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 a RNA, comprising the steps of
A) providing a plasmid DNA encoding the RNA by
A1) linearizing the plasmid DNA in a linearization reaction;
A2) optionally terminating the linearization reaction; and
A3) diafiltering and/or concentrating and/or purifying the linearization reaction comprising linearized plasmid DNA by one or more steps of tangential flow filtration TFF) using a TFF membrane cassette;
B) transcribing the linearized DNA to yield a solution comprising a transcribed RNA; and
C) diafiltering and/or concentrating and/or purifying the solution comprising the transcribed RNA by one or more steps of TFF, optionally a TFF membrane cassette.
2. The method according to claim 1 , wherein step C) comprises at least one diafiltration step using TFF and/or at least one concentration step using TFF.
3. The method according to claim 2 , wherein the at least one diafiltration step using TFF in step C) comprises diafiltration with an aqueous salt solution.
4. The method according to claim 3 , wherein the aqueous salt solution is a NaCl solution.
5. The method according to claim 2 , wherein the at least one diafiltration step using TFF of step C) comprises diafiltration with water.
6. The method according to claim 1 , wherein the method does not comprise a step of phenol/chloroform extraction and/or DNA and/or RNA precipitation.
7. The method according to claim 1 , wherein the method does not comprise a step of using a TFF hollow fiber membrane.
8. The method according to claim 1 , wherein the at least one or more steps of TFF comprises using a TFF membrane with a molecular weight cutoff of 500 kDa.
9. The method according to claim 1 , wherein the at least one or more steps of TFF comprises using a TFF membrane comprising at least one of polyethersulfone (PES), modified polyethersulfone (mPES), a cellulose derivative membrane or combinations thereof.
10. The method according to claim 1 , wherein the at least one or more steps of TFF comprises using a TFF membrane comprising a cellulose derivative membrane with a molecular weight cutoff of about 100 kDa.
11. The method according to claim 1 , wherein the method comprises in step C) at least one further purification method before or after the one or more steps of TFF.
12. The method according to claim 1 , wherein the method comprises in step C) the steps:
C1) optionally terminating transcription;
C2) diafiltering and/or concentrating and/or purifying the solution comprising the transcribed RNA by one or more steps of TFF;
C3) purifying the RNA by a further purification method, wherein said further purification method does not comprise a step of phenol/chloroform extraction and/or DNA and/or RNA precipitation; and
C4) diafiltering and/or concentrating and/or purifying the solution comprising the transcribed RNA obtained after step C3) by one or more steps of TFF.
13. The method according to claim 12 , wherein step C2) comprises at least one step of diafiltration using TFF with water and/or diafiltration with an aqueous salt solution.
14. The method according to claim 12 , wherein step C4) comprises at least one first diafiltration step using TFF.
15. The method according to claim 14 , wherein step C4) comprises at least one second diafiltration step using TFF.
16. The method according to claim 14 , wherein the at least one first diafiltration step using TFF in step C4) comprises diafiltration with an aqueous salt solution.
17. The method according to claim 15 , wherein the second diafiltration step using TFF of step C4) comprises diafiltration with water.
18. A method for producing and purifying RNA, comprising the steps of
A) providing DNA encoding the RNA, comprising a step of TFF;
B) transcribing the DNA to yield a solution comprising transcribed RNA; and
C) diafiltrating and/or concentrating and/or purifying the solution comprising transcribed RNA by one or more steps of tangential flow filtration (TFF),
D) carrying out at least one additional purification step before or after the one or more steps of TFF of step C) by means of reversed phase chromatography,
wherein the TFF employed in step A), step C), and optionally that in step D), is a TFF membrane cassette.
19. The method of claim 18 , wherein n in step D) organic solvents are used in the reversed phase chromatography.
20. The method of claim 18 , wherein spermidine and/or organic solvents are removed by said method for producing and purifying RNA.
21. The method of claim 18 , wherein salts, short oligonucleotides, proteins, spermidine, and organic solvents are removed by said method for producing and purifying RNA.
22. The method of claim 18 , wherein the produced RNA has a higher purity and/or integrity than the transcribed RNA provided in step B).
23. The method according to claim 1 , wherein step C) comprises one or more steps of TFF using a TFF membrane cassette.
24. The method according to claim 18 , wherein step D) comprises one or more steps of TFF using a TFF membrane cassette.