Methods for purification of messenger rna

Abstract

The present invention provides, among other things, methods of purifying messenger RNA (mRNA) including the steps of (a) precipitating mRNA from an impure preparation; (b) subjecting the impure preparation comprising precipitated mRNA to a purification process involving membrane filtration such that the precipitated mRNA is captured by a membrane; and (c) eluting the captured precipitated mRNA from the membrane by re-solubilizing the mRNA, thereby resulting in a purified mRNA solution. In some embodiments, a purification process involving membrane filtration suitable for the present invention is tangential flow filtration.

1 . A method of purifying messenger RNA (mRNA), comprising
 (a) precipitating mRNA from an impure preparation;   (b) subjecting the impure preparation comprising precipitated mRNA to a purification process involving membrane filtration such that the precipitated mRNA is captured by a membrane; and   (c) eluting the captured precipitated mRNA from the membrane by re-solubilizing the mRNA, thereby resulting in a purified mRNA solution.   
     
     
         2 . The method of  claim 1 , wherein the purification process involving membrane filtration is tangential flow filtration. 
     
     
         3 . (canceled) 
     
     
         4 . The method of  claim 1 , wherein the step of precipitating mRNA comprises treating the impure preparation with a solution comprising a reagent selected from the group consisting of lithium chloride, potassium chloride, guanidinium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, ammonium acetate and combinations thereof. 
     
     
         5 . The method of  claim 4 , wherein the reagent is guanidinium thiocyanate. 
     
     
         6 - 8 . (canceled) 
     
     
         9 . The method of  claim 1 , wherein the step of precipitating mRNA further comprises a step of treating the impure preparation with absolute ethanol. 
     
     
         10 . The method of  claim 1 , wherein the membrane is selected from the group consisting of polyethersulfone (mPES) (not modified), polyethersulfone (mPES) hollow fiber membrane, polyvinylidene fluoride (PVDF), cellulose acetate, nitrocellulose, MCE (mixed cellulose esters), ultra-high MW polyethylene (UPE), polyfluorotetraethylene (PTFE), nylon, and combination thereof. 
     
     
         11 . The method of  claim 1 , wherein the method further comprises washing the captured precipitated mRNA before eluting. 
     
     
         12 - 13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein the eluting step comprises re-solubilizing the captured precipitated mRNA with RNAse-free water. 
     
     
         15 - 17  (canceled) 
     
     
         18 . The method of  claim 1 , wherein the mRNA is in vitro synthesized and the impure preparation comprises an in vitro mRNA synthesis reaction mixture. 
     
     
         19 . The method of  claim 18 , wherein the impure preparation comprises prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis. 
     
     
         20 . The method of  claim 19 , wherein the purified mRNA solution contains less than 1% of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis. 
     
     
         21 - 24 . (canceled) 
     
     
         25 . The method of  claim 20 , wherein the prematurely aborted RNA sequences comprise less than 15 bases. 
     
     
         26 - 27 . (canceled) 
     
     
         28 . The method of  claim 1 , wherein the mRNA is purified at a scale of or greater than 1 gram, 10 gram, 100 gram, 1 kg, 10 kg, or 100 kg per batch. 
     
     
         29 . The method of  claim 1 , wherein the mRNA is purified before a cap and tail are added to the mRNA. 
     
     
         30 . The method of  claim 1 , wherein the mRNA is purified after a cap and tail are added to the mRNA. 
     
     
         31 . The method of  claim 1 , wherein the mRNA is purified after a cap is added. 
     
     
         32 - 33 . (canceled) 
     
     
         34 . The method of  claim 1 , wherein the mRNA comprises one or more modifications to enhance stability. 
     
     
         35 . The method of  claim 34 , wherein the one or more modifications comprises modified nucleotide and/or modified sugar phosphate backbones. 
     
     
         36 . The method  claim 1 , wherein the mRNA is unmodified. 
     
     
         37 - 39 . (canceled) 
     
     
         40 . A method of purifying messenger RNA (mRNA), comprising
 (a) precipitating mRNA from an impure preparation;   (b) subjecting the impure preparation comprising precipitated mRNA to tangential flow filtration such that the precipitated mRNA is captured by a filtration membrane while impurities are discarded through permeation; and   (c) eluting the captured precipitated mRNA by re-solubilizing the precipitated mRNA, resulting in a purified mRNA solution.   
     
     
         41 - 52 . (canceled)