A nucleic acid lipid nanoparticle lyophilized formulation, and a preparation method and application thereof

Abstract

The present application relates to the technical field of medicine, and particularly relates to a nucleic acid lipid nanoparticle lyophilized preparation and a preparation method and application thereof.The preparation method of the nucleic acid lipid nanoparticle lyophilized preparation comprises the following steps: preparing a nucleic acid-containing solution; preparing a nucleic acid-containing lipid nanoparticle; preparing a buffer containing the lipid nanoparticle and a lyophilization protective agent; freeze-drying the buffer containing the lipid nanoparticle and the lyophilization protective agent; and the lipid nanoparticle comprises a protonatable cationic lipid, DSPC, cholesterol and DMG-PEG2000.The preparation method of the nucleic acid lipid nanoparticle lyophilized preparation provided in the present application has small changes in particle size and encapsulation efficiency before and after lyophilization, and the in-vivo immune response of the preparation after freeze-thawing is not significantly different from that of the non-lyophilized mRNA vaccine, and the cost and efficiency of mRNA vaccine storage and transportation are significantly reduced.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a lyophilized formulation of nucleic acid lipid nanoparticles, its preparation method, and its application. Background Technology

[0002] In recent years, messenger RNA-lipid nanoparticles (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 offer numerous advantages, including high efficacy, safety, short production cycles, and low cost. The approval of two mRNA-LNPs vaccines, BNT162b2 and mRNA-1273, in 2020 effectively reduced the risk of COVID-19 infection in humans, demonstrating the advantages of mRNA-LNPs technology in vaccine development for outbreak-related infectious diseases.

[0003] However, under mild conditions (4°C or room temperature), the particle size of mRNA-LNPs increases significantly after one week, and their biological activity (RNA expression) in solution decreases. To ensure the stability and efficacy of mRNA-LNP drugs such as the COVID-19 mRNA vaccine BNT162b2 (developed in collaboration with Pfizer and BioNTech), these 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 after thawing to room temperature is extremely limited, remaining stable for only 2 hours. Such stringent cold chain requirements pose significant challenges to global logistics, distribution, and storage facilities, especially in regions with limited resources or insufficient cold chain infrastructure, which greatly restricts the large-scale application and widespread use of such nucleic acid drugs.

[0004] To address the physical and chemical stability challenges of lipid nanoparticles (LNPs) during long-term storage, freeze-drying can be employed to convert them into a dry powder that can be easily stored at 4°C for extended periods, restoring activity simply through rehydration before administration. However, given the complex structure of lipid nanoparticles and their high sensitivity to freeze-drying processes, especially when they contain nucleic acid macromolecules (such as mRNA and DNA), the pressure and stress applied during freeze-drying can potentially damage their active structure. Furthermore, the protonable lipid components within LNPs increase internal electrostatic interactions, significantly impacting their physical properties and chemical stability.

[0005] Wang Haomeng et al. used mRNA encoding the SARS-CoV-2 spike protein as a model to study the formulation of lyophilization protectants and the lyophilization process of nucleic acid-lipid nanoparticles. This study, based on adding 10% lyophilization protectant to mRNA-LNPs, screened sucrose, trehalose, mannitol, glucose, and lactose as lyophilization protectants for nucleic acid-lipid nanoparticles. Experimental data showed that the proportion of lyophilization protectant components significantly affected the particle size of the mRNA-LNP lyophilized formulation. A gentle, multi-step heating method in the freeze-drying process was more beneficial for protecting the mRNA-LNP structure, improving mRNA encapsulation efficiency and integrity, and enhancing the stability of the lyophilized formulation, such as three-step heating. Hu Yong et al.'s research showed that when using the same lyophilization protectant formulation, low-temperature freeze-drying was more effective than room-temperature freeze-drying. Furthermore, their proposed temperature-controlled program method, through slow cooling, pre-freezing, and ultra-low temperature drying, significantly improved the particle size change before and after freeze-drying, and the particle size gradually decreased with increasing protectant concentration.

