Preparation method of lipid nanoparticle freeze-drying preparation

Abstract

According to the preparation method of the LNP freeze-dried preparation, the particle size difference before and after LNP freeze-drying can be reduced and the product quality can be improved by adding salt into an LNP solution, adjusting the pH value or carrying out curing treatment and the like.

Description

[0001] This application is a divisional application of patent application No. CN202510713470.0 (the original application has an application date of May 30, 2025, and the invention is named: Preparation method of a lipid nanoparticle lyophilized preparation). TECHNICAL FIELD

[0002] The present application belongs to the technical field of pharmaceutical preparations, and relates to a preparation method of a lipid nanoparticle lyophilized preparation. BACKGROUND

[0003] Due to the fact that many diseases often originate from genetic differences, messenger RNA (mRNA) related therapies have become a potential method for treating a variety of diseases. Many commercially available mRNA vaccines currently use lipid nanoparticles (LNP) as a delivery carrier. Lipid nanoparticles are composed of one or more phospholipid bilayers to form a spherical vesicular structure, and are an effective non-viral delivery carrier. However, the lipid nanoparticles in the prior art have defects in stability, which puts high requirements on their production and supply chain, resulting in increased costs.

[0004] Although the freeze-drying technology has been used in the post-processing process in commercial mRNA vaccine products, it is still difficult to well solve the problem of poor stability. For example, studies have shown that cytomegalovirus (CMV) mRNA-lipid nanoparticle (mRNA-LNP) products can be successfully stored for 6 months at 4℃ and 25℃ after freeze-drying, and the particle size may be in a wide range of 60-150 nm. Although the product in this particle size range can induce a strong immune response in non-human primates (NHPs), the antibody titers produced by mRNA-LNP in mice are very relevant to the difference in particle size, that is, the difference in particle size can have an uncertain impact on the immune effect that mRNA-LNP can induce, and thus may have an adverse impact on the drug efficacy. Therefore, it is necessary to reduce the particle size difference of mRNA-LNP products.

[0005] For LNP lyophilized formulation products, the particle size regulation of lipid nanoparticles is crucial, because it can affect the drug encapsulation efficiency, delivery, distribution and clearance, further affecting safety, efficacy, immunogenicity and overall effectiveness. Studies have shown that by adjusting the process and formulation of the preparation, the particle size of LNP can be affected. For example, within the average particle size range of 50-150 nm, by adjusting the volume flow rate of ethanol and water, the self-assembly behavior of lipid molecules in the aqueous phase can be affected, so that the particle size and distribution of lipid nanoparticles can be adjusted. By changing the molar percentage of polyethylene glycol (PEG) lipid, the particle size can also be changed. This is because the PEG lipid as a component of LNPs can stabilize the lipid nanoparticles and prevent particle aggregation due to its hydrophilicity. Although the above methods can affect the particle size, the adjustment of the formulation and process may change the uptake mechanism of the lipid nanoparticles and the kinetics of the lipid nanoparticles into cells. Therefore, the method of improving the particle size difference by adjusting the process and formulation is risky. SUMMARY

[0006] The present application provides a preparation method of a lipid nanoparticle lyophilized formulation, which can effectively reduce the particle size difference of LNP samples before and after lyophilization and ensure sample stability by adding salt to the LNP solution, changing the pH value of the LNP solution or maturing the LNP solution without changing the formulation and preparation process of the LNP.

[0007] To achieve this purpose, the present application adopts the following technical solutions:

[0008] The present application provides a preparation method of a lipid nanoparticle lyophilized formulation, which comprises the following steps:

[0009] 1) preparing a lipid nanoparticle solution;

[0010] 2) mixing the lipid nanoparticle solution obtained in step 1) with a salt to obtain an intermediate solution;

[0011] or, dialyzing the lipid nanoparticle solution obtained in step 1), then adjusting the pH value, and then mixing with a protective agent to obtain an intermediate solution;

[0012] or, maturing the lipid nanoparticle solution obtained in step 1), then dialyzing, and then mixing with a protective agent to obtain an intermediate solution;

[0013] 3) freeze-drying the intermediate solution obtained in step 2) to obtain the lipid nanoparticle lyophilized formulation.

[0014] The application effectively reduces the particle size difference of the LNP sample before and after freeze-drying and improves the stability of the sample by adding salt, adjusting the pH value or aging the LNP solution without changing the original prescription and preparation process of the LNP.

[0015] Preferably, the salt in step 2) includes any one or a combination of at least two of chloride, sulfate, carbonate, phosphate or nitrate.

[0016] Preferably, the salt in step 2) is sodium chloride.

[0017] Preferably, the concentration of the salt in the intermediate solution in step 2) is 0.5-6 mg / mL (for example, it can be 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, etc.).

[0018] Preferably, the pH value in step 2) is adjusted to 8-10 (for example, it can be 8, 8.5, 9, 9.5, 10, etc.).

[0019] Preferably, the adjusting agent used for adjusting the pH value in step 2) includes tromethamine and hydrochloric acid.

[0020] Preferably, the method of aging in step 2) is to store the lipid nanoparticle solution at 2-8°C (for example, it can be 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, etc.) for 1-15 days (for example, it can be 1 day, 2 days, 5 days, 8 days, 10 days, 12 days, 15 days, etc.).

[0021] Preferably, the method of aging in step 2) is to store the lipid nanoparticle solution at 2-5°C for 5-15 days.

[0022] Preferably, the protective agent in step 2) is selected from any one or a combination of at least two of sucrose, lactose, mannitol, maltose and trehalose, preferably sucrose.

[0023] Preferably, the protective agent in step 2) includes sucrose.

