A method of preparing a lipid nanoparticle lyophilized formulation

Abstract

The application provides a preparation method of a lipid nanoparticle (LNP) freeze-dried preparation. By adding a salt to an LNP solution, adjusting pH or performing a curing treatment, the particle size difference before and after LNP freeze-drying can be reduced, and the product quality is improved.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical formulation technology and relates to a method for preparing a lyophilized lipid nanoparticle formulation. Background Technology

[0002] Since many diseases often stem from genetic differences, messenger RNA (mRNA)-related therapies have emerged as potential treatments for a wide range of conditions. Many commercially available mRNA vaccines utilize lipid nanoparticles (LNPs) as delivery carriers. Lipid nanoparticles, composed of spherical vesicles formed from one or more phospholipid bilayers, are an effective non-viral delivery vector. However, existing lipid nanoparticle technologies suffer from stability issues, placing high demands on their production and supply chain, leading to increased costs.

[0003] Although freeze-drying technology has been used in the post-processing of commercial mRNA vaccine products, it remains a challenge to effectively address the issue of poor stability. For example, studies have shown that cytomegalovirus (CMV) mRNA-lipid nanoparticles (mRNA-LNPs), after freeze-drying, can be successfully stored for 6 months at 4°C and 25°C, respectively. Their particle size can range widely from 60 to 150 nm. While this product within this size range can elicit a strong immune response in non-human primates (NHPs), the antibody titer produced by mRNA-LNPs in mice is highly correlated with particle size variation. In other words, particle size variation can have an uncertain impact on the immune response induced by mRNA-LNPs, potentially adversely affecting drug efficacy. Therefore, reducing the particle size variation of mRNA-LNP products is essential.

[0004] For lyophilized lipid nanoparticle (LNP) formulations, particle size control is crucial as it influences drug encapsulation efficiency, delivery, distribution, and clearance, further impacting safety, potency, immunogenicity, and overall efficacy. Studies have shown that adjusting the formulation process and formulation can affect LNP particle size. For example, within an average particle size range of 50–150 nm, adjusting the volumetric flow rates of ethanol and water can affect the self-assembly behavior of lipid molecules in the aqueous phase, thereby regulating the particle size and distribution of lipid nanoparticles. Changing the molar percentage of polyethylene glycol (PEG) lipids can also alter particle size. This is because PEG lipids, as components of LNPs, possess hydrophilic properties that stabilize lipid nanoparticles and prevent aggregation. While these methods can influence particle size, adjustments to formulation and process may alter the uptake mechanism of lipid nanoparticles and their kinetics in cell entry. Therefore, methods that improve particle size variation through process and formulation adjustments carry significant risks. Summary of the Invention

[0005] This invention provides a method for preparing a lyophilized lipid nanoparticle formulation. Without changing the formulation and preparation process of LNP, the particle size difference of the LNP sample before and after lyophilization can be effectively reduced by adding salt to the LNP solution, changing the pH value of the LNP solution, or aging the LNP solution, and the stability of the sample can be guaranteed.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] This invention provides a method for preparing a lyophilized lipid nanoparticle formulation, the method comprising the following steps:

[0008] 1) Preparation of lipid nanoparticle solution;

[0009] 2) Mix the lipid nanoparticle solution obtained in step 1) with salt to obtain an intermediate solution;

[0010] Alternatively, the lipid nanoparticle solution obtained in step 1) can be dialyzed, the pH value adjusted, and then mixed with a protective agent to obtain an intermediate solution.

[0011] Alternatively, the lipid nanoparticle solution obtained in step 1) can be aged, then dialyzed, and then mixed with a protective agent to obtain an intermediate solution.

[0012] 3) Freeze-dry the intermediate solution obtained in step 2) to obtain the lyophilized lipid nanoparticle formulation.

[0013] This invention, without altering the original LNP formulation and preparation process, effectively reduces the particle size difference of LNP samples before and after freeze-drying and improves sample stability by adding salt, adjusting pH, or aging the LNP solution.

[0014] Preferably, the salt in step 2) includes any one or a combination of at least two of chlorides, sulfates, carbonates, phosphates or nitrates.

[0015] Preferably, the salt in step 2) is sodium chloride.

[0016] Preferably, the salt concentration in the intermediate solution in step 2) is 0.5-6 mg / mL (e.g., 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, etc.).

[0017] Preferably, step 2) involves adjusting the pH value to 8-10 (e.g., 8, 8.5, 9, 9.5, 10, etc.).

[0018] Preferably, the pH adjuster used in step 2) includes tromethamine and hydrochloric acid.

[0019] Preferably, the ripening method in step 2) is to store the lipid nanoparticle solution at 2~8℃ (e.g., 2℃, 3℃, 4℃, 5℃, 6℃, 7℃, 8℃, etc.) for 1~15 days (e.g., 1 day, 2 days, 5 days, 8 days, 10 days, 12 days, 15 days, etc.).

[0020] Preferably, the ripening method in step 2) is to store the lipid nanoparticle solution at 2-5°C for 5-15 days.

[0021] Preferably, the protective agent in step 2) is selected from any one or a combination of at least two of the group consisting of sucrose, lactose, mannitol, maltose and trehalose, with sucrose being preferred.

[0022] Preferably, the protective agent in step 2) includes sucrose.

