Method for ethanol-free mRNA purification

Abstract

Provided herein are methods of purifying messenger RNA (mRNA) by subjecting a formulation comprising in vitro synthesized mRNA to one or more steps of enzymatic digestion with a proteolytic enzyme, and optionally further steps of oligomeric dT affinity chromatography. Also provided are mRNA purified by the methods described herein.

Description

Background Art

[0001] The therapeutic agent based on messenger RNA (mRNA) has been established as an effective way for treating a variety of diseases. mRNA molecules encode treatment-related proteins (such as antigens for vaccination), which are produced in subjects after administration. However, mRNA molecules are relatively large, have high negative charges, and are easily degraded. In addition, the presence of pollutants remaining after mRNA purification can lead to undesirable immune responses. Therefore, it is important to develop effective mRNA purification strategies to ensure that the highly purified, safe mRNA products suitable for treatment and prevention purposes are obtained. Common mRNA purification strategies use corrosive or flammable solvents (such as ethanol), which are not desired for large-scale production due to the presence of safety challenges. Therefore, it is necessary to produce a large-scale and rapid mRNA purification scheme without flammable solvents for the highly purified, safe mRNA molecules that can be accepted for therapeutic purposes. Summary of the invention

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

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

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

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

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

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

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

[0009] In certain embodiments, the TFF uses about 100 kDa to about 300 kDa filters.

[0010] In certain embodiments, the mRNA contained in the preparation obtained from step (a) is concentrated using the 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, prior to step (b), the preparation is subjected to a capping step to produce capped mRNA.

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

[0014] In certain embodiments, the formulation 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 a poly(A) polymerase to produce mRNA with a poly(A) tail.

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

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

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

[0022] In certain embodiments, the TFF uses about 50 kDa to about 300 kDa filters.

[0023] In certain embodiments, the mRNA contained in the preparation obtained from step (b) is concentrated using the 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, the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; (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.

[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 mRNA with a poly(A) tail.

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

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

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

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

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

[0033] In certain embodiments, the proteolytic enzyme is inactivated with a reducing agent, optionally wherein the reducing agent is DTT.

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

[0035] In certain embodiments of any of the methods described above, no precipitation step is performed.

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

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

[0038] In certain embodiments of any of the methods described above, the formulation is subjected to a step of frontal filtration after capping.

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

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

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

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

[0043] In another aspect, the present disclosure provides mRNA obtainable by the above method. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The foregoing and other features and advantages of the present disclosure will be more fully understood from the following detailed description of illustrative embodiments, taken in conjunction with the accompanying drawings.

[0045] Figure 1 A flow chart outlining exemplary steps of an ethanol-free purification method is shown.The ethanol-free purification method can use both enzymatic digestion and affinity chromatography (enzymatic / affinity method; left panel) or only enzymatic digestion (holoenzymatic method; right panel).

[0046] Figure 2Images of silver-stained SDS-PAGE gels of proteins present at different steps of an ethanol-free purification process using an enzyme / affinity method are shown. Samples were taken at different points after capping. The enzyme / affinity method was performed without (panel A) or with (panel B) an intermediate forward filtration step before affinity chromatography. In both panels, lane 1 corresponds to the product obtained immediately after capping, lane 3 corresponds to the affinity chromatography eluate, lane 4 corresponds to the TFF eluate, and lane 5 corresponds to the product obtained after the final forward filtration. MW: molecular weight marker. The size of the molecular weight marker is shown on the right in kDa. SU: subunit. In panel A, lane 2 corresponds to the affinity chromatography flow-through. In panel B, lane 1' corresponds to the product obtained after the intermediate step of forward filtration (performed after capping and before affinity chromatography). The proteins used for capping are shown together with RNase A. It was observed that when the affinity chromatography step was performed directly after capping, the affinity chromatography did not completely remove guanylyltransferase (GuaT). In contrast, after the affinity chromatography step, when an intermediate step of forward filtration is performed, the RNA product is enzyme-free.

[0047] Figure 3 Images of silver-stained SDS-PAGE gels of IVT reactions treated with different proteolytic enzyme conditions are shown. Lane 1: treated with 0.55 U / mg RNA of Serine Proteolytic Enzyme (Arctic Zymes), 2 h incubation; Lane 2: treated with 0.05 U / mg RNA of Serine Proteolytic Enzyme (Arctic Zymes), 2 h incubation; Lane 3: treated with 0.025 U / mg RNA of Proteolytic Enzyme K (Sigma), 30 min incubation.

[0048] Figure 4 Images of silver-stained SDS-PAGE gels showing proteins present at different steps of the ethanol-free purification process are shown. Specifically, samples were taken after in vitro transcription (lane: IVT), after treatment with protease K (Sigma) (lane: P), after TFF and before capping (lane: TFF) or after capping (lane: Capping). Diafiltration was performed using Tris NaCl buffer in the presence (condition 1) or absence (condition 2) of DTT.

[0049] Figure 5A graph depicting the integrity of purified mRNA and intermediates over time at +5°C is shown. Samples were aliquoted after IVT (white bars), after capping (black bars), and after complete mRNA purification using an enzyme / affinity method (dashed bars), stored at +5°C, and analyzed for mRNA integrity by capillary electrophoresis (CE) at different time points. The integrity of the mRNA in the IVT reaction was stable at +5°C for at least 21 days, and the integrity did not significantly decrease over time. The integrity of the mRNA obtained after capping was >85% at 21 days, and the integrity did not significantly decrease over time. The purified mRNA was stable at +5°C for at least 3 months, and the integrity did not significantly decrease over time. The advantages of having RNA stable at +5°C notably include the ease of storing the product (i.e., in a cold room instead of a -20°C freezer) and the absence of thawing constraints in downstream processes.

[0050] Figure 6 Photographs showing the effect of sodium citrate or EDTA on the precipitate formed after large-scale in vitro transcription of mRNA are shown. Turbidity and therefore the presence of precipitate is measured by A of a preparation containing a precipitate formed after large-scale in vitro transcription of mRNA under different treatment conditions at about 37°C for 5 minutes. 600 nm Sample 1: untreated; 2: 10 mM sodium citrate; 3: 25 mM sodium citrate; 4: 50 mM sodium citrate; 5: 25 mM EDTA.

[0051] Figure 7 A graph depicting mRNA integrity after capping (Capping) and after oligo dT affinity chromatography (XTO) for large-scale mRNA purification as determined by capillary electrophoresis is shown. Results for a 10 g batch and the average of 3 replicates for a 20 g batch are shown. mRNA integrity increases after completing the affinity chromatography step.

[0052] Figure 8 Figures depicting the amount of (A) residual plasmid DNA and (B) percentage of RNA of double-stranded RNA (dsRNA) after the first tangential flow filtration step (TFF1) and after oligo dT affinity chromatography (XTO) for large-scale mRNA purification as determined by qPCR and ELISA, respectively. Results for a 10 g batch and 3 independent replicates (Replicate 1, Replicate 2, Replicate 3) of a 20 g batch are shown. For both 10 g and 20 g batch sizes, high purity mRNA samples were obtained after both TFF and oligo dT affinity chromatography.

[0053] Fig. 9Figures depicting the contour map of the mRNA concentration of mRNA construct 2 after incubation for 2h in (A) NaCl or (B) KCl are shown. The presence of precipitate was measured using UV-Vis 600nm absorbance values ​​by UV spectroscopy, and expressed in absorbance units (AU). Negative AU values ​​are equivalent to 0 absorbance, and indicate a clear solution. At least 0.05 AU values ​​indicate the risk of precipitate formation; When the AU value is at least 0.3, the naked eye can see the precipitate in a turbid form. When compared with NaCl at the same mRNA concentration, the use of KCl reduces the formation of precipitate at high salt concentrations.

[0054] Fig.10 Shown are graphs depicting contour plots of salt concentration compared to mRNA concentration of mRNA construct 3 after 2 h incubation in (A) NaCl or (B) KCl. The presence of precipitate was measured by UV spectroscopy using UV-Vis 600 nm absorbance values ​​and expressed in absorbance units (AU) as described above for Fig. 9 The use of KCl reduced precipitate formation at high salt concentrations when compared to NaCl at the same mRNA concentration.

[0055] Fig.11 Shown are graphs depicting contour plots of salt concentration compared to mRNA concentration of mRNA construct 4 after 2 h incubation in (A) NaCl or (B) KCl. The presence of precipitate was measured by UV spectroscopy using UV-Vis 600 nm absorbance values ​​and expressed in absorbance units (AU) as described above for Fig. 9 In contrast to NaCl, no precipitate was observed when KCl was used under any of the conditions evaluated. DETAILED DESCRIPTION

[0056] The present disclosure relates in particular to methods for purifying mRNA by subjecting a preparation comprising in vitro synthesized mRNA to one or more steps of enzymatic digestion with a proteolytic enzyme and optionally a further step of oligo dT affinity chromatography.

[0057] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this disclosure should have the meaning commonly understood by those of ordinary skill in the art. Although methods and materials similar to or equivalent to those described herein can also be used in the practice or test of this disclosure, exemplary methods and materials are described below. In the event of a conflict, this specification including the definition shall prevail. Usually, the nomenclature and technology used in conjunction with cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicine and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization as described herein are those nomenclatures and technologies well known and commonly used in the art. Enzymatic reactions and purification techniques are carried out as usually realized in this area or as described herein according to the manufacturer's instructions. Further, unless the context requires otherwise, singular terms should include plural numbers, and plural terms should include singular numbers. Throughout the present specification and examples, the words "have" and "comprise" or variations such as "has / having", "comprises / comprising" should be understood to mean the inclusion of the stated integer or groups of integers but not the exclusion of any other integer or groups of integers. All publications and other references mentioned herein are incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents forms part of the common general knowledge in the art.

[0058] It should be noted that the term "a" or "an" entity refers to one or more of the entity; for example, "nucleotide sequence" should be understood to mean one or more nucleotide sequences. Therefore, the terms "a / an", "one or more", and "at least one" can be used interchangeably herein.

[0059] In addition, "and / or" where used herein should be regarded as a specific disclosure of each of the two specified features or components with or without the other. Therefore, the term "and / or" as used in phrases such as "A and / or B" is intended 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 cover 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).

[0060] It should be understood that wherever aspects are described herein with the language "comprising," similar aspects described in the form of "consisting of" and / or "consisting essentially of" are also provided.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure relates. For example, 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 Oxford Dictionary Of Biochemistry And Molecular Biology, Revised ed., 2000, Oxford University Press can provide a general dictionary of many of the terms used in the present disclosure for one of ordinary skill.

[0062] Units, prefixes and symbols are all expressed in a form acceptable to the International System of Units (SI). Numerical ranges include numbers that define the range. The headings provided herein are not limitations on the various aspects of the present disclosure. Therefore, the terms of the definitions immediately below are more fully defined by reference to the specification (in its entirety).

[0063] The term "approximately" or "about" is used herein to mean approximately, roughly, approximately, or about. When the term "approximately" is used in conjunction with a numerical range, it defines the range by extending the boundary above and below the numerical value described. In general, the term "approximately" can limit the numerical value to a change of, for example, 10% above and below (higher or lower) above and below the specified value. In some embodiments, the term represents 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 indicated numerical value. In some embodiments, "approximately" represents a deviation of ±10% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±5% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±4% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±3% from the indicated numerical value. In some embodiments, "approximately" represents a deviation of ±2% from the indicated numerical value. In some embodiments, "about" means a deviation of ±1% from the indicated value. In some embodiments, "about" means a deviation of ±0.9% from the indicated value. In some embodiments, "about" means a deviation of ±0.8% from the indicated value. In some embodiments, "about" means a deviation of ±0.7% from the indicated value. In some embodiments, "about" means a deviation of ±0.6% from the indicated value. In some embodiments, "about" means a deviation of ±0.5% from the indicated value. In some embodiments, "about" means a deviation of ±0.4% from the indicated value. In some embodiments, "about" means a deviation of ±0.3% from the indicated value. In some embodiments, "about" means a deviation of ±0.1% from the indicated value. In some embodiments, "about" means a deviation of ±0.05% from the indicated value. In some embodiments, "about" means a deviation of ±0.01% from the indicated value.

[0064] As used herein, the term "batch" refers to the number or amount of mRNA purified at one time (e.g., purified according to a single manufacturing instruction during the same manufacturing cycle). A batch can refer to the amount of mRNA purified in a single purification run.

[0065] As used herein, the term "subjecting" or "contacting" or "incubating" refers to mixing two or more components so that those components can interact (e.g., incubating a preparation containing in vitro synthesized mRNA with a proteolytic enzyme). The two or more components can be incubated for any time sufficient to produce the desired effect.

[0066] As used herein, "purification method" refers to a method for obtaining a target molecule from a mixture (e.g., a solution or suspension) comprising 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 the method is performed is enhanced or increased compared to the concentration of the target molecule in the mixture before the method is performed. Purification of the target molecule can also be referred to as enriching 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.

[0067] More specifically, the present invention relates to a method for purifying mRNA, the method comprising: (a) subjecting a preparation comprising mRNA to enzymatic digestion with a proteolytic enzyme; and (b) subjecting the preparation obtained from step (a) to oligo dT affinity chromatography.

[0068] As used herein, the term "messenger RNA" or "mRNA" refers to a polynucleotide encoding at least one polypeptide. An mRNA may contain one or more coding regions and non-coding regions. The coding region may alternatively be referred to as an open reading frame (ORF).

