Nucleic acid lipid nanoparticle freeze-drying preparation as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a nucleic acid lipid nanoparticle freeze-drying preparation as well as a preparation method and application thereof. The preparation method of the nucleic acid lipid nanoparticle freeze-dried preparation comprises the following steps: preparing a nucleic acid-containing solution; preparing lipid nanoparticles containing nucleic acid; preparing a buffer solution containing lipid nanoparticles and a freeze-drying protective agent; freeze-drying the buffer solution containing the lipid nanoparticles and the freeze-drying protective agent; the lipid nanoparticles comprise cationic lipid capable of being protonated, DSPC (Distearoyl Phosphate Chloride), cholesterol and DMG-PEG2000 (Dimethyl Glycol-Polyethylene Glycol 2000). According to the preparation method of the nucleic acid lipid nanoparticle freeze-dried preparation provided by the invention, the change of the particle size and the wrapping rate before and after freeze-drying is small, the in-vivo immune response of the freeze-dried and re-thawed preparation is high and has no significant difference from that of an unfreeze-dried mRNA vaccine, and the storage and transportation cost and efficiency of the mRNA vaccine are significantly reduced.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a nucleic acid lipid nanoparticle freeze-dried preparation and a preparation method and application thereof. Background Art

[0002] In the past few years, messenger RNA-lipid nanoparticle (mRNA-LNPs) technology has attracted increasing research and commercial attention due to its potential in addressing infectious disease prevention and cancer treatment. Compared with traditional vaccines, mRNA-LNPs vaccines have many advantages, such as high efficiency, safety, short production cycle and low cost. In 2020, the approval of two mRNA-LNPs vaccines, BNT162b2 and mRNA-1273, effectively reduced the risk of human infection with COVID-19, indicating that mRNA-LNPs technology has certain advantages in the research and development of vaccines for infectious diseases.

[0003] However, after one week under mild conditions (4°C or room temperature), the particle size of mRNA-LNPs increases significantly and the biological activity (RNA expression) in solution decreases. In order to ensure the stability and efficacy of mRNA-LNPs drugs such as the COVID-19 mRNA vaccine BNT162b2 (co-developed by Pfizer and BioNTech), such drugs require extreme low temperature conditions during transportation and storage. For example, the BNT162b2 vaccine must be stored in an ultra-low temperature environment of -80°C to -60°C, and its shelf life is extremely limited after thawing to room temperature, and it can only remain stable within 2 hours. Such strict cold chain requirements pose huge challenges to logistics, distribution and storage facilities around the world, especially in areas with limited resources or insufficient cold chain infrastructure, which greatly restricts the large-scale application and popularization of such nucleic acid drugs.

[0004] To solve the physical and chemical stability problems faced by LNPs during long-term storage, freeze-drying can be used to convert them into dry powders that can be easily stored at 4°C for a long time. The activity can be restored by rehydrating before administration. However, given the complex structure of lipid nanoparticles and their high sensitivity to freeze-drying processes, especially when containing nucleic acid macromolecules (such as mRNA and DNA), the pressure and stress applied during freeze-drying are likely to damage their active structures. In addition, the protonable lipid components contained in LNPs increase the electrostatic interactions within the particles, which has a significant impact on the physical properties and chemical stability of LNPs.

[0005] Wang Haomeng et al. used the mRNA encoding the SARS-CoV-2 spike protein as a model to study the freeze-drying protective agent formula and freeze-drying process of nucleic acid-lipid nanoparticles. Based on the addition of 10% freeze-drying protective agent to mRNA-LNPs, the study screened the formula of sucrose, trehalose, mannitol, glucose and lactose as freeze-drying protective agents for nucleic acid-lipid nanoparticles. Experimental data showed that the proportion of freeze-drying protective agent components had a significant effect on the particle size of mRNA-LNPs freeze-dried preparations. The gentle and multi-step heating method in the freeze-drying process was more conducive to protecting the mRNA-LNPs structure, improving the mRNA encapsulation rate and integrity, and improving the stability of the freeze-dried preparation, such as three-step heating. The study by Hu Yong et al. showed that when the same freeze-drying protective agent formula was used, the freeze-drying effect of low-temperature freeze-drying was better than that of room-temperature freeze-drying. In addition, the programmed temperature control method proposed by them significantly improved the change in particle size before and after freeze-drying through slow cooling, pre-freezing and ultra-low temperature drying, and the particle size gradually decreased with the increase of protective agent concentration.

