Separation and purification method of pseudouridine

Abstract

The invention relates to a method for separating and purifying pseudouridine, which comprises the following steps: (1) sequentially dissolving and filtering a pseudouridine crude product to obtain a pseudouridine solution; and (2) loading the pseudouridine solution obtained in the step (1) to a chromatographic column filled with polymer microspheres for chromatography, eluting the pseudouridine solution by using an acidic aqueous solution as a flowing phase, collecting the pseudouridine solution with a target peak value in sections, and collecting component solutions meeting requirements. According to the separation and purification method provided by the invention, a high-purity and high-yield pseudouridine product can be obtained, the effect is stable, the operation is simple, the used stationary phase can be repeatedly utilized, the use amount of the mobile phase is greatly reduced, and the separation and purification method has good economic benefits and environmental benefits.

Description

Technical Field

[0001] This invention relates to the field of aminoglycoside purification technology, specifically to a method for separating and purifying pseudouridine. Background Technology

[0002] Pseudoruriline is a C-glycosylpyrimidine composed of uracil, with a β-D-furanofuranosyl residue linked at the C-5 position. It is the C-glycosyl isomer of the pyrimidine nucleoside uridine and functions as a basic metabolite. Pseudoruriline is present in all species and all classes of RNA except mRNA, and is produced by the catalysis of uridine residues by pseudouridine synthase. As an important component of mRNA-based COVID-19 vaccines, pseudouridine can protect synthesized mRNA from immune system attack and can also improve molecular stability and protein yield. For this reason, researchers have replaced uridine with pseudouridine and its analogues in mRNA to address the problem of mRNA drugs being easily recognized and cleared by the immune system, causing immune side effects. As an RNA metabolite, pseudouridine is excreted solely through the kidneys, thus it can be used clinically to monitor tumor development and progression, and for the diagnosis and treatment of kidney disease. Furthermore, β-pseudoruriline, found in tRNA of bacteria, archaea, and eukaryotes, has been shown to have the potential to reduce radiation-induced chromosomal aberrations in human lymphocytes.

[0003] Currently, existing research on pseudouridine mainly focuses on production methods and product applications, while there are few reports on the isolation and purification of pseudouridine.

[0004] CN118271296A discloses a method for preparing and applying pseudouridine. The method involves passing crude pseudouridine through a rapid silica gel column using a 4:1 volume ratio of petroleum ether and ethyl acetate as eluent. After column chromatography, the product is rotary evaporated, and then dissolved in a 3:1 volume ratio of petroleum ether and ethyl acetate to induce crystallization. However, this method requires a high amount of organic solvent, is cumbersome, and is neither environmentally friendly nor economically viable.

[0005] Therefore, providing a method for the separation and purification of pseudouridine is a technical problem that needs to be solved in this field. Summary of the Invention

[0006] To address the above problems, the present invention aims to provide a method for the separation and purification of pseudouridine. Compared with the prior art, the separation and purification method provided by the present invention can obtain pseudouridine products with high purity and high yield, and the effect is stable, the operation is simple, the stationary phase can be reused, and the amount of mobile phase used is greatly reduced, which has good economic and environmental benefits.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] This invention provides a method for isolating and purifying pseudouridine, the method comprising the following steps:

[0009] (1) The crude pseudouridine product was dissolved and filtered sequentially to obtain a pseudouridine solution;

[0010] (2) The pseudouridine solution obtained in step (1) is loaded onto a chromatography column packed with polymer microspheres for chromatography. An acidic aqueous solution is used as the mobile phase to elute the pseudouridine solution. The pseudouridine solution at the target peak is collected in segments, and the component solutions that meet the requirements are summarized.

[0011] The separation and purification method provided by this invention significantly reduces the amount of eluent used in the elution step, and only requires an acidic aqueous solution as the eluent in the elution stage, which can significantly reduce the amount of organic solvent used. The mobile phase used is safe and pollution-free, and the method has low cost, short purification cycle, and the obtained product has high purity and yield. Therefore, the separation and purification method has good economic and environmental benefits and is conducive to industrial application.

