MRNA-LNP preparation and freeze-drying method thereof

Abstract

The invention discloses an mRNA-LNP preparation and a freeze-drying method thereof.The freeze-drying method comprises the following steps that mRNA-LNP nanoparticles and a freeze-drying protective agent composition are taken and mixed, a mixture is obtained, the mixture is pre-frozen, and a frozen body is obtained; under the condition that the temperature is lower than the eutectic point temperature of the frozen body, sublimating and drying the frozen body to obtain an initial product; and carrying out desorption drying on the initial product to obtain the mRNA-LNP preparation. According to the method, the mRNA-LNP sample with stable nano particle size, quite high nucleic acid encapsulation efficiency and biological activity is prepared by adopting ultralow-temperature pre-freezing, slow desorption drying and a low-temperature end-point freeze-drying curve, and the mRNA-LNP sample can be stored for a long time at normal temperature.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an mRNA-LNP preparation and its freeze-drying method. Background Technology

[0002] mRNA-LNP vaccines, as a new generation of biomedical technology, have shown great potential in areas such as infectious disease prevention and cancer treatment. However, their stability has always been a technical bottleneck restricting their large-scale clinical application. Existing commercially available mRNA-LNP vaccines typically require storage and transportation at ultra-low temperatures of -60°C or even -80°C, which not only significantly increases costs but also limits their widespread application in resource-constrained areas.

[0003] Freeze-drying technology is an effective means of improving the stability of biological agents by removing moisture to reduce hydrolysis and other degradation reactions. Currently, research has explored the application of freeze-drying technology to mRNA-LNP vaccines. For example, Regeneron Biotech developed the world's first freeze-dried mRNA COVID-19 vaccine, which can be stably stored at 4°C and 25°C. Moderna has also converted its cytomegalovirus vaccine mRNA-1647 from a liquid formulation to a freeze-dried formulation, which has been reported to be stable for 18 months under refrigeration.

[0004] However, existing mRNA-LNP lyophilization technologies still have significant limitations. Traditional lyophilization processes typically use pre-freezing at -45°C or below for about 3 hours, which is insufficient for protecting the thermally unstable LNP structure. More importantly, existing technologies often use high concentrations of lyophilization protectants (such as a 17% sugar combination), which, while stabilizing the LNP structure to some extent, increases osmotic pressure, potentially affecting in vivo transfection efficiency. It also prolongs the lyophilization time (usually 40-100 hours), increasing energy consumption and production costs.

[0005] Studies have shown that the stability bottleneck of the mRNA-LNP system mainly lies in the mRNA molecule itself. The long, single-stranded mRNA chain makes it more susceptible to hydrolysis and degradation than smaller, double-stranded RNA molecules (such as siRNA). Although the internal environment of the LNP can protect the mRNA to some extent, phase transitions and ice crystal formation during freeze-drying can still lead to mRNA damage and LNP structural disruption. Summary of the Invention

[0006] The purpose of this invention is to provide an mRNA-LNP formulation and its freeze-drying method. This freeze-drying method can effectively protect the structural integrity of mRNA-LNP, while improving production efficiency and reducing costs. The prepared mRNA-LNP formulation has stable nanoparticle size, high nucleic acid encapsulation rate and high biological activity. It can be stored at room temperature for a long time and exhibits good stability at 37°C, which can fully meet the requirements of conventional cold chain storage and transportation.

[0007] To achieve the above objectives, a specific embodiment of the present invention provides the following technical solution:

[0008] A lyophilization method for an mRNA-LNP formulation, the lyophilization method comprising the following steps:

[0009] Mix the mRNA-LNP nanoparticles and the lyophilization protectant composition to obtain a mixture:

[0010] The mixture is pre-frozen to obtain a frozen body;

[0011] The frozen body was sublimated and dried under conditions below the eutectic point temperature to obtain the initial product.

[0012] The initial product was analyzed and dried to obtain the mRNA-LNP formulation.

[0013] In one or more embodiments of the present invention, the sublimation drying step is carried out at a temperature 10°C-15°C below the eutectic point temperature of the frozen body, the sublimation drying time is 24h-36h, and the vacuum degree is 1Pa-30Pa.

