Method for producing nucleic acid, method for removing impurities, and nucleic acid having few impurities

Abstract

The present invention is a method for producing a nucleic acid, comprising: a step (A) for synthesizing a nucleic acid in which an amino group in a base moiety is protected by an acyl-based protecting group; step B, contacting the nucleic acid obtained in the step A with a nucleophilic reagent under a neutral or acidic pH condition; and step C, performing deprotection on the protecting group of the nucleic acid obtained in the step B. According to the present invention, it is possible to reduce the content of by-products (B) that cannot be removed by conventional techniques, and to produce high-purity nucleic acids.

Description

Technical Field

[0001] The present invention relates to a method for producing nucleic acid, a method for removing impurities, and nucleic acid with less impurities. Background Art

[0002] In the past, the pharmacologically active ingredients of marketed drugs were mainly low molecular weight compounds, but recently, drugs containing medium molecular weight pharmacologically active ingredients such as peptides and nucleic acids have gradually been marketed in large quantities. In particular, nucleic acid drugs have a characteristic mechanism of action that is not found in other seed drugs, such as being able to specifically exert their effects by forming base pairs with target mRNA, and therefore are currently highly anticipated seed drugs for future development.

[0003] Nucleic acids, which are raw materials for nucleic acid medicines, are generally produced by chemical synthesis. As a document introducing such a method for producing nucleic acids, for example, the following Patent Document 1 can be cited.

[0004] Patent Document 1 relates to a method for deprotecting an oligonucleotide, and describes, for example, contacting a solid-phase synthesized oligonucleotide with an organic amine or the like to cleave the protecting group without removing the oligonucleotide from the solid phase.

[0005] In terms of pharmaceuticals, the types and amounts of impurities are strictly controlled under various restrictions to ensure their safety. In the manufacture of bulk drugs, the management of impurities is also strict, and it is even more desirable to create a technology to remove impurities generated in the manufacture of nucleic acids as bulk drugs.

[0006] As methods for removing impurities and purifying nucleic acids, reverse phase column (RPC), reverse phase high performance liquid chromatography (RPHPLC), anion exchange chromatography (AEX), polyacrylamide gel electrophoresis (PAGE), and the like are known.

[0007] In addition, Patent Document 2 describes a method for purification of oligonucleotides, which uses a mixed mode matrix comprising a strong anion exchange ligand, a strong cation exchange ligand, and a hydrophobic ligand to remove impurities including non-complexed oligonucleotides and failed sequences (claim 1, paragraph 0006, etc.).

[0008] Patent document 3 describes a method for purification of oligonucleotides, which uses hydrophobic interaction chromatography (HIC) to remove product-related impurities such as n-1 impurities, P=O impurities, abasic impurities, CNEt impurities, and N+1 impurities (claim 1, paragraph 0006, etc.).

[0009] Patent Document 4 describes a method for purification of oligonucleotides, which uses a two-phase mobile phase-stationary phase liquid-liquid chromatography method to remove other oligonucleotides such as shortmers and longmers (Claim 1, paragraph 0003).

[0010] Patent Document 5 describes a method for purification of oligonucleotides, which uses a titratable anion exchange composition to remove impurities such as short oligonucleotides (claim 1, paragraph 0012, etc.).

[0011] Patent Document 6 describes a method for purification of oligonucleotides using a porous carrier to remove impurities (claim 1, etc.).

[0012] Non-Patent Document 1 describes the control and removal of non-oligonucleotide product-related impurities (PRI) in the production of therapeutic oligonucleotides.

[0013] Prior art literature

[0014] Patent Literature

[0015] Patent Document 1: Japanese Patent No. 4705716

[0016] Patent Document 2: Japanese Patent Application No. 2022-539327

[0017] Patent Document 3: Japanese Patent Application No. 2019-518759

[0018] Patent Document 4: Japanese Patent Application No. 2014-525254

[0019] Patent Document 5: Japanese Patent Application No. 2005-520547

[0020] Patent Document 6: Japanese Patent Application Laid-Open No. 7-227284

[0021] Non-patent literature

[0022] Non-patent literature 1: Org. Process Res. Dev., 2022, 26, 1130-1144 Summary of the invention

[0023] Problem that the invention aims to solve

[0024] The inventors of the present application have found that there is a problem that in the production of nucleic acids, nucleic acids (byproducts (B)) with a mass 34 daltons greater than that of the target nucleic acid (A) are contained as impurities in the nucleic acid (A). The molecular weight and physical properties of the byproducts (B) are very similar to those of the nucleic acid (A), and therefore, they cannot be removed even by using conventional purification methods such as Patent Documents 1 to 6 and Non-Patent Document 1.

[0025] Therefore, the technical problem of the present invention is to provide a method for producing nucleic acid by removing byproduct (B) and reducing the content of impurities. The technical problem of the present invention is also to provide a method for removing byproduct (B) and a nucleic acid with less byproduct (B).

[0026] Solutions for solving problems

[0027] The present inventors have conducted intensive research and found that after nucleic acid synthesis, the content of by-product (B) can be significantly reduced by treating the synthesized nucleic acid with a nucleophilic reagent while the amino group of the base portion is protected by an acyl protecting group, followed by deprotection. Based on this finding, the present inventors have completed the present invention shown below.