[0023] Preferably, in step 2), based on 100 mL of the system after pH adjustment or aging for dialysis, the amount of each protective agent is independently 6-18 g (e.g., 6 g, 8 g, 10 g, 12 g, 14 g, 16 g, 18 g, etc.).

[0024] Preferably, the lipid nanoparticle solution in step 1) comprises: lipids, buffer solution and protectant; and further comprises therapeutic or preventive agents.

[0025] Preferably, step 1) of preparing the lipid nanoparticle solution includes: mixing a therapeutic agent or preventive agent and a buffer solution for encapsulation to obtain an aqueous phase; mixing 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 with a buffer solution for ultrafiltration for ultrafiltration, and then mixing with a protective agent to obtain a lipid nanoparticle solution.

[0026] Preferably, the organic solvent includes ethanol.

[0027] Preferably, the lipids include cationic lipids, neutral lipids, accessory lipids, and long-circulating lipids.

[0028] Preferably, the cationic lipid is selected from any one or a combination of at least two of the following compounds:

[0029] (1) A compound of formula I, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G1 is C 1～6 Alkylene; G2 is C 2～8 Alkylene; G3 is C 1～3 Alkylene; L1 is C 6～15 Straight-chain alkyl; L2 is C12～25 Branched alkyl groups;

[0030]

[0031] Formula I

[0032] (2) A compound of formula II, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G1 is C 2~8 Alkylene; G2 is C 2~8 Alkylene; L1 is -C(O)O- or -OC(O)-; L2 is -C(O)O- or -OC(O)-; R1 is C 6~25 Straight-chain or branched alkyl; R2 is C 6~25 Straight-chain or branched alkyl; G3 is HO(CH2)2- or HO(CH2)3-; G4 is HO(CH2)2- or HO(CH2)3-; L is (CH2)2-, -(CH2)3- or -(CH2)4-;

[0033]

[0034] Formula II

[0035] (3) A compound of formula III, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein: G1 is C 1~6 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~20 Straight-chain or branched alkyl; R2 is C 12~25 Branched alkyl; G3 is: HO(CH2)2N(CH3)(CH2)2-, HO(CH2)2N(CH2CH3)(CH2)2-, (HO(CH2)2)2N(CH2)2-, CH3O(CH2)2N(CH3)(C H2)2-, (CH3)2N(CH2)3SC(O)O(CH2)2-, (CH3)2N(CH2)3SC(O)-, CH3NH(CH2)2N(CH3)(CH2)2- or CH3CH2NH(CH2)2-;

[0036]

[0037] Formula III

[0038] (4) A compound of formula IV, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G1 is C 1~8 Alkylene; G2 is C 2~8 Alkylene; R1 is C 6~25 Straight-chain or branched alkyl; R2 is C 12~25Straight-chain or branched alkyl group; G3 is: HO(CH2)2N(R3)CH2CH(OH)CH2-, where R3 is -CH3, -CH2CH3 or -CH2CH2OH. ;

[0039]

[0040] Formula IV

[0041] (5) A compound of formula V, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein G 1 and G 2 Each is independently unsubstituted C6-C 10 Alkylene; G 3 For unsubstituted C1-C 12 Alkylene; R 1 and R 2 Each independently is C6-C 24 Alkyl or C6-C 24 alkenyl; R 3 -H, -OR 5 -CN, -C(=O)OR 4 -OC(=O)R 4 Or –NR 5 C(=O)R 4 ;R 4 For C1-C 12 hydrocarbon group; and R 5 It is a -H or C1-C6 hydrocarbon group;

[0042]

[0043] Formula V

[0044] (6) A compound of formula VI, or its N-oxide, solvate, pharmaceutically acceptable salt or stereoisomer, wherein R4 is -(CH2). n Q or -(CH2) n CHQR; Q represents -OR, -OH, or -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 heterocyclic rings; each R is independently selected from C 1-3 Alkyl, C2-3 Alkenyl or H; X is independently selected from -F, -Cl, -Br or -I; n is 1, 2 or 3;

[0045]

[0046] Style VI

[0047] (7) Compounds of formula VII, or their N-oxides, solvates, pharmaceutically acceptable salts, or stereoisomers,

[0048]

[0049] Equation VII

[0050] Preferably, the cationic lipid is selected from...

[0051] , ,

[0052] , and Any one of the groups or a combination of at least two of them.

[0053] Preferably, the neutral lipid is selected from any one or a combination of at least two of the group consisting of 1,2-distearyl-sn-glycerol-3-phosphate choline (DSPC), 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC), 1,2-dimyristoyl-sn-glycerol-3-phosphate choline (DMPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), and 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE).

[0054] Preferably, the auxiliary 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 their derivatives.

[0055] Preferably, the long-circulating lipid is selected from any one or a combination of at least two of the group consisting of distearylphosphatidylethanolamine polyethylene glycol 2000 (DSPE-PEG2000), dimyristoylglycerol-3-methoxy polyethylene glycol 2000 (DMG-PEG2000), and methoxy polyethylene glycol bis(tetradecylacetamide) (ALC-0159).

[0056] Preferably, the molar ratio of the cationic lipid, neutral lipid, auxiliary lipid, and long-circulating lipid is (30~60):(5~15):(30~50):(0.5~5).

