Strong anion exchange filler for purifying oligonucleotide and preparation method thereof

Abstract

The invention discloses a strong anion exchange filler for purifying oligonucleotide and a preparation method of the strong anion exchange filler, and belongs to the technical field of biochemical purification. The preparation method comprises the following steps: carrying out PEG (Polyethylene Glycol) hydrophilic modification on PMMA (Polymethyl Methacrylate) mesoporous microspheres with the particle size of 15 microns and the pore diameter of # imgabs0 #, substituting with sulfoxide halide, and aminating with a sulfur trioxide trimethylamine compound. The HNmicro-15Q filler prepared by the method has excellent adsorption performance, and the oligonucleotide load of the HNmicro-15Q filler is up to 38.4 mg / mL; meanwhile, excellent alkali resistance is achieved, and the load decline rate is only 2.6% when an oligonucleotide and BSA alternate test is carried out; moreover, the anion exchange filler has excellent pressure resistance and good separation performance for oligonucleotides with different base pairs, and is a strong anion exchange filler with huge potential and practical significance.

Description

Technical Field

[0001] The present application relates to a strong anion exchange filler for purifying oligonucleotides and a preparation method thereof, belonging to the technical field of biochemical purification. Background Art

[0002] Synthetic oligonucleotides are short single-stranded DNA sequences composed of multiple nucleotide units linked together by phosphodiester bridges. They are primarily used in antisense drug therapy, but also have a wide range of applications in molecular biology and other life sciences. Synthetic oligonucleotides are generally generated by phosphoramidite chemistry on a solid support consisting of cross-linked polystyrene beads. In each coupling cycle, a small portion of the oligonucleotide chain will not be extended, resulting in full-length (n) and truncated ineffective sequences (n-1, n-2). In addition, there are byproducts of small molecule impurities during the cleavage and deprotection processes. Since high-purity synthetic oligonucleotides are a prerequisite for therapeutic and other applications, they need to be purified.

[0003] Most purification procedures are based on gel electrophoresis, reverse phase chromatography (RP-HPLC) or anion exchange chromatography (IE-HPLC). Gel electrophoresis is only suitable for small-scale oligonucleotide purification and has a narrow scope of application and is not suitable for large-scale production. Anion exchange high performance liquid chromatography (IE-HPLC) is based on the number of charged groups (phosphates) in the oligonucleotide backbone. Anion exchange chromatography involves the use of a salt gradient mobile phase on a quaternary ammonium stationary phase, and has excellent resolution for purifications below 40 bases, especially small amounts. The main advantage of using IE-HPLC over RP-HPLC is in the purification of oligonucleotides with significant secondary structure, which are often found in sequences with high GC content. IE-HPLC is very effective for such oligonucleotides, but due to the highly alkaline pH of the mobile phase, this disrupts hydrogen bonds and thus the secondary structure. This technology can be combined with RP-HPLC to add a second dimension of protection to the separation process. Based on the above, it is particularly necessary to develop anion exchange fillers with high alkaline resistance to meet the needs. Summary of the invention

[0004] The present application provides a method for preparing a strong anion exchange filler for purifying oligonucleotides. The strong anion exchange filler prepared by the method has high loading capacity, alkali resistance and excellent separation performance.

[0005] In order to solve the above problems, the technical solutions adopted in this application are as follows:

[0006] The present application provides a method for preparing a strong anion exchange filler for purifying oligonucleotides, the preparation method comprising the following steps:

[0007] S1. Mixing the continuous phase and the dispersed phase to synthesize mesoporous PMMA microspheres;

[0008] S2. The mesoporous PMMA microspheres of step S1 are hydrophilically modified to obtain PMMA-PEG microspheres;

[0009] S3. The PMMA-PEG microspheres of step S2 are mixed with sulfoxide halide to obtain chlorine balls;

[0010] S4. The chlorine balls obtained in step S3 are mixed with a tertiary amine compound and water to obtain HNmicro-15Q, i.e., a 15 μm strong anion exchange filler.

[0011] Furthermore, in step S1, the volume ratio of the continuous phase to the dispersed phase is 30:2.9.

[0012] Furthermore, in step S1, the continuous phase includes a 4% PVA aqueous solution, and the dispersed phase includes methyl methacrylate, divinylbenzene, toluene, and azobisisobutyronitrile.

[0013] Furthermore, the volume ratio of methyl methacrylate, divinylbenzene and toluene is 2:0.1:0.8.

[0014] Furthermore, in step S1, the mixing specifically includes: reacting at 160 rpm and 60°C for 1 hour, rising to 70°C and reacting for another 4 hours, washing the obtained microspheres three times with ethanol and deionized water respectively, soaking them with 95% ethanol, and shaking them in a shaker for 6 hours to remove the porogen, thereby obtaining 15 μm mesoporous PMMA microspheres.

