Nucleic acid production method, method for removing impurities, and nucleic acid having less 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

Method for producing nucleic acid, method for removing impurities, and nucleic acid with low impurities

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

[0002] Traditionally, the pharmacologically active ingredients of pharmaceuticals on the market have been exclusively low-molecular-weight compounds, but recently, many pharmaceuticals containing medium-molecular-weight pharmacologically active ingredients such as peptides and nucleic acids have also been marketed. In particular, nucleic acid drugs have a unique mechanism of action not seen in other drug discovery seeds, such as the ability to act specifically by forming base pairs with target mRNA, and therefore are currently a drug discovery seed with great expectations for future development.

[0003] Nucleic acids, which are raw materials for nucleic acid drugs, are generally produced by chemical synthesis. Patent Document 1 below is an example of a document that introduces such a method for producing nucleic acids. Patent Document 1 relates to a method for deprotecting oligonucleotides, and introduces a method in which a solid-phase synthesized oligonucleotide is brought into contact with an organic amine or the like to cleave the protecting group without detaching the oligonucleotide from the solid phase.

[0004] To ensure the safety of pharmaceuticals, the types and amounts of impurities are subject to strict quality control under various regulations. Impurity control is also strict in the manufacture of drug substances, and the development of technology to remove impurities generated during the manufacture of nucleic acids, which are used as drug substances, is all the more desirable.

[0005] Known methods for purifying nucleic acids by removing impurities include reversed-phase cartridge (RPC), reversed-phase high-performance liquid chromatography (RP HPLC), anion exchange chromatography (AEX), polyacrylamide gel electrophoresis (PAGE), etc. Patent Document 2 describes the purification of oligonucleotides (claim 1,

[0006] , etc.) using a mixed-mode matrix containing strong anion exchange ligands, strong cation exchange ligands, and hydrophobic ligands to remove impurities including uncomplexed oligonucleotides and failure sequences.

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

[0006] , etc.) using 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. Patent Document 4 describes the purification of oligonucleotides (claim 1,

[0003] ) using two-phase mobile phase-stationary phase liquid-liquid chromatography to remove other oligonucleotides such as shortmers and longmers.

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

[0012] , etc.) using a titratable anion exchange composition to remove impurities such as short oligonucleotides. Patent Document 6 describes the purification of oligonucleotides (claim 1, etc.) using a porous carrier to remove impurities. Non-Patent Document 1 describes the control and removal of non-oligonucleotide product-related impurities (PRIs) in the production of therapeutic oligonucleotides.

[0008] Patent No. 4705716 Publication Special Publication No. 2022-539327 Publication Special Publication No. 2019-518759 Publication Special Publication No. 2014-525254 Publication Special Publication No. 2005-520547 Publication Japanese Unexamined Patent Publication No. Hei 7-227284

[0009] Org. Process Res. Dev., 2022, 26, 1130-1144

[0010] The present inventors have discovered that in the production of nucleic acids, there is a problem in that nucleic acid (A) contains a nucleic acid (by-product (B)) that has a mass 34 daltons larger than that of the target nucleic acid (A) as an impurity. Because by-product (B) has a molecular weight and physical properties very similar to those of nucleic acid (A), it cannot be removed even using conventionally known purification methods such as those described in Patent Documents 1 to 6 and Non-Patent Document 1. Therefore, an 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. Another object of the present invention is to provide a method for removing by-product (B) and nucleic acids that contain little by-product (B).

[0011] As a result of extensive research, the present inventors have found that the content of by-product (B) can be significantly reduced by treating the synthesized nucleic acid with a nucleophilic agent in a state in which the amino group of the base moiety is protected with an acyl-based protecting group, followed by deprotection. Based on this finding, the present inventors have completed the present invention described below.

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

[0013] [5] A method for removing nucleic acid impurities 34 Daltons larger than the nucleic acid from a target nucleic acid, the method comprising contacting a nucleic acid, the amino group of which is protected with an acyl-based protecting group, with a nucleophile under neutral or acidic pH conditions, and subsequently deprotecting the protecting group of the nucleic acid. [6] The removal method according to [5], wherein the pH is 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 of [5] to [7], wherein the nucleic acid is contacted with the nucleophile at about 5 to about 80°C for about 0.25 hours or more. [9] A nucleic acid, wherein the ratio of detected ion intensity during mass spectrometry of a nucleic acid to a nucleic acid impurity that may be contained in the nucleic acid and has a mass 34 Daltons larger than the nucleic acid is 0.50 or less relative to 100 of the nucleic acid.