[0057] The values ​​mentioned above (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.; (0.5~5) can be, for example, 0.5, 1, 2, 3, 4, 5, etc.

[0058] Preferably, the therapeutic or preventative agent is a nucleic acid.

[0059] Preferably, the nucleic acid is ribonucleic acid (RNA).

[0060] Preferably, the nucleic acid is deoxyribonucleic acid (DNA).

[0061] Preferably, the RNA is selected from any one or a combination of at least two of the following groups: 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).

[0062] Preferably, the RNA is mRNA.

[0063] Preferably, the buffer solution used for encapsulation is a citrate buffer solution.

[0064] Preferably, the pH of the citrate buffer solution is 3 to 5 (e.g., it can be 3, 3.5, 4, 4.5, 5, etc.).

[0065] Preferably, the concentration of the therapeutic or preventative agent in the aqueous phase is 0.2-1.5 mg / mL (e.g., 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.).

[0066] Preferably, the buffer solution for ultrafiltration is selected from any one of the group consisting of tromethamine-hydrochloride / tromethamine (Tris-HCl / Tris) buffer, Duchenne phosphate (DPBS) buffer, and phosphate buffer, or a combination of at least two of them.

[0067] Preferably, the pH of the buffer solution used for ultrafiltration is 3 to 10 (e.g., it can be 3, 4, 5, 6, 7, 8, 9, 10, etc.).

[0068] Preferably, the volume ratio of the lipid nanoparticle intermediate solution to the buffer solution for ultrafiltration is 1:2 to 1:15 (e.g., it 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.).

[0069] Preferably, the protective agent is selected from any one of the group consisting of sucrose, trehalose, and maltose, or a combination of at least two of them.

[0070] Compared with the prior art, the beneficial effects of the present invention are reflected in the following: by adjusting the physicochemical conditions of the non-freeze-dried sample before the freeze-drying process, the particle size difference between the freeze-dried sample and the non-freeze-dried sample is reduced, while keeping the lipid composition (formula) and microfluidic control preparation parameters (process) unchanged, which can effectively avoid changing the uptake mechanism and efficacy. Attached Figure Description

[0071] To more clearly illustrate the technical solutions in the specific embodiments of this disclosure or the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of this disclosure. For those skilled in the art, various changes made within the spirit and scope of this disclosure can also be regarded as the scope intended by the accompanying drawings of this disclosure.

[0072] Figure 1 This is a graph showing the difference in particle size of samples before and after freeze-drying for Comparative Example 1-1, Example 1-1, and Example 1-3. Comparative Example 1-1 is an LNP solution without sodium chloride, Example 1-1 is an LNP solution with 1 mg / mL sodium chloride, and Example 1-3 is an LNP solution with 5 mg / mL sodium chloride.

[0073] Figure 2 This is a graph showing the difference in encapsulation efficiency of samples before and after freeze-drying for Comparative Example 1-1, Example 1-1, and Example 1-3. Comparative Example 1-1 is an LNP solution without sodium chloride, Example 1-1 is an LNP solution with 1 mg / mL sodium chloride, and Example 1-3 is an LNP solution with 5 mg / mL sodium chloride.

[0074] Figure 3The figures show the particle size differences of Comparative Example 2-1, Example 2-4, Comparative Example 1-1, and Example 2-1 before and after lyophilization. Comparative Example 2-1 and Comparative Example 1-1 are LNP solutions prepared using 1 / 2 DPBS or Tris / Tris-HCl as buffer and then directly lyophilized. Examples 2-4 and Example 2-1 are lyophilized using 1 / 2 DPBS or Tris / Tris-HCl as buffer and then adjusted to pH 8.0.

[0075] Figure 4 This is a graph showing the difference in encapsulation efficiency of samples before and after lyophilization in Comparative Example 2-1, Example 2-4, Comparative Example 1-1, and Example 2-1. Comparative Example 2-1 and Comparative Example 1-1 are LNP solutions prepared using 1 / 2 DPBS or Tris / Tris-HCl as buffer and then directly lyophilized. Examples 2-4 and Example 2-1 are lyophilized using 1 / 2 DPBS or Tris / Tris-HCl as buffer and then adjusted to pH 8.0.

[0076] Figure 5 This is a particle size difference graph before and after lyophilization of Comparative Examples 2-1, 3-9, 3-10, 3-4, 3-7, 3-8, 3-5, 3-9, 3-10, 3-6, 3-11, and 3-12, using 1 / 2 DPBS as buffer. Comparative Example 2-1 represents 0 days of undried processing; Comparative Example 3-9 represents 0 days of undried processing followed by dialysis and the addition of 8.7% (m / V) sucrose; Comparative Example 3-10 represents 0 days of undried processing followed by dialysis and the addition of 15% (m / V) sucrose; and Comparative Example 3-4 represents 5 days of undried processing. Examples 3-7 involved aging for 5 days, dialysis, and the addition of 8.7% (m / V) sucrose; Examples 3-8 involved aging for 5 days, dialysis, and the addition of 15% (m / V) sucrose; Comparative Examples 3-5 involved aging for 10 days; Examples 3-9 involved aging for 10 days, dialysis, and the addition of 8.7% (m / V) sucrose; Examples 3-10 involved aging for 10 days, dialysis, and the addition of 15% (m / V) sucrose; Comparative Examples 3-6 involved aging for 15 days; Examples 3-11 involved aging for 15 days, dialysis, and the addition of 8.7% (m / V) sucrose; and Examples 3-12 involved aging for 15 days, dialysis, and the addition of 15% (m / V) sucrose.

