Method for producing nucleic acids, method for removing impurities, and nucleic acids with fewer impurities

Abstract

The present invention pertains to: a nucleic acid production method comprising a step A for synthesizing a nucleic acid in which an amino group of a base part thereof is protected by an acyl-based protecting group, a step B for bringing the nucleic acid obtained in step A into contact with a nucleophilic agent in a neutral or acidic pH condition, and a step C for deprotecting the protecting group of the nucleic acid obtained in step B; and the like. The present invention makes it possible to produce a highly pure nucleic acid by reducing the contained amount of by-products (B) that cannot be removed by conventional technologies.

Description

Technical Field

[0001] The present invention relates to a method for producing nucleic acids, a method for removing impurities, and nucleic acids with few impurities.

Background Art

[0002] Conventionally, the pharmacologically active ingredients of marketed pharmaceuticals have exclusively been low-molecular-weight compounds, but recently, many pharmaceuticals containing medium-molecular-weight pharmacologically active ingredients such as peptides and nucleic acids have come to be marketed. In particular, nucleic acid pharmaceuticals have characteristic mechanisms of action not found in other drug discovery leads, such as the ability to act specifically by forming base pairs with target mRNA, and are thus drug discovery leads that are highly anticipated for future development.

[0003] Nucleic acids, which are raw materials for nucleic acid pharmaceuticals, are generally produced by chemical synthesis. For example, the following Patent Document 1 can be cited as a document introducing the production method of nucleic acids. Patent Document 1 relates to a method for deprotecting oligonucleotides, and introduces contacting a solid-phase synthesized oligonucleotide with an organic amine or the like to cleave a protecting group without detaching the oligonucleotide from the solid phase.

[0004] For pharmaceuticals, the types and amounts of impurities are strictly quality-controlled under various regulations from the perspective of ensuring their safety. Similarly, impurity management is strict in the production of active pharmaceutical ingredients, and the creation of technologies for removing impurities generated in the production of nucleic acids used as active pharmaceutical ingredients is even more desirable.

[0005] As methods for purifying nucleic acids by removing impurities, reverse-phase cartridges (RPC), reverse-phase high-performance liquid chromatography (RP HPLC), anion-exchange chromatography (AEX), polyacrylamide gel electrophoresis (PAGE), etc. are known. Furthermore, Patent Document 2 describes a method for purifying oligonucleotides (Claim 1, 2) that uses a mixed-mode matrix containing a strong anion-exchange ligand, a strong cation-exchange ligand, and a hydrophobic ligand to remove impurities including uncomplexed oligonucleotides and failure sequences.

[0006] (etc.) is listed.

[0006] Patent Document 3 describes the purification of oligonucleotides (Claim 1,

[0006] , etc.) by removing product-related impurities such as n-1 impurities, P=O impurities, debasement impurities, CNEt impurities, and N+1 impurities using hydrophobic interaction chromatography (HIC). Patent Document 4 describes a method for purifying oligonucleotides (Claim 1,

[0003] ) that removes other oligonucleotides such as shorter and longer using a two-phase mobile-stationary-liquid chromatography system.

[0007] Patent Document 5 describes the purification of oligonucleotides (claims 1,

[0012] , etc.) by removing impurities such as short oligonucleotides using a titrable anion exchange composition. Patent document 6 describes the purification of oligonucleotides (claim 1, etc.) by removing impurities using a porous carrier. Non-patent document 1 describes the control and removal of non-oligonucleotide product-related impurities (PRIs) in the manufacture of therapeutic oligonucleotides. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] Patent No. 4705716 [Patent Document 2] Special Publication No. 2022-539327 [Patent Document 3] Special Publication No. 2019-518759 [Patent Document 4] Special Publication No. 2014-525254 [Patent Document 5] Special Publication No. 2005-520547 [Patent Document 6] Japanese Patent Application Publication No. 7-227284 [Non-patent literature]

[0009] [Non-Patent Document 1] Org. Process Res. Dev., 2022, 26, 1130-1144 [Overview of the project] [Problems that the invention aims to solve]

[0010] The inventors of this application have found that in the production of nucleic acids, a nucleic acid (by-product (B)) with a mass 34 daltons greater than the target nucleic acid (A) is included as an impurity in nucleic acid (A). Because by-product (B) has a molecular weight and physical properties very similar to nucleic acid (A), it cannot be removed even by using conventionally known purification methods such as those described in Patent Documents 1-6 and Non-Patent Document 1. Therefore, the object of the present invention is to provide a method for producing nucleic acids that removes by-product (B) and reduces the content of impurities. Furthermore, the object is to provide a method for removing by-product (B) and nucleic acids that contain a small amount of by-product (B). [Means for solving the problem]

[0011] As a result of diligent research, the inventors discovered that, after nucleic acid synthesis, treating the synthesized nucleic acid with a nucleophile while the amino group of the base portion is protected with an acyl protecting group, followed by deprotection, significantly reduces the content of by-product (B). Based on this finding, the inventors have completed the present invention as described below.

