Method for purifying RNA

Patent metadata

Field	Value
Publication number	US-11279923-B2
Application number	US-201716464152-A
Country	US
Kind code	B2
Filing date	Nov 28, 2017
Priority date	Nov 28, 2016
Publication date	Mar 22, 2022
Grant date	Mar 22, 2022

Abstract

The present invention relates to methods for purifying RNA by chromatography under high salt conditions, e.g. by hydrophobic interaction chromatography.

First claim

The invention claimed is:

1. A method for purifying RNA, comprising the steps of:
a) applying a sample containing in vitro transcribed RNA in an equilibration buffer having a high salt concentration to a support material that binds the RNA under said high salt concentration, wherein the support material comprises a binding ligand consisting of hydroxyl or sulfate groups and, wherein the equilibration buffer has said high salt concentration of at least about 150 mM;
b) optionally washing the support material with a washing buffer having a high salt concentration of at least about 150 mM; and
c) eluting the RNA from the support material with an elution solution.

2. The method according to claim 1 , wherein the equilibration buffer and/or the washing buffer has a salt concentration of 500 mM to 3 M.

3. The method according to claim 1 , wherein the equilibration buffer and/or the washing buffer comprises sodium chloride or ammonium sulfate.

4. The method according to claim 1 , wherein the equilibration buffer and/or the washing buffer comprises at least about 2 M NaCl.

5. The method according to claim 4 , wherein the equilibration buffer and/or the washing buffer comprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl.

6. The method according to claim 1 , wherein the equilibration buffer and the washing buffer have the same composition and the same pH.

7. The method according to claim 1 , wherein the support material is a monolithic support material.

8. The method according to claim 1 , wherein the support material is a methacrylate polymer.

9. The method according to claim 1 , wherein the RNA is eluted by gradually decreasing the salt concentration of the elution solution.

10. The method according to claim 1 , wherein the elution solution does not contain a salt.

11. The method according to claim 1 , wherein the elution solution comprises 20 mM HEPES-NaOH, pH 7.0.

12. The method according to claim 1 , wherein the method comprises the steps of:
a) transcribing RNA from a template DNA in vitro;
b) applying a sample containing the in vitro transcribed RNA in an equilibration buffer having a high salt concentration of at least about 150 mM to a support material capable of binding the RNA under high salt conditions, wherein the support material comprises a binding ligand consisting of hydroxyl or sulfate groups;
c) washing the support material with a washing buffer having a high salt concentration of at least about 150 mM; and
d) eluting the RNA from the support material with an elution solution.

13. The method according to claim 12 , further comprising a step a1) of degrading the template DNA.

14. The method according to claim 13 , wherein the template DNA is degraded by treatment with DNase.

15. The method according to claim 12 , further comprising a step a2) of subjecting the in vitro transcribed RNA to a reverse phase-HPLC step.

16. The method according to claim 12 , wherein said RNA is suitable for preparing a pharmaceutical composition.

17. The method according to claim 2 , wherein the RNA is a mRNA.

18. A method for preparing a pharmaceutical composition comprising:
a) transcribing RNA from a template DNA in vitro;
b) applying a sample containing the in vitro transcribed RNA in an equilibration buffer having a high salt concentration of at least about 150 mM to a support material capable of binding the RNA under high salt conditions, wherein the support material comprises a binding ligand consisting of hydroxyl or sulfate groups;
c) washing the support material with a washing buffer having a high salt concentration of at least about 150 mM; and
d) eluting the washed RNA from the support material with an elution solution; and
e) formulating the eluted RNA into a pharmaceutical composition.

19. The method according to claim 18 , wherein the equilibration buffer and/or the washing buffer comprise about 500 mM to 3 M NaCl.

20. The method according to claim 17 , wherein the binding ligand contains hydroxyl groups.

21. The method according to claim 17 , wherein the binding ligand contains sulfate groups.

Assignees

• Curevac Ag

Inventors

• Funkner Andreas
• Sewing Stefanie
• Strobel Isabel
• Mutzke Thorsten

Classifications

• C12N15/1006 Primary

  by means of a solid support carrier, e.g. particles, polymers · CPC title

• C12N15/101

  by chromatography, e.g. electrophoresis, ion-exchange, reverse phase · CPC title