An RNA room-temperature preservation stabilizer, an RNA product, a preparation method and an application thereof

Abstract

The present invention belongs to the field of biotechnology, and discloses an RNA room-temperature storage stabilizer, an RNA product, a preparation method and an application thereof. The RNA room-temperature storage stabilizer provided by the present invention comprises functionalized polystyrene microspheres. The RNA room-temperature storage stabilizer has good adsorption capacity for RNA, and the functional groups thereon can interact with the groups in RNA, causing the RNA molecules to rotate, twist, wind and fold to form a specific three-dimensional structure, playing a role in stabilizing the RNA structure. Without introducing ultra-low temperature equipment and other protective reagents, long-term stable storage of RNA at room temperature can be achieved; moreover, the addition of water can disrupt the binding between the functionalized polystyrene microspheres and RNA, realizing the complete separation of RNA and the stabilizer. It has the advantages of low cost, simple operation and high biosafety, and has good application prospects.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to an RNA room temperature storage stabilizer, an RNA product, a preparation method and an application thereof. Background Art

[0002] RNA is a long-chain molecule formed by the condensation of ribonucleotides through phosphodiester bonds, and is widely present in biological cells, some viruses and viroids. Among them, mRNA is responsible for transmitting genetic information during protein synthesis and directly guiding protein synthesis, and can be applied to fields such as vaccines (infectious vaccines and tumor vaccines), protein replacement therapy, cell engineering, and gene editing - by transferring mRNA into the cells of patients (or recipients), enabling it to successfully translate the specified protein and trigger effective humoral and cellular immune responses to achieve the purpose of preventing diseases or changing specific disease states. Compared with traditional drugs and vaccines, mRNA vaccines can design and encode any antigen according to the unique properties of diseases and regulate the expression of the selected antigen, so as to quickly respond to the mutations of pathogenic microorganisms and provide more timely and reliable protection. However, the stability defect of the mRNA itself makes it inevitably degraded during storage and transportation, resulting in structural changes and quality reduction, and further affecting the biological activity of mRNA.

[0003] At present, generally, ultra-low temperature storage, addition of protective agents, or a combination of both methods are used to store mRNA. However, in ultra-low temperature storage, mRNA is generally stored in an ultra-low temperature refrigerator at -80°C or even liquid nitrogen to minimize its degradation rate, which poses high requirements for transportation and application scenarios, resulting in problems such as increased energy consumption, elevated transportation costs, and safety. The introduction of protective agents may reduce the biological safety of mRNA drugs or vaccines, with relatively large limitations. Summary of the Invention

[0004] The first object of the present invention is to solve the problems existing in the storage of existing mRNA, such as high requirements for storage conditions, high storage costs, and low biological safety, and to provide an RNA room temperature storage stabilizer for obtaining a simple, effective, economical, practical and highly biologically safe method for long-term stable storage and transportation of mRNA.

[0005] The second object of the present invention is to provide a preparation method of the above RNA room temperature storage stabilizer.

[0006] The third object of the present invention is to provide an RNA product.

[0007] The fourth object of the present invention is to provide a preparation method of an RNA product.

[0008] The fifth object of the present invention is to provide the application of the above RNA room temperature preservation stabilizer and / or the preparation method of the RNA product in the production of mRNA drugs and / or vaccines.

[0009] Specifically, the RNA room temperature preservation stabilizer provided by the present invention includes functionalized polystyrene microspheres.

[0010] In some specific embodiments, the functionalized polystyrene microspheres are polystyrene microspheres modified with functional groups, and the functional groups are selected from one or more of C1-C20 chloroalkyl groups, chlorophenyl groups, C1-C20 hydroxy-substituted alkyl groups, hydroxy-substituted phenyl groups, C1-C20 carboxyl-substituted alkyl groups, carboxylic acid-substituted phenyl groups, C1-C20 amino-substituted alkyl groups, amino-substituted phenyl groups, and nitrogen-containing heterocycles.

[0011] In some specific embodiments, the functionalized polystyrene microspheres are selected from one or more of chloromethyl-modified polystyrene microspheres, hydroxy-modified polystyrene microspheres, carboxyl-modified polystyrene microspheres, and nitrogen-containing five-membered heterocycle-modified polystyrene microspheres.

[0012] In some specific embodiments, the average particle size of the functionalized polystyrene microspheres is 0.1-200 μm.

[0013] In some specific embodiments, the average particle size of the functionalized polystyrene microspheres is 1-2 μm.

[0014] The preparation method of the above RNA room temperature preservation stabilizer provided by the present invention includes: S1. Taking a polymerization monomer, a functionalized modifier, an initiator, and optionally polystyrene particles for a polymerization reaction to obtain the functionalized polystyrene microspheres; S2. Taking the functionalized polystyrene microspheres for nuclease treatment to obtain the functionalized polystyrene microspheres.

[0015] In some specific embodiments, in step S1, the polymerization monomer is selected from one or more of styrene, styrene-propylene, and divinylbenzene.

[0016] In some specific embodiments, in step S1, the functionalized modifier is selected from one or more of chloroolefins, enols, unsaturated fatty acids, and olefinated pyrrolidones.

