Method for producing and purifying RNA, comprising at least one step of tangential flow filtration
The present invention relates to method for producing and purifying RNA comprising the steps of providing DNA encoding the RNA; transcription of the DNA into RNA; and conditioning and/or purifying of the solution comprising transcribed RNA by one or more steps of tangential flow filtration (TFF).
1. A method for producing formulated purified RNA, comprising the steps of:
A1) providing a plasmid DNA comprising a RNA polymerase promoter sequence, a RNA coding sequence, and a restriction endonuclease cleavage site, wherein the coding sequence for said RNA is no greater than 15,000 nucleotides in length;
A2) linearizing the plasmid DNA with a restriction endonuclease to produce linearized DNA;
A3) ultrafiltering the linearized DNA by tangential flow filtration (TFF), to produce purified linearized DNA;
B1) transcribing the purified linearized DNA to produce transcribed RNA;
B2) treating the transcribed RNA with a DNase;
C1) performing at least one step of TFF using a TFF membrane cassette having a molecular weight cutoff (MWCO) of less than or equal to 500 kDa;
C2) further purifying the RNA by a step filtration or chromatography;
C3) performing at least one further step of TFF using a TFF membrane cassette to produce purified RNA; and
D) formulating the purified RNA to produce the formulated purified RNA.
2. The method of claim 1 , wherein the coding sequence for said RNA is 500 to 10,000 nucleotides in length.
3. The method of claim 1 , wherein the restriction endonuclease is a type II restriction endonuclease.
4. The method of claim 3 , wherein the restriction endonuclease is SapI.
5. The method of claim 1 , wherein the RNA polymerase promoter sequence is a T7 promoter sequence.
6. The method of claim 1 , wherein the TFF membrane cassette of step C1) has a MWCO of about 100 kDa to 300 kDa.
7. The method of claim 1 , wherein the TFF membrane cassette of step C1) has a MWCO of about 300 kDa.
8. The method of claim 7 , wherein the TFF membrane cassette of step C1) has a membrane comprising cellulose or a cellulose derivative.
9. The method of claim 1 , wherein providing the plasmid DNA comprises purifying the plasmid DNA.
10. The method of claim 9 , wherein the method comprises at least one step of DNA or RNA purification using anion exchange chromatography.
11. The method of claim 1 , wherein step B1) transcribing the purified linearized DNA is performed in the presence of spermidine.
12. The method of claim 11 , wherein transcribing the purified linearized DNA is performed in the presence of 0.1 mM to 10 mM spermidine.
13. The method of claim 12 , wherein step C1) comprises ultrafiltering the transcribed RNA by at least one step of TFF with an aqueous salt solution using a TFF membrane cassette having a MWCO of less than or equal to 500 kDa, thereby reducing the level of spermidine associated with the transcribed RNA.
14. The method of claim 13 , wherein the aqueous salt solution comprises EDTA.
15. The method of claim 14 , wherein step D) comprises formulating the purified RNA by complexing the purified RNA with a cationic compound, to obtain the formulated purified RNA.
16. The method of claim 15 , wherein said RNA is a mRNA that is 500 to 10,000 nucleotides in length.
17. The method of claim 15 , wherein said RNA is a replicon RNA.
18. The method of claim 15 , further comprising adding EDTA to the transcribed RNA prior to step C1).
19. The method of claim 18 , wherein step B1) comprises transcribing the purified linearized DNA in the presence of a cap analog to produce a transcribed capped RNA.
20. The method of claim 19 , wherein step B1) comprises transcribing the purified linearized DNA in the presence of a cap analog, nucleoside triphosphates (NTPs), T7 polymerase, spermidine, salts, and a buffer to produce a transcribed capped RNA.
21. The method of claim 20 , wherein step B1) comprises transcribing the purified linearized DNA in the presence of a cap analog, nucleoside triphosphates (NTPs), T7 polymerase, spermidine, salts, and a HEPES or Tris buffer to produce a transcribed capped RNA.
22. The method of claim 21 , wherein step B1) comprises transcribing the purified linearized DNA in the presence of a modified nucleotide to produce transcribed capped RNA having the modified nucleotide and wherein the modified nucleotide is 1-methyl-pseudouridine.
23. The method of claim 1 , wherein step C2) comprises performing a filtration of the RNA.
24. The method of claim 1 , wherein the method does not comprises any step of phenol or chloroform extraction of the RNA.
25. The method of claim 24 , wherein the method does not comprises any step of RNA precipitation.
26. The method of claim 1 , wherein step C2) comprises purifying the RNA by at least one step of chromatography.
27. The method of claim 26 , wherein step C2) comprises purifying the RNA by at least one step of anion exchange chromatography.
