Ethanol-free mRNA purification method

Abstract

Provided herein is a method for purifying messenger RNA (mRNA) by subjecting a preparation containing in vitro synthesized mRNA to one or more steps of enzymatic digestion with proteinase and optionally further by an oligo dT affinity chromatography step. Also provided is mRNA purified by the method described herein.

Description

Background Art

[0001] Messenger RNA (mRNA)-based therapeutic agents have been established as an effective means in the treatment of various diseases. mRNA molecules encode therapeutic-related proteins, such as antigens for vaccination, and are produced in the body of a subject by administration. However, mRNA molecules are relatively large, highly negatively charged, and easily degraded. In addition, the presence of contaminants remaining after mRNA purification can lead to unwanted immune responses. Therefore, it is important to develop an effective mRNA purification strategy to ensure a high-purity and safe mRNA product suitable for therapeutic and prophylactic use. General mRNA purification strategies use the use of caustic or flammable solvents such as ethanol, which is undesirable for mass production due to safety issues. Therefore, there is a need for a large-scale and rapid mRNA purification scheme that does not use flammable solvents and results in high-purity and safe mRNA molecules acceptable for therapeutic use.

Summary of the Invention

Means for Solving the Problems

[0002] In one aspect, the present disclosure provides a method for purifying messenger RNA (mRNA), comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a proteinase; and (b) subjecting the preparation obtained from step (a) to oligo dT affinity chromatography.

[0003] In certain embodiments, the proteinase comprises a serine protease.

[0004] In certain embodiments, the preparation containing in vitro synthesized mRNA is incubated with the proteinase at about 37° C. for at least 30 minutes.

[0005] In certain embodiments, the mRNA-proteinase mixture is stirred.

[0006] In certain embodiments, the protease is inactivated with a reducing agent, and optionally, the reducing agent is dithiothreitol (DTT).

[0007] In certain embodiments, DTT is added to a concentration of at least about 20 mM.

[0008] In certain embodiments, the preparation obtained from step (a) is subjected to a tangential flow filtration (TFF) step prior to step (b).

[0009] In certain embodiments, TFF uses a filter of about 100 kDa to about 300 kDa.

[0010] In certain embodiments, the mRNA contained in the preparation obtained from step (a) is concentrated using TFF.

[0011] In certain embodiments, the mRNA contained in the preparation obtained from step (a) is concentrated to at least about 5 mg / mL.

[0012] In certain embodiments, the preparation is subjected to a capping step prior to step (b) to produce capped mRNA.

[0013] In certain embodiments, the preparation is incubated with 2'O-methyltransferase and guanylyltransferase.

[0014] In certain embodiments, the preparation is incubated with an RNase inhibitor.

[0015] In certain embodiments, the preparation is incubated with GTP and S-adenosylmethionine.

[0016] In certain embodiments, capping is performed at about 37 °C for at least 30 minutes, optionally with stirring.

[0017] In certain embodiments, the preparation is subjected to a tailing step with poly(A) polymerase to produce poly(A)-tailed mRNA.

[0018] In certain embodiments, the preparation is incubated with one or more capping enzymes and poly(A) polymerase to produce capped and poly(A)-tailed mRNA.

[0019] In certain embodiments, the preparation is incubated with one or more capping enzymes and subsequently incubated with poly(A) polymerase to produce capped and poly(A)-tailed mRNA.

[0020] In certain embodiments, oligo(dT) affinity chromatography uses an oligo(dT) 25 chromatography resin.

[0021] In certain embodiments, the preparation obtained from step (b) is subjected to a TFF step.

[0022] In certain embodiments, TFF uses a filter of about 50 kDa to about 300 kDa.

[0023] In certain embodiments, the mRNA contained in the preparation obtained from step (b) is concentrated using TFF.

[0024] In certain embodiments, the mRNA contained in the preparation obtained from step (b) is concentrated to at least about 2 mg / mL.

[0025] In one aspect, the present disclosure provides a method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to a tangential flow filtration (TFF) step; (c) subjecting the preparation obtained from step (b) to oligo dT affinity chromatography; and (d) subjecting the preparation obtained from step (c) to a TFF step.

[0026] In certain embodiments, the preparation obtained from step (b) is incubated with one or more capping enzymes to produce capped mRNA.

[0027] In certain embodiments, the preparation obtained from step (b) is incubated with poly(A) polymerase to produce poly(A)-tailed mRNA.

[0028] In certain embodiments, the preparation obtained from step (b) is incubated with one or more capping enzymes and poly(A) polymerase to produce capped and poly(A)-tailed mRNA.

[0029] In one aspect, the present disclosure provides a method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to a first enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to a second enzymatic digestion with a protease; and (c) subjecting the preparation obtained from step (b) to a TFF step.

[0030] In certain embodiments, the protease comprises a serine protease.

[0031] In certain embodiments, the first enzymatic digestion and / or the second enzymatic digestion is performed at about 37 °C for at least 30 minutes.

[0032] In certain embodiments, the first enzymatic digestion and / or the second enzymatic digestion is stirred.

[0033] In certain embodiments, the protease is inactivated with a reducing agent, and optionally, the reducing agent is DTT.

[0034] In certain embodiments, DTT is added to a concentration of at least about 20 mM.

[0035] In certain embodiments of the methods described above, the precipitation step is not performed.

[0036] In certain embodiments of any of the above methods, at least about 0.5 grams of mRNA is purified.

[0037] In certain embodiments of any of the above methods, from about 0.5 grams to about 100 grams of mRNA is purified.

[0038] In certain embodiments of the methods described above, the preparation is subjected to a pre-filtration step after capping.

[0039] In certain embodiments of the methods described above, the preparation is subjected to a pre-filtration step prior to the oligo dT affinity chromatography step.

[0040] In certain embodiments of the methods described above, the preparation is subjected to a pre-filtration step after capping and prior to the oligo dT affinity chromatography step.

[0041] In certain embodiments, the filter used for pre-filtration is a 0.2 μm filter.

[0042] In one aspect, the present disclosure provides a method for manufacturing mRNA, comprising synthesizing mRNA in vitro and purifying the in vitro synthesized mRNA using the methods described above.

[0043] In another aspect, the present disclosure provides mRNA obtainable by the above methods.

[0044] The above and other features and advantages of the present disclosure will be more particularly understood from the following detailed description of exemplary embodiments in conjunction with the accompanying drawings.

Brief Description of the Drawings

[0045]

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Mode for Carrying Out the Invention

[0046] The present disclosure particularly relates to a method for purifying mRNA by subjecting a preparation consisting of in vitro synthesized mRNA to one or more steps of enzymatic digestion with proteinase and optionally further subjecting it to an oligo dT affinity chromatography step.

[0047] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings commonly understood by one of ordinary skill in the art. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control. Generally, the nomenclature used in connection with, and the laboratory procedures of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal chemistry, and pharmaceutical chemistry, as well as protein and nucleic acid chemistry and hybridization described herein and in the techniques they are used in, are those well known and commonly employed in the art. Enzyme reactions and purification techniques are performed according to manufacturers' specifications, either as commonly practiced in the art or as described herein. Further, unless the context dictates otherwise, the singular terms shall include the plural and the plural terms shall include the singular. Throughout this specification and the embodiments, the words "have" and "comprise" or variations thereof, such as "has", "having", "comprises" or "comprising", are to be understood to mean the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although many references are cited herein, this citation does not constitute an admission that any of these references forms part of the common general knowledge in the art.

[0048] It should be noted that the term "a" or "an" entity refers to one or more of that entity. For example, it is understood that a "nucleotide sequence" represents one or more nucleotide sequences. Thus, the terms "a" (or "an"), "one or more" and "at least one" may be used interchangeably herein.

[0049] Furthermore, as used herein, "and / or" shall be regarded as a specific disclosure of each of the two recited features or components, regardless of the presence or absence of the other. Thus, the term "and / or" as used in phrases such as "A and / or B" is intended herein to include "A and B", "A or B", "A" (alone), and "B" (alone). Similarly, the term "and / or" as used in phrases such as "A, B and / or C" is intended to encompass each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0050] It is understood that whenever an aspect is described herein in the language of "comprising", a similar aspect described in the terms of "consisting of" and / or "consisting essentially of" is also provided in other respects.

[0051] Unless defined otherwise, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press may provide the skilled artisan with general dictionaries for many of the terms used in this disclosure.

[0052] Units, prefixes, and symbols are shown in the form approved by the International System of Units (SI). Numerical ranges include the numbers defining the range. The headings provided in this specification do not limit the various aspects of the present disclosure. Accordingly, the terms defined immediately below are more fully defined by reference to the entire specification.

[0053] The terms "about" or "approximately" are used herein to mean about, generally, around, or within range. When the term "about" is used with a numerical range, it modifies that range by extending the boundaries above and below the recited numerical values. Generally, the term "about" can modify the numerical values above and below the recited value by, for example, a difference of up to or down to (higher or lower) 10 percent. In some embodiments, the term can indicate a deviation of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, or ±0.01% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±10% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±5% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±4% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±3% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±2% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±1% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.9% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.8% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.7% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.6% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.5% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.4% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.3% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.1% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.05% from the recited numerical value. In some embodiments, "about" indicates a deviation of ±0.01% from the recited numerical value.

[0054] As used herein, the term "batch" refers to the amount or quantity of mRNA purified at one time, e.g., that purified according to a single manufacturing order during the same manufacturing cycle. A batch can refer to the amount of mRNA purified in a single purification.

[0055] As used herein, the terms "exposed to", or "contacted with", or "incubated with" refer to the mixing of two or more components such that they can interact with each other (e.g., incubating a preparation containing in vitro synthesized mRNA with a protease). The two or more components can be incubated for a time sufficient to effect the desired result.

[0056] As used herein, a "purification method" is a method for obtaining a target molecule from a mixture, such as a solution or suspension, containing the target molecule to be purified and components other than the target molecule, wherein the concentration of the target molecule in the solution obtained after carrying out the method is enhanced or increased as compared to the concentration of the target molecule in the mixture before carrying out the method. Purification of the target molecule can also be referred to as concentration of the target molecule by removing or at least substantially removing components other than the target molecule. In the context of the present invention, the target molecule is an mRNA molecule.

[0057] More specifically, the present invention is a method for purifying mRNA, comprising (a) subjecting a preparation consisting of mRNA to enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to oligo dT affinity chromatography and a method comprising the same.

[0058] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide encoding at least one polypeptide. The mRNA can include one or more coding regions and non-coding regions. The coding region is alternatively referred to as an open reading frame (ORF).

[0059] As used herein, mRNA encompasses both modified RNA and unmodified RNA. In some embodiments, the mRNA may contain at least one chemical modification. In some embodiments, the mRNA may typically contain one or more modifications that improve RNA stability. The modified mRNA provided herein may include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, the mRNA is 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 analogs or derivatives of modified nucleotides, purines, and pyrimidines, such as 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, pseudouracil (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-pseudouracil, queuosine, β-D-mannosyl-queuosine, wybutoxosine, and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine, and inosine, etc.

[0060] In some embodiments, the mRNA may comprise at least one chemical modification including, but not limited to, pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0061] In some embodiments, the chemical modification is selected from the group consisting of pseudouridine, N1-methylpseudouridine, 5-methylcytosine, 5-methoxyuridine, and any combination thereof. In some embodiments, the chemical modification comprises N1-methylpseudouridine.

[0062] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the mRNA are chemically modified.

[0063] In some embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the uracil nucleotides in the ORF are chemically modified.

[0064] The preparation of such analogs is described, for example, in 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, and U.S. Patent No. 5,132,418.

[0065] mRNA can be synthesized in a cell-free environment, for example, by in vitro transcription (IVT). IVT is a process that enables the template-directed synthesis of ribonucleic acid (RNA), such as mRNA. In vitro transcription (IVT) is typically performed in a buffer system that may contain a promoter, a pool of ribonucleotide triphosphates, DTT, and magnesium ions, as well as a linear or circular DNA template containing an appropriate RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or an RNAse inhibitor. The exact conditions will vary depending on the specific application. Thus, in some embodiments, the production of mRNA comprises the step of performing IVT by mixing (i) a DNA template containing an appropriate promoter and (ii) an RNA polymerase to generate an impurity preparation consisting of full-length mRNA that is subjected to the purification methods disclosed herein. The presence of these reagents can be referred to as impurities that can be purified or removed to provide a clean and homogeneous mRNA suitable for therapeutic use, as they are not desirable in the final mRNA product.

[0066] In some embodiments, the DNA template is a linear DNA template. In some embodiments, the DNA template is a circular DNA template. In some embodiments, the DNA template is a PCR-amplified DNA molecule. The present invention is not limited with respect to the RNA polymerase used for transcription. For example, in some embodiments, T7, T3, or SP6 type RNA polymerases can be used. In some embodiments, the polymerase is T7 polymerase. In some embodiments, the polymerase is SP6 polymerase. In some embodiments, in vitro transcription further includes mixing a pool of ribonucleotide triphosphates.

[0067] In some embodiments, the DNA template to be transcribed can be optimized to promote more efficient transcription and / or translation. For example, the DNA template can be optimized with respect to cis-regulatory elements (e.g., TATA box, termination signals, and protein binding sites), artificial recombination sites, chi sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slip sites, and / or other elements related to transcription; the DNA template can be optimized with respect to cryptic splice sites, mRNA secondary structure, stable free energy of the mRNA, repetitive sequences, mRNA instability motifs, and / or other elements related to mRNA processing and stability; the DNA template can be optimized with respect to codon usage bias, codon adaptation, internal chi sites, ribosome binding sites (e.g., IRES), premature poly(A) sites, Shine-Dalgarno (SD) sequences, and / or other elements related to translation; and / or the DNA template can be optimized with respect to codon context, codon-anticodon interactions, translation pause sites, and / or other elements related to protein folding. For example, there is GeneOptimizer from ThermoFisher and OptimumGene® described in U.S. Patent Application Publication No. 20110081708, the contents of which are incorporated herein by reference in their entirety.

[0068] The inventors have found that precipitates can form in the in vitro transcription process. In some examples, most of the synthesized mRNA is present in this precipitate, and low concentrations of mRNA are present in the supernatant (see Example 10). Surprisingly, this precipitate could not be redissolved by dilution with water, dilution with buffer, or lowering the pH of the preparation.