[0015] Furthermore, in step S1, the mixing specifically includes: the dispersed phase is 2mL methyl methacrylate, 0.1mL divinylbenzene, 0.8mL toluene, and 0.04g azobisisobutyronitrile; the continuous phase is 30mL 4% PVA aqueous solution, the continuous phase and the dispersed phase are mixed, reacted at 160rpm and 60°C for 1h, and then reacted at 70°C for another 4h. The obtained microspheres are washed three times with ethanol and deionized water respectively, soaked with 95% ethanol, and shaken in a shaker for 6h to remove the porogen to obtain 15μm mesoporous PMMA-DVB microspheres.

[0016] Furthermore, in step S2, the specific steps of the hydrophilic modification include: reacting a hydroxyl polymer, olefinic acid, N,N′-dicyclohexylcarbodiimide (DCC), and 4-N,N-dimethylpyridine (DMAP) in CH2Cl2 at room temperature overnight, and refluxing the reaction product with the mesoporous PMMA microspheres and SnCl4 in step S1 under nitrogen protection for 48 hours to obtain PMMA-PEG microspheres.

[0017] Furthermore, the mass ratio of the hydroxy polymer, olefinic acid, N,N'-dicyclohexylcarbodiimide, and 4-N,N-dimethylpyridine is 2:0.6:0.6:0.12; and the mass ratio of the mesoporous PMMA microspheres to SnCl4 is 5:2.

[0018] Furthermore, the hydroxy polymer includes one or any combination of PEG 600, PEG 2000, PEG 4000, and PEG 6000. Furthermore, the hydroxy polymer is PEG 4000.

[0019] Further, the enoic acid includes one or any combination of 4-pentenoic acid, but-3-enoic acid, and 4-methylpent-4-enoic acid. Further, the enoic acid is 4-pentenoic acid.

[0020] Furthermore, in step S2, the specific steps of the hydrophilic modification include: reacting PEG 4000, 4-pentenoic acid, N,N′-dicyclohexylcarbodiimide (DCC), and 4-N,N-dimethylpyridine (DMAP) in CH2Cl2 at room temperature overnight, and refluxing the reaction product with the mesoporous PMMA microspheres and SnCl4 in step S1 under nitrogen protection for 48 hours to obtain PMMA-PEG microspheres.

[0021] Furthermore, in step S2, the specific steps of the hydrophilic modification include: dissolving PEG-4000 in CH2Cl2, adding 4-pentenoic acid and 4-N,N-dimethylpyridine (DMAP), stirring at room temperature for 5 minutes, and then adding DCC (N,N-dicyclohexyl carbonate diimide) dissolved in CH2Cl2; stirring at room temperature overnight, filtering and removing the solvent, dissolving the remaining product in CH2Cl2, extracting with saturated NaHCO3 aqueous solution, drying with Na2SO4, and then extracting with ether, repeating the process twice to obtain a white solid; adding the obtained white solid, mesoporous PMMA microspheres, and SnCl4 to 10mL of toluene, reflux under nitrogen protection for 48h; washing three times with ethanol, filtering with a sand core funnel to obtain PMMA-PEG microspheres.

[0022] Furthermore, in step S2, the specific steps of the hydrophilic modification include: dissolving 2g PEG-4000 in 10mL of CH2Cl2, adding 0.6g 4-pentenoic acid and 0.12g DMAP (4-N,N-dimethylpyridine), stirring at room temperature for 5min, and then adding 0.6g DCC (N,N-dicyclohexyl carbonate diimide) dissolved in 5mL CH2Cl2; stirring at room temperature overnight, filtering and removing the solvent, the remaining product is dissolved in CH2Cl2, extracted with saturated NaHCO3 aqueous solution, dried with Na2SO4, and then extracted with ether, and the process is repeated twice to obtain a white solid; the obtained white solid, 5g mesoporous PMMA microspheres, and 2g SnCl4 are added to 10mL toluene, and refluxed under nitrogen protection for 48h; washed with ethanol three times, filtered with a sand core funnel, and PMMA-PEG microspheres are obtained.

[0023] Further, in step S3, the sulfoxide halogenide includes one or any combination of sulfoxide chloride and sulfoxide bromide. Further, the sulfoxide halogenide is sulfoxide chloride.

[0024] Furthermore, in step S3, the PMMA-PEG microspheres are mixed with the halogenated sulfoxide under the following conditions: a rotation speed of 160 rpm, a temperature of 50° C., and a time of 16 h.

[0025] Furthermore, in step S3, the specific steps of mixing the PMMA-PEG microspheres with thionyl chloride include: mixing PMMA-PEG microspheres, thionyl chloride, and tetrahydropyran and ultrasonically dispersing them, reacting at 160 rpm and 50° C. for 16 hours, washing with 50% ethanol aqueous solution for 3 times, and filtering with a sand core funnel to obtain chloride balls.

[0026] Furthermore, the mass ratio of the PMMA-PEG microspheres, sulfoxide halide and tetrahydropyran is 5:0.2:10.

[0027] Furthermore, in step S3, the specific steps of mixing the PMMA-PEG microspheres with sulfoxide halides include: mixing 5 g of PMMA-PEG microspheres, 0.2 g of sulfoxide chloride, and 10 g of tetrahydropyran and ultrasonically dispersing them, reacting at 160 rpm and 50° C. for 16 h, washing three times with 50% ethanol aqueous solution, and filtering with a sand core funnel to obtain chloride balls.

