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 method for purifying mRNA, comprising steps of:
providing a suspension comprising precipitated mRNA; and
centrifuging the suspension in a centrifuge comprising a porous substrate such that the precipitated mRNA is captured on the porous substrate, thereby purifying contaminants from the mRNA.
2 . The method of claim 1 , comprising purifying at least about 10 grams, 25 grams, 50 grams, 100 grams, or 1 kilogram mRNA
wherein the total purified mRNA is recovered in an amount that results in a yield of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, or 95%; and/or
the total purified mRNA is substantially free of prematurely aborted RNA sequences and/or enzyme reagents used in in vitro synthesis.
3 - 7 . (canceled)
8 . The method of claim 1 , further comprising a step of first producing the suspension of precipitated mRNA by providing a solution comprising mRNA and adding to the solution one or more agents that promote precipitation of mRNA.
9 . The method of claim 1 , wherein the suspension comprising precipitated mRNA comprises at least one filtration aid that is a dispersant.
10 - 12 . (canceled)
13 . The method of claim 1 , wherein the speed of the centrifuging of the mRNA suspension is between about 2000 RPM and about 4000 RPM, about 1000 RPM and about 5000 RPM, about 2000 RPM and about 4000 RPM, about 2000 RPM and about 3000 RPM, or about 2500 RPM and about 3500 RPM.
14 - 17 . (canceled)
18 . The method of claim 1 , further comprising adding one or more agents to the suspension that denature proteins and/or keep proteins soluble in an aqueous medium.
19 - 20 . (canceled)
21 . The method of claim 1 , further comprising a step of washing the purified mRNA composition with a solvent.
22 - 26 . (canceled)
27 . The method of claim 1 , further comprising a step of drying the captured mRNA.
28 - 30 . (canceled)
31 . The method of claim 27 , wherein the dried purified mRNA is collected and stored at a temperature of or below about 0° C. for a time period of at least about one week to about two years, and wherein the dried purified mRNA is stored as a solid.
32 - 34 . (canceled)
35 . The method of claim 31 , wherein the dried purified mRNA is reconstituted following storage.
36 . The method of claim 31 , wherein the dried purified mRNA has substantially the same integrity as prior to storage.
37 - 60 . (canceled)
61 . The method of claim 1 , wherein the purified mRNA is substantially free of prematurely aborted RNA sequences, DNA templates, and/or enzyme reagents used in in vitro synthesis of the single mRNA species.
62 - 72 . (canceled)
73 . A method for purifying mRNA, said method comprising
providing a composition comprising mRNA purified according to the method of claim 1 ; and
subjecting the provided composition to further purification comprising dialysis, diafiltration, and/or ultrafiltration of a solution comprising the purified mRNA.
74 . (canceled)
75 . The method of claim 73 , said method comprising
providing the obtained composition comprising purified mRNA; and
subjecting the provided composition to further purification by
precipitating said mRNA to provide a suspension comprising precipitated mRNA; and
centrifuging the suspension in a centrifuge comprising a porous substrate such that the precipitated mRNA is captured on the porous substrate, thereby purifying contaminants from the mRNA.
76 . The method of claim 75 , said method further comprising
providing a composition comprising mRNA purified according to the method of claim 75 ; and
subjecting the provided composition to further purification comprising dialysis, diafiltration, and/or ultrafiltration of a solution comprising the purified mRNA.
77 - 82 . (canceled)
83 . The method of claim 1 , wherein the mRNA comprises one or more nucleotide modifications.
84 . The method of claim 83 , wherein the one or more nucleotide modifications comprises modified sugars, modified bases, and/or modified sugar phosphate backbones.
85 - 98 . (canceled)
99 . A composition comprising dried purified mRNA, wherein said mRNA is obtained by a method comprising
providing a suspension comprising precipitated mRNA;
centrifuging the suspension in a centrifuge comprising a porous substrate such that the precipitated mRNA is captured on the porous substrate, thereby purifying contaminants from the mRNA;
washing the purified mRNA composition with a solvent; and
drying the captured mRNA.
100 - 110 . (canceled)
111 . A composition comprising purified mRNA according to the method of claim 1 .
112 . (canceled)
113 . A method for treating a disease or disorder comprising administering to a subject in need thereof the composition of claim 111 .