[0012] [1] Step A, which involves synthesizing nucleic acids in which the amino group of the base is protected by an acyl protecting group. Step B involves contacting the nucleic acid obtained in step A with a nucleophile under neutral or acidic pH conditions, and A method for producing nucleic acids, comprising step C for deprotecting the protecting group of the nucleic acid obtained in step B. [2] The production method according to [1], wherein the pH is from about 3 to about 8. [3] The production method according to [1] or [2], wherein the nucleophile is water and / or alcohol. [4] The production method according to any one of [1] to [3], wherein the nucleophile is contacted for about 0.25 hours or more at about 5 to about 80°C.

[0013] [5] A method for removing a nucleic acid impurity having a mass 34 daltons larger than that of the nucleic acid from the target nucleic acid, comprising contacting a nucleic acid in which the amino group of the salt base is protected with an acyl-based protecting group with a nucleophile under neutral or acidic pH conditions, and then deprotecting the protecting group of the nucleic acid. [6] The removal method according to [5], wherein the pH is from about 3 to about 8. [7] The removal method according to [5] or [6], wherein the nucleophile is water and / or alcohol. [8] The removal method according to any one of [5] to [7], wherein the nucleophile is contacted for about 0.25 hours or more at about 5 to about 80°C. [9] A nucleic acid, wherein the detection ion intensity ratio in mass spectrometry of a nucleic acid impurity having a mass 34 daltons larger than that of the nucleic acid that may be contained in the nucleic acid is 0.50 or less with respect to 100 of the nucleic acid. [Effect of the Invention]

[0014] 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 the prior art and produce a nucleic acid with high purity. [Embodiments for Carrying Out the Invention]

[0015] Various matters related to the method for producing a nucleic acid of the present invention will be described in detail below. However, the following description is an exemplification for explaining the present invention, and the present invention is not intended to be particularly limited only to this description range. As used in this specification and the claims, the term "about" means a numerical value within the range of ±10% of the numerical value indicated by about, preferably within the range of ±5%, more preferably within the range of ±2%, and particularly preferably within the range of ±1%.

[0016] 1. Method for producing nucleic acids The present invention provides a method for producing nucleic acids, comprising: step A, synthesizing nucleic acids in which the amino group of the base portion is protected with an acyl protecting group; step B, contacting the nucleic acid obtained in step A with a nucleophile under neutral to acidic pH conditions; and step C, deprotecting the protecting group of the nucleic acid obtained in step B.

[0017] (nucleic acid) The nucleic acids in the present invention have a structure in which nucleosides are linked by phosphate diester bonds, thiophosphate ester bonds, or amide phosphate ester bonds, and are preferably oligonucleotides. The length of the oligonucleotide is not particularly limited, but is for example 10 to 100 nucleotides, and particularly preferably 12 to 60 nucleotides. The nucleic acids used in this invention are single-stranded, with a sugar of ribose or deoxyribose, and a base selected from adenine (A), thymine (T), uracil (U), guanine (G), and cytosine (C). It is desirable that the nucleic acids used in this invention contain a purine base such as adenine. In this specification, the unit "base length" may be replaced with the unit "mer" or "nucleotide."

[0018] Examples of nucleic acids in the present invention include DNA or RNA. Unless otherwise specified, DNA has a deoxyribose ring as its sugar moiety and bases selected from adenine, thymine, guanine, and cytosine. Unless otherwise specified, RNA has a ribose ring as its sugar moiety and bases selected from adenine, uracil, guanine, and cytosine. The single-stranded RNA or DNA synthesized by this invention can be used as is as an antisense, CpG oligo, or aptamer. Alternatively, by forming base pairs (annealing) with RNA or DNA having complementary base sequences, double-stranded RNA or DNA can be produced and used as siRNA, miRNA, decoy, or HDO (hetero-double-stranded nucleic acid).

[0019] (Modification of nucleic acids) The nucleic acid in the present invention may, for example, have its base portion modified by a substituent. Examples of substituents include halogen groups, acyl groups, alkyl groups, arylalkyl groups, alkoxy groups, hydroxyl groups, amino groups, monoalkylamino groups, dialkylamino groups, carboxyl groups, cyano groups, and nitro groups. Examples of such modified bases include 8-bromoadenyl group, 8-bromoguanyl group, 5-bromocytosyl group, 5-bromouracil group, 5-iodouracil group, 5-iodocytosyl group, 5-fluorouracil group, 5-methylcytosyl group (mC), 8-oxoguanyl group, and hypoxanthinyl group.

