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, wherein the purification process involving membrane filtration is tangential flow filtration.
2. 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.
3. The method of claim 2 , wherein the reagent is guanidinium thiocyanate.
4. The method of claim 1 , wherein the step of
precipitating mRNA further comprises a step of treating the impure preparation with absolute ethanol.
5. 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, wherein the purified mRNA solution is dialyzed with 1 mM sodium citrate using a 100 kDa molecular weight cut-off (MWCO) membrane.
6. The method of claim 1 , wherein the mRNA is in vitro synthesized and the impure preparation comprises an in vitro mRNA synthesis reaction mixture.
7. The method of claim 1 , wherein the purified mRNA solution contains less than 1% of prematurely aborted RNA sequences and/or enzyme reagents used in in vitrosynthesis.
8. The method of claim 1 , wherein the purified mRNA solution is substantially free of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis.
9. 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.
10. The method of claim 1 , wherein the mRNA is purified both before and after a cap and/or tail are added to the mRNA.
11. The method of claim 1 , wherein the mRNA comprises one or more modifications to enhance stability.
12. The method of claim 1 , wherein the mRNA is unmodified.
13. The method of claim 1 , wherein the purified mRNA has an integrity of or greater than 95%.
14. 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.
15. 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 14 .
16. A batch of purified mRNA manufactured according to the method of claim 15 , comprising 5 gram or more of a single mRNA species suitable for administration to a human subject.
17. The batch of claim 16 , wherein the batch is substantially free of prematurely aborted RNA sequences, DNA templates, and/or enzyme reagents used in in vitro synthesis of the single mRNA species.
18. A composition comprising in vitro synthesized messenger RNA, wherein the composition comprises less than 5% of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis.