Methods for purification of messenger RNA
The present invention provides, among other things, methods of purifying messenger RNA (mRNA) including the steps of subjecting an impure preparation comprising in vitro synthesized rnRNA to a denaturing condition, and purifying the rnRNA from the impure preparation from step (a) by tangential flow filtration, wherein the mRNA purified from step (b) is substantially free of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis.
1. A method of purifying messenger RNA (mRNA), comprising:
(a) providing an impure preparation comprising in vitro synthesized mRNA and prematurely aborted RNA sequences;
(b) subjecting the impure preparation comprising in vitro synthesized mRNA to a denaturing condition; and
(c) subjecting the treated impure preparation from step (b) to tangential flow filtration, thereby purifying the mRNA.
2. The method of claim 1 , wherein achieving the denaturing condition of step (b) comprises heating and/or adding one or more denaturing agents to the impure preparation.
3. The method of claim 2 , wherein the one or more denaturing agents is selected from the group consisting of urea, guanidinium thiocyanate, KCl, sodium dodecyl sulfate, sarcosyl, and combinations thereof.
4. The method of claim 1 , wherein the tangential flow filtration is performed using only aqueous solvents.
5. The method of claim 1 , wherein the mRNA purified from step (c) contains less than 1% of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis.
6. The method of claim 1 , wherein the mRNA purified from step (c) contains undetectable amounts of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis as determined by agarose gel electrophoresis or chromatographic methods.
7. The method of claim 5 , wherein the prematurely aborted RNA sequences comprise about 8-12 bases.
8. The method of claim 5 , wherein the enzyme reagents used in in vitro synthesis comprise RNA polymerase, DNAse I, pyrophosphatase, and/or RNAse inhibitor.
9. The method of claim 1 , wherein the in vitro synthesized mRNA is greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, or 5 kb in length.
10. The method of claim 1 , wherein the in vitro synthesized mRNA comprises one or more modifications to enhance stability.
11. The method of claim 10 , wherein the one or more modifications are selected from modified nucleotide, modified sugar phosphate backbones, 5′ and/or 3′ untranslated region.
12. The method of claim 1 , wherein the mRNA purified from step (c) has an integrity greater than 95%.
13. A method for manufacturing messenger RNA (mRNA) comprising:
synthesizing mRNA in vitro; and
purifying the in vitro synthesized mRNA using a method according to claim 1 .
14. A method of purifying messenger RNA (mRNA), comprising
(a) providing an impure preparation comprising in vitro synthesized mRNA and enzyme reagents;
(b) treating the impure preparation comprising in vitro synthesized mRNA with a denaturing agent under conditions that facilitates separation of the enzyme reagents from the mRNA; and
(c) subjecting the treated impure preparation from step (b) to tangential flow filtration, thereby purifying the mRNA;
wherein the mRNA purified from step (c) is substantially free of enzyme reagents selected from the group consisting of RNA polymerase, DNAse I, pyrophosphatase, and RNAse inhibitor.
15. A method of manufacturing messenger RNA (mRNA) comprising:
(a) synthesizing mRNA in vitro to provide an impure preparation of mRNA;
(b) adding a 5′ cap to the mRNA; and
(c) purifying the mRNA;
wherein purifying the mRNA comprises:
(i) subjecting the impure preparation of mRNA to a denaturing condition; and
(ii) subjecting the treated impure preparation from step (b)(i) to tangential flow filtration.