Methods for purification of messenger RNA
The present invention relates, in part, to methods for large-scale purification of mRNA. The method includes, at least, steps of forming an mRNA slurry, stirring the slurry, and vacuum or pressure filtering the slurry.
1. A method of large scale purification of mRNA, comprising steps of:
providing a solution comprising at least 250 mg of in vitro synthesized mRNA;
adding one or more agents that promote precipitation of the mRNA, thereby obtaining a slurry;
stirring the slurry in an agitated filter dryer at a speed between 50 revolutions per minute RPM and 500 revolutions per minute underpressure to the slurry and/or a vacuum to the slurry sufficient to direct the slurry's mother liquor through the filter, thereby obtaining a precipitate-containing composition; and
washing the precipitate-containing composition with stiffing and under pressure, thereby yielding a purified mRNA precipitate,
wherein the purified mRNA has reduced impurity of enzyme reagents used in in vitro synthesis as compared to the otherwise same mRNA purified by tangential flow filtration and has an integrity greater than about 95% determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining.
2. The method of claim 1 , wherein the method purifies at least about 2.5 grams mRNA in one batch.
3. The method of claim 1 , wherein the pressure is between about 5 pounds per square inch and about 100 pounds per square inch.
4. The method of claim 1 , further comprising adding one or more agents that denatures proteins and/or keeps proteins soluble in an aqueous medium.
5. The method of claim 1 , further comprising a step of drying the purified mRNA precipitate, wherein the purified mRNA precipitate is dried by continuing to provide pressure and/or vacuum such that precipitated mRNA is obtained as a cake of precipitate.
6. The method of claim 1 , wherein the slurry comprises at least one dispersant.
7. The method of claim 6 , further comprising a step of drying the purified mRNA precipitate which is included in a cake along with the dispersant or comprising one or more steps for separating the dispersant from the purified mRNA precipitate.
8. The method of claim 1 , wherein the one or more agents that promote precipitation of mRNA are a chaotropic salt and an alcohol.
9. The method of claim 1 , wherein the mRNA is contacted with the one or more agents that promote precipitation of mRNA for a total amount of about one minute to about one hour.
10. The method of claim 1 , further comprising a step of solubilizing the purified mRNA precipitate in an aqueous medium, thereby obtaining a solution comprising purified mRNA.
11. The method of claim 1 , wherein the mRNA encodes cystic fibrosis transmembrane conductance regulator protein (CFTR).
12. The method of claim 1 , wherein the mRNA encodes ornithine transcarbamylase (OTC).
13. A composition comprising a purified mRNA precipitate prepared by the method of claim 1 .
14. A pharmaceutical composition comprising the composition of claim 13 and at least one pharmaceutically-acceptable excipient.
15. A method for treating a disease or disorder comprising administering to a subject in need thereof the pharmaceutical composition of claim 14 .
16. The method of claim 1 , wherein the method purifies at least 5 grams mRNA in one batch.
17. The method of claim 1 , wherein the method purifies at least 10 grams mRNA in one batch.
18. The method of claim 1 , wherein the total purified mRNA is recovered in an amount that results in a yield of at least about 80%.
19. The method of claim 1 , wherein the total purified mRNA is recovered in an amount that results in a yield of at least about 95%.
20. The method of claim 1 , wherein the purified mRNA has an integrity greater than about 98% determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis with silver staining.
21. The method of claim 1 , wherein the slurry comprises at least one filtration aid.