[0069] The inventors attempted to add 10 mM, 25 mM, or 50 mM sodium citrate to preparations containing mRNA synthesized in vitro. A significant decrease in turbidity was observed after the addition of 10 mM sodium citrate, and complete redissolution of the precipitate was observed after the addition of 25 mM or 50 mM sodium citrate. Thus, in some embodiments, after the mRNA has been subjected to enzymatic digestion with proteinase, sodium citrate is added to the preparation containing mRNA synthesized in vitro. In some embodiments, sodium citrate is added to the preparation containing mRNA synthesized in vitro at a concentration of at least about 10 mM, optionally at least about 25 mM. In certain embodiments, sodium citrate is added to the preparation containing mRNA synthesized in vitro at a concentration of about 25 mM. This preparation can be incubated with sodium citrate for at least about 5 minutes. Before adding sodium citrate, the preparation is optionally maintained at about 37 °C (e.g., 35 - 37 °C). Alternatively, the preparation is optionally held at about 35 °C to about 37 °C for at least about 5 minutes after the addition of sodium citrate.

[0070] The inventors tested the addition of EDTA to preparations containing in vitro synthesized mRNA and also observed complete redissolution of the precipitate with 25 mM EDTA. Thus, in some embodiments, after the mRNA has undergone enzymatic digestion by proteinase, EDTA is added to the preparation containing in vitro synthesized mRNA. In some embodiments, EDTA is added to the preparation containing in vitro synthesized mRNA at a concentration of at least about 10 mM, optionally at least about 25 mM. In certain embodiments, EDTA is added to the preparation containing in vitro synthesized mRNA at a concentration of about 25 mM. This preparation can be incubated with EDTA for at least about 5 minutes. Prior to adding EDTA, the preparation is optionally maintained at about 37°C (e.g., 35 - 37°C). Alternatively, the preparation is optionally maintained at about 35°C to about 37°C for at least about 5 minutes after the addition of EDTA.

[0071] As used herein, the terms "shortmer" or "truncated RNA sequence" refer to incomplete products of an mRNA synthesis reaction (e.g., an in vitro synthesis reaction). For various reasons, RNA polymerase does not necessarily complete the transcription of a DNA template completely; for example, RNA synthesis terminates prematurely. Possible causes for premature termination of RNA synthesis include the quality of the DNA template, polymerase terminator sequences specific to the polymerase present in the template, degraded buffer, temperature, ribonucleotide depletion, and mRNA secondary structure. A truncated RNA sequence can be of any length if it is shorter than the intended length of the desired transcript. For example, a truncated mRNA sequence can 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, less than 10 bases, or less.

[0072] Double-stranded RNA (dsRNA) can also be formed during in vitro synthesis of mRNA (for example, when the 3' end of the full-length RNA transcript hybridizes to itself). Since dsRNA can have immunostimulatory effects, this is undesirable. Removing dsRNA contamination from the mRNA product is difficult because the size and essential nature of dsRNA are similar. RNase III (also known as RNase C) can be used to digest dsRNA in the presence of the desired mRNA product to facilitate its removal. However, since RNase III also digests the mRNA product of interest, the proportion of intact mRNA often decreases, and additional method steps are required to remove RNAse III from the mRNA preparation.

[0073] As used herein, the term "substantially" refers to a qualitative condition indicating the entire or nearly entire range or degree of a particular feature or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to a state of completion and / or fruition or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical events.

[0074] As used herein, the term "substantially free" refers to a state in which the substance to be removed (e.g., an interrupted RNA sequence) is relatively little or not present at all. For example, "substantially free of interrupted RNA sequences" means that the interrupted RNA sequences are present at a level of 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 impurities. Alternatively, "substantially free of interrupted RNA sequences" means that the interrupted RNA sequences are present at a level of about 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.

[0075] In one embodiment, a method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to oligo dT affinity chromatography is provided.

[0076] The present invention can be used for purifying mRNA encoding any protein. Usually, the present invention is used for purifying a single mRNA species. That is, a preparation consisting of mRNA contains mRNA derived from a single gene, a single synthesis reaction or a single expression construct. In contrast, total mRNA purified from cells contains multiple mRNA species. In some embodiments, mRNA provided by an in vitro transcription reaction may be desirable, while other sources of mRNA, including wild-type mRNA produced from bacteria, fungi, plants, and / or animals, are also contemplated as being within the scope of the present invention.

[0077] As used herein, the term "in vitro" refers to events that occur not within a multicellular organism but in an artificial environment, such as in a test tube or reaction vessel, in a cell culture, etc.

[0078] 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 lengths 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 more than 15 kb. In some embodiments, the present invention is used to purify mRNAs that generally contain one or more modifications that enhance stability. In some embodiments, the one or more modifications are selected from modified nucleotides and modified sugar phosphate backbones as described herein. In some embodiments, the present invention is used to purify unmodified in vitro synthesized mRNAs.

[0079] Typically, an mRNA has a 5' cap structure. The 5' cap of an mRNA can provide resistance to nucleases found in most eukaryotic cells and can enhance translation efficiency. Several types of 5' caps are known. The 7-methylguanosine cap (also referred to as "m7G" or "cap-0") contains guanosine linked to the first transcribed nucleotide through a 5'-5'-triphosphate bond.

[0080] The term "capping" refers to the addition of a 5' cap structure to mRNA. The 5' cap is typically added as follows: First, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates; subsequently, guanosine triphosphate (GTP) is added to the terminal phosphate by guanylyl transferase, generating a 5'5'5 triphosphate bond; subsequently, the 7-nitrogen of guanine is methylated by a 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. Additional cap structures are described in U.S. Patent Application Publication No. 2016 / 0032356 and U.S. Patent Application Publication No. 2018 / 0125989, which are incorporated herein by reference.

[0081] 5'-Capping can be completed simultaneously during the IVT reaction using a cap analog and incorporated as the first "base" of the nascent RNA strand. The cap analog can be a cap 0, cap 1, cap 2, m6Am, or a cap analog. As an example, to generate a 5'-guanosine cap structure, the following chemical RNA cap analogs can be used according to the manufacturer's instructions: 3'-O-Me-m7G(5')ppp(5')G (ARCA cap); G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G; m7G(5')ppp(5')(2'OMeA)pG; m7G(5')ppp(5')(2'OMeA)pU; m7G(5')ppp(5')(2'OMeG)pG (New England BioLabs, Ipswich, MA; TriLink Biotechnologies).

[0082] Alternatively, capping of the mRNA can be performed post-transcriptionally (i.e., after IVT). For example, vaccinia virus capping enzyme can be used to generate the cap 0 structure: m7G(5’)ppp(5’)G. Both vaccinia virus capping enzyme and 2’-O-methyl-transferase can be used to generate the cap 1 structure to produce m7G(5’)ppp(5’)G-2’-O-methyl. The cap 2 structure is generated from the cap 1 structure, and subsequently 2’-O-methylation of the second nucleotide from the 5’-end can be performed using 2’-O-methyl-transferase. The cap 3 structure is generated from the cap 2 structure, and subsequently 2’-O-methylation of the third nucleotide from the 5’-end can be performed using 2’-O-methyl-transferase.

[0083] In some embodiments, the mRNA comprises a 5’ cap selected from the group consisting of 3’-O-Me-m7G(5’)ppp(5’)G (ARCA cap), G(5’)ppp(5’)A, G(5’)ppp(5’)G, m7G(5’)ppp(5’)A, m7G(5’)ppp(5’)G, m7G(5’)ppp(5’)(2’OMeA)pG, m7G(5’)ppp(5’)(2’OMeA)pU, and m7G(5’)ppp(5’)(2’OMeG)pG. In certain embodiments, the mRNA comprises the following 5’ caps.

Chemical formula

[0084] “Capping enzyme” refers to an enzyme that catalyzes the attachment of a 5’ cap to the mRNA. It is also possible to use a single capping enzyme that contains the enzymatic activities necessary for capping. Alternatively, two or more enzymes with different enzymatic activities, such as 2’O-methyltransferase and guanylyltransferase, can be used. Capping can occur in the presence of an RNAse inhibitor, S-adenosylmethionine which is a methyl donor, and / or GTP.

[0085] In some embodiments, the mRNA comprises a 5' and / or 3' untranslated region (UTR). In some embodiments, the 5'UTR comprises one or more elements that affect the stability or translation of the mRNA, such as an iron-responsive element. Exemplary 5' and / or 3' UTR sequences may be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histone, and citric acid cycle enzymes) to enhance the stability of the sense mRNA molecule. For example, the 5'UTR sequence may comprise a partial sequence of the CMV immediate early 1 (IE1) gene or a fragment thereof to improve nuclease resistance and / or improve the half-life of the polynucleotide. Inclusion of a sequence encoding human growth hormone (hGH) or a fragment thereof at the 3' end or untranslated region of the mRNA is also contemplated.

[0086] In some embodiments, the 5'UTR can be about 50 to 500 nucleotides in length. In some embodiments, the 3'UTR comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the mRNA located in the cell, or one or more binding sites for miRNAs. In some embodiments, the 3'UTR can be 50 to 500 nucleotides or longer in length.

[0087] Typically, mRNA contains a poly(A) tail. As used herein, the terms "poly(A) sequence", "poly(A) tail", and "poly(A) portion" refer to the sequence of adenosine nucleotides at the 3' end of an mRNA molecule. The poly(A) tail can confer stability to the mRNA and protect the mRNA from exonucleolytic degradation. The poly(A) tail can enhance translation. In some embodiments, the poly(A) tail is essentially a homopolymer. For example, a poly(A) tail of 100 adenosine nucleotides can essentially have a length of 100 nucleotides. If the mRNA contains a 3'UTR, the poly(A) tail can be directly downstream of the coding region (i.e., before the 3'UTR) or downstream of the 3'UTR. The poly(A) tail can contain 10 to 500 adenosine monophosphates. For example, the poly(A) tail can contain at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, or 500 adenosine monophosphates. In some embodiments, the poly(A) tail contains 20 to 300 adenosine monophosphates. In some embodiments, the poly(A) tail contains 50 to 250 adenosine monophosphates. In some embodiments, the poly(A) tail contains 75 to 225 adenosine monophosphates. In some embodiments, the poly(A) tail contains 90 to 200 adenosine monophosphates. In some embodiments, the poly(A) tail contains 100 to 150 adenosine monophosphates. In some embodiments, the poly(A) tail contains at least 100 adenosine monophosphates. In some embodiments, the poly(A) tail contains at least 150 adenosine monophosphates. In some embodiments, the poly(A) tail contains at least 200 adenosine monophosphates. In some embodiments, the poly(A) tail contains at least 250 adenosine monophosphates.In some embodiments, the poly(A) tail comprises at least 300 adenosine monophosphates. In some embodiments, the poly(A) tail comprises at least 350 adenosine monophosphates. In some embodiments, the poly(A) tail comprises at least 400 adenosine monophosphates. In some embodiments, the poly(A) tail comprises at least 450 adenosine monophosphates. In certain embodiments, the poly(A) tail can be interrupted by at least one nucleotide different from an adenosine nucleotide (e.g., a nucleotide that is not an adenosine nucleotide). For example, a 100-adenosine nucleotide poly(A) tail can have a length greater than 100 nucleotides (including 100 adenosine nucleotides and at least one nucleotide or stretch of nucleotides different from an adenosine nucleotide).

[0088] In some embodiments, the poly(A) tail of a nucleic acid is obtained from a DNA template during in vitro transcription of RNA. In certain embodiments, the poly(A) tail is obtained in vitro by general chemical synthesis methods without being transcribed from a DNA template. In various embodiments, the poly(A) tail is generated by enzymatic polyadenylation of RNA (after in vitro transcription of RNA) using a commercially available polyadenylation kit and corresponding protocol or alternatively by using immobilized poly(A) polymerase using, for example, the methods and means described in WO 2016 / 174271 pamphlet.

[0089] The nucleic acid can contain a poly(A) tail obtained by enzymatic polyadenylation, and most of the nucleic acid molecule contains about 100 (+ / -20) to about 500 (+ / -50) or about 250 (+ / -20) adenosine nucleotides.

[0090] In some embodiments, the nucleic acid can contain a poly(A) tail derived from template DNA, as described, for example, in WO 2016 / 091391 pamphlet, and can additionally contain at least one further poly(A) tail generated by enzymatic polyadenylation.

[0091] As used herein, the term "protease" refers to a protease that catalyzes the hydrolysis of a protein by splitting the protein into smaller peptide fractions, i.e., polypeptides or amino acids. In other words, a protease enzymatically digests a protein or polypeptide. Examples of proteases or proteinases include, but are not limited to, serine proteases (e.g., proteinase K), cysteine proteases, threonine proteases, aspartic proteases, glutamic acid proteases, metalloproteases, and asparagine peptide lyases.

[0092] "Isolated," "isolated," "purified," "purified," "concentrated," and "concentrated," when used with respect to the mRNA of interest, indicate that at some point the mRNA of interest has been separated, concentrated, sorted, etc. from or with respect to other biological materials or chemical components, such as components of an IVT reaction, such that the proportion of the mRNA of interest is higher compared to other biological materials, chemical components, contaminants, or active agents such as enzymes. The isolated mRNA can be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 65%, about 70%, about 75%, 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 about 99% or more of the other components to which it was initially bound. In some embodiments, the isolated mRNA is at a purity of 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 greater than about 99%. As used herein, a substance is "pure" if it is substantially free of other components. Components for isolating mRNA include, but are not limited to, dsRNA, plasmid DNA, enzymes, and endotoxins. As used herein, excipients (buffers, water, etc.) should not be included in the calculation of the percentage purity of the isolated substance.

[0093] 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 the acceptable level of residual plasmid DNA. In some embodiments, the residual plasmid DNA in the purified mRNA of the present invention 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 is less than about 1 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 2 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 3 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 4 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 5 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 6 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 7 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 8 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 9 pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 10 pg / mg.

[0094] The inventors have shown that the methods provided herein can efficiently remove residual plasmid DNA from large batches of mRNA. In some embodiments, in vitro synthesized mRNA is purified on a scale of about 10 grams or more per batch, and the residual plasmid DNA in the purified mRNA is less than about 0.5 pg / mg (e.g., less than about 0.2 pg / mg). In some embodiments, in vitro synthesized mRNA is purified on a scale of about 20 grams or more per batch, and the residual plasmid DNA in the purified mRNA is less than about 0.5 pg / mg (e.g., less than about 0.2 pg / mg). In some embodiments, the residual plasmid DNA is evaluated by methods in the art, for example, by use of qPCR.

