Method for preparing cryoprotectants and lyophilized nucleic acid-lipid nanoformulations

Abstract

The invention belongs to the field of pharmaceutical preparations, and provides a cryoprotectant and a preparation method of a freeze-dried nucleic acid-lipid nano preparation. The cryoprotectant provided by the invention can have a good protective effect on a preparation for freeze-drying treatment, and is especially suitable for freeze-drying mRNA-LNP. According to the preparation method of the freeze-dried nucleic acid-lipid nano preparation, interference in the freeze-drying process can be reduced, good quality attributes of finished products can be kept, amplification and batch-to-batch consistency of the products can be achieved, mRNA-LNP vaccine activity can be maintained, convenience of mRNA-LNP in transportation, storage and circulation is improved, and cost is greatly saved.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical preparations, and particularly relates to a preparation method of a cryoprotectant and a freeze-dried nucleic acid-lipid nano preparation. Background Art

[0002] In recent years, mRNA vaccines (mRNA-LNP) based on lipid nanoparticle (LNP) delivery systems have demonstrated the advantages of fast development speed and high protection efficiency that traditional vaccines do not have in the prevention of respiratory diseases and the treatment of tumors, attracting widespread attention from researchers.

[0003] However, the key components in mRNA and LNP are sensitive to conditions such as oxygen, water, pH, and temperature, and require ultra-low temperature cold chain transportation and storage. For example, the Moderna and BioNTech / Pfizer new crown vaccines need to be stored at -15 to -25°C and -60 to -90°C respectively, and have a shelf life of less than 6 months. This seriously hinders their global circulation and distribution, especially in some low-income countries with very poor infrastructure.

[0004] Therefore, how to improve the stability of mRNA-LNP preparations is the key to improving the accessibility of vaccines, among which freeze-drying has been widely used in biomacromolecule products. Freeze-drying is to first cool down and freeze a substance containing a large amount of water into a solid, and then sublimate the solid water molecules directly into water vapor under vacuum and appropriate heating conditions and extract them. It can effectively protect the biological activity of heat-sensitive substances and reduce the impact of oxygen molecules on biological activity. It is a promising direction to solve the current storage and transportation problems of mRNA vaccines.

[0005] At present, the process of freeze-dried mRNA-LNP vaccines faces many challenges. Problems such as low vaccine product quality, poor stability, poor consistency between batches, and difficulty in scale-up are common, which seriously hinder the marketing of freeze-dried vaccines. Summary of the invention

[0006] In order to solve the above problems existing in the prior art, the first aspect of the present invention provides a cryoprotectant, wherein the cryoprotectant contains disaccharides, and the concentration of the disaccharides is 10%-20%; the disaccharides at least include sucrose.

[0007] The second aspect of the present invention provides a method for preparing a freeze-dried nucleic acid-lipid nanoformulation, the method comprising mixing nucleic acid-lipid nanoparticles with the cryoprotectant provided by the first aspect of the present invention to form a frozen mixture, and then freeze-drying the frozen mixture.

[0008] The cryoprotectant provided by the present invention can have a good protective effect on preparations used for freeze-drying treatment, and is particularly suitable for the preparation of freeze-dried mRNA-LNP. The preparation method of the freeze-dried nucleic acid-lipid nanoformulation provided by the present invention can reduce the interference of the freeze-drying process to maintain good quality attributes of the finished product, achieve the scalability and batch consistency of the product, maintain the activity of the mRNA-LNP vaccine, improve the convenience of mRNA-LNP in transportation, storage and circulation, and greatly save costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is a freeze-drying machine loading chart for Example 8-4 at a freeze-drying scale of 2000 bottles / batch.

[0010] Figure 2 It is a flow cytometry graph of the expression of B5 antigen in HEK293 cells after transfection of Comparative Example 9, Example 9-1 and Example 9-2.

[0011] Figure 3 The flow cytometry results show that HEK293 cells express preF monomer and preF trimer after being transfected with HEK293 cells in Comparative Example 10, Example 10-1 and Example 10-2.

[0012] Figure 4 The specific binding antibody titer levels for the four antigens A27, L1, A33 and B5 in the mouse sera of Comparative Example 11, Example 11-1 and Example 11-2 3 and 6 weeks after immunization of the mice.

[0013] Figure 5 These are the ELISPOT test results of IFN-γ cytokine in Comparative Example 11, Example 11-1 and Example 11-2 after 8 weeks of immunization of mice.

[0014] Figure 6 The titer levels of specific binding antibodies against preF antigen in the mouse sera of Comparative Example 12, Example 12-1 and Example 12-2 3 and 6 weeks after immunization of the mice.

[0015] Figure 7 These are the ELISPOT test results of IFN-γ cytokine in Comparative Example 12, Example 12-1 and Example 12-2 after 8 weeks of immunization of mice. DETAILED DESCRIPTION

[0016] To make the technical solution, purpose and advantages of the present invention clearer, the present invention is further described in detail below through specific implementation examples. It should be understood that the specific implementation methods described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0017] A first aspect of the present invention provides a cryoprotectant, wherein the cryoprotectant contains disaccharides, and the disaccharides include at least sucrose.

[0018] In a preferred embodiment, the cryoprotectant further contains other disaccharides, such as trehalose, maltose and the like.

[0019] According to the first aspect of the present invention, the concentration of disaccharide in the cryoprotectant is 10%-20% (w / v), such as any one of 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or a range between two thereof, such as 14%-16%, 13%-17%, 12%-18%, 11%-19%, 12%-16%, 11%-18%, 13%-17%, etc., preferably 15% (w / v).

[0020] In a preferred embodiment, the cryoprotectant contains sucrose, trehalose, and maltose.

[0021] In another preferred embodiment, the cryoprotectant contains sucrose and trehalose.

[0022] Preferably, the total concentration of sucrose and trehalose is 15% (w / v).

[0023] Further preferably, the concentration of sucrose is 6%-12%, for example, any one of 6%, 7%, 8%, 9%, 10%, 11%, 12% or a range between two thereof, for example, 8%-10%, 7%-10%, 7%-11%, 6%-10%, 6%-11%, etc., preferably 9% (w / v); the concentration of trehalose is 3%-7%, for example, any one of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7% or a range between two thereof, for example, 5%-7%, 5.5%-6.5%, 4.5%-6.5%, 3.5%-6.5%, 4%-7%, etc., preferably 6% (w / v).

[0024] More preferably, the cryoprotectant contains sucrose and trehalose, wherein the concentration of sucrose is 9% and the concentration of trehalose is 6%.

[0025] According to the first aspect of the present invention, the buffer used in the cryoprotectant is Tris buffer or PBS buffer.

[0026] In a preferred embodiment, the concentration of the Tris buffer is 5mM-100mM, for example, a range between any one or two of 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, for example, 5mM-10mM, 10mM-20mM, 20mM-30mM, 10mM-50mM, 20mM-80mM, 30mM-70mM, 40mM-90mM, etc.; preferably 10mM.

[0027] The concentration of the PBS buffer is 5mM-100mM, for example, a range between any one or two of 5mM, 10mM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 55mM, 60mM, 65mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM, 100mM, for example, 5mM-10mM, 10mM-20mM, 20mM-30mM, 10mM-50mM, 20mM-80mM, 30mM-70mM, 40mM-90mM, etc.

