MRNA (messenger ribonucleic acid) vaccine freeze-drying protective agent and preparation method thereof

Abstract

The invention relates to an mRNA vaccine freeze-drying protective agent and a preparation method thereof, and belongs to the technical field of biology. The mRNA vaccine freeze-drying protective agent comprises cane sugar with the final concentration of 10-15%, trehalose with the final concentration of 0-0.5% and glycine with the final concentration of 0-3 mM. The composition further comprises sodium acetate with the final concentration of 20 mM, His with the final concentration of 10 mM to 20 mM or HEPES with the final concentration of 10 mM to 20 mM. The solvent of the mRNA vaccine freeze-drying protective agent is water for injection. The mRNA vaccine freeze-drying protective agent is simple in component, solves the problems of extremely low-temperature transportation and storage of the mRNA vaccine, solves the problem that mRNA is easy to degrade, and has the advantages of low cost, easiness in obtaining, no toxicity and the like.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a freeze-drying protectant for mRNA vaccines and its preparation method. Background Technology

[0002] In recent years, mRNA vaccines have become a core technological approach for treating emerging infectious diseases (such as COVID-19 and influenza) and cancer due to their advantages such as short development cycles and strong immunogenicity. mRNA vaccines encapsulate mRNA encoding the target antigen into lipid particles via a lipid delivery system, enabling delivery into host cells and inducing a specific immune response. The key to this technology lies in maintaining the structural integrity and biological activity of the mRNA molecule, preventing degradation or inactivation during production, storage, and transportation. However, mRNA molecules themselves are physicochemically unstable and easily degraded by factors such as temperature, humidity, and oxidation. Liquid mRNA vaccines, in particular, require storage and transportation at -80°C or -20°C, leading to extremely high logistics costs and the risk of vaccine activity reduction due to cold chain disruptions. Even under low-temperature conditions, liquid mRNA vaccines have a short shelf life, making it difficult to meet the practical needs of long-term storage, transportation, and emergency reserves. Therefore, developing stable storage technologies for mRNA vaccines and reducing their dependence on the cold chain has become a key bottleneck in promoting the industrialization and large-scale application of mRNA vaccines.

[0003] Freeze-drying technology removes moisture through low-temperature freezing and vacuum sublimation, significantly reducing the metabolic activity and degradation rate of biological products. It is a commonly used stabilization method in the biopharmaceutical field. However, when traditional freeze-drying technology is directly applied to mRNA vaccines, it faces several challenges:

[0004] (1) Low-temperature freezing and water sublimation during the freeze-drying process can cause damage to the secondary / tertiary structure of mRNA molecules, leading to nucleic acid chain breakage or aggregation, resulting in a significant decrease in biological activity after reconstitution;

[0005] (2) The delivery system used for mRNA vaccines is prone to particle size increase or breakage during the reconstitution process after freeze-drying, which directly leads to mRNA leakage and degradation.

[0006] (3) Traditional freeze-drying protectants (such as single sugars) cannot simultaneously meet multiple requirements such as mRNA structural stability, vaccine morphology maintenance, and rapid reconstitution, and are prone to problems such as insufficient protective efficiency or poor clarity after reconstitution.

[0007] (4) Existing freeze-drying process parameters (such as pre-freezing temperature, sublimation rate, and drying time) are mostly designed based on protein or inactivated vaccines, and their products have relatively high water content, which affects the thermal stability of mRNA vaccines.

[0008] Currently, the application of mRNA vaccines places higher demands on their storage conditions, transportation costs, and shelf life. Existing technologies cannot achieve long-term stable storage of mRNA vaccines at 2-8℃ or room temperature, limiting their widespread application in areas with weak cold chain infrastructure. Furthermore, traditional freeze-drying formulations and processes have poor compatibility with mRNA vaccines of different antigen sequences, requiring repeated development of adaptation solutions, leading to increased R&D costs and extended development cycles. Therefore, developing a freeze-drying technology that adapts to the characteristics of mRNA vaccines, effectively maintains the structural integrity of mRNA and the stability of the lipid delivery system, reduces cold chain dependence, and has a simple and controllable process is crucial for addressing the many shortcomings of existing freeze-drying technologies in mRNA vaccine applications. This has significant practical and industrial value for promoting the industrialization of mRNA vaccines and expanding their application scope. Therefore, freeze-drying of mRNA vaccines focuses on two aspects: 1. Exploring freeze-drying protectants; 2. Exploring freeze-drying processes. Overcoming the shortcomings of existing technologies is a pressing issue in the field of biotechnology. Summary of the Invention

[0009] The purpose of this invention is to overcome the shortcomings of the prior art and provide a freeze-drying protectant for mRNA vaccines and its preparation method.