[0028] [1] A method for producing a nucleic acid, comprising: step A, synthesizing a nucleic acid in which the amino group of the base portion is protected by an acyl protecting group; step B, contacting the nucleic acid obtained in step A with a nucleophilic reagent under neutral or acidic pH conditions; and step C, deprotecting the protecting group of the nucleic acid obtained in step B.

[0029] [2] The production method according to [1], wherein the pH is about 3 to about 8.

[0030] [3] The production method according to [1] or [2], wherein the nucleophilic reagent is water and / or alcohol.

[0031] [4] The production method according to any one of [1] to [3], wherein the contact with the nucleophilic reagent is performed at about 5°C to about 80°C for about 0.25 hours or more.

[0032] [5] A method for removing nucleic acid impurities, wherein a nucleic acid whose amino group at the base portion is protected by an acyl protecting group is contacted with a nucleophilic reagent under neutral or acidic pH conditions, and then the protecting group of the nucleic acid is deprotected to obtain a target nucleic acid, thereby removing nucleic acid impurities larger than 34 daltons from the target nucleic acid.

[0033] [6] The removal method according to [5], wherein the pH is about 3 to about 8.

[0034] [7] The removal method according to [5] or [6], wherein the nucleophilic agent is water and / or alcohol.

[0035] [8] The removal method according to any one of [5] to [7], wherein the contact with the nucleophilic agent is performed at about 5°C to about 80°C for about 0.25 hours or more.

[0036] [9] A nucleic acid, wherein the ratio of detected ion intensities of the nucleic acid to nucleic acid impurities in mass spectrometry analysis is 0.50 or less relative to 100 of the nucleic acid, and the nucleic acid impurities are nucleic acid impurities contained in the nucleic acid and have a mass greater than that of the nucleic acid by 34 Daltons.

[0037] Effects of the Invention

[0038] The method for producing a nucleic acid of the present invention can reduce the content of a by-product (B) that cannot be removed by conventional techniques, and produce a nucleic acid with high purity. DETAILED DESCRIPTION

[0039] Hereinafter, each item of the method for producing a nucleic acid of the present invention will be described in detail. However, the following description is an example for describing the present invention, and is not intended to limit the present invention to the scope of the description.

[0040] The term "about" as used in this specification and claims refers to a value within ±10% of the value expressed as about, preferably within ±5%, more preferably within ±2%, and particularly preferably within ±1%.

[0041] 1. Method for producing nucleic acid

[0042] The method for producing nucleic acid of the present invention is a method for producing nucleic acid comprising the following steps: step A, synthesizing a nucleic acid in which the amino group of the base part is protected by an acyl-based protecting group; step B, contacting the nucleic acid obtained in step A with a nucleophilic reagent under neutral to acidic pH conditions; and step C, deprotecting the protecting group of the nucleic acid obtained in step B.

[0043] (Nucleic Acids)

[0044] The nucleic acid in the present invention has a structure in which nucleosides are linked by phosphodiester bonds, phosphorothioate bonds or phosphoamidoester bonds, and is preferably an oligonucleotide. The length of the oligonucleotide is not particularly limited, and is, for example, 10 to 100 bases in length, and is particularly preferably 12 to 60 bases in length.

[0045] As for the nucleic acid in the present invention, the synthesized structure is a single chain, the sugar is ribose or deoxyribose, and the base is selected from adenine (A), thymine (T), uracil (U), guanine (G), and cytosine (C). Ideally, the nucleic acid in the present invention contains purine bases such as adenine. In this specification, the unit of "base length" can be replaced by the unit of "mer" or the unit of "nucleotide".

[0046] Examples of the nucleic acid in the present invention include DNA and RNA.

[0047] Unless otherwise specified, the sugar moiety of DNA is a deoxyribose ring, and the base moiety is selected from adenine, thymine, guanine, and cytosine. Unless otherwise specified, the sugar moiety of RNA is a ribose ring, and the base moiety is selected from adenine, uracil, guanine, and cytosine.

[0048] The single-stranded RNA or DNA synthesized by the present invention can be directly used as an antisense, CpG oligonucleotide or aptamer, or can be used as siRNA, miRNA, bait, HDO (heteroduplex nucleic acid) by forming base pairs (annealing) between single-stranded RNA or DNA with complementary base sequences to produce double-stranded RNA or DNA.

[0049] (Nucleic Acid Modification)

[0050] The nucleic acid in the present invention may be, for example, a nucleic acid whose base moiety is modified by a substituent. As the substituent, for example, halogen, acyl, alkyl, arylalkyl, alkoxy, hydroxyl, amino, monoalkylamino, dialkylamino, carboxyl, cyano, nitro, etc. may be cited. As the modified base, for example, 8-bromoadenyl, 8-bromoguanyl, 5-bromocytosine, 5-bromouracil, 5-iodouracil, 5-iodocytosine, 5-fluorouracil, 5-methylcytosine (mC), 8-oxoguaninyl, hypoxanthine, etc. may be cited.