[0028] Furthermore, in step S4, the tertiary amine compound includes one or any combination of trimethylamine, trimethylamine dihydrate, trimethylamine hydrochloride, sulfur trioxide trimethylamine complex. Furthermore, the tertiary amine compound is sulfur trioxide trimethylamine complex.

[0029] Furthermore, in step S4, the conditions for mixing the chlorine ball with the tertiary amine compound and water include: temperature of 40° C. and time of 12 hours.

[0030] Furthermore, in step S4, the specific steps of mixing the chlorine ball with the tertiary amine compound and water include: mixing the chlorine ball, sulfur trioxide trimethylamine complex and water and ultrasonically dispersing them, reacting them at 160 rpm and 40° C. for 12 hours, washing them with 50% ethanol aqueous solution for 3 times, and filtering them with a sand core funnel to obtain HNmicro-15Q.

[0031] Furthermore, in step S4, the specific steps of mixing the chlorine ball with the tertiary amine compound and water include: mixing 5g of chlorine ball, 15g of sulfur trioxide trimethylamine complex, and 10g of water and ultrasonically dispersing, reacting at 160rpm and 40°C for 12h, washing 3 times with 50% ethanol aqueous solution, and filtering with a sand core funnel to obtain HNmicro-15Q.

[0032] Furthermore, the mass ratio of the chlorine ball, the tertiary amine compound and water is 1:3:2.

[0033] The present application also provides a strong anion exchange filler for purifying oligonucleotides, and the strong anion exchange filler is prepared using the aforementioned preparation method.

[0034] Beneficial effects of this application:

[0035] (1) The present application provides a strong anion exchange filler for purifying oligonucleotides. By connecting strong anion exchange groups, such as tertiary amines, etc., to interact with the negatively charged groups carried by the oligonucleotide chains, a salt concentration gradient is used to gradually reduce the interaction between the oligonucleotides and the cationic phase of the chromatographic column. Longer oligonucleotides with higher charges are eluted and separated from impurities with shorter chains, so that the obtained strong anion exchange filler has excellent adsorption properties, and the oligonucleotide loading capacity is as high as 38.4 mg / mL.

[0036] (2) The purification of oligonucleotides by the strong anion exchange filler provided in the present application is generally carried out in an alkaline environment with a high salt concentration. The PMMA polymer as a filler matrix itself has alkali resistance. In the present application, an additional PEG hydrophilic layer is coated to prevent alkaline substances from contacting the filler and protect the ester groups in the polymer from being destroyed. The prepared strong anion exchange filler has excellent alkali resistance and can maintain excellent and stable performance under high alkaline conditions (pH = 14). The oligonucleotide and BSA alternating test loading decrease rate is only 2.6%.

[0037] (3) The strong anion exchange filler in the present application has a good effect on the separation of oligonucleotide sequences with different base numbers, especially for full-length oligonucleotide sequences and truncated invalid sequences (n-1, n-2). BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the BET test diagram of HNmicro-15Q in Example 2 of the present application.

[0039] Figure 2 This is a test chart of the oligonucleotide loading capacity of the HNmicro-15Q 1mL prepacked column in Example 3 of the present application.

[0040] Figure 3 This is the linear elution diagram of the HNmicro-15Q 1 mL prepacked column in Example 6 of the present application. DETAILED DESCRIPTION

[0041] The present application is further described below in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and cannot be used to limit the protection scope of the present application.

[0042] Before introducing the embodiments of the present application, the relevant terms involved in the embodiments of the present application are explained as follows:

[0043] Oligonucleotide: A linear polynucleotide fragment composed of 2 to 10 nucleotide residues connected by phosphodiester bonds. Oligonucleotides can be used as primers and gene probes for DNA synthesis, and are widely used in gene chips, electrophoresis, fluorescence in situ hybridization and other processes. Because oligonucleotides can be linked to their complementary pairs, they play the role of code recognizers in nucleic acid hybridization technology and are used to detect specific DNA or RNA sequences.

[0044] Deionized water: Deionized water is pure water obtained by removing ionic impurities in water through a specific treatment process, such as ion exchange resin or reverse osmosis membrane.

[0045] Ion exchange filler: It is composed of three parts: polymer matrix, charged group and counter ion. Its core principle is to use the difference in electrostatic force between ions in the sample and the charged groups on the filler for separation. According to the charged nature of the functional group, ion exchange fillers can be divided into cation exchange fillers and anion exchange fillers. The charged groups of cation exchange fillers are negatively charged, while the charged groups of anion exchange fillers are positively charged.

[0046] BET test: It is based on the theoretical framework proposed by three scientists, Brunauer, Emmett and Teller. The theory elaborates on the phenomenon of multi-molecular layer adsorption, and establishes the mathematical relationship between the single-layer adsorption amount and the multi-layer adsorption amount through the BET equation. The specific surface area of ​​the material is calculated by measuring the adsorption amount of gas on the solid surface.