[0020] The nucleic acids in this invention may, for example, have modifications at the 2' and 5' positions of their sugar moiety, or cross-linking modifications. Specific examples of 2'-position modifications include, for example, 2'-F, 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), etc. Specific examples of 5'-position modifications include, for example, 5'-methyl (5'-Me), 5'-cyclopropylene (5'-CP), etc. Specific examples of cross-linking modifications include those in which a cross-linking structure is introduced between the 2' and 4' positions, such as 2',4'-BNA / LNA, 2',4'-BNACOC, 2',4'-BNANC, ENA, AmNA, scpBNA, cEt, GuNA, etc. The nucleic acids in this invention can be modified at the level of a few nucleotides (e.g., 1 to 3 nucleotides) or at the level of all nucleotides.

[0021] (Methods for synthesizing nucleic acids) (1) Process A In step A, nucleic acids are synthesized in which the amino group of the base portion is protected by an acyl protecting group. Examples of bases having an amino group include adenine, guanine, cytosine, or bases modified with substituents. Examples of acyl protecting groups include benzoyl group, isobutyryl group, acetyl group, phenoxyacetyl group, isopropylphenoxyacetyl group, tert-butylphenoxyacetyl group, etc., with benzoyl group, acetyl group, or isobutyryl group being preferred. Furthermore, in step A, the phosphate diester bond, thiophosphate bond, or amide phosphate bond in the nucleic acid may be protected with a protecting group. Examples of protecting groups for phosphate diester bonds, thiophosphate bonds, or amide phosphate bonds include those commonly used in conventional methods, such as 2-cyanoethyl.

[0022] The method for synthesizing nucleic acids in step A is not limited to this, and examples include solid-phase synthesis methods and liquid-phase synthesis methods. A solid-phase synthesis method or a liquid-phase synthesis method is preferred, and a solid-phase synthesis method is particularly preferred. The details of a solid-phase synthesis method are described below as an example.

[0023] (Solid phase synthesis method) The solid-phase synthesis method in the present invention can be carried out according to conventional methods unless otherwise specified herein. Examples of solid-phase synthesis methods include the H-phosphonate method, the phosphoester method, and the phosphoramidite method, with the phosphoramidite method being particularly preferred. In step A, the solid-phase synthesis method using the phosphoramidite method can be carried out, for example, by performing the following steps (a) to (e) in order. (a) Deprotection step: The protecting group of the hydroxyl group at the 5' position of the nucleoside supported on the solid phase via a linker is removed. (b) Coupling step: Condensation occurs 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 using an activator. (c) Oxidation / Sulfurization process: Using an oxidizing agent or sulfurizing agent, the phosphite ester bonds between nucleosides (including those with protecting groups) are converted to phosphate diester bonds (including those with protecting groups) or thiophosphate ester bonds (including those with protecting groups). (d) Capping step: In this step, a capping agent is used to attach a protecting group to the hydroxyl group at the 5' position of the nucleoside supported on the solid phase that was unreacted in step (b). The capping step is to prevent unreacted material from step (b) from participating in subsequent reactions in steps (a) to (d). (e) Cyanoethyl group removal step: After repeating steps (a) to (d) a desired number of times, the protective group is removed from the phosphate diester bond or thiophosphate ester bond between nucleosides without removing the nucleic acid of the desired chain length produced from the solid phase.

[0024] Steps (a) to (e) are illustrated below using structural formulas. Note that the structural formula below is an example of a deoxyribose ring (with a hydrogen atom bonded at the 2' position), but the same applies when using a ribose ring (with a hydroxyl group at the 2' position) or a modified version thereof (for example, one in which the 2' position is O-methoxyethylated (2'-MOE), O-methylated (2'-OMe), or fluorinated (2'-F)).

[0025] Process (a): JPEG0007897322000001.jpg29159[In the formula, DMTr represents a 4,4'-dimethoxytrityl group. Base PG The symbols independently represent adenine, thymine, uracil, guanine, cytosine, or modified versions thereof, and the amino group of the base portion is protected by an acyl protecting group. The ● portion represents a solid support bonded to a linker. Examples of solid-phase supports used in solid-phase synthesis include glass beads, resin beads, and silica gel, with glass beads or resin beads being preferred. Examples of linkers include 3-aminopropyl, succinyl, 2,2'-diethanolsulfonyl, and long-chain alkylamino (LCAA). In addition to the 4,4'-dimethoxytrityl group, other groups such as the 4-methoxytrityl group can also be used as protecting groups for the 5'-position hydroxyl group of nucleosides, nucleotide chains, nucleoside phosphoramidites, etc., that are supported on a solid phase.

