Methods for purification of messenger RNA

Abstract

To provide methods for purifying high quality messenger RNA (mRNA) suitable for clinical use, without using any caustic or flammable solvents.SOLUTION: Provided is a method of purifying messenger RNA (mRNA) comprising: a) precipitating the mRNA in a suspension comprising a high molar salt solution and an amphiphilic polymer to provide precipitated mRNA; b) capturing the precipitated mRNA; c) washing the precipitated mRNA captured in step b) with a wash solution to purify the precipitated mRNA; and d) solubilizing the precipitated mRNA from step c) to obtain a purified mRNA composition.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims priority to U.S. Provisional Application No. 62 / 848,412, filed May 15, 2019, and U.S. Provisional Application No. 62 / 891,781, filed Aug. 26, 2019, each of which is hereby incorporated by reference in its entirety for all purposes.

Background Art

[0002] Messenger RNA (mRNA) therapy is becoming an increasingly important approach for the treatment of various diseases. mRNA therapy involves the administration of a pharmaceutical product containing in vitro transcribed (IVT) and highly purified messenger RNA (mRNA) to patients in need of treatment, and the production of proteins encoded by the mRNA in the patient's body. Thus, there is a need for efficient and large - scale production of high - purity mRNA products suitable for therapeutic use.

[0003] Conventionally, mRNA produced from in vitro transcription has been purified using commercially available chromatography systems, such as HPLC, and / or by extraction into an organic mixture (phenol:chloroform:isoamyl alcohol) followed by ethanol precipitation. However, the use of column systems is expensive and difficult, and the use of caustic or flammable solvents in mRNA extraction can present safety and cost challenges, especially in large - scale applications.

[0004] There is currently no safe and cost - effective method for producing high - purity mRNA acceptable for therapeutic use.

Summary of the Invention

Means for Solving the Problems

[0005] The present invention provides, inter alia, a very efficient and cost-effective method for purifying messenger RNA (mRNA). The present invention is based in part on the surprising discovery of a method for purifying mRNA using a small amount of volatile organic compounds or without using volatile organic compounds, which results in highly intact and highly pure mRNA. Thus, in one aspect, the present invention provides a more effective, reliable, and safe method for purifying mRNA that can be used for therapeutic applications in large-scale manufacturing processes without using any caustic or flammable solvents.

[0006] In some aspects, the present invention provides a method for purifying messenger RNA (mRNA), comprising: a) precipitating the mRNA in a suspension comprising a high molar salt solution and an amphiphilic polymer to provide a precipitated mRNA; b) capturing the precipitated mRNA; c) washing the precipitated mRNA captured in step b) with a washing solution to purify the precipitated mRNA; and d) solubilizing the precipitated mRNA from step c) with a solubilizing solution to obtain a purified mRNA composition.

[0007] In some embodiments, the purified mRNA composition is substantially free of contaminants including short abortive RNA species, long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzyme, residual solvent, and / or residual salts. In some embodiments, the purified mRNA composition is substantially free of contaminants including short abortive RNA species. For example , The purified mRNA composition has less than about 1% short-chain defective RNA species. In some embodiments, the purified mRNA composition is substantially free of contaminants containing long-chain defective RNA species. For example, the purified mRNA composition has about 55% or less long-chain defective / degraded species as determined by capillary gel electrophoresis (CGE). In some embodiments, the purified mRNA composition is substantially free of contaminants containing double-stranded RNA (dsRNA). For example, the purified mRNA composition has less than 1% double-stranded RNA. In some embodiments, the purified mRNA composition is substantially free of contaminants containing residual plasmid DNA. For example, the purified mRNA composition has 10 pg / mg or less residual plasmid DNA. In some embodiments, the purified mRNA composition is substantially free of contaminants containing residual in vitro transcription enzymes. For example, for 15 μg of the purified mRNA composition, it is less than 0.3 ng of polymerase. For 15 μg of the purified mRNA composition, it is less than 0.3 ng of cap enzyme. For 15 μg of the purified mRNA composition, it is less than 0.3 ng of tail enzyme. In some embodiments, the purified mRNA composition is substantially free of contaminants containing residual solvents. In some embodiments, the purified mRNA composition is substantially free of contaminants containing residual salts.

[0008] In some embodiments, the purified mRNA contains 10 pg / mg or less of residual plasmid DNA relative to the purified mRNA.

[0009] In some embodiments, the amphiphilic polymer in the suspension is selected from pluronic (registered trademark), polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), polyethers such as polypropylene glycol (PPG) or polypropylene oxide, or combinations thereof. In some embodiments, the amphiphilic polymer in the suspension is pluronic. In some embodiments, the amphiphilic polymer in the suspension is polyvinylpyrrolidone. In some embodiments, the amphiphilic polymer in the suspension is polyvinyl alcohol. In some embodiments, the amphiphilic polymer in the suspension is polyethylene glycol ether (PEG). In some embodiments, the amphiphilic polymer in the suspension is a polyether. In some embodiments, the amphiphilic polymer in the suspension is polypropylene glycol (PPG). In some embodiments, the amphiphilic polymer in the suspension is polypropylene oxide.

[0010] In some embodiments, the suspension does not contain an organic solvent. In some embodiments, the amphiphilic polymer in the suspension is PEG. In some embodiments, the suspension does not contain an organic solvent and the mRNA is precipitated using polyethylene glycol (PEG). In some embodiments, the suspension contains PEG for precipitating the mRNA. In some embodiments, the suspension contains PEG at a concentration of about 10 weight / volume% to about 100 weight / volume%.

[0011] In some embodiments, the suspension contains PEG at a concentration of about 50 weight / volume%.

[0012] In some embodiments, the suspension contains a final concentration of PEG of less than 25 wt / vol%. In some embodiments, the suspension contains a final concentration of PEG of about 5 wt / vol% to 20 wt / vol%. In certain embodiments, the suspension contains a final concentration of PEG of about 10 wt / vol% to 15 wt / vol%, for example 12 wt / vol%. In some embodiments, the molecular weight of PEG is from about 2000 to about 10000 g / mol. In some embodiments, the molecular weight of PEG is from about 4000 to about 8000 g / mol. In some embodiments, the molecular weight of PEG is about 6000 g / mol (e.g., PEG-6000). As shown in the examples, a final concentration of PEG (e.g., PEG-6000) having a molecular weight of about 6000 g / mol in a suspension of about 12 wt / vol% ensures effective purification and non- always provides a pure mRNA sample.

[0013] In some embodiments, the suspension does not contain an organic solvent and contains triethylene glycol (TEG). In some embodiments, the suspension contains TEG to precipitate mRNA. In some embodiments, the suspension contains TEG at a concentration of about 10 wt / vol% to about 100 wt / vol%.

[0014] In some embodiments, the suspension contains TEG at a concentration of about 50 wt / vol%.

[0015] In some embodiments, the suspension does not contain an organic solvent and contains triethylene glycol monomethyl ether (MTEG). In some embodiments, the suspension contains MTEG to precipitate mRNA. In some embodiments, the suspension contains MTEG at a concentration of about 10 wt / vol% to about 100 wt / vol%.

[0016] In some embodiments, the suspension contains MTEG at a concentration of about 50 wt / vol%.

[0017] In some embodiments, the suspension contains MTEG at a final concentration of about 15 wt / vol% to about 45 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 20 wt / vol% to about 40 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 20 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 25 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 30 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 35 wt / vol%.

[0018] In some embodiments, the high molar salt solution contains guanidinium thiocyanate (GSCN). In some embodiments, GSCN is at a final concentration of about 2 - 4 M. In some embodiments, GSCN is at a final concentration of 2.5 - 3 M. In certain embodiments, GSCN is at a final concentration of about 2.7 M.

[0019] In some embodiments, the amphiphilic polymer in the washing solution is selected from Pluronic, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), polyethers such as polypropylene glycol (PPG) or polypropylene oxide, or combinations thereof. In some embodiments, the amphiphilic polymer in the washing solution is Pluronic. In some embodiments, the amphiphilic polymer in the washing solution is polyvinylpyrrolidone. In some embodiments, the amphiphilic polymer in the washing solution is polyvinyl alcohol. In some embodiments, the amphiphilic polymer in the washing solution is polyethylene glycol ether (PEG). In some embodiments, the amphiphilic polymer in the washing solution is a polyether. In some embodiments, the amphiphilic polymer in the washing solution is polypropylene glycol (PPG). In some embodiments, the amphiphilic polymer in the washing solution is polypropylene oxide.

[0020] In some embodiments, the cleaning solution does not contain an organic solvent. In some embodiments, the cleaning solution does not contain an organic solvent and contains polyethylene glycol (PEG). In some embodiments, the PEG used in the cleaning solution has a viscosity of 90 centistrokes or less. In some embodiments, the PEG used in the cleaning solution has a viscosity of 80 centistrokes or less. In some embodiments, the PEG used in the cleaning solution has a viscosity of 70 centistrokes or less. In some embodiments, the PEG used in the cleaning solution has a viscosity of 60 centistrokes or less. In some embodiments, the PEG used in the cleaning solution has a viscosity of 50 centistrokes or less In some embodiments, the PEG used in the cleaning solution has a viscosity of 40 centistrokes or less. In some embodiments, the PEG used in the cleaning solution has a viscosity of 30 centistrokes or less. In some embodiments, the PEG used in the cleaning solution has a viscosity of 20 centistrokes or less. In some embodiments, the PEG used in the cleaning solution has a viscosity of 10 centistrokes or less. A PEG suitable for use in the cleaning solution has a viscosity of about 90 centistrokes at 25 °C, such as PEG-400. Thus, in certain embodiments, the PEG used in the cleaning solution is PEG-400.

[0021] In some embodiments, the cleaning solution does not contain an organic solvent and contains triethylene glycol (TEG). In some embodiments, the solvent contains THG.

[0022] In some embodiments, the cleaning solution does not contain an organic solvent and contains triethylene glycol monomethyl ether (MTEG). In some embodiments, the solvent contains MTEG. In some embodiments, MTEG is present in the cleaning solution at a concentration of about 90 wt / vol% to about 100 wt / vol%. In certain embodiments, MTEG is present in the cleaning solution at a concentration of about 95 wt / vol%.

[0023] As shown in the examples, MTEG is suitable for use in washing solutions and effectively washes the precipitated mRNA while keeping it in the precipitated form. MTEG has a viscosity of about 7 centistokes at room temperature. Thus, MTEG enables very efficient purification and recovery of mRNA regardless of the purification process used (e.g., flow filtration, depth filtration, or centrifugation).

[0024] In some embodiments, the amphiphilic polymer in the washing solution is PEG. In certain embodiments, the molecular weight of PEG in the washing solution is from about 200 g / mol to about 600 g / mol. In certain embodiments, the PEG in the washing solution has a molecular weight of about 400 g / mol (e.g., PEG-400).

[0025] In some embodiments, PEG is present in the washing solution at a concentration of from about 10 weight / volume % to about 100 weight / volume %.

[0026] In some embodiments, PEG is present in the washing solution at a concentration of from about 50 to about 90 weight / volume %. In some embodiments, PEG is present in the washing solution at a concentration of from about 90 weight / volume % to about 100 weight / volume %.

[0027] In some embodiments, PEG is present in the washing solution at a concentration of about 90 weight / volume %. In certain embodiments, the PEG in the washing solution has a molecular weight of about 400 g / mol (e.g., PEG-400). In some embodiments, the PEG in the washing solution has a molecular weight of about 400 g / mol (e.g., PEG-400) and a concentration of from about 90 weight / volume % to about 100 weight / volume %. In certain embodiments, the PEG in the washing solution has a molecular weight of about 400 g / mol (e.g., PEG-400) and is present in the washing solution at a concentration of about 90 weight / volume %.

[0028] In some embodiments, the molecular weight of PEG in the suspension is from about 200 to about 40,000 g / mol. In some embodiments, the molecular weight of PEG in the washing solution is from about 200 to about 40,000 g / mol. In some embodiments, the molecular weight of PEG in both the suspension and the washing solution is from about 200 to about 40,000 g / mol. In certain embodiments, the molecular weight of PEG in the suspension is from about 2,000 g / mol to about 10,000 g / mol, and the molecular weight of PEG in the washing solution is from about 200 g / mol to about 600 g / mol. In certain embodiments, the PEG in the suspension is PEG-6000, and the PEG in the washing solution is PEG-400.

[0029] In some embodiments, the PEG in the suspension is linear. In some embodiments, the PEG in the suspension is branched. In some embodiments, the PEG in the suspension is Y-shaped. In some embodiments, the PEG in the suspension is multi-arm shaped.

[0030] In some embodiments, the PEG in the washing solution is linear. In some embodiments, the PEG in the washing solution is branched. In some embodiments, the PEG in the washing solution is Y-shaped. In some embodiments, the PEG in the washing solution is multi-arm shaped.

[0031] In some embodiments, the PEG in both the suspension and the washing solution is linear. In some embodiments, the PEG in both the suspension and the washing solution is branched. In some embodiments, the PEG in both the suspension and the washing solution is Y-shaped. In some embodiments, the PEG in both the suspension and the washing solution is multi-arm shaped.

[0032] In some embodiments, the suspension comprises a PEG selected from triethylene glycol, tetraethylene glycol, PEG200, PEG300, PEG400, PEG600, PEG1,000, PEG1,500, PEG2,000, PEG3,000, PEG3,350, PEG4,000, PEG6,000, PEG8,000, PEG10,000, PEG20,000, PEG35,000, and PEG40,000. In some embodiments, the suspension comprises triethylene glycol. In some embodiments, the suspension comprises tetraethylene glycol. In some embodiments, the suspension comprises PEG200. In some embodiments, the suspension comprises PEG300. In some embodiments, the suspension comprises PEG400. In some embodiments, the suspension comprises PEG600. In some embodiments, the suspension comprises PEG1,000. In some embodiments, the suspension comprises PEG1,500. In some embodiments, the suspension comprises PEG2,000. In some embodiments, the suspension comprises PEG3,000. In some embodiments, the suspension comprises PEG3,350. In some embodiments, the suspension comprises PEG4,000. In some embodiments, the suspension comprises PEG6,000. In some embodiments, the suspension comprises PEG8,000. In some embodiments, the suspension comprises PEG10,000. In some embodiments, the suspension comprises PEG20,000. In some embodiments, the suspension comprises PEG35,000. In some embodiments, the suspension comprises PEG40,000.

[0033] In some embodiments, the suspension comprises PEG6,000.

[0034] In some embodiments, the suspension does not comprise PEG6,000.

[0035] In some embodiments, the suspension is PEG400.

[0036] In some embodiments, the suspension is PEG150.

[0037] In some embodiments, the suspension comprises a mixture of one or more PEG polymers.

[0038] In some embodiments, the mixture of PEG polymers in the suspension comprises polymers having distinct molecular weights.

[0039] In some embodiments, the mixture of PEG polymers in the suspension comprises polymers having distinct geometric shapes.

[0040] In some embodiments, the suspension is aqueous.

[0041] In some embodiments, the suspension has volatile organic compounds that constitute less than about 50% of the total volume of the suspension. For example, in some embodiments, the suspension has volatile organic compounds that constitute less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1%, 0.5%, 0.01% of the total volume of the suspension. Thus, in some embodiments, the suspension has volatile organic compounds that constitute less than about 50% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 45% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 40% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 35% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 30% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 25% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 20% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 15% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 10% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 5% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 2% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 1% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 0.5% of the total volume of the suspension. In some embodiments, the suspension has volatile organic compounds that constitute less than 0.1% of the total volume of the suspension. Many volatile organic compounds are known in the art and include, for example, ethanol, isopropyl alcohol, and benzyl alcohol.

[0042] In some embodiments, the suspension is free of volatile organic compounds.

[0043] In some embodiments, the suspension does not contain alcohol.

[0044] In some embodiments, the suspension does not contain ethanol. In some embodiments, the suspension does not contain isopropyl alcohol. In some embodiments, the suspension does not contain benzyl alcohol.

[0045] In some embodiments, the suspension contains a non-aqueous component. In some embodiments, the non-aqueous component of the suspension is ethanol. In some embodiments, the non-aqueous component of the suspension is isopropyl alcohol. In some embodiments, the non-aqueous component of the suspension is benzyl alcohol.

[0046] In some embodiments, the cleaning solution contains a PEG selected from triethylene glycol, tetraethylene glycol, PEG200, PEG300, PEG400, PEG600, PEG1,000, PEG1,500, PEG2,000, PEG3,000, PEG3,350, PEG4,000, PEG6,000, PEG8,000, PEG10,000, PEG20,000, PEG35,000, and PEG40,000. In some embodiments, the cleaning solution contains triethylene glycol. In some embodiments, the cleaning solution contains tetraethylene glycol. In some embodiments, the wash The cleaning solution contains PEG200. In some embodiments, the cleaning solution contains PEG300. In some embodiments, the cleaning solution contains PEG400. In some embodiments, the cleaning solution contains PEG600. In some embodiments, the cleaning solution contains PEG1,000. In some embodiments, the cleaning solution contains PEG1,500. In some embodiments, the cleaning solution contains PEG2,000. In some embodiments, the cleaning solution contains PEG3,000. In some embodiments, the cleaning solution contains PEG3,350. In some embodiments, the cleaning solution contains PEG4,000. In some embodiments, the cleaning solution contains PEG6,000. In some embodiments, the cleaning solution contains PEG8,000. In some embodiments, the cleaning solution contains PEG10,000. In some embodiments, the cleaning solution contains PEG20,000. In some embodiments, the cleaning solution contains PEG35,000. In some embodiments, the cleaning solution contains PEG40,000.

[0047] In some embodiments, the cleaning solution contains PEG6,000.

[0048] In some embodiments, the cleaning solution does not contain PEG6,000.

[0049] In some embodiments, the cleaning solution is PEG400.

[0050] In some embodiments, the cleaning solution contains a mixture of one or more PEG polymers.

[0051] In some embodiments, the mixture of PEG polymers in the cleaning solution contains polymers having distinct molecular weights.

[0052] In some embodiments, the mixture of PEG polymers in the cleaning solution contains polymers having distinct geometric shapes.

[0053] In some embodiments, the cleaning solution is aqueous.

[0054] In some embodiments, the cleaning solution does not contain volatile organic compounds.

[0055] In some embodiments, the cleaning solution does not contain alcohol.

[0056] In some embodiments, the cleaning solution has volatile organic compounds that make up less than about 50% of the total volume of the cleaning solution. For example, in some embodiments, the cleaning solution has volatile organic compounds that make up less than about 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 2%, 1%, 0.5%, 0.01% of the total volume of the cleaning solution. Thus, in some embodiments, the cleaning solution has volatile organic compounds that make up less than about 50% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 45% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 40% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 35% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 30% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 25% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 20% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 15% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 10% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that make up less than 5% of the total volume of the cleaning solution. In some embodiments, the cleaning sol The liquid has volatile organic compounds that constitute less than 2% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that constitute less than 1% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that constitute less than 0.5% of the total volume of the cleaning solution. In some embodiments, the cleaning solution has volatile organic compounds that constitute less than 0.1% of the total volume of the cleaning solution. Many volatile organic compounds are known in the art and include, for example, ethanol, isopropyl alcohol, and benzyl alcohol.

[0057] In some embodiments, the cleaning solution does not contain ethanol. In some embodiments, the cleaning solution does not contain isopropyl alcohol. In some embodiments, the cleaning solution does not contain benzyl alcohol.

