METHOD FOR PRODUCING mRNA

Abstract

The present invention provides, for example, a method for producing mRNA having a high capping rate and a method for improving the capping rate of mRNA.　Provided is a method for producing mRNA coding for a target protein, said method comprising the following step: (a) an IVT reaction step for preparing a solution containing at least template DNA designed so as to be capable of transferring mRNA coding for the target protein, a capping reagent, NTP, RNA polymerase, and Mg2+, and transferring, from the template DNA, the mRNA coding for the target protein, where the ratio of molarity of Mg2+ to molarity of NTP (molarity of Mg2+ / molarity of NTP) is 0.30-0.75.

Description

mRNA production method

[0001] The present invention relates to a method for producing mRNA, a method for improving the capping rate of mRNA, a method for removing and / or reducing impurities in mRNA, and the like.

[0002] Messenger RNA (mRNA) medicines are capable of producing target proteins within cells, and research and development is underway for a variety of medical applications, including vaccines, enzyme replacement therapy, genome editing, etc. For mRNA to express (translate) a target protein within cells, it is important that the mRNA has a cap structure at its 5' end (Non-Patent Document 1).

[0003] As a method for synthesizing mRNA having a cap structure at the 5'-end, an in vitro transcription reaction (IVT reaction) using the CleanCap (registered trademark) reagent (hereinafter sometimes referred to as "capping reagent") is known (Patent Document 1, Non-Patent Document 2). This reaction using a capping reagent can incorporate a cap structure into the 5'-end of mRNA simultaneously with transcription from template DNA to mRNA, and can synthesize mRNA having a cap structure in a single step, making it a highly practical method for producing mRNA.

[0004] However, IVT reactions using capping reagents are known to by-produce uncapped mRNA in addition to the target capped mRNA, and the mRNA obtained after the IVT reaction contains a certain amount of uncapped mRNA (Patent Document 1, Non-Patent Document 2). For example, Patent Document 1 reports that when mRNA obtained after the IVT reaction is treated with antarctic phosphatase, an enzyme that dephosphorylates 5'-triphosphate, 5'-diphosphate, and 5'-monophosphate of mRNA to 5'-OH, and then analyzed by LC-MS, a certain amount of 5'-OH is observed, confirming the presence of uncapped mRNA (Patent Document 1). This 5'-triphosphate mRNA is known to be immunogenic (Non-Patent Document 3).

[0005] Non-Patent Document 4 reports that the capping rate can be improved by using a modified T7 RNA polymerase in an IVT reaction using a capping reagent, but this method requires enzyme modification.

[0006] Linear plasmid DNA, which serves as a template for the transcription reaction, becomes an impurity in mRNA, so it is typically fragmented using a DNA-degrading enzyme (DNase) after the IVT reaction. After the DNA fragmentation process is complete, the enzymatic reaction is stopped, and mRNA is isolated by various purification methods. After the enzymatic reaction is stopped, the sample is stored at low temperature until the purification process to prevent degradation of the mRNA due to storage at room temperature and a decrease in mRNA purity. Salting-out precipitation is known as one method for isolating mRNA (Non-Patent Document 2). Purification methods such as salting-out precipitation can remove raw materials such as proteins and nucleoside triphosphates (NTPs) and fragmented DNA during the IVT reaction, allowing for the purification of mRNA. However, the NTPs and capping reagents used as raw materials are not completely removed, and a certain amount of them remain in the resulting mRNA.

[0007] WO2017 / 053297

[0008] Nucleic Acids Research, 2016, 44(16), 7511-7526. Current Protocols e39, Volume 1. Science 314, 5801, 994-997. bioRxiv:10.1101 / 2022.09.01.506264.

[0009] To obtain mRNA with a high capping rate, which is expected to reduce immunogenicity and lead to improved intracellular protein expression and pharmacological activity, it is necessary to increase the amount of capping reagent in the IVT (in vitro transcription) reaction or modify enzymes such as T7 RNA polymerase. However, these methods have problems in terms of production cost and complexity. Therefore, the present inventors have discovered a new problem: improving capping efficiency without requiring complicated procedures such as modifying the enzyme used in the IVT reaction or increasing the amount of capping reagent added. Furthermore, in order to establish a high-quality and efficient method for producing mRNA, it is desirable to develop a method for purifying mRNA with fewer impurities such as NTPs and capping reagents.

[0010] Furthermore, there is a problem that if an mRNA solution that has undergone an IVT reaction and subsequent DNase enzymatic treatment is stored at room temperature until the mRNA is isolated and purified, mRNA degradation progresses and purity decreases. In order to establish a high-quality and efficient mRNA production process, it is desirable to develop a production method that can suppress mRNA degradation and side reactions even when the reaction solution after the IVT reaction and DNase enzymatic treatment is stored at room temperature without being stored at low temperature, and that maintains high mRNA purity.

[0011] Therefore, an object of the present invention is to provide a method for producing mRNA with a high capping rate and a method for improving the capping rate of mRNA. Another object of the present invention is to provide a method for removing and / or reducing mRNA impurities such as NTPs and capping reagents. A further object of the present invention is to provide a method for enabling stable storage of mRNA at room temperature.

[0012] The present inventors have conducted extensive research and have found that in an IVT reaction using a capping reagent, Mg ２＋ Adjusting the ratio of Mg concentration to NTP concentration, and / or ２＋It has been found that by setting the concentration of the capping reagent below a predetermined concentration, the efficiency of capping of mRNA can be improved. At the same time, it has been found that even when the concentration of the capping reagent is set below a predetermined concentration, the capping rate does not decrease significantly and a high capping rate can be maintained. Furthermore, it has been found that in the purification of mRNA after IVT reaction and DNase treatment, the impurities NTP and / or capping reagent can be removed and / or reduced by adding citric acid to the mRNA solution and performing the purification, and the present invention has been completed.

[0013] That is, the present invention provides the following: [1] A method for producing mRNA encoding a protein of interest, comprising the steps of: (a) transcribing a template DNA designed to be capable of transcribing mRNA encoding the protein of interest, a capping reagent, NTP, RNA polymerase, and Mg; ２＋ an IVT reaction step of preparing a solution containing at least ２＋ The ratio of molar concentrations (Mg ２＋ [2] The method for producing mRNA according to [1], wherein the capping reagent is a compound represented by the following formula or a salt thereof:

[0014]

[0015] [3] The method for producing mRNA according to [1] or [2], wherein the concentration of the capping reagent in the IVT reaction is 3.8 mM or less. [4] The method for producing mRNA according to [1] or [2], wherein the concentration of the capping reagent in the IVT reaction is 3.8 mM or less. ２＋ [5] The method for producing mRNA according to any one of [1] to [3], wherein the concentration of Mg in the IVT reaction is 20.0 mM or less. ２＋[6] The method for producing mRNA according to any one of [1] to [3], wherein the NTP concentration in the IVT reaction is 12 to 40 mM. [7] The method for producing mRNA according to any one of [1] to [6], wherein the capping rate of the mRNA obtained by the IVT reaction is improved. [8] The method for producing mRNA according to any one of [1] to [6], wherein the capping rate of the mRNA obtained by the IVT reaction is 80% or higher. [9] The method for producing mRNA according to any one of [1] to [8], wherein the IVT reaction conditions are: (1) a transcription reaction time of 30 minutes to 24 hours, and (2) a temperature of 30 to 60°C.

[10] The method for producing mRNA according to any one of [1] to [9], further comprising the following steps: (b) reacting the mRNA solution obtained in step (a) with DNase.

[11] Ca in the mRNA solution in the step (b) ２＋

[10] The method for producing mRNA according to

[10] , wherein the concentration of citric acid in the mRNA solution obtained in step (b) is 2 mM or more.

[12] The method for producing mRNA according to any one of [1] to

[11] , further comprising the steps of: (c) adding citric acid to the mRNA solution obtained in step (b), and (d) salting out or column purifying the mRNA solution obtained in step (c).

[13] The method for producing mRNA according to

[12] , wherein the citric acid concentration in the mRNA solution in step (c) is 10 to 200 mM.

[14] The method for producing mRNA according to

[12] or

[13] , wherein the citric acid concentration in the mRNA solution in step (c) is 50 to 140 mM.

[15] The method for producing mRNA according to

[12] to

[14] , wherein the ratio of the molar concentration of citric acid to the molar concentration of divalent metal ions in the mRNA solution in step (c) (molar concentration of citric acid / molar concentration of divalent metal ions) is 2 to 13.

[16] The method for producing mRNA according to any one of

[12] to

[15] , characterized in that the mRNA solution obtained in step (c) is stored at room temperature for 8 hours or more, and then step (d) is carried out.

[17] The method for producing mRNA according to any one of

[12] to

[16] , characterized in that step (d) is salting out using an ammonium salt.

[18] The method for producing mRNA according to

[17] , characterized in that the ammonium salt is ammonium acetate.

[19] The ratio of Mg to the molar concentration of NTP is 1:1. ２＋ The ratio of molar concentrations (Mg ２＋

[20] A method for improving the capping rate of mRNA, characterized in that the IVT reaction is carried out under conditions where the molar concentration of Mg in the IVT reaction is 0.30 to 1.0. ２＋

[21] The method for improving the capping rate of mRNA according to

[19] , wherein the capping reagent concentration in the IVT reaction is 20.0 mM or less.

[22] A method for removing and / or reducing impurities during mRNA production, comprising the following steps: (a) obtaining an mRNA solution by an IVT reaction, (b) reacting the mRNA solution obtained in step (a) with DNase, (c) adding citric acid to the mRNA solution obtained in step (b), and (d) salting out or column purifying the mRNA solution obtained in step (c).

[23] The method for removing and / or reducing impurities during mRNA production according to

[22] , wherein the citric acid concentration in the mRNA solution in step (c) is 50 to 140 mM.

[24] The method for removing and / or reducing impurities during mRNA production according to

[22] or

[23] , wherein the step (d) is salting out using an ammonium salt.

[25] The method for removing and / or reducing impurities during mRNA production according to

[24] , wherein the ammonium salt is ammonium acetate.

[0016] According to the present invention, Mg in the IVT reaction solution ２＋ Adjusting the ratio of Mg concentration to NTP concentration, and / or ２＋ By reducing the concentration of the capping reagent to a predetermined concentration or less, the efficiency of capping of mRNA is improved. Furthermore, even when the concentration of the capping reagent is reduced to a predetermined concentration or less, the capping rate does not decrease significantly, and a high capping rate is maintained. Furthermore, the present invention achieves the effect of removing and / or reducing impurities such as NTP and / or capping reagent by adding citric acid to mRNA during mRNA purification.