[0006] While studies have demonstrated that conventional cryoprotectants can effectively prevent particle aggregation during freezing, a key issue remains: even with these cryoprotectants, lyophilized formulations prepared through this process may still become ineffective. Changes in the particle size of lipid nanoparticles before and after lyophilization are crucial for the stability and bioactivity of mRNA vaccines. Increased particle size may reduce the stability of mRNA vaccines, as larger particles are more likely to aggregate, affecting their distribution and delivery efficiency in vivo. Furthermore, reduced encapsulation efficiency means that more mRNA may leak out during or after lyophilization, which can decrease the efficacy and stability of mRNA vaccines. Therefore, effectively controlling the particle size and encapsulation efficiency changes in mRNA-LNP lyophilized formulations is a critical technical problem that urgently needs to be solved. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a lyophilized formulation of nucleic acid lipid nanoparticles, its preparation method, and its application.

[0008] According to the research findings of this invention, the main solutions to realize the commercialization potential of mRNA-LNP lyophilized formulations include at least one of the following aspects: (1) selecting suitable protonable lipid components; (2) adding suitable exogenous components, such as lyophilization protectants, to mRNA-LNPs; (3) optimizing the formulation process; and (4) optimizing the freeze-drying procedure. The lyophilized formulation preparation method provided by this invention can effectively control the changes in particle size and encapsulation efficiency of nucleic acid lipid nanoparticles before and after lyophilization. The particle size change can be controlled within approximately 10 nm, and the encapsulation efficiency change can be controlled within 10%. Furthermore, the encapsulation efficiency of the lyophilized 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 method for preparing the lyophilized formulation of nucleic acid lipid nanoparticles provided by the present invention comprises:

[0010] 1) Prepare a solution containing nucleic acid.

[0011] 2) Preparation of lipid nanoparticles containing nucleic acids.

[0012] 3) Prepare a buffer solution containing lipid nanoparticles and a lyophilization protectant.

[0013] 4) Freeze-dry the buffer solution containing lipid nanoparticles and freeze-drying protectant.

[0014] 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:

[0015]

[0016] Formula 1.

[0017] The process for preparing lyophilized nucleic acid lipid nanoparticle formulations provided by this invention employs a novel lipid nanoparticle formulation and adds a lyophilization protectant. The particle size and encapsulation rate change little before and after lyophilization, and the cell transfection efficiency of the lyophilized and rehydrated formulation is not significantly different from that of the unlyophilized mRNA vaccine. Furthermore, it exhibits a high in vivo immune response and significantly reduces the cost and efficiency of storage and transportation.

[0018] In some embodiments, the molar ratio of protonable cationic lipid:DSPC:cholesterol:DMG-PEG2000 in the lipid nanoparticles is 35:14~18:45~48:2~3, preferably 35:16:46.5:2.5. Using the above-mentioned protonable cationic lipid and its ratio with DSPC, cholesterol, and DMG-PEG2000 allows for precise regulation of the lipid nanoparticle functionality and its potential effect in achieving efficient mRNA delivery. It is suitable for mRNA lyophilized formulations, resulting in lyophilized formulations with minimal particle size variation and an encapsulation efficiency of over 70%.

[0019] In some embodiments, the freeze-drying protectant comprises sucrose and trehalose in a mass ratio of 2 to 5:1, and any one selected from mannitol or poloxamer. This invention has found that using a specific three-component freeze-drying protectant in the above-mentioned proportions significantly improves the freeze-drying protection effect.

[0020] In some embodiments, the freeze-drying protectant is composed of sucrose and trehalose in a mass ratio of 4.2 to 4.8:1, preferably 4.3 to 4.5:1, and mannitol or poloxamer, for example, in a mass ratio of 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, and any value therebetween; more preferably, the freeze-drying protectant is composed of sucrose and trehalose in a mass ratio of 4.3 to 4.5:1, and mannitol or poloxamer at a mass concentration of 0.01 to 0.05%.