[0058] In some embodiments, the cleaning solution contains a non-aqueous component. In some embodiments, the non-aqueous component of the cleaning solution is ethanol. In some embodiments, the non-aqueous component of the cleaning solution is isopropyl alcohol. In some embodiments, the non-aqueous component of the cleaning solution is benzyl alcohol.

[0059] In some embodiments, both the suspension and the wash buffer are aqueous. In some embodiments, both the suspension and the wash buffer contain PEG. In some embodiments, both the suspension and the wash buffer are aqueous and contain the same PEG. In some embodiments, both the suspension and the wash buffer are aqueous, the suspension contains a first PEG, and the wash buffer contains a second PEG different from the first PEG. In some embodiments, the molecular weight of the PEG in the suspension is from about 2000 to about 10000 g / mol, and the molecular weight of the PEG in the wash buffer is from about 200 to 600 g / mol. In some embodiments, the molecular weight of the PEG in the suspension is from about 4000 to about 8000 g / mol, and the molecular weight of the PEG in the wash buffer is from about 300 to 500 g / mol. In some embodiments, the molecular weight of the PEG in the suspension is about 6000 g / mol (e.g., PEG-6000), and the molecular weight of the PEG in the wash buffer is about 400 g / mol (e.g., PEG-400).

[0060] In some embodiments, the capture of the precipitated mRNA occurs on a filter. In some embodiments, the filter is selected from a microfiltration filter or an ultrafiltration filter. In some embodiments, the microfiltration filter has a pore size of 0.05 μm to 1.0 μm. For example, in some embodiments, the microfiltration filter has a pore size of 0.05 μm, 0.10 μm, 0.20 μm, 0.3 μm, 0.4 μm, 0.5 μm, 0.6 μm, 0.7 μm, 0.8 μm, 0.9 μm, or 1.0 μm. Thus, in some embodiments, the microfiltration filter has a pore size of 0.05 μm. In some embodiments, the microfiltration filter has a pore size of 0.10 μm. In some embodiments, the microfiltration filter has a pore size of 0.20 μm. In some embodiments, the microfiltration filter has a pore size of 0.30 μm. In some embodiments, the microfiltration filter has a pore size of 0.40 μm. In some embodiments, the microfiltration filter has a pore size of 0.50 μm. In some embodiments, the microfiltration filter has a pore size of 0.60 μm. In some embodiments, the microfiltration filter has a pore size of 0.70 μm. In some embodiments, the microfiltration filter has a pore size of 0.80 μm. In some embodiments, the microfiltration filter has a pore size of 0.90 μm. In some embodiments, the microfiltration filter has a pore size of 1.0 μm.

[0061] In some embodiments, the filter will have a nominal molecular weight limit (NMKL) of 100 kDa to 1,000 kDa In some embodiments, the filter will have an NMWL of 200 kDa to 700 kDa. In some embodiments, the filter will have an NMWL of 200 kDa to 500 kDa. In some embodiments, the filter has an NMWL of 300 kDa. In some embodiments, the filter has an NMWL of 500 kDa.

[0062] In some embodiments, the microfiltration filter has a nominal molecular weight limit (NMKL) greater than 1,000 kilodaltons (kDa). In some embodiments, the ultrafiltration filter has a pore size less than 0.05 μm. In some embodiments, the ultrafiltration filter has a nominal molecular weight limit (NMWL) of about 1 kDa to 1,000 kDa. For example, in some embodiments, the ultrafiltration filter has an NMWL of 1 kDa, 5 kDa, 10 kDa, 15 kDa, 20 kDa, 25 kDa, 50 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, 500 kDa, 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, or 1,000 kDa. Thus, in some embodiments, the ultrafiltration filter has an NMWL of 1 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 5 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 10 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 15 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 20 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 25 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 50 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 100 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 150 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 200 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 250 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 300 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 350 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 400 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 450 kDa. In some embodiments, the ultrafiltration filter has an NMWL of 500 kDa.In some embodiments, the ultrafiltration filter has a NMWL of 550 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 600 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 650 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 700 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 750 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 800 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 850 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 900 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 950 kDa. In some embodiments, the ultrafiltration filter has a NMWL of 1,000 kDa.

[0063] In some embodiments, tangential flow filtration (TFF) or diafiltration is used to purify the mRNA precipitated in step c). Thus, in some embodiments, TFF is used to purify the mRNA precipitated in step c). In some embodiments, diafiltration is used to purify the mRNA precipitated in step c).

[0064] In some embodiments, a filter aid is used.

[0065] In some embodiments, the filter aid is cellulose-based. In certain embodiments, the cell The low ash filter aid is added to the suspension at a mass ratio of precipitated mRNA to filter aid of 1:10. In some embodiments, the cellulose-based filter aid comprises purified cellulose fibers having a length of about 5 to about 500 μm. In some embodiments, the length of the cellulose fibers is about 10 to about 100 μm. In some embodiments, the cellulose fibers are about 20 μm, 30 μm, 40 μm, or 50 μm in length. In certain embodiments, the cellulose-based filter aid comprises purified cellulose fibers having a length of about 20 μm (e.g., Solka-Floc® or Sigmacell Cellulose 20).

[0066] In some embodiments, the filter aid comprises diatomaceous earth and / or volcanic ash. In some embodiments, the filter aid comprises diatomaceous earth. In some embodiments, the filter aid comprises volcanic ash. In some embodiments, the filter aid comprises diatomaceous earth and volcanic ash.

[0067] In some embodiments, the solubilization solution is selected from water, Tris-EDTA (TE), sodium citrate, or combinations thereof. In some embodiments, the solubilization solution is water. In some embodiments, the solubilization solution is TE. In some embodiments, the solubilization solution is sodium citrate.

[0068] In some embodiments, the yield of purified mRNA is about 50% to about 100%.

[0069] In some embodiments, the yield of purified mRNA is about 70% to about 99%.

[0070] In some embodiments, the yield of purified mRNA is about 90 to about 99%. In certain embodiments, the yield of purified mRNA is greater than about 93%, e.g., greater than about 94%, particularly greater than about 95%.

[0071] In some embodiments, the purity of the purified mRNA is about 60% to about 100%.

[0072] In some embodiments, the purity of the purified mRNA is about 80% to 99%.

[0073] In some embodiments, the purity of the purified mRNA is about 90% to about 99%.

[0074] In some embodiments, the method does not include a chromatography step.

[0075] In some embodiments, the precipitated mRNA is centrifuged to obtain an mRNA pellet.

[0076] In some embodiments, the mRNA pellet is resuspended in a buffer.

[0077] In some embodiments, the buffer is selected from water, TE, sodium citrate, or combinations thereof. In some embodiments, the buffer is water. In some embodiments, the buffer is TE. In some embodiments, the buffer is sodium citrate.

[0078] In some embodiments, the precipitated mRNA comprises at least 100 mg, 1 g, 10 g, 100 g, 1 kg, 10 kg, 100 kg, 1 metric ton, or 10 metric tons of mRNA, or any amount therebetween. Thus, in some embodiments, the precipitated mRNA comprises at least 100 mg. In some embodiments, the precipitated mRNA comprises at least 1 g. In some embodiments, the precipitated mRNA comprises at least 10 g. In some embodiments, the precipitated mRNA comprises at least 1 It contains 00 g. In some embodiments, the precipitated mRNA contains at least 1 kg. In some embodiments, the precipitated mRNA contains at least 10 kg. In some embodiments, the precipitated mRNA contains at least 100 kg. In some embodiments, the precipitated mRNA contains at least 1 metric ton. In some embodiments, the precipitated mRNA contains at least 10 metric tons.

[0079] In some embodiments, the precipitated mRNA contains more than 1 kg of mRNA.

[0080] In some embodiments, the method does not contain ethanol.

[0081] In some aspects, the present invention provides a method for purifying messenger RNA (mRNA), comprising: a) precipitating mRNA in a guanidinium thiocyanate (GSCN) solution containing PEG; b) centrifuging the solution of step a) to produce an mRNA pellet; c) resuspending the mRNA pellet in a buffer; d) capturing the mRNA on a filter; e) washing the mRNA of step d) with a PEG composition; and f) solubilizing the washed mRNA to obtain an mRNA composition substantially free of contaminants.

[0082] In some embodiments, the invention is a method of producing mRNA, comprising: (a) performing in vitro transcription (IVT) by mixing (i) a DNA template comprising a promoter and (ii) an RNA polymerase to generate an impure preparation comprising full-length mRNA; (b) providing a high molar salt and an amphiphilic polymer to a suspension to precipitate the full-length mRNA and provide the full-length mRNA precipitated in the suspension; (c) capturing the precipitated full-length mRNA by applying the suspension to a filter; (d) washing the precipitated full-length mRNA of step (c) with an aqueous solvent to obtain a purified full-length mRNA in an aqueous solution; and (e) solubilizing the precipitated mRNA from step (d) to obtain a purified mRNA composition, wherein the purified full-length mRNA in the aqueous solution obtained from step (d) is substantially free of (i) the DNA template comprising the promoter and (ii) the RNA polymerase.

[0083] In some embodiments, in step (a), the RNA polymerase is SP6 polymerase.

[0084] In some embodiments, the purified full-length mRNA in the aqueous solution obtained from step (e) is also substantially free of (v) double-stranded RNA (dsRNA).

[0085] In some embodiments, the suspension comprises PEG (e.g., PEG-6000) having a molecular weight of about 6000 g / mol at a final concentration of about 5 wt / vol% to 20 wt / vol%, and GSCN at a final concentration of about 2 to 4 M. In certain embodiments, the suspension comprises PEG (e.g., PEG-6000) having a molecular weight of about 6000 g / mol at a final concentration of about 10 wt / vol%, 11 wt / vol%, 12 wt / vol%, 13 wt / vol%, 14 wt / vol%, or 15 wt / vol%, and GSCN at a final concentration of about 2.5 to 3 M. As shown in the following examples, polymer-induced precipitation with a final concentration of less than 20% PEG in the suspension resulted in a high-purity mRNA sample after purification. Furthermore, a final concentration of about 12% PEG (e.g., a ratio of 1 of 50% PEG-6000) and a final GSCN concentration of 2.7 M in the suspension achieved very effective purification of the mRNA.

[0086] In some embodiments, MTEG can be used in place of PEG to provide a suspension of precipitated mRNA. In certain embodiments, MTEG is used for this purpose at a final concentration of about 15 wt / vol% to about 45 wt / vol%. In some embodiments, The suspension contains MTEG at a final concentration of about 20 wt / vol% to about 40 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 20 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 25 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 30 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of about 35 wt / vol%. In some embodiments, the suspension contains MTEG at a final concentration of less than 35 wt / vol%. The remaining conditions used for MTEG-induced precipitation are the same as those used for PEG-induced precipitation. As shown in the examples, suspensions containing mRNA, GSCN, and MTEG with a final concentration of MTEG less than 35 wt / vol% ensured efficient recovery of mRNA without undesirable precipitation of process enzymes. Particularly suitable for efficient recovery of mRNA without undesirable precipitation of process enzymes is a suspension containing mRNA, GSCN, and MTEG with MTEG at a final concentration of about 25%, in addition to a filter aid (e.g., a cellulose-based filter aid) at a mass ratio to the precipitated mRNA of about 10:1.

[0087] The following drawings are for illustrative purposes only and are not limiting.

Brief Description of the Drawings

[0088]

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DETAILED DESCRIPTION OF THE INVENTION

[0089] Definitions For the present invention to be more readily understood, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0090] Terms such as "above", "at least", "greater than", etc., for example, "at least one" means at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more are included, but it is understood that the scope is not limited to these. Also included are any larger numbers or fractions in between.

[0091] Conversely, the term "less than" includes each value that is less than the specified value. For example, "100 nucleotides or less" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Also included are any smaller numbers or fractions in between.

[0092] The term "plurality" includes, but is not limited to, "at least two", "two or more", "at least a second", etc., at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more, and any greater number or fraction therebetween is also included.

[0093] Approximately or about: As used herein, the term "approximately" or "about" applied to one or more values of interest refers to a value similar to the reference value presented. In certain embodiments, the term "approximately" or "about" refers to within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the recited value. Unless otherwise clear from the context, all numerical values provided herein are modified by the term "approximately" or "about".

[0094] Batch: As used herein, the term "batch" refers to the quantity or amount of mRNA that is purified at one time, e.g., purified according to a single manufacturing sequence during the same manufacturing cycle. A batch can refer to the amount of mRNA purified in a single reaction.

[0095] Biologically active: As used herein, the phrase "biologically active" refers to the characteristic of any agent that is active in a biological system, particularly in a living organism. For example, an agent that has a biological effect on a living organism when administered to that organism is considered to be biologically active.

[0096] Comprising: As used herein, the term "comprising", or variations such as "comprise" or "comprises", is understood to imply the inclusion of the stated element, integer or step, or group of elements, integers or steps, but not the exclusion of other elements, integers or steps, or group of elements, integers or steps.

[0097] dsRNA: As used herein, the term "dsRNA" refers to the production of complementary RNA sequences during an in vitro transcription (IVT) reaction. Complementary RNA sequences can be made for various reasons, including, for example, short truncated transcripts that can hybridize to the complementary sequence of a nascent RNA strand, short truncated transcripts that act as primers for RNA-dependent DNA-dependent RNA transcription, and the potential for RNA polymerase template reversal.

[0098] Expression: As used herein, "expression" of a nucleic acid sequence refers to the translation of the mRNA into a polypeptide (e.g., the heavy or light chain of an antibody), the assembly of multiple polypeptides (e.g., the heavy or light chain of an antibody) into an intact protein (e.g., an antibody), and / or the post-translational modification of the polypeptide or fully assembled protein (e.g., an antibody). In this application, the terms "expression" and "production", as well as grammatical synonyms, are used interchangeably. Peptide or fully assembled protein (e.g., an antibody).

[0099] Functional: As used herein, a "functional" biomolecule is a biomolecule in a form that exhibits the properties and / or activities by which it is characterized.

[0100] Improve, increase, or decrease: As used herein, "improve", "increase", or "decrease", or grammatical synonyms thereof, refer to a value as compared to a baseline measurement, e.g., a measurement in the same individual prior to the initiation of treatment described herein, or a measurement in a control subject (or control subjects) in the absence of the treatment described herein. A "control subject" is a subject that has the same disease form as the treated subject and is approximately the same age as the treated subject.

[0101] Impurity: As used herein, the term "impurity" refers to a substance in a limited amount in a liquid, gas, or solid that is different from the chemical composition of the target substance or compound. Impurities are also referred to as "contaminants".

[0102] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, under cell culture, etc., rather than within a multicellular organism.

[0103] In vivo: As used herein, the term "in vivo" refers to events that occur within multicellular organisms such as humans and non-human animals. In the context of cell-based systems, this term can be used to refer to events that occur within living cells (e.g., as contrasted with in vitro systems).

[0104] Isolated: As used herein, the term "isolated" means (1) separated from at least some of the components with which it was associated when first produced (regardless of whether in nature and / or an experimental environment), and / or (2) refers to a substance and / or entity that has been artificially produced, prepared, and / or manufactured. Isolated substances and / or entities can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, the isolated agent is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" when it substantially does not contain other components. As used herein, the calculation of the percent purity of an isolated substance and / or entity should not include excipients (e.g., buffers, solvents, water, etc.).

[0105] Messenger RNA (mRNA): As used herein, the term "messenger RNA (mRNA)" refers to a polynucleotide that encodes at least one polypeptide. The mRNA used herein encompasses both modified RNA and unmodified RNA. The mRNA can include one or more coding regions and non-coding regions.

[0106] Integrity of mRNA: As used herein, the term "integrity of mRNA" generally refers to the quality of mRNA. In some embodiments, the integrity of mRNA refers to the proportion of mRNA that is not degraded after the purification process. The integrity of mRNA can be determined by methods well known in the art , for example, using RNA agarose gel electrophoresis (e.g., Ausubel et al., John Weley & Sons, Inc., 1997, Current Protocols in Molecular Biology).

[0107] Nucleic acid: As used herein, the term "nucleic acid" in its broadest sense refers to any compound and / or substance that can be incorporated into or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and / or substance that can be incorporated into or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain containing individual nucleic acid residues. In some embodiments, "nucleic acid" includes RNA, as well as single-stranded and / or double-stranded DNA and / or cDNA. Further, the terms "nucleic acid", "DNA", "RNA", and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, so-called "peptide nucleic acids", which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered to be within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and / or encode the same amino acid sequence. Nucleotide sequences encoding proteins and / or RNAs may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems, optionally purified, and chemically synthesized, etc. Optionally, for example, in the case of chemically synthesized molecules, nucleic acids can include nucleoside analogs such as chemically modified bases or sugars, analogs having backbone modifications, etc. Nucleic acids are presented in the 5' to 3' direction unless otherwise indicated.In some embodiments, the nucleic acid is a natural nucleoside (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine), a nucleoside analogue (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), a chemically modified base, a biologically modified base (e.g., a methylated base), an intercalated base, a modified sugar (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose), and / or a modified phosphate group (e.g., phosphorothioate and 5'-N-phosphoramidite linkages), or comprises the same. In some embodiments, the invention is specifically directed to "unmodified nucleic acids," which means nucleic acids (e.g., polynucleotides and residues containing nucleotides and / or nucleosides) that are not chemically modified to facilitate or achieve delivery. In some embodiments, the invention is specifically directed to "unmodified nucleic acids," which means nucleic acids (e.g., polynucleotides and residues containing nucleotides and / or nucleosides) that are not chemically modified to facilitate or achieve delivery.

[0108] Precipitate: As used herein, the term "precipitation" (or any grammatically equivalent thereof) refers to the formation of a solid in solution. When used in connection with mRNA, the term "precipitation" refers to the formation of an insoluble or solid form of mRNA in a liquid.

[0109] Prematurely aborted RNA sequences: As used herein, the terms "prematurely aborted RNA sequence," "short abortive RNA species," "shortmer," and "long abortive RNA species" refer to incomplete products of an mRNA synthesis reaction (e.g., an in vitro synthesis reaction). For various reasons, RNA polymerase does not always complete the transcription of a DNA template. For example, RNA synthesis is prematurely terminated. Possible causes of premature termination of RNA synthesis include the quality of the DNA template, polymerase transcription termination sequences specific to the polymerase present in the template, lysis buffer, temperature, ribonucleotide depletion, and mRNA secondary structure. Prematurely aborted RNA sequences can be of any length shorter than the intended length of the desired transcript. For example, a prematurely aborted mRNA sequence may be less than 1000 bases, less than 500 bases, less than 100 bases, less than 50 bases, less than 40 bases, less than 30 bases, less than 20 bases, less than 15 bases, or less than 10 bases. Salts: As used herein, the term "salt" means an ionic compound that results from, or can result from, a neutralization reaction between an acid and a base.

[0110] Substantially: As used herein, the term "substantially" refers to a qualitative state that exhibits all or almost all of the scope or degree of a desired feature or characteristic. One of ordinary skill in the biological arts understands that biological and chemical phenomena rarely, if ever, achieve completion and / or come to completion, or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the inherent lack of perfection that is characteristic of many biological and chemical phenomena.

[0111]

[0112] ​Substantially free of: As used herein, the term "substantially free of" refers to a state where the amount of the substance to be removed (e.g., premature termination RNA sequence) is relatively small or absent. For example, "substantially free of premature termination RNA sequence" means that the premature termination RNA sequence is present at levels less than approximately 5%, 4%, 3%, 2%, 1.0%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% or less (w / w) of the impurities. Alternatively, "substantially free of premature termination RNA sequence" means that the premature termination RNA sequence is present at levels less than approximately 100 ng, 90 ng, 80 ng, 70 ng, 60 ng, 50 ng, 40 ng, 30 ng, 20 ng, 10 ng, 1 ng, 500 pg, 100 pg, 50 pg, 10 pg or less.