[0017] Mg in IVT reactions ２＋ 1 is a graph showing the relationship between the concentration and the capping rate in the IVT reaction. ２＋ 1 is a graph showing the relationship between the Mg concentration / NTP concentration ratio and the capping rate in the IVT reaction. ２＋ 1 is a graph showing the relationship between the concentration and the capping rate in the IVT reaction. ２＋1 is a diagram showing the relationship between the concentration / NTP concentration ratio and the capping rate. 2 is a diagram showing the relationship between the NTP concentration and the capping rate in the IVT reaction. 3 is a diagram showing the relationship between the Mg concentration / NTP concentration ratio and the capping rate in the IVT reaction. ２＋ 1 is a graph showing the relationship between the Mg concentration / NTP concentration ratio and the capping rate in the IVT reaction. ２＋ 1 is a graph showing the relationship between the concentration and the capping rate in the IVT reaction. ２＋ 1 is a graph showing the relationship between the Mg concentration / NTP concentration ratio and the capping rate in the IVT reaction. ２＋ 1 is a graph showing the relationship between the concentration and the capping rate in the IVT reaction. ２＋ 1 is a graph showing the relationship between the Mg concentration / NTP concentration ratio and the capping rate in the IVT reaction. ２＋ 1 is a graph showing the relationship between the concentration and the capping rate in the IVT reaction. ２＋ 1 is a diagram showing the relationship between the concentration / NTP concentration ratio and the capping rate. 2 is a diagram showing the relationship between the NTP concentration and the capping rate in the IVT reaction. 3 is a diagram showing the relationship between the Mg concentration / NTP concentration ratio and the capping rate in the IVT reaction. ２＋ 1 is a diagram showing the relationship between the concentration / NTP concentration ratio and the capping rate. 2 is a diagram showing the relationship between the NTP concentration and the capping rate in the IVT reaction. 3 is a diagram showing the relationship between the Mg concentration / NTP concentration ratio and the capping rate in the IVT reaction. ２＋ FIG. 10 is a graph showing the relationship between the concentration / NTP concentration ratio and the capping rate.

[0018] In this specification, the term "process" refers not only to an independent process, but also to processes that cannot be clearly distinguished from other processes, as long as the intended purpose of the process is achieved. Furthermore, the content or concentration of each component in a composition refers to the sum of the total amount or concentration of the multiple substances present in the composition, unless otherwise specified, when multiple substances corresponding to each component are present in the composition. Furthermore, the upper and lower limits of the numerical ranges described in this specification can be arbitrarily selected and combined from the numerical values ​​exemplified as numerical ranges.

[0019] Hereinafter, embodiments of the present invention will be described in detail.

[0020] 1. IVT Reaction The present invention relates to the Mg concentration relative to the NTP concentration. ２＋ Concentration ratio (Mg ２＋The present invention also provides a method for producing mRNA, which comprises a step of carrying out an IVT reaction under predetermined conditions (Mg concentration / NTP concentration). ２＋ Adjusting the concentration / NTP concentration ratio and / or Mg ２＋ The present invention provides a method for improving the capping rate of mRNA by reducing the concentration to a predetermined concentration or less.

[0021] The IVT reaction is a transcription synthesis reaction carried out in a test tube (in vitro), and the IVT reaction using a capping reagent is widely used for the production of mRNA (Patent Document 1, Non-Patent Document 2). The IVT reaction usually requires template DNA, a capping reagent, NTP as a substrate, transcriptase (RNA polymerase), Mg ２＋ Various ions such as these or their salts, buffer solutions, etc. are used, and reducing agents (DTT, etc.), polyamines (spermidine, etc.), RNase inhibitors, various enzymes (pyrophosphatase, etc.) may also be used as needed. In the method for producing mRNA of the present invention, in addition to those specifically stated, methods that those skilled in the art can use for IVT reactions can be used in appropriate combination.

[0022] The template DNA that can be used in the mRNA production method of the present invention is not particularly limited as long as it allows the IVT reaction to proceed, but it may have a promoter sequence and a transcription target sequence downstream (3' side) thereof. Examples of such template DNA include circular or linear plasmid DNA, PCR products, and chemically synthesized oligo-DNA, and preferably a double-stranded linear plasmid. A linear plasmid can be obtained, for example, by digesting circular plasmid DNA with a restriction enzyme to linearize it.

[0023] The promoter sequence in the template DNA is a sequence that is recognized and bound by the RNA polymerase used. Therefore, a promoter sequence that can be recognized by the RNA polymerase used is appropriately selected; for example, a T7 promoter can be used when using T7 RNA polymerase. Furthermore, the transcription target sequence in the template DNA serves as a template sequence for the target mRNA. That is, the transcription target sequence includes a sequence that functions as an mRNA, and may be composed of, for example, from the 5' end, a Cap structure, a 5' untranslated region (5'-UTR), an ORF encoding the target protein, a 3' untranslated region (3'-UTR), and poly(A), and may be appropriately modified depending on the purpose. The transcription target sequence may contain, in addition to the sequence encoding the target protein (i.e., the ORF), a nucleotide sequence that has the activity of regulating translation efficiency. Examples of such sequences include an untranslated region (e.g., a human β-globin sequence) and a Kozak sequence. Furthermore, the template DNA may have a terminator sequence downstream of the sequence to be transcribed, which controls the termination of transcription.

[0024] The capping reagent in the present invention is a reagent that adds 7-methylguanylic acid ( ７ｍ The capping reagent refers to an oligonucleotide primer that introduces a cap structure having a 7-methylguanosine cap structure (G). Examples of capping reagents include a cap-containing initiation oligonucleotide primer having the following 7-methylguanosine cap structure or a salt thereof.

[0025]

[0026] [In the formula, B １ From B ３ are each independently a natural, modified, or non-natural nucleoside base; M is 0 or 1; L is 0 or 1; q1 is 1, q2 is 0 or 1, and q3 is 1; R １ is H or methyl, R ２ and R ３ are independently H, OH, alkyl, O-alkyl, or halogen; １ From X６ each independently is O or S; １ From Y ６ each independently represents OH, SH, BH ３ , aryl, alkyl, O-alkyl or O-aryl; Z ０ From Z ８ each independently represents O, S, NH, CH ２ , C (halogen) ２ or CH (halogen), and R ４ From R ６ each independently is H, OH, or OMe.

[0027] The capping reagent that can be used in the mRNA production method of the present invention is not particularly limited as long as it allows the IVT reaction to proceed. For example, the capping reagent described in "Abstracts of Oral Presentations at the 44th International Symposium on Nucleic Acid Chemistry and the 1st Annual Meeting of the Japanese Society for Nucleic Acid Chemistry" can be used. ７ｍ GpppA ｍ p.g., ７ｍ G ３’ｍ pppA ｍ p.g., ７ｍ G ３’ｍ pppA ｍ p.g. ｍ p.g., ７ｍ GpppG ｍ p.g., ７ｍ G ３’ｍ pppG ｍ p.g., ７ｍ GpppC ｍ p.g., ７ｍ G ３’ｍ pppC ｍ p.g., ７ｍ GpppA ｍ pC, ７ｍ GpppU ｍ pG and ７ｍ GpppU ｍ A capping reagent such as pU can be used. Preferably, the capping reagent is at least one compound selected from the group consisting of compounds represented by the following formulas or a salt thereof.

[0028]

[0029]

[0030]

[0031]

[0032] More preferably, it is a compound represented by the following formula or a salt thereof:

[0033]

[0034] The capping reagent that can be used in the mRNA production method of the present invention is not particularly limited as long as it allows the IVT reaction to proceed, and may be used in the form of a salt. Examples of such salts include metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (e.g., calcium salt and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (e.g., ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzyl-phenethylamine salt, piperazine salt, tetramethylammonium salt, ... Examples of suitable salts include amine salts, such as organic salts like ethylammonium salts and tris(hydroxymethyl)aminomethane salts; hydrohalides, such as hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic salts, such as nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates, such as methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates, such as benzenesulfonate and p-toluenesulfonate; organic salts, such as acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts, such as glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. The salt form of the capping reagent is preferably an alkali metal salt, more preferably a sodium salt. These salts can be prepared by known methods.

[0035] Capping reagents add a modified structure called a Cap structure to the 5' end of mRNA, which is important for protein translation in eukaryotes. The presence of this Cap structure allows mRNA to avoid recognition by the immune system and enable stable translation in vivo. A known natural Cap structure is one with N7-methylguanosine at the 5' position, but the Cap structure added may vary depending on the type of Capping reagent used in the IVT reaction. In this specification, the Cap structure obtained when the Capping reagent of the above formula is used may be referred to as a Clean Cap structure. Furthermore, in this specification, the percentage of mRNAs having a Cap structure at their 5' end among the mRNAs obtained in the IVT reaction is referred to as the Capping rate. The Capping rate is calculated from the peak area ratio obtained from HPLC analysis under the following conditions:

[0036] [HPLC analysis conditions] Detector: Diode array detector (measurement wavelength: 260 nm) Column: ACQUITY Premier Oligonucleotide C18 (2.1 mm × 50 mm, 1.7 μm, Waters Corporation) Column temperature: Constant temperature around 65 ° C. Mobile phase A: (water / HFIP / TEA) mixture containing 2.5 (w / v) % MeOH Mobile phase B: (water / hexafluoroisopropyl alcohol (HFIP) / triethylamine (TEA)) mixture / methanol mixture (7 / 3 (v / v)) The composition of the (water / HFIP / TEA) mixture is (1000 / 10 / 0.8 (v / v / v)). Flow rate: 0.4 mL / min Concentration gradient of mobile phase: As shown in Table A.

[0037]

[0038] The RNA polymerase that can be used in the mRNA production method of the present invention is not particularly limited as long as it allows the IVT reaction to proceed, and for example, phage RNA polymerase can be used. Examples of phage RNA polymerases include T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, K1-5 RNA polymerase, K1E RNA polymerase, K1F RNA polymerase, and K11 RNA polymerase, with T7 RNA polymerase being preferred. The phage RNA polymerase used may also be a mutant that has been appropriately modified. The concentration of the RNA polymerase is not particularly limited, and commercially available RNA polymerases can be used at their recommended concentrations, for example, 0.1 to 50 units / μL, and preferably 1 to 15 units / μL.

[0039] Mg that can be used in the present invention ２＋ The magnesium source is not particularly limited as long as it allows the IVT reaction to proceed, and can be used in the form of various magnesium salts. Examples of magnesium salts include magnesium halides (e.g., magnesium chloride, magnesium bromide, etc.), magnesium carbonate, magnesium sulfate, magnesium nitrate, and magnesium acetate. Preferred magnesium salts include magnesium halides (e.g., magnesium chloride, magnesium bromide, etc.), magnesium carbonate, magnesium sulfate, magnesium nitrate, magnesium acetate, etc. ２＋ The source is magnesium chloride or magnesium acetate. Mg ２＋ The sources may be used alone or in combination of two or more.