[0006] Although studies have shown that conventional cryoprotectants can effectively prevent particles from aggregating during the freezing process, they also point out a key problem: even with the addition of these antifreeze additives, the freeze-dried preparations prepared by this process may still fail. The change in particle size of lipid nanoparticles before and after freeze-drying is crucial to the stability and biological activity of mRNA vaccines. An increase in particle size may reduce the stability of mRNA vaccines because larger particles may be more likely to aggregate, affecting their distribution and delivery efficiency in the body. In addition, a decrease in the encapsulation rate means that more mRNA may leak out during or after freeze-drying, which will reduce the effectiveness and stability of the mRNA vaccine. Therefore, effectively controlling the changes in particle size and encapsulation rate of mRNA-LNPs freeze-dried preparations is a key technical issue that needs to be solved urgently. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a nucleic acid lipid nanoparticle freeze-dried preparation and a preparation method and application thereof.

[0008] According to the present invention, the main solution to realize the commercialization potential of mRNA-LNPs freeze-dried preparations includes at least one of the following aspects: ⑴ Selecting suitable protonable lipid components; ⑵ Adding suitable exogenous components to mRNA-LNPs, such as freeze-drying protectants; ⑶ Optimizing the preparation process; ⑷ Optimizing the freeze-drying procedure. The freeze-dried preparation preparation method provided by the present invention can effectively control the changes in particle size and encapsulation rate of nucleic acid lipid nanoparticles before and after freeze-drying, wherein the particle size change can be controlled within about 10nm, and the encapsulation rate change can be controlled within 10%; and the encapsulation rate of the freeze-dried nucleic acid lipid nanoparticles does not change significantly within 6 months at 2-8°C, thereby extending the shelf life of the nucleic acid lipid nanoparticles and facilitating transportation and storage.

[0009] In a first aspect, the present invention provides a method for preparing a lyophilized nucleic acid lipid nanoparticle preparation, comprising: 1) Prepare a solution containing nucleic acid.

[0010] 2) Preparation of lipid nanoparticles containing nucleic acids.

[0011] 3) Prepare a buffer solution containing lipid nanoparticles and a lyoprotectant.

[0012] 4) freeze-drying the buffer solution containing the lipid nanoparticles and the lyoprotectant.

[0013] The lipid nanoparticles contain protonable cationic lipids, DSPC, cholesterol and DMG-PEG2000; the structural formula of the protonable cationic lipids is shown in Formula 1: Formula 1.

[0014] The process for preparing a freeze-dried preparation of nucleic acid lipid nanoparticles provided by the present invention adopts a new lipid nanoparticle formula and adds a freeze-drying protectant. The particle size and encapsulation rate change little before and after freeze-drying, and the cell transfection efficiency of the preparation after freeze-drying and rehydration is not significantly different from that of the unfreeze-dried mRNA vaccine, and the in vivo immune response is high, which significantly reduces the cost and efficiency of storage and transportation.

[0015] In some embodiments, in the lipid nanoparticles, the molar ratio of protonatable cationic lipid: DSPC: cholesterol: DMG-PEG2000 is 35: 14-18: 45-48: 2-3, preferably 35: 16: 46.5: 2.5. The use of the above-mentioned protonatable cationic lipid and its ratio with DSPC, cholesterol and DMG-PEG2000 can achieve fine regulation of the functionality of lipid nanoparticles and its potential effect in achieving efficient mRNA delivery. It is suitable for mRNA lyophilized preparations, and the particle size change of the obtained lyophilized preparation is small and the encapsulation rate is above 70%.

[0016] In some embodiments, the lyoprotectant comprises sucrose and trehalose in a mass ratio of 2 to 5:1, and any one selected from mannitol or poloxamer. The present invention finds that the lyoprotectant with the specific three components and the above ratio significantly improves the lyoprotective effect.