[0021] In some embodiments, the total weight percentage of sucrose and trehalose in the freeze-drying protectant is 10-15% of the formulation. This study also found that a sucrose and trehalose content of 10-15% results in better freeze-drying performance.

[0022] Preferably, in step 1), a lyophilization protectant solution is incorporated when preparing the nucleic acid-containing solution; preferably, the volume percentage of the lyophilization protectant solution in the nucleic acid-containing solution is 45-55%, more preferably 50%. Pre-adding the lyophilization protectant of the present invention to the nucleic acid solution can significantly reduce particle size variation to less than 10 nm; the encapsulation rate is also significantly improved.

[0023] Further preferably, the nucleic acid-containing solution prepared in step 1) and / or the buffer solution containing lipid nanoparticles and lyophilization protectant prepared in step 3) contain 8-9% sucrose, 1.8-2.4% trehalose, and 0.01-0.05% mannitol or 0.01-0.5% poloxamer.

[0024] Preferably, it contains 8-9% sucrose, 1.8-2.4% trehalose, and 0.02-0.05% mannitol or 0.08-0.12% poloxamer by mass.

[0025] For example, it preferably contains 8.8% sucrose, 2.0% trehalose, and 0.04% mannitol or 0.1% poloxamer by mass.

[0026] In this invention, a formulation method with a higher concentration is adopted. While the freeze-drying protection effect is excellent, the stability and immunogenicity of the formulation are also good, which can be better used to prepare freeze-dried formulations of mRNA vaccines.

[0027] In some implementations, in step 3), the solution of the lyophilization protectant uses Tris-HCl buffer as a solvent.

[0028] In some embodiments, the concentration of the Tris-HCl buffer is 30 to 100 mM, for example, it can be 30, 40, 50, 60, 70, 80, 90, 100 mM and any value in between, preferably 50 mM.

[0029] In some implementations, in step 1), citrate buffer is used as a solvent in the lyophilization protectant solution.

[0030] In some embodiments, in step 1), the concentration of citrate buffer used in the lyophilization protectant solution can be 30, 40, 50, 60, 70, 80, 90, or 100 mM, preferably 50 mM.

[0031] In some embodiments, the method for preparing lyophilized nucleic acid lipid nanoparticle formulations uses mRNA as the nucleic acid.

[0032] In this invention, the freeze-drying process typically includes the following steps: First, pre-freezing, where the sample to be treated is rapidly cooled to a temperature below its eutectic point and held for a certain period of time, thereby completely freezing the sample and forming a stable ice crystal structure. The pre-freezing temperature and time are determined based on the characteristics of the sample. Next is main drying, where the pressure is reduced under vacuum, causing the frozen water to sublimate directly from the solid to the gaseous state. At this stage, precise control of temperature and vacuum is required to prevent the sample from overheating or collapsing, in order to effectively remove moisture. Then comes desorption drying, where the temperature is further increased and vacuum is maintained to remove unsublimated bound water from the sample until a dried finished product is obtained.

[0033] In some embodiments, the method for preparing lyophilized nucleic acid lipid nanoparticle formulations includes, in which the lyophilization process comprises:

[0034] 1) After cooling to -60℃ to -40℃, maintain constant temperature freezing for 2 to 5 hours for pre-freezing. Preferably, the temperature is lowered at a rate of 0.5℃ to 1.0℃ per minute until it reaches -45℃.

[0035] 2) Drying at -50℃ to -10℃ for 18 to 36 hours under vacuum conditions; drying at 2℃ to 8℃ for 4 to 10 hours; and drying at 10℃ to 20℃ for 1 to 4 hours.

[0036] Secondly, the present invention provides a lyophilized formulation of nucleic acid lipid nanoparticles prepared by the aforementioned preparation method.

[0037] Thirdly, the present invention provides the application of the aforementioned nucleic acid lipid nanoparticle freeze-dried formulation in the preparation of mRNA vaccines.

