A method for purifying an oligonucleotide

Abstract

The application discloses a purification method of oligonucleotide, which comprises the following steps: loading the crude oligonucleotide into an anion exchange column which is equilibrated with a phosphate buffer solution, washing the anion exchange column with the mixture of the phosphate buffer solution and the phosphate buffer solution containing alkali metal halide after equilibrating the anion exchange column with the phosphate buffer solution, equilibrating the anion exchange column with the phosphate buffer solution again, then washing the anion exchange column with an acidic aqueous solution to remove the hydroxyl protecting group of the terminal nucleoside of the oligonucleotide, and equilibrating the anion exchange column with the phosphate buffer solution, finally eluting the target oligonucleotide with the phosphate buffer solution containing alkali metal halide, and collecting the eluent containing the target oligonucleotide. The purification method of the application can efficiently and stably obtain high-purity oligonucleotide without using organic solvents, and is suitable for the purification of oligonucleotide in large-scale commercial process.

Description

Technical Field

[0001] This invention relates to a method for purifying oligonucleotides. Background Technology

[0002] Oligonucleotides have a wide range of biological and biochemical applications. For example, they can be used in drug discovery and development, in diagnostic testing, as research agents (such as primers or probes in polymerase chain reaction (PCR), as antisense agents in target validation, as competitive inhibitors of transcription factors, as ribozymes, as aptamers, as stimulants of the immune system, or as biotherapeutic agents for treating diseases. Due to the significant progress made in oligonucleotide drug research in recent years, an increasing number of oligonucleotide drugs are entering clinical trials. Therefore, rapid, inexpensive, and effective methods for developing GMP-quality oligonucleotides are becoming increasingly important.

[0003] Nusinersen sodium is an 18-base single-chain phosphate-thioester oligonucleotide, an antisense oligonucleotide (ASO) drug used to treat spinal muscular atrophy (SMA, a rare neuromuscular disease). It contains an 18-base oligonucleotide with the sequence 5'-TCA CTT TCA TAA TGC TGG-3', with each nucleoside having a MOE (ethoxyethyl) substitution at the 2' position and a methyl group introduced at the 5' position of all cytosine bases. The thiophosphate esterification process involves replacing non-bonded oxygen atoms with sulfur atoms in the internucleotide bonds.

[0004] Currently, commonly used oligonucleotide separation techniques include using reversed-phase high-performance liquid chromatography (RP-HPLC) to purify oligonucleotides (e.g., Krotz et al., Organic Process Research & Development 2003, 7, 47-52). This method mainly involves the following steps: reversed-phase purification, concentration and desalting, removal of DMT (4,4'-dimethoxytriphenylmethyl), and further desalting. This method requires a significant amount of operating time, a large amount of organic reagents as the mobile phase, has high site requirements, and generates a large amount of organic waste liquid. Furthermore, it is not effective in removing common impurities, such as impurities with missing nucleotides in the oligomer (called nx impurities, with one missing nucleotide referred to as n-1), impurities with phosphodiester bonds instead of the desired thiophosphate bonds (called P=O impurities), and impurities with excess nucleotides in the oligomer (called n+x impurities, with one excess nucleotide referred to as n+1), and the yield is low. To reduce the number of purification steps and thus operation time, and also to achieve a higher overall yield, CellGene Corporation disclosed in prior art CN 111148519 a method for loading crude oligonucleotides containing cleaved 5'-hydroxyl groups of terminal nucleosides protected by a 4,4'-dimethoxytriphenylmethyl (DMT) protecting group onto an anion exchange column (Q Sepharose FF column medium). After elution and on-column DMT removal, the oligonucleotides were washed with a mixture of 25 mM sodium hydroxide solution and 2 M sodium chloride solution to obtain oligonucleotides of the target purity. While this method significantly reduces operation time, the use of high pH buffers may lead to oligonucleotide degradation, producing byproducts such as P=O impurities, n-1 impurities, and C hydrolysis to U. Hofmeister AG disclosed in prior art CN114051499 a method using Source 30Q as the anion exchange column medium and a mixture of 25mM sodium phosphate buffer, 0.5-2M sodium chloride solution, and 10% acetonitrile as the eluent to obtain oligonucleotides of the target purity. While this method solves the potential instability issue associated with using high-pH buffers for ion exchange purification of oligonucleotides, the addition of 10% acetonitrile to the mobile phase significantly increases the amount of waste requiring treatment and places higher demands on the plant.

