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 and purifying RNA, comprising the steps of:
A) providing DNA encoding the RNA;
B) transcribing the DNA to yield a solution comprising transcribed RNA; and
C) purifying the transcribed RNA by performing: (i) at least one step of reversed phase chromatography; and (ii) at least one step of tangential flow filtration (TFF) using a TFF membrane cassette.
2. The method of claim 1 , wherein the at least one step of TFF is performed before the at least one step of reversed phase chromatography.
3. The method of claim 1 , wherein the at least one step of TFF is performed after the at least one step of reversed phase chromatography.
4. The method of claim 1 , wherein C) purifying the transcribed RNA comprises at least two steps of TFF.
5. The method of claim 4 , wherein at least one step of TFF is performed before the at least one step of reversed phase chromatography and at least one step of TFF is performed after the at least one step of reversed phase chromatography.
6. The method of claim 4 , wherein the at least two steps of TFF are both using a TFF membrane cassette.
7. The method of claim 1 , wherein the TFF membrane cassette comprises a TFF membrane comprising at least one of polyethersulfone (PES), modified polyethersulfone (mPES), a cellulose derivative membrane, or a combination thereof.
8. The method of claim 1 , wherein the reversed phase chromatography is reversed phase high performance liquid chromatography (RP-HPLC).
9. The method of claim 1 , wherein the reversed phase chromatography is performed using at least one organic solvent.
10. The method of claim 1 , wherein the at least one step of reversed phase chromatography comprises chromatography using, as a stationary phase, a polystyrenedivinylbenzene.
11. The method of claim 10 , wherein the at least one step of reversed phase chromatography comprises chromatography using, as a stationary phase, a porous non-alkylated polystyrenedivinylbenzene.
12. The method of claim 1 , wherein the at least one step of reversed phase chromatography comprises chromatography using, as a stationary phase, a silica gel.
13. The method of claim 12 , wherein the at least one step of reversed phase chromatography comprises chromatography using, as a stationary phase, a silica gel modified with octadecyl containing residues.
14. The method of claim 1 , wherein A) providing DNA comprises providing linearized DNA.
15. The method of claim 14 , further comprising linearizing the DNA.
16. The method of claim 1 , further comprising purifying the DNA before said transcribing step B).
17. The method of claim 16 , wherein purifying the DNA comprises at least one step of TFF.
18. The method of claim 16 , wherein purifying the DNA comprises at least one step of phenol/chloroform extraction and/or DNA precipitation.
19. The method of claim 1 , wherein performing at least one step of TFF comprises performing at least one diafiltration step of the RNA using TFF and/or at least one concentration step of the RNA using TFF.
20. The method of claim 19 , wherein performing at least one diafiltration step is with an aqueous salt solution.
21. The method of claim 20 , wherein the aqueous salt solution is a NaCl solution or an organic salt solution.
22. The method of claim 1 , wherein performing at least one step of TFF comprises using a TFF membrane with a molecular weight cutoff of ≤500 kDa.
23. The method of claim 22 , wherein performing at least one step of TFF comprises using a TFF membrane with a molecular weight cutoff of about 100 kDa.
24. The method of claim 1 , further comprising treating the solution with DNAse after said transcribing.
25. The method of claim 1 , wherein the RNA is a mRNA.
26. A method for producing and purifying RNA, comprising the steps of
A) providing linearized DNA encoding the RNA;
B) transcribing the linearized DNA to yield a solution comprising transcribed RNA; and
C) purifying the transcribed RNA by performing, in order from (i) to (iii):
(i) at least one step of diafiltration using tangential flow filtration (TFF);
(ii) at least one step of purification using reversed phase high performance liquid chromatography (RP-HPLC); and
(iii) at least a second step of diafiltration using TFF wherein at least one of said TFF steps is performed using a TFF membrane cassette.
27. The method of claim 26 , wherein said first and second step of diafiltration are performed using a TFF membrane cassette having a TFF membrane with a molecular weight cutoff of ≤500 kDa.
28. The method of claim 26 , wherein the RP-HPLC comprises, as a stationary phase, a polystyrenedivinylbenzene.
29. The method of claim 26 , wherein the RP-HPLC comprises, as a stationary phase, a silica gel.
30. A method for purifying RNA by performing, in order from (i) to (ii):
(i) at least one step of RNA purification using high performance liquid chromatography (HPLC) in the presence of at least one organic solvent; and
(ii) at least a step of diafiltration using a tangential flow filtration (TFF) membrane cassette with a molecular weight cutoff of ≤500 kDa to remove the at least one organic solvent and to obtain purified RNA.
31. The method of claim 30 , further comprising, prior to step (i):
A) providing linearized DNA encoding the RNA; and
B) transcribing the linearized DNA to yield a solution comprising transcribed RNA.
32. The method of claim 31 , further comprising treating the solution with DNAse after said transcribing.
33. The method of claim 31 , wherein the solution comprising transcribed RNA comprises spermidine.
34. The method of claim 33 , wherein the solution comprising transcribed RNA comprises 0.1 mM to about 10 mM spermidine.
35. The method of claim 33 , wherein the method produces purified RNA with a reduced level of spermidine.
36. The method of claim 30 , wherein the at least a step of diafiltration comprises using a TFF membrane cassette with a molecular weight cutoff of about 100 kDa.
37. The method of claim 30 , wherein at least one organic solvent comprises acetonitrile, methanol, ethanol, 1-propanol, 2-propanol or acetone.
38. The method of claim 37 , wherein at least one organic solvent comprises ethanol.
39. The method of claim 38 , wherein performing at least one diafiltration step is with an aqueous salt solution.
40. The method of claim 39 , wherein the aqueous salt solution is a NaCl solution.
41. The method of claim 39 , wherein the RNA is a mRNA.
42. The method of claim 41 , wherein the mRNA comprises a modified nucleotide.
43. The method of claim 42 , wherein the modified nucleotide is 1-methyl-pseudouridine.
44. The method of claim 1 , wherein the mRNA comprises a modified nucleotide.
45. The method of claim 44 , wherein the modified nucleotide is 1-methyl-pseudouridine.