[0017] In some specific embodiments, in step S1, the initiator is an azo initiator and / or a peroxide initiator.

[0018] In some specific embodiments, in step S1, the average particle size of the polystyrene particles is 0.01-1 μm.

[0019] In some specific embodiments, in step S1, the input mass ratio of the polymerization monomer, the functionalized modifier, the initiator, and the optional polystyrene particles is (5 - 20):(0.1 - 20):(0.1 - 10):(0.001 - 20).

[0020] In some specific embodiments, in step S1, the temperature of the polymerization reaction is 70 - 90°C, and the time is 1 - 48 h.

[0021] In some specific embodiments, the ribonuclease treatment specifically includes: mixing the functionalized polystyrene microspheres with RNase-free water and then performing centrifugal column purification to obtain the RNA room-temperature storage stabilizer.

[0022] In some specific embodiments, the centrifugal speed for the centrifugal column purification is 5000 - 1000 rpm, and the time is 5 - 30 min.

[0023] The RNA product provided by the present invention includes the above RNA room-temperature storage stabilizer.

[0024] The preparation method of the RNA product provided by the present invention includes: mixing RNA with the RNA room-temperature storage stabilizer and then performing freeze-drying treatment to obtain the RNA product.

[0025] In some specific embodiments, the input mass ratio of the RNA to the RNA room-temperature storage stabilizer is 1:(10 - 1000).

[0026] In some specific embodiments, the freeze-drying treatment includes a pre-freezing stage, a primary drying stage, and a secondary drying stage. The temperature of the pre-freezing stage is -60 - -50°C, and the time is 5 - 15 h; the temperature of the primary drying stage is -45 - -40°C, the vacuum degree is not higher than 10 Pa, and the time is 1 - 5 h; the secondary drying stage includes an alternating heating period and a constant-temperature period. The starting temperature of the secondary drying stage is -35 - -30°C, the final temperature is 20 - 25°C, the vacuum degree is not higher than 10 Pa, the total time is 40 - 60 h, the heating rate of the heating period is 0.5 - 2°C / min, and the time is 25 - 40 min.

[0027] The present invention also provides the application of the above-mentioned RNA room-temperature storage stabilizer, RNA product, and / or the preparation method of the RNA product in the production of mRNA drugs and / or vaccines.

[0028] Beneficial effects:

[0029] The present invention provides an RNA room-temperature storage stabilizer, which includes functionalized polystyrene microspheres. These functionalized polystyrene microspheres have good adsorption ability for RNA, and the functional groups on them can interact with the groups in RNA, causing the RNA molecules to rotate, twist, wind, and fold to form a specific three-dimensional structure, playing a role in stabilizing the RNA structure. Without introducing ultra-low temperature equipment and other protective reagents, long-term stable storage of RNA at room temperature can be achieved. Moreover, the addition of water can disrupt the binding between the functionalized polystyrene microspheres and RNA, realizing the complete separation of RNA and the stabilizer. It has the advantages of low cost, simple operation, and high biological safety, and has good application prospects. Description of the Drawings

[0030] Figure 1 It is the infrared spectrum of the PS-Cl microspheres prepared in Preparation Example 1 of the present invention;

[0031] Figure 2 It is the SEM image (200μm) of the PS-Cl microspheres prepared in Preparation Example 1 of the present invention;

[0032] Figure 3 It is the infrared spectrum of the PS-OH microspheres prepared in Preparation Example 2 of the present invention;

[0033] Figure 4 It is the SEM image (5.0μm) of the PS-OH microspheres prepared in Preparation Example 2 of the present invention;

[0034] Figure 5 It is the infrared spectrum of the PS-COOH microspheres prepared in Preparation Example 3 of the present invention;

[0035] Figure 6 It is the SEM image (5.0μm) of the PS-COOH microspheres prepared in Preparation Example 3 of the present invention;

[0036] Figure 7 It is the infrared spectrum of the PS-N microspheres prepared in Preparation Example 4 of the present invention;

[0037] Figure 8 It is the SEM image (5.0μm) of the PS-N microspheres prepared in Preparation Example 4 of the present invention;

[0038] Figure 9 It is the experimental result graph of the electrophoresis test of the RNA product stored at 25°C for 7 days in the test example of the present invention;

[0039] Figure 10 It is the experimental result graph of the electrophoresis test of the RNA product stored at 25°C for 21 days in the test example of the present invention;

[0040] Figure 11 This is the experimental result graph of the electrophoresis test on the RNA product after storing it at 37°C for 7 days in the test example of the present invention;

[0041] Figure 12 This is the experimental result graph of the electrophoresis test on the RNA product after storing it at 37°C for 21 days in the test example of the present invention;

[0042] Figure 13 This is the experimental result graph of the electrophoresis test on the RNA product after storing it at 60°C for 1 day in the test example of the present invention;

[0043] Figure 14 This is the experimental result graph of the electrophoresis test on the RNA product after storing it at 60°C for 5 days in the test example of the present invention;

[0044] Figure 15 This is the experimental result graph of the electrophoresis test on the RNA product after storing it at 60°C for 14 days in the test example of the present invention. Detailed implementation mode