[0040] The NTP that can be used in the present invention is not particularly limited as long as it allows the IVT reaction to proceed. Examples of suitable NTPs include natural, unmodified nucleic acids such as adenosine triphosphate (ATP), guanosine triphosphate (GTP), cytidine triphosphate (CTP), and uridine triphosphate (UTP), as well as various nucleoside triphosphates (modified nucleic acids) obtained by chemically modifying these nucleotides. Examples of modified nucleic acids include triphosphates of adenosine, guanosine, uridine, pseudouridine, and cytidine derivatives. Examples of triphosphates of adenosine derivatives include 6'-methyladenosine triphosphate, etc.; examples of triphosphates of guanosine derivatives include 2'-fluoroguanosine triphosphate, etc.; examples of triphosphates of uridine derivatives include 5-methyluridine triphosphate and 5-methoxyuridine triphosphate, etc.; examples of triphosphates of pseudouridine derivatives include pseudouridine triphosphate and N-methylpseudouridine triphosphate, etc.; and examples of triphosphates of cytidine derivatives include 5-methylcytidine triphosphate. Preferred modified nucleic acids are pseudouridine triphosphate, N-methylpseudouridine triphosphate, 5-methyluridine triphosphate, 5-methoxyuridine triphosphate, and 5-methylcytidine triphosphate. For each of these NTPs, only unmodified nucleic acids may be used, or one or more modified nucleic acids may be used, or an unmodified nucleic acid may be used in combination with one or more modified nucleic acids.

[0041] The buffer solution that can be used in the present invention is not particularly limited as long as it allows the IVT reaction to proceed, and examples thereof include Tris buffer solution, MOPS buffer solution, and HEPES buffer solution.

[0042] The method for producing mRNA of the present invention is characterized in that Mg is ２＋ By adjusting the Mg concentration / NTP concentration ratio, the mRNA capping rate can be improved. In this specification, "improvement" means that the mRNA capping rate is improved compared to the mRNA capping rate under the formulation conditions of Comparative Example a1 described in Non-Patent Document 2 described below. In this specification, the Mg concentration / NTP concentration ratio is ２＋ The concentration ratio is (Mg ２＋The value is expressed as the sum of the molar concentrations of each NTP used in the IVT reaction (Mg ２＋ concentration / NTP concentration," "Mg / NTP ratio," or "Mg ２＋ This is sometimes referred to as the "concentration / NTP concentration ratio."

[0043] Mg ２＋ The concentration / NTP concentration ratio can be, for example, 0.10 to 1.0, 0.20 to 0.80, or 0.30 to 1.0, preferably 0.30 to 0.75, 0.30 to 0.74, 0.30 to 0.73, 0.30 to 0.72, or 0.30 to 0.71, more preferably 0.35 to 0.70 or 0.35 to 0.65, and even more preferably 0.35 to 0.60. Alternatively, it can be 1.0 or less, preferably 0.90 or less, 0.80 or less, 0.75 or less, 0.74 or less, 0.73 or less, 0.72 or less, 0.71 or less, 0.70 or less, 0.65 or less, or 0.60 or less, more preferably 0.55 or less. Alternatively, it can be 0.10 or more, preferably 0.20 or more, 0.25 or more, or 0.30 or more, more preferably 0.35 or more. Mg ２＋ When the concentration / NTP concentration ratio is within these ranges, the capping rate of the resulting mRNA tends to be improved.

[0044] As used herein, the term "NTP concentration" refers to the sum of the molar concentrations of each NTP used in an IVT reaction. The sum of the concentrations of each NTP for each corresponding base may be the same or different. That is, when only unmodified nucleic acids are used, the concentrations of ATP, GTP, CTP, and UTP may be the same or different. When CTP and 5-methylcytidine triphosphate are used as NTPs corresponding to cytidine bases, the sum of the concentrations of CTP and 5-methylcytidine triphosphate may be the same or different from the concentrations of each NTP (ATP, GTP, or UTP) corresponding to other bases. The NTP concentration (i.e., the sum of the molar concentrations of each NTP) in the present invention is not particularly limited, but can be, for example, 4 to 60 mM or 8 to 50 mM, preferably 12 to 40 mM or 12 to 30 mM, more preferably 16 to 30 mM, and even more preferably 20 to 30 mM. Alternatively, the NTP concentration can be 60 mM or less or 50 mM or less, preferably 40 mM or less, 35 mM or less, more preferably 30 mM or less. Alternatively, the NTP concentration can be 4 mM or more or 8 mM or more, preferably 12 mM or more, more preferably 16 mM or more, and even more preferably 20 mM or more. ２＋ Because it is linked to the concentration / NTP concentration ratio, Mg ２＋ When the concentration is constant, as the NTP concentration increases, Mg ２＋ The ratio of Mg concentration to NTP concentration decreases. ２＋ The concentration / NTP concentration ratio is in an increasing relationship.

[0045] The method for producing mRNA of the present invention is characterized in that Mg is not added to the IVT reaction. ２＋ By setting the concentration below a certain concentration, the capping rate of mRNA can be improved. ２＋The concentration is not particularly limited, but can be, for example, 8.0 to 50.0 mM or 10.0 to 40.0 mM, preferably 10.0 to 30.0 mM or 10.0 to 20.0 mM, more preferably 10.0 to 15.0 mM or 10.0 to 14.9 mM, and even more preferably 10.0 to 12.5 mM. Alternatively, it can be 50.0 mM or less, preferably 40.0 mM or less, 35.0 mM or less, 30.0 mM or less, 25.0 mM or less, or 20.0 mM or less, more preferably 15.0 mM or less, 14.9 mM or less, or 12.5 mM or less, and even more preferably 11.5 mM or less, or 10.5 mM or less. ２＋ When the concentration is within these ranges, the capping rate of the resulting mRNA tends to be improved. ２＋ The concentration is Mg ２＋ Since it is linked to the concentration / NTP concentration ratio, when the NTP concentration is constant, Mg ２＋ As the concentration increases, Mg ２＋ The concentration / NTP concentration ratio increases, and Mg ２＋ As the concentration decreases, Mg ２＋ The concentration / NTP concentration ratio is in a decreasing relationship.

[0046] As mentioned above, Mg in the IVT reaction ２＋ Adjustment of concentration / NTP concentration ratio and / or Mg ２＋ The capping rate improved by reducing the concentration to a predetermined concentration or less can be maintained or the degree of reduction can be alleviated even if the capping reagent concentration in the IVT reaction is reduced to a predetermined concentration or less. That is, since the capping reagent concentration in the IVT reaction can affect the capping rate of mRNA, reducing the capping reagent concentration tends to reduce the capping rate. However, in the mRNA production method of the present invention, the Mg concentration in the IVT reaction ２＋ Adjustment of concentration / NTP concentration ratio and / or Mg ２＋By setting the concentration at or below a predetermined concentration, it is possible to maintain the mRNA capping rate or mitigate the degree of its reduction, even when the capping reagent concentration is set at or below the predetermined concentration. The capping reagent concentration is also a factor in increasing the production cost of mRNA, so reducing the capping reagent concentration is useful from the perspectives of production cost and production efficiency. The capping reagent concentration in the mRNA production method of the present invention is not particularly limited, but can be, for example, 0.5 mM to 8.0 mM, preferably 0.5 mM to 7.0 mM or 0.5 mM to 6.0 mM, more preferably 1.0 mM to 5.0 mM or 1.5 mM to 4.0 mM, and even more preferably 2.0 mM to 3.8 mM. Alternatively, it is preferably 5.0 mM or less, 4.0 mM or less, or 3.8 mM or less, more preferably 3.5 mM or less, and even more preferably 3.0 mM or less. Alternatively, it is preferably 0.1 mM or more, 0.5 mM or more, or 1.0 mM or more, more preferably 1.5 mM or more, and even more preferably 2.0 mM or more.

[0047] As used herein, the phrase "maintaining the mRNA capping rate or mitigating the degree of its reduction" refers to the mRNA capping rate being equal to or greater than a predetermined value when the capping reagent concentration is set to a predetermined concentration or less. The mRNA capping rate when the capping reagent concentration is set to a predetermined concentration or less is, for example, 70% or more, preferably 75% or more or 80% or more, more preferably 83% or more, 85% or more, or 88% or more, and even more preferably 90% or more.

[0048] In the mRNA production method of the present invention, the capping rate of the mRNA obtained by the IVT reaction is not particularly limited, but is, for example, 70% or more, preferably 75% or more or 80% or more, more preferably 83% or more, 85% or more or 88% or more, and even more preferably 90% or more.

[0049] In the mRNA production method of the present invention, the purity of the mRNA obtained by the IVT reaction is not particularly limited, but is, for example, 70% or more, preferably 75% or more or 80% or more, more preferably 83% or more, 85% or more or 88% or more, and even more preferably 90% or more.

[0050] In the mRNA production method of the present invention, the reaction time in the IVT reaction is not particularly limited, but is, for example, 30 minutes to 24 hours, preferably 1 hour to 12 hours, more preferably 2 hours to 8 hours, and even more preferably 3 hours to 6 hours.

[0051] In the mRNA production method of the present invention, the reaction temperature in the IVT reaction is not particularly limited, but is, for example, 30°C to 60°C, preferably 33°C to 50°C, more preferably 34°C to 40°C, and even more preferably 35°C to 39°C.

[0052] 2. DNase Treatment The present invention provides a method for producing mRNA, which further comprises a step of reacting the mRNA solution obtained by the IVT reaction with DNase.

[0053] The template DNA used in the IVT reaction becomes an impurity in the resulting mRNA solution, and therefore may be fragmented with DNase after the IVT reaction. In the mRNA production method of the present invention, methods that those skilled in the art can use for DNase reactions can be used in appropriate combinations, in addition to those specifically specified. DNases that can be used in the mRNA production method of the present invention are not particularly limited as long as they can degrade template DNA, and examples include DNase I. The concentration of DNase used in the DNase treatment is not particularly limited, but commercially available RNA polymerases can be used at their recommended concentrations, for example, 0.100 to 0.500 units / μL, preferably in the range of 0.125 to 0.250 units / μL. Depending on the type of DNase, the activity of Ca ２＋ In order to request ２＋ That is, the mRNA solution obtained in the IVT reaction can be treated with DNase I and Ca ２＋ can be added to degrade the template DNA.

[0054] Ca that can be used for DNase treatment ２＋The calcium source is not particularly limited as long as it allows the enzyme reaction to proceed, and can be used in the form of, for example, various calcium salts. Examples of calcium salts include calcium halides (e.g., calcium chloride and calcium bromide), calcium carbonate, calcium sulfate, calcium nitrate, and calcium acetate. Calcium chloride is preferred. ２＋ The sources may be used alone or in combination of two or more.