[0077] Figure 6This is a graph showing the particle size difference before and after lyophilization of Comparative Examples 1-1, 3-7, 3-8, 3-1, 3-1, 3-2, 3-2, 3-3, 3-4, 3-3, 3-5, and 3-6, using Tris / Tris-HCl as buffer. Comparative Example 1-1 represents unripened samples - 0 days (0d); Comparative Example 3-7 represents unripened samples - 0d - dialysis - addition of 8.7% (m / V) sucrose; Comparative Example 3-8 represents unripened samples - 0d - dialysis - addition of 15% (m / V) sucrose; and Comparative Example 3-1 represents ripened samples - Example 3-1: 5-day maturation, dialysis, and addition of 8.7% (m / V) sucrose; Example 3-2: 5-day maturation, dialysis, and addition of 15% (m / V) sucrose; Comparative Example 3-2: 10-day maturation; Example 3-3: 10-day maturation, dialysis, and addition of 8.7% (m / V) sucrose; Example 3-4: 10-day maturation, dialysis, and addition of 15% (m / V) sucrose; Comparative Example 3-3: 15-day maturation; Example 3-5: 15-day maturation, dialysis, and addition of 8.7% (m / V) sucrose; Example 3-6: 15-day maturation, dialysis, and addition of 15% (m / V) sucrose.

[0078] Figure 7 The figures show the particle size differences before and after lyophilization of Comparative Example 4-2, Example 3-9, and Example 3-10, which used 1 / 2 DPBS as buffer. Comparative Example 4-2 involved two ultrafiltrations, Example 3-9 involved aging-10d-dialysis-addition of 8.7% (m / V) sucrose, and Example 3-10 involved aging-10d-dialysis-addition of 15% (m / V) sucrose.

[0079] Figure 8 The graph shows the particle size difference before and after lyophilization of Comparative Example 4-1, Example 3-3, and Example 3-4, which used Tris / Tris-HCl as buffer. Comparative Example 4-1 involved two ultrafiltrations, Example 3-3 involved aging-10d-dialysis-addition of 8.7% (m / V) sucrose, and Example 3-4 involved aging-10d-dialysis-addition of 15% (m / V) sucrose. Detailed Implementation

[0080] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the described embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0081] The present invention will be further described below with reference to embodiments. However, the present invention 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 specified are conventional conditions in the industry. In the specific embodiments of the present invention, the raw materials used are all commercially available. Unless otherwise stated, percentages in the context are weight percentages, and all temperatures are given in degrees Celsius. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0082] The therapeutic or preventive agent used in all embodiments is mRNA, but other therapeutic or preventive agents may be used instead.

[0083] Example 1-1 Preparation of LNP lyophilized formulation (adding salt to LNP solution)

[0084] 1.1 Preparation of LNP solution

[0085] (1) Aqueous phase preparation

[0086] The mRNA stock solution was dissolved in pH 4.0 citrate buffer to prepare an aqueous phase with a concentration of 0.789 mg / mL.

[0087] (2) Organic phase preparation

[0088] The cationic lipid YK009 (Yuekang Kaiyue), the neutral lipid DSPC (Nippon Seika), the auxiliary lipid chol (Nippon Seika), and the long-circulating lipid mPEG-DMG (Sinobang Bio) were dissolved in anhydrous ethanol at a mass ratio of 49:10:39.5:1.5 to prepare an organic phase.

[0089] (3) Encapsulation

[0090] The organic and aqueous phases were rapidly mixed and encapsulated in a microfluidic device (manufacturer: Precision NanoSystems) to prepare an LNP intermediate solution. The encapsulation method involved an organic-to-aqueous phase influent ratio of 1:3, a total influent flow rate of 12 mL / min, and the chip model was Ignite NxGen.

[0091] (4) Ultrafiltration

[0092] The LNP intermediate solution was diluted with 8 times its volume of Tris / Tris-HCl buffer (tromethamine / tromethamine-hydrochloride buffer), and then concentrated manually by ultrafiltration. The ultrafiltration membrane was Hydrosart (Sartorius), with a pore size of 300 kDa, a main pump flow rate of 400 mL / min, and a transmembrane pressure (TMP) of 0.1 bar. Ultrafiltration was stopped when the volume was concentrated to approximately 100 mL.

[0093] (5) Add protective agent

[0094] Using the LNP sample ultrafiltered with Tris / Tris-HCl buffer, sucrose was added at a rate of 8.7 g per 100 mL of solution (denoted as 8.7%, m / V) to obtain the LNP solution.

[0095] 1.2 Add salt to LNP solution

[0096] Sodium chloride was added to the LNP solution prepared in 1.1 until the final concentration of sodium chloride in the system was 1 mg / mL, thus obtaining an intermediate solution.