[0095] In some embodiments, the method according to the invention removes RNA sequences interrupted in more than about 80%, more than about 85%, more than about 90%, more than about 95%, more than about 96%, more than about 97%, more than about 98%, more than about 99% or substantially all of the way. In some embodiments, the mRNA composition is substantially free of RNA sequences interrupted in the middle. In some embodiments, the mRNA composition contains less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2% or less than about 1%) of RNA sequences interrupted in the middle. In some embodiments, the mRNA composition contains less than about 1% (e.g., less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2% or less than about 0.1%) of RNA sequences interrupted in the middle. In some embodiments, the mRNA isolated according to the method of the invention contains undetectable interrupted RNA sequences as determined, for example, by high performance liquid chromatography (HPLC) (e.g., shoulders or separate peaks), ethidium bromide, Coomassie staining, capillary electrophoresis (CE) or glyoxal gel electrophoresis (e.g., the presence of a separate lower band). In some embodiments, the length of the interrupted RNA sequence is less than 1000 bases, less than 500 bases, less than 100 bases, less than 90 bases, less than 80 bases, less than 70 bases, less than 60 bases, less than 50 bases, less than 40 bases, less than 30 bases, less than 20 bases or less than 10 bases. In some embodiments, the shortmer is detected or quantified after addition of a 5'-cap and / or 3'-polyA tail. In some embodiments, the interrupted RNA transcript contains less than 15 bases (e.g., less than 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4 or 3 bases). In some embodiments, the interrupted RNA transcript contains about 8-15, 8-14, 8-13, 8-12, 8-11 or 8-10 bases.

[0096] The inventors have shown that double-stranded RNA (dsRNA) can also be efficiently removed from large batches of mRNA using the methods provided herein. In some embodiments, the purified mRNA contains less than about 1% (e.g., less than about 0.5%, less than about 0.2% or less than about 0.1%) dsRNA. In some embodiments, the purified mRNA contains less than about 0.1% (e.g., less than about 0.05%, less than about 0.03%, less than about 0.025% or less than about 0.01%) dsRNA. In some embodiments, the dsRNA levels are evaluated by methods in the art such as ELISA or dot blot using the anti-dsRNA monoclonal antibody J2. In some embodiments, the method does not include the step of contacting the preparation with RNase III.

[0097] In some embodiments, the in vitro synthesized mRNA is purified on a scale of about 10 grams or more per batch, and the purified mRNA composition contains less than about 0.1% dsRNA. In some embodiments, the in vitro synthesized mRNA is purified on a scale of about 10 grams or more per batch, and the purified mRNA composition contains less than about 0.01% dsRNA. In some embodiments, the in vitro synthesized mRNA is purified on a scale of about 20 grams or more per batch, and the purified mRNA composition contains less than about 0.025% dsRNA.

[0098] In some embodiments, the in vitro synthesized mRNA is purified on a scale of about 10 grams or more per batch, and the purified mRNA composition contains less than about 0.1% dsRNA, and the purified mRNA has a completeness of about 85% or more. In some embodiments, the in vitro synthesized mRNA is purified on a scale of about 10 grams or more per batch, and the purified mRNA composition contains less than about 0.01% dsRNA, and the purified mRNA has a completeness of about 85% or more. In some embodiments, the in vitro synthesized mRNA is purified on a scale of about 20 grams or more per batch, and the purified mRNA composition contains less than about 0.025% dsRNA, and the purified mRNA has a completeness of about 85% or more.

[0099] In some embodiments, the mRNA purified according to the methods provided herein is substantially free of enzyme reagents used in IVT, including but not limited to RNA polymerase, DNAse I, pyrophosphatase, and / or RNase inhibitors. In some embodiments, the purified mRNA according to the present invention contains less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of the enzyme reagents used in in vitro synthesis. In some embodiments, the purified mRNA contains less than about 1% (e.g., less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1%) of the enzyme reagents used in IVT. In some embodiments, the purified mRNA contains undetectable enzyme reagents used in IVT, as determined by, for example, silver staining, gel electrophoresis, HPLC, ultra-high performance liquid chromatography (UPLC), and / or CE, ethidium bromide, and / or Coomassie staining.

[0100] In various embodiments, the mRNA purified according to the methods provided herein maintains high integrity. As used herein, the term "mRNA integrity" generally refers to the quality of the mRNA after purification. The integrity of mRNA can be determined using methods well known in the art, such as, for example, RNA agarose gel electrophoresis. In some embodiments, mRNA integrity refers to the percentage of mRNA that has not been degraded after the purification process. In some embodiments, the integrity of mRNA can be determined by the banding pattern of RNA agarose gel electrophoresis. In some embodiments, the purified mRNA of the present invention shows little or no banding compared to the reference band of RNA agarose gel electrophoresis. In some embodiments, the purified mRNA of the present invention has an integrity of greater than about 60%, 70%, 80%, 90% or 95% (e.g., greater than about 96%, 97%, 98%, 99% or more). In some embodiments, the purified mRNA of the present invention has an integrity of greater than 98%. In some embodiments, the purified mRNA of the present invention has an integrity of greater than 99%. In some embodiments, the purified mRNA of the present invention has an integrity of approximately 100%.

[0101] In some embodiments, the mRNA synthesized in vitro is purified on a scale of about 10 grams or more per batch, and the purified mRNA has an integrity of greater than about 80% (e.g., at least about 85%). In some embodiments, the mRNA synthesized in vitro is purified on a scale of about 20 grams or more per batch, and the purified mRNA has an integrity of greater than about 80% (e.g., at least about 85%).

[0102] In some embodiments, the purified mRNA is evaluated with respect to one or more of the following characteristics: appearance, identity, quantity, concentration, presence of impurities, microbiological assessment, pH level, and activity. In some embodiments, an acceptable appearance comprises a clear, colorless solution essentially free of visible particulates. 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% to 110% (e.g., 0.9 to 1.1 mg / mL).

[0103] In some embodiments, evaluating the purity of the mRNA includes evaluating the integrity of the mRNA and evaluating residual plasmid DNA. In some embodiments, an acceptable level of integrity of the mRNA is evaluated by agarose gel electrophoresis. The gel is analyzed to determine whether the banding pattern and apparent nucleotide length match the analytical reference standards. Additional methods for evaluating the integrity of the RNA include, for example, evaluation of the purified mRNA using capillary gel electrophoresis (CGE). In some embodiments, an acceptable purity of the purified mRNA as determined by CGE is that the purified mRNA composition has species with interrupted / degraded lengths of about 40% or less.

[0104] In some embodiments, microbiological tests are performed on the purified mRNA, including, for example, the evaluation of endotoxins in bacteria. In some embodiments, the endotoxins in bacteria are <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 endotoxins in the purified mRNA are <0.5 EU / mL. In some embodiments, the endotoxins in the purified mRNA are <0.4 EU / mL. In some embodiments, the endotoxins in the purified mRNA are <0.3 EU / mL. In some embodiments, the endotoxins in the purified mRNA are <0.2 EU / mL. In some embodiments, the endotoxins in the purified mRNA are <0.1 EU / mL.

[0105] In some embodiments, the pH of the purified mRNA is evaluated. In some embodiments, the acceptable pH of the purified mRNA is 5-8. 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 pH of this purified mRNA is about 7. In some embodiments, the pH of this purified mRNA is about 7. In some embodiments, the pH of this purified mRNA is about 8.

[0106] In some embodiments, the translation fidelity of the purified mRNA is evaluated. The translation fidelity can be evaluated by various methods, including, for example, transfection and Western blot analysis. The acceptable characteristics of the purified mRNA include the band formation pattern on a Western blot that migrates with a molecular weight similar to that of the standard. In some embodiments, the purified mRNA is evaluated for permeability. In some embodiments, the acceptable characteristics of the purified mRNA include a permeability that is about 50% - 150% of the standard.

[0107] The purified mRNA is also evaluated for cap percentage and poly(A) tail length. In some embodiments, an acceptable cap percentage includes % area: NLT90 for cap 1. In some embodiments, an acceptable poly(A) tail length is 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 poly(A) tail length is about 100 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 200 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 250 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 300 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 350 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 400 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 450 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 500 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 550 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 600 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 650 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 700 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 750 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 800 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 850 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 900 nucleotides. In some embodiments, an acceptable poly(A) tail length is about 950 nucleotides.In some embodiments, the acceptable poly(A) tail length is about 1000 nucleotides. In some embodiments, the acceptable poly(A) tail length is about 1100 nucleotides. In some embodiments, the acceptable poly(A) tail length is about 1200 nucleotides. In some embodiments, the acceptable poly(A) tail length is about 1300 nucleotides. In some embodiments, the acceptable poly(A) tail length is about 1400 nucleotides. In some embodiments, the acceptable poly(A) tail length is about 1500 nucleotides.

[0108] In some embodiments, the mRNA synthesized in vitro is purified on a scale of about 10 grams or more per batch, and the purified mRNA has a cap rate of at least about 90%. In some embodiments, the mRNA synthesized in vitro is purified on a scale of about 20 grams or more per batch, and the purified mRNA has a cap rate of at least about 90%.

[0109] In some embodiments, the mRNA synthesized in vitro is purified on a scale of about 10 grams or more per batch and has a poly(A) tail length of at least about 100 nucleotides. In some embodiments, the mRNA synthesized in vitro is purified on a scale of about 20 grams or more per batch and has a poly(A) tail length of at least about 100 nucleotides.

[0110] A variety of 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, the synthesized mRNA is characterized prior to capping or tailing. In some embodiments, the synthesized mRNA is characterized after capping and tailing.

[0111] The preparation containing the mRNA provided in step (a) is an impure preparation (i.e., contains impurities). As used herein, the term "impurity" refers to a substance present in a limited amount of liquid, gas, or solid that differs from the chemical composition of the material or compound of interest. Impurities are also referred to as contaminants. The mRNA purification strategies described herein can remove a number of impurities associated with IVT-produced mRNA, such as IVT enzymes (i.e., RNA polymerases), free NTPs, buffer components (i.e., salts), mRNA fragments (e.g., truncated sequences), and tail-less mRNA. The mRNA purification strategies described herein can also remove nucleic acid contaminants such as double-stranded RNA and residual plasmid DNA. Thus, the mRNA purification strategies described herein can be used to provide a purified mRNA preparation suitable for therapeutic use.

[0112] As used herein, the term "affinity chromatography" or "affinity purification" refers to a separation method based on specific binding interactions between a ligand immobilized or bound to a solid support and its binding partner. When a complex mixture is passed through a column, molecules having a specific binding affinity for the ligand bind. After the other sample components are washed away, the bound molecules are removed from the solid support and, as a result, are purified from the original mixture. Each specific affinity system requires a unique set of conditions known to those skilled in the art.

[0113] As used herein, the term "oligo dT" refers to a single-stranded sequence of deoxythymidine (dT). Oligo dT can bind to the poly(A) tail of an mRNA molecule and can be used as a method for purifying mRNA. Oligo dT can be of any length suitable for hybridizing to the poly(A) tail, for example, 12 - 50 mer. Multiple types of oligo dT ligands are known in the art and are commercially available. For example, oligo(dT) 25 is a homogeneous mixture of 25-mer deoxythymidine, while oligo(dT) 12~18 is a mixture of 12-mer to 18-mer deoxythymidine.

[0114] In some embodiments, the method of purifying mRNA does not include the step of applying the preparation to a primary amino solid phase. The term "primary amino solid phase" refers to a solid phase suitable for performing chromatography, on the surface of which primary amino ligands are predominantly or exclusively present.

[0115] I. Proteinase treatment In some embodiments, one or more proteinases can be used to enzymatically digest the proteins used in in vitro transcription, DNase treatment, capping and / or tailing reactions to facilitate the purification of mRNA. In some embodiments, the proteinases include serine proteases (e.g., proteinase K, chymotrypsin and chymotrypsin-like serine proteases, trypsin and trypsin-like serine proteases, elastase and elastase-like serine proteases, subtilisin and subtilisin-like serine proteases), cysteine proteases, threonine-like proteases, asparagine proteases, metalloproteases, and asparagine peptide lyases.

[0116] In some embodiments, the protease is a serine protease. In some embodiments, the protease is Proteinase K. Proteinase K is a serine protease that exhibits a very broad cleavage specificity and cleaves peptide bonds adjacent to the carboxyl groups of aliphatic and aromatic amino acids. By subjecting a preparation containing mRNA to enzymatic digestion using a protease such as a serine protease, enzymatic degradation and inactivation can be advantageously achieved. Proteinase treatment significantly facilitates downstream mRNA purification. Short peptide fragments of IVT enzymes can be more easily purified by filtration (i.e., TFF) and / or chromatography-based purification (e.g., affinity chromatography such as oligo dT chromatography). In some embodiments, the serine protease is Proteinase K from Engyodontium album. In some embodiments, Proteinase K is a recombinant protein. In some embodiments, Proteinase K is heat labile. In some embodiments, Proteinase K is selected from among the variants described in U.S. Patent Application Publication No. 10633644 B1.

[0117] In some embodiments, a preparation containing in vitro synthesized mRNA is subjected to enzymatic digestion with a protease, thereby obtaining an mRNA-protease mixture. In some embodiments, a preparation containing in vitro synthesized mRNA is incubated with a protease at about 25°C to about 40°C for at least 30 minutes. In some embodiments, a preparation containing in vitro synthesized mRNA is incubated with a protease at about 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C for at least 30 minutes. In some embodiments, a preparation containing in vitro synthesized mRNA is incubated with a protease at about 37°C for at least 30 minutes. In some embodiments, a preparation containing in vitro synthesized mRNA is incubated with a protease at about 25°C to about 40°C for about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 135 minutes, about 150 minutes, about 165 minutes, about 180 minutes, about 195 minutes, about 210 minutes, about 225 minutes, or about 240 minutes. In some embodiments, a preparation containing in vitro synthesized mRNA is incubated with a protease at about 37°C for about 30 minutes, about 45 minutes, about 60 minutes, about 75 minutes, about 90 minutes, about 105 minutes, about 120 minutes, about 135 minutes, about 150 minutes, about 165 minutes, about 180 minutes, about 195 minutes, about 210 minutes, about 225 minutes, or about 240 minutes.

