Nucleic acid-containing lipid nanoparticles with a low void ratio and method for producing the same

Abstract

This invention discloses a lipid nanoparticle composition for encapsulating nucleic acids and a method for producing the same. In this composition, the ratio of empty lipid nanoparticles (those not encapsulating nucleic acids) to the total number of lipid nanoparticles, i.e., the empty particle rate, is 10% or less. By the production method of this invention, after the lipid nanoparticles have sufficiently fused, the morphology and structure of the particles become more uniform, and the particles become more stable. Compared to particles produced by conventional methods, the nucleic acid-containing lipid nanoparticles of this invention are essentially all effective particles that encapsulate nucleic acids, which is advantageous for improving nucleic acid delivery efficiency, reducing the risk of inflammatory adverse reactions due to lipid components, and improving the efficacy and safety of nucleic acid drugs.

Description

Technical Field

[0001] Cross-reference to Related Applications This invention claims the priority of Chinese Patent Application No. 202310626158.9 filed on May 30, 2023 and Chinese Patent Application No. 202311497985.9 filed on November 10, 2023, and the entire contents of these two applications are incorporated herein by reference.

[0002] This invention belongs to the technical field of biomedicine. In particular, this invention relates to nucleic acid-containing lipid nanoparticles with a low airborne particle rate and a method for producing the same.

Background Art

[0003] Half a century ago, Friedmann and Roblin proposed that dysfunction caused by genetic diseases can be treated by introducing functional genes. Conventional drug targets are usually proteins, and nucleic acid drugs produce therapeutic effects by regulating gene expression. Thus, the method of introducing foreign nucleic acids into cells to remove defective genes is a very effective treatment means with high specificity and a sustainable therapeutic effect. Thirty years ago, nucleic acid drugs achieved extraordinary research progress, and some in vitro nucleic acid drugs and in vivo nucleic acid drugs for the prevention or treatment of genetic diseases such as infectious diseases, cancer, muscle, and retinal dystrophy have been approved or are in the late-stage development stage clinically.

[0004] Nucleic acid molecules have low stability and are easily degraded in vivo by nucleases. Furthermore, because nucleic acid molecules carry a large negative charge, they cannot penetrate the cell membrane and enter the cytoplasm to exert their therapeutic effects. Therefore, special delivery techniques are required to deliver nucleic acid drugs into cells. Currently, commonly used nucleic acid delivery techniques include lipid nanoparticles (LNPs), electroporation, protamine, cationic nanoemulsions, cationic lipid complexes, lipid polymer complexes, polymer nanoparticles, and inorganic nanoparticles. Electroporation involves creating cell membrane voids with high-voltage pulses to directly deliver nucleic acids to human cells; many of the remaining methods either protect nucleic acids from degradation or promote fusion of nucleic acids with the cell membrane to improve delivery efficiency. For example, protamine is a naturally occurring cationic protein that can complex electrically charged nucleic acids with nanoscale nucleic acid particles, thereby protecting them from degradation by serum nucleases. However, the strong binding between protamine and nucleic acids affects the intracellular protein expression efficiency of nucleic acid drugs using this technique.

[0005] Nucleic acid drugs, particularly mRNA vaccines, have played a crucial role during the burst and transmission of novel coronaviruses and have attracted widespread attention. Both the mRNA vaccines already on the market and the earlier RNA drug Onpattro® are delivered using LNPs. LNPs are the most clinically advanced non-viral gene delivery system. Compared to other types of nucleic acid drug delivery systems, LNPs offer numerous advantages, including high nucleic acid encapsulation rates, high potential for efficient transfection into cells, strong tissue permeability, and low cytotoxicity and immunogenicity. These advantages make LNPs a superior nucleic acid delivery system.

[0006] LNPs are generally formed by rapidly mixing lipids in an organic phase with nucleic acids in an aqueous phase, followed by self-assembly, and then removing the organic solvent by dialysis or ultrafiltration and replacing it with a buffer solution. A typical method for producing LNPs and the particle formation mechanism is as follows: (1) Mixing process: When lipids are dissolved in a solvent and nucleic acids are dissolved in an acidic (e.g., pH 4.0) buffer solution and the two phases are mixed, the system pH is significantly lower than the pKa of the ionizable lipids. As a result, the ionizable cationic lipids become positively charged after protonation, creating an electrostatic attraction with the negatively charged nucleic acids. On the other hand, hydrophobic interactions also occur due to the low water solubility of the lipids. These act as driving forces, causing the lipids to encapsulate the nucleic acids and self-assemble to form lipid nanoparticles. Observations using a low-temperature transmission electron microscope have shown that under these conditions, at least two forms of LNPs exist simultaneously: vesicle-like particles containing a two-layer structure and particles with a high electron-density structure. (2) Dialysis or ultrafiltration process: In the dialysis or ultrafiltration process, when the mixed solution is replaced with a physiological pH (e.g., pH 7.4) buffer, the ionizable cationic lipids are deprotonated, most of the positive charge is lost, the electrostatic repulsion between particles decreases, the ionizable cationic lipids move inward to form hydrophobic amorphous nuclei, the bilayer structure of the particles is broken, fusion between particles occurs to compensate for the decrease in the ratio of surface lipids to core lipids, and the size of the fused LNPs increases (Kulkarni JA, et al. On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA. ACS Nano. 2018 May 22;12(5):4787-4795). Various literatures report the existence of various forms of LNPs after dialysis. Examples include spherical high-electron-density structures, or non-spherical high-electron-density structures containing vesicles of various size ratios. These suggest that differences in formulation and manufacturing process factors can lead to variations in the physicochemical properties of the final LNP product after fusion. Furthermore, it has already been proven that changes in the physicochemical properties (composition and structure) of LNPs affect their stability and safety.

[0007] The instability of LNPs and the need for cryogenic storage limit the storage, transport, and circulation of nucleic acid drugs, particularly mRNA vaccines, impacting cost and access. Two novel coronavirus mRNA vaccines already on the market, BNT162b2 and mRNA-1273, require storage at -80 to -60°C and -25 to -15°C, respectively, with a shelf life of only six months. During long-term storage of LNPs, particle aggregation and sedimentation, oxidative hydrolysis of mRNA, and irreversible changes in phase state after freeze-thaw cycles can occur. In a neutron scattering evaluation of the thermal stability of Pfizer-BioNtech's COVID-19 mRNA vaccine, changes in the molecular vibration spectrum during the freeze-thaw cycle indicated a two-stage thawing process when the vaccine was first warmed from a low-temperature frozen state, indicating a two-phase morphology. Upon warming after refreezing, a single-stage thawing process was observed, indicating a single-phase morphology. In other words, after a single freeze-thaw cycle, the biphasic morphology of Pfizer-BioNtech's COVID-19 mRNA vaccine irreversibly changed to a monophasic morphology with significantly increased molecular fluidity and flexibility, resulting in irreversible adverse effects on the vaccine's long-term storage stability (Mamontov E, et al. Melting and Re-Freezing Leads to Irreversible Changes in the Morphology and Molecular-Level Dynamics of Pfizer-BioNTech COVID-19 Vaccine. Medicina(Kaunas). 2021 Dec 9;57(12):1343).

[0008] With the widespread vaccination of COVID-19 mRNA vaccines during this pandemic, the safety of nucleic acid drugs has also attracted considerable attention. A large-scale study involving tens of millions of people has for the first time quantified the risk of cardiac adverse events (myocarditis, pericarditis, arrhythmias) associated with COVID-19 vaccination. The study found that vaccination with mRNA and adenovirus vaccines in adults was associated with an increased risk of myocarditis. In men under 40 years of age, the number of excess myocarditis events per million people after a second dose of mRNA-1273 was higher than after SARS-CoV-2 infection and significantly higher than that after mRNA vaccine BNT162b2 and adenovirus vaccine ChAdOx1. Differences in the incidence of cardiac-related adverse events among different mRNA vaccines may be related to differences in in vivo kinetics due to the components and structure of mRNA LNPs (Patone M, et al. Risk of Myocarditis After Sequential Doses of COVID-19 Vaccine and SARS-CoV-2 Infection by Age and Sex. Circulation. 2022 Sep 6;146(10):743-754).

[0009] Compared to conventional vaccines such as inactivated vaccines and subunit vaccines, mRNA vaccines can promote very potent humoral and cellular immune responses. Studies have shown that empty LNPs (Lapse-celled nanoparticles) themselves, which do not encapsulate nucleic acids, can stimulate specific pathways in the immune system, leading to specific activation of the immune system—an advantage of LNPs as vaccine delivery carriers. However, at the same time, the inflammatory nature of LNPs also causes typical inflammatory side effects such as pain, swelling, and fever. As vaccine-related technologies advance, there is a need to find a balance between the benefits of LNP immune responses and the adverse inflammatory reactions (Ndeupen S, et al. The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory. iScience. 2021 Dec 17;24(12):103479). The ratio of empty LNPs in LNPs is a crucial characteristic to note for nucleic acid delivery carriers. Studies have shown that conventionally produced LNPs may contain 40%–80% empty particles (Li S, et al. Payload distribution and capacity of mRNA lipid nanoparticles. Nat Commun. 2022 Sep 23;13(1):5561). Empty LNPs cannot deliver nucleic acids into cells and instead increase the risk of inflammatory adverse reactions, impacting the safety of nucleic acid drugs.

[0010] Therefore, there remains a need to provide lipid nanoparticles with improved safety, stability, and efficacy for the delivery of therapeutic or prophylactic nucleic acid drugs. [Overview of the Initiative]

[0011] One object of the present invention is to provide a lipid nanoparticle composition with a low empty particle ratio for delivering therapeutic or preventive nucleic acid drugs.

[0012] Another object of the present invention is to provide a method for producing lipid nanoparticle compositions with a low empty particle ratio for the delivery of therapeutic or preventive nucleic acid pharmaceuticals.

[0013] The technical problem that this invention aims to solve is to provide a lipid nanoparticle composition with a low void ratio for encapsulating nucleic acids.

[0014] Another technical problem that the present invention aims to solve is to provide a lipid nanoparticle composition having a low void ratio and improved thermal stability for encapsulating nucleic acids.

[0015] Another technical problem that the present invention aims to solve is to provide a lipid nanoparticle composition for encapsulating nucleic acids that has a low void ratio, improved thermal stability, and improved safety.

[0016] Another technical problem that the present invention aims to solve is to provide a method for producing a nucleic acid-containing lipid nanoparticle composition having one or more of the aforementioned advantages.

[0017] To solve the aforementioned technical problems, the inventors conducted research on the manufacturing process of nucleic acid-containing lipid nanoparticles and found that by increasing the degree of particle fusion, nucleic acid-containing lipid nanoparticles with a low empty particle ratio can be obtained. The obtained nucleic acid-containing lipid nanoparticles have improved safety, stability, and efficacy, thereby meeting the requirements of the prior art.

[0018] In a first embodiment of the present invention, a lipid nanoparticle composition for encapsulating nucleic acids is provided, characterized in that the empty particle ratio of the lipid nanoparticles is 10% or less, where the empty particle ratio means the ratio of the number of empty lipid nanoparticles that do not encapsulate nucleic acids to the total number of lipid nanoparticles in the composition. In some embodiments, the lipid nanoparticles have an empty particle ratio of 9% or less, preferably 8.5% or less, more preferably 6% or less, even more preferably 3% or less, or any value within the above range, for example, 2%, 3%, 4%, 5%, 6%, 7%, 7.1%, 7.6%, 7.7%, 7.9%, 8%, 8.3%, or 9%.

[0019] In some embodiments, the lipid nanoparticles have an average particle diameter of 50 nm to 150 nm, preferably 70 nm to 120 nm, more preferably 90 nm to 110 nm, or any value within the above range, for example, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 101 nm, 102 nm, 103 nm, 104 nm, 105 nm, 106 nm, 107 nm, 108 nm, 109 nm, 110 nm, 111 nm, 112 nm, 113 nm, 114 nm, 115 nm, 116 nm, 117 nm, 118 nm, 119 nm, or 120 nm.

[0020] In some embodiments, the encapsulation rate of lipid nanoparticles is 80% or more, preferably 85% or more, and more preferably 90% or more.

[0021] In some embodiments, a lipid nanoparticle composition encapsulating nucleic acids is provided, where the lipid nanoparticles are: (1) nucleic acids, and (2) A lipid component comprising ionizable cationic lipids, structural lipids, helper lipids, and surfactants.

[0022] In some embodiments, the nucleic acid is selected from mRNA, small interfering RNA (siRNA), DNA, plasmid, antisense oligonucleotide, ribozyme, etc., and the nucleic acid encodes a target therapeutic product and is complexed with one or more lipids, encapsulated with one or more lipids, or associated with one or more lipids to form lipid nanoparticles.

[0023] In some embodiments, the nucleic acid is selected from mRNA, which comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any one nucleotide sequence of SEQ ID NO: 1-6, or which comprises an amino acid sequence whose encoded coronavirus antigen has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of SEQ ID NO: 7, or which comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any one nucleotide sequence of SEQ ID NO: 8-26. Preferably, the mRNA comprises the nucleotide sequence of SEQ ID NO: 1, and more preferably, the mRNA is the nucleotide sequence of SEQ ID NO: 1.

[0024] In some embodiments, the lipid component comprises ionizable cationic lipids, structural lipids, helper lipids, and surfactants, with a total molar content of 100% in mole percentages (mol%). In some embodiments, the lipid component comprises 20-60 mol% ionizable cationic lipids, 25-55 mol% structural lipids, 2-25 mol% helper lipids, and 0.5-15 mol% surfactants.

[0025] In some embodiments, the ionizable cationic lipid is selected from SM-102 (CAS number: 2089251-47-6), ALC-0315 (CAS number: 2036272-55-4), Dlin-MC3-DMA (CAS number: 1224606-06-7), DODMA (CAS number: 104162-47-2), C12-200 (CAS number: 1220890-25-4), and DlinDMA (CAS number: 871258-12-7).

[0026] In some embodiments, the structural lipid is selected from cholesterol or cholesterol derivatives.

[0027] In some embodiments, the helper lipid is selected from distearoyl phosphatidylcholine (DSPC), dioleoyl phosphatidylethanolamine (DOPE), dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylglycerol (DOPG), and dioleoyl phosphatidylserine (DOPS).

[0028] In some embodiments, the surfactant is selected from mPEG-DMG-2K (CAS number: 160743-62-4), ALC-0159 (CAS number: 1849616-42-7), polyethylene glycolylated distearoyl phosphatidylethanolamine (PEG-DSPE), methoxypolyethylene glycol bis(tetradecylpropylamine) (DTDA-PEG2000), and vitamin E polyethylene glycol succinate (TPGS).

[0029] In some embodiments, the lipid component of the lipid nanoparticle composition comprises 20-50 mol% of ionizable cationic lipids, preferably 30-50 mol% or 40-50 mol% of ionizable cationic lipids. For example, the lipid component may include 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, or 50 mol% of ionizable cationic lipids (e.g., SM-102 or Dlin-MC3-DMA). In some embodiments, the lipid component comprises 50 mol% of SM-102.

