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, a step of centrifuging an mRNA suspension in a centrifuge comprising a porous substrate at a speed sufficient to remove process contaminants and to precipitate purified mRNA composition onto the porous substrate.
1. A composition comprising at least 10 grams of dried purified mRNA, wherein said mRNA is obtained by a method for large-scale purification of mRNA, comprising the steps of:
providing a suspension comprising precipitated mRNA manufactured by in vitro synthesis; and
centrifuging the suspension at a speed of about 1000 to 5000 RPM in a centrifuge comprising a porous substrate such that the precipitated mRNA is captured on the porous substrate, and contaminants are removed from the precipitated mRNA to provide captured purified mRNA;
washing the captured purified mRNA composition with a solvent; and
drying the captured purified mRNA, thereby obtaining dried purified mRNA in the form of a solid,
wherein the purified mRNA yield is at least about 80% and is substantially free of prematurely aborted RNA sequences, DNA templates, and one or more enzyme reagents used in in vitro synthesis;
wherein the dried purified mRNA is collected and stored as a solid at a temperature of or below about 0° C. for a period of at least about one week to about two years; and
wherein the dried purified mRNA has substantially the same integrity as prior to storage.
2. The composition of claim 1 , wherein said suspension comprising precipitated mRNA manufactured by in vitro synthesis comprises at least about 25 grams, 50 grams, 100 grams, or 1 kilogram of precipitated mRNA.
3. The composition of claim 1 , wherein the enzyme reagents used in in vitro synthesis comprise T7 RNA polymerase, SP6 RNA polymerase, DNAse I, pyrophosphatase, and/or RNAse inhibitor.
4. The composition of claim 1 , wherein the mRNA encodes cystic fibrosis transmembrane receptor (CFTR).
5. The composition of claim 1 , wherein the mRNA encodes ornithine transcarbamylase (OTC).
6. The composition of claim 1 , wherein the washing of the captured purified mRNA occurs via centrifugation.
7. The composition of claim 1 , wherein the drying of the captured purified mRNA occurs via centrifugation.
8. The composition of claim 1 , wherein said dried purified mRNA is obtained by a method further comprising collecting the dried purified mRNA from the porous substrate while the centrifuge is centrifuging.
9. The composition of claim 1 , wherein the dried purified mRNA can be sectioned into manageable pieces.