[0051] The nucleic acid in the present invention can be, for example, a nucleic acid whose sugar part has been modified at the 2' position, the 5' position, or the cross-linked modification. Specific examples of the 2' position modification include: 2'-F, 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), etc. Specific examples of the 5' position modification include: 5'-methyl (5'-Me), 5'-cyclopropene (5'-CP), etc. Specific examples of the cross-linked modification include modifications that introduce a cross-linked structure between the 2' position and the 4' position, such as: 2',4'-BNA / LNA, 2',4'-BNACOC, 2',4'-BNANC, ENA, AmNA, scpBNA, cEt, GuNA, etc.

[0052] The nucleic acid of the present invention may be modified in several nucleotide units (eg, 1 to 3 nucleotides) or all nucleotide units.

[0053] (Nucleic Acid Synthesis Method)

[0054] (1) Step A

[0055] In step A, a nucleic acid in which the amino group of the base moiety is protected by an acyl-based protecting group is synthesized.

[0056] Examples of the amino group-containing base include adenine, guanine, cytosine, or bases modified with substituents thereof. Examples of the acyl protecting group include benzoyl, isobutyryl, acetyl, phenoxyacetyl, isopropylphenoxyacetyl, tert-butylphenoxyacetyl, and the like, and preferably benzoyl, acetyl, or isobutyryl, and the like.

[0057] Furthermore, the phosphodiester bond, phosphorothioate bond or phosphoamidoester bond in the nucleic acid may be protected by a protecting group in step A. Examples of the protecting group for the phosphodiester bond, phosphorothioate bond or phosphoamidoester bond include those used in conventional methods, such as 2-cyanoethyl.

[0058] The method for synthesizing the nucleic acid in step A is not limited, and examples thereof include solid phase synthesis method, liquid phase synthesis method, etc. Solid phase synthesis method or liquid phase synthesis method is preferred, and solid phase synthesis method is particularly preferred. The details of the solid phase synthesis method are described below as an example.

[0059] (Solid Phase Synthesis Method)

[0060] The solid phase synthesis method in the present invention can be carried out according to a conventional method unless otherwise specified in the present specification. Examples of the solid phase synthesis method include the H-phosphonate method, the phosphate method, and the phosphoramidite method, and the phosphoramidite method is particularly preferred.

[0061] In step A, the solid phase synthesis method using the phosphoramidite method can be performed, for example, by sequentially performing the following steps (a) to (e).

[0062] (a) Deprotection step: removing the protecting group of the 5'-hydroxyl group of the nucleoside supported on the solid phase via a linker.

[0063] (b) Coupling step: using an activating agent, condensation is effected between the phosphorus atom bonded to the 3'-hydroxyl group of the nucleoside phosphoramidite and the 5'-hydroxyl group of the nucleoside supported on the solid phase.

[0064] (c) Oxidation / sulfurization step: using an oxidizing agent or a sulfurizing agent, the phosphite bond between nucleosides (including the case where a protecting group is bonded) is converted into a phosphodiester bond (including the case where a protecting group is bonded) or a phosphorothioate bond (including the case where a protecting group is bonded).

[0065] (d) Capping step: Using a capping agent, a protecting group is bonded to the 5' hydroxyl group of the nucleoside supported on the solid phase that has not reacted in step (b). The capping step is a step for preventing the unreacted product of step (b) from participating in the subsequent reactions of steps (a) to (d).

[0066] (e) Cyanoethyl removal step: After repeating steps (a) to (d) a desired number of times, the protecting group is removed from the phosphodiester bond or phosphorothioate bond between nucleosides without releasing the produced nucleic acid of the desired chain length from the solid phase.

[0067] Steps (a) to (e) are exemplified below using structural formulas.

[0068] It should be noted that the following structural formula is an example of a deoxyribose ring (a hydrogen atom is bonded to the 2' position), but the same is true for a ribose ring (a hydroxyl group is bonded to the 2' position) or a substance obtained by modifying it (for example, a substance obtained by O-methoxyethylation (2'-MOE), O-methylation (2'-OMe) or fluorination (2'-F) at the 2' position).

[0069] Step (a):

[0070]

[0071] [In the formula, DMTr represents 4,4'-dimethoxytrityl. Base PG

[0047] independently represents adenine, thymine, uracil, guanine, cytosine or modified forms thereof, wherein the amino group of the base moiety is protected by an acyl protecting group.

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[0091] [0

[0072] Examples of the solid phase support in solid phase synthesis include glass beads, resin beads, and silica gel, and glass beads and resin beads are preferred.

[0073] Examples of the linker include a 3-aminopropyl group, a succinyl group, a 2,2′-diethanolsulfonyl group, and a long-chain alkylamino group (LCAA).

[0074] As the protecting group of the 5'-hydroxyl group of the nucleoside, nucleotide chain, nucleoside phosphoramidite, etc. supported on the solid phase, for example, 4-methoxytrityl group can be used in addition to 4,4'-dimethoxytrityl group.

