Purification method of oligonucleotide

Abstract

The invention discloses an oligonucleotide purification method which comprises the following steps: loading an oligonucleotide crude product onto an anion exchange column, balancing by using a buffer solution, and then carrying out gradient elution on target oligonucleotide with a hydroxyl protecting group by using a mixture of the buffer solution and a buffer solution containing alkali metal halide, collecting elution fractions containing target oligonucleotides, carrying out ultrafiltration desalination, loading the elution fractions onto the anion exchange column again, balancing the elution fractions with a buffer solution, washing the anion exchange column with an acidic aqueous solution to remove hydroxyl protecting groups of the oligonucleotides, balancing the elution fractions with the buffer solution, and collecting the target oligonucleotides. And carrying out gradient elution on the target oligonucleotide with the hydroxyl protecting group removed by using a buffer solution and a buffer solution containing alkali metal halide. The oligonucleotide obtained by the purification method is high in purity, low in cost and suitable for large-scale production.

Description

Technical Field

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

[0002] Oligonucleotides are a general term for a class of short-chain nucleotide compounds, typically composed of 20 or more nucleotides. Based on their structure and function, they are classified into antisense nucleic acids (ASO), small interfering RNA (siRNA), microRNA (miRNA), and nucleic acid aptamers. Due to their advantages such as short development cycles, high specificity, and low toxicity, their market demand and market size are continuously expanding. Currently, there are 20 marketed oligonucleotide drugs worldwide used to treat diseases such as Duchenne muscular dystrophy, spinal muscular atrophy, and familial amyloid polyneuropathy.

[0003] Oligonucleotides are synthesized stepwise, with a monomer added to the newly formed oligonucleotide chain at regular intervals. In each reaction cycle, the oligonucleotide chain is chain-extended almost quantitatively, but a small number of reaction sites remain incompletely reacted. Therefore, after 20 or more cycles of synthesis, the resulting product typically contains small amounts of oligonucleotide impurity chains of varying lengths. These include impurities with missing nucleotides (called nx impurities, n-1 if one nucleotide is missing), impurities with phosphodiester bonds instead of the desired thiophosphate bonds (called P=O impurities), and impurities with extra nucleotides (called n+x impurities, n+1 if one nucleotide is extra). Therefore, downstream oligonucleotide purification methods are crucial for obtaining high-purity products.

[0004] Currently, commonly used purification methods include gel electrophoresis and chromatography. Gel electrophoresis is suitable for the purification of small-scale oligonucleotides, but it cannot be scaled up and has poor practicality. Chromatography is widely used in the field of oligonucleotide purification due to its advantages such as high loading capacity and industrial scale-up capability. Existing chromatographic processes are generally divided into two types. One type targets oligonucleotides retaining protecting groups such as 5'-4,4'-dimethoxytriphenylmethyl (DMT), using hydrophobic or reversed-phase columns for purification. After elution, deprotection is performed, followed by purification using a column with small-particle-size anion exchange packing. The other type targets oligonucleotides with protecting groups such as 5'-4,4'-dimethoxytriphenylmethyl (DMT), using anion exchange columns for preliminary purification (crude purity) followed by purification using a reversed-phase column. Both chromatographic purification methods are two-step processes. Although the product purity meets the standard, the use of two types of packing materials and the introduction of an organic phase increase the cost and difficulty of the purification process. Existing technologies also employ a "one-step" chromatography method for oligonucleotides. For example, Hofmeister AG disclosed in prior art CN114051499A a method using a Source 30Q anion exchange column as the medium and a mixture of 25 mM sodium phosphate buffer, 0.5–2 M sodium chloride solution, and 10% acetonitrile as the eluent to obtain oligonucleotides of the target purity. Although this method uses a single-packed column, the addition of acetonitrile as the eluent in the mobile phase results in higher costs for waste treatment and more stringent plant requirements. Prior art CN117143165A is similar to prior art CN114051499A, but it improves the mobile phase elution process and does not add acetonitrile to the mobile phase, thus reducing waste treatment costs and being more environmentally friendly. However, both existing technologies share the same problem: after removing pre-eluting impurities (short-fragment ineffective sequences, i.e., nx impurities) before removing DMT and other protecting groups, the protecting groups are directly removed on the column with an acidic solution, followed by another elution to obtain the target oligonucleotide. Although this process is simple to operate, it overlooks an important point: it only involves the pre-eluting impurity step and does not address the post-eluting impurities in the oligonucleotide (long-fragment sequences, branched-chain compound impurities, i.e., n+x impurities, and other impurities with strong retention on the column due to DMT and other hydroxyl protecting groups). As a result, when eluting the target oligonucleotide after removing DMT and other hydroxyl protecting groups, these post-eluting impurities will co-elute with the target oligonucleotide, leading to a higher proportion of post-eluting impurities in the final product.

[0005] Therefore, there is an urgent need in this field to develop a new method for purifying oligonucleotides that can effectively remove both pre-elution and post-elution impurities simultaneously. Summary of the Invention

[0006] To address the problems existing in current oligonucleotide purification methods, this invention provides a method for purifying oligonucleotides, comprising the following steps:

[0007] (a) Loading crude oligonucleotides onto an anion exchange column, 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 the buffer solution, then elute the target oligonucleotide with hydroxyl protecting groups using a gradient of the buffer solution and the buffer solution containing alkali metal halides, collect the elution fraction containing the target oligonucleotide with hydroxyl protecting groups, and perform ultrafiltration desalting to obtain a concentrate.

[0009] (c) Wash the anion exchange column with an alcohol-containing salt solution;

[0010] (d) Load the concentrate obtained in step (b) onto the anion exchange column;

[0011] (e) Equilibrate the anion exchange column with the buffer solution and then wash the anion exchange column with an acidic aqueous solution to remove the hydroxyl protecting group of the oligonucleotide;

[0012] (f) Equilibrate the anion exchange column with the buffer solution, then elute the target oligonucleotide (with the hydroxyl protecting group removed) with a gradient of the buffer solution and the buffer solution containing the alkali metal halide. Collect the elution fraction containing the target oligonucleotide, desalt it by ultrafiltration, and remove water to obtain the oligonucleotide.