[0005] Therefore, there is a need to develop oligonucleotide purification methods that can effectively remove impurities from oligonucleotides, and that are efficient, stable, environmentally friendly, and suitable for large-scale commercial processes. Summary of the Invention

[0006] The purpose of this invention is to provide a method for purifying oligonucleotides, the purification method comprising the following steps:

[0007] (a) Loading crude oligonucleotides onto an anion exchange column equilibrated with phosphate buffer, wherein the 5'-hydroxyl group of the terminal nucleoside of the oligonucleotides in the crude oligonucleotides has a hydroxyl protecting group;

[0008] (b) Equilibrate the anion exchange column with phosphate buffer solution, and then wash the anion exchange column with a mixture of phosphate buffer solution and phosphate buffer solution containing alkali metal halide;

[0009] (c) Equilibrate the anion exchange column with phosphate buffer solution, and then wash the anion exchange column with acidic aqueous solution to remove the 5'-hydroxy protecting group of the terminal nucleoside of the oligonucleotide;

[0010] (d) Equilibrate the anion exchange column with phosphate buffer solution, then elute the target oligonucleotide that has removed the 5'-hydroxy protecting group with phosphate buffer solution containing alkali metal halide, and collect the eluent containing the target oligonucleotide.

[0011] In another preferred embodiment, the nucleotide sequence of the oligonucleotide is shown in SEQ ID No. 1.

[0012] In another preferred embodiment, the crude oligonucleotide contains invalid sequence (nx) impurities, co-eluted n-1 impurities, P=O impurities, and n+1 impurities, and wherein the UV purity of the oligonucleotide is greater than or equal to 70%.

[0013] In another preferred embodiment, the crude oligonucleotide is loaded onto an anion exchange column in solution form, wherein the solvent of the solution is an ammonia solution with a concentration of less than 13% or an ammonia solution with a volume content of less than 10% ethanol.

[0014] In another preferred embodiment, the 5'-hydroxy protecting group of the terminal nucleoside of the oligonucleotide is 4,4'-dimethoxytriphenylmethyl.

[0015] In another preferred embodiment, the resin of the anion exchange column is a strong basic anion exchange resin containing quaternary ammonium groups, with a particle size greater than or equal to 15 μm, more preferably 30 μm.

[0016] In another preferred embodiment, in steps (a), (b) and (c), the phosphate buffer used to equilibrate the anion exchange column is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or mixtures thereof.

[0017] In another preferred embodiment, in steps (a), (b) and (c), the phosphate buffer used to equilibrate the anion exchange column has a phosphate content of 10 mM to 100 mM and a pH of 6.5 to 8.5. More preferably, the phosphate buffer has a phosphate content of 15 mM to 50 mM and a pH of 7.0 to 8.0.

[0018] In another preferred embodiment, in steps (b) and (d), the phosphate in the alkali metal halide-containing phosphate buffer solution is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or mixtures thereof.

[0019] In another preferred embodiment, in steps (b) and (d), the alkali metal halide in the phosphate buffer solution containing the alkali metal halide is selected from sodium chloride or sodium bromide, preferably sodium chloride.

[0020] In another preferred embodiment, in steps (b) and (d), the phosphate content in the alkali metal halide-containing phosphate buffer solution is 10 mM to 100 mM, more preferably 15 mM to 50 mM, and the pH of the alkali metal halide-containing phosphate buffer solution is 6.5 to 8.5, more preferably 7.0 to 8.0, and / or

[0021] In another preferred embodiment, in steps (b) and (d), the mass concentration of the alkali metal halide in the phosphate buffer solution containing the alkali metal halide is 1.0 M to 3.0 M, more preferably 1.0 M to 2.5 M.

[0022] In another preferred embodiment, in step (b), the volume ratio of the phosphate buffer solution to the phosphate buffer solution containing the alkali metal halide in the mixture is 6:1 to 2:1.

[0023] In another preferred embodiment, in step (c), the acid in the acidic aqueous solution is selected from hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, haloacetic acid, and preferably acetic acid.

[0024] In another preferred embodiment, the haloacetic acid is selected from trichloroacetic acid, trifluoroacetic acid, or dichloroacetic acid.

[0025] In another preferred embodiment, in step (c), the volume concentration of acid in the acidic aqueous solution is 50% to 95%, more preferably 70% to 90%.

[0026] In another preferred embodiment, in step (b), the amount of the mixture of phosphate buffer solution and phosphate buffer solution containing alkali metal halide is 5 to 12 times the column volume.

[0027] In another preferred embodiment, in step (c), the flow rate of the acidic aqueous solution during the washing of the anion exchange column is 100 cm / h to 400 cm / h, and the amount used is 7 to 22 times the column volume (CV).

[0028] In another preferred embodiment, in step (d), the amount of the alkali metal halide phosphate buffer is 20 to 50 times, more preferably 25 to 45 times, the column volume.