[0012] Preferably, the crude pseudouridine product described in step (1) is dissolved in an acidic aqueous solution.

[0013] Preferably, the volume percentage of the acidic aqueous solution used for dissolution is 0.05-0.15%, for example, it can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14% or 0.15%, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0014] Preferably, the concentration of crude pseudouridine in the solution obtained by dissolution is 20-25 mg / mL, for example, it can be 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL or 25 mg / mL, but is not limited to the listed values, and other unlisted values ​​within the range are also applicable.

[0015] Preferably, the filtration accuracy in step (1) is 0.2-2μm, for example, it can be 0.2μm, 0.4μm, 0.6μm, 0.8μm, 1μm, 1.2μm, 1.4μm, 1.6μm, 1.8μm or 2μm, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0016] Preferably, the polymer microspheres in step (2) are monodisperse microspheres with a porous structure.

[0017] Preferably, the polymer microspheres include PS-DVB microspheres, i.e., polystyrene-divinylbenzene polymer microspheres. The PS-DVB polymer microspheres can be any PS-DVB microspheres commonly used in the art, such as PS10-100 or PS30-100 produced by Suzhou Nanomicro Technology Co., Ltd.

[0018] Preferably, the pore size of the polymer microspheres is [missing information]. or Any one or at least two of them, preferably or Any one or at least two of them.

[0019] Preferably, the polymer microspheres have a particle size of 10 μm and / or 30 μm.

[0020] In this invention, by optimizing and controlling the type, pore size, and particle size of polymer microspheres, it is possible to make them highly targeted when used as chromatographic packing material, so that impurities are basically present in the later stage of elution, and pseudouridine can achieve higher purity.

[0021] Preferably, the volume percentage of the acidic aqueous solution used for elution in step (2) is 0.05-0.15%, for example, it can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14% or 0.15%, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0022] In this invention, by optimally controlling the volume percentage of the acidic aqueous solution, it is possible to avoid the diffusion of impurities when the volume percentage is too high or too low, thereby ensuring the separation effect and improving the purity of pseudouridine.

[0023] Preferably, the amount of acidic aqueous solution used in the elution in step (2) is 3-5 column volumes, for example, it can be 3, 4 or 5, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0024] In this invention, using an acidic aqueous solution as the mobile phase for elution achieves excellent elution results for pseudouridine. This is because pseudouridine is polar and has a relatively weak retention capacity. By using an acidic aqueous solution to adjust the pH value, this invention reduces the interaction between pseudouridine and the stationary phase, thereby increasing the retention time and improving the separation and purification effect. Compared to using a combination of acidic aqueous solution and organic solvent, this invention achieves better separation results using only an acidic aqueous solution. This is because the addition of organic solvent further weakens the retention capacity of pseudouridine. Therefore, this invention not only provides better separation results but also significantly reduces the amount of mobile phase and organic solvent used, making it more economical and environmentally friendly.

[0025] Preferably, the elution flow rate is 0.5-1 mL / min, for example, it can be 0.5 mL / min, 0.6 mL / min, 0.7 mL / min, 0.8 mL / min, 0.9 mL / min or 1 mL / min, but is not limited to the listed values, and other unlisted values ​​within the range are also applicable.

[0026] In this invention, by optimally controlling the elution flow rate, it is possible to avoid excessively low flow rates leading to excessively long purification times, and to avoid excessively high flow rates leading to excessive operating pressure, which would affect normal operation.

[0027] Preferably, the pre-column pretreatment of the chromatography column is performed before the sample loading in step (2).

[0028] Preferably, the pre-column pretreatment includes: first removing impurities from the chromatography column, and then equilibrating the chromatography column.

[0029] Preferably, the mobile phase used for the impurity removal process comprises an organic solvent or a combination of an organic solvent and an acidic aqueous solution.

[0030] Preferably, the volume percentage of organic solvent in the mobile phase used for the impurity removal treatment is 50-100%, for example, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, but is not limited to the listed values, and other unlisted values ​​within the range are also applicable.