[0028] The cryoprotectant provided in the first aspect of the present invention is particularly suitable for the preparation of freeze-dried poxvirus mRNA POX-LNP, freeze-dried luciferase-expressing Luc-LNP, and freeze-dried RSV-LNP expressing respiratory syncytial virus prefusion F protein.

[0029] The second aspect of the present invention provides a method for preparing a freeze-dried nucleic acid-lipid nanoformulation, the method comprising mixing nucleic acid-lipid nanoparticles with the cryoprotectant provided by the first aspect of the present invention to form a frozen mixture, and then freeze-drying the frozen mixture.

[0030] According to the second aspect of the present invention, in the frozen mixture, the concentration of mRNA is 0.05-0.5 mg / mL, for example, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, any one or two of them, for example, 0.1-0.2 mg / mL, 0.15-0.35 mg / mL, 0.3-0.5 mg / mL, 0.35-0 .45mg / mL, 0.1-0.25mg / mL, 0.05-0.15mg / mL, 0.3-0.5mg / mL, etc.; preferably 0.1mg / mL; the concentration of disaccharide in the cryoprotectant is 5-15% (w / v), for example, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, any one or a range between two, for example 9%-11%, 9%-12%, 8%-11%, 8%-12%, 7%-13%, 6%-14%, etc., preferably 10% (w / v).

[0031] According to the second aspect of the present invention, the freeze-drying process includes a pre-freezing stage; in the pre-freezing stage, the pre-freezing cooling rate is less than 1.0°C / min, for example, less than 0.1°C / min, 0.2°C / min, 0.3°C / min, 0.4°C / min, 0.5°C / min, 0.6°C / min, 0.7°C / min, 0.8°C / min, 0.9°C / min, 1.0°C / min, preferably 0.1-0.4°C / min, and further preferably 0.4°C / min.

[0032] In the pre-freezing stage, the pre-freezing temperature (freeze dryer shelf temperature) is below 20°C, for example, below 20°C, 15°C, 10°C, 0°C, -10°C, -15°C, -20°C, -25°C, -30°C, preferably below -10°C, and more preferably -20°C.

[0033] In the pre-freezing stage, the temperature is lowered to -40 to -60°C (preferably -50°C) and then maintained for at least 3 hours, preferably 4 hours.

[0034] According to the second aspect of the present invention, the freeze-drying process includes a sublimation drying stage; the sublimation drying stage includes: maintaining at -40°C to -25°C for 15-50 hours (preferably -30°C for 20 hours), and the vacuum pressure is controlled to be no higher than 3Pa throughout the process.

[0035] According to the second aspect of the present invention, the freeze-drying process includes a desorption drying stage; the desorption drying stage includes: maintaining at 20°C to 30°C for 3-10 hours (preferably maintaining at 25°C for 4 hours), and the vacuum pressure is controlled to be no higher than 3Pa throughout the process.

[0036] The method provided in the second aspect of the present invention is particularly suitable for preparing freeze-dried poxvirus mRNA POX-LNP, freeze-dried Luc-LNP expressing luciferase, and freeze-dried RSV-LNP expressing respiratory syncytial virus prefusion F protein.

[0037] The freeze-dried nucleic acid-lipid nanoformulation prepared by the method provided by the second aspect of the present invention also falls within the protection scope of the present invention.

[0038] The various reagents, materials, etc. used in the following examples, unless otherwise specified, are all products that can be obtained from commercial channels; the various tests and detection methods used in the following examples, unless otherwise specified, are all conventional tests and detection methods in the art and can be obtained from textbooks, reference books or academic journals.

[0039] Example 1: Acquisition of poxvirus mRNA POX-LNP preparation and composition of cryoprotectant.

[0040] Acquisition of poxvirus mRNA POX-LNP preparation: Poxvirus POX mRNA (3100nt) disclosed in patent CN116286913B was used as a model mRNA molecule, and a POX mRNA aqueous solution with an mRNA concentration of 0.137 mg / mL was prepared using a 50mM citric acid-sodium citrate buffer at pH = 4.0. ALC-0315, DSPC, cholesterol and ALC-0159 were dissolved in anhydrous ethanol at a molar ratio of 47.5:10:40.7:1.8 to obtain an organic phase solution with a total lipid concentration of 16mM. The aqueous phase solution and the organic phase solution were subjected to the mRNA embedding process operation at a flow rate ratio of 3:1 (aqueous phase 3, organic phase 1) and a total flow rate of 60mL / min, and the outflowing mRNA-LNP complex liquid was collected. The effluent was ultrafiltrated using 10 mM Tris buffer at pH 7.4 to remove ethanol and concentrate the POX-LNP complex to a concentration of 0.5-1.0 mg / mL. The filtrate was sterilized and filtered using a 0.22 μm filter membrane to collect the filtrate to obtain a POX-LNP preparation.

[0041] Parameter detection of POX-LNP preparations: The particle size distribution and PDI of POX-LNP preparations were analyzed using a Malvern particle size analyzer. The mRNA encapsulation efficiency and mRNA concentration of POX-LNP preparations were detected using the RiboGreen (InvitrogenTM, R11490) nucleic acid dye method.

[0042] According to the formulation in Table 1, each disaccharide cryoprotectant (sucrose, trehalose and maltose) was first dissolved in 10 mM Tris buffer to prepare a disaccharide cryoprotectant mother solution with a disaccharide concentration of 40% (w / v). The cryoprotectant mother solution and 10 mM Tris buffer were then added to the POX-LNP preparation mother solution to adjust the disaccharide concentration in the cryoprotectant to 10% (w / v), and the mRNA concentration was adjusted to 0.1 mg / mL.

[0043] Table 1 Cryoprotectant formulation

[0044]

[0045] The POX-LNP preparation after adding the cryoprotectant was placed in a -80°C refrigerator for 12 hours, and then allowed to thaw at room temperature. After the solution was completely melted, the particle size and PDI of the thawed POX-LNP preparation were detected by a Malvern particle size analyzer, and the mRNA encapsulation rate of the thawed POX LNP preparation was detected by the RiboGreen nucleic acid dye method. The results are shown in Table 2 (Comparative Example 1 is POX-LNP that has not been frozen).

[0046] Table 2 Particle size, PDI and mRNA encapsulation efficiency of POX-LNP before and after freezing

[0047] Group Particle size (nm) PDI Encapsulation rate (%) Comparative Example 1 73.40 0.07 97.18 Example 1-1 75.98 0.09 97.12 Example 1-2 95.17 0.12 94.62 Examples 1-3 92.42 0.12 95.24 Examples 1-4 76.47 0.11 97.40 Examples 1-5 75.08 0.11 97.67

[0048] It can be seen that after freezing and thawing at -80°C, compared with Example 1-2 and Example 1-3, the particle size, PDI and encapsulation efficiency of Example 1-1, Example 1-4 and Example 1-5 changed less (all compared with Comparative Example 1), indicating that compared with single-component trehalose (Example 1-2) and maltose (Example 1-3), the cryoprotectant containing sucrose can effectively protect POX-LNP nanoparticles from the impact of the freezing process.

