RNA room-temperature preservation stabilizer, RNA product, preparation method, and use

Abstract

An RNA room-temperature preservation stabilizer, an RNA product, a preparation method, and a use. The RNA room-temperature preservation stabilizer comprises functionalized polystyrene microspheres. The RNA room-temperature preservation stabilizer has good adsorption capacity for an RNA. A functional group on the RNA room-temperature preservation stabilizer can interact with a group in the RNA, so that the RNA molecule undergoes rotation, bending, twisting, and folding to form a specific three-dimensional structure, thereby achieving the effect of stabilizing the structure of the RNA. Long-term stable preservation of the RNA at room temperature can be realized without introducing ultra-low temperature equipment and other protective reagents. Moreover, the addition of water can destroy the binding between the functionalized polystyrene microspheres and the RNA, thereby achieving complete separation of the RNA from the stabilizer. The present invention has the advantages of low cost, simple operation, and high biological safety and has good application prospects.

Description

RNA room temperature storage stabilizer, RNA product and preparation method and application TECHNICAL FIELD

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

[0002] RNA is a long-chain molecule composed of ribonucleotides condensed by phosphodiester bonds, and widely exists in biological cells, some viruses and viroids. Among them, mRNA can be applied to the fields of vaccines (infectious vaccines and tumor vaccines), protein replacement therapy, cell engineering and gene editing by transferring mRNA into the cells of patients (or recipients) to successfully translate the specified proteins 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 be designed to code any antigen according to the unique properties of diseases and regulate the expression of selected antigens, so as to quickly respond to the variation of pathogenic microorganisms and provide more timely and reliable protection. However, the structural stability defects of mRNA itself lead to inevitable degradation during storage and transportation, resulting in structural changes, quality reduction and further affecting the biological activity of mRNA.

[0003] At present, mRNA is generally stored by using the methods of ultra-low temperature preservation, additional protective agent or a combination of both. However, in the ultra-low temperature preservation, mRNA is generally stored in an ultra-low temperature refrigerator at-80 DEG C or even liquid nitrogen to minimize the degradation rate, which puts forward higher requirements for transportation and application scenarios, resulting in increased energy consumption, higher transportation cost and safety problems; and the introduction of protective agents may reduce the biological safety of mRNA drugs or vaccines, which has great limitations.

[0004] SUMMARY

[0005] The first object of the present application is to solve the problems of high storage condition requirement, high storage cost and low biological safety in the existing mRNA storage, and to obtain a simple and effective, economical and practical and high biological safety method for realizing long-term stable storage and transportation of mRNA, and a RNA room temperature storage stabilizer is provided.

[0006] The second object of the present application is to provide a preparation method of the above RNA room temperature storage stabilizer.

[0007] The third object of the present application is to provide a RNA product.

[0008] A fourth object of the present application is to provide a method for preparing an RNA product.

[0009] A fifth object of the present application is to provide an application of the above RNA room temperature storage stabilizer and / or the method for preparing an RNA product in mRNA drug and / or vaccine production.

[0010] Specifically, the RNA room temperature storage stabilizer provided by the present application comprises functionalized polystyrene microspheres.

[0011] In some specific embodiments, the functionalized polystyrene microspheres are polystyrene microspheres modified with a functional group selected from one or more of C1-C20 chloroalkyl, C6-C20 chlorophenyl, C1-C20 hydroxy-substituted alkyl, C6-C20 hydroxy-substituted phenyl, C2-C20 carboxy-substituted alkyl, C7-C20 carboxylic acid-substituted phenyl, C1-C20 amino-substituted alkyl, C6-C20 amino-substituted phenyl, and C4-C20 nitrogen-containing heterocycle.

[0012] In some specific embodiments, 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 heterocycle-modified polystyrene microspheres.

[0013] In some specific embodiments, the functionalized polystyrene microspheres have an average particle size of 0.1-200 μm.

[0014] In some specific embodiments, the functionalized polystyrene microspheres have an average particle size of 1-2 μm.

[0015] The method for preparing the above RNA room temperature storage stabilizer provided by the present application comprises: S1, taking a polymerization monomer, a functionalization modifier, an initiator, and optionally polystyrene particles to perform a polymerization reaction to obtain the functionalized polystyrene microspheres; and S2, taking the functionalized polystyrene microspheres to perform a de-nucleic acid enzyme treatment to obtain the RNA room temperature storage stabilizer.