[0097] 1.3 Preparation of LNP lyophilized formulations

[0098] The newly prepared intermediate solution from step 1.2 was poured into Schott vials. The vials were then placed on pre-cooled freeze-drying shelves in a LYO 0.5 freeze dryer (Dongfulong) and kept at -45°C for 4 hours to ensure uniform freezing of all vials. During the drying phase, the pressure inside the drying chamber was reduced to 5 Pa, and the freeze-drying shelf temperature was maintained at -45°C. The samples were then dried for 60 hours. To effectively remove any residual moisture, the shelf temperature was gradually increased to 25°C over 2 hours after the drying process ended, and maintained for 6 hours. After stoppering, freeze-drying was completed to obtain the LNP lyophilized formulation.

[0099] Examples 1-2

[0100] The only difference between this embodiment and Embodiment 1-1 is that sodium chloride is added in step 1.2 until the final concentration of sodium chloride in the system is 3 mg / mL. Otherwise, the same applies as in Embodiment 1-1.

[0101] Examples 1-3

[0102] The only difference between this embodiment and Embodiment 1-1 is that sodium chloride is added in step 1.2 until the final concentration of sodium chloride in the system is 5 mg / mL. Otherwise, the same applies as in Embodiment 1-1.

[0103] Comparative Example 1-1

[0104] This comparative example uses the conventional method of preparing LNP lyophilized formulation with existing technology, that is, the prepared LNP solution is not treated and is directly lyophilized. The only 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 in step 1.3. Other aspects are the same as in Example 1-1.

[0105] Example 2-1 Preparation of LNP lyophilized formulation (adjusting the pH of LNP solution)

[0106] 2.1 Preparation of LNP solution

[0107] The experimental procedure is the same as 1.1 in Example 1-1.

[0108] 2.2 Adjusting the pH of the LNP solution

[0109] The LNP solution prepared in section 2.1 was dialyzed with Tris solution (12 h). The pH of the Tris-base solution was adjusted by adding an appropriate amount of HCl to a final pH of 8. Sucrose was then added at a rate of 8.7 g per 100 mL of solution (denoted as 8.7%, m / V).

[0110] 2.3 Preparation of LNP lyophilized formulations

[0111] The experimental procedure is the same as 1.3 in Example 1-1.

[0112] Example 2-2

[0113] The only difference between this embodiment and Embodiment 2-1 is that the final pH value is controlled to be 9 in step 2.2; otherwise, the same applies as in Embodiment 2-1.

[0114] Example 2-3

[0115] The only difference between this embodiment and Embodiment 2-1 is that the final pH value is controlled to be 10 in step 2.2, while the rest is the same as in Embodiment 2-1.

[0116] Examples 2-4

[0117] The only difference between this embodiment and embodiment 2-1 is that in step 2.1, step (4) of ultrafiltration, 8 times the volume of Tris / Tris-HCl buffer (tromethamine / tromethamine-hydrochloric acid buffer) is replaced with 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) for dilution. The rest is the same as in embodiment 2-1.

[0118] Examples 2-5

[0119] The only difference between this embodiment and embodiment 2-2 is that in step 2.1, step (4) of ultrafiltration, 8 times the volume of Tris / Tris-HCl buffer (tromethamine / tromethamine-hydrochloric acid buffer) is replaced with 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) for dilution. The rest is the same as in embodiment 2-2.

[0120] Examples 2-6

[0121] The only difference between this embodiment and embodiments 2-3 is that in step 2.1, step (4) of ultrafiltration, 8 times the volume of Tris / Tris-HCl buffer (tromethamine / tromethamine-hydrochloric acid buffer) is replaced with 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline). The rest is the same as in embodiments 2-3.

[0122] Comparative Example 2-1

[0123] This comparative example uses the conventional method of preparing LNP lyophilized formulation with existing technology, that is, the prepared LNP solution is not treated and is directly lyophilized. The only difference between this example and Examples 2-4 is that step 2.2 is not performed, and the LNP solution prepared in step 2.1 is directly subjected to the freeze-drying in step 2.3. Other aspects are the same as in Examples 1-4.

[0124] Example 3-1 Preparation of LNP lyophilized formulation (LNP solution maturation)

[0125] 3.1 Preparation of LNP solution

[0126] The experimental procedure is the same as 1.1 in Example 1-1.

[0127] 3.2 LNP solution aging

[0128] The LNP solution prepared in 3.1 was aged (i.e., stored at 5°C with an allowable temperature fluctuation range of 2~8°C) for 5 days. The aged LNP solution was dialyzed, and then sucrose was added at a rate of 8.7 g per 100 mL of solution (denoted as 8.7%, m / V).

[0129] 3.3 Preparation of LNP lyophilized formulations

[0130] The experimental procedure is the same as 1.3 in Example 1-1.

[0131] Example 3-2

[0132] The only difference between this embodiment and embodiment 3-1 is that the amount of sucrose added in step 3.2 is 15%, while the rest is the same as in embodiment 3-1.

[0133] Example 3-3

[0134] The only difference between this embodiment and embodiment 3-1 is that the maturation period in step 3.2 is 10 days; otherwise, refer to embodiment 3-1.

[0135] Examples 3-4

[0136] The only difference between this embodiment and embodiment 3-1 is that the maturation period in step 3.2 is 10 days and the amount of sucrose added is 15%. Other aspects are the same as in embodiment 3-1.

[0137] Examples 3-5

[0138] The only difference between this embodiment and embodiment 3-1 is that the maturation period in step 3.2 is 15 days; otherwise, refer to embodiment 3-1.