[0049] Example 2: Composition of poxvirus mRNA POX-LNP cryoprotectant.

[0050] 1. Screening of cryoprotectant concentration

[0051] The POX-LNP preparation was prepared according to the method of Example 1, and then different concentrations of sucrose were added to the POX-LNP preparation as a cryoprotectant (dissolved in 10mM Tris) according to the formula in Table 3, and the mRNA concentration was adjusted to 0.1mg / mL to obtain Examples 2-1 to 2-7. POX-LNP without cryoprotectant was used as Comparative Example 2 to detect the particle size, PDI and mRNA encapsulation efficiency of each example and comparative example. The glass transition temperature (Tg') and collapse temperature (Tc) of each example were detected using a differential scanning calorimeter (DSC) and a freeze-drying microscope, respectively.

[0052] Table 3 Sucrose concentration and glass transition temperature and collapse temperature of each group

[0053] Group Sucrose concentration (%, w / v) Tg'(℃) Tc(℃) Comparative Example 2 0 N / A N / A Example 2-1 10 -34.1 -29.5 Example 2-2 12 -34.0 -29.6 Example 2-3 14 -33.7 -29.4 Embodiment 2-4 15 -33.4 -29.8 Embodiment 2-5 16 -33.3 -29.7 Embodiment 2-6 18 -33.1 -30.5 Embodiment 2-7 20 -33.0 -31.7

[0054] It can be seen that after adding sucrose of different concentrations, the glass transition temperature of each embodiment is distributed in the range of -33.0 to -34.1°C, and the collapse temperature is distributed in the range of -29.4 to -31.7°C.

[0055] Take 0.5 mL of each sample of Example 2-1 to Example 2-7 and Comparative Example 2 and put it into a 3 mL vial for freeze drying (referred to as "freeze drying"). Freeze drying procedure: Pre-freezing stage: reduce the temperature of the freeze dryer partition from room temperature to -50°C at a cooling rate of 1°C / min, and maintain at -50°C for 4 hours; sublimation drying stage: maintain at -30°C for 20 hours, and control the vacuum pressure at 3Pa throughout the process; analytical drying stage: maintain at 25°C for 4 hours, and control the vacuum pressure at 3Pa throughout the process.

[0056] The freeze-dried samples of Example 2-1 to Example 2-7 and Comparative Example 2 were taken respectively to observe the freeze-dried cake morphology. 0.5 mL of nuclease-free water was added for reconstitution, the bottle was gently swirled to eliminate bubbles, the state of the reconstituted solution was observed, and samples were taken to detect the particle size, PDI and mRNA encapsulation efficiency of POX-LNP after freeze-drying and reconstitution. The results are shown in Table 4.

[0057] Table 4 Particle size, PDI and mRNA encapsulation efficiency of each POX-LNP before and after freeze-drying

[0058]

[0059]

[0060] By observing the morphology of the freeze-dried cake and the reconstituted solution, only a small amount of white powder was visible at the bottom of the bottle in Comparative Example 2 without a cryoprotectant. After reconstitution with water, the solution became turbid and a large number of insoluble particles were visible. However, Examples 2-1 to 2-7 all formed complete white solid cakes, which could be quickly reconstituted after adding water. The solutions were translucent and had a light blue opalescent luster.

[0061] According to the results of particle size, PDI and encapsulation efficiency, the particle size of comparative example 2 increased to 323 nm after freeze-drying, the PDI increased to 0.36, and the mRNA encapsulation efficiency decreased to about 20%, indicating that its LNP structure has been completely destroyed. In each embodiment, Examples 2-4 have better overall performance. After freeze-drying, the particle size increased to about 120 nm, the PDI increased to about 0.10, and the mRNA encapsulation efficiency decreased to about 80%, indicating that a cryoprotectant concentration of 15% (w / v) can effectively alleviate the damage of POX-LNP during the freeze-drying process.

[0062] 2. Screening of cryoprotectant composition ratio

[0063] The POX-LNP preparation was prepared according to the method of Example 1, and then the total protective agent concentration was controlled to 15% (w / v) according to the formula in Table 5, and different composition ratios of cryoprotectants (dissolved in 10 mM Tris) were added to the POX-LNP preparation. The mRNA concentration was adjusted to 0.1 mg / mL to obtain Examples 2-4, 2-8 to 2-17, and the particle size, PDI and mRNA encapsulation efficiency of each example were detected.

[0064] Table 5 Cryoprotectant prescriptions for each group

[0065]

[0066]

[0067] The freeze-dried product was reconstituted with water according to the above method, and the particle size, PDI and mRNA encapsulation efficiency after freeze-drying and reconstitution were tested. The results are shown in Table 6.

[0068] Table 6 Particle size, PDI and mRNA encapsulation efficiency of each POX-LNP before and after freeze-drying

[0069]

[0070]

[0071] The results of comprehensive particle size, PDI and encapsulation rate show that the overall performance of Examples 2-13 to 2-17 is not as good as that of Examples 2-8 to 2-12, indicating that the freeze-drying protection effect of the binary protective agent prescription using sucrose-maltose is not as good as that of the binary protective agent prescription using sucrose-trehalose. Comparing the data of Examples 2-8 to 2-12 with Example 2-4, it can be seen that when the total sugar concentration is controlled to be 15% (w / v) and the added concentration of trehalose is 1-6%, there is no significant effect on the particle size and mRNA encapsulation rate of POX-LNP after freeze-drying, and only PDI increases slightly (not exceeding 0.15); when the trehalose concentration rises to 7.5% (w / v), the particle size and PDI of POX-LNP after freeze-drying increase significantly, and the mRNA encapsulation rate decreases significantly. In summary, 15% (w / v) sucrose-trehalose binary cryoprotectant is preferably used for freeze-drying of POX-LNP, wherein the sucrose concentration ranges from 9-15% (w / v) and the trehalose concentration ranges from 0-6% (w / v).

[0072] Example 3: Pre-freezing cooling rate of freeze-dried poxvirus mRNA POX-LNP.

[0073] POX-LNP preparations dispersed in 10 mM Tris buffer (pH = 7.4) were prepared according to the method in Example 1, and the cryoprotectant (dissolved in 10 mM Tris) as shown in Table 7 was added to each POX-LNP preparation, the mRNA concentration was adjusted to 0.1 mg / mL, and the particle size, PDI and mRNA encapsulation efficiency were detected.

[0074] Take 0.5 mL of POX-LNP preparation into a 3 mL vial for freeze drying. Freeze drying procedure: Pre-freezing stage: gradually reduce the temperature of the freeze dryer plate from room temperature to -50 ° C according to the cooling rate shown in Table 7, and maintain it at -50 ° C for 4 hours; sublimation drying stage: maintain at -30 ° C for 20 hours, and the vacuum pressure is controlled at 3 Pa throughout the process; analytical drying stage: maintain at 25 ° C for 4 hours, and the vacuum pressure is controlled at 3 Pa throughout the process.

[0075] Table 7 Cryoprotectant prescription and pre-freezing cooling rate

[0076]

[0077]

[0078] After the freeze-dried cakes of the above examples were reconstituted with water, the particle size, PDI and mRNA encapsulation efficiency after freeze-drying were tested. The results are shown in Table 8.