[0045] The RNA room-temperature storage stabilizer provided by the present invention specifically includes functionalized polystyrene microspheres. The functionalized polystyrene microspheres are specifically polystyrene microspheres modified with functional groups, and the functional groups specifically include one or more of C1-C20 chloroalkyl groups, C6-C20 chlorophenyl groups, C1-C20 hydroxy-substituted alkyl groups, C6-C20 hydroxy-substituted phenyl groups, C2-C20 carboxy-substituted alkyl groups, C7-C20 carboxylic acid-substituted phenyl groups, C1-C20 amino-substituted alkyl groups, C6-C20 amino-substituted phenyl groups, and C4-C20 nitrogen-containing heterocycles. Among them, specific examples of the C1-C20 chloroalkyl groups include, but are not limited to: -CH2Cl, -CH2CH(Cl)CH3, or -(CH2)5CH(Cl)CH3. Specific examples of the C6-C20 chlorophenyl groups include, but are not limited to: 1-chloromethylphenyl, 2-chloromethylphenyl, or 3-chloroethylphenyl. Specific examples of the C1-C20 hydroxy-substituted alkyl groups include, but are not limited to: -CH2OH, -CH2CH2OH, -CH2CH(OH)CH3, or -(CH2)5CH(OH)CH3. Specific examples of the C6-C20 hydroxy-substituted phenyl groups include, but are not limited to: 1-hydroxyphenyl, 2-hydroxyphenyl, or 3-hydroxyphenyl. Specific examples of the C2-C20 carboxy-substituted alkyl groups include, but are not limited to: -CH2COOH, -(CH2) 12COOH or -(CH2)8CH(COOH)CH3. Specific examples of the C7-C20 carboxylic acid-substituted phenyl groups include, but are not limited to: 1-carboxyphenyl, 2-carboxyphenyl, or 1-carboxyethylphenyl. Specific examples of the C1-C20 amino-substituted alkyl groups include, but are not limited to: -CH2NH3, -CH2NH3, -CH2CH2NH3, -CH2CH(NH3)CH3, or -(CH2)5CH(NH3)CH3. Specific examples of the C6-C20 amino-substituted phenyl groups include, but are not limited to: 1-aminophenyl, 2-aminophenyl, or 1-aminomethylphenyl. The C4-C20 nitrogen-containing heterocycle is preferably a nitrogen-containing five-membered heterocycle, more preferably an N-ethyl-2-pyrrolidone group or a 1-(propyl)pyrrolidin-2-one group.

[0046] In the present invention, the functionalized polystyrene microspheres are preferably one or more of chloromethyl-modified polystyrene microspheres, hydroxyl-modified polystyrene microspheres, carboxyl-modified polystyrene microspheres, and nitrogen-containing five-membered heterocycle-modified polystyrene microspheres. In some specific embodiments, the functionalized polystyrene microspheres are more preferably nitrogen-containing five-membered heterocycle-modified polystyrene microspheres. At this time, the nitrogen-containing five-membered heterocycle-modified polystyrene microspheres are rich in functional groups and have a five-membered heterocyclic structure similar to ribose. While having a more ideal effect of stabilizing the RNA structure, it can also slow down the cleavage of ribonuclease on RNA, and has a better protection and stabilization function for RNA.

[0047] In the present invention, the average particle size of the functionalized polystyrene microspheres is preferably 0.1-200 μm, such as 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 15.6 μm, 25 μm, 50 μm, 78 μm, 90 μm, 99 μm, 100 μm, or any value therebetween. In some specific embodiments, the average particle size of the functionalized polystyrene microspheres is more preferably 20-90 μm.

[0048] The preparation method of the above RNA room temperature storage stabilizer provided by the present invention specifically includes: S1. Taking a polymerization monomer, a functionalized modifier, an initiator, and optionally polystyrene particles for a polymerization reaction to obtain the functionalized polystyrene microspheres; S2. Taking the functionalized polystyrene microspheres for a nuclease removal treatment to obtain the functionalized polystyrene microspheres.

[0049] In the present invention, the functionalizing modifier in step S1 is a type of compound that can react with the polymerization monomer and thereby introduce functional groups into the polystyrene microspheres during the polymerization of polystyrene. It can be adaptively selected according to the expected functionalized polystyrene microspheres and is not particularly limited thereto. Specific examples include, but are not limited to, one or more of chloroolefins, enols, unsaturated fatty acids, and olefinated pyrrolidones. Among them, the chloroolefin can specifically be, but is not limited to, one or more of 2-chlorostyrene, 3-chlorostyrene, chloromethylstyrene, 1-chloropropene, and 3-chloropropene. The enol can specifically be, but is not limited to, one or more of 2-propen-1-ol, 3-buten-1-ol, 2-hydroxystyrene, and 3-hydroxystyrene. Specific examples of the unsaturated fatty acid include, but are not limited to, one or more of oleic acid, linoleic acid, linolenic acid, and arachidonic acid. The olefinated pyrrolidone can specifically be, but is not limited to, N-vinyl-2-pyrrolidone and / or 1-(allyl)pyrrolidin-2-one.