[0016] In some specific embodiments, in step S1, the polymerization monomer is selected from one or more of styrene, phenylacrylate, and divinylbenzene.

[0017] In some specific embodiments, in step S1, the functionalization modifier is selected from one or more of a chloroalkene, an enol, an unsaturated fatty acid, and an olefinated pyrrolidone.

[0018] In some specific embodiments, in step S1, the initiator is an azo initiator and / or a peroxide initiator.

[0019] In some specific embodiments, the polystyrene particles have an average particle size of 0.01-1 μm in step S1.

[0020] In some specific embodiments, the mass ratio of the polymerized monomer, the functional modification agent, the initiator and the polystyrene particles is (5-20):(0.1-20):(0.1-10):(0.001-20) in step S1.

[0021] In some specific embodiments, the polymerization reaction is performed at a temperature of 70-90℃ for 1-48 h in step S1.

[0022] In some specific embodiments, the RNase removal process specifically comprises: mixing the functionalized polystyrene microspheres with RNase-free water, and then performing centrifugal column purification to obtain the RNA room temperature storage stabilizer.

[0023] In some specific embodiments, the centrifugal column purification is performed at a centrifugal speed of 5000-1000 rpm for 5-30 min.

[0024] The RNA product provided by the present application comprises the above RNA room temperature storage stabilizer.

[0025] The preparation method of the RNA product provided by the present application comprises: mixing RNA with the RNA room temperature storage stabilizer, and then performing freeze-drying treatment to obtain the RNA product.

[0026] In some specific embodiments, the mass ratio of the RNA and the RNA room temperature storage stabilizer is 1:(10-1000).

[0027] In some specific embodiments, the freeze-drying treatment comprises a pre-freezing stage, a first drying stage and a second drying stage, the pre-freezing stage is performed at a temperature of -60 to -50℃ for 5-15 h, the first drying stage is performed at a temperature of -45 to -40℃, a vacuum degree of no more than 10 Pa, and a time of 1-5 h, and the second drying stage comprises an alternating temperature rising period and a constant temperature period, the second drying stage is performed at a starting temperature of -35 to -30℃, a final temperature of 20-25℃, a vacuum degree of no more than 10 Pa, and a total time of 40-60 h, the temperature rising rate of the temperature rising period is 0.5-2℃ / min, and the time is 25-40 min.

[0028] The present application also provides the use of the above-mentioned RNA room temperature storage stabilizer, RNA product and / or preparation method of the RNA product in the production of mRNA drugs and / or vaccines. Advantages:

[0029] The application provides an RNA room temperature storage stabilizer, which comprises functionalized polystyrene microspheres, the functionalized polystyrene microspheres have good adsorption capacity for RNA, and functionalized groups on the functionalized polystyrene microspheres can interact with groups in the RNA, so that the RNA molecules are rotated, twisted, wound and folded to form a specific three-dimensional structure, thereby playing a role in stabilizing the RNA structure, and long-term stable storage of the RNA at room temperature can be realized without introducing an ultralow-temperature device and other protective reagents; and the addition of water can destroy the combination between the functionalized polystyrene microspheres and the RNA, so that the RNA and the stabilizer are completely separated, and the application has the advantages of low cost, simple operation, high biological safety and good application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0030] Fig. 1 is an infrared spectrum of the PS-Cl microspheres prepared in Preparation Example 1 of the application;

[0031] Fig. 2 is an SEM image (200 μm) of the PS-Cl microspheres prepared in Preparation Example 1 of the application;

[0032] Fig. 3 is an infrared spectrum of the PS-OH microspheres prepared in Preparation Example 2 of the application;

[0033] Fig. 4 is an SEM image (5.0 μm) of the PS-OH microspheres prepared in Preparation Example 2 of the application;

[0034] Fig. 5 is an infrared spectrum of the PS-COOH microspheres prepared in Preparation Example 3 of the application;

[0035] Fig. 6 is an SEM image (5.0 μm) of the PS-COOH microspheres prepared in Preparation Example 3 of the application;

[0036] Fig. 7 is an infrared spectrum of the PS-N microspheres prepared in Preparation Example 4 of the application;