[0029] In steps (a), (b), (c) and (d), the amount of phosphate buffer solution used to equilibrate the anion exchange column is 1 to 15 times the column volume, more preferably 3 to 10 times.

[0030] In another preferred embodiment, the purification method further includes desalting and freeze-drying. Attached Figure Description

[0031] Figure 1 This is the UPLC chromatogram of the oligonucleotide shown in SEQ ID NO.1 obtained in Example 1 of this invention. The chromatogram shows that the oligonucleotide shown in SEQ ID NO.1 prepared using the purification method of this invention has high chromatographic purity.

[0032] Figure 2 This is the MS spectrum of the oligonucleotide shown in SEQ ID NO.1 obtained in Example 1 of this invention. The spectrum shows that the oligonucleotide shown in SEQ ID NO.1 prepared using the purification method of this invention has high mass spectrometry purity. Detailed implementation method:

[0033] In existing ion exchange analysis and purification techniques for oligonucleotides, elution solvents with a certain proportion of organic solvent or high pH values ​​are typically used to make the oligonucleotides more open (by disrupting hydrogen bonds or secondary structures between oligonucleotides), thus making it easier to remove impurities. However, the use of organic solvents generates a large amount of organic solvent waste liquid, and the use of high pH values ​​(pH 10-12) may lead to oligonucleotide degradation, producing byproducts such as P=O impurities, n-1 impurities, and C hydrolysis into U (Ullman, JS; McCarthy, BJ Biochim. Biophys. Acta 1973, 294, 396-494; Germann, MW; Pon, RT; van de Sande, J Hanal. Biochem. 1987, 165, 399-405). The inventors of this application have discovered through extensive research that using a mild sodium phosphate buffer (pH 6.5-8.5) is more effective. Column purification of oligonucleotides (nucleotide sequences as shown in SEQ ID NO.1) can yield high-purity oligonucleotide products without using large amounts of organic solvents and without impurities that may be generated in a high pH environment.

[0034] The method for purifying oligonucleotides of the present invention includes the following steps:

[0035] (a) Loading a solution of crude oligonucleotides onto an anion exchange column equilibrated with phosphate buffer, wherein the 5'-hydroxyl group of the terminal nucleoside of the oligonucleotides in the crude oligonucleotides has a hydroxyl protecting group;

[0036] (b) Equilibrate the anion exchange column with phosphate buffer, and then wash the anion exchange column with a mixture of phosphate buffer and phosphate buffer containing alkali metal halide; (c) Equilibrate the anion exchange column with phosphate buffer, and then wash the anion exchange column with acidic aqueous solution to remove the 5'-hydroxy protecting group of the terminal nucleoside of the oligonucleotide; (d) Equilibrate the anion exchange column with phosphate buffer, and then elute the target oligonucleotide with the 5'-hydroxy protecting group removed with phosphate buffer containing alkali metal halide, and collect the eluent containing the target oligonucleotide.

[0037] The purification method of this invention uses commercially available Cytiva FineLINE series purification columns or Hanbang Technology dynamic axial preparative compression columns, employing conventional, commercially available anion exchange resins, which can be obtained from Cytiva, Tosoh Bioscience, Bio-Rad, Merck, or Suzhou Nanomicro Technology. Typical resins are Cytiva's Source 30Q resin or Suzhou Nanomicro Technology's NanoQ-30L resin.

[0038] In this invention, the sequence shown in SEQ ID NO.1 is 5'-TCACTTTCATAATGCTGG-3', wherein the 5'-hydroxyl group of the terminal nucleoside has a DMT protecting group, the 2' position of each nucleoside is substituted with MOE (ethoxyethyl), a methyl group is introduced at the 5' position of all cytosine bases, and the non-bonded oxygen in each internucleotide bond is replaced with a sulfur atom. This sequence can also be represented as '5'-DMT-Ts Me CsAs Me CsTsTsTs Me CsAsTsAsAsTsGs Me CsTsGsG-3'.

[0039] In some embodiments, crude oligonucleotides loaded onto an anion exchange column are loaded in the form of an ammonia solution. The ammonia solution is obtained by directly concentrating an oligonucleotide-containing solution obtained through cleavage and deprotection (using 25–28% concentrated ammonia as a solvent), with a pH of approximately 7.5–10.5. The oligonucleotide content in the crude oligonucleotide solution is 1.0–5.0% by mass, for example, 1.5–4.5%, 2.0–4.0%. In some embodiments, the total oligonucleotide content in the solution of crude oligonucleotides loaded onto the column is 500–1000 OD / mL, preferably 600–900 OD / mL (e.g., detected by a wavelength of 260 nm).