[0118] In some embodiments, the protease is at a concentration of at least about 0.02 U / mg RNA. In some embodiments, the protease is at a concentration of at least about 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, or 0.5 U / mg RNA. In some embodiments, the protease is at a concentration of 0.02 - 0.6 U / mg RNA. In some embodiments, the protease is at a concentration of 0.02 - 0.03 U / mg RNA. In some embodiments, the protease is at a concentration of about 0.5 - 0.6 U / mg RNA. In some embodiments, the protease is at a concentration of about 0.55 U / mg RNA. In some embodiments, a preparation containing in vitro synthesized mRNA is incubated with a protease at a concentration of 0.02 - 0.6 U / mg RNA at about 37°C for at least about 30 minutes.

[0119] In some embodiments, the mRNA-protease mixture (i.e., a preparation containing in vitro synthesized mRNA and protease) is agitated. As used herein, the term "agitated" means any action suitable for mixing, for example, a mixture composed of a preparation containing in vitro synthesized mRNA and protease. Exemplary devices for achieving agitation are known to those of skill in the art and include shakers, mixers (e.g., vortex mixers or static mixers), magnetic stirrers (including stir bars), and stir bars, which are available in different sizes depending on the volume of the mixture. Agitation can be carried out for a time sufficient to achieve complete mixing. Agitation enhances the overall effectiveness of protease treatment, particularly for large-scale mRNA purification of at least about 1 gram.

[0120] In some embodiments, the mRNA purification described herein includes one step of enzymatic digestion with a protease, followed by an affinity chromatography step (e.g., oligo dT). Additional steps such as a TFF step, a capping step, a tailing step, etc. may be present between the step of enzymatic digestion with a protease and the step of affinity chromatography.

[0121] In one aspect, the present specification provides a method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to a TFF step; (c) subjecting the preparation obtained from step (b) to oligo dT affinity chromatography; and (d) subjecting the preparation obtained from step (c) to a TFF step.

[0122] In other embodiments, the mRNA purification described herein includes a first step of enzymatic digestion with a protease, followed by a second step of enzymatic digestion with a protease. As provided herein, at least one additional step such as a TFF step, a capping step, a tailing step, and / or a pre-filtration step occurs between the first and second steps of enzymatic digestion with a protease. In some implementations, the first step of enzymatic digestion is performed following the TFF step, and then the capping and / or tailing steps are performed. In some embodiments, the first step of enzymatic digestion is performed following the TFF step, then followed by the capping and / or tailing steps and finally the pre-filtration step. In some embodiments where the first and second steps of enzymatic digestion with a protease are performed, the affinity chromatography step is not performed.

[0123] In one aspect, the present specification provides a method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to a first enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to a second enzymatic digestion with a protease; and (c) subjecting the preparation obtained from step (b) to a tangential flow filtration (TFF) step.

[0124] In another aspect, the present specification provides a method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to a TFF step; (c) subjecting the preparation obtained from step (b) to enzymatic digestion with a protease; and (d) subjecting the preparation obtained from step (c) to a TFF step.

[0125] In another aspect, the present disclosure provides a method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to an incubation step with a first protease to generate a first mRNA-protease mixture; (b) subjecting the first mRNA-protease mixture to a first TFF step to generate a first filtered mRNA mixture; (c) subjecting the first filtered mRNA mixture to an incubation step with a second protease to generate a second mRNA-protease mixture; and (d) subjecting the second mRNA-protease mixture to a second TFF step to generate purified mRNA.

[0126] In any of the methods provided herein, a precipitation step is not performed (e.g., a preparation containing in vitro synthesized mRNA does not undergo a precipitation step with ethanol).

[0127] Particularly excellent advantages provided by the present invention are that mRNA synthesized in vitro, particularly mRNA, can be purified on a large scale or a commercial scale. For example, in some embodiments, the mRNA synthesized in vitro is purified on a scale of about 100 milligrams, about 1 gram, about 10 grams, about 50 grams, about 100 grams or more per batch.

[0128] In a particular embodiment, the mRNA synthesized in vitro is purified on a scale of 10 grams per batch. In a particular embodiment, the mRNA synthesized in vitro is purified on a scale of 20 grams per batch. In a particular embodiment, the mRNA synthesized in vitro is purified on a scale of 25 grams per batch. In a particular embodiment, the mRNA synthesized in vitro is purified on a scale of 50 grams per batch. In another particular embodiment, the mRNA synthesized in vitro is purified on a scale of 100 grams or more per batch.

[0129] In any of the methods provided herein, at least about 0.5 grams of mRNA is purified. In some embodiments, from about 0.5 grams to about 100 grams of mRNA is purified. In some embodiments, about 0.5 grams, about 1 gram, about 5 grams, about 10 grams, about 15 grams, about 20 grams, about 25 grams, about 30 grams, about 35 grams, about 40 grams, about 45 grams, about 50 grams, about 55 grams, about 60 grams, about 65 grams, about 70 grams, about 75 grams, about 80 grams, about 85 grams, about 90 grams, about 95 grams or about 100 grams of mRNA is purified. In some embodiments, at least 0.5 grams, at least 1 gram, at least 5 grams, at least 10 grams, at least 15 grams, at least 20 grams, at least 25 grams, at least 30 grams, at least 35 grams, at least 40 grams, at least 45 grams, at least 50 grams, at least 55 grams, at least 60 grams, at least 65 grams, at least 70 grams, at least 75 grams, at least 80 grams, at least 85 grams, at least 90 grams, at least 95 grams or at least 100 grams of mRNA is purified.

[0130] In further embodiments, the serine protease is inactivated after incubation with a preparation containing mRNA transcribed in vitro. In some embodiments, the serine protease is inactivated by heating. In some embodiments, the serine protease is inactivated by heating the preparation to at least 55°C. In some embodiments, the serine protease is inactivated with a chelating agent such as EDTA. In some embodiments, the serine protease is inactivated with a serine protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF). In some embodiments, the serine protease is inactivated with a reducing agent. Non-limiting examples of reducing agents include dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP). In certain embodiments, DTT is added to a concentration of at least about 20 mM. In certain embodiments, DTT is added to a concentration of about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM or about 100 mM.

[0131] II. Tangential Flow Filtration (TFF) Tangential flow filtration (TFF), also called cross-flow filtration, is a type of filtration in which the material being filtered passes tangentially across the filter rather than through it. In TFF, the unwanted permeate passes through the filter while the desired concentrate (e.g., mRNA) flows along the filter and is captured or retained by a downstream filter or membrane. In contrast, in frontal filtration, the direction of flow is perpendicular to the filter surface when the material being filtered passes through the filter.

[0132] In an embodiment of the mRNA purification method described herein, an mRNA-containing sample (e.g., a preparation containing in vitro synthesized mRNA or an mRNA-protease mixture) is subjected to at least one TFF step (i.e., a first TFF step). In some embodiments, the sample containing mRNA is subjected to a second TFF step.

[0133] In some embodiments, the mRNA-protease mixture is subjected to at least the first TFF step prior to the oligo dT affinity chromatography step. In some embodiments, the mRNA-protease mixture is subjected to at least the first TFF step after the first step of enzymatic digestion with protease and before the second step of enzymatic digestion with protease.

[0134] In one embodiment, the TFF step includes a filter of about 50 kDa to about 300 kDa. In some embodiments, the TFF step includes a 50 kDa filter, 60 kDa filter, 70 kDa filter, 80 kDa filter, 90 kDa filter, 100 kDa filter, 110 kDa filter, 120 kDa filter, 130 kDa filter, 140 kDa filter, 150 kDa filter, 160 kDa filter, 170 kDa filter, 180 kDa filter, 190 kDa filter, 200 kDa filter, 210 kDa filter, 220 kDa filter, 230 kDa filter, 240 kDa filter, 250 kDa filter, 260 kDa filter, 270 kDa filter, 280 kDa filter, 290 kDa filter or 300 kDa filter. In some embodiments, the TFF step includes a filter of about 100 kDa to about 300 kDa. In some embodiments, the TFF step includes a filter of about 50 kDa to about 100 kDa. The kDa filter size means the molecular weight cut-off (MWCO).

[0135] Suitable membranes for TFF can be made of any material. Suitable membrane materials include, but are not limited to, polyethersulfone (PES) (unmodified), modified polyethersulfone (mPES; such as mPES hollow fiber membranes), polyvinylidene fluoride (PVDF), cellulose acetate, nitrocellulose cellulose acetate, nitrocellulose, mixed cellulose ester (MCE), ultra-high molecular weight polyethylene (UPE), polytetrafluoroethylene (PTFE), nylon, polysulfone, polyethersulfone, polyacrylonitrile, polypropylene, polyvinyl chloride, and any combination thereof. In one embodiment, the membrane is cellulose. In one embodiment, the membrane is a hollow fiber membrane of polyethersulfone (mPES).

[0136] The membranes suitable for the present invention can have various surface areas. In some embodiments, the suitable membranes have a surface area large enough to promote large-scale production of mRNA. For example, suitable membranes can have a surface area of about 1,500 cm 2 , 2,000 cm 2 , 2,500 cm 2 , 3,000 cm 2 , 3,500 cm 2 , 4,000 cm 2 , 4,500 cm 2 , 5000 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 more than 50 m 2 in surface area.

[0137] In some embodiments, the mRNA - protease mixture is further subjected to a first TFF step before the oligo dT affinity chromatography step, and the TFF step includes a filter of about 100 kDa to about 300 kDa.

[0138] In some embodiments, the mRNA - protease mixture is concentrated in at least a first TFF step. In some embodiments, the mRNA - protease mixture is diafiltered in at least a first TFF step. In one embodiment, the mRNA - protease mixture is concentrated prior to diafiltration, thereby reducing the volume required for diafiltration.

[0139] In some embodiments, the mRNA - protease mixture is concentrated to at least about 5 mg / mL. In some embodiments, the mRNA - protease mixture is concentrated to at least about 10 mg / mL. In some embodiments, the mRNA - protease mixture is concentrated to about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL or about 20 mg / mL. In some embodiments, the mRNA - protease mixture is concentrated to about 10 - 12 mg / mL. In some embodiments, the mRNA - protease mixture is concentrated to about 12 mg / mL or less.

[0140] In some embodiments, the mRNA (i.e., affinity - purified mRNA) contained in the preparation obtained from oligo - dT affinity chromatography is subjected to a TFF step (e.g., a second TFF step). In some embodiments, the preparation that has been subjected to two separate steps of enzymatic digestion with protease is further subjected to a TFF step (e.g., a second TFF step).

[0141] In some embodiments, the second TFF step includes a filter of about 50 kDa to about 300 kDa. In some embodiments, the second TFF step includes a filter of about 50 kDa to about 100 kDa.

[0142] In some embodiments, the affinity-purified mRNA is concentrated in the TFF process. In some embodiments, the affinity-purified mRNA is diafiltered in the TFF process. In some embodiments, the affinity-purified mRNA is concentrated prior to diafiltration, thereby advantageously reducing the volume required for diafiltration.

[0143] In some embodiments, the affinity-purified mRNA is concentrated to at least about 0.5 mg / mL. In some embodiments, the affinity-purified mRNA is concentrated to at least about 1 mg / mL. In some embodiments, the affinity-purified mRNA is concentrated to at least about 2 mg / mL. In some embodiments, the affinity-purified mRNA is concentrated to at least about 5 mg / mL.

[0144] In some embodiments, the affinity-purified mRNA is concentrated to about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, or about 10 mg / mL.

[0145] In some embodiments, the affinity-purified mRNA is diluted to less than about 2 mg / mL after diafiltration. In some embodiments, the affinity-purified mRNA is diluted to less than about 1 mg / mL after diafiltration. In some embodiments, the affinity-purified mRNA is diluted to less than about 0.5 mg / mL after diafiltration.

[0146] In a typical TFF process, at least three process variables are important: transmembrane pressure (TMP), feed rate, and permeate flow rate. TMP is the force that drives fluid through the filter and transports permeable molecules. In some embodiments, the TMP is 1 to 30 pounds per square inch (psi) or less. In some embodiments, the TMP is about 5 to about 15 psi. In some embodiments, the TMP is about 7 to about 9 psi. In some embodiments, the TMP is about 1 psi, about 2 psi, about 3 psi, about 4 psi, about 5 psi, about 6 psi, about 7 psi, about 8 psi, about 9 psi, about 10 psi, about 11 psi, about 12 psi, about 13 psi, about 14 psi, about 15 psi, about 16 psi, about 17 psi, about 18 psi, about 19 psi, about 20 psi, about 21 psi, about 22 psi, about 23 psi, about 24 psi, about 25 psi, about 26 psi, about 27 psi, about 28 psi, about 29 psi, or about 30 psi. 1 psi is equivalent to about 0.07 bar. Thus, in some embodiments, the TMP is about 0.07 or more and about 2.07 bar or less. In some embodiments, the TMP is about 0.34 bar to about 1.03 psi. In some embodiments, the TMP is about 0.48 bar to about 0.62 bar.

[0147] The feed rate (also referred to as the cross-flow rate) indicates the rate of flow of the solution passing through the filter through the feed channel. The feed rate determines the force to sweep away the molecules that clog or contaminate the filter and restrict the flow of the filtrate. In some embodiments, the feed rate is from 1 to 1000 mL / min. In some embodiments, the feed rate is from 50 to 800 mL / min. In some embodiments, the feed rate is from 50 to 750 mL / min. In some embodiments, the feed rate is from 50 to 300 mL / min. In some embodiments, the feed rate is from 50 to 200 mL / min. In some embodiments, the feed rate is from 75 to 200 mL / min. In some embodiments, the feed rate is from 100 to 200 mL / min. In some embodiments, the feed rate is from 125 to 175 mL / min. In some embodiments, the feed rate is 130 mL / min. In some embodiments, the feed rate is from 60 mL / min to 220 mL / min. In some embodiments, the feed rate is 60 mL / min or more. In some embodiments, the feed rate is 100 mL / min or more. In some embodiments, the feed rate is 150 mL / min or more. In some embodiments, the feed rate is 200 mL / min or more. In some embodiments, the feed rate is 220 mL / min or more.