[0113] All technical and scientific terms used herein, unless otherwise defined, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs and are commonly used in the technical field to which this application belongs, and such techniques are incorporated by reference in their entirety. In case of conflict, the present specification including the definitions will prevail.

[0114] The present invention particularly provides an improved method for purifying mRNA without using alcohol in the purification process.

[0115] Various aspects of the present invention are described in detail in the following sections. The use of the sections is not meant to limit the present invention. Each section can be applied to any aspect of the present invention. In this application, the use of "or" means "and / or" unless otherwise stated.

[0116] Purification method The use of caustic or flammable solvents in mRNA purification can present safety and cost challenges, especially in large-scale preparations. The present invention relates to a method for purifying mRNA without using caustic or flammable solvents. The methods provided herein enable efficient capture, washing, and high-yield isolation of mRNA produced on a scale sufficient to meet most clinical and commercial needs. Accordingly, the present disclosure provides a path for mRNA replacement therapies, enabling them to be a viable and successful alternative to more traditional enzyme replacement therapies and biological therapies currently available.

[0117] To be a viable and successful alternative, a method for mRNA purification needs to be confirmed as being safe, cost-effective, robust, scalable, and equipped with large-scale manufacturing capabilities to meet all clinical and commercial needs. A suitable mRNA purification method is safe, cost-effective, and easily scalable while also providing an equivalent or better product compared to currently available industry-standard mRNA purification methods. In particular, the methods provided herein eliminate the use of caustic or flammable solvents and result in high post-purification mRNA yields, maintenance of the integrity of the purified mRNA, and removal of process-related contaminants (e.g., prematurely terminated RNA sequences (short truncated RNA species or "shortmers"), long truncated RNA species, double-stranded RNA (dsRNA), plasmid DNA, residual solvents, residual salts, and residual in vitro transcription enzymes).

[0118] The methods provided herein are applicable on a wide range of scales. For example, as further discussed herein, the provided methods enable purification on various scales such as from less than 100 mg to more than 1 kg. Further, the data provided herein indicate that the present invention is a capable (and lower cost) alternative to currently available methods that rely on the use of flammable solvents such as alcohol for mRNA purification. The mRNA purification methods provided herein are suitable for various uses, including, for example, experimental, clinical, or commercial use. Further, the present invention has the important additional benefit of scalability not available with industry standard methods and kits. Finally, the methods disclosed herein are extremely cost - efficient compared to current processes such as filtration methods that include alcohol solvents and / or chromatography. See, for example, WO2011 / 068810, WO2012 / 075040, WO2014 / 152659, WO2014 / 152673, WO2014 / 152966, WO2015 / 164773, WO2016 / 004318, US62 / 420,413, and PCT / US16 / 57044.

[0119] Accordingly, the methods described herein are advantageous for the purification of mRNA, including large amounts of mRNA (e.g., any batch size or loading volume described herein). The described purification methods can provide mRNA having a therapeutically acceptable high level of integrity and purity and minimal loss of full - length mRNA for purification.

[0120] The method of purifying mRNA described herein includes precipitating the mRNA, capturing the precipitated mRNA, and washing the captured precipitated mRNA to obtain a purified mRNA composition substantially free of contaminants. Each of these steps is described in detail in the subsequent sections.

[0121] Precipitation of mRNA A method for purifying mRNA includes a step of precipitating mRNA in a suspension containing a high molar salt solution and an amphiphilic polymer, a step of capturing the mRNA, and a step of washing the mRNA, thereby obtaining mRNA substantially free of contaminants.

[0122] The methods described herein are suitable for the purification of mRNA in a suspension containing the provided mRNA (e.g., an in vitro synthesis reaction mixture). The suspension can have various contaminants such as, for example, plasmid DNA and enzymes.

[0123] In one embodiment, a salt (e.g., a chaotropic salt such as guanidine thiocyanate (GSCN)) is added to the suspension containing mRNA to denature and solubilize contaminating proteins. Thus, in one embodiment, GSCN is in the high molar solution in the suspension. Thereafter, an amphiphilic polymer is added to selectively precipitate the mRNA. After mRNA precipitation, the resulting precipitated mRNA is captured using a filter or membrane and washed to obtain a precipitate free of contaminants such as, for example, short-chain incomplete RNA species, long-chain truncated RNA species, dsRNA, plasmid DNA, residual in vitro transcription enzyme, residual salt, and residual solvent. Subsequent dissolution of the precipitated mRNA in water yields a purified mRNA composition.

[0124] In some embodiments, one agent that promotes mRNA precipitation comprises guanidine thiocyanate (e.g., a solution comprising about 1-5 M guanidine thiocyanate). For example, the solution may comprise about 1 M, 1.5 M, 2.0 M, 2.5 M, 3.0 M, 3.5 M, 4.0 M, 4.5 M, or about 5 M GSCN. Examples of suitable GSCN buffers include, for example, an aqueous solution comprising 4 M guanidine thiocyanate, 25 mM sodium citrate pH 6.5, 0.5% sodium N-lauroyl sarcosinate. Further examples of GSCN buffers are aqueous solutions containing 5 M GSCN in 10 mM dithiothreitol (DTT) buffer. In some embodiments, the GSCN has a final concentration of 2-4 M. In some embodiments, the GSCN (e.g., 5 M GSCN-10 mM DTT buffer) has a final concentration of 2.5-3 M. In certain embodiments, the GSCN has a final concentration of about 2.7 M.

[0125] Many amphiphilic polymers are known in the art. In some embodiments, the amphiphilic polymers used in the methods herein include Pluronics, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), or combinations thereof. In some embodiments, the amphiphilic polymer is selected from one or more of the following: PEG triethylene glycol, tetraethylene glycol, PEG200, PEG300, PEG400, PEG600, PEG1,000, PEG1,500, PEG2,000, PEG3,000, PEG3,350, PEG4,000, PEG6,000, PEG8,000, PEG10,000, PEG20,000, PEG35,000, and PEG40,000, or combinations thereof. In some embodiments, the amphiphilic polymer comprises a mixture of PEG polymers of two or more molecular weights. For example, in some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 molecular weight PEG polymers constitute the amphiphilic polymer. Thus, in some embodiments, the PEG solution comprises a mixture of one or more PEG polymers. In some embodiments, the mixture of PEG polymers comprises polymers having distinct molecular weights.

[0126] In some embodiments, to precipitate mRNA in suspension, it comprises one or more amphiphilic polymers. In some embodiments, to precipitate mRNA in suspension, it comprises a PEG polymer. Various types of PEG polymers are recognized in the art, and some of them have distinct geometric shapes. PEG polymers suitable for the methods herein include, for example, PEG polymers having a linear, branched, Y-shaped, or multi-arm shape. In some embodiments, the PEG is in a suspension comprising one or more PEGs of distinct geometric shapes. In some embodiments, precipitation of mRNA can be achieved by precipitating the mRNA using PEG-6000. In some embodiments, precipitation of mRNA can be achieved by precipitating the mRNA using PEG-400. In some embodiments, precipitation of mRNA can be achieved by precipitating the mRNA using triethylene glycol (TEG). In some embodiments, precipitation of mRNA can be achieved by precipitating the mRNA using triethylene glycol monomethyl ether (MTEG). In some embodiments, precipitation of mRNA can be achieved by using tert-butyl-TEG-O-propionate to precipitate the mRNA. In some embodiments, precipitation of mRNA can be achieved by using TEG-dimethacrylate to precipitate the mRNA. In some embodiments, precipitation of mRNA can be achieved by using TEG-dimethyl ether to precipitate the mRNA. In some embodiments, precipitation of mRNA can be achieved by using TEG-divinyl ether to precipitate the mRNA In some embodiments, precipitation of mRNA can be achieved by precipitating mRNA using TEG - monobutyl ether. In some embodiments, precipitation of mRNA can be achieved by precipitating mRNA using TEG - methyl ether methacrylate. In some embodiments, precipitation of mRNA can be achieved by precipitating mRNA using TEG - monodecyl ether. In some embodiments, precipitation of mRNA can be achieved by precipitating mRNA using TEG - dibenzoate. Any one of these PEG - or TEG - based reagents can be used in combination with guanidinium thiocyanate to precipitate mRNA. The structures of each of these reagents are shown in Table A.

Table A

[0127] In some embodiments, to precipitate mRNA in a suspension, a PEG polymer is included, and the PEG polymer includes a PEG - modified lipid. In some embodiments, the PEG - modified lipid is 1,2 - dimyristoyl - sn - glycerol, methoxypolyethylene glycol (DMG - PEG - 2K). In some embodiments, the PEG - modified lipid is a DOPA - PEG conjugate. In some embodiments, the PEG - modified lipid is a poloxamer - PEG conjugate. In some embodiments, the PEG - modified lipid includes DOTAP. In some embodiments, the PEG - modified lipid includes cholesterol.

[0128] In some embodiments, the mRNA precipitates in a suspension containing an amphiphilic polymer. In some embodiments, the mRNA precipitates in a suspension containing any of the aforementioned PEG reagents. In some embodiments, the PEG is present in the suspension at a concentration of about 10 wt / vol% to about 100 wt / vol%. For example, in some embodiments, the PEG is present in the suspension at about 5 wt / vol%, 10 wt / vol%, 15 wt / vol%, 20 wt / vol%, 25 wt / vol%, 30 wt / vol%, 35 wt / vol%, 40 wt / vol%, 45 wt / vol%, 50 wt / vol%, 55 wt / vol%, 60 wt / vol%, 65 wt / vol%, 70 wt / vol%, 75 wt / vol%, 80 wt / vol%, 85 wt / vol%, 90 wt / vol%, 95 wt / vol%, 100 wt / vol% concentration, and any value therebetween. In some embodiments, the PEG is present in the suspension at a concentration of about 5 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 6 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 7 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 8 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 9 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 10 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 12 wt / vol%. In some embodiments, the PEG is present in the suspension at about 15 wt / vol%. In some embodiments, the PEG is present in the suspension at about 18 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 20 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 25 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 30 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 35 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 40 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 45 wt / vol%. In some embodiments, the PEG is present in the suspension at a concentration of about 50 wt / vol%.In some embodiments, the PEG is present in the suspension at a concentration of about 55 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 60 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 65 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 70 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 75 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 80 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 85 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 90 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 95 weight / volume %. In some embodiments, the PEG is present in the suspension at a concentration of about 100 weight / volume %.

[0129] In some embodiments, precipitating the mRNA in the suspension comprises a volume:volume ratio of PEG to the total mRNA suspension volume of about 0.1 to about 5.0. For example, in some embodiments, the PEG is present in the mRNA suspension at about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.25, 1.5, 1.75, 2.0, 2.25, 2.5, 2.75, 3.0, 3.25, 3.5, 3.75, 4.0, 4.25 , 4.5, 4.75, 5.0 are present in a volume:volume ratio. Thus, in some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.1. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.2. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.3. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.4. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.5. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.6. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.7. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.8. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 0.9. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 1.0. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 1.25. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 1.5. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 1.75. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 2.0. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 2.25. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 2.5. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 2.75. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 3.0. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 3.25. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 3.5. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 3.75. In some embodiments, PEG is present in the mRNA suspension at a volume:volume ratio of about 4.0.In some embodiments, the PEG is present in the mRNA suspension at a volume:volume ratio of about 4.25. In some embodiments, the PEG is present in the mRNA suspension at a volume:volume ratio of about 4.50. In some embodiments, the PEG is present in the mRNA suspension at a volume:volume ratio of about 4.75. In some embodiments, the PEG is present in the mRNA suspension at a volume:volume ratio of about 5.0. In certain embodiments, the PEG is present in the mRNA suspension at a volume:volume ratio of about 1.0, about 1.5, or about 2.0.

[0130] In some embodiments, the reaction volume for mRNA precipitation contains GSCN and PEG. In certain embodiments, the reaction volume for mRNA precipitation contains GSCN and PEG having a molecular weight of about 4000 to about 8000 g / mol, such as about 6000 g / mol (e.g., PEG-6000). The GSCN is typically at a final concentration of 2M to 4M. The PEG is typically at a final concentration of about 10% to about 20% (weight / volume).

[0131] In some embodiments, the method of purifying mRNA does not include alcohol.

[0132] In some embodiments, a non-aqueous solvent (e.g., alcohol) is added to precipitate the mRNA. In some embodiments, the solvent may be isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethanol, methanol, denatonium, and combinations thereof. In an embodiment, the solvent is an alcohol solvent (e.g., methanol, ethanol, or isopropanol). In an embodiment, the solvent is a ketone solvent (e.g., acetone, methyl ethyl ketone, or methyl isobutyl ketone). In some embodiments, the non-aqueous solvent is mixed with an amphiphilic solution.

[0133] In some embodiments, an aqueous solution is added to precipitate the mRNA. In some embodiments, the aqueous solution contains a polymer. In some embodiments, the aqueous solution contains a PEG polymer.

[0134] In some embodiments, the method further comprises the step of adding one or more agents (e.g., RNA polymerase and DNase I added post-transcriptionally to remove DNA template) that denature the protein and / or maintain the soluble protein in the aqueous medium. In some embodiments, the one or more agents that denature the protein and / or maintain the soluble protein in the aqueous medium are salts, such as chaotropic salts.

[0135] In some embodiments, the precipitation step comprises the use of a chaotropic salt (e.g., guanidine thiocyanate) and / or an amphiphilic polymer (e.g., polyethylene glycol or an aqueous solution of polyethylene glycol) and / or an alcohol solvent (e.g., an aqueous solution of alcohol such as absolute ethanol or aqueous ethanol solution). Thus, in some embodiments, the precipitation step comprises the use of a chaotropic salt and an amphiphilic polymer such as GSCN and PEG, respectively.

[0136] In some embodiments, the agent that promotes precipitation of mRNA comprises or results from a denaturing agent. As used herein, the term "denatured state" refers to any chemical or physical state that can cause denaturation. Exemplary denaturing conditions include, but are not limited to, the use of chemical reagents, high temperature, extreme pH, etc. In some embodiments, the denatured state is achieved by adding one or more denaturing agents to an impure preparation containing the mRNA to be purified. In some embodiments, the denaturing agent suitable for the present invention is a protein and / or DNA denaturing agent. In some embodiments, the denaturing agent can be 1) an enzyme (such as serine protease or DNase), 2) an acid, 3) a solvent, 4) a cross-linking agent, 5) a chaotropic agent, 6) a reducing agent, and / or 7) a high ionic strength via a high salt concentration. In some embodiments, a particular agent can fall into one or more of these categories.

[0137] In some embodiments, one or more enzymes may be used as denaturing agents to degrade the proteins and DNA templates used in mRNA synthesis. In some embodiments, suitable enzymes include, but are not limited to, serine proteases such as chymotrypsin and chymotrypsin-like serine proteases, trypsin and trypsin-like serine proteases, elastase and elastase-like serine proteases, subtilisin and subtilisin-like serine proteases, and combinations thereof, deoxyribonucleases (DNases) such as deoxyribonuclease I, II and / or IV, restriction enzymes such as EcoRI, EcoRII, BamHI, HindIII, SpeI, SphI, StuI, XbaI, and combinations thereof.

[0138] In some embodiments, an acid may be used as a denaturing agent. In some embodiments, suitable acids may be acetic acid, formic acid, oxalic acid, citric acid, benzoic acid, chloroacetic acid, dichloroacetic acid, trichloroacetic acid, ascorbic acid, sulfosalicylic acid, and combinations thereof.

[0139] In some embodiments, a solvent may be used as a denaturing agent. In some embodiments, the solvent does not contain caustic or flammable agents. In some embodiments, the solvent does not contain ethanol. In some embodiments, the solvent does not contain isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethanol, methanol, denatonium, and combinations thereof. In some embodiments, the solvent does not contain an alcohol solvent (e.g., methanol, ethanol, or isopropanol). In some embodiments, the solvent does not contain a ketone solvent (e.g., acetone, methyl ethyl ketone, or methyl isobutyl ketone).

[0140] In some embodiments, the solvent can be used as a denaturant. In some embodiments, the solvent can be ethanol. In some embodiments, the solvent is isopropyl alcohol, acetone, methyl ethyl ketone, methyl isobutyl ketone, ethanol, methanol, sodium dodecyl sulfate, and combinations thereof. In some embodiments, the solvent is an alcohol solvent (e.g., methanol, ethanol, or isopropanol). In some embodiments, the solvent is a ketone solvent (e.g., acetone, methyl ethyl ketone, or methyl isobutyl ketone).

[0141] In some embodiments, the chaotropic agent can be used as a denaturant. A chaotropic agent is a substance that disrupts the structure of macromolecules such as proteins and nucleic acids by interfering with non-covalent forces such as hydrogen bonds and van der Waals forces. In some embodiments, the chaotropic agent may be urea, thiourea, guanidinium chloride, guanidinium thiocyanate, guanidinium isothiocyanate, lithium acetate, magnesium chloride, sodium dodecyl sulfate, lithium perchlorate, and combinations thereof.

[0142] In some embodiments, the reducing agent can be used as a denaturant. A reducing agent is a compound that provides electrons to another species and is itself oxidized. In some embodiments, the reducing agent may be lithium aluminum hydride, sodium amalgam, diborane, sodium borohydride, sulfite, diisobutylaluminum hydride, phosphite ester, carbon monoxide, 2-mercaptoethanol, dithiothreitol, or tris(2-carboxyethyl)phosphine, and combinations thereof.

[0143] In some embodiments, one or more of pH, heat, and / or heavy metals (such as lead, mercury, or cadmium) can also be used as denaturants to provide a denatured state. Extreme pH is known to denature proteins. Although the backbone of the protein chain is neutral, the amino acid residues that make up the protein often contain acidic and basic groups. These groups are usually charged and can form salt bridges with groups of opposite charge. Thus, extreme pH can change the charges of these acidic and basic groups and break the salt bridges.

[0144] In some embodiments, less drastic changes in pH can also affect the activity and solubility of proteins. Like individual amino acids, proteins have an isoelectric point at which the number of negative charges equals the number of positive charges. This is often the point of minimum water solubility. At the isoelectric pH, there is no net charge on the molecule. Individual molecules tend to approach each other, coagulate, and precipitate from the solution. At pH values above or below the isoelectric point, the molecules have a net negative or positive charge, respectively. Thus, when protein molecules approach each other, they have the same overall charge and repel each other.

[0145] In some embodiments, heat can be used as a denaturant. Heat can supply kinetic energy to protein molecules and cause their atoms to vibrate more rapidly. In some embodiments, this breaks relatively weak forces such as hydrogen bonds and hydrophobic interactions. Heat is also used for sterilization because it denatures and destroys enzymes in bacteria.

[0146] In some embodiments, salts of metal ions such as mercury(II), lead(II), and silver can be used as denaturants because of their ability to form strong bonds with disulfide groups and the carboxylate ions of acidic amino acids. Thus, both disulfide bridges and salt bonds are broken, and the protein precipitates from the solution as an insoluble metal protein salt.