[0040] In some embodiments, the crude oligonucleotides loaded onto the anion exchange column contain impurities including invalid sequence (nx) impurities, co-eluted n-1 impurities, P=O impurities, and n+1 impurities. In some embodiments, the UV purity (e.g., detected by a 260 nm wavelength) of the crude oligonucleotides loaded onto the anion exchange column is greater than or equal to 70%, for example, 70–93%, 85–90%, or 70–85%.

[0041] The phosphate used to prepare the buffer solution of the present invention may be selected from alkali metal phosphates or mixtures of alkali metal phosphates, such as sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or mixtures thereof. In some embodiments, the pH of the buffer solution used in steps (a), (b), (c), and (d) of the equilibrium ion exchange column is preferably 6.5 to 8.5, more preferably 7.0 to 8.0, wherein the total content of phosphates (sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate) is preferably 10 mM to 100 mM, more preferably 15 mM to 50 mM. In some embodiments, the volume of the buffer solution used in steps (a), (b), (c), and (d) of the equilibrium ion exchange column is preferably 1 to 15 times the column volume (CV), more preferably 3 to 10 times the column volume (CV).

[0042] In some embodiments, the pH of the phosphate buffer solution containing alkali metal halides in steps (b) and (d) is preferably 6.5–8.5, more preferably 7.0–8.0, wherein the total content of phosphates (sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate) is preferably 10 mM–100 mM, more preferably 15 mM–50 mM. In some embodiments, the alkali metal halide in the phosphate buffer solution containing alkali metal halides in steps (b) and (d) is sodium chloride. The concentration of sodium chloride in the phosphate buffer solution is preferably 1.0 M–3.0 M, more preferably 1.0 M–2.5 M.

[0043] The purpose of washing the anion exchange column with a mixture of phosphate buffer and phosphate buffer containing alkali metal halide in step (b) is to remove oligonucleotides without protecting groups, such as invalid sequence (nx) impurities.

[0044] The acid used in step (c) of this invention can be an inorganic acid or an organic acid, such as hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, or haloacetic acid (e.g., trichloroacetic acid, trifluoroacetic acid, dichloroacetic acid, etc.). In some embodiments, the acidic aqueous solution is an aqueous solution of acetic acid, preferably with a volume concentration of 50% to 95%, more preferably 70% to 90%.

[0045] Steps (a) to (d) of the oligonucleotide purification method of the present invention can all be carried out at room temperature. "Room temperature" in this document refers to 4°C to 40°C, for example, 15°C to 35°C, 20°C to 30°C, or 20°C to 25°C.

[0046] In some embodiments, the eluent containing the target oligonucleotide collected in step (d) is desalted by tangential flow filtration and then lyophilized to obtain a solid oligonucleotide. Tangential flow filtration and lyophilization can be performed in a manner conventional in the art.

[0047] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0048] The oligonucleotide purification method of this invention does not use elution solvents containing added organic solvents or high-pH elution solvents; high purity can still be obtained by using only mild sodium phosphate buffer (pH 6.5–8.5). Oligonucleotides. They have less environmental impact, lower cost, and are more suitable for industrial production.

[0049] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0050] Example 1

[0051] abbreviation:

[0052] MOE = 2-Methoxyethyl

[0053] NMI = N-methylimidazolium

[0054] PADS = Phenylacetyl disulfide

[0055] Tol = Toluene

[0056] A = 2'-MOE adenosine

[0057] T = 2'-MOE thymidine

[0058] Me C=2'-MOE 5-methylcytidine

[0059] G=2'-MOE guanosine

[0060] DCA = dichloroacetic acid

[0061] DCI = 4,5-Dicyanoimidazole

[0062] DEA = Diethylamine

[0063] ACN = Acetonitrile

[0064] Ac₂O = Acetic anhydride

[0065] CV = Column Volume

[0066] (1.1)5'-DMT-Ts Me CsAs Me CsTsTsTs Me CsAsTsAsAsTsGs Me Synthesis method of CsTsGsG-3'

[0067] target compound used oligopilot plus 100 was synthesized at a scale of 2 mmol as “DMT-on”.

[0068] in" s "Represents a thiophosphate bridging bond."

