METHODS FOR PURIFICATION OF MESSENGER RNA
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)