[0079] Table 8 Particle size, PDI and mRNA encapsulation efficiency of each POX-LNP before and after freeze-drying

[0080]

[0081]

[0082] The results of particle size, PDI and mRNA encapsulation rate showed that when the pre-freezing cooling rate was gradually slowed down from 1℃ / min to 0.4℃ / min, the particle size of POX-LNP after freeze-drying could be reduced from 121nm to 107nm, PDI had no significant change, and mRNA encapsulation rate could be increased from 80% to 83%; when the pre-freezing cooling rate continued to be gradually slowed down from 0.4℃ / min to 0.1℃ / min, the particle size, PDI and mRNA encapsulation rate of POX-LNP after freeze-drying had no significant change. When the cooling rate was the same, no matter which formula was used as the cryoprotectant, 15% sucrose (w / v), 12% sucrose + 3% trehalose, 9% sucrose + 6% trehalose (w / v), the particle size and mRNA encapsulation rate of the above three POX-LNPs after freeze-drying had no significant difference, indicating that the pre-freezing cooling rate had the same influence trend on the POX-LNPs containing the above three cryoprotectants. In summary, the pre-freezing cooling rate is preferably 0.1-0.4°C / min, which can effectively slow down the effect of the freeze-drying process on POX-LNP. In order to reduce the total freeze-drying time and save process energy consumption, the more preferred pre-freezing cooling rate is 0.4°C / min.

[0083] Example 4: Freeze-dried poxvirus mRNA POX-LNP pre-frozen into the machine temperature.

[0084] POX-LNP preparations dispersed in 10 mM Tris buffer (pH = 7.4) were prepared according to the method in Example 1, and their particle size, PDI and mRNA encapsulation efficiency were detected. Cryoprotectants (dissolved in 10 mM Tris) were added according to the following Table 9, and the mRNA concentration was adjusted to 0.1 mg / mL.

[0085] Take 0.5mL of POX-LNP preparation into a 3mL vial for freeze drying. Freeze drying procedure: Pre-freezing stage: As shown in Table 9 below, first adjust the freeze dryer shelf temperature to the preset temperature, put the freeze-dried sample into the freeze dryer and maintain it at this temperature for 20 minutes, then reduce the temperature to -50°C at a cooling rate of 0.4°C / min, and continue to maintain it at -50°C for 4 hours; sublimation drying stage: maintain at -30°C for 20 hours, and control the vacuum pressure at 3Pa throughout the process; analytical drying stage: maintain at 25°C for 4 hours, and control the vacuum pressure at 3Pa throughout the process.

[0086] Table 9 Cryoprotectant prescription and freeze dryer preset temperature

[0087]

[0088]

[0089] After the freeze-dried cakes of the above examples were reconstituted with water, the particle size, PDI and mRNA encapsulation efficiency of the freeze-dried samples of each example were tested. The results are shown in Table 10.

[0090] Table 10 Particle size, PDI and mRNA encapsulation efficiency of each POX-LNP before and after freeze-drying

[0091]

[0092] The results of particle size, PDI and mRNA encapsulation rate show that after the pre-freezing temperature of the POX-LNP sample is reduced from 20℃ to -20℃, whether 15% sucrose, 12% sucrose + 3% trehalose or 9% sucrose + 6% trehalose (w / v) is used as a cryoprotectant, the particle size of the freeze-dried POX-LNP can be reduced from 108nm to 96nm, PDI has no significant change, and the mRNA encapsulation rate can be increased from 83% to 88%, which effectively alleviates the effect of the freeze-drying process on POX-LNP; while further reducing the pre-freezing temperature to -30℃ did not further improve the sample, and its particle size and PDI were slightly higher than those of the sample at -20℃, while the encapsulation rate was lower than that of the sample at -20℃. Therefore, -20℃ is preferred as the pre-freezing temperature of the POX-LNP sample.

[0093] Example 5: Long-term, accelerated, and high temperature stability of freeze-dried poxvirus mRNA POX-LNPs.

[0094] POX-LNP preparations dispersed in 10 mM Tris buffer (pH = 7.4) were prepared according to the method in Example 1, and their particle size, PDI and mRNA encapsulation efficiency were detected, and cryoprotectants of different prescriptions were added thereto to adjust the mRNA concentration to 0.1 mg / mL. Example 5-1: Add 15% sucrose (w / v); Example 5-2: Add 12% sucrose + 3% trehalose (w / v); Example 5-3: Add 9% sucrose + 6% trehalose (w / v).

[0095] Referring to the freeze-drying procedure in Example 4, the pre-freeze temperature of -20°C was used, and the pre-freeze cooling rate of 0.4°C / min was used to freeze-dry each embodiment. After the freeze-drying was completed, the freeze-dried cake was re-dissolved with water, and the particle size, PDI, mRNA encapsulation efficiency and mRNA integrity of each embodiment sample after freeze-drying were detected. And each freeze-dried sample was stored in a 2-8°C, 25°C and 37°C incubator to investigate its long-term, accelerated and high temperature stability. Long-term experimental sampling time points: 0, 30, 60, 90, 180, 270, 360 days, accelerated experimental sampling time points: 0, 14, 30, 60, 90, 180, 270, 360 days, high temperature experimental sampling time points: 0, 14, 30, 60, 90, 180 days. The stability data of Examples 5-1, 5-2 and 5-3 are shown in Tables 11, 12 and 13, respectively.

[0096] Table 11 Long-term, accelerated, and high temperature stability data of Example 5-1.

[0097]

[0098]

[0099] Table 12 Long-term, accelerated, and high temperature stability data of Example 5-2.

[0100]

[0101]

[0102] Table 13 Long-term, accelerated, and high temperature stability data for Example 5-3.

[0103]

[0104] Based on the above data, it can be seen that after storage at 2-8°C for 360 days, the particle size, PDI, mRNA encapsulation rate and mRNA integrity of each embodiment of Examples 5-1 to 5-3 did not change significantly, and the long-term stability of the preparation was excellent. After storage at 25°C accelerated conditions for 360 days, the particle size of Example 5-1 group increased to about 126nm, PDI did not change significantly, mRNA encapsulation rate decreased to about 81%, and mRNA integrity decreased to about 53%; the particle size of Example 5-2 group increased to about 109nm, PDI did not change significantly, mRNA encapsulation rate decreased to about 84%, and mRNA integrity decreased to about 59%; the particle size of Example 5-3 group increased to about 100nm, PDI and mRNA encapsulation rate did not change significantly, and mRNA integrity decreased to about 64%. After being stored at 37°C for 180 days, the particle size of Example 5-1 increased significantly to about 314 nm, the PDI increased to 0.46, the mRNA encapsulation rate decreased significantly to about 37%, and the mRNA was completely degraded; the particle size of Example 5-2 increased to about 157 nm, the PDI did not change significantly, the mRNA encapsulation rate decreased to about 74%, and the mRNA integrity decreased to about 22%; the particle size of Example 5-3 increased to about 120 nm, the PDI did not change significantly, the mRNA encapsulation rate decreased slightly to about 82%, and the mRNA integrity decreased to about 19%. In summary, the freeze-dried POX-LNP prepared by the cryoprotectant and freeze-drying process provided by the present invention has good stability, especially the best stability when 9% sucrose + 6% trehalose (w / v) is used as a cryoprotectant in Example 5-3.