[0037] Fig. 8 is an SEM image (5.0 μm) of the PS-N microspheres prepared in Preparation Example 4 of the application;

[0038] Fig. 9 is an experimental result diagram of electrophoresis test of the RNA product after storage at 25 DEG C for 7 days in the test example of the application;

[0039] Fig. 10 is an experimental result diagram of electrophoresis test of the RNA product after storage at 25 DEG C for 21 days in the test example of the application;

[0040] Fig. 11 is an experimental result diagram of electrophoresis test of the RNA product after storage at 37 DEG C for 7 days in the test example of the application;

[0041] Fig. 12 is an experimental result diagram of electrophoresis test of the RNA product after storage at 37 DEG C for 21 days in the test example of the application;

[0042] Figure 13 is a graph showing the results of the electrophoresis test of the RNA product stored at 60°C for 1 day in the test example of the present application;

[0043] Figure 14 is a graph showing the results of the electrophoresis test of the RNA product stored at 60°C for 5 days in the test example of the present application;

[0044] Figure 15 is a graph showing the results of the electrophoresis test of the RNA product stored at 60°C for 14 days in the test example of the present application. DETAILED DESCRIPTION

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

[0046] In the present application, 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 functional groups rich on the nitrogen-containing five-membered heterocycle-modified polystyrene microspheres have a five-membered heterocycle structure similar to ribose, which can slow down the cleavage of RNA by ribonuclease while having a more ideal stable RNA structure, and has a better protection and stabilization function for RNA.

[0047] In the present application, 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, 150 μm, 200 μm, or any value therebetween. In some specific embodiments, the average particle size of the functionalized polystyrene microspheres is more preferably 1-2 μm.

[0048] The preparation method of the above RNA room temperature storage stabilizer provided by the present application specifically comprises: S1, taking a polymerization monomer, a functionalization modifier, an initiator, and optionally polystyrene particles to perform a polymerization reaction to obtain the functionalized polystyrene microspheres; S2, taking the functionalized polystyrene microspheres to perform a ribozyme treatment to obtain the RNA room temperature storage stabilizer.

[0049] In the present application, the functionalization modifier in step S1 is a type of compound that can introduce a functional group into the polystyrene microspheres during the reaction with the polymerization monomer and the polystyrene polymerization process, which can be selected according to the desired functionalized polystyrene microspheres, and is not particularly limited, and specific examples include but are not limited to one or more of chlorinated olefins, enols, unsaturated fatty acids, and olefinated pyrrolidones. Among them, the chlorinated olefins can be but are not limited to one or more of 2-chlorostyrene, 3-chlorostyrene, chloromethylstyrene, 1-chloropropene, and 3-chloropropene. The enols can be but are 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 acids include but are not limited to one or more of oleic acid, linoleic acid, linolenic acid, and arachidonic acid. The olefinated pyrrolidones can be but are not limited to N-vinyl-2-pyrrolidone and / or 1-(allyl)pyrrolidine-2-one.

[0050] In the present application, the initiator in step S1 is a kind of compound commonly used in existing polystyrene polymerization, which can decompose to generate free radicals and initiate polymerization of the polymerization monomer, and is not particularly limited, and specific examples include but are not limited to: azo initiator and / or peroxide initiator. Among them, the azo initiator can be but is not limited to one or more of azobisisobutyronitrile, azobisisoheptyl nitrile, dimethyl azobis-2-ethylhexanoate and azoisobutyryl cyan formamide. The peroxide initiator can 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 application, the addition of the polystyrene particles in step S1 is beneficial to the control of the microspherical 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 application, the mass ratio of the polymerization monomer, the functionalization modifier, the initiator and the 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 application, the conditions of the polymerization reaction in step S1 include a temperature of preferably 70-90℃, such as 70℃, 71.5℃, 72℃, 73℃, 75℃, 78℃, 80℃, 81℃, 84℃, 88℃, 90℃ or any value therebetween; and a time of preferably 1-48 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 application, the RNase-free enzyme treatment in step S2 specifically includes: after the functionalized polystyrene microspheres are mixed with RNase-free water, centrifugal column purification is performed to obtain the RNA room temperature storage stabilizer.

