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 purified RNA, comprising the steps of:
A1) providing plasmid DNA encoding a RNA of 500 to 10000 nucleotides in length;
A2) linearizing the DNA with a restriction endonuclease to produce linearized DNA;
B) transcribing the linearized DNA to yield transcribed RNA, wherein said transcribing is in a solution comprising: nucleoside triphosphates (NTPs); T7 polymerase, spermidine, salts and a HEPES or TRIS buffer; and
C) purifying the transcribed RNA by performing at least one step of tangential flow filtration (TFF) using a TFF membrane cassette, thereby producing purified RNA, wherein the method comprises at least one step of DNA or RNA purification using chromatography.
2. The method of claim 1 , wherein the at least one step of DNA or RNA purification using chromatography comprises using anion exchange chromatography.
3. The method of claim 1 , wherein at least one step of TFF is performed after the 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 the at least two steps of TFF are both using a TFF membrane cassette.
6. The method of claim 1 , wherein the TFF membrane cassette comprises a cellulose-based TFF membrane.
7. The method of claim 6 , wherein C) purifying the transcribed RNA comprises performing at least one step of TFF with an aqueous salt solution.
8. The method of claim 7 , wherein the aqueous salt solution is a NaCl solution or an organic salt solution.
9. The method of claim 7 , wherein performing at least one step of TFF comprises using a TFF membrane with a molecular weight cutoff of ≤500 kDa.
10. The method of claim 1 , further comprising treating the solution with DNAse after said transcribing.
11. The method of claim 1 , wherein the RNA is a mRNA.
12. The method of claim 9 , wherein the transcribing is in a buffer comprising 0.1 mM to 10 mM spermidine.
13. The method of claim 12 , wherein the method produces purified RNA with a reduced level of spermidine relative to the level of spermidine in step B.
14. The method of claim 11 , wherein the mRNA comprises a modified nucleotide.
15. The method of claim 14 , wherein the modified nucleotide is 1-methyl-pseudouridine.
16. The method of claim 13 , wherein the transcribing is in a buffer comprising a HEPES buffer.
17. The method of claim 13 , wherein performing at least one step of TFF comprises using a TFF membrane with a molecular weight cutoff of about 100 kDa to 300 kDa.
18. The method of claim 13 , wherein the membrane cassette of step C2) has a MWCO of about 300 kDa.
19. The method of claim 13 , wherein the RNA is 500 to 5000 nucleotides in length.
20. The method of claim 19 , wherein the transcribing is in a buffer comprising a cap analog to produce a capped RNA.
21. The method of claim 20 , wherein the capped RNA is a CAP1 capped RNA.
22. The method of claim 21 , wherein the restriction endonuclease is a type II restriction endonuclease.
23. The method of claim 17 , wherein the method further comprises formulating the purified RNA.
24. The method of claim 22 , further comprising a filling step.
25. The method of claim 17 , wherein the TFF membrane cassette comprises a stabilized cellulose-based TFF membrane.
26. The method of claim 23 , wherein formulating the purified RNA comprises complexing the RNA with a cationic compound.