[0047] Linear elution: Linear elution refers to the elution of sample components by gradually increasing the polarity of the mobile phase during chromatography. This method achieves separation based on the difference in interaction between different components and the stationary phase and mobile phase.

[0048] The present application provides a method for preparing a strong anion exchange filler for purifying oligonucleotides, comprising the following steps:

[0049] S01. The continuous phase and the dispersed phase are mixed to synthesize mesoporous PMMA microspheres;

[0050] In step S01, the continuous phase refers to the liquid phase with a higher content in the emulsion or two-phase aqueous system, which exists as a medium for dispersing other substances; the dispersed phase refers to the substance dispersed in the continuous medium, usually in the form of fine particles or droplets.

[0051] In an embodiment of the present application, the continuous phase can be 30 mL of a 4% PVA aqueous solution, which is a transparent liquid formed by dissolving polyvinyl alcohol (PVA) in water and can be prepared by a known method; the dispersed phase can be 2 mL of methyl methacrylate (MMA), 0.1 mL of divinylbenzene (DVB), 0.8 mL of toluene (porogen), and 0.04 g of azobisisobutyronitrile (AIBN), wherein methyl methacrylate can be a commercially available product or prepared by a known method. In an embodiment of the present application, the PVA aqueous solution is prepared by the acetone cyanohydrin method.

[0052] In the embodiment of the present application, the continuous phase and the dispersed phase are mixed, and the specific process for synthesizing mesoporous PMMA microspheres is: react at 160rpm and 60°C for 1h, and then react for another 4h after the temperature rises to 70°C. The obtained microspheres are washed three times with ethanol and deionized water respectively, immersed in 95% ethanol, and shaken in a shaker for 6h.

[0053] S02. The mesoporous PMMA microspheres of step S01 are hydrophilically modified to obtain PMMA-PEG microspheres.

[0054] In step S02, the hydrophilic modification is to react a hydroxyl polymer, olefinic acid, N,N'-dicyclohexylcarbodiimide (DCC), and 4-N,N-dimethylpyridine (DMAP) in CH2Cl2 at room temperature overnight, and the reaction product is refluxed with the mesoporous PMMA microspheres and SnCl4 in step S01 under nitrogen protection for 48 hours to obtain PMMA-PEG microspheres.

[0055] The hydroxy polymer may be one of PEG 600, PEG 2000, PEG 4000, and PEG 6000, or any combination thereof.

[0056] In the embodiment of the present application, the hydroxyl polymer is PEG 4000. PEG 4000 forms hydrogen bonds with water molecules through the hydroxyl groups on its surface, thereby increasing the hydrophilicity of the material surface. PEG 4000 can also serve as a linker to help form a stable network structure.

[0057] The enoic acid may be one or any combination of 4-pentenoic acid, but-3-enoic acid, and 4-methylpent-4-enoic acid.

[0058] In the embodiment of the present application, the enoic acid is 4-pentenoic acid, which can provide a reactive carboxyl group and undergo an esterification reaction with PEG to form a polymer chain.

[0059] The N,N′-dicyclohexylcarbodiimide (DCC) is a bifunctional crosslinking agent, which is commonly used in reactions such as esterification and amidation, and can also be used in the synthesis of anhydrides, aldehydes, ketones, and isocyanates.

[0060] In the examples of the present application, the N,N'-dicyclohexylcarbodiimide (DCC) is used as a coupling agent to connect PEG and 4-pentenoic acid to form a copolymer.

[0061] In the present application example, the 4-N, N-dimethylpyridine (DMAP) is a super strong nucleophilic acylation catalyst. The resonance energy of the electron-donating dimethylamino group and the parent ring (pyridine ring) in its structure strongly activates the nitrogen atom on the ring for nucleophilic substitution, significantly catalyzing the acylation / esterification reaction of alcohols and amines / acids with high steric hindrance and low reactivity. The role of DMAP in the present application example is to promote the reaction between PEG (polyethylene glycol) and 4-pentenoic acid, forming a specific chemical bond, thereby connecting to the PMMA microspheres. The presence of DMAP can accelerate this process and improve the efficiency of the reaction and the yield of the product.

[0062] In the embodiment of the present application, the specific implementation steps of step S02 include: dissolving 2g PEG-4000 in 10mL of CH2Cl2, adding 0.6g 4-pentenoic acid and 0.12g 4-N,N-dimethylpyridine (DMAP), stirring at room temperature for 5min, and then adding 0.6g DCC (N,N-dicyclohexyl carbonate diimide) dissolved in 5mL CH2Cl2; stirring at room temperature overnight, filtering and removing the solvent, the remaining product is dissolved in CH2Cl2, extracted with saturated NaHCO3 aqueous solution, dried with Na2SO4, and then extracted with ether, and the process is repeated twice to obtain a white solid; the obtained white solid, 5g mesoporous PMMA microspheres, and 2gSnCl4 are added to 10mL toluene, and refluxed under nitrogen protection for 48h; washed with ethanol three times, filtered with a sand core funnel, and PMMA-PEG microspheres are obtained.