[0024] Preferably, the amount of the protective agent in step 2) is independently 6-18 g (for example, it can be 6 g, 8 g, 10 g, 12 g, 14 g, 16 g, 18 g, etc.) for each, based on the system of 100 mL after the pH value is adjusted or aged and dialyzed.

[0025] Preferably, the lipid nanoparticle solution in step 1) includes lipids, buffers and protective agents; and further includes therapeutic agents or prophylactic agents.

[0026] Preferably, the preparing the lipid nanoparticle solution of step 1) comprises: mixing the therapeutic or prophylactic agent, the buffer for encapsulation to obtain an aqueous phase; mixing the lipid and the organic solvent to obtain an organic phase; mixing the aqueous phase and the organic phase for encapsulation to obtain a lipid nanoparticle intermediate solution; mixing the lipid nanoparticle intermediate solution and the buffer for ultrafiltration for ultrafiltration, and then mixing with the protective agent to obtain the lipid nanoparticle solution.

[0027] Preferably, the organic solvent comprises ethanol.

[0028] Preferably, the lipid comprises a cationic lipid, a neutral lipid, a helper lipid, and a long-circulating lipid.

[0029] Preferably, the cationic lipid is selected from any one or a combination of at least two of the following compounds:

[0030] (1) a compound of the structure of Formula I, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1～6 alkylene; G2is C 2～8 alkylene; G3is C 1～3 alkylene; L1is C 6～15 linear alkyl; L2is C 12～25 branched alkyl;

[0031]

[0032] Formula I

[0033] (2) a compound of the structure of Formula II, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 2~8 alkylene; G2is C 2~8 alkylene; L1is -C(O)O- or -OC(O)-; L2is -C(O)O- or -OC(O)-; R1is C 6~25 linear or branched alkyl; R2is C 6~25 linear or branched alkyl; G3is HO(CH2)2- or HO(CH2)3-; G4is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3-, or -(CH2)4-;

[0034]

[0035] Formula II

[0036] (3) a compound of the structure of Formula III, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: G1is C 1~6 alkylene; G2is C 2~8alkylene; R1is C 6~20 linear or branched alkyl; R2is C 12~25 branched alkyl; G3is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-;

[0037]

[0038] Formula III

[0039] (4) a compound of the structure of Formula IV, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1~8 alkylene; G2is C 2~8 alkylene; R1is C 6~25 linear or branched alkyl; R2is C 12~25 linear or branched alkyl; G3is: HO(CH2)2N(R3)CH2CH(OH)CH2-, wherein R3is -CH3, -CH2CH3, or -CH2CH2OH ;

[0040]

[0041] Formula IV

[0042] (5) a compound of the structure of Formula V, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein, G 1 and G 2 are each independently unsubstituted C6-C 10 alkylene; G 3 is unsubstituted C1-C 12 alkylene; R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is -H, -OR 5 , -CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 ; R 4 is C1-C 12hydrocarbyl; and R 5 is -H or C1-C6 hydrocarbyl;

[0043]

[0044] Formula V

[0045] (6) a compound of the structure of Formula VI, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R4is -(CH2) n Q or -(CH2) n CHQR; Q is -OR, -OH, -0(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8, or a heterocycle; each R is independently selected from C 1-3 alkyl, C 2-3 alkenyl, or H; each X is independently selected from -F, -Cl, -Br, or -I; n is 1, 2, or 3;

[0046]

[0047] Formula VI

[0048] (7) a compound of the structure of Formula VII, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof,

[0049]

[0050] Formula VII

[0051] Preferably, the cationic lipid is selected from any one of, or a combination of at least two of, the group consisting of

[0052] , ,

[0053] , and

[0054] ​Preferably, the neutral lipid is selected from any one or a combination of at least two of the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE).

[0055] Preferably, the helper lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, vitamin E, and DC-Cholesterol or derivatives thereof.

[0056] Preferably, the long-circulating lipid is selected from any one or a combination of at least two of the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycero-3-methoxypolyethylene glycol 2000 (DMG-PEG2000), and methoxypolyethylene glycol bismethylidodecylacetamide (ALC-0159).

[0057] Preferably, the molar ratio of the cationic lipid, the neutral lipid, the helper lipid, and the long-circulating lipid is (30-60):(5-15):(30-50):(0.5-5).

[0058] The above numerical value (30-60) can be, for example, 30, 35, 40, 45, 50, 55, 60, etc.; (5-15) can be, for example, 5, 8, 10, 12, 15, etc.; (30-50) can be, for example, 30, 35, 40, 45, 50, etc.; and (0.5-5) can be, for example, 0.5, 1, 2, 3, 4, 5, etc.

[0059] Preferably, the therapeutic or prophylactic agent is a nucleic acid.

[0060] Preferably, the nucleic acid is a ribonucleic acid (RNA).

[0061] Preferably, the nucleic acid is a deoxyribonucleic acid (DNA).

[0062] Preferably, the RNA is selected from any one or a combination of at least two of the group consisting of messenger RNA (mRNA), small interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), Dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), small activating RNA (saRNA), small guide RNA (sgRNA), and transfer RNA (tRNA).

[0063] Preferably, the RNA is mRNA.

[0064] Preferably, the buffer for encapsulation is a citric acid buffer.

[0065] Preferably, the pH of the citric acid buffer is 3-5 (e.g., can be 3, 3.5, 4, 4.5, 5, etc.).

[0066] Preferably, the concentration of the therapeutic or prophylactic agent in the aqueous phase is 0.2-1.5 mg / mL (e.g., can be 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.789 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, etc.).