[0031] In this invention, the acidic aqueous solution and organic solvent used for dissolution, impurity removal, equilibration, elution, and regeneration in the same batch separation and purification method are exactly the same, generally with the acidic aqueous solution as phase A and the organic solvent as phase B.

[0032] Preferably, the mobile phase used in the equilibration process comprises an acidic aqueous solution.

[0033] Preferably, the volume percentage of the acidic aqueous solution used in the equilibration treatment is 0.05-0.15%, for example, it can be 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14% or 0.15%, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0034] Preferably, the chromatography column is regenerated after elution in step (2).

[0035] Preferably, the mobile phase used for regeneration comprises an organic solvent or a combination of an organic solvent and an acidic aqueous solution.

[0036] Preferably, the volume percentage of organic solvent in the mobile phase used for regeneration is 50-100%, for example, it can be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100%, but is not limited to the listed values. Other unlisted values ​​within the range are also applicable.

[0037] Preferably, the acidic aqueous solution contains any one or a combination of at least two of acetic acid, trifluoroacetic acid, or formic acid.

[0038] Preferably, the organic solvent includes any one or a combination of at least two of methanol, ethanol, acetonitrile, acetone or isopropanol.

[0039] As a preferred embodiment of the present invention, the separation and purification method includes the following steps:

[0040] (1) Dissolve the crude pseudouridine at a concentration of 20-25 mg / mL in an acidic aqueous solution with a volume percentage of 0.05-0.15%, and then filter it with a filtration precision of 0.2-2 μm to obtain a pseudouridine solution.

[0041] (2) An organic solvent or a combination of an organic solvent and an acidic aqueous solution is used as the mobile phase to remove impurities from the chromatography column. The volume percentage of the organic solvent in the mobile phase is controlled to be 50-100%. Then, the chromatography column is equilibrated with an acidic aqueous solution with a volume percentage of 0.05-0.15%. The pseudouridine solution obtained in step (1) is then loaded onto a chromatography column packed with polymer microspheres for chromatography. The polymer microspheres are monodisperse PS-DVB microspheres with a porous structure, and the pore size of the polymer microspheres is [not specified]. or The pseudouridine solution is eluted for 3-5 column volumes using any one or at least two of the following methods, with a particle size of 10 μm and / or 30 μm. An acidic aqueous solution with a volume percentage of 0.05-0.15% is used as the mobile phase. The pseudouridine solution is collected in fractions at a flow rate of 0.5-1 mL / min. The fractions of pseudouridine solution with the target peak are collected and the fractions that meet the requirements are summarized.

[0042] The column is regenerated using an organic solvent or a combination of an organic solvent and an acidic aqueous solution as the mobile phase. The volume percentage of the organic solvent in the mobile phase used for regeneration is 50-100%.

[0043] The acidic aqueous solution contains any one or a combination of at least two of acetic acid, trifluoroacetic acid, or formic acid, and the organic solvent contains any one or a combination of at least two of methanol, ethanol, acetonitrile, acetone, or isopropanol.

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] (1) The separation and purification method provided by this invention only requires an acidic aqueous solution as the mobile phase in the elution step, which is safer and less polluting. Only 3-5 column volumes of mobile phase are needed to achieve high purity and yield of pseudouridine. The purity of pseudouridine obtained by this invention can reach over 99.83%, and over 99.926% under optimal conditions. The yield of pseudouridine can reach over 67.11%, and over 69.58% under optimal conditions. Furthermore, the separation and purification effect is stable and the operation is simple and convenient.

[0046] (2) The separation and purification method provided by the present invention does not require the use of organic solvents in the elution stage, and the amount of mobile phase used in the elution stage is also significantly reduced. The purification cycle is shorter, the stationary phase can be reused, and the production cost can be significantly reduced. It has high economic and environmental benefits and is conducive to industrial application. Attached Figure Description

[0047] Figure 1 This is a structural diagram of the pseudouridine described in Example 1 of the present invention;

[0048] Figure 2 This is the high-performance liquid chromatography (HPLC) chromatogram of the crude pseudouridine product before separation and purification in Example 1 of the present invention;