[0014] In one or more embodiments of the present invention, the pre-freezing step is performed at a temperature of -60°C to -80°C for 6-8 hours.

[0015] In one or more embodiments of the present invention, the cooling rate in the pre-freezing step is 2℃ / min-5℃ / min.

[0016] In one or more embodiments of the present invention, the temperature in the analytical drying step is 20℃-25℃, the time is 12h-20h, and the vacuum degree is 1Pa-10Pa.

[0017] In one or more embodiments of the present invention, in the analytical drying step, the temperature is increased to 20°C-25°C at a heating rate of less than or equal to 0.15°C / min.

[0018] In one or more embodiments of the present invention, the lyophilization protectant composition comprises a sugar, an osmotic pressure maintainer, and a buffer solution, wherein the concentration of the sugar is 4%-10% (w / v) and the concentration of the osmotic pressure maintainer is 0.1%-1% (w / v).

[0019] In one or more embodiments of the present invention, the carbohydrate is at least one selected from sucrose and galactose; and / or,

[0020] The osmotic pressure maintaining agent is calcium chloride; and / or,

[0021] The buffer solution is a Tris buffer with a concentration of 10mM-20mM and a pH of 7.2-7.6.

[0022] Another specific embodiment of the present invention provides the following technical solution:

[0023] An mRNA-LNP formulation was prepared by the above-described method.

[0024] In one or more embodiments of the present invention, the mRNA-LNP formulation has a particle size of 65nm-95nm, a PDI < 0.3, and a potential of -10 to 10mV.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] 1. Significantly improved stability: The freeze-dried mRNA-LNP vaccine can be stably stored for at least 18 months at 2-8℃. After reconstitution, the particle size is 65-95, PDI < 0.3, potential is -10 to 10mV, and encapsulation efficiency is > 90%.

[0027] 2. Significantly improved production efficiency: The total freeze-drying time is shortened from 40-100 hours in the traditional process to 40-65 hours, and energy consumption is reduced by more than 30%.

[0028] 3. Better preservation of biological activity: In vitro experiments show that the transfection efficiency of lyophilized and reconstituted mRNA-LNP is stable, and animal experiments show that the immunogenicity is comparable to that of the fresh liquid formulation.

[0029] 4. Significantly reduced storage and transportation costs: It enables long-term stable storage under conventional refrigeration conditions (2-8℃), eliminating the dependence on ultra-low temperature cold chain. Detailed Implementation

[0030] To enable those skilled in the art to better understand the technical solutions in this disclosure, the technical solutions in the embodiments of this disclosure are described clearly and completely below. Obviously, the described embodiments are only some embodiments of this disclosure, and not all embodiments. Based on the embodiments in this disclosure, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this disclosure.

[0031] Existing lyophilization methods use high concentrations of cryoprotectants to prevent particle breakage and aggregation during freezing. While this protects the sample's particle size and encapsulation efficiency, it increases the sample's osmotic pressure, leading to reduced in vivo transfection efficiency. Furthermore, the excessively high content of excipients used in lyophilization results in long freezing times and increased energy consumption. Additionally, while the encapsulation efficiency of samples obtained from lyophilized reconstitution is acceptable (around 80%), the particle size is relatively high (160 nm), which is unfavorable for cell and animal experiments. Current research indicates that standard cryoprotectants (such as sugars) cannot reliably stabilize lipid nanoparticles or microparticles during freezing.

[0032] This invention employs ultra-low temperature pre-freezing + slow desorption drying + low-temperature endpoint freeze-drying curve to prepare samples with stable nanoparticle size, high nucleic acid encapsulation efficiency, and high bioactivity, which can be stored at room temperature for extended periods, thus solving the technical problems of poor stability and demanding storage conditions of mRNA-LNP vaccines. The freeze-drying method for mRNA-LNP formulations in this invention overcomes the bottleneck of LNP freeze-drying technology: by combining ultra-low temperature pre-freezing and slow desorption drying, the technical challenge of easy structural damage to mRNA-LNP during freeze-drying is solved; low-temperature storage is achieved: enabling mRNA-LNP vaccines to be stored for extended periods under conventional refrigeration conditions of 2-8℃, eliminating dependence on ultra-low temperature cold chains below -60℃; and production efficiency is improved: reducing the total freeze-drying time from 40-100 hours to 40-65 hours.