[0067] Preferably, the buffer for ultrafiltration is selected from any one or a combination of at least two of the group consisting of tromethamine-hydrochloric acid / tromethamine (Tris-HCl / Tris) buffer, Dulbecco's phosphate buffered saline (DPBS) buffer, and phosphate buffer.

[0068] Preferably, the pH of the buffer for ultrafiltration is 3-10 (e.g., can be 3, 4, 5, 6, 7, 8, 9, 10, etc.).

[0069] Preferably, the volume ratio of the lipid nanoparticle intermediate solution and the buffer for ultrafiltration is 1:2-1:15 (e.g., can be 1:2, 1:3, 1:4, 1:4.5, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, etc.).

[0070] Preferably, the protective agent is selected from any one or a combination of at least two of the group consisting of sucrose, trehalose, and maltose.

[0071] Compared with the prior art, the beneficial effects of the present application are embodied in: by adjusting the physicochemical conditions of the non-frozen sample before the freeze-drying process, reducing the particle size difference between the freeze-dried sample and the non-frozen sample while keeping the lipid composition (formula) and microfluidic preparation parameters (process) unchanged, which can effectively avoid changing the uptake mechanism and drug efficacy. BRIEF DESCRIPTION OF DRAWINGS

[0072] In order to more clearly illustrate the technical solutions in the specific embodiments of the present disclosure or the prior art, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present disclosure, and various changes made by those of ordinary skill in the art within the spirit and scope of the present disclosure can also be considered as the scope intended by the drawings of the present disclosure.

[0073] Figure 1 is a sample particle size difference chart before and after freeze-drying of Comparative Example 1-1, Example 1-1, and Example 1-3, wherein Comparative Example 1-1 is an LNP solution without sodium chloride, Example 1-1 is added with 1 mg / mL sodium chloride, and Example 1-3 is added with 5 mg / mL sodium chloride.

[0074] Figure 2 is a sample encapsulation efficiency difference chart before and after freeze-drying of Comparative Example 1-1, Example 1-1, and Example 1-3, wherein Comparative Example 1-1 is an LNP solution without sodium chloride, Example 1-1 is added with 1 mg / mL sodium chloride, and Example 1-3 is added with 5 mg / mL sodium chloride.

[0075] Figure 3 is a sample particle size difference chart before and after freeze-drying of Comparative Example 2-1, Example 2-4, Comparative Example 1-1, and Example 2-1, wherein Comparative Example 2-1 and Comparative Example 1-1 are directly freeze-dried after preparing LNP solutions using 1 / 2 DPBS or Tris / Tris-HCl as a buffer, and Example 2-4 and Example 2-1 are freeze-dried after adjusting the pH to 8.0 after using 1 / 2 DPBS or Tris / Tris-HCl as a buffer.

[0076] Figure 4 is a sample encapsulation efficiency difference chart before and after freeze-drying of Comparative Example 2-1, Example 2-4, Comparative Example 1-1, and Example 2-1, wherein Comparative Example 2-1 and Comparative Example 1-1 are directly freeze-dried after preparing LNP solutions using 1 / 2 DPBS or Tris / Tris-HCl as a buffer, and Example 2-4 and Example 2-1 are freeze-dried after adjusting the pH to 8.0 after using 1 / 2 DPBS or Tris / Tris-HCl as a buffer.

[0077] Figure 5Figure 1 is a graph of particle size difference before and after lyophilization of samples of Comparative Example 2-1, Comparative Example 3-9, Comparative Example 3-10, Comparative Example 3-4, Example 3-7, Example 3-8, Comparative Example 3-5, Example 3-9, Example 3-10, Comparative Example 3-6, Example 3-11, Example 3-12 using 1 / 2 DPBS as the buffer, wherein Comparative Example 2-1 is unripened-0 days (0d), Comparative Example 3-9 is unripened-0d-dialysis-8.7% (m / V) sucrose, Comparative Example 3-10 is unripened-0d-dialysis-15% (m / V) sucrose, Comparative Example 3-4 is ripened-5d, Example 3-7 is ripened-5d-dialysis-8.7% (m / V) sucrose, Example 3-8 is ripened-5d-dialysis-15% (m / V) sucrose, Comparative Example 3-5 is ripened-10d, Example 3-9 is ripened-10d-dialysis-8.7% (m / V) sucrose, Example 3-10 is ripened-10d-dialysis-15% (m / V) sucrose, Comparative Example 3-6 is ripened-15d, Example 3-11 is ripened-15d-dialysis-8.7% (m / V) sucrose, and Example 3-12 is ripened-15d-dialysis-15% (m / V) sucrose.

[0078] Figure 6 Figure 2 is a graph of particle size difference before and after lyophilization of samples of Comparative Example 1-1, Comparative Example 3-7, Comparative Example 3-8, Comparative Example 3-1, Example 3-1, Example 3-2, Comparative Example 3-2, Example 3-3, Example 3-4, Comparative Example 3-3, Example 3-5, Example 3-6 using Tris / Tris-HCl as the buffer, wherein Comparative Example 1-1 is unripened-0 days (0d), Comparative Example 3-7 is unripened-0d-dialysis-8.7% (m / V) sucrose, Comparative Example 3-8 is unripened-0d-dialysis-15% (m / V) sucrose, Comparative Example 3-1 is ripened-5d, Example 3-1 is ripened-5d-dialysis-8.7% (m / V) sucrose, Example 3-2 is ripened-5d-dialysis-15% (m / V) sucrose, Comparative Example 3-2 is ripened-10d, Example 3-3 is ripened-10d-dialysis-8.7% (m / V) sucrose, Example 3-4 is ripened-10d-dialysis-15% (m / V) sucrose, Comparative Example 3-3 is ripened-15d, Example 3-5 is ripened-15d-dialysis-8.7% (m / V) sucrose, and Example 3-6 is ripened-15d-dialysis-15% (m / V) sucrose.