[0075] Step (b):

[0076]

[0077] [wherein, DMTr, base PG 、●Part has the same meaning as above]

[0078] Examples of the activating agent used in step (b) include 1H-tetrazole, 5-ethylthiotetrazole, 4,5-dichloroimidazole, 4,5-dicyanoimidazole, benzotriazole trifluoromethanesulfonate, imidazole trifluoromethanesulfonate, pyridine trifluoromethanesulfonate, N,N-diisopropylethylamine, 2,4,6-trimethylpyridine / N-methylimidazole, 5-(benzylthio)-1H-tetrazole, and 5-[3,5-bis(trifluoromethyl)phenyl]tetrazole. Step (c):

[0079]

[0080] [wherein, DMTr, base PG 、●part has the same meaning as above. X represents an oxygen atom or a sulfur atom]

[0081] Examples of the oxidizing agent in step (c) include meta-chloroperbenzoic acid, metaperiodate, hydrogen peroxide, iodine, (1S)-(+)-(10-camphorsulfonic)oxazine, and examples of the sulfiding agent include 3-[(N,N-dimethylaminomethylene)amino]-3H-1,2,4-dithiazole-5-thione (DDTT), 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent), 3H-1,2-benzodithiol-3-one, bisphenylacetyl disulfide (PADS), tetraethylthiuram disulfide (TETD), 3-amino-1,2,4-dithiazole-5-thione (ADTT), and examples of the reaction solvent in step (c) include dichloromethane, acetonitrile, pyridine, 3-methylpyridine, water, tetrahydrofuran, or any combination thereof.

[0082] Step (d):

[0083]

[0084] [wherein, DMTr, base PG 、●Part has the same meaning as above]

[0085] Examples of the capping agent in step (d) include acetic anhydride, phenoxyacetic anhydride, etc. Examples of the reaction solvent in step (d) include acetonitrile, pyridine, 2,6-lutidine, tetrahydrofuran, and any mixture thereof.

[0086] Step (e):

[0087]

[0088] [wherein, base PG , ●, X have the same meanings as above. R represents 4,4'-dimethoxytrityl or a hydrogen atom. n represents an integer greater than 0]

[0089] Examples of the cyanoethyl group-removing agent in step (e) include triethylamine, diethylamine, 1,8-diazabicyclo[5.4.0]-7-undecene, etc. The cyanoethyl group-removing agent may be diluted with acetonitrile, toluene, etc. before use.

[0090] (2) Step B

[0091] In step B, the nucleic acid obtained in step A is contacted with a nucleophilic reagent under neutral or acidic pH conditions. Step B can reduce the content of byproducts (B) that cannot be removed by conventional techniques, and produce nucleic acids with high purity.

[0092] As nucleophilic reagent, there is no particular limitation, and examples thereof include: water, alcohol, thiol, amine, halide ion, cyanide ion, etc. Specifically, examples thereof include: water, methanol, ethanol, n-propanol, isopropanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, ethylene glycol, glycerol, phenol, methyl mercaptan, ethyl mercaptan, dodecanethiol, dithiothreitol, methylamine, dimethylamine, ethylamine, diethylamine, ethylenediamine, aniline, pyrrolidine, piperidine, piperazine, morpholine, etc. It is preferably selected from water, alcohol, thiol, more preferably water and / or alcohol, and further preferably water, primary alcohol and secondary alcohol, for example, selected from water, methanol, ethanol, isopropanol, n-butanol, and from the perspective of ease of acquisition and operation, it is further preferably selected from water, methanol, ethanol, and most preferably water. Liquid nucleophilic reagents can be used as solvents.

[0093] Step B is carried out under neutral (eg, pH 6-8) or acidic (eg, pH 6 or below) pH conditions, preferably under pH conditions of about 3-8, and more preferably under pH conditions of about 4-7.

[0094] The contact amount of the nucleophilic reagent and the synthetic product in step B is not particularly limited. From the viewpoint of production efficiency, it is preferably 1 mL to 1000 mL, more preferably 5 mL to 100 mL, and particularly preferably 10 mL to 50 mL relative to 1 mmol of the nucleic acid. There is a tendency that the effect of the present invention is higher as the contact time of the nucleophilic reagent and the synthetic product is longer, for example, it is 0.25 hours or more, preferably 0.5 hours or more, more preferably 1 hour or more, and further preferably 2 hours or more. From the viewpoint of production efficiency, it is ideally within 24 hours.

[0095] The temperature during the contact between the nucleophile and the synthetic product in step B is not particularly limited, and is, for example, in the range of 5°C to 80°C, more preferably 10°C to 50°C, and particularly preferably 15°C to 30°C from the viewpoint of ease of operation.

[0096] The material of the liquid contact surface of the container used when contacting with the nucleophilic reagent is not particularly limited, and for example, SUS316, SUS316L, SUS304, glass, acrylic resin, polyethylene, polypropylene, polystyrene, PET, PTFE, PFA, ETFE, FEP, etc. can be listed, preferably selected from glass, acrylic resin, polyethylene, polypropylene, polystyrene, PET, PTFE, PFA, and more preferably selected from glass, acrylic resin, polyethylene, polypropylene, PTFE, PFA. By implementing the operation of contacting with the nucleophilic reagent in a non-metallic container, even in the case where the target oligonucleotide has a thiophosphate, the generation of desulfurized impurities (substances formed by the thiophosphate bond of the target oligonucleotide being changed to a phosphodiester bond) in the cleavage and deprotection steps caused by the dissolution of metal ions from the reaction container can be suppressed.