[0050] In the present invention, the initiator in step S1 is a type of compound commonly used in the existing polystyrene polymerization, limited to being able to decompose to generate free radicals and initiate the polymerization reaction of the polymerization monomer, and is not particularly limited thereto. Specific examples include, but are not limited to, azo initiators and / or peroxy initiators. Among them, the azo initiator can specifically be, but is not limited to, one or more of azobisisobutyronitrile, azobisisoheptonitrile, dimethyl azobisisobutyrate, and azoisobutyronitrile formamide. The peroxy initiator can specifically be, but is not limited to, one or more of benzoyl peroxide, tert-butyl peroxy-2-ethylhexanoate, and tert-butyl peroxybenzoate.

[0051] In the present invention, the addition of the polystyrene particles in step S1 is beneficial to the control of the microsphere shape and particle size, thereby obtaining functionalized polystyrene microspheres with better storage stability for RNA; the particle size of the polystyrene particles is preferably 0.01 - 1 μm, such as 0.01 μm, 0.03 μm, 0.05 μm, 0.07 μm, 0.1 μm, 0.25 μm, 0.4 μm, 0.6 μm, 0.8 μm, 1 μm, or any value therebetween.

[0052] In the present invention, the input mass ratio of the polymerization monomer, functionalizing modifier, initiator, and optional polystyrene particles in step S1 is preferably (5 - 20):(0.1 - 20):(0.1 - 10):(0.001 - 20), such as 5:0.1:0.1:0.001, 5:13:7:19, 16:3:7:20, 20:0.1:0.1:19, 20:20:10:20, or any value therebetween.

[0053] In the present invention, the conditions for the polymerization reaction in step S1 include that the temperature is preferably 70 to 90 °C, such as 70 °C, 71.5 °C, 72 °C, 73 °C, 75 °C, 78 °C, 80 °C, 81 °C, 84 °C, 88 °C, 90 °C or any value therebetween; the time is preferably 1 to 48 h, such as 1 h, 4 h, 10 h, 12 h, 15 h, 18 h, 24 h, 30 h, 38 h, 48 h or any value therebetween.

[0054] In the present invention, the ribonuclease treatment in step S2 specifically includes: mixing the functionalized polystyrene microspheres with RNase-free water and then performing centrifugal column purification to obtain the RNA room temperature storage stabilizer.

[0055] In some specific embodiments, the conditions for the centrifugal column purification include that the centrifugation speed is preferably 5000 to 1000 rpm, such as 5000, 5100, 5500, 6000, 7000, 8000, 9000 or any value therebetween; the time is preferably 5 to 30 min, such as 5 min, 7 min, 10 min, 13 min, 15 min, 20 min, 25 min, 27 min, 30 min or any value therebetween.

[0056] The RNA product provided by the present invention includes the above-mentioned RNA room temperature storage stabilizer; by compounding the RNA room temperature storage stabilizer with RNA, the RNA product can be given good room temperature storage stability.

[0057] The preparation method of the above-mentioned RNA product provided by the present invention specifically includes: mixing RNA with the RNA room temperature storage stabilizer and then performing freeze-drying treatment to obtain the RNA product.

[0058] In the present invention, the input mass ratio of the RNA to the RNA room temperature storage stabilizer is preferably 1:(10 to 1000), such as 1:10, 1:50, 1:100, 1:125, 1:500, 1:1000 or any value therebetween.

[0059] In the present invention, the freeze-drying treatment can be a method and conditions conventionally used in the preparation of existing technology RNA products, and no special limitation is imposed on such methods, as long as dehydration and drying can be achieved.

[0060] In the present invention, the lyophilization treatment can also be a process designed by the inventors of the present invention based on a profound understanding of the changes of the above-mentioned RNA room-temperature storage stabilizer, RNA, and water molecules during the treatment through creative labor. This process specifically includes three stages: a pre-freezing stage, a primary drying stage, and a secondary drying stage. Among them, the temperature in the pre-freezing stage is preferably -60 to -50 °C, such as -60 °C, -58 °C, -54 °C, -52 °C, -51 °C, -50 °C, or any value between them; the time is preferably 5 to 15 h, such as 5 h, 5.8 h, 6 h, 7 h, 9 h, 10 h, 12 h, 15 h, or any value between them. The temperature in the primary drying stage is preferably -45 to -40 °C, such as -45 °C, -44.8 °C, -43 °C, -41.5 °C, -40 °C, or any value between them; the vacuum degree is preferably not higher than 10 Pa, more preferably 0 to 9 Pa, such as 0 Pa, 0.001 Pa, 0.1 Pa, 0.5 Pa, 1 Pa, 3 Pa, 5 Pa, 7 Pa, 9 Pa, or any value between them; the time is preferably 1 to 5 h, such as 1 h, 1.3 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 5 h, or any value between them. The secondary drying stage includes a heating period and a constant-temperature period that alternate. The starting temperature of the secondary drying stage is preferably -35 to -30 °C, such as -35 °C, -34.8 °C, -33 °C, -31 °C, -30 °C, or any value between them; the final temperature is preferably 20 to 25 °C, such as 20 °C, 21 °C, 23 °C, 24 °C, 25 °C, or any value between them; the vacuum degree is preferably not higher than 10 Pa, such as 0 Pa, 0.001 Pa, 0.3 Pa, 1.5 Pa, 3.2 Pa, 5 Pa, 9 Pa, or any value between them; the total time is preferably 40 to 60 h, 40 h, 41 h, 43 h, 45 h, 50 h, 51 h, 56 h, 59 h, 60 h, or any value between them; the heating rate in the heating period is preferably 0.5 to 2 °C / min, such as 0.5 °C / min, 0.6 °C / min, 0.8 °C / min, 1 °C / min, 1.5 °C / min, 1.8 °C / min, 2 °C / min, or any value between them; the time is preferably 25 to 40 min, such as 25 min, 25.6 min, 28 min, 30 min, 33 min, 35 min, 37.6 min, 38 min, 40 min, or any value between them. At this time, the method of segmented lyophilization supplemented by intermittent heating can effectively reduce the damage to the RNA structure during the lyophilization process and achieve a better binding effect between the functionalized polystyrene microspheres and RNA, further improving the room-temperature storage stability of the RNA product.