[0010] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0011] A lyophilized protectant for mRNA vaccines, comprising sucrose at a final concentration of 10-15%, trehalose at a final concentration of 0-0.5%, and glycine at a final concentration of 0-3 mM; further comprising sodium acetate at a final concentration of 20 mM, His at a final concentration of 10 mM-20 mM, or HEPES at a final concentration of 10 mM-20 mM.

[0012] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0013] Furthermore, preferably, the mRNA vaccine includes a rabies mRNA vaccine and a COVID-19 mRNA vaccine.

[0014] Furthermore, preferably, the mRNA vaccine freeze-drying protectant comprises sucrose at a final concentration of 12%, trehalose at a final concentration of 0.5%, glycine at a final concentration of 3mM, and sodium acetate at a concentration of 20mM.

[0015] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0016] This invention also provides a method for preparing the mRNA vaccine freeze-drying protectant, comprising the following steps:

[0017] Sucrose, trehalose, glycine, sodium acetate, His, and HEPES are added to water for injection to the target concentration to obtain the lyophilized protective agent for mRNA vaccines.

[0018] Furthermore, preferably, the preparation method of the mRNA vaccine freeze-drying protectant includes the following steps:

[0019] First, prepare 50% sucrose stock solution, 1% trehalose stock solution, 1M glycine stock solution, 1M NaAC stock solution, 1M HEPES stock solution, and 1M His stock solution. Then, add each prepared stock solution to water for injection to bring the sucrose, trehalose, glycine, sodium acetate, His, and HEPES to the target concentration, thus obtaining the mRNA vaccine freeze-dried protective agent.

[0020] The present invention also provides the application of the aforementioned mRNA vaccine freeze-drying protectant in the preparation of mRNA vaccines.

[0021] Furthermore, preferably, the freeze-drying process is as follows:

[0022] Pre-freeze at -45℃ for 2 hours;

[0023] Sublimation drying at -40℃ under a vacuum of 0.35 mbar for 5 hours;

[0024] Sublimation drying at a vacuum of 0.2 mbar and -36°C for 8 hours;

[0025] Sublimation drying at -30℃ under vacuum of 0.2 mbar for 8 hours;

[0026] Sublimation drying at a vacuum of 0.1 mbar and -28°C for 8 hours;

[0027] Sublimation drying at -25°C for 3 hours under vacuum of 0.1 mbar;

[0028] Sublimation drying at -15℃ under vacuum of 0.1 mbar for 10 h;

[0029] Desorption drying at a vacuum of 0.01 mbar and -25°C for 6 hours;

[0030] Desorption drying at a vacuum of 0.01 mbar and -28°C for 5 hours;

[0031] Desorption drying was performed at a vacuum of 0.01 mbar and -25°C for 6 hours.

[0032] In the freeze-drying protectant for mRNA vaccines of this invention, the final concentrations of sucrose and trehalose are both mass percentages.

[0033] In this invention, His: histidine; HEPES: 4-hydroxyethylpiperazine ethanesulfonic acid; Tris is not used because the purity of mRNA after reconstitution of the lyophilized vaccine is lower than that of other buffers; the solvent used is water for injection.

[0034] The stock solutions for the six types of lyophilized protective agents used in mRNA vaccines are prepared as follows:

[0035] Preparation of 50% sucrose stock solution (1L): Weigh 500g of sucrose powder, add about 800mL of water for injection, stir until completely dissolved, and bring the volume up to 1000mL with water for injection. After mixing, filter through a 0.22μm filter membrane for sterilization and store at 4℃ protected from light.

[0036] Preparation of 1% trehalose stock solution (1L): Weigh 11.05g of trehalose dihydrate powder, add about 800mL of water for injection, stir until completely dissolved, bring the volume up to 1000mL with water for injection, mix well, filter through a 0.22μm filter membrane for sterilization, and store at 4℃ protected from light.

[0037] Preparation of 1M glycine solution stock solution (1L): Weigh 75.07g of powder (molecular weight 75.07), add about 800mL of water for injection, stir until completely dissolved, and bring the volume up to 1000mL with water for injection. After mixing, filter through a 0.22μm filter membrane for sterilization and store at 4℃ protected from light.

[0038] Preparation of 1M NaAC solution stock solution (1L): Weigh 136.08g of sodium acetate trihydrate powder (molecular weight 136.08), add about 800mL of water for injection, stir until completely dissolved, slowly add acetic acid solution dropwise while stirring, calibrate to the required pH with a pH meter, and bring the volume to 1000mL with water for injection. After mixing, filter through a 0.22μm filter membrane for sterilization and store at 4℃ protected from light.