[0038] The beneficial effects of the present invention include at least the following:

[0039] (1) The novel lipid nanoparticle formulation of the present invention, after the addition of a lyophilization protectant, can result in minimal changes in particle size and encapsulation rate before and after lyophilization, and is suitable for preparing lyophilized nucleic acid lipid nanoparticle formulations.

[0040] (2) The present invention screened a better freeze-drying protectant formulation. The inventors made a surprising discovery that when the ratio of sucrose to trehalose is controlled at 4.3~4.5:1, the addition of appropriate mannitol or poloxamer can significantly control the particle size variation to about 10nm and the encapsulation rate variation to within 10%.

[0041] (3) This invention proposes a new lyophilization formulation method based on a novel lipid nanoparticle formulation. A lyophilization protectant solution is added to the mRNA stock solution, and an approximately proportional lyophilization protectant solution is added to the prepared mRNA-LNPs stock solution. This can reduce the particle size of the lyophilized formulation, and reduce the range of particle size change before and after lyophilization, as well as the range of encapsulation rate change.

[0042] (4) The respiratory syncytial virus mRNA vaccine prepared based on the freeze-drying process of the present invention rehydrates rapidly, with high total nucleic acid content, high encapsulation rate and high nucleic acid integrity. In addition, the in vivo immune response of the freeze-dried and rehydrated formulation is not significantly different from that of the unfreeze-dried mRNA vaccine. Furthermore, by reducing or even removing the water-containing microenvironment in the system through freeze-drying, the nucleic acid hydrolysis reaction catalyzed by nucleases can be inhibited, which significantly improves the long-term stability of nucleic acid lipid nanoparticles. Attached Figure Description

[0043] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0044] Figure 1 This is a process optimization roadmap for mRNA-LNPs lyophilized formulations provided in this embodiment of the invention.

[0045] Figure 2 The synthesis process of protonable cationic lipid MH-017 provided in the embodiments of the present invention.

[0046] Figure 3 The nuclear magnetic resonance spectrum of the protonable cationic lipid MH-017 provided in this embodiment of the invention.

[0047] Figure 4 Appearance comparison of FM08 in Example 3 and FM16 in Example 4 provided for embodiments of the present invention.

[0048] Figure 5Particle size data of the optimized formulation provided in this embodiment of the invention when stored at 2-8°C.

[0049] Figure 6 Encapsulation rate data of the optimized formulation provided in this embodiment of the invention when stored at 2-8°C.

[0050] Figure 7 Integrity data of the optimized formulation provided in this embodiment of the invention when stored at 2-8°C.

[0051] Figure 8 A comparison of the levels of neutralizing antibodies induced by the optimized formulation and the liquid formulation provided in this embodiment of the invention. Detailed Implementation

[0052] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings, taking the RSV F protein mRNA vaccine as an example. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0053] In this embodiment of the invention, the protonable cationic lipid MH-017 has the following structure:

[0054] .

[0055] The synthesis method and size characterization of the protonable cationic lipid MH-017 are as follows: Figures 2-3 As shown.

[0056] The DSPC is a non-cationic lipid with CAS number 816-94-4, and its structure is shown below:

[0057] .

[0058] The DMG-PEG2000 is a PEG lipid with CAS number 160743-62-4, and its structure is shown below:

[0059] .

[0060] The cholesterol in question has the CAS number 57-88-5 and its structure is shown below:

[0061] .

[0062] In this embodiment of the invention, the preparation steps of the mRNA stock solution are as follows:

[0063] 1) Synthesis of RSV F protein mRNA:

[0064] Nanjing GenScript Biotechnology Co., Ltd. was commissioned to synthesize plasmid DNA using the DNA sequence encoding RSV F protein (SEQ ID No: 1). After preparing a linearized DNA template by enzyme digestion, RSV F mRNA was synthesized according to the following system:

[0065]

[0066] After incubating at 37°C for 3-4 hours, add DNAase I and incubate for another 0.5 hours to complete the reaction.