[0061] The present invention also provides the application of the above-mentioned RNA room-temperature storage stabilizer, RNA product and / or the preparation method of the RNA product in the production of mRNA drugs and / or vaccines.

[0062] The embodiments of the present invention are described in detail below. The examples are intended to explain the present invention and should not be construed as limiting the present invention. For those not specified in the embodiments, the techniques or conditions described in the literature in the art or in accordance with the product specifications are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0063] Synthesis Example

[0064] This synthesis example is used to illustrate the preparation of polystyrene microspheres, which specifically includes: accurately sucking 9.8 mL of styrene, 15 mL of absolute ethanol, and 125 mL of deionized water into a round-bottom flask, adding 652 mg of polyvinylpyrrolidone (PVP K-30, weight-average molecular weight of 58,000) and 153.2 mg of benzoyl peroxide (BPO), vacuum degassing and rapidly stirring to form an emulsion; then, under a nitrogen atmosphere, reacting in an oil bath at 73 °C for 6 h; after the reaction is completed and cooled to room temperature, the obtained emulsion is washed repeatedly with absolute ethanol 10 times, then washed with deionized water 8 times, and vacuum dried at 60 °C for 48 h to obtain polystyrene microspheres, denoted as PS microspheres.

[0065] Preparation Example 1

[0066] This preparation example is used to illustrate the preparation of chloromethyl-modified polystyrene microspheres, which specifically includes: accurately sucking 17.6 mL of styrene, 2 mL of divinylbenzene, 5 mL of chloromethylstyrene, and 250 mL of deionized water into a round-bottom flask, adding 2.0 g of polyvinyl alcohol (viscosity of 6.0 mPa·s) and 500 mg of BPO, vacuum degassing and rapidly stirring to form an emulsion; then, under a nitrogen atmosphere, reacting in an oil bath at 82 °C for 4 h; after the reaction is completed and cooled to room temperature, the obtained emulsion is washed repeatedly with absolute ethanol 10 times, then washed with deionized water 8 times, and vacuum dried at 60 °C for 48 h to obtain chloromethyl-modified polystyrene microspheres, denoted as PS-Cl microspheres.

[0067] The SEM image and infrared spectrum of the PS-Cl microspheres prepared in this preparation example are as Figure 1 and 2 shown. The PS-Cl microspheres are spherical with a smooth surface, and the diameter is 38 - 82 μm; and strong absorption peaks appear near 2930 - 2850 cm -1 and 700 - 750 cm -1 nearby.

[0068] Preparation Example 2

[0069] This preparation example is used to illustrate the preparation of hydroxyl-modified polystyrene microspheres, which specifically includes: accurately pipetting 9.0 mL of styrene, 50 μL of divinylbenzene, 1 mL of 3-buten-1-ol, 22 mL of absolute ethanol, and 19.5 mL of deionized water into a round-bottom flask, adding 1.0 g of PS microspheres provided by the synthesis example, 3.0 g of polyethylene glycol 4000, and ultrasonically dispersing for 30 min; then adding 0.182 g of ammonium persulfate, stirring thoroughly, degassing under vacuum, and quickly stirring until it becomes an emulsion; subsequently, under a nitrogen atmosphere, reacting in an oil bath at 70 °C for 12 h; after the reaction is completed and cooled to room temperature, the obtained emulsion is repeatedly washed 10 times with absolute ethanol, then washed 8 times with deionized water, and dried in vacuum at 60 °C for 48 h to obtain hydroxyl-modified polystyrene microspheres, denoted as PS-OH microspheres.

[0070] The SEM image and infrared spectrum of the PS-OH microspheres prepared in this preparation example are as Figure 3 and 4 shown. The PS-OH microspheres are spherical with certain defects on the surface, and the diameter is 2 - 3.5 μm; and strong absorption peaks appear near 3650 - 3600 cm -1 , 1500 - 1450 cm -1 and 769 - 659 cm -1 .