[0017] In some embodiments, the lyoprotectant is composed of sucrose and trehalose in a mass ratio of 4.2 to 4.8:1, preferably 4.3-4.5:1, and mannitol or poloxamer, for example, a mass ratio of 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, etc. and any value therebetween; more preferably, the lyoprotectant is composed of sucrose and trehalose in a mass ratio of 4.3-4.5:1, and a mass concentration of 0.01-0.05% mannitol or a mass concentration of 0.01-0.5% poloxamer.

[0018] In some embodiments, the total weight percentage of sucrose and trehalose in the lyoprotectant is 10-15%. The present invention also found that the freeze-drying effect is better when the proportion of sucrose and trehalose is 10-15%.

[0019] Preferably, in step 1), a lyoprotectant solution is added when preparing the solution containing nucleic acid; preferably, the volume percentage of the lyoprotectant solution in the solution containing nucleic acid is 45-55%, preferably 50%. Adding the lyoprotectant of the present invention in advance to the nucleic acid solution can significantly reduce the change in particle size to less than 10nm; the encapsulation rate is also significantly improved.

[0020] Further preferably, the nucleic acid-containing solution prepared in step 1) and / or the buffer containing lipid nanoparticles and lyoprotectant prepared in step 3) contains sucrose with a mass concentration of 8-9%, trehalose with a mass concentration of 1.8-2.4%, and mannitol with a mass concentration of 0.01-0.05% or poloxamer with a mass concentration of 0.01-0.5%.

[0021] Preferably, it contains sucrose with a mass concentration of 8-9%, trehalose with a mass concentration of 1.8-2.4%, and mannitol with a mass concentration of 0.02-0.05% or poloxamer with a mass concentration of 0.08-0.12%.

[0022] Exemplarily, it is preferred to contain sucrose with a mass concentration of 8.8%, trehalose with a mass concentration of 2.0%, and mannitol with a mass concentration of 0.04% or poloxamer with a mass concentration of 0.1%.

[0023] In the present invention, a preparation method with a higher concentration is adopted, and the freeze-drying protection effect is excellent. At the same time, the stability and immunogenicity of the preparation are good, and it can be better used to prepare mRNA vaccine freeze-dried preparations.

[0024] In some embodiments, in step 3), the solution of the lyoprotectant uses Tris-HCl buffer as a solvent.

[0025] In some embodiments, the concentration of the Tris-HCl buffer is 30-100 mM, for example, 30, 40, 50, 60, 70, 80, 90, 100 mM and any value therebetween, preferably 50 mM.

[0026] In some embodiments, in step 1), citrate buffer is used as a solvent in the lyoprotectant solution.

[0027] In some embodiments, in step 1), the concentration of the citric acid buffer used in the lyoprotectant solution can be 30, 40, 50, 60, 70, 80, 90, 100 mM, preferably 50 mM.

[0028] In some embodiments, in the method for preparing a lyophilized nucleic acid lipid nanoparticle preparation, the nucleic acid is mRNA.

[0029] In the present invention, the freeze-drying process generally includes the following steps: first, pre-freezing, rapidly cooling the sample to be processed to a temperature below its eutectic point, and maintaining it for a certain period of time, so that the sample is completely frozen and a stable ice crystal structure is formed. The pre-freezing temperature and time are determined according to the characteristics of the sample. Then comes the main drying, reducing the pressure in a vacuum environment to cause the frozen water to sublime directly from a solid state to a gaseous state. At this stage, the temperature and vacuum degree need to be precisely controlled to prevent the sample from overheating or collapsing, so as to effectively remove the moisture. Then comes the analytical drying, further increasing the temperature and maintaining the vacuum to remove the unsublimated bound water in the sample until a dry finished product is obtained.

[0030] In some embodiments, the method for preparing a lyophilized nucleic acid lipid nanoparticle preparation, wherein the freeze-drying comprises: 1) After cooling to -60°C to -40°C, maintain constant temperature freezing for 2 to 5 hours for pre-freezing. Preferably, the temperature is lowered at a rate of 0.5°C to 1.0°C per minute to -45°C.