[0148] The flow rate of the permeate is the rate at which the permeate is removed from the system. When the feed rate is constant, increasing the permeate water flow rate increases the pressure across the filter and improves the filtration rate, but also increases the risk of filter clogging or fouling. The principles, theories, and apparatuses used in TFF are described in Michaels et al., “Tangential Flow Filtration” in Separations Technology, Pharmaceutical and Biotechnology Applications (W.P. Olson, ed., Interpharm Press, Inc., Buffalo Grove, Ill. 1995). See also U.S. Patent Nos. 5,256,294 and 5,490,937 for high performance tangential flow filtration (HP-TFF), which is an improvement over TFF. In some embodiments, the flow rate is from 1 to 100 mL / min. In some embodiments, the flow rate is from 10 to 100 mL / min. In some embodiments, the flow rate is from 10 to 90 mL / min. In some embodiments, the flow rate is from 10 to 80 mL / min. In some embodiments, the flow rate is from 10 to 70 mL / min. In some embodiments, the flow rate is from 10 to 60 mL / min. In some embodiments, the flow rate is from 10 to 50 mL / min. In some embodiments, the flow rate is from 10 to 40 mL / min. In some embodiments, the flow rate is from 15 to 60 mL / min. In some embodiments, the flow rate is from 20 to 40 mL / min. In some embodiments, the flow rate is 10 mL / min, 15 mL / min, 20 mL / min, 25 mL / min, 30 mL / min, 35 mL / min, 40 mL / min, 45 mL / min, 50 mL / min, 55 mL / min, 60 mL / min, 65 mL / min, 70 mL / min, 75 mL / min, 80 mL / min, 85 mL / min, 90 mL / min, 95 mL / min, or 100 mL / min.

[0149] Any combination of the various process variables described herein can be used. In some embodiments, TFF is performed at a feed rate of approximately 100 - 200 mL / min (e.g., approximately 100 - 180 mL / min, 100 - 160 mL / min, 100 - 140 mL / min, 110 - 190 mL / min, 110 - 170 mL / min, or 110 - 150 mL / min) and / or at a feed rate of approximately 10 - 50 mL / min (e.g., approximately 10 - 40 mL / min, 10 - 30 mL / min, 20 - 50 mL / min, or 20 - 40 mL / min). In some embodiments, TFF is performed at a feed rate of approximately 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mL / min and / or at a feed rate of approximately 10, 20, 30, 40, or 50 mL / min. In other embodiments, TFF is performed at a feed rate of approximately 500, 750 mL / min, 1, 2, 3, 4, or 5 L / min and / or at a feed rate of approximately 100, 200, 250, 500, 750 mL / min, or 1 L / min.

[0150] In some embodiments, a filtration step is further performed after the affinity chromatography step or after the second enzymatic digestion with proteinase. In some embodiments, sterile filtration or pre-filtration is performed. In some embodiments, the filtration is performed with a filter having a pore size of 0.2 μm or 0.22 μm. In some embodiments, the filtration is performed with a polyethersulfone (PES) filter.

[0151] In some embodiments, the pre-filtration step is performed after capping. In some embodiments, the pre-filtration step is performed after the TFF step. In some embodiments, the pre-filtration step is performed after capping and before oligo dT affinity chromatography. In some embodiments, the pre-filtration step is performed after oligo dT affinity chromatography. In some embodiments, the pre-filtration step is performed after the second TFF step. In some embodiments, the pre-filtration is performed with a filter having a pore size of 0.2 μm or 0.22 μm. In some embodiments, the pre-filtration uses a polyethersulfone (PES) filter.

[0152] III. Affinity Chromatography In one embodiment, mRNA from the impurity preparation is purified by oligo-dT chromatography. Oligo-dT is a single-stranded sequence of deoxythymidine (dT) and can be used in affinity chromatography to purify mRNA containing stretches of poly(A) such as the poly(A) tail. In one embodiment, the oligo-dT is a homogeneous mixture of deoxythymidine of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 mers such as oligo(dT) 12 , oligo(dT) 14 , oligo(dT) 18 , oligo(dT) 20 , oligo(dT) 23 or oligo(dT) 25 . In another embodiment, the oligo-dT is a mixture of two or more oligo(dT) ligands of different lengths such as oligo(dT) 12~18 . The oligo-dT can be immobilized on a chromatography resin or other type of solid support. In particular, the oligo-dT can be bound to a monolithic matrix, membrane or beads. The oligo-dT can be bound to any of the following supports: borosilicate glass, agarose, sepharose, magnetic beads, polystyrene, polyacrylamide, membrane, silica, semiconductor material, silicon, organic polymer, ceramic, glass, metal, plastic polycarbonate, polyethylene, polyethylene glycol terephthalate, polymethyl methacrylate, polypropylene, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone or soda lime glass. In one embodiment, the oligo-dT is bound to cross-linked poly(styrene-divinylbenzene) (PSDVB). In one embodiment, the oligo-dT is bound to sepharose. In one embodiment, the oligo-dT is bound to agarose. In one embodiment, the oligo-dT is bound to cellulose. In one embodiment, the oligo-dT is bound to poly(glycidyl methacrylate-co-ethylenedimethacrylate).

[0153] In some embodiments, the oligo dT is attached to the solid support via a linker, such as a carbon linker, disposed between the oligo dT molecule and the solid support. The linker can be included at various positions within the oligo dT and / or the support or on the oligo dT and / or the support. The selection of the linker is within the ability of one skilled in the art. Suitable linkers include alkyl and aryl groups containing heteroalkyl and heteroaryl, and substituted derivatives thereof. In some examples, the linker can be amino acid-based and / or contain an amide bond. Examples of linkers are amino derivatives, thiol derivatives, aldehyde derivatives, formyl derivatives, azide derivatives (click chemistry), biotin derivatives, alkyne derivatives, hydroxyl derivatives, activated hydroxyl or derivatives, carboxylate derivatives, activated carboxylate derivatives, activated carbonates, activated esters, NHS esters (succinimidyl), NHS carbonates (succinimidyl), imide esters or derivatives, cyanogen bromide derivatives, maleimide derivatives, haloacteyl derivatives, iodoacetamide / iodoacetyl derivatives, epoxy derivatives, streptavidin derivatives, tresyl derivatives, diene / conjugated diene derivatives (Diels-Alder type reaction), alkene derivatives, substituted phosphate derivatives, bromohydrin / halohydrin, substituted disulfides, pyridyl-disulfide derivatives, aryl azides, acyl azides, azlactones, hydrazide derivatives, halobenzene derivatives, nucleoside derivatives, branched / polyfunctional linkers, dendrimer functionality and / or nucleoside derivatives; or any combination thereof. In one embodiment, the linker contains an amide bond. Non-limiting examples of carbon linkers include linkers of the formula C x wherein X is any integer from 5 to 50. In some embodiments, the carbon linker is C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 or C 15It is. In some embodiments, the linker contains 5'-hexylamine. In some embodiments, the oligo dT is attached to the support via an amide bond. In some embodiments, the oligo dT containing a 5' amino group such as 5'-hexylamine is conjugated to the N-hydroxysuccinimidyl (NHS) ester attached to the support.

[0154] In one embodiment, the oligo dT chromatography resin has the following structure:

Chemical formula

[0155] In one embodiment, the oligo dT chromatography resin has the following structure:

Chemical formula

[0156] In one embodiment, the oligo dT chromatography resin has the following structure:

Chemical formula

[0157] In one embodiment, the oligo dT chromatography resin has the following structure:

Chemical formula

[0158] In one embodiment, the oligo dT affinity purification involves oligo(dT) 18 or oligo(dT) 25 chromatography resin. The purification mechanism involves hybridization of the poly(A) tail of mRNA with oligo dT. Contaminating impurities in preparations such as proteins, unreacted nucleotides, plasmid DNA, cap analogs, partial transcripts, dsRNA by-products, enzymes, etc. lack a poly(A) portion and do not bind.

[0159] The sample loading buffer can be used to load the mRNA preparation onto an oligo dT substrate (oligo dT immobilized on a chromatography resin or other type of solid support). Oligo dT affinity purification is typically performed by raising the salt concentration of the sample loading buffer to about 250 mM. Under low conductivity conditions, the mutual electrostatic repulsion between the oligo dT substrate and the backbone of mRNA is strong enough to prevent the formation of hydrogen bonds (required for hybridization of the poly(A) tail of mRNA with oligo dT), so a certain amount of salt is required in the loading buffer. Raising the salt concentration of the loading buffer has the theoretical advantage of suppressing non-specific binding of contaminants to the oligo dT substrate. However, at high salt concentrations, the mRNA product can precipitate (especially when the mRNA is present at high concentrations in the sample), which can inhibit the flow of the mRNA preparation on the oligo dT substrate. This is particularly problematic when purifying large amounts of mRNA (e.g., at least about 10 μg, 20 μg or 100 μg) in a single batch, as the mRNA is usually present at high concentrations (e.g., > 0.5 mg / ml) in the oligo dT loading buffer, especially when the batch size of the mRNA is large.

[0160] The adverse effects associated with the high salt concentration of the oligo dT loading buffer can be mitigated by minimizing the time the mRNA sample is exposed to high salt conditions. Thus, in some embodiments, prior to the preparation being applied to the oligo dT substrate, salt (e.g., sodium chloride) is added to a preparation containing at least about 0.5 mg / ml of mRNA to a final salt concentration of 400 mM to about 1200 mM within about 2 hours (e.g., within about 1 hour). In some embodiments, salt (e.g., sodium chloride) is added to a preparation containing about 0.5 mg / ml to about 2.0 mg / ml of mRNA to a final salt concentration of about 600 mM to about 1000 mM by about 2 hours prior to applying the preparation to the oligo dT substrate. For example, in some embodiments, salt (e.g., sodium chloride) is added to a preparation containing about 2.0 mg / ml of mRNA to a final salt concentration of about 800 mM by about 2 hours prior to applying the preparation to the oligo dT substrate.

[0161] It is common to add sodium chloride to the oligo dT loading buffer to increase the ionic strength. The inventors have found that adding approximately 800 mM sodium chloride at a concentration of approximately 2.0 mg / ml to a 20 g scale batch of mRNA is suitable for oligo dT purification in a specific mRNA preparation. However, it has been revealed that adding sodium chloride at a final concentration of 800 mM to a 1 g batch of another mRNA before loading onto the oligo dT substrate causes the mRNA to precipitate, clog the column, and reduce the yield of purified mRNA. The inventors have clarified that if precipitation of mRNA is observed after adding sodium chloride to the oligo dT loading buffer, this precipitation can be at least partially prevented by replacing sodium chloride with potassium chloride (see Example 11). Therefore, when precipitation of mRNA is observed, by using potassium chloride instead of sodium chloride, a high concentration of salt can be used in the oligo dT loading buffer even at a high mRNA concentration (to suppress non-specific binding of contaminants), enabling a more rapid purification process using a small amount of buffer for the purification of a large batch of mRNA, and reducing the adverse effects of mRNA precipitation. Since both sodium chloride and potassium chloride are neutral salts and have similar properties, this is a surprising discovery.

[0162] Accordingly, in some embodiments, the step of oligo dT affinity chromatography comprises loading a preparation comprising at least about 0.5 mg / ml of mRNA and at least about 500 mM of potassium chloride onto an oligo dT substrate. For example, in some embodiments, the preparation comprises from about 0.5 mg / ml to about 2.0 mg / ml of mRNA (e.g., about 1.0 mg / ml). In some embodiments, the preparation comprises from about 500 mM to about 1000 mM of potassium chloride (e.g., from about 600 mM to about 900 mM of potassium chloride).

[0163] Unbound contaminants can be removed in the washing step, and poly(A) mRNA can be eluted from the resin using a low ionic strength buffer or a solution of an oligonucleotide that binds competitively.

[0164] In one embodiment, a preparation containing in vitro synthesized mRNA is diluted prior to oligo dT affinity chromatography. In some embodiments, the dilution is performed after the capping step. In some embodiments, the dilution is at least 2-fold. In some embodiments, the dilution is about 2-fold, about 3-fold, about 4-fold or about 5-fold.

[0165] In some embodiments, the method comprises subjecting the mRNA preparation to one step of oligo dT affinity chromatography and does not include additional affinity chromatography steps.

[0166] IV. Capping and Tailing of Messenger RNA In one embodiment, the method of purifying mRNA described herein includes a capping step. In some embodiments, a poly(A) tailing reaction step is also performed.

[0167] In some embodiments, the mRNA is incubated with one or more capping enzymes to produce capped mRNA. Capping can be performed at any point in the purification process. In one embodiment, capping is performed simultaneously with IVT. In another embodiment, capping is performed after IVT. In one embodiment, capping is performed prior to subjecting a preparation comprising mRNA synthesized in vitro to enzymatic digestion with proteinase (step (a)). In one embodiment, capping is performed on purified in vitro transcribed mRNA. In one embodiment, capping is performed after the first step of enzymatic digestion with proteinase. In one embodiment, capping is performed after a preparation comprising mRNA synthesized in vitro has been subjected to the TFF step. In one embodiment, capping is performed after a preparation comprising mRNA synthesized in vitro has been subjected to the steps of enzymatic digestion with proteinase and TFF. In one embodiment, capping is performed prior to the affinity chromatography step. In one embodiment, capping is performed prior to subjecting the preparation obtained from step (a) to oligo dT affinity chromatography (step (b)) in the method provided herein. In one embodiment, if the method includes two steps of enzymatic digestion with proteinase, capping is performed prior to the second step of enzymatic digestion with proteinase.

[0168] In some embodiments, the mRNA is incubated with 2’O-methyltransferase and guanylyltransferase. In some embodiments, the mRNA is incubated with an RNase inhibitor. In some embodiments, the mRNA is incubated with GTP and S-adenosylmethionine. In some embodiments, the mRNA is incubated with 2’O-methyltransferase, guanylyltransferase, GTP and S-adenosylmethionine. In some embodiments, the mRNA is incubated with 2’O-methyltransferase, guanylyltransferase, RNase inhibitor, GTP and S-adenosylmethionine.

[0169] In some embodiments, capping is performed at about 25°C to about 37°C for at least about 30 minutes. In some embodiments, the mRNA is incubated with one or more capping enzymes at about 25°C to about 37°C for at least about 60 minutes. In some embodiments, the mRNA is incubated with one or more capping enzymes at about 25°C to about 37°C for at least about 90 minutes. In some embodiments, capping is performed at about 25°C to about 37°C for at least about 120 minutes.

[0170] In some embodiments, capping is performed while stirring the mRNA-containing mixture.

[0171] In some embodiments, the mRNA lacks a poly(A) tail after the first IVT reaction with RNA polymerase and needs to be tailed.