[0071] Preparation Example 3

[0072] This preparation example is used to illustrate the preparation of carboxyl-modified polystyrene microspheres, which specifically includes: accurately pipetting 20 mL of styrene, 0.2 mL of divinylbenzene, 2.0 mL of linoleic acid, and 75 mL of absolute ethanol into a round-bottom flask, adding 1.5 g of PS microspheres provided by the synthesis example, 7.0 g of polyethylene glycol 4000, and 6.0 g of BPO, and ultrasonically dispersing for 30 min; degassing under vacuum and quickly stirring until it becomes an emulsion; subsequently, under a nitrogen atmosphere, reacting in an oil bath at 70 °C for 10 h; after the reaction is completed and cooled to room temperature, the obtained emulsion is repeatedly washed 10 times with absolute ethanol, then washed 8 times with deionized water, and dried in vacuum at 60 °C for 48 h to obtain carboxyl-modified polystyrene microspheres, denoted as PS-COOH microspheres.

[0073] The SEM image and infrared spectrum of the PS-COOH microspheres prepared in this preparation example are as Figure 5 and 6 shown. The PS-COOH microspheres are spherical with a smooth surface, and the diameter is 2 - 3 μm; and strong absorption peaks appear near 3300 - 2500 cm -1 , 1720 - 1210 cm -1 and 750 - 720 cm -1 .

[0074] Preparation Example 4

[0075] This preparation example is used to illustrate the preparation of nitrogen-containing five-membered heterocyclic modified polystyrene microspheres, which specifically includes: accurately sucking 9.0 mL of styrene, 15 mL of absolute ethanol, and 125 mL of deionized water into a round-bottom flask, adding 652 mg of polyvinylpyrrolidone (PVP K-30, weight-average molecular weight of 58000) and 122.5 mg of AIBN, vacuum degassing and rapidly stirring to form an emulsion; then, under a nitrogen atmosphere, reacting in an oil bath at 73 °C for 15 h, and then adding 1 mL of N-vinyl-2-pyrrolidone and continuing the reaction for 3 h; after the reaction is completed and cooled to room temperature, the obtained emulsion is repeatedly washed 10 times with absolute ethanol, then washed 8 times with deionized water, and dried in vacuo at 60 °C for 48 h to obtain nitrogen-containing five-membered heterocyclic modified polystyrene microspheres, denoted as PS-N microspheres.

[0076] The SEM image and infrared spectrum of the PS-N microspheres prepared in this preparation example are as Figure 7 and 8 shown. The PS-N microspheres are smooth spherical in shape with a diameter of 1 - 1.5 μm; and strong absorption peaks appear near 3100 - 2750 cm -1 , 1750 - 1370 cm -1 and 750 - 650 cm -1 .

[0077] Example 1

[0078] This example is used to illustrate a preparation method of an RNA product, and the storage method specifically includes:

[0079] S1. Pretreatment: (1) Take 3.5 mg of the PS-Cl microspheres provided in Preparation Example 1 and perform deenzyming treatment by the centrifugal column method, repeat the washing 6 times, vortex for 5 min each time; centrifuge at 7000 rpm for 30 s, add RNase-free water to the precipitate to a final volume of 200 μL to obtain an RNA room temperature storage stabilizer; (2) In a sterile and enzyme-free workbench, add 10 μg of IVT RNA to 200 μL of the RNA room temperature storage stabilizer to obtain an RNA sample, fully mix and transfer it to a vial, and semi-cork it.

[0080] S2. Freeze-drying: Place the semi-corked vial on the freeze-drying chamber shelf of a four-ring vacuum freeze-dryer (LGJ-S30 capping type) at room temperature, and perform freeze-drying treatment according to the procedure shown in Table 1 to obtain an RNA product.

[0081] S3. Transfer the vial containing the RNA product to the glove box transition room, evacuate and perform nitrogen replacement, place it overnight and then transfer it to the operation room, and wax-seal the vial cap.

[0082] Among them, the IVT RNA used in step S1 of this embodiment is synthesized by PCR reaction with primers of eGFP nucleotide sequence using pUC19 plasmid DNA as a template and then in vitro transcription with T7 polymerase. Its length is 850 nt.

[0083] Table 1.

[0084]

[0085] Example 2

[0086] The preparation method of the RNA product provided in this embodiment is basically the same as that of Example 1. The difference is that in step S1, PS-OH microspheres provided in Preparation Example 2 with equal mass are used to replace the PS-Cl microspheres provided in Preparation Example 1, and other conditions are the same, obtaining the RNA product.

[0087] Example 3

[0088] The preparation method of the RNA product provided in this embodiment is basically the same as that of Example 1. The difference is that in step S1, PS-COOH microspheres provided in Preparation Example 3 with equal mass are used to replace the PS-Cl microspheres provided in Preparation Example 1, and other conditions are the same, obtaining the RNA product.

[0089] Example 4

[0090] The preparation method of the RNA product provided in this embodiment is basically the same as that of Example 1. The difference is that in step S1, PS-N microspheres provided in Preparation Example 4 with equal mass are used to replace the PS-Cl microspheres provided in Preparation Example 1, and other conditions are the same, obtaining the RNA product.