[0013] In steps (a), (b), (d), (e), and (f), the pH of the buffer solution is 7-11.

[0014] In another preferred embodiment, in steps (a), (b), (d), (e), and (f), the buffer solution is selected from phosphate buffer solution, acetate buffer solution, carbonate buffer solution, ethylenediaminetetraacetic acid buffer solution, or a mixture thereof, preferably phosphate buffer solution or acetate buffer solution.

[0015] In another preferred embodiment, the salt content in the buffer solution is 10 mM to 100 mM, more preferably 15 mM to 50 mM.

[0016] In another preferred embodiment, the pH of the buffer solution is 7.5-9.0. In yet another more preferred embodiment, the pH of the buffer solution is 7.5-8.5.

[0017] In another preferred embodiment, the buffer solution is a phosphate buffer solution with a pH of 7.5 to 9.0.

[0018] In another preferred embodiment, the buffer solution is an acetate buffer solution with a pH of 7.5 to 9.0.

[0019] In another preferred embodiment, in steps (b), (e), and (f), the amount of buffer solution used to equilibrate the anion exchange column is 3 to 8 times the column volume, more preferably 4 to 6 times.

[0020] In another preferred embodiment, in step (b), the alkali metal halide is selected from sodium chloride, sodium bromide or sodium iodide, preferably sodium bromide.

[0021] In another preferred embodiment, in step (b), the molar concentration of the alkali metal halide in the 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 amount of the mixture of the buffer solution and the buffer solution containing the alkali metal halide is 15 to 50 times the column volume.

[0023] In another preferred embodiment, in step (b), the volume ratio of the buffer solution to the buffer solution containing the alkali metal halide in the mixture of the buffer solution and the buffer solution containing the alkali metal halide is 1:1 to 2, more preferably 1:1.2 to 1.8.

[0024] In another preferred embodiment, in steps (b) and (f), the ultrafiltration membrane package used for ultrafiltration desalination is selected from regenerated cellulose and / or polyethersulfone.

[0025] In another preferred embodiment, in steps (b) and (f), the size of the ultrafiltration membrane pack is 1 KD to 3 KD, preferably 2 KD.

[0026] In another preferred embodiment, in step (c), the alcohol is selected from ethanol, methanol, isopropanol, and more preferably, isopropanol.

[0027] In another preferred embodiment, in step (c), the volume content of alcohol in the alcohol-containing salt solution is 15-95%, more preferably 25-40%.

[0028] In another preferred embodiment, in step (c), the salt is sodium chloride or sodium bromide, preferably sodium chloride.

[0029] In another preferred embodiment, in step (c), the molar concentration of the salt solution is 0.5 M to 4 M, more preferably 1 M to 2 M.

[0030] In another preferred embodiment, in step (e), the acid in the acidic aqueous solution is selected from carbonic acid, formic acid, acetic acid, haloacetic acid, and preferably acetic acid.

[0031] In another more preferred embodiment, in step (e), the acidic aqueous solution is an aqueous solution of acetic acid, wherein the volume content of acetic acid is 60% to 90%, preferably 70% to 90%.

[0032] In another preferred embodiment, in step (e), the amount of the acidic aqueous solution is 5 to 25 times the column volume, more preferably 5 to 15 times.

[0033] In another preferred embodiment, in step (f), the alkali metal halide is selected from sodium chloride, sodium bromide or sodium iodide, preferably sodium chloride.

[0034] In another preferred embodiment, in step (f), the molar concentration of the alkali metal halide in the buffer solution is 1.0 M to 3.0 M, more preferably 1.0 M to 2.0 M.

[0035] In another preferred embodiment, in step (f), the amount of the mixture of the buffer solution and the buffer solution containing the alkali metal halide is 15 to 50 times the column volume.

[0036] In another preferred embodiment, in step (f), the volume ratio of the buffer solution to the buffer solution containing the alkali metal halide in the mixture of the buffer solution and the buffer solution containing the alkali metal halide is 1:4 to 10, more preferably 1:5 to 8.

[0037] In another preferred embodiment, water removal is carried out by freeze-drying.

[0038] In another preferred embodiment, the nucleotide sequence of the oligonucleotide is shown in SEQ ID NO.1.

[0039] In another preferred embodiment, the crude oligonucleotide contains one or more of the following: failed sequence nx impurities, n+x impurities, and P=O impurities.

[0040] In another preferred embodiment, the UV purity of the crude oligonucleotide is not less than 75%.

[0041] In another preferred embodiment, the hydroxyl protecting group is 4,4'-dimethoxytriphenylmethyl.

[0042] In another preferred embodiment, the resin of the anion exchange column is a strongly basic anion exchange resin with a particle size greater than or equal to 15 μm to 100 μm, more preferably, the particle size is 20 to 50 μm.

[0043] In another preferred embodiment, in steps (a) and (d), the anion exchange column is equilibrated with the buffer solution before loading the oligonucleotide crude product or the concentrate onto the anion exchange column.

[0044] In another preferred embodiment, in steps (a) and (d), before loading the crude oligonucleotide or the concentrate onto the anion exchange column, the amount of buffer solution used to equilibrate the anion exchange column is 3 to 8 times, more preferably 4 to 6 times, the column volume. Attached Figure Description

[0045] Figure 1 This is a flowchart of the purification method for crude oligonucleotides of the present invention.

[0046] Figure 2 This is an ultra-high performance liquid chromatography (UPLC) chromatogram of the crude oligonucleotides used in the embodiments and comparative examples of this invention.

[0047] Figure 3 This is the UPLC spectrum of the final product oligonucleotide obtained in Example 1 of the present invention.

[0048] Figure 4 This is the UPLC spectrum of the final product oligonucleotide obtained in Example 2 of the present invention.