[0147] In some embodiments, high concentrations of salts (high salinity) can also be used as denaturants. High concentrations of salts are known to precipitate both proteins and nucleic acids from aqueous solutions. In some embodiments, the high concentration of salt can be from 1 M to 10 M. In some embodiments, the high concentration of salt can be from 2 M to 9 M. In some embodiments, the high concentration of salt can be from 2 M to 8 M. In some embodiments, the high concentration of salt can be from 2 M to 5 M. In some embodiments, the high concentration of salt can be at a concentration higher than 1 M. In some embodiments, the high concentration of salt can be at a concentration higher than 2 M. In some embodiments, the high concentration of salt can be at a concentration higher than 3 M. In some embodiments, the high concentration of salt can be at a concentration higher than 4 M. In some embodiments, the high concentration of salt can be at a concentration higher than 5 M. In some embodiments, the high concentration of salt can be at a concentration higher than 6 M. In some embodiments, the high concentration of salt can be at a concentration higher than 7 M. In some embodiments, the high concentration of salt can be at a concentration higher than 8 M. In some embodiments, a single salt is used as the denaturant. In some embodiments, two or more salts are used as the denaturant.

[0148] In some embodiments, the salt used as the denaturant can be a calcium salt, an iron salt, a magnesium salt, a potassium salt, a sodium salt, or a combination thereof. In some embodiments, exemplary specific salts suitable for use as denaturants include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), lithium chloride (LiCl), calcium chloride (CaCl 2 2), potassium bromide (KBr), sodium bromide (NaBr), and lithium bromide (LiBr). In some embodiments, the denaturant to which the crude preparation is subjected is potassium chloride (KCl). In some embodiments, KCl is added such that the resulting KCl concentration is about 1 M or higher. In some embodiments, KCl is added such that the resulting KCl concentration is about 2 M or higher, 3 M or higher, 4 M or higher, or 5 M or higher.

[0149] In some embodiments, the method does not include a chromatography step. In some embodiments, the precipitated mRNA is centrifuged to obtain an mRNA pellet. The mRNA pellet is then resuspended in a buffer such as water, TE, sodium citrate, or a combination thereof. Thus, in some embodiments, the mRNA pellet is resuspended in water. In some embodiments, the mRNA pellet is resuspended in TE. In some embodiments, the mRNA pellet is resuspended in sodium citrate.

[0150] In some embodiments, the mRNA is precipitated in a suspension comprising GSCN at a final concentration of about 2-4 M, PEG having a molecular weight of about 4000-8000 g / mol, for example, about 6000 g / mol (e.g., PEG-6000) at a final concentration of about 5%-about 20% (weight / volume), and a filter aid (e.g., a cellulose-based filter aid) at a mass ratio to the precipitated mRNA of about 2:1, about 5:1, about 10:1 or about 15:1. In some embodiments, the mRNA is precipitated in a suspension comprising GSCN at a final concentration of about 2.5-3 M, PEG having a molecular weight of about 6000 g / mol (e.g., PEG-6000) at a final concentration of about 10%-about 15% (weight / volume), and a filter aid (e.g., a cellulose-based filter aid) at a mass ratio to the precipitated mRNA of about 10:1. In certain embodiments, the mRNA is precipitated in a suspension comprising GSCN at a final concentration of about 2.7 M, PEG having a molecular weight of about 6000 g / mol (e.g., PEG-6000) at a final concentration of about 12% (weight / volume), and a filter aid (e.g., a cellulose-based filter aid, e.g., Solka-Floc) at a mass ratio to the precipitated mRNA of about 10:1. As shown in the examples, suspensions containing mRNA, salts and PEG at these concentrations achieve very effective purification of the mRNA without precipitating process enzymes.

[0151] In some embodiments, MTEG can be used instead of PEG to provide a suspension of precipitated mRNA. In certain embodiments, MTEG is used for this purpose at a final concentration of about 15 weight / volume % to about 45 weight / volume %. In some embodiments, the suspension comprises MTEG at a final concentration of about 20 weight / volume % to about 40 weight / volume %. In some embodiments, the suspension comprises MTEG at a final concentration of about 20 weight / volume %. In some embodiments, the suspension comprises MTEG at a final concentration of about 25 weight / volume %. In some embodiments, the suspension comprises MTEG at a final concentration of about 30 weight / volume %. In some embodiments, the suspension comprises MTEG at a final concentration of about 35 weight / volume %. In some embodiments, the suspension comprises MTEG at a final concentration of less than 35 weight / volume %. The remaining conditions used for MTEG-induced precipitation are the same as those used for PEG-induced precipitation. As shown in the examples, suspensions comprising mRNA, GSCN, and MTEG with a final concentration of MTEG less than 35 weight / volume % ensured efficient recovery of mRNA without undesirable precipitation of process enzymes. Particularly suitable for efficient recovery of mRNA without undesirable precipitation of process enzymes is a suspension comprising mRNA, GSCN, and MTEG with a filtration aid (e.g., a cellulose-based filtration aid) at a mass ratio to the precipitated mRNA of about 10:1 and having a final concentration of about 25% MTEG.

[0152] For example, GSCN can be provided as a solution of 4-8M (e.g., in 10 mM DTT buffer), and then this is combined with mRNA and MTEG to prepare a suspension of precipitated mRNA. In some embodiments, the suspension comprises precipitated mRNA, a chaotropic salt such as GSCN, and MTEG in a volume ratio of 1:2-3:1-2. In some embodiments, the suspension comprises precipitated mRNA, a chaotropic salt such as GSCN, and MTEG in a volume ratio of 1:2-2.5:1-2. In some embodiments, the suspension comprises precipitated mRNA, a chaotropic salt such as GSCN, and MTEG in a volume ratio of 1:2.3:1-2. In certain embodiments, the suspension comprises precipitated mRNA, GSCN, and MTEG in a ratio of 1:2.3:2. In certain embodiments, the suspension comprises precipitated mRNA, GSCN, and MTEG in a volume ratio of 1:2.3:1.7. In certain embodiments, the suspension comprises precipitated mRNA, GSCN, and MTEG in a ratio of 1:2.3:1. As shown in the examples, suspensions containing mRNA, GSCN, and MTEG in volume ratios of 1:2.3:1, 1:2.3:1.7, and 1:2.3:2 are particularly suitable for the polymer-induced purification method in combination with a MTEG washing solution with a final concentration of about 95%, and this combination of steps ensures efficient recovery of mRNA without unwanted precipitation in the process.

[0153] Capture of mRNA Another step in the method for purifying mRNA described herein involves capturing the mRNA. Various methods for capturing mRNA are known in the art. In some embodiments, an impure preparation containing precipitated mRNA is subjected to a purification process involving membrane filtration such that the precipitated mRNA is captured or retained by a membrane or filter. Thus, in some embodiments, the impure preparation is subjected to membrane filtration after precipitation without pretreatment to remove insoluble substances.

[0154] To capture or retain precipitated mRNA, various types of membrane filtration may be used. Typically, membrane filtration involves using one or more inserted permeable membranes to separate solids from fluids. Membrane filtration can also be used to filter particles from gaseous samples. Generally speaking, there are two main forms of membrane filtration: passive filtration that proceeds only by solution diffusion, and active filtration that uses positive or negative pressure (i.e., vacuum) to force a liquid or gas through the membrane. Typically, membrane filtration involves the steps of loading, washing, and elution.

[0155] The capture of mRNA on a filter involves loading a solution containing precipitated mRNA onto the membrane or filter. This step is usually referred to as the loading step. The loading step involves loading the feedstock (e.g., an impure preparation containing precipitated mRNA) onto the membrane or filter and forcing it through by positive or negative pressure, such that the retentate is captured or retained on the membrane. As used herein, the term "retentate" refers to any non-permeable solute and / or insoluble material retained by the membrane. According to the present invention, precipitated mRNA is captured by the membrane as the retentate. As used herein, the term "membrane" or "filter" refers to any porous layer or sheet material. In this application, the term "membrane" is used interchangeably with "filter".

[0156] In some embodiments, a suitable membrane has a pore size appropriate for capturing or retaining precipitated mRNA, while allowing impurities (including soluble impurities and / or insoluble substances smaller than the pore size) to pass through as permeate. In some embodiments, a suitable membrane has an average pore size of about 0.10 μm, 0.20 μm, 0.22 μm, 0.24 μm, 0.26 μm, 0.28 μm, 0.30 μm, 0.40 μm, 0.5 μm, or 1.0 μm or greater. In certain embodiments, a suitable membrane has an average pore size of about 0.22 μm. In some embodiments, a suitable membrane has an average pore size of about 1.0 μm, 1.5 μm, 2.0 μm, 2.5 μm, 3.0 μm, 3.5 μm, 4.0 μm, 4.5 μm, 5.0 μm, 5.5 μm, 6.0 μm, 6.5 μm, 7.0 μm, 7.5 μm, 8.0 μm, 8.5 μm, 9.0 μm, 9.5 μm, and 10 μm. In some embodiments, for example, a suitable membrane for use in normal flow filtration (NFF) or depth filtration has an average pore size of about 5 μm to about 8 μm. In some embodiments, a suitable membrane for use in NFF or depth filtration has an average pore size of about 7 μm. In some embodiments, for example, a suitable membrane for use in centrifugal filtration has an average pore size of about 0.5 μm to about 2.0 μm. In some embodiments, a suitable membrane for use in centrifugal filtration is about 1 μm. In some embodiments, a suitable pore size for retaining precipitated mRNA may be determined by the nominal molecular weight limit (NMKL) of the precipitated mRNA, also referred to as the molecular weight cut off (MWCO). Typically, a membrane with a pore size smaller than the NMWL or MWCO of the precipitated mRNA is used. In some embodiments, a membrane having a pore size less than 2 to 6 (e.g., 2, 3, 4, 5, or 6) times the NMWL or MWCO of the precipitated mRNA is used.In some embodiments, membranes suitable for the present invention can have a pore size of about 100 kilodaltons (kDa), 300 kDa, 500 kDa, 1,000 kDa, 1,500 kDa, 2,000 kDa, 2,500 kDa, 3,000 kDa, 3,500 kDa, 4,000 kDa, 4,500 kDa, 5,000 kDa, 5,500 kDa, 6,000 kDa, 6,500 kDa, 7,000 kDa, 7,500 kDa, 8,000 kDa, 8,500 kDa, 9,000 kDa, 9,500 kDa, or 10,000 kDa or greater. In some embodiments, the membrane has a pore size that is larger than the NMWL and MWCO of the mRNA, but smaller than the NMWL and MWCO of the precipitated mRNA. Thus, in certain embodiments, the present invention provides for precipitating mRNA in a suspension comprising a high molar salt solution and a PEG polymer to provide precipitated mRNA in the suspension, and capturing the precipitated mRNA on a filter having a pore size that is larger than the NMWL and MWCO of the mRNA, but smaller than the NMWL and MWCO of the precipitated mRNA, and washing the captured and precipitated mRNA to obtain a purified mRNA composition substantially free of contaminants, and provides a method for purifying mRNA.

[0157] The membranes suitable for the present invention may be made from any material. Exemplary membrane materials include, but are not limited to, polyethersulfone (mPES) (unmodified), polyethersulfone (mPES) hollow fiber membranes, polyvinylidene fluoride (PVDF), cellulose acetate , nitrocellulose, MCE (mixed cellulose ester), ultra-high MW polyethylene (UPE), polytetrafluoroethylene (PTFE), nylon, polysulfone, polyethersulfone, polyacrylonitrile, polypropylene, polyvinyl chloride, and combinations thereof. In certain embodiments, the membrane is a polypropylene filter having an average pore size of about 1.0 μm.

[0158] Membranes suitable for the present invention may have various surface areas. In some embodiments, suitable membranes have a surface area sufficient to promote large-scale production of mRNA. For example, suitable membranes may have a surface area of about 2,000 cm 2 , 2,500 cm 2 , 3,000 cm 2 , 3,500 cm 2 , 4,000 cm 2 , 4,500 cm 2 , 5,000 cm 2 , 7,500 cm 2 , 10,000 cm 2 , 5 m 2 , 10 m 2 , 12 m 2 , 15 m 2 , 20 m 2 , 24 m 2 , 25 m 2 , 30 m 2 , or 50 m 2 or more.

[0159] Membrane filtration can be carried out in various forms to capture the precipitated mRNA. In some embodiments, membrane filtration is carried out as part of tangential flow filtration (TFF). In some embodiments, membrane filtration includes normal flow filtration (NFF) or depth filtration. In some embodiments, membrane filtration includes centrifugal filtration.

[0160] Filter aids (including dispersants) In some embodiments, in the methods described herein, filter aids are used. Filter aids can be used when purifying precipitated mRNA using a filtration centrifuge. The filter aid can assist in retaining the precipitated mRNA on the filter of the filtration centrifuge and in removing the retained mRNA from the surface of the filter of the filtration centrifuge.

[0161] In some embodiments, the filter aid is a dispersant. In some embodiments, the filter aid comprises one or more of ash, clay, diatomaceous earth, perlite, glass beads, plastic beads, polymers, polypropylene beads, polystyrene beads, salts (e.g., cellulose salts), sand, volcanic ash, diatomaceous earth and / or sugar. In some embodiments, the dispersant is beads. In some embodiments, the precipitated mRNA composition does not contain a dispersant.

[0162] In some embodiments, the step of adding one or more agents to promote precipitation of mRNA is carried out in the absence of any dispersant.

[0163] In some embodiments, the step of adding one or more agents to promote precipitation of mRNA is carried out in the presence of at least one dispersant.

[0164] In some embodiments, a dispersant is added to the slurry obtained after adding one or more agents to promote precipitation of mRNA.

[0165] Thus, in some embodiments, the purification method may further include one or more steps for separating the dispersant from the purified mRNA precipitate, such as washing and drying the cake. The method may further include solubilizing and eluting the purified mRNA from the cake using an aqueous medium, such as water, while filtering the dispersant. In embodiments, the precipitation step and the drying step can be carried out simultaneously.

[0166] In embodiments, the filter aid is cellulose. In embodiments, the cellulose filter aid is powdered cellulose fibers (e.g., Solka-Floc® or Sigmacell Cellulose 20). In embodiments, the cellulose filter aid is So It is a powdered cellulose fiber such as lka-Floc (registered trademark) 200NF or Sigmacell Cellulose Type 20 (20 μm). In some embodiments, the filter aid is volcanic ash. In some embodiments, the filter aid is diatomaceous earth.

[0167] In some embodiments, the precipitated mRNA and the filter aid (e.g., powdered cellulose fiber such as Solka Floc) are in a mass ratio of 1:2, 1:5, 1:10 or 1:15. In certain embodiments, the precipitated mRNA and the filter aid (e.g., powdered cellulose fiber such as Solka Floc) are in a mass ratio of 1:10.

[0168] Washing of the captured mRNA The method of purifying mRNA also includes washing the captured insoluble mRNA before eluting to remove impurities retained on the membrane.

[0169] In some embodiments, the amphiphilic polymer is used in the washing step. In some embodiments, the amphiphilic polymer is polyethylene glycol (PEG). Thus, in some embodiments, a PEG solution (“PEG washing solution”) is used to wash the captured mRNA. The PEG washing solution includes triethylene glycol, tetraethylene glycol, PEG200, PEG300, PEG400, PEG600, PEG1,000, PEG1,500, PEG2,000, PEG3,000, PEG3,350, PEG4,000, PEG6,000, PEG8,000, PEG10,000, PEG20,000, PEG35,000, and PEG40,000, or combinations thereof. In some embodiments, the PEG washing solution includes triethylene glycol. In some embodiments, the PEG washing solution includes tetraethylene glycol. In some embodiments, the PEG washing solution includes PEG200. In some embodiments, the PEG solution includes PEG300. In some embodiments, the washing PEG washing solution includes PEG400. In some embodiments, the PEG washing solution includes PEG600. In some embodiments, the PEG washing solution includes PEG1,000. In some embodiments, the PEG washing solution includes PEG1,500. In some embodiments, the PEG washing solution includes PEG2,000. In some embodiments, the PEG washing solution includes PEG3,000. In some embodiments, the PEG washing solution includes PEG3,350. In some embodiments, the PEG washing solution includes PEG4,000. In some embodiments, the PEG washing solution includes PEG6,000. In some embodiments, the PEG washing solution includes PEG8,000. In some embodiments, the PEG washing solution includes PEG10,000. In some embodiments, the PEG washing solution includes PEG20,000. In some embodiments, the PEG washing solution includes PEG35,000. In some embodiments, the PEG washing solution includes PEG40,000. In some embodiments, washing the precipitated mRNA includes one or more washes with PEG having a viscosity of 90 centistokes or less.In some embodiments, the PEG used to wash the precipitated mRNA has a viscosity of 80 centistokes or less. In some embodiments, the PEG used to wash the precipitated mRNA has a viscosity of 70 centistokes or less. In some embodiments, the PEG used to wash the precipitated mRNA has a viscosity of 60 centistokes or less. In some embodiments, the PEG used to wash the precipitated mRNA has a viscosity of 50 centistokes or less. In some embodiments, the PEG used to wash the precipitated mRNA has a viscosity of 40 centistokes or less. In some embodiments, the PEG used to wash the precipitated mRNA has a viscosity of 30 centistokes or less. In some embodiments, the PEG used to wash the precipitated mRNA has a viscosity of 20 centistokes or less. In some embodiments, the PEG used to wash the precipitated mRNA has a viscosity of 10 centistokes. It has the following viscosity. In some embodiments, the washing of the precipitated mRNA can be achieved using triethylene glycol (TEG). In some embodiments, the washing of the precipitated mRNA can be achieved using triethylene glycol monomethyl ether (MTEG). In some embodiments, the washing of the precipitated mRNA can be achieved using tert-butyl-TEG-O-propionate. In some embodiments, the washing of the precipitated mRNA can be achieved using TEG-dimethacrylate. In some embodiments, the washing of the precipitated mRNA can be achieved using TEG-dimethyl ether. In some embodiments, the washing of the precipitated mRNA can be achieved using TEG-divinyl ether. In some embodiments, the washing of the precipitated mRNA can be achieved using TEG-monobutyl. In some embodiments, the washing of the precipitated mRNA can be achieved using TEG-methyl ether methacrylate. In some embodiments, the washing of the precipitated mRNA can be achieved using TEG-monodecyl ether. In some embodiments, the washing of the precipitated mRNA can be achieved using TEG-dibenzoate. The structure of each of these reagents is shown in Table A above.

[0170] The viscosity of the liquid solution can be measured at room temperature (e.g., about 18 - 25 °C) using methods well known in the art, for example, using a viscometer.

[0171] In some embodiments, the PEG in the PEG washing solution comprises a PEG-modified lipid. In some embodiments, the PEG in the PEG washing solution is a PEG-modified lipid, 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol (DMG-PEG-2K). In some embodiments, the PEG-modified lipid is a DOPA-PEG conjugate. In some embodiments, the PEG-modified lipid is a poloxamer-PEG conjugate. In some embodiments, the PEG-modified lipid comprises DOTAP. In some embodiments, the PEG-modified lipid comprises cholesterol.

[0172] In some embodiments, the PEG washing solution comprises a mixture of two or more molecular weight PEG polymers. For example, in some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 molecular weight PEG polymers constitute the PEG washing solution. Thus, in some embodiments, the PEG washing solution comprises a mixture of one or more PEG polymers. In some embodiments, the mixture of PEG polymers comprises polymers having distinct molecular weights.

[0173] The PEG used in the PEG washing solution can have various geometric shapes. For example, suitable PEG polymers include PEG polymers having a linear, branched, Y-shaped, or multi-arm shape. In some embodiments, the PEG is in a suspension comprising one or more PEGs of distinct geometric shapes.