[0049] Figure 3 This is the high-performance liquid chromatography (HPLC) chromatogram of the pseudouridine product after separation and purification in Example 1 of the present invention. Detailed Implementation

[0050] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0051] Example 1

[0052] This embodiment provides a method for separating and purifying pseudouridine, the method comprising the following steps:

[0053] (1) The crude pseudouridine (initial purity 91.93%, structure diagram of pseudouridine as shown in the figure) was prepared. Figure 1 (As shown) Dissolved in an aqueous solution of acetic acid with a volume percentage of 0.1% at a concentration of 25 mg / mL, and then filtered. The filter membrane used for filtration has a precision of 0.45 μm to obtain a pseudouridine solution.

[0054] (2) A 4.6 mm chromatographic column was used. The mobile phase, consisting of a combination of acetonitrile and an aqueous acetic acid solution, was used to remove impurities. The volume percentage of acetonitrile in the mobile phase was controlled to be 80%. Then, the column was equilibrated with an aqueous acetic acid solution containing 0.1% acetic acid. The pseudouridine solution obtained in step (1) was loaded onto PS-DVB microspheres (monodisperse PS10-100 microspheres with a porous structure produced by Suzhou Nanomicro Technology Co., Ltd., with a pore size of [missing information - likely a pore size]. Chromatography was performed in a 10 μm particle size column using an aqueous acetic acid solution with a volume percentage of 0.1% as the mobile phase. The pseudouridine solution was eluted for 4 column volumes at a flow rate of 0.75 mL / min. The pseudouridine solution at the target peak was collected in fractions, and the fractions that met the requirements were summarized.

[0055] The chromatography column was regenerated using a combination of acetonitrile and aqueous acetic acid as the mobile phase, with the organic solvent comprising 80% by volume in the mobile phase used for regeneration.

[0056] Taking Example 1 as an example, the crude pseudouridine product before separation and purification was analyzed by high performance liquid chromatography, and the results are as follows: Figure 2 As shown, the purified pseudouridine product was analyzed by high-performance liquid chromatography (HPLC), and the results are as follows. Figure 3 As shown, from Figure 2 and Figure 3 The comparison shows that the separation and purification method provided by the present invention can effectively separate and remove impurities in pseudouridine.

[0057] Example 2

[0058] This embodiment provides a method for separating and purifying pseudouridine, the method comprising the following steps:

[0059] (1) The crude pseudouridine (initial purity of 91.93%) was dissolved in a 0.05% formic acid aqueous solution at a concentration of 20 mg / mL, and then filtered. The filter membrane used for filtration had a precision of 0.2 μm to obtain a pseudouridine solution.

[0060] (2) A 4.6 mm chromatographic column was used. A combination of methanol and formic acid aqueous solution was used as the mobile phase to remove impurities from the column. The volume percentage of methanol in the mobile phase was controlled to be 60%. Then, the column was equilibrated with a 0.05% volume percentage formic acid aqueous solution. The pseudouridine solution obtained in step (1) was loaded onto PS-DVB microspheres (monodisperse PS30-100 with a porous structure produced by Suzhou Nanomicro Technology Co., Ltd., with a pore size of...). Chromatography was performed in a 30 μm particle size chromatography column. A 0.05% formic acid aqueous solution was used as the mobile phase. The pseudouridine solution was eluted for 4 column volumes at a flow rate of 0.5 mL / min. The pseudouridine solution at the target peak was collected in fractions, and the components that met the requirements were summarized.

[0061] The chromatography column was regenerated using a combination of methanol and formic acid aqueous solution as the mobile phase, with the organic solvent comprising 60% by volume in the mobile phase used for regeneration.

[0062] Example 3

[0063] This embodiment provides a method for separating and purifying pseudouridine, the method comprising the following steps:

[0064] (1) The crude pseudouridine (initial purity of 91.93%) was dissolved in an aqueous solution of acetic acid with a volume percentage of 0.15% at a concentration of 25 mg / mL, and then filtered. The filter membrane used for filtration had a precision of 1.5 μm to obtain a pseudouridine solution.