[0079] Figure 7Figure 1 is a graph showing the particle size difference before and after freeze-drying of the sample of Comparative Example 4-2, Example 3-9, and Example 3-10, wherein Comparative Example 4-2 is twice ultrafiltration, Example 3-9 is maturation-10d-dialysis-addition of 8.7% (m / V) sucrose, and Example 3-10 is maturation-10d-dialysis-addition of 15% (m / V) sucrose, using 1 / 2 DPBS as the buffer.

[0080] Figure 8 Figure 2 is a graph showing the particle size difference before and after freeze-drying of the sample of Comparative Example 4-1, Example 3-3, and Example 3-4, wherein Comparative Example 4-1 is twice ultrafiltration, Example 3-3 is maturation-10d-dialysis-addition of 8.7% (m / V) sucrose, and Example 3-4 is maturation-10d-dialysis-addition of 15% (m / V) sucrose, using Tris / Tris-HCl as the buffer. DETAILED DESCRIPTION

[0081] In order to make the objectives, technical solutions, and advantages of the embodiments of the present application clearer, the technical solutions of the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are part of, rather than all of, the embodiments of the present application. Based on the described embodiments of the present application, all other embodiments obtained by a person of ordinary skill in the art without any inventive effort fall within the scope of protection of the present application.

[0082] The present application will be further described below in conjunction with the embodiments. However, the present application is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific use, and the implementation conditions not mentioned are the conventional conditions in the industry. In the specific embodiments of the present application, the raw materials used can be obtained through marketing. Unless otherwise specified, the percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in each embodiment of the present application can be combined with each other as long as they do not conflict with each other.

[0083] The therapeutic or prophylactic agent used in all the embodiments is mRNA, which can also be replaced by other therapeutic or prophylactic agents.

[0084] Example 1-1 Preparation of LNP freeze-dried preparation (addition of salt in LNP solution)

[0085] 1.1 Preparation of LNP solution

[0086] (1) Preparation of aqueous phase

[0087] The mRNA stock solution was dissolved in a pH 4.0 citric acid buffer to prepare the aqueous phase, and the concentration of the aqueous phase was 0.789 mg / mL.

[0088] (2) Preparation of organic phase

[0089] Cationic lipid YK009 (Yokoyama), neutral lipid DSPC (NOF Corporation), helper lipid chol (NOF Corporation), long-circulating lipid mPEG-DMG (SinoBiologics) were dissolved in anhydrous ethanol according to the mass ratio of 49:10:39.5:1.5 to prepare the organic phase.

[0090] (3) Encapsulation

[0091] The organic phase and the aqueous phase were rapidly mixed in a microfluidic device (manufacturer: Precision NanoSystems) to prepare the LNP intermediate solution. The encapsulation method was that the flow rate ratio of the organic phase and the aqueous phase was 1:3, the total flow rate was 12 mL / min, and the chip model was Ignite NxGen.

[0092] (4) Ultrafiltration

[0093] The above LNP intermediate solution was diluted by adding 8 times its volume of Tris / Tris-HCl buffer (Tris / Tris-HCl buffer), and concentrated by manual ultrafiltration. The ultrafiltration membrane was Hydrosart (Sartorius), the membrane pore size was 300KD, the main pump flow rate was 400 mL / min, and the transmembrane pressure (TMP) was 0.1 bar. When the concentration was about 100 mL, the ultrafiltration was stopped.

[0094] (5) Add a protective agent

[0095] The LNP sample after ultrafiltration with Tris / Tris-HCl buffer was added with sucrose (denoted as 8.7%, m / V) according to the amount of 8.7 g of sucrose per 100 mL of solution to obtain the LNP solution.

[0096] 1.2 Add salt to the LNP solution

[0097] Sodium chloride was added to the LNP solution prepared in 1.1 to a final concentration of 1 mg / mL of sodium chloride in the system to obtain an intermediate solution.

[0098] 1.3 Preparation of LNP freeze-dried preparation

[0099] The freshly prepared intermediate solution from 1.2 was filled into Schott vials. The vials were then placed on the pre-cooled freeze-drying shelves of a freeze-dryer (LYO 0.5, Dongfeng) and kept at -45 °C for 4 h to ensure uniform freezing of all vials. Upon entering the drying phase, the pressure in the drying chamber was reduced to 5 Pa and the shelf temperature was kept at -45 °C. The samples were then dried for 60 h. To effectively remove any residual moisture, the shelf temperature was gradually increased to 25 °C over a period of 2 h after the end of the drying process and kept at this temperature for 6 h. The freeze-drying was completed after plugging, resulting in a LNP lyophilized formulation.

[0100] Example 1-2

[0101] The difference between this example and Example 1-1 is that in step 1.2, sodium chloride was added to the system to a final concentration of 3 mg / mL, and the other steps refer to Example 1-1.

[0102] Example 1-3

[0103] The difference between this example and Example 1-1 is that in step 1.2, sodium chloride was added to the system to a final concentration of 5 mg / mL, and the other steps refer to Example 1-1.

[0104] Comparative Example 1-1

[0105] This comparative example uses the conventional method of preparing a LNP lyophilized formulation in the prior art, i.e. the prepared LNP solution is not treated and is directly freeze-dried. The difference between this example and Example 1-1 is that step 1.2 is not performed, and the LNP solution prepared in step 1.1 is directly subjected to the freeze-drying of step 1.3, and the other steps refer to Example 1-1.

