein said lysate is treated with said high salt agent for at least six hours at approximately four degrees Celsius.
- 8. The process of claim 1, wherein said isolated plasmid DNA is pharmaceutical-grade plasmid DNA suitable for administration to humans.
- 9. The process of claim 1, wherein at least 100 milligrams of said isolated plasmid DNA is obtained.
- 10. A process for isolating plasmid DNA comprising the steps of:
(a) resuspending cells in approximately 50 mM of Tris at a pH of about 8.0 and approximately 10 mM EDTA(Na)2; (b) lysing cells containing said plasmid DNA with a lysis agent comprising an approximately equal volume of 0.2N sodium hydroxide in 1% SDS, thereby forming a lysate; (c) treating said lysate with a high salt agent comprises a mixture of 1M potassium acetate and 7M ammonium acetate at a pH between 7.0 and 9.0, thereby forming a high salt solution; and (c) purifying said treated solution to provide isolated plasmid DNA.
- 11. The process of claim 10, wherein said isolated plasmid DNA is pharmaceutical-grade plasmid DNA suitable for administration to humans.
- 12. The process of claim 10, wherein at least 100 milligrams of said isolated plasmid DNA is obtained.
- 13. In the process of claim 10 for isolating plasmid DNA from lysate of a cell containing said plasmid DNA, wherein the improvement comprises treating said lysate with a high salt agent capable of precipitating non-plasmid DNA cellular components contained in said lysate.
- 14. The process of claim 13, wherein said high salt agent is capable of precipitating a significant portion of any RNA molecules from said cells.
- 15. The process of claim 13, wherein said high salt agent comprises one or more salts at a pH above approximately 5.5.
- 16. The process of claim 15, wherein said high salt agent comprises a mixture of 1M potassium acetate and 7M ammonium acetate at a pH between 7.0 and 9.0.
- 17. The process of claim 16, wherein said lysate is treated with said high salt agent for at least six hours at approximately four degrees Celsius.
- 18. A process for isolating plasmid DNA comprising the steps of:
(a) lysing cells containing said plasmid DNA with a lysis agent, thereby forming a lysate; and (b) purifying said lysate with anion exchange chromatography using a step gradient, thereby producing isolated plasmid DNA (The process of claim 18, wherein the isolated plasmid DNA is enriched with at least 80% supercoiled plasmid DNA).
- 19. The process of claim 18, wherein said isolated plasmid DNA is pharmaceutical-grade plasmid DNA suitable for administration to humans.
- 20. The process of claim 18, wherein at least 100 milligrams of said isolated plasmid DNA is obtained.
- 21. The process of claim 18, wherein said anion exchange chromatography is performed with a resin having a particle size of 20-40 microns.
- 22. The process of claim 22, wherein said anion exchange chromatography has a plasmid DNA binding capacity of about 1.5 mg of plasmid per mL of resin.
- 23. The process of claim 18, wherein said anion exchange chromatography is performed with a Fractogel EMD TMAE(650-S) resin.
- 24. The process of claim 18 for isolating plasmid DNA from lysate of a cell containing said plasmid DNA, wherein the improvement comprises purifying said lysate with anion exchange chromatography using a step gradient, thereby producing isolated plasmid DNA enriched with at least 80% supercoiled plasmid DNA.
- 25. The process of claim 24, wherein said anion exchange chromatography is performed with a resin having a particle size of 20-40 microns.
- 26. The process of claim 24, wherein said anion exchange chromatography has a plasmid DNA binding capacity of about 1.5 mg of plasmid per mL of resin.
- 27. The process of claim 24, wherein said anion exchange chromatography is performed with a Fractogel EMD TMAE(S) resin.
- 28. A process for isolating plasmid DNA comprising the steps of:
(a) lysing cells containing said plasmid DNA with a lysis agent, thereby forming a lysate; and (b) using hydrophobic interaction chromatography to purify said lysate, thereby providing isolated plasmid DNA.
- 29. The process of claim 27, wherein said hydrophobic interaction chromatography is performed with at least 1.6M ammonium sulfate.
- 30. The process of claim 28, wherein said hydrophobic interaction chromatography is performed with an Octyl Sepharose 4 FF resin.
- 31. The process of claim 28, wherein said isolated plasmid DNA is pharmaceutical-grade plasmid DNA suitable for administration to humans.
- 32. The process of claim 28, wherein at least 100 milligrams of said isolated plasmid DNA is obtained.