[0033] A specific embodiment of the present invention provides a lyophilization method for mRNA-LNP formulations, which specifically includes the following steps:

[0034] Step 1: Mix the mRNA-LNP nanoparticles and the lyophilization protectant composition to obtain a mixture.

[0035] Specifically, the lyophilization protectant composition includes a sugar, an osmotic pressure maintainer, and a buffer solution. The concentration of the sugar is 4%-10% (w / v), and the sugar is a monosaccharide, specifically at least one of sucrose and galactose. The concentration of the osmotic pressure maintainer is 0.1%-1% (w / v), specifically calcium chloride. The buffer solution is a 10mM-20mM tris(hydroxymethyl)aminomethane buffer (Tris buffer) with a pH of 7.2-7.6.

[0036] The lyophilization protectant composition used in this invention has a significantly lower concentration of sugar substances compared to the 17%-20% high concentration of sugar protectants in the prior art, which alleviates the osmotic pressure problem. Combined with component optimization, it effectively ensures its protective effect on mRNA-LNP nanoparticles.

[0037] Step 2: Pre-freeze the mixture to obtain a frozen body.

[0038] Specifically, in this step, the temperature is lowered to -60℃ to -80℃ at a rate of 2℃ / min to 5℃ / min, and pre-frozen for 6-8 hours. Pre-freezing the mixture in an ultra-low temperature environment ensures complete freezing of the mixture through lower temperatures and longer pre-freezing times, forming a uniform and fine ice crystal structure, which lays the foundation for the subsequent sublimation and drying steps.

[0039] Step 3: Under conditions below the eutectic point temperature of the frozen body, the frozen body is sublimated and dried to obtain the initial product.

[0040] Specifically, sublimation drying is performed at temperatures below the eutectic point of the frozen body to avoid the destruction of the LNP structure caused by the melting of ice crystals. The eutectic point of the frozen body is around -26℃, and sublimation drying is carried out at temperatures 10℃-15℃ below -26℃, such as -36℃, -39℃, and -41℃, for a duration of 24-36 hours and a vacuum level of 1Pa-30Pa.

[0041] Step 4: The initial product is desorbed and dried to obtain the mRNA-LNP formulation.

[0042] Specifically, in this step, the temperature is increased to 20℃-25℃ at a heating rate of less than or equal to 0.15℃ / min, and the desorption and drying time is 12h-20h, with a vacuum degree of 1Pa-10Pa. The heating rate of ≤0.15℃ / min in this step is 1 / 3 to 1 / 2 of that in traditional processes, and the final desorption and drying temperature does not exceed 25℃, which is lower than the 30℃ required for traditional virus and protein freeze-drying. By controlling the slow heating and lower temperature conditions, the heat-sensitive mRNA and liposome structures can be effectively protected.

[0043] Another specific embodiment of the present invention provides an mRNA-LNP formulation obtained by the above preparation method.

[0044] Specifically, the mRNA-LNP formulation prepared by the above method has a particle size of 65nm-95nm, can be stably stored for at least 18 months under 2℃-8℃ conditions, and after reconstitution, the particle size is 65nm-95nm, PDI < 0.3, potential -10mV to 10mV, and encapsulation efficiency > 90%.

[0045] The present invention will be further described in detail below with reference to specific embodiments.

[0046] The preparation of the mRNA-LNP nanoparticles used in the embodiments and comparative examples of this invention is as follows:

[0047] Respiratory syncytial virus mRNA (manufacturer: Aimei Explorer Life Science R&D Co., Ltd., product code mRNA-2307) and varicella-zoster virus mRNA (manufacturer: Aimei Explorer Life Science R&D Co., Ltd., product code mRNA-2308) were processed to prepare 2307-mRNA-LNP nanoparticle solutions and 2308-mRNA-LNP nanoparticle solutions, respectively: 2 mg of mRNA was dissolved in 15 mL of sodium acetate buffer with a pH of 4.7, designated as phase A. The lipid compounds were dissolved in 5 mL of anhydrous ethanol, with a total concentration of 10 mg / mL, designated as phase B. The types and molar ratios of lipid compounds in phase B were D-Lin-MC3-DMA (1,2-dioleoyloxy-3-dimethylaminopropane):DSPC (distearate phosphatidylcholine):cholesterol:mPEG-DMG-2K (methoxy-polyethylene glycol-dimyristoyl-glycerol) = 50:10:38.5:1.5.