[0055] Ca in DNase treatment ２＋ The concentration is not particularly limited, but can be, for example, 0.5 to 40 mM, preferably 1.0 to 20 mM or 1.5 to 15 mM, more preferably 1.7 to 10 mM or 2.0 to 6.0 mM, and even more preferably 2.2 to 5.0 mM.

[0056] 3. Addition of Citric Acid The present invention provides a method for producing mRNA, which includes the step of adding citric acid to the mRNA solution obtained by the DNase treatment. The present invention also provides a method for removing and / or reducing impurities in the mRNA solution by adding citric acid to the mRNA solution obtained by the DNase treatment and / or adjusting the ratio of the citric acid concentration to the divalent metal ion concentration.

[0057] The storage stability of the mRNA solution can be improved by adding citric acid to the mRNA solution obtained by the DNase treatment and / or adjusting the ratio of the citric acid concentration to the divalent metal ion concentration. In this specification, the ratio of the citric acid concentration to the divalent metal ion concentration refers to a value expressed as (molar concentration of citric acid) / (sum of molar concentrations of divalent metal ions), and may be simply referred to as "citric acid concentration / divalent metal ion concentration" or "citric acid concentration / divalent metal ion concentration ratio." In addition, in this specification, Mg ２＋ and Ca ２＋ The ratio of citric acid concentration to Mg concentration is (molar concentration of citric acid) / (Mg ２＋ and Ca ２＋ The molar concentration of citric acid is expressed as the sum of the molar concentrations of citric acid and Mg ２＋ and Ca ２＋ "Concentration" or "Citric acid concentration / Mg２＋ and Ca ２＋ This is sometimes called the "concentration ratio."

[0058] As used herein, the term "divalent metal ion concentration" refers to the sum of the molar concentrations of divalent metal ions contained in the mRNA solution to which citric acid is added. Examples of such divalent metal ions include those used in the IVT reaction and DNase treatment, such as Mg ２＋ , Ca ２＋ , Zn ２＋ , and Fe ２＋ Preferably, Mg ２＋ and Ca ２＋ In addition, in this specification, "Mg ２＋ and Ca ２＋ "Concentration" refers to Mg ２＋ and Ca ２＋ The sum of the molar concentrations of

[0059] The citric acid source that can be used in the present invention is not particularly limited, and can be used in the form of various salts, anhydrides, or hydrates. Specific examples include anhydrous citric acid (anhydrous citric acid), citric acid monohydrate, anhydrous sodium citrate (anhydrous sodium citrate), and sodium citrate dihydrate. Citric acid monohydrate, sodium citrate dihydrate, or a combination thereof is preferred. One citric acid source may be used alone, or two or more may be used in combination.

[0060] The mRNA production method of the present invention can be used to store the mRNA solution at room temperature or in a refrigerator, for example, by adding citric acid and / or adjusting the citric acid concentration / divalent metal ion concentration ratio, until the mRNA solution is subjected to a subsequent purification step such as salting out. Alternatively, the storage time of the mRNA solution can be extended. Room temperature may be, for example, 15 to 35°C, preferably 20 to 30°C or 25 to 30°C, and more preferably 25°C. The storage time of the mRNA solution is not particularly limited, but may be, for example, 2 hours or more, 4 hours or more, 8 hours or more, 16 hours or more, 24 hours or more, 48 hours or more, 65 hours or more, 72 hours or more, 96 hours or more, 120 hours or more, or 196 hours or more at room temperature. Alternatively, the mRNA solution may be stored at room temperature for 196 hours or less, 120 hours or less, 96 hours or less, 72 hours or less, 65 hours or less, 48 ​​hours or less, 24 hours or less, 16 hours or less, 8 hours or less, 4 hours or less, or 2 hours or less.

[0061] The mRNA of the present invention is not particularly limited as long as it is obtained by the production method described herein, but for example, the length of the mRNA is 0.5 to 10 kilobases, preferably 0.5 to 7 kilobases, and more preferably 1 to 5 kilobases.

[0062] The concentration of citric acid in the mRNA solution after the addition of citric acid is not particularly limited, but can be, for example, 10 to 200 mM, preferably 15 to 180 mM or 35 to 170 mM, more preferably 45 to 150 mM, and even more preferably 50 to 140 mM. Alternatively, it can be 200 mM or less, preferably 190 mM or less, 180 mM or less, 170 mM or less, or 160 mM or less, more preferably 150 mM or less, 140 mM or 130 mM or less, and even more preferably 120 mM or less. Alternatively, it can be 10 mM or more, preferably 15 mM or more or 35 mM or more, more preferably 50 mM or more, and even more preferably 60 mM or more. When the concentration of citric acid in the mRNA solution after the addition of citric acid is within these numerical ranges, degradation of mRNA in the mRNA solution is suppressed, and the storage stability of the mRNA solution tends to be improved.

[0063] The ratio of citric acid concentration to divalent metal ion concentration in the mRNA solution after the addition of citric acid is not particularly limited, but can be, for example, 1 to 15, preferably 1.5 to 15 or 2 to 13, more preferably 2.5 to 12, and even more preferably 2.5 to 11. Alternatively, it can be 1.5 or more, preferably 2.0 or more, and more preferably 2.5 or more. Citric acid concentration / Mg ２＋ When the concentration ratio is within these numerical ranges, the degradation of mRNA in the mRNA solution is suppressed, and the storage stability of the mRNA solution tends to improve.

[0064] Citric acid concentration / Mg in mRNA solution after addition of citric acid ２＋ and Ca ２＋ The concentration ratio is not particularly limited, but can be, for example, 1.0 to 15, preferably 1.5 to 15 or 2.0 to 13, more preferably 2.5 to 12, and even more preferably 2.5 to 11. Alternatively, it can be 1.5 or more, preferably 2.0 or more, and more preferably 2.5 or more. Citric acid concentration / Mg ２＋ When the concentration ratio is within these numerical ranges, the degradation of mRNA in the mRNA solution is suppressed, and the storage stability of the mRNA solution tends to improve.

[0065] 4. Purification The present invention provides a method for producing mRNA, which includes a step of further purifying the mRNA solution obtained by adding citric acid. The present invention also provides a method for removing and / or reducing impurities in the mRNA solution by further purifying the mRNA solution obtained by adding citric acid.

[0066] The purification step can be performed mainly to remove impurities in the mRNA solution, i.e., components other than the target mRNA remaining in the mRNA solution. Impurities include template DNA (including those fragmented by DNase treatment), Capping reagents, NTPs, residues from the IVT reaction such as transcriptase, double-stranded RNA (dsRNA) generated by side reactions of the enzyme reaction, RNA-DNA complexes, DNase, citric acid, glycerol as an enzyme storage solution, buffer substrates such as HEPES or Tris, and Mg ２＋ or Ca ２＋These include, but are not limited to, various ions such as ions and their salts. Impurities in an mRNA solution affect the purity of the resulting mRNA. In particular, these impurities can cause side effects such as inflammatory reactions in the body. Therefore, when mRNA is used in pharmaceuticals such as vaccines, removing and / or reducing impurities in the mRNA solution can reduce unintended natural immune responses, promote the expression of the target protein encoded by the mRNA, and enable the pharmaceutical to achieve the desired effect. Among these impurities, capping reagents and NTPs tend to remain in the mRNA solution even after purification, which can affect the purity of the mRNA. The mRNA production method of the present invention can remove and / or reduce capping reagents and NTPs.

[0067] In the present invention, the method for purifying mRNA is not particularly limited, and any purification method that can be used for producing mRNA can be used appropriately depending on the purpose. Examples of such purification methods include, but are not limited to, filtration such as coarse filtration, microfiltration, and ultrafiltration, salting out, reverse-phase ion exchange, anion exchange (AEX), size exclusion chromatography (SEC), hydrophobic interaction chromatography (HIC), column purification such as affinity chromatography using poly(dT) as a capture agent, and bead purification using oligo(dT) as a capture agent. One purification method may be used alone, or multiple purification methods may be used in combination as appropriate for mRNA purification.

[0068] Salting out is preferably performed in combination with adding citric acid after DNase treatment and / or adjusting the citric acid concentration / divalent metal concentration ratio. ２＋By performing salting out treatment after adjusting the concentration ratio, impurities in the mRNA solution can be more effectively removed. The salt used for salting out is not particularly limited and can be appropriately selected depending on the target mRNA, and examples thereof include sodium salts, lithium salts, magnesium salts, calcium salts, and ammonium salts. Examples of sodium salts include sodium acetate and sodium chloride, examples of lithium salts include lithium chloride, examples of ammonium salts include ammonium salts of organic acids, specifically ammonium acetate, and examples of magnesium salts include magnesium chloride. Preferably, ammonium acetate or lithium chloride can be used, and more preferably, ammonium acetate can be used.

[0069] In the mRNA production method of the present invention, the purity of the purified mRNA is not particularly limited, but is, for example, 70% or more, preferably 75% or more or 80% or more, more preferably 83% or more, 85% or more, 88% or more, or 90% or more, and even more preferably 93% or more.

[0070] In the mRNA production method of the present invention, the mass concentration of NTP remaining in the mRNA solution after purification may be, for example, 5% by mass or less for each base, preferably 3% by mass or less or 2% by mass or less, more preferably 1% by mass or less. Furthermore, the mass concentration of the capping reagent remaining in the mRNA solution after purification may be 25% by mass or less, preferably 20% by mass or less or 17% by mass or less, more preferably 10% by mass or less.

[0071] In the mRNA production method of the present invention, the sum of the mass concentrations of the Capping reagent and each NTP remaining in the mRNA solution after purification may be 30% by mass or less, preferably 25% by mass or less, 20% by mass or less, or 15% by mass or less, more preferably 10% by mass or less. Furthermore, in the mRNA production method of the present invention, the sum of the mass concentrations of the Capping reagent and each NTP remaining in the mRNA solution after purification may be reduced by 15% or more, preferably 20% or more, 25% or more, 30% or more, or 40%, more preferably 50% or more, 60% or more, and even more preferably 70% or more.

[0072] The purified mRNA solution may be further processed depending on the purpose, or may be used as is depending on the purpose. For example, when the obtained mRNA solution is used in a pharmaceutical such as a vaccine, the mRNA can be encapsulated in a carrier molecule and used. Examples of such carrier molecules include lipid particles, preferably lipid particles composed of cationic lipids, sterol lipids, amphipathic lipids, and PEG lipids. Examples of such lipid particles include the lipid particles described in International Publication WO2021 / 251453.

[0073] Examples of cationic lipids include (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl diacetate, 3-dimethylaminopropyl(9Z,12Z)-octacosa-19,22-dien-11-yl carbonate, and (7R,9Z)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-dien-7,29-diyl acetate, which are described in International Publication No. 2015 / 005253. Examples include (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, MC3: (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate, ALC-0315: [(4-hydroxybutyl)azanediyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoic acid ester), and SM-102: heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoic acid ester.