[0097] The contact with the nucleophilic reagent in step B may be carried out in the reaction column used in the synthesis, or in another container transferred after being removed from the reaction column.

[0098] (3) Step C

[0099] In step C, the protecting group of the nucleic acid obtained in step B is deprotected.

[0100] When the method for synthesizing nucleic acid in step A is a solid phase synthesis method, step C (a step of deprotecting the protecting group) may be a step of simultaneously cleaving the nucleic acid from the solid phase support.

[0101] After the above steps (a) to (e) and step B in step A, the following step (f) may be performed as step C.

[0102] (f) Cleavage / deprotection step: The nucleic acid of the desired length produced up to step (e) and step B is cleaved from the solid support using a cleavage / deprotection agent to remove the protecting groups such as the base part and the 2'-hydroxyl group.

[0103] Step (f):

[0104]

[0105] [wherein, base PG , ●, X have the same meanings as above. The base independently represents adenine, thymine, uracil, guanine, cytosine or a modified substance thereof from which the protecting group has been removed. R represents a 4,4'-dimethoxytrityl group or a hydrogen atom. n represents an integer greater than 0] (reaction solution in the cleavage / deprotection step)

[0106] Examples of the cleavage / deprotection agent include alkaline substances such as concentrated ammonia water, methylamine, and sodium hydroxide. The ammonia concentration of the concentrated ammonia water is preferably 20 to 30% by weight, more preferably 25 to 30% by weight. The amount of the cleavage / deprotection agent used is preferably 10 mL to 1000 mL relative to 1 mmol of the nucleic acid supported on the solid phase carrier, and more preferably 50 mL to 500 mL relative to 1 mmol of the nucleic acid.

[0107] The cleavage / deprotection agent can be diluted with water, methanol, ethanol, isopropanol, 1-propanol, 1-butanol, 2-butanol, acetonitrile, tetrahydrofuran, 1,2-dimethoxyethane, dimethyl sulfoxide, etc., preferably with water, ethanol or isopropanol. The reaction solution in the cleavage / deprotection step is preferably alkaline, more preferably pH 10 to 14.

[0108] The reaction temperature of step (f) is, for example, 5 to 75° C., preferably 15 to 60° C. The reaction time of step (f) is, for example, 48 hours or less, preferably 24 hours or less.

[0109] The nucleic acid is cleaved from the solid phase support by the cleavage / deprotection agent, thereby removing the protecting group bonded to the nucleic acid.

[0110] Steps (a) to (f) of the solid phase synthesis method can be performed using a commercially available nucleic acid automatic synthesizer, etc. In addition, according to the above steps, a nucleic acid is produced such that a nucleotide chain extends from the 3' carbon atom to the 5' carbon atom.

[0111] (4) Separation and purification steps

[0112] The produced nucleic acid can be purified by commonly used separation and purification methods, such as reverse phase chromatography, reverse phase ion pair chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, etc. In addition, salts contained in the solution as impurities can be removed by ultrafiltration, and the oligonucleic acid solution can be powdered by freeze drying.

[0113] (By-product (B))

[0114] The method for producing nucleic acid of the present invention can reduce the content of byproduct (B) that cannot be removed by the prior art, and produce nucleic acid with high purity. Specifically, the ratio of the detected ion intensity during mass spectrometry analysis of nucleic acid impurities (byproduct (B)) having a mass greater than that of the target nucleic acid (A) by 34 daltons is 0.50 or less, preferably 0.10 or less relative to nucleic acid (A) 100.

[0115] The inventor of the present application presumes that the byproduct (B) is generated in the synthesis operation, mainly in the step (a), by a mutation to a higher polarity on the adenine base contained in its sequence. In addition, the byproduct (B) is close to the physical properties such as molecular weight and polarity of the target nucleic acid (A), and it is difficult to remove the separation and purification techniques commonly used in nucleic acid manufacturing such as anion exchange chromatography. Therefore, it is ideal to reduce the content of the byproduct (B) in step B and step C before the separation and purification step.

[0116] The quality analysis of the produced nucleic acid can be performed by combining a commonly performed analysis method for nucleic acid, such as reverse phase chromatography, reverse phase ion pair chromatography, ion exchange chromatography, etc. as a separation method, and a UV detector, mass spectrometer, etc. as a detector. The amount of byproducts (impurities) in nucleic acid can be calculated based on the area ratio of the detected peaks when a UV detector is used, and based on the detected ion intensity ratio when a mass spectrometer is used. As a method for calculating the ion intensity ratio, a method of converting the detected polyvalent ion group of nucleic acid into the mass of a molecule (neutral species) or the m / z of a monovalent ion (a dimensionless quantity obtained by dividing the relative mass obtained by dividing the mass of the ion by the unified atomic mass unit by the number of charges of the ion) by a deconvolution operation and then calculating, a method of extracting the signal intensity of a specific polyvalent ion and calculating, etc.