[0049] Figure 5 This is the UPLC spectrum of the final product oligonucleotide obtained in Comparative Example 1 of this invention.

[0050] Figure 6 This is the UPLC spectrum of the final product oligonucleotide obtained in Comparative Example 2 of this invention.

[0051] Figure 7 This is the UPLC spectrum of the final product oligonucleotide obtained in Comparative Example 3 of this invention. Detailed Implementation

[0052] To address some problems in existing oligonucleotide purification methods, such as the inability to effectively remove pre-eluting and post-eluting impurities simultaneously, the low column loading capacity that cannot meet the requirements for industrial scale-up production, the high preparation cost due to the use of columns with different packing materials, and the need to use organic solvents, etc. Through in-depth research, the inventors of this application unexpectedly discovered that by using the same anion exchange column and performing column chromatography purification in stages, the first step involved purifying crude oligonucleotides with 5'-hydroxy protecting groups using an anion exchange column. This removed most of the pre-elution and post-elution impurities. The elution fraction containing oligonucleotides with 5'-hydroxy protecting groups was collected, desalted by ultrafiltration, and the resulting concentrate was loaded back onto the anion exchange column (after eluting the oligonucleotides with 5'-hydroxy protecting groups, the anion exchange column was washed with an alcohol-containing salt solution to thoroughly remove impurities). After removing the 5'-hydroxy protecting groups using an acidic aqueous solution, the target oligonucleotide was eluted. The elution fraction containing the target oligonucleotide was collected, desalted by ultrafiltration, and water was removed to obtain a high-purity target oligonucleotide solid. Based on this, the present invention was completed.

[0053] The purification method for crude oligonucleotides of the present invention includes the following steps: (a) loading the crude oligonucleotides onto an anion exchange column, wherein the 5'-hydroxyl group of the terminal nucleoside of the oligonucleotides in the crude oligonucleotides is protected by a hydroxyl group; (b) equilibrating the anion exchange column with the buffer solution, then eluting the target oligonucleotides with the hydroxyl group using a gradient of the buffer solution and the buffer solution containing an alkali metal halide, collecting the elution fraction containing the target oligonucleotides with the hydroxyl group, and performing ultrafiltration desalting to obtain a concentrate; (c) washing the anion exchange column with an alcohol-containing salt solution; (d) loading the concentrate obtained in step (b) onto the anion exchange column; (e) equilibrating the anion exchange column with the buffer solution. (a) Wash the anion exchange column with an acidic aqueous solution to remove the hydroxyl protecting group of the oligonucleotide; (f) Equilibrate the anion exchange column with the buffer solution, and then elute the target oligonucleotide with the removed hydroxyl protecting group by gradient elution with a mixture of the buffer solution and the buffer solution containing an alkali metal halide. Collect the elution fraction containing the target oligonucleotide, desalt it by ultrafiltration, and remove water to obtain the oligonucleotide. In steps (a), (b), (d), (e) and (f), the pH of the buffer solution is 7-11.

[0054] In the purification method of the above-mentioned crude oligonucleotide, steps (a), (b) and (c) perform column chromatography purification of oligonucleotides with hydroxyl protecting groups, which is called "first column chromatography purification". Steps (d), (e) and (f) deprotect and elute oligonucleotides with hydroxyl protecting groups on an anion exchange column to obtain the target oligonucleotide, which is called "second column chromatography purification".

[0055] In the purification method for the above-mentioned crude oligonucleotides, the composition and components of the buffer solution in steps (a), (b), (d), (e), and (f) are preferably the same. The buffer solution is a commonly used buffer solution in the art.

[0056] The purification method of this invention uses an anion exchange column that can be a commercially available Cytiva FineLINE series purification column or a dynamic axial preparative compression column from Hanbang Technology, employing conventional, commercially available anion exchange resins that can be obtained from Cytiva, Tosoh Bioscience, Bio-Rad, Merck, or Suzhou Nanomicro Technology. Typical resins include Cytiva's Source 30Q resin or Suzhou Nanomicro Technology's NanoQ-30L resin.

[0057] The 5'-hydroxy protecting group of the oligonucleotide of the present invention is a common acid-sensitive hydroxy protecting group in the art. In a specific embodiment of the present invention, the nucleotide sequence of the oligonucleotide to be purified is shown in SEQ ID NO.1, and its sequence 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.

[0058] In some embodiments, the crude oligonucleotides loaded onto the anion exchange column are loaded in solution form. The crude oligonucleotides can be loaded in solution form. For example, the solvent of the solution is ammonia solution with a mass concentration of less than 10% or a mixture of ethanol and ammonia solution, wherein the volume content of ethanol is less than 10% and the mass concentration of ammonia solution is less than 10%. In some embodiments, the total oligonucleotide content in the solution of the 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).

[0059] In the purification method of this invention, the crude oligonucleotide loaded onto the anion exchange column contains common impurities generated during the synthesis process, such as n+x impurities, nx impurities, P=O impurities, etc. (nx impurities, also known as short failing sequences, have weak retention on the exchange column and are usually eluted before the target oligonucleotide; they are called "pre-eluted impurities." n+x impurities, also known as long failing sequences, have strong retention on the exchange column and are usually eluted after the target oligonucleotide; they are called "post-eluted impurities." When x=1, i.e., n-1 and n+1 impurities, as well as P=O impurities, have sequences close to the target oligonucleotide sequence and are easily co-eluted; therefore, they are also called "co-eluted impurities.") The UV purity (e.g., detected by a 260 nm wavelength) of the crude oligonucleotide loaded onto the anion exchange column is preferably not less than 70%, for example, 70-93%, 85-90%, or 70-85%. Crude products with lower UV purity can be purified by pre-purification or by improving the synthesis method before being purified using the method of this invention.