[0067] 2) Affinity chromatography purification of RSV F protein mRNA:

[0068] The synthesized mRNA solution was purified using a chromatography system. The synthesized mRNA solution was diluted 5-fold with chromatography buffer (500 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, pH=7.0). The OligodT chromatography column was then equilibrated with chromatography buffer to a final volume of 5. Sample loading was performed at a flow rate of 30 cm / h. After loading, the column was washed with washing buffer (10 mM Tris, 50 mM NaCl, 1 mM EDTA, pH=7.0) to remove impurities. After washing until UV A260 and conductivity equilibration, the sample was eluted with water for injection. The elution peak was collected to obtain the stock mRNA solution, and the mRNA concentration was adjusted to 200 μg / ml.

[0069] Example 1

[0070] Step 1: Prepare mRNA stock solution

[0071] The prepared mRNA stock solution was diluted with 50mM citrate buffer at a volume ratio of 1:1 to a concentration of 100μg / ml.

[0072] Step 2: Prepare lipid mixture and mRNA encapsulation

[0073] A mixed solution with a total lipid concentration of 16 mM was prepared by preparing protonable cationic lipids MH017:DSPC:cholesterol:DMG-PEG2000 in a molar ratio of 35:16:46.5:2.5 (solvent anhydrous ethanol).

[0074] The mRNA stock solution prepared in the first step and the prepared lipid mixture were encapsulated on a nanomedicine manufacturing device at a nitrogen / phosphorus ratio (N / P) of 6:1. After ultrafiltration and liquid exchange, the solution was concentrated to obtain RSV F mRNA-LNPs stock solution with an mRNA content of 200 μg / ml.

[0075] Step 3: Prepare RSV F mRNA-LNPs liquid formulation containing lyophilization protectant.

[0076] 1) Preparation of lyophilization protectant solution

[0077] 100 ml of lyophilization protectant solution was prepared using 50 mM Tris-HCl buffer as solvent. The lyophilization protectant solution contained 17.6% sucrose, 4.0% trehalose, and 0.08% mannitol by weight, respectively. The prepared lyophilization protectant solutions were sterilized by filtration through a 0.22 μm sterile filter membrane.

[0078] 2) Preparation of RSV F mRNA-LNPs liquid formulation containing lyophilization protectant

[0079] Take the RSV F mRNA-LNPs stock solution prepared in step 2, add the lyophilization protectant solution at a volume ratio of 1:1 and mix them to make the mRNA content 100 μg / ml, and prepare RSV F mRNA-LNPs liquid formulation containing lyophilization protectant.

[0080] Step 4: Freeze-drying to prepare lyophilized formulations

[0081] The RSV F mRNA-LNPs liquid formulation containing the above-mentioned lyophilization protectant was freeze-dried, and the specific freeze-drying parameters are shown in Table 1.

[0082] Table 1. Freeze-drying process parameter settings

[0083]

[0084] Example 2

[0085] 1) Prepare lyophilization protectant solutions with different components

[0086] 100 ml of lyophilization protectant solution was prepared using 50 mM citrate buffer as solvent. The component contents (weight percentage) of sucrose, trehalose, and mannitol in the lyophilization protectant solution were 17.6%, 4.0%, and 0.08%, respectively. The prepared lyophilization protectant solutions were sterilized by filtration through a 0.22 μm sterile filter membrane.

[0087] 2) Improved RSV F mRNA-LNPs liquid formulation method

[0088] Step 1: Adding a lyophilization protectant to the mRNA stock solution

[0089] The prepared lyophilization protectant solution was incorporated into the prepared mRNA stock solution at a volume ratio of 1:1 to achieve an mRNA content of 100 μg / ml.

[0090] Step 2: Prepare lipid mixture and mRNA encapsulation

[0091] A lipid mixture solution was prepared according to a molar ratio of protonable cationic lipid MH017:DSPC:cholesterol:DMG-PEG2000 of 35:16:46.5:2.5.

[0092] The mRNA stock solution containing the lyophilization protectant solution prepared in the first step was encapsulated with the lipid mixture prepared in the second step at a nitrogen / phosphorus ratio (N / P) of 6:1 on a nanomedicine manufacturing device. After ultrafiltration and liquid exchange, the solution was concentrated to obtain RSV F protein mRNA-LNPs stock solution encapsulated with the lyophilization protectant, with an mRNA content of 200 μg / ml.