[0031] 2) Drying under vacuum conditions at -50°C to -10°C for 18 to 36 hours; drying at 2°C to 8°C for 4 to 10 hours; and drying at 10°C to 20°C for 1 to 4 hours.

[0032] In a second aspect, the present invention provides a lyophilized preparation of nucleic acid lipid nanoparticles obtained by the preparation method.

[0033] In a third aspect, the present invention provides the use of the nucleic acid lipid nanoparticle freeze-dried preparation in the preparation of mRNA vaccines.

[0034] The beneficial effects of the present invention include at least the following: (1) After adding a lyophilization protectant, the novel lipid nanoparticle formula of the present invention can make the particle size and encapsulation rate change less before and after lyophilization, and is suitable for preparing nucleic acid lipid nanoparticle lyophilized preparations.

[0035] (2) The present invention screened out a better freeze-drying protective agent formula. The inventor surprisingly found that when the ratio of sucrose to trehalose is controlled at 4.3-4.5:1, the additional addition of appropriate mannitol or poloxamer can significantly control the particle size change to about 10 nm, and the encapsulation rate change can be controlled within 10%.

[0036] ⑶ The present invention proposes a new freeze-dried preparation method based on a new lipid nanoparticle formula. A freeze-dried protective agent solution is added to the mRNA stock solution, and a freeze-dried protective agent solution of an approximate proportion is added to the prepared mRNA-LNPs stock solution. The particle size of the freeze-dried preparation can be reduced, and the range of particle size variation before and after freeze-drying is reduced, and the range of variation of the encapsulation rate is reduced.

[0037] ⑷ The respiratory syncytial virus mRNA vaccine prepared based on the freeze-drying process of the present invention reconstitutes rapidly, and has high total nucleic acid content, encapsulation rate and nucleic acid integrity. In addition, there is no significant difference in the in vivo immune response of the freeze-dried and reconstituted preparation compared with the non-freeze-dried mRNA vaccine. Moreover, by reducing or even removing the aqueous microenvironment in the system through freeze-drying, the nucleic acid hydrolysis reaction catalyzed by nuclease can be inhibited, thereby significantly improving the long-term stability of nucleic acid lipid nanoparticles. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 A process optimization roadmap for the mRNA-LNPs freeze-dried preparation provided in an embodiment of the present invention.

[0040] Figure 2 The invention provides a synthesis process of the protonable cationic lipid MH-017.

[0041] Figure 3 This is the nuclear magnetic resonance spectrum of the protonable cationic lipid MH-017 provided in the embodiments of the present invention.

[0042] Figure 4 This is an appearance comparison of FM08 in Example 3 and FM16 in Example 4 provided in the embodiments of the present invention.

[0043] Figure 5The particle size data of the preparation after process optimization provided in the embodiments of the present invention stored at 2-8°C.

[0044] Figure 6 The encapsulation rate data of the preparation after process optimization provided in the embodiment of the present invention stored at 2-8°C.

[0045] Figure 7 The integrity data of the preparation after process optimization provided in the embodiments of the present invention when stored at 2-8°C.

[0046] Figure 8 Comparison of the neutralizing antibody levels induced by the process-optimized preparation and the liquid preparation provided in the embodiments of the present invention. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention, taking RSV F protein mRNA vaccine as an example. Obviously, the described embodiments are part of the embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention.

[0048] In the embodiment of the present invention, the protonable cationic lipid MH-017 has a structure as shown below: .

[0049] The synthesis method and size characterization of the protonatable cationic lipid MH-017 are as follows Figure 2-Figure 3 shown.

[0050] The DSPC is a non-cationic lipid with a CAS number of 816-94-4, and its structure is shown below: .

[0051] The DMG-PEG2000 is a PEG lipid with a CAS number of 160743-62-4, and its structure is shown below: .

[0052] The CAS number of the cholesterol is 57-88-5, and its structure is shown below: .

[0053] In the embodiment of the present invention, the steps for preparing the mRNA stock solution are as follows: 1) Synthesis of RSV F protein mRNA: We commissioned Nanjing GenScript Biotechnology Co., Ltd. to synthesize plasmid DNA using the DNA sequence encoding RSV F protein (SEQ ID No: 1), and then prepared the linearized DNA template by enzyme digestion, and synthesized RSV F mRNA according to the following system:

[0054] After incubation at 37°C for 3-4 hours, DNAase I was added and incubated for another 0.5 hour to terminate the reaction.