[0063] Among them, CH2Cl2 dichloromethane is a commonly used organic solvent, which is used to dissolve PEG, 4-pentenoic acid, DMAP, DCC and other substances, and acts as a solvent in the reaction process; saturated NaHCO3 aqueous solution is used to extract the product from the organic phase, because many organic molecules are insoluble in an aqueous environment; Na2SO4 is used as a desiccant to remove moisture from the extract and prevent subsequent reactions from being affected by moisture; ether is used to extract the product and transfer the product from the aqueous phase to the organic phase for further processing and purification; SnCl4 tin tetrachloride may act as a catalyst or cross-linking agent in this reaction, which helps to form a stable PMMA-PEG microsphere structure; toluene is used as the solvent and reflux medium of the reaction, which helps to maintain the reaction temperature and promote the reaction; ethanol is used to wash the product to remove any residual impurities or catalysts.

[0064] S03. Mix the PMMA-PEG microspheres of step S02 with sulfoxide halide to obtain chlorine balls.

[0065] In step S03, the sulfoxide halide is one or any combination of sulfoxide chloride and sulfoxide bromide.

[0066] In the embodiment of the present application, the sulfoxide halide is sulfoxide chloride. As a strong chlorinating agent, sulfoxide chloride can react with specific groups on PMMA-PEG microspheres to introduce chlorine atoms, thereby changing the chemical properties of the microsphere surface. Through the chlorination reaction, the active sites on the microsphere surface can be increased, providing more possibilities for subsequent functionalization, such as connecting biomolecules or other functional polymers.

[0067] In the embodiment of the present application, the specific process of mixing PMMA-PEG microspheres with sulfoxide halide to obtain chlorine balls includes: 5g PMMA-PEG microspheres, 0.2g thionyl chloride, and 10g tetrahydropyran are mixed and ultrasonically dispersed, and reacted at 160rpm and 50°C for 16h. Wash with 50% ethanol aqueous solution for 3 times, and filter with a sand core funnel to obtain chlorine balls.

[0068] Among them, tetrahydropyran may be used as a solvent in this reaction to provide a suitable reaction environment, which helps to improve the selectivity and efficiency of the reaction; 50% ethanol aqueous solution is used to wash the microspheres after the reaction to remove unreacted reagents and by-products, and at the same time help stabilize the structure of the microspheres.

[0069] S04. The chlorine balls obtained in step S03 are mixed with a tertiary amine compound and water to obtain HNmicro-15Q, i.e., a 15 μm strong anion exchange filler.

[0070] In step S04, the tertiary amine compound may be one or any combination of trimethylamine, trimethylamine dihydrate, trimethylamine hydrochloride, and sulfur trioxide trimethylamine complex.

[0071] In the embodiment of the present application, the tertiary amine compound is a sulfur trioxide trimethylamine complex, which may be used as a modifier in this reaction, reacting with the chlorine atoms on the surface of the chlorine ball to introduce new functional groups. This complex usually has strong reactivity and selectivity. Through the reaction with the chlorine ball, the sulfur trioxide trimethylamine complex can change the chemical properties of the surface of the chlorine ball, making it more suitable for subsequent applications or further functionalization. This modification is of great significance for improving the performance and application range of the material.

[0072] In the embodiment of the present application, the specific process of mixing chlorine balls with tertiary amine compounds and water to obtain HNmicro-15Q includes: mixing 5g of chlorine balls, 15g of sulfur trioxide trimethylamine complex, and 10g of water and ultrasonically dispersing them, reacting at 160rpm and 40°C for 12h, washing 3 times with 50% ethanol aqueous solution, and filtering with a sand core funnel to obtain HNmicro-15Q.

[0073] The following are specific embodiments of the present application.

[0074] Example 1

[0075] A method for preparing a strong anion exchange filler for purifying oligonucleotides, the specific steps are as follows:

[0076] (1) The continuous phase and the dispersed phase were mixed and reacted at 160 rpm and 60° C. for 1 h. The temperature was raised to 70° C. and then the reaction was continued for 4 h. The obtained microspheres were washed three times with ethanol and deionized water respectively, soaked with 95% ethanol, and shaken in a shaker for 6 h to remove the porogen, thereby obtaining 15 μm mesoporous PMMA microspheres. Wherein:

[0077] Continuous phase (aqueous phase): 30 mL 4% PVA aqueous solution;

[0078] Dispersed phase (oil phase): 2 mL methyl methacrylate (MMA), 0.1 mL divinylbenzene (DVB), 0.8 mL toluene (porogen), 0.04 g azobisisobutyronitrile (AIBN).

[0079] (2) 2 g PEG (Mn 4000) was dissolved in 10 mL CH2Cl2, and then 0.6 g 4-pentenoic acid and 0.12 g 4-N, N-dimethylpyridine (DMAP) were added. After stirring at room temperature for 5 min, 0.6 g DCC (N, N-dicyclohexyl carbonate diimide) dissolved in 5 mL CH2Cl2 was added; the mixture was stirred at room temperature overnight, filtered and the solvent was removed, the remaining product was dissolved in CH2Cl2, extracted with saturated NaHCO3 aqueous solution, dried with Na2SO4, and extracted with ether. The process was repeated twice to obtain a white solid; the obtained white solid, 5 g mesoporous PMMA microspheres, and 2 g SnCl4 were added to 10 mL toluene, and refluxed under nitrogen protection for 48 h; washed with ethanol three times, and filtered with a sand core funnel to obtain PMMA-PEG microspheres.