[0093] Step 3: Incorporating freeze-drying protectant into the mRNA-LNPs vaccine

[0094] Take the RSV F protein mRNA-LNPs stock solution prepared in the second step and add the lyophilization protectant solution from Example 1 at a volume ratio of 1:1 to make the mRNA content 100 μg / ml, and prepare a modified RSV F mRNA-LNPs liquid formulation.

[0095] 3) Freeze-drying to prepare lyophilized formulations

[0096] Freeze-drying was performed according to the freeze-drying process in Example 1.

[0097] Example 3

[0098] Step 1: Prepare mRNA stock solution

[0099] The prepared mRNA stock solution was diluted with 50mM citrate buffer at a volume ratio of 1:1 to a concentration of 100μg / ml.

[0100] Step 2: Prepare lipid mixture and mRNA encapsulation

[0101] A lipid mixture solution was prepared according to a molar ratio of protonable cationic lipid MH017:DSPC:cholesterol:DMG-PEG2000 of 35:16:46.5:2.5.

[0102] The mRNA stock solution prepared in the first step and the prepared lipid mixture were encapsulated on a nanomedicine manufacturing device at a nitrogen / phosphorus ratio (N / P) of 6:1. After ultrafiltration and liquid exchange, the solution was concentrated to obtain RSV F mRNA-LNPs stock solution with an mRNA content of 200 μg / ml.

[0103] Step 3: Prepare a liquid formulation of RSV F protein mRNA-LNPs containing a lyophilization protectant.

[0104] 1) Prepare lyophilization protectant solutions with different components

[0105] Using 50mM Tris-HCl buffer as solvent, prepare 100ml of each lyophilization protectant solution containing different components such as sucrose, trehalose, mannitol or poloxamer. The content (weight percentage) of each component in each lyophilization protectant solution is shown in Table 2. The prepared lyophilization protectant solutions are sterilized by filtration through a 0.22μm sterile filter membrane.

[0106] Table 2 Different lyophilization protectant solutions

[0107]

[0108] 2) Preparation of RSV F mRNA-LNPs liquid formulation containing lyophilization protectant

[0109] Take the RSV F mRNA-LNPs stock solution prepared in step 2, and add the lyophilization protectant solution in Table 2 at a volume ratio of 1:1 to mix them so that the mRNA content is 100 μg / ml, and prepare RSV F protein mRNA-LNPs liquid formulation containing lyophilization protectant, as shown in Table 3.

[0110] Table 3. Liquid formulations of RSV F protein mRNA-LNPs containing lyophilization protectants

[0111]

[0112] Step 4: Freeze-drying to prepare lyophilized formulations

[0113] The RSV F mRNA-LNPs liquid formulation containing the above-mentioned lyophilization protectant was freeze-dried, and the specific freeze-drying parameters are shown in Table 4.

[0114] Table 4 Freeze-drying process parameter settings

[0115]

[0116] After lyophilization, the product was collected, reconstituted with an equal volume of the original reconstitution solvent, and processed using Ribo Green. TMThe encapsulation efficiency of nanoparticles was determined, and the particle size and PDI potential of nanoparticles were measured using a Malvern NS90Z nanoparticle size and potential analyzer. The experimental results are shown in Table 5.

[0117] Table 5 Changes in key indicators of mRNA-LNPs before and after freeze-drying

[0118]

[0119] Based on the data above, compared with formulations FM01, FM02 and FM03, the addition of 0.04% mannitol or 0.1% poloxamer to the freeze-drying protectant can reduce the changes in particle size and encapsulation rate before and after freeze-drying. When sucrose and trehalose are used as freeze-drying 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.