[0055] 2) Affinity chromatography purification of RSV F protein mRNA: The synthesized mRNA solution was purified using a chromatography system. The synthesized mRNA solution was diluted 5 times with chromatography buffer (500mM NaCl, 10mMTris-HC1, 1mM EDTA, pH=7.0), and then the OligodT chromatography column was equilibrated with chromatography buffer. After equilibration for 5 column volumes, the sample was loaded at a flow rate of 30cm / h. After loading, the column was rinsed with a wash buffer (10mM Tris, 50mM NaCl, 1mM EDTA, pH=7.0) to remove impurities. After rinsing until the UV A260 and conductivity were balanced, the sample was eluted with injection water, the elution peak was collected to obtain the mRNA stock solution, and the mRNA content was adjusted to 200μg / ml.

[0056] Example 1 Step 1: Prepare mRNA stock solution The prepared mRNA stock solution was diluted with 50 mM citrate buffer at a volume ratio of 1:1 to a concentration of 100 μg / ml.

[0057] Step 2: Preparation of lipid mixed solution and mRNA encapsulation The protonable cationic lipid MH017: DSPC: cholesterol: DMG-PEG2000 was prepared into a mixed solution with a total lipid concentration of 16 mM according to the molar ratio of protonable cationic lipid MH017: DSPC: cholesterol: DMG-PEG2000 of 35:16:46.5:2.5 (solvent: anhydrous ethanol).

[0058] The mRNA stock solution prepared in the first step and the prepared lipid mixed solution were encapsulated on a nanomedicine manufacturing device at a nitrogen / phosphorus ratio (N / P) of 6:1, and then ultrafiltration was performed to replace the liquid and concentrated to obtain RSV F mRNA-LNPs stock solution, in which the mRNA content was 200 μg / ml.

[0059] Step 3: Preparation of RSV F mRNA-LNPs liquid formulation containing lyophilized protective agent 1) Prepare lyophilization protective agent solution 100 ml of freeze-dried protective agent solution was prepared with 50 mM Tris-HCl buffer as solvent, and the component contents (weight percentage) of sucrose, trehalose and mannitol in the freeze-dried protective agent solution were 17.6%, 4.0% and 0.08% respectively. Each freeze-dried protective agent solution was sterilized by filtration through a 0.22 μm sterile filter membrane.

[0060] 2) Preparation of RSV F mRNA-LNPs liquid formulation containing lyophilized protective agent The RSV F mRNA-LNPs stock solution prepared in the second step was added with a lyophilization protectant solution at a volume ratio of 1:1 and mixed to make the mRNA content 100 μg / ml, thereby preparing a RSV F mRNA-LNPs liquid preparation containing a lyophilization protectant.

[0061] Step 4: Freeze drying to prepare freeze-dried preparations The RSV F mRNA-LNPs liquid preparation containing the lyoprotectant was freeze-dried, and the specific freeze-drying parameters were set as shown in Table 1.

[0062] Table 1 Freeze-drying process parameter settings

[0063] Example 2 1) Prepare freeze-dried protective agent solutions of different components 100 ml of freeze-dried protective agent solution was prepared with 50 mM citric acid buffer as solvent, and the component contents (weight percentage) of sucrose, trehalose and mannitol in the freeze-dried protective agent solution were 17.6%, 4.0% and 0.08% respectively. Each of the prepared freeze-dried protective agent solutions was sterilized by filtration through a 0.22 μm sterile filter membrane.

[0064] 2) Improved RSV F mRNA-LNPs liquid formulation method Step 1: Add lyophilization protectant to mRNA stock solution The prepared lyophilized protective agent solution was added to the prepared mRNA stock solution at a volume ratio of 1:1, so that the mRNA content was 100 μg / ml.

[0065] Step 2: Preparation of lipid mixed solution and mRNA encapsulation A lipid mixed solution was prepared according to the molar ratio of protonable cationic lipid MH017: DSPC: cholesterol: DMG-PEG2000 of 35:16:46.5:2.5.