[0014] The method for producing nucleic acid of the present invention can reduce the content of by-product (B), which cannot be removed by conventional techniques, and produce nucleic acid with high purity.

[0015] Various matters relating to the method for producing nucleic acids of the present invention are described in detail below. However, the following description is an example for explaining the present invention, and is not intended to specifically limit the present invention to the scope of this description. The term "about" used in this specification and claims means a numerical value within a range of ±10% of the numerical value indicated by "about," preferably a numerical value within a range of ±5%, more preferably a numerical value within a range of ±2%, and particularly preferably a numerical value within a range of ±1%.

[0016] 1. Method for Producing Nucleic Acid The method for producing nucleic acid of the present invention comprises: step A of synthesizing a nucleic acid in which the amino group of the base moiety is protected with an acyl-based protecting group; step B of contacting the nucleic acid obtained in step A with a nucleophilic agent under neutral to acidic pH conditions; and step C of deprotecting the protecting group of the nucleic acid obtained in step B.

[0017] (Nucleic Acid) The nucleic acid of the present invention has a structure in which nucleosides are linked by phosphodiester bonds, thiophosphate bonds, or phosphoramidate bonds, and is preferably an oligonucleotide. The length of the oligonucleotide is not particularly limited, but is, for example, 10 to 100 bases long, particularly preferably 12 to 60 bases long. The nucleic acid of the present invention is synthesized as a single-stranded chain, with the sugar being ribose or deoxyribose and the base being selected from adenine (A), thymine (T), uracil (U), guanine (G), and cytosine (C). The nucleic acid of the present invention desirably contains a purine base such as adenine. In this specification, the unit of "base length" may be replaced with the unit of "mer" or "nucleotide."

[0018] Examples of nucleic acids in the present invention include DNA and RNA. 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. The single-stranded RNA or DNA synthesized according to the present invention can be used as an antisense, CpG oligo, or aptamer as is, but can also be used as siRNA, miRNA, decoy, or HDO (heteroduplex nucleic acid) by forming base pairs (annealing) between strands having complementary base sequences to produce double-stranded RNA or DNA.

[0019] (Modification of Nucleic Acid) The nucleic acid of the present invention may be one in which the base moiety is modified with a substituent. Examples of the substituent include a halogen group, an acyl group, an alkyl group, an arylalkyl group, an alkoxy group, a hydroxy group, an amino group, a monoalkylamino group, a dialkylamino group, a carboxy group, a cyano group, and a nitro group. Examples of the modified base include an 8-bromoadenyl group, an 8-bromoguanyl group, a 5-bromocytosyl group, a 5-bromouracil group, a 5-iodouracil group, a 5-iodocytosyl group, a 5-fluorouracil group, a 5-methylcytosyl group (mC), an 8-oxoguanyl group, and a hypoxanthinyl group.

[0020] The nucleic acids of the present invention may be modified, for example, at the 2'- or 5'-position of the sugar moiety, or may be crosslinked. Specific examples of modifications at the 2'-position include 2'-F, 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-MOE), and the like. Specific examples of modifications at the 5'-position include 5'-methyl (5'-Me), 5'-cyclopropylene (5'-CP), and the like. Specific examples of the crosslinked modifications include those in which a crosslinked 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, and the like. The nucleic acids of the present invention can be modified at several nucleotide units (e.g., 1 to 3 nucleotides) or at all nucleotide units.

[0021] (Method for Synthesizing Nucleic Acid) (1) Step A In Step A, a nucleic acid is synthesized in which the amino group in the base moiety is protected with an acyl protecting group. Examples of bases having an amino group include adenine, guanine, cytosine, and bases obtained by modifying these groups with a substituent. Examples of acyl protecting groups include a benzoyl group, an isobutyryl group, an acetyl group, a phenoxyacetyl group, an isopropylphenoxyacetyl group, and a tert-butylphenoxyacetyl group, and preferred examples include a benzoyl group, an acetyl group, or an isobutyryl group. Furthermore, in Step A, the phosphodiester bond, thiophosphate bond, or amide phosphoric acid ester bond in the nucleic acid may be protected with a protecting group. Examples of protecting groups for the phosphodiester bond, thiophosphate bond, or amide phosphoric acid ester bond include protecting groups commonly used in conventional methods, such as 2-cyanoethyl.