[0048] A microfluidic approach was used, with a total flow rate of 20 mL / min and a flow rate ratio of 3:1 for phase A and phase B, which were then mixed to form nanoparticles. The prepared nanoparticle solution was purified using tangential flow technology with 20 mM Tris-HCl-calcium chloride buffer (calcium chloride concentration 0.1% (w / v), pH 7.4). Finally, the solution was filtered through a 0.22 μm aqueous filter membrane to obtain an mRNA-LNP nanoparticle solution with a concentration of 1 mg / mL.

[0049] Example 1

[0050] The specific lyophilization method for the mRNA-LNP formulation in this embodiment is as follows:

[0051] The 2307-mRNA-LNP nanoparticle solution, sucrose, calcium chloride, and 20 mM Tris buffer were mixed to obtain a mixture sample to be lyophilized, with a sucrose concentration of 8% (w / v), a calcium chloride concentration of 0.1% (w / v), and a mRNA-LNP nanoparticle concentration of 100 μg / ml.

[0052] The mixture sample was placed in a freeze dryer at room temperature and cooled to -60°C at a rate of 2°C / min for 360 min. Then, it was heated to -38°C over 30 min and subjected to sublimation drying for 2040 min, maintaining a vacuum of 5 Pa. Next, it was heated to 23°C over 720 min and subjected to desorption drying for 240 min, maintaining a vacuum of 5 Pa. The resulting mRNA-LNP formulation was obtained.

[0053] In this embodiment, the particle size, PDI, potential, and encapsulation efficiency of the mRNA-LNP nanoparticles before and after freeze-drying are shown in Table 1.

[0054] Table 1. Particle size, PDI, potential, and encapsulation efficiency of mRNA-LNP nanoparticles before and after freeze-drying in Example 1.

[0055]

[0056] Example 2

[0057] The specific lyophilization method for the mRNA-LNP formulation in this embodiment is as follows:

[0058] The 2308-mRNA-LNP nanoparticle solution, sucrose, calcium chloride, and 20 mM Tris buffer were mixed to obtain a sucrose concentration of 8% (w / v), a calcium chloride concentration of 0.1% (w / v), and a mRNA-LNP nanoparticle concentration of 100 μg / ml, thus obtaining the mixture sample to be lyophilized.

[0059] The mixture sample was placed in a freeze dryer at room temperature and cooled to -65°C at a rate of 3°C / min for 420 min. Then, it was heated to -38°C over 30 min and subjected to sublimation drying for 1680 min under a vacuum of 5 Pa. Next, it was heated to 20°C over 600 min and subjected to desorption drying for 300 min under a vacuum of 5 Pa. The resulting mRNA-LNP formulation was obtained.

[0060] In this embodiment, the particle size, PDI, potential, and encapsulation efficiency of the mRNA-LNP nanoparticles before and after freeze-drying are shown in Table 2.

[0061] Table 2. Particle size, PDI, potential, and encapsulation efficiency of mRNA-LNP nanoparticles before and after freeze-drying in Example 2.

[0062]

[0063] Example 3

[0064] The specific lyophilization method for the mRNA-LNP formulation in this embodiment is as follows:

[0065] The 2308-mRNA-LNP nanoparticle solution, sucrose, calcium chloride, and 20 mM Tris buffer were mixed to obtain a sucrose concentration of 8% (w / v), a calcium chloride concentration of 0.1% (w / v), and a mRNA-LNP nanoparticle concentration of 100 μg / ml, thus obtaining the mixture sample to be lyophilized.

[0066] The mixture sample was placed in a freeze dryer at room temperature and cooled to -70°C at a rate of 5°C / min for 480 min. Then, it was heated to -40°C over 30 min and subjected to sublimation drying for 2160 min under a vacuum of 20 Pa. Next, it was heated to 25°C over 720 min and subjected to desorption drying for 360 min under a vacuum of 10 Pa. The resulting mRNA-LNP formulation was obtained.