[0030] In yet another embodiment, the lipid component of the lipid nanoparticle composition comprises 50-60 mol% of ionizable cationic lipids. For example, the lipid component may include 51 mol%, 52 mol%, 53 mol%, 54 mol%, 55 mol%, 56 mol%, 57 mol%, 58 mol%, 59 mol%, or 60 mol% of ionizable cationic lipids (e.g., SM-102 or Dlin-MC3-DMA).

[0031] In some embodiments, the lipid component of the lipid nanoparticle composition contains 2-25 mol% of helper lipids, preferably 2-20 mol% of helper lipids, and more preferably 2-15 mol% of helper lipids. For example, the lipid component may include 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% of helper lipids (e.g., DSPC). In some embodiments, the lipid component contains 2-15 mol% of DSPC. In some embodiments, the lipid component contains 10 mol% of DSPC.

[0032] In some embodiments, the lipid component of the lipid nanoparticle composition contains 25-55 mol% structural lipids, preferably 30-40 mol% structural lipids. For example, the lipid component may include 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, or 40 mol% structural lipids (e.g., cholesterol). In some embodiments, the lipid component contains 38.5 mol% structural lipids. In some embodiments, the lipid component contains 38.5 mol% cholesterol.

[0033] In some embodiments, the lipid component of the lipid nanoparticle composition contains 0.5-15 mol% of a surfactant, preferably 0.5-10 mol% or 0.5-5 mol% of a surfactant, more preferably 1-2 mol% of a surfactant. For example, the lipid component may contain 1 mol%, 1.5 mol%, or 2 mol% of a surfactant (e.g., mPEG-DMG-2K). In some embodiments, the lipid component contains 1.5 mol% of mPEG-DMG-2K.

[0034] In some embodiments, the lipid component of the lipid nanoparticle composition comprises 50 mol% ionizable cationic lipid, 10 mol% helper lipid, 38.5 mol% structural lipid, and 1.5 mol% surfactant.

[0035] In some embodiments, the lipid components of the lipid nanoparticle composition include 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% mPEG-DMG-2K.

[0036] In some embodiments, the lipid nanoparticles have an N:P ratio of about 2:1 to about 30:1, preferably about 2:1 to about 15:1, more preferably about 2:1 to about 10:1, and more preferably about 3:1 to about 6:1. In some embodiments, the lipid nanoparticles have an N:P ratio of about 6:1. In some embodiments, the lipid nanoparticles have an N:P ratio of about 3:1.

[0037] In some embodiments, the lipid nanoparticle composition has a weight ratio of ionizable cationic lipid components to nucleic acids of about 5:1 to about 100:1, preferably about 5:1 to about 50:1, preferably about 5:1 to about 30:1, and more preferably about 10:1 to about 20:1. In some embodiments, the lipid nanoparticle composition has a weight ratio of ionizable cationic lipid components to nucleic acids of about 20:1. In some embodiments, the lipid nanoparticle composition has a weight ratio of ionizable cationic lipid components to nucleic acids of about 10:1.

[0038] In some embodiments, the lipid nanoparticle composition further comprises a buffer solution of the finished product.

[0039] In some embodiments, the finished buffer solution includes a buffering agent and / or a cryoprotectant.

[0040] In some embodiments, the buffer may be selected from acetates, formates, carbonates, phosphates, borates, succinates, glucons, lactates, citric acid, glycine, leucine, barbiturates, phthalates, tromethamine (Tris), triethylamine, ammonium salts, and combinations thereof. The content / concentration of the buffer is determined based on the acid-base properties of the specific type of buffer in order to set the pH value of the buffer to 7-8.

[0041] In some embodiments, the cryoprotectant may be selected from sugars, polyols, polymers, surfactants, amino acids, and salts, where the sugar may be selected from lactose, sucrose, trehalose, galactose, and the like.

[0042] In some embodiments, the amount of the cryoprotectant is 1-50% w / w of the composition, for example, 2-50% w / w, or 4-45% w / w, or 6-12% w / w, preferably 6-10% w / w, and most preferably 7-9% w / w.

[0043] In some embodiments, the final buffer solution contains tromethamine, sodium acetate, and sucrose, and has a pH of 7-8.

[0044] In some embodiments, the tromethamine content is selected from 10-30 mmol / L, preferably 15-25 mmol / L, preferably 15-20 mmol / L, for example, the tromethamine content is selected from 15 mmol / L, 15.5 mmol / L, 16 mmol / L, 16.5 mmol / L, 17 mmol / L, 17.5 mmol / L, 18 mmol / L, 18.5 mmol / L, 19 mmol / L, 19.5 mmol / L, 20 mmol / L, 20.5 mmol / L, 21 mmol / L, 21.5 mmol / L, 22 mmol / L, 22.5 mmol / L, 23 mmol / L, 23.5 mmol / L, 24 mmol / L, 24.5 mmol / L, and 25 mmol / L, with 20 mmol / L being the most preferred.

[0045] In some embodiments, the sodium acetate content is selected from 0-20 mmol / L, preferably 5-11 mmol / L, for example, the sodium acetate content is selected from 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 10.6 mmol / L, 10.7 mmol / L, 10.8 mmol / L, 10.9 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L, 12.5 mmol / L, and 13 mmol / L, with 10.7 mmol / L being the most preferred.

[0046] In some embodiments, the sucrose content is selected from 5-15%, preferably 7.5-10%, more preferably 7.5-9%L, for example, from 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 9%, 9.5%, and 10%, with most preferably 8.7%.

[0047] In some embodiments, the composition comprises lipid nanoparticles encapsulating nucleic acids and a buffer solution of the finished product.

[0048] In some embodiments, the composition comprises nucleic acid-encapsulated lipid nanoparticles, a final buffer for the product, and a cryoprotectant.

[0049] In some embodiments, the composition comprises nucleic acid-encapsulated lipid nanoparticles, tromethamine, sodium acetate, and sucrose.

[0050] In some embodiments, the composition comprises nucleic acid-encapsulated lipid nanoparticles, 20 mmol / L trometamol, 10.7 mmol / L sodium acetate, and 8.7% sucrose, the pH of the composition is 7.0-8.0, the lipid nanoparticles contain nucleic acid, which is mRNA at a concentration of 100 μg / ml, and the lipid component comprises 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% mPEG-DMG-2K, and the empty particle ratio of the composition is 10% or less.

[0051] A second aspect of the present invention provides a method for producing a lipid nanoparticle composition containing nucleic acids according to the first aspect, comprising the steps of (1) preparing precursor lipid nanoparticles to obtain a buffer system containing precursor lipid nanoparticles, and (2) replacing the buffer system containing precursor lipid nanoparticles with a neutral buffer system to obtain a finished lipid nanoparticle composition, wherein the content of the organic solvent in the buffer system containing precursor lipid nanoparticles is reduced to 5% (w / w) or less when the pH value of the system becomes 0.5 units or more lower than the pKa of the lipid nanoparticles.

[0052] Precursor lipid nanoparticles can be produced by methods known in the art for producing lipid nanoparticles.

[0053] In some embodiments, precursor lipid nanoparticles can be prepared by a method comprising the steps of: dissolving lipid components (ionizable cationic lipids, helper lipids, structural lipids, and surfactants) in an organic solvent to form a lipid phase; dissolving nucleic acids in an acidic buffer to form an aqueous phase; and mixing the two phases of the lipid phase and the aqueous phase, so that the lipids encapsulate the nucleic acids and form precursor lipid nanoparticles.

[0054] In some embodiments, precursor lipid nanoparticles can be prepared by a method comprising the steps of: dissolving lipid components (ionizable cationic lipids, helper lipids, structural lipids, and surfactants) in an organic solvent to form a lipid phase; mixing the lipid phase with an acidic buffer to form empty lipid nanoparticles; dissolving nucleic acids in an acidic buffer to form an aqueous phase; and mixing the empty lipid nanoparticles with the nucleic acid-containing aqueous phase so that the lipids encapsulate the nucleic acids and form precursor lipid nanoparticles.

[0055] In the above embodiment, the organic solvent is selected from C1-C4 lower alcohols, and is preferably ethanol.

[0056] In the above embodiment, the total concentration of lipid components in the lipid phase (ionizable cationic lipids, structural lipids, helper lipids, and surfactants) is 10-15 mg / ml.

[0057] In the above embodiment, the nucleic acid concentration in the aqueous phase is 0.01-1 mg / ml, preferably 0.05-0.5 mg / ml, and more preferably 0.1-0.2 mg / ml.

[0058] In the embodiments described above, the acidic buffer comprises a buffer selected from acetates, formates, carbonates, phosphates, borates, succinates, glucons, lactates, citric acid, glycine, leucine, barbiturates, phthalates, tromethamine (Tris), triethylamine, ammonium salts, and combinations thereof. The content / concentration of the buffer is determined based on the acid-base properties of the specific type of buffer such that the pH of the acidic buffer is lower than the pKa of the lipid nanoparticles. In some embodiments, the acidic buffer further comprises an osmotic regulator selected from sodium chloride, potassium chloride, and the like.

[0059] In some embodiments, the acidic buffer contains citric acid and sodium chloride. Preferably, the acidic buffer contains 10-20 mM citric acid and 120-140 mM sodium chloride. In some embodiments, the acidic buffer contains 10 mM citric acid and 130 mM sodium chloride, with a pH of 3.9-4.1, for example, pH 4.0. In some embodiments, the acidic buffer contains 20 mM citric acid and 130 mM sodium chloride, with a pH of 3.9-4.1, for example, pH 4.0. Preferably, in some embodiments, the acidic buffer contains 10 mM citric acid and 130 mM sodium chloride, with a pH of 4.0, or 20 mM citric acid and 130 mM sodium chloride, with a pH of 4.0.

[0060] In the above embodiment, the two phases, the lipid phase and the aqueous phase, are mixed in a volume ratio of 1:2-1:9, preferably 1:2-1:5, and preferably 1:3.

[0061] In the above embodiment, the mixing is performed through a microfluidic mixer, which is selected from a staggered herringbone mixer (SHM), a T-junction mixer, and a microfluidic hydrodynamic focusing (MHF).

[0062] In the above embodiment, the resulting precursor lipid nanoparticles are prepared to have a particle size of 20-150 nm, preferably 30-100 nm, more preferably 40-90 nm, and even more preferably 50-80 nm.

[0063] In the above embodiment, the obtained precursor lipid nanoparticles are prepared so that the particle dispersion coefficient PDI is 0.01-0.3, preferably 0.01-0.2, and more preferably 0.01-0.15.

[0064] In the above embodiment, the encapsulation rate of the manufactured precursor lipid nanoparticles is 50-100%, preferably 80-100%, and more preferably 90-100%.

[0065] After producing precursor lipid nanoparticles, the buffer system containing the precursor lipid nanoparticles is replaced with a neutral buffer system to produce the finished lipid nanoparticle composition.

[0066] In some embodiments, the buffer system replacement includes a two-step buffer system replacement process.

[0067] In some embodiments, the two-stage buffer system substitution step includes substitution using an acidic or neutral buffer in the first stage, and substitution using a neutral buffer in the second stage. In this step, the pH of the system is maintained below the pKa of the lipid nanoparticles in the first stage, and the organic solvent content in the system is reduced to a certain level (e.g., 5% or less) by substitution with an acidic or neutral buffer, ensuring that there is little residual organic solvent in the system when the particles fuse in the second stage. In the second stage, particle fusion begins when the pH of the system rises to near the pKa of the lipid nanoparticles by substitution with a neutral buffer, and particle fusion is more complete at lower organic solvent content. When the pH of the system exceeds the pKa of the lipid nanoparticles, particle fusion is completed, and thermodynamically stable particles (i.e., finished lipid nanoparticles) are formed.

[0068] In the above embodiments, the acidic buffer comprises a buffer selected from acetates, formates, carbonates, phosphates, borates, succinates, glucons, lactates, citric acid, glycine, leucine, barbiturates, phthalates, tromethamine (Tris), triethylamine, ammonium salts, and combinations thereof. The buffer content / concentration is determined based on the acid-base properties of the specific type of buffer such that the pH of the acidic buffer is lower than the pKa of the lipid nanoparticles. In some embodiments, the lipid nanoparticles have a pKa of 6.0-7.0, and the acidic buffer has a pH lower than the pKa of the lipid nanoparticles, for example, pH 3.0-5.0, preferably 4.0-5.0.

[0069] In some embodiments, the acidic buffer further comprises an osmotic regulator selected from sodium chloride, potassium chloride, and the like.

[0070] In some embodiments, the acidic buffer contains citric acid and sodium chloride. Preferably, the acidic buffer contains 10-20 mM citric acid and 120-140 mM sodium chloride. In some embodiments, the acidic buffer contains 10 mM citric acid and 130 mM sodium chloride, with a pH of 3.9-4.1, for example, pH 4.0. Preferably, in some embodiments, the acidic buffer contains 10 mM citric acid and 130 mM sodium chloride, with a pH of 4.0.

[0071] In the above embodiments, the neutral buffer comprises a buffer selected from acetates, formates, carbonates, phosphates, borates, succinates, glucons, lactates, citric acid, glycine, leucine, barbiturates, phthalates, tromethamine (Tris), triethylamine, ammonium salts, and combinations thereof. The buffer content / concentration is determined based on the acid-base properties of the specific type of buffer such that the pH of the neutral buffer is higher than the pKa of the lipid nanoparticles. In some embodiments, if the lipid nanoparticles have a pKa of 6.0-7.0, the neutral buffer has a pH higher than the pKa of the lipid nanoparticles, for example, pH 7.0-8.0.

[0072] In some embodiments, the neutral buffer further comprises a cryoprotectant. The cryoprotectant may be selected from substances such as sugars, polyols, polymers, surfactants, amino acids, and salts, and the sugars may be selected from lactose, sucrose, trehalose, galactose, and the like.

[0073] In some embodiments, the amount of the cryoprotectant is 1% w / w–50% w / w of the buffer, for example, 2% w / w–50% w / w, or 4% w / w–45% w / w, or 6% w / w–12% w / w, preferably 6% w / w–10% w / w, most preferably 7% w / w–9% w / w.

[0074] In some embodiments, the neutral buffer solution contains tromethamine, sodium acetate, and sucrose, and has a pH of 7-8.

[0075] In some embodiments, the tromethamine content is selected from 10-30 mmol / L, preferably 15-25 mmol / L, preferably 15-20 mmol / L, for example, the tromethamine content is selected from 15 mmol / L, 15.5 mmol / L, 16 mmol / L, 16.5 mmol / L, 17 mmol / L, 17.5 mmol / L, 18 mmol / L, 18.5 mmol / L, 19 mmol / L, 19.5 mmol / L, 20 mmol / L, 20.5 mmol / L, 21 mmol / L, 21.5 mmol / L, 22 mmol / L, 22.5 mmol / L, 23 mmol / L, 23.5 mmol / L, 24 mmol / L, 24.5 mmol / L, and 25 mmol / L, with 20 mmol / L being the most preferred.