[0022] The method for synthesizing nucleic acid in step A is not limited, and examples thereof include solid-phase synthesis and liquid-phase synthesis. Solid-phase synthesis or liquid-phase synthesis is preferred, and solid-phase synthesis is particularly preferred. The solid-phase synthesis is described in detail below as an example.

[0023] (Solid-Phase Synthesis Method) The solid-phase synthesis method of the present invention can be carried out according to a conventional method unless otherwise specified in this specification. 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 sequentially carrying out the following steps (a) to (e): (a) Deprotection step: Removing the protecting group of the hydroxyl group at the 5' position of the nucleoside supported on the solid phase via a linker. (b) Coupling step: Using an activating agent, condensation is carried out between the phosphorus atom bound to the 3' hydroxyl group of the nucleoside phosphoramidite and the 5' hydroxyl group of the nucleoside supported on the solid phase. (c) Oxidation / Sulfurization Step: Using an oxidizing or sulfurizing agent, the internucleoside phosphite bond (including those with a protecting group attached) is converted to a phosphodiester bond (including those with a protecting group attached) or a thiophosphate bond (including those with a protecting group attached). (d) Capping Step: Using a capping agent, a protecting group is attached to the 5'-hydroxyl group of the nucleoside supported on the solid phase that was unreacted in step (b). The capping step is intended to prevent the 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) the desired number of times, the protecting group is removed from the internucleoside phosphodiester bond or thiophosphate bond without detaching 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 attached to the 2'-position), but the same applies when a ribose ring (with a hydroxyl group at the 2'-position) or a modified ribose ring (for example, one in which the 2'-position is O-methoxyethylated (2'-MOE), O-methylated (2'-OMe), or fluorinated (2'-F)) is used.

[0025] Step (a): [In the formula, DMTr represents a 4,4'-dimethoxytrityl group. ＰＧThe symbols independently represent adenine, thymine, uracil, guanine, cytosine, or modified versions thereof, and the amino group in the base moiety is protected with an acyl-based protecting group. The symbol ● represents a solid-phase support bound 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, a 4-methoxytrityl group can also be used as a protecting group for the 5'-hydroxyl group of solid-phase-supported nucleosides, nucleotide chains, nucleoside phosphoramidites, and the like.

[0026] Step (b):

[0027] [In the formula, DMTr, Base ＰＧ , and the ● portion has the same meaning as defined above.] Examples of the activating agent 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] Step (c):

[0029] [In the formula, DMTr, Base ＰＧThe ● portion has the same meaning as defined above. X represents an oxygen atom or a sulfur atom.] Examples of the oxidizing agent in step (c) include metachloroperbenzoic acid, metaperiodate, hydrogen peroxide, iodine, (1S)-(+)-(10-camphorsulfonyl)oxaziridine, and examples of the sulfurizing agent 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), 3-amino-1,2,4-dithiazole-5-thione (ADTT), and the like. Examples of the reaction solvent in step (c) include dichloromethane, acetonitrile, pyridine, 3-picoline, water, tetrahydrofuran, and any combination of these solvents.

[0030] Step (d):

[0031] [In the formula, DMTr, Base ＰＧ The ● portion has the same meaning as defined above.] Examples of the capping agent used in step (d) include acetic anhydride, phenoxyacetic anhydride, etc. Examples of the reaction solvent used in step (d) include acetonitrile, pyridine, 2,6-lutidine, tetrahydrofuran, or a mixture of these in any combination.

[0032] Step (e):

[0033] [In the formula, Base ＰＧ , ●, and X are as defined above. R represents a 4,4'-dimethoxytrityl group or a hydrogen atom. n represents an integer of 0 or greater.] Examples of the cyanoethyl group-removing agent in step (e) include triethylamine, diethylamine, and 1,8-diazabicyclo[5.4.0]-7-undecene. The cyanoethyl group-removing agent can also be used after diluting with, for example, acetonitrile, toluene, or the like.

[0034] (2) Step B In step B, the nucleic acid obtained in step A is contacted with a nucleophilic agent 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 highly pure nucleic acid.