[0069] As used herein, mRNA encompasses both modified RNA and unmodified RNA. In some embodiments, mRNA may include at least one chemical modification. In some embodiments, mRNA may contain one or more modifications that typically enhance RNA stability. Modified mRNA as provided herein may include, for example, backbone modifications, sugar modifications, or base modifications. In some embodiments, mRNA can be synthesized from naturally occurring nucleotides and / or nucleotide analogs (modified nucleotides), including but not limited to purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)) and modified nucleotide analogs or derivatives of purines and pyrimidines, such as, for example, 1-methyl-adenine, 2-methyl-adenine, 2-methylthio-N-6-isopentenyl-adenine, N6-methyladenine, N6-isopentenyl adenine, 2-thiocytosine, 3-methylcytosine, 4-acetylcytosine, 5-methylcytosine, 2,6-diaminopurine, 1-methylguanine, 2-methylguanine, 2,2-dimethylguanine, 7-methylguanine, inosine, 1-methylinosine, pseudouracil (5-uracil ), dihydrouracil, 2-thiouracil, 4-thiouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-(carboxyhydroxymethyl)-uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyluracil, 5-methyl-2-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5-methoxyaminomethyl-2-thiouracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxyuracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 1-methyl-pseudouracil, braided glycoside, β-D-mannosyl-braided glycoside, white butoxy glycoside and phosphoramidates, phosphorothioates, peptide nucleotides, methylphosphonates, 7-deazaguanosine, 5-methylcytosine and inosine.

[0070] 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-thiol-1-methyl-1-deaza-pseudouridine, 2-thiol-1-methylpseudouridine, 2-thiol-5-aza-uridine, 2-thiol-dihydropseudouridine, 2-thiol-dihydrouridine, 2-thiol-pseudouridine, 4-methoxy-2-thiol-pseudouridine, 4-methoxy-pseudouridine, 4-thiol-1-methylpseudouridine, 4-thiol-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methyluridine, 5-methoxyuridine, and 2'-O-methyluridine.

[0071] 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.

[0072] 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.

[0073] 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 an ORF are chemically modified.

[0074] The preparation of such analogs is described, for example, in U.S. Pat. No. 4,373,071, U.S. Pat. No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S. Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679, U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418.

[0075] mRNA can be synthesized in a cell-free environment, for example, by in vitro transcription (IVT). IVT is a process that allows template-directed synthesis of ribonucleic acid (RNA) (e.g., mRNA). In vitro transcription (IVT) is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNase I, pyrophosphatase, and / or RNase inhibitors. The exact conditions will vary depending on the specific application. Therefore, in some embodiments, making mRNA includes the following steps: performing IVT by mixing (i) a DNA template containing a suitable promoter and (ii) an RNA polymerase to produce an impure preparation containing full-length mRNA, and then subjecting the preparation to a purification method disclosed herein. The presence of these reagents is undesirable in the final mRNA product, and therefore these reagents can be referred to as impurities that can be purified or removed to provide a clean and / or homogeneous mRNA suitable for therapeutic use.

[0076] 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 DNA molecule amplified by PCR. With regard to the RNA polymerase used for transcription, the present invention is not limited. For example, in some embodiments, T7, T3 or SP6 type RNA polymerases can be used. In some embodiments, the polymerase is a T7 polymerase. In some embodiments, the polymerase is an SP6 polymerase. In some embodiments, the in vitro transcription further comprises a mixed ribonucleotide triphosphate pool.

[0077] 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 the following items: cis-regulatory elements (e.g., TATA boxes, termination signals, and protein binding sites), artificial recombination sites, x sites, CpG dinucleotide content, negative CpG islands, GC content, polymerase slippage sites, and / or other elements associated with transcription; the DNA template can be optimized with respect to the following items: cryptic splicing sites, mRNA secondary structures, stable free energy of mRNA, repeat sequences, mRNA unstable motifs, and / or other elements associated with mRNA processing and stability; the DNA template can be optimized with respect to the following items: codon usage bias, codon adaptability, internal x sites, ribosome binding sites (e.g., IRES), premature poly (A) sites, Shine-Dalgarno (SD) sequences, and / or other elements associated with translation; and / or the DNA template can be optimized with respect to the following items: codon background, codon-anticodon interactions, translation pause sites, and / or other elements associated with protein folding. Optimization methods known in the art can be used in the present invention, such as GeneOptimizer and OptimumGene from ThermoFisher. TM , described in US20110081708 (the contents of which are incorporated herein by reference in their entirety).

[0078] The inventors have found that a precipitate can be formed during the in vitro transcription process. In some cases, most of the synthesized mRNA is present in this precipitate, while a low concentration of mRNA is present in the supernatant (see Example 10). Unexpectedly, the precipitate cannot be redissolved by dilution in water, by dilution in a buffer, or by lowering the pH of the formulation.

[0079] The inventors tested adding 10mM, 25mM or 50mM sodium citrate to a preparation comprising in vitro synthesized mRNA. After adding 10mM sodium citrate, a significant reduction in turbidity was observed, and the precipitate was completely redissolved after adding 25mM or 50mM sodium citrate. Therefore, in some embodiments, after mRNA is subjected to enzymatic digestion with a proteolytic enzyme, sodium citrate is added to a preparation comprising in vitro synthesized mRNA. In some embodiments, sodium citrate is added to a preparation comprising in vitro synthesized mRNA at a concentration of at least about 10mM, optionally at least about 25mM. In a specific embodiment, sodium citrate is added to a preparation comprising in vitro synthesized mRNA at a concentration of about 25mM. The preparation can be incubated with sodium citrate for at least about five minutes. Before adding sodium citrate, the preparation is optionally at about 37°C (e.g., 35°C-37°C). Alternatively, after the sodium citrate is added, the formulation is optionally maintained at about 35°C to about 37°C for at least about 5 minutes.

[0080] The inventors have also tested the addition of EDTA to a preparation comprising an in vitro synthesized mRNA, and observed that the precipitate was completely re-dissolved with 25mM EDTA. Therefore, in some embodiments, after mRNA is subjected to enzymatic digestion with a proteolytic enzyme, EDTA is added to the preparation comprising in vitro synthesized mRNA. In certain embodiments, EDTA is added to the preparation comprising in vitro synthesized mRNA at a concentration of at least about 10mM, optionally at least about 25mM. In a specific embodiment, EDTA is added to the preparation comprising in vitro synthesized mRNA at a concentration of about 25mM. The preparation can be incubated with EDTA for at least about five minutes. Before adding EDTA, the preparation is optionally at about 37°C (e.g., 35°C-37°C). Alternatively, after adding EDTA, the preparation is optionally kept at about 35°C to about 37°C for at least about 5 minutes.

[0081] As used herein, the term "short polymer" or "prematurely aborted RNA sequence" refers to an incomplete product of an mRNA synthesis reaction (e.g., an in vitro synthesis reaction). For a variety of reasons, RNA polymerase does not always complete the transcription of a DNA template; for example, RNA synthesis terminates prematurely. Possible causes for premature termination of RNA synthesis include the quality of the DNA template, the polymerase terminator sequence of a specific polymerase present in the template, degraded buffer, temperature, depletion of ribonucleotides, and mRNA secondary structure. A prematurely aborted RNA sequence can be any length less than the expected length of the desired transcription product. For example, a prematurely aborted 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.

[0082] Double-stranded RNA (dsRNA) can also be formed during the in vitro synthesis of mRNA (e.g., when the 3' end of the full-length RNA transcript hybridizes with itself). This is undesirable because dsRNA may have an immunostimulatory effect. Due to the similar size and intrinsic characteristics of dsRNA contaminants, it is challenging to remove dsRNA contaminants from mRNA products. RNase III (also referred to as RNase C) can be used to digest dsRNA in the presence of a desired mRNA product to facilitate its removal. However, RNase III can also digest the desired mRNA product, so the percentage of complete mRNA is often reduced, and additional method steps are required to remove RNase III from mRNA preparations.

[0083] As used herein, the term "substantially" refers to the qualitative condition of exhibiting all or nearly all ranges or degrees of a feature or characteristic of interest. It will be appreciated by those of ordinary skill in the biological field that biological and chemical phenomena rarely, if ever, complete and / or proceed to completion or achieve or avoid absolute results. Therefore, the term "substantially" is used herein to capture the potential lack of completion inherent in many biological and chemical phenomena.

[0084] As used herein, the term "substantially free of" refers to a state in which there is a relatively small amount or no substance to be removed (e.g., RNA sequences of premature abortion). For example, "substantially free of RNA sequences of premature abortion" means that the RNA sequences of premature abortion are present at a level of impurities less than about 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). Alternatively, "substantially free of RNA sequences of premature abortion" means that the RNA sequences of premature abortion are present at a level of less than about 100ng, 90ng, 80ng, 70ng, 60ng, 50ng, 40ng, 30ng, 20ng, 10ng, 1ng, 500pg, 100pg, 50pg, 10pg or less.

[0085] In one embodiment, a method for purifying mRNA is provided, the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; and (b) subjecting the preparation obtained from step (a) to oligo dT affinity chromatography.

[0086] The present invention can be used for purifying the mRNA of any protein encoding.Typically, the present invention is used for purifying single mRNA species, that is, the preparation comprising mRNA contains the mRNA derived from a single gene, a single synthesis reaction or a single expression construct.By contrast, the total mRNA purified from cells contains a variety of mRNA species.Although it may be necessary in some embodiments to provide mRNA from in vitro transcription reaction, mRNA from other sources, including wild-type mRNA produced by bacteria, fungi, plants and / or animals is considered within the scope of the present invention.

[0087] As used herein, the term "in vitro" refers to events that occur in an artificial environment (eg, in a test tube or reaction vessel, in cell culture, etc.) rather than within a multicellular organism.

[0088] According to various embodiments, the present invention is used to purify in vitro synthesized mRNA with various lengths. In certain embodiments, the present invention is used to purify in vitro synthesized mRNA with a length of less than about 1 kb, 1.5 kb, 2 kb, 2.5 kb, 3 kb, 3.5 kb, 4 kb, 4.5 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb or 15 kb. In certain embodiments, the present invention is used to purify mRNA containing one or more modifications that typically enhance stability. In certain embodiments, one or more modifications are selected from modified nucleotides (such as those described herein) and modified sugar-phosphate backbones. In certain embodiments, the present invention is used to purify unmodified in vitro synthesized mRNA.

[0089] Typically, mRNA contains a 5' cap structure. The mRNA 5' cap can provide resistance to nucleases found in most eukaryotic cells and promote translation efficiency. Several types of 5' caps are known. The 7-methylguanosine cap (also known as "m7G" or "cap 0") contains a guanosine linked to the first transcribed nucleotide via a 5'-5'-triphosphate bond.

[0090] The term "capping" refers to the addition of a 5' cap structure to an mRNA. The 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one terminal phosphate group from the 5' nucleotide, leaving two terminal phosphates; then guanosine triphosphate (GTP) is added to the terminal phosphate via a guanylyltransferase to produce a 5'5'5 triphosphate bond; then, the 7-nitrogen of guanine is methylated by a methyltransferase. 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. Publication No. US2016 / 0032356 and U.S. Publication No. US2018 / 0125989, which are incorporated herein by reference.

[0091] During the IVT reaction, 5'-capping can be accomplished concomitantly using a cap analog that can be incorporated as the first "base" of the nascent RNA strand. The cap analog can be cap 0, cap 1, cap 2, m6Am, or a cap analog. As an example, the following chemical RNA cap analogs can be used to generate a 5'-guanosine cap structure 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).

[0092] Alternatively, capping of the mRNA can be performed post-transcriptionally (i.e., after IVT). For example, a vaccinia capping enzyme can be used to generate the Cap 0 structure: m7G(5')ppp(5')G. The Cap 1 structure can be generated using both the vaccinia capping enzyme and a 2'-O methyltransferase to generate: m7G(5')ppp(5')G-2'-O-methyl. The Cap 2 structure can be generated from the Cap 1 structure, and then the 5' third to last nucleotide is 2'-O-methylated using a 2'-O methyl-transferase. The Cap 3 structure can be generated from the Cap 2 structure, and then the 5' fourth to last nucleotide is 2'-O-methylated using a 2'-O methyl-transferase.

[0093] 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' cap:

[0094] "Capping enzyme" refers to an enzyme that catalyzes the attachment of a 5' cap to an mRNA. A single capping enzyme comprising the enzymatic activity necessary for capping can be used. Alternatively, two or more enzymes with different enzymatic activities can be used, such as a 2'O-methyltransferase and a guanylyltransferase. Capping can occur in the presence of an RNase inhibitor, the methyl donor S-adenosylmethionine, and / or GTP.

[0095] In certain embodiments, mRNA includes 5' and / or 3' untranslated region (UTR). In certain embodiments, 5'UTR includes one or more elements affecting the stability or translation of mRNA, such as iron response elements. Exemplary 5' and / or 3'UTR sequences can be derived from stable mRNA molecules (e.g., globin, actin, GAPDH, tubulin, histone and citric acid cycle enzymes), to increase the stability of sense mRNA molecules. For example, 5'UTR sequences can include CMV early 1 (IE1) gene or its fragment of partial sequence, to improve the nuclease resistance of polynucleotides and / or improve the half-life of polynucleotides. It is also contemplated that the sequence encoding human growth hormone (hGH) or its fragment is included in the 3' end or untranslated region of mRNA.

[0096] In some embodiments, the length of the 5'UTR may be between about 50 and 500 nucleotides. In some embodiments, the 3'UTR includes one or more of the following: a polyadenylation signal, a binding site for a protein that affects the positional stability of the mRNA in the cell, or one or more binding sites for a miRNA. In some embodiments, the length of the 3'UTR may be between 50 and 500 nucleotides or longer.

[0097] Typically, mRNA comprises poly (A) tail. As used herein, the term "poly (A) sequence", "poly (A) tail" and "poly (A) part" refers to the adenosine nucleotide sequence at the 3' end of the mRNA molecule. Poly (A) tail can give stability to mRNA and protect it from exonuclease degradation. Poly (A) tail can enhance translation. In certain embodiments, poly (A) tail is substantially homopolymeric. For example, poly (A) tail of 100 adenosine nucleotides can substantially have a length of 100 nucleotides. When mRNA comprises 3'UTR, poly (A) tail can be directly located downstream of the coding region (that is, before 3'UTR) or downstream of 3'UTR. 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 contains at least 300 adenosine monophosphates. In some embodiments, the poly (A) tail contains at least 350 adenosine monophosphates. In some embodiments, the poly (A) tail contains at least 400 adenosine monophosphates. In some embodiments, the poly (A) tail contains at least 450 adenosine monophosphates. In certain embodiments, the poly (A) tail may 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 poly(A) tail of 100 adenosine nucleotides may have a length of more than 100 nucleotides (including 100 adenosine nucleotides and at least one nucleotide or stretch of nucleotides other than adenosine nucleotides).