[0060] The buffer solutions used in the purification method for oligonucleotides of the present invention include, but are not limited to, phosphate buffer solutions, acetate buffer solutions, carbonate buffer solutions, and ethylenediaminetetraacetic acid (EDTA) buffer solutions. These buffer solutions can be prepared according to conventional methods in the art. In some embodiments, the buffer solution is a phosphate buffer solution. During the preparation of the phosphate buffer solution, the phosphate used can 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 a specific embodiment of the present invention, the phosphate buffer solution contains sodium dihydrogen phosphate and disodium hydrogen phosphate, or potassium dihydrogen phosphate and dipotassium hydrogen phosphate, with a total phosphate content of 15 mM to 30 mM, and a pH of 7.5 to 9.0. In some embodiments, the buffer solution is a sodium acetate buffer solution with a concentration of 15 to 25 mM, more preferably 20 mM.

[0061] In some embodiments, the alkali metal halide in the buffer solution containing the alkali metal halide used to elute the target oligonucleotide in steps (b) and (f) may be the same or different. In one specific embodiment of the invention, in step (b), the alkali metal halide is sodium bromide. In one specific embodiment of the invention, in step (f), the alkali metal halide is sodium chloride. The concentration of the alkali metal halide in the buffer solution is preferably 1.0 M to 3.0 M, more preferably 1.5 M to 2.5 M. In one specific embodiment of the invention, in step (b), the buffer solution containing the alkali metal halide is a buffer solution containing 2.0 M sodium bromide and 20 mM sodium phosphate (pH 8). In one specific embodiment of the invention, in step (f), the buffer solution containing the alkali metal halide is a buffer solution containing 1.0 M sodium chloride and 20 mM sodium phosphate (pH 8).

[0062] In the oligonucleotide purification method of the present invention, step (b) involves equilibrating the anion exchange column with the buffer solution and washing the anion exchange column with a mixture of the buffer solution and a buffer solution containing an alkali metal halide. This process removes most of the pre-elution impurities from the crude oligonucleotide, yielding an elution fraction containing the target product. In a specific embodiment of the present invention, the gradient elution procedure in step (b) is as follows: first, elution is performed with a mixture of 0.5–2 CV 95% buffer solution and 5% alkali metal halide-containing buffer solution; then, elution is performed with a mixture of 25–35 CV 40% buffer solution and 60% alkali metal halide-containing buffer solution. The buffer solution is either 15–25 mM sodium phosphate buffer or 15–25 mM sodium acetate buffer, and the alkali metal halide is NaBr.

[0063] In step (b), ultrafiltration desalination can be performed according to conventional methods in the art. In some specific embodiments of the invention, the criterion for the end of ultrafiltration is a permeation conductivity of approximately 10 mS / cm.

[0064] Step (c) involves washing the anion exchange column with an alcohol-containing salt solution to remove residual post-elution impurities. The washed anion exchange column can then be reused. The alcohol includes, but is not limited to, ethanol, methanol, and isopropanol, and the salt solution includes, but is not limited to, sodium chloride and sodium bromide (aqueous) solutions. In some specific embodiments of the invention, the alcohol-containing salt solution is an aqueous NaCl solution containing isopropanol, wherein the volume content of isopropanol is 30%, and the concentration of the NaCl solution is 2M. In some specific embodiments of the invention, the anion exchange column is washed with an alcohol-containing salt solution containing 4-6 CVs.

[0065] In step (e), the acid used to prepare the acidic aqueous solution for removing the 5'-hydroxy protecting group can be an inorganic or organic acid. For example, carbonic acid, formic acid, acetic acid, and haloacetic acids (e.g., trichloroacetic acid, trifluoroacetic acid, dichloroacetic acid, etc.) can all be used in this invention. In a specific embodiment of this invention, 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%.

[0066] Step (f) involves equilibrating the anion exchange column with the buffer solution and washing the anion exchange column with a mixture of the buffer solution and a buffer solution containing an alkali metal halide. This process removes most of the pre-elution impurities from the oligonucleotides that have lost their hydroxyl protecting groups, yielding an elution fraction containing the target product. In a specific embodiment of the invention, the gradient elution procedure in step (f) is as follows: first, elution with a mixture of 0.5–2 CV of 95% buffer solution and 5% of alkali metal halide buffer solution; then, elution with a mixture of 20–30 CV of 10% buffer solution and 90% of alkali metal halide buffer solution; and finally, elution with a mixture of 0.5–2 CV of 100% alkali metal halide buffer solution. The buffer solution is either 15–25 mM sodium phosphate buffer or 15–25 mM sodium acetate buffer, and the alkali metal halide is NaCl.

[0067] In step (f), ultrafiltration desalination can be performed according to conventional methods in the art. In some specific embodiments of the invention, the criterion for the end of ultrafiltration is a permeation conductivity of approximately 50 mS / cm.

[0068] 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℃ to 40℃, for example, 15℃ to 35℃, 20℃ to 30℃, or 20℃ to 25℃.

[0069] Compared with the prior art, the present invention has at least the following advantages:

[0070] 1) The use of the same anion exchange column enables the purification of oligonucleotides by two column chromatography processes. This method has low operating costs and a large anion exchange column capacity, which can be used to achieve the purpose of scale-up production.

[0071] 2) In the purification method of this invention, during the first column chromatography purification process, the target oligonucleotide with hydroxyl protecting groups is eluted using a gradient of a mixture of buffer solution and a buffer solution containing alkali metal halides, and the target product is collected, effectively removing pre-elution and post-elution impurities. During the second column chromatography purification process, the oligonucleotide with high purity and hydroxyl protecting groups is deprotected, and then the oligonucleotide is eluted again using a gradient of a mixture of buffer solution and a buffer solution containing alkali metal halides, further effectively removing pre-elution and post-elution impurities. Therefore, the final oligonucleotide obtained has very high purity, with a UPLC purity of over 99%, and no impurities greater than 0.3%.

[0072] The present invention will be further described in detail below with reference to specific implementation examples. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0073] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0074] In the following examples, the ultra-high performance liquid chromatography used for purity determination was Waters Acquity Premier SQD2.