[0076] In some embodiments, the sodium acetate content is selected from 0-20 mmol / L, preferably 5-11 mmol / L, for example, the sodium acetate content is selected from 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 10.6 mmol / L, 10.7 mmol / L, 10.8 mmol / L, 10.9 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L, 12.5 mmol / L, and 13 mmol / L, with 10.7 mmol / L being the most preferred.

[0077] In some embodiments, the sucrose content is selected from 5%–15%, preferably 7.5%–10%, more preferably 7.5%–9%, ​​for example, from 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.5%, and 10%, with 8.7% being the most preferred.

[0078] In some embodiments, the neutral buffer contains 20 mM Tris, 10.7 mM NaOAc, and 8.7% sucrose, and has a pH of 7.4–7.6. Preferably, in some embodiments, the neutral buffer contains 20 mM Tris, 10.7 mM NaOAc, and 8.7% sucrose, and has a pH of 7.6.

[0079] The above-mentioned neutral buffer component remains in the finished product and is also called the finished product buffer.

[0080] In some embodiments, the substitution step is selected from one or more of tangential flow filtration, membrane dialysis, column dialysis, and dilution.

[0081] In some embodiments, the two-stage buffer system replacement step includes dialysis using an acidic buffer or a neutral buffer in the first stage, and dialysis using a neutral buffer in the second stage.

[0082] In some embodiments, the two-step buffer replacement step includes dilution with an acidic buffer in the first step and dialyzing with a neutral buffer in the second step.

[0083] In some embodiments, the two-step buffer system replacement step may be repeated multiple times, that is, the step of increasing the pH and the step of decreasing the pH may be repeated alternately multiple times.

[0084] In the two-stage buffer system substitution step of the above embodiment, at the end of the first stage, the organic solvent content in the buffer system containing precursor lipid nanoparticles is reduced to 5% (w / w) or less, preferably less than 3% (w / w).

[0085] In the two-stage buffer system substitution process of the above embodiment, at the end of the first stage, the pH of the buffer system containing the precursor lipid nanoparticles is 0.5 units or more lower than the pKa of the lipid nanoparticles. For example, in some embodiments, if the lipid nanoparticles have a pKa of 6.0-7.0, at the end of the first stage of the two-stage buffer system substitution process, the pH of the buffer system is 0.5 units or more lower than the pKa of the lipid nanoparticles, for example, 5.0-6.0.

[0086] In the two-stage buffer system substitution step of the above embodiment, at the end of the second stage, the organic solvent content in the buffer system containing the lipid nanoparticles of the finished product is reduced to less than 1% (w / w), preferably less than 0.1% (w / w).

[0087] In the two-stage buffer system replacement process of the above embodiment, at the end of the second stage, the pH of the buffer system containing the finished lipid nanoparticles is 0.5 units or higher than the pKa of the lipid nanoparticles. For example, in some embodiments, if the lipid nanoparticles have a pKa of 6.0-7.0, at the end of the second stage of the two-stage buffer system replacement process, the pH of the buffer system is 0.5 units or higher than the pKa of the lipid nanoparticles, for example, 7.0-8.0.

[0088] In some embodiments, the buffer system substitution includes a two-stage buffer dialysis step. In some embodiments, the two-stage buffer dialysis step includes substitution with an acidic buffer in the first stage and substitution with a neutral buffer in the second stage, or substitution with one type of neutral buffer in the first stage and substitution with another neutral buffer in the second stage. The acidic buffer and neutral buffer are as described above. In this step, the first-stage substitution reduces the organic solvent content in the system to a certain level (e.g., 5% or less) when the pH value of the system is 0.5 units or more lower than the pKa of the lipid nanoparticles, ensuring that there is little residual organic solvent in the system when the particles fuse. The second-stage substitution causes particle fusion to begin when the pH value of the system rises to near the pKa of the lipid nanoparticles, and particle fusion is more complete at low organic solvent content. When the pH value of the system exceeds the pKa of the lipid nanoparticles, particle fusion is completed, and thermodynamically stable particles (i.e., finished lipid nanoparticles) are formed.

[0089] In some embodiments, the buffer system substitution is performed using only one neutral buffer (a one-step buffer dialysis process). The neutral buffer is as described above. In this process, only one type of neutral buffer is used, but by controlling the pH value and buffering capacity of the neutral buffer, when the pH value of the system is 0.5 units or more lower than the pKa of the lipid nanoparticles during the dialysis process, the organic solvent content in the system is reduced to a certain level (e.g., 5% or less) (i.e., first-step substitution), dialysis is continued using the neutral buffer (second-step substitution), and when the pH value of the system rises to near the pKa of the lipid nanoparticles, particle fusion begins. At lower organic solvent content, particle fusion is more complete, and when the pH value of the system exceeds the pKa of the lipid nanoparticles, particle fusion is completed, and thermodynamically stable particles (i.e., finished lipid nanoparticles) are formed.

[0090] In one specific embodiment, the method for producing a nucleic acid-encapsulated lipid nanoparticle composition according to the present invention includes: (1) a step of dissolving lipid components (ionizable cationic lipids, helper lipids, structural lipids, and surfactants) in an organic solvent to form a lipid phase; (2) a step of dissolving nucleic acids in an acidic buffer to form an aqueous phase; (3) a step of mixing the two phases of the lipid phase and the aqueous phase to form lipid precursor nanoparticles in which the lipids encapsulate the nucleic acids; and (4) a step of substituting the organic solvent-acidic buffer system containing the lipid precursor nanoparticles obtained in step (3) with a neutral buffer in a two-step substitution to obtain a finished lipid nanoparticle composition, wherein the first step involves substitution with an acidic buffer or a neutral buffer, and the second step involves substitution with a neutral buffer. In the first step, the pH value of the system is maintained below the pKa of the lipid nanoparticles, and the content of the organic solvent in the system is reduced to a certain extent by substitution with an acidic buffer or a neutral buffer, and in the second step, it is ensured that there is little residual organic solvent in the system when the particles fuse. In the second stage, particle fusion begins when the pH of the system rises to near the pKa of the lipid nanoparticles by substitution with a neutral buffer. Particle fusion is more complete with a low content of organic solvent, and when the pH of the system exceeds the pKa of the lipid nanoparticles, particle fusion is completed, and thermodynamically stable particles (i.e., finished lipid nanoparticles) are formed.

[0091] In some embodiments, the organic solvent in step (1) is selected from C1-C4 lower alcohols, and is preferably ethanol.

[0092] In some embodiments, the total concentration of lipid components (ionizable cationic lipids, structural lipids, helper lipids, and surfactants) in the lipid phase in step (1) is 10-15 mg / ml.

[0093] In some embodiments, the nucleic acid concentration in the aqueous phase in step (2) is 0.01–1 mg / ml, preferably 0.05–0.5 mg / ml, and more preferably 0.1–0.2 mg / ml.

[0094] In some embodiments, the acidic buffer in step (2) or step (4) includes a buffer selected from acetates, formates, carbonates, phosphates, borates, succinates, glucons, lactates, citric acid, glycine, leucine, barbiturates, phthalates, tromethamine (Tris), triethylamine, ammonium salts, and combinations thereof. The buffer content / concentration is determined based on the acid-base properties of the specific type of buffer such that the pH of the acidic buffer is lower than the pKa of the lipid nanoparticles. In some embodiments, if the lipid nanoparticles have a pKa of 6.0-7.0, the acidic buffer in step (2) or step (4) has a pH value lower than the pKa of the lipid nanoparticles, for example, 3.0-5.0, preferably 4.0-5.0.

[0095] In some embodiments, the acidic buffer in step (2) or step (4) further comprises an osmotic regulator selected from sodium chloride, potassium chloride, and the like.

[0096] In some embodiments, the acidic buffer in step (2) or step (4) comprises citric acid and sodium chloride. Preferably, the buffer comprises 10–20 mM citric acid and 120–140 mM sodium chloride.

[0097] In some embodiments, the acidic buffer in step (2) or step (4) comprises 10 mM citric acid and 130 mM sodium chloride, with a pH of 3.9–4.1, for example, pH 4.0. In some embodiments, the acidic buffer in step (2) or step (4) comprises 20 mM citric acid and 130 mM sodium chloride, with a pH of 3.9–4.1, for example, pH 4.0. Preferably, in some embodiments, the acidic buffer in step (2) or step (4) comprises 10 mM citric acid and 130 mM sodium chloride, with a pH of 4.0; or comprises 20 mM citric acid and 130 mM sodium chloride, with a pH of 4.0.

[0098] In some embodiments, the mixing of the lipid phase and the aqueous phase in step (3) is in a volume ratio of 1:2-1:9, preferably 1:2-1:5, and preferably 1:3.

[0099] In some embodiments, step (3) involves mixing the lipid phase and the aqueous phase using a microfluidic mixer, which is selected from a staggered herringbone mixer (SHM), a T-junction mixer, or a microfluidic hydrodynamic focusing (MHF).

[0100] In some embodiments, the precursor lipid nanoparticles in step (3) have a particle size of 20-150 nm, preferably 30-100 nm, more preferably 40-90 nm, and even more preferably 50-80 nm.

[0101] In some embodiments, the precursor lipid nanoparticles in step (3) have a particle dispersion coefficient PDI of 0.01-0.3, preferably 0.01-0.2, and more preferably 0.01-0.15.

[0102] In some embodiments, the encapsulation rate of precursor lipid nanoparticles in step (3) is 50-100%, preferably 80-100%, and more preferably 90-100%.

[0103] In some embodiments, the substitution step in step (4) is selected from one or more of tangential flow filtration, membrane dialysis, column dialysis, and dilution.

[0104] In some embodiments, in the two-step substitution step in step (4), at the end of the first step, the organic solvent content in the buffer system containing precursor lipid nanoparticles is reduced to 5% (w / w) or less, preferably less than 3% (w / w).

[0105] In some embodiments, in the two-step substitution step in step (4), at the end of the first step, the pH of the buffer system containing the precursor lipid nanoparticles is 0.5 units or more lower than the pKa of the lipid nanoparticles. For example, in some embodiments, if the lipid nanoparticles have a pKa of 6.0-7.0, at the end of the first step of the two-step substitution step in step (4), the pH of the buffer system is 0.5 units or more lower than the pKa of the lipid nanoparticles, for example, pH 5.0-6.0.

[0106] In some embodiments, at the end of the second step of the two-step substitution process in step (4), the organic solvent content in the buffer system containing the finished lipid nanoparticles is reduced to less than 1% (w / w), preferably less than 0.1% (w / w).

[0107] In some embodiments, at the end of the second stage of the two-step substitution process in step (4), the pH of the buffer system containing the finished lipid nanoparticles is 0.5 units or more higher than the pKa of the lipid nanoparticles. For example, in some embodiments, if the lipid nanoparticles have a pKa of 6.0-7.0, at the end of the second stage of the two-step substitution process, the pH of the buffer system is 0.5 units or more higher than the pKa of the lipid nanoparticles, for example, 7.0-8.0.

[0108] In some embodiments, the two-step substitution step in step (4) may be repeated multiple times, that is, the step of increasing the pH and the step of decreasing the pH may be repeated alternately multiple times. By repeatedly raising and lowering the pKa of the lipid nanoparticles, the particle fusion effect can be enhanced.

[0109] In some embodiments, the neutral buffer in step (4) includes a buffer selected from acetates, formates, carbonates, phosphates, borates, succinates, glucons, lactates, citric acid, glycine, leucine, barbiturates, phthalates, tromethamine (Tris), triethylamine, ammonium salts, and combinations thereof. The buffer content / concentration is determined based on the acid-base properties of the specific type of buffer such that the pH of the neutral buffer is higher than the pKa of the lipid nanoparticles. In some embodiments, if the lipid nanoparticles have a pKa of 6.0-7.0, the neutral buffer has a pH higher than the pKa of the lipid nanoparticles, for example, pH 7.0-8.0.

[0110] In some embodiments, the neutral buffer in step (4) further comprises a cryoprotectant. The cryoprotectant may be selected from substances such as sugars, polyols, polymers, surfactants, amino acids, and salts, and the sugars may be selected from lactose, sucrose, trehalose, galactose, and the like.

[0111] In some embodiments, the amount of the cryoprotectant is 1% w / w–50% w / w of the buffer, for example, 2% w / w–50% w / w, or 4% w / w–45% w / w, or 6% w / w–12% w / w, preferably 6% w / w–10% w / w, most preferably 7% w / w–9% w / w.

[0112] In some embodiments, the neutral buffer solution contains tromethamine, sodium acetate, and sucrose, and has a pH of 7-8.

[0113] In some embodiments, the tromethamine content is selected from 10-30 mmol / L, preferably 15-25 mmol / L, preferably 15-20 mmol / L, for example, the tromethamine content is selected from 15 mmol / L, 15.5 mmol / L, 16 mmol / L, 16.5 mmol / L, 17 mmol / L, 17.5 mmol / L, 18 mmol / L, 18.5 mmol / L, 19 mmol / L, 19.5 mmol / L, 20 mmol / L, 20.5 mmol / L, 21 mmol / L, 21.5 mmol / L, 22 mmol / L, 22.5 mmol / L, 23 mmol / L, 23.5 mmol / L, 24 mmol / L, 24.5 mmol / L, and 25 mmol / L, with 20 mmol / L being the most preferred.

[0114] In some embodiments, the sodium acetate content is selected from 0-20 mmol / L, preferably 5-11 mmol / L, for example, the sodium acetate content is selected from 5 mmol / L, 5.5 mmol / L, 6 mmol / L, 6.5 mmol / L, 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 8.5 mmol / L, 9 mmol / L, 9.5 mmol / L, 10 mmol / L, 10.5 mmol / L, 10.6 mmol / L, 10.7 mmol / L, 10.8 mmol / L, 10.9 mmol / L, 11 mmol / L, 11.5 mmol / L, 12 mmol / L, 12.5 mmol / L, and 13 mmol / L, with 10.7 mmol / L being the most preferred.

[0115] In some embodiments, the sucrose content is selected from 5%–15%, preferably 7.5%–10%, more preferably 7.5%–9%, ​​for example, from 7.6%, 7.7%, 7.8%, 7.9%, 8.0%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.5%, and 10%, with 8.7% being the most preferred.

[0116] In some embodiments, the neutral buffer in step (4) contains 20 mM Tris, 10.7 mM NaOAc, and 8.7% sucrose, and has a pH of 7.4–7.6. Preferably, in some embodiments, the neutral buffer in step (4) contains 20 mM Tris, 10.7 mM NaOAc, and 8.7% sucrose, and has a pH of 7.6.

[0117] The neutral buffer component in step (4) remains in the finished product and is also called the finished product buffer.

[0118] In some embodiments, the above manufacturing method optionally includes steps (5) concentration, dilution, sterilization filtration, sterile filling, and combinations thereof. The steps of concentration, dilution, and sterile filling are used to ensure that the nucleic acid drug reaches specifications and doses suitable for administration, and the step of sterilization filtration is used to ensure that the microbiological properties of the nucleic acid drug meet requirements.

