Method for producing rna

Abstract

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; and 
   b) in vitro transcription of 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; 
 iv) determining the purity of the RNA or the purified RNA; 
 v) determining the pH of a sample comprising the RNA or the purified RNA; 
 vi) determining the osmolality of a sample comprising the RNA or the purified RNA; 
 vii) determining the presence and/or the amount of the template DNA in a sample comprising the RNA or the purified RNA; and 
 viii) determining the presence and/or the amount of an organic solvent in 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 presence and/or the amount of RNA; the presence and/or the amount of protein; the presence and/or the amount of endotoxin; the presence and/or the amount of bacterial DNA; and/or the presence and/or the amount of ribonuclease. 
     
     
         6 . The method according to  claim 5 , wherein the presence and/or the amount of bacterial DNA is determined by using a PCR method. 
     
     
         7 . The method according to  claim 6 , wherein the presence and/or the amount of bacterial DNA is determined using a universal primer pair for bacterial DNA. 
     
     
         8 . The method according to  claim 6 , wherein the presence and/or the amount of  E. coli  DNA is determined using a primer pair specific for  E. coli  DNA. 
     
     
         9 . The method according to  claim 9 , 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/or 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 presence and/or the amount of protein; the presence and/or the amount of endotoxin; the presence and/or the amount of bacterial DNA; the presence and/or the amount of plasmid DNA; and/or the presence and/or the amount of organic solvent. 
     
     
         14 . The method according to  claim 13 , wherein the presence and/or the amount of bacterial DNA is determined by using a PCR method. 
     
     
         15 . The method according to  claim 14 , wherein the presence and/or the amount of bacterial DNA is determined using a universal primer pair for bacterial DNA. 
     
     
         16 . The method according to  claim 14 , wherein the presence and/or 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; and isolating the template DNA from the bacteria. 
     
     
         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.