[0074] Examples of sterol lipids include cholesterol, sitosterol, ergosterol, campesterol, stigmasterol, and brassicasterol.

[0075] Examples of amphipathic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine, and dioleoylphosphatidylethanolamine.

[0076] Examples of PEG lipids include 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol and / or N-[methoxy poly(ethylene glycol) 2000]carbamoyl]-1,2-dimyristyloxypropyl-3-amine, ALC-0159 (methoxypolyethylene glycol (2000)-N,N-ditetradecylacetamide), and the like.

[0077] The method for producing nucleic acid-lipid particles encapsulating mRNA is not particularly limited as long as it allows the production of nucleic acid-lipid particles, and can be produced by, for example, the thin film method, reverse phase evaporation method, ethanol injection method, ether injection method, dehydration-rehydration method, surfactant dialysis method, hydration method, freeze-thaw method, etc. More specifically, they can be produced by the ethanol injection method described below. Specific examples of methods for producing nucleic acid-lipid particles are described below, but these descriptions are for illustrative purposes only and do not limit the present invention in any way.

[0078] Step 1: A lipid such as a cationic lipid, a sterol lipid, an amphipathic lipid, or a PEG lipid is dissolved in ethanol to obtain a lipid ethanol solution.

[0079] Step 2: The mRNA is dissolved in a buffer solution of pH 3 to 6, or the mRNA solution is subjected to solvent exchange using a buffer solution of pH 3 to 6 by a method such as ultrafiltration or dialysis to obtain an aqueous mRNA solution.

[0080] Step 3: The lipid ethanol solution and mRNA aqueous solution obtained in Steps 1 and 2 above are pumped at a volume ratio of 1:20 to 1:1 using a pump (e.g., a gear pump), and the two solutions are mixed in a channel (preferably in a microchannel such as a NanoAssemblr) to obtain a crude dispersion of nucleic acid-lipid particles. At this time, the channels for the two solutions are connected to one channel by an appropriate connector (e.g., a T-connector or a Y-connector, but not limited to these).

[0081] Step 4: Ethanol and free nucleic acids contained in the crude dispersion of nucleic acid-lipid particles are removed by methods such as ultrafiltration or diafiltration using a desired buffer solution to obtain stable nucleic acid-lipid particles. In step 4, the mRNA solution is replaced with the desired buffer solution by using an arbitrary amount of buffer solution. The buffer solution can be appropriately selected from phosphate buffer, Tris buffer, histidine buffer, etc. The buffer concentration may be, for example, 0.1 to 100 mM, preferably 0.1 to 50 mM, and more preferably 0.1 to 20 mM. Specific values ​​for the buffer concentration are, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50. An excipient may be added to the buffer solution. Examples of excipients include sucrose, maltose, glucose, glycerol, sorbitol, and trehalose. The concentration of the solution to which the excipient is added can be appropriately selected. For example, the concentration may be 10 to 500 mM, and preferably 100 mM, 200 mM, 300 mM, or 400 mM.

[0082] If necessary, a dilution step with an appropriate solvent may be inserted between step 3 and step 4. In this case, the crude dispersion of nucleic acid-lipid particles is diluted with an appropriately selected buffer solution such as phosphate buffer, Tris buffer, or histidine buffer.

[0083] The total lipid concentration of the lipid ethanol solution in step 1 may be, for example, in the range of 0.1 to 100 mM, preferably 5 to 50 mM, and more preferably 5 to 30 mM. Specific values ​​for the concentration of the lipid ethanol solution in step 1 are, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mM, and preferably 5, 10, 20, or 30 mM. Here, the total lipid concentration is the sum of the moles of all lipids divided by the volume of the solution.

[0084] The concentration of the aqueous mRNA solution in step 2 may be, for example, in the range of 0.01 mg / mL to 100 mg / mL, preferably 0.1 mg / mL to 10 mg / mL, and more preferably 0.1 mg / mL to 1 mg / mL. Specific values ​​for the concentration of the aqueous mRNA solution in step 2 include, for example, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 mg / mL, preferably 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mg / mL, and more preferably 0.1, 0.2, 0.3, 0.4, or 0.5 mg / mL.

[0085] An example of the buffer for the aqueous mRNA solution in step 2 is a citrate buffer.

[0086] The flow rate of the solution flowing through the flow channel in step 3 may be 10 to 1000 mL / min, preferably 30 to 300 mL / min. Specifically, the flow rate is preferably 30, 40, 50, 60, 70, 80, 90, 100, 200, or 300 mL / min.

[0087] The ultrafiltration in step 4 can be performed using tangential flow filtration (TFF). By appropriately changing the pore size of the filter, substances having a desired molecular weight can be concentrated or purified. Furthermore, by performing ultrafiltration using a desired buffer in step 4, the buffer can be replaced with a buffer. In this case, for example, a buffer such as a phosphate buffer, a Tris buffer, or a histidine buffer can be used. These buffers may further contain sucrose, an isotonic agent, or the like.

[0088] Examples of nucleic acid-lipid particles include nucleic acid-lipid particles containing components in any molar ratio selected from the group consisting of the following (a) to (i): (a) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 10:48:40:2, (b) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 10:38:50:2, (c) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 10:33:55:2, (d) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 10:28:60:2, (e) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 15:33:50:2, (f) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 10:48.5:40:1.5, (g) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 10:47.5:40:2.5 (h) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 12.5:41:45:1.5, (i) amphipathic lipid:sterol lipid:cationic lipid:PEG lipid = 17.5:21:60:1.5.

[0089] In the nucleic acid-lipid particles, the ratio (N / P) of the number of cationic lipid molecules (N) to the number of phosphorus atoms (P) derived from the nucleic acid is preferably about 2.0 to 15.0, more preferably about 2.0 to 12.0, even more preferably 2.0 to 9.0, and even more preferably 3.0 to 9.0. The lower limit of the N / P ratio is preferably 2.0, more preferably 2.5, and even more preferably 3.0, and the upper limit is preferably 15.0, more preferably 12.0, and even more preferably 9.0.

[0090] As used herein, the encapsulation rate of nucleic acid-lipid particles is calculated by the following formula: {[amount of mRNA in the presence of surfactant] - [amount of mRNA in the absence of surfactant]} / [amount of mRNA in the presence of surfactant]} x 100 (%). The encapsulation rate is preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. A preferred range for the encapsulation rate is 80% to 100%, more preferably 90% to 100%, and even more preferably 95% to 100%.

[0091] The particles of the present invention may have an average particle size of 30 to 300 nm, preferably 30 to 200 nm, more preferably 30 to 150 nm, and even more preferably 30 to 100 nm. The average particle size can be obtained by measuring the volume average particle size based on the principle of dynamic light scattering using an instrument such as Zeta Potential / Particle Sizer NICOMP (registered trademark) or 380ZLS (PARTICLE SIZING SYSTEMS).

[0092] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0093] Test Example 1 IVT Reaction (a1 to a3) To produce mRNA having the sequence of SEQ ID NO: 19 in International Publication WO2021 / 251453 (wherein C is cytosine and U is uracil), linear plasmid DNA was prepared as template DNA by the following method. A DNA fragment (SEQ ID NO: 17 in International Publication WO2021 / 251453) containing a sequence in which the T7 promoter sequence, human β-globin 5'-UTR sequence, KOZAK sequence, SARS-CoV-2 RBD optimized, and human β-globin 3'-UTR sequence were linked in order was artificially synthesized and introduced into the plasmid. 1 ng of the plasmid was dissolved in nuclease-free water (69 μL), and the solution was diluted with 5x SuperFi Green Buffer (20 μL, ThermoFisher Scientific catalog #12357-010), 2 mM dNTP mix (8 μL, Takara Bio Inc. catalog #4030), 50 μM sense primer 2 (1 μL, SEQ ID NO: 13 in International Publication WO2021 / 251453), 50 μM antisense primer 2 (1 μL, SEQ ID NO: 14 in International Publication WO2021 / 251453), and Platinum SuperFi DNA Polymerase (1 μL, ThermoFisher Scientific SARS-CoV-2 RBD optimized DNA was amplified by adding a PCR product (catalog #12357-010) and incubating at 98°C for 30 seconds, followed by 20 cycles of 98°C for 5 seconds, 60°C for 10 seconds, and 72°C for 1 minute, followed by further incubation at 72°C for 1 minute (SEQ ID NO: 18 in International Publication WO2021 / 251453). The template DNA was cleaved with restriction enzymes NheI and HindIII and then introduced into a plasmid cleaved with the same restriction enzymes to create a circular template plasmid (pUCKIVT1-RBD optimized). The plasmid was cleaved with restriction enzyme BspQI, and the DNA was purified by isopropanol precipitation to prepare linear plasmid DNA.

[0094] Using this linear plasmid DNA as template DNA, an IVT reaction was carried out according to Non-Patent Document 2 (Current Protocols e39, Volume 1). Specifically, various reagents were mixed as shown in Table 1 to a total volume of 900 μL. After incubation at 37°C for 2 hours, 10×DNase I Reaction Buffer (120 μL, NEB catalog # B0303) and DNase I (2000 units / mL, 180 μL, NEB catalog # M0303) were added and the mixture was incubated at 37°C for 20 minutes. This was mixed with 8M LiCl solution (600 μL, Sigma-Aldrich catalog #L7026) and allowed to stand at -20°C for 1.5 hours. After centrifugation (4°C, 15,000 rpm, 30 minutes) using a centrifuge (himac catalog #CF15R), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 15,000 rpm, 10 minutes). The supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water to obtain an mRNA solution. The resulting mRNA was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was the desired length.

[0095]

[0096] The above reaction is shown as a1 in Table 4. In a similar manner, Mg(OAc) ２ An IVT reaction was performed in which the final concentration of each of the following was changed from 16.5 mM to the values ​​indicated as a2 and a3 in Table 4. The resulting mRNA was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) and confirmed to be of the desired length.

[0097] In addition, Mg in Table 4 ２＋ The Mg concentration / NTP concentration ratio was calculated using the following formula: ２＋ Concentration / NTP concentration ratio=Mg(OAc) ２Final concentration (mM) / (ATP+GTP+CTP+UTP final concentration (mM))

[0098] Furthermore, the term "natural" in the Nucleotide column of Table 4 means that the NTP used was unmodified (ATP, GTP, CTP, UTP).