28. A method for producing formulated purified RNA, comprising the steps of:
A1) providing a plasmid DNA comprising a RNA polymerase promoter sequence, a RNA coding sequence, and a restriction endonuclease cleavage site, wherein the coding sequence for said RNA is no greater than 15,000 nucleotides in length;
A2) linearizing the plasmid DNA with a restriction endonuclease to produce linearized DNA;
A3) ultrafiltering the linearized DNA by tangential flow filtration (TFF), to produce purified linearized DNA;
B1) transcribing the purified linearized DNA to produce transcribed RNA;
B2) treating the transcribed RNA with a DNase;
C1) performing at least one step of TFF using a TFF membrane cassette having a molecular weight cutoff (MWCO) of less than or equal to 500 kDa;
C2) further purifying the RNA by a step filtration or chromatography to produce purified RNA; and
D) formulating the purified RNA to produce the formulated purified RNA.
29. The method of claim 28 , further comprising performing at least one further step of TFF on the formulated purified RNA.
30. The method of claim 28 , wherein the coding sequence for said RNA is 500 to 10,000 nucleotides in length.
31. The method of claim 28 , wherein the restriction endonuclease is a type II restriction endonuclease.
32. The method of claim 31 , wherein the restriction endonuclease is SapI.
33. The method of claim 28 , wherein the RNA polymerase promoter sequence is a T7 promoter sequence.
34. The method of claim 28 , wherein the TFF membrane cassette of step C1) has a MWCO of about 100 kDa to 300 kDa.
35. The method of claim 28 , wherein the TFF membrane cassette of step C1) has a MWCO of about 300 kDa.
36. The method of claim 34 , wherein the TFF membrane cassette of step C1) has a membrane comprising cellulose or a cellulose derivative.
37. The method of claim 28 , wherein providing the plasmid DNA comprises purifying the plasmid DNA.
38. The method of claim 37 , wherein the method comprises at least one step of DNA or RNA purification using anion exchange chromatography.
39. The method of claim 28 , wherein step B1) transcribing the purified linearized DNA is performed in the presence of spermidine.
40. The method of claim 39 , wherein transcribing the purified linearized DNA is performed in the presence of 0.1 mM to 10 mM spermidine.
41. The method of claim 40 , wherein step C1) comprises ultrafiltering the transcribed RNA by at least one step of TFF with an aqueous salt solution using a TFF membrane cassette having a MWCO of less than or equal to 500 kDa, thereby reducing the level of spermidine associated with the transcribed RNA.
42. The method of claim 41 , wherein the aqueous salt solution comprises EDTA.
43. The method of claim 42 , wherein step D) comprises formulating the purified RNA by complexing the purified RNA with a cationic compound, to obtain the formulated purified RNA.
44. The method of claim 43 , wherein said RNA is a mRNA that is 500 to 10,000 nucleotides in length.
45. The method of claim 43 , wherein said RNA is a replicon RNA.
46. The method of claim 43 , further comprising adding EDTA to the transcribed RNA prior to step C1).
47. The method of claim 46 , wherein step B1) comprises transcribing the purified linearized DNA in the presence of a cap analog to produce a transcribed capped RNA.
48. The method of claim 47 , wherein step B1) comprises transcribing the purified linearized DNA in the presence of a cap analog, nucleoside triphosphates (NTPs), T7 polymerase, spermidine, salts, and a buffer to produce a transcribed capped RNA.
49. The method of claim 48 , wherein step B1) comprises transcribing the purified linearized DNA in the presence of a cap analog, nucleoside triphosphates (NTPs), T7 polymerase, spermidine, salts, and a HEPES or Tris buffer to produce a transcribed capped RNA.
50. The method of claim 49 , wherein step B1) comprises transcribing the purified linearized DNA in the presence of a modified nucleotide to produce transcribed capped RNA having the modified nucleotide and wherein the modified nucleotide is 1-methyl-pseudouridine.
51. The method of claim 28 , wherein step C2) comprises performing a filtration of the RNA.
52. The method of claim 28 , wherein the method does not comprise any step of phenol or chloroform extraction of the RNA.
53. The method of claim 52 , wherein the method does not comprise any step of RNA precipitation.
54. The method of claim 28 , wherein step C2) comprises purifying the RNA by at least one step of chromatography.
55. The method of claim 54 , wherein step C2) comprises purifying the RNA by at least one step of anion exchange chromatography.