[0105] Example 6: Universality of freeze-drying process

[0106] In addition to using POX mRNA, Luc mRNA (Novozyme, DD4511, 1921nt) expressing luciferase and RSV mRNA (1875nt) expressing prefusion F protein (preF) of respiratory syncytial virus were used respectively. POX-LNP, Luc-LNP and RSV-LNP preparations dispersed in 10mM Tris buffer (pH=7.4) were prepared according to the method in Example 1, and their particle size, PDI and mRNA encapsulation efficiency were detected. 9% sucrose + 6% trehalose (w / v) were added as cryoprotectants, and the mRNA concentration was adjusted to 0.1mg / mL. Example 6-1: POX-LNP; Example 6-2: Luc-LNP; Example 6-3: RSV-LNP.

[0107] The freeze-dried cakes of each example were reconstituted with water according to the freeze-drying procedure in Example 5, and the particle size, PDI and mRNA encapsulation efficiency after freeze-drying were tested. The results are shown in Table 14.

[0108] Table 14 Particle size, PDI and mRNA encapsulation efficiency of each mRNA-LNP before and after freeze-drying

[0109]

[0110] The comprehensive performance of particle size, PDI and mRNA encapsulation rate shows that even if different mRNA molecules are used, after freeze-drying using the same freeze-drying procedure, the physical and chemical properties of each embodiment are similar and there is no significant difference. After freeze-drying, the particle size distribution range of each mRNA-LNP is 94-100nm, the PDI is less than 0.15, and the mRNA encapsulation rate distribution range is 86-90%. This shows that the freeze-drying procedure is universal for mRNA-LNP preparations using different mRNA molecules.

[0111] Example 7: Long-term, accelerated, and high temperature stability of lyophilized Luc-LNP and lyophilized RSV-LNP

[0112] The above-mentioned lyophilized samples of Example 6-2 and Example 6-3 were stored in thermostats at 2-8°C, 25°C and 37°C, respectively, to investigate their long-term, accelerated and high temperature stability. The particle size, PDI, mRNA encapsulation efficiency and mRNA integrity were tested after regular sampling and re-dissolution with water. The sampling time points for long-term experiments were: 0, 30, 60, 90, 180, 270, 360 days, the sampling time points for accelerated experiments were: 0, 14, 30, 60, 90, 180, 270, 360 days, and the sampling time points for high temperature experiments were: 0, 14, 30, 60, 90, 180 days. The stability data of Examples 6-2 and 6-3 are shown in Tables 15 and 16, respectively.

[0113] Table 15 Long-term, accelerated, and high temperature stability data for Example 6-2.

[0114]

[0115] Table 16 Long-term, accelerated, and high temperature stability data for Example 6-3.

[0116]

[0117]

[0118] From the above data, it can be seen that after storage at 2-8°C for 360 days, the particle size, PDI, mRNA encapsulation rate and mRNA integrity of Example 6-2 and Example 6-3 did not change significantly, and the formulation stability performance was excellent. After storage at 25°C accelerated conditions for 360 days, the particle size of Example 6-2 group increased to about 103nm, PDI and mRNA encapsulation rate did not change significantly, and mRNA integrity decreased to about 62%; the particle size of Example 6-3 group increased to about 108nm, PDI and mRNA encapsulation rate did not change significantly, and mRNA integrity decreased to about 58%. After storage at 37°C high temperature conditions for 180 days, the particle size of Example 6-2 group increased to about 150nm, mRNA encapsulation rate decreased to about 50%, and mRNA integrity decreased to about 37%; the particle size of Example 6-3 group increased to about 157nm, mRNA encapsulation rate decreased to about 61%, and mRNA integrity decreased to about 22%. In summary, it can be seen that the freeze-dried Luc-LNP and freeze-dried RSV-LNP prepared by using the cryoprotectant and freeze-drying process provided by the present invention both have good stability.

[0119] Example 8: Pilot scale-up and process stability of freeze-dried poxvirus mRNA POX-LNP freeze-drying process

[0120] 1. Pilot scale-up of freeze-dried poxvirus mRNA POX-LNP freeze-drying process

[0121] POX-LNP preparations dispersed in 10 mM Tris buffer (pH = 7.4) were prepared according to the method in Example 1, and their particle size, PDI and mRNA encapsulation efficiency were detected. 9% sucrose + 6% trehalose (w / v) were added as cryoprotectants, and the mRNA concentration was adjusted to 0.1 mg / mL.

[0122] Referring to the freeze-drying procedure in Example 4, the pre-freeze temperature was -20°C and the pre-freeze cooling rate was 0.4°C / min, and each example was freeze-dried. After the freeze-drying was completed, the freeze-dried cake was reconstituted with water, and the particle size, PDI, mRNA encapsulation efficiency and mRNA integrity of each example sample after freeze-drying were tested. Example 8-1: 20 bottles / batch; Example 8-2: 100 bottles / batch; Example 8-3: 500 bottles / batch; Example 8-4: 2000 bottles / batch. Figure 1 This is a diagram of the installed capacity of the freeze dryer of Example 8-4.

[0123] After the freeze-dried cakes of the above examples were reconstituted with water, the particle size, PDI and mRNA encapsulation efficiency of the freeze-dried samples of each example were tested. The results are shown in Table 17.

[0124] Table 17 Particle size, PDI and mRNA encapsulation efficiency of each POX-LNP after freeze-drying

[0125]

[0126] The comprehensive results of particle size, PDI and mRNA encapsulation rate show that at each freeze-drying batch scale, there is no obvious difference in the particle size, PDI and mRNA encapsulation rate of POX-LNP after freeze-drying. The particle size distribution is in the range of 92-97nm, the PDI is less than 0.15, and the mRNA encapsulation rate is in the range of 85%-88%. The freeze-drying process shows good process scale-up performance. Even if the freeze-drying scale is gradually increased from 20 bottles / batch to 2000 bottles / batch, the physical and chemical properties of freeze-dried POX-LNP will not deteriorate.

[0127] 2. Stability and batch-to-batch consistency of freeze-dried poxvirus mRNA POX-LNP

[0128] Referring to Example 8-4, the freeze-drying scale of 2000 bottles / batch was maintained, and two batches of freeze-dried POX-LNP were repeatedly prepared to obtain Examples 8-5 and 8-6. After the freeze-dried cakes of the above Examples 8-5 and 8-6 were reconstituted with water, the particle size, PDI and mRNA encapsulation efficiency of the freeze-dried samples of each example were tested, and the results are shown in Table 18.

[0129] Table 18 Particle size, PDI and mRNA encapsulation efficiency of each POX-LNP after freeze-drying

[0130]

[0131]

[0132] The comprehensive results of particle size, PDI and mRNA encapsulation rate showed that there was no obvious difference in particle size, PDI and mRNA encapsulation rate among the three batches of freeze-dried POX-LNP. The particle size distribution after freeze-drying was about 92-96nm, PDI was less than 0.15, and the encapsulation rate ranged from about 86-88%, indicating that the freeze-drying process had excellent process stability and batch consistency at the scale of 2000 bottles / batch.