[0039] Preparation of 1M HEPES stock solution (pH 7.0) (1L): Weigh 238.3g of HEPES powder (molecular weight 238.3), add about 800mL of water for injection, stir until completely dissolved, slowly add 1M NaOH solution dropwise while stirring, calibrate the pH to 7.0 with a pH meter, and bring the volume to 1000mL with water for injection. After mixing, filter through a 0.22μm filter membrane for sterilization and store at 4℃ protected from light.

[0040] Preparation of 1M HEPES stock solution (pH 7.5) (1L): Weigh 238.3g of HEPES powder (molecular weight 238.3), add about 800mL of water for injection, stir until completely dissolved, slowly add 1M NaOH solution dropwise while stirring, calibrate the pH to 7.5 with a pH meter, and bring the volume to 1000mL with water for injection. After mixing, filter through a 0.22μm filter membrane for sterilization and store at 4℃ protected from light.

[0041] Preparation of 1M His solution stock solution (1L): Weigh 209.63g L-histidine hydrochloride monohydrate, place it in a beaker, add about 800mL of water for injection, and stir magnetically until completely dissolved. Insert the electrode into the dissolved solution and slowly add 1mol / L NaOH solution dropwise while stirring to prevent local pH from becoming too high, until the solution pH stabilizes at 7.0. Then transfer the solution to a volumetric flask of the corresponding size, wash the beaker 2-3 times with a small amount of water for injection, transfer all the washing solution into the volumetric flask, and make up to 1000mL. After mixing, filter through a 0.22μm filter membrane for sterilization and store at 4℃ protected from light.

[0042] Taking 100mL of lyophilized protective agent for mRNA vaccines as an example:

[0043] For example, 20 mL, 25 mL, and 30 mL of 50% sucrose stock solution were added to prepare 100 mL of lyophilized protective agent for mRNA vaccines, resulting in final sucrose concentrations of 10%, 12.5%, and 15%, respectively.

[0044] Adding 50 mL of 1% trehalose stock solution to 100 mL of lyophilized protective agent for mRNA vaccine will result in a final trehalose concentration of 0.5%.

[0045] If 0.3 mL of 1 M glycine stock solution is added to prepare 100 mL of lyophilized protective agent for mRNA vaccine, the final concentration of glycine will be 3 mM.

[0046] If 2 mL of 1 M NaAC stock solution is added to prepare 100 mL of lyophilized protective agent for mRNA vaccine, the final NaAC concentration will be 20 mM.

[0047] If 2 mL of 1 M HEPES stock solution is added to prepare 100 mL of mRNA vaccine freeze-drying protectant, the final HEPES concentration will be 20 mM.

[0048] If 2 mL of 1 M His solution stock solution is added to prepare 100 mL of lyophilized protective agent for mRNA vaccine, the final His concentration will be 20 mM.

[0049] In this invention, the final concentration of the mRNA vaccine encapsulation solution is 60 μg / mL.

[0050] Compared with the prior art, the beneficial effects of this invention are as follows:

[0051] (1) The mRNA active ingredient protected by the freeze-drying protectant of the mRNA vaccine of the present invention has a longer storage time, which solves the problem of ultra-low temperature transportation and storage of mRNA vaccines and solves the problem of easy degradation of mRNA.

[0052] (2) The mRNA vaccine freeze-drying protectant of the present invention has simple components and has the advantages of low cost, easy availability and non-toxicity, which greatly reduces the cost of raw materials.

[0053] (3) The mRNA vaccine freeze-drying protectant and freeze-drying technology of the present invention can store the mRNA vaccine at 25°C for up to six months.

[0054] (4) Compared with traditional freeze-drying technology, the freeze-dried mRNA vaccine of the present invention improves the integrity of the mRNA by 5%-10%. Attached Figure Description

[0055] Figure 1 Based on the freeze-drying profile process; Figure 2 The morphology of samples 1-10 placed at 37℃ for 7 days under the basic freeze-drying curve;

[0056] Figure 3 The expression of immunoblot in samples 1-10 under the basic freeze-drying curve;

[0057] Figure 4 The expression of immunoblot in samples 1-10 after optimization of the freeze-drying process;

[0058] Figure 5 A diagram showing the stable placement of the freeze-drying process after optimization.

[0059] Figure 6 To assess the stability of the lyophilized process after optimization, bioactive immunoblotting was performed.

[0060] Figure 7 Detection of the bioactivity of lyophilization protectants 11-18 using Western blot;

[0061] Figure 8 The bioactivity of lyophilized samples 19-24 and their stability after lyophilization process optimization was measured by immunoblotting.

[0062] Figure 9 The results of mRNA molecule integrity were used to measure the stability of 1-10 samples before and after lyophilization curve optimization.