[0172] In some embodiments, the mRNA is incubated with poly(A) polymerase to produce poly(A)-tailed mRNA. In some embodiments, the poly(A) polymerase is a poly(A) polymerase derived from E. coli.

[0173] In some embodiments, the mRNA is co-incubated with one or more capping enzymes and poly(A) polymerase to produce capped and poly(A)-tailed mRNA. In other embodiments, the mRNA is incubated with one or more capping enzymes followed by incubation of the mRNA with poly(A) polymerase to produce capped and poly(A)-tailed mRNA.

[0174] In some embodiments, tailing is performed at about 25°C to about 37°C for at least about 30 minutes. In some embodiments, tailing is performed at about 25°C to about 37°C for at least about 60 minutes. In some embodiments, tailing is performed at about 25°C to about 37°C for at least about 90 minutes. In some embodiments, tailing is performed at about 25°C to about 37°C for at least about 120 minutes.

[0175] In some embodiments, after capping, a front filtration step is performed. In some embodiments, when tailing is performed simultaneously with or after capping, the front filtration step is performed after capping and tailing.

[0176] In one aspect, provided herein is a method for manufacturing mRNA, the method comprising synthesizing mRNA in vitro and purifying the in vitro synthesized mRNA using the methods described herein.

[0177] In some embodiments, the manufacture of mRNA includes methods for the large-scale production of full-length mRNA molecules. In some embodiments, the manufacture of mRNA includes methods for producing a composition enriched in full-length mRNA molecules having a length greater than 500 nucleotides. In some embodiments, at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.01%, 99.05%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the purified mRNA molecules are full-length mRNA molecules. In some embodiments, the composition or batch contains at least 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1 g, 5 g, 10 g, 25 g, 50 g, 75 g, 100 g or more of mRNA.

[0178] In another aspect, provided is mRNA obtained by the methods described herein.

[0179] In another aspect, a composition comprising purified mRNA obtained by the methods described herein is provided. In some embodiments, the composition further comprises at least one pharmaceutically acceptable excipient.

[0180] In another aspect, a method of treating a disease or disorder is provided, comprising administering to a subject in need thereof a purified mRNA or a composition comprising purified mRNA obtained by the methods described herein.

[0181] In another aspect, a composition comprising purified mRNA for use in therapy or purified mRNA obtained by the methods described herein is provided.

[0182] The present invention includes the following embodiments.

[0183] Embodiment 1. A method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a protease; and (b) subjecting the preparation obtained from step (a) to oligo dT affinity chromatography.

[0184] Embodiment 2. The method according to Embodiment 1, wherein the protease comprises a serine protease.

[0185] Embodiment 3. The method according to Embodiment 1 or 2, wherein the preparation containing in vitro synthesized mRNA is incubated with the protease at about 37 °C for at least 30 minutes.

[0186] Embodiment 4. The method according to any one of Embodiments 1 to 3, wherein the mRNA-protease mixture is stirred.

[0187] Embodiment 5. The method according to any one of Embodiments 1 to 4, wherein the protease is inactivated with a reducing agent, and optionally, the reducing agent is DTT.

[0188] Embodiment 6. The method according to any one of Embodiment 5, wherein DTT is added to a concentration of at least about 20 mM.

[0189] Embodiment 7. The method according to any one of Embodiments 1 to 6, wherein the preparation obtained from step (a) is subjected to a TFF step before step (b).

[0190] Embodiment 8. The method according to Embodiment 7, wherein TFF uses a filter of about 100 kDa to about 300 kDa.

[0191] Embodiment 9. The method according to Embodiment 7 or 8, wherein the mRNA contained in the preparation obtained from step (a) is concentrated using TFF.

[0192] Embodiment 10. The method according to Embodiment 9, wherein the mRNA contained in the preparation obtained from step (a) is concentrated to at least about 5 mg / mL.

[0193] Embodiment 11. The method according to any one of Embodiments 1 to 10, wherein sodium citrate is added to the preparation obtained from step (a).

[0194] Embodiment 12. The method according to Embodiment 11, wherein sodium citrate is added to a concentration of at least about 10 mM, optionally at least about 25 mM.

[0195] Embodiment 13. The method according to Embodiment 11, wherein sodium citrate is added to a concentration of about 25 mM.

[0196] Embodiment 14. The method according to any one of Embodiments 11 to 13, wherein the preparation is held at about 35°C to about 37°C for at least about 5 minutes after the addition of sodium citrate.

[0197] Embodiment 15. The method according to any one of Embodiments 1 to 10, wherein EDTA is added to the preparation obtained from step (a).

[0198] Embodiment 16. The method according to embodiment 15, wherein EDTA is added at a concentration of at least about 10 mM, optionally up to at least about 25 mM.

[0199] Embodiment 17. The method according to embodiment 15, wherein EDTA is added up to a concentration of about 25 mM.

[0200] Embodiment 18. The method according to any one of embodiments 15 - 17, wherein the preparation is maintained at about 35°C to about 37°C for at least about 5 minutes after the addition of EDTA.

[0201] Embodiment 19. The method according to any one of embodiments 1 - 18, wherein the preparation is subjected to a capping step prior to step (b) to produce capped mRNA.

[0202] Embodiment 20. The method according to embodiment 19, wherein the preparation is incubated with 2'O - methyltransferase and guanylyltransferase.

[0203] Embodiment 21. The method according to embodiment 19 or 20, wherein the preparation is incubated with an RNase inhibitor.

[0204] Embodiment 22. The method according to any one of embodiments 19 - 21, wherein the preparation is incubated with GTP and S - adenosylmethionine.

[0205] Embodiment 23. The method according to any one of embodiments 19 - 22, wherein capping is performed at about 37°C for at least 30 minutes, optionally with stirring.

[0206] Embodiment 24. The method according to any one of embodiments 1 - 23, wherein the preparation is subjected to a tailing step with poly(A) polymerase to produce poly(A) - tailed mRNA.

[0207] Embodiment 25. The method according to any one of Embodiments 1 to 23, wherein the preparation is incubated with one or more capping enzymes and poly(A) polymerase to produce capped and poly(A)-tailed mRNA.

[0208] Embodiment 26. The method according to any one of Embodiments 1 to 23, wherein the preparation is incubated with one or more capping enzymes and subsequently incubated with poly(A) polymerase to produce capped and poly(A)-tailed mRNA.

[0209] Embodiment 27. Oligo dT affinity chromatography uses oligo(dT) 25 chromatography resin. The method according to any one of Embodiments 1 to 26.

[0210] Embodiment 28. The preparation obtained from step (b) is subjected to the TFF step. The method according to any one of Embodiments 1 to 27.

[0211] Embodiment 29. TFF uses a filter of about 50 kDa to about 300 kDa. The method according to Embodiment 28.

[0212] Embodiment 30. The mRNA contained in the preparation obtained from step (b) is concentrated using the TFF step. The method according to Embodiment 28 or 29.

[0213] Embodiment 31. The mRNA contained in the preparation obtained from step (b) is concentrated to at least about 2 mg / mL. The method according to Embodiment 30.

[0214] Embodiment 32. The preparation is subjected to a pre-filtration step after the capping step. The method according to any one of Embodiments 19 to 31.

[0215] Embodiment 33. The preparation is subjected to a pre-filtration step after the capping step and before step (b). The method according to any one of Embodiments 19 to 31.

[0216] Embodiment 34. The method according to any one of Embodiments 28 to 33, further comprising a front filtration step after the TFF step.

[0217] Embodiment 35. A method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to a TFF step; (c) subjecting the preparation obtained from step (b) to oligo dT affinity chromatography; and (d) subjecting the preparation obtained from step (c) to a TFF step.

[0218] Embodiment 36. The method according to Embodiment 35, wherein the preparation obtained from step (b) is incubated with one or more capping enzymes to produce capped mRNA.

[0219] Embodiment 37. The method according to Embodiment 35 or 36, wherein the preparation obtained from step (b) is incubated with poly(A) polymerase to produce poly(A)-tailed mRNA.

[0220] Embodiment 38. The method according to any one of Embodiments 35 to 37, wherein the preparation obtained from step (b) is incubated with one or more capping enzymes and poly(A) polymerase to produce capped and poly(A)-tailed mRNA.

[0221] Embodiment 39. The method according to any one of Embodiments 35 to 38, wherein the preparation is subjected to a front filtration step before step (c).

[0222] Embodiment 40. The method according to any one of Embodiments 36 to 38, wherein the preparation is subjected to a front filtration step after capping and before step (c).

[0223] Embodiment 41. The method according to any one of Embodiments 35 to 40, further comprising a front filtration step after step (d).

[0224] Embodiment 42. Front filtration is the method according to any one of Embodiments 32 to 34 and 39 to 41, including a 0.2 μm filter.

[0225] Embodiment 43. A method for purifying messenger RNA (mRNA), comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to a tangential flow filtration (TFF) step; (c) subjecting the preparation obtained from step (b) to a capping step; (d) subjecting the preparation obtained from step (c) to a front filtration step; (e) subjecting the preparation obtained from step (d) to oligo dT affinity chromatography; and (f) subjecting the preparation obtained from step (e) to a TFF step.

[0226] Embodiment 44. The method according to Embodiment 43, further comprising a front filtration step after step (f).

[0227] Embodiment 45. Front filtration is the method according to Embodiment 43 or 44, including a 0.2 μm filter.

[0228] Embodiment 46. Front filtration is the method according to any one of Embodiments 32 to 34, 39 to 42, and 43 to 45, which reduces the guanylyl transferase (GuaT) content.

[0229] Embodiment 47. The oligo dT affinity chromatography according to any one of the previous embodiments, including adding salt to the preparation to a final concentration of about 400 mM to about 1200 mM up to 2 hours before the preparation is applied to the oligo dT substrate.

[0230] Embodiment 48. The method according to Embodiment 47, wherein the salt is added to the preparation to a final concentration of about 500 mM to about 1000 mM.

[0231] Embodiment 49. The method according to any one of Embodiments 47 to 48, wherein the salt is added to the preparation up to a final concentration of about 800 mM.

[0232] Embodiment 50. The method according to any one of Embodiments 47 to 49, wherein the preparation contains at least about 0.5 mg / ml of mRNA.

[0233] Embodiment 51. The method according to Embodiment 50, wherein the preparation contains about 0.5 mg / ml to about 2.0 mg / ml of mRNA.

[0234] Embodiment 52. The method according to Embodiment 51, wherein the preparation contains about 1.0 mg / ml of mRNA.

[0235] Embodiment 53. The method according to any one of Embodiments 47 to 52, wherein the salt is sodium chloride.

[0236] Embodiment 54. The method according to any one of Embodiments 47 to 52, wherein the salt is potassium chloride.

[0237] Embodiment 55. The method according to any one of Embodiments 47 to 52 and 54, wherein the oligo dT affinity chromatography includes loading a preparation containing at least about 0.5 mg / ml of mRNA and at least about 500 mM of potassium chloride onto an oligo dT substrate.

[0238] Embodiment 56. The method according to Embodiment 55, wherein the preparation contains about 500 mM to about 1000 mM of potassium chloride.

[0239] Embodiment 57. The method according to Embodiment 56, wherein the preparation contains about 600 mM to about 900 mM of potassium chloride.

[0240] Embodiment 58. A method for purifying mRNA, comprising: (a) subjecting a preparation containing in vitro synthesized mRNA to a first enzymatic digestion with a protease; (b) subjecting the preparation obtained from step (a) to a second enzymatic digestion with a protease; and (c) subjecting the preparation obtained from step (b) to a TFF step.

[0241] Embodiment 59. The method according to Embodiment 58, wherein the protease comprises a serine protease.

[0242] Embodiment 60. The method according to Embodiment 58 or 59, wherein the first enzymatic digestion and / or the second enzymatic digestion is carried out at about 37 °C for at least 30 minutes.

[0243] Embodiment 61. The method according to any one of Embodiments 58 to 60, wherein the first enzymatic digestion and / or the second enzymatic digestion is stirred.

[0244] Embodiment 62. The method according to any one of Embodiments 58 to 61, wherein the protease is inactivated with a reducing agent, and optionally, the reducing agent is dithiothreitol (DTT).

[0245] Embodiment 63. The method according to Embodiment 62, wherein DTT is added to a concentration of at least about 20 mM.

[0246] Embodiment 64. The method according to any one of the previous embodiments, wherein a precipitation step is not performed.

[0247] Embodiment 65. The method according to any one of the previous embodiments, wherein at least about 0.5 grams of mRNA is purified.

[0248] Embodiment 66. The method according to any one of the previous embodiments, wherein about 0.5 grams to about 100 grams of mRNA is purified.

[0249] [[ID=3)] Embodiment 67. The mRNA synthesized in vitro is purified on a scale of about 100 milligrams, about 1 gram, about 10 grams, about 20 grams, about 50 grams, about 100 grams or more per batch, and optionally, the mRNA is purified on a scale of about 10 grams or more per batch, according to any one of the previous embodiments.

[0250] Embodiment 68. The residual plasmid DNA in the purified mRNA is less than about 1 pg / mg, according to any one of the previous embodiments.

[0251] Embodiment 69. The residual plasmid DNA in the purified mRNA is less than about 0.5 pg / mg, according to the method described in Embodiment 68.

[0252] Embodiment 70. The residual plasmid DNA in the purified mRNA is about 0.2 pg / mg or less, according to the method described in Embodiment 69.

[0253] Embodiment 71. The mRNA synthesized in vitro is purified on a scale of about 10 grams or more per batch, and the residual plasmid DNA in the purified mRNA is about 0.2 pg / mg or less, according to any one of the previous embodiments.

[0254] The mRNA synthesized in vitro is purified on a scale of about 20 grams or more per batch, and the residual plasmid DNA in the purified mRNA is about 0.2 pg / mg or less, according to the method described in Embodiment 72.

[0255] Embodiment 73. Remove about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more or substantially all of the interrupted RNA sequences in the middle, according to any one of the previous embodiments.

[0256] Embodiment 74. The method according to any one of the previous embodiments, wherein the purified mRNA contains less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of RNA sequences interrupted in the middle.

[0257] Embodiment 75. The method according to embodiment 74, wherein the purified mRNA contains less than about 1% (e.g., less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2%, or less than about 0.1%) of RNA sequences interrupted in the middle.