[0133] Example 9: Evaluation of in vitro immune activity of freeze-dried poxvirus mRNA POX-LNP

[0134] 5 μg POX mRNA was transfected into each well. 6 HEK293 cells were added with the POX-LNP preparations of Comparative Example 8, Example 8-1 or 8-2, respectively. After 24 hours of culture, the cells were peeled off the surface of the six-well plate with PBS containing 3% FBS, and then stained with live cell green fluorescent dye (Invitrogen, L34966A, 1:1000) to distinguish live cells from dead cells. The cells were fixed with intracellular fixation buffer (Invitrogen, 00-8222-49), permeabilized with a permeabilizer (Invitrogen, 008333-56), and blocked with CD16 / CD32 monoclonal antibody (Invitrogen, 14-0161-85, 1:1000). Finally, cells were stained with a monoclonal antibody (Henxy Biotech, Shanghai, China, RAB-2442) that specifically recognizes B5 (1:200 dilution), and then anti-human IgG-AF488 (Invitrogen, A-11013, 1:500) was added as a secondary antibody. Cells were collected on an Attune NxT acoustic focusing flow cytometer (Thermo Fisher Scientific), and data were analyzed using FlowJo10.8.1. The data results are shown in Tables 19 and Figure 2 .

[0135] Comparative Example 9: empty LNP; Example 9-1: POX-LNP preparation containing 9% sucrose + 6% trehalose (w / v) (not freeze-dried); Example 9-2: freeze-dried POX-LNP preparation containing 9% sucrose + 6% trehalose (w / v) (stored at 2-8°C for 180 days).

[0136] Table 19 B5 antigen cell positive rate of each example

[0137] Group B5 antigen cell positive rate (%) Comparative Example 9 0.5 Example 9-1 97.4 Example 9-2 96.7

[0138] From the above data, it can be seen that after transfection of HEK 293 cells in Example 9-1 and Example 9-2, both expressed B5 protein, and the positive cell rates were similar, 97.4% and 96.7%, respectively, indicating that the POX-LNP preparation containing 9% sucrose + 6% trehalose (w / v), after freeze-drying and storage at 2-8°C for 180 days, did not have a significant effect on its in vitro immune activity.

[0139] Example 10: Evaluation of in vitro immune activity of freeze-dried respiratory syncytial virus mRNA RSV-LNP

[0140] 5 μg of RSV mRNA was transfected into each well. 6 HEK293 cells were added with the POX-LNP preparations of Comparative Example 10, Example 10-1, and Example 10-2, respectively. After culturing for 24 hours, the cells were peeled off the surface of the six-well plate with PBS containing 3% FBS, and then stained with live cell green fluorescent dye (Invitrogen, L34966A, 1:1000) to distinguish live cells from dead cells. The cells were stained with human monoclonal antibody D25 (Cambridge, 01-07-0120) that recognizes the antigenic site Φ of the pre-F protein and human monoclonal antibody AM14 (Cambridge, 01-07-0119) that recognizes the trimer structure of pre-F, and then the cell surface was stained with anti-human IgG-AF488 (Invitrogen, A-11013, 1:500) and post-F specific mouse monoclonal antibody 4D7-AF647 (self-prepared). Cells were collected on an Attune NxT acoustic focusing flow cytometer (Thermo Fisher Scientific) and the data were analyzed using FlowJo10.8.1. The data results are shown in Tables 20 and Figure 3 .

[0141] Comparative Example 10: empty LNP; Example 10-1: RSV-LNP preparation containing 9% sucrose + 6% trehalose (w / v) (not freeze-dried); Example 10-2: freeze-dried RSV-LNP preparation containing 9% sucrose + 6% trehalose (w / v) (stored at 2-8°C for 180 days).

[0142] Table 20 RSV related antigen cell positive rate of each example

[0143] Group PreF antigen cell positive rate (%) PreF trimer positive rate (%) Comparative Example 10 0.12 0.55 Example 10-1 97.1 70.6 Example 10-2 95.5 67.3

[0144] The results of the in vitro transfection test showed that after transfection of HEK 293 cells with Example 10-1, the expressed F protein mainly showed pre-F conformation (positive staining with D25 antibody) and trimer conformation (positive staining with AM14 antibody), with the proportions of 97.1% and 70.6%, respectively. After transfection with Example 10-2, HEK 293 cells expressed F protein, which also mainly showed pre-F conformation and trimer conformation, with the proportions of 95.5% and 67.3%, respectively. The positive rates of antigen expression after in vitro transfection of cells in the two groups of examples were comparable, indicating that the RSV-LNP preparation containing 9% sucrose + 6% trehalose (w / v), after freeze-drying and storage at 2-8°C for 180 days, did not significantly affect its in vitro immune activity.

[0145] Example 11: Evaluation of in vivo immune activity of freeze-dried poxvirus mRNA POX-LNP

[0146] 1. Immunization Program

[0147] 6-8 week old Balb / c mice, 5 in each group, were divided into three groups: Comparative Example 11: normal saline; Example 11-1: POX-LNP preparation containing 9% sucrose + 6% trehalose (w / v) (not freeze-dried); Example 11-2: freeze-dried POX-LNP preparation containing 9% sucrose + 6% trehalose (w / v) (stored at 2-8°C for 180 days); the entire experimental period was carried out in the SPF-grade mouse house of Bichai Experimental Animal Co., Ltd.

[0148] The day of the first immunization of mice was defined as day 0, the day before immunization was defined as day -1, the day after immunization was defined as day 1, and so on. The prepared vaccine was injected intramuscularly into mice on day 0 and day 28, with a dose of 5 μg per mouse each time.

[0149] Blood samples were collected from mice twice, at -7 days and 21 and 42 days after the first immunization, and the serum was separated and stored in a -80°C refrigerator for antigen-specific antibody detection. The spleen of mice was taken 56 days after the first immunization for cellular immunity evaluation.

[0150] 2. Antigen-specific antibody titer detection

[0151] The titer of antigen-specific antibody IgG in mouse serum during the entire immunization cycle was detected by ELISA. The specific steps are as follows: VACV A27 (CUSABIO, CSB-EP31812OVAI), L1 (CUSABIO, CSB-EP324949VAA1), A33 (CUSABIO, CSB-EP300755VAA1), and B5 (CUSABIO, CSB-EP321891VAA1) four antigens were diluted to 1 μg / mL with 1×ELISA coating solution (SoLarbio, C1055), and the above dilution was added to a 96-well ELISA plate (Corning, 9018) at 100 μL / well, and placed at 4°C overnight for coating. The liquid in the well plate was discarded, washed with PBST 3 times, and then 200 μL / well of 5% skim milk powder prepared with PBST was added, and placed at 37°C for 1 hour for blocking. Then wash the plate 3 times with PBST. During the blocking period, the mouse serum was diluted with PBST containing 2% skim milk powder, and the serum before immunization, 21 days after the first immunization, and 42 days after the first immunization was diluted from 1:100, and then 3-fold gradient dilution was performed, for a total of 8 gradients. Add the above diluted mouse serum to the blocked and washed well plate, 100 μL / well. Place at 37°C for 1 hour, and then wash the plate 5 times with PBST. Add Goat anti-mouse IgG-HRP (SouthernBiotech, 1031-05) diluted 1:400 with PBST containing 2% skim milk powder, 100 μL / well. Place at 37°C for 1 hour, and then wash the plate 5 times with PBST. Add TMB colorimetric solution (InvitrogenTM, 002023), 100 μL / well, and let stand at room temperature for 5 minutes. Add ELISA stop solution (New Saimei, E40500), 50 μL / well. The detection was performed at 450 / 620 nm wavelength using an ELISA reader (Thermo Fisher Scientific, Varioskan LUX), and the serum antibody titer was finally determined based on 2.1 times the pre-immunization mouse serum reading as the cut-off value. The lower limit of antibody titer detection in the experiment was 100. The test results are shown in Tables 21 and Figure 4 .