[0098] In some embodiments, the poly (A) tail of the nucleic acid is obtained from the DNA template during RNA in vitro transcription. In certain embodiments, the poly (A) tail is obtained in vitro by conventional chemical synthesis methods without transcription from a DNA template. In various embodiments, the poly (A) tail is produced by enzymatic polyadenylation of the RNA (after RNA in vitro transcription) using a commercially available polyadenylation kit and corresponding protocols, or alternatively, by using an immobilized poly (A) polymerase, for example using methods and means as described in WO 2016 / 174271.

[0099] The nucleic acid may comprise a poly (A) tail obtained by enzymatic polyadenylation, wherein most nucleic acid molecules comprise from about 100 (+ / - 20) to about 500 (+ / - 50) or about 250 (+ / - 20) adenosine nucleotides.

[0100] In some embodiments, the nucleic acid may include a poly(A) tail derived from the template DNA, and may further include at least one additional poly(A) tail produced by enzymatic polyadenylation, for example as described in WO 2016 / 091391.

[0101] As used herein, the term "proteolytic enzyme" refers to a protease that catalyzes the hydrolytic decomposition of a protein by cleaving the protein into smaller peptide fractions (i.e., polypeptides or amino acids). In other words, a proteolytic enzyme enzymatically digests a protein or polypeptide. Examples of proteolytic enzymes or proteases include, but are not limited to, serine proteases (e.g., proteolytic enzyme K), cysteine ​​proteases, threonine proteases, aspartic proteases, glutamic proteases, metalloproteases, and asparagine peptide lyases.

[0102] "Isolated," "isolated," "purified," "purified," "enriched," and "enriched," when applied to an mRNA of interest, indicate that the mRNA of interest is separated, enriched, sorted, etc., from or relative to other biological materials or chemical components (such as components of an IVT reaction) at a certain point in time to produce a higher proportion of the mRNA of interest compared to other biological materials, chemical components, contaminants, or active agents (such as enzymes). The isolated mRNA can be 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 more than about 99% separated from the other components with which it was initially associated. In some embodiments, the isolated mRNA is about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. Components from which mRNA can be isolated include dsRNA, plasmid DNA, enzymes, and endotoxins, among others. As used herein, calculations of the purity percentage of an isolated substance should not include excipients (e.g., buffer, water, etc.).

[0103] In some embodiments, less than 10pg / mg (e.g., less than 10pg / mg, less than 9pg / mg, less than 8pg / mg, less than 7pg / mg, less than 6pg / mg, less than 5pg / mg, less than 4pg / mg, less than 3pg / mg, less than 2pg / mg, or less than 1pg / mg) is an 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 1pg / mg, less than about 2pg / mg, less than about 3pg / mg, less than about 4pg / mg, less than about 5pg / mg, less than about 6pg / mg, less than about 7pg / mg, less than about 8pg / mg, less than about 9pg / mg, less than about 10pg / mg, less than about 11pg / mg, or less than about 12pg / mg. Therefore, the residual plasmid DNA in the purified mRNA is less than about 1pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 2pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 3pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 4pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 5pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 6pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 7pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 8pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 9pg / mg. In some embodiments, the residual plasmid DNA in the purified mRNA is less than about 10pg / mg.

[0104] The inventors have demonstrated that the methods provided herein can be used to effectively remove residual plasmid DNA from large quantities of mRNA. In some embodiments, the in vitro synthesized mRNA is purified at a scale of or greater than about 10 grams per batch, and the residual plasmid DNA in the purified mRNA is less than about 0.5pg / mg (e.g., less than about 0.2pg / mg). In some embodiments, the in vitro synthesized mRNA is purified at a scale of or greater than about 20 grams per batch, and the residual plasmid DNA in the purified mRNA is less than about 0.5pg / mg (e.g., less than about 0.2pg / mg). In some embodiments, the residual plasmid DNA is evaluated by methods in the art (e.g., by using qPCR).

[0105] In some embodiments, more than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or substantially all RNA sequences of premature abortion are removed according to the method of the present invention. In some embodiments, the mRNA composition is substantially free of RNA sequences of premature abortion. In some embodiments, the mRNA composition contains less than about 5% (e.g., less than about 4%, 3%, 2%, or 1%) RNA sequences of premature abortion. In some embodiments, the mRNA composition contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) RNA sequences of premature abortion. In some embodiments, the mRNA isolated according to the methods of the present invention contains undetectable premature abortion RNA sequences, as determined by, for example, high performance liquid chromatography (HPLC) (e.g., shoulder or separation peak), ethidium bromide, Coomassie staining, capillary electrophoresis (CE) or glyoxal gel electrophoresis (e.g., presence of a separate low band). In some embodiments, the length of the premature abortion 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, short polymers are detected or quantified after adding a 5'-cap and / or a 3'-poly A tail. In some embodiments, the RNA transcript of premature abortion comprises 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 RNA transcript of premature abortion contains about 8-15, 8-14, 8-13, 8-12, 8-11, or 8-10 bases.

[0106] The inventors have shown that double-stranded RNA (dsRNA) can also be effectively removed from large quantities 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%, 0.2%, or 0.1%) dsRNA. In some embodiments, the purified mRNA contains less than about 0.1% (e.g., less than about 0.05%, 0.03%, 0.025%, or 0.01%) dsRNA. In some embodiments, dsRNA levels are assessed by methods of the art (e.g., ELISA or dot blots using anti-dsRNA monoclonal antibody J2). In some embodiments, the method does not include a step of contacting the preparation with RNase III.

[0107] In some embodiments, the in vitro synthesized mRNA is purified at 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 at 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 at a scale of about 20 grams or more per batch, and the purified mRNA composition contains less than about 0.025% dsRNA.

[0108] In some embodiments, the in vitro synthesized mRNA is purified at 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 greater than about 85% integrity. In some embodiments, the in vitro synthesized mRNA is purified at 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 greater than about 85% integrity. In some embodiments, the in vitro synthesized mRNA is purified at 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 greater than about 85% integrity.

[0110] In some embodiments, the mRNA purified according to the methods provided herein is substantially free of enzyme reagents used for IVT, including but not limited to RNA polymerase, DNase I, pyrophosphatase and / or RNase inhibitors. In some embodiments, the mRNA purified according to the present invention contains less than about 5% (e.g., less than about 4%, 3%, 2% or 1%) of enzyme reagents used for IVT. In some embodiments, the purified mRNA contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) of enzyme reagents used for IVT. In some embodiments, the purified mRNA contains no detectable 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.

[0111] In various embodiments, the mRNA purified according to the methods provided herein maintains a high degree of integrity. As used herein, the term "mRNA integrity" generally refers to the quality of the purified mRNA. Methods well known in the art can be used, such as by RNA agarose gel electrophoresis to determine mRNA integrity. In some embodiments, mRNA integrity refers to the percentage of mRNA that is not degraded after the purification process. In some embodiments, mRNA integrity can be determined by the band pattern of RNA agarose gel electrophoresis. In some embodiments, compared with the reference band of RNA agarose gel electrophoresis, the purified mRNA of the present invention shows very few bands or no bands. In some embodiments, the purified mRNA of the present invention has an integrity 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 greater than 98%. In some embodiments, the purified mRNA of the present invention has an integrity greater than 99%. In some embodiments, the purified mRNA of the present invention has an integrity of about 100%.

[0112] In some embodiments, the in vitro synthesized mRNA is purified at 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 in vitro synthesized mRNA is purified at 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%).

[0113] In some embodiments, the following one or more characteristics of the purified mRNA are evaluated: appearance, identity, quantity, concentration, presence of impurities, microbial evaluation, pH level and activity. In some embodiments, an acceptable appearance includes a clear, colorless solution, substantially free of visible particles. In some embodiments, the identity of the mRNA is evaluated by a sequencing method. In some embodiments, the concentration is evaluated by a suitable method (such as UV spectrophotometry). In some embodiments, a suitable concentration is between about 90% and 110% of the nominal value (e.g., 0.9-1.1 mg / mL).

[0114] In certain embodiments, the purity of the mRNA is assessed including assessing mRNA integrity and assessing residual plasmid DNA. In certain embodiments, acceptable mRNA integrity levels are assessed by agarose gel electrophoresis. The gel is analyzed to determine whether the band pattern and apparent nucleotide length are consistent with analytical reference standards. Other methods for assessing RNA integrity include, for example, assessing purified mRNA using capillary gel electrophoresis (CGE). In certain embodiments, the acceptable purity of the purified mRNA determined by CGE is that the purified mRNA composition has no more than about 40% long abortion / degradation material.

[0115] In some embodiments, the purified mRNA is subjected to microbial testing, including, for example, assessing bacterial endotoxins. In some embodiments, bacterial endotoxins are <0.5EU / mL, <0.4EU / mL, <0.3EU / mL, <0.2EU / mL, or <0.1EU / mL. Therefore, in some embodiments, the bacterial endotoxins in the purified mRNA are <0.5EU / mL. In some embodiments, the bacterial endotoxins in the purified mRNA are <0.4EU / mL. In some embodiments, the bacterial endotoxins in the purified mRNA are <0.3EU / mL. In some embodiments, the bacterial endotoxins in the purified mRNA are <0.2EU / mL. In some embodiments, the bacterial endotoxins in the purified mRNA are <0.1EU / mL.

[0116] In some embodiments, the pH of the purified mRNA is assessed. In some embodiments, the acceptable pH of the purified mRNA is between 5 and 8. Thus, in some embodiments, the pH of the purified mRNA is about 5. In some embodiments, the pH of the purified mRNA is about 6. In some embodiments, the pH of the purified mRNA is about 7. In some embodiments, the pH of the purified mRNA is about 7. In some embodiments, the pH of the purified mRNA is about 8.

[0117] In some embodiments, the translation fidelity of the purified mRNA is assessed. Translation fidelity can be assessed by various methods, and includes, for example, transfection and Western blot analysis. Acceptable features of the purified mRNA include a band pattern of molecular weights migrating on a Western blot similar to a reference standard. In some embodiments, the conductivity of the purified mRNA is assessed. In some embodiments, the acceptable features of the purified mRNA include a conductivity of between about 50% and 150% of a reference standard.

[0118] The cap percentage and poly (A) tail length of purified mRNA are also assessed. In certain embodiments, acceptable cap percentage includes cap 1, area %: NLT90. In certain embodiments, acceptable poly (A) tail length is about 100-1500 nucleotides (e.g., 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400 or 1500 nucleotides). Therefore, in certain embodiments, acceptable poly (A) tail length is about 100 nucleotides. In certain embodiments, acceptable poly (A) tail length is about 200 nucleotides. In certain embodiments, acceptable poly (A) tail length is about 250 nucleotides. In certain embodiments, acceptable poly (A) tail length is about 300 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 350 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 400 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 450 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 500 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 550 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 600 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 650 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 700 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 750 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 800 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 850 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 900 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 950 nucleotides. In some embodiments, the length of the poly (A) tail of an acceptable amount is about 1000 nucleotides. In some embodiments, the length of the poly (A) tail is about 1100 nucleotides. In some embodiments, the length of the poly (A) tail is about 1200 nucleotides. In some embodiments, the length of the poly (A) tail is about 1300 nucleotides. In some embodiments, the length of the poly (A) tail is about 1400 nucleotides. In some embodiments, the length of the poly (A) tail is about 1500 nucleotides.

[0119] In some embodiments, the in vitro synthesized mRNA is purified at a scale of about 10 grams or more per batch, and the purified mRNA has a cap percentage of at least about 90%. In some embodiments, the in vitro synthesized mRNA is purified at a scale of about 20 grams or more per batch, and the purified mRNA has a cap percentage of at least about 90%.

[0120] In some embodiments, the in vitro synthesized mRNA is purified at 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 in vitro synthesized mRNA is purified at a scale of about 20 grams or more per batch and has a poly (A) tail length of at least about 100 nucleotides.

[0121] Various methods for detecting and quantifying mRNA purity are known in the art. For example, such methods include blotting, capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver staining, spectroscopy, ultraviolet (UV) or UPLC or a combination thereof. In certain embodiments, before gel electrophoresis ("glyoxal gel electrophoresis"), the mRNA is first denatured by glyoxal dye. In certain embodiments, the synthesized mRNA is characterized before capping or tailing. In certain embodiments, the synthesized mRNA is characterized after capping and tailing.

[0122] The preparation containing mRNA provided in step (a) is an impure preparation (i.e., it contains impurities). As used herein, the term "impurity" refers to a substance that has a different chemical composition from the target material or compound in a limited amount of liquid, gas or solid. Impurities are also referred to as contaminants. The mRNA purification strategy described herein is capable of removing many impurities associated with the mRNA produced by IVT, such as IVT enzymes (i.e., RNA polymerases), free NTPs, buffer components (i.e., salts), mRNA fragments (e.g., prematurely aborted sequences), and untailed mRNA. The mRNA purification strategy described herein is also capable of removing nucleic acid contaminants, such as double-stranded RNA and residual plasmid DNA. Therefore, the mRNA purification strategy described herein can be used to provide a purified mRNA preparation suitable for therapeutic use.

[0123] As used herein, the term "affinity chromatography" or "affinity purification" refers to a separation method based on a specific binding interaction between a ligand immobilized or coupled to a solid support and its binding partner. As the complex mixture passes through the column, molecules with specific binding affinity for the ligand bind. After washing away other sample components, the bound molecules are removed from the solid support, resulting in their purification from the original mixture. Each specific affinity system requires its own set of conditions known to those skilled in the art.

[0124] As used herein, the term "oligo dT" refers to a single-stranded sequence of deoxythymidine (dT). Oligo dT binds to the poly (A) tail of an mRNA molecule and can be used as a method for purifying mRNA. Oligo dT can have any length suitable for hybridizing to the poly (A) tail, such as 12 to 50-mers. Various types of oligo dT ligands are known in the art and are commercially available. For example, oligo (dT) 25 It is a homogeneous mixture of 25-mer deoxythymidine, while oligo(dT) 12-18 It is a mixture of 12-mer to 18-mer deoxythymidine.