[0066] The mRNA stock solution mixed with the lyophilized protective agent solution prepared in the first step and the lipid mixed solution prepared in the second step were encapsulated on a nanomedicine manufacturing device at a nitrogen / phosphorus ratio (N / P) of 6:1, and then ultrafiltration was performed to exchange the liquid and concentrate to obtain the RSV F protein mRNA-LNPs stock solution coated with the lyophilized protective agent, in which the mRNA content was 200 μg / ml.

[0067] Step 3: Adding lyophilized protective agent to mRNA-LNPs vaccine The RSV F protein mRNA-LNPs stock solution prepared in the second step was added with the lyophilized protective agent solution in Example 1 at a volume ratio of 1:1 to make the mRNA content 100 μg / ml to prepare an improved RSV F mRNA-LNPs liquid preparation.

[0068] 3) Freeze drying and preparation of freeze-dried preparations Freeze-drying was carried out according to the freeze-drying process in Example 1.

[0069] Example 3 Step 1: Prepare mRNA stock solution The prepared mRNA stock solution was diluted with 50 mM citrate buffer at a volume ratio of 1:1 to a concentration of 100 μg / ml.

[0070] Step 2: Preparation of lipid mixed solution and mRNA encapsulation A lipid mixed solution was prepared according to the molar ratio of protonable cationic lipid MH017: DSPC: cholesterol: DMG-PEG2000 of 35:16:46.5:2.5.

[0071] The mRNA stock solution prepared in the first step and the prepared lipid mixed solution were encapsulated on a nanomedicine manufacturing device at a nitrogen / phosphorus ratio (N / P) of 6:1, and then ultrafiltration was performed to replace the liquid and concentrated to obtain RSV F mRNA-LNPs stock solution, in which the mRNA content was 200 μg / ml.

[0072] Step 3: Preparation of RSV F protein mRNA-LNPs liquid formulation containing lyophilized protective agent 1) Prepare freeze-dried protective agent solutions of different components Using 50 mM Tris-HCl buffer as solvent, 100 ml of lyophilized protective agent solutions containing different components such as sucrose, trehalose, mannitol or poloxamer were prepared. The content (weight percentage) of each component of each lyophilized protective agent solution was as shown in Table 2. The prepared lyophilized protective agent solutions were sterilized by filtration through a 0.22 μm sterile filter membrane.

[0073] Table 2 Different lyophilization protective agent solutions

[0074] 2) Preparation of RSV F mRNA-LNPs liquid formulation containing lyophilized protective agent The RSV F mRNA-LNPs stock solution prepared in the second step was taken, and the lyophilization protective agent solutions in Table 2 were added at a volume ratio of 1:1 to mix so that the mRNA content was 100 μg / ml, and the RSV F protein mRNA-LNPs liquid preparation containing lyophilization protective agent was prepared, as shown in Table 3.

[0075] Table 3 RSV F protein mRNA-LNPs liquid formulation containing lyophilized protective agent

[0076] Step 4: Freeze drying to prepare freeze-dried preparations The RSV F mRNA-LNPs liquid preparation containing the lyoprotectant was freeze-dried, and the specific freeze-drying parameters were set as shown in Table 4.

[0077] Table 4 Freeze-drying process parameter settings

[0078] After lyophilization, the product was collected and reconstituted with an equal volume of resolvent as before lyophilization, and Ribo Green TM The nanoparticle encapsulation efficiency was determined, and the nanoparticle size and PDI potential were determined by Malvern NS90Z nanoparticle size and potential analyzer. The experimental results are shown in Table 5.

[0079] Table 5 Changes of main indicators of mRNA-LNPs before and after freeze-drying

[0080] From the above data, compared with preparations FM01, FM02 and FM03, when 0.04% mannitol or 0.1% poloxamer is added to the lyophilization protectant, the changes in particle size and encapsulation rate before and after lyophilization can be reduced. When sucrose and trehalose are used as lyophilization protectants in ratios of 4.4:1, 4:1 and 2:1, the changes in particle size and encapsulation rate are significantly better than those in ratios of 1:1 and 6:1.