[0152] Table 21 Specific antibody titers of the four antigens A27, L1, A33, and B5 in each example

[0153]

[0154] The results of the immune response detection in mice after vaccination showed that Example 11-1 and Example 11-2 both produced specific binding antibodies to the four antigens after the first immunization of mice. The GMTs of the A27 specific binding antibody were 125 and 374, respectively (p=0.14), the GMTs of the L1 specific binding antibody were 12570 and 19507, respectively (p=0.49), the GMTs of the A33 specific binding antibody were 3363 and 1740, respectively (p=0.31), and the GMTs of the B5 specific binding were 300 and 100, respectively (p=0.06). There were no significant differences in the binding antibodies to the four antigens between Example 11-1 and Example 11-2 (p<0.05 was considered to be significantly different).

[0155] After the second immunization, the serum binding antibodies of the two vaccine groups were significantly increased. The A27-specific binding antibody GMTs in the sera of mice immunized with Example 11-1 and Example 11-2 were 15659 and 37710 (p=0.20), the L1-specific binding antibody GMTs were 3054498 and 1580036 (p=0.09), the A33-specific binding antibody GMTs were 1018166 and 817324 (p=0.67), and the B5-specific binding antibody GMTs were 58520 and 46976 (p=0.86). There were no significant differences in the binding antibodies to the four antigens between Example 11-1 and Example 11-2.

[0156] The above results indicate that freeze-dried POX-LNPs stored at 2-8°C for 180 days can induce specific binding antibody responses of equal intensity in mice compared to freshly prepared non-freeze-dried POX-LNPs.

[0157] 3. Cellular Immune Response Detection

[0158] Add 100 μL / well of 35% ethanol to the ELISPOT plate (MiLLipore, MSIPS4W10). Discard the liquid after 1 minute and wash 5 times with sterile deionized water at 200 μL / well. Dilute the IFN-γ capture antibody (MABTECH, 3321-2H) with PBS to 15 μg / mL, add 100 μL / well to the above plate, and incubate at 4°C overnight. After washing with PBS the next day, add RPMI1640 (Gibco, 61870127) + 10% FBS (Gibco, 10091148) culture medium and incubate at room temperature for 30 minutes. Take the spleen of the mouse and prepare a single cell suspension, which is counted after red blood cell lysis and washing with PBS. Discard the culture medium in the ELISPOT plate, take an appropriate amount of cells and add them, and at the same time add the poxvirus vaccine expression antigen peptide library, with a final concentration of 2.5 μg / mL. After the well plate was placed in a 37°C cell culture incubator for 36 hours, the cells were discarded and washed with PBS, and then the diluted (1:1000) detection antibody (MABTECH, 3321-2H) was added and incubated at room temperature for 2 hours. After completion, it was washed again with PBS and diluted (1:1000) streptavidin-HRP (MABTECH, 3321-2H) was added and incubated at room temperature for 1 hour. After washing with PBS, TMB colorimetric solution (MABTECH, 3651-10) was added and washed with deionized water after the spots were obvious. After drying at room temperature, the well plate was photographed and the spots in the wells were counted using a CTL enzyme-linked immunospot analyzer (CTL, S6UniversaL). The results are shown in Tables 22 and Figure 5 .

[0159] Table 22 Spot number of each embodiment

[0160] Group Number of spots Comparative Example 11 2 Example 11-1 658 Example 11-2 816

[0161] The results of the cellular immune detection on the 56th day showed that there was no obvious spot formation in the spleen cells of the comparative example 11 after stimulation by the peptide library, while obvious spots were produced in the vaccine immunization groups of the examples 11-1 and 11-2 after stimulation by the peptide library, and both the examples 11-1 and 11-2 produced good T cell responses in mice, and the average number of cells secreting IFN-γ per million cells was 658 and 816 (p=0.51), respectively, with no significant difference between the two groups. This indicates that the freeze-dried POX-LNPs stored at 2-8°C for 180 days can induce the same intensity of T cell immune response in mice compared to the freshly prepared non-freeze-dried POX-LNPs.

[0162] Example 12: Evaluation of in vivo immune activity of freeze-dried respiratory syncytial virus mRNA RSV-LNP

[0163] 1. Immunization Program

[0164] 6-8 week old Balb / c mice, 5 in each group, were divided into three groups: Comparative Example 12: normal saline; Example 12-1: RSV-LNP preparation containing 9% sucrose + 6% trehalose (w / v) (not freeze-dried); Example 12-2: freeze-dried RSV-LNP preparation containing 9% sucrose + 6% trehalose (w / v) (stored at 2-8°C for 180 days); the entire experimental period was carried out in an SPF mouse room.

[0165] The day of the first immunization of mice was defined as day 0, the day before immunization was defined as day -1, the day after immunization was defined as day 1, and so on. The prepared vaccine was injected intramuscularly into mice on day 0 and day 28, with a dose of 5 μg per mouse each time.

[0166] Blood samples were collected from mice twice, at -7 days and 21 and 42 days after the first immunization, and the serum was separated and stored in a -80℃ refrigerator for antigen-specific antibody detection. The spleen of mice was taken 56 days after the first immunization for cellular immunity evaluation.

[0167] 2. Antigen-specific antibody titer detection

[0168] The ELISA method was used to detect the titer of antigen-specific antibody IgG in mouse serum during the entire immunization cycle. The specific steps are as follows: dilute the preF protein to 1 μg / mL with 1×ELISA coating solution (SoLarbio, C1055), add the above dilution to a 96-well ELISA plate (Corning, 9018) at 100 μL / well, and place it at 4°C overnight for coating. Discard the liquid in the well plate, wash it 3 times with PBST, then add 200 μL / well of 5% skim milk powder prepared with PBST, and place it at 37°C for 1 hour for blocking. Then wash the plate 3 times with PBST. During the blocking period, dilute the mouse serum with PBST containing 2% skim milk powder, and dilute the serum before immunization, 21 days after the first immunization, and 42 days from 1:100, respectively, and then perform a 3-fold gradient dilution, for a total of 8 gradients. Add the above diluted mouse serum to the blocked and washed well plate, 100 μL / well. Place at 37°C for 1 hour, then wash the plate 5 times with PBST. Add 100 μL / well of Goat anti-mouse IgG-HRP (Southern Biotech, 1031-05) diluted 1:400 with PBST containing 2% skim milk powder. Place at 37°C for 1 hour, then wash the plate 5 times with PBST. Add TMB colorimetric solution (InvitrogenTM, 002023), 100 μL / well, and let stand at room temperature for 5 minutes. Add ELISA stop solution (New Cyme, E40500), 50 μL / well. Detect at a wavelength of 450 / 620nm using an enzyme reader (Thermo Fisher, Varioskan LUX), and finally determine the serum antibody titer based on 2.1 times the pre-immune mouse serum reading as the cut-off value. The lower limit of antibody titer detection in the experiment was 100, and the test results are shown in Tables 23 and Figure 6 .