[0125] In some embodiments, the method of purifying mRNA does not comprise 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 chromatography which carries predominantly or exclusively primary amino ligands on its surface or surfaces. I. Proteolytic enzyme treatment

[0126] In some embodiments, one or more proteolytic enzymes can be used to enzymatically digest proteins used in in vitro transcription, DNA enzyme treatment, capping and / or tailing reactions and facilitate purification of mRNA. In some embodiments, proteolytic enzymes include serine proteases (e.g., protease 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 proteases, aspartic acid proteases, glutamic acid proteases, metalloproteases, and asparagine peptide cleavage enzymes.

[0127] In some embodiments, the proteolytic enzyme is a serine protease. In some embodiments, the proteolytic enzyme is proteolytic enzyme K. Proteolytic enzyme K is a serine protease that exhibits a very wide range of cleavage specificity, and it cleaves peptide bonds adjacent to the carboxyl groups of aliphatic and aromatic amino acids. By subjecting the preparation comprising mRNA to enzymatic digestion with a proteolytic enzyme (such as a serine protease), degradation and inactivation of the enzyme can be advantageously achieved. Proteolytic enzyme treatment greatly promotes downstream mRNA purification. It is easier to purify and remove shorter peptide fragments of the IVT enzyme using filtration (i.e., TFF) and / or chromatography-based purification (e.g., affinity chromatography, such as oligo dT chromatography). In some embodiments, the serine proteolytic enzyme is a proteolytic enzyme K from Engyodontium album. In some embodiments, proteolytic enzyme K is a recombinant protein. In some embodiments, proteolytic enzyme K is heat-resistant. In some embodiments, proteolytic enzyme K is selected from the variants described in U.S. Patent No. US10633644 B1.

[0128] In some embodiments, the preparation comprising in vitro synthesized mRNA is subjected to enzymatic digestion with a proteolytic enzyme to obtain an mRNA-proteolytic enzyme mixture. In some embodiments, the preparation comprising in vitro synthesized mRNA is incubated with a proteolytic enzyme at about 25°C to about 40°C for at least 30 minutes. In some embodiments, the preparation comprising in vitro synthesized mRNA is incubated with a proteolytic enzyme 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, the preparation comprising in vitro synthesized mRNA is incubated with a proteolytic enzyme at about 37°C for at least about 30 minutes. In some embodiments, the preparation comprising in vitro synthesized mRNA is incubated with a proteolytic enzyme 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, the preparation comprising in vitro synthesized mRNA is incubated with a proteolytic enzyme 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.

[0129] In some embodiments, the concentration of the proteolytic enzyme is at least about 0.02U / mg RNA. In some embodiments, the concentration of the proteolytic enzyme is 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 0.5U / mg RNA. In some embodiments, the concentration of the proteolytic enzyme is 0.02-0.6U / mg RNA. In some embodiments, the concentration of the proteolytic enzyme is 0.02-0.03U / mg RNA. In some embodiments, the concentration of the proteolytic enzyme is about 0.5-0.6U / mg RNA. In some embodiments, the concentration of the proteolytic enzyme is about 0.55U / mg RNA. In some embodiments, the preparation comprising in vitro synthesized mRNA is incubated at about 37°C for at least about 30 minutes with a proteolytic enzyme having a concentration of 0.02-0.6U / mg RNA.

[0130] In certain embodiments, the mRNA-proteolytic enzyme mixture (i.e., the preparation of the mRNA synthesized in vitro and the proteolytic enzyme) is stirred. As used herein, the term "stirred" means any effect suitable for mixing a mixture (e.g., consisting of a preparation of the mRNA synthesized in vitro and a proteolytic enzyme). Exemplary devices for achieving stirring are known to the technician, and include an oscillator, a mixer (e.g., a vortex mixer or a static mixer), a magnetic stirrer (including a stirring rod) and a stirring rod, which are available in different sizes depending on the volume of the mixture to be mixed. Stirring can be carried out to achieve a time sufficient to thoroughly mix. Stirring enhances the overall efficacy of the proteolytic enzyme process, particularly for at least about 1 gram of large-scale mRNA purification.

[0131] In some embodiments, the mRNA purification described herein includes a step of enzymatic digestion with a proteolytic enzyme, followed by an affinity chromatography step (e.g., oligo dT). Additional steps, such as a TFF step, a capping step, and / or a tailing step, may be present between the step of enzymatic digestion with a proteolytic enzyme and the affinity chromatography step.

[0132] In one aspect, provided herein is a method for purifying mRNA, the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; (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.

[0133] In other embodiments, mRNA purification as described herein comprises the first step of enzymatic digestion with proteolytic enzyme, followed by the second step of enzymatic digestion with proteolytic enzyme.At least one other step occurs between the first step and the second step of enzymatic digestion with proteolytic enzyme, such as TFF step, capping step, tailing step and / or forward filtration step, as provided herein.In certain embodiments, the first step of enzymatic digestion is followed by the step of TFF, and then the step of capping and / or tailing.In certain embodiments, the first step of enzymatic digestion is followed by the step of TFF, and then the step of capping and / or tailing, and then the step of forward filtration.In certain embodiments, in the case of the first step and the second step of enzymatic digestion with proteolytic enzyme, affinity chromatography step is not carried out.

[0134] In one aspect, the present invention provides a method for purifying mRNA, comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to a first enzymatic digestion with a proteolytic enzyme; (b) subjecting the preparation obtained from step (a) to a second enzymatic digestion with a proteolytic enzyme; and (c) subjecting the preparation obtained from step (b) to a TFF step.

[0135] On the other hand, provided herein is a method for purifying mRNA, the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; (b) subjecting the preparation obtained from step (a) to a TFF step; (c) subjecting the preparation obtained from step (b) to an enzymatic digestion with a proteolytic enzyme; and (d) subjecting the preparation obtained from step (c) to a TFF step.

[0136] On the other hand, the present disclosure provides a method for purifying mRNA, the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to a first proteolytic enzyme incubation step, thereby producing a first mRNA-proteolytic enzyme mixture; (b) subjecting the first mRNA-proteolytic enzyme mixture to a first step of TFF, thereby producing a first filtered mRNA mixture; (c) subjecting the first filtered mRNA mixture to a second proteolytic enzyme incubation step, thereby producing a second mRNA-proteolytic enzyme mixture; and (d) subjecting the second mRNA-proteolytic enzyme mixture to a second step of TFF, thereby producing purified mRNA.

[0137] In any of the methods provided herein, a precipitation step is not performed (eg, a preparation comprising in vitro synthesized mRNA is not subjected to an ethanol-mediated precipitation step).

[0138] The special advantage provided by the present invention is that can purify mRNA, particularly in vitro synthesized mRNA with large-scale or commercial scale.For example, in some embodiments, in vitro synthesized mRNA is purified with each batch being or greater than about 100 milligrams, 1 gram, 10 grams, 50 grams, 100 grams or larger scale.

[0139] In a specific embodiment, the in vitro synthesized mRNA is purified on a scale of 10 grams per batch. In a specific embodiment, the in vitro synthesized mRNA is purified on a scale of 20 grams per batch. In a specific embodiment, the in vitro synthesized mRNA is purified on a scale of 25 grams per batch. In a specific embodiment, the in vitro synthesized mRNA is purified on a scale of 50 grams per batch. In another specific embodiment, the in vitro synthesized mRNA is purified on a scale of 100 grams or more per batch.

[0140] In any of the methods provided herein, at least about 0.5 grams of mRNA are purified. In some embodiments, at least about 0.5 grams to about 100 grams of mRNA are 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 are 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 are purified.

[0141] In other embodiments, the serine protease is inactivated after incubation with a preparation comprising in vitro transcribed mRNA. In certain embodiments, the serine protease is inactivated by heating. In certain embodiments, the serine protease is inactivated by heating the preparation to at least 55°C. In certain embodiments, the serine protease is inactivated with a chelating agent such as EDTA. In certain embodiments, the serine protease is inactivated with a serine protease inhibitor such as phenylmethylsulfonyl fluoride (PMSF). In certain embodiments, the serine protease is inactivated with a reducing agent. Non-limiting examples of reducing agents include dithiothreitol (DTT) and tri-2-carboxyethylphosphine hydrochloride (TCEP). In certain embodiments, the 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. II. Tangential Flow Filtration (TFF)

[0142] Tangential flow filtration (TFF), also known as cross-flow filtration, is a type of filtration in which the material to be filtered passes tangentially over the filter rather than through it. In TFF, the undesired permeate passes through the filter, while the desired retentate (e.g., mRNA) passes along the filter and is captured or retained on a downstream filter or membrane. In contrast, in forward filtration, the flow direction is perpendicular to the filter surface as the material to be filtered passes through the filter.

[0143] In embodiments of the mRNA purification methods described herein, a sample containing mRNA (e.g., a preparation containing in vitro synthesized mRNA or an mRNA-proteolytic enzyme mixture) is subjected to at least one TFF step (i.e., a first TFF step). In some embodiments, a sample containing mRNA is subjected to a second TFF step.

[0144] In some embodiments, the mRNA-proteolytic enzyme mixture is subjected to at least a first TFF step prior to the oligo dT affinity chromatography step. In some embodiments, the mRNA-proteolytic enzyme mixture is subjected to at least a first TFF step after a first step of enzymatic digestion with a proteolytic enzyme and prior to a second step of enzymatic digestion with a proteolytic enzyme.

[0145] In an embodiment, the TFF step includes about 50kDa to about 300kDa filter. In certain embodiments, the TFF step includes about 50kDa filter, 60kDa filter, 70kDa filter, 80kDa filter, 90kDa filter, 100kDa filter, 110kDa filter, 120kDa filter, 130kDa filter, 140kDa filter, 150kDa filter, 160kDa filter, 170kDa filter, 180kDa filter, 190kDa filter, 200kDa filter, 210kDa filter, 220kDa filter, 230kDa filter, 240kDa filter, 250kDa filter, 260kDa filter, 270kDa filter, 280kDa filter, 290kDa filter or 300kDa filter. In certain embodiments, the TFF step includes about 100kDa to about 300kDa filter. In certain embodiments, the TFF step includes about 50kDa to about 100kDa filter. kDa filter size refers to the molecular weight cut-off (MWCO).

[0146] 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 membrane), polyvinylidene fluoride (PVDF), cellulose acetate, cellulose nitrate, mixed cellulose ester (MCE), ultra-high molecular weight polyethylene (UPE), polyfluorotetraethylene (PTFE), nylon, polysulfone, polyethersulfone, polyacrylonitrile, polypropylene, polyvinyl chloride and any combination thereof. In one embodiment, membrane is cellulose. In one embodiment, membrane is polyethersulfone (mPES) hollow fiber membrane.

[0147] Suitable membranes for use in the present invention can have different surface areas. In some embodiments, suitable membranes have a surface area large enough to facilitate large-scale production of mRNA. For example, suitable membranes can have a surface area of ​​about 1,500 cm or greater. 2 、2,000cm 2 、2,500cm 2 、3,000cm 2 、3,500cm 2 、4,000cm 2 、4,500cm 2 、5,000cm 2 、7,500cm 2 、10,000cm 2 、5m 2 、10m 2 、12m 2 、15m 2 、20m 2 、24m 2 、25m 2 、30m 2 , or 50m 2 surface area.

[0148] In some embodiments, prior to the oligo dT affinity chromatography step, the mRNA-proteolytic enzyme mixture is further subjected to a first TFF step, wherein the TFF step comprises an about 100 kDa to about 300 kDa filter.

[0149] In some embodiments, the mRNA-proteolytic enzyme mixture is concentrated with at least a first TFF step. In some embodiments, the mRNA-proteolytic enzyme mixture is diafiltered with at least a first TFF step. In one embodiment, the mRNA-proteolytic enzyme mixture is concentrated before diafiltration to reduce the volume required for diafiltration.

[0150] In certain embodiments, mRNA-proteolytic enzyme mixture is concentrated to at least about 5mg / mL.In certain embodiments, mRNA-proteolytic enzyme mixture is concentrated to at least about 10mg / mL.In certain embodiments, mRNA-proteolytic enzyme mixture is concentrated to about 5mg / mL, about 6mg / mL, about 7mg / mL, about 8mg / mL, about 9mg / mL, about 10mg / mL, about 11mg / mL, about 12mg / mL, about 13mg / mL, about 14mg / mL, about 15mg / mL, about 16mg / mL, about 17mg / mL, about 18mg / mL, about 19mg / mL or about 20mg / mL.In certain embodiments, mRNA-proteolytic enzyme mixture is concentrated to about 10-12mg / mL.In certain embodiments, mRNA-proteolytic enzyme mixture is concentrated to no more than about 12mg / mL.

[0151] In some embodiments, the mRNA contained in the preparation obtained from the oligo dT affinity chromatography (i.e., affinity purified mRNA) 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 a proteolytic enzyme is further subjected to a TFF step (e.g., a second TFF step).

[0152] In some embodiments, the second TFF step comprises an about 50 kDa to about 300 kDa filter. In some embodiments, the second TFF step comprises an about 50 kDa to about 100 kDa filter.

[0153] In some embodiments, the affinity purified mRNA is concentrated using a TFF step. In some embodiments, the affinity purified mRNA is diafiltered using a TFF step. In some embodiments, the affinity purified mRNA is concentrated prior to diafiltration, as this advantageously reduces the volume required for diafiltration.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] In a typical TFF process, the following at least three process variables are important: transmembrane pressure (TMP), feed rate and flow rate of permeate. TMP is the force that drives fluid to carry permeable molecules through the filter. In certain embodiments, TMP is between 1 and 30 pounds force per square inch (psi), including end value. In certain embodiments, TMP is from about 5 to about 15psi. In certain embodiments, TMP is from about 7 to about 9psi. In certain embodiments, TMP is about 1psi, about 2psi, about 3psi, about 4psi, about 5psi, about 6psi, about 7psi, about 8psi, about 9psi, about 10psi, about 11psi, about 12psi, about 13psi, about 14psi, about 15psi, about 16psi, about 17psi, about 18psi, about 19psi, about 20psi, about 21psi, about 22psi, about 23psi, about 24psi, about 25psi, about 26psi, about 27psi, about 28psi, about 29psi or about 30psi. 1 psi is equivalent to about 0.07 bar. Thus, in some embodiments, the TMP is between about 0.07 and about 2.07 psi, inclusive. In some embodiments, the TMP is from about 0.34 bar to about 1.03 psi. In some embodiments, the TMP is from about 0.48 bar to about 0.62 bar.