[0119] The important steps in the method for producing the nucleic acid-containing lipid nanoparticle composition according to the present invention are the preparation of precursor lipid nanoparticles (lipid phase-aqueous phase mixing) and buffer replacement. The formation of thermodynamically stable nucleic acid-containing lipid nanoparticles depends on the particle fusion process in the buffer system replacement process. Reducing the organic solvent content in the system to 5% (w / w) or less before particle fusion is advantageous for sufficient particle fusion, thereby obtaining thermodynamically stable lipid nanoparticles. Advantageously, the content of the organic solvent in the buffer system containing the precursor lipid nanoparticles is reduced to 5% (w / w) or less by buffer system replacement when the pH value of the system is 0.5 units or more lower than the pKa of the lipid nanoparticles. [Effects of the Invention]

[0120] This invention provides a novel method for improving the stability and pharmacokinetics of nucleic acid drugs by obtaining nucleic acid-encapsulated lipid nanoparticles with a low void ratio by increasing the degree of particle fusion during the manufacturing process. The morphology and structure of well-fused particles become more uniform, the particles become more stable, and the prepared mRNA LNPs can be stored for at least 6 months under refrigerated conditions of 2-8°C and at least 12 months under frozen conditions of -15°C--25°C. Its stability is significantly superior to that of commercially available drugs, making it convenient for storage, transport, and distribution. Compared to particles prepared by conventional methods, the nucleic acid-containing lipid nanoparticles with a low void ratio according to this invention are almost entirely effective particles encapsulating nucleic acids, which is advantageous for improving nucleic acid delivery efficiency, reducing the risk of inflammatory adverse reactions due to lipid components, and improving the efficacy and safety of nucleic acid drugs. Furthermore, such nucleic acid-containing lipid nanoparticles with a low void ratio tend to remain at the injection site, reducing their distribution in the blood and non-target organs (e.g., the heart), thereby reducing the risk of adverse reactions due to non-target organ distribution.

[0121] Explanation of terms As used herein and in the claims, the following terms have the meanings set forth below unless otherwise specified. Where not expressly defined herein, terms should be understood to have the meanings well known in the art. Furthermore, terms used herein are solely for the purpose of describing specific embodiments and should be understood not to limit the scope of protection of the invention in any way.

[0122] Ionizable cationic lipids As used herein, the term "ionizable cationic lipid" refers to a special type of lipid that possesses ionizable properties. Under acidic conditions lower than pKa, it becomes positively charged, allowing it to complex with negatively charged RNA, encapsulating and protecting the RNA within LNPs. Under physiological conditions higher than pKa (neutral), it becomes uncharged, electrically neutralizing LNPs and conferring low toxicity, thereby transporting RNA into the cell via the endosomal pathway. Within acidic endosomes, the ionizable cationic lipid regains its positive charge and interacts with negatively charged phospholipids on the endosomal membrane, disrupting the endosome and enabling endosomal escape and the release of RNA into the cytoplasm.

[0123] pKa of lipid nanoparticles As used herein, the term "pKa of lipid nanoparticles" represents the apparent ionization constant of lipid nanoparticles containing a specific ionizable cationic lipid. The pKa value of lipid nanoparticles containing a specific ionizable cationic lipid is related to the tolerance at the injection site, safety in the body, stability, and intracellular delivery effect of the lipid nanoparticles. The pKa value of lipid nanoparticles containing a specific ionizable cationic lipid can be measured using a sodium 6-(p-toluidino)-2-naphthalenesulfonate (TNS) probe, which is commonly reported in the literature. In titration, the pH value corresponding to the point at which the TNS fluorescence signal abruptly changes with a change in pH is the pKa value of the lipid nanoparticle containing the ionizable cationic lipid.

[0124] Pre-lipid nanoparticles As used herein, the term "precursor lipid nanoparticles" refers to intermediate lipid nanoparticles present in the preparation process of LNPs containing ionizable cationic lipids. These are formed by self-assembly after rapid mixing of lipids in the organic phase with nucleic acids in the aqueous phase. After mixing, because the pH of the system is significantly lower than the pKa of the ionizable cationic lipids, the ionizable cationic lipids are protonated and become positively charged, creating an electrostatic attraction with the negatively charged nucleic acids. Simultaneously, due to the low water solubility of the lipids, hydrophobic interactions also occur. These driving forces cause the lipids to encapsulate the nucleic acids, undergoing self-assembly and forming lipid nanoparticles. In subsequent dialysis or ultrafiltration processes, the precursor lipid nanoparticles fuse in response to changes in the pH of the system, forming the finished lipid nanoparticles.

[0125] N:P ratio As used herein, the term "N:P ratio" represents the molar ratio of protonable nitrogen elements in ionizable cationic lipids to phosphate groups in mRNA. The N:P ratio is determined by the amino group (N) in the ionizable cationic lipid. + ) and the cation charge and phosphate group (PO4) in the nucleic acid skeleton - This describes the ratio between the anionic charge of LNPs and the N:P ratio, which is the basis for the electrostatic interaction between ionizable cationic lipids and nucleic acids. The N:P ratio is an important prescribing factor for LNPs and affects the physicochemical properties of LNPs and drug release in the body.

[0126] Inclusion rate As used herein, the term "encapsulation rate" refers to the proportion of nucleic acids encapsulated within lipid nanoparticles relative to the total amount of nucleic acids. The encapsulation rate is usually detected by fluorescence spectrophotometry. Taking RNA as an example, first, the number of RNA molecules released outside the lipid nanoparticles in the LNP-RNA solution is detected using RiboGreen fluorescent dye. Then, the lipid nanoparticle structure is disrupted with Triton-100 to release the RNA into the external solution, and the total number of RNA molecules in that solution is detected. The difference between these two values ​​is then used to determine the encapsulation rate, which is the number of RNA molecules encapsulated within the LNP particles.

[0127] Empty particle rate As used herein, the term "empty particle ratio" represents the ratio of lipid nanoparticles that do not encapsulate nucleic acids to the total number of lipid nanoparticles. In this invention, the empty particle ratio is detected using a flow nanoanalyzer. First, the total number of lipid nanoparticles in the sample is detected using flow nanoanalyzer technology. Next, ribonuclease is added to the sample to remove free nucleic acids, and then a membrane-permeable nucleic acid dye is added. This dye permeates the LNP membrane and binds to the nucleic acids inside the LNPs, generating fluorescence. This allows the number of lipid nanoparticles encapsulating nucleic acids to be obtained, and the ratio of LNPs that do not encapsulate nucleic acids, i.e., the empty particle ratio, is calculated.

[0128] nucleic acid The terms “nucleic acid” or “nucleic acid molecule” have meanings recognized and understood by those skilled in the art. The term “nucleic acid” as used herein refers to a polymer containing at least two single-stranded or double-stranded deoxyribonucleotides or ribonucleotides, and includes DNA and RNA. DNA may be, for example, antisense molecules, plasmid DNA, pre-condensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. RNA may be siRNA, asymmetric interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include synthetic, naturally occurring, naturally occurring, known nucleotide analogs, or nucleic acids containing modified skeletal residues or bindings that have binding properties similar to a reference nucleic acid. Examples of such analogs include, non-limitingly, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, the term encompasses nucleic acids containing known analogs of native nucleotides having similar binding properties to the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence inherently includes not only the explicitly stated sequence but also its conservedly modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences. In particular, degenerate codon substitution can be achieved by constructing sequences in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or a deoxyinosine residue (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). A "nucleotide" contains a sugar deoxyribose (DNA) or sugar ribose (RNA), a base, and a phosphate group.Nucleotides are linked together through phosphate groups. "Bases" include, but are not limited to, purines and pyrimidines, which further include, the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, as well as synthetic derivatives of purines and pyrimidines, which include modifications that introduce novel reactive groups such as amines, alcohols, thiols, carboxylates, and alkyl halides.

[0129] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, etc. Plasmid DNA, cDNA, and antisense DNA are preferred, and plasmid DNA is more preferred. Circular DNA such as plasmid DNA may be digested with restriction enzymes or the like to obtain linear DNA.

[0130] The types of RNA that can be used in the present invention are not particularly limited and can be appropriately selected depending on the purpose of use. Examples include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, and ribosomal RNA, with siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon being preferred.

[0131] The origin of the nucleic acids that can be used in the present invention is not particularly limited, and can be appropriately selected depending on the purpose, such as using commercially available nucleic acids or preparing them by referring to methods disclosed in the prior art. In some embodiments, the mRNA used in the present invention is prepared by referring to the method described in Chinese Patent Application 202111445859.X filed on November 30, 2021, and the entirety of the disclosures of the above patent application is incorporated herein for all purposes.

[0132] In this invention, numerical ranges and parameter approximations are described over a wide range, but all numerical values ​​shown in the specific examples are described as accurately as possible. However, any numerical value inherently contains a certain degree of error due to the standard deviation in each measurement. Furthermore, it should be understood that all ranges disclosed herein encompass all and any subranges contained therein. For example, when described herein, the range "1-10" encompasses all and any subranges contained between the minimum value of 1 and the maximum value of 10 (including the endpoints), that is, all subranges starting with a minimum value of 1 or a value greater than 1, e.g., 1-6.1, and all subranges ending with a maximum value of 10 or a value less than 10, e.g., 5.5-10. In addition, it should be understood that all references described as "incorporated herein" are incorporated in their entirety.

[0133] For the sake of brevity in this specification, some quantitative data do not explicitly use the term “approximately.” However, whether or not the term “approximately” is explicitly used, it should be understood that each numerical value given herein includes not only the actual value (specific value) but also equivalents and approximations of that specific value resulting from experimental and / or measurement conditions, as reasonably inferred by a person skilled in the art. Such approximations are preferably within the ranges of ±20%, ±15%, ±10%, ±8%, ±6%, ±5%, ±4%, ±3%, ±2%, and ±1% of the specific value.

[0134] As used herein, the singular form includes the plural form of the referent unless the context clearly and explicitly limits it to a single referent. Furthermore, unless otherwise clearly specified in the context, the term "or" may be used interchangeably with the term "and / or".

[0135] Those skilled in the art will understand that particle size can be determined using any suitable existing or future method (including, but not limited to, gravity sedimentation, sieving, microscopic observation, or laser particle size analyzers). If the nanoparticles disclosed herein include multiple particles, it will also be understood that not all particles necessarily have to have the same particle size; as long as their average particle size falls within the above range, they are also included within the scope of this specification. In some specific embodiments, particle size is measured by a laser particle size analyzer.

[0136] Other embodiments of the present invention include:

[0137] 1. A lipid nanoparticle composition for encapsulating nucleic acids, characterized in that the ratio of the number of empty lipid nanoparticles that do not encapsulate nucleic acids to the total number of lipid nanoparticles, i.e., the empty particle rate, is 10% or less, preferably 9% or less, more preferably 8.5% or less, more preferably 6% or less, even more preferably 3% or less, or any value within the above range, for example, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 7.1%, 7.6%, 7.7%, 7.9%, 8.3%.

[0138] 2. The composition according to Embodiment 1, characterized in that the average particle size of the lipid nanoparticles is 50 nm to 150 nm, preferably 70 nm to 120 nm, more preferably 90 nm to 110 nm, or any value within the above range, for example, the average particle size is 96 nm, 96 nm, 97 nm, 98 nm, 99 nm, 100 nm, 101 nm, 102 nm, 103 nm, 104 nm, 105 nm, 106 nm, 107 nm, 109 nm, 110 nm, 111 nm, 112 nm, 113 nm, 114 nm, 115 nm, 116 nm, 117 nm, 119 nm, or 120 nm.

[0139] 3. The composition according to Embodiment 1, characterized in that the encapsulation rate of lipid nanoparticles is 80% or more, preferably 85% or more, and more preferably 90% or more.

[0140] 4. The composition according to Embodiment 1, characterized in that the lipid nanoparticles comprise (1) nucleic acids and (2) lipid components including ionizable cationic lipids, structural lipids, helper lipids and surfactants.

[0141] 5. The composition according to Embodiment 4, characterized in that the lipid component comprises 20-60 mol% of ionizable cationic lipids, 25-55 mol% of structural lipids, 2-25 mol% of helper lipids, and 0.5-15 mol% of surfactants.

[0142] 6. The composition according to Embodiment 5, characterized in that the ionizable cationic lipid is selected from SM-102, ALC-0315, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA; the structural lipid is selected from cholesterol or a cholesterol derivative; the auxiliary lipid is selected from DSPC, DOPE, DOPC, DOPG, and DOPS; and the surfactant is selected from mPEG-DMG-2K, ALC-0159, PEG-DSPE, DTDA-PEG2000, and TPGS.

[0143] 7. The composition according to Embodiment 5, characterized in that the N:P ratio of the lipid nanoparticles is about 2:1 to about 30:1, preferably about 2:1 to about 15:1, more preferably about 2:1 to about 10:1, and more preferably about 3:1 to about 6:1.

[0144] 8. The composition according to Embodiment 4, characterized in that the weight ratio of ionizable cationic lipid components to nucleic acids is about 5:1 to about 100:1, preferably about 5:1 to about 50:1, preferably about 5:1 to about 30:1, and more preferably about 10:1 to about 20:1.

[0145] 9. The composition according to Embodiment 1, characterized in that the lipid nanoparticle composition further comprises a buffer solution containing a buffering agent and / or cryoprotectant.

[0146] 10. The buffer is selected from acetate, formate, carbonate, phosphate, borate, succinate, gluconate, lactate, citric acid, glycine, leucine, barbituric acid, phthalic acid, tromethamine (Tris), triethylamine, ammonium salt, or a combination thereof, and the pH of the buffer is 7-8, or

[0147] The composition according to Embodiment 9, characterized in that the cryoprotectant is selected from substances such as sugars / polyols, polymers, surfactants, amino acids, and salts, and the sugar is selected from lactose, sucrose, trehalose, galactose, and the like.

[0148] 11. The composition according to Embodiment 9, characterized in that the buffer solution of the finished product contains tromethamine, sodium acetate, and sucrose, and the pH of the buffer solution of the finished product is 7-8.

[0149] 12. The nucleic acid is selected from one type of mRNA, and the mRNA contains a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with one of the nucleotide sequences of sequence number (SEQ ID NO): 1-6, preferably sequence number (SEQ ID NO): 1, or

[0150] The composition according to Embodiment 1, characterized in that it is selected from one type of mRNA and contains an amino acid sequence in which the coronavirus antigen encoded by the mRNA has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of Sequence ID No: 7.

[0151] 13. The composition according to Embodiment 12, characterized in that the composition comprises nucleic acid-encapsulated lipid nanoparticles, 20 mmol / L tromethamine, 10.7 mmol / L sodium acetate, and 8.7% sucrose, has a pH of 7.0-8.0, has an mRNA concentration of 100 μg / ml, and the lipid nanoparticles consist of 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% mPEG-DMG-2K, and has an empty particle ratio of 10% or less.