[0065] (2) A 4.6 mm chromatographic column was used, with ethanol as the mobile phase for impurity removal. The column was then equilibrated with a 0.15% (v / v) acetic acid aqueous solution. The pseudouridine solution obtained in step (1) was loaded onto a PS-DVB microsphere packing system (monodisperse PS10-100 with a porous structure, manufactured by Suzhou Nanomicro Technology Co., Ltd., with a pore size of [missing information - likely a pore size]. Chromatography was performed in a 10 μm particle size column. An aqueous acetic acid solution with a volume percentage of 0.15% was used as the mobile phase. The pseudouridine solution was eluted for 5 column volumes at a flow rate of 1 mL / min. The pseudouridine solution at the target peak was collected in fractions, and the fractions that met the requirements were summarized.

[0066] The chromatography column was regenerated using ethanol as the mobile phase.

[0067] Example 4

[0068] This embodiment provides a method for separating and purifying pseudouridine, the method comprising the following steps:

[0069] (1) The crude pseudouridine (initial purity of 91.93%) was dissolved in a 0.1% trifluoroacetic acid aqueous solution at a concentration of 25 mg / mL, and then filtered. The filter membrane used for filtration had a precision of 0.45 μm to obtain a pseudouridine solution.

[0070] (2) A 4.6 mm chromatographic column was used. The mobile phase, consisting of a combination of acetonitrile and trifluoroacetic acid aqueous solution, was used to remove impurities. The volume percentage of acetonitrile in the mobile phase was controlled to be 80%. Then, the column was equilibrated with a 0.1% volume percentage of trifluoroacetic acid aqueous solution. The pseudouridine solution obtained in step (1) was loaded onto PS-DVB microspheres (monodisperse PS10-100 with a porous structure produced by Suzhou Nanomicro Technology Co., Ltd., with a pore size of...). Chromatography was performed in a 10 μm particle size column. A 0.1% trifluoroacetic acid aqueous solution was used as the mobile phase. The pseudouridine solution was eluted at a flow rate of 0.75 mL / min for 4 column volumes. The pseudouridine solution at the target peak was collected in fractions, and the components that met the requirements were summarized.

[0071] The chromatography column was regenerated using a combination of acetonitrile and trifluoroacetic acid aqueous solution as the mobile phase, with the organic solvent comprising 80% by volume in the mobile phase used for regeneration.

[0072] Example 5

[0073] This embodiment provides a method for separating and purifying pseudouridine. The only difference between this method and that in Example 1 is that the polymer microspheres in step (2) are replaced with PS40-130.

[0074] Example 6

[0075] This embodiment provides a method for separating and purifying pseudouridine. The only difference between this method and that in Example 1 is that the polymer microspheres in step (2) are replaced with PS10-300.

[0076] Example 7

[0077] This embodiment provides a method for separating and purifying pseudouridine. The only difference between this method and that in Example 1 is that the volume percentage of the acetic acid aqueous solution used for elution in step (2) is 0.01%.

[0078] Example 8

[0079] This embodiment provides a method for separating and purifying pseudouridine. The only difference between this method and that in Example 1 is that the volume percentage of the acetic acid aqueous solution used for elution in step (2) is 0.25%.

[0080] Comparative Example 1

[0081] This comparative example provides a method for separating and purifying pseudouridine. The only difference from Example 1 is that the mobile phase used for elution in step (2) is an aqueous solution of acetic acid and acetonitrile, with acetonitrile accounting for 10%, and the same column volume is used for isogradient elution as in Example 1.

[0082] Taking Example 1 and Comparative Example 1 as examples, although Comparative Example 1 can reduce the consumption of acetic acid by about 0.32 L / kg of crude product compared to Example 1, it consumes 120 L / kg of crude product more acetonitrile. Based on the market price of acetonitrile of 9,000 yuan / ton and the market price of acetic acid of 3,700 yuan / ton, the cost of the reagent can be saved by about 847.64 yuan / kg of crude product.