[0063] Figure 10 The results of mRNA molecule integrity were used to assess the stability of samples from 11 to 24 days after lyophilization curve optimization. Detailed Implementation

[0064] The present invention will now be described in further detail with reference to the embodiments.

[0065] Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be construed as limiting the scope of the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed in accordance with the techniques or conditions described in the literature in the field or according to the product instructions. Materials or equipment whose manufacturers are not specified are all conventional products that can be obtained by purchase.

[0066] Unless otherwise stated, all percentages in this invention are mass percentages.

[0067] Example 1

[0068] A lyophilized protectant for mRNA vaccines, wherein the lyophilized protectant comprises sucrose at a final concentration of 15% and sodium acetate at a final concentration of 20 mM;

[0069] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0070] The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

[0071] The preparation method of the mRNA vaccine lyophilization protectant includes the following steps:

[0072] Adding sucrose and sodium acetate to water for injection to the target concentration yields the lyophilized protective agent for mRNA vaccines.

[0073] Example 2

[0074] A lyophilized protectant for mRNA vaccines, wherein the lyophilized protectant comprises sucrose at a final concentration of 10%, trehalose at a final concentration of 0.5%, and glycine at a final concentration of 3mM.

[0075] This also includes His at a final concentration of 10 mM;

[0076] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0077] The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

[0078] The preparation method of the mRNA vaccine lyophilization protectant includes the following steps:

[0079] Sucrose, trehalose, glycine, and His are added to water for injection to the target concentration to obtain the lyophilized protective agent for mRNA vaccines.

[0080] Example 3

[0081] A lyophilized protectant for mRNA vaccines, wherein the lyophilized protectant comprises sucrose at a final concentration of 12%, trehalose at a final concentration of 0.3%, and glycine at a final concentration of 2 mM;

[0082] This also includes His with a final concentration of 15 mM;

[0083] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0084] The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

[0085] The preparation method of the mRNA vaccine lyophilization protectant includes the following steps:

[0086] First, prepare a 50% sucrose stock solution, a 1% trehalose stock solution, a 1M glycine stock solution, and a 1M His stock solution. Then, add each of the prepared stock solutions to water for injection to bring the sucrose, trehalose, glycine, and His to the target concentrations, thus obtaining the mRNA vaccine freeze-dried protectant.

[0087] Example 4

[0088] A lyophilized protectant for mRNA vaccines, wherein the lyophilized protectant comprises sucrose at a final concentration of 15%, trehalose at a final concentration of 0.5%, and glycine at a final concentration of 3 mM.

[0089] This also includes His at a final concentration of 20 mM;

[0090] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0091] The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

[0092] The preparation method of the mRNA vaccine lyophilization protectant includes the following steps:

[0093] First, prepare a 50% sucrose stock solution, a 1% trehalose stock solution, a 1M glycine stock solution, and a 1M His stock solution. Then, add each of the prepared stock solutions to water for injection to bring the sucrose, trehalose, glycine, and His to the target concentrations, thus obtaining the mRNA vaccine freeze-dried protectant.

[0094] Example 5

[0095] A freeze-drying protectant for mRNA vaccines, wherein the freeze-drying protectant comprises sucrose at a final concentration of 10%, trehalose at a final concentration of 0.1%, and glycine at a final concentration of 1 mM;

[0096] It also includes HEPES at a final concentration of 10 mM;

[0097] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0098] The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

[0099] The preparation method of the mRNA vaccine lyophilization protectant includes the following steps:

[0100] First, prepare 50% sucrose stock solution, 1% trehalose stock solution, 1M glycine stock solution, and 1M HEPES stock solution. Then, add each prepared stock solution to water for injection to bring the sucrose, trehalose, glycine, and HEPES to the target concentration, thus obtaining the mRNA vaccine freeze-dried protective agent.

[0101] Example 6

[0102] A lyophilized protectant for mRNA vaccines, wherein the lyophilized protectant comprises sucrose at a final concentration of 12%, trehalose at a final concentration of 0.4%, and glycine at a final concentration of 2mM.

[0103] It also includes HEPES at a final concentration of 15 mM;

[0104] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0105] The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

[0106] The preparation method of the mRNA vaccine lyophilization protectant includes the following steps:

[0107] First, prepare 50% sucrose stock solution, 1% trehalose stock solution, 1M glycine stock solution, and 1M HEPES stock solution. Then, add each prepared stock solution to water for injection to bring the sucrose, trehalose, glycine, and HEPES to the target concentration, thus obtaining the mRNA vaccine freeze-dried protective agent.