[0152] 14. The process includes: (1) dissolving lipid components in an organic solvent to form a lipid phase; (2) dissolving nucleic acids in an acidic buffer to form an aqueous phase; (3) mixing the two phases, the lipid phase and the aqueous phase, to form lipid precursor nanoparticles in which the lipids encapsulate the nucleic acids; and (4) replacing the organic solvent-acidic buffer system containing the lipid precursor nanoparticles obtained in step (3) with a neutral buffer by two-step substitution to obtain a finished lipid nanoparticle composition.

[0153] A method for producing the lipid nanoparticle composition, characterized in that, in the two-step substitution, the first step is substitution with an acidic buffer or a neutral buffer, and the second step is substitution with a neutral buffer.

[0154] 15. The manufacturing method according to Embodiment 14, characterized in that, in the two-step substitution step in step (4), at the end of the first step, the organic solvent content in the buffer system containing precursor lipid nanoparticles is reduced to 5% (w / w) or less, preferably less than 3% (w / w).

[0155] 16. The manufacturing method according to Embodiment 14, characterized in that, in the two-step substitution step in step (4), at the end of the first step, the pH of the buffer system containing the precursor lipid nanoparticles becomes 0.5 units or more lower than the pKa of the lipid nanoparticles.

[0156] 17. The manufacturing method according to Embodiment 14, characterized in that, in the two-stage substitution step in step (4), at the end of the second stage, the content of organic solvent in the buffer system containing lipid nanoparticles of the finished product is reduced to less than 1% (w / w), preferably less than 0.1% (w / w).

[0157] 18. The manufacturing method according to Embodiment 14, characterized in that, in the two-stage substitution step in step (4), at the end of the second stage, the pH of the buffer system containing the lipid nanoparticles of the finished product is 0.5 units or more higher than the pKa of the lipid nanoparticles.

[0158] 19. The manufacturing method according to Embodiment 14, characterized in that the manufacturing method optionally includes step (5) concentration, dilution, sterilization filtration, sterile filling, and combinations thereof.

[0159] 20. The manufacturing method according to Embodiment 14, characterized in that the organic solvent in step (1) is selected from C1-C4 lower alcohols, and is preferably ethanol.

[0160] 21. The manufacturing method according to Embodiment 14, characterized in that the total concentration of lipid components in the lipid phase in step (1) is 10-15 mg / ml.

[0161] 22. The manufacturing method according to Embodiment 14, characterized in that the nucleic acid concentration in the aqueous phase in step (2) is 0.01-1 mg / ml, preferably 0.05-0.5 mg / ml, and more preferably 0.1-0.2 mg / ml.

[0162] 23. The manufacturing method according to Embodiment 14, characterized in that the acidic buffer in step (2) or step (4) contains a buffer selected from acetate, formate, carbonate, phosphate, borate, succinate, gluconate, lactate, citric acid, glycine, leucine, barbituric acid, phthalic acid, tromethamine (Tris), triethylamine salt, sodium chloride, potassium chloride, or a combination thereof, and the pH of the acidic buffer is lower than the pKa of the lipid nanoparticles.

[0163] 24. The manufacturing method according to Embodiment 14, characterized in that the acidic buffer in step (2) or step (4) further comprises an osmotic pressure adjusting agent selected from sodium chloride, potassium chloride, and the like.

[0164] 25. The manufacturing method according to Embodiment 14, characterized in that, when the lipid nanoparticles have a pKa of 6.0-7.0, the pH value of the acidic buffer in step (2) or step (4) is lower than the pKa of the lipid nanoparticles, for example, 3.0-5.0, preferably 4.0-5.0.

[0165] 26. The manufacturing method according to Embodiment 14, characterized in that the acidic buffer in step (2) or step (4) contains citric acid and sodium chloride, preferably containing 10-20 mM citric acid and 120-140 mM sodium chloride.

[0166] 27. The manufacturing method according to Embodiment 14, characterized in that the mixing of the two phases, the lipid phase and the aqueous phase, in step (3) is in a volume ratio of 1:2-1:9, preferably 1:2-1:5, and preferably 1:3.

[0167] 28. The manufacturing method according to Embodiment 14, characterized in that the substitution step in step (4) is selected from one or more of tangential flow filtration, membrane dialysis, column dialysis, and dilution.

[0168] 29. The manufacturing method according to Embodiment 14, characterized in that the two-step substitution step in step (4) may be repeated multiple times, that is, the step of raising the pH and the step of lowering the pH may be repeated alternately multiple times, thereby repeatedly raising and lowering the pKa of lipid nanoparticles and reducing the void particle ratio.

[0169] 30. The manufacturing method according to Embodiment 14, characterized in that the neutral buffer in step (4) contains a buffer selected from acetate, formate, carbonate, phosphate, borate, succinate, gluconate, lactate, citric acid, glycine, leucine, barbituric acid, phthalic acid, tromethamine (Tris), triethylamine, ammonium salt, sucrose, and combinations thereof, and the pH of the neutral buffer is higher than the pKa of the lipid nanoparticles.

[0170] 31. The manufacturing method according to Embodiment 14, characterized in that when the lipid nanoparticles have a pKa of 6.0-7.0, the pH of the neutral buffer in step (4) becomes higher than the pKa of the lipid nanoparticles, for example, pH 7.0-8.0. [Brief explanation of the drawing]

[0171] [Figure 1] Figure 1 shows the structure of the linearized plasmid template of the T7 promoter in Preparation Example 1. [Figure 2] Figure 2 shows the curve of fluorescence intensity against pH change when the pKa value of lipid nanoparticles containing SM-102 was measured using the TNS method. [Figure 3] Figure 3 shows the curves of changes in particle size and PDI with respect to pH value for lipid nanoparticles containing SM-102 during a two-stage dialysis process. [Figure 4] Figure 4 shows curves illustrating how the particle size of different cationic lipid LNPs changes with the dialysis factor during the buffer replacement process. [Figure 5] Figure 5 shows curves illustrating how the PDI of different cationic lipid LNPs changes with the dialysis factor during the buffer replacement process. [Figure 6] Figure 6 shows a curve illustrating how the particle size of LNPs changes during the reverse dialysis process. [Figure 7] Figure 7 shows fluorescence nanoflow cytometer diagrams of intermediates in the LNP manufacturing process after two-phase mixing (A), after pH 4.0 buffer dialysis (B), and after pH 7.6 buffer dialysis (C), according to Embodiment 1-1. The horizontal axis SS-H represents the peak height of the scattering signal, the vertical axis FITC-A represents the peak area of ​​the fluorescence signal, P1 represents the proportion of nucleic acid-encapsulated LNPs, and P2 represents the proportion of empty particles (empty LNPs). [Figure 8] Figure 8 shows low-temperature transmission electron microscope images of intermediates in the LNP manufacturing process after two-phase mixing, pH 4.0 buffer dialysis, and pH 7.6 buffer dialysis, according to Embodiment 1-1. [Figure 9]Figure 9 shows a typical low-temperature transmission electron microscope image of the finished lipid nanoparticles according to Embodiment 1-1. [Figure 10] Figure 10 shows a typical low-temperature transmission electron microscope image of the finished lipid nanoparticles produced by Comparative Example 1-1. [Figure 11] Figure 11 shows the pseudotype virus-neutralizing antibody activity of immunoserum obtained after immunizing mice with lipid nanoparticle compositions prepared according to Embodiment 1-1 and Comparative Example 1-1. [Figure 12] Figure 12 shows a comparison of the in vitro cell transfection efficacy of luciferase mRNA-LNPs produced by Embodiment 1-1 and Comparative Example 1-1. [Figure 13] Figure 13 shows the tissue distribution results in mice after administration of lipid nanoparticles produced according to Embodiment 1-1 and Comparative Example 1-1. [Modes for carrying out the invention]

[0172] To further understand the present invention, specific embodiments of the invention will be described in detail below with reference to examples. However, it should be understood that these descriptions are for the purpose of further illustrating the features and advantages of the present invention and do not limit the invention in any way.

[0173] Preparation Example 1: Preparation of mRNA An exemplary method for producing mRNA used in this invention is as follows.

[0174] 1. The IVT reaction system was prepared according to the instructions for the IVT kit (E131, Novoprotein). Specifically, 10x concentration Transcription Buffer, ATP, GTP, CTP, 1-N-Me-Pseudo UTP (Catalog No.: WA0992, Hongene BioTech), 5' cap analog m7G(5')ppp(5')(2'OMeA)pG (Catalog No.: GAGNH23C2L1B, Hongene BioTech), water for injection, a linear plasmid template of the T7 promoter (custom prepared by GenScript Biotech Co., Ltd., see Figure 1 for structure), and Enzyme Mix were mixed. The selected sequences are as follows:

[0175] [Table 0]

[0176] 2. Allow the mixed reaction system to react at 37°C for 40 minutes; 3. The reaction was stopped by adding DNase I in the appropriate ratio.

[0177] After synthesizing S protein mRNA in vitro, it was concentrated and purified by hydrophobic chromatography and ultrafiltration to obtain high-purity mRNA (>95%). Experimental results showed that this process yielded high-purity mRNA that could be used in subsequent experiments. Note: Internal standard (25nt, Agilent, RNA 6000 Nano Kit (reorder-no. 5067-1511)).

[0178] Example 1: Preparation of lipid nanoparticles by two-step dialysis method This example illustrates an example of a method for producing lipid nanoparticles with a low void ratio according to the present invention. Here, precursor lipid nanoparticles were subjected to two-step dialysis in pH 4.0 buffer and pH 7.6 buffer to obtain the finished lipid nanoparticles. The specific preparation method is as follows.

[0179] 1. Preparation of precursor lipid nanoparticles 1.1 Preparation of the lipid phase: Four types of lipids were precisely weighed so that their molar ratios were SM-102:mPEG-DMG-2K:DSPC:cholesterol = 50%:1.5%:10%:38.5%, dissolved in anhydrous ethanol, and a lipid phase with a total lipid concentration of 12 mg / ml was obtained.

[0180] 1.2 Preparation of the aqueous phase: The mRNA stock solution obtained in Preparation Example 1 was diluted with 10 mM citrate-130 mM sodium chloride buffer at pH 4.0 to obtain an aqueous phase with an mRNA concentration of 0.18 mg / ml.

[0181] 1.3 Mixing: The lipid phase and aqueous phase were delivered using a syringe pump and mixed in a microfluidic mixer at a volume ratio of 1:3. This caused the lipids to encapsulate the mRNA, forming a precursor lipid nanoparticle suspension.

[0182] 2. Preparation of finished lipid nanoparticles: 2.1 Dialysis with pH 4.0 buffer solution (first stage): A precursor lipid nanoparticle suspension was collected, and a pH 4.0 10 mM citrate-130 mM sodium chloride buffer was used as the first-stage dialysate. Isovolume dialysis was performed 1-5 times by tangential flow filtration (TFF) to reduce the ethanol content in the system, and the drug solution after dialyzing with pH 4.0 buffer was obtained.

[0183] 2.2 Dialysis with pH 7.6 buffer (second stage): The drug solution obtained after dialysis with the pH 4.0 buffer solution was collected, and a 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution with a pH of 7.6 was used as the second-stage dialysate. Equivolute dialysis was performed 3-6 times using TFF, and as the pH value increased, the particles fused together, forming stable, finished lipid nanoparticles.

[0184] In the method described above, during the first stage (dialysis with pH 4.0 buffer), the pH of the system was maintained below the pKa of the lipid nanoparticles, and the ethanol content was gradually reduced to a constant level. This ensured that the ethanol content in the system remained low when particle fusion occurred in the second stage.

[0185] In the second stage (dialysis with pH 7.6 buffer), particle fusion began when the system's pH rose to near the pKa of the lipid nanoparticles, and the lower the ethanol content, the more complete the particle fusion. When the system's pH exceeded the pKa of the lipid nanoparticles, particle fusion was completed, and thermodynamically stable, finished lipid nanoparticles were formed.

[0186] Some specific embodiments of Example 1 are shown in Table 1.

[0187] [Table 1]

[0188] Comparative Example 1: Production of lipid nanoparticles by a one-step dialysis method This comparative example describes a conventional method for producing lipid nanoparticles. Here, precursor lipid nanoparticles were subjected to a single-step dialysis in a pH 7.6 buffer solution to obtain the finished lipid nanoparticles. The specific preparation method is as follows.

[0189] 1. Precursor lipid nanoparticles were prepared in the same manner as in Example 1.

[0190] 2. Preparation of finished lipid nanoparticles: A suspension of precursor lipid nanoparticles was collected, and a 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution at pH 7.6 or a higher concentration buffer was used as the dialysate. Equivolute dialysis was performed 5-8 times by tangential flow filtration (TFF), and the particles fused as the pH value increased, forming the finished lipid nanoparticles.

[0191] In this method, a one-step dialysis process using a pH 7.6 buffer was performed. When the pH of the system rose to near the pKa of the lipid nanoparticles, particle fusion began, resulting in the formation of finished lipid nanoparticles with high ethanol content, insufficient particle fusion, and thermodynamic instability.

[0192] Table 2 shows some specific embodiments of Comparative Example 1.

[0193] [Table 2]

[0194] Example 2: Preparation of lipid nanoparticles by a two-step process of dilution and dialysis. This example illustrates an alternative method for producing lipid nanoparticles with a low void ratio according to the present invention. Here, precursor lipid nanoparticles were diluted with pH 4.0 buffer and then dialyzed with pH 7.6 buffer to obtain the finished lipid nanoparticles. The specific preparation method is as follows.

[0195] 1. Preparation of precursor lipid nanoparticles: The same method as in Example 1 was used.

[0196] 2. Preparation of finished lipid nanoparticles 2.1 Dilution with pH 4.0 buffer: A suspension of precursor lipid nanoparticles was collected and diluted 10-fold with 10 mM citrate-130 mM sodium chloride buffer at pH 4.0 to reduce the ethanol content in the system without increasing the pH value, thereby obtaining the drug solution after dilution with pH 4.0 buffer.

[0197] 2.2 Dialysis with pH 7.6 buffer: The drug solution obtained above, after being diluted with pH 4.0 buffer, was collected and first concentrated 10-fold using tangential flow filtration (TFF). Then, using a pH 7.6 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution as the dialysate, isovolume dialysis was performed 4-6 times. As the pH value increased, the particles fused together, forming stable, finished lipid nanoparticles.

[0198] In the method described above, after the first step (dilution with pH 4.0 buffer), the pH of the system was lower than the pKa of the lipid nanoparticles, and the ethanol content was reduced to a certain level. This ensured that the ethanol content in the system was low when particle fusion occurred in the second step.

[0199] In the second stage (dialysis with pH 7.6 buffer), particle fusion began when the system's pH rose to near the pKa of the lipid nanoparticles, and the lower the ethanol content, the more complete the particle fusion. When the system's pH exceeded the pKa of the lipid nanoparticles, particle fusion was completed, and thermodynamically stable, finished lipid nanoparticles were formed.

[0200] Some specific embodiments of Example 2 are shown in Table 3.