[0083] Comparative Example 2

[0084] This comparative example provides a method for separating and purifying pseudouridine. The only difference between this method and Example 4 is that the mobile phase used for elution in step (2) is trifluoroacetic acid aqueous solution and acetonitrile, with acetonitrile accounting for 10%, and the column volume is the same as in Example 4 for isogradient elution.

[0085] Taking Example 4 and Comparative Example 2 as examples, although Comparative Example 2 can reduce the consumption of trifluoroacetic acid by about 0.32 L / kg of crude product compared to Example 4, it consumes 120 L / kg of crude product more acetonitrile. Based on the market price of acetonitrile of 9,000 yuan / ton and the market price of trifluoroacetic acid of 31,000 yuan / ton, it can still save 834.11 yuan / kg of crude product in reagent costs.

[0086] The purity of the pseudouridine products obtained from Examples 1-8 and Comparative Examples 1-2 was determined using a Shimadzu analytical instrument. The analytical method was as follows: mobile phase A: 0.2% ammonium acetate solution; mobile phase B: acetonitrile; mobile phase A: mobile phase B = 95:5 (v / v); flow rate: 1 mL / min; isocratic elution for 10 min. The results are shown in Table 1. The yields of the pseudouridine products obtained from Examples 1-8 and Comparative Examples 1-2 were calculated, and the results are shown in Table 1.

[0087] Table 1

[0088]

[0089]

[0090] In Table 1, " / " indicates that the data is not available.

[0091] The following points can be observed from the data in Table 1:

[0092] (1) As can be seen from the data in Examples 1-8, the separation and purification method provided by the present invention can achieve a purity of more than 99.83% for the collected pseudouridine, and more than 99.926% under better conditions. The yield of the obtained pseudouridine can reach more than 67.11%, and more than 69.58% under better conditions.

[0093] (2) Comparing the data of Example 1 and Example 5-6, it can be seen that the only difference between Example 5-6 and Example 1 is that the pore size and particle size of the polymer microspheres used are not within the preferred range of the present invention. The purity and recovery rate of pseudouridine in Example 1 are significantly higher than those in Example 5-6. It can be seen that the present invention can further promote the separation and purification of pseudouridine and improve its purity and yield by optimizing the control of the pore size and particle size of polymer microspheres.

[0094] (3) A comprehensive comparison of the data from Example 1 and Examples 7-8 shows that the only difference between Example 7-8 and Example 1 is that the volume percentage of the acidic aqueous solution is not within the preferred range of the present invention. The purity and yield of pseudouridine in Example 1 are significantly higher than those in Example 7-8. Therefore, the present invention can further promote the separation and purification of pseudouridine and improve its purity and yield by optimally controlling the volume percentage of the acidic aqueous solution.

[0095] (4) A comprehensive comparison of Example 1 and Comparative Examples 1-2 shows that the elution using a mobile phase composed of acidic aqueous solution and organic solvent in Comparative Examples 1-2 not only fails to obtain pseudouridine with a purity >99.7%, but also requires an additional amount of organic solvent. Therefore, the separation and purification method provided by this invention not only achieves good separation and purification of pseudouridine, but also reduces the amount of organic solvent used, saving processing costs.

[0096] In summary, the separation and purification method provided by this invention can obtain high-purity, high-yield pseudouridine products, and has stable effects, simple operation, reusable stationary phase, and greatly reduced mobile phase consumption, thus having good economic and environmental benefits.

[0097] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for isolating and purifying pseudouridine, characterized in that, The separation and purification method includes the following steps: (1) The crude pseudouridine product was dissolved and filtered sequentially to obtain a pseudouridine solution; (2) The pseudouridine solution obtained in step (1) is loaded onto a chromatography column packed with polymer microspheres for chromatography. An acidic aqueous solution is used as the mobile phase to elute the pseudouridine solution. The pseudouridine solution at the target peak is collected in segments, and the component solutions that meet the requirements are summarized.

2. The separation and purification method according to claim 1, characterized in that, In step (1), the crude pseudouridine is dissolved in an acidic aqueous solution; Preferably, the volume percentage of the acidic aqueous solution used for dissolution is 0.05-0.15%; Preferably, the concentration of crude pseudouridine in the solution obtained by dissolution is 20-25 mg / mL.