[0158] The feed rate (also referred to as the cross-flow velocity) is the rate at which the solution flows through the feed channel and through the filter. The feed rate determines the force of flushing away the molecules that could originally clog or contaminate the filter and thereby limit the flow of the filtrate. In some embodiments, the feed rate is between 1 and 1000 mL / min. In some embodiments, the feed rate is between 50 and 800 mL / min. In some embodiments, the feed rate is between 50 and 750 mL / min. In some embodiments, the feed rate is between 50 and 300 mL / min. In some embodiments, the feed rate is between 50 and 200 mL / min. In some embodiments, the feed rate is between 75 and 200 mL / min. In some embodiments, the feed rate is between 100 and 200 mL / min. In some embodiments, the feed rate is between 125 and 175 mL / min. In some embodiments, the feed rate is 130 mL / min. In some embodiments, the feed rate is between 60 mL / min and 220 mL / min. In some embodiments, the feed rate is 60 mL / min or greater. In some embodiments, the feed rate is 100 mL / min or greater. In some embodiments, the feed rate is 150 mL / min or greater. In some embodiments, the feed rate is 200 mL / min or greater. In some embodiments, the feed rate is 220 mL / min or greater.

[0159] The flow rate of permeate is the rate of removing permeate from the system. For constant feed rate, increasing the permeate flow rate can increase the pressure through the filter, resulting in enhanced filtration rate, while also potentially increasing the risk of filter clogging or contamination. The principle, theory and device of TFF are described in Michaels et al., "Tangential Flow Filtration" in Separations Technology [" tangential flow filtration " in separation technology], Pharmaceutical and Biotechnology Applications [medicine and biotechnology applications] (WP Olson edited, Interpharm Press, Inc. [International Pharmaceutical Company Press Company], Buffalo Grove, 111.1995). Regarding the description of high efficiency tangential flow filtration (HP-TFF) (it represents the improvement of TFF), also refer to U.S. Patent number 5,256,294 and 5,490,937. In certain embodiments, the flow rate is between 1 and 100mL / minute. In certain embodiments, the flow rate is between 10 and 100mL / minute. In certain embodiments, the flow rate is between 10 and 90mL / minute. In some embodiments, the flow rate is between 10 and 80mL / minutes. In some embodiments, the flow rate is between 10 and 70mL / minutes. In some embodiments, the flow rate is between 10 and 60mL / minutes. In some embodiments, the flow rate is between 10 and 50mL / minutes. In some embodiments, the flow rate is between 10 and 40mL / minutes. In some embodiments, the flow rate is between 15 and 60mL / minutes. In some embodiments, the flow rate is between 20 and 40mL / minutes. In some embodiments, the flow rate is 10mL / minutes, 15mL / minutes, 20mL / minutes, 25mL / minutes, 30mL / minutes, 35mL / minutes, 40mL / minutes, 45mL / minutes, 50mL / minutes, 55mL / minutes, 60mL / minutes, 65mL / minutes, 70mL / minutes, 75mL / minutes, 80mL / minutes, 85mL / minutes, 90mL / minutes, 95mL / minutes or 100mL / minutes.

[0160] Any combination of various process variables described herein can be used. In certain embodiments, TFF is carried out with a feed rate of about 100-200 mL / minute (e.g., about 100-180 mL / minute, 100-160 mL / minute, 100-140 mL / minute, 110-190 mL / minute, 110-170 mL / minute, or 110-150 mL / minute) and / or a flow rate of about 10-50 mL / minute (e.g., about 10-40 mL / minute, 10-30 mL / minute, 20-50 mL / minute, or 20-40 mL / minute). In certain embodiments, TFF is carried out with a feed rate of about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or 200 mL / minute and / or a flow rate of about 10, 20, 30, 40, or 50 mL / minute. In other embodiments, TFF is performed at a feed rate of about 500, 750 mL / min, 1, 2, 3, 4, or 5 L / min and / or a flow rate of about 100, 200, 250, 500, 750 mL / min, or 1 L / min.

[0161] In some embodiments, a filtration step is further performed after the affinity chromatography step or after a second enzymatic digestion with a proteolytic enzyme. In some embodiments, sterile filtration or forward filtration is performed. In some embodiments, filtration is performed on a filter with a pore size of 0.2 or 0.22 μm. In some embodiments, filtration is performed on a polyethersulfone (PES) filter.

[0162] In some embodiments, a forward filtration step is performed after capping. In some embodiments, a forward filtration step is performed after the step of TFF. In some embodiments, a forward filtration step is performed after capping and before oligo dT affinity chromatography. In some embodiments, a forward filtration step is performed after oligo dT affinity chromatography. In some embodiments, a forward filtration step is performed after the second step of TFF. In some embodiments, forward filtration is performed on a filter with a pore size of 0.2 or 0.22 μm. In some embodiments, forward filtration uses a polyethersulfone (PES) filter. III. Affinity chromatography

[0163] In one embodiment, mRNA from an impure preparation is purified by oligo dT chromatography. Oligo dT is a single stranded sequence of deoxythymidine (dT) and can be used, for example, in affinity chromatography to purify mRNA containing a stretch of poly(A), such as a poly(A) tail. In one embodiment, oligo dT is a homogenous mixture of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30-mer deoxythymidine, 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 . Oligo dT can be fixed on a chromatographic resin or other type of solid support. In particular, oligo dT can be combined with a monolithic matrix, a membrane or a bead. Oligo dT can be combined with one of the following supports: borosilicate glass, agarose, sepharose, magnetic beads, polystyrene, polyacrylamide, membrane, silica, semiconductor material, silicon, organic polymer, ceramic, glass, metal, plastic polycarbonate, polycarbonate, polyethylene, polyethylene terephthalate, polymethyl methacrylate, polypropylene, polyvinyl acetate, polyvinyl chloride, polyvinyl pyrrolidone or soda lime glass. In one embodiment, oligo dT is combined with cross-linked poly (styrene-divinylbenzene) (PSDVB). In one embodiment, oligo dT is combined with agarose gel. In one embodiment, oligo dT is combined with agarose. In one embodiment, oligo dT is combined with cellulose. In one embodiment, oligo dT is combined with poly (glycidyl methacrylate-co-ethylene glycol dimethacrylate).

[0164] In certain embodiments, oligodT is attached to a solid support via a joint (such as a carbon joint), which is located between the oligodT molecule and the solid support. The joint can be included in multiple positions within or on oligodT and / or the support. The selection of the joint is within the capabilities of the technician. Suitable joints include alkyl and aryl groups (including heteroalkyl and heteroaryl), and substituted derivatives of these. In some cases, the joint can be based on amino acids and / or contain amide bonds. Examples of joints are: amino derivatives, thiol derivatives, aldehyde derivatives, formyl derivatives, azide derivatives (click chemistry), biotin derivatives, alkyne derivatives, hydroxy derivatives, activated hydroxy or derivatives, carboxylate derivatives, activated carboxylate derivatives, activated carbonates, activated esters, nhs esters (succinimidyl), nhs carbonates (succinimidyl), imidoesters or derivatives, cyanogen bromide derivatives, maleimide derivatives, haloacetyl derivatives, iodoacetamide / iodoacetamide Acyl derivatives, epoxide derivatives, streptavidin derivatives, trifluoroethanesulfonyl derivatives, diene / conjugated diene derivatives (Diels-Alder type reaction), olefin derivatives, substituted phosphate derivatives, bromohydrins / halohydrins, substituted disulfides, pyridyl-disulfide derivatives, aryl azides, acyl azides, azlactones, hydrazide derivatives, halogenated benzene derivatives, nucleoside derivatives, branched / multifunctional linkers, dendrimer functional groups and / or nucleoside derivatives; or any combination thereof. In one embodiment, the linker comprises an amide bond. Non-limiting examples of carbon linkers include formula C x wherein x is any integer between 5 and 50. In some embodiments, the carbon linker is C6, C7, C8, C9, C 10 , C 11 , C 12 , C 13 , C 14 or C 15 In some embodiments, the linker comprises 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 an N-hydroxysuccinimidyl (NHS) ester bound to the support.

[0165] In an embodiment, the oligo dT chromatography resin comprises the following structure: wherein n is an integer selected from the group consisting of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30.

[0166] In an embodiment, the oligo dT chromatography resin comprises the following structure:

[0167] In an embodiment, the oligo dT chromatography resin comprises the following structure: wherein n is an integer selected from the group consisting of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. PSDVB is polystyrene divinylbenzene.

[0168] In an embodiment, the oligo dT chromatography resin comprises the following structure:

[0169] In an embodiment, oligo dT affinity purification comprises oligo(dT) 18 or oligo(dT) 25 Chromatographic resin. The mechanism of purification involves hybridization of the poly(A) tail of the mRNA with oligo dT. Contaminant impurities in the preparation (such as proteins, unreacted nucleotides, plasmid DNA, cap analogs, partial transcripts, dsRNA byproducts and enzymes) lack the poly(A) portion and do not bind.

[0170] Sample loading buffer can be used for mRNA preparation to be loaded into oligodT substrate (oligodT fixed on chromatographic resin or another type of solid support). OligodT affinity purification is typically carried out in sample loading buffer with a salt concentration up to about 250mM. Some salts are needed in loading buffer because under low conductivity conditions, the mutual electrostatic repulsion between oligodT substrate and mRNA skeleton is very strong and is enough to prevent the formation of hydrogen bonds (and the formation of hydrogen bonds is required for the hybridization of the poly (A) tails of mRNA and oligodT). Increasing the concentration of salt in loading buffer has the theoretical benefit of suppressing the non-specific binding of pollutants to oligodT substrates. However, high salt concentration can cause the precipitation of mRNA product (particularly when mRNA is present in sample with high concentration), which can interfere with the flow of mRNA preparation on oligodT substrate. This is particularly problematic for large quantities of mRNA because when purifying large quantities of mRNA (e.g., at least about 10 g, 20 g, or 100 g) in a single batch, the mRNA will typically be present in the oligo dT loading buffer at a high concentration (e.g., greater than 0.5 mg / ml).

[0171] The negative effect associated with having high salt concentration in oligodT loading buffer can be reduced by minimizing the time period of mRNA sample exposure to high salt conditions.Therefore, in some embodiments, no more than about 2 hours (for example, no more than about 1 hour) before preparation is applied to oligodT substrate, salt (for example, sodium chloride) is added to the preparation comprising at least about 0.5mg / mlmRNA to a final salt concentration of 400mM to about 1200mM.In certain embodiments, no more than about 2 hours before preparation is applied to oligodT substrate, salt (for example, sodium chloride) is added to the preparation comprising about 0.5mg / ml to about 2.0mg / ml mRNA to a final salt concentration of about 600mM to about 1000mM.For example, in some embodiments, no more than about 2 hours before preparation is applied to oligodT substrate, salt (for example, sodium chloride) is added to the preparation comprising about 2.0mg / ml mRNA to a final salt concentration of about 800mM.

[0172] Sodium chloride is usually added to oligo dT loading buffer to increase its ionic strength. The inventors found that 800mM sodium chloride is added to the mRNA of 20g scale batches with a concentration of about 2.0mg / ml and is suitable for carrying out oligo dT purification to specific mRNA preparations. However, it is found that before being loaded on oligo dT substrates, adding sodium chloride to a final concentration of 800mM in the different mRNAs of 1g batches can cause mRNA precipitation, which causes the productivity of the reduction of the mRNA of column blocking and purification. The inventors found that in the case of observing mRNA precipitation after adding sodium chloride to oligo dT loading buffer, by replacing sodium chloride with potassium chloride, this precipitation (referring to Example 11) can be at least partially prevented. Therefore, in the case of observing mRNA precipitation, using potassium chloride to replace sodium chloride can allow the use of high salt concentration (to suppress the non-specific binding of pollutants) in oligo dT loading buffer in the case of high concentration mRNA (making it possible to use smaller buffer volume to purify large quantities of mRNA, for faster purification process), while reducing the negative effect of mRNA precipitation. This was an unexpected finding because sodium chloride and potassium chloride are both neutral salts with similar properties.

[0173] Thus, in some embodiments, the oligo dT affinity chromatography step comprises loading a formulation comprising at least about 0.5 mg / ml mRNA and at least about 500 mM potassium chloride onto an oligo dT substrate. For example, in some embodiments, the formulation comprises about 0.5 mg / ml to about 2.0 mg / ml mRNA (e.g., about 1.0 mg / ml). In some embodiments, the formulation comprises about 500 mM to about 1000 mM potassium chloride (e.g., about 600 mM to about 900 mM potassium chloride).

[0174] Washing steps remove unbound contaminants, and the poly(A) mRNA can then be eluted from the resin using a low ionic strength buffer or a competitive binding oligonucleotide solution.

[0175] In an embodiment, the preparation comprising in vitro synthesized mRNA is diluted prior to oligo dT affinity chromatography. In some embodiments, the dilution occurs 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.

[0176] In some embodiments, the method comprises subjecting the mRNA preparation to an oligo dT affinity chromatography step, and does not comprise any additional affinity chromatography steps. IV. Messenger RNA Capping and Tailing

[0177] In one embodiment, the method for purifying mRNA described herein includes a capping step. In some embodiments, a poly (A) tailing step is also performed.

