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 mRNA on a preparative scale comprising the following steps:
a) providing a template DNA comprising a nucleic acid sequence encoding an mRNA;
b) in vitro transcribing the template DNA in order to obtain a composition comprising the mRNA; and
c) purifying a preparative quantity of the mRNA obtained in step b) by purification steps comprising, at least:
i) oligo dT-based affinity purification; and
ii) core bead chromatography,
thereby producing a preparative quantity of purified mRNA.
2. The method of claim 1 , wherein the oligo dT-based affinity purification is performed before the core bead chromatography.
3. The method of claim 1 , wherein the core bead chromatography is performed before the oligo dT-based affinity purification.
4. The method of claim 1 , wherein the core bead chromatography is performed using a core bead flow-through chromatography medium.
5. The method of claim 1 , wherein the core bead chromatography is performed on a liquid chromatography system.
6. The method of claim 1 , wherein the core bead chromatography is a core bead flow-through chromatography.
7. The method of claim 1 , wherein the core bead chromatography is performed in a way that the template DNA and proteins are captured in the beads, and RNA products flow through.
8. The method of claim 1 , 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/or
IV) determining the purity of the template DNA.
9. The method of claim 1 , wherein the following steps are used to assess the quality of the mRNA obtained in steps b) or c):
i) determining the concentration of the mRNA or the purified mRNA in a sample;
ii) determining the integrity of the mRNA or the purified mRNA;
iii) determining the identity of the mRNA or the purified mRNA;
iv) determining the purity of the mRNA or the purified mRNA;
v) determining the pH of a sample comprising the mRNA or the purified mRNA;
vi) determining the osmolality of a sample comprising the mRNA or the purified mRNA;
vii) determining the presence and/or the amount of the template DNA in a sample comprising the mRNA or the purified mRNA; and/or
viii) determining the presence and/or the amount of an organic solvent in a sample comprising the mRNA or the purified mRNA.
10. The method of claim 1 , wherein the purified mRNA obtain in step c) comprises 1 to 5 grams of mRNA.
11. The method of claim 1 , wherein step a) comprises synthesis of the template DNA.
12. The method of claim 1 , wherein the template DNA is a DNA plasmid.
13. The method of claim 1 , wherein the in vitro transcription in step b) is carried out in presence of modified nucleotides.
14. The method of claim 1 , wherein the purification in step c) further comprises a step of RP-HPLC.
15. The method of claim 1 , wherein the purification in step c) further comprises a precipitation step.
16. The method of claim 15 , wherein the precipitation step is an alcoholic precipitation step or a LiCl precipitation step.
17. The method of claim 1 , wherein the purification in step c) comprises a further chromatographic step selected from the group consisting of anion exchange chromatography, affinity chromatography, and hydroxyapatite chromatography.
18. The method according to claim 1 , wherein the mRNA obtained in step b) or the purified mRNA obtained in step c) is lyophilized.