[0075] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0076] abbreviation:

[0077] MOE = 2-methoxyethyl

[0078] NMI = N-methylimidazolium

[0079] PADS = Phenylacetyl disulfide

[0080] Tol = Toluene

[0081] A = 2'-MOE adenosine

[0082] T = 2'-MOE thymidine

[0083] Me C = 2'-MOE 5-methylcytidine

[0084] G = 2'-MOE guanosine

[0085] DCA = dichloroacetic acid

[0086] DCI = 4,5-dicyanimidazolium

[0087] DEA = diethylamine

[0088] ACN = Acetonitrile

[0089] Ac₂O = Acetic anhydride

[0090] CV = Column Volume

[0091] The following examples and comparative examples illustrate the method for purifying oligonucleotides of the present invention, using the target oligonucleotide with the following structure as an example: 5'-DMT-Ts Me CsAs Me CsTsTsTs Me CsAsTsAsAsTsGs Me CsTsGsG-3'. Where "s" represents the thiophosphate bridging bond.

[0092] Crude oligonucleotides (also known as "crude oligonucleotides") can be synthesized using methods common in the art. The crude oligonucleotides used in the following examples and comparative examples of this invention were synthesized in the laboratory by Shanghai Aurite Biotechnology Co., Ltd. using a standard phosphoramidite chemistry method. Oligonucleotides were produced on a solid-phase scale at a scale of 2.0 mmol using ÄKTA™ oligopilot plus 100 and Primer Support Unylinker (NittoPhase LH Unylinker 300) ("DMT-on"). Typically, 1.8 equivalents of 2'-MOE-phosphoramidite were 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 crude oligonucleotide solution was concentrated. Synthetic parameters are shown in Table 1 below.

[0093] Table 1

[0094]

[0095] The purity of the obtained crude oligonucleotides is shown in the figure. Figure 2(UPLC diagram).

[0096] Example 1

[0097] The first column chromatography purification process is as follows:

[0098] The anion exchange column was equilibrated with 20 mM sodium phosphate buffer (pH 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 (containing Source 30Q packing material, 1.0 cm inner diameter, approximately 18 cm height, and approximately 14.137 mL column volume) at a loading rate of approximately 800 OD / mL. The purification equipment used was an ÄKTA™ Pure 150. After the crude oligonucleotides were loaded onto the anion exchange column, the column was first equilibrated with 20 mM sodium phosphate buffer for 5 CVs. Then, the oligonucleotides with DMT protecting groups on the anion exchange column were eluted with a mixed solution of 31 CVs (the mixed solution consisted of 20 mM sodium phosphate buffer (pH 8) and 20 mM sodium phosphate buffer containing 2.0 M sodium bromide (pH 8), eluted according to a specific gradient). The elution fraction containing the target oligonucleotides with DMT was collected. The anion exchange column was then washed with 30% isopropanol + 2 M sodium chloride aqueous solution for 5 CVs. After washing, the anion exchange column was equilibrated with 20 mM sodium phosphate buffer for 5 CVs. The specific elution gradient is shown in Table 2.

[0099] The collected elution fractions containing the target oligonucleotides with DMT were combined based on the results of parallel tube analysis, and then desalted by ultrafiltration. The ultrafiltration membrane pack was made of Kobot RC material with a pore size of 2 KD and an area of ​​0.11 m². 2 The standard for the end of ultrafiltration is a permeation conductivity of approximately 10 mS / cm.

[0100] The second column chromatography purification process is as follows:

[0101] The concentrated solution obtained from ultrafiltration desalting was loaded onto an anion exchange column at a loading capacity of 800 OD / ml. After loading, the anion exchange column was equilibrated with 20 mM sodium phosphate buffer for 5 CVs. The column was then washed with 80% acetic acid aqueous solution (10 CVs) to remove the 4,4'-dimethoxytriphenylmethyl (DMT) protecting group from the 5'-hydroxyl terminus of the oligonucleotide. The column was equilibrated with 20 mM sodium phosphate buffer for 10 CVs, and then eluted with a gradient of 20 mM sodium phosphate buffer and 20 mM sodium phosphate buffer containing 1.0 M sodium chloride (pH 8) at a specific ratio (total volume 27 CVs) to remove the DMT-removed oligonucleotides from the column. The specific elution gradient is shown in Table 3. The elution fraction containing the target oligonucleotide was collected. The collected elution fractions were then combined according to the analytical results and sent to IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry) (instrument: Waters Acquity Premier-SQD2; analytical column: Waters ACQUITY Premier BEH C18 (2.1×150mm, 1.7 μm), detection wavelength 260 nm) for analysis. Qualified samples were combined and subjected to ultrafiltration desalting. The ultrafiltration membrane pack could be made of Kebotan RC material with a pore size of 2 KD and an area of ​​0.11 m². 2 The process continues until the conductivity at the through end is less than 50 μS / cm.

[0102] The sample obtained from ultrafiltration desalting was then transferred to a lyophilizer for lyophilization: loading temperature was 20-30℃; pre-freezing temperature was -50℃ to -30℃, and vacuum degree was 5-60 Pa; drying was carried out at -20℃ to 30℃ and 50 Pa for 48-72 hours to obtain the target oligonucleotide with the target nucleic acid sequence as a solid. Its ultra-high performance liquid chromatogram is shown below. Figure 3 .

[0103] from Figure 3 It can be observed that after two-step column chromatography purification, the final product has a very high chromatographic purity, reaching over 99%.