[0069] Using standard phosphorus amide chemistry method, Oligonucleotides were produced on the solid phase at a scale of 2.0 mmol using oligopilot plus 100 and PrimerSupport Unylinker (NittoPhase LH Unylinker 300). 1.8 equivalents of 2'-MOE-phosphoramide were typically used. Other reagents (dichloroacetic acid, 1-methylimidazole, 4,5-dicyanimidazole, acetic anhydride, phenylacetyl disulfide, pyridine, triethylamine) were used as is from commercially available sources. Detailed synthetic parameters and reagent lists are shown in Table 1 below. Cleavage and deprotection were achieved using 25–28% ammonium hydroxide, and the resulting solution was concentrated to obtain crude oligonucleotides. Synthetic parameters are shown in Table 1 below.

[0070] Table 1

[0071]

[0072]

[0073] (1.2) Purification Method: The anion exchange column was equilibrated with 20 mM sodium phosphate buffer (pH 7.5, prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate) for 5 CVs. The crude oligonucleotide solution prepared in 1.1 was then loaded onto the anion exchange column (with Source 30Q column media, inner diameter 3.5 cm, column height approximately 18 cm, and column volume approximately 173 mL) to achieve a column loading (i.e., sample loading) of approximately 800 OD / mL. The purification equipment used was... Pure 150. After the crude oligonucleotides were loaded onto the anion exchange column, the column was equilibrated with 20 mM sodium phosphate buffer for 3 CVs, then washed with a mixture of 85% 20 mM sodium phosphate buffer (pH 7.5) and 15% buffer containing 2.5 M sodium chloride and 20 mM sodium phosphate (pH 7.5) for 7 CVs, and then washed with 20 mM sodium phosphate buffer for 6 CVs.

[0074] The anion exchange column was washed with 80% acetic acid aqueous solution (13 CVs) until the pH of the eluent was approximately 3 or lower, allowing the 5'-hydroxyl terminus of the oligonucleotide to be deprotected by the 4,4'-dimethoxytriphenylmethyl (DMT) protecting group. The anion exchange column was equilibrated with 20 mM sodium phosphate buffer (8 CVs), and then washed with a buffer containing 2.5 M sodium chloride and 20 mM sodium phosphate (pH 7.5) (40 CVs) to elute the target oligonucleotide with the deprotected hydroxyl protecting group. The eluent containing the target oligonucleotide with the deprotected hydroxyl protecting group was collected and detected by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry) (instrument: Waters Acquity Premier-SQD2; column: Waters ACQUITY Premier BEH C18 (2.1 × 150 mm, 1.7 μm), detection wavelength 260 nm). The specific purification parameters are shown in Table 2 below.

[0075] Table 2

[0076]

[0077]

[0078] IP-HPLC-UV-MS analysis showed that the purity of the collected eluent was 99.15%.

[0079] (1.3) Ultrafiltration / Percolation Method: Purified oligonucleotide eluates from two purification batches (each at a scale of 1 mmol) were combined and subjected to a regenerated cellulose cassette (molecular weight cutoff of 3000 DA; membrane area of ​​0.1 m²). 2 )of The Flux 6 system desalinates via ultrafiltration / diafiltration. It is initially loaded at a concentration of approximately 50 OD / mL and operates at a rate of approximately 5 L / min / m³. 2 The solution is desalted by tangential flow rate, and then percolated with water for 6-8 exchanges until the conductivity of the permeate (permeate) is less than 50 μS / cm and the concentration of desalted oligonucleotides obtained in the retention solution is at least 250 OD / mL.

[0080] Concentration was achieved through freeze-drying: the ultrafiltration sample was freeze-dried on a Genesis EL-35 freeze dryer at a loading temperature of 20–30°C; the pre-freezing temperature was -50°C to -30°C, and a vacuum was applied at 100–600 mTorr; the sample was then dried at -20°C to 30°C and 20–150 mTorr for 48–72 hours to obtain a solid product containing the target oligonucleotide with the nucleic acid sequence shown in SEQ ID NO.1.

[0081] Example 2

[0082] According to Example 1, 5'-DMT-Ts Me CsAs Me CsTsTsTs Me CsAsTsAsAsTsGs Me The method for synthesizing CsTsGsG-3' yielded a crude oligonucleotide solution.

[0083] The purification method is as follows:

[0084] The anion exchange column was equilibrated with 50 mM sodium phosphate buffer (pH 7.8, prepared from disodium hydrogen phosphate and sodium dihydrogen phosphate) for 5 CVs. The crude oligonucleotide solution was then loaded onto the anion exchange column (using Source 30Q column media, 3.5 cm inner diameter, approximately 18 cm column height, and approximately 173 mL column volume) to achieve a column loading (i.e., loading) of approximately 800 OD / mL. After loading the crude oligonucleotides onto the anion exchange column, it was equilibrated with 50 mM sodium phosphate buffer for 3 CVs, then washed with a 10 CV mixed solution (85% 50 mM sodium phosphate buffer (pH 7.8) and 15% buffer containing 2.0 M sodium chloride and 50 mM sodium phosphate (pH 7.8)), and finally washed with 50 mM sodium phosphate buffer for 6 CVs.