[0174] In some embodiments, the PEG in the cleaning solution is present at a concentration of about 10 weight / volume % to about 100 weight / volume %. For example, in some embodiments, the PEG is present in the cleaning solution at a concentration of about 10 weight / volume %, 15 weight / volume %, 20 weight / volume %, 25 weight / volume %, 30 weight / volume %, 35 weight / volume %, 40 weight / volume %, 45 weight / volume %, 50 weight / volume %, 55 weight / volume %, 60 weight / volume %, 65 weight / volume %, 70 weight / volume %, 75 weight / volume %, 80 weight / volume %, 85 weight / volume %, 90 weight / volume %, 95 weight / volume %, 100 weight / volume %, and any value therebetween. In some embodiments, the PEG is present in the cleaning solution at a concentration of about 10 weight / volume %. In some embodiments, the PEG is present in the cleaning solution at about 15 weight / volume %. In some embodiments, the PEG is present in the cleaning solution at a concentration of about 20 weight / volume %. Some In an embodiment, PEG is present in the washing solution at a concentration of about 25 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 30 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 35 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 40 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 45 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 50 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 55 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 60 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 65 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 70 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 75 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 80 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 85 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 90 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 95 wt / vol%. In some embodiments, PEG is present in the washing solution at a concentration of about 100 wt / vol%.

[0175] In some embodiments, the washing buffer contains PEG-400 at a concentration of about 80 - 100%. Thus, in some embodiments, the washing buffer contains PEG-400 at a concentration of about 80%. In some embodiments, the washing buffer contains PEG-400 at a concentration of about 85%. In some embodiments, the washing buffer contains PEG-400 at a concentration of about 90%. In some embodiments, the washing buffer contains PEG-400 at a concentration of about 95%. In some embodiments, the washing buffer contains PEG-400 at a concentration of about 100%.

[0176] In some embodiments, the precipitated mRNA is washed in a solution containing an amphiphilic polymer. In some embodiments, the amphiphilic polymer is PEG. The precipitated mRNA can be washed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more than 10 times. Thus, in some embodiments, the precipitated mRNA is washed once with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed twice with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed three times with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed four times with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed five times with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed six times with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed seven times with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed eight times with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed nine times with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed ten times with a solution containing a PEG polymer. In some embodiments, the precipitated mRNA is washed more than ten times with a solution containing a PEG polymer.

[0177] In some embodiments, the wash solution used to wash the captured mRNA is aqueous. Thus, in some embodiments, the wash solution does not contain an alcohol such as ethanol, isopropyl alcohol, or benzyl alcohol.

[0178] In some embodiments, the PEG wash solution contains a non-aqueous component such as, for example, ethanol, isopropyl alcohol, or benzyl alcohol.

[0179] In some embodiments, the washing step involves an amphiphilic polymer (e.g., polyethylene glycol It includes a plurality of rinsing cycles using a solution containing a [[CALL]]. In some embodiments, the washing step includes a plurality of rinsings using a solution containing one or more distinct amphiphilic polymers. In some embodiments, the washing step may be performed by a plurality of rinsing cycles using a solution containing about 10% to about 100% amphiphilic polymer. In certain embodiments, the plurality of rinsing cycles consists of 2 cycles, 3 cycles, 4 cycles, 5 cycles, 6 cycles, 7 cycles, 8 cycles, 9 cycles, 10 cycles, or more than 10 cycles.

[0180] In some embodiments, PEG is present in the washing solution at a concentration of about 90 to about 100 weight / volume %. In certain embodiments, PEG (e.g., PEG-400) is present in the washing solution at a concentration of about 90 weight / volume %. As shown in the examples, the final concentration of PEG having a molecular weight of about 400 g / mol (e.g., PEG-400) at about 90 weight / volume % to about 100 weight / volume % is particularly suitable for the washing step because this washing solution results in a high-yield and high-purity mRNA sample.

[0181] In some embodiments, MTEG is present in the washing solution at a concentration of about 75 weight / volume % to about 95 weight / volume %. In some embodiments, MTEG is present in the washing solution at a concentration of about 75 weight / volume %, about 80 weight / volume %, about 85 weight / volume %, about 90 weight / volume %, or about 95 weight / volume %. In some embodiments, MTEG is present in the washing solution at a concentration of about 90 weight / volume % to about 100 weight / volume %. In certain embodiments, MTEG is present in the washing solution at a concentration of about 95 weight / volume %. As shown in the examples, the final concentration of MTEG at about 90 or about 95 weight / volume % is particularly suitable for the washing step because these final concentrations achieve a high-efficiency recovery of mRNA without precipitating the process enzymes.

[0182] Elution or collection Typically, the captured or retained mRNA can be eluted or collected by re-solubilizing the precipitated mRNA into solution. For example, the captured mRNA may be eluted with RNase-free water. In certain embodiments, eluting the captured mRNA involves recirculating RNase-free water. For example, RNase-free water can be circulated for about 5 - 30 minutes (e.g., about 5 - 25 minutes, about 5 - 20 minutes, or about 5 - 15 minutes). In certain embodiments, RNase-free water is recirculated for about 5 - 10 minutes (e.g., about 5, 6, 7, 8, 9, or 10 minutes). Other buffers such as TE and / or sodium citrate can be used to re-solubilize the mRNA. The term "elute" can be used in connection with a purification process involving, for example, depth filtration, while the term "collect" can be used in connection with a purification process involving centrifugation.

[0183] In some embodiments, the re-solubilized mRNA may be dialyzed into a desired formulation at a desired concentration. Various formulations can be used for dialysis. In some embodiments, the purified mRNA solution is dialyzed against 1 mM sodium citrate. In some embodiments, the purified mRNA solution is dialyzed against sodium acetate, ammonium carbonate, ammonium bicarbonate, pyridinium acetate, pyridinium formate, ammonium acetate, urea, potassium chloride, etc. Depending on the size of the target mRNA, a dialysis membrane with an appropriate molecular weight cut-off (MWCO) can be used. For example, suitable dialysis membranes can have an MWCO of about 50 kDa, 60 kDa, 70 kDa, 80 kDa, 90 kDa, 100 kDa, 150 kDa, 200 kDa, 250 kDa, 300 kDa, 350 kDa, 400 kDa, 450 kDa, or 500 kDa.

[0184] Scale and Recovery Yield Certain advantages provided by the present invention are that mRNA, particularly mRNA synthesized in vitro NA is the ability to be purified on a large scale or a commercial scale. For example, in some embodiments, in vitro synthesized mRNA is purified on a scale of about 100 milligrams, 1 gram, 10 grams, 50 grams, 100 grams, 200 grams, 300 grams, 400 grams, 500 grams, 600 grams, 700 grams, 800 grams, 900 grams, 1 kg, 5 kg, 10 kg, 50 kg, 100 kg, 1 metric ton, 10 metric tons or more per batch. In embodiments, in vitro synthesized mRNA is purified on a scale of about 1 kg or more.

[0185] In one particular embodiment, in vitro synthesized mRNA is purified on a scale of 10 grams per batch. In one particular embodiment, in vitro synthesized mRNA is purified on a scale of 20 grams per batch. In one particular embodiment, in vitro synthesized mRNA is purified on a scale of 25 grams per batch. In one particular embodiment, in vitro synthesized mRNA is purified on a scale of 50 grams per batch. In another particular embodiment, in vitro synthesized mRNA is purified on a scale of 100 grams per batch. In yet another particular embodiment, in vitro synthesized mRNA is purified on a scale of 1 kg per batch. In yet another particular embodiment, in vitro synthesized mRNA is purified on a scale of 10 kg per batch. In yet another particular embodiment, in vitro synthesized mRNA is purified on a scale of 100 kg per batch. In yet another particular embodiment, in vitro synthesized mRNA is purified on a scale of 1,000 kg per batch. In yet another particular embodiment, in vitro synthesized mRNA is purified on a scale of 10,000 kg per batch.

[0186] In some embodiments, the mRNA is purified on a scale of 1 gram, 5 grams, 10 grams, 15 grams, 20 grams, 25 grams, 30 grams, 35 grams, 40 grams, 45 grams, 50 grams, 75 grams, 100 grams, 150 grams, 200 grams, 250 grams, 300 grams, 350 grams, 400 grams, 450 grams, 500 grams, 550 grams, 600 grams, 650 grams, 700 grams, 750 grams, 800 grams, 850 grams, 900 grams, 950 grams, 1 kg, 2.5 kg, 5 kg, 7.5 kg, 10 kg, 25 kg, 50 kg, 75 kg, 100 kg or more per batch.

[0187] In some embodiments, the solution containing mRNA comprises at least 1 gram, 10 grams, 100 grams, 1 kilogram, 10 kilograms, 100 kilograms, 1 metric ton, 10 metric tons, or more of mRNA, or any amount therebetween. In some embodiments, the methods described herein are used to purify an amount of mRNA that is at least about 250 mg of mRNA. In one embodiment, the methods described herein are used to purify an amount of mRNA that is at least about 250 mg of mRNA, about 500 mg of mRNA, about 750 mg of mRNA, about 1000 mg of mRNA, about 1500 mg of mRNA, about 2000 mg of mRNA, or about 2500 mg of mRNA. In embodiments, the methods described herein are used to purify an amount of mRNA that is at least about 250 mg of mRNA to about 500 g of mRNA. In embodiments, the methods described herein are used to purify an amount of mRNA that is at least about 500 mg of mRNA to about 250 g of mRNA, about 500 mg of mRNA to about 100 g of mRNA, about 500 mg of mRNA to about 50 g of mRNA, about 500 mg of mRNA to about 25 g of mRNA, about 500 mg of mRNA to about 10 g of mRNA, or about 500 mg of mRNA to about 5 g of mRNA. In embodiments, the methods described herein are used to purify an amount of mRNA that is at least about 100 mg of mRNA to about 10 g of mRNA, about 100 mg of mRNA to about 5 g of mRNA, or about 100 mg of mRNA to about 1 g of mRNA.

[0188] In some embodiments, the methods described herein provide a recovery amount (or yield) of purified mRNA that is at least about 40%, 45%, 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 97%, about 98%, about 99%, or about 100%. Thus, in some embodiments, the recovery amount of purified mRNA is about 40%. In some embodiments, the recovery amount of purified mRNA is about 45%. In some embodiments, the recovery amount of purified mRNA is about 50%. In some embodiments, the recovery amount of purified mRNA is about 55%. In some embodiments, the recovery amount of purified mRNA is about 60%. In some embodiments, the recovery amount of purified mRNA is about 65%. In some embodiments, the recovery amount of purified mRNA is about 70%. In some embodiments, the recovery amount of purified mRNA is about 75%. In some embodiments, the recovery amount of purified mRNA is about 75%. In some embodiments, the recovery amount of purified mRNA is about 80%. In some embodiments, the recovery amount of purified mRNA is about 85%. In some embodiments, the recovery amount of purified mRNA is about 90%. In some embodiments, the recovery amount of purified mRNA is about 91%. In some embodiments, the recovery amount of purified mRNA is about 92%. In some embodiments, the recovery amount of purified mRNA is about 93%. In some embodiments, the recovery amount of purified mRNA is about 94%. In some embodiments, the recovery amount of purified mRNA is about 95%. In some embodiments, the recovery amount of purified mRNA is about 96%. In some embodiments, the recovery amount of purified mRNA is about 97%. In some embodiments, the recovery amount of purified mRNA is about 98%. In some embodiments, the recovery amount of purified mRNA is about 99%. In some embodiments, the recovery amount of purified mRNA is about 100%.

[0189] In certain embodiments, the recovered amount of purified mRNA is greater than about 80%, or greater than about 90%, for example, about 90% to 100%.

[0190] Characterization of Purified mRNA The mRNA purification methods provided herein result in a purified mRNA composition that is substantially free of contaminants including short-chain truncated RNA species, long-chain truncated RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcription enzyme, residual solvent, and / or residual salts.

[0191] The methods described herein result in purified mRNA having a purity of about 60% to about 100%. Thus, in some embodiments, the purified mRNA has a purity of about 60%. In some embodiments, the purified mRNA has a purity of about 65%. In some embodiments, the purified mRNA has a purity of about 70%. In some embodiments, the purified mRNA has a purity of about 75%. In some embodiments, the purified mRNA has a purity of about 80%. In some embodiments, the purified mRNA has a purity of about 85%. In some embodiments, the purified mRNA has a purity of about 90%. In some embodiments, the purified mRNA has a purity of about 91%. In some embodiments, the purified mRNA has a purity of about 92%. In some embodiments, the purified mRNA has a purity of about 93%. In some embodiments, the purified mRNA has a purity of about 94%. In some embodiments, the purified mRNA has a purity of about 95%. In some embodiments, the purified mRNA has a purity of about 96%. In some embodiments, the purified mRNA has a purity of about 97%. In some embodiments, the purified mRNA has a purity of about 98%. In some embodiments, the purified mRNA has a purity of about 99%. In some embodiments, the purified mRNA has a purity of about 100% pure degree.

[0192] In some embodiments, the mRNA generated by the methods disclosed herein has less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, and / or less than 0.1% impurities other than full-length mRNA. Impurities include IVT contaminants such as proteins, enzymes, DNA templates, free nucleotides, residual solvents, residual salts, double-stranded RNA (dsRNA), premature termination RNA sequences (“shortmers” or short truncated RNA species), and / or long truncated RNA species. In some embodiments, the purified mRNA is substantially free of process enzymes.

[0193] In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 1 pg / mg, less than about 2 pg / mg, less than about 3 pg / mg, less than about 4 pg / mg, less than about 5 pg / mg, less than about 6 pg / mg, less than about 7 pg / mg, less than about 8 pg / mg, less than about 9 pg / mg, less than about 10 pg / mg, less than about 11 pg / mg, or less than about 12 pg / mg. Thus, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 1 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 2 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 3 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 4 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 5 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 6 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 7 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 8 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 9 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 10 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 11 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA using the purification method described herein is less than about 12 pg / mg.

[0194] In some embodiments, the invention removes or eliminates premature termination RNA sequences (also known as "shortmers"). In some embodiments, the method according to the invention removes greater than about 90%, greater than 95%, greater than 96%, greater than 97%, greater than 98%, greater than 99%, or substantially all premature termination RNA sequences. In some embodiments, the mRNA purified according to the invention is substantially free of premature termination RNA sequences. In some embodiments, the mRNA purified according to the invention contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) premature termination RNA sequences. In some embodiments, the mRNA purified according to the invention contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) premature termination RNA sequences. In some embodiments, the mRNA purified according to the invention is, for example, by high performance liquid chromatography (HPLC) (e.g., shoulder or separate peak), ethidium bromide, Coomassie staining, capillary electrophoresis, or glyoxal gel electrophoresis (e.g., presence of distinct lower band) It contains undetectable prematurely abortive RNA sequences determined thereby. As used herein, the terms "shortmer", "short abortive RNA species", "prematurely abortive RNA sequences", or "long abortive RNA species" refer to any transcript that is shorter than full length. In some embodiments, a "shortmer", "short abortive RNA species", or "prematurely abortive RNA sequences" is less than 100 nucleotides in length, less than 90 nucleotides in length, less than 80 nucleotides in length, less than 70 nucleotides in length, less than 60 nucleotides in length, less than 50 nucleotides in length, less than 40 nucleotides in length, less than 30 nucleotides in length, less than 20 nucleotides in length, or less than 10 nucleotides in length. In some embodiments, shortmers are detected or quantified after addition of a 5'-cap and / or a 3'-polyA tail. In some embodiments, the prematurely abortive RNA transcript is composed of less than 15 bases (e.g., less than 14 bases, 13 bases, 12 bases, 11 bases, 10 bases, 9 bases, 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, or 3 bases). In some embodiments, the prematurely abortive RNA transcript contains about 8 to 15 bases, 8 to 14 bases, 8 to 13 bases, 8 to 12 bases, 8 to 11 bases, or 8 to 10 bases.

[0195] In some embodiments, the method according to the present invention removes or eliminates advanced enzyme reagents used in in vitro synthesis, including but not limited to T7 RNA polymerase, DNAse I, pyrophosphatase, and / or RNase inhibitors. In some embodiments, the present invention is particularly effective in removing T7 RNA polymerase. In some embodiments, the method according to the present invention removes more than about 90%, more than 95%, more than 96%, more than 97%, more than 98%, more than 99%, or substantially all of the enzyme reagents used in in vitro synthesis. In some embodiments, the mRNA purified according to the present invention is substantially free of enzyme reagents used in in vitro synthesis. In some embodiments, the mRNA purified according to the present invention contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) of the enzyme reagents used in in vitro synthesis. In some embodiments, the mRNA purified according to the present invention contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of the enzyme reagents used in in vitro synthesis. In some embodiments, the mRNA purified according to the present invention contains undetectable enzyme reagents used in in vitro synthesis, including, for example, those determined by silver staining, gel electrophoresis, high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), and / or capillary electrophoresis, ethidium bromide and / or Coomassie staining.

[0196] In various embodiments, the mRNA purified using the methods described herein maintains a high degree of integrity. As used herein, the term "mRNA integrity" generally refers to the quality of the mRNA after purification. mRNA integrity can be determined by methods well known in the art, such as RNA agarose gel electrophoresis. In some embodiments, mRNA integrity can be determined by the band pattern of RNA agarose gel electrophoresis. In some embodiments, the mRNA purified according to the present invention shows little or no banding compared to the reference band of RNA agarose gel electrophoresis. In some embodiments, the mRNA purified according to the present invention has an integrity of greater than about 95% (e.g., about 96%, 97%, 98%, 99% or more). In some embodiments, the mRNA purified according to the present invention has an integrity of greater than 98%. In some embodiments, the mRNA purified according to the present invention has an integrity of greater than 99%. In some embodiments, the mRNA purified according to the present invention has an integrity of approximately 100%. In some embodiments, the methods described herein are such that the composition has an increased activity, for example, at least 2-fold, 3-fold, 4-fold, 5-fold, or more, compared to a composition with a low percentage of full-length mR NA molecules.

[0197] In some embodiments, the purified mRNA is evaluated for one or more of appearance, identity, quantity, concentration, presence of impurities, microbiological assessment, pH level, and activity. In some embodiments, an acceptable appearance includes a clear, colorless solution that is essentially free of visible particles. In some embodiments, the identity of the mRNA is evaluated by a sequencing method. In some embodiments, the concentration is evaluated by a suitable method such as UV spectrophotometry. In some embodiments, a suitable concentration is nominally about 90% - 110% (0.9 - 1.1 mg / mL).