[0080] (3) 5 g of PMMA-PEG microspheres, 0.2 g of thionyl chloride, and 10 g of tetrahydropyran were mixed and ultrasonically dispersed, reacted at 160 rpm and 50° C. for 16 h, washed three times with 50% ethanol aqueous solution, and filtered with a sand core funnel to obtain chloride balls.

[0081] (4) 5 g of chlorine ball, 15 g of sulfur trioxide trimethylamine complex, and 10 g of water were mixed and ultrasonically dispersed, reacted at 160 rpm and 40° C. for 12 h, washed three times with 50% ethanol aqueous solution, and filtered with a sand core funnel to obtain HNmicro-15Q.

[0082] Example 2 BET test

[0083] (1) Sample preparation

[0084] Place the HNmicro-15Q filler sample to be tested into the sample tube, ensuring that the sample is evenly distributed and the amount is appropriate, generally 30-500 mg, depending on the specific surface area of ​​the sample.

[0085] (2) Degassing treatment

[0086] Install the sample tube containing the sample into the degassing station, align the port and tighten the screws to ensure sealing; use the heating pack on the sample tube, set the file information and degassing temperature and other parameters; turn on the vacuum pump, heat the sample, and vacuum degas the sample to remove the gas adsorbed on the surface of the material. This step is crucial to ensure the accuracy of the test results.

[0087] (3) Sample weighing

[0088] After degassing, turn off the heating power supply, wait for the sample to cool to room temperature, and then backfill with helium to normal pressure. Remove the sample tube and immediately cover it with a rubber stopper. Use an analytical balance to weigh to 0.1 mg, and record the weight of the helium-filled sample tube, stopper, and filling rod (i.e., the gross weight of the sample tube). Calculate the actual mass of the sample by the reduction method.

[0089] (4) Adsorption and desorption test

[0090] Load the weighed sample tube to the analysis station and add liquid nitrogen to the Dewar flask; input the sample mass into the analysis file, set the test parameters, and start the adsorption and desorption test process; during the test, nitrogen is used as the adsorbate, helium or hydrogen is used as the carrier gas, and the two gases are mixed in a certain proportion, and flow through the solid sample after reaching the specified relative pressure of 0.05-0.35; when the sample tube is placed in liquid nitrogen for insulation, the sample physically adsorbs the nitrogen in the mixed gas; when the liquid nitrogen is taken away, the sample tube is back to room temperature, and the adsorbed nitrogen is desorbed. Finally, a known volume of pure nitrogen is injected into the mixed gas to obtain a calibration peak. According to the peak area of ​​the calibration peak and the desorption peak, the adsorption amount of the sample at the relative pressure can be calculated.

[0091] (5) Data processing

[0092] According to the adsorption and desorption data, the specific surface area of ​​the sample is calculated using the BET formula. The applicable range of the BET formula is p / p0=0.05~0.35, because within this range the BET equation is consistent with the actual adsorption process and the graph linearity is also very good.

[0093] Figure 1 This is the BET test graph of HNmicro-15Q filler. The most probable pore size of this filler is 43nm, indicating that this filler has a pore size of The specific surface area of ​​the filler is as high as 570m 2 / g, with abundant binding sites, which is beneficial for subsequent modification.

[0094] Example 3 Loading Test

[0095] The column was packed with HNmicro-15Q filler (1 mL), and the loading capacity test was performed at a flow rate of 0.5 mL / min.

[0096] The specific steps are as follows:

[0097] (1) Column Packing

[0098] Load the HNmicro-15Q filler into the column, making sure that the filler is evenly distributed and there are no bubbles. The column volume is 1 mL, which means that the volume of the filler is also 1 mL.

[0099] (2) Solution preparation

[0100] The solution preparation method is as follows:

[0101] Balance solution: 10 mM NaOH (pH 11.0), used to adjust the pH environment of the filler and remove possible impurities;

[0102] Loading solution: 10 mg / mL oligonucleotide, as the sample to be separated or purified.

[0103] Washing solution: 10 mM NaOH (pH 11.0), used to remove unadsorbed or non-specifically adsorbed substances.

[0104] Elution buffer: A mixed solution containing 1M NaCl and 10mM NaOH, pH 11.0, used to elute the oligonucleotides adsorbed on the filler.

[0105] (3) Balance

[0106] The equilibration solution (10 mM NaOH, pH 11.0) was passed through the column at a flow rate of 0.5 mL / min until the pH value of the effluent was the same as that of the equilibration solution.

[0107] (4) Sample loading

[0108] Prepare an oligonucleotide solution with a concentration of 10 mgg / mL as the loading solution.