[0026] Process (b): JPEG0007897322000002.jpg46148

[0027] [In the formula, DMTr, Base PG The part marked with ● is equivalent to the above. Examples of activators used in step (b) include 1H-tetrazole, 5-ethylthiotetrazole, 4,5-dichloroimidazole, 4,5-dicyanoimidazole, benzotriazole triflate, imidazole triflate, pyridinium triflate, N,N-diisopropylethylamine, 2,4,6-collidine / N-methylimidazole, 5-(benzylthio)-1H-tetrazole, and 5-[3,5-bis(trifluoromethyl)phenyl]tetrazole.

[0028] Process (c): JPEG0007897322000003.jpg46133

[0029] [In the formula, DMTr, Base PG The part marked with ● has the same meaning as above. X represents an oxygen atom or a sulfur atom. Examples of oxidizing agents for step (c) include metachloroperbenzoic acid, metaperiodate, hydrogen peroxide, iodine, (1S)-(+)-(10-camphorsulfonyl)oxaziridine, and examples of sulfiding agents include 3-[(N,N-dimethylaminomethylidene)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), and 3-amino-1,2,4-dithiazole-5-thione (ADTT). Examples of reaction solvents for step (c) include dichloromethane, acetonitrile, pyridine, 3-picoline, water, tetrahydrofuran, or solvents obtained by mixing these in any combination.

[0030] Process (d): JPEG0007897322000004.jpg29159

[0031] [In the formula, DMTr, Base PG The part marked with ● is equivalent to the above. Examples of capping agents in step (d) include acetic anhydride and phenoxyacetic anhydride. Examples of reaction solvents in step (d) include acetonitrile, pyridine, 2,6-lutidine, tetrahydrofuran, or solvents obtained by mixing these in any combination.

[0032] Process (e): JPEG0007897322000005.jpg51170

[0033] [In the formula, Base PG ● and X are equivalent to those described above. R represents a 4,4'-dimethoxytrityl group or a hydrogen atom. n represents a non-negative integer. Examples of cyanoethyl group removers for step (e) include triethylamine, diethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene. These cyanoethyl group removers can also be used after being diluted with, for example, acetonitrile or toluene.

[0034] (2) Process B In step B, the nucleic acid obtained in step A is contacted with a nucleophile under neutral or acidic pH conditions. Step B reduces the content of by-product (B), which cannot be removed by conventional techniques, and enables the production of high-purity nucleic acid.

[0035] The nucleophile is not particularly limited, but examples include water, alcohol, thiols, amines, halide ions, cyanide ions, etc. Specifically, examples include water, methanol, ethanol, n-propyl alcohol, isopropyl alcohol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, decanol, undecanol, dodecanol, ethylene glycol, glycerol, phenol, methanethiol, ethanethiol, dodecanethiol, dithiothreitol, methylamine, dimethylamine, ethylamine, diethylamine, ethylenediamine, aniline, pyrrolidine, piperidine, piperazine, morpholine, etc. Preferably selected from water, alcohol, and thiols, more preferably water and / or alcohol, even more preferably water, primary alcohol and secondary alcohol, for example, selected from water, methanol, ethanol, isopropyl alcohol and n-butyl alcohol, even more preferably selected from water, methanol and ethanol due to ease of availability and handling, and most preferably water. The liquid nucleophile may be used as a solvent.

[0036] Step B is carried out under neutral (e.g., pH 6-8) or acidic (e.g., pH 6 or lower) pH conditions, preferably under pH conditions of about 3-8, and more preferably under pH conditions of about 4-7. The amount of nucleophile and synthetic product in contact in step B is not particularly limited, but from the viewpoint of production efficiency, it is preferably 1 mL to 1000 mL, more preferably 5 mL to 100 mL, and especially preferably 10 mL to 50 mL per 1 mmol of nucleic acid. The longer the contact time between the nucleophile and synthetic product, the greater the effect of the present invention tends to be, for example, 0.25 hours or more, preferably 0.5 hours or more, more preferably 1 hour or more, and even more preferably 2 hours or more, and it is desirable to be within 24 hours from the viewpoint of production efficiency. The temperature during contact between the nucleophile and the compound in step B is not particularly limited, but 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.