3. The separation and purification method according to claim 1 or 2, characterized in that, The filtration accuracy in step (1) is 0.2-2 μm.

4. The separation and purification method according to any one of claims 1-3, characterized in that, The polymer microspheres in step (2) are monodisperse microspheres with a porous structure; Preferably, the polymer microspheres comprise PS-DVB microspheres; Preferably, the pore size of the polymer microspheres is [missing information]. or Any one or at least two of them; Preferably, the polymer microspheres have a particle size of 10 μm and / or 30 μm.

5. The separation and purification method according to any one of claims 1-4, characterized in that, The volume percentage of the acidic aqueous solution used for elution in step (2) is 0.05-0.15%.

6. The separation and purification method according to any one of claims 1-5, characterized in that, In step (2), the amount of acidic aqueous solution used in the elution is 3-5 column volumes; Preferably, the elution flow rate is 0.5-1 mL / min.

7. The separation and purification method according to any one of claims 1-6, characterized in that, Step (2) involves performing pre-column pretreatment of the chromatography column before sample loading; Preferably, the pre-column pretreatment includes: first removing impurities from the chromatography column, and then equilibrating the chromatography column; Preferably, the mobile phase used for the impurity removal treatment comprises an organic solvent or a combination of an organic solvent and an acidic aqueous solution; Preferably, the volume percentage of organic solvent in the mobile phase used for the impurity removal treatment is 50-100%. Preferably, the mobile phase used in the equilibrium treatment comprises an acidic aqueous solution; Preferably, the volume percentage of the acidic aqueous solution used in the equilibration treatment is 0.05-0.15%.

8. The separation and purification method according to any one of claims 1-7, characterized in that, After elution as described in step (2), the chromatography column is regenerated; Preferably, the mobile phase used for regeneration includes an organic solvent or a combination of an organic solvent and an acidic aqueous solution; Preferably, the volume percentage of organic solvent in the mobile phase used for regeneration is 50-100%.

9. The separation and purification method according to any one of claims 1-8, characterized in that, The acidic aqueous solution contains any one or a combination of at least two of acetic acid, trifluoroacetic acid, or formic acid. Preferably, the organic solvent includes any one or a combination of at least two of methanol, ethanol, acetonitrile, acetone or isopropanol.

10. The separation and purification method according to any one of claims 1-9, characterized in that, The separation and purification method includes the following steps: (1) Dissolve the crude pseudouridine at a concentration of 20-25 mg / mL in an acidic aqueous solution with a volume percentage of 0.05-0.15%, and then filter it with a filtration precision of 0.2-2 μm to obtain a pseudouridine solution. (2) An organic solvent or a combination of an organic solvent and an acidic aqueous solution is used as the mobile phase to remove impurities from the chromatography column. The volume percentage of the organic solvent in the mobile phase is controlled to be 50-100%. Then, the chromatography column is equilibrated with an acidic aqueous solution with a volume percentage of 0.05-0.15%. The pseudouridine solution obtained in step (1) is then loaded onto a chromatography column packed with polymer microspheres for chromatography. The polymer microspheres are monodisperse PS-DVB microspheres with a porous structure, and the pore size of the polymer microspheres is [not specified]. or The pseudouridine solution is eluted for 3-5 column volumes using any one or at least two of the following methods, with a particle size of 10 μm and / or 30 μm. An acidic aqueous solution with a volume percentage of 0.05-0.15% is used as the mobile phase. The pseudouridine solution is collected in fractions at a flow rate of 0.5-1 mL / min. The fractions of pseudouridine solution with the target peak are collected and the fractions that meet the requirements are summarized. The column is regenerated using an organic solvent or a combination of an organic solvent and an acidic aqueous solution as the mobile phase. The volume percentage of the organic solvent in the mobile phase used for regeneration is 50-100%. The acidic aqueous solution contains any one or a combination of at least two of acetic acid, trifluoroacetic acid, or formic acid, and the organic solvent contains any one or a combination of at least two of methanol, ethanol, acetonitrile, acetone, or isopropanol.

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