[0106] Example 2-1 Preparation of a LNP lyophilized formulation (adjusting the pH of the LNP solution)

[0107] 2.1 Preparation of a LNP solution

[0108] The experimental steps are the same as in 1.1 of Example 1-1.

[0109] 2.2 Adjusting the pH of the LNP solution

[0110] The LNP solution prepared in 2.1 was dialyzed against a Tris solution (12 h). The pH was adjusted by adding an appropriate amount of HC1 to the Tris-base solution to a final pH of 8. Then, sucrose was added in an amount of 8.7 g per 100 mL of solution (denoted as 8.7%, m / V).

[0111] 2.3 Preparation of a LNP lyophilized formulation

[0112] The experimental procedure was the same as 1.3 in Example 1-1.

[0113] Example 2-2

[0114] The difference between this example and Example 2-1 is that in step 2.2, the final pH value is controlled at 9, and the other steps are the same as in Example 2-1.

[0115] Example 2-3

[0116] The difference between this example and Example 2-1 is that in step 2.2, the final pH value is controlled at 10, and the other steps are the same as in Example 2-1.

[0117] Example 2-4

[0118] The difference between this example and Example 2-1 is that in step 2.1, in the fourth step of ultrafiltration, 8 volumes of Tris / Tris-HCl buffer (Trometamol / Trometamol-HCl buffer) are replaced by 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) for dilution, and the other steps are the same as in Example 2-1.

[0119] Example 2-5

[0120] The difference between this example and Example 2-2 is that in step 2.1, in the fourth step of ultrafiltration, 8 volumes of Tris / Tris-HCl buffer (Trometamol / Trometamol-HCl buffer) are replaced by 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) for dilution, and the other steps are the same as in Example 2-2.

[0121] Example 2-6

[0122] The difference between this example and Example 2-3 is that in step 2.1, in the fourth step of ultrafiltration, 8 volumes of Tris / Tris-HCl buffer (Trometamol / Trometamol-HCl buffer) are replaced by 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) for dilution, and the other steps are the same as in Example 2-3.

[0123] Comparative Example 2-1

[0124] The comparative example uses the conventional method of preparing the LNP lyophilized preparation of the prior art, i.e. the prepared LNP solution is not treated and is directly lyophilized, and the difference from Examples 2-4 is only that step 2.2 is not performed and the LNP solution prepared in step 2.1 is directly subjected to the freeze-drying of step 2.3, and the other steps are as in Examples 1-4.

[0125] Example 3-1 Preparation of LNP lyophilized preparation (maturation of LNP solution)

[0126] 3.1 Preparation of LNP solution

[0127] The experimental procedure is as in 1.1 of Example 1-1.

[0128] 3.2 Maturation of LNP solution

[0129] The LNP solution prepared in 3.1 is matured (i.e. stored at 5°C, allowing temperature fluctuations in the range of 2-8°C), and the maturation period is 5 days. The matured LNP solution is dialyzed, and sucrose is added in an amount of 8.7 g per 100 mL of solution (denoted as 8.7%, m / V).

[0130] 3.3 Preparation of LNP lyophilized preparation

[0131] The experimental procedure is as in 1.3 of Example 1-1.

[0132] Example 3-2

[0133] The difference between this example and Example 3-1 is only that the amount of sucrose added in step 3.2 is 15%, and the other steps are as in Example 3-1.

[0134] Example 3-3

[0135] The difference between this example and Example 3-1 is only that the maturation period in step 3.2 is 10 days, and the other steps are as in Example 3-1.

[0136] Example 3-4

[0137] The difference between this example and Example 3-1 is only that the maturation period in step 3.2 is 10 days and the amount of sucrose added is 15%, and the other steps are as in Example 3-1.

[0138] Example 3-5

[0139] The difference between this example and Example 3-1 is only that the maturation period in step 3.2 is 15 days, and the other steps are as in Example 3-1.

[0140] Example 3-6

[0141] The difference between this example and Example 3-1 is only that the aging time in step 3.2 is 15 days, and the amount of sucrose added is 15%, and the rest is according to Example 3-1.

[0142] Comparative Example 3-1

[0143] The difference between this comparative example and Example 3-1 is only that after aging in step 3.2, no dialysis is performed, and no sucrose is added, and the rest is according to Example 3-1.

[0144] Comparative Example 3-2

[0145] The difference between this comparative example and Example 3-1 is only that the aging time in step 3.2 is 10 days, and after aging, no dialysis is performed, and no sucrose is added, and the rest is according to Example 3-1.

[0146] Comparative Example 3-3

[0147] The difference between this comparative example and Example 3-1 is only that the aging time is 15 days, and after aging, no dialysis is performed, and no sucrose is added, and the rest is according to Example 3-1.

[0148] Examples 3-7 to 3-12

[0149] Examples 3-7 to 3-12 differ from Examples 3-1 to 3-6 only in that in step 3.1 of Examples 3-1 to 3-6, the 8-fold volume of Tris / Tris-HCl buffer used in the ultrafiltration of step (4) is replaced by 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) for dilution, to obtain Examples 3-7 to 3-12, and the rest is according to Examples 3-1 to 3-6, respectively.

[0150] Comparative Examples 3-4 to 3-6

[0151] Comparative Examples 3-4 to 3-6 differ from Comparative Examples 3-1 to 3-3 only in that in step 3.1 of Comparative Examples 3-1 to 3-3, the 8-fold volume of Tris / Tris-HCl buffer used in the ultrafiltration of step (4) is replaced by 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) for dilution, to obtain Comparative Examples 3-4 to 3-6, and the rest is according to Comparative Examples 3-1 to 3-3, respectively.