[0085] The anion exchange column was washed with 85% aqueous acetic acid (16 CVs) until the pH of the eluent was approximately 3 or lower, allowing the 5'-hydroxyl terminus of the oligonucleotide to be deprotected by the 4,4'-dimethoxytriphenylmethyl (DMT) protecting group. The anion exchange column was equilibrated with 50 mM sodium phosphate buffer (8 CVs), and then washed with a buffer containing 2.0 M sodium chloride and 50 mM sodium phosphate (pH 7.8) (30 CVs). The eluent containing the target oligonucleotide with the deprotected hydroxyl group was collected to elute the target oligonucleotide. The collected eluent was analyzed by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry). Specific purification parameters are shown in Table 3 below.

[0086] Table 3

[0087]

[0088] IP-HPLC-UV-MS analysis showed that the purity of the collected eluent was 98.22%.

[0089] Following the ultrafiltration / percolation method of Example 1, a solid product of the target oligonucleotide having the nucleic acid sequence shown in SEQ ID NO.1 was obtained.

[0090] Example 3

[0091] According to Example 1, 5'-DMT-Ts Me CsAs Me CsTsTsTs Me CsAsTsAsAsTsGs Me The method for synthesizing CsTsGsG-3' yielded a crude oligonucleotide solution.

[0092] The purification method is as follows:

[0093] The anion exchange column was equilibrated with 20 mM potassium phosphate buffer (pH 7.2, prepared from dipotassium hydrogen phosphate and potassium dihydrogen phosphate) for 5 CVs. The crude oligonucleotide solution was then loaded onto the anion exchange column (with NanoQ-30L column media, 3.5 cm inner diameter, approximately 18 cm column height, and approximately 173 mL column volume) to achieve a column loading (i.e., loading) of approximately 800 OD / mL. After loading the crude oligonucleotides onto the anion exchange column, it was equilibrated with 20 mM potassium phosphate buffer for 3 CVs, then washed with a 10 CV mixed solution (a mixture of 70% 20 mM potassium phosphate buffer (pH 7.2) and 30% buffer containing 1 M sodium chloride and 20 mM potassium phosphate (pH 7.2)), and finally washed with 20 mM sodium phosphate buffer for 5 CVs.

[0094] The anion exchange column was washed with 78% acetic acid aqueous solution (13 CVs) until the pH of the eluent was approximately 3 or lower, allowing the 5'-hydroxyl terminus of the oligonucleotide to be deprotected by the 4,4'-dimethoxytriphenylmethyl (DMT) protecting group. The anion exchange column was equilibrated with 20 mM sodium phosphate buffer (10 CVs), and then washed with a buffer containing 1 M sodium chloride and 20 mM sodium phosphate (pH 7.2) (35 CVs) to elute the target oligonucleotide with the deprotected hydroxyl protecting group. The eluent containing the deprotected target oligonucleotide was collected and analyzed by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry). Specific purification parameters are shown in Table 4 below.

[0095] Table 4

[0096]

[0097] The purity of the collected eluent was 97.83% as determined by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry).

[0098] Following the ultrafiltration / percolation method of Example 1, a solid product of the target oligonucleotide having the nucleic acid sequence shown in SEQ ID NO.1 was obtained.

[0099] Comparative Example 1

[0100] Purification procedure: The crude oligonucleotide solution prepared in Example 1 was loaded onto an anion exchange column (with Source 30Q column media, inner diameter 3.5 cm, bed height 18 cm, and column volume 173 mL) using 25 mM NaOH solution (pH 11) for 5 CVs to achieve a column loading of approximately 800 OD / mL. The purification equipment used was... Pure 150. After the crude oligonucleotides were loaded onto the anion exchange column, they were equilibrated with 25 mM NaOH solution (pH 11) for 5 CVs, followed by washing with a 15 CV mixed solution (the mixed solution consisted of 75% 25 mM NaOH solution and 25% solution containing 25 mM NaOH and 2.5 M sodium chloride (pH 11)), and then washed with NaOH solution (pH 11) for 5 CVs.