[0037] The material of the wetted surface of the container used when contacting the nucleophile is not particularly limited, but examples include SUS316, SUS316L, SUS304, glass, acrylic resin, polyethylene, polypropylene, polystyrene, PET, PTFE, PFA, ETFE, FEP, etc. Preferably selected from glass, acrylic resin, polyethylene, polypropylene, polystyrene, PET, PTFE, and PFA, and more preferably selected from glass, acrylic resin, polyethylene, polypropylene, PTFE, and PFA. By carrying out the operation of contacting the nucleophile in a non-metallic container, even if the target oligonucleotide has a thiophosphate ester, it is possible to suppress the generation of desulfurized impurities (where the thiophosphate ester bond of the target oligonucleotide is changed to a phosphate diester bond) during the cleavage and deprotection process due to the elution of metal ions from the reaction vessel. Contact with the nucleophile in step B can be carried out either in the reaction column used for synthesis, or in a separate container to which the reaction column has been removed and the mixture transferred.

[0038] (3) Process C In step C, the protecting group of the nucleic acid obtained in step B is deprotected. If the nucleic acid synthesis method in step A is a solid-phase synthesis method, then step C (the step of deprotecting the protecting group) can simultaneously be the nucleic acid cleavage step from the solid support. Following steps (a) to (e) of process A and process B, the following step (f) can be performed as process C. (f) Excavation and deprotection process: The nucleic acid of the desired chain length produced in process (e) and up to process B is excised from the solid support using an excision and deprotection agent, and the base portion and protecting groups such as the hydroxyl group at the 2' position are removed.

[0039] Process (f): JPEG0007897322000006.jpg54170

[0040] [In the formula, Base PG● and X are synonymous with those described above. Base independently represents adenine, thymine, uracil, guanine, cytosine, or modified versions thereof, with the protecting group removed. R represents a 4,4'-dimethoxytrityl group or a hydrogen atom. n represents a non-negative integer.

[0041] (Reaction solution in the excision and deprotection process) Examples of cleavage and deprotection agents include basic substances such as concentrated ammonia water, methylamine, and sodium hydroxide. The ammonia concentration of the concentrated ammonia water is preferably 20-30% by weight, and more preferably 25-30% by weight. The amount of cleavage and deprotection agent used is preferably 10 mL to 1000 mL per 1 mmol of nucleic acid supported on the solid phase carrier, and more preferably 50 mL to 500 mL per 1 mmol of nucleic acid. The cleavage and deprotection agent can also be used diluted with, for example, water, methanol, ethanol, isopropyl alcohol, 1-propyl alcohol, 1-butanol, 2-butanol, acetonitrile, tetrahydrofuran, 1,2-dimethoxyethane, dimethyl sulfoxide, etc., and is preferably diluted with water, ethanol, or isopropyl alcohol. The reaction solution in the cleavage and deprotection step is preferably basic, and more preferably pH 10 to 14.

[0042] The reaction temperature in step (f) is, for example, in the range of 5 to 75°C, preferably in the range of 15 to 60°C. The reaction time in step (f) is, for example, 48 hours or less, preferably 24 hours or less. The cleavage and deprotection agent cleaves nucleic acids from the solid support and removes any protecting groups bound to them. Steps (a) to (f) of the solid-phase synthesis method described above can be carried out using commercially available automated nucleic acid synthesizers. Furthermore, nucleic acids are produced in such a way that the nucleotide chain extends from the 3' carbon atom to the 5' carbon atom according to the above steps.

[0043] (4) Separation and purification process The manufactured nucleic acids can be purified using commonly used separation and purification methods, such as reverse-phase chromatography, reverse-phase ion-pair chromatography, ion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. Furthermore, it is possible to remove salts, which are impurities in the solution, by ultrafiltration, and the oligonucleotide solution can be powdered by freeze-drying.

[0044] (By-product (B)) The nucleic acid production method of the present invention can produce highly pure nucleic acids by reducing the content of by-product (B), which cannot be removed by conventional techniques. Specifically, the detection ion intensity ratio during mass spectrometry of nucleic acid impurities (by-product (B)) with a mass 34 Daltons greater than that of the target nucleic acid (A) is 0.50 or less, preferably 0.10 or less, per 100 units of nucleic acid (A). The inventors of this invention estimate that by-product (B) is generated during the synthesis process, mainly during step (a) above, by mutations occurring on the adenine bases in its sequence that result in higher polarity. Furthermore, by-product (B) and the target nucleic acid (A) have similar physical properties such as molecular weight and polarity, making removal difficult using separation and purification techniques commonly used in nucleic acid production, such as anion exchange chromatography. Therefore, it is desirable to reduce the content of by-product (B) in steps B and C before the separation and purification steps.