[0067] In this embodiment, the particle size, PDI, potential, and encapsulation efficiency of the mRNA-LNP nanoparticles before and after freeze-drying are shown in Table 3.

[0068] Table 3. Particle size, PDI, potential, and encapsulation efficiency of mRNA-LNP nanoparticles before and after freeze-drying in Example 3.

[0069]

[0070] Example 4

[0071] The specific lyophilization method for the mRNA-LNP formulation in this embodiment is as follows:

[0072] The 2308-mRNA-LNP nanoparticle solution, sucrose, calcium chloride, and 20 mM Tris buffer were mixed to obtain a sucrose concentration of 8% (w / v), a calcium chloride concentration of 0.1% (w / v), and a mRNA-LNP nanoparticle concentration of 100 μg / ml, thus obtaining the mixture sample to be lyophilized.

[0073] The mixture sample was placed in a freeze dryer at room temperature and cooled to -80°C at a rate of 5°C / min for 420 min. Then, it was heated to -41°C over 30 min and subjected to sublimation drying for 2160 min, maintaining a vacuum of 30 Pa. Next, it was heated to 22°C over 500 min and subjected to desorption drying for 300 min, maintaining a vacuum of 10 Pa. The resulting mRNA-LNP formulation was obtained.

[0074] In this embodiment, the particle size, PDI, potential, and encapsulation efficiency of the mRNA-LNP nanoparticles before and after freeze-drying are shown in Table 4.

[0075] Table 4. Particle size, PDI, potential, and encapsulation efficiency of mRNA-LNP nanoparticles before and after freeze-drying in Example 4.

[0076]

[0077] Example 5

[0078] The specific lyophilization method for the mRNA-LNP formulation in this embodiment is as follows:

[0079] The 2308-mRNA-LNP nanoparticle solution, sucrose, calcium chloride, and 20 mM Tris buffer were mixed to obtain a sucrose concentration of 8% (w / v), a calcium chloride concentration of 0.1% (w / v), and a mRNA-LNP nanoparticle concentration of 100 μg / ml, thus obtaining the mixture sample to be lyophilized.

[0080] The mixture sample was placed in a freeze dryer at room temperature and cooled to -70°C at a rate of 5°C / min for 450 min. Then, it was heated to -40°C over 30 min and subjected to sublimation drying for 2100 min under a vacuum of 20 Pa. Finally, it was heated to 25°C over 720 min and subjected to desorption drying for 350 min under a vacuum of 10 Pa. The resulting mRNA-LNP formulation was obtained.

[0081] Comparative Example 1

[0082] The specific lyophilization method for the mRNA-LNP formulation in this comparative example is as follows:

[0083] The 2308-mRNA-LNP nanoparticle solution, sucrose, calcium chloride, and 20mM Tris buffer were mixed to obtain a sucrose concentration of 8% (w / v), a calcium chloride concentration of 0.1% (w / v), and a mRNA-LNP nanoparticle concentration of 100ug / ml, thus obtaining the mixture sample to be lyophilized.

[0084] The mixture sample was placed in a freeze dryer at -40°C for 150 min. Then, the temperature was raised to -38°C over 30 min for sublimation drying for 1680 min, maintaining a vacuum of 5 Pa. Next, the temperature was raised to 28°C over 60 min for desorption drying for 800 min, maintaining a vacuum of 5 Pa. The resulting mRNA-LNP formulation was obtained.

[0085] Table 5 shows the particle size, PDI, potential, and encapsulation efficiency of the mRNA-LNP nanoparticles before and after freeze-drying in this comparative example.

[0086] Table 5. Particle size, PDI, potential, and encapsulation efficiency of mRNA-LNP nanoparticles before and after lyophilization in Comparative Example 1.

[0087]

[0088] Comparative Example 2

[0089] The specific lyophilization method for the mRNA-LNP formulation in this comparative example is as follows:

[0090] The 2308-mRNA-LNP nanoparticle solution, sucrose, calcium chloride, and 20mM Tris buffer were mixed to obtain a sucrose concentration of 8% (w / v), a calcium chloride concentration of 0.1% (w / v), and a mRNA-LNP nanoparticle concentration of 100ug / ml, thus obtaining the mixture sample to be lyophilized.