[0101] The anion exchange column was washed with 80% acetic acid aqueous solution (14 CVs) until the pH of the resulting eluent was approximately 3 or lower, causing the 5'-hydroxyl terminus of the crude oligonucleotide to lose the 4,4'-dimethoxytriphenylmethyl (DMT) protecting group, forming a deprotected oligonucleotide. The anion exchange column was then equilibrated with 25 mM NaOH solution (pH 11) to readjust the pH to 11 (10 CVs of NaOH solution). The column was then washed with an aqueous solution containing 25 mM NaOH and 2.5 M sodium chloride (pH 11) (35 times the column volume) to elute the target oligonucleotide with the deprotected hydroxyl group. The eluent containing the deprotected target oligonucleotide was collected and analyzed by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry). Specific purification parameters are shown in Table 5 below.

[0102] Table 5

[0103]

[0104]

[0105] The purity of the collected eluent containing the target oligonucleotide was 96.19% as determined by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry).

[0106] Following the ultrafiltration / percolation method of Example 1, a solid product of the target oligonucleotide having the nucleic acid sequence shown in SEQ ID NO.1 was obtained.

[0107] Comparative Example 2

[0108] The anion exchange column was equilibrated with 20 mM sodium phosphate buffer (pH 8.5) containing 10% ACN for 5 CVs. The crude oligonucleotide solution prepared in Example 1 was then loaded onto the anion exchange column (with Source30Q column media, 3.5 cm inner diameter, approximately 18 cm column height, and approximately 173 mL column volume) to achieve a column loading of approximately 800 OD / mL. The purification equipment used was… Pure 150. After the crude oligonucleotides were loaded onto the anion exchange column, the column was washed with a mixture of 8 CV buffers (pH 8.5) containing 20 mM sodium phosphate and 17% buffers containing 10% ACN, 20 mM sodium phosphate, and 2.5 M sodium chloride.

[0109] The anion exchange column was washed with 80% aqueous acetic acid (washing for 16 CVs) until the pH of the resulting eluent was approximately 3 or lower, allowing the 5'-hydroxyl terminus of the oligonucleotide to lose its 4,4'-dimethoxytriphenylmethyl (DMT) protecting group, forming a dehydroxylated oligonucleotide. The anion exchange column was then equilibrated with 20 mM sodium phosphate buffer (pH 8.5) containing 10% ACN (7 times the column volume). The column was then equilibrated with a solution containing 20 mM sodium phosphate (pH 8.5) containing 10% ACN and a buffer containing 10% ACN, 20 mM sodium phosphate, and 2.5 M sodium chloride (pH 8.5). 8.5) Wash the anion exchange column (using an elution buffer at a volume 35 times the column volume) to elute the target oligonucleotide with the hydroxyl protecting group removed. Collect the eluent containing the target oligonucleotide with the hydroxyl protecting group removed. Analyze the collected eluent by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry). Specific purification parameters are shown in Table 6 below.

[0110] Table 6.

[0111]

[0112]

[0113] The purity of the collected eluent containing the target oligonucleotide was 97.32% when detected by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry).

[0114] Following the ultrafiltration / percolation method of Example 1, a solid product of the target oligonucleotide having the nucleic acid sequence shown in SEQ ID NO.1 was obtained.

[0115] The purity, yield, and impurity levels of the products obtained from Examples 1-3 and Comparative Examples 1-2 are shown in Table 7 below.

[0116] Table 7

[0117] Instance number HPLC purity Purification yield P=O impurity content n-1 Impurity content n+1 impurity content Example 1 99.15％ 85.00％ 0.94％ 0.32％ 0.14％ Example 2 98.22％ 83.90％ 0.96％ 0.36％ 0.12％ Example 3 97.83％ 86.23％ 1.00％ 0.41％ 0.16％ Comparative Example 1 96.19％ 80.15％ 1.26％ 0.98％ 0.41％ Comparative Example 2 97.32％ 78.89％ 1.46％ 0.57％ 0.25％

[0118] As shown in Table 7 above, compared with Comparative Examples 1-2, the oligonucleotide products shown in SEQ ID NO.1 prepared using the purification method of the present invention have fewer P=O impurities, n-1 impurities, and n+1 impurities, and the yield is higher.

[0119] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for purifying oligonucleotides, characterized in that, The purification method includes the following steps: (a) Loading crude oligonucleotides onto an anion exchange column equilibrated with phosphate buffer, wherein the 5'-hydroxyl group of the terminal nucleoside of the oligonucleotides in the crude oligonucleotides has a hydroxyl protecting group; (b) Equilibrate the anion exchange column with phosphate buffer solution, and then wash the anion exchange column with a mixture of phosphate buffer solution and phosphate buffer solution containing alkali metal halide; (c) Equilibrate the anion exchange column with phosphate buffer solution, and then wash the anion exchange column with acidic aqueous solution to remove the 5'-hydroxy protecting group of the terminal nucleoside of the oligonucleotide; (d) Equilibrate the anion exchange column with phosphate buffer solution, then elute the target oligonucleotide to remove the 5'-hydroxy protecting group with phosphate buffer solution containing alkali metal halides, and collect the eluent containing the target oligonucleotide. The pH of the phosphate buffer solution is 7.0-8.