[0178] 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 occurs with IVT. In another embodiment, capping occurs after IVT. In one embodiment, capping occurs before step (a) of subjecting a preparation containing in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme. In one embodiment, capping is performed on purified in vitro transcribed mRNA. In one embodiment, capping is performed after a first step of enzymatic digestion with a proteolytic enzyme. In one embodiment, capping is performed after a preparation containing in vitro synthesized mRNA has been subjected to a step of TFF. In one embodiment, capping is performed after a preparation containing in vitro synthesized mRNA has been subjected to an enzymatic digestion with a proteolytic enzyme and a TFF step. In one embodiment, capping occurs before the step of affinity chromatography. In one embodiment, capping occurs before step (b) of subjecting the preparation obtained from step (a) to oligo dT affinity chromatography in the methods provided herein. In one embodiment, when the method comprises two steps of enzymatic digestion with a proteolytic enzyme, capping occurs before the second step of enzymatic digestion with a proteolytic enzyme.

[0179] 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.

[0180] 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.

[0181] In some embodiments, capping is performed while the mixture containing the mRNA is stirred.

[0182] In some embodiments, the mRNA lacks a poly(A) tail after an initial IVT reaction with an RNA polymerase and must be tailed.

[0183] In some embodiments, the mRNA is incubated with a poly(A) polymerase to produce an mRNA with a poly(A) tail. In some embodiments, the poly(A) polymerase is an E. coli poly(A) polymerase.

[0184] In some embodiments, the mRNA is co-incubated with one or more capping enzymes and poly (A) polymerase to produce capped mRNA with a poly (A) tail. In other embodiments, the mRNA is incubated with one or more capping enzymes, and then the mRNA is incubated with a poly (A) polymerase to produce capped mRNA with a poly (A) tail.

[0185] 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.

[0186] In some embodiments, the step of forward filtering is performed after capping. In some embodiments, when tailing is performed concomitantly with or after capping, the step of forward filtering is performed after capping and tailing.

[0187] In one aspect, provided herein is a method for producing mRNA, the method comprising: synthesizing mRNA in vitro; and purifying the in vitro synthesized mRNA using the method described herein.

[0188] In some embodiments, manufacturing mRNA includes methods for large-scale production of full-length mRNA molecules. In some embodiments, manufacturing mRNA includes methods for producing compositions rich in full-length mRNA molecules, the lengths of these full-length mRNA molecules are 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 comprises 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 mRNA.

[0189] In another aspect, there is provided an mRNA obtainable by the methods described herein.

[0190] 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.

[0191] In another aspect, a method for treating a disease or disorder is provided, the method comprising administering to a subject in need thereof a purified mRNA obtained by the method described herein or a composition comprising the purified mRNA obtained by the method described herein.

[0192] In another aspect, a purified mRNA obtained by the methods described herein or a composition comprising the purified mRNA obtained by the methods described herein for use in therapy is provided.

[0193] The present invention includes the following embodiments.

[0194] Example 1. A method for purifying mRNA, the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; and (b) subjecting the preparation obtained from step (a) to oligo dT affinity chromatography.

[0195] Embodiment 2. The method according to embodiment 1, wherein the proteolytic enzyme comprises a serine protease.

[0196] Embodiment 3. The method according to embodiment 1 or 2, wherein the preparation comprising in vitro synthesized mRNA is incubated with the proteolytic enzyme at about 37° C. for at least 30 minutes.

[0197] Embodiment 4. The method according to any one of embodiments 1-3, wherein the mRNA-proteolytic enzyme mixture is stirred.

[0198] Embodiment 5. The method according to any one of embodiments 1-4, wherein the proteolytic enzyme is inactivated with a reducing agent, optionally wherein the reducing agent is DTT.

[0199] Embodiment 6. The method according to any one of Embodiment 5, wherein the DTT is added to a concentration of at least about 20 mM.

[0200] Embodiment 7. The method according to any one of embodiments 1-6, wherein before step (b), the preparation obtained from step (a) is subjected to a TFF step.

[0201] Embodiment 8. The method of embodiment 7, wherein the TFF uses an about 100 kDa to about 300 kDa filter.

[0202] Embodiment 9. The method according to embodiment 7 or 8, wherein the mRNA contained in the preparation obtained from step (a) is concentrated using the TFF.

[0203] 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.

[0204] Embodiment 11. The method according to any one of embodiments 1-10, wherein sodium citrate is added to the formulation obtained from step (a).

[0205] Embodiment 12. The method of embodiment 11, wherein sodium citrate is added at a concentration of at least about 10 mM, optionally at least about 25 mM.

[0206] Embodiment 13. The method of embodiment 11, wherein sodium citrate is added at a concentration of about 25 mM.

[0207] Embodiment 14. The method of any one of embodiments 11-13, wherein after adding the sodium citrate, the formulation is maintained at about 35°C to about 37°C for at least about 5 minutes.

[0208] Embodiment 15. The method according to any one of embodiments 1-10, wherein EDTA is added to the preparation obtained from step (a).

[0209] Embodiment 16. The method of embodiment 15, wherein EDTA is added at a concentration of at least about 10 mM, optionally at least about 25 mM.

[0210] Embodiment 17. The method of embodiment 15, wherein EDTA is added at a concentration of about 25 mM.

[0211] Embodiment 18. The method of any one of embodiments 15-17, wherein after adding EDTA, the formulation is maintained at about 35°C to about 37°C for at least about 5 minutes.

[0212] Embodiment 19. The method according to any one of embodiments 1-18, wherein prior to step (b), the preparation is subjected to a capping step to produce capped mRNA.

[0213] Embodiment 20. The method of embodiment 19, wherein the preparation is incubated with 2'O-methyltransferase and guanylyltransferase.

[0214] Embodiment 21. The method of embodiment 19 or 20, wherein the preparation is incubated with an RNase inhibitor.

[0215] Embodiment 22. The method according to any one of embodiments 19-21, wherein the preparation is incubated with GTP and S-adenosylmethionine.

[0216] Embodiment 23. The method of any one of embodiments 19-22, wherein capping is performed at about 37°C for at least 30 minutes, optionally under stirring.

[0217] Embodiment 24. The method according to any one of embodiments 1-23, wherein the preparation is subjected to a tailing step with a poly(A) polymerase to produce an mRNA with a poly(A) tail.

[0218] Embodiment 25. The method according to any one of embodiments 1-23, wherein the preparation is incubated with one or more capping enzymes and a poly(A) polymerase to produce a capped mRNA with a poly(A) tail.

[0219] Embodiment 26. The method according to any one of embodiments 1-23, wherein the preparation is incubated with one or more capping enzymes and then the mRNA is incubated with a poly(A) polymerase to produce a capped mRNA with a poly(A) tail.

[0220] Embodiment 27. The method according to any one of embodiments 1-26, wherein the oligo dT affinity chromatography uses oligo (dT) 25 Chromatographic resins.

[0221] Embodiment 28. The method according to any one of embodiments 1-27, wherein the preparation obtained from step (b) is subjected to a TFF step.

[0222] Embodiment 29. The method of embodiment 28, wherein the TFF uses an about 50 kDa to about 300 kDa filter.

[0223] Embodiment 30. The method according to embodiment 28 or 29, wherein the mRNA contained in the preparation obtained from step (b) is concentrated using the TFF step.

[0224] Embodiment 31. The method according to embodiment 30, wherein the mRNA contained in the preparation obtained from step (b) is concentrated to at least about 2 mg / mL.

[0225] Embodiment 32. The method according to any one of embodiments 19-31, wherein the preparation is subjected to a forward filtration step after the capping step.

[0226] Embodiment 33. The method according to any one of embodiments 19-31, wherein after the capping step and before step (b), the preparation is subjected to a step of forward filtration.

[0227] Embodiment 34. The method according to any one of embodiments 28-33, further comprising a step of forward filtration after the step of TFF.

[0228] Example 35. A method for purifying mRNA, the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; (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.

[0229] 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.

[0230] Embodiment 37. A method according to embodiment 35 or 36, wherein the preparation obtained from step (b) is incubated with poly(A) polymerase to produce mRNA with a poly(A) tail.

[0231] Embodiment 38. The method according to any one of embodiments 35-37, wherein the preparation obtained from step (b) is co-incubated with one or more capping enzymes and poly(A) polymerase to produce capped mRNA with a poly(A) tail.

[0232] Embodiment 39. The method according to any one of embodiments 35-38, wherein prior to step (c), the preparation is subjected to a forward filtration step.

[0233] Embodiment 40. The method according to any one of embodiments 36-38, wherein after capping and before step (c), the preparation is subjected to a step of forward filtration.

[0234] Embodiment 41. The method according to any one of embodiments 35-40, further comprising a step of forward filtration after step (d).

[0235] Embodiment 42. The method of any one of embodiments 32-34 and 39-41, wherein the forward filtration comprises a 0.2 μm filter.

[0236] Embodiment 43. A method for purifying messenger RNA (mRNA), the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; (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 forward 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.

[0237] Embodiment 44. The method according to embodiment 43, wherein the method further comprises a step of forward filtration after step (f).

[0238] Embodiment 45. The method of Embodiment 43 or 44, wherein the forward filtration comprises a 0.2 μm filter.

[0239] Embodiment 46. The method of any one of embodiments 32-34, 39-42, and 43-45, wherein the forward filtration reduces the guanylyltransferase (GuaT) content.

[0240] Embodiment 47. The method according to any one of the preceding embodiments, wherein the oligo dT affinity chromatography comprises adding salt to the preparation to a final concentration of about 400 mM to about 1200 mM no more than 2 hours before applying the preparation to the oligo dT substrate.

[0241] Embodiment 48. The method of embodiment 47, wherein the salt is added to the formulation to a final concentration of about 500 mM to about 1000 mM.

[0242] Embodiment 49. The method of any one of embodiments 47-48, wherein the salt is added to the formulation to a final concentration of about 800 mM.

[0243] Embodiment 50. The method of any one of embodiments 47-49, wherein the formulation comprises at least about 0.5 mg / ml mRNA.

[0244] Embodiment 51. The method of embodiment 50, wherein the formulation comprises about 0.5 mg / ml to about 2.0 mg / ml mRNA.

[0245] Embodiment 52. The method of embodiment 51, wherein the formulation comprises about 1.0 mg / ml mRNA.

[0246] Embodiment 53. The method of any one of embodiments 47-52, wherein the salt is sodium chloride.

[0247] Embodiment 54. The method of any one of embodiments 47-52, wherein the salt is potassium chloride.

[0248] Embodiment 55. The method of any one of embodiments 47-52 and 54, wherein the oligo dT affinity chromatography comprises loading a formulation comprising at least about 0.5 mg / ml mRNA and at least about 500 mM potassium chloride onto the oligo dT substrate.

[0249] Embodiment 56. The method of embodiment 55, wherein the formulation comprises about 500 mM to about 1000 mM potassium chloride.

[0250] Embodiment 57. The method of embodiment 56, wherein the formulation comprises about 600 mM to about 900 mM potassium chloride.

[0251] Example 58. A method for purifying mRNA, the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to a first enzymatic digestion with a proteolytic enzyme; (b) subjecting the preparation obtained from step (a) to a second enzymatic digestion with a proteolytic enzyme; and (c) subjecting the preparation obtained from step (b) to a TFF step.

[0252] Embodiment 59. The method of embodiment 58, wherein the proteolytic enzyme comprises a serine protease.

[0253] Embodiment 60. The method of embodiment 58 or 59, wherein the first enzymatic digestion and / or the second enzymatic digestion occurs at about 37°C for at least 30 minutes.

[0254] Embodiment 61. The method of any one of embodiments 58-60, wherein the first enzymatic digestion and / or the second enzymatic digestion is agitated.

[0255] Embodiment 62. The method of any one of embodiments 58-61, wherein the proteolytic enzyme is inactivated with a reducing agent, optionally wherein the reducing agent is dithiothreitol (DTT).

[0256] Embodiment 63. The method of embodiment 62, wherein the DTT is added to a concentration of at least about 20 mM.

[0257] Embodiment 64. The method of any preceding embodiment, wherein no precipitation step is performed.

[0258] Embodiment 65. A method according to any of the preceding embodiments, wherein at least about 0.5 grams of mRNA is purified.

[0259] Embodiment 66. The method of any one of the preceding embodiments, wherein about 0.5 grams to about 100 grams of mRNA is purified.

[0260] Embodiment 67. A method according to any of the preceding embodiments, wherein the in vitro synthesized mRNA is purified at a scale of about 100 mg, 1 g, 10 g, 20 g, 50 g, 100 g or more per batch, optionally wherein the mRNA is purified at a scale of about 10 g per batch or more.

[0261] Embodiment 68. A method according to any one of the preceding embodiments, wherein the residual plasmid DNA in the purified mRNA is less than about 1 pg / mg.

[0262] Embodiment 69. A method according to embodiment 68, wherein the residual plasmid DNA in the purified mRNA is less than about 0.5 pg / mg.

[0263] Embodiment 70. A method according to embodiment 69, wherein the residual plasmid DNA in the purified mRNA is less than or equal to about 0.2 pg / mg.

[0264] Embodiment 71. A method according to any of the preceding embodiments, wherein the in vitro synthesized mRNA is purified in batches of about 10 grams or more, and the residual plasmid DNA in the purified mRNA is less than or equal to about 0.2 pg / mg.

[0265] A method according to Example 72, wherein the in vitro synthesized mRNA is purified in a batch size of or greater than about 20 grams, and the residual plasmid DNA in the purified mRNA is less than or equal to about 0.2 pg / mg.

[0266] Embodiment 73. A method according to any of the preceding embodiments, wherein the method removes more than about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or substantially all of the RNA sequences for premature abortion.

[0267] Embodiment 74. A method according to any of the preceding embodiments, wherein the purified mRNA contains less than about 5% (e.g., less than about 4%, 3%, 2% or 1%) premature abortion RNA sequences.

[0268] Embodiment 75. A method according to embodiment 74, wherein the purified mRNA contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) of RNA sequences for premature abortion.

[0269] Embodiment 76. The method of any one of the preceding embodiments, wherein the purified mRNA contains less than about 1% (e.g., less than about 0.5%, 0.2% or 0.1%) dsRNA.

[0270] Embodiment 77. A method according to embodiment 76, wherein the purified mRNA contains less than about 0.1% (e.g., less than about 0.050%, 0.030%, 0.025% or 0.010%) dsRNA.

[0271] Embodiment 78. The method according to any one of the preceding embodiments, wherein the in vitro synthesized mRNA is purified in batches of about 10 grams or more, and the purified mRNA contains less than about 0.01% dsRNA.