[0045] The quality of manufactured nucleic acids can be analyzed using commonly used nucleic acid analysis methods, such as reverse-phase chromatography, reverse-phase ion-pair chromatography, and ion-exchange chromatography as separation methods, and UV detectors and mass spectrometers as detectors. The amount of by-products (impurities) in the nucleic acid can be calculated from the area ratio of detected peaks when using a UV detector, or from the detected ion intensity ratio when using a mass spectrometer. Methods for calculating the ion intensity ratio include deconvolution of the detected polyvalent ions of nucleic acid to convert them into the mass of molecules (neutral species) or the m / z of monovalent ions (a dimensionless quantity obtained by dividing the relative mass obtained by dividing the ion mass by the number of ions' charges), or by extracting the signal intensity of specific polyvalent ions and calculating it.

[0046] 2. Method for removing impurities The present invention provides a method for removing impurities, which involves contacting a nucleic acid in which the amino group of the base portion is protected with an acyl protecting group with a nucleophile under neutral to acidic pH conditions, and then deprotecting the protecting group of the nucleic acid, thereby removing a nucleic acid impurity (by-product (B)) whose mass is 34 daltons greater than that of the nucleic acid. The method for removing impurities according to the present invention can be carried out by performing steps B and C described above.

[0047] 3. Nucleic acids with few impurities. The nucleic acid of the present invention is a nucleic acid in which the ratio of the detected ion intensity during mass spectrometry of the nucleic acid and a nucleic acid impurity that has a mass 34 Daltons greater than the nucleic acid that may be contained in the nucleic acid is 0.50 or less per 100 units of the nucleic acid. The nucleic acid with low impurities according to the present invention can be produced using the nucleic acid production method of the present invention described above. [Examples]

[0048] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. Unless otherwise specified, the DNA in the following examples has a deoxyribose ring as the sugar moiety and a base moiety selected from adenine, thymine, guanine, and cytosine. Unless otherwise specified, the LNA(2',4'-BNA) in the examples has a structure in which the oxygen atom at the 2' position and the carbon atom at the 4' position of the ribose ring are bridged (-CH2-) as the sugar moiety, and a base moiety selected from adenine, thymine, guanine, and 5-methylcytosine. In the production example and comparative production example, a benzoyl group was used as the protecting group for the 6-position amino group of adenine, an isobutyryl group as the protecting group for the 2-position amino group of guanine, and a benzoyl group as the protecting group for the 4-position amino groups of cytosine and 5-methylcytosine.

[0049] [Manufacturing Example 1] (Consideration of nucleophile species) Using a SUS316L reaction column packed with a solid support and the OligoPilot® plus100 nucleic acid synthesizer, phosphoramidites were sequentially condensed onto nucleosides bound to the solid support via a linker, and finally, a triethylamine / acetonitrile (1:1) mixed solution was passed through to synthesize the target oligonucleotide (FLP). Next, the solid support to which the target oligonucleotide was bound was immersed in a glass test tube in 20 mL of one of the nucleophiles listed in Table 1 per 1 mmol of solid support at 20°C for 3 hours and shaken. Subsequently, the solid support was immersed in a 28 wt% aqueous ammonia / ethanol (4:1) mixed solution at 60°C for 8 hours to cleave the target oligonucleotide from the solid support and deprotect the protecting group.

[0050] After the above-mentioned excision and deprotection, the mixed solution was diluted with a water / ethanol (4:1) mixture to obtain a crude solution of the target oligonucleotide (Examples 1-5). The solid support, linker, and target oligonucleotide mentioned above are as follows. Solid-phase support, linker: Primer Support (registered trademark) 5G Unylinker 350 (Cytiva) 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 ID 1: 16mer; ^ indicates a phosphothioate bond. Each nucleoside unit with (L) next to the base has the same structure as LNA (2',4'-BNA), and other nucleoside units have the same structural units as DNA. mC indicates the base is methylcytosine.)

[0051] [Comparative Manufacturing Example 1] Using a solid support to which the target oligonucleotide synthesized in Production Example 1 was bound, the same procedure as in Production Example 1 was performed under the same conditions as in Production Example 1, except that the line "Next, the solid support to which the target oligonucleotide was bound was immersed in 20 mL of any nucleophile listed in Table 1 at 20°C for 3 hours at 20°C per 1 mmol of solid support and shaken. Then, the solid support was placed in a 28 wt% aqueous ammonia / ethanol (4:1) mixed solution" was changed to "Next, the solid support to which the target oligonucleotide was bound was placed in a 28 wt% aqueous ammonia / ethanol (4:1) mixed solution" to obtain a crude solution of the target oligonucleotide (Comparative Example 1).