[0055] In some specific embodiments, the conditions of the centrifugal column purification include a centrifugal speed of preferably 5000-10000 rpm, such as 5000 rpm, 5100 rpm, 5500 rpm, 6000 rpm, 7000 rpm, 8000 rpm, 9000 rpm, or any value therebetween; and a time of preferably 5-30 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 application comprises the above-mentioned RNA room temperature storage stabilizer; by compounding the RNA room temperature storage stabilizer with the RNA, the RNA product can be endowed with good room temperature storage stability.

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

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

[0059] In the present application, the freeze-drying treatment can be the method and condition commonly used in the preparation of the prior art RNA product, and such method is not particularly limited, as long as it can realize dehydration and drying.

[0060] In the present application, the freeze-drying process can also be a process designed by the inventors of the present application based on their deep understanding of the changes of the above-mentioned RNA room temperature storage stabilizer, RNA and water molecules during the process, after creative labor, which specifically comprises three stages of pre-freezing stage, first drying stage and second drying stage. The temperature of the pre-freezing stage is preferably -60 to -50℃, such as -60℃, -58℃, -54℃, -52℃, -51℃, -50℃ or any value between them, and the time is preferably 5 to 15h, such as 5h, 5.8h, 6h, 7h, 9h, 10h, 12h, 15h or any value between them. The temperature of the first drying stage is preferably -45 to -40℃, such as -45℃, -44.8℃, -43℃, -41.5℃, -40℃ or any value between them; the vacuum degree is preferably not higher than 10Pa, more preferably 0 to 9Pa, such as 0Pa, 0.001Pa, 0.1Pa, 0.5Pa, 1Pa, 3Pa, 5Pa, 7Pa, 9Pa or any value between them; the time is preferably 1 to 5h, such as 1h, 1.3h, 1.5h, 2h, 2.5h, 3h, 4h, 5h or any value between them. The second drying stage includes alternating temperature rising period and constant temperature period, the initial temperature of the second drying stage is preferably -35 to -30℃, such as -35℃, -34.8℃, -33℃, -31℃, -30℃ or any value between them; the final temperature is preferably 20 to 25℃, such as 20℃, 21℃, 23℃, 24℃, 25℃ or any value between them; the vacuum degree is preferably not higher than 10Pa, such as 0Pa, 0.001Pa, 0.3Pa, 1.5Pa, 3.2Pa, 5Pa, 9Pa or any value between them; the total time is preferably 40 to 60h, 40h, 41h, 43h, 45h, 50h, 51h, 56h, 59h, 60h or any value between them; the temperature rising rate of the temperature rising period is preferably 0.5 to 2℃ / min, such as 0.5℃ / min, 0.6℃ / min, 0.8℃ / min, 1℃ / min, 1.5℃ / min, 1.8℃ / min, 2℃ / min or any value between them; the time is preferably 25 to 40min, such as 25min, 25.6min, 28min, 30min, 33min, 35min, 37.6min, 38min, 40min or any value between them. At this time, the method of segmented freeze-drying supplemented by intermittent temperature rising can effectively reduce the damage of the freeze-drying process to the structure of RNA, and achieve better combination effect of functionalized polystyrene microspheres and RNA, further improve the room temperature storage stability of RNA products.

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

[0062] Embodiments of the present application are described in detail below, examples of which are intended to explain the present application and cannot be understood as a limitation of the present application. If specific techniques or conditions are not mentioned in the embodiments, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the manufacturer of the reagent or instrument is not mentioned, it is a conventional product that can be obtained by purchase.

[0063] Synthesis Example

[0064] The synthesis example is used to illustrate the preparation of polystyrene microspheres, specifically including: accurately taking 9.8 mL of styrene, 15 mL of anhydrous 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 153.2 mg of benzoyl peroxide (BPO), vacuum degassing and rapid stirring to emulsion; then under a nitrogen atmosphere, reacting in an oil bath at 73°C for 6h; after the reaction is completed, cooling to room temperature, the obtained emulsion is repeatedly washed with anhydrous ethanol for 10 times, then continues to be washed with deionized water for 8 times, and vacuum dried at 60°C for 48h to obtain polystyrene microspheres, which are recorded as PS microspheres.