[0081] Example 4 Evaluation of the applicability of the improved formulation method to RSV F mRNA-LNPs freeze-dried formulation 1) Prepare freeze-dried protective agent solutions of different components Using 50 mM citric acid buffer as solvent, 100 ml of lyophilized protective agents of different formulations were prepared, including sucrose, trehalose, and either mannitol or poloxamer. The contents (weight percentage) of the components of the lyophilized protective agent solutions were as shown in Table 6. The prepared lyophilized protective agent solutions were sterilized by filtration through a 0.22 μm sterile filter membrane.

[0082] Table 6 Different lyophilization protective agent solutions

[0083] 2) Improved RSV F mRNA-LNPs liquid formulation method Step 1: Add lyophilization protectant to mRNA stock solution The lyophilized protective agent solutions in Table 6 were added to the prepared mRNA stock solutions at a volume ratio of 1:1, so that the mRNA content was 100 μg / ml.

[0084] Step 2: Preparation of lipid mixed solution and mRNA encapsulation A lipid mixed solution was prepared according to the molar ratio of protonable cationic lipid MH017: DSPC: cholesterol: DMG-PEG2000 of 35:16:46.5:2.5.

[0085] The mRNA stock solution mixed with the lyophilized protective agent solution prepared in the first step and the lipid mixed solution prepared in the second step were encapsulated on a nanomedicine manufacturing device at a nitrogen / phosphorus ratio (N / P) of 6:1, and then ultrafiltration was performed to exchange the liquid and concentrate to obtain the RSV F protein mRNA-LNPs stock solution coated with the lyophilized protective agent, in which the mRNA content was 200 μg / ml.

[0086] Step 3: Adding lyophilized protective agent to mRNA-LNPs vaccine The RSV F mRNA-LNPs stock solution prepared in the second step was added with the lyophilized protective agent solutions LP04 to LP09 in Table 2 of Example 3 at a volume ratio of 1:1 to make the mRNA content 100 μg / ml to prepare an improved RSV F protein mRNA-LNPs liquid preparation, as shown in Table 7.

[0087] Table 7 Improved RSV F protein mRNA-LNPs liquid formulation

[0088] 3) Freeze drying and preparation of freeze-dried preparations The freeze-drying process was followed in Example 1. After drying, the product was collected and reconstituted with an equal volume of ultrapure water as before freeze-drying. TM The nanoparticle encapsulation efficiency was determined, and the nanoparticle size and PDI potential were determined by Malvern NS90Z nanoparticle size and potential analyzer. The experimental results are shown in Table 8.

[0089] Table 8 Changes of main indicators of mRNA-LNPs before and after freeze-drying

[0090] The process optimization route for preparing the mRNA-LNPs freeze-dried preparation provided in this example is different from the unoptimized process route in Example 3. Figure 1 As shown. From the above data, it can be seen that compared with freeze-drying without encapsulating the lyophilization protectant of the present invention in the nucleic acid lipid nanoparticles, adding the lyophilization protectant of the present invention during the encapsulation process of the nucleic acid lipid nanoparticles can further reduce the particle size of the nucleic acid lipid nanoparticles before and after freeze-drying, and the variation range is significantly narrower than that of the preparation method in Example 3, and the particle size variation can be controlled at about 10nm; and the downward trend of the encapsulation rate is smaller, and the variation range is significantly narrower than that of the preparation method in Example 3, and the encapsulation rate is not less than 80%, among which the particle size variation of preparations FM16 and FM17 is smaller. The appearance comparison before (lyophilized preparation FM08 in Example 3) and after (lyophilized preparation FM16 in Example 4) freeze-drying process optimization is as shown in the following figure. Figure 4 shown.

[0091] Example 5 Stability evaluation of lyophilized preparations Long-term stability storage conditions: 2-8°C, 6 months. The particle size, encapsulation efficiency and mRNA integrity of the preparations FM16 and FM17 of Example 4 were measured at 2-8°C for 0, 1, 3 and 6 months. The data results are shown in Table 9 and the trend analysis is shown in Table 9. Figures 5 to 7 .