[0152] Comparative Example 3-7

[0153] The present comparative example differs from Example 3-1 only in that no maturation is performed in step 3.2, and the amount of sucrose added is 15%, otherwise refer to Example 3-1.

[0154] Comparative Example 3-8

[0155] The present comparative example differs from Example 3-1 only in that no maturation is performed in step 3.2, and the amount of sucrose added is 15%, otherwise refer to Example 3-1.

[0156] Comparative Example 3-9~Comparative Example 3-10

[0157] Comparative Example 3-9~Comparative Example 3-10 differ from Comparative Example 3-7~Comparative Example 3-8 only in that the 8-fold volume of Tris / Tris-HCl buffer used in step 3.1, step (4) of Comparative Example 3-7~Comparative Example 3-8 is replaced by 1 / 2 volume of DPBS (Dulbecco’s Phosphate-Buffered Saline) for dilution to obtain Comparative Example 3-9~Comparative Example 3-10, respectively, otherwise refer to Comparative Example 3-7~Comparative Example 3-8, respectively.

[0158] Preparation of LNP lyophilized formulation (two times ultrafiltration of LNP solution)

[0159] The present comparative example adopts a method (A preparation method for mRNA-LNPs with improved properties. J Control Release. 2023 Dec;364:632-643.) in the prior art that can reduce the particle size difference of LNP lyophilized formulation, i.e., the prepared LNP solution is subjected to two times ultrafiltration before lyophilization.

[0160] 4.1. Preparation of LNP solution

[0161] The experimental steps are the same as 1.1 in Example 1-1.

[0162] 4.2. Two times ultrafiltration of LNP solution

[0163] The LNP solution prepared in 4.1 is subjected to ultrafiltration again (tangential flow filtration, TFF), and then sucrose is added in an amount of 8.7 g sucrose per 100 mL solution (denoted as 8.7%, m / V).

[0164] 4.3. Preparation of LNP lyophilized formulation

[0165] The experimental steps are the same as 1.3 in Example 1-1.

[0166] Comparative Example 4-2

[0167] The difference between this comparative example and Comparative Example 4-1 is that in step 4.1 (4), the 8-fold volume of Tris / Tris-HCl buffer is replaced by 1 / 2-fold volume of DPBS (Dulbecco's Phosphate-Buffered Saline) for dilution, and the other steps refer to Comparative Example 4-1.

[0168] Test Example 1: Lipid Nanoparticle (LNP) sample property detection before and after freeze-drying

[0169] The particle size, dispersity, encapsulation efficiency and integrity of the LNP samples before freeze-drying and after reconstitution of the LNP freeze-drying preparation prepared in all the above examples and comparative examples were detected respectively, and the influence of different preparation methods on the properties of freeze-dried products was evaluated.

[0170] (1) Particle size and dispersity

[0171] The particle size and particle size distribution of lipid nanoparticles are related to their encapsulation efficiency and stability, directly affect the behavior of liposomes in the body tissue, and are important parameters for evaluating their biochemistry, biophysics and drug delivery system, and affect the in vivo biodistribution and pharmacokinetics of the product. Therefore, particle size and particle size distribution are important characterization indicators of LNP product stability.

[0172] The detection method is as follows:

[0173] Particle size and particle size distribution coefficient (PDI): dynamic light scattering method (DLS) was used to detect particle size and PDI on a nano-laser particle size analyzer (manufacturer: MALVERN; model: ZSU3305) at 25 ± 1°C. 60 μL of sample was taken and placed in a quartz micro-sample cell for detection. The detection parameters were set as follows:

[0174] Temperature: 25°C; Equilibrium time: 30 s; Material: Liposome; Dispersion system: Water; Detection mode: Automatic.

[0175] Particle size and PDI are important characteristics of liposomes. During freeze-thawing, there is a fusion process of lipids, and small particles of lipids are fused into liposomes with slightly larger particle size. Within a certain range, the smaller the particle size, the better the product quality and process. PDI represents the distribution of particle size, and the smaller the value, the more uniform the size of the liposome particles.

[0176] (2) Encapsulation efficiency

[0177] Stability of mRNA drug is a major challenge for drug delivery. The main responsibility of the delivery vehicle is to avoid the degradation of mRNA by RNA cleavage enzymes in vivo. Encapsulation efficiency is a key quality attribute of liposomes, which refers to the percentage of drug content encapsulated in the lipid bilayer relative to the total drug amount. The higher the encapsulation efficiency, the greater the proportion of mRNA encapsulated by liposomes. Higher encapsulation efficiency means a higher proportion of encapsulated mRNA, which is easily degraded. Therefore, encapsulation efficiency is an important characterization index of LNP product stability.

[0178] Detection method:

[0179] The Quant-it Ribogreen RNA quantification assay kit (Thermo Fisher Scientific, UK) was used to determine the encapsulation efficiency of lipid nanoparticles. The specific method is as follows:

[0180] The sample was diluted to 2.8 μg / mL, and a part was mixed with an equal volume (50 μL) of Triton X-100, and emulsified thoroughly, for determining the total mRNA concentration, and another part was mixed with an equal volume (50 μL) of TE (Tris-EDTA), for determining the concentration of free unencapsulated mRNA. After incubating the samples added with TE (Tris-EDTA) and Triton X-100 respectively at 37°C for a certain period of time, 100 μL of diluted 200 times Ribogreen reagent was added, and after centrifugation to remove bubbles, the multifunctional enzyme marker was used to measure the fluorescence value at an excitation wavelength of 485 nm and an emission wavelength of 528 nm, and the plate was read.