[0117] 2. Methods for removing impurities

[0118] The impurity removal method of the present invention is a method for removing nucleic acid impurities (byproduct (B)), wherein a nucleic acid whose amino group of the base portion is protected by an acyl protecting group is brought into contact with a nucleophilic reagent under neutral to acidic pH conditions, and then the protecting group of the nucleic acid is deprotected to obtain a target nucleic acid, thereby removing nucleic acid impurities having a mass greater than 34 Daltons than the nucleic acid from the target nucleic acid.

[0119] The impurity removal method of the present invention can be implemented by performing the above-mentioned steps B and C.

[0120] 3. Nucleic acid with few impurities

[0121] The nucleic acid with few impurities of the present invention is a nucleic acid in which the ratio of the detected ion intensity during mass spectrometry analysis between the nucleic acid and the nucleic acid impurity is 0.50 or less relative to the nucleic acid 100, and the nucleic acid impurity is a nucleic acid impurity contained in the nucleic acid and has a mass 34 Daltons greater than that of the nucleic acid.

[0122] The nucleic acid with few impurities of the present invention can be produced using the above-mentioned method for producing a nucleic acid of the present invention.

[0123] Example

[0124] The present invention will be described in further detail below with reference to Examples and the like, but the present invention is not limited to these Examples and the like.

[0125] Unless otherwise specified, the sugar portion of the DNA in the following examples is a deoxyribose ring, and the base portion is selected from adenine, thymine, guanine, and cytosine. Unless otherwise specified, the sugar portion of the LNA (2', 4'-BNA) in the examples has a structure in which the oxygen atom at the 2' position of the ribose ring and the carbon atom at the 4' position are cross-linked (-CH2-), and the base portion is selected from adenine, thymine, guanine, and 5-methylcytosine. In the manufacturing examples and comparative manufacturing examples, a benzoyl group is used as a protecting group for the amino group at the 6th position of adenine, an isobutyryl group is used as a protecting group for the amino group at the 2nd position of guanine, and a benzoyl group is used as a protecting group for the amino group at the 4th position of cytosine and 5-methylcytosine.

[0126] [Production Example 1]

[0127] (Study on types of nucleophiles)

[0128] Using a SUS316L reaction column filled with a solid phase carrier and a nucleic acid synthesis device OligoPilot (registered trademark) plus100, phosphoramidites were sequentially condensed on the nucleoside bonded to the solid phase carrier via a linker, and finally a triethylamine / acetonitrile (1:1) mixed solution was passed to synthesize the target oligonucleotide (FLP).

[0129] Next, the solid phase support bonded with the target oligonucleotide was immersed in 20 mL of any nucleophilic reagent listed in Table 1 per 1 mmol of the solid phase support in a glass test tube at 20° C. for 3 hours and shaken. Then, the solid phase support was immersed in a 28 wt % ammonia aqueous solution / ethanol (4:1) mixed solution at 60° C. for 8 hours to perform cleavage of the target oligonucleotide from the solid phase support and deprotection of the protecting group.

[0130] After the above cleavage / deprotection, the mixed solution was diluted with a water / ethanol (4:1) mixed solution to obtain a crude solution of the target oligonucleotide (Examples 1 to 5).

[0131] It should be noted that the above-mentioned solid phase carrier, linker, and target oligonucleotide are as follows.

[0132] Solid phase support, linker: Primer Support (registered trademark) 5G Unylinker 350 (Cytiva).

[0133] Target oligonucleotide (FLP): 5'-T(L)^G(L)^C^T^A^G^C^A^G^A^T^G^C^T^A(L)^mC(L)-3' (Sequence number 1: 16mer, ^ represents a phosphorothioate bond. In addition, each nucleoside unit with (L) recorded next to the base has the same structure as LNA (2',4'-BNA), and the other nucleoside units represent the same structural units as DNA. mC indicates that the base is methylcytosine).

[0134] [Comparative Production Example 1]

[0135] Using the solid phase support bonded with the target oligonucleotide synthesized in Preparation Example 1, the following substitution was made: “Next, immerse the solid phase support bonded with the target oligonucleotide in 20 mL of any nucleophilic reagent listed in Table 1 per 1 mmol of the solid phase support in a glass test tube at 20°C for 3 hours and shaken. Then, the solid phase support was immersed in a 28 wt % ammonia solution / ethanol (4:1) mixed solution” was changed to “Next, immerse the solid phase support bonded with the target oligonucleotide in a 28 wt % ammonia solution / ethanol (4:1) mixed solution”. Except for this, the same operation was performed under the same conditions as in Preparation Example 1 to obtain a crude solution of the target oligonucleotide (Comparison Example 1).

[0136] [Production Example 2]

[0137] (Comparative Study of Contact Conditions)

[0138] The crude solution of the target oligonucleotide was obtained by performing the same operation as in Preparation Example 1 except that "immersing in 20 mL of any nucleophilic reagent listed in Table 1 for 3 hours at 20°C and shaking for each 1 mmol of the solid phase carrier loading" was changed to "immersing in water and shaking under the conditions of any contact temperature, water contact amount per unit solid phase carrier loading, and contact time listed in Table 2".

[0139] [Comparative Production Example 2]

[0140] The same operation as in Comparative Production Example 1 was carried out using the solid phase support to which the target oligonucleotide was bound and synthesized in Production Example 2, thereby obtaining a crude solution of the target oligonucleotide (Comparative Example 2).