[0108] Example 7

[0109] A lyophilized protectant for mRNA vaccines, wherein the lyophilized protectant comprises sucrose at a final concentration of 11%, trehalose at a final concentration of 0.2%, and glycine at a final concentration of 1.5 mM;

[0110] It also includes HEPES at a final concentration of 20 mM;

[0111] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0112] The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

[0113] The preparation method of the mRNA vaccine lyophilization protectant includes the following steps:

[0114] First, prepare 50% sucrose stock solution, 1% trehalose stock solution, 1M glycine stock solution, and 1M HEPES stock solution. Then, add each prepared stock solution to water for injection to bring the sucrose, trehalose, glycine, and HEPES to the target concentration, thus obtaining the mRNA vaccine freeze-dried protective agent.

[0115] Example 8

[0116] A freeze-drying protectant for mRNA vaccines, wherein the freeze-drying protectant comprises sucrose at a final concentration of 12%, trehalose at a final concentration of 0.5%, glycine at a final concentration of 3 mM, and sodium acetate at a concentration of 20 mM.

[0117] The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

[0118] The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

[0119] The preparation method of the mRNA vaccine lyophilization protectant includes the following steps:

[0120] First, prepare a 50% sucrose stock solution, a 1% trehalose stock solution, a 1M glycine stock solution, and a 1M NaAC stock solution. Then, add each of the prepared stock solutions to water for injection to bring the sucrose, trehalose, glycine, and NaAC to the target concentrations, thus obtaining the mRNA vaccine freeze-dried protective agent.

[0121] Application Examples

[0122] I. Preparation of Nucleic Acid-Lipid Nanoparticles

[0123] The mRNA molecule encoding the rabies virus G protein is bound to the rabies virus N protein to form a nucleocapsid complex, which is then encapsulated in a lipid delivery system to obtain the encapsulation stock solution; the specific operation is as follows:

[0124] Preparation of stock solution of mRNA encoding rabies virus G protein:

[0125] Taking a 100mL reaction system as an example, the specific reaction system is as follows:

[0126] 10×T7 RNA polymerase buffer 10mL;

[0127] 3mL each of ATP, CTP, GTP, and N1-Me-pUTP;

[0128] CAP GAG 3mL;

[0129] T7 RNA polymerase 1.5 mL;

[0130] Pyrophosphatase 0.2 mL;

[0131] RNase inhibitor 2.5 mL;

[0132] 5 mg of linearized DNA template;

[0133] Add RNase-free water to a final volume of 100 mL;

[0134] The above system was mixed evenly and reacted at 37℃ for 3-4 hours. After the reaction was completed, 2.5 mL of DNase I was added and incubated for another 30 minutes. The mRNA stock solution was obtained after purification by Oligo dT.

[0135] The nucleotide sequence of the linearized DNA template is as follows:

[0136]

[0137] The 1300 ng / μL mRNA stock solution was diluted to a final concentration of 900 μg / mL in an aqueous buffer, and the rabies N protein was diluted to a final concentration of 225 μg / mL in an aqueous buffer. The two were bound at 37°C for 30 min, and then concentrated by ultrafiltration and the buffer was changed to pH 5.0 NaAC (10 mM) containing 0.001% trehalose buffer to obtain the nucleocapsid complex, which was used as the aqueous solution.

[0138] The aqueous buffer solution consists of: Tris pH 7.2 (10 mM), NaCl (50 mM), KCl (10 mM), MgCl2 (10 mM), DTT (5 mM), and glycerol (0.5%).

[0139] Lipids DHA-1, DSPC, cholesterol, DOTAP-CL, and mPEG-DTA-1-2K were respectively prepared into mother liquors using anhydrous ethanol to obtain DHA-1 mother liquor with a mass percentage concentration of 20%, DSPC mother liquor with a mass percentage concentration of 10%, cholesterol mother liquor with a mass percentage concentration of 3%, DOTAP-CL mother liquor with a mass percentage concentration of 10%, and mPEG-DTA-1-2K mother liquor with a mass percentage concentration of 10%.

[0140] Preparation of the organic phase solution: Taking the preparation of 0.6 mL of organic phase solution as an example, take 100 μL of the above-mentioned 20% DHA-1 stock solution, 40 μL of 10% DSPC stock solution, 300 μL of 3% cholesterol stock solution, 30 μL of 10% DOTAP-CL stock solution, and 18 μL of 10% mPEG-DTA-1-2K stock solution, and add 112 μL of anhydrous ethanol. Mix well to obtain the organic phase solution; that is, the molar percentages of DHA-1, DSPC, cholesterol, DOTAP-CL, and mPEG-DTA-1-2K are 43.9%: 8.51%: 39.13%: 7.22%: 1.24%.