- 33. The process of claim 28, wherein said plasmid DNA is not precipitated and wherein said process involves no linear gradients and uses no organic solvents.
- 34. The process of claim 28, wherein said isolated plasmid DNA is substantially free of endotoxins and host cell chromosomal DNA.
- 35. The process of claim 28, wherein said plasmid DNA is not exposed to acidic pH or elevated temperatures.
- 36. The process of claim 28, wherein said isolated plasmid DNA is produced in a yield of at least 60%.
- 37. The process of claim 28, wherein said hydrophobic interaction chromatography is performed in an aqueous solution containing a high concentration of salt.
- 38. The process of claim 37, wherein said salt is ammonium sulfate.
- 39. The process of claim 28, wherein said cells are recombinant E. coli cells.
- 40. In a process for isolating plasmid DNA from a lysate of a cell containing said plasmid DNA, wherein the improvement comprises using hydrophobic interaction chromatography to purify said lysate, thereby providing isolated plasmid DNA.
- 41. The process of claim 42, wherein said hydrophobic interaction chromatography is performed with at least 1.6M ammonium sulfate.
- 42. The process of claim 42, wherein said hydrophobic interaction chromatography is performed with an Octyl Sepharose 4 FF resin.
- 43. The process of claim 42, wherein said hydrophobic interaction chromatography is performed in an aqueous solution containing a high concentration of salt.
- 44. The process of claim 42, wherein said salt is ammonium sulfate.
- 45. A device for isolating plasmid DNA from cells containing said plasmid DNA, comprising:
(a) a means for providing fast cell resuspension in a semi-continuous mode; (b) a means for providing mixing and cell lysis in a continuous flow mode; and (c) a means for providing chilling and mixing to denature and precipitate chromosomal DNA, protein, and RNA.
- 46. The device of claim 47, wherein said means for providing mixing and cell lysis in a continuous flow mode comprises an impeller mixer.
- 47. The device of claim 47, wherein said means for providing mixing and cell lysis in a continuous flow mode comprises an in-line static mixer.
- 48. The device of claim 47, wherein said means for providing mixing and cell lysis in a continuous flow mode comprises a lysis coil.
- 49. The device of claim 47, further comprising a means for performing hydrophobic interaction chromatography.
- 50. The device of claim 47, wherein said means for providing chilling and mixing to denature and precipitate chromosomal DNA, protein, and RNA comprises a chilled jacketed tank.
- 51. A process for isolating plasmid DNA, comprising the steps of:
(1) fermenting cells containing said plasmid DNA, harvesting said cells, and washing said cells; (2) exposing said cells to an alkaline lysis and neutralization agent, thereby forming a lysate; (3) performing centrifugation and filtration on said lysate; (4) treating said lysate with RNase at about 37 degrees Celsius for about one hour; (5) filtrating said lysate and diluting said lysate with 2 volumes of WFI. (6) passing said lysate through a Q Sepharose HP resin, a DEAE 650-S resin, and a Phenyl 650-S resin; and (7) filtrating the eluate from step 6 to yield the final product of isolated plasmid DNA.
- 52. A process for isolating plasmid DNA, comprising the steps of:
(a) fermenting cells containing said plasmid DNA, harvesting said cells, and washing said cells; (b) exposing said cells to an alkaline lysis and neutralization agent, thereby forming a lysate; (c) performing centrifugation or filtration on said lysate and performing a 1.5 volume dilution with WFI on said lysate; (d) exposing said lysate to an anionic change resin; (e) washing the nicked and/or relaxed circular plasmid, as well as residual RNA, off of the resin with about 0.6M NaCl; (f) eluting the plasmid DNA off of the resin with about 1.9M ammonium sulfite; (g) passing the eluate through a hydrophobic interaction chromatography resin; and (h) filtrating the eluate to yield a final product of isolated plasmid DNA.
RELATED APPLICATIONS
[0001] This application claims priority to co-pending U.S. patent application Ser. No. 08/887,673, filed Jul. 3, 1997, which in turn, claims priority to U.S. Provisional Patent Application Ser. No. 60/022,157, filed Jul. 19, 1996. Both applications are hereby incorporated by reference as if fully set forth herein.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60022157 |
Jul 1996 |
US |
Continuations (1)
|
Number |
Date |
Country |
Parent |
08887673 |
Jul 1997 |
US |
Child |
09774284 |
Jan 2001 |
US |