[0035] The nucleophile is not particularly limited, but examples thereof include water, alcohol, thiol, amine, halide ion, cyanide ion, etc. Specific 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. Preferred examples include water, alcohol, and thiol, more preferably water and / or alcohol, even more preferably water, primary alcohol, and secondary alcohol, such as water, methanol, ethanol, isopropyl alcohol, and n-butyl alcohol. From the viewpoint of availability and ease of handling, water, methanol, and ethanol are even more preferred, and water is most preferred. A 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 less) pH conditions, preferably a pH of about 3-8, and more preferably a pH of about 4-7. The amount of contact between the nucleophile and the synthesized product in step B is not particularly limited, but is preferably 1 mL to 1000 mL, more preferably 5 mL to 100 mL, and particularly preferably 10 mL to 50 mL per 1 mmol of nucleic acid, from the viewpoint of production efficiency and the like. The longer the contact time between the nucleophile and the synthesized product, the more enhanced the effects of the present invention tend to be; for example, it is 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 from the viewpoint of production efficiency and the like, it is desirably within 24 hours. The temperature during contact between the nucleophile and the synthesized product in step B is not particularly limited, but is, for example, a temperature within 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 and the like.

[0037] The material of the liquid-contacting surface of the container used when contacting with the nucleophile is not particularly limited, but examples thereof 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, more preferably selected from glass, acrylic resin, polyethylene, polypropylene, PTFE, and PFA. By carrying out the contacting operation with the nucleophile in a non-metallic container, even if the target oligonucleotide has a thiophosphate, it is possible to suppress the generation of desulfurized impurities (those in which the thiophosphate ester bond of the target oligonucleotide has been changed to a phosphate diester bond) during the cleavage and deprotection steps due to the elution of metal ions from the reaction container. The contact with the nucleophile in step B can be carried out in the reaction column used for synthesis, or in a separate container transferred after removal from the reaction column.

[0038] (3) Step C In step C, the protecting groups of the nucleic acid obtained in step B are deprotected. When the method for synthesizing nucleic acid in step A is a solid-phase synthesis method, step C (the step of deprotecting the protecting groups) can simultaneously be a step of excising the nucleic acid from the solid-phase support. Following the above steps (a) to (e) and step B of step A, the following step (f) can be carried out as step C. (f) Excision / deprotection step: The nucleic acid of the desired chain length produced in steps (e) and B is excised from the solid-phase support using an excision / deprotection agent, and protecting groups such as those at the base moiety and the 2'-position hydroxyl group are removed.

[0039] Step (f):

[0040] [In the formula, Base ＰＧ , ●, and X are as defined above. Base independently represents adenine, thymine, uracil, guanine, cytosine from which the protecting group has been removed, or a modified version of any of these. R represents a 4,4'-dimethoxytrityl group or a hydrogen atom. n represents an integer of 0 or greater.]

[0041] (Reaction Solution in the Cleavage / Deprotection Step) Examples of the cleavage / deprotection agent include basic substances such as concentrated aqueous ammonia, methylamine, and sodium hydroxide. The ammonia concentration of concentrated aqueous ammonia 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 1,000 mL per 1 mmol of nucleic acid supported on the solid phase carrier, more preferably 50 mL to 500 mL per 1 mmol of nucleic acid. The cleavage / deprotection agent can also be diluted with, for example, water, methanol, ethanol, isopropyl alcohol, 1-propyl alcohol, 1-butanol, 2-butanol, acetonitrile, tetrahydrofuran, 1,2-dimethoxyethane, dimethyl sulfoxide, or the like, and is preferably diluted with water, ethanol, or isopropyl alcohol. The reaction solution in the cleavage / deprotection step is preferably basic, more preferably having a pH of 10 to 14.

[0042] The reaction temperature in step (f) is, for example, within the range of 5 to 75°C, preferably within 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 nucleic acid is cleaved from the solid support using a cleavage / deprotection agent, and the protecting groups bound to the nucleic acid are removed. Steps (a) to (f) of the above solid-phase synthesis method can be performed using a commercially available automated nucleic acid synthesizer or the like. Furthermore, according to the above steps, nucleic acid is produced so that the nucleotide chain elongates from the 3' carbon atom to the 5' carbon atom.

[0043] (4) Separation and Purification Step The produced nucleic acid can be purified by commonly used separation and purification means, such as reverse phase chromatography, reverse phase ion pair chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, etc. Furthermore, salts, which are impurities contained in the solution, can be removed by ultrafiltration or the like, and the oligonucleic acid solution can be powdered by freeze-drying.