[0104] Table 2 First column chromatography purification

[0105]

[0106] Table 3 Second column chromatography purification

[0107]

[0108] Example 2

[0109] The first column chromatography purification process is as follows:

[0110] The anion exchange column was equilibrated with 20 mM sodium acetate buffer (pH 8, prepared from sodium acetate) for 5 CVs. The crude oligonucleotide solution was then loaded onto the anion exchange column (packed with NanoQ 30L column packing material, 1.0 cm inner diameter, approximately 18 cm height, and approximately 14.137 mL column volume). The first step of anion purification was performed with a loading of approximately 800 OD / mL. The purification equipment used was an ÄKTA™ Pure 150. After the crude oligonucleotides were loaded onto the anion exchange column, the column was first equilibrated with 20 mM sodium acetate buffer for 5 CVs. Then, the oligonucleotides with DMT protecting groups on the anion exchange column were eluted with a mixed solution of 31 CVs (the mixed solution consisted of 20 mM sodium acetate buffer (pH 8) and 20 mM sodium acetate buffer (pH 8) containing 2.0 M sodium bromide, eluted according to a specific gradient). The column was then washed with 30% ethanol + 1.5 M sodium bromide buffer for 5 CVs, and the elution fraction containing the target oligonucleotides with DMT protecting groups was collected. After washing, the column was equilibrated with 20 mM sodium acetate buffer for 5 CVs. The specific elution gradient is shown in Table 4.

[0111] The collected elution fractions containing target oligonucleotides with DMT were combined based on the results of parallel tube analysis, and then desalted by ultrafiltration. The ultrafiltration membrane packing could be made of Kobot RC material with a pore size of 2 KD and an area of ​​0.11 m². 2 The standard for the end of ultrafiltration is a permeation conductivity of approximately 10 mS / cm.

[0112] The second column chromatography purification process is as follows:

[0113] The concentrated solution obtained from ultrafiltration desalting was loaded onto an anion exchange column at a loading capacity of 800 OD / ml. After loading, the anion exchange column was equilibrated with 20 mM sodium acetate buffer for 5 CVs. The column was then washed with 80% acetic acid aqueous solution (10 CVs) to remove the DMT protecting group from the 5'-hydroxyl terminus of the oligonucleotide. The column was equilibrated with 20 mM sodium acetate buffer for 10 CVs. Then, the target oligonucleotides without the DMT protecting group were eluted from the column (total volume 27 CVs) using a gradient of 20 mM sodium acetate buffer and 20 mM sodium acetate buffer containing 1.0 M sodium chloride (pH 8). The elution fraction containing the target oligonucleotides was collected. The specific elution gradient is shown in Table 5.

[0114] The collected elution fractions were combined according to the detection results and sent to IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry) for analysis (instrument: Waters Acquity Premier-SQD2; analytical column: Waters ACQUITY Premier BEH C18 (2.1×150mm, 1.7 μm), detection wavelength 260 nm). Qualified samples were then combined and subjected to ultrafiltration desalination. The ultrafiltration membrane was selected from Kebaite RC material, with a pore size of 2 KD and an area of ​​0.11 m². 2 The process continues until the conductivity at the through end is less than 50 μS / cm.

[0115] The sample obtained from ultrafiltration desalting was then transferred to a lyophilizer for lyophilization: loading temperature was 20-30℃; pre-freezing temperature was -50℃ to -30℃, and vacuum degree was 5-60 Pa; drying was carried out at -20℃ to 30℃ and 50 Pa for 48-72 hours to obtain the target oligonucleotide with the target nucleic acid sequence as a solid. Its ultra-high performance liquid chromatogram is shown below. Figure 4 .

[0116] from Figure 4 As can be observed, after two purification steps, the final product has a very high chromatographic purity, reaching over 99%.

[0117] Table 4 First column chromatography purification

[0118]

[0119] Table 5 Second column chromatography purification

[0120]

[0121] Comparative Example 1

[0122] Hofmeister AG discloses in prior art CN114051499A a method for eluting oligonucleotides to the target purity using a Source 30Q anion exchange column as the medium and a mixture of 25 mM sodium phosphate buffer, 0.5–2 M sodium chloride solution, and 10% acetonitrile as the eluent. This comparative example purifies crude oligonucleotides according to the method disclosed in this prior art.

[0123] Purification procedure: The anion exchange column was equilibrated with phase A (10% ACN + 25 mM sodium phosphate buffer (pH 8.5)) for 5 CVs. The crude oligonucleotide solution was then loaded onto the anion exchange column (with Source 30Q column media, 1.0 cm inner diameter, approximately 18 cm height, and approximately 14.13 mL column volume) to achieve a column loading of approximately 800 OD / mL. The purification equipment used was an ÄKTA™ Pure 150. After the crude oligonucleotides were loaded onto the anion exchange column, the column was equilibrated with two column volumes of phase A. Then, linear elution was performed using a mixture of three column volumes of phase A and phase B (10% ACN + 25 mM sodium phosphate buffer (pH 8.5) + 2M MeCN) (starting from 100% A, 0% B phase and ending with 0% A phase, 100% B phase). The anion exchange column was then washed with a mixture of five column volumes of phase B and phase A, and finally equilibrated with two column volumes of phase A.

[0124] The anion exchange column was washed with 80% acetic acid aqueous solution (for 20 column volumes) to remove the 4,4'-dimethoxytriphenylmethyl (DMT) protecting group from the 5'-hydroxyl terminus of the oligonucleotide, forming a dehydroxylated oligonucleotide. The anion exchange column was equilibrated with 5 column volumes of phase A, followed by linear elution with a mixture of 20 column volumes of phases A and B (starting from 100% phase A, 0% phase B and ending with 0% phase A, 100% phase B). This was then followed by isocratic elution with 2 column volumes of phase B to complete the elution. The elution fraction of the target oligonucleotide with the dehydroxylated protecting group was collected and analyzed by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry). The purity of the product is shown in the figure. Figure 5 .

[0125] Comparative Example 2

[0126] The prior art, Krotz AH, McElroy B, Scozzari AN, et al. Controlled detritylation of antisense oligonucleotides[J]. Organic process research & development, 2003, 7(1): 47-52, describes the purification of oligonucleotides using reversed-phase high-performance liquid chromatography (RP-HPLC), which mainly includes the following steps: reversed-phase column purification, ultrafiltration desalting and concentration, de-DMT (4,4'-dimethoxytriphenylmethyl) removal and purification on an anion exchange column, and further ultrafiltration desalting and concentration. Comparative Example 2 describes the purification of crude oligonucleotides according to the method disclosed in this prior art.