[0141] The aqueous and organic phase solutions were mixed at a volume ratio of 3:1 using nanofluidic technology, and then concentrated and replaced using a tangential flow filtration system. The mixture was then prepared in a 10 mM NaAC (pH 6.2) buffer solution containing 0.001% trehalose to obtain the encapsulation stock solution.

[0142] The specific method for concentration and liquid replacement is as follows: First, the mixture obtained through nano-microfluidic technology is diluted 30-35 times by volume in an aqueous solution containing 0.001% trehalose and 10mM NaAC (pH 6.2). Then, the mixture is concentrated and replaced with ultrafiltration through a 100KD hollow fiber column to the original volume before dilution (e.g., 50mL of mixture, diluted 30 times to 1500mL, and finally replaced with ultrafiltration to 50mL) to obtain the encapsulated stock solution.

[0143] II. Preparation of Lyophilization Protectant

[0144] Regarding the selection of lyophilization protectants, protectants 1-24 were tested, with Tris (pH 7.2), NaAC (pH 6.2), His (pH 7.0), and HEPES (pH 7.0-7.5) buffer solutions as the solvents, respectively. The final concentrations of each protectant are shown in Table 1. This invention uses formulations 1-10 of the sample lyophilization protectants to optimize the lyophilization curve. Then, the glycine content in lyophilization protectant formulations 11-18 was adjusted and optimized; and the lyophilization buffer solution in lyophilization protectant formulations 19-24 was further adjusted.

[0145] Table 1. Formulation of freeze-drying protectant

[0146]

[0147]

[0148]

[0149] Note: % represents mass percentage.

[0150] III. Sample Preparation

[0151] The encapsulation stock solution from step one was subjected to mRNA concentration determination using the Ribogreen method. The solution was then diluted with the lyophilization protectant from step two according to formulations 1-24 to obtain a formulation with an mRNA content of 60 μg / mL, thus obtaining samples 1-24.

[0152] IV. Sample freeze-drying

[0153] The samples 1-10 obtained in step 3 above were filled and partially stoppered at a filling volume of 0.55 mL / bottle. The freeze dryer was turned on, and when the plate temperature dropped to 4°C, the filled and partially stoppered samples 1-10 were evenly placed into the same plate of the freeze dryer and freeze-dried according to the set freeze-drying program (see Table 2).

[0154] Table 2. Basic freeze-drying process

[0155]

[0156] After the freeze-drying process is complete, full plugging is performed under vacuum conditions. After full plugging, the vacuum is broken, the product is removed from the chamber, and capped. The freeze-drying curve is shown below. Figure 1 . Figure 1 The reason there are two product temperatures is that there are two temperature probes in the freeze dryer, and the two temperature probes correspond to two product temperatures. This setting in the freeze dryer is to ensure the temperature uniformity at different locations in the freeze dryer.

[0157] V. Detection of indexes in freeze-dried samples

[0158] Key parameters of samples prepared using lyophilization protectants formulations 1-10 before and after lyophilization were measured: appearance, reconstitution time, particle size, polymer dispersibility index (PDI), potential, encapsulation efficiency, mRNA molecular integrity (CE), water content, and transfection. The results are shown in Table 3. Figure 3 Meanwhile, when freeze-dried products 1-10 were placed at 37℃ for 7 days, it was found that their finished product shape was significantly shrunken, such as... Figure 2 As shown.

[0159] Note: 1) Reconstitution time refers to the time required from the addition of 0.55 mL of water for injection to the complete dissolution of the lyophilized preparation sample into a liquid state;

[0160] 2) Particle size, potential, and PDI detection: Particle size, potential, and PDI were detected using the standard method of the nanoparticle size analyzer (Anton Paar). The sample volume was 750-1000 μL, and the detection temperature was 20-25℃.

[0161] 3) Encapsulation efficiency: Ribogreen RNA reagent was used to bind to free mRNA and total mRNA after Triton X-100 membrane permeation, respectively. The content of free mRNA and total mRNA after membrane permeation was determined by fluorescence analysis. The encapsulation efficiency was calculated as (total mRNA detected after membrane permeation - total free mRNA detected) / total mRNA detected after membrane permeation × 100%.

[0162] 4) mRNA molecular integrity (CE) detection: Take 100 μl of the encapsulation solution and add 900 μL of 2% Triton X-100 solution to break the membrane. Then add 500 μL of 8M lithium chloride solution to extract mRNA (Merk, catalog number: L7026). After dissolving in RNase-free water, the integrity of the mRNA was detected by an Agilent 7100 instrument.

[0163] The method for preparing 100mL of 2% Triton X-100 solution is as follows: take 98mL of sterile enzyme-free water, add 2mL of Triton X-100 solution, and mix thoroughly to obtain 2% Triton X-100 solution.