[0120] Example 4: Suitability evaluation of the improved formulation method for RSV F mRNA-LNPs lyophilized formulation

[0121] 1) Prepare lyophilization protectant solutions with different components

[0122] Using 50 mM citrate buffer as solvent, 100 ml of different formulations of lyophilization protectants containing sucrose, trehalose, and either mannitol or poloxamer were prepared. The content (weight percentage) of each component in the lyophilization protectant solution is shown in Table 6. Each lyophilization protectant solution was sterilized by filtration through a 0.22 μm sterile filter membrane.

[0123] Table 6 Different lyophilization protectant solutions

[0124]

[0125] 2) Improved RSV F mRNA-LNPs liquid formulation method

[0126] Step 1: Adding a lyophilization protectant to the mRNA stock solution

[0127] The freeze-drying protectant solutions listed in Table 6 above were added to the prepared mRNA stock solution at a volume ratio of 1:1 to make the mRNA content 100 μg / ml.

[0128] Step 2: Prepare lipid mixture and mRNA encapsulation

[0129] A lipid mixture solution was prepared according to a molar ratio of protonable cationic lipid MH017:DSPC:cholesterol:DMG-PEG2000 of 35:16:46.5:2.5.

[0130] The mRNA stock solution containing the lyophilization protectant solution prepared in the first step was encapsulated with the lipid mixture prepared in the second step at a nitrogen / phosphorus ratio (N / P) of 6:1 on a nanomedicine manufacturing device. After ultrafiltration and liquid exchange, the solution was concentrated to obtain RSV F protein mRNA-LNPs stock solution encapsulated with the lyophilization protectant, with an mRNA content of 200 μg / ml.

[0131] Step 3: Incorporating freeze-drying protectant into the mRNA-LNPs vaccine

[0132] Take the RSV F mRNA-LNPs stock solution prepared in the second step and add the freeze-drying protectant solutions LP04~LP09 from Table 2 of Example 3 at a volume ratio of 1:1 to make the mRNA content 100 μg / ml, and prepare the modified RSV F protein mRNA-LNPs liquid formulation as shown in Table 7.

[0133] Table 7. Modified RSV F protein mRNA-LNPs liquid formulation

[0134]

[0135] 3) Freeze-drying to prepare lyophilized formulations

[0136] The freeze-drying process was performed according to Example 1. After drying, the product was collected and reconstituted with Ribo Green using an equal volume of ultrapure water as before freeze-drying. TM The encapsulation efficiency of nanoparticles was determined, and the particle size and PDI potential of nanoparticles were measured using a Malvern NS90Z nanoparticle size and potential analyzer. The experimental results are shown in Table 8.

[0137] Table 8 Changes in key indicators of mRNA-LNPs before and after freeze-drying

[0138]

[0139] The optimized process route for preparing the mRNA-LNPs lyophilized formulation provided in this embodiment is different from the unoptimized process route in Example 3. Figure 1 As shown in the above data, compared to lyophilizing 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 can further reduce the particle size of the nucleic acid lipid nanoparticles before and after lyophilization. The variation range is significantly smaller than that of the preparation method in Example 3, and the particle size change can be controlled within about 10 nm. Furthermore, the decreasing trend of the encapsulation rate is smaller, with a significantly smaller variation range than that of the preparation method in Example 3, and the encapsulation rate is not less than 80%. The particle size changes of formulations FM16 and FM17 are even smaller. The appearance comparison before lyophilization process optimization (lyophilized formulation FM08 in Example 3) and after lyophilization (lyophilized formulation FM16 in Example 4) is shown below. Figure 4 As shown.

[0140] Example 5: Stability evaluation of lyophilized formulation

[0141] Long-term stability storage conditions: 2-8℃, 6 months. The particle size, encapsulation efficiency, and mRNA integrity of formulations FM16 and FM17 from Example 4 were determined after 0, 1, 3, and 6 months of storage at 2-8℃. The data are shown in Table 9, and trend analysis is provided below. Figures 5 to 7 .

