Methods for purification of messenger rna
AssigneeTranslate Bio, Inc.
InventorsJonathan ABYSALH, Frank DEROSA, Cameron SMITH, Jorel E. VARGAS
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Abstract
The present invention relates, in part, to methods, systems and processes for large-scale purification of mRNA using a filtering centrifuge operating at lower gravitational forces. The invention also relates to compositions of purified mRNA and uses thereof.
Claims151
A method for purifying messenger RNA (mRNA), the method comprising the steps of:
- I. precipitating mRNA from a solution comprising one or more protein and/or short abortive transcript contaminants from manufacturing the mRNA to provide a suspension comprising precipitated mRNA;
- II. loading the suspension comprising the precipitated mRNA into a filtering centrifuge comprising a filter wherein the precipitated mRNA is retained by the filter;
- III. washing the retained precipitated mRNA by adding a wash buffer to the filtering centrifuge;
- and
- IV. recovering the retained precipitated mRNA from the filter;
- wherein the filtering centrifuge is operated during loading step (b) and washing step (c) at a centrifuge speed that exerts a gravitational (g) force of less than 1300 g.
The method of any one of the preceding claims, wherein the filtering centrifuge is operated at the same centrifuge speed during loading step (b) and washing step (c).
The method of any one of the preceding claims, wherein the recovering the retained precipitated mRNA from the filter comprises the steps of:
- i. solubilising the retained precipitated mRNA;
- and
- ii. collecting the solubilised mRNA.
The method of any one of the preceding claims, wherein precipitating the mRNA comprises adding one or more agents that promote precipitation of mRNA, for example one or more of an alcohol, an amphiphilic polymer, a buffer, a salt, and/or a surfactant.
The method of any one of the preceding claims, wherein the suspension comprises precipitated mRNA, a salt and MTEG.
The method of any one of the preceding claims, wherein step (a) further comprises adding at least one filtration aid to the suspension comprising precipitated mRNA.
The method of any one of the preceding claims, wherein the suspension comprises at least 100 mg, 1 g, 10 g, 100 g, 250 g, 500 g, 1 kg, 10 kg, 100 kg, one metric ton, or ten metric tons, of mRNA or any amount there between.
The method of any one of the preceding claims, wherein the filter comprises a porous substrate.
The method of any one of the preceding claims, wherein the filter is a microfiltration membrane or ultrafiltration membrane.
The method of any one of the preceding claims, wherein the filter has an average pore size is about 0.5 micron or greater, about 0.75 micron or greater, about 1 micron or greater, about 2 microns or greater, about 3 microns or greater, about 4 microns or greater, or about 5 microns or greater.
The method of any one of the preceding claims, wherein the volume of wash buffer for washing the retained precipitated mRNA is between about 0.5 L/g mRNA and about 8 L/g mRNA.
The method of any one of the preceding claims, wherein the volume of wash buffer for washing the retained precipitated mRNA is between about 0.5 L/g mRNA and about 1.5 L/g mRNA.
The method of any one of the preceding claims, wherein the volume of wash buffer for washing the retained precipitated mRNA is about 0.5 L/g mRNA.
The method of any of the preceding claims, wherein the wash buffer comprises one or more of an alcohol, an amphiphilic polymer, a buffer, a salt, and/or a surfactant.
The method of any one of the proceeding claims, wherein the recovering the retained mRNA occurs while the filtering centrifuge is in operation.
The method of any one of the preceding claims, wherein the filtering centrifuge is a continuous centrifuge and/or the filtering centrifuge is orientated vertically or horizontally or the centrifuge is an inverted horizontal centrifuge.
The method of any one of the preceding claims, wherein the filtering centrifuge comprises a sample feed port and/or a sample discharge port.
The method of any one of the preceding claims, method of any one of the preceding claims, wherein the mRNA suspension is loaded into the filtering centrifuge at a rate of about 1 liter/min to about 60 liter/min, e.g., at a rate of about 5 liter/min to about 45 liter/min.