[0091] Example 5

[0092] The preparation method of the RNA product provided in this embodiment is basically the same as that of Example 1. The difference is that in step S1, epoxy group-modified polystyrene microspheres (Zhiyi, brand number PSEP) are used to replace the PS-Cl microspheres provided in Preparation Example 1, and other conditions are the same, obtaining the RNA product.

[0093] Example 6

[0094] The preparation method of the RNA product provided in this embodiment is basically the same as that of Example 1. The difference is that in step S1, the addition amount of IVT RNA is 1 μg, and other conditions are the same, obtaining the RNA product.

[0095] Example 7

[0096] The preparation method of the RNA product provided in this embodiment is basically the same as that in Example 1, except that in step S1, the amount of IVT RNA added is 200 μg, and other conditions are the same to obtain an RNA product.

[0097] Example 8

[0098] The preparation method of the RNA product provided in this embodiment is basically the same as that in Example 1, except that in step S2, the freeze-drying conditions are different, as shown in Table 2, to obtain the RNA product.

[0099] Table 2.

[0100]

[0101] Example 9

[0102] The preparation method of the RNA product provided in this embodiment is basically the same as that in Example 1, except that in step S2, the half-stoppered vial is directly placed at -70°C for freeze drying for 63 hours, and other conditions are the same to obtain the RNA product.

[0103] Comparative Example

[0104] This comparative example provides a method for preparing an RNA product, which specifically comprises:

[0105] S1. Pretreatment: Add 10 μg of IVT RNA to 200 μL of RNase-free water to obtain RNA sample. After thorough mixing, transfer to a vial and half-stopper it.

[0106] S2. Freeze drying: Place the half-stoppered vial on the freeze drying chamber shelf of a four-ring vacuum freeze dryer (LGJ-S30 capping type) at room temperature, and perform freeze drying according to the same procedure as in Example 1 (ie, Table 1) to obtain an RNA product.

[0107] S3. Transfer the vials containing RNA products to the transition room of the glove box, evacuate and replace with nitrogen, leave them overnight and move them to the operating room, and seal the vial caps with wax.

[0108] Test Case

[0109] This test example is used to illustrate the storage stability of the RNA products provided in the above embodiments and comparative examples. The RNA product was taken out after being stored at 25, 37 and 60°C for a period of time. In a sterile enzyme-free operating table, 100 μL of RNase-free water was taken to rehydrate the RNA product, shaken and mixed for 15 minutes, centrifuged at 7000 rpm for 4 minutes, and the supernatant was taken. The supernatant was filtered using a 0.22 μm RNase-free filter membrane. The RNA product with trehalose added as a protective agent was used as a positive quality control group. The preparation of the RNA product specifically includes: in a sterile enzyme-free operating table, 10 μg of IVT RNA was added to 200 μL of 1.72% (w:v) trehalose RNase-free aqueous solution to obtain an RNA sample, which was fully mixed and transferred to a vial and half-stoppered; the half-stoppered vial was placed on the freeze-drying chamber shelf of a four-ring vacuum freeze dryer (LGJ-S30 capping type) at room temperature, and freeze-dried according to the same procedure as in Example 1 (i.e., Table 1) to obtain an RNA product. And the detection was performed according to the following method:

[0110] (1) Capillary electrophoresis detection: 1 μL of the supernatant was taken and tested using an automatic capillary electrophoresis instrument (Agilent Fragment Analyzer 5200) to obtain the percentage of normal IVT RNA concentration to the total RNA concentration. The results are shown in Table 3.

[0111] Table 3.

[0112]

[0113] Note: -- indicates that normal RNA bands can no longer be detected and the RNA is completely degraded.

[0114] It can be seen from the test results that, compared with the comparative example, the RNA products provided by Examples 1 to 9 of the present invention and the positive quality control group can still maintain good RNA integrity after being stored at 37° C. for 21 days.

[0115] (2) Agarose gel electrophoresis detection: (i) Mix 0.5 g of agarose with 50 mL of 1×TAE solution and heat in a microwave oven until completely dissolved to obtain 1% agarose gel; after cooling for a few seconds, pour into an electrophoresis mold, cool into gel, remove the comb, put into a TAE electrophoresis tank, and pour in 1×TAE solution until the wells are filled with liquid and the solution covers the agarose gel;

[0116] (ii) Take 500 mg of sucrose, 500 μL of water, and 5 μL of 10,000× Gel Red to obtain the Loading Dye loading buffer; take 3 μL of Ladder (TaKaRa, catalog number 3427A), add 15 μL of RNase-free water, and then add 2 μL of the Loading Dye loading buffer to obtain the Ladder electrophoresis loading solution;

[0117] (iii) Take 500 ng of the supernatant, 2 μL of the Loading Dye, and add RNase-free water to a final volume of 20 μL to obtain the RNA electrophoresis loading solution;

[0118] (iv) Add the Ladder electrophoresis loading solution and the RNA electrophoresis loading solution to the agarose gel wells in the electrophoresis tank at one time, cover the lid, and perform electrophoresis at 120 V for 30 min; then transfer the gel to a Bio-Rad instrument for photography, and the results are as Figures 9 - 15 shown.