[0099] [Test Example 2] IVT reaction (b1 to b10, c1 to c4, and d1 to d7) In order to produce mRNA having the sequence of SEQ ID NO: 19 in International Publication WO2021 / 251453 (wherein C is 5-methylcytosine and U is 5-methyluracil), 100 mM 5-Me-CTP (Hongene catalog #R3-029) and 100 mM 5-Me-UTP (Hongene catalog #R5-104) were used instead of the CTP and UTP used in Test Example 1, and the parameters described in Tables 5, 6, and 7 were changed to the final concentrations described in the tables instead of the final concentrations described in Test Example 1, and the IVT reaction was performed in the same manner as in Test Example 1. Note that for parameters not described in Tables 5, 6, and 7, the same values ​​as in Table 1 were used.

[0100] The resulting mRNA was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was the desired length.

[0101] In addition, Mg in Tables 5, 6, and 7 ２＋ The Mg concentration / NTP concentration ratio was calculated using the following formula: ２＋ Concentration / NTP concentration ratio=Mg(OAc) ２ Final concentration / (ATP+GTP+5-Me-CTP+5-Me-UTP final concentration (mM))

[0102] Furthermore, the description of "modified" in the Nucleotide column of Tables 5, 6 and 7 indicates that the NTPs used were ATP, GTP, 5-Me-CTP and 5-Me-UTP.

[0103] [Test Example 3] IVT reaction (e1 to e16, f1 to f16, g1 to g10, h1 to h9, i1 to i11, j1 to j5, k1 to k13, and l1 to l7)

[0104] To produce mRNA having the sequence of SEQ ID NO: 19 in International Publication WO2021 / 251453 (wherein C represents 5-methylcytosine and U represents 5-methyluracil), the same linear plasmid DNA as in Test Example 1 was used as template DNA and various reagents were mixed as listed in Table 2 to a total volume of 1000 μL. After incubation at 37°C for 4 hours, DNase I (1 U / μL, 25.0 μL, Promega catalog #M610A) was added and incubated at 37°C for 1 hour. 10 M ammonium acetate solution (342 μL, Nippon Gene catalog #319-90191) was added and the mixture was left to stand overnight at -20°C. The mixture was centrifuged (4°C, 15,000 rpm, 30 minutes) using a centrifuge (Himac catalog #CF15R), and the supernatant was discarded. 70% ethanol was added, followed by centrifugation (4°C, 15,000 rpm, 10 minutes). The supernatant was discarded, and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water to obtain an mRNA solution. The reaction conditions are listed as e4 in Table 8.

[0105]

[0106] The resulting mRNA was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was the desired length.

[0107] IVT reactions were performed in the same manner as the above IVT reactions, except that the parameters listed in Tables 8 to 15 were set to the values ​​listed in Tables 8 to 15 instead of the final concentrations listed in Table 2 (e1 to e16 in Table 8, f1 to f16 in Table 9, g1 to g10 in Table 10, h1 to h9 in Table 11, i1 to i11 in Table 12, j1 to j5 in Table 13, k1 to k13 in Table 14, and l1 to l7 in Table 15). Parameters not listed in Tables 8 to 15 were set to the same final concentrations as in Table 2. The resulting mRNA was analyzed using the LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) and confirmed to be the desired length.

[0108] In addition, Mg in Tables 8 to 15 ２＋ The concentration / NTP concentration ratio was calculated using the following formula: (Formula) Mg ２＋ Concentration / NTP concentration ratio=MgCl ２ Final concentration / (ATP+GTP+5-Me-CTP+5-Me-UTP final concentration (mM)) Furthermore, the description of "modification" in the Nucleotide column of Tables 8 to 15 indicates that the NTPs used were ATP, GTP, 5-Me-CTP, and 5-Me-UTP.

[0109] [Test Example 4] Measurement of Capping Rate The capping rate was measured according to the literature (Anal Bioanal Chem (2016) 408:5021-5030). The mRNA solutions obtained by the IVT reactions of Test Examples 1 to 3 were purified using an RNeasy Midi kit (Qiagen catalog #75144) according to the kit's instructions. Nuclease-free water was used for elution. A volume of the purified mRNA solution equivalent to 72 μg of mRNA was taken, and an appropriate amount of nuclease-free water and rCutSmart buffer (10x, 10 μL, New England BioLabs catalog #B6004) were added and mixed. Finally, Quick CIP (5 U / μL, 18 μL, New England BioLabs catalog #M0525) was added and mixed by pipetting, bringing the total volume to 100 μL. This solution was incubated at 37 ° C for 1 hour, and then left to stand at 75 ° C for 15 minutes. Ammonium acetate solution (7.5 M, 100 μL) was added and mixed, and the mixture was left to stand at -20 ° C for 2 hours or more. The mixture was centrifuged (4°C, 14,000 rpm, 20 minutes) using a centrifuge (Eppendorf catalog #5430R), the supernatant was discarded, 70% ethanol was added, and the mixture was centrifuged (4°C, 14,000 rpm, 10 minutes), the supernatant was discarded, and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water (101 μL).

[0110] To the entire solution obtained, RNase H reaction buffer (10x, 13 μL, New England BioLabs catalog #B0297) and a probe solution (100 μM, 6 μL) that hybridizes to the 5' end of mRNA were added and mixed. This solution was heated at 95°C for 5 minutes, 65°C for 5 minutes, 55°C for 5 minutes, and 40°C for 5 minutes. A 120μL suspension of magnetic beads (Dynabeads MyOne Streptavidin C1 (Thermo Fisher Scientific catalog #65002) was washed sequentially with 0.1M sodium hydroxide, 0.05M sodium chloride solution, and 0.1M sodium chloride solution, and then suspended in the same amount of 0.1M sodium chloride solution as the initially weighed bead suspension) was added to a microtube and allowed to stand on a magnetic stand for 30 seconds, after which the supernatant was removed. The RNA solution prepared above through the heating operation was added to the tube containing the beads, and after shaking and stirring for 30 minutes, RNase H (5U / μL, 10μL, New England BioLabs catalog #M0297) was added, mixed by pipetting, and allowed to stand at 37 ° C for 3 hours. The beads were then placed on a magnetic stand for 30 seconds, the supernatant was removed, and 100 μL of reaction wash solution (5 mM Tris-HCl, 0.5 mM EDTA, 1 M sodium chloride solution) was added and mixed by pipetting. After placing the beads on a magnetic stand for 30 seconds, the supernatant was removed. The washing procedure with reaction wash solution was repeated two more times, and then 100 μL of nuclease-free water was added and mixed by pipetting. After placing the beads on a magnetic stand for 30 seconds, the supernatant was removed. The washing procedure with nuclease-free water was repeated two more times. After that, 50 μL of nuclease-free water heated to 80 ° C was added to the beads from which the supernatant had been removed, and the mixture was mixed by pipetting. The beads were then placed on a magnetic stand for 30 seconds, and the supernatant was transferred to a microtube.To the beads from which the supernatant had been removed, 50 μL of nuclease-free water preheated to 80°C was added again and mixed by pipetting. This solution was left to stand at 80°C for 3 minutes and then placed on a magnetic stand for 30 seconds. The supernatant was transferred to the same microtube as before, and the resulting solution was used as the sample solution.

[0111] The obtained sample solution was analyzed by HPLC under the following conditions to measure the percentage of mRNA having a clean cap structure at the 5' end (capping rate) among the mRNA obtained by the IVT reactions of Test Examples 1 to 3. The capping rate (%) was calculated using the following formula: Capping rate = (area of ​​peaks derived from mRNA capped at the 5' end) / (total area of ​​peaks detected from the time of solvent peak detection to 25 minutes after injection of the sample solution) × 100 (%).

[0112] The peak derived from the 5'-end capped mRNA was observed between 18.3 and 21.3 minutes in terms of retention time.

[0113] The obtained sample solution was also analyzed by HPLC under the same conditions as for the capping rate measurement to measure the proportion of mRNA (5'OH form) having a hydroxyl group (OH group) at the 5' end of the mRNA in the obtained sample solution. The 5'OH form refers to a molecule that does not have a triphosphate structure, diphosphate structure, monophosphate structure, or clean cap structure at the 5' end of the mRNA, is transcribed from the first letter, and has a hydroxyl group at the 5' end. Note that the above-mentioned process for preparing the sample solution includes phosphatase treatment, which converts mRNA having a triphosphate structure, diphosphate structure, or monophosphate structure at the 5' end to a 5'OH form. Therefore, the proportion of 5'OH form measured here represents the sum of the molecular species of mRNA having 5'triphosphate, 5'diphosphate, 5'monophosphate, and 5'OH form among the mRNA obtained by the IVT reactions of Test Examples 1 to 3. The proportion (%) of 5'OH form was calculated using the following formula: (Formula) Percentage of 5'-OH Form = (area of ​​peak derived from 5'-OH form) / (total area of ​​peaks detected from the time point after the solvent peak is detected until 25 minutes after the injection of the sample solution) × 100 (%)

[0114] The peak derived from 5'OH mRNA was observed between 12.1 and 14.6 minutes in terms of retention time.

[0115] [HPLC analysis conditions] Detector: Diode array detector (measurement wavelength: 260 nm) Column: ACQUITY Premier Oligonucleotide C18 2.1 mm x 50 mm, 1.7 μm, Waters Corporation) Column temperature: Constant temperature around 65 ° C. Mobile phase A: (water / HFIP / TEA) mixture containing 2.5 (w / v) % MeOH Mobile phase B: (water / HFIP / TEA) mixture / methanol mixture (7 / 3 (v / v)) The composition of the (water / HFIP / TEA) mixture is (1000 / 10 / 0.8 (v / v / v)). Flow rate: 0.4 mL / min Concentration gradient of mobile phase: The concentration gradient was controlled as shown in Table 3.

[0116]

[0117] The analytical results of Test Examples 1 to 4 are shown in Tables 4 to 15 below and in FIGS. 1 to 8.

[0118]

[0119] From the results in Table 4, as compared with a1 (comparative example) described in Non-Patent Document 2, as shown in Examples a2 and a3, Mg(OAc) ２ and Mg ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio. The results are shown in Figures 1-1 and 1-2.

[0120]

[0121] From the results in Table 5, compared with b2 (comparison example) using the concentration described in Non-Patent Document 2, Mg(OAc) ２ and Mg ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio. The results are shown in Figures 2-1 and 2-2.

[0122]

[0123] From the results in Table 6, compared with c1 (b2, Comparative Example) using the concentration described in Non-Patent Document 2, the final concentration of NTP was increased, and Mg ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio. The results are shown in Figures 3-1 and 3-2.

[0124]

[0125] From the results in Table 7, Mg(OAc) ２ and the final concentration of NTP, ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio.

[0126]

[0127] From the results in Table 8, MgCl ２ and Mg ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio. The results are shown in Figures 4-1 and 4-2.

[0128]

[0129] From the results in Table 9, MgCl ２ and Mg ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio. The results are shown in Figures 5-1 and 5-2.