[0258] Embodiment 76. The method according to any one of the previous embodiments, wherein the purified mRNA contains less than about 1% (e.g., less than about 0.5%, less than about 0.2%, or less than about 0.1%) of dsRNA.

[0259] Embodiment 77. The method according to embodiment 76, wherein the purified mRNA contains less than about 0.1% (e.g., less than about 0.050%, less than about 0.030%, less than about 0.025%, or less than about 0.010%) of dsRNA.

[0260] Embodiment 78. The method according to any one of the previous embodiments, wherein the mRNA synthesized in vitro is purified on a scale of about 10 grams or more per batch, and the purified mRNA contains less than about 0.01% of dsRNA.

[0261] Embodiment 79. The method according to embodiment XX, wherein the mRNA synthesized in vitro is purified on a scale of about 20 grams or more per batch, and the purified mRNA contains less than about 0.025% of dsRNA.

[0262] Embodiment 80. The method according to any one of the previous embodiments, wherein the purified mRNA contains less than about 5% (e.g., less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of the enzyme reagents used in in vitro synthesis.

[0263] Embodiment 81. The method according to embodiment 80, wherein the purified mRNA contains less than about 1% (e.g., less than about 0.9%, less than about 0.8%, less than about 0.7%, less than about 0.6%, less than about 0.5%, less than about 0.4%, less than about 0.3%, less than about 0.2% or less than about 0.1%) of the enzyme reagents used in in vitro synthesis.

[0264] Embodiment 82. The method according to any one of the previous embodiments, wherein the purified mRNA contains undetectable enzyme reagents used in in vitro synthesis, as determined by, for example, silver staining, gel electrophoresis, HPLC, ultra-high performance liquid chromatography (UPLC) and / or CE, ethidium bromide and / or Coomassie staining.

[0265] Embodiment 83. The method according to any one of the previous embodiments, wherein the mRNA synthesized in vitro is purified on a scale of about 10 grams or more per batch, and the purified mRNA contains undetectable enzyme reagents used in in vitro synthesis, as determined by silver staining.

[0266] Embodiment 84. The method according to embodiment 83, wherein the mRNA synthesized in vitro is purified on a scale of about 20 grams or more per batch, and the purified mRNA contains undetectable enzyme reagents used in in vitro synthesis, as detected by silver staining.

[0267] Embodiment 85. The method according to any one of the previous embodiments, wherein the purified mRNA has a completeness of more than about 60%, about 70%, about 80%, about 90% or about 95% (e.g., more than about 96%, more than about 97%, more than about 98%, more than about 99% or more).

[0268] Embodiment 86. The method according to any one of the previous embodiments, wherein the purified mRNA has a completeness of more than about 80%.

[0269] Embodiment 87. The method according to embodiment 86, wherein the purified mRNA has a completeness of more than about 85%.

[0270] Embodiment 88. The method according to any one of the previous embodiments, wherein the mRNA synthesized in vitro is purified on a scale of about 10 grams or more per batch, and the purified mRNA has a integrity of more than about 80% (for example, at least about 85%).

[0271] Embodiment 89. The method according to embodiment 88, wherein the mRNA synthesized in vitro is purified on a scale of about 20 grams or more per batch, and the purified mRNA has a integrity of more than about 80% (for example, at least about 85%).

[0272] Embodiment 90. The method according to any one of the previous embodiments, which does not include the step of contacting the preparation with RNase III.

[0273] Embodiment 91. The method according to any one of the previous embodiments, which does not include the step of adding the preparation to a primary amino solid phase.

[0274] Embodiment 92. The method according to any one of the previous embodiments, which does not include any additional affinity chromatography steps.

[0275] Embodiment 93. The method according to any one of the previous embodiments, which does not include any additional filtration steps.

[0276] Embodiment 94. The method according to any one of the previous embodiments, which does not include any additional purification steps.

[0277] Embodiment 95. The method according to any one of the previous embodiments, wherein the purified mRNA is suitable for therapeutic use.

[0278] Embodiment 96. A method for producing mRNA, comprising the step of synthesizing mRNA in vitro and the step of purifying the mRNA synthesized in vitro using the method according to any one of the previous embodiments.

[0279] Embodiment 97. mRNA that can be obtained by the method according to any one of the previous embodiments.

Example

[0280] The foregoing description of specific embodiments is to fully clarify the general nature of the present disclosure, so that others can, without departing from the general concepts of the present disclosure and without undue experimentation, by applying knowledge within the scope of the skills of those skilled in the art, easily modify and / or adapt such specific embodiments to various applications. Therefore, such adapted and modified forms are intended to be within the meaning and scope of equivalents of the disclosed embodiments based on the teachings and guidance presented herein. It should be understood that the expressions or terms herein are for illustrative purposes rather than limiting, as they are to be interpreted by those skilled in the art in light of the teachings and guidance.

[0281] The methods described below were performed in a nuclease-free environment: a) All single-use components (e.g., bottles, pipette tips, tubes, etc.), assemblies, and devices were made nuclease-free through decontamination by gamma-ray irradiation treatment or sodium hydroxide treatment with sufficient contact time, and then rinsed with nuclease-free water. b) Buffers were prepared from powders or concentrated commercial solutions in sterile containers and finally filtered through a 0.22 μm vacuum filter under a biosafety laminar flow. c) Surfaces were decontaminated with commercial RNase, cleaning solutions, and wet tissues.

[0282] Example 1: RNA Synthesis IVT Reaction Conditions In the following examples, mRNA was transcribed in vitro by standard methods. Briefly, for each gram of transcribed mRNA, a reaction mixture containing a linear double-stranded DNA plasmid with an RNA polymerase-specific promoter, an RNA polymerase (e.g., SP6 polymerase or T7 polymerase), an RNase inhibitor, pyrophosphatase, NTPs, DTT, and a reaction buffer was prepared with RNase-free water and then incubated at 37°C for the specified time. The reaction was then quenched by the addition of DNase I and DNase I buffer to facilitate digestion of the double-stranded DNA template in preparation for purification. One gram of in vitro transcribed mRNA was subjected to various purification methods.

[0283] Capping After synthesizing mRNA as described above, the in vitro transcribed mRNA was enzymatically modified by the addition of a 5’ N 7 -methylguanylate cap 0 structure using guanylyltransferase and the addition of a methyl group at the 2’O position of the penultimate nucleotide to yield a cap 1 structure using 2’O-methyltransferase. Capping was performed immediately before the affinity chromatography step when using the enzyme / affinity method and immediately before the second step of enzymatic digestion with proteinase when using the complete enzyme method.

[0284] Tailing Unless otherwise specified, the mRNA transcribed by IVT includes a template tailed to a linear plasmid that tails the mRNA as part of the IVT reaction, or the 3’ end is tailed in a subsequent enzymatic step. When tailing is performed as part of the IVT reaction, poly(T) or similar tailing characteristics are incorporated into the pDNA template so that a poly(A) tail or a similar appropriate tail is formed on the mRNA as part of the IVT process. Alternatively, a poly(A) tail can be enzymatically added to the 3’ end of the IVT-produced mRNA after the IVT reaction, for example, using poly(A) polymerase.

[0285] Example 2: Purification method with ethanol (comparative example) Purification of the IVT reaction product using ethanol-based precipitation was carried out using the method summarized in Table 8 of WO 2020 / 041793 pamphlet (see also Examples 1, 2 and 4 incorporated herein by reference), using an initial amount of 1 g of the transcribed mRNA.

[0286] Example 3: Enzymatic digestion and affinity chromatography method Enzymatic digestion and filtration Serine protease (550 units; ArcticZymes ref.71600-110) was added directly to the mixture and the solution was stirred at 37 °C for 2 hours. The reaction was quenched by dilution with DTT solution to a final concentration of 55 mM DTT.

[0287] The entire amount was passed through a Kr2i TFF system (manufactured by Repligen) equipped with a 100 kDa modified polyethersulfone (mPES) hollow fiber (manufactured by Repligen, lumen 0.5 mm, height 41.5 cm) for 700 s -1It was introduced at a shear rate of. The TMP was maintained at 7 - 9 psi (about 0.48 bar - about 0.62 bar), and the conductivity was monitored. First, the product was concentrated to 10 - 12 mg / mL, then dialyzed and filtered successively with 50 mM Tris, 150 mM NaCl, 55 mM DTT, pH 8, then 50 mM Tris, 150 mM NaCl, pH 8, and finally with water for injection. All the product was collected, the fibers were washed with water for injection, and the product was pooled. The mRNA yield after concentration and diafiltration was at least 90%. The mRNA yield did not change when using a 300 kDa filter instead of a 100 kDa filter (i.e., at least 90%).

[0288] The concentration was adjusted to 4 g / L before performing the capping reaction (described above).

[0289] Affinity Chromatography and Filtration After capping, the reaction solution was diluted 4 - fold with an injection (loading) buffer (50 mM sodium phosphate, 1070 mM sodium chloride, 5 mM EDTA, pH 7), and oligo(dT) 25 was injected into an AKTA(™) Avant 150 equipped with a HiScale(™) 50 / 40 column (GE Healthcare) packed with the resin. Thus, the final concentration of sodium chloride in the mRNA preparation solution for injection was 800 mM. The column volume was 0.433 L, and it was operated at 244 - 422 cm / hour. After injection, the column was washed 3 times each with 50 mM sodium phosphate, 800 mM NaCl, 5 mM EDTA, pH 7, 50 mM sodium phosphate, 400 mM NaCl, 5 mM EDTA, pH 7, and 50 mM sodium phosphate, 5 mM EDTA, pH 7, and eluted with nuclease - free water. The only peak was collected into a sterile bottle and then transferred to a Kr2i TFF system equipped with a 50 kDa mPES hollow fiber (Repligen), 0.5 mm inner lumen, 41.5 cm height, at 700 s -1It was transferred at a shear rate of. The TMP was maintained at 7 - 9 psi (about 0.48 bar - about 0.62 bar), and the conductivity was monitored. The product was first concentrated to 5 mg / mL and then diafiltered with nuclease - free water for injection. The product was recovered, the fibers were washed twice, and then the recovered fraction and the wash fraction were pooled. After adjusting the concentration to 1 g / L, it was filtered through a pre - gamma - irradiated PES 0.22 μm filter.

[0290] Example 4: Complete enzymatic method First enzymatic digestion and filtration Serine protease (550 units; ArcticZymes ref.71600 - 110) was added directly to the mixture, and the solution was stirred at 37 °C for 2 hours. The reaction was quenched by dilution with DTT solution to a final concentration of 55 mM DTT.

[0291] The entire amount was introduced into a Kr2i TFF system (manufactured by Repligen) equipped with 100 kDa mPES hollow fibers (manufactured by Repligen, inner lumen 0.5 mm, height 41.5 cm) at a shear rate of 700 s -1 It was introduced at a shear rate of. The TMP was maintained at 7 - 9 psi (about 0.48 bar - about 0.62 bar), and the conductivity was monitored. First, the product was concentrated to 10 - 12 mg / mL, then diafiltered with 50 mM Tris, 150 mM NaCl, 55 mM DTT, pH 8, then with 50 mM Tris, 150 mM NaCl, pH 8, and finally with water for injection. The entire product was recovered, the fibers were rinsed with water for injection and pooled with the product. The concentration was adjusted to 4 g / L before performing the capping reaction (described above).

[0292] Second enzymatic digestion and filtration After capping, serine protease (550 units; ArcticZymes ref.71600 - 110) was added directly to the mixture, and the solution was stirred at 37 °C for 2 hours. The reaction was quenched by dilution with DTT solution to a final concentration of 55 mM DTT.

[0293] This entire volume was introduced into a Kr2i TFF system (manufactured by Repligen) equipped with a 100 kDa modified polyethersulfone (mPES) hollow fiber (manufactured by Repligen, inner lumen 0.5 mm, height 41.5 cm) at a shear rate of 700 s -1 for 700 s. A TMP of 7 - 9 psi (about 0.48 bar - about 0.62 bar) was maintained while monitoring the conductivity. The product was first concentrated to at least 2 mg / mL and then diafiltered against 50 mM Tris, 150 mM NaCl, 55 mM DTT, pH 8, then 50 mM Tris, 150 mM NaCl, pH 8, and finally against water for injection. The entire product was recovered, the fibers were rinsed twice with water for injection and pooled with the product. After adjusting the concentration to 1 g / L, it was filtered through a pre - gamma - irradiated PES 0.22 μm filter. The finished product was stored at - 20 °C until formulation.

[0294] Example 5: Analysis of Purified mRNA The quality of the purified mRNA was evaluated by several metrics such as capillary gel electrophoresis (CGE) to measure mRNA integrity, capping rate (cap 1), poly(A) tail length, and known techniques to measure the total mRNA length (see Tables 1 - 2). The electrophoretograms of mRNA purified by the enzyme / affinity method and the complete enzyme method were as expected. The absence of a significant shoulder (data not shown) indicated that these methods did not adversely affect the quality and integrity of the mRNA. The integrity of mRNA at 5 °C was evaluated over time by capillary electrophoresis (CE).

[0295] The presence of proteins (e.g., IVT enzymes, proteases, capping enzymes) was analyzed in samples taken at various stages of the method. Samples were loaded onto a 4 - 15% Criterion™ TGX™ SDS - PAGE gel (Bio - Rad) and the gel was silver stained. Samples were treated with RNAse A to remove RNA before loading onto the gel.

[0296] Results and Discussion The steps of the enzyme / affinity method and the complete enzyme method are summarized in Figure 1. In the enzyme / affinity method and the complete enzyme method, after the IVT / DNase step, serine protease was used to digest the enzymes involved in the enzyme step, and the resulting peptides were removed from the mixture by TFF. In the enzyme / affinity method, after the capping step, a resin supporting the poly dT ligand was used to capture the mRNA by affinity with the polyA tail. The enzyme / affinity method uses an orthogonal purification strategy: that is, first removing impurities from the mixture and then using one of the characteristics of mRNA (for example, the polyA tail) to capture the mRNA.

[0297] mRNA on a 1g scale was purified using three methods, and the results are summarized in Table 1 below. The mRNA obtained by the enzyme / affinity method and the complete enzyme method had characteristics equivalent to those of the mRNA obtained by the ethanol method. The size and tail length of the mRNA were consistent with the predictions. The cap rate was also as expected, indicating that the enzyme / affinity method and the complete enzyme method did not have an adverse effect on the capping reaction.