[0109] At a flow rate of 0.5 mL / min, pass the sample solution through the chromatographic column to allow the oligonucleotide to fully contact and adsorb the filler. Record the sample volume.

[0110] (5) Washing

[0111] The equilibration solution (10 mM NaOH, pH 11.0) was continued to be passed through the column at a flow rate of 0.5 mL / min until the effluent no longer contained impurities.

[0112] (6) Elution

[0113] The elution process was started by passing the eluent (1 M NaCl / 10 mM NaOH, pH 11.0) through the column at a flow rate of 0.5 mL / min.

[0114] Collect the eluate and monitor its absorbance or conductivity and other indicators to determine the elution endpoint.

[0115] Figure 2 This is a test chart of the oligonucleotide loading capacity of the HNmicro-15Q 1mL prepacked column.

[0116] The loading capacity is calculated by the following formula:

[0117] Loading capacity = OD洗脱液 / OD 上样液 ×k×V 洗脱液 (mg / mL), where k is the extinction coefficient of the corresponding oligonucleotide;

[0118] In this example, the oligonucleotide loading capacity is as high as 38.4 mg / mL, and the filler has excellent adsorption performance.

[0119] Example 4 Alkali resistance test

[0120] Oligonucleotide and BSA were tested for alternating loading. The test process was the same as in Example 3.

[0121] After 3 oligonucleotide loading tests, 2 BSA loading tests were performed, followed by 1 oligonucleotide loading test.

[0122] The results of the alternating loading test of oligonucleotides and BSA are shown in Table 1. The loading decrease rate is only 2.6%, and the filler has excellent alkali resistance.

[0123] Table 1 Oligonucleotide and BSA alternating loading test results (unit: mg / mL)

[0124] Sample name Nucleotide 1 Nucleotide 2 Nucleotide 3 BSA 1 BSA 2 Nucleotide 4 Drop rate HNmicro-15Q 38.2 37.5 38.9 58.3 38.4 37.2 2.60％

[0125] Example 5: Pressure Withstand Effect Test

[0126] The specific steps are as follows:

[0127] (1) Column Packing

[0128] Load the HNmicro-15Q filler into the column, making sure that the filler is evenly distributed and there are no bubbles. The column volume is 1 mL, which means that the volume of the filler is also 1 mL.

[0129] (2) Solution preparation

[0130] The solution preparation method is as follows:

[0131] Balance solution: 10 mM NaOH (pH 11.0), used to adjust the pH environment of the filler and remove possible impurities;

[0132] Loading solution: 10 mg / mL oligonucleotide, as the sample to be separated or purified.

[0133] Washing solution: 10 mM NaOH (pH 11.0), used to remove unadsorbed or non-specifically adsorbed substances.

[0134] Elution buffer: A mixed solution containing 1 M NaCl and 10 mM NaOH, pH 11.0, used to elute the oligonucleotides adsorbed on the filler.

[0135] (3) Install the chromatographic column

[0136] A chromatographic column filled with HNmicro-15Q filler was connected to a high pressure liquid chromatography (HPLC) system.

[0137] (4) Set flow rate and pressure

[0138] Set the flow rate of the HPLC system to a predetermined value. Start with a lower pressure and gradually increase the pressure to evaluate the pressure resistance of the packing at different pressures.

[0139] (5) Perform a withstand voltage test

[0140] At each pressure point, maintain a period of time (e.g. 10 minutes), observe the effluent from the chromatographic column, and record any abnormalities, such as a sudden drop in pressure, turbidity of the effluent, etc. If the packing shows signs of instability or damage at a certain pressure point, record the pressure value as the pressure limit of the packing.

[0141] (6) Repeat the test

[0142] In order to obtain more accurate results, the pressure resistance test can be repeated multiple times and the average value can be taken as the pressure resistance limit of the filler.

[0143] The pressure flow rate test results are shown in Table 2, and the results show that the filler has excellent pressure resistance.

[0144] Table 2 Pressure and flow rate data table

[0145] Flow rate (mL / min) 1 2 3 4 5 6 7 Pressure(Mpa) 0.196 0.363 0.525 0.675 0.816 0.943 1.080

[0146] Example 6 Linear elution test

[0147] The specific steps are as follows:

[0148] (1) Preparation of pre-packed columns

[0149] Ensure that the HNmicro-15Q 1mL prepacked column has been properly installed and connected to the HPLC system. Check that the connection of the column is tight and leak-free.

[0150] (2) Solution preparation

[0151] The solution preparation method is as follows:

[0152] Balance solution: 10 mM NaOH (pH 11.0), used to adjust the pH environment of the filler and remove possible impurities;

[0153] Loading solution: 10 mg / mL oligonucleotide, as the sample to be separated or purified.

[0154] Washing solution: 10 mM NaOH (pH 11.0), used to remove unadsorbed or non-specifically adsorbed substances.

[0155] Elution buffer: A mixed solution containing 1 M NaCl and 10 mM NaOH, pH 11.0, used to elute the oligonucleotides adsorbed on the filler.

[0156] (3) Balancing the chromatographic column

[0157] A chromatographic column filled with HNmicro-15Q filler was connected to a high pressure liquid chromatography (HPLC) system.