[0065] Preparation Example 1

[0066] The preparation example is used to illustrate the preparation of chloromethyl-modified polystyrene microspheres, specifically including: accurately taking 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 rapid stirring to emulsion; then under a nitrogen atmosphere, reacting in an oil bath at 82°C for 4h; after the reaction is completed, cooling to room temperature, the obtained emulsion is repeatedly washed with anhydrous ethanol for 10 times, then continues to be washed with deionized water for 8 times, and vacuum dried at 60°C for 48h to obtain chloromethyl-modified polystyrene microspheres, which are recorded as PS-Cl microspheres.

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

[0068] Preparation Example 2

[0069] This preparation example is used to illustrate the preparation of a hydroxyl modified polystyrene microsphere, specifically comprising: accurately pipetting 9.0 mL of styrene, 50 μL of divinylbenzene, 1 mL of 3-buten-1-ol, 22 mL of anhydrous 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 ultrasonic dispersion for 30 min; then adding 0.182 g of ammonium persulfate and stirring thoroughly, vacuum degassing and fast stirring to emulsion; then under nitrogen atmosphere, reacting in an oil bath at 70°C for 12 h; after the reaction is completed, cooling to room temperature, the obtained emulsion is repeatedly washed with anhydrous ethanol for 10 times, then continues to be washed with deionized water for 8 times, and vacuum dried at 60°C for 48 h to obtain a hydroxyl modified polystyrene microsphere, denoted as PS-OH microsphere.

[0070] The SEM image and infrared spectrum of the PS-OH microsphere obtained by this preparation example are shown in Figures 3 and 4, the PS-OH microsphere is in a spherical shape with a certain defect 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 a carboxyl modified polystyrene microsphere, specifically comprising: accurately pipetting 20 mL of styrene, 0.2 mL of divinylbenzene, 2.0 mL of linoleic acid and 75 mL of anhydrous 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 ultrasonic dispersion for 30 min; vacuum degassing and fast stirring to emulsion; then under nitrogen atmosphere, reacting in an oil bath at 70°C for 10 h; after the reaction is completed, cooling to room temperature, the obtained emulsion is repeatedly washed with anhydrous ethanol for 10 times, then continues to be washed with deionized water for 8 times, and vacuum dried at 60°C for 48 h to obtain a carboxyl modified polystyrene microsphere, denoted as PS-COOH microsphere.

[0073] The SEM image and infrared spectrum of the PS-COOH microsphere obtained by this preparation example are shown in Figures 5 and 6, the PS-COOH microsphere is in a spherical shape 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 a nitrogen-containing five-membered heterocyclic ring modified polystyrene microsphere, which specifically comprises: accurately taking 9.0 mL of styrene, 15 mL of anhydrous 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 rapid stirring to emulsion; then under the atmosphere of nitrogen, reacting in an oil bath at 73℃ for 15h, and then adding 1 mL of N-vinyl-2-pyrrolidone to continue reacting for 3h; after the reaction is completed, cooling to room temperature, the obtained emulsion is repeatedly washed with anhydrous ethanol for 10 times, then deionized water is used to continue washing for 8 times, and vacuum drying at 60℃ for 48h to obtain a nitrogen-containing five-membered heterocyclic ring modified polystyrene microsphere, which is recorded as PS-N microsphere.

[0076] The SEM image and infrared spectrum of the PS-N microsphere obtained by this preparation example are shown in Figures 7 and 8, the PS-N microsphere is a spherical ball with smooth surface, the diameter is 1-1.5μm; and strong absorption peaks appear near 3100-2750cm -1 , 1750-1370cm -1 and 750-650cm -1 .

[0077] Example 1

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

[0079] S1, pretreatment: (1) 3.5mg of PS-Cl microspheres provided by the preparation example 1 are subjected to de-enzyme treatment by centrifugal column method, repeated washing for 6 times, each time vortexing for 5min; centrifuging at 7000rpm for 30s, adding RNase-free water to the precipitate to a final volume of 200μL to obtain an RNA room temperature storage stabilizer; (2) in a sterile and enzyme-free operation table, 10μg of IVT RNA is added to 200μL of the RNA room temperature storage stabilizer to obtain an RNA sample, which is transferred to a flask after mixing and half-capping.

[0080] S2, freeze-drying: the half-capped flask is placed on the freeze-drying shelf of the four-ring vacuum freeze-drying machine (LGJ-S30 pressure cover type) at room temperature, and the freeze-drying treatment is carried out according to the program shown in Table 1 to obtain an RNA product.