[0044] (By-product (B)) The nucleic acid production method of the present invention reduces the content of by-product (B), which cannot be removed by conventional techniques, and thereby enables the production of highly pure nucleic acids. Specifically, the detected ion intensity ratio of a nucleic acid impurity (by-product (B)) with a mass 34 daltons larger than that of the target nucleic acid (A) in mass spectrometry is 0.50 or less, preferably 0.10 or less, relative to 100 of nucleic acid (A). The inventors of the present invention believe that by-product (B) is generated during the synthesis procedure, primarily in step (a), by mutation of the adenine base contained in the sequence, resulting in a higher polarity. Furthermore, by-product (B) and the target nucleic acid (A) have similar physical properties, such as molecular weight and polarity, making their 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 prior to the separation and purification step.

[0045] The quality of the produced nucleic acid can be analyzed using a combination of commonly used nucleic acid analytical techniques, such as a separation technique such as reverse-phase chromatography, reverse-phase ion-pair chromatography, or ion-exchange chromatography, combined with a UV detector and a mass spectrometer. The amount of by-products (impurities) in the nucleic acid can be calculated from the area ratio of the detected peaks when a UV detector is used, or from the ion intensity ratio when a mass spectrometer is used. The ion intensity ratio can be calculated by deconvoluting the detected multivalent ion group of the nucleic acid to the mass of the molecule (neutral species) or the m / z of the monovalent ion (a dimensionless quantity obtained by dividing the relative mass obtained by dividing the mass of the ion in unified atomic mass units by the charge number of the ion), or by extracting and calculating the signal intensity of a specific multivalent ion.

[0046] 2. Method for Removing Impurities The method for removing impurities of the present invention is a method for removing a nucleic acid impurity (by-product (B)) having a mass 34 daltons larger than that of a target nucleic acid, from the target nucleic acid by contacting a nucleic acid in which the amino group of the base moiety is protected with an acyl-based protecting group with a nucleophile under neutral to acidic pH conditions, and then deprotecting the protecting group of the nucleic acid. The method for removing impurities of the present invention can be carried out by carrying out the above-mentioned steps B and C.

[0047] 3. Nucleic Acid with Few Impurities The nucleic acid with few impurities of the present invention is a nucleic acid in which the ratio of detected ion intensity in mass spectrometry between a nucleic acid and a nucleic acid impurity that may be contained in the nucleic acid and has a mass 34 daltons larger than that of the nucleic acid is 0.50 or less relative to 100 of the nucleic acid. 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.

[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 sugar moiety of the DNA in the following examples is a deoxyribose ring, and the base moiety is selected from adenine, thymine, guanine, and cytosine. Unless otherwise specified, the sugar moiety of LNA (2',4'-BNA) in the examples is a ribose ring in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged (-CH２ -) structure, and the base moiety is selected from adenine, thymine, guanine, and 5-methylcytosine. In the Production Examples and Comparative Production Examples, a benzoyl group was used as the protecting group for the 6-amino group of adenine, an isobutyryl group was used as the protecting group for the 2-amino group of guanine, and a benzoyl group was used as the protecting group for the 4-amino group of cytosine and 5-methylcytosine.

[0049] [Production Example 1] (Study on Nucleophile Type) Using a SUS316L reaction column packed with a solid support and an OligoPilot® plus 100 nucleic acid synthesizer, phosphoramidites were sequentially condensed with nucleosides linked to the solid support via linkers, 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 20 mL of one of the nucleophiles listed in Table 1 per 1 mmol of solid support in a glass test tube and shaken at 20°C for 3 hours. The solid support was then 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 groups.

[0050] After the cleavage and 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). The solid support, linker, and target oligonucleotide mentioned above were as follows: Solid support, linker: Primer Support (registered trademark) 5G Unilinker 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' (SEQ ID NO: 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 unit as DNA. mC indicates that the base is methylcytosine.)