[0198] In some embodiments, the evaluation of mRNA purity includes the evaluation of mRNA integrity, the evaluation of residual plasmid DNA, and the evaluation of residual solvents. In some embodiments, an acceptable level of mRNA integrity is evaluated by agarose gel electrophoresis. The gel is analyzed to determine whether the band pattern and apparent nucleotide length match the analytical reference standard. Additional methods for evaluating RNA integrity include, for example, the evaluation of purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, an acceptable purity of the purified mRNA determined by CGE is that the purified mRNA composition has about 55% or less long abortive / degraded species. In some embodiments, residual plasmid DNA is evaluated by methods in the art, such as the use of qPCR. In some embodiments, less than 10 pg / mg (e.g., less than 10 pg / mg, less than 9 pg / mg, less than 8 pg / mg, less than 7 pg / mg, less than 6 pg / mg, less than 5 pg / mg, less than 4 pg / mg, less than 3 pg / mg, less than 2 pg / mg, or less than 1 pg / mg) is an acceptable level of residual plasmid DNA. In some embodiments, an acceptable residual solvent level is 10,000 ppm or less, 9,000 ppm or less, 8,000 ppm or less, 7,000 ppm or less, 6,000 ppm or less, 5,000 ppm or less, 4,000 ppm or less, 3,000 ppm or less, 2,000 ppm or less, 1,000 ppm or less. Thus, in some embodiments, an acceptable residual solvent level is 10,000 ppm or less. In some embodiments, an acceptable residual solvent level is 9,000 ppm or less. In some embodiments, an acceptable residual solvent level is 8,000 ppm or less. In some embodiments, an acceptable residual solvent level is 7,000 ppm or less. In some embodiments, an acceptable residual solvent level is 6,000 ppm or less. In some embodiments, an acceptable residual solvent level is 5,000 ppm or less. In some embodiments, an acceptable residual solvent level is 4,000 ppm or less.In some embodiments, the acceptable residual solvent level is 3,000 ppm or less. In some embodiments, the acceptable residual solvent level is 2,000 ppm or less. In some embodiments, the acceptable residual solvent level is 1,000 ppm or less.

[0199] In some embodiments, the microbiological test is performed on the purified mRNA, which includes, for example, the evaluation of bacterial endotoxins. In some embodiments, the bacterial endotoxin is <0.5 EU / mL, <0.4 EU / mL, <0.3 EU / mL, <0.2 EU / mL, or <0.1 EU / mL. Thus, in some embodiments, the bacterial endotoxin in the purified mRNA is <0.5 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.4 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.3 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.2 EU / mL. In some embodiments, the purified mRNA has a bacterial endotoxin of <0.2 EU / mL. In some embodiments, the bacterial endotoxin in the purified mRNA is <0.1 EU / mL. In some embodiments, the purified mRNA is 1 CFU / 10 mL or less, 1 CFU / 25 mL or less, 1 CFU / 50 mL or less, 1 CFU / 75 mL or less, or 1 CFU / 100 mL or less. Thus, in some embodiments, the purified mRNA is 1 CFU / 10 mL or less. In some embodiments, the purified mRNA is 1 CFU / 25 mL or less. In some embodiments, the purified mRNA is 1 CFU / 50 mL or less. In some embodiments, the purified mRNA is 1 CFR / 75 mL or less. In some embodiments, the purified mRNA has 1 CFU / 100 mL.

[0200] In some embodiments, the pH of the purified mRNA is evaluated. In some embodiments, the acceptable pH of the purified mRNA is between 5 and 8. Thus, in some embodiments, the purified mRNA has a pH of about 5. In some embodiments, the purified mRNA has a pH of about 6. In some embodiments, the purified mRNA has a pH of about 7. In some embodiments, the purified mRNA has a pH of about 7. In some embodiments, the purified mRNA has a pH of about 8.

[0201] In some embodiments, the translational fidelity of the purified mRNA is evaluated. The translational fidelity can be evaluated by various methods, including, for example, transfection and Western blot analysis. Acceptable properties of the purified mRNA include a band pattern on a Western blot that migrates with a molecular weight similar to that of a reference standard.

[0202] In some embodiments, the purified mRNA is evaluated for conductance. In some embodiments, acceptable properties of the purified mRNA include a conductance that is about 50% to 150% of that of a reference standard.

[0203] The purified mRNA is also evaluated for cap percentage and polyA tail length. In some embodiments, acceptable cap percentages include Cap1, % area: NLT90. In some embodiments, acceptable polyA tail lengths are from about 100 to 1500 nucleotides (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000, 1100, 1200, 1300, 1400, or 1500 nucleotides). Thus, in some embodiments, an acceptable polyA tail length is about 100 nucleotides. In some embodiments, the polyA tail length is about 200 nucleotides. In some embodiments, the polyA tail length is about 250 nucleotides. In some embodiments, the polyA tail length is about 300 nucleotides. In some embodiments, the polyA tail length is about 350 nucleotides. In some embodiments, the polyA tail length is about 400 nucleotides. In some embodiments, the polyA tail length is about 450 nucleotides. In some embodiments, the polyA tail length is about 500 nucleotides. In some embodiments, the polyA tail length is about 550 nucleotides. In some embodiments, the polyA tail length is about 600 nucleotides. In some embodiments, the polyA tail length is about 650 nucleotides. In some embodiments, the polyA tail length is about 700 nucleotides. In some embodiments, the polyA tail length is about 750 nucleotides. In some embodiments, the polyA tail length is about 800 nucleotides. In some embodiments, the polyA tail length is about 850 nucleotides. In some embodiments, the polyA tail length is about 900 nucleotides. In some embodiments, the polyA tail length is about 950 nucleotides. In some embodiments, the polyA tail length is about 1000 nucleotides. In some embodiments, the polyA tail length is about 1100 nucleotides. In some embodiments, the polyA tail length is about 1200 It is a nucleotide. In some embodiments, the polyA tail length is about 1300 nucleotides. In some embodiments, the polyA tail length is about 1400 nucleotides. In some embodiments, the polyA tail length is about 1500 nucleotides.

[0204] In some embodiments, the purified mRNA is also evaluated for any residual PEG. In some embodiments, the purified mRNA has less than 10 ng PEG / mg purified mRNA to less than 1000 ng PEG / mg mRNA. Thus, in some embodiments, the purified mRNA has less than about 10 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 100 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 250 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 500 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 750 ng PEG / mg purified mRNA. In some embodiments, the purified mRNA has less than about 1000 ng PEG / mg purified mRNA.

[0205] Various methods for detecting and quantifying mRNA purity are known in the art. For example, such methods include blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV), or UPLC, or combinations thereof. In some embodiments, the mRNA is first denatured with glyoxal dye prior to gel electrophoresis ("glyoxal gel electrophoresis"). In some embodiments, synthetic mRNA is characterized prior to capping or tailing. In some embodiments, synthetic mRNA is characterized after capping and tailing.

[0206] Nucleic acids suitable for the described method Any kind of nucleic acid can be purified using the methods described herein. In some embodiments, the nucleic acid is in vitro transcribed (IVT) mRNA. Briefly, IVT typically involves a buffer system that may contain a promoter, a pool of ribonucleotide triphosphates, DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor, and is carried out using a linear or circular DNA template. In some embodiments, the IVT reaction includes a two-step process, the first step including in vitro transcription of mRNA followed by a purification step, and the second step including capping and tailing of the in vitro transcribed mRNA followed by a second purification step. In some embodiments, the IVT reaction is a one-step process that results in in vitro transcription of capped and tailed mRNA. For example, in some embodiments, in vitro transcription produces capped and tailed mRNA, which is then purified. This is achieved, for example, by using a plasmid that contains a polyT region and / or CleanCap®. The exact conditions vary depending on the particular application. The presence of these reagents is not desirable in the final product according to some embodiments and can therefore be referred to as impurities, and a preparation containing one or more of these impurities can be referred to as an impure preparation. Thus, in one aspect, the invention provides a method comprising: (a) performing in vitro transcription (IVT) by mixing (i) a DNA template comprising a promoter and (ii) an RNA polymerase to produce an impure preparation comprising full-length mRNA; (b) providing a high molar salt and an amphiphilic polymer in a suspension to precipitate the full-length mRNA and provide the full-length mRNA precipitated in the suspension; (c) capturing the precipitated full-length mRNA by applying the suspension to a filter; (d) washing the precipitated full-length mRNA from step (c) with an aqueous solvent to obtain purified full-length mRNA in an aqueous solution; and (e) solubilizing the precipitated mRNA from step (d) to obtain a purified mRNA composition. Provided is a method for producing mRNA, comprising: a step of obtaining a purified full-length mRNA in the aqueous solution obtained from step (d), wherein the purified full-length mRNA is substantially free of (i) the DNA template containing the promoter and (ii) RNA polymerase.

[0207] In some embodiments, in step (a), the DNA template is a linear DNA template. In some embodiments, in step (a), the polymerase is SP6 polymerase. In some embodiments, in step (a), the mixing further comprises mixing a pool of ribonucleotide triphosphates. In some embodiments, in step (a), the mixing further comprises an RNase inhibitor, such as an RNase I inhibitor, RNase A, RNase B, and RNase C.

[0208] In some embodiments, in step (b), the high molar salt is GSCN. In some embodiments, in step (b), the high molar salt comprises GSCN. In some embodiments, in step (b), the amphiphilic polymer comprises a PEG polymer. In some embodiments, in step (b), the amphiphilic polymer comprises MTEG. In certain embodiments, in step (b), the high molar salt comprises GSCN and the amphiphilic polymer comprises a PEG or MTEG having a molecular weight of about 6000 g / mol (e.g., PEG-6000).

[0209] In some embodiments, in step (c), the filter has a MWCO that is smaller than the precipitated full-length mRNA but larger than the full-length mRNA. In some embodiments, in step (c), the filter is a depth-type filter. In some embodiments, in step (c), the filter is used together with centrifugation.

[0210] In some embodiments, in step (d), the aqueous solvent comprises an amphiphilic polymer. In some embodiments, in step (d), the amphiphilic polymer in the aqueous solvent is the same as the amphiphilic polymer used in step (b). In some embodiments, in step (d), the amphiphilic polymer in the aqueous solvent is different from the amphiphilic polymer used in step (b). In some embodiments, in step (d), the amphiphilic polymer comprises a PEG polymer. In some embodiments, in step (d), the PEG polymer in the aqueous solvent is the same as the PEG polymer used in step (b). In some embodiments, in step (d), the PEG polymer in the aqueous solvent is different from the PEG polymer used in step (b).

[0211] In some embodiments, the purified full-length mRNA in the aqueous solution obtained from step (e) also substantially does not contain (iv) pre-aborted RNA sequences. In some embodiments, the pre-aborted RNA sequences comprise shortmers. In some embodiments, the purified full-length mRNA in the aqueous solution obtained from step (e) also substantially does not contain (v) double-stranded RNA (dsRNA). In some embodiments, the purified full-length mRNA in the aqueous solution obtained from step (e) also substantially does not contain (iv) pre-aborted RNA sequences and (v) double-stranded RNA (dsRNA).

[0212] In some embodiments, in step (a), the RNA polymerase is SP6 polymerase, and the purified full-length mRNA in the aqueous solution obtained from step (e) also substantially does not contain (v) double-stranded RNA (dsRNA).

[0213] In some embodiments, this production method generates high-purity mRNA without using chromatography. In some embodiments, this production method generates high-purity mRNA without using an alcohol-based solvent. In some embodiments, this production method generates high-purity mRNA without using chromatography and an alcohol-based solvent.

[0214] According to various embodiments, the present invention is used to purify in vitro synthesized mRNAs of various lengths. In some embodiments, the present invention is used to purify in vitro synthesized mRNAs having a length of 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, or greater than 15 kb. In some embodiments, the present invention is used to purify mRNAs that typically contain one or more modifications that enhance stability. In some embodiments, the one or more modifications are selected from modified nucleotides, modified sugar phosphate backbones, 5' and / or 3' untranslated regions. In some embodiments, the present invention is used to purify unmodified in vitro synthesized mRNAs.

[0215] Typically, mRNA is modified to enhance its stability. The modification of mRNA can include, for example, the modification of RNA nucleotides. The modified mRNA according to the present invention can thus include, for example, backbone modification, sugar modification, or base modification. In some embodiments, antibody-encoding mRNA (e.g., heavy-chain and light-chain encoding mRNA) can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N6-isopentenyl-adenine, N6-methyl-adenine, N6-isopentenyl-adenine, 2-thio-cytosine, 3-methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 2,6-diaminopurine, 1-methyl-guanine, 2-methyl-guanine, 2,2-dimethyl-guanine, 7-methyl-guanine, inosine, 1-methyl-inosine, pseudouridine (5-uracil), dihydro-uracil, 2-thio-uracil, 4-thio-uracil, 5-carboxymethylaminomethyl-2-thio-uracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluoro-uracil, 5-bromo-uracil, 5-carboxymethylaminomethyl-uracil, 5-methyl-2-thio-uracil, 5-methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy-uracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouridine, queosine, beta-D-mannosyl-queosine, wybutoxosine, and phosphoramidite, phosphorothioate, peptide nucleotide, methylphosphonate, 7-deazaguanosine, 5-methylcytosine, and inosine, etc. are included.The preparation of such analogs is known to those skilled in the art, for example, from U.S. Patent No. 4,373,071, U.S. Patent No. 4,401,796, U.S. Patent No. 4,415,732, U.S. Patent No. 4,458,066, U.S. Patent No. 4,500,707, U.S. Patent No. 4,668,777, U.S. Patent No. 4,973,679, U.S. Patent No. 5,047,524, U.S. Patent No. 5,132,418, U.S. Patent No. 5,153,319, U.S. Patent No. 5,262,530, and No. 5,700,642, the disclosures of which are incorporated herein by reference in their entireties.

[0216] Typically, mRNA synthesis involves the addition of a "cap" to the N-terminus (5') and a "tail" to the C-terminus (3'). The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" serves to protect the mRNA from exonucleolytic degradation.

[0217] Thus, in some embodiments, the m RNA purified using the methods described herein includes a 5' cap structure. The 5' cap is typically added as follows: First, RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide leaving two terminal phosphates, then guanosine triphosphate (GTP) is added to the terminal phosphate via guanylyl transferase to form a 5'5'5 triphosphate bond, and then the 7-nitrogen of guanine is methylated by methyl transferase. Examples of cap structures include, but are not limited to, m7G(5')ppp(5'(A, G(5')ppp(5')A, and G(5')ppp(5')G.

[0218] In some embodiments, the mRNA provided from the in vitro transcription reaction is desired, but other sources of mRNA, including mRNA produced from bacteria, fungi, plants, and / or animals, are intended to be within the scope of the present invention.

[0219] In some embodiments, the mRNA for purification in the methods described herein includes 5' and / or 3' untranslated regions. In some embodiments, the 5' untranslated region includes one or more elements that affect mRNA stability or translation, such as iron-responsive elements. In some embodiments, the 5' untranslated region can be about 50 to 500 nucleotides in length.

[0220] In some embodiments, the 3' untranslated region includes one or more of a polyadenylation signal, a binding site for a protein that affects the positional stability of mRNA within the cell, or a binding site for one or more miRNAs. In some embodiments, the 3' untranslated region can be 50 to 500 nucleotides in length or longer.

[0221] The present invention can be used to purify mRNA encoding any protein. Non-limiting examples of mRNA purified using the methods described herein are presented in the following section.

Examples

[0222] Example 1. Synthesis of mRNA IVT Reaction Conditions In the following examples, unless otherwise stated, mRNA was synthesized via in vitro transcription (IVT) using either T7 polymerase or SP6 polymerase. Briefly, in the SP6 polymerase IVT reaction, for each gram of transcribed mRNA, a reaction containing an RNA polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, 5 mM NTPs, 10 mM DTT, and reaction buffer (10x - 250 mM Tris-HCl, pH 7.5, 20 mM spermidine, 50 mM NaCl) and 20 mg of linearized double-stranded DNA plasmid was prepared with RNase-free water and then incubated at 37°C for 60 minutes. Next, the reaction was treated with DNase I and DNase I buffer (10x - 100 mM Tris-HCl, 5 mM MgCl 2 and 25 mM CaCl 2、quenched by the addition of pH 7.6 to facilitate digestion of double-stranded DNA templates in the preparation for purification. The final reaction volume was 204 mL.

[0223] 5' cap Unless otherwise stated, IVT-transcribed mRNA was either capped as part of the IVT reaction by including a cap structure or was capped at its 5' end in a subsequent enzymatic step. For capping as part of the IVT reaction, a cap analog can be incorporated as the first "base" of the nascent RNA strand. Cap analogs can be Cap0, Cap1, Cap2, m6 A m or non-natural caps. Alternatively, uncapped and purified in vitro transcribed (IVT) mRNA can be enzymatically modified after IVT to include a cap, for example, by addition of a 5'N -methylguanylate cap 0 structure using guanylate transferase and by addition of a methyl group at the 2'O position of the penultimate nucleotide to yield a Cap1 structure using 2'O-methyltransferase as described in Fechter, P.; Brownlee, G.G. “Recognition of mRNA cap structures by viral and 7 cellular proteins” J. Gen. Virology 2005, 86, 1239 - 1249.

[0224] 3' tail Unless otherwise stated, the IVT-transcribed mRNA is tailed at its 3'-end by including a tail template in the linearized plasmid, which tails the mRNA either as part of the IVT reaction or in a subsequent enzymatic step. For tailing as part of the IVT reaction, a polyA tail or a similar suitable tail is formed on the mRNA as part of the IVT process by incorporating a polyT or similar tailing function into the pDNA template. Alternatively, the polyA tail can be enzymatically added to the 3'-end of the IVT-produced mRNA after the IVT reaction, for example, using polyA polymerase.

[0225] Example 2. Purification of mRNA via polymer-induced precipitation without VOCs of mRNA This example shows that a polymer can be used instead of a volatile organic compound (VOC) such as ethanol during the mRNA precipitation step of mRNA purification. Such a polymer-induced precipitation method provides final yield and purity levels suitable for therapeutic use.

[0226] As described in Example 1, three 5 mg batches of CFTR mRNA were synthesized via IVT synthesis and each batch was precipitated under three different conditions described below.

[0227] Condition 1: Ethanol and GSCN (experimental control) One volume of mRNA was mixed with 2.3 volumes of 5 M GSCN-10 mM DTT buffer to a final concentration of 2 M GSCN. Then, 1.7 volumes of 100% ethanol were added to the suspension to a final ethanol concentration of 34%.

[0228] Condition 2: PEG-6000 and GSCN (polymer-induced precipitation without ethanol) One volume of mRNA was mixed with 2.3 volumes of 5 M GSCN-10 mM DTT buffer to a final concentration of 2 M GSCN. Then, 1.7 volumes of 50% PEG-6000 were added to the suspension to a final PEG-6000 concentration of 17%.

[0229] Condition 3: PEG-6000 and NaCl (polymer-induced precipitation without ethanol) The precipitation step was carried out under the conditions described in Schmitz et al., Notes & Tips, Anal Biochem. (2006) 311 - 313. One volume of mRNA was mixed with NaCl to obtain a final concentration of NaCl of 500 mM. Then, 50% PEG-6000 was added to the suspension to give a final concentration of PEG-6000 of 19%.

[0230] The mRNA samples precipitated under each condition were captured on a Qiagen RNeasy maxi column, washed twice with 10 ml of 80% ethanol, and dissolved in 5 ml of RNase-free water. The concentration of the dissolved mRNA samples was quantified by a NanoDrop2000 spectrophotometer using the absorbance at 260 nm. The yields for each precipitation condition are shown in Figure 1. Furthermore, the presence of residual process enzymes was detected via silver staining described below, and the results are shown in Figure 2.