[0201] [Table 3]

[0202] Comparative Example 2: Preparation of lipid nanoparticles by a two-step process of dilution followed by dialysis. This comparative example describes another conventional method for producing lipid nanoparticles. Here, precursor lipid nanoparticles were diluted in a pH 7.6 buffer solution and then dialyzed to obtain the final lipid nanoparticles. The specific preparation method is as follows.

[0203] 1. Preparation of precursor lipid nanoparticles: The same method as in Example 1 was used.

[0204] 2. Preparation of finished lipid nanoparticles 2.1 Dilution with pH 7.6 buffer: A suspension of precursor lipid nanoparticles was collected and diluted three-fold with a pH 7.6 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution. As the ethanol content decreased and the pH of the system increased, the drug solution after dilution with pH 7.6 buffer was obtained.

[0205] 2.2 Dialysis with pH 7.6 buffer: The drug solution diluted with the pH 7.6 buffer obtained above was collected and first concentrated 3-fold using tangential flow filtration (TFF). Then, using a pH 7.6 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution as the dialysate, isovolume dialysis was performed 4-6 times. As the pH value increased, the particles fused together, and stable, finished lipid nanoparticles were formed.

[0206] In the method described above, the pH of the system increased after the first step (dilution with pH 7.6 buffer). In the second step (liquid dialysis with pH 7.6 buffer), particle fusion began when the pH of the system rapidly increased to near the pKa of the lipid nanoparticles. However, due to the high ethanol content, particle fusion was insufficient, resulting in the formation of thermodynamically unstable finished lipid nanoparticles.

[0207] Some specific embodiments of Comparative Example 2 are shown in Table 4.

[0208] [Table 4]

[0209] Example 3: Preparation of lipid nanoparticles by a one-step dialysis process (improvement of the buffering capacity of the acidic buffer solution) This example describes a method for producing lipid nanoparticles with a low void ratio according to the present invention. Here, precursor lipid nanoparticles were subjected to a one-step dialysis in a pH 7.6 buffer solution to obtain the finished lipid nanoparticles. The specific preparation method is as follows.

[0210] 1. Preparation of precursor lipid nanoparticles 1.1 Preparation of the lipid phase: The same method as in Example 1 was used.

[0211] 1.2 Preparation of aqueous phase: The mRNA stock solution was diluted with 20 mM citrate-130 mM sodium chloride buffer at pH 4.0 to obtain an aqueous phase with an mRNA concentration of 0.18 mg / ml.

[0212] 1.3 Mixing: The method was the same as in Example 1.

[0213] 2. Preparation of the finished lipid nanoparticles: The same method as in Comparative Example 1 was used.

[0214] In this method, by employing a higher concentration citrate buffer system during the preparation of precursor lipid nanoparticles, the rate of increase in the pH value of the system during the buffer system replacement process by one-step dialysis was reduced. As a result, the ethanol content in the system was reduced to an acceptable level at the time of particle fusion, allowing the particles to fuse sufficiently and forming thermodynamically stable finished lipid nanoparticles. The one-step dialysis process in this embodiment can be divided into two stages. In the first stage, the pH value of the system was lower than the pKa of the lipid nanoparticles, and the ethanol content was reduced to a certain level, ensuring that the ethanol content in the system was low when particle fusion occurred in the second stage. In the second stage, when the pH value of the system rose to near the pKa of the lipid nanoparticles, particle fusion began, and due to the low ethanol content, particle fusion was more complete. When the pH value of the system exceeded the pKa of the lipid nanoparticles, particle fusion was completed, and thermodynamically stable finished lipid nanoparticles were formed.

[0215] Some specific embodiments of Example 3 are shown in Table 5.

[0216] [Table 5]

[0217] Example 4 Preparation of precursor lipid nanoparticles by post-loading step This example describes an alternative method for preparing precursor lipid nanoparticles according to the present invention. Unlike the standard preparation method in Example 1, which involves directly mixing the lipid phase and the aqueous phase containing nucleic acids, in the post-loading step, the lipid phase and an acidic buffer without nucleic acids are first mixed to form empty lipid nanoparticles, which are then mixed with the aqueous phase containing nucleic acids, allowing the lipids to encapsulate the nucleic acids and form precursor lipid nanoparticles. The specific preparation method is as follows.

[0218] 1.1 Preparation of the lipid phase: Four types of lipids were precisely weighed so that the molar ratio was SM-102:mPEG-DMG-2K:DSPC:cholesterol = 50%:1.5%:10%:38.5%, dissolved in anhydrous ethanol, and a lipid phase with a total lipid concentration of 12 mg / ml was obtained.

[0219] 1.2 Preparation of empty lipid nanoparticles: Using a syringe pump, the lipid phase and 10 mM citrate-130 mM sodium chloride buffer at pH 4.0 were delivered and mixed in a microfluidic mixer in a volume ratio of 1:3 to form an empty lipid nanoparticle suspension.

[0220] 1.3 Preparation of the aqueous phase: The mRNA stock solution obtained in Preparation Example 1 was diluted with 10 mM citrate-130 mM sodium chloride buffer at pH 4.0 to obtain an aqueous phase with an mRNA concentration of 0.18 mg / ml.

[0221] 1.4 Preparation of Precursor Lipid Nanoparticles: An empty lipid nanoparticle suspension and an aqueous phase were delivered using a syringe pump and mixed in a microfluidic mixer in a volume ratio of 4:3. This allowed the lipids to encapsulate mRNA, forming a precursor lipid nanoparticle suspension.

[0222] Example 5: Investigation of the relationship between particle fusion pH value and the pKa of lipid nanoparticles The fusion process of lipid nanoparticles is closely related to the charge state of LNPs containing ionizable cationic lipids. For ionizable cationic lipids, the charge ratio at different pH values ​​follows the Henderson-Hasselbalch equation.

[0223]

number

[0224] Here, [BH + ] represents the concentration of positively charged, ionizable cationic lipids, and [B] represents the concentration of neutral, ionizable cationic lipids.

[0225] In this example, using the ionizable cationic lipid SM-102 as an example, the relationship between the pH value at the start and end of particle fusion during the buffer system substitution process and the pKa of LNPs containing the ionizable cationic lipid was investigated based on changes in particle size. Furthermore, the changes in particle size during the buffer system substitution process were compared for LNPs containing the cationic lipid DOTAP, the ionizable cationic lipid SM-102, and Dlin-MC3-DMA, respectively, to clarify whether different types of cationic lipids affect the particle fusion process.

[0226] 1. pKa measurement of LNPs containing ionizable cationic lipids The pKa of LNPs containing ionizable cationic lipids was measured using a 6-(p-tolidine amino)-2-naphthalene sulfonate sodium salt (TNS) fluorescent probe, which is commonly reported in the literature. TNS is nonfluorescent in aqueous solution, but exhibits strong fluorescence when bound to cationic lipids and entering a hydrophobic environment. As the pH value decreased, the interaction between negatively charged TNS and positively charged ionizable cationic lipids increased, leading to an enhancement of the fluorescence signal. The fluorescence intensity-pH curve was fitted with a Boltzmann function, and the pH value at the peak position after differentiating the fitting result was defined as the pKa of LNPs containing ionizable cationic lipids. The specific measurement method is as follows.

[0227] 1.1 Preparation of SM-102 lipid nanoparticles: The preparation method was the same as in Example 1.

[0228] 1.2 pKa measurement of SM-102 lipid nanoparticles Sample solutions with different pH gradients were prepared by adding 1 ml of buffer solution (citrate, phosphate, or borate buffer) with a different pH gradient to a centrifuge tube, and then adding 50 μl of SM-102 lipid nanoparticle dispersion. 200 μl of each sample solution with a different pH gradient was dispensed into a 96-well plate, and 30 μl (300 μM) of the prepared TNS-DMSO reagent was added to each well. The plate was left to stand for 5 minutes under light protection. The 96-well plate was placed in a microplate reader, the fluorescence mode was selected, and the excitation wavelength was set to 321 nm and the emission wavelength to 445 nm for measurement. The fluorescence intensity-pH value curve was fitted with a Boltzmann function, and the pH value at the peak position (peak center) after differentiating the fitting result was 6.52, which was the pKa value of the lipid nanoparticles containing SM-102. The fitting curve is shown in Figure 2.

[0229] 2. Relationship between particle fusion pH value and the pKa of ionizable cationic lipids Precursor lipid nanoparticles were prepared according to the method described in Example 1, and finished lipid nanoparticles were prepared by a two-step dialysis method. During the pH increase process, the particle fusion process of ionizable cationic lipids and the relationship between the pH value and the pKa of ionizable cationic lipid LNPs when fusion occurs were explained using particle size as an indicator.

[0230] The aforementioned two-stage dialysis process is as follows:

[0231] SM-102 precursor lipid nanoparticles were collected and first subjected to triple isovolume dialysis with a pH 4.0 10 mM citrate-130 mM sodium chloride buffer, followed by dialysis with a pH 7.6 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution. During the pH increase process, samples were taken at pH 4.0, 4.5, 5.0, 5.5, 6.0, 6.2, 6.8, 7.2, and 7.4, and particle size and PDI were measured. After dialysis, lipid nanoparticles of the finished SM-102 product were collected, the pH was adjusted to 8.0 and 8.5 with a sodium hydroxide solution, and samples were taken to measure particle size and PDI. The results are shown in Table 6 and Figure 3.

[0232] [Table 6]

[0233] During the dialysis process, when the pH value rose to 6.0, no significant change in particle size was observed, and the particles had not yet begun to fuse. When the pH value rose to around the pKa of SM-102 (6.2 and 6.8), the particle size and PDI increased significantly, indicating that the particles were fusing. When the pH value rose to 7.2 or higher, the PDI decreased, and the particle size and PDI were maintained at stable levels, indicating that particle fusion was complete.

[0234] Therefore, in the two-stage substitution process, the objective of the first stage is to reduce the concentration of the organic solvent in the system, and it is necessary to maintain pH ≤ pKa -0.5. At this point, the particles do not clearly begin to fuse. The objective of the second stage is to fuse the particles to form the finished lipid nanoparticles, and it is necessary to maintain pH ≥ pKa +0.5 at the end of the process. At this point, particle fusion is completed and a steady state is reached.

[0235] 3. Particle changes of different cationic lipid LNPs during the buffer replacement process. Precursor lipid nanoparticles were prepared using the ionizable cationic lipids SM-102, Dlin-MC3-DMA, and the permanently charged cationic lipid DOTAP, according to the method described in Example 1. Finished lipid nanoparticles were then prepared using a two-step dialysis process. During the pH increase process, the particle fusion process was investigated using particle size as an indicator.

[0236] Precursor lipid nanoparticles were prepared using the cationic lipid DOTAP, the ionizable cationic lipid SM-102, or Dlin-MC3-DMA, respectively, according to the method described in Example 1. First, isovolume dialysis was performed three times with a pH 4.0 10 mM citrate-130 mM sodium chloride buffer to remove ethanol, and then dialyzed with a pH 7.6 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution. Samples were taken during the pH increase process, and the ethanol content, particle size, and PDI were measured. The results are shown in Table 7 and Figure 4-5.

[0237] [Table 7]

[0238] The following was found from the changes in particle size and PDI during the buffer system substitution process described above.

[0239] (1) Lipid nanoparticles containing the ionizable cationic lipids SM-102 and Dlin-MC3-DMA fused at pH 6.36 and 6.40, respectively, resulting in increased particle size and PDI. Subsequently, fusion was completed at pH 7.29 and 7.30, respectively, with a significant increase in particle size and a significant decrease in PDI. After particle fusion was complete, both particle size and PDI were maintained at stable levels.

[0240] (2) Lipid nanoparticles containing the cationic lipid DOTAP remained charged within the pH range of the process, and particle size and PDI steadily increased. No particle fusion process due to changes in charge state occurred during the buffer system substitution process.

[0241] These results indicate that, for permanently charged cationic lipid LNPs, a particle fusion process due to a change in charge state does not occur during the buffer system substitution process, whereas for ionizable cationic lipid LNPs, a particle fusion process generally exists, and fusion occurs when the pH value reaches near pKa.

[0242] Example 6: Summary of the physicochemical properties of lipid nanoparticles from different examples and comparative examples. 1. Method for measuring encapsulation rate: The nucleic acid encapsulation rate of finished lipid nanoparticles was measured using the Ribogreen® fluorescence kit. After diluting the sample with TE buffer, it was added to a 96-well plate made of black polystyrene. TE buffer or an equal volume of Triton X-100 was added, and after incubation, Ribogreen® fluorescence reagent diluted with TE was added to the sample. Fluorescence intensity was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. The ratio of fluorescence intensity between the sample diluted with TE and the sample destroyed with Triton X-100 represented the percentage of free mRNA.

[0243] 2. Particle size measurement method: The particle size and polydispersity coefficient (PDI) of lipid nanoparticles were measured using a Malvern Zetasizer Nano ZS laser particle size distribution analyzer.

[0244] 3. Comparison of Particle Physicochemical Properties: During the buffer system substitution process, the degree of particle fusion is affected by the organic solvent content at the time of fusion, which in turn affects important physicochemical properties such as particle size, PDI, and encapsulation rate of the finished lipid nanoparticles, as well as storage stability. For different examples and comparative examples, important physicochemical properties and residual organic solvents before and after particle fusion during the buffer system substitution process were statistically compared. The results of the comparison of major physicochemical properties of particles before and after fusion are shown in Table 8.

[0245] [Table 8]

[0246] As is clear from the table above, compared to the comparative method, the manufacturing method of the present invention allows particle fusion to proceed more completely (larger particle size after fusion) by reducing the ethanol content to a low level (<5%) before particle fusion.

[0247] Example 7: Effect of ethanol content on particle fusion Precursor lipid nanoparticles were prepared according to the method described in Example 1, and 0.5 ml of the suspension was taken. This was injected into 5 ml G50 dextran gel columns pre-equilibrated with pH 7.6 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solutions containing different ethanol content, and then eluted using the corresponding equilibration buffer. When the eluate turned milky white, the samples were collected, and the pH value and particle size of the finished lipid nanoparticles prepared by dextran gel column dialysis were measured to investigate the effect of ethanol content on particle fusion during the dextran gel column dialysis process. The results are shown in Table 9.

[0248] [Table 9]

[0249] During particle fusion, when the ethanol content was ≤5%, the degree of particle size increase was similar, and the particle size after dialysis was 105 nm or larger, indicating complete particle fusion. When the ethanol content was ≥10%, the degree of particle size increase was small, and the particle size after dialysis was 90 nm or smaller, indicating insufficient particle fusion.

[0250] Example 8: Reverse Dialysis The reverse dialysis method further demonstrated the conclusion that steady and quasi-steady states exist in the fusion process. The specific method for performing reverse dialysis is as follows.

[0251] First, precursor lipid nanoparticles were prepared according to the method described in Example 1. The precursor lipid nanoparticle suspension was collected and subjected to 5-fold isovolume osmosis with a pH 7.6 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution, followed by 3-fold isovolume reverse dialysis with a pH 4.0 10 mM citrate-130 mM sodium chloride buffer, and finally 3-fold isovolume dialysis with a pH 7.6 20 mM Tris-10.7 mM NaOAc-8.7% sucrose solution.

