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 a plasmid DNA encoding a RNA, wherein said RNA is 500 to 10000 nucleotides in length;
A2) linearizing the plasmid DNA 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; and
C1) ultrafiltering the transcribed RNA by 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, to produce the purified RNA.
2. The method of claim 1 , wherein the TFF membrane cassette has a MWCO of about 100 kDa to 300 kDa.
3. The method of claim 1 , wherein the TFF membrane cassette has a MWCO of about 300 kDa.
4. The method of claim 1 , wherein the TFF membrane cassette has a membrane comprising cellulose or a cellulose derivative.
5. The method of claim 1 , wherein the method comprises at least one step of DNA or RNA purification using anion exchange chromatography.
6. A method for producing purified RNA, comprising the steps of:
A1) providing a plasmid DNA encoding a RNA, wherein said RNA is 500 to 10000 nucleotides in length;
A2) linearizing the plasmid DNA to produce linearized DNA;
A3) ultrafiltering the linearized DNA by tangential flow filtration (TFF) to produce a purified linearized DNA;
B1) transcribing the purified linearized DNA in the presence of a cap analog to produce a transcribed capped RNA;
B2) treating the transcribed capped RNA with a DNase;
C1) ultrafiltering the transcribed capped RNA by at least one step of TFF with an aqueous salt solution using a TFF membrane cassette having a molecular weight cutoff (MWCO) of less than or equal to 500 kDa; and
C2) performing at least one further filtration using TFF, to obtain the purified RNA.
7. The method of claim 6 , wherein providing the plasmid DNA comprises purifying the plasmid DNA.
8. The method of claim 6 , wherein the TFF membrane cassette of step C1) has a MWCO of about 100 kDa to 300 kDa.
9. The method of claim 6 , wherein the TFF membrane cassette of step C1) has a MWCO of about 300 kDa.
10. The method of claim 6 , wherein the TFF membrane cassette comprises cellulose or a cellulose derivative.
11. The method of claim 7 , wherein the method comprises at least one step of DNA or RNA purification using anion exchange chromatography.
12. The method of claim 6 , wherein transcribing the purified linearized DNA is performed 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.
13. The method of claim 7 , wherein the method further comprises formulating the purified RNA.
14. The method of claim 13 , further comprising a filling step.
15. The method of claim 6 , wherein transcribing the purified linearized DNA is performed in the presence of spermidine.
16. The method of claim 15 , wherein transcribing the purified linearized DNA is performed in the presence of 0.1 mM to 10 mM spermidine.
17. The method of claim 16 , wherein step C1) produces RNA with a reduced level of spermidine relative to the level of spermidine in the transcribing step.
18. The method of claim 17 , comprising treating the transcribed capped RNA with DNaseI in the presence of CaCl 2 , following by addition of EDTA.
19. The method of claim 6 , wherein the TFF of step C2) is with a TFF membrane cassette.
20. The method of claim 19 , wherein the membrane cassette of step C2) has a MWCO of about 100 kDa to 300 kDa.
21. The method of claim 19 , wherein the membrane cassette of step C2) has a MWCO of about 300 kDa.
22. The method of claim 7 , wherein the method comprises at least one step of DNA or RNA purification using anion exchange chromatography.
23. The method of claim 6 , wherein the method comprises at least one further step of RNA purification or RNA concentration using TFF.
24. The method of claim 6 , wherein the method does not include a step for organic solvent extraction or alcohol precipitation of the transcribed capped RNA.
25. The method of claim 6 , wherein the method does not include a step for organic solvent extraction or alcohol precipitation of the plasmid DNA or the linearized DNA.
26. The method of claim 25 , wherein the transcribed capped RNA comprises a Cap1 5′ cap and a Poly(A) sequence of 50 to 250 nucleotides.
27. The method of claim 26 , wherein the transcribed capped RNA comprises a modified nucleotide, which is 1-methyl-pseudouridine.
28. The method of claim 27 , wherein linearizing the plasmid DNA is with a type II restriction endonuclease.
29. A method for producing a formulated purified mRNA, comprising the steps of:
A1) providing a plasmid DNA encoding a mRNA, wherein said mRNA is 500 to 5000 nucleotides in length and comprises a Poly(A) sequence of 50 to 250 nucleotides;
A2) linearizing the plasmid DNA to produce linearized DNA;
A3) ultrafiltering the linearized DNA by tangential flow filtration (TFF) to produce a purified linearized DNA;
B1) 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 mRNA;
B2) treating the transcribed capped mRNA with a DNase followed by addition of EDTA;
C1) ultrafiltering the transcribed capped mRNA by at least one step of TFF with an aqueous salt solution using a TFF membrane cassette having a molecular weight cutoff (MWCO) of less than or equal to 500 kDa, thereby reducing the level of spermidine associated with the transcribed capped mRNA;
C2) performing at least one further filtration using TFF, to obtain the purified mRNA; and
D) formulating the purified mRNA by complexing the purified mRNA with a cationic compound, to obtain a formulated purified mRNA.
30. The method of claim 29 , wherein the TFF membrane cassette has a MWCO of about 300 kDa and comprises a stabilized cellulose-based TFF membrane.