[0127] The crude oligonucleotide solution was loaded onto a reversed-phase C18 column (Unisil C18, 10-120, SS 20 * 250 mm, column volume 78.5 mL) using 5 column volumes of mobile phase A1 (50 mM TEAA aqueous solution, pH=6.5). Mobile phase B1 was pure acetonitrile with a loading of approximately 200 OD / mL. The purification equipment used was a Boyun Bio Prep100. After the crude oligonucleotide was loaded onto the reversed-phase C18 column, it was eluted isocratically with a mixture of 2 column volumes of 90% A1 and 10% B1 mobile phases to equilibrate. Then, 20 column volumes of linear elution were performed, linearly changing the mobile phase ratio from 90% A1 + 10% B1 to 10% A1 + 90% B1. Finally, elution was performed isocratically with a mixture of 2 column volumes of 10% A1 and 90% B1 mobile phases to terminate the elution. The elution fraction containing the target oligonucleotide with the DMT protecting group was collected and concentrated by ultrafiltration desalting to allow for re-loading. In the prior art, deprotection is performed in solution. In this comparative example, to improve sample purity, deprotection was performed on an anion exchange column, followed by elution.

[0128] The concentrate obtained from ultrafiltration desalting was loaded onto an anion exchange column (with Source 30Q column media, 1.0 cm inner diameter, approximately 18 cm column height, and approximately 14.13 mL column volume) at a loading of 800 OD / mL. The purification equipment was an ÄKTA™ Pure 150. The column was washed with 5 column volumes of A2 mobile phase (20 mM sodium phosphate buffer, pH 7.5). The anion exchange column was washed with 80% acetic acid aqueous solution (10 column volumes) to remove the DMT protecting group from the target oligonucleotide. The column was then equilibrated with 10 column volumes of A2 mobile phase. Linear elution was then initiated with 100% A2 mobile phase and 0% B2 mobile phase (20 mM sodium phosphate + 1.0 M sodium chloride, pH = 7.5). After 20 column volumes, the ratio of A2 and B2 mobile phases became 50 / 50. Another 1 column volume of linear elution was performed, reducing the A2 mobile phase ratio to 0% and the B2 mobile phase ratio to 100%. Finally, 2 column volumes of 100% B2 mobile phase were used to complete the elution. The eluent containing the target oligonucleotide was collected and analyzed by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry). The purity of the product is shown in the figure. Figure 6 .

[0129] Comparative Example 3

[0130] Existing technology CN1155887A provides a method for purifying oligonucleotides using a hydrophobic chromatography (HIC) column + anion exchange column. First, the HIC column is used to remove oligonucleotides without DMT, then the triphenylmethyl group is removed, followed by purification using a DEAD-5PW anion exchange column, and finally ultrafiltration and lyophilization. This comparative example purifies crude oligonucleotides according to the method disclosed in this prior art.

[0131] The crude oligonucleotide solution (adjusted to 0.5M ammonium sulfate solution with 1M ammonium sulfate) was loaded onto a hydrophobic column (UniHR Phenyl 30L, 1.0 * 18.0 cm, column volume 14.13 mL) using a mobile phase A1 (0.5M ammonium sulfate + 50 mM Tris, pH=8.5) with a loading of approximately 400 OD / mL. The purification equipment used was an ÄKTA™ Pure 150. After the oligonucleotide solution was loaded onto a hydrophobic chromatographic column, it was equilibrated with 5 column volumes of A1 mobile phase. Then, linear elution was performed with a mixture of 5 column volumes of A1 and B1 mobile phase (50 mM ammonium sulfate + 50 mM Tris, pH=8.5), with the ratio of A1 to B1 mobile phase varying from 100% A1, 0% B1 to 0% A1, 100% B1. Finally, 5 column volumes of 100% B1 mobile phase were eluted isocratically. The eluent containing the target oligonucleotide with the DMT protecting group was collected, desalted, and concentrated by ultrafiltration in preparation for the second purification step.

[0132] The concentrated solution obtained by ultrafiltration desalting was loaded onto an anion exchange column (with Source 30Q column media, inner diameter of 1.0 cm, column height of approximately 18 cm, and column volume of approximately 14.13 mL) at a loading of 800 OD / mL. The column was washed with 5 column volumes of A2 mobile phase (50 mM Tris-HCl, pH=7.2). The anion exchange column was washed with 80% acetic acid aqueous solution (10 column volumes) to remove the DMT protecting group from the target oligonucleotide. The column was then equilibrated with 10 column volumes of A2 mobile phase, followed by linear elution with a 30 column volume mixture of A2 and B2 mobile phases (50 mM Tris-HCl + 2M NaCl, pH=7.2). The ratio of A2 to B2 mobile phase was changed from 100% A2, 0% B2 to 0% A2, 100% B2. Isocratic elution was then performed for 2 column volumes with 100% B2 mobile phase. The elution fraction containing the target oligonucleotide was collected and detected by IP-HPLC-UV-MS (i.e., ion-pair reversed-phase liquid chromatography-mass spectrometry).

[0133] The purity, yield, and impurity levels of the products from the five cases mentioned above are summarized in Table 6 below.