[0142] Table 9. Results of long-term storage stability evaluation of formulations FM16 and FM17 at 2-8℃

[0143]

[0144] Example 6 Immunogenicity evaluation of lyophilized formulation

[0145] BALB / c mice were used in a comparative study with the lyophilized formulations FM16 and FM17 and the liquid formulations described in Example 4. Mice were randomly divided into two groups and injected intramuscularly with 10 μg of each formulation. A second immunization with the same dose was administered 21 days after the initial immunization. Serum was collected 35 days after the initial immunization to detect the level of neutralizing antibodies against RSV. Results are as follows: Figure 8 As shown, the level of neutralizing antibodies induced by the optimized lyophilized formulation was not significantly different from that of the liquid formulation.

[0146] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a lyophilized formulation of nucleic acid lipid nanoparticles, characterized in that, include: 1) Prepare a solution containing nucleic acids; 2) Preparation of lipid nanoparticles containing nucleic acids; 3) Prepare a buffer solution containing lipid nanoparticles and a lyophilization protectant; 4) Freeze-dry the buffer solution containing lipid nanoparticles and a freeze-drying protectant; The nucleic acid-containing solution prepared in step 1) and the buffer solution containing lipid nanoparticles and lyophilization protectant prepared in step 3) contain 8-9% sucrose, 1.8-2.4% trehalose, and 0.02-0.05% mannitol or 0.08-0.12% poloxamer; the nucleic acid is mRNA. The freeze-drying includes: 1) After cooling to -60℃ to -40℃, maintain constant temperature freezing for 2 to 5 hours for pre-freezing, and cool down at a rate of 0.5℃ to 1.0℃ per minute until -45℃ is reached; 2) Drying at -50℃ to -10℃ for 18 to 36 hours under vacuum conditions; drying at 2℃ to 8℃ for 4 to 10 hours; and drying at 10℃ to 20℃ for 1 to 4 hours. The lipid nanoparticles contain protonable cationic lipids, DSPC, cholesterol, and DMG-PEG2000; the molar ratio of the protonable cationic lipids:DSPC:cholesterol:DMG-PEG2000 is 35:14~18:45~48:2~3; the structural formula of the protonable cationic lipids is shown in Formula 1. Formula 1.

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

5.

3. The method for preparing the lyophilized formulation of nucleic acid lipid nanoparticles according to claim 1, characterized in that, The mass ratio of sucrose to trehalose in the freeze-drying protectant is 4.2~4.8:

1.

4. The method for preparing the lyophilized formulation of nucleic acid lipid nanoparticles according to claim 3, characterized in that, The mass ratio of sucrose to trehalose in the freeze-drying protectant is 4.3~4.5:

1.

5. The method for preparing the lyophilized formulation of nucleic acid lipid nanoparticles according to claim 4, characterized in that, The total weight percentage of sucrose and trehalose in the freeze-drying protectant is 10-15% of the formulation.

6. The method for preparing the lyophilized formulation of nucleic acid lipid nanoparticles according to claim 1, characterized in that, In step 1), the volume percentage of the lyophilization protectant solution in the nucleic acid-containing solution is 45-55%.

7. The method for preparing the lyophilized formulation of nucleic acid lipid nanoparticles according to claim 6, characterized in that, In step 1), the volume percentage of the lyophilization protectant solution in the nucleic acid-containing solution is 50%.

8. The method for preparing the lyophilized formulation of nucleic acid lipid nanoparticles according to claims 1-7, characterized in that, The freeze-drying includes: 1) After cooling to -60℃ to -40℃, maintain constant temperature freezing for 2 to 5 hours for pre-freezing, and cool down at a rate of 0.5℃ to 1.0℃ per minute until -45℃ is reached; 2) Drying at -50℃ to -10℃ for 18 to 36 hours under vacuum conditions; drying at 2℃ to 8℃ for 4 to 10 hours; and drying at 10℃ to 20℃ for 1 to 4 hours.

9. The lyophilized formulation of nucleic acid lipid nanoparticles prepared by the preparation method according to any one of claims 1-8.

10. The use of the lyophilized nucleic acid lipid nanoparticle formulation of claim 9 in the preparation of mRNA vaccines.

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