[0272] Embodiment 79. The method of embodiment XX, wherein the in vitro synthesized mRNA is purified in batches of about 20 grams or more, and the purified mRNA contains less than about 0.025% dsRNA.

[0273] Embodiment 80. A method according to any of the preceding embodiments, wherein the purified mRNA contains less than about 5% (e.g., less than about 4%, 3%, 2% or 1%) of an enzymatic reagent used in the in vitro synthesis.

[0274] Embodiment 81. A method according to embodiment 80, wherein the purified mRNA contains less than about 1% (e.g., less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% or 0.1%) of an enzyme reagent used in in vitro synthesis.

[0275] Embodiment 82. A method according to any of the preceding embodiments, wherein the purified mRNA contains no detectable enzymatic 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.

[0276] Embodiment 83. A method according to any of the preceding embodiments, wherein the in vitro synthesized mRNA is purified in batches of about 10 grams or greater, and the purified mRNA contains no detectable amount of enzymatic reagents used in the in vitro synthesis, as determined by silver staining.

[0277] Embodiment 84. A method according to embodiment 83, wherein the in vitro synthesized mRNA is purified in batches of about 20 grams or greater, and the purified mRNA contains no detectable amount of enzymatic reagents used in the in vitro synthesis, as determined by silver staining.

[0278] Embodiment 85. A method according to any of the preceding embodiments, wherein the purified mRNA has greater than about 60%, 70%, 80%, 90% or 95% (e.g., greater than about 96%, 97%, 98%, 99% or more) integrity.

[0279] Embodiment 86. A method according to any one of the preceding embodiments, wherein the purified mRNA has greater than about 80% integrity.

[0280] Embodiment 87. A method according to Embodiment 86, wherein the purified mRNA has an integrity greater than about 85%.

[0281] Embodiment 88. A method according to any of the preceding embodiments, wherein the in vitro synthesized mRNA is purified in batches of about 10 grams or more, and the purified mRNA has an integrity of greater than about 80% (e.g., at least about 85%).

[0282] Embodiment 89. A method according to embodiment 88, wherein the in vitro synthesized mRNA is purified in batches of about 20 grams or more, and the purified mRNA has an integrity of greater than about 80% (e.g., at least about 85%).

[0283] Embodiment 90. A method according to any one of the preceding embodiments, wherein the method does not comprise the step of contacting the preparation with RNase III.

[0284] Embodiment 91. The method according to any one of the preceding embodiments, wherein the method does not include the step of applying the formulation to a primary amino solid phase.

[0285] Embodiment 92. A method according to any one of the preceding embodiments, wherein the method does not comprise any additional affinity chromatography step.

[0286] Embodiment 93. A method according to any of the preceding embodiments, wherein the method does not comprise any additional filtration step.

[0287] Embodiment 94. A method according to any of the preceding embodiments, wherein the method does not comprise any additional purification steps.

[0288] Embodiment 95. A method according to any one of the preceding embodiments, wherein the purified mRNA is suitable for therapeutic use.

[0289] Embodiment 96. A method for producing mRNA, the method comprising: synthesizing mRNA in vitro; and purifying the in vitro synthesized mRNA using the method according to any one of the preceding embodiments.

[0290] Embodiment 97. An mRNA which can be obtained by the method according to any one of the preceding embodiments. Examples

[0291] The foregoing description of specific embodiments will fully disclose the general nature of the present disclosure, and others can easily modify and / or adjust such particular embodiments for a variety of applications by applying knowledge within the technical scope of the art without undue experimentation and without departing from the overall concept of the present disclosure. Therefore, based on the teachings and guidance given herein, such adjustments and modifications are intended to be included in the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the words or terms in this article are for the purpose of description rather than limitation, and therefore the terms or words of this specification will be interpreted by those skilled in the art based on the teachings and guidance.

[0292] The method described below is performed in a nuclease-free environment, wherein: a) all single-use parts (e.g., bottles, pipette tips, tubes, etc.), components and equipment are decontaminated by RNase via gamma irradiation or by sodium hydroxide treatment with sufficient contact time and then rinsed with RNase-free water; b) buffers are prepared from powders or concentrated commercial solutions in sterile vessels and under biosafety laminar flow and finally filtered on a 0.22 μm vacuum filter; and c) surfaces are decontaminated with commercial RNase, cleaning solutions and wipes. Example 1: RNA synthesis

[0293] IVT reaction conditions

[0294] For the following examples, mRNA was transcribed in vitro by standard methods. In brief, for each gram of transcribed mRNA, a reaction comprising the following was prepared with RNase-free water: a linearized double-stranded DNA plasmid with an RNA polymerase-specific promoter, an RNA polymerase (e.g., SP6 polymerase or T7 polymerase), an RNase inhibitor, pyrophosphatase, NTP, DTT, and a reaction buffer, and then incubated at 37°C for a specified time. The reaction was then quenched by adding 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.

[0295] Cap

[0296] After mRNA synthesis as described above, the in vitro transcribed mRNA was enzymatically modified by adding 5'N 7 The 2'-methylguanylate cap 0 structure is added, and the cap 1 structure is generated by adding a methyl group at the 2'O position of the penultimate nucleotide using a 2'O-methyltransferase as described in Fechter, P. and Brownlee, GG "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249. When the enzyme / affinity method is performed, the capping is performed immediately before the affinity chromatography step, while when the holoenzyme method is performed, the capping is performed immediately before the second step of enzymatic digestion with a proteolytic enzyme.

[0297] Add tail Unless otherwise stated, IVT-transcribed mRNAs are tailed at their 3' end by including a tailing template in the linearized plasmid that tails the mRNA as part of the IVT reaction, or in a subsequent enzymatic step. For tailing as part of the IVT reaction, a poly (T) or similar tailing feature is incorporated into the pDNA template so that a poly (A) tail or 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-generated mRNA following the IVT reaction, for example using a poly (A) polymerase. Example 2: Ethanol-based purification method (comparative example)

[0298] Using an initial amount of 1 g of transcribed mRNA, purification of the IVT reaction was performed using ethanol-based precipitation using the method summarized in Table 8 of WO 2020 / 041793 (see also Examples 1, 2, and 4, incorporated herein by reference). Example 3: Enzyme digestion and affinity chromatography method

[0299] Enzyme digestion and filtration

[0300] Serine protease (550 units; Arctic Enzyme reference number 71600-110) was added directly to the mixture, and the solution was stirred for 2 hours at 37° C. The reaction was quenched by diluting with DTT solution to a final concentration of 55 mM DTT.

[0301] The entire volume was then introduced into a 100 kDa modified polyethersulfone (mPES) hollow fiber (Repligen) (0.5 mm lumen, 41.5 cm height) at a shear rate of 700 s. -1 The Kr2i TFF system (Ripley King) of 1000nm was used. TMP was maintained between 7 and 9 psi (about 0.48 bar to about 0.62 bar), and conductivity was monitored. The product was initially concentrated to 10-12 mg / mL, then 50 mM Tris, 150 mM NaCl, 55 mM DTT (pH 8) diafiltration, then 50 mM Tris, 150 mM NaCl (pH 8) diafiltration and finally diafiltration with water for injection. The whole product was harvested, and the fiber was rinsed with water for injection and merged with the product. The mRNA yield after concentration and diafiltration was at least 90%. When a 300 kDa filter was used to replace a 100 kDa filter, the mRNA yield was constant (that is, at least 90%).

[0302] The concentration was adjusted to 4 g / L and then the capping reaction was performed (as described above).

[0303] Affinity chromatography and filtration

[0304] After capping, the reaction was diluted 4-fold with injection (loading) buffer (50 mM sodium phosphate, 1070 mM sodium chloride, 5 mM EDTA, pH 7) and injected into a 4-well plate equipped with oligo(dT)-filled 25 Resin HiScale TM AKTA 50 / 40 column (GE Healthcare) TM Avant 150. Therefore, the final concentration of sodium chloride in the mRNA preparation for injection is 800mM. The column volume is 0.433L and runs between 244 and 422cm / h. After injection, the column is washed three times with 50mM sodium phosphate, 800mM NaCl, 5mM EDTA (pH 7); 50mM sodium phosphate, 400mM NaCl, 5mM EDTA (pH 7); 50mM sodium phosphate, 5mM EDTA (pH 7), and then eluted with nuclease-free water. The only peak is collected in a sterile bottle and then transferred to a shear rate of 700s equipped with a 50kDa mPES hollow fiber (Ripligen) (0.5mm inner cavity, 41.5cm height). -1 The Kr2i TFF system was used. TMP was maintained between 7 and 9 psi (about 0.48 bar to about 0.62 bar) and conductivity was monitored. The product was initially concentrated to 5 mg / mL and then diafiltered with nuclease-free water for injection. The product was harvested and the fiber was rinsed twice and then the harvest and rinse fractions were combined. The concentration was adjusted to 1 g / L and then filtered on a PES 0.22 μm filter that had previously been subjected to gamma radiation. Example 4: Holoenzyme method

[0305] First enzyme digestion and filtration

[0306] Serine protease (550 units; Arctic Enzyme reference number 71600-110) was added directly to the mixture, and the solution was stirred for 2 hours at 37° C. The reaction was quenched by diluting with DTT solution to a final concentration of 55 mM DTT.

[0307] The entire volume was then introduced into a 100 kDa mPES hollow fiber (RipleyKin) (0.5 mm lumen, 41.5 cm height) at a shear rate of 700 s. -1The Kr2i TFF system (Ripley King) of 100 μ L was used. TMP was maintained between 7 to 9 psi (about 0.48 bar to about 0.62 bar), and conductivity was monitored. The product was initially concentrated to 10-12 mg / mL, then 50 mM Tris, 150 mM NaCl, 55 mM DTT (pH 8) diafiltration, followed by 50 mM Tris, 150 mM NaCl (pH 8) diafiltration and finally diafiltration with water for injection. The whole product was harvested, and the fiber was rinsed with water for injection and merged with the product. The concentration was adjusted to 4 g / L, and then capping reaction (as described above) was carried out.

[0308] Second enzyme digestion and filtration

[0309] After capping, serine protease (550 units; Arctic Enzyme Ref. 71600-110) was added directly to the mixture and the solution was stirred for 2 hours at 37° C. The reaction was quenched by diluting with DTT solution to a final concentration of 55 mM DTT.

[0310] The entire volume was then introduced into a 100 kDa modified polyethersulfone (mPES) hollow fiber (Repligen) (0.5 mm lumen, 41.5 cm height) at a shear rate of 700 s. -1 The Kr2i TFF system (Ripley King) of 100 μ L was used. TMP was maintained between 7 and 9 psi (about 0.48 bar to about 0.62 bar), and conductivity was monitored. The product was initially concentrated to at least 2 mg / mL, then 50 mM Tris, 150 mM NaCl, 55 mM DTT (pH 8) diafiltration, then 50 mM Tris, 150 mM NaCl (pH 8) diafiltration and finally diafiltration with water for injection. The whole product was harvested, and the fiber was rinsed twice with water for injection and merged with the product. The concentration was adjusted to 1 g / L, then filtered on a PES 0.22 μm filter that had previously undergone gamma radiation. The product was finally stored at -20 ° C until preparation. Example 5: Analysis of purified mRNA

[0311] The quality of the purified mRNA was assessed by several metrics, including measurement of mRNA integrity by capillary gel electrophoresis (CGE), and measurement of the percentage of capping (cap 1), poly (A) tail length, and total mRNA length by known techniques (see Tables 1-2). The electropherograms of mRNA purified by the enzyme / affinity method and the holoenzyme method were consistent with expectations. No significant shoulder peaks were observed (data not shown), indicating that these methods did not adversely affect mRNA quality and integrity. mRNA integrity over time at 5°C was evaluated by capillary electrophoresis (CE).

[0312] Samples collected at various stages of the method are analyzed for the presence of proteins (e.g., IVT enzymes, proteolytic enzymes, or capping enzymes). Samples are loaded onto 4%-15% Criterion TM TGX TM The samples were loaded onto SDS-PAGE gels (Bio-Rad) and the gels were silver stained. Before loading onto the gels, the samples were treated with RNase A to remove RNA. Results and discussion

[0313] The steps for the enzyme / affinity method and the holoenzyme method are summarized in Figure 1 In the enzyme / affinity method and the holoenzyme method, a serine protease is used after the IVT / DNAse step to digest the enzymes involved in the enzymatic step, and the resulting peptides are then eliminated from the mixture by TFF. For the enzyme / affinity method, after the capping step, the resin carrying the poly-dT ligand is used to capture the mRNA because of its affinity for the poly-A tail. The enzyme / affinity method uses an orthogonal purification strategy: first eliminate the impurities from the mixture, then use one of its properties (e.g., the poly-A tail) to capture the mRNA.

[0314] The mRNA of 1 g scale was purified by all three methods, and the results are summarized in Table 1 below. The mRNA obtained with the enzyme / affinity method and the holoenzyme method had comparable characteristics to the mRNA obtained from the ethanol method. The mRNA size and tail length were consistent with expectations. The cap percentage was also consistent with expectations, indicating that the enzyme / affinity method and the holoenzyme method did not adversely affect the capping reaction. Table 1. Results for 1 g scale batches of mRNA purified by ethanol-based, enzyme / affinity and holoenzyme methods. NA: Not applicable

[0315] The cycle times for the ethanol method, enzyme / affinity method, and holoenzyme method were compared. For the ethanol method, the total time for the IVT reaction and purification steps was about 10 hours and 45 minutes. The same steps in the enzyme / affinity method and holoenzyme method each took about 9 hours and 45 minutes. The total time for the capping / purification / filtration / redistribution steps of the ethanol method was about 11 hours and 45 minutes, while the total times for the same steps for the enzyme / affinity method and holoenzyme method were 10 hours and 30 minutes and 10 hours and 45 minutes, respectively. Example 6: Evaluation of residual protein after capping

[0316] After in vitro transcription and enzymatic digestion to remove contaminating proteins, additional proteins were introduced into the mRNA product for capping. The presence of proteins introduced into the capping reaction was evaluated at different points downstream of the capping reaction using silver-stained SDS-PAGE gels. It was observed that although RNase inhibitors and 2'O-methyltransferase were no longer detectable after affinity chromatography, both subunits of guanylyltransferase (GuaT) were still present (see Figure 2 , lane 4 of panel A). However, GuaT was no longer detectable after forward filtration on a 0.22 μm PES filter (see Figure 2 , lane 5 of the panel). The incorporation of an intermediate forward filtration step on a 0.22 μm PES filter significantly reduced the amount of contaminating GuaT protein from the affinity chromatography step (see Figure 2 , lane 1' of panel B). The use of an intermediate forward filtration step (i.e., prior to affinity chromatography) advantageously reduces the protein load applied to the affinity chromatography column and carried over to downstream steps (e.g., TFF). The incorporation of a forward filtration step prior to affinity chromatography was evaluated at 1 g and 10 g scales and produced similar results. Example 7: Evaluation of Enzyme Digestion Conditions

[0317] Various concentrations of protease K (Sigma, Ref. P2308) were tested and protein digestion was assessed using silver stained SDS-PAGE gels.