[0231] The data show that the three precipitation conditions resulted in similar mRNA yields after purification. For precipitation conditions 1 and 2, as shown in Figure 2, there were no observable process enzymes by silver staining, and a very pure mRNA sample was obtained. However, polymer-induced precipitation via PEG-6000 and NaCl (condition 3) gave samples showing bands consistent with the presence of process enzymes, which require additional purification steps to be acceptable for therapeutic use. The data herein support that mRNA precipitation by condition 2 can achieve sufficient yields and purity levels for use in the purification of mRNA for therapeutic applications. mRNA purity - Residual process enzyme detection (silver staining)

[0232] mRNA purity - Residual process enzyme detection (silver staining) For residual IVT enzymes and optionally cap and / or tail enzymes, mRNA purity was evaluated by silver staining. Specifically, each of the following residual process enzymes can be detected using this approach: RNA polymerase, RNA inhibitors, pyrophosphatase, guanylyl transferase (GuaT), 2'-OM, and polyA polymerase, as well as RNase I, an enzyme used as part of the silver staining gel preparation. Specifically, silver staining gels were run according to the Invitrogen kit using the following pre-staining sample preparations. 15.5 μl of 1 mg / mL RNA was treated with 4 μl of RNase I (100 U / mL, Invitrogen) at 37 °C for 30 minutes. Samples were prepared in Invitrogen LDS loading buffer with a reducing reagent and finally loaded onto a 10% Bis-Tris gel. Electrophoresis was performed at 200 V for 35 minutes. Gels were stained using the Silver Quest staining kit and developed for 8 minutes. Samples containing purified mRNA were considered to be substantially free of specific process enzymes if no bands for the specific process enzymes were visible.

[0233] Example 3. Testing the range of polymer ratios in polymer-induced precipitation of mRNA This example shows, according to various embodiments, that different ratios of polymers can be used for polymer-induced precipitation of mRNA to purify mRNA suitable for therapeutic use.

[0234] Seven 5 mg batches of CFTR mRNA were synthesized by IVT synthesis as described above, and each batch was precipitated under different conditions shown in Table 1 below. [Table 1]

[0235] The mRNA samples precipitated under each condition were captured with a Qiagen RNeasy maxi column, washed twice with 10 ml of 80% ethanol, and dissolved in 5 ml of RNase-free water. The concentration of the dissolved mRNA samples was quantified using a NanoDrop2000 spectrophotometer by measuring the absorbance at 260 nm. The yields of each precipitation condition are shown in Figure 3. Furthermore, the presence of residual process enzymes was detected via silver staining as described in Example 2, and the results are shown in Figure 4.

[0236] The data indicate that all six conditions of purified mRNA using polymer-induced precipitation of polymeric mRNA resulted in higher yields of mRNA after purification, which was similar to the conventional ethanol precipitation method (Figure 3). Furthermore, mRNA samples purified via polymer-induced precipitation with a final PEG concentration of less than 20% (or a ratio of 50% PEG-6000 of less than 2.0) resulted in very pure mRNA samples without observable process enzymes by silver staining as shown in Figure 4 (lanes 5-8). However, samples containing mRNA purified via polymer-induced precipitation with a final PEG-6000 concentration greater than 20% showed residual process enzymes (lane 9). Interestingly, polymer-induced precipitation with 24% PEG-6000 resulted in more residual process enzymes in the purified sample than the sample using 22% PEG-6000 polymer-induced precipitation. Without wishing to be bound by any particular theory, a higher % of PEG-6000 is thought to promote the precipitation of both nucleic acids and process enzymes.

[0237] Collectively, the data herein further support that mRNA purification via polymer-induced precipitation is a viable method for purifying mRNA to achieve sufficient yields and purity levels for therapeutic use. Polymer-induced precipitation with a final PEG-6000 concentration of 7-24% results in efficient mRNA precipitation and recovery. For the remaining examples, a final PEG-6000 concentration of 12% was used for polymer-induced precipitation.

[0238] Example 4. Ethanol-free purification of mRNA and effect of different polymers in the washing process. This example shows that in both the mRNA precipitation and washing steps, polymers can be used without using VOCs such as ethanol to purify mRNA at yields and purity levels suitable for therapeutic applications.

[0239] As described in Example 1, twelve 5 mg batches of CFTR mRNA were synthesized via IVT synthesis and 5' caps and 3' polyA tails were added. The twelve resulting 5 mg batches of IVT mRNA were each precipitated via polymer-induced precipitation. For each 5 mg batch, 5 M GSCN-10 mM DTT buffer was added to a final concentration of 2.7 M GSCN. Then, 50% PEG-6000 was added to the suspension to a final concentration of 12% PEG-6000. The precipitated mRNA samples were captured on Qiagen RNeasy maxi columns and washed twice with 10 ml of one of the following polymer wash buffers listed in Table 2 below instead of 80% ethanol. No VOCs or alcohols were used in the purification of the IVT-synthesized mRNA. Specifically, no VOCs or alcohols were used in the precipitation or washing steps. [Table 2]

[0240] The twelve samples were dissolved in 5 ml of RNase-free water and the concentration was quantified by a NanoDrop2000 spectrophotometer using the absorbance at 260 nm. The yields for each precipitation condition are shown in Figure 5. Further, the presence of residual process enzymes was detected via the silver staining described in Example 2 and the results are shown in Figure 6.

[0241] As shown in Figure 5, a strong correlation was observed between the percentage of polymer in the wash buffer and the amount of mRNA recovered. Without wishing to be bound by any particular theory, the addition of water to these wash buffers is thought to result in solubilization of the mRNA during these washing steps.

[0242] Overall, surprisingly, the data show that mRNA can be purified without any ethanol through polymer-induced precipitation followed by polymer washing, achieving surprising yields and purity levels suitable for therapeutic use. A wash buffer containing PEG-400 at a final concentration of 90-100% resulted in high yields as shown in Figure 5 and high-purity mRNA samples without observable process enzymes by silver staining as shown in Figure 6 (lanes 3-4). PEG-400 can be modified for use in different systems at different scales because it has the lowest viscosity among the tested buffers. Taken together, these data demonstrate that ethanol-free mRNA purification via polymer-induced precipitation and polymer washing described herein can efficiently purify high-quality mRNA with yield recovery rates, integrity profiles, purity, and functionality equal to or better than those of industry-standard mRNA purification methods using VOCs such as alcohol containing ethanol. Furthermore, the present invention has significant additional benefits in scalability and safety, which are not available with existing industry-standard methods using VOCs such as alcohol containing ethanol.

[0243] Example 5. Ethanol-free Purification and Analysis of OTC and CFTR mRNA This example demonstrates that mRNA can be purified using the above-described ethanol-free mRNA purification method regardless of the construct size or nucleotide composition. Furthermore, the purified mRNA by the method described in this specification results in high yields, purity, and integrity.

[0244] A 5 mg batch of OTC mRNA (≈1400 nt) and a 5 mg batch of CFTR mRNA (≈4600 nt) were synthesized via IVT synthesis as described in Example 1. The resulting IVT mRNA samples were precipitated via polymer-induced precipitation. For each 5 mg batch, 5 M GSCN-10 mM DTT buffer was added to a final concentration of 2.7 M GSCN. Next, 50% PEG-6000 was added to the suspension to a final concentration of 12% PEG-6000. The precipitated mRNA samples were captured on a Qiagen RNeasy maxi column and washed twice with 10 mL of 90% PEG-400 wash buffer. The washed samples were dissolved in 5 ml of RNase-free water, and the buffer was exchanged to ultrapure water using a 100 kD Amicon spin column and concentrated to 2 mg / ml.

[0245] Next, the purified and concentrated IVT mRNA was capped and tailed via the enzymatic processes described in Example 1. The mRNA with 5’ cap and 3’ tail was purified via ethanol-free polymer-induced precipitation and polymer washing. Specifically, for each batch precipitation step, 5 M GSCN-10 mM DTT buffer was added to a final concentration of 2.7 M GSCN. Next, 50% PEG-6000 was added to the suspension to a final concentration of 12% PEG-6000. The precipitated mRNA samples were captured on a Qiagen RNeasy maxi column and washed twice with 10 mL of 90% PEG-400 wash buffer. The washed samples were dissolved in 5 ml of RNase-free water, and the buffer was exchanged to ultrapure water using a 100 kD Amicon spin column and concentrated to 1 mg / ml.

[0246] As shown in Figure 7, the yield of each final mRNA product was determined. The data showed that the mRNA yields of the 5 mg scale OTC and CFTR mRNA were 80% and 78% respectively. These values were within or above the range of yields of industry-standard mRNA purification methods.

[0247] The purity and integrity of the final mRNA products were analyzed as described below. Integrity and poly(A) tail length were evaluated using capillary electrophoresis as shown in Figure 8. The results showed that the final products from the purification of both OTC and CFTR mRNAs on a 5 mg scale had distinct peaks similar to the peak of the control (10 grams of OTC of current ethanol). Furthermore, the tail lengths of both constructs were within the target range of 500 nt (OTC = 468 nt and CFTR = 649 nt). The presence of residual process enzymes evaluated via silver staining was not detected in the purification of either mRNA construct on a 5 mg scale. Purity was further confirmed via dsRNA J2 dot blot as described below. As shown in Figure 10, the results showed that dsRNA was not detected in the final products of either construct. Finally, ELISA was used to confirm that PEG was completely removed during the dialysis process as shown in Table 3. [Table 3]

[0248] Collectively, the data showed that ethanol-free purification of mRNA via the polymer-induced precipitation and polymer washing described herein is applicable to the purification of mRNAs of different lengths and constructs. The mRNA purified by the method described herein meets or exceeds the large-scale mRNA lots to date with respect to the important disclosure characteristics described above. Thus, the high yield, integrity, and purity levels of the method described herein are suitable for therapeutic use.

[0249] Analysis of the Integrity of Purified mRNA RNA Integrity Analysis (Fragment Analyzer - Capillary Electrophoresis) The integrity and tail length of RNA were evaluated using a CE Fragment Analyzer and a commercially available RNA detection kit. Analysis of the integrity peak profile and size shift of the tail length was performed on the raw data as well as the normalized data set.

[0250] mRNA Cap Species Analysis (HPLC / MS) The cap species present in the final purified mRNA product were quantified using the chromatography method described in U.S. Patent No. 9,970,047. This method can accurately quantify uncapped mRNA as a percentage of total mRNA. This method can also quantify the amounts of specific cap structures, such as the amounts of CapG, Cap0, and Cap1, and can be reported as a percentage of total mRNA.

[0251] dsRNA Detection (J2 Dot Blot) The presence of dsRNA in individual mRNA samples was measured using the J2 anti-dsRNA dot blot previously described by Kariko et al, Nucleic Acids Research, 2011.39, No.21. Briefly, either 200 ng of RNA or 25 ng of dsRNA control was blotted onto supercharged Nytran. The blot was dried, blocked with 5% non-fat dry milk, and then probed with 1 μg of J2 antibody per blot. The blot was washed and probed with HRP-conjugated donkey anti-mouse and then washed again. The blot was detected with ECL and Western blot detection reagents, and the image was captured on film. Samples containing purified mRNA were considered to be substantially free of dsRNA if each blot did not show a visibly darker color compared to a control lacking dsDNA.

[0252] PEG Quantification / Detection ELISA (Abcam Kit) ​The presence of various molecular weight PEG species in the mRNA sample was determined using a PEG-ELISA kit from Abcam. Briefly, a competitive inhibition ELISA that detects PEG in samples as low as 10 ng / mL and can accurately quantify high molecular weight PEG at that level was used. The mRNA samples were purified using standard ethanol-based precipitation methods, as well as the following ethanol-free methods without solvent, at 1 / 10 dilution, and at 1 / 100 dilution. The limits of detection were determined for 10 ng / mg of RNA, 100 ng / mg of RNA, and 1 μg / mg of RNA at solvent-free concentration, at 1 / 10 dilution, and at 1 / 100 dilution, respectively.

[0253] Example 6.1 Purification of Ethanol-Free mRNA on 1 Gram and 10 Gram Scales This example demonstrates that the ethanol-free mRNA purification method described above can be used to purify mRNA at the scale and quality required for therapeutic applications. The mRNA purified at 1 gram and 10 gram scales according to the methods described herein resulted in high yields, purity, and integrity, demonstrating the scalability of the method.

[0254] Via the IVT synthesis described in Example 1, OTC mRNA was synthesized on a 1-gram scale and a 10-gram scale, and CFTR mRNA was synthesized on a 10-gram scale. The obtained IVT mRNA samples were precipitated via polymer-induced precipitation. For each mRNA sample, 5M GSCN-10mM DTT buffer was added to a final concentration of 2.7M GSCN. Then, 50% PEG-6000 was added to the suspension to a final concentration of 12% PEG-6000. Solka-Floc cellulose-based filter aid was added to the precipitated mRNA at a ratio of 10 to 1 by weight ratio of filter aid to RNA and mixed well. Using the filter aid, the mRNA sample precipitated on a 1-gram scale was captured with a 0.22-μm polyethersulfone (PES) vacuum filter flask. Using the filter aid, the mRNA sample precipitated on a 10-gram scale was captured with an H300P filtration centrifuge equipped with a 1-μm polypropylene filter bag. Then, the 1-gram and 10-gram mRNA samples were washed twice with 1L or 10L of 90% PEG-400 washing buffer. The washed and precipitated mRNA was removed from the filter by filtering manually or with an H300P filtration centrifuge equipped with a 1-μm polypropylene filter bag. Then, the washed and precipitated mRNA was solubilized with 1L or 10L of RNase-free water, and the buffer was exchanged to ultrapure water using a 100kD spectrum TFF column (mPES) and concentrated to 2mg / ml.

[0255] Next, the IVT mRNA purified and concentrated by the enzymatic reaction described in Example 1 was capped and tailed. The mRNA with 5' cap and 3' tail was purified via ethanol-free polymer-induced precipitation and polymer washing. For each, 5M GSCN-10mM DTT buffer was added to a final concentration of 2.7M GSCN. Then, 50% PEG-6000 was added to the suspension to a final concentration of 12% PEG-6000. Solka-Floc cellulose-based filter aid was added to the precipitated mRNA at a ratio of 10 to 1 by weight ratio of filter aid to RNA and mixed well. The precipitated mRNA was washed twice with 1L or 10L of 90% PEG-400 buffer. The washed mRNA samples were removed from the filter by filtering manually or with an H300P filtration centrifuge equipped with a 1μm polypropylene filter bag. The mRNA samples were dissolved in 1L or 10L of RNase-free water, the buffer was exchanged to ultrapure water using a 100kD spectral TFF column (mPES), and concentrated to 1mg / ml.

[0256] As shown in Figure 7, the yields of each final mRNA product were determined. The data demonstrated that the yields of mRNA for the 1 gram scale and 10 gram scale of OTC mRNA and the 10 gram scale of CFTR mRNA were all over 80% and as high as 93%. These values were within or above the range of yields of industry-standard mRNA purification methods.

[0257] The purity and integrity of the final mRNA products were analyzed as described above. Integrity and polyA tail length were evaluated using capillary electrophoresis as shown in Figure 8. The results showed that the final products from the purification of 1 gram and 10 gram scales of OTC and CFTR mRNA had distinct peaks similar to the peak of the control (current 10 gram ethanol of OTC). For the 10 gram scale purified mRNA, the cap species were quantified using the HPLC-MS assay described in Example 2. Furthermore, the tail lengths of both constructs were within the target range (OTC at 1 gram = 306 nt, OCT at 10 grams = 158 nt, CFTR at 10 grams = 712 nt). The presence of residual process enzymes evaluated via silver staining was not detected in either the 1 gram scale or 10 gram scale purification of both mRNA constructs, as shown in Figure 9. Purity was further confirmed via dsRNA J2 dot blot as described in Example 5. As shown in Figure 10, the results showed that dsRNA was not detected in the final products at both the 1 gram scale and 10 gram scale. Finally, ELISA was used to confirm that PEG was completely removed during the dialysis process.

[0258] Collectively, the data demonstrate the scalability of ethanol-free purification of mRNA via polymer-induced precipitation and polymer washing for purifying mRNA at the required scale and quality for clinical therapeutic use. The mRNA purified at 1 gram and 10 gram scales by the methods described herein meet or exceed previous large-scale mRNA lots with respect to the important disclosure characteristics described above, demonstrating the suitability of the mRNA manufacturing and methods of use in therapeutics.

[0259] The present invention can be used to purify mRNA encoding any protein. Non-limiting examples of mRNA purified using the method are described.

[0260] Example 7: Purification of Ethanol-Free mRNA in the Washing Process and the Effect of MTEG This example shows that the amphiphilic polymer MTEG can be used during the washing process without using VOCs such as ethanol to purify mRNA at a yield and purity level suitable for therapeutic use.

[0261] Table 4 shows the properties of MTEG compared to the polymers tested in the previous examples. Despite having a molecular weight similar to TEG, MTEG has a much lower viscosity due to the presence of methyl groups. This means that MTEG has a viscosity much closer to water, which means that the use of pumps is easier. Furthermore, while this is classified as "safe" by the US Food and Drug Administration (FDA), TEG, PEG-400, and PEG-6000 (50%) are classified as "generally recognized as safe" (GRAS). [Table 4]

[0262] As described in Example 1, five 5 mg batches of CFTR mRNA were synthesized via IVT synthesis and a 5' cap and 3' polyA tail were added. The five resulting 5 mg IVT mRNA batches were each precipitated via polymer-induced precipitation. The volume ratio of mRNA, GSCN (5M GSCN - 10 mM DTT buffer), and MTEG (100 wt / vol%) in the precipitation reaction was 1:2.3:1. The precipitated mRNA samples were captured on Qiagen RNeasy maxi columns and washed twice with 2.5 ml of one of the following MTEG wash buffers listed in Table 5 below instead of 80% ethanol. No VOCs or alcohols were used for the purification of IVT-synthesized mRNA. Specifically, no VOCs or alcohols were used in the precipitation or washing steps. [Table 5]

[0263] Five samples were dissolved in 5 mL of RNase-free water, and the concentration was quantified by a NanoDrop 2000 spectrophotometer using the absorbance at 260 nm. The concentration of the recovered RNA is shown in Figure 11.

[0264] As shown in Figure 11, a strong correlation was observed between the proportion of MTEG in the wash buffer and the amount of recovered mRNA. Without wishing to be bound by any particular theory, as outlined above, the addition of water to these wash buffers is thought to result in the solubilization of mRNA during these washing steps. As shown in Figure 11, the wash buffer containing 90 - 95% MTEG by final concentration resulted in a very high yield that was only close to the theoretically achievable 100% recovery level. The purity and recovery rate were equivalent to the washing conditions using 80% ethanol. In the remaining examples, a 95% MTEG concentration was selected for use as the wash buffer.

[0265] These data further demonstrate that mRNA can be purified using a process that is completely ethanol-free.

[0266] Example 8: Purification of ethanol-free mRNA and the effect of MTEG in the washing step. This example shows that the amphiphilic polymer MTEG can be used in both the mRNA precipitation step and the washing step without using VOCs such as ethanol to purify mRNA at a yield and purity level suitable for therapeutic use.

[0267] As described in Example 1, five 5 mg batches of CFTR mRNA were synthesized via IVT synthesis and a 5’ cap and 3’ polyA tail were added. Four of the resulting 5 mg IVT mRNA batches were each precipitated via polymer-induced precipitation. For each 5 mg batch, mRNA, 5M GSCN-10mM DTT buffer, and MTEG were combined in the volumes presented in Table 6 below to form a suspension of the precipitated mRNA. The precipitated mRNA samples were captured on a Qiagen RNeasy maxi column and washed twice with 2.5 ml of 95% MTEG. No VOC or alcohol was used for the purification of the IVT synthesized mRNA. Specifically, no VOC or alcohol was used in the precipitation or washing steps. As an experimental control, one batch of mRNA was precipitated with ethanol at a volume ratio of mRNA, GSCN, and ethanol (100 weight / volume%) of 1:2.3:1.7 as described in Example 2 and washed twice with 2 ml of 80% ethanol.