[0130]

[0131] From the results in Table 10, MgCl ２ It was revealed that by adjusting the final concentration of the capping reagent to 10 mM, a high capping rate of 87% or more was achieved even when the final concentration of the capping reagent was reduced from 5 mM to 1 mM.

[0132]

[0133] From the results in Table 11, MgCl ２ and the final concentration of NTP, and the Mg concentration ２＋It was revealed that by reducing the / NTP concentration ratio, the capping rate increased and the amount of 5'OH bodies decreased.

[0134]

[0135] From the results in Table 12, the final concentration of NTP was increased, and Mg ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio.

[0136]

[0137] From the results in Table 13, MgCl ２ and Mg ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio. The results are shown in Figures 6-1 and 6-2.

[0138]

[0139] From the results in Table 14, MgCl ２ and NTP concentration, and Mg ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio. The results are shown in Figures 7-1 and 7-2.

[0140]

[0141] From the results in Table 15, MgCl ２ and the final concentration of NTP, and ２＋ It was revealed that the capping rate increased and the amount of 5'OH was reduced by decreasing the concentration / NTP concentration ratio. The results are shown in Figures 8-1 and 8-2.

[0142] Test Example 5: Addition of citric acid and salting-out treatment (1) IVT reaction (1-1) IVT reaction-1 To produce mRNA having the sequence of SEQ ID NO: 19 in International Publication WO2021 / 251453 (wherein C is 5-methylcytosine and U is 5-methyluracil), the same linear plasmid DNA as in Test Example 1 was used as template DNA, and various reagents were mixed as shown in Table 16 to a total volume of 5000 μL. After incubation at 37°C for 4 hours, DNase I (1 U / μL, 125 μL, Promega catalog #M610A) was added and incubated at 37°C for 1 hour. The resulting solution was designated DNase treatment solution 1 and immediately used in the salting-out treatment described in Test Example 5 (2-2).

[0143]

[0144] (1-2) IVT reaction-2 MgCl ２ An IVT reaction was carried out in the same manner as in Test Example 5 (1-1), except that the final concentration of was 10.0 mM instead of 8.0 mM as described in Table 16. The resulting solution was designated as DNase treatment solution 2 and was immediately used in the salting-out treatment described in Test Example 5 (2-2).

[0145] (1-3) IVT reaction-3 MgCl ２ An IVT reaction was carried out in the same manner as in Test Example 5 (1-1), except that the final concentration of was 30.0 mM instead of 8.0 mM as described in Table 16. The resulting solution was designated as DNase treatment solution 3 and immediately used in the salting-out treatment described in Test Example 5 (2-2).

[0146] (2) Salting-out Treatment of mRNA (2-1) Preparation of Citrate Buffer Sodium citrate hydrate (2.676 g, FUJIFILM Wako, catalog #197-18855) and citric acid hydrate (0.189 g, FUJIFILM Wako, catalog #032-25245) were dissolved in nuclease-free water (7.135 g).

[0147] (2-2) Salting-out Treatment DNase treatment solutions 1, 2 and 3 were dispensed in 900 μL aliquots and subjected to salting-out treatment under the salting-out conditions A, B or C in Table 17 to obtain mRNA.

[0148]

[0149] The resulting mRNA was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was the desired length.

[0150] (3) Quantification of the Amounts of Residual Capping Reagent and Residual NTP Contained in the mRNA Obtained After Salting-Out The mRNA solution obtained by the salting-out treatment described in Test Example 5 (2-2) was analyzed under the following HPLC conditions to quantify the amounts of residual ATP, residual GTP, residual 5-Me-CTP, residual 5-Me-UTP, and residual capping reagent (CleanCap AG) contained in the mRNA solution. A standard curve was prepared by diluting 100 mM ATP (Hongene catalog #R1331), 100 mM GTP (Hongene catalog #R2331), 100 mM 5-Me-CTP (Hongene catalog #R3-029), 100 mM 5-Me-UTP (Hongene catalog #R5-104), and CleanCap AG (TriLink catalog #N-7113) with 0.1 M triethylamine acetate (pH 7.0). The mRNA solution obtained in Test Example 5 (2-2) was diluted to 100 ng / μL with 0.1 M triethylamine acetate (pH 7.0) and used for HPLC measurement.

[0151] [HPLC analysis conditions] Detector: Diode array detector (measurement wavelength: 260 nm) Column: YMC-UltraHT Hydrosphere C18 (2.0 mm × 100 mm, 2 μm, YMC) Column temperature: Constant temperature around 40°C Mobile phase A: 0.1 M triethylamine acetate (pH 7.0) Mobile phase B: 0.1 M triethylamine acetate (pH 7.0) / acetonitrile mixture (1:1) Flow rate: 0.4 mL / min Mobile phase gradient: The gradient was controlled as shown in Table 18.

[0152]

[0153] The amounts of residual ATP, residual GTP, residual 5-Me-CTP, residual 5-Me-UTP, and residual capping reagent (CleanCap AG) contained in the mRNA obtained by the salting-out treatment in Test Example 5 (2-2) were measured by the above-mentioned HPLC, and the results of these residual substance amounts (%) are shown in Table 19. The residual substance amounts (%) shown in Table 19 were calculated using the following formula: (Formula) Residual substance amount (%) = (free form concentration of various substances measured by HPLC (ng / μL)) / (concentration (ng / μL) of the mRNA solution obtained by the salting-out treatment in Test Example 5 (2-2) injected into HPLC) × 100

[0154] The "Total" value for the amount of residual substance shown in Table 19 represents the sum of the amounts of remaining ATP, GTP, 5-Me-CTP, 5-Me-UTP, and CleanCap AG, and was calculated using the following formula: Total amount of residual substance (%) = ATP remaining amount (%) + GTP remaining amount (%) + 5-Me-CTP remaining amount (%) + 5-Me-UTP remaining amount (%) + CleanCap AG remaining amount (%). Note that "-" in Table 19 indicates a value below the lower limit of quantitation.

[0155]

[0156] The results in Table 19 demonstrate that the addition of citric acid before salting out with ammonium acetate as a post-treatment after the IVT reaction and DNase treatment can reduce the amount of residual CleanCap AG and NTP contained in the salted out mRNA.

[0157] Test Example 6: Addition of citric acid and salting-out treatment (1) IVT reaction (1-1) IVT reaction-1 To produce mRNA having the sequence of SEQ ID NO: 19 in International Publication WO2021 / 251453 (wherein C is 5-methylcytosine and U is 5-methyluracil), the same linear plasmid DNA as in Test Example 1 was used as template DNA and various reagents were mixed as shown in Table 20 to make a total of 200 g. After incubation at 37°C for 4 hours, DNase I (1 U / μL, 5.00 g, Hongene catalog #ON-109) was added and the mixture was incubated at 37°C for 1 hour. The resulting solution was designated DNase treatment solution 4 and immediately used in the salting-out treatment described in Test Example 6 (2-2).

[0158]

[0159] The (±)-dithiothreitol solution in the table represents a solution prepared by adding 7.71 g of (±)-dithiothreitol to 42.29 g of water for injection.

[0160] (1-2) IVT Reaction-2 To produce mRNA having the sequence of SEQ ID NO: 19 in International Publication WO2021 / 251453 (wherein C represents 5-methylcytosine and U represents 5-methyluracil), the same linear plasmid DNA as in Test Example 1 was used as template DNA and various reagents were mixed as shown in Table 21 to make a total of 200 g. After incubation at 37°C for 4 hours, DNase I (1 U / μL, 2.80 g, Hongene catalog #ON-109) was added and the mixture was incubated at 37°C for 1 hour. The resulting solution was designated DNase treatment solution 5 and immediately used in the salting-out treatment described in Test Example 6 (2-2).

[0161]

[0162] The (±)-dithiothreitol solution in the table represents a solution prepared by adding 7.71 g of (±)-dithiothreitol to 42.29 g of water for injection.

[0163] (2) Salting-out Treatment of mRNA (2-1) Preparation of Citrate Buffer Sodium citrate hydrate (26.76 g, FUJIFILM Wako, Catalog #197-18855) and citric acid hydrate (1.89 g, FUJIFILM Wako, Catalog #032-25245) were dissolved in nuclease-free water to a total of 100 g.

[0164] (2-2) Salting-out Treatment DNase treatment solutions 4 and 5 were dispensed in 1.0 mL aliquots and subjected to salting-out treatment under the salting-out conditions D, E, F, or G in Table 22 to obtain mRNA.

[0165]

[0166] The resulting mRNA was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010) to confirm that it was the desired length.

[0167] (3) Quantification of the Amounts of Residual Capping Reagent and Residual NTP Contained in the mRNA Obtained After Salting Out The mRNA solution obtained in Test Example 6 (2-2) was analyzed by HPLC in the same manner as in Test Example 5 (3), and the amounts of residual ATP, residual GTP, residual 5-Me-CTP, residual 5-Me-UTP, and residual capping reagent (CleanCap AG) contained in the mRNA solution were quantified. The results of the amounts of these residual substances (%) are shown in Table 23. In Table 23, "-" indicates a value below the lower limit of quantitation.

[0168]

[0169] The results in Table 23 demonstrate that the addition of citric acid before salting out with ammonium acetate as a post-treatment after the IVT reaction and DNase treatment can reduce the amount of residual CleanCap AG and NTP contained in the salted out mRNA.

[0170] Test Example 7 Addition of Citric Acid Various reagents were mixed as shown in Table 24-1 to prepare three types of 1.33 M citric acid solutions with different compositions.

[0171]

[0172] To produce mRNA having the sequence of SEQ ID NO: 19 in International Publication WO2021 / 251453 (wherein C is 5-methylcytosine and U is 5-methyluracil), the same linear plasmid DNA as in Test Example 1 was used as template DNA, and various reagents were mixed as shown in Table 24-2 to make a total volume of 4000 μL. After incubation at 37 ° C for 4 hours, DNase I (1 U / μL, 100 μL, Promega catalog #M610A) was added and incubated at 37 ° C for 1 hour. 360 μL of the DNase-treated solution was dispensed into 9 tubes, and each tube was mixed with 40 μL of one of the three citrate buffers and nuclease-free water to achieve the values ​​listed in Table 25. The mixture was then left to stand at room temperature under air conditioning for 65 hours or more. This mixture was mixed with 10 M ammonium acetate solution (133 μL, Nippon Gene catalog #319-90191) and allowed to stand at room temperature under air conditioning for 1 hour. After centrifugation (4°C, 14,000 rpm, 20 minutes) using a centrifuge, the supernatant was discarded, 70% ethanol (500 μL) was added, and the mixture was centrifuged (4°C, 14,000 rpm, 10 minutes). The supernatant was discarded and the mixture was air-dried. The resulting residue was dissolved in nuclease-free water to obtain an mRNA solution. The resulting mRNA solution was analyzed using the LabChip GX Touch Standard RNA Reagent Kit to confirm that it was the desired length. Furthermore, the area ratio of the main peak having the desired length was analyzed to determine the mRNA purity. The results of the mRNA purity are shown in Table 25. The DNase I solution used contained no CaCl ２ That is, after adding the citrate buffer, each mRNA solution contains 10 mM of Mg ２＋ is 9.7 mM, Ca ２＋ Each of these is contained at 0.2 mM, for a total of 9.9 mM as divalent metal ions.