[0181] (3) Integrity

[0182] Although mRNA is encapsulated and protected by liposomes, mRNA may still be degraded during the preparation, transportation and long-term storage of the preparation, which directly affects the expression of nucleic acid drugs in vivo. Therefore, evaluating the integrity of mRNA is very important for judging the quality of the product. Therefore, it is also an important characterization index of LNP product stability.

[0183] Detection method:

[0184] The specific method is as follows: mRNA in mRNA-LNP is extracted using a kit, and after dilution and denaturation at 65°C, the prepared sample is placed on ice. In addition, gel buffer solution is prepared, and the prepared sample solution, gel buffer solution and ribonuclease-free water are sequentially added to the sample plate, and the mRNA integrity of the sample is determined by PA800 Plus gel electrophoresis principle.

[0185] Experimental results:

[0186] The particle size, particle size distribution coefficient (PDI), encapsulation efficiency and integrity results of each sample in all examples and comparative examples are shown in Tables 1-4, respectively. The particle size statistical method adopts two-way ANOVA, Bonferroni Post-hoc Tests, and 3 samples are taken for each sampling for particle size and PDI, and the average value is calculated.

[0187] Table 1 Particle size data of samples before and after freeze-drying

[0188]

[0189] Table 2 Polydispersity Index (PDI) of particle size distribution of samples before and after freeze-drying

[0190]

[0191] Table 3 Encapsulation efficiency data of samples before and after freeze-drying

[0192]

[0193] Table 4 Integrity data of samples before and after freeze-drying

[0194]

[0195] Analysis of experimental results:

[0196] 1. After adding salt to the LNP solution and then freeze-drying, the difference in particle size of the sample before and after freeze-drying can be effectively reduced, and the stability of the LNP freeze-dried product can be ensured.

[0197] As can be seen from the data in Table 1, using the method of adding salt to the LNP solution and then freeze-drying (Examples 1-1~1-3), compared with the conventional method of freeze-drying without adding salt (Comparative Example 1-1), the difference in particle size of the sample before and after freeze-drying is significantly reduced. The average particle size difference is 48.4 nm when the LNP solution is freeze-dried without NaCl, while the average particle size difference is only 16.1 nm, 13.0 nm and 11.9 nm when the LNP solution is freeze-dried after adding 1 mg / mL, 3 mg / mL and 5 mg / mL NaCl, Figure 1 ).

[0198] As can be seen from Tables 2, 3 and 4, compared with freeze-drying of the LNP solution, freeze-drying after adding salt to the LNP solution has consistent changes in particle size distribution, encapsulation efficiency Figure 2 and integrity of the freeze-dried product.

[0199] The method of adding salt to the LNP solution and then freeze-drying in the present application can significantly reduce the particle size difference before and after freeze-drying, and can ensure the stability of the LNP freeze-dried product.

[0200] 2. Adjusting the pH value of the LNP solution to 8-10 and then freeze-drying can effectively reduce the particle size difference before and after freeze-drying, and can ensure the stability of the LNP freeze-dried product.

[0201] As can be seen from Table 1, adjusting the pH value of the LNP solution to 8-10 and then freeze-drying (Examples 2-1 to 2-6) can significantly reduce the particle size difference before and after freeze-drying compared with the conventional method of freeze-drying the LNP solution without adjusting the pH value (Comparative Example 2-1). The average particle size difference is 40.3 nm when freeze-drying the LNP solution without adjusting the pH value, while the average particle size difference is only 4.1 nm, 2.2 nm and 0.7 nm when freeze-drying the LNP solution with the pH value adjusted to 8, 9 and 10, respectively. Figure 3 ).

[0202] As can be seen from Tables 2, 3 and 4, adjusting the pH value of the LNP solution to 8-10 and then freeze-drying has basically the same changes in product particle size distribution, encapsulation efficiency and integrity compared with freeze-drying the LNP solution without adjusting the pH value. Figure 4

[0203] The method of adjusting the pH value of the LNP solution to 8-10 and then freeze-drying in the present application can significantly reduce the particle size difference before and after freeze-drying, and can ensure the stability of the LNP freeze-dried product.

[0204] 3. Maturing the LNP solution, then dialyzing, then adding a protective agent, and then freeze-drying can effectively reduce the particle size difference before and after freeze-drying, and can ensure the stability of the LNP freeze-dried product.

[0205] As can be seen from Table 1, maturing the LNP solution, then dialyzing, then adding a protective agent, and then freeze-drying (Examples 3-1 to 3-12) can significantly reduce the particle size difference before and after freeze-drying compared with the conventional method of freeze-drying the LNP solution without maturing (Comparative Example 1-1 and Comparative Example 2-1). Figure 5 and Figure 6 ).

[0206] As can be seen from Tables 2, 3 and 4, maturing the LNP solution, then dialyzing, then adding a protective agent, and then freeze-drying have the same changes in product particle size distribution, encapsulation efficiency and integrity compared with freeze-drying the LNP solution without maturing.

[0207] The method of maturing the LNP solution, then dialyzing, then adding a protective agent, and then freeze-drying in the present application can significantly reduce the particle size difference before and after freeze-drying, and can ensure the stability of the LNP freeze-dried product. ​

[0208] And, compared with the method of reducing the particle size difference of LNP freeze-dried preparation in the prior art (comparative example 4-1), i.e. twice ultrafiltration of LNP, the LNP solution is dialyzed after maturation, then a protective agent is added, and then freeze-dried (examples 3-1~3-12), the particle size difference of the sample before and after freeze-drying is significantly reduced Figure 7 and Figure 8 )。

[0209] The above examples are only used to illustrate the technical solutions of the present disclosure, but not to limit it; although the present disclosure has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some or all of the technical features can be replaced by equivalents. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present disclosure shall be included in the protection scope of the claims of the present disclosure.