[0164] 5) Transfection: Take 100 μL of the reconstituted sample and add it to 293T cells (5 × 10⁶ cells) that have been seeded 24 hours in advance. 5 Cells were cultured in 6-well plates (cells / well) with DMEM solution containing 10% fetal bovine serum by volume. After 24 hours, 500 μL of cell protein extraction buffer (Merk, catalog number: FNN0021) was added. After protein extraction, the expression of G proteins was detected by Western blotting.

[0165] 6) Moisture content test: The moisture content was tested according to the coulometric method of the 2025 edition of the Chinese Pharmacopoeia.

[0166] Table 3. Detection results of key indicators for freeze-dried samples 1-10

[0167]

[0168] The transfection-WB test results are shown below. Figure 3 This indicates that the current basic freeze-drying curve significantly affects the biological activity of mRNA vaccine products after freeze-drying, and that the expression of the target protein is affected when detected by immunoblotting after cell transfection.

[0169] VI. Optimization of freeze-drying process

[0170] Samples 1-10 after freeze-drying showed high water content and severe lipid shrinkage, indicating that the high water content significantly affected the biological activity. The freeze-drying process caused some structural damage. While sucrose and trehalose in the freeze-drying protectants provided some protection and acted as excipients, the sugar content affected the eutectic point temperature (i.e., the time and temperature control from pre-freezing to sublimation). Therefore, the freeze-drying curves were readjusted. The freeze-drying program is shown in Table 4, and the freeze-drying curves are shown in [Table 4]. Figure 4 .

[0171] Table 4. Adjusted mRNA vaccine freeze-drying procedure

[0172]

[0173] The optimized lyophilization process was used to lyophilize samples prepared with lyophilization protectants of formulations 1-10. Key indicators after lyophilization included: appearance, reconstitution time, particle size, polymer dispersibility index (PDI), potential, encapsulation efficiency, mRNA molecular integrity (CE), water content, and transfection. The test results are shown in Table 5. Figure 4 .

[0174] Table 5. Detection of key indicators for samples 1-10 after freeze-drying process optimization

[0175]

[0176] The transfection-WB test results are shown below. Figure 4 .Will Figure 4 and Figure 3 In comparison, using the same lyophilization protectant but with different lyophilization curves, the optimized lyophilization curve did not affect the biological activity. Both pre- and post-lyophilized samples transfected into 293T cells showed expression of the target protein, while the sample with the more dynamic baseline lyophilization curve exhibited compromised biological activity. Figure 3 ).

[0177] Meanwhile, samples 1-10, after being freeze-dried, were placed at 37°C for 7 days, and their morphology did not change significantly (see...). Figure 5 Meanwhile, its mRNA integrity remained above 80% (see Table 6), and its biological activity did not show a significant trend of change (see Table 6). Figure 6 ).

[0178] Table 6. Bioactivity of samples 1-10 after lyophilization process optimization

[0179]

[0180] The results showed that, before and after the freeze-drying process optimization, the extended freeze-drying time significantly reduced the water content. After being stored at 37℃, the morphology remained well preserved, and the biological activity and mRNA molecule integrity were maintained well even after 7 days at 37℃. This indicates that the optimized freeze-drying process, with its reduced water content and slow gradient sublimation during the lipid delivery system, maintained the integrity of the lipid structure, minimizing the impact on mRNA integrity and thus benefiting the bioexpression efficiency of the mRNA-lipid delivery system particles. In conclusion, the optimized freeze-drying process plays a positive role in ensuring the biological activity and quality stability of the mRNA-lipid delivery system.

[0181] Based on this, the glycine content in the lyophilization protectant was adjusted from 1 mM to 3 mM (i.e., lyophilization protectant formulations 11-18). The lyophilization curve parameters are the same as in Table 4. Simultaneously, lyophilized samples 11-18 were tested (see Table 7). Immunoblotting analysis of bioactivity is shown in [Table 7]. Figure 7 The comprehensive freeze-drying protectant formulations 1-18 use glycine at a concentration range of 1-3 mM.

[0182] Table 7. Detection of key indicators for samples 11-18 after freeze-drying process optimization

[0183]

[0184] The transfection-WB test results are shown below. Figure 7This indicates that when the glycine concentration is 3%, the trehalose concentration range can be 0.5%-1.0%, and the sucrose concentration range can be 10%-12%.

[0185] IV. Optimization of buffer solution in lyophilization protectant

[0186] For samples 1-18, NaAC and Tris buffers were selected as the lyophilization protectants. The concentrations and pH of His and HEPES buffers were simultaneously optimized. Specific lyophilization protectants are listed in Table 1 (samples 19-24). The optimized lyophilization process was used. Key indicators were measured for these six lyophilized samples (samples 19-24), and the results are shown in Table 8. Furthermore, after placing the samples at 37℃ for 7 days, there was no significant difference in their biological activity. Figure 8 In summary, the results indicate that the concentrations of His and HEPES buffers and the pH value did not significantly affect the physicochemical properties after lyophilization.