[0092] Table 9 Results of long-term storage stability evaluation of formulations FM16 and FM17 at 2-8°C

[0093] Example 6 Evaluation of Immunogenicity of Lyophilized Formulations BALB / c mice were randomly divided into two groups and injected intramuscularly with 10ug of FM16 and FM17 in Example 4 and the liquid preparations for comparison. The mice were given a second immunization of the same dose 21 days after the first immunization. Serum was collected 35 days after the first immunization to detect the level of neutralizing antibody response to RSV. The results are shown in Figure 8 As shown, the neutralizing antibody level induced by the optimized lyophilized preparation was not significantly different from that of the liquid preparation.

[0094] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a lyophilized nucleic acid lipid nanoparticle preparation, characterized in that: include: 1) preparing a solution containing nucleic acid; 2) Preparation of lipid nanoparticles containing nucleic acids; 3) preparing a buffer solution containing lipid nanoparticles and a lyoprotectant; 4) freeze-drying the buffer solution containing lipid nanoparticles and lyoprotectant; The lipid nanoparticles contain protonable cationic lipids, DSPC, cholesterol and DMG-PEG2000; the structural formula of the protonable cationic lipids is shown in Formula 1: Formula 1.

2. The method for preparing the lyophilized nucleic acid lipid nanoparticle preparation according to claim 1, characterized in that: In the lipid nanoparticles, the molar ratio of protonable cationic lipid: DSPC: cholesterol: DMG-PEG2000 is 35:14-18:45-48:2-3, preferably 35:16:46.5:2.

5.

3. The method for preparing the freeze-dried nucleic acid lipid nanoparticle preparation according to claim 1 or 2, characterized in that: The lyoprotectant comprises sucrose and trehalose in a mass ratio of 2 to 5:1, and any one selected from mannitol or poloxamer; Preferably, the lyoprotectant is composed of sucrose and trehalose in a mass ratio of 4.2-4.8:1, preferably 4.3-4.5:1, and mannitol or poloxamer.

4. The method for preparing the freeze-dried nucleic acid lipid nanoparticle preparation according to claim 3, characterized in that: The total weight percentage of sucrose and trehalose in the freeze-drying protective agent is 10-15% of the preparation.

5. The method for preparing the freeze-dried nucleic acid lipid nanoparticle preparation according to any one of claims 1 to 4, characterized in that: In step 1), when preparing the solution containing nucleic acid, a lyoprotectant solution is added; preferably, the volume percentage of the lyoprotectant solution in the solution containing nucleic acid is 45-55%, preferably 50%.

6. The method for preparing the lyophilized nucleic acid lipid nanoparticle preparation according to claim 5, characterized in that: The nucleic acid-containing solution prepared in step 1) and / or the buffer containing lipid nanoparticles and lyophilization protectants prepared in step 3) contain sucrose with a mass concentration of 8-9%, trehalose with a mass concentration of 1.8-2.4%, and mannitol with a mass concentration of 0.01-0.05% or poloxamer with a mass concentration of 0.01-0.5%; Preferably, it contains sucrose with a mass concentration of 8-9%, trehalose with a mass concentration of 1.8-2.4%, and mannitol with a mass concentration of 0.02-0.05% or poloxamer with a mass concentration of 0.08-0.12%.

7. The method for preparing the freeze-dried nucleic acid lipid nanoparticle preparation according to claim 1, characterized in that: The nucleic acid is mRNA.

8. The method for preparing the freeze-dried nucleic acid lipid nanoparticle preparation according to claims 1-7, characterized in that: The freeze drying comprises: 1) Cooling to -60°C to -40°C and then maintaining constant temperature freezing for 2 to 5 hours for pre-freezing. Preferably, cooling at a rate of 0.5°C to 1.0°C per minute to -45°C; 2) Drying under vacuum conditions at -50°C to -10°C for 18 to 36 hours; drying at 2°C to 8°C for 4 to 10 hours; and drying at 10°C to 20°C for 1 to 4 hours.

9. A lyophilized nucleic acid lipid nanoparticle preparation obtained by the preparation method according to any one of claims 1 to 8.

10. Use of the nucleic acid lipid nanoparticle freeze-dried preparation according to claim 9 in the preparation of mRNA vaccines.

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