[0134] Table 6

[0135]

[0136] As shown in Table 6 above, Comparative Example 1, using the one-step purification method for oligonucleotides disclosed in existing technology CN114051499A, yielded a product with relatively low levels of pre-eluting impurities (also known as "pre-impurities," short fragments of ineffective sequences), but relatively high levels of post-eluting impurities (also known as "post-impurities," long fragments of sequences, branched compounds, etc.). Comparative Example 2 is a commonly used method for purifying oligonucleotides, employing reversed-phase high-performance liquid chromatography (HPLC) in the first step and anion exchange column in the second step. The overall content of pre-eluting, post-eluting, and co-eluting impurities was relatively low. Comparative Example 3 is a two-step purification method using a hydrophobic chromatography column followed by anion exchange column purification. Since the first step of hydrophobic chromatography can only achieve baseline separation of pre-eluting impurities and cannot effectively separate post-eluting impurities, its impurity levels are comparable to the one-step method in Comparative Example 1. Furthermore, the hydrophobic chromatography column has only half the loading capacity of the anion exchange column, further limiting the application of this purification method in large-scale oligonucleotide purification. Examples 1 and 2 employ the method of this invention to purify oligonucleotides. The first step involves column chromatography purification of crude oligonucleotides containing DMT. By equilibration and gradient elution, some pre-eluting impurities are removed, while stronger post-eluting impurities remain on the column. These are then removed from the column by a mixture of alcohol and salt solution. Therefore, in the second step of column chromatography purification, after the removal of the protecting group, the target oligonucleotide is further eluted by gradient elution. The resulting sample has a high purity, which can reach or even surpass the purification effect of the reversed-phase chromatography column + anion exchange column purification mode. Moreover, the oligonucleotide purification method of this invention does not have the limitations of low loading capacity of reversed-phase chromatography columns and medium loading capacity of hydrophobic chromatography columns, thus having certain advantages in large-scale production.

[0137] 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 preparation method includes the following steps: (a) Loading crude oligonucleotides onto an anion exchange column, 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 the buffer solution, then elute the target oligonucleotide with hydroxyl protecting groups using a gradient of the buffer solution and the buffer solution containing alkali metal halides, collect the elution fraction containing the target oligonucleotide with hydroxyl protecting groups, and perform ultrafiltration desalting to obtain a concentrate. (c) Wash the anion exchange column with an alcohol-containing salt solution; (d) Load the concentrate obtained in step (b) onto the anion exchange column; (e) Equilibrate the anion exchange column with the buffer solution and then wash the anion exchange column with an acidic aqueous solution to remove the hydroxyl protecting group of the oligonucleotide; (f) Equilibrate the anion exchange column with the buffer solution, then elute the target oligonucleotide with a gradient of the buffer solution and the buffer solution containing an alkali metal halide to remove the hydroxyl protecting group. Collect the elution fraction containing the target oligonucleotide, desalt it by ultrafiltration, and remove water to obtain the oligonucleotide. In steps (a), (b), (d), (e), and (f), the pH of the buffer solution is 7 to 11.

2. The method for purifying oligonucleotides according to claim 1, characterized in that, In steps (a), (b), (d), (e), and (f), the buffer solution satisfies one or more of the following characteristics: (i) It is selected from phosphate buffer, acetate buffer, carbonate buffer, ethylenediaminetetraacetic acid buffer or mixtures thereof, preferably phosphate buffer or acetate buffer; (ii) The salt content is 10 mM to 100 mM, more preferably 15 mM to 50 mM. (iii) Its pH is 7.5-9.0; (iv) The amount of buffer solution used to equilibrate the anion exchange column is 3 to 8 times the column volume.

3. The method for purifying oligonucleotides according to claim 1, characterized in that, Step (b) satisfies one or more of the following characteristics: (i) The alkali metal halide is selected from sodium chloride, sodium bromide or sodium iodide, preferably sodium bromide; (ii) The molar concentration of the alkali metal halide in the buffer solution containing the alkali metal halide is 1.0 M to 3.0 M, more preferably 1.0 M to 2.5 M; (iii) The amount of the mixture of the buffer solution and the buffer solution containing the alkali metal halide is 15 to 50 times the column volume.

4. The method for purifying oligonucleotides according to claim 1, characterized in that, In steps (b) and (f), the ultrafiltration membrane package used for ultrafiltration desalination meets one or more of the following characteristics: (i) Its materials are selected from regenerated cellulose and / or polyethersulfone; (ii) Its size is 1 KD to 3 KD, preferably 2 KD.

5. The method for purifying oligonucleotides according to claim 1, characterized in that, Step (c) satisfies one or more of the following characteristics: (i) The alcohol is selected from ethanol, methanol, isopropanol, and more preferably isopropanol; (ii) The volume content of alcohol in the alcohol-containing salt solution is 15-5%, more preferably 25-40%; (iii) The salt is sodium chloride or sodium bromide, preferably sodium chloride; (iv) The molar concentration of the salt solution is 0.5 M to 4 M, more preferably 1 M to 2 M.

6. The method for purifying oligonucleotides according to claim 1, characterized in that, The acidic aqueous solution in step (e) satisfies one or more of the following characteristics: (i) The acid in the acidic aqueous solution is selected from carbonic acid, formic acid, acetic acid, haloacetic acid, preferably acetic acid. (ii) The amount of the acidic aqueous solution used is 5 to 25 times the column volume, more preferably 5 to 15 times.

7. The method for purifying oligonucleotides according to claim 1, characterized in that, The buffer solution containing the alkali metal halide in step (f) satisfies one or more of the following characteristics: (i) The alkali metal halide is selected from sodium chloride, sodium bromide or sodium iodide, preferably sodium chloride; (ii) The molar concentration of the alkali metal halide in the buffer solution is 1.0 M to 3.0 M, more preferably 1.0 M to 2.0 M; (iii) The amount of the mixture of the buffer solution and the buffer solution containing the alkali metal halide is 15 to 50 times the column volume.

8. The method for purifying oligonucleotides according to claim 1, characterized in that, The crude oligonucleotide product has one or more of the following characteristics: (i) Contains one or more of the following: failure sequence nx impurities, n+x impurities, and P=O impurities. (ii) Its UV purity is not less than 75%, (iii) The hydroxyl protecting group is 4,4'-dimethoxytriphenylmethyl. (iv) The nucleotide sequence of the oligonucleotide is shown in SEQ ID NO.

1.

9. The method for purifying oligonucleotides according to claim 1, characterized in that, The resin of the anion exchange column is a strongly basic anion exchange resin with a particle size greater than or equal to 15 μm, more preferably 20~50 μm.

10. The method for purifying oligonucleotides according to claim 1, characterized in that, Before loading the crude oligonucleotide or the concentrate onto the anion exchange column, the anion exchange column is equilibrated with the buffer solution.

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