[0052] [Manufacturing Example 2] (Comparative study of contact conditions) Crude solutions of the target oligonucleotides were obtained by performing the same procedure under the same conditions as in Production Example 1, except that the instruction "Immerse in 20 mL of any nucleophile listed in Table 1 per 1 mmol of solid support at 20°C for 3 hours and shake" was changed to "Immerse in water and shake under any of the contact temperature, water contact amount per solid support, and contact time conditions listed in Table 2" was changed.

[0053] [Comparative Manufacturing Example 2] Using a solid support to which the target oligonucleotide synthesized in Production Example 2 was bound, a crude solution of the target oligonucleotide was obtained by performing the same procedure under the same conditions as in Comparative Production Example 1 (Comparative Example 2).

[0054] [Manufacturing Example 3] (Consideration of contact time) Using a SUS316L reaction column packed with a solid support and an OligoPilot® plus100 nucleic acid synthesizer, phosphoramidites were sequentially condensed onto nucleosides bound to the solid support via a linker. Finally, a triethylamine / acetonitrile (1:1) mixed solution was passed through to synthesize the target oligonucleotide (FLP). After that, 960 mL of water per 1 mmol of solid support was passed through the reaction column for 4 minutes to bring the solid support into contact with water. Next, the solid support to which the target oligonucleotide was bound was immersed in a 28% by weight aqueous ammonia / ethanol (4:1) mixed solution in a glass test tube at 60°C for 19 hours to cleave the target oligonucleotide from the solid support and deprotect the protecting group.

[0055] After the above-mentioned excision and deprotection, the mixed solution was diluted with a water / ethanol (4:1) mixture to obtain a crude solution of the target oligonucleotide (Reference Example 1). The solid support, linker, and target oligonucleotide mentioned above are as follows. Solid-phase support, linker: Primer Support (registered trademark) 5G Unylinker 350 (Cytiva) Target oligonucleotide (FLP): 5'-C^T^A^G^C^A^G^A^T^G^C^T-3' (SEQ ID NO: 2: 12mer DNA, ^ indicates a phosphothioate bond)

[0056] [Comparative Manufacturing Example 3] A crude solution of the target oligonucleotide was obtained by performing the same procedure under the same conditions as in Production Example 3, except that the phrase "After synthesizing the target oligonucleotide (FLP), the solid support was brought into contact with water by passing 960 mL of water per 1 mmol of solid support through the reaction column for 4 minutes" was changed to "The target oligonucleotide (FLP) was synthesized."

[0057] [Manufacturing Example 4] (Purification of target oligonucleotides) A crude solution obtained under the same conditions and procedures as in Production Example 1 (using water as a nucleophile, Example 13) and a crude solution obtained under the same conditions and procedures as in Comparative Production Example 1 (Comparative Example 4) were prepared. Each of the above crude solutions was preparatively purified using anion exchange chromatography under the following conditions to obtain the purified oligonucleotide-containing solution (Example 13, Comparative Example 4). Preparative 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

[0058] [Test Example 1: Measurement of By-product (B) amount] For each oligonucleotide produced in Production Examples 1-4 and Comparative Production Examples 1 and 2, the crude solution (and the purified oligonucleotide-containing solution in Production Example 4) was measured by ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) under the following conditions. The amount of the target oligonucleotide (FLP) and the amount of by-product (B), which was 34 Daltons more in mass than the target oligonucleotide, were determined, and the by-product (B) content (ionic strength of by-product / ionic strength of target oligonucleotide (FLP) (%)) was calculated. Note that, as used herein, by-product (B) is common in that it is 34 Daltons more in mass than the target oligonucleotide, but it is a different substance depending on the type of target oligonucleotide. Measurement conditions: UHPLC instrument; Vanquish UHPLC system, mass spectrometer; Q Exactive, column; Waters ACQUITY UPLC Oligonucleotide BEH C18, 130 Å, 1.7 μm, 2.1 mm × 100 mm, UV detection; 260 nm, Buffer A; 100 mM HFIP, 8 mM TEA in H2O:MeCN=98:2, Buffer B; H2O:MeCN=50:50, temperature; 60 °C, ionization method; ESI, detection mode; negative, spray voltage; 2.5 kV, capillary temperature; 250 °C The results measured above are shown in Tables 1-4 below.