[0091] The mixture sample was placed in a freeze dryer at room temperature and cooled to -65°C at a rate of 3°C / min for 420 min. Then, it was heated to -38°C over 30 min and subjected to sublimation drying for 1680 min, maintaining a vacuum of 5 Pa. Next, it was heated to 20°C over 60 min and subjected to desorption drying for 300 min, maintaining a vacuum of 5 Pa. The resulting mRNA-LNP formulation was obtained.

[0092] Table 6 shows the particle size, PDI, potential, and encapsulation efficiency of the mRNA-LNP nanoparticles before and after freeze-drying in this comparative example.

[0093] Table 6. Particle size, PDI, potential, and encapsulation efficiency of mRNA-LNP nanoparticles before and after lyophilization in Comparative Example 2.

[0094]

[0095] Comparative Example 3

[0096] The specific lyophilization method for the mRNA-LNP formulation in this comparative example is as follows:

[0097] The 2308-mRNA-LNP nanoparticle solution, sucrose, calcium chloride, and 20mM Tris buffer were mixed to obtain a sucrose concentration of 8% (w / v), a calcium chloride concentration of 0.1% (w / v), and a mRNA-LNP nanoparticle concentration of 100ug / ml, thus obtaining the mixture sample to be lyophilized.

[0098] The mixture sample was placed in a freeze dryer at room temperature and cooled to -65°C at a rate of 3°C / min for 420 min. Then, it was heated to -38°C over 30 min and subjected to sublimation drying for 1680 min, maintaining a vacuum of 5 Pa. Next, it was heated to 35°C over 600 min and subjected to desorption drying for 300 min, maintaining a vacuum of 5 Pa. The resulting mRNA-LNP formulation was obtained.

[0099] Table 7 shows the particle size, PDI, potential, and encapsulation efficiency of the mRNA-LNP nanoparticles before and after freeze-drying in this comparative example.

[0100] Table 7. Particle size, PDI, potential, and encapsulation efficiency of mRNA-LNP nanoparticles before and after lyophilization in Comparative Example 3.

[0101]

[0102] As can be seen from the above, in the embodiments of the present invention, all indicators of the mRNA-LNP nanoparticles before and after freeze-drying are within the quality standard range (quality standard: particle size 65nm-95nm, PDI < 0.3, potential -10 to 10mV, encapsulation efficiency > 90%). In contrast, in the comparative examples, the encapsulation efficiency decreased by more than or equal to 7% after freeze-drying, and the LNP encapsulation efficiency was less than 90%, which is outside the quality standard range. Furthermore, in the embodiments, the encapsulation efficiency decreased by less than or equal to 4% after freeze-drying, resulting in better product quality indicators compared to the comparative examples. The final residual moisture content of the mRNA-LNP freeze-dried formulation in the embodiments of the present invention was reduced to below 1.5%, and the freeze-dried product was a white, loose cake-like substance.

[0103] The mRNA-LNP formulations in each example were reconstituted using sterile water for injection. The results showed that the particle size of the reconstituted mRNA-LNP nanoparticles was in the range of 65nm-95nm, the reconstitution time was <10 seconds, the PDI was <0.3, the potential was -10 to 10mV, and the encapsulation efficiency was >90%, all of which met the quality standard range.

[0104] In addition, the in vitro biological activity of the mRNA vaccine was evaluated by in vitro cell transfection assay (ELISA method), that is, by the expression of protein on HEK293 cells. The mixture sample before lyophilization and the product after lyophilization and reconstitution were tested respectively, and the results are shown in Table 8.

[0105] Table 8 Results of in vitro cell transfection experiments

[0106]

[0107] As can be seen from Table 8, the protein expression efficiency of the mRNA-LNP formulation after reconstitution in the embodiments of the present invention is not significantly different from that of the original solution before lyophilization.

[0108] The mixture sample before lyophilization (liquid dosage form) and the lyophilized mRNA-LNP formulation (lyophilized dosage form) were taken and placed at 37℃ for 3 days and 7 days respectively for accelerated stability testing. Various indicators of mRNA-LNP nanoparticles were measured. The results are shown in Tables 9 and 10.