0. The phosphate in the phosphate buffer solution is selected from sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, or mixtures thereof. The alkali metal halide in the phosphate buffer solution containing alkali metal halides is selected from sodium chloride or sodium bromide.

2. The method for purifying oligonucleotides according to claim 1, characterized in that, The nucleotide sequence of the oligonucleotide is shown in SEQ ID NO.

1.

3. The method for purifying oligonucleotides according to claim 1 or 2, characterized in that, The crude oligonucleotide contains ineffective sequence nx impurities, co-eluted n-1 impurities, P=O impurities, and n+1 impurities, and the UV purity of the oligonucleotides is greater than or equal to 70%. The 5'-hydroxy protecting group of the terminal nucleoside of the oligonucleotide is 4,4'-dimethoxytriphenylmethyl.

4. The method for purifying oligonucleotides according to claim 1 or 2, characterized in that, The resin of the anion exchange column is a strong basic anion exchange resin containing quaternary ammonium groups, with a particle size greater than or equal to 15 μm.

5. The method for purifying oligonucleotides according to claim 4, characterized in that, The resin in the anion exchange column has a particle size of 30 μm.

6. The method for purifying oligonucleotides according to claim 1 or 2, characterized in that, In steps (a), (b), (c) and (d), the phosphate content in the phosphate buffer solution is 10 mM to 100 mM.

7. The method for purifying oligonucleotides according to claim 1 or 2, characterized in that, In steps (a), (b), (c) and (d), the phosphate content in the phosphate buffer solution is 15 mM to 50 mM.

8. The method for purifying oligonucleotides according to claim 1, characterized in that, The alkali metal halide in the phosphate buffer solution containing alkali metal halides is selected from sodium chloride.

9. The method for purifying oligonucleotides according to claim 1, characterized in that, The phosphate buffer solution containing alkali metal halides has a phosphate content of 10 mM to 100 mM and a pH of 7.0 to 8.

0. The phosphate buffer solution containing alkali metal halides has a mass concentration of 1.0 M to 3.0 M for the alkali metal halides.

10. The method for purifying oligonucleotides according to claim 1, characterized in that, The phosphate buffer solution containing alkali metal halides has a phosphate content of 15 mM to 50 mM and a pH of 7.0 to 8.

0. The phosphate buffer solution containing alkali metal halides has a mass concentration of 1.0 M to 2.5 M for the alkali metal halides.

11. The method for purifying oligonucleotides according to claim 1 or 2, characterized in that, In step (b), the volume ratio of the phosphate buffer solution to the phosphate buffer solution containing alkali metal halide in the mixture is 6:1 to 2:

1.

12. The method for purifying oligonucleotides according to claim 1 or 2, characterized in that, In step (c), the acid in the acidic aqueous solution is selected from hydrochloric acid, sulfuric acid, phosphoric acid, nitric acid, formic acid, acetic acid, and haloacetic acid. In step (c), the volume content of acid in the acidic aqueous solution is 50% to 95%.

13. The method for purifying oligonucleotides according to claim 12, characterized in that, In step (c), the acid in the acidic aqueous solution is selected from acetic acid. In step (c), the volume content of acid in the acidic aqueous solution is 70% to 90%.

14. The method for purifying oligonucleotides according to claim 1 or 2, characterized in that, In step (b), the mixture of phosphate buffer solution and phosphate buffer solution containing alkali metal halide is used in an amount of 5 to 12 times the column volume. In step (c), the flow rate of the acidic aqueous solution during washing of the anion exchange column is 100 cm / h to 400 cm / h, and the amount used is 7 to 22 times the column volume (CV). In step (d), the amount of phosphate buffer containing alkali metal halides is 20 to 50 times the column volume. In steps (a), (b), (c), and (d), the amount of phosphate buffer solution used to equilibrate the anion exchange column is 1 to 15 times the column volume.

15. The method for purifying oligonucleotides according to claim 14, characterized in that, In steps (a), (b), (c) and (d), the amount of phosphate buffer solution used to equilibrate the anion exchange column is 3 to 10 times the column volume.

16. The method for purifying oligonucleotides according to claim 14, characterized in that, In step (d), the amount of phosphate buffer containing alkali metal halides is 25 to 45 times the column volume. The amount of phosphate buffer solution used to balance the anion exchange column is 3 to 10 times the column volume.

Images