[0059] [Test Example 2: Measurement of the amount of target oligonucleotide (FLP)] For each sample solution of the crude oligonucleotide and the purified oligonucleotide-containing solution prepared in Production Example 4, ultra-high-performance liquid chromatography (UHPLC) measurements were performed under the following conditions, and the purity of the target oligonucleotide (FLP) (peak area of ​​target oligonucleotide (FLP) / total peak area (%)) was calculated. Measurement conditions: UHPLC instrument; ACQUITY UPLC, column; Waters ACQUITY UPLC (registered trademark) Oligonucleotide BEH C18, 130 Å, 1.7 μm, 2.1 mm × 100 mm, UV detection; 260 nm, Buffer A; 100 mM HFIP, 8 mM TEA in H2O:MeCN=98:2, Buffer B; H2O:MeCN=50:50, temperature; 60°C Furthermore, for each sample solution of the crude oligonucleotide and the purified oligonucleotide-containing solution produced in Production Example 4, the absorbance was measured using a spectrophotometer after dilution with water, and the yield (the ratio of the actually measured OD value to the theoretically obtainable maximum OD value) was calculated from the obtained OD values. The results measured above are shown in Table 4 below.

[0060] [Table 1]

[0061] [Table 2]

[0062] [Table 3]

[0063] [Table 4]

[0064] The percentage of by-product (B) / FLP listed in Tables 1-4 above represents the percentage of by-product (B) when FLP (Full Length Product: target oligonucleotide) is set to 100%.

[0065] As shown in Table 1, samples that were in contact with specific nucleophiles according to the present invention (water, methanol, ethanol, isopropyl alcohol, n-butanol) (Examples 1-5) showed a significantly lower content of by-product (B) than samples that were not in contact with nucleophiles (Comparative Example 1). In this case, in Example 2, where methanol was used as the nucleophile, it was confirmed that 3.20% (percentage relative to 100% of the target oligonucleotide) of an alkoxy byproduct was formed, in which the adenine base of the target oligonucleotide was substituted with a methoxy group. The alkoxy byproduct is considered to be separable from the target oligonucleotide by reverse-phase chromatography.

[0066] As shown in Table 2, the amount of water used in contact with water according to the present invention did not affect the content of by-product (B) (Examples 7 and 8). Furthermore, it was shown that contact with water for 0.5 hours (Example 9) resulted in a significantly lower content of by-product (B) compared to the case where there was no contact with water (Comparative Example 2). The reduction rate of by-product (B) was also greater when contacted for 0.5 hours (Example 9 in Table 2) than when contacted for only 4 minutes (Reference Example 1 in Table 3), and even greater when contacted for 2 hours (Example 10 in Table 2). These results indicate that the effect of reducing the content of by-product (B) is enhanced with increasing contact time.

[0067] Furthermore, it was shown that when the contact temperature with water was 30°C (Example 11 in Table 2), the by-product (B) decreased more rapidly than when the contact temperature was 15°C (Example 6 in Table 2) or 20°C (Example 9 in Table 2). These results indicate that the effect of reducing the content of the impurity is enhanced as the contact temperature increases. As shown in Table 4, contact with water according to the present invention was demonstrated to be more effective in reducing the content of by-product (B) than in conventional preparative purification operations. Furthermore, it was confirmed that the presence or absence of contact with water did not affect the purity or yield of FLP. [Industrial applicability]

[0068] This invention contributes to the production of high-quality nucleic acid drugs with low impurity content.

Claims

1. Step A, which involves synthesizing nucleic acids in which the amino group of the base is protected by an acyl protecting group, and the phosphate diester bond, thiophosphate ester bond, or amide phosphate ester bond is not protected by a protecting group. Step B involves contacting the nucleic acid obtained in step A with a nucleophile, which is water, a primary alcohol, and / or a secondary alcohol, under pH conditions of approximately 3 to approximately 8, and A method for producing nucleic acids, comprising step C for deprotecting the protecting group of the nucleic acid obtained in step B.

2. The method for producing a product according to claim 1, wherein the nucleophile is water, ethanol, methanol, isopropyl alcohol and / or n-butanol.

3. The manufacturing method according to claim 1 or 2, wherein the nucleophile is brought into contact with the nucleophile at a temperature of approximately 5 to approximately 80°C for approximately 0.25 hours or more.

4. A method for removing nucleic acid impurities from a target nucleic acid, wherein the amino group of the base portion is protected by an acyl protecting group, and the phosphate diester bond, thiophosphate ester bond, or amide phosphate ester bond is not protected by a protecting group, by contacting the nucleic acid with a nucleophile which is water, a primary alcohol and / or a secondary alcohol under pH conditions of about 3 to about 8, and then deprotecting the protecting group of the nucleic acid.

5. The removal method according to claim 4, wherein the nucleophile is water, ethanol, methanol, isopropyl alcohol and / or n-butanol.

6. The removal method according to claim 4 or 5, comprising contacting the nucleophile with the nucleophile at a temperature of approximately 5 to approximately 80°C for approximately 0.25 hours or more.

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