[0141] [Production Example 3]

[0142] (Contact Time Study)

[0143] Using a SUS316L reaction column filled with a solid phase carrier and a nucleic acid synthesis device OligoPilot (registered trademark) plus100, phosphoramidites were sequentially condensed on the nucleosides bonded to the solid phase carrier via a linker, and finally a triethylamine / acetonitrile (1:1) mixed solution was introduced to synthesize the target oligonucleotide (FLP), and then 960 mL of water per 1 mmol of solid phase carrier loading was introduced into the reaction column over 4 minutes to bring the solid phase carrier into contact with water.

[0144] Next, the solid support to which the target oligonucleotide was bound was immersed in a 28 wt % aqueous ammonia solution / ethanol (4:1) mixed solution at 60° C. for 19 hours in a glass test tube to cleave the target oligonucleotide from the solid support and deprotect the protecting group.

[0145] After the above cleavage / deprotection, the mixed solution was diluted with a water / ethanol (4:1) mixed solution to obtain a crude solution of the target oligonucleotide (Reference Example 1).

[0146] It should be noted that the above-mentioned solid phase carrier, linker, and target oligonucleotide are as follows.

[0147] Solid phase support, linker: Primer Support (registered trademark) 5G Unylinker 350 (Cytiva).

[0148] Target oligonucleotide (FLP): 5'-C^T^A^G^C^A^G^A^T^G^C^T-3' (SEQ ID NO. 2: 12mer DNA, ^ represents a phosphorothioate bond).

[0149] [Comparative Production Example 3]

[0150] The method of "synthesizing the target oligonucleotide (FLP) and then passing 960 mL of water per 1 mmol of the solid phase carrier loading into the reaction column over 4 minutes to bring the solid phase carrier into contact with water" was changed to "synthesizing the target oligonucleotide (FLP)". Except for this, the same operation was performed under the same conditions as in Manufacturing Example 3 to obtain a crude solution of the target oligonucleotide (Comparative Example 3).

[0151] [Production Example 4]

[0152] (Purification of target oligonucleotide)

[0153] A crude solution obtained under the same conditions and the same operation as in Production Example 1 (a crude solution using water as a nucleophile, Example 13) and a crude solution obtained under the same conditions and the same operation as in Comparative Production Example 1 (Comparative Example 4) were prepared.

[0154] The crude liquids were separated and purified using anion exchange chromatography under the following conditions to obtain purified oligonucleotide-containing liquids (Example 13, Comparative Example 4).

[0155] Separation and purification conditions: ion exchange resin; BioPro IEX SmartSep Q20 (YMC), column volume; 7 mL, column temperature; room temperature, mobile phase A: 10 mM sodium hydroxide aqueous solution / methanol (85:15) mixed solution, mobile phase B: 10 mM sodium hydroxide·2 M sodium chloride aqueous solution / methanol (85:15) mixed solution.

[0156] [Test Example 1: Measurement of the amount of by-product (B)]

[0157] For the crude liquid of each oligonucleotide produced in Production Examples 1 to 4 and Comparative Production Examples 1 and 2 (Production Example 4 also for the purified oligonucleotide-containing liquid), the measurement using ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) was carried out under the following conditions to obtain the amount of the target oligonucleotide (FLP) and the amount of the byproduct (B) whose mass is increased by 34 Daltons compared to the target oligonucleotide, and the content of the byproduct (B) was calculated (ionic strength of the byproduct / ionic strength of the target oligonucleotide (FLP) (%)). It should be noted that the byproduct (B) in this specification is common in that the mass is increased by 34 Daltons compared to the target oligonucleotide, but it is a different substance for each type of the target oligonucleotide.

[0158] Assay conditions: UHPLC device; Vanquish UHPLC system, mass spectrometer; Q Exactive, column; Waters ACQUITY UPLC Oligonucleotide BEH C18, 1.7μm, 2.1mm×100mm, UV detection; 260nm, buffer A; 100mM HFIP, 8mM TEA in H2O:MeCN=98:2, buffer B; H2O:MeCN=50:50, temperature; 60℃, ionization method; ESI, detection mode; negative, spray voltage; 2.5|kV|, capillary temperature; 250℃.

[0159] The results of the above measurements are shown in Tables 1 to 4 below.

[0160] [Test Example 2: Determination of the amount of target oligonucleotide (FLP)]

[0161] The crude liquid of the oligonucleotide produced in Production Example 4 and each sample solution of the purified liquid containing the oligonucleotide were measured using ultra-high performance liquid chromatography (UHPLC) under the following conditions to calculate the purity of the target oligonucleotide (FLP) (peak area of ​​the target oligonucleotide (FLP) / total area of ​​all peaks (%)).

[0162] Measurement conditions: UHPLC device; ACQUITY UPLC, column; Waters ACQUITY UPLC (registered trademark) Oligonucleotide BEH C18, 1.7μm, 2.1mm×100mm, UV detection; 260nm, buffer A; 100mM HFIP, 8mM TEA in H2O:MeCN=98:2, buffer B; H2O:MeCN=50:50, temperature; 60℃.