[0139] Examples 3-6

[0140] The only difference between this embodiment and embodiment 3-1 is that the maturation period in step 3.2 is 15 days and the amount of sucrose added is 15%. Other aspects are the same as in embodiment 3-1.

[0141] Comparative Example 3-1

[0142] The only difference between this comparative example and Example 3-1 is that, after maturation in step 3.2, dialysis is not performed and sucrose is not added; otherwise, the process is the same as in Example 3-1.

[0143] Comparative Example 3-2

[0144] The only difference between this comparative example and Example 3-1 is that the maturation period in step 3.2 is 10 days, no dialysis is performed after maturation, and no sucrose is added. Otherwise, the same applies as in Example 3-1.

[0145] Comparative Example 3-3

[0146] The only difference between this comparative example and Example 3-1 is that the maturation period is 15 days, no dialysis is performed after maturation, and no sucrose is added. Otherwise, it is the same as Example 3-1.

[0147] Examples 3-7 to 3-12

[0148] The only difference between Examples 3-7 to 3-12 and Examples 3-1 to 3-6 is that the 8-fold volume of Tris / Tris-HCl buffer (tromethamine / tromethamine-hydrochloride buffer) used in step 3.1 (4) of ultrafiltration in Examples 3-1 to 3-6 is replaced with 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) to obtain Examples 3-7 to 3-12. The other steps are the same as in Examples 3-1 to 3-6.

[0149] Comparative Examples 3-4 to 3-6

[0150] The only difference between Comparative Examples 3-4 to 3-6 and Comparative Examples 3-1 to 3-3 is that the 8-fold volume of Tris / Tris-HCl buffer used in step 3.1 (4) of the ultrafiltration in Comparative Examples 3-1 to 3-3 was replaced with 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) to obtain Comparative Examples 3-4 to 3-6. The other steps were the same as those in Comparative Examples 3-1 to 3-3.

[0151] Comparative Examples 3-7

[0152] The only difference between this comparative example and Example 3-1 is that the ripening process is not performed in step 3.2; otherwise, the process is the same as in Example 3-1.

[0153] Comparative Examples 3-8

[0154] The only difference between this comparative example and Example 3-1 is that no ripening is performed in step 3.2, and the amount of sucrose added is 15%. Otherwise, the same applies as in Example 3-1.

[0155] Comparative Examples 3-9 to 3-10

[0156] The only difference between Comparative Examples 3-9 to 3-10 and Comparative Examples 3-7 to 3-8 is that the 8-fold volume of Tris / Tris-HCl buffer used in step 3.1 (4) of the ultrafiltration in Comparative Examples 3-7 to 3-8 was replaced with 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline) to obtain Comparative Examples 3-9 to 3-10. The other steps were the same as those in Comparative Examples 3-7 to 3-8.

[0157] Comparative Example 4-1: Preparation of LNP lyophilized formulation (LNP solution ultrafiltration twice)

[0158] This comparative example uses a method in the prior art that can reduce the particle size difference of LNP lyophilized formulations (A preparation method for mRNA-LNPs with improved properties. J Control Release. 2023 Dec;364:632-643.), which involves subjecting the prepared LNP solution to ultrafiltration twice before lyophilization.

[0159] 4.1. Preparation of LNP solution

[0160] The experimental procedure is the same as 1.1 in Example 1-1.

[0161] 4.2. Ultrafiltration of LNP solution twice

[0162] The LNP solution prepared in 4.1 was subjected to ultrafiltration again (tangential flow filtration, TFF), and then sucrose was added at a rate of 8.7 g per 100 mL of solution (denoted as 8.7%, m / V).

[0163] 4.3. Preparation of LNP lyophilized formulations

[0164] The experimental procedure is the same as 1.3 in Example 1-1.

[0165] Comparative Example 4-2

[0166] The only difference between this comparative example and comparative example 4-1 is that in step 4.1 (4) ultrafiltration, 8 times the volume of Tris / Tris-HCl buffer (tromethamine / tromethamine-hydrochloric acid buffer) is replaced with 1 / 2 volume of DPBS (Dulbecco's Phosphate-Buffered Saline). The rest is the same as comparative example 4-1.

[0167] Test Example 1: Determination of Sample Properties Before and After Lyophilization of Lipid Nanoparticles (LNPs)

[0168] The particle size, dispersibility, encapsulation efficiency, and integrity of the LNP samples prepared in all the above examples and comparative examples before and after reconstitution of the LNP lyophilized formulation were tested to evaluate the impact of different preparation methods on the properties of the lyophilized products.

[0169] (1) Particle size and dispersion

[0170] The particle size and size distribution of lipid nanoparticles are related to their encapsulation efficiency and stability, directly affecting the behavior of liposomes in body tissues. They are important parameters for evaluating their biochemical, biophysical, and drug delivery systems, influencing the in vivo biodistribution and pharmacokinetic properties of the product. Therefore, particle size and size distribution are important characterization indicators of the stability of LNP products.

[0171] The detection method is as follows:

[0172] Particle size and particle size distribution index (PDI): Particle size and PDI were measured using dynamic light scattering (DLS) with a nano-laser particle size analyzer (manufacturer: MALVERN; model: ZSU3305) at 25 ± 1℃. A 60 μL sample was placed in a quartz micro-sample cell for analysis. The detection parameters were set as follows:

[0173] Temperature: 25℃; Equilibrium time: 30 s; Material: Liposomes; Dispersion system: Water; Detection mode: Automatic.