[0187] Table 8. Detection of key indicators for samples 19-24 after freeze-drying process optimization

[0188]

[0189] The transfection-WB test results are shown below. Figure 8 This indicates that, with the freeze-drying protectants (sucrose, trehalose, and glycine) remaining unchanged, the composition of the buffer solution was adjusted when comparing freeze-drying protectant formulations 19-24 and 11-18, demonstrating that His and HEPES buffer solutions also have good freeze-drying protection effects in freeze-drying protectants.

[0190] V. Physical and Chemical Stability Study

[0191] The intended storage temperature for the freeze-dried vaccine using the mRNA-lipid delivery system is 2-8℃. Accelerated stability studies were conducted on the freeze-dried samples, and the integrity of the mRNA molecules was tested. The results showed that freeze-drying protectants 1-24 exhibited good stability. Furthermore, after optimization of the freeze-drying process parameters, the stability of samples 1-10 was better than before parameter optimization. The stability trend is shown below. Figure 9 Stability was also assessed for samples adjusted for glycine and buffer (the glycine-adjusted lyophilizers were numbered 11-18, and the buffer-adjusted lyophilizers were numbered 19-24). After two weeks of storage at 37°C, the mRNA molecules remained intact in all samples. Figure 10 ).

[0192] VI. Lyophilization Applications of Other mRNA Vaccines

[0193] When the lyophilization protectant formulation No. 11 was applied to the novel coronavirus mRNA vaccine, the lyophilized sample could be stored stably at 25°C for up to 6 months.

[0194] Table 9

[0195]

[0196] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A freeze-drying protectant for mRNA vaccines, characterized in that, The mRNA vaccine freeze-drying protectant includes sucrose at a final concentration of 10-15%, trehalose at a final concentration of 0-0.5%, and glycine at a final concentration of 0-3mM; it also includes sodium acetate at a final concentration of 20mM, His at a final concentration of 10mM-20mM, or HEPES at a final concentration of 10mM-20mM. The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

2. The mRNA vaccine freeze-drying protectant according to claim 1, characterized in that, The mRNA vaccines mentioned include rabies mRNA vaccines and COVID-19 mRNA vaccines.

3. The mRNA vaccine freeze-drying protectant according to claim 1, characterized in that, The mRNA vaccine freeze-drying protectant includes sucrose at a final concentration of 12%, trehalose at a final concentration of 0.5%, glycine at a final concentration of 3mM, and sodium acetate at a concentration of 20mM. The solvent for the mRNA vaccine freeze-drying protectant is water for injection.

4. The method for preparing the mRNA vaccine freeze-drying protectant according to any one of claims 1 to 3, characterized in that, Includes the following steps: Sucrose, trehalose, glycine, sodium acetate, His, and HEPES are added to water for injection to the target concentration to obtain the lyophilized protective agent for mRNA vaccines.

5. The method for preparing the mRNA vaccine freeze-drying protectant according to claim 4, characterized in that: First, prepare 50% sucrose stock solution, 1% trehalose stock solution, 1M glycine stock solution, 1M NaAC stock solution, 1M HEPES stock solution, and 1M His stock solution. Then, add each prepared stock solution to water for injection to bring the sucrose, trehalose, glycine, sodium acetate, His, and HEPES to the target concentrations, thus obtaining the mRNA vaccine freeze-dried protective agent.

6. The use of the mRNA vaccine freeze-drying protectant according to any one of claims 1 to 3 in the preparation of mRNA vaccines.

7. The application of the mRNA vaccine lyophilization protectant according to claim 6 in the preparation of mRNA vaccines, characterized in that, The freeze-drying process is as follows: Pre-freeze at -45℃ for 2 hours; Sublimation drying at -40℃ under a vacuum of 0.35 mbar for 5 hours; Sublimation drying at a vacuum of 0.2 mbar and -36°C for 8 hours; Sublimation drying at -30℃ under vacuum of 0.2 mbar for 8 hours; Sublimation drying at a vacuum of 0.1 mbar and -28°C for 8 hours; Sublimation drying at -25°C for 3 hours under vacuum of 0.1 mbar; Sublimation drying at -15℃ under vacuum of 0.1 mbar for 10 h; Desorption drying at a vacuum of 0.01 mbar and -25°C for 6 hours; Desorption drying at a vacuum of 0.01 mbar and -28°C for 5 hours; Desorption drying was performed at a vacuum of 0.01 mbar and -25°C for 6 hours.

Images