Methods for purification of messenger rna

Abstract

The present invention provides, among other things, methods for purifying high quality messenger RNA (mRNA) suitable for clinical use, without using any caustic or flammable solvents. The present invention is, in part, based on surprising discovery that mRNA can be successfully purified by selective precipitation and washing without using ethanol while maintaining integrity and high purity of mRNA. Thus, the present invention provides an effective, reliable, and safer method of purifying RNA from large scale manufacturing process therapeutic applications without using any caustic or inflammable solvents.

1 . A method of purifying messenger RNA (mRNA) comprising:
 a) precipitating the mRNA in a suspension comprising a high molar salt solution and an amphiphilic polymer to provide precipitated mRNA;   b) capturing the precipitated mRNA;   c) washing the precipitated mRNA captured in step b) with a wash solution to purify the precipitated mRNA; and   d) solubilizing the precipitated mRNA from step c) to obtain a purified mRNA composition.   
     
     
         2 .- 3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the amphiphilic polymer is selected from pluronics, polyvinyl pyrrolidone, polyvinyl alcohol, triethylene glycol monomethyl ether (MTEG), polyethylene glycol (PEG), or combinations thereof. 
     
     
         5 .- 7 . (canceled) 
     
     
         8 . The method of  claim 1 , wherein the high molar salt solution comprises guanidinium thiocyanate (GSCN). 
     
     
         9 . The method of  claim 1 , wherein a wash solution comprising an amphiphilic polymer is used for washing the mRNA in step c). 
     
     
         10 . The method of  claim 9 , wherein the amphiphilic polymer is selected from pluronics, polyvinyl pyrrolidone, polyvinyl alcohol, triethylene glycol monomethyl ether (MTEG), polyethylene glycol (PEG), or combinations thereof. 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 10 , wherein the amphiphilic polymer is present in the wash solution at about 50 to about 90% weight/volume concentration. 
     
     
         13 .- 23 . (canceled) 
     
     
         24 . The method of  claim 1 , wherein the wash solution is aqueous. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 1 , wherein the wash solution is free of ethanol, isopropyl alcohol, or benzyl alcohol. 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 1 , wherein the wash solution comprises ethanol, isopropyl alcohol, or benzyl alcohol. 
     
     
         29 . The method of  claim 1 , wherein the capturing the precipitated mRNA occurs on a microfiltration or ultrafiltration filter. 
     
     
         30 .- 35 . (canceled) 
     
     
         36 . The method of  claim 1 , wherein a filter aid is used. 
     
     
         37 . The method of  claim 36 , wherein the filter aid is cellulose-based, or comprises diatomaceous earth and/or volcanic ash. 
     
     
         38 .- 43 . (canceled) 
     
     
         44 . The method of  claim 1 , wherein the yield of the purified mRNA is about 70% to about 99%. 
     
     
         45 .- 55 . (canceled) 
     
     
         56 . The method of  claim 1 , wherein the precipitated mRNA comprises about 100 mg, 1 g, 10 g, 100 g, 1 kg, 10 kg, 100 kg, one metric ton, or ten metric tons, of mRNA or any amount there between. 
     
     
         57 .- 64 . (canceled) 
     
     
         65 . A method of manufacturing mRNA comprising the steps of:
 a) performing in vitro transcription (IVT) by mixing (i) a DNA template comprising a promoter and (ii) an RNA polymerase to generate an impure preparation comprising full-length mRNA;   b) providing high molar salt and an amphiphilic polymer to the suspension to precipitate full-length mRNA and provide precipitated full-length mRNA in the suspension;   c) capturing the precipitated full-length mRNA by applying the suspension to a filter;   d) washing the precipitated full-length mRNA of step (c) with an aqueous solvent to obtain a purified full-length mRNA in an aqueous solution; and   e) solubilizing the precipitated mRNA from step (d) to obtain a purified mRNA composition wherein the purified full-length mRNA in the aqueous solution provided from step (d) is substantially free of (i) the DNA template comprising a promoter and (ii) the RNA polymerase.   
     
     
         66 . The method of  claim 65 , wherein in step (a) the RNA polymerase is SP6 polymerase. 
     
     
         67 .- 77 . (canceled) 
     
     
         78 . The method of  claim 65 , wherein the suspension further comprises a filter aid at a mass ratio with the precipitated mRNA of 2:1; 5:1; 10:1 or 15:1. 
     
     
         79 .- 93 . (canceled) 
     
     
         94 . A purified mRNA composition, wherein the purified mRNA composition is substantially free of contaminants comprising abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzymes, residual solvent and/or residual salt. 
     
     
         95 . The composition of  claim 94 , wherein the residual plasmid DNA is 10 pg/mg or less. 
     
     
         96 . The composition of  claim 94 , wherein the composition comprises less than 1% double-stranded RNA.