[0174] Particle size and PDI (partial particle size distribution) are important characteristics of liposomes. During freeze-thaw cycles, lipids fuse, with smaller particles coalescing into slightly larger liposomes. Within a certain range, smaller particle size indicates better product quality and processing. PDI represents the particle size distribution; a lower PDI value indicates more uniform liposome particle size.

[0175] (2) Encapsulation rate

[0176] The stability of mRNA drugs is a major challenge in drug delivery. The primary responsibility of the delivery vector is to prevent mRNA degradation by RNA-cutting enzymes in vivo. Encapsulation efficiency is a key quality attribute of liposomes, referring to the percentage of drug content encapsulated within the lipid bilayer relative to the total drug dosage. A higher encapsulation efficiency indicates a greater proportion of mRNA encapsulated by the liposomes. A higher encapsulation efficiency means a higher proportion of protected mRNA, while unencapsulated mRNA is more easily degraded. Therefore, encapsulation efficiency is an important indicator of the stability of LNP products.

[0177] Detection method:

[0178] The encapsulation efficiency of lipid nanoparticles was determined using the Quant-it Ribogreen RNA Quantification Kit (Thermo Fisher Scientific, UK). The specific method is as follows:

[0179] The sample was diluted to 2.8 μg / mL. A portion was mixed with an equal volume (50 μL) of Triton X-100 and thoroughly demulsified for determining the total mRNA concentration. The other portion was mixed with an equal volume (50 μL) of TE (Tris-EDTA) for determining the concentration of free, unencapsulated mRNA. The samples with added TE (Tris-EDTA) and Triton X-100 were incubated at 37°C for a certain time. Then, 100 μL of Ribogreen reagent diluted 200-fold was added. After centrifugation to remove air bubbles, fluorescence was measured using a multi-plate reader at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. The plate was then read.

[0180] (3) Integrity

[0181] Although mRNA is protected by liposome encapsulation, it can still degrade during formulation preparation, transportation, and long-term storage, directly affecting the expression efficacy of nucleic acid drugs in vivo. Therefore, evaluating mRNA integrity is crucial for assessing product quality and has become an important indicator of LNP product stability.

[0182] Detection method:

[0183] The specific method is as follows: mRNA is extracted from mRNA-LNP using a kit, diluted, and denatured at 65 °C. After preparation, the sample is placed on ice. Separately, gel buffer is prepared, and the prepared sample solution, gel buffer, and ribonuclease-free water are added sequentially to the sample dish. The integrity of the sample mRNA is determined using the PA800 Plus gel electrophoresis principle.

[0184] Experimental results:

[0185] The particle size, particle size distribution coefficient (PDI), encapsulation efficiency, and integrity results for each sample in all examples and comparative examples are shown in Tables 1-4. The particle size statistics were performed using two-way ANOVA and Bonferroni post-hoc tests. For each particle size and PDI test, three samples were taken, and the average value was calculated.

[0186] Table 1. Particle size data of samples before and after freeze-drying

[0187]

[0188] Table 2. Polydispersity Index (PDI) of particle size distribution of samples before and after freeze-drying.

[0189]

[0190] Table 3 Encapsulation efficiency data of samples before and after freeze-drying

[0191]

[0192] Table 4. Sample integrity data before and after freeze-drying

[0193]

[0194] Analysis of experimental results:

[0195] 1. Adding salt to the LNP solution before freeze-drying can effectively reduce the particle size difference of the sample before and after freeze-drying, and also ensure the stability of the LNP freeze-dried product.

[0196] As can be seen from the data in Table 1, the method of adding salt to LNP solution before freeze-drying (Examples 1-1 to 1-3) significantly reduces the particle size difference of samples before and after freeze-drying compared to the conventional method of direct freeze-drying without salt (Comparative Example 1-1). The average particle size difference was 48.4 nm when LNP solution was freeze-dried without NaCl, while the average particle size differences were only 16.1 nm, 13.0 nm, and 11.9 nm after adding 1 mg / mL, 3 mg / mL, and 5 mg / mL NaCl to the LNP solution, respectively. Figure 1 ).

[0197] As can be seen from Tables 2, 3, and 4, compared with direct freeze-drying of LNP solution, adding salt to LNP solution before freeze-drying results in lower particle size distribution and encapsulation efficiency of the freeze-dried product. Figure 2 The changes in integrity are consistent with the changes in the overall structure.

[0198] This indicates that the method of adding salt to the LNP solution before freeze-drying in this invention can significantly reduce the particle size difference of the product before and after freeze-drying, and also ensure the stability of the LNP freeze-dried product.

[0199] 2. Adjusting the pH of the LNP solution to 8-10 before freeze-drying can effectively reduce the particle size difference of the sample before and after freeze-drying, and also ensure the stability of the LNP freeze-dried product.

[0200] As can be seen from the data in Table 1, adjusting the pH of the LNP solution to 8-10 before freeze-drying (Examples 2-1-2-6) significantly reduced the particle size difference before and after freeze-drying compared to the conventional method of directly freeze-drying the LNP solution without adjusting the pH (Comparative Example 2-1). Direct freeze-drying of the LNP solution without pH adjustment resulted in an average particle size difference of 40.3 nm, while adjusting the pH of the LNP solution to 8, 9, and 10 resulted in average particle size differences of only 4.1 nm, 2.2 nm, and 0.7 nm, respectively. Figure 3 ).

