Method for purifying rna

Abstract

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

1 . Method for purifying RNA, comprising the steps of:
 a) applying a sample containing RNA in an equilibration buffer having a high salt concentration to a support material capable of binding the RNA under high salt conditions, wherein the support comprises hydroxyl or sulfate groups;   b) optionally washing the support material with a washing buffer having a high salt concentration; and   c) eluting the nucleic acid from the support material with an elution solution.   
     
     
         2 . Method according to  claim 1 , wherein the RNA is obtained by RNA in vitro transcription. 
     
     
         3 . Method according to  claim 1  or  2 , wherein the equilibration buffer and/or the washing buffer has a salt concentration of 50 mM to 5M. 
     
     
         4 . Method according to any one of the preceding claims, wherein the equilibration buffer and/or the washing buffer comprises sodium chloride or ammonium sulfate. 
     
     
         5 . Method according to any one of the preceding claims, wherein the equilibration buffer and/or the washing buffer comprises 2 M NaCl. 
     
     
         6 . Method according to any one of the preceding claims, wherein the equilibration buffer and/or the washing buffer comprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl. 
     
     
         7 . Method according to any one of the preceding claims, wherein the equilibration buffer and the washing buffer have the same composition and the same pH. 
     
     
         8 . Method according to any one of the preceding claims, wherein the support material is a monolithic support material. 
     
     
         9 . Method according to any one of the preceding claims, wherein the support material is a methacrylate polymer. 
     
     
         10 . Method according to any one of the preceding claims, wherein the RNA is eluted by gradually decreasing the salt concentration. 
     
     
         11 . Method according to any one of the preceding claims, wherein the elution solution does not contain a salt. 
     
     
         12 . Method according to any one of the preceding claims, wherein the elution solution comprises 20 mM HEPES-NaOH, pH 7.0. 
     
     
         13 . Method for purifying in vitro transcribed RNA, comprising 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 to a support material capable of binding the RNA under high salt conditions, wherein the support material comprises hydroxyl or sulfate groups;   c) washing the support material with a washing buffer having a high salt concentration; and   d) eluting the RNA from the support material with an elution solution.   
     
     
         14 . Method according to  claim 13 , further comprising a step a1) of degrading the template DNA. 
     
     
         15 . Method according to  claim 14 , wherein the template DNA is degraded by treatment with DNase. 
     
     
         16 . Method according to any one of  claims 13  to  15 , further comprising a step a2) of subjecting the in vitro transcribed RNA to an RP-HPLC step. 
     
     
         17 . Method according to any one of  claims 13  to  16 , further comprising a step e) of preparing a pharmaceutical composition comprising said RNA. 
     
     
         18 . Method according to any one of  claims 13  to  17 , wherein the equilibration buffer and/or the washing buffer has a salt concentration of 50 mM to 5M. 
     
     
         19 . Method according to any one of  claims 13  to  18 , wherein the equilibration buffer and/or the washing buffer comprises sodium chloride or ammonium sulfate. 
     
     
         20 . Method according to any one of  claims 13  to  19 , wherein the equilibration buffer and/or the washing buffer comprises 2 M NaCl. 
     
     
         21 . Method according to any one of  claims 13  to  20 , wherein the equilibration buffer and/or the washing buffer comprises 20 mM HEPES-NaOH, pH 7.0, 2 M NaCl. 
     
     
         22 . Method according to any one of  claims 13  to  21 , wherein the equilibration buffer and the washing buffer have the same composition and the same pH. 
     
     
         23 . Method according to any one of  claims 13  to  22 , wherein the support material is a monolithic support material. 
     
     
         24 . Method according to any one of  claims 13  to  23 , wherein the support material is a methacrylate polymer. 
     
     
         25 . Method according to any one of  claims 13  to  24 , wherein the RNA is eluted by gradually decreasing the salt concentration. 
     
     
         26 . Method according to any one of  claims 13  to  25 , wherein the elution solution does not contain a salt. 
     
     
         27 . Method according to any one of  claims 13  to  26 , wherein the elution buffer comprises 20 mM HEPES-NaOH, pH 7.0. 
     
     
         28 . Use of hydrophobic interaction chromatography for the purification of RNA obtained by RNA in vitro transcription.