[0163] Furthermore, each sample solution of the crude oligonucleotide prepared in Preparation Example 4 and the purified oligonucleotide-containing liquid was diluted with water and the absorbance was measured using a spectrophotometer. The yield (the ratio of the OD value actually measured to the maximum OD value that can be obtained theoretically) was calculated based on the OD value obtained.

[0164] The results of the above measurements are shown in Table 4 below.

[0165] [Table 1]

[0166] Types of Nucleophiles By-product (B) amount / FLP amount (%) Example 1 water 0.04 Example 2 Methanol 0.02 Example 3 Ethanol 0.09 Example 4 Isopropyl alcohol 0.46 Example 5 n-Butanol 0.26 Comparative Example 1 none 0.99

[0167] [Table 2]

[0168]

[0169]

[0170] [Table 3]

[0171] Whether there is water supply By-product (B) amount / FLP amount (%) Comparative Example 3 none 1.11 Reference Example 1 have 0.82

[0172] [Table 4]

[0173]

[0174] The by-product (B) amount / FLP amount (%) described in Tables 1 to 4 above is the ratio (%) of the by-product (B) when FLP (full-length product: target oligonucleotide) is set to 100 (%).

[0175] As shown in Table 1, the samples (Examples 1 to 5) contacted with the specific nucleophilic reagent of the present invention (water, methanol, ethanol, isopropanol, and n-butanol) have significantly lower content of the by-product (B) than the sample (Comparative Example 1) not contacted with the nucleophilic reagent.

[0176] At this time, it was confirmed that in Example 2, in which the nucleophile was methanol, 3.20% (ratio when the target oligonucleotide was 100%) of the alkoxy byproduct in which the adenine base of the target oligonucleotide was substituted with a methoxy group was generated. It is believed that the alkoxy byproduct can be separated from the target oligonucleotide by reverse phase chromatography.

[0177] As shown in Table 2, regarding the contact with water in the present invention, it was shown that the amount of water in contact had no influence on the content of the by-product (B) (Examples 7 and 8).

[0178] In addition, it is shown that by contacting with water for 0.5 hours (Example 9), the content of byproduct (B) is significantly reduced compared with the case of not contacting with water (Comparative Example 2). In addition, it is shown that compared with the case of only contacting with water for 4 minutes (Reference Example 1 of Table 3), the reduction rate of byproduct (B) under the case of contacting for 0.5 hours (Example 9 of Table 2) is large, and the reduction rate of byproduct (B) under the case of contacting for 2 hours (Example 10 of Table 2) is greater. It is shown from the above results that as the contact time increases, the effect of reducing the content of byproduct (B) is enhanced.

[0179] In addition, it is shown that the by-product (B) is reduced more rapidly when the contact temperature with water is 30°C (Example 11 in Table 2) than when the contact temperature is 15°C (Example 6 in Table 2) or 20°C (Example 9 in Table 2). The above results show that the effect of reducing the content of the impurity increases with the increase in the contact temperature.

[0180] As shown in Table 4, it is shown that the contact with water of the present invention is more effective in reducing the content of the by-product (B) than the conventional separation and purification operation. In addition, it was confirmed that the presence or absence of contact with water has no effect on the purity and yield of FLP.

[0181] Industrial Applicability

[0182] The present invention is useful for producing high-quality nucleic acid medicines with low impurity content.

Claims

1. A method for producing a nucleic acid, comprising: Step A, synthesizing a nucleic acid in which the amino group of the base part is protected by an acyl protecting group; Step B, contacting the nucleic acid obtained in step A with a nucleophilic reagent under neutral or acidic pH conditions; as well as Step C, deprotecting the protecting group of the nucleic acid obtained in step B.

2. The manufacturing method according to claim 1, wherein: The pH is about 3 to about 8.

3. The manufacturing method according to claim 1 or 2, wherein: The nucleophile is water and / or alcohol.

4. The production method according to any one of claims 1 to 3, wherein The contacting with the nucleophile is carried out at a temperature of about 5°C to about 80°C for more than about 0.25 hours.

5. A method for removing nucleic acid impurities, wherein: The target nucleic acid is obtained by contacting a nucleic acid whose amino group in the base part is protected by an acyl protecting group with a nucleophilic reagent under neutral or acidic pH conditions, and then deprotecting the protecting group of the nucleic acid, thereby removing nucleic acid impurities having a mass greater than 34 Daltons than the nucleic acid from the target nucleic acid.

6. The removal method according to claim 5, wherein: The pH is about 3 to about 8.

7. The removal method according to claim 5 or 6, wherein: The nucleophile is water and / or alcohol.

8. The removal method according to any one of claims 5 to 7, wherein: The contacting with the nucleophile is carried out at a temperature of about 5°C to about 80°C for more than about 0.25 hours.

9. A nucleic acid, wherein The ratio of detected ion intensities in mass spectrometry analysis between nucleic acid and nucleic acid impurities is 0.50 or less relative to 100 of the nucleic acid, and the nucleic acid impurities are nucleic acid impurities contained in the nucleic acid and having a mass 34 daltons greater than that of the nucleic acid.

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