[0051] Comparative Production Example 1 Using the solid phase carrier bound to the target oligonucleotide synthesized in Production Example 1, the same procedure as in Production Example 1 was carried out under the same conditions as in Production Example 1, except that the procedure was changed from "The solid phase carrier bound to the target oligonucleotide was then immersed in 20 mL of any of the nucleophiles listed in Table 1 per 1 mmol of the amount carried on the solid phase carrier in a glass test tube at 20°C for 3 hours and shaken. The solid phase carrier was then immersed in a 28 wt% aqueous ammonia / ethanol (4:1) mixed solution" to "The solid phase carrier bound to the target oligonucleotide was then immersed in a 28 wt% aqueous ammonia / ethanol (4:1) mixed solution," to obtain a crude solution of the target oligonucleotide (Comparative Example 1).

[0052] [Preparation Example 2] (Comparison of Contact Conditions) A crude solution of the target oligonucleotide was obtained by carrying out the same operation and conditions as in Preparation Example 1, except that "the solid phase carrier was immersed in 20 mL of any of the nucleophiles listed in Table 1 per 1 mmol of the amount carried on the solid phase carrier and the mixture was shaken at 20°C for 3 hours." was changed to "the solid phase carrier was immersed in water and shaken under the conditions of any of the contact temperatures, water contact amounts per amount carried on the solid phase carrier, and contact times listed in Table 2." (Examples 6 to 12).

[0053] Comparative Production Example 2 A crude solution of the target oligonucleotide was obtained by carrying out the same operations under the same conditions as in Comparative Production Example 1 using the solid phase support to which the target oligonucleotide synthesized in Production Example 2 was bound (Comparative Example 2).

[0054] [Production Example 3] (Contact Time Study) Using a SUS316L reaction column packed with a solid phase carrier and an OligoPilot (registered trademark) plus 100 nucleic acid synthesizer, phosphoramidites were sequentially condensed with nucleosides linked to the solid phase carrier via linkers, and finally, a triethylamine / acetonitrile (1:1) mixed solution was passed through to synthesize the target oligonucleotide (FLP). Then, 960 mL of water per 1 mmol of solid phase carrier loaded was passed through the reaction column for 4 minutes to contact the solid phase carrier with water. Next, the solid phase carrier to which the target oligonucleotide was bound was immersed in a 28 wt % 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 phase carrier and deprotect the protecting group.

[0055] After the cleavage and 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). The solid phase support, linker, and target oligonucleotide are as follows: Solid phase support, linker: Primer Support (registered trademark) 5G Unilinker 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 Production Example 3 The same operations and conditions as in Production Example 3 were carried out, except that "After synthesizing the target oligonucleotide (FLP), 960 mL of water per 1 mmol of the amount carried on the solid phase carrier was passed through the reaction column over 4 minutes to bring the solid phase carrier into contact with water." was changed to "The target oligonucleotide (FLP) was synthesized." A crude solution of the target oligonucleotide was obtained (Comparative Example 3).

[0057] [Production Example 4] (Purification of target oligonucleotide) A crude liquid (using water as a nucleophile, Example 13) obtained under the same conditions and procedures as in Production Example 1 and a crude liquid (Comparative Example 4) obtained under the same conditions and procedures as in Comparative Production Example 1 were prepared. Each of the crude liquids was purified by anion exchange chromatography under the following conditions to obtain a purified oligonucleotide-containing liquid (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 aqueous sodium hydroxide / methanol (85:15) mixed solution, mobile phase B: 10 mM aqueous sodium hydroxide / 2 M aqueous sodium chloride / methanol (85:15) mixed solution

[0058] Test Example 1: Measurement of the amount of by-product (B) The crude solutions of each oligonucleotide produced in Production Examples 1 to 4 and Comparative Production Examples 1 and 2 (and the purified oligonucleotide-containing solution in Production Example 4) were measured by ultra-high performance liquid chromatography mass spectrometry (UHPLC-MS) under the conditions below, and the amount of the target oligonucleotide (FLP) and the amount of by-product (B), which had a mass 34 Daltons greater than that of the target oligonucleotide, were determined, and the content of by-product (B) (ionic strength of by-product / ionic strength (%) of target oligonucleotide (FLP)) was calculated. Note that, although the by-product (B) in this specification has in common the fact that it has a mass 34 Daltons greater than that of the target oligonucleotide, it is a different substance depending on the type of target oligonucleotide. Measurement conditions: UHPLC apparatus: 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 H ２ O:MeCN=98:2, BufferB;H ２ O:MeCN=50:50, temperature: 60°C, ionization method: ESI, detection mode: negative, spray voltage: 2.5|kV|, capillary temperature: 250°C. The results of the above measurements are shown in Tables 1 to 4 below.