[0119] From Figure 9 and 10 it can be seen that after storing at 25 °C for 7 d and 21 d, the bands of the comparative example showed slight downward diffusion, indicating that some RNA was degraded, while the bands of Examples 1 to 4 of the present invention were clearly visible and there was no downward diffusion, indicating that the RNA room temperature storage stabilizer has good protection and stabilization effects on the freeze-drying of RNA and the storage process at 25 °C.

[0120] From Figure 11 and 12 it can be seen that after storing at 37 °C for 7 d and 21 d, the bands of the comparative example showed slight downward diffusion, indicating that some RNA was degraded, while the bands of Examples 1 to 4 of the present invention were clearly visible and there was no downward diffusion, indicating that the RNA room temperature storage stabilizer has good protection and stabilization effects on the freeze-drying of RNA and the storage process at 37 °C.

[0121] From Figures 13 - 15 it can be seen that after storing at 60 °C for 1 d and 5 d, the bands of the comparative example showed slight and obvious downward diffusion respectively, indicating that RNA was degraded at 5 d, while the bands of Examples 1 to 4 of the present invention did not show significant degradation; after storing at 60 °C for 14 d, the bands of the comparative example completely disappeared, while the bands of Examples 1 to 4 of the present invention were still clearly visible and only partially degraded, indicating that the RNA room temperature storage stabilizer has good protection and stabilization effects on the freeze-drying of RNA and the storage process at 60 °C.

[0122] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

Claims

1. A method for preparing an RNA product, characterized in that, The preparation method includes: mixing RNA with an RNA room-temperature storage stabilizer and then performing freeze-drying treatment to obtain the RNA product; wherein, the RNA room-temperature storage stabilizer includes functionalized polystyrene microspheres, and the functionalized polystyrene microspheres are selected from one or more of chloromethyl-modified polystyrene microspheres, hydroxyl-modified polystyrene microspheres, carboxyl-modified polystyrene microspheres, and nitrogen-containing five-membered heterocyclic ring-modified polystyrene microspheres.

2. The preparation method of the RNA product according to claim 1, wherein The average particle size of the functionalized polystyrene microspheres is 0.1 - 200 μm.

3. The preparation method of the RNA product according to claim 1, characterized in that, The average particle size of the functionalized polystyrene microspheres is 1 - 2 μm.

4. The preparation method of the RNA product according to claim 1, characterized in that, The preparation method of the functionalized polystyrene microspheres includes: S1. Taking a polymerization monomer, a functionalization modifier, an initiator, and optionally polystyrene particles for polymerization reaction to obtain the functionalized polystyrene microspheres; S2. Taking the functionalized polystyrene microspheres for ribonuclease treatment to obtain the functionalized polystyrene microspheres.

5. The preparation method of the RNA product according to claim 4, wherein In step S1, the polymerization monomer is selected from one or more of styrene, styrene propylene, and divinylbenzene.

6. The preparation method of the RNA product according to claim 4, wherein In step S1, the functionalization modifier is selected from one or more of chloroolefins, enols, unsaturated fatty acids, and olefinated pyrrolidones.

7. The method for preparing an RNA product according to claim 4, wherein In step S1, the initiator is an azo initiator and / or a peroxide initiator.

8. The preparation method of the RNA product according to claim 4, wherein In step S1, the average particle size of the polystyrene particles is 0.01 - 1 μm.

9. The preparation method of the RNA product according to claim 4, wherein In step S1, the input mass ratio of the polymerization monomer, functionalization modifier, initiator, and polystyrene particles is (5 - 20):(0.1 - 20):(0.1 - 10):(0.001 - 20).

10. The preparation method of the RNA product according to claim 4, wherein, In step S1, the temperature of the polymerization reaction is 70 - 90 °C, and the time is 1 - 48 h.

11. The preparation method of the RNA product according to claim 4, characterized in that, In step S2, the ribonuclease treatment specifically includes: mixing the functionalized polystyrene microspheres with RNase-free water and then performing centrifugal column purification to obtain the RNA room-temperature storage stabilizer.

12. The method for preparing an RNA product according to claim 11, wherein In step S2, the centrifugation speed for the centrifugal column purification is 5000 - 1000 rpm, and the time is 5 - 30 min.

13. The preparation method of the RNA product according to claim 1, characterized in that, The input mass ratio of the RNA to the RNA room-temperature storage stabilizer is 1:(10 - 1000).

14. The preparation method of the RNA product according to claim 1, characterized in that, The freeze-drying treatment includes a pre-freezing stage, a primary drying stage, and a secondary drying stage. The temperature of the pre-freezing stage is -60 - -50 °C, and the time is 5 - 15 h; the temperature of the primary drying stage is -45 - -40 °C, the vacuum degree is not higher than 10 Pa, and the time is 1 - 5 h; the secondary drying stage includes an alternating heating period and a constant temperature period. The starting temperature of the secondary drying stage is -35 - -30 °C, the final temperature is 20 - 25 °C, the vacuum degree is not higher than 10 Pa, the total time is 40 - 60 h, the heating rate of the heating period is 0.5 - 2 °C / min, and the time is 25 - 40 min.

15. Application of the preparation method of the RNA product according to claims 1 - 14 in the production of mRNA drugs and / or vaccines.

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