The method of any one of the preceding claims, wherein the manufacturing the mRNA comprises in vitro transcription (IVT) synthesis of the mRNA.
The method of any one of the preceding claims, wherein the mRNA is or greater than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, or 20 kb in length.
The method of any one of the preceding claims, wherein the mRNA comprises one or more nucleotide modifications.
The method of any one of the preceding claims, wherein the recovery of purified mRNA is at least 10 g, 20 g, 50 g, 100 g, 250 g, 500 g, 1 kg, 5 kg, 10 kg, 50 kg, or 100 kg per single batch.
The method of any one of the preceding claims, wherein the total purified mRNA is recovered in an amount that results in a yield of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or about 100%.
The method of any one of the preceding claims, wherein the purity of the purified mRNA is between about 60% and about 100%.
The method of any one of the preceding claims, wherein the purified mRNA has an integrity of at least about 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99%.
The method of any one of the preceding claims, wherein the purified mRNA has a clinical grade purity without further purification.
The method of any one of the preceding claims, wherein the purified mRNA has integrity of 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater as determined by capillary electrophoresis.
The method of any one of the preceding claims, wherein one or more protein and/or short abortive transcript contaminants include enzyme reagents used in IVT mRNA synthesis.
The method of any one of the preceding claims, wherein the method also removes long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA residual solvent and/or residual salt.
The method of any one of the preceding claims, wherein the short abortive transcript contaminants comprise less than 15 bases.
The method of any one of preceding claims, wherein the short abortive transcript contaminants comprise about 8-12 bases.
The method of the preceding claims, wherein the method also removes RNAse inhibitor.
A process for purifying mRNA, the process comprising the steps of:
- I. providing a suspension comprising precipitated mRNA in a first vessel, wherein the precipitated mRNA comprises one or more protein and/or short abortive transcript contaminants from manufacturing the mRNA;
- II. providing a wash buffer in a second vessel;
- III. transferring the content of the first vessel into a filtering centrifuge comprising a filter, wherein the transferring occurs at a rate of about 5 liter/min/m2 to about 25 liter/min/m2 (with respect to the surface area of the filter of the filtering centrifuge) while the filtering centrifuge is in operation at a first centrifuge speed such that the precipitated mRNA is retained on the filter of said filtering centrifuge;
- IV. transferring the content of the second vessel into the filtering centrifuge, wherein the transferring occurs at a rate of about 5 liter/min/m2 to about 25 liter/min/m2 (with respect to the surface area of the filter of the filtering centrifuge)while the filtering centrifuge remains in operation at the first centrifuge speed, thereby washing the precipitated mRNA retained on the filter of said filtering centrifuge with the wash buffer;
- and
- V. recovering the washed precipitated mRNA from the filter of said filtering centrifuge.
A system for purifying mRNA, wherein the system comprises:
- I. a first vessel for receiving precipitated mRNA;
- II. a second vessel for receiving wash buffer;
- III. a third vessel for receiving the washed precipitated mRNA and/or an aqueous medium for solubilising precipitated mRNA;
- IV. a filtering centrifuge comprising:
- i. a filter, wherein the filter is arranged and dimensioned to retain precipitated mRNA and/or a filtration aid, and to let pass solubilised mRNA;
- ii. a sample feed port;
- and
- iii. a sample discharge port;
- V. a fourth vessel for receiving purified mRNA, wherein said vessel is connected to the sample discharge port of the filtering centrifuge;
- VI. a pump configured to direct flow through the system at a rate of about 5 liter/min/m2to about 25 liter/min/m2 with respect to the surface area of the filter of the filtering centrifuge (e.g. about 15 liter/min/m2);
- wherein the first vessel, the second vessel and the third vessel are operably linked to an input of the pump, and wherein the sample feed port of the filtering centrifuge is connected to an output of the pump;
- and
- VII. one or more valves configured to preclude simultaneous flow from the first, second and third vessels.
A composition comprising mRNA, amphiphilic polymer and a filtration aid at relative concentrations of about 1:1:10 in a sterile, RNase-free container.
Dependencies are inferred from the claim text.