[0059] Test Example 2: Measurement of the amount of target oligonucleotide (FLP) Each sample solution of the crude solution of the oligonucleotide produced in Production Example 4 and the purified oligonucleotide-containing solution was measured by ultra-high performance liquid chromatography (UHPLC) under the following conditions, and the purity of the target oligonucleotide (FLP) (peak area of ​​target oligonucleotide (FLP) / total area of ​​all peaks (%)) was calculated. Measurement conditions: UHPLC apparatus: 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 H２ O:MeCN=98:2, BufferB;H ２ O:MeCN=50:50, temperature: 60°C. Furthermore, each sample solution of the crude oligonucleotide solution and the purified oligonucleotide-containing solution prepared in Preparation Example 4 was diluted with water and then subjected to spectrophotometric measurement of absorbance, and the yield (the ratio of the actually measured OD value to the theoretically achievable maximum OD value) was calculated from the obtained OD value. The results of the above measurements are shown in Table 4 below.

[0060]

[0061]

[0062]

[0063]

[0064] The amount of by-product (B) / amount of FLP (%) shown in Tables 1 to 4 above is the ratio (%) of by-product (B) when FLP (Full Length Product: target oligonucleotide) is taken as 100 (%).

[0065] As shown in Table 1, the samples (Examples 1 to 5) contacted with specific nucleophiles according to the present invention (water, methanol, ethanol, isopropyl alcohol, n-butanol) showed a significantly lower content of by-product (B) than the sample not contacted with a nucleophile (Comparative Example 1). In this case, in the case of Example 2, in which the nucleophile was methanol, it was confirmed that 3.20% (proportion when the target oligonucleotide is taken as 100%) of alkoxy by-products in which the adenine bases of the target oligonucleotide were substituted with methoxy groups were produced. It is believed that the alkoxy by-products can be separated from the target oligonucleotide by reverse-phase chromatography.

[0066] As shown in Table 2, in the case of contact with water according to the present invention, it was shown that the amount of water used for contact does not affect the content of by-product (B) (Examples 7 and 8). It was also shown that contact with water for 0.5 hours (Example 9) significantly reduced the content of by-product (B) compared to no contact with water (Comparative Example 2). It was also shown that the reduction rate of by-product (B) was greater when the sample was contacted with water for 0.5 hours (Example 9 in Table 2) than when the sample was contacted with water for only 4 minutes (Reference Example 1 in Table 3), and even greater when the sample was contacted for 2 hours (Example 10 in Table 2). These results demonstrate that the effect of reducing the content of by-product (B) is enhanced with an increase in 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). The above results demonstrate that the effect of reducing the content of the impurity increases with increasing contact temperature. As shown in Table 4, it was demonstrated that contact with water according to the present invention is more effective in reducing the content of by-product (B) than conventional preparative separation and purification procedures. It was also confirmed that the presence or absence of contact with water does not affect the FLP purity or yield.

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

Claims

1. Step A is for synthesizing a nucleic acid in which the amino group of the base portion is protected with an acyl-based protecting group; Step B, in which the nucleic acid obtained in step A is contacted with a nucleophile under neutral or acidic pH conditions; and A method for producing a nucleic acid, comprising step C of deprotecting the protecting group of the nucleic acid obtained in step B.

2. The process of claim 1, wherein the pH is from about 3 to about 8.

3. The process according to claim 1 or 2, wherein the nucleophile is water and / or an alcohol.

4. The process according to claim 1 or 2, wherein the contact with the nucleophile is carried out at about 5 to about 80° C. for about 0.25 hours or more.

5. A method for removing nucleic acid impurities having a mass 34 daltons larger than that of a target nucleic acid by contacting a nucleic acid in which the amino group of the base portion 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 method of claim 5, wherein the pH is from about 3 to about 8.

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

8. The method according to claim 5 or 6, wherein the contact with the nucleophile is carried out at about 5 to about 80° C. for about 0.25 hours or more.

9. A nucleic acid, in which the ratio of detected ion intensity during mass spectrometry between a nucleic acid and a nucleic acid impurity having a mass 34 daltons larger than that of the nucleic acid, which may be contained in the nucleic acid, is 0.50 or less relative to 100 of the nucleic acid.