Claims

1. A method of preparing a lipid nanoparticle lyophilized formulation, characterized in that, The preparation method comprises the following steps: 1) preparing a lipid nanoparticle solution; 2) mixing the lipid nanoparticle solution obtained in step 1) and a salt to obtain an intermediate solution; 3) freeze-drying the intermediate solution obtained in step 2) to obtain the lipid nanoparticle lyophilized preparation.

2. The production method according to claim 1, characterized by, The salt in step 2) is selected from any one or a combination of at least two of the group consisting of chloride, sulfate, carbonate, phosphate and nitrate; The concentration of the salt in the intermediate solution is 0.5-6 mg / mL.

3. The preparation method according to claim 1, characterized in that, The lipid nanoparticle solution in step 1) comprises: lipids, a buffer and a protective agent; and further comprises a therapeutic agent or a prophylactic agent.

4. The production method according to claim 3, characterized by, The preparation of the lipid nanoparticle solution in step 1) comprises: mixing the therapeutic agent or the prophylactic agent and a buffer for encapsulation to obtain an aqueous phase; mixing the lipids and an organic solvent to obtain an organic phase; mixing the aqueous phase and the organic phase for encapsulation to obtain a lipid nanoparticle intermediate solution; mixing the lipid nanoparticle intermediate solution and a buffer for ultrafiltration for ultrafiltration, and then mixing with the protective agent to obtain the lipid nanoparticle solution.

5. The preparation method according to claim 3, characterized in that, The lipids comprise cationic lipids, neutral lipids, helper lipids and long-circulating lipids; The cationic lipids are selected from any one or a combination of at least two of the group consisting of the following compounds: (1) a compound of the structure of Formula I, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1～6 alkylene; G2is C 2～8 alkylene; G3is C 1～3 alkylene; L1is C 6～15 straight chain alkyl; L2is C 12～25 branched chain alkyl; Formula I (2) a compound of the structure of Formula II, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 2~8 alkylene; G2is C 2~8 alkylene; L1is -C(O)O- or -OC(O)-; L2is -C(O)O- or -OC(O)-; R1is C 6~25 straight or branched chain alkyl; R2is C 6~25 straight or branched chain alkyl; G3is HO(CH2)2- or HO(CH2)3-; G4is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3-, or -(CH2)4-. Formula II (3) a compound of the structure of Formula III, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein: G1is C 1~6 alkylene; G2is C 2~8 alkylene; R1is C 6~20 linear or branched alkyl; R2is C 12~25 branched alkyl; G3is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(CH2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2-, or CH3CH2NH(CH2)2-; Formula III (4) a compound of the structure of Formula IV, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G1is C 1~8 alkylene; G2is C 2~8 alkylene; R1is C 6~25 straight or branched chain alkyl; R2is C 12~25 straight or branched chain alkyl; G3is: HO(CH2)2N(R3)CH2CH(OH)CH2- wherein R3is -CH3, -CH2CH3, or -CH2CH2OH; Formula IV (5) a compound of the structure of Formula V, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein G 1 and G 2 each independently is unsubstituted C6-C 10 alkylene; G 3 is unsubstituted C1-C 12 alkylene; R 1 and R 2 each independently is C6-C 24 alkyl or C6-C 24 alkenyl; R 3 is -H, -OR 5 , -CN, -C(=O)OR 4 , -OC(=O)R 4 , or -NR 5 C(=O)R 4 ; R 4 is C1-C 12 alkyl; and R 5 is -H or C1-C6 alkyl; Formula V (6) a compound of the structure of Formula VI, or an N-oxide, solvate, pharmaceutically acceptable salt, or stereoisomer thereof, wherein R4is -(CH2) n Q or -(CH2) n CHQR; Q is -OR, -OH, -O(CH2) n N(R)2, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, -N(R)S(O)2R, -N(H)S(O)2R, -N(R)C(O)N(R)2, -N(H)C(O)N(R)2, -N(H)C(O)N(H)(R), -N(R)C(S)N(R)2, -N(H)C(S)N(R)2, -N(H)C(S)N(H)(R), -N(R)S(O)2R8, or a heterocycle; each R is independently selected from C 1-3 alkyl, C 2-3 alkenyl, or -H; each X is independently selected from -F, -Cl, -Br, or -I; n is 1, 2, or 3; Formula VI (7) a compound of the structure of Formula VII, or an N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer thereof, Formula VII; The neutral lipids are selected from any one or a combination of at least two of the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, 1,2-dimyristoyl-sn-glycero-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; The helper lipids are selected from any one or a combination of at least two of the group consisting of cholesterol, vitamin E and DC-cholesterol or derivatives thereof; The long-circulating lipids are selected from any one or a combination of at least two of the group consisting of distearoylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycero-3-methoxypolyethylene glycol 2000 and methoxypolyethylene glycol ditetradecylamido; The molar ratio of the cationic lipids, neutral lipids, helper lipids and long-circulating lipids is (30-60):(5-15):(30-50):(0.5-5).

6. The preparation method according to claim 3, characterized in that, The therapeutic agent or the prophylactic agent is a nucleic acid; The protective agent is selected from any one or a combination of at least two of the group consisting of sucrose, trehalose and maltose.

7. The preparation method according to claim 4, characterized in that, The buffer for encapsulation is a citric acid buffer; the pH of the citric acid buffer is 3-5; The buffer for ultrafiltration is selected from any one or a combination of at least two of the group consisting of tromethamine-hydrochloric acid / tromethamine buffer, Dulbecco's phosphate buffer and phosphate buffer; The pH of the buffer for ultrafiltration is 3-10.

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