[0201] As can be seen from Tables 2, 3, and 4, adjusting the pH of the LNP solution to 8-10 before freeze-drying resulted in better particle size distribution and encapsulation efficiency compared to directly freeze-drying the LNP solution without pH adjustment. Figure 4 The changes in integrity are basically consistent with those in other aspects.

[0202] This indicates that the method of adjusting the pH of the LNP solution to 8-10 before freeze-drying in this invention can significantly reduce the particle size difference of the product before and after freeze-drying, and also ensure the stability of the LNP freeze-dried product.

[0203] 3. After the LNP solution is aged, it is dialyzed, then a protective agent is added, and then it is lyophilized. This can effectively reduce the particle size difference of the sample before and after lyophilization, and ensure the stability of the LNP lyophilized product.

[0204] As can be seen from the data in Table 1, the LNP solution was aged, dialyzed, then a protective agent was added, and then freeze-dried (Examples 3-1 to 3-12). Compared with the conventional method of directly freeze-drying the LNP solution without aging (Comparative Example 1-1, Comparative Example 2-1), the particle size difference of the samples before and after freeze-drying was significantly reduced. Figure 5 and Figure 6 ).

[0205] As can be seen from Tables 2, 3 and 4, compared with direct freeze-drying after LNP solution aging, the changes in particle size distribution, encapsulation efficiency and integrity of the product are consistent after LNP solution aging followed by dialysis, addition of a protective agent and then freeze-drying.

[0206] This indicates that the LNP solution in this invention is aged, dialyzed, then protected, and then freeze-dried, which can significantly reduce the particle size difference before and after freeze-drying and ensure the stability of the LNP freeze-dried product.

[0207] Furthermore, compared with existing methods for reducing particle size differences in LNP lyophilized formulations (Comparative Example 4-1), which involves two ultrafiltrations of LNP, the LNP solution is aged, dialyzed, and then a protective agent is added before lyophilization (Examples 3-1 to 3-12). This significantly reduces the particle size difference of the samples before and after lyophilization. Figure 7 and Figure 8 ).

[0208] The above embodiments are merely illustrative of the technical solutions of this disclosure and are not intended to limit it. Although this disclosure 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 or all of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this disclosure should be included within the scope of protection of the claims of this disclosure.

Claims

1. A method for preparing a lyophilized lipid nanoparticle formulation, characterized in that, The preparation method includes the following steps: 1) Preparation of lipid nanoparticle solution; the lipid nanoparticle solution includes: lipids, buffer solution and protectant; and also includes therapeutic or preventive agents; the lipids include cationic lipids, neutral lipids, accessory lipids and long-circulating lipids in a mass ratio of (45~55):(5~15):(35~45):(0.5~3); the therapeutic or preventive agent is nucleic acid; 2) The lipid nanoparticle solution obtained in step 1) is aged, then dialyzed, and then mixed with a protective agent to obtain an intermediate solution; the aging method is to store the lipid nanoparticle solution at 2~8℃ for 10~15 days; the protective agent is selected from any one or a combination of at least two of the group consisting of sucrose, lactose, mannitol, maltose and trehalose. 3) Freeze-dry the intermediate solution obtained in step 2) to obtain the lyophilized lipid nanoparticle formulation; The cationic lipids are selected from 、 、 , and Any one or at least two of the constituent elements; The neutral lipid is selected from any one or a combination of at least two of the following groups: 1,2-distearatel-sn-glycerol-3-phosphocholine, 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine, 1,2-dimyristoyl-sn-glycerol-phosphocholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine. The auxiliary lipid is selected from any one or a combination of at least two of the group consisting of cholesterol, vitamin E and DC-cholesterol. The long-circulating lipid is selected from any one or a combination of at least two of the group consisting of distearylphosphatidylethanolamine polyethylene glycol 2000, dimyristoylglycerol-3-methoxy polyethylene glycol 2000, and methoxy polyethylene glycol bistetradecylacetamide.

2. The preparation method according to claim 1, characterized in that, In step 2), based on a dialysis system of 100 mL after aging, the amount of each protective agent is independently 6-18 g.

3. The preparation method according to claim 1, characterized in that, Step 1) The preparation of the lipid nanoparticle solution includes: mixing a therapeutic agent or preventive agent and a buffer solution for encapsulation to obtain an aqueous phase; mixing 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 with a buffer solution for ultrafiltration for ultrafiltration, and then mixing with a protective agent to obtain a lipid nanoparticle solution.

4. The preparation method according to claim 1, characterized in that, Step 1) The protective agent is selected from any one of the group consisting of sucrose, trehalose and maltose, or a combination of at least two of them.

5. The preparation method according to claim 1, characterized in that, The buffer solution used for encapsulation is a citrate buffer solution; the pH of the citrate buffer solution is 3-5; The buffer solution used for ultrafiltration is selected from any one or a combination of at least two of the group consisting of tromethamine-hydrochloride / tromethamine buffer, Durex phosphate buffer, and phosphate buffer. The pH of the buffer solution used for ultrafiltration is 3 to 10.

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