[0081] S3, the flask containing the RNA product is transferred to the glove box transition room, vacuumized and replaced with nitrogen, and placed overnight before being moved to the operation room. The flask cap is wax-sealed.

[0082] The IVT RNA used in step S1 in the embodiment is synthesized by in vitro transcription using pUC19 plasmid DNA as a template and primers of the eGFP nucleotide sequence, and has a length of 850 nt.

[0083] Table 1. Freeze-drying program

[0084] Example 2

[0085] The preparation method of the RNA product provided in the embodiment is basically the same as that in Example 1, except that the PS-OH microspheres provided in Preparation Example 2 are used instead of the PS-Cl microspheres provided in Preparation Example 1 in step S1, and other conditions are the same, to obtain the RNA product.

[0086] Example 3

[0087] The preparation method of the RNA product provided in the embodiment is basically the same as that in Example 1, except that the PS-COOH microspheres provided in Preparation Example 3 are used instead of the PS-Cl microspheres provided in Preparation Example 1 in step S1, and other conditions are the same, to obtain the RNA product.

[0088] Example 4

[0089] The preparation method of the RNA product provided in the embodiment is basically the same as that in Example 1, except that the PS-N microspheres provided in Preparation Example 4 are used instead of the PS-Cl microspheres provided in Preparation Example 1 in step S1, and other conditions are the same, to obtain the RNA product.

[0090] Example 5

[0091] The preparation method of the RNA product provided in the embodiment is basically the same as that in Example 1, except that the epoxy-modified polystyrene microspheres (Zhiyi, model number PSEP) are used instead of the PS-Cl microspheres provided in Preparation Example 1 in step S1, and other conditions are the same, to obtain the RNA product. PSEP) are used instead of the PS-Cl microspheres provided in Preparation Example 1 in step S1, and other conditions are the same, to obtain the RNA product.

[0092] Example 6

[0093] The preparation method of the RNA product provided in the embodiment is basically the same as that in Example 1, except that the addition amount of IVT RNA is 1 μg in step S1, and other conditions are the same, to obtain the RNA product.

[0094] Example 7

[0095] The preparation method of the RNA product provided in this example is basically the same as that in Example 1, except that in step S1, the addition amount of IVT RNA is 200 μg, and other conditions are the same, to obtain the RNA product.

[0096] Example 8

[0097] The preparation method of the RNA product provided in this example is basically the same as that in Example 1, except that in step S2, the conditions of freeze-drying are different, and are specifically shown in Table 2, to obtain the RNA product.

[0098] Table 2. Freeze-drying program

[0099] Example 9

[0100] The preparation method of the RNA product provided in this example is basically the same as that in Example 1, except that in step S2, the semi-closed flask is directly placed in a-70℃ freeze dryer for 63h, and other conditions are the same, to obtain the RNA product.

[0101] Comparative Example

[0102] The comparative example provides a preparation method of an RNA product, which specifically comprises:

[0103] S1, pretreatment: 10 μg of IVT RNA is added to 200 μL of RNase-free water to obtain an RNA sample, which is mixed thoroughly and then transferred to a flask, which is semi-closed.

[0104] S2, freeze-drying: the semi-closed flask is placed on the freeze-drying shelf of a four-ring vacuum freeze dryer (LGJ-S30 compression cover type) at room temperature, and freeze-drying treatment is performed according to the same procedure as in Example 1 (i.e., Table 1), to obtain the RNA product.

[0105] S3, the flask containing the RNA product is transferred to a glove box transition room, vacuumed and replaced with nitrogen, and then placed overnight in the operation room, and the flask cap is wax-sealed.

[0106] Test Example

[0107] The test example is used to illustrate the storage stability of the RNA products provided by the above examples and comparative examples. After the RNA products are stored at 25, 37 and 60°C for a period of time, 100 μL of RNase-free water is taken to rehydrate the RNA products in a sterile and enzyme-free operation table, shaken and mixed for 15 min, centrifuged at 7000 rpm for 4 min, and the supernatant is taken and filtered with a 0.22 μm RNase-free filter membrane. The RNA product with trehalose as a protective agent is used as a positive quality control group. The preparation of the RNA product specifically includes: in a sterile and enzyme-free operation table, 10 μg of IVT RNA is added to 200 μL of RNase-free water solution with a concentration of 1.72% (w:v) trehalose to obtain an RNA sample, which is mixed thoroughly and then transferred to a flask and half-capped; the half-capped flask is placed on the freeze-drying shelf of a four-ring vacuum freeze dryer (LGJ-S30 pressure cover type) at room temperature, and the freeze-drying process is carried out according to the same procedure as in Example 1 (i.e., Table 1) to obtain the RNA product. The detection is carried out according to the following method:

[0108] (1) Capillary electrophoresis detection: 1 μL of the supernatant is detected by using a full-automatic capillary electrophoresis instrument (Agilent Fragment Analyzer 5200) to obtain the percentage of normal IVT RNA concentration in the total RNA concentration, and the results are shown in Table 3.

[0109] Table 3. Storage stability of RNA products

[0110] Note: -- indicates that the band of normal RNA has been detected, and the degradation is complete

[0111] From the test results, compared with the comparative examples, the RNA products provided by Examples 1-9 of the application and the positive quality control group can still maintain good integrity after being stored at 37°C for 21 days.

[0112] (2) Agarose gel electrophoresis detection: (i) 0.5 g of agarose is mixed with 50 mL of 1 × TAE solution, heated in a microwave oven at high fire until completely dissolved to obtain 1% agarose gel; after cooling for a few seconds, pour into an electrophoresis mold, cool into a gel, pull out the comb, and place in a TAE electrophoresis tank, pour 1 × TAE solution to immerse the hole with liquid and the solution covers the agarose gel;

[0113] (ii) Take 500 mg of sucrose, 500 μL of water and 5 μL of 10000x GeL Red to obtain Loading Dye loading buffer; take 3 μL of Ladder (TaKaRa, item No. 3427A), add 15 μL of RNase-free water, and then add 2 μL of Loading Dye loading buffer to obtain Ladder electrophoresis loading solution;

[0114] (iii) Take 500 ng of supernatant, 2 μL of Loading Dye, and add RNase-free water to a final volume of 20 μL to obtain RNA electrophoresis loading solution;

[0115] (iv) Take the Ladder electrophoresis loading solution and the RNA electrophoresis loading solution and add them to the agarose gel hole of the electrophoresis tank at a time, cover the cover, and perform electrophoresis at 120 V for 30 min; then transfer the gel to a Bio-Rad instrument for photographing, and the results are shown in Figures 9-15.

[0116] As can be seen from Figures 9 and 10, after storage at 25℃ for 7d and 21d, the bands of the comparative example appear to be slightly dispersed downward, indicating that part of the RNA is degraded, while the bands of Examples 1-4 of the present application are clearly visible and do not appear to be dispersed downward, indicating that the RNA room temperature storage stabilizer has a good protective and stabilizing effect on the freeze-drying and storage process of the RNA at 25℃.

[0117] As can be seen from Figures 11 and 12, after storage at 37℃ for 7d and 21d, the bands of the comparative example appear to be slightly dispersed downward, indicating that part of the RNA is degraded, while the bands of Examples 1-4 of the present application are clearly visible and do not appear to be dispersed downward, indicating that the RNA room temperature storage stabilizer has a good protective and stabilizing effect on the freeze-drying and storage process of the RNA at 37℃.

[0118] As can be seen from Figures 13-15, after storage at 60℃ for 1d and 5d, the bands of the comparative example appear to be slightly and obviously dispersed downward, respectively, indicating that the RNA is degraded at 5d, while the bands of Examples 1-4 of the present application do not significantly degrade; after storage at 60℃ for 14d, the bands of the comparative example completely disappear, while the bands of Examples 1-4 of the present application are still clearly visible and only partially degraded, indicating that the RNA room temperature storage stabilizer has a good protective and stabilizing effect on the freeze-drying and storage process of the RNA at 60℃.

[0119] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above embodiments without departing from the principles and purposes of the present application within the scope of the present application.

Claims

1. An RNA room temperature storage stabilizer, characterized by, The RNA room temperature storage stabilizer comprises functionalized polystyrene microspheres.

2. The RNA room temperature storage stabilizer of claim 1, wherein, The functionalized polystyrene microspheres are polystyrene microspheres modified with functional groups selected from one or more of C1-C20 chloroalkyl, C6-C20 chlorophenyl, C1-C20 hydroxy-substituted alkyl, C6-C20 hydroxy-substituted phenyl, C2-C20 carboxy-substituted alkyl, C7-C20 carboxylic acid-substituted phenyl, C1-C20 amino-substituted alkyl, C6-C20 amino-substituted phenyl, and C4-C20 nitrogen-containing heterocycle.