Table 6

[0268] Five samples were dissolved in 5 mL of RNase-free water and the concentration was quantified by a NanoDrop2000 spectrophotometer using the absorbance at 260 nm. The purity of the mRNA samples generated from the precipitation and washing steps observed using silver staining is shown in Figure 12 (lanes 6-10). Precipitation and washing using MTEG achieved very efficient mRNA recovery and purification of mRNA. Addition of 1-2 volumes of MTEG to 1 volume of mRNA and 2.3 volumes of GSCN showed comparable levels of purity and yield. As shown in Figure 12, addition of 2.5 volumes of MTEG to 1 volume of mRNA and 2.3 volumes of GSCN resulted in faint additional bands on the gel. This may suggest that higher MTEG concentrations result in precipitation of proteins from the IVT reaction.

[0269] Collectively, these data support that mRNA purification using MTEG in both the precipitation and wash buffer is a viable method for purifying mRNA that achieves sufficient yields and purity levels for therapeutic use.

[0270] Collectively, these data demonstrate that MTEG mRNA purification can efficiently purify high-quality mRNA that yields comparable or better recovery rates, integrity profiles, and purity than industry-standard mRNA purification methods using VOCs such as alcohols containing ethanol. MTEG can replace both ethanol and high molecular weight polymers such as PEG-6000 during precipitation. Furthermore, MTEG can replace low molecular weight polymers such as ethanol or PEG-400 in the wash step and is the most versatile polymer for use in ethanol-free purification. Additionally, as outlined above, MTEG-based purification, like other polymer-based ethanol-free methods described herein, has significant additional benefits in scalability and safety, which are not available with existing industry-standard methods using VOCs such as alcohols containing ethanol.

[0271] Example 9: MTEG Polymer-Induced Precipitation and MTEG Wash Buffer Applicable to Purification Using Depth Filtration and Centrifugation. This example demonstrates that due to its low viscosity and associated excellent handling properties, MTEG is surprisingly versatile and can be utilized in various ethanol-free mRNA purification methods at various scales, with recovery yields exceeding 90%.

[0272] Samples were prepared according to the above examples and purified using either depth filtration (DF) or centrifugation.

[0273] Depth Filtration Purification For smaller batches, 7.5 g of OTC mRNA was synthesized via IVT synthesis, and a 5' cap and 3' polyA tail were added as described in Example 1 and the scaling reaction conditions. The mRNA was precipitated using the same MTEG polymer-induced precipitation as shown in Example 7. Thus, the volume ratio of mRNA, GSCN (5 M GSCN - 10 mM DTT buffer), and MTEG (100 wt / vol%) in the precipitation reaction was 1:2.3:1. The suspension was mixed at 60 Hz in a 60 L Lee vessel equipped with an impeller attached to the bottom, and then loaded onto a 0.11 m 2 depth filter with a loading capacity of 68 g / m 2 at a flow rate of 60 L / min / m 2 . The precipitated and retained mRNA was washed using 90% of MTEG at a flow rate of 60 L / min / m 2 .

[0274] For larger batches, 15 g of CFTR mRNA was synthesized via IVT synthesis, and a 5' cap and 3' polyA tail were added as described in Example 1 and the scaling reaction conditions. The mRNA was precipitated using the same MTEG polymer-induced precipitation as shown in Example 7. Thus, the volume ratio of mRNA, GSCN (5 M GSCN - 10 mM DTT buffer), and MTEG (100 wt / vol%) in the precipitation reaction was 1:2.3:1. The suspension was mixed at 60 Hz in a 60 L Lee vessel equipped with an impeller attached to the bottom, and then loaded onto a 0.11 m 2 depth filter with a loading capacity of 68 g / m 2 at a flow rate of 60 L / min / m 2 . The precipitated and retained mRNA was washed using 95% of MTEG at a flow rate of 60 L / min / m 2 . When the pressure increased during washing, the flow rate decreased to 30 L / min / m 2 . The filtration process was repeated on the same 0.11 m 2 depth filter.

[0275] Centrifugation purification 15 g of CFTR mRNA was synthesized via in vitro transcription (IVT) and a 5' cap and 3' polyA tail were added as described in Example 1 and the scaling reaction conditions. The mRNA was precipitated using the same MTEG polymer-induced precipitation as shown in Example 7. Thus, the volume ratio of mRNA, GSCN (5 M GSCN - 10 mM DTT buffer), and MTEG (100 wt / vol%) in the precipitation reaction was 1:2.3:1. The suspension was mixed at 60 Hz in a 60 L Lee vessel equipped with an impeller attached to the bottom, and cellulose filter aid was added at a mass ratio of 1:10 mRNA:filter aid. The suspension was loaded into a filtration centrifuge and washed with 95% MTEG.

[0276] For both purification strategies, the final mRNA yield was quantified using absorbance at 280 nm by a NanoDrop 2000 spectrophotometer. The % recovery of RNA is shown in Table 7 below. Furthermore, capillary electrophoresis (CE) smear analysis was used to evaluate the integrity of the mRNA, and silver staining analysis was used to evaluate the purity of the mRNA to detect residual process enzymes.

[0277] Results For batch sizes of 7.5 g and above, the use of MTEG as the precipitation polymer and wash buffer component ensured a very high % recovery of mRNA. As shown in Table 7, using the same precipitation protocol, MTEG can be used as both the precipitation buffer and the wash buffer in both centrifuge-based purification processes and filter membrane or filter cartridge-based purification processes, such as depth filtration (DF). The use of centrifugation resulted in almost 100% recovery of the mRNA (see Table 7 below). [Table 7]

[0278] Furthermore, the use of MTEG in both the precipitation and washing steps maintained the integrity and purity of the mRNA. As shown in Figure 13, the final OTC integrity (CE) was approximately 92%. Additionally, no residual process enzymes were detected on the silver stain (see Figure 14). For the 15 g CFTR sample after depth filtration, the integrity was approximately 73% (see Figure 15), and the purity was very high considering the absence of process enzymes on the silver stain (Figure 16). Finally, for the 15 g CFTR sample after centrifugation, not only was the yield 100%, but the integrity was also approximately 82% (Figure 17), and the silver stain showed no residual process enzymes (Figure 18). Thus, MTEG is a highly suitable polymer for purifying mRNA using polymer-induced precipitation without the use of ethanol.

[0279] Equivalents and Ranges One of ordinary skill in the art will recognize many equivalents to the specific embodiments of the invention described herein or will be able to ascertain such equivalents using methods not departing from routine experimental work. The scope of the invention is not intended to be limited to the above description, but rather is as set forth in the following claims.

Claims

1. 1. A method for purifying messenger RNA (mRNA), comprising: a) precipitating said mRNA in a suspension comprising a high molar salt solution and an amphipathic polymer to provide a precipitated mRNA; b) capturing the precipitated mRNA; and c) washing the precipitated mRNA captured in step b) with a wash solution to purify the precipitated mRNA; d) solubilizing the precipitated mRNA from step c) to obtain a purified mRNA composition.

2. 2. The method of claim 1, wherein the purified mRNA composition is substantially free of contaminants including short abortive RNA species, long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptase, residual solvents, and / or residual salts.

3. 3. The method of claim 2, wherein the residual plasmid DNA is less than or equal to 10 pg / mg.

4. 2. The method of claim 1, wherein the amphiphilic polymer is selected from pluronics, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), or a combination thereof.

5. The method of claim 4 , wherein the amphiphilic polymer is PEG.

6. The method of claim 5, wherein the suspension comprises PEG at a concentration of about 10% to about 100% weight / volume.

7. The method of claim 6 , wherein the suspension comprises PEG at a concentration of about 50% weight / volume.

8. 10. The method of any one of the preceding claims, wherein the high molar salt solution comprises guanidinium thiocyanate (GSCN).

9. 10. The method according to any one of the preceding claims, wherein a washing solution comprising an amphiphilic polymer is used to wash the mRNA in step c).

10. The method of claim 9 , wherein the amphiphilic polymer comprises PEG.

11. The method of claim 10, wherein PEG is present in the cleaning solution at a concentration of about 10% to about 100% weight / volume.

12. The method of claim 10, wherein PEG is present in the cleaning solution at a concentration of about 50 to about 90% weight / volume.

13. 13. The method of claim 12, wherein the PEG is present in the cleaning solution at a concentration of about 90% weight / volume.

14. The method according to any one of claims 5 to 13, wherein the molecular weight of PEG is about 200 to 40,000 g / mol.

15. 14. The PEG is linear, branched, Y-shaped, or multi-armed. The method described above.

16. The method of claim 15 , wherein the PEG is linear.

17. 17. The method of claim 16, wherein the PEG solution comprises a PEG selected from triethylene glycol, tetraethylene glycol, PEG 200, PEG 300, PEG 400, PEG 600, PEG 1,000, PEG 1,500, PEG 2,000, PEG 3,000, PEG 3,350, PEG 4,000, PEG 6,000, PEG 8,000, PEG 10,000, PEG 20,000, PEG 35,000, and PEG 40,000.

18. 18. The method of claim 17, wherein the PEG solution comprises PEG 6,000.

19. The method of any one of claims 5 to 17, wherein the PEG solution does not contain PEG 6,000.

20. 18. The method of claim 17, wherein the PEG is PEG 400.

21. The method of any one of claims 5 to 20, wherein the PEG solution comprises a mixture of one or more PEG polymers.

22. 22. The method of claim 21, wherein the mixture of PEG polymers comprises polymers having distinct molecular weights.

23. 23. The method of claim 22, wherein the mixture of PEG polymers comprises polymers having distinct geometric shapes.

24. 10. The method of any one of the preceding claims, wherein the cleaning solution is aqueous.

25. 25. The method of claim 24, wherein the cleaning solution is alcohol-free.

26. 26. The method of claim 25, wherein the cleaning solution does not contain ethanol, isopropyl alcohol, or benzyl alcohol.

27. The method of any one of claims 5 to 23, wherein the PEG solution comprises a non-aqueous component.

28. 28. The method of claim 27, wherein the non-aqueous component is ethanol, isopropyl alcohol, or benzyl alcohol.

29. 13. The method of any one of the preceding claims, wherein capturing the precipitated mRNA occurs on a filter.

30. 30. The method of claim 29, wherein the filter is selected from a microfiltration filter or an ultrafiltration filter.

31. 31. The method of claim 30, wherein the microfiltration filter has a pore size of 0.05 μm to 1.0 μm.

32. 32. The method of claim 31, wherein the microfiltration filter has a nominal molecular weight limit (NMWL) of greater than 1,000 kilodaltons (kDa).

33. 31. The method of claim 30, wherein the ultrafiltration filter has a pore size of less than 0.05 μm.

34. 34. The method of claim 33, wherein the ultrafiltration filter has a NMWL of about 1 kDa to 1,000 kDa.

35. 10. The method according to any one of the preceding claims, wherein tangential flow filtration (TFF) or diafiltration is used to purify the precipitated mRNA in step c).

36. 10. The method according to any one of the preceding claims, wherein a filter aid is used.

37. 37. The method of claim 36, wherein the filter aid is cellulosic.

38. 38. The method of claim 37, wherein the filter aid comprises diatomaceous earth and / or volcanic ash.

39. 13. The method according to any one of the preceding claims, wherein the method does not include a chromatography step.

40. 2. The method of any one of the preceding claims, wherein the precipitated mRNA is centrifuged to obtain an mRNA pellet.

41. 41. The method of claim 40, wherein the mRNA pellet is resuspended in a buffer.

42. 42. The method of claim 41, wherein the buffer is selected from water, Tris-EDTA (TE), sodium citrate, or a combination thereof.

43. 10. The method of any one of the preceding claims, wherein the yield of purified mRNA is from about 50% to about 100%.

44. 44. The method of claim 43, wherein the yield of purified mRNA is from about 70% to about 99%.

45. 45. The method of claim 44, wherein the yield of purified mRNA is about 90 to about 99%.

46. 10. The method of any one of the preceding claims, wherein the purity of the purified mRNA is from about 60% to about 100%.

47. 47. The method of claim 46, wherein the purity of the purified mRNA is about 80% to 99%.

48. 48. The method of claim 47, wherein the purity of the purified mRNA is from about 90% to about 99%.

49. 1. A method for purifying messenger RNA (mRNA), comprising: a) precipitating the mRNA in a suspension comprising a high molar salt solution and an amphipathic polymer; b) capturing the mRNA on a filter; c) washing the mRNA of step b) with a PEG solution to purify it substantially free of contaminants; and obtaining an mRNA composition comprising the above-mentioned.

50. 50. The method of claim 49, wherein the yield of purified mRNA is from about 50% to about 100%.

51. 51. The method of claim 50, wherein the yield of purified mRNA is from about 70% to about 99%.

52. 52. The method of claim 51, wherein the yield of purified mRNA is about 90 to about 99%.

53. 53. The method of any one of claims 49 to 52, wherein the purity of the purified mRNA is from about 60% to about 100%.

54. 54. The method of claim 53, wherein the purity of the purified mRNA is about 80% to 99%.

55. 55. The method of claim 54, wherein the purity of the purified mRNA is from about 90% to about 99%.

56. 56. The method of any one of claims 49-55, wherein the precipitated mRNA comprises at least 100 mg, 1 g, 10 g, 100 g, 1 kg, 10 kg, 100 kg, 1 metric ton, or 1 metric ton of mRNA, or any amount therebetween.

57. 56. The method of claim 55, wherein the precipitated mRNA comprises more than 1 kg of mRNA.

58. 58. The method of any one of claims 49 to 57, wherein the amphiphilic polymer is selected from pluronics, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol (PEG), or a combination thereof.

59. 59. The method of claim 58, wherein the amphiphilic polymer is PEG.

60. 60. The method of any one of claims 49 to 59, wherein the high molar salt solution comprises guanidinium thiocyanate (GSCN).

61. 61. The method of any one of claims 49 to 60, wherein the method is ethanol-free.

62. 62. The method of any one of claims 49-61, wherein the purified mRNA composition is substantially free of contaminants including short abortive RNA species, long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptase, residual solvents, and / or residual salts.

63. 1. A method for purifying messenger RNA (mRNA), comprising: a) precipitating the mRNA in a guanidine thiocyanate (GSCN) solution containing PEG; b) centrifuging the solution of step a) to produce an mRNA pellet; c) resuspending the mRNA pellet in a buffer; d) capturing the mRNA on a filter; e) washing the mRNA of step d) with a PEG solution; f) solubilizing the washed mRNA of step e) to obtain mRNA that is substantially free of contaminants. and obtaining the NA composition.

64. 64. The method of claim 63, wherein the purified mRNA composition is substantially free of contaminants including short abortive RNA species, long abortive RNA species, double-stranded RNA (dsRNA), residual plasmid DNA, residual in vitro transcriptase, residual solvents and / or residual salts.

65. 1. A method for producing mRNA, comprising: a) performing in vitro transcription (IVT) by mixing (i) a DNA template containing a promoter and (ii) an RNA polymerase to generate an impure preparation containing full-length mRNA; b) providing a high molar salt and an amphipathic polymer to the suspension to precipitate full-length mRNA and provide precipitated full-length mRNA in said suspension; c) capturing the precipitated full-length mRNA by applying the suspension to a filter; d) washing the precipitated full-length mRNA of step (c) with an aqueous solvent to obtain purified full-length mRNA in an aqueous solution; e) solubilizing the precipitated mRNA from step (d) to obtain a purified mRNA composition, wherein the purified full-length mRNA in the aqueous solution obtained from step (d) is substantially free of (i) the DNA template, including a promoter, and (ii) the RNA polymerase.

66. 66. The method of claim 65, wherein in step (a), the RNA polymerase is SP6 polymerase.

67. 67. The method of claim 65 or 66, wherein the purified full-length mRNA in the aqueous solution resulting from step (e) is also substantially free of (v) double-stranded RNA (dsRNA).

68. 66. The method of any one of claims 1, 49 and 65, wherein the amphiphilic polymer comprises MTEG.

69. 69. The method of claim 68, wherein the suspension comprises precipitated mRNA, the high molar salt solution, and MTEG, the MTEG being at a final concentration of about 15% to about 45% weight / volume.

70. 70. The method of claim 69, wherein the suspension comprises precipitated mRNA, the high molar salt solution, and MTEG, the MTEG being at a final concentration of about 20% to about 40% weight / volume.

71. 71. The method of claim 70, wherein the suspension comprises precipitated mRNA, the high molar salt solution, and MTEG, the MTEG being at a final concentration of about 20%, about 25%, about 30%, or about 35% weight / volume.

72. 66. The method of any one of claims 1, 49 and 65, wherein the suspension comprises precipitated mRNA, a high molar salt solution, and PEG or MTEG.

73. 66. The method of claim 5, 59, or 65, wherein the high molar salt is at a final concentration of about 2-4 M and PEG is at a final concentration of about 5% to about 20% weight / volume.

74. 74. The method of claim 73, wherein the high molar salt is at a final concentration of about 2.5 to 3 M and the PEG is at a final concentration of about 10% to about 15% weight / volume.

75. 75. The method of claim 74, wherein the high molar salt is at a final concentration of about 2.7 M and the PEG is at a final concentration of about 12% weight / volume.

76. 76. The method of any one of claims 69 to 75, wherein the high molar salt solution comprises GCSN.

77. 77. The method of any one of claims 72 to 76, wherein the PEG has a molecular weight of about 6000 g / mol (e.g., PEG-6000).

78. 78. The method of any one of claims 69-77, wherein the suspension further comprises a filter aid in a mass ratio with the precipitated mRNA of 2:1, 5:1, 10:1, or 15:

1.

79. 79. The method of claim 78, wherein the filter aid is in a mass ratio with the precipitated mRNA of 10:

1.

80. 80. The method of claim 78 or 79, wherein the filter aid is cellulosic.

81. The method of claim 9 , wherein the amphiphilic polymer comprises MTEG.

82. 82. The method of claim 81, wherein MTEG is present in the cleaning solution at a concentration of about 75%, about 80%, about 85%, about 90%, or about 95% weight / volume.

83. 83. The method of claim 82, wherein the MTEG is present in the cleaning solution at a concentration of about 90% to 100% weight / volume.

84. 84. The method of claim 83, wherein the MTEG is present in the cleaning solution at a concentration of about 95% weight / volume.

85. 64. The method of claim 63, step a), wherein the GSCN is at a final concentration of about 2-4 M and the PEG is at a final concentration of about 5% to about 20% weight / volume.

86. 86. The method of claim 85, wherein the GSCN is at a final concentration of about 2.7 M and the PEG is at a final concentration of about 12% weight / volume.

87. 87. The method of claim 85 or 86, wherein the PEG has a molecular weight of about 6000 g / mol (e.g., PEG-6000).

88. 88. The method of any one of claims 85-87, wherein the solution further comprises a filter aid in a mass ratio with the precipitated mRNA of 2:1, 5:1, 10:1, or 15:

1.

89. 89. The method of claim 88, wherein the filter aid is in a mass ratio with the precipitated mRNA of 10:

1.

90. 90. The method of claim 88 or 89, wherein the filter aid is cellulosic.

91. 64. The method of claim 63, wherein the PEG in the PEG solution is at a concentration of about 50 to about 95% weight / volume.

92. The PEG in the PEG solution has a concentration of about 90 to about 100% weight / volume. Item 64. The method according to item 63, step e).

93. 91. The method of claim 90, wherein the PEG in the PEG solution is at a concentration of about 90% weight / volume.

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