[0252] During the preparation of the finished lipid nanoparticles by reverse dialysis, the system changed from acidic to neutral and then back to acidic and finally neutral. The initial process from pH 4.0 to pH 7.6 was similar to that of the preparation of the finished lipid nanoparticles by the one-step dialysis method in Comparative Example 1, with the first stage ending at isovolume dialysis 1x. The pH value of the system was below the pKa of the lipid nanoparticles containing ionizable cationic lipids, and the ethanol content was 8%, exceeding the acceptable limit. The second stage ended at isovolume dialysis 2x or higher. The pH value of the system rose above the pKa of the lipid nanoparticles containing ionizable cationic lipids, and during the pH increase process, the particles fused to form the finished lipid nanoparticles. However, due to the high ethanol content in the system, the fusion between particles was insufficient, and a steady state was not reached. After removing ethanol with dialysis 5x, the particle size of the obtained particles was 80 nm. Reverse dialysis (from pH 7.6 to pH 4.0) allowed the empty LNPs and unstable LNPs to fuse again, reaching a steady state, and the resulting particle size was 110 nm. In the second step of the pH change from 4.0 to 7.6, the particle size of the obtained particles was 117 nm, which was almost consistent with the value after reverse dialysis. The changes in particle size and PDI during the process are shown in Table 10 and Figure 6.

[0253] [Table 10]

[0254] The results showed that when the ethanol content of the system was high (>5%) at the time particle fusion occurred, particle fusion was insufficient. The resulting quasi-steady state particles fused further under low ethanol content (<0.1%) to become steady state particles. This suggests that quasi-steady state particles may undergo significant changes in their physicochemical properties (such as particle size) during storage.

[0255] Example 9 Detection of nucleic acid encapsulation rate by fluorescence nanoflow cytometry Using fluorescence nanoflow cytometry, the ratio of nucleic acid-encapsulated LNPs to empty LNPs was measured, and the particle fusion status during the process and the ratio of nucleic acid-encapsulated LNPs to empty LNPs in products obtained by different manufacturing methods were compared and investigated.

[0256] The basic principle of measuring the nucleic acid encapsulation rate by fluorescence nanoflow cytometry is as follows.

[0257] After adding a membrane-permeable nucleic acid dye to the sample, the dye permeated through the LNP membrane and bound to the internal mRNA, generating fluorescence. From this fluorescence signal and scattering signal, the ratio of nucleic acid-encapsulated LNP to empty LNP in the sample was calculated. Specific test method: After collecting the measurement sample, adding ribonuclease and incubating to remove free mRNA, adding a nucleic acid dye and incubating, and then using a nanoflow cytometer to measure the scattered light signal of individual LNPs. Through the fluorescence labeling of the dye, empty LNPs and nucleic acid-encapsulated LNPs were quickly identified, and the ratio of nucleic acid-encapsulated LNPs to empty LNPs was calculated.

[0258] Using the fluorescence nanoflow cytometry method, the ratio change of nucleic acid-encapsulated LNP to empty LNP in the intermediate of the LNP production process of Embodiment 1-1 was measured. The results are shown in Table 11 and Figure 7. After mixing the lipid phase and the aqueous phase, and after dialysis with a pH 4.0 buffer solution, the ratio of nucleic acid-encapsulated LNP was about 20%. After dialysis with a pH 7.6 buffer solution, since particle fusion reached a steady state, there was basically no empty LNP in the system, and the ratio of particles encapsulating mRNA reached more than 90%.

[0259]

Table 11

[0260] Using the fluorescence nanoflow cytometry method, the ratio of nucleic acid-encapsulated LNP to empty LNP was measured for the lipid nanoparticles of the finished products obtained from different examples and comparative examples. The results are shown in Table 12. In the examples, the ratio of empty LNP was all 10% or less, indicating that particle fusion proceeded sufficiently, the empty particle rate was low, and it was confirmed that a steady state had been reached.

[0261]

Table 12

[0262] Example 10 Morphological evaluation by cryo-TEM We evaluated the morphology of lipid nanoparticles using a cryo-TEM (low-temperature transmission electron microscope) and compared and examined the changes in particle morphology during the manufacturing process and the product morphology obtained by different manufacturing methods.

[0263] Figure 8 shows a low-temperature transmission electron microscope image of the intermediate in the LNP manufacturing process of Embodiment 1-1. After mixing the lipid phase and the aqueous phase, and after pH 4.0 buffer dialysis, small-particle-size lipid nanoparticles were present in the system. After pH 7.6 buffer dialysis, the particles fused to form large, uniform lipid nanoparticles. The low-temperature transmission electron microscope results indicate the presence of a particle fusion process in LNP manufacturing, and this result is consistent with the particle size and fluorescence nanoflow cytometry results.

[0264] Figure 9 shows a low-temperature transmission electron microscope image of the finished lipid nanoparticles obtained in Embodiment 1-1. The particle size was uniform and the morphology was consistent. On the other hand, the low-temperature transmission electron microscope image of the finished lipid nanoparticles obtained in Comparative Example 1-1 (Figure 10) showed a variety of particle morphologies, with numerous initial fused LNPs or empty LNPs present. Such particles represent a thermodynamically unstable system, and there was a possibility that the physicochemical properties of the particles would change during the storage process.

[0265] Example 11 Preparation of lipid nanoparticles containing different types of nucleic acids Lipid nanoparticles could be used to deliver various genetic materials such as siRNA, pDNA, and mRNA. Using the method of Embodiment 1-1 of the present invention, lipid nanoparticles of siRNA (conventional siRNA, GenePharm) and pDNA (pVAX.1, GenScript Biotech Co., Ltd.) were prepared, and the physicochemical properties and nucleic acid encapsulation rates of the obtained products are shown in Table 13. As the results in the table show, the product had a uniform particle size, a high encapsulation rate, and a low empty particle rate.

[0266] [Table 13]

[0267] The sequence of the above siRNA (patisiran sodium, MedChemExpress) is: RNA (SEQ ID NO: 28, AUGGAA-Um-ACUCUUGGU-Um-AC-dT-dT), binding RNA (SEQ ID NO: 29, G-Um-A-ACm-Cm-AAGAG-Um-A-Um-Um-Cm-Cm-A-Um-dT-dT) (1:1) sodium salt. Here, m represents 2'-OMe modification and dT represents thymidine nucleotide.

[0268] The above pDNA (pVAX.1) was Invitrogen™, catalog number: V26020.

[0269] Using the method of Embodiment 1-1 of the present invention, the mRNA stock solution obtained in Preparation Example 1 was replaced with mRNA represented by other sequences in the present invention (mRNA represented by SEQ ID NO: 2-7 or SEQ ID NO: 8-26), and all other operations were carried out in the same manner as in Embodiment 1-1. In all cases, products with ideal characteristics such as uniform particle size, high encapsulation rate, and low empty particle rate were obtained.

[0270] Example 12: Study on the storage stability of intermediates in the manufacturing process of lipid nanoparticles. The intermediates from the manufacturing process of Embodiment 1-1 were stored at 2-8°C for two months, and their particle size was measured. The results showed that particles that had fully fused (after pH 7.6 buffer dialysis) were highly stable, and no significant changes were observed in either the average particle size or the PDI. However, particles that had not fully fused (after mixing and after pH 7.6 buffer dialysis) showed a clear increase in particle size and PDI. The results are shown in Table 14.

[0271] [Table 14]

[0272] Example 13: Study of freeze-thaw stability of mRNA-LNPs In the storage, transportation, and use of lipid nanoparticles, differences in the stability of lipid nanoparticles prepared by different manufacturing methods occur. For mRNA-LNPs stored by freezing, when the finished lipid nanoparticles prepared according to the manufacturing methods of Example 1-1 and Comparative Example 1-1 were repeatedly frozen and thawed (-20°C ± 5°C / room temperature), changes in physicochemical properties were investigated. The results are shown in Table 15.

[0273]

Table 15

[0274] The mRNA-LNP prepared in Example 1-1 showed a significantly smaller degree of increase in particle size and PDI after 30 freeze-thaw cycles compared to Comparative Example 1-1, indicating that the freeze-thaw stability of the lipid nanoparticles prepared in Example 1-1 is more excellent.

[0275] Stability Study of Example 14 mRNA-LNP at Freezing, Refrigeration, and Room Temperature The lipid nanoparticle composition (mRNA-LNP) prepared in Example 1-1 was stored under freezing (-20°C ± 5°C), refrigeration (5°C ± 3°C), and room temperature (25°C ± 2°C) conditions, respectively, sampled at different time points, and the particle size, PDI, mRNA purity, and biological activity were analyzed. The stability results are shown in Table 16.

[0276] mRNA purity detection method: mRNA was extracted and purified using the PureLink RNA Mini Kit (Invitrogen) kit, and the purified mRNA was concentrated to a concentration of 200 ng / μl using a 30KD ultrafiltration centrifugal tube (Merck). After analysis and confirmation by a Nanodrop ultraviolet spectrophotometer (Thermo), the purity of mRNA was analyzed and evaluated using an Agilent 2100 bioanalyzer.

[0277] Biological activity detection method: Healthy, well-developed 6-8 week old BALB / c mice were selected, sorted by body weight, and randomly divided into groups (10 mice / group, half male and half female). Each animal was immunized twice at 7-day intervals with a dose of 5 μg. Serum was collected 7 days after the final dose, and the titer of anti-S protein-binding antibody in each mouse serum sample was measured by ELISA. The geometric mean of antibody titers for all test mice was calculated.

[0278] [Table 16]

[0279] The results showed that the lipid nanoparticle composition (mRNA-LNP) exhibited no significant changes in either the physicochemical properties or biological activity of the particles even after storage under refrigerated conditions (5°C ± 3°C) for 6 months, under room temperature conditions (25°C ± 2°C) for 14 days, and under frozen conditions (-20°C ± 5°C) for 12 months, demonstrating superior thermal stability compared to similar products.

[0280] Example 15: Efficacy testing of pseudotyped virus-neutralizing antibodies Lipid nanoparticles prepared in Embodiment 1-1 and Comparative Example 1-1 were used. BALB / c mice (6-8 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd) were immunized by intramuscular injection using two different immunization programs (5 mice / group, female). The two immunization programs were as follows: (1) Two immunizations at 7-day intervals, serum collected 14 days after the final dose, with a dose of 10 μg. (2) Two immunizations at 21-day intervals, serum collected 7 days after the final dose, with a dose of 3 μg. After immunization, mouse serum was collected and diluted in DMEM complete medium (containing hydroxylone, 1% bispecific antibody, 10% FBS, 1% non-essential amino acids, and 1% HEPES), with the initial dilution being 30-fold, followed by subsequent 3-fold dilutions. This was then stored in 650 TCID. 50SARS-CoV-2 WT strain and BA.4 / 5 pseudotype virus strain (Zhongke Guobang (Beijing) Inspection and Testing Co., Ltd.) were co-incubated at 37°C for 1 hour each, then 2x10 4 After adding individual Vero cells and culturing for 20-28 hours, the supernatant was discarded, luciferase detection reagent (Perkin Elmer) was added, and the fluorescence value was read. The titer of the 50% neutralizing antibody was calculated according to the Reed-Muench method. The results are shown in Figure 11. The results showed that in both immunization programs, the immunoserum of Embodiment 1-1 exhibited superior neutralizing activity against both the WT strain and the BA.4 / 5 pseudotype virus compared to Comparative Example 1-1.

[0281] Example 16: In vitro cell transfection efficacy testing Luciferase mRNA-LNP was prepared by replacing the mRNA of SEQ ID NO:1 with the RNA sequence encoding luciferase (SEQ ID NO:27) using the methods of Embodiment 1-1 and Comparative Example 1-1 of the present invention. Luciferase mRNA-LNP was co-incubated with HEK293 T cells (ATCC) to create four groups: 50 ng mRNA / well, 100 ng mRNA / well, 200 ng mRNA / well, and 400 ng mRNA / well (96-well plate). After 24 hours of incubation, luciferase expression was quantitatively detected by adding a luciferase detection reagent (Promega) (n=3). A two-sided unpaired t-test was used for data analysis, with * indicating P<0.05, ** indicating P<0.01, *** indicating P<0.001, and ns indicating no significant difference. The results are shown in Figure 12. The results clearly showed that the in vitro cell transfection efficacy of luciferase mRNA-LNP prepared by the method of Embodiment 1-1 was superior to that of luciferase mRNA-LNP prepared by the method of Comparative Example 1-1.

[0282] Example 17: Animal tissue distribution test C57BL / 6J mice (6-8 weeks old, Beijing Vital River Laboratory Animal Technology Co., Ltd.) were administered 5 μg of lipid nanoparticles prepared in Embodiment 1-1 and Comparative Example 1-1, respectively, by single intramuscular injection (6 mice / group, half male and half female). Whole mouse blood (orbital blood collection, EDTA anticoagulation) was collected 2, 24, and 96 hours after administration, perfused with physiological saline, and then the mouse hearts and local muscle tissue were excised. RNA was extracted using the PureLink RNA Mini Kit (Thermo Fisher Scientific), and the RNA concentration was measured by ultraviolet spectrophotometric analysis (Nanodrop instrument, Thermo Scientific). Next, RNA was reverse transcribed into cDNA using the PrimeScript® RT Master Mix kit (TaKaRa). Taq Pro HS Universal U+ Probe Master Mix (Vazyme) and primers PRD-F6 (SEQ ID NO: 30, AGCGTGCTCTATAACTCGGC), PRD-R6 (SEQ ID NO: 31, TCGGACCTCATCGCCTCTAA), and PRD-P6 (SEQ ID NO: 32, ACGGCGTGAGCCCCACAAAG) were added, and qPCR was performed using a LightCycler 480 real-time PCR instrument to quantitatively detect mRNA levels in blood and tissue. Unpaired two-sided t-tests were used for data analysis, with * indicating P<0.05, ** indicating P<0.01, *** indicating P<0.001, and ns indicating no significant difference. The results are shown in Figure 12. AUC (Area Under Curve) results up to 96 hours after administration showed that the samples prepared in Embodiment 1-1 had low levels of mRNA distribution in the heart and blood, which reduced the likelihood of myocarditis and systemic adverse reactions.

[0283] The following shows the relevant sequences related to the present invention.

[0284] SEQ ID NO:1

[0285]

[0286] SEQ ID NO:2

[0287]

[0288] SEQ ID NO:3

[0289]

[0290] SEQ ID NO:4

[0291] SEQ ID NO:5

[0292]

[0293] SEQ ID NO:6

[0294]

[0295] SEQ ID NO:7

[0296] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNY R YRLFRKSNLKPFERDISTEIYQAGSTPCNGV Q GFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQ GVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGG FNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGITITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLD PP EAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSSGNCDVVI GIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0297] (In the sequences described in the specification, the underlined parts indicate the relevant mutation sites for the WT virus strain.)