[0109] Table 9 Accelerated stability tests of lyophilized formulations in Examples 1, 3, and 4

[0110]

[0111] Table 10 Accelerated stability tests of liquid dosage forms in Examples 2 and 5

[0112]

[0113] As can be seen from Tables 9 and 10, the protein concentration of liquid formulation samples decreased to varying degrees after accelerated testing, while the protein concentration of lyophilized formulation samples remained stable (within a certain range) with minimal changes after accelerated testing. Therefore, lyophilized formulations have an advantage over liquid formulations during long-term storage.

[0114] In summary, the lyophilization method for mRNA-LNP formulations provided in this invention has the following advantages:

[0115] 1. Special freeze-drying method: This invention forms a stable freeze-drying protectant system by using appropriate freeze-drying protectants and freeze-drying curves. This system can more effectively protect the structure and activity of mRNA and liposomes during the freeze-drying process, thereby improving the stability of mRNA-LNP vaccines.

[0116] 2. Extended shelf life: The lyophilization protectant in this invention can improve the stability of mRNA-LNP, thus extending its shelf life, making it easier to store and transport, and significantly improving the convenience and efficiency of its application.

[0117] 3. Improved bioavailability: The freeze-drying protectant system of the present invention can not only protect the structure and activity of mRNA and liposomes during the freeze-drying process, but also avoid affecting their activity, thereby improving their bioavailability.

[0118] 4. Improved reproducibility: The optimization of the freeze-drying protectant system of this invention is based on scientific theoretical basis, which can ensure the stability and reproducibility of the protective effect, and is of great significance for large-scale production and clinical application.

[0119] It will be apparent to those skilled in the art that this disclosure is not limited to the details of the exemplary embodiments described above, and that this disclosure can be implemented in other specific forms without departing from the spirit or essential characteristics of this disclosure. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of this disclosure is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within this disclosure.

[0120] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A lyophilization method for an mRNA-LNP formulation, characterized in that, The freeze-drying method includes the following steps: Mix the mRNA-LNP nanoparticles and the lyophilization protectant composition to obtain a mixture: The mixture is pre-frozen to obtain a frozen body; The frozen body was sublimated and dried under conditions below the eutectic point temperature to obtain the initial product. The initial product was analyzed and dried to obtain the mRNA-LNP formulation.

2. The lyophilization method for the mRNA-LNP formulation according to claim 1, characterized in that, In the sublimation drying step, sublimation drying is carried out at a temperature 10℃-15℃ lower than the eutectic point temperature of the frozen body, the sublimation drying time is 24h-36h, and the vacuum degree is 1Pa-30Pa.

3. The lyophilization method for the mRNA-LNP formulation according to claim 1, characterized in that, In the pre-freezing step, the temperature is -60℃ to -80℃ and the time is 6h to 8h.

4. The lyophilization method for the mRNA-LNP formulation according to claim 3, characterized in that, In the pre-freezing step, the cooling rate is 2℃ / min-5℃ / min.

5. The lyophilization method for the mRNA-LNP formulation according to claim 1, characterized in that, In the analytical drying step, the temperature is 20℃-25℃, the time is 12h-20h, and the vacuum degree is 1Pa-10Pa.

6. The lyophilization method for the mRNA-LNP formulation according to claim 5, characterized in that, In the analytical drying step, the temperature is increased to 20℃-25℃ at a heating rate of less than or equal to 0.15℃ / min.

7. The lyophilization method for the mRNA-LNP formulation according to claim 1, characterized in that, The lyophilization protectant composition includes a sugar, an osmotic pressure maintainer, and a buffer solution, wherein the concentration of the sugar is 4%-10% (w / v) and the concentration of the osmotic pressure maintainer is 0.1%-1% (w / v).

8. The lyophilization method for the mRNA-LNP formulation according to claim 7, characterized in that, The carbohydrate is at least one of sucrose and galactose; and / or, The osmotic pressure maintaining agent is calcium chloride; and / or, The buffer solution is a Tris buffer with a concentration of 10mM-20mM and a pH of 7.2-7.

6.

9. An mRNA-LNP formulation, characterized in that, It is prepared by the preparation method according to any one of claims 1-8.

10. The mRNA-LNP formulation according to claim 9, characterized in that, The mRNA-LNP formulation has a particle size of 65nm-95nm, a PDI < 0.3, and a potential of -10 to 10mV.

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