Method for producing RNA
The present invention relates to a method for producing RNA. In particular, the present invention relates to a method for producing RNA, which is scalable and provides RNA at a high purity. The present invention provides a method for producing RNA under GMP and/or cGMP-compliant conditions. The invention further provides specific processes for use as a quality control in the manufacturing of a template DNA and/or in a method for producing RNA, in particular by in vitro transcription.
1. A method for producing purified RNA comprising the following steps:
a) providing a template DNA comprising a nucleic acid sequence encoding an RNA sequence, wherein the following steps are used to control the quality of the template DNA provided in step a):
I) determining the concentration of the template DNA in a sample;
II) determining the integrity of the template DNA;
III) determining the identity of the template DNA; and
IV) determining the purity of the template DNA by determining in a sample comprising the template DNA: the presence of RNA; the presence of protein; the presence of endotoxin; the presence of bacterial DNA; and the presence of ribonuclease;
b) in vitro transcribing the template DNA in order to obtain a composition comprising the RNA; and
c) purifying the RNA by at least one purification step selected from the group consisting of a precipitation step and a chromatographic step to obtain purified RNA, wherein the following steps are used to assess the quality of the RNA obtained in step b or the purified RNA obtained in step c:
i) determining the concentration of the RNA or the purified RNA in a sample;
ii) determining the integrity of the RNA or the purified RNA;
iii) determining the identity of the RNA or the purified RNA, by determining the length of the RNA; by digesting the RNA with a ribonuclease; by determining the length of a cDNA obtained by reverse transcription (RT)-PCR using the RNA as a template; and by determining the sequence of a cDNA obtained by RT or RT-PCR using the RNA as a template;
iv) determining the purity of the RNA or the purified RNA, by determining in a sample comprising the RNA the presence of protein; the presence of endotoxin; the presence of bacterial DNA; the presence of plasmid DNA; and the presence of organic solvent;
v) determining the pH of a sample comprising the RNA or the purified RNA; and
vi) determining the osmolality of a sample comprising the RNA or the purified RNA.
2. The method according to claim 1 , wherein the purified RNA obtain in step c) comprises 1 to 5 grams of RNA.
3. The method according to claim 1 , wherein the concentration of the template DNA provided in step a) is determined by photometric measurement.
4. The method according to claim 1 , wherein the identity of the nucleic acid sequence encoding the RNA sequence, provided in step a) is determined by using at least one step selected from polymerase chain reaction (PCR), restriction analysis or sequence analysis.
5. The method according to claim 1 , wherein the purity of the template DNA provided in step a) is determined by determining in a sample comprising the template DNA the amount of RNA; the amount of protein; the amount of endotoxin; and the amount of bacterial DNA.
6. The method according to claim 5 , wherein the amount of bacterial DNA is determined by using a PCR method.
7. The method according to claim 6 , wherein the amount of bacterial DNA is determined using a universal primer pair for bacterial DNA.
8. The method according to claim 6 , wherein the amount of E. coli DNA is determined using a primer pair specific for E. coli DNA.
9. The method according to claim 8 , wherein the primer pair is specific for the E. coli uidA gene.
10. The method according to claim 1 , wherein the concentration of the RNA obtained in step b) or the purified RNA obtained in step c) is determined by photometric measurement.
11. The method according to claim 1 , wherein the integrity of the RNA obtained in step b) or the purified RNA obtained in step c) is determined by determining the percentage of full-length RNA.
12. The method according to claim 1 , wherein the identity of the RNA obtained in step b) or the purified RNA obtained in step c) is determined by determining the length of the RNA; by digesting the RNA with a ribonuclease; by determining the length of a cDNA obtained by reverse transcription (RT)-PCR using the RNA as a template; by oligonucleotide mapping; by determining the sequence of the RNA by RNA sequencing; and by determining the sequence of a cDNA obtained by RT or RT-PCR using the RNA as a template.
13. The method according to claim 1 , wherein the purity of the RNA obtained in step b) or the purified RNA obtained in step c) is determined by determining in a sample comprising the RNA the amount of protein; the amount of endotoxin; the amount of bacterial DNA; and the amount of plasmid DNA.
14. The method according to claim 13 , wherein the amount of bacterial DNA is determined by using a PCR method.
15. The method according to claim 14 , wherein the amount of bacterial DNA is determined using a universal primer pair for bacterial DNA.
16. The method according to claim 14 , wherein the amount of E. coli DNA is determined using a primer pair specific for E. coli DNA.
17. The method according to claim 16 , wherein the primer pair is specific for the E. coli uidA gene.
18. The method according to claim 1 , wherein step a) comprises selecting an RNA sequence.
19. The method according to claim 1 , wherein step a) comprises synthesis of the template DNA.
20. The method according to claim 1 , wherein the template DNA is a DNA plasmid.
21. The method according to claim 20 , further comprising, prior to step a) culturing bacteria comprising the plasmid under selective conditions; isolating the template DNA from the bacteria and linearization of the template DNA;
wherein the purity of the template DNA provided in step a) is determined by determining in a sample comprising the template DNA the amount of RNA; the amount of protein; the amount of endotoxin; the amount of bacterial DNA; and the presence of ribonuclease;
wherein the identity of the RNA obtained in step b) or the purified RNA obtained in step c) is determined by determining the length of the RNA; by digesting the RNA with a ribonuclease; by determining the length of a cDNA obtained by reverse transcription (RT)-PCR using the RNA as a template; and by determining the sequence of a cDNA obtained by RT or RT-PCR using the RNA as a template;
wherein the chromatographic step comprises HPLC; and
wherein the purity of the RNA obtained in step c) is determined by determining in a sample comprising the RNA the amount of protein; the presence of endotoxin; the amount of bacterial DNA; the amount of plasmid DNA; and the presence of organic solvent.
22. The method according to claim 20 , wherein step a) comprises linearization of the template DNA.
23. The method according to claim 1 , wherein the in vitro transcription in step b) is carried out in presence of modified nucleotides.
24. The method according to claim 1 , wherein the in vitro transcription in step b) is carried out in presence of a cap analog.
25. The method according to claim 1 , wherein the precipitation step is an alcoholic precipitation step or a LiCl precipitation step.
26. The method according to claim 1 , wherein the chromatographic step is selected from the group consisting of HPLC, anion exchange chromatography, affinity chromatography, hydroxyapatite chromatography and core bead chromatography.
27. The method according to claim 1 , wherein the RNA obtained in step b) is purified by at least one first and at least one second purification step.
28. The method according to claim 27 , wherein the at least one first purification step comprises a precipitation step and the at least one second purification step comprises a chromatographic step.
29. The method according to claim 27 , wherein the at least one first purification step comprises a LiCl precipitation step and the at least one second purification step comprises a step of RP-HPLC.
30. The method according to claim 1 , wherein the RNA obtained in step b) or the purified RNA obtained in step c) is lyophilized.
31. The method according to claim 1 , wherein the presence of protein in the template DNA and the purified RNA is not determined by a Bradford assay.
32. The method according to claim 1 , wherein the presence of protein in the template DNA or the purified RNA is determined by a Bichinonic Acid (BCA) assay.
33. The method according to claim 31 , further comprising determining the amount of protein in the template DNA or the purified RNA by the BCA assay.
34. The method according to claim 1 , wherein the amount of protein in the purified RNA is determined by a Bichinonic Acid (BCA) assay.