[0318] like Figure 3 As shown, the digestion obtained with 0.025 U / mg RNA of proteinase K (Sigma) after 30 min incubation was comparable to the digestion obtained with 0.55 U / mg RNA of serine proteinase (Arctic Enzyme) after 2 h incubation (compare Figure 3 In contrast, the digestion obtained with 0.05 U / mg RNA of serine protease (Arctic Enzyme) after 2 h of incubation was incomplete (see Figure 3 Lane 2). Therefore, when using proteolytic enzyme K (Sigma), the duration of enzymatic digestion can be reduced to 1 / 4, while the amount of proteolytic enzyme used is also reduced to 1 / 22. Downstream capping is comparable between all three conditions. These results verify the use of proteolytic enzymes from different providers. When using proteolytic enzyme K, both the enzyme concentration and the duration of enzymatic digestion can be significantly reduced. This advantageously further reduces the cycle time of the method. Example 8: Evaluation of buffer conditions TFF was performed after 30 min of enzymatic digestion with 0.025 U / mg RNA of protease K (Sigma). Different diafiltration buffer conditions were evaluated. In particular, diafiltration was performed using 50 mM Tris, 150 mM NaCl buffer in the presence or absence of DTT (55 mM). The profiles were similar between the buffer conditions (see Figure 4 , comparing conditions 1 and 2). Furthermore, capping was comparable when either buffer was used (i.e., Cap 1 was 93.1% when using Tris NaCl DTT buffer and 95.5% when using Tris NaCl buffer). Therefore, the presence of DTT in the buffer is not required. Example 9: Scale-up

[0319] The mRNA was purified at 10 g and 20 g scale using the enzyme / affinity method and the results are summarized in Table 2 below. Scale-up of the purification was performed successfully. The analytical profile was acceptable. Residual GuaT was eliminated using an intermediate forward filtration with a 0.2 μm PES filter.

[0320] Assessing the integrity of mRNA purified by enzymatic / affinity methods over time, e.g. Figure 5 As displayed. Table 2. Results for 10 g and 20 g scale batches of mRNA purified by the enzyme / affinity method. NA: Not available

[0321] The mRNA was also successfully purified on a 100 g scale by the enzyme / affinity method. The 100 g batch was found to meet all acceptance criteria.

[0322] Overall, the enzyme / affinity method and the whole enzyme method result in a high yield of isolated purified mRNA, and the yield is similar or better than that of the method based on ethanol. In addition, the total time of the enzyme / affinity method and the whole enzyme method is reduced compared with the ethanol method.

[0323] The enzyme / affinity and holoenzyme methods advantageously do not require ethanol, which is highly flammable and hazardous to use when conducting commercial-scale mRNA production. Example 10: Process Optimization - mRNA Dissolution

[0324] Although no precipitate was observed during mRNA purification with the first mRNA construct used in the previous example (hereinafter referred to as construct 1), the formation of precipitate could be observed during in vitro transcription of the second construct (construct 2). This suggests that in some cases, precipitate may form during large-scale purification of mRNA, which may have a negative impact on downstream steps, particularly downstream filtration, as the precipitate may clog the filter.

[0325] First, the precipitate is characterized to determine whether it contains in vitro transcribed mRNA. In both the supernatant and the precipitate fractions, ion pair reversed phase ultra-high performance liquid chromatography (RP-IP-UPLC-UV) and capillary electrophoresis are used to evaluate the mRNA amount and integrity. Before RP-IP-UPLC-UV, the supernatant and the precipitate are separated, and the precipitate is washed in water without RNase and redissolved with 1mM HCl. The chromatographic peak corresponding to mRNA shows that the surface area of ​​the supernatant is 0.825, and the surface area of ​​the precipitate is 8.416, corresponding to the mRNA concentration of 0.34mg / mL and 3.21mg / mL, respectively. Here, it is found that about 90% of the detected mRNA is in the precipitate. Therefore, very high amounts of mRNA may be precipitated from the preparation during in vitro transcription. In contrast, precipitation does not negatively affect mRNA integrity.

[0326] Considering the need to re-dissolve the precipitate for downstream purification, several conditions were evaluated and based on the solution at A 600 nm The presence of precipitate was determined by the absorbance value at 400 nm (Nanodrop).

[0327] Diluting the precipitate in RNase-free water or in Tris buffer (50 mM Tris, 150 mM NaCl, pH 7.5) did not redissolve the precipitate. Lowering the pH of the solution from about 7 to 4 with 500 mM citric acid solution also did not redissolve the precipitate. (Data not shown.)

[0328] Unexpectedly, the precipitate was successfully resolubilized by adding sodium citrate or EDTA to the formulation, as shown in Figure 6 A 5 min incubation time was sufficient to allow complete dissolution at 35 °C, indicating that the precipitate formation is not irreversible and that it can be quickly re-dissolved.

[0329] Therefore, the addition of sodium citrate or EDTA to preparations comprising in vitro transcribed mRNA advantageously allows any precipitated mRNA to be resolubilized prior to downstream filtration steps. Example 11: Process Optimization - Oligo dT Loading Buffer

[0330] The mRNA (construct 1) purified with 10g or 20g batch scale was subjected to additional analysis. Specifically, the presence of mRNA integrity and dsRNA and residual plasmid DNA was evaluated immediately after capping or the first TFF step and also immediately after the affinity chromatography step. The presence of dsRNA was detected by ELISA using J2 monoclonal antibody 1. The presence of residual DNA was detected by qPCR.

[0331] TFF and affinity chromatography were performed as described above in Example 3. The oligo dT resin was loaded with a formulation containing 2 mg / mL mRNA, 800 mM NaCl, 30 mM Tris, 8 mM EDTA, pH 7.4. Washing was performed with the same solution but containing only 100 mM NaCl.

[0332] 95%-100% of mRNA is recovered after affinity chromatography. In addition, mRNA integrity is increased ( Figure 7 ), and after TFF and affinity chromatography, low levels of residual plasmid DNA contamination and dsDNA contamination were observed for all batches ( Figure 8 ).

[0333] Therefore, large-scale purification of mRNA yields highly pure mRNA with excellent quality attributes after the affinity chromatography step.

[0334] When conditions were adjusted for loading onto oligo dT resin as described above, precipitation was unexpectedly observed for two other mRNA constructs, which resulted in column plugging.

[0335] To determine the effect of NaCl in the loading buffer on precipitate formation, different concentrations of NaCl or KCl were evaluated as a function of mRNA concentration for three different mRNA constructs (Constructs 2-4). Specifically, after incubation of mRNA with salt for 2 h, the precipitate was visualized by UV (NanoDop) using UV-Vis A 600nm The precipitation was monitored by absorbance values ​​and the results were presented on a contour plot ( Figure 9-11 ). Negative AU values ​​correspond to an absorbance of 0 and indicate a clear solution. An AU value of at least 0.05 indicates a risk of precipitate formation; at AU values ​​of at least 0.3, the precipitate is visible to the naked eye in a turbid form.

[0336] like Figure 9-11 As shown, the formation of precipitates is reduced over a wider range of KCl concentrations compared to NaCl concentrations at a given mRNA concentration. In particular, for these three constructs, the mRNA does not precipitate at higher KCl concentrations. Therefore, in cases where precipitation occurs prior to affinity chromatography, KCl represents an advantageous alternative to NaCl because higher concentrations can be used.

[0337] Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice disclosed herein. The specification and examples are intended to be considered exemplary only, with the true scope and spirit of the disclosure being indicated by the following claims.

[0338] All patents and publications cited herein are incorporated by reference in their entirety.

Claims

1. A method for purifying messenger RNA (mRNA), the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; as well as (b) subjecting the preparation obtained from step (a) to oligo dT affinity chromatography.

2. The method of claim 1, wherein the proteolytic enzyme comprises a serine protease.

3. The method of claim 1 or 2, wherein the preparation comprising in vitro synthesized mRNA is incubated with the proteolytic enzyme at about 37°C for at least 30 minutes.

4. The method of any one of claims 1-3, wherein the proteolytic enzyme is inactivated with a reducing agent, optionally wherein the reducing agent is dithiothreitol (DTT).

5. The method of any one of claims 1 to 4, wherein prior to step (b), the preparation obtained from step (a) is subjected to a tangential flow filtration (TFF) step.

6. The method of claim 5, wherein the TFF uses about 100 kDa to about 300 kDa filters.

7. The method of any one of claims 1 to 6, wherein prior to step (b), the preparation is subjected to a capping step to produce capped mRNA.

8. The method of claim 7, wherein capping is performed at about 37°C for at least 30 minutes, optionally under stirring.

9. The method of any one of claims 1 to 8, wherein the preparation is subjected to a tailing step with a poly(A) polymerase to produce mRNA with a poly(A) tail.

10. The method according to any one of claims 1 to 9, wherein the preparation obtained from step (b) is subjected to a TFF step.

11. The method of claim 10, wherein the TFF uses about 50 kDa to about 300 kDa filters.

12. The method of any one of claims 7 to 11, wherein after the capping step the preparation is subjected to a step of forward filtration.

13. A method for purifying messenger RNA (mRNA), the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; (b) subjecting the preparation obtained from step (a) to a step of tangential flow filtration (TFF); (c) subjecting the preparation obtained from step (b) to oligo dT affinity chromatography; as well as (d) subjecting the preparation obtained from step (c) to a TFF step.

14. The method of claim 13, wherein the proteolytic enzyme comprises a serine protease.

15. The method of claim 12 or 13, wherein the preparation comprising in vitro synthesized mRNA is incubated with the proteolytic enzyme at about 37°C for at least 30 minutes.

16. The method of any one of claims 13-15, wherein the proteolytic enzyme is inactivated with a reducing agent, optionally wherein the reducing agent is dithiothreitol (DTT).

17. The method of any one of claims 13-16, wherein the TFF of step (b) uses an about 100 kDa to about 300 kDa filter.

18. The method of any one of claims 13-17, wherein prior to step (c), the preparation is subjected to a capping step to produce capped mRNA.

19. The method of claim 18, wherein capping is performed at about 37°C for at least 30 minutes, optionally under stirring.

20. The method of any one of claims 13-19, wherein the preparation is subjected to a tailing step with a poly(A) polymerase to produce mRNA with a poly(A) tail.

21. The method of claim 20, wherein tailing is performed before step (c), optionally concomitantly with capping or after capping.

22. The method of any one of claims 13-20, wherein the TFF of step (d) uses an about 50 kDa to about 300 kDa filter.

23. The method of any one of claims 1 to 22, wherein no precipitation step is performed.

24. The method of any one of claims 1-23, wherein at least about 0.5 grams of mRNA is purified.

25. The method of any one of claims 18-24, wherein after the capping step the preparation is subjected to a step of forward filtration.

26. A method for purifying messenger RNA (mRNA), the method comprising: (a) subjecting a preparation comprising in vitro synthesized mRNA to enzymatic digestion with a proteolytic enzyme; (b) subjecting the preparation obtained from step (a) to a step of tangential flow filtration (TFF); (c) subjecting the preparation obtained from step (b) to a capping step; (d) subjecting the preparation obtained from step (c) to a forward filtration step; (e) subjecting the preparation obtained from step (d) to oligo dT affinity chromatography; as well as (f) subjecting the preparation obtained from step (e) to a TFF step.

27. The method of claim 26, wherein after step (f), the preparation is subjected to a forward filtration step.

28. The method of any one of claims 12 or 25-27, wherein the forward filtration uses a 0.2 μm filter.

29. The method of any one of claims 12 or 25-28, wherein the forward filtration reduces the guanylyltransferase (GuaT) content.

30. The method of any of the preceding claims, wherein the in vitro synthesized mRNA is purified in batches of or greater than about 100 mg, 1 gram, 10 grams, 20 grams, 50 grams, 100 grams or more.

31. The method of any of the preceding claims, wherein the in vitro synthesized mRNA is purified in batches of about 10 grams or more, and: (i) the residual plasmid DNA in the purified mRNA is equal to or less than about 0.2 pg / mg; (ii) the purified mRNA contains less than about 0.1% dsRNA; (iii) wherein the purified mRNA contains less than about 1% of an enzymatic reagent used for in vitro synthesis; and / or (iv) the purified mRNA has greater than about 80% integrity.

32. The method of any of the preceding claims, wherein the in vitro synthesized mRNA is purified in batches of about 20 grams or more, and: (i) the residual plasmid DNA in the purified mRNA is equal to or less than about 0.2 pg / mg; (ii) the purified mRNA contains less than about 0.1% dsRNA; (iii) wherein the purified mRNA contains less than about 1% of an enzymatic reagent used for in vitro synthesis; and / or (iv) the purified mRNA has greater than about 80% integrity.

33. The method of any of the preceding claims, wherein the purified mRNA is suitable for therapeutic use.

34. The method of any one of the preceding claims, wherein the method does not comprise any additional affinity chromatography step.

35. A method for producing messenger RNA (mRNA), the method comprising: In vitro synthesis of mRNA; as well as The in vitro synthesized mRNA is purified using the method of any of the preceding claims.

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