[0169] Table 23 RSV antigen-specific antibody titer of each example

[0170]

[0171]

[0172] The results of the immune response test in mice after vaccination showed that after the first immunization, Example 12-1 and Example 12-2 significantly induced mice to produce antibodies against preF compared with Comparative Example 12, and the antibody titers were comparable, with GMT (geometric mean titer) of 72900 and 58520 (p=0.35), respectively. After the second immunization, the serum antibody titers of mice in the two vaccine groups were further improved, with GMTs of 4740109 and 9163493 (p=0.09) for Example 12-1 and Example 12-2, respectively, and there was no significant difference between the two groups, indicating that freeze-dried RSV-LNPs stored at 2-8°C for 180 days can induce specific binding antibody responses of equal intensity in mice compared to freshly prepared non-freeze-dried RSV-LNPs.

[0173] 3. Cellular Immune Response Detection

[0174] Add 35% ethanol 50 μL / well to the ELISPOT plate (MiLLipore, MSIPS4W10). Discard the liquid after 1 minute and wash 5 times with sterile deionized water at 200 μL / well. Dilute the IFN-γ capture antibody (MABTECH, 3321-2H) with PBS to 15 μg / mL, add 100 μL / well to the above plate, and incubate at 4°C overnight. After washing with PBS the next day, add RPMI1640 (Gibco, 61870127) + 10% FBS (Gibco, 10091148) culture medium and incubate at room temperature for 30 minutes. Take the mouse spleen and prepare a single cell suspension, which is counted after red blood cell lysis and washing with PBS. Discard the culture medium in the ELISPOT plate, take an appropriate amount of cells and add them, and at the same time add the RSV vaccine expression antigen peptide library, with a final concentration of 2.5 μg / mL. After the well plate was placed in a 37°C cell culture incubator for 36 hours, the cells were discarded and washed with PBS, and then diluted (1:1000) detection antibody (MABTECH, 3321-2H) was added and incubated at room temperature for 2 hours. After completion, it was washed again with PBS and diluted (1:1000) streptavidin-HRP (MABTECH, 3321-2H) was added and incubated at room temperature for 1 hour. After washing with PBS, TMB colorimetric solution (MABTECH, 3651-10) was added and washed with deionized water after the spots were obvious. After drying at room temperature, the well plate was photographed and the spots in the wells were counted using a CTL enzyme-linked immunospot analyzer (CTL, S6UniversaL). The results are shown in Tables 24 and Figure 7 .

[0175] Table 24 Spot number of each embodiment

[0176] Group Number of spots Comparative Example 12 3 Example 12-1 1508 Example 12-2 2095

[0177] The results of cellular immunity detection on the 56th day showed that there was no obvious spot formation in the spleen cells of the control group 12 after stimulation with the RSV peptide library, while obvious spots were produced in the vaccine immunization groups of Examples 12-1 and 12-2 after stimulation with the RSV peptide library, and the number of spots in Example 12-2 was slightly more than that in Example 12-1. The average number of cells secreting IFN-γ per million cells was 2095 and 1508, respectively (p=0.02). This indicates that RSV-LNPs that have been freeze-dried and stored at 2-8°C for 180 days can induce a slightly stronger T cell immune response in mice than freshly prepared non-freeze-dried RSV-LNPs.

[0178] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cryoprotectant, characterized in that The cryoprotectant contains disaccharides, the concentration of which is 10%-20%; the disaccharides at least include sucrose.

2. The cryoprotectant according to claim 1, characterized in that: The disaccharide further comprises trehalose and / or maltose.

3. The cryoprotectant according to claim 1 or 2, characterized in that: The concentration of disaccharide in the cryoprotectant is any one of 14%-16%, 13%-17%, 12%-18%, 11%-19%, 12%-16%, 11%-18%, 13%-17%; Preferably, the concentration of disaccharide in the cryoprotectant is 15%.

4. The cryoprotectant according to claim 3, characterized in that: The cryoprotectant contains sucrose and trehalose; Preferably, the total concentration of sucrose and trehalose is 15%; Preferably, the concentration of sucrose is any one of 6%-12%, 8%-10%, 7%-10%, 7%-11%, 6%-10%, 6%-11%, preferably 9%; Preferably, the concentration of trehalose is any one of 3%-7%, 5%-7%, 5.5%-6.5%, 4.5%-6.5%, 3.5%-6.5%, 4%-7%, preferably 6%.

5. A method for preparing a freeze-dried nucleic acid-lipid nanoformulation, characterized in that: The method comprises mixing the nucleic acid-lipid nanoparticles with the cryoprotectant according to any one of claims 1 to 4 to form a freezing mixture, and then freeze-drying the freezing mixture.

6. The method according to claim 5, characterized in that: In the frozen mixture, the concentration of mRNA is any one of 0.05-0.5 mg / mL, 0.1-0.2 mg / mL, 0.15-0.35 mg / mL, 0.3-0.5 mg / mL, 0.35-0.45 mg / mL, 0.1-0.25 mg / mL, 0.05-0.15 mg / mL, 0.3-0.5 mg / mL; preferably 0.1 mg / mL; In the freezing mixture, the concentration of disaccharides in the cryoprotectant is any one of 5-15%, 9%-11%, 9%-12%, 8%-11%, 8%-12%, 7%-13%, 6%-14%; preferably 10% (w / v).

7. The method according to claim 5 or 6, characterized in that: The freeze-drying process includes a pre-freezing stage; In the pre-freezing stage, the pre-freezing temperature reduction rate is less than 1.0°C / min; Preferably, in the pre-freezing stage, the pre-freezing cooling rate is 0.1-0.4°C / min; Further preferably, in the pre-freezing stage, the pre-freezing cooling rate is 0.4°C / min.

8. The method according to claim 7, characterized in that: In the pre-freezing stage, the pre-freezing temperature is lower than 20°C; Preferably, in the pre-freezing stage, the pre-freezing temperature is lower than -10°C; Further preferably, in the pre-freezing stage, the pre-freezing temperature is -20°C; Preferably, in the pre-freezing stage, the temperature is lowered to -40 to -60°C, preferably -50°C, and then maintained for at least 3 hours, preferably 4 hours.

9. The method according to claim 8, characterized in that: The freeze-drying process includes a sublimation drying stage; The sublimation drying stage includes: maintaining at -40°C to -25°C for 15-50 hours, preferably maintaining at -30°C for 20 hours; and the vacuum pressure is controlled to be no higher than 3Pa during the whole process.

10. The method according to claim 9, characterized in that: The freeze-drying process includes an analytical drying stage; The analytical drying stage includes: maintaining at 20° C. to 30° C. for 3-10 hours, preferably maintaining at 25° C. for 4 hours; and the vacuum pressure is controlled to be no higher than 3 Pa during the whole process.

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