[0298] SEQ ID NO.8 (SF-1-BA.5 BA.5 S protein natural sequence)

[0299]

[0300] SEQ ID NO.9 (SF-2-BA.5 codon optimized sequence)

[0301]

[0302] SEQ ID NO.10 (SF-3-BA.5 codon-optimized sequence)

[0303]

[0304] SEQ ID NO.11 (SF-4-BA.5 codon-optimized sequence)

[0305]

[0306] SEQ ID NO.12 (SF-5-BA.5 codon-optimized sequence)

[0307]

[0308] SEQ ID NO.13 (SF-6-XBB-1 codon-optimized sequence)

[0309]

[0310] SEQ ID NO.14 (SF-7-XBB-2 codon optimized sequence)

[0311]

[0312] SEQ ID NO.15 (SF-8-BA.2.75-1 Codon-Optimized Sequence)

[0313]

[0314] SEQ ID NO.16 (SF-9-BA.2.75-2 Codon-Optimized Sequence)

[0315]

[0316] SEQ ID NO.17 (SF-10-BQ.1-01 codon-optimized sequence)

[0317]

[0318] SEQ ID NO.18 (SF-11-BQ.1-02 Codon-Optimized Sequence)

[0319]

[0320] SEQ ID NO.19 (SF-12-BF.7-01 Codon-Optimized Sequence)

[0321]

[0322] SEQ ID NO.20 (SF-13-BF.7-02 Codon-Optimized Sequence)

[0323]

[0324] SEQ ID NO.21 (SF-14-Ch.1.1-01 Codon-Optimized Sequence)

[0325]

[0326] SEQ ID NO.22 (SF-15-XBB.1.5 codon-optimized sequence)

[0327]

[0328] SEQ ID NO.23 (SF-16-XBB.1.16 codon-optimized sequence)

[0329]

[0330] SEQ ID NO.24 (SF-17-BA.5 codon-optimized sequence)

[0331]

[0332] SEQ ID NO.25 (SF-18-BA.5 codon-optimized sequence)

[0333]

[0334] SEQ ID NO.26 (SF-19-BA.5 codon-optimized sequence)

[0335]

[0336] SEQ IND NO:27 Luciferase RNA sequence

[0337]

[0338] Note: The T and U in the above sequence may be interchanged, and all or some of them may be replaced with modified bases, such as 1-methyl pseudouridi or pseudouridi.

[0339] SEQ ID NO:28

[0340] AUGGAAUmACUCUUGGUUmACdTdT

[0341] SEQ ID NO:29

[0342] GUmAACmCmAAGAGUmAUmUmCmCmAUmdTdT

[0343] SEQ ID NO:30

[0344] AGCGTGCTCTATAACTCGGC

[0345] SEQ ID NO:31

[0346] TCGGACCTCATCGCCTCTAA

[0347] SEQ ID NO:32

[0348] ACGGCGTGAGCCCCACAAAG

Claims

1. A lipid nanoparticle composition for encapsulating nucleic acids, A lipid nanoparticle composition for encapsulating nucleic acids, wherein the empty particle ratio of the lipid nanoparticles is 10% or less, preferably 9% or less, and more preferably 8.5% or less, and the empty particle ratio refers to the ratio of the number of empty lipid nanoparticles that do not encapsulate nucleic acids to the total number of lipid nanoparticles in the composition.

2. The composition according to claim 1, wherein the average particle size of the lipid nanoparticles is 50 nm to 150 nm, preferably 70 nm to 120 nm, and more preferably 90 nm to 110 nm.

3. The composition according to claim 1, wherein the encapsulation rate of the lipid nanoparticles is 80% or more, preferably 85% or more, and more preferably 90% or more.

4. The aforementioned lipid nanoparticles (1) Nucleic acids, and (2) Lipid components including ionizable cationic lipids, helper lipids, structural lipids and surfactants A composition according to any one of claims 1 to 3, comprising

5. The composition according to claim 4, wherein the lipid component comprises, with the total molar content of the lipid component being 100%, 20-60 mol% of ionizable cationic lipids, 25-55 mol% of structural lipids, 2-25 mol% of helper lipids, and 0.5-15 mol% of surfactants.

6. The cationic lipid is selected from SM-102, ALC-0315, Dlin-MC3-DMA, DODMA, C12-200, and DlinDMA, and / or The structural lipid is selected from cholesterol or a cholesterol derivative, and / or The helper lipid is selected from DSPC, DOPE, DOPC, DOPG, and DOPS, and / or The composition according to claim 4 or 5, wherein the surfactant is selected from mPEG-DMG-2K, ALC-0159, PEG-DSPE, DTDA-PEG2000, and TPGS.

7. The composition according to claim 4 or 5, wherein the N:P ratio of the lipid nanoparticles is about 2:1 to about 30:1, preferably about 2:1 to about 15:1, more preferably about 2:1 to about 10:1, and even more preferably about 3:1 to about 6:

1.

8. The composition according to claim 4, wherein the weight ratio of ionizable cationic lipids to nucleic acids is about 5:1 to about 100:1, preferably about 5:1 to about 50:1, more preferably about 5:1 to about 30:1, and even more preferably about 10:1 to about 20:

1.

9. The composition according to claim 4, wherein the lipid nanoparticle composition further comprises a buffer of the finished product, and the buffer of the finished product comprises a buffering agent and / or a cryoprotectant.

10. The buffer is selected from acetate, formate, carbonate, phosphate, borate, succinate, gluconate, lactate, citric acid, glycine, leucine, barbiturate, phthalate, tromethamine (Tris), triethylamine, ammonium salt, and combinations thereof, and the pH value of the buffer is 7-8, and / or The composition according to claim 9, wherein the cryoprotectant is selected from sugars, polyols, polymers, surfactants, amino acids, and salts, and the sugar is selected from lactose, sucrose, trehalose, and galactose.

11. The composition according to claim 9, wherein the buffer solution of the finished product contains tromethamine, sodium acetate, and sucrose, and the pH value of the buffer solution is 7-8.

12. The nucleic acid is selected from mRNA, and the mRNA contains a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any one nucleotide sequence of sequence number ID NO: 1-6, or The coronavirus antigen it codes for contains an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with the amino acid sequence of Sequence ID No. 7, or It includes a nucleotide sequence that has at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100% identity with any one nucleotide sequence with Sequence ID No. 8-26. Preferably, the mRNA comprises the nucleotide sequence of sequence number (SEQ ID NO): 1, and more preferably, the mRNA comprises the nucleotide sequence of sequence number (SEQ ID NO): 1, according to claim 1.

13. It contains nucleic acid-encapsulated lipid nanoparticles, tromethamine, sodium acetate, and sucrose, however, The pH of the aforementioned composition is 7.0-8.

0. The nucleic acid is mRNA, and its concentration is 100 μg / ml, and The composition according to claim 1, wherein the lipid components of the lipid nanoparticles comprise 50 mol% SM-102, 10 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% mPEG-DMG-2K.

14. A method for producing a lipid nanoparticle composition according to any one of claims 1 to 13, A step of preparing precursor lipid nanoparticles to obtain a buffer system containing precursor lipid nanoparticles, The process includes the step of replacing a buffer system containing precursor lipid nanoparticles with a neutral buffer system to obtain the finished lipid nanoparticle composition. A method for producing a lipid nanoparticle composition, wherein, by the aforementioned buffer system substitution, the content of the organic solvent in the buffer system containing the precursor lipid nanoparticles is reduced to 5% (w / w) or less, preferably less than 3% (w / w), when the pH value of the system is 0.5 units or more lower than the pKa of the lipid nanoparticles.

15. The manufacturing method according to claim 14, wherein the buffer system substitution includes a two-stage buffer system substitution step, preferably comprising a step in which the buffer system is substituted with an acidic buffer or a neutral buffer in the first stage, and a step in which it is substituted with a neutral buffer in the second stage.

16. The manufacturing method according to claim 15, wherein the substitution step is selected from one or more of tangential flow filtration, membrane dialysis, column dialysis, and dilution.

17. At the end of the first stage, the pH value of the buffer system containing the precursor lipid nanoparticles is 0.5 units or more lower than the pKa of the lipid nanoparticles, the organic solvent content is reduced to 5% (w / w) or less, preferably less than 3% (w / w), and / or The manufacturing method according to claim 15, wherein at the end of the second stage, the pH value of the buffer system containing the lipid nanoparticles of the finished product is 0.5 units or more higher than the pKa of the lipid nanoparticles, and / or the organic solvent content in the buffer system containing the lipid nanoparticles of the finished product is reduced to less than 1% (w / w), preferably less than 0.1% (w / w).

18. The manufacturing method according to claim 15, wherein the pH value of the acidic buffer is less than the pKa of the lipid nanoparticles, and the buffer comprises a buffer selected from acetate, formate, carbonate, phosphate, borate, succinate, gluconate, lactate, citric acid, glycine, leucine, barbituric acid, phthalic acid, tromethamine (Tris), triethylamine, ammonium salt, and combinations thereof, and an optional osmotic pressure regulator selected from sodium chloride and potassium chloride, preferably the buffer comprises citric acid and sodium chloride.

19. The pH value of the neutral buffer is higher than the pKa of the lipid nanoparticles, and the buffer comprises a buffer selected from acetates, formates, carbonates, phosphates, borates, succinates, glucons, lactates, citric acid, glycine, leucine, barbiturates, phthalates, tromethamine (Tris), triethylamine, ammonium salts and combinations thereof, and an optional cryoprotectant selected from sugars, polyols, polymers, surfactants, amino acids and salts. Preferably, the manufacturing method according to claim 15, wherein the buffer solution comprises tromethamine, sodium acetate, and sucrose.

20. The manufacturing method according to any one of claims 14 to 19, wherein the pKa of the lipid nanoparticles is 6.0-7.

0.

21. A step of forming a lipid phase by dissolving lipid components, including ionizable cationic lipids, helper lipids, structural lipids, and surfactants, in an organic solvent; a step of forming an aqueous phase by dissolving nucleic acids in an acidic buffer; and a step of mixing the two phases, the lipid phase and the aqueous phase, so that the lipids encapsulate the nucleic acids and form precursor lipid nanoparticles, or The manufacturing method according to claim 14, comprising the steps of: dissolving lipid components including ionizable cationic lipids, helper lipids, structural lipids and surfactants in an organic solvent to form a lipid phase; mixing the lipid phase with an acidic buffer to form empty lipid nanoparticles; dissolving nucleic acids in an acidic buffer to form an aqueous phase; and mixing the empty lipid nanoparticles with the aqueous phase containing nucleic acids to cause the lipids to encapsulate the nucleic acids and form precursor lipid nanoparticles.

22. (1) A step of dissolving lipid components in an organic solvent to form an organic phase, (2) A step of dissolving nucleic acids in an acidic buffer to form an aqueous phase. (3) A step in which two phases, an organic phase and an aqueous phase, are mixed, and then lipids encapsulate nucleic acids to form precursor lipid nanoparticles, and (4) The process includes a step of replacing the organic solvent-acid buffer system containing the precursor lipid nanoparticles obtained in step (3) with a neutral buffer system by two-step substitution to obtain the finished lipid nanoparticle composition. A method for producing a lipid nanoparticle composition containing nucleic acids according to any one of claims 1 to 13, wherein the nucleic acids are replaced with an acidic buffer or a neutral buffer in the first step and with a neutral buffer in the second step.

23. The manufacturing method according to claim 22, wherein in step (4), at the end of the first step, the pH value of the buffer system containing the precursor lipid nanoparticles is 0.5 units or more lower than the pKa of the lipid nanoparticles, and / or the organic solvent content in the buffer system containing the precursor lipid nanoparticles is reduced to 5% (w / w) or less, preferably less than 3% (w / w).

24. The manufacturing method according to claim 22, wherein in step (4), at the end of the second stage, the pH value of the buffer system containing the lipid nanoparticles of the finished product is 0.5 units or more higher than the pKa of the lipid nanoparticles, and / or the organic solvent content in the buffer system containing the lipid nanoparticles of the finished product is reduced to less than 1% (w / w), preferably less than 0.1% (w / w).

25. The manufacturing method according to claim 22, which optionally includes step (5) concentration, dilution, sterilization filtration, sterile filling, or a combination thereof.

26. The manufacturing method according to claim 22, wherein the organic solvent in step (1) is selected from C1-C4 lower alcohols, preferably ethanol.

27. The manufacturing method according to claim 22, wherein the total concentration of lipid components in the organic phase during step (1) is 10-15 mg / ml.

28. The manufacturing method according to claim 22, wherein the nucleic acid concentration in the aqueous phase during step (2) is 0.01-1 mg / ml, preferably 0.05-0.5 mg / ml, and more preferably 0.1-0.2 mg / ml.

29. The manufacturing method according to claim 22, wherein the acidic buffer in step (2) or step (4) comprises a buffer selected from acetate, formate, carbonate, phosphate, borate, succinate, gluconate, lactate, citric acid, glycine, leucine, barbituric acid, phthalic acid, tromethamine (Tris), triethylamine, ammonium salt, and combinations thereof, and the pH value of the acidic buffer is less than the pKa of the lipid nanoparticles.

30. The manufacturing method according to claim 29, wherein the pKa of the lipid nanoparticles is 6.0-7.0, and the pH value of the acidic buffer in step (2) or step (4) is 3.0-5.0, preferably 4.0-5.

0.

31. The manufacturing method according to claim 22, wherein the acidic buffer in step (2) or step (4) further comprises an osmotic pressure regulator, and the osmotic pressure regulator is selected from sodium chloride and potassium chloride.

32. The manufacturing method according to claim 31, wherein the acidic buffer in step (2) or step (4) comprises citric acid and sodium chloride, preferably 10-20 mM citric acid and 120-140 mM sodium chloride.

33. The manufacturing method according to claim 22, wherein the volume ratio of the two-phase mixture of the organic phase and the aqueous phase in step (3) is 1:2-1:9, preferably 1:2-1:5, and more preferably 1:

3.

34. The manufacturing method according to claim 22, wherein the substitution step in step (4) is selected from one or more of tangential flow filtration, membrane dialysis, column dialysis, and dilution.

35. The manufacturing method according to claim 22, wherein the two-stage substitution step in step (4) is repeated multiple times.

36. The manufacturing method according to claim 22, wherein the neutral buffer in step (4) comprises a buffer selected from acetate, formate, carbonate, phosphate, borate, succinate, gluconate, lactate, citric acid, glycine, leucine, barbituric acid, phthalic acid, tromethamine (Tris), triethylamine, ammonium salt, and combinations thereof, and the pH value of the neutral buffer is higher than the pKa of the lipid nanoparticles.

37. The manufacturing method according to claim 36, wherein the pKa of the lipid nanoparticles is 6.0-7.0, and the pH value of the neutral buffer in step (4) is 7.0-8.

0.

38. The manufacturing method according to claim 36, wherein the neutral buffer in step (4) further comprises a cryoprotectant selected from sugars, polyols, polymers, surfactants, amino acids, and salts, and the sugar is selected from lactose, sucrose, trehalose, and galactose.

Images