[0298]

Table 1

[0299] The cycle times of the ethanol method, the enzyme / affinity method, and the complete enzyme method were compared. In the ethanol method, the total time for the IVT reaction and the purification step was about 10 hours and 45 minutes. In the enzyme / affinity method and the complete enzyme method, the same steps each required about 9 hours and 45 minutes. The total time for the capping / purification / filtration / redistribution steps in the ethanol method was about 11 hours and 45 minutes, while the times for the same steps in the enzyme / affinity method and the complete enzyme method were 10 hours and 30 minutes and 10 hours and 45 minutes, respectively.

[0300] Example 6: Evaluation of Residual Protein after Capping After in vitro transcription and enzymatic digestion to remove contaminating proteins, proteins are further introduced into the mRNA product for capping. The presence of the proteins introduced in the capping reaction was evaluated at various time points downstream of the capping reaction using silver-stained SDS-PAGE gels. As a result of affinity chromatography, the RNase inhibitor and 2’O-methyltransferase were no longer detected, but a subunit of guanylyltransferase (GuaT) was detected (see lane 4 in panel A of Figure 2). However, GuaT could no longer be detected after pre-filtration through a 0.22 μm PES filter (see lane 5 in the panel of Figure 2). Incorporating an intermediate pre-filtration step with a 0.22 μm PES filter significantly reduced the amount of contaminating GuaT protein from the affinity chromatography step (see lane 1’ in panel B of Figure 2). Using an intermediate pre-filtration step prior to affinity chromatography reduced the protein load on the affinity chromatography column and the protein load carried over to downstream steps (such as TFF). Incorporating a pre-filtration step prior to affinity chromatography was evaluated on both 1 g and 10 g scales, and similar results were obtained.

[0301] Example 7: Evaluation of Enzymatic Digestion Conditions Various concentrations of proteinase K (Sigma, reference number P2308) were tested, and protein digestion was evaluated using silver-stained SDS-PAGE gels.

[0302] As shown in Figure 3, the digest obtained by incubating with proteinase K (Sigma) at 0.025 U / mg RNA for 30 minutes was comparable to the digest obtained by incubating with serine protease (ArcticZymes) at 0.55 U / mg RNA for 2 hours (compare lanes 1 and 3 in Figure 3). On the other hand, digestion after incubating serine protease (ArcticZymes) at 0.05 U / mg RNA for 2 hours was incomplete (see lane 2 in Figure 3). Thus, using proteinase K (Sigma) allowed the enzyme digestion time to be shortened by a factor of 4 and simultaneously reduced the amount of proteinase used to 1 / 22. Downstream capping was equivalent under all three conditions. These results validate the use of proteinases from various vendors. When using proteinase K, both the enzyme concentration and the enzyme digestion time can be significantly reduced. This advantageously further shortens the cycle time of the method.

[0303] Example 8: Evaluation of Buffer Conditions After 30 minutes of enzyme digestion with proteinase K (Sigma) at 0.025 U / mg RNA, TFF was performed. Various buffer conditions for diafiltration were evaluated. In particular, for diafiltration in the presence or absence of DTT (55 mM), a 50 mM Tris, 150 mM NaCl buffer was used. The profiles were similar between buffer conditions (see Figure 4, compare conditions 1 and 2). Furthermore, capping was equivalent regardless of the buffer used (i.e., 93.1% of cap 1 when using Tris NaCl DTT buffer and 95.5% of cap 1 when using Tris NaCl buffer). Thus, the presence of DTT in the buffer was not required.

[0304] Example 9: Scale-up The mRNA of 10 g and 20 g scales was purified by the enzymatic / affinity method, and the results were summarized in Table 2 below. The scale-up of the purification was successful. The analysis profiles were within the acceptable range. Residual GuaT was removed by intermediate front filtration with a 0.2 μm PES filter.

[0305] As shown in Figure 5, the time-dependent change in the integrity of the mRNA purified by the enzymatic / affinity method was evaluated.

[0306] [Table 2]

[0307] The mRNA of 100 g scale was also successfully purified by the enzymatic / affinity method. The 100 g batch was found to meet all the acceptance criteria.

[0308] Overall, the enzymatic / affinity method and the complete enzymatic method have a high yield of isolated and purified mRNA, and the yield is equal to or higher than that of the ethanol-based method. Furthermore, the overall time of the enzymatic / affinity method and the complete enzymatic method is shortened compared to the ethanol method.

[0309] The enzymatic / affinity method and the complete enzymatic method advantageously do not require ethanol, which is highly flammable and operationally dangerous, when performing mRNA production on a commercial scale.

[0310] Example 10: Process Optimization - mRNA Solubilization No precipitate was observed during the mRNA purification of the first mRNA construct (hereinafter referred to as construct 1) used in the previous example, but the formation of a precipitate can be observed during the in vitro transcription of the second construct (construct 2). This means that in some cases, a precipitate can be formed during the large-scale purification of mRNA, and the precipitate can clog the filter, which can have an adverse effect on the downstream process, especially the downstream filtration.

[0311] The precipitate was characterized to determine whether it was initially composed of in vitro transcribed mRNA. The amount and integrity of the mRNA were analyzed in the supernatant and precipitate fractions using ion pair reverse phase ultra-high performance liquid chromatography (RP-IP-UPLC-UV) and capillary electrophoresis, respectively. Prior to performing RP-IP-UPLC-UV, the supernatant and precipitate were separated, the precipitate was washed with RNase-free water and redissolved in 1 mM HCl. The chromatographic peak corresponding to the mRNA showed a surface area of 0.825 in the supernatant and 8.416 in the precipitate, and the mRNA concentrations corresponded to 0.34 mg / mL and 3.21 mg / mL, respectively. Here, approximately 90% of the detected mRNA was contained in the precipitate. Thus, a very large amount of mRNA can precipitate from the preparation during in vitro transcription. In contrast, precipitation did not adversely affect the integrity of the mRNA.

[0312] From the perspective of redissolving the precipitate for downstream purification, several conditions were evaluated, and the presence of the precipitate was measured based on the absorbance value (Nanodrop) of the solution at A 600nm .

[0313] The precipitate did not redissolve when diluted with RNase-free water or Tris buffer (50 mM Tris, 150 mM NaCl, pH 7.5). The precipitate also did not redissolve when the pH of the solution was lowered from approximately 7 to 4 with a 500 mM citric acid solution. (Data not shown.)

[0314] Surprisingly, as shown in Figure 6, the precipitate was successfully redissolved by adding sodium citrate or EDTA to the preparation. Incubation at 35 °C for 5 minutes was sufficient for complete dissolution. This indicates that the formation of the precipitate is not irreversible and can be rapidly redissolved.

[0315] Thus, by adding sodium citrate or EDTA to a preparation containing in vitro transcribed mRNA, the precipitated mRNA can be advantageously redissolved prior to performing the downstream filtration step.

[0316] Example 11: Process Optimization - Oligo dT Loading Buffer Further analysis was performed on mRNA purified at a batch scale of 10 g or 20 g (Construct 1). Specifically, the integrity of the mRNA, the presence of dsRNA and residual plasmid DNA were evaluated directly after capping or the first TFF step and after the affinity chromatography step. The presence or absence of dsRNA was detected by ELISA using the J2 monoclonal antibody I. The presence of residual DNA was detected by qPCR.

[0317] TFF and affinity chromatography were performed as described in Example 3 above. A preparation of 2 mg / mL mRNA, 800 mM NaCl, 30 mM Tris, 8 mM EDTA, pH 7.4 was loaded onto the oligo dT resin. Washing was performed with the same solution containing only 100 mM NaCl.

[0318] After affinity chromatography, 95% - 100% of the mRNA was recovered. Furthermore, the integrity of the mRNA was improved (Figure 7), and it was observed that the contamination of residual plasmid DNA and dsDNA was at a low level in all batches after TFF and affinity chromatography (Figure 8).

[0319] Thus, by purifying mRNA on a large scale, high-purity mRNA with excellent quality characteristics can be obtained after the affinity chromatography step.

[0320] When the loading conditions onto the oligo dT resin were adjusted as described above, precipitation was unexpectedly observed in two other mRNA constructs, causing clogging of the column.

[0321] To examine the effect of NaCl in the loading buffer on the formation of precipitates, various concentrations of NaCl or KCl were evaluated as a function of mRNA concentration for three different mRNA constructs (Constructs 2 - 4). Specifically, the mRNA was incubated with the salt for 2 hours, and then UV-Vis A 600nmThe absorbance values (Nanodrop) were used to monitor the precipitation by UV, and the results were shown as contour plots (Figs. 9 - 11). If the AU value was negative, the absorbance was equal to 0, indicating that the solution was clear. If the AU value was at least 0.05, it indicated the risk of precipitate formation, and if the AU value was at least 0.3, the precipitate could be visually confirmed in a cloudy form.

[0322] As shown in Figs. 9 - 11, at a given mRNA concentration, the formation of precipitate was suppressed in a wider range of KCl concentration compared to NaCl concentration. In particular, for these three constructs, even at high KCl concentrations, the mRNA did not precipitate. Therefore, it is advantageous that KCl can be used at a higher concentration instead of NaCl if precipitation occurs before affinity chromatography.

[0323] Other embodiments of the present disclosure will be apparent to those skilled in the art from the consideration of the present specification and the practice of the disclosure disclosed herein. The specification and examples are intended to be considered as illustrative only, and the true scope and spirit of the present disclosure are indicated by the following claims.

[0324] All patents and publications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method for purifying messenger RNA (mRNA), (a) A step of subjecting a preparation containing mRNA synthesized in vitro to enzymatic digestion with proteinase, (b) A step of subjecting the preparation obtained from step (a) to oligo dT affinity chromatography, (c) A step of subjecting the preparation obtained from step (b) to a tangential flow filtration (TFF) step. A method that includes this.

2. i) The proteinase includes serine protease; ii) The preparation containing the in vitro synthesized mRNA is incubated with the proteinase at approximately 37°C for at least 30 minutes; iii) The proteinase is inactivated with a reducing agent, optionally the reducing agent being dithiothreitol (DTT); and / or iv) The method according to claim 1, wherein the preparation obtained from step (a) is subjected to a TFF step before step (b), and optionally the TFF uses a filter of about 100 kDa to about 300 kDa.

3. i) The preparation is subjected to a capping step prior to step (b) to produce capped mRNA, and optionally, the capping is carried out at approximately 37°C for at least 30 minutes with agitation; ii) The preparation is subjected to a tailing step with poly(A) polymerase to produce poly(A)-tailed mRNA; and / or iii) The method according to claim 1 or 2, wherein the TFF in step (c) uses a filter of about 50 kDa to about 300 kDa.

4. The prepared material is subjected to a front filtration step after the capping step, and optionally, i) The front filtration shall use a 0.2 μm filter; and / or ii) The above-mentioned front filtration reduces the guanylyltransferase (GuaT) content. The method according to claim 3.

5. A method for purifying messenger RNA (mRNA), (a) A step of subjecting a preparation containing mRNA synthesized in vitro to enzymatic digestion with proteinase, (b) A step of subjecting the preparation obtained from step (a) to a tangential flow filtration (TFF) step, (c) A step of subjecting the preparation obtained from step (b) to oligo dT affinity chromatography, (d) A step of subjecting the preparation obtained from step (c) to the TFF step A method that includes this.

6. i) The proteinase includes serine protease; and / or ii) The method of claim 5, wherein the preparation comprising in vitro synthesized mRNA is incubated with the proteinase at approximately 37°C for at least 30 minutes.

7. i) The proteinase is inactivated with a reducing agent, optionally the reducing agent being dithiothreitol (DTT); and / or ii) The method according to claim 5 or 6, wherein the TFF in step (b) is a filter of about 100 kDa to about 300 kDa.

8. The method according to claim 5 or 6, wherein the preparation is subjected to a capping step prior to step (c) to produce capped mRNA, and optionally the capping is carried out at approximately 37°C for at least 30 minutes with stirring.

9. i) The preparation is subjected to a tailing step with poly(A) polymerase to produce poly(A)-tailed mRNA, optionally, the tailing is performed before step (c), optionally simultaneously with or after capping; and / or ii) The method according to claim 5 or 6, wherein the TFF in step (d) is a filter of about 50 kDa to about 300 kDa.

10. i) The precipitation step is not performed; and / or ii) The method according to any one of claims 1, 2, 5, and 6, wherein at least about 0.5 grams of mRNA is purified.

11. The prepared material is subjected to a front filtration step after the capping step, and optionally, i) The front filtration shall use a 0.2 μm filter; and / or ii) The method according to claim 8, wherein the front filtration reduces the guanylyltransferase (GuaT) content.

12. A method for purifying messenger RNA (mRNA), (a) A step of subjecting a preparation containing mRNA synthesized in vitro to enzymatic digestion with proteinase, (b) A step of subjecting the preparation obtained from step (a) to a tangential flow filtration (TFF) step, (c) A step of subjecting the preparation obtained from step (b) to a capping step, (d) A step of subjecting the preparation obtained from step (c) to a step of front filtration, (e) A step of subjecting the preparation obtained from step (d) to oligo dT affinity chromatography, (f) A step of subjecting the preparation obtained from step (e) to the TFF step A method that includes this.

13. a) The preparation is subjected to a front filtration step after step (f); and / or b) The method according to claim 12, wherein the front filtration is i) using a 0.2 μm filter and / or ii) reducing the guanylate transferase (GuaT) content.

14. A) The mRNA synthesized in vitro is purified in batches of approximately 100 milligrams, 1 gram, 10 grams, 20 grams, 50 grams, 100 grams or more; B) The mRNA synthesized in vitro is purified on a scale of approximately 10 grams or more per batch or approximately 20 grams or more per batch, and (i) The residual plasmid DNA in the purified mRNA is approximately 0.2 pg / mg or less. (ii) The purified mRNA contains less than 0.1% dsRNA, (iii) The purified mRNA contains less than 1% of enzyme reagents used in in vitro synthesis, and / or (iv) The purified mRNA has an integrity of more than 80%; (C) The purified mRNA is suitable for therapeutic use; and / or (D) The method according to any one of claims 1, 2, 5, 6, 12, and 13, which does not include any additional affinity chromatography steps.

15. A method for producing messenger RNA (mRNA), The process of synthesizing mRNA in vitro, A step of purifying the in vitro synthesized mRNA using the method according to any one of claims 1, 2, 5, 6, 12, and 13. A method that includes this.

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