[0173]

[0174]

[0175] The results in Table 25 confirmed that adding a citric acid solution to the solution after the IVT reaction and DNase treatment tended to improve the storage stability of the mRNA solution at room temperature. In particular, when the citrate buffer concentration was 36 mM or higher, the purity of the mRNA was maintained at a high level, and the mRNA was particularly stable when stored. These results showed that adding a citric acid solution to the DNase-treated reaction solution prevented a decrease in mRNA purity and enabled long-term storage at room temperature.

[0176] Test Example 8 IVT Reaction To produce mRNA having the sequence of SEQ ID NO: 2, linear plasmid DNA was prepared by replacing SARS-CoV-2 RBD optimized with the sequence of SARS-CoV-2 RBD optimized2 (SEQ ID NO: 1) in the linear plasmid DNA sequence used in Test Example 1. This linear plasmid DNA was used as template DNA and mixed with various reagents as listed in Table 26 to a total volume of 50 g. After incubation at 37°C for 4 hours, 0.67 g of the DNase reaction additive solution listed in Table 27 and DNase I (1 U / μL, 1.25 g, Hongene Biotech, catalog #ON-109) were added so that the final calcium chloride concentration was 2 mM or higher, and the mixture was incubated at 37°C for 1 hour. 4.45 g of the citrate buffer shown in Table 28 was mixed and allowed to stand at room temperature overnight.

[0177]

[0178]

[0179]

[0180] The mixture was mixed with 18.47 g of ammonium acetate solution A listed in Table 29 and stirred at room temperature for 1 hour. After centrifugation (room temperature, 4000 x g, 30 minutes) using a centrifuge, the supernatant was discarded. 75.3 g of ammonium acetate solution B listed in Table 30 was added, followed by centrifugation (room temperature, 4000 x g, 30 minutes). The supernatant was discarded, and the resulting residue was dissolved in 75.3 g of water for injection. The resulting solution was passed through an activated carbon filter (Millistak pod+ CR40 media) previously passed through with water for injection, and then concentrated by ultrafiltration using a tangential flow filtration membrane (PALL CSUM100T001). After diafiltration with the formulation listed in Table 32, mRNA was obtained.

[0181]

[0182]

[0183]

[0184]

[0185] The obtained mRNA was analyzed using LabChip GX Touch Standard RNA Reagent Kit (PerkinElmer catalog #CLS960010), and the mRNA purity of the main peak was 94.1%.

[0186]

[0187] In the table, C in SEQ ID NO: 2 represents 5-methylcytosine, and U represents 5-methyluracil. In addition, the 5' end of SEQ ID NO: 2 contains 7-methylguanylic acid ( ７ｍ A Cap structure having a cap group (G) has been introduced.

[0188] Test Example 9 Preparation of a formulation filled with a dispersion of mRNA-encapsulated nucleic acid lipid particles The lipid solution was prepared by mixing 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)pentatriaconta-9,26-diene-7,29-diyl diacetate (cationic lipid), 1,2-distearoyl-sn-glycero-3-phosphocholine (PEG ２０００ -DMG) to DSPC:cholesterol:cationic lipid:PEG ２０００ The total lipid concentration was prepared by dissolving in ethanol at a molar ratio of -DMG=12.5:41:45:1.5.

[0189] The mRNA concentration of the mRNA solution was adjusted by adding mRNA to a citrate buffer solution (20 mM, pH 4.0).

[0190] The formulation buffer was prepared by adding L-histidine and sucrose to water for injection to prepare a 300 mM sucrose, 10 mM histidine buffer solution (pH 7.0).

[0191] The prepared lipid solution and mRNA solution were mixed in the microchannel at a volume ratio of 1:3 to generate a crude dispersion of nucleic acid-lipid particles, which was then diluted with 300 mM sucrose and 10 mM histidine buffer (pH 7.0).

[0192] The drug solution prepared by the mixing operation was concentrated using a tangential flow filtration device, and then the ethanol was removed using 300 mM sucrose and 10 mM histidine buffer (pH 7.0), to obtain a dispersion of purified mRNA-encapsulated nucleic acid-lipid particles.

[0193] The purified mRNA-encapsulated nucleic acid-lipid particles were diluted with 300 mM sucrose and 10 mM histidine buffer (pH 7.0) to an mRNA concentration of 0.1 mg / mL. The diluted solution was filtered through a filter with a pore size of 0.2 μm.

[0194] The sterile-filtered drug solution was filled into a washed and sterilized glass vial, and the vial was then stoppered with a washed and sterilized rubber stopper. The stoppered vial was then capped using a capping machine to obtain a formulation filled with a dispersion of mRNA-encapsulated nucleic acid-lipid particles.

[0195] The mRNA encapsulation rate of the resulting formulation was measured using the Quant-iT RiboGreen RNA Assay kit. The mRNA in the nucleic acid-lipid particle dispersion was quantified in the presence and absence of Triton X-100 surfactant, and the mRNA encapsulation rate was calculated by comparing the values ​​in the absence (no RNA) and presence (total RNA) of Triton X-100. The mRNA encapsulation rate (%) was 95.4%.

[0196] The particle size was measured by measuring the mean particle size and size distribution of the nucleic acid-lipid particles using a particle size analyzer (DLS). The results were reported as the mean particle size and polydispersity index (PDI). The particle size was 104 nm, and the PDI was 0.1.

[0197] The mRNA production method of the present invention, the method for improving the capping rate of mRNA, the method for removing and / or reducing impurities in mRNA, etc., enable the production of mRNA with a high capping rate and purity, and are therefore industrially useful.

[0198] SEQ ID NO: 1: SARS-CoV-2 RBD optimized2 SEQ ID NO: 2: SARS-CoV-2 RBD optimized2 mRNA

Claims

1. A method for producing an mRNA encoding a protein of interest, comprising the steps of: (a) preparing a template DNA designed to be capable of transcribing an mRNA encoding the protein of interest, a capping reagent, NTP, RNA polymerase, and Mg; ２＋ and an IVT reaction step of transcribing mRNA encoding a target protein from a template DNA by preparing a solution containing at least ２＋ The ratio of molar concentrations (Mg ２＋ The condition is that the molar concentration of NTP / molar concentration of NTP is 0.30 to 0.

75.

2. The method for producing mRNA according to claim 1, wherein the capping reagent is a compound represented by the following formula or a salt thereof:

3. A method for producing mRNA described in claim 1 or 2, wherein the concentration of the capping reagent in the IVT reaction is 3.8 mM or less.

4. Mg in IVT reactions ２＋ The method for producing mRNA according to any one of claims 1 to 3, wherein the concentration is 20.0 mM or less.

5. Mg in IVT reactions ２＋ The method for producing mRNA according to any one of claims 1 to 3, wherein the concentration is 15.0 mM or less.

6. The method for producing mRNA described in any one of claims 1 to 5, wherein the NTP concentration in the IVT reaction is 12 to 40 mM.

7. A method for producing mRNA described in any one of claims 1 to 6, in which the capping rate of mRNA obtained by IVT reaction is improved.

8. The method for producing mRNA described in any one of claims 1 to 6, wherein the capping rate of the mRNA obtained by the IVT reaction is 80% or more.

9. The method for producing mRNA according to any one of claims 1 to 8, further comprising the following IVT reaction conditions: (1) a transcription reaction time of 30 minutes to 24 hours, and (2) a temperature of 30 to 60°C.

10. The method for producing mRNA according to any one of claims 1 to 9, further comprising the following steps: (b) reacting the mRNA solution obtained in step (a) with DNase.

11. Ca in the mRNA solution in the step (b) ２＋ The method for producing mRNA according to claim 10, characterized in that the concentration is 2 mM or more.

12. The method for producing mRNA according to any one of claims 1 to 11, further comprising the steps of: (c) adding citric acid to the mRNA solution obtained in step (b); and (d) subjecting the mRNA solution obtained in step (c) to salting out or column purification.

13. The method for producing mRNA according to claim 12, characterized in that the concentration of citric acid in the mRNA solution in step (c) is 10 to 200 mM.

14. The method for producing mRNA according to claim 12 or 13, characterized in that the concentration of citric acid in the mRNA solution in step (c) is 50 to 140 mM.

15. A method for producing mRNA described in claims 12 to 14, wherein the ratio of the molar concentration of citric acid to the molar concentration of divalent metal ions in the mRNA solution in step (c) (molar concentration of citric acid / molar concentration of divalent metal ions) is 2 to 13.

16. A method for producing mRNA described in any one of claims 12 to 15, characterized in that the mRNA solution obtained in step (c) is stored at room temperature for 8 hours or more, and then step (d) is carried out.

17. The method for producing mRNA according to any one of claims 12 to 16, characterized in that the step (d) is salting out using an ammonium salt.

18. The method for producing mRNA according to claim 17, characterized in that the ammonium salt is ammonium acetate.

19. Mg vs. molar concentration of NTP ２＋ The ratio of molar concentrations (Mg ２＋ The method for improving the capping rate of mRNA comprises carrying out an IVT reaction under conditions in which the molar concentration of mRNA / the molar concentration of NTP is 0.30 to 1.

0.

20. Furthermore, Mg in IVT reactions ２＋ The method for improving the capping rate of mRNA according to claim 19, wherein the concentration is 20.0 mM or less.

21. A method for improving the capping rate of mRNA described in claim 19 or 20, further comprising the step of: a concentration of the capping reagent in the IVT reaction being 3.8 mM or less.

22. A method for removing and / or reducing impurities during mRNA production, comprising the steps of: (a) obtaining an mRNA solution by an IVT reaction; (b) reacting the mRNA solution obtained in step (a) with DNase; (c) adding citric acid to the mRNA solution obtained in step (b); and (d) salting out or column purifying the mRNA solution obtained in step (c).

23. A method for removing and / or reducing impurities during mRNA production according to claim 22, characterized in that the citric acid concentration in the mRNA solution in step (c) is 50 to 140 mM.

24. A method for removing and / or reducing impurities during the production of mRNA described in claim 22 or 23, characterized in that the (d) step is salting out using an ammonium salt.

25. A method for removing and / or reducing impurities during mRNA production as described in claim 24, characterized in that the ammonium salt is ammonium acetate.

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