[0158] (4) Sample loading

[0159] The test solution is a mixed solution of oligonucleotides of 16, 19, and 20 base pairs. Use an injector to inject the sample into the chromatographic column and record the amount of sample loaded.

[0160] (5) Set up the elution program

[0161] A linear elution program was set up in the HPLC system, including parameters such as the initial ratio of the eluent, the final ratio, the elution time, and the flow rate. The eluent was set to gradually increase from 10% to 90%, the elution time was 60 minutes, and the flow rate was 0.5 mL / min.

[0162] (6) Elution

[0163] Start the HPLC system and start the elution process. Observe the chromatogram and record the retention time and peak area of ​​each component.

[0164] (7) Data analysis

[0165] The separation and purity of each component were analyzed based on the chromatogram. If necessary, the elution procedure could be further optimized to improve the separation effect.

[0166] Figure 3 This is the linear elution profile of the HNmicro-15Q 1mL prepacked column. The results show that the filler has excellent separation performance for oligonucleotides with different base pairs.

[0167] The above is only a preferred implementation of the present application. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present application. These improvements and modifications should also be regarded as the scope of protection of the present application.

Claims

1. A method for preparing a strong anion exchange filler for purifying oligonucleotides, characterized in that: The preparation method comprises the following steps: S1. Mixing the continuous phase and the dispersed phase to synthesize mesoporous PMMA microspheres; S2. The mesoporous PMMA microspheres of step S1 are hydrophilically modified to obtain PMMA-PEG microspheres; S3. The PMMA-PEG microspheres, halogenated sulfoxide and tetrahydropyran of step S2 are mixed to obtain chlorine balls; S4. The chlorine balls obtained in step S3 are mixed with a tertiary amine compound and water to obtain a strong anion exchange filler.

2. The method for preparing a strong anion exchange filler for purifying oligonucleotides according to claim 1, characterized in that: In step S1, the volume ratio of the continuous phase to the dispersed phase is 30:2.9; the continuous phase includes a 4% PVA aqueous solution, the dispersed phase includes methyl methacrylate, divinylbenzene, toluene, and azobisisobutyronitrile, and the volume ratio of methyl methacrylate, divinylbenzene, and toluene is 2:0.1:0.8; the mixing conditions of the continuous phase and the dispersed phase are stirring and reacting at 60-70°C for 4-5 hours.

3. The method for preparing a strong anion exchange filler for purifying oligonucleotides according to claim 1, characterized in that: In step S2, the specific steps of the hydrophilic modification include: reacting a hydroxyl polymer, olefinic acid, N,N′-dicyclohexylcarbodiimide, and 4-N,N-dimethylpyridine in CH2Cl2 at room temperature overnight, and refluxing the reaction product with the mesoporous PMMA microspheres and SnCl4 in step S1 for 48 hours under nitrogen protection to obtain PMMA-PEG microspheres.

4. The method for preparing a strong anion exchange filler for purifying oligonucleotides according to claim 3, characterized in that: The hydroxy polymer is one or more of PEG 600, PEG 2000, PEG 4000, and PEG 6000; the enoic acid is one or more of 4-pentenoic acid, but-3-enoic acid, and 4-methylpent-4-enoic acid.

5. The method for preparing a strong anion exchange filler for purifying oligonucleotides according to claim 3, characterized in that: The mass ratio of the hydroxy polymer, olefinic acid, N,N′-dicyclohexylcarbodiimide and 4-N,N-dimethylpyridine is 2:0.6:0.6:0.12; the mass ratio of the mesoporous PMMA microspheres and SnCl4 is 5:

2.

6. The method for preparing a strong anion exchange filler for purifying oligonucleotides according to claim 1, characterized in that: In step S3, the sulfoxide halide is one or more of sulfoxide chloride and sulfoxide bromide.

7. The method for preparing a strong anion exchange filler for purifying oligonucleotides according to claim 1, characterized in that: In step S3, the PMMA-PEG microspheres, sulfoxide halides and tetrahydropyran are mixed under the following conditions: a rotation speed of 160 rpm, a temperature of 50° C. and a time of 16 h; and a mass ratio of the PMMA-PEG microspheres, sulfoxide halides and tetrahydropyran is 5:0.2:

10.

8. The method for preparing a strong anion exchange filler for purifying oligonucleotides according to claim 1, characterized in that: In step S4, the tertiary amine compound is one or more of trimethylamine, trimethylamine dihydrate, trimethylamine hydrochloride, and sulfur trioxide-trimethylamine complex.

9. The method for preparing a strong anion exchange filler for purifying oligonucleotides according to claim 1, characterized in that: In step S4, the conditions for mixing the chlorine ball, the tertiary amine compound and water are: temperature 40° C., time 12 hours; and the mass ratio of the chlorine ball, the tertiary amine compound and water is 1:3:

2.

10. A strong anion exchange filler for purifying oligonucleotides, characterized in that: The strong anion exchange filler is prepared by the preparation method described in any one of claims 1 to 9.

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