3. The RNA room temperature storage stabilizer of claim 1, wherein 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 heterocycle-modified polystyrene microspheres.

4. The RNA room temperature storage stabilizer of claim 1, wherein The functionalized polystyrene microspheres have an average particle size of 0.1-200 μm.

5. The RNA room temperature storage stabilizer of claim 1, wherein The functionalized polystyrene microspheres have an average particle size of 1-2 μm.

6. The method for preparing the RNA room temperature storage stabilizer according to claim 1, characterized by, The preparation method comprises: S1, taking a polymerization monomer, a functionalization modifier, an initiator, and optionally polystyrene particles to perform a polymerization reaction to obtain the functionalized polystyrene microspheres; and S2, taking the functionalized polystyrene microspheres to perform a de-nucleic enzyme treatment to obtain the RNA room temperature storage stabilizer.

7. The method for preparing an RNA room temperature storage stabilizer according to claim 6, characterized by, In step S1, the polymerization monomer is selected from one or more of styrene, phenylacrylate, and divinylbenzene.

8. The method for preparing an RNA room temperature storage stabilizer according to claim 6, characterized by, In step S1, the functionalization modifier is selected from one or more of a chloroalkene, an enol, an unsaturated fatty acid, and an olefinated pyrrolidone.

9. The method for preparing an RNA room temperature storage stabilizer according to claim 6, characterized by, In step S1, the initiator is an azo initiator and / or a peroxide initiator.

10. The method for preparing an RNA room temperature storage stabilizer according to claim 6, characterized by, In step S1, the polystyrene particles have an average particle size of 0.01-1 μm.

11. The method of claim 6, wherein the RNA room temperature storage stabilizer is prepared by, In step S1, the input mass ratio of the polymerization monomer, the functionalization modifier, the initiator, and the polystyrene particles is (5-20):(0.1-20):(0.1-10):(0.001-20).

12. The method of claim 6, wherein the RNA room temperature storage stabilizer is prepared by, In step S1, the polymerization reaction has a temperature of 70-90 °C and a time of 1-48 h.

13. The method for preparing the RNA room temperature storage stabilizer according to claim 6, characterized in that, In step S2, the de-nucleic enzyme treatment specifically comprises: taking the functionalized polystyrene microspheres and mixing them with RNase-free water, and then performing centrifugal column purification to obtain the RNA room temperature storage stabilizer.

14. The method of claim 13, wherein the RNA room temperature storage stabilizer is prepared by, In step S2, the centrifugal column purification has a centrifugal speed of 5000-1000 rpm and a time of 5-30 min.

15. An RNA product, characterized in that, The RNA room temperature storage stabilizer of claim 1.

16. A method of producing the RNA product of claim 15, characterized in that, The preparation method comprises: taking RNA and mixing it with the RNA room temperature storage stabilizer, and then performing freeze-drying treatment to obtain the RNA product.

17. The method of claim 16, wherein the RNA product is prepared by a method comprising: The input mass ratio of the RNA and the RNA room temperature storage stabilizer is 1:(10-1000).

18. The method of claim 16, wherein the RNA product is prepared by a method comprising: The freeze-drying process comprises a pre-freezing stage, a primary drying stage and a secondary drying stage, the pre-freezing stage has a temperature of -60 to -50 DEG C and a time of 5 to 15 h; the primary drying stage has a temperature of -45 to -40 DEG C, a vacuum degree of not higher than 10 Pa and a time of 1 to 5 h; the secondary drying stage comprises alternately performed temperature rising periods and constant temperature periods, the secondary drying stage has a starting temperature of -35 to -30 DEG C, a final temperature of 20 to 25 DEG C, a vacuum degree of not higher than 10 Pa and a total time of 40 to 60 h, the temperature rising rate of the temperature rising periods is 0.5 to 2 DEG C / min and the time is 25 to 40 min.

19. Use of the RNA room temperature storage stabilizer of claim 1, the RNA product of claim 15 and / or the method of preparing the RNA product of claim 16 in the production of mRNA drugs and / or vaccines.

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