Lyoprotectant for nucleic acid-lipid nanoparticles, formula of lyophilized formulation, and preparation method

Abstract

Disclosed in the present invention is a lyoprotectant for nucleic acid-lipid nanoparticles. The lyoprotectant of the present invention is formed by combining sucrose or trehalose with polyvinylpyrrolidone in a specific ratio, and provides a good lyoprotective effect for the nucleic acid-lipid nanoparticles, ensuring that there is no significant difference in particle size, uniformity, encapsulation efficiency, nucleic acid integrity, and expression performance of the nucleic acid-lipid nanoparticles before and after lyophilization. Furthermore, by adjusting the salt concentration in a mixed solution of the nucleic acid-lipid nanoparticles and the lyoprotectant, the lyoprotective effect on the nucleic acid-lipid nanoparticles is further improved, so that the performance of lyophilized nucleic acid-lipid nanoparticles is closer to that before lyophilization.

Description

A lyophilization protectant for nucleic acid-lipid nanoparticles, a lyophilization formulation and a preparation method Technical Field

[0001] This invention relates to the field of nucleic acid drug technology, and more specifically, to a lyophilization protectant for nucleic acid-lipid nanoparticles, a lyophilization formulation, and a preparation method thereof. Background Technology

[0002] With the tremendous success of mRNA vaccines in the COVID-19 pandemic, mRNA therapy has been recognized as a novel treatment approach with significant clinical value and promise. Beyond preventative vaccines, mRNA-based therapies are increasingly being used in therapeutic vaccines, therapeutic drugs, and other fields.

[0003] Because naked mRNA faces hydrolysis by nucleases in vivo, and its negative charge and large size make it difficult for cells to take up, a suitable delivery system is needed. Currently, lipid nanoparticles (LNPs) are the most important and effective nucleic acid delivery carriers in clinical practice. Typically, LNPs consist of four lipid components: ionizable lipids, neutral accessory phospholipids (such as DSPC, DOPE, etc.), cholesterol lipids, and polyethylene glycol esters (PEG-Lipid). Among these, ionizable lipids are considered the most important component of LNPs, directly determining the encapsulation and delivery efficiency of mRNA.

[0004] Because mRNA itself is relatively unstable and easily inactivated by enzymatic hydrolysis, oxidation, or other processes, and because ionizable lipids and helper phospholipids in lipid nanoparticles are also susceptible to slow oxidation and hydrolysis due to factors such as pH, dissolved oxygen, ions, and temperature, mRNA-LNPs exhibit poor stability in solution or under high-temperature conditions. Therefore, to extend the shelf life of mRNA-LNPs, extremely low temperatures are generally required for storage and transportation. For example, the mRNA COVID-19 vaccine COMIRNATY requires storage and transportation at -90℃ to -60℃, while the mRNA COVID-19 vaccine SPIKEVAX and the respiratory syncytial virus (RSV) vaccine mRESVIA require storage and transportation at -50℃ to -15℃ and -40℃ to -15℃, respectively. These extremely low storage and transportation temperatures significantly increase the clinical cost of using mRNA vaccines or drugs.

[0005] Of the factors affecting the stability of mRNA-LNPs mentioned above, the vast majority are due to the presence of moisture. Freeze-drying can remove moisture from the formulation, making it an effective way to extend shelf life and lower storage and transportation barriers for products requiring ultra-low temperature freezing and transportation. While there are existing reports on freeze-drying protectants for mRNA-LNPs, no protectant currently offers good protection and can guarantee long-term stability of the freeze-dried product. Summary of the Invention

[0006] The purpose of this invention is to provide a freeze-drying protectant for nucleic acid-lipid nanoparticles. The freeze-drying protectant of this invention is composed of sucrose or trehalose and povidone in a specific ratio, which has a good freeze-drying protective effect on nucleic acid-lipid nanoparticles, ensuring that there are no significant differences in particle size, uniformity, encapsulation efficiency, nucleic acid integrity, and expression performance before and after freeze-drying.

[0007] Another object of the present invention is to provide the use of lyophilization protectants in the preparation of lyophilized formulations of nucleic acid-lipid nanoparticles.

[0008] Another object of the present invention is to provide a nucleic acid-lipid nanoparticle freeze-dried composition.

[0009] Another objective of this invention is to provide a method for preparing a lyophilized formulation of nucleic acid-lipid nanoparticles and the resulting lyophilized formulation.

[0010] The above-mentioned objective of the present invention is achieved by the following solution:

[0011] A lyophilization protectant for nucleic acid-lipid nanoparticles, comprising sucrose and povidone in a mass ratio of 10 to 1:1, or trehalose and povidone in a mass ratio of 10 to 1:1;

[0012] The molecular weight of povidone ranges from 2,000 to 668,000.

[0013] Preferably, the molecular weight of polyvinylpyrrolidone is 4,000 to 58,000.

[0014] Preferably, the mass ratio of sucrose to povidone is 7 to 1:1.

[0015] Preferably, the mass ratio of sucrose to povidone is 5 to 1:1.

[0016] Preferably, the mass ratio of sucrose to povidone is 7:1, 5:1, or 3:1.

[0017] Preferably, the mass ratio of trehalose to povidone is 7 to 1:1.

[0018] Preferably, the mass ratio of trehalose to povidone is 5 to 1:1.

[0019] Preferably, the mass ratio of trehalose to povidone is 7:1, 5:1, or 3:1.

[0020] Preferably, the K value of povidone is 10 to 32.

[0021] Preferably, the K value of povidone is 12, 15, 17, 25 or 30.

[0022] Preferably, the K value of povidone is 12 or 15.

[0023] Preferably, the K value of povidone is 12 or 17.

[0024] Preferably, the K value of povidone is 12 or 25.

[0025] Preferably, the K value of povidone is 12 or 30.

[0026] Preferably, the K value of povidone is 15 or 17.

[0027] Preferably, the K value of povidone is 15 or 25.

[0028] Preferably, the K value of povidone is 15 or 30.

[0029] Preferably, the K value of povidone is 17 or 25.

[0030] Preferably, the K value of povidone is 17 or 30.

[0031] Preferably, the K value of povidone is 25 or 30.

[0032] Preferably, the povidone is one or more of povidone C12, povidone C15, povidone C17, povidone C25, povidone C30, povidone K12, povidone K15, povidone K17, povidone K25, povidone K29, povidone K30 or povidone K32.

[0033] Preferably, the povidone is one or more of povidone C12, povidone C15, povidone C17, povidone C25, povidone K12, povidone K15, povidone K17, povidone K25 or povidone K29.

[0034] Preferably, the povidone is one or more of povidone C12, povidone C15, povidone C17, povidone K12, povidone K15 or povidone K17.

[0035] Preferably, the povidone is one or more of povidone C12, povidone C15, povidone K12 or povidone K15.

[0036] Preferably, the polyvinylpyrrolidone is polyvinylpyrrolidone C12.

[0037] Preferably, the povidone is one or more of povidone C12, povidone C15 or povidone C17.

[0038] Preferably, the povidone is one or more of povidone C12, povidone C15, or povidone C25.

[0039] Preferably, the polyvinylpyrrolidone is one or more of polyvinylpyrrolidone C12, polyvinylpyrrolidone C15, or polyvinylpyrrolidone C30.

[0040] Preferably, the povidone is one or more of povidone C12, povidone C17, or povidone C25.

[0041] Preferably, the povidone is one or more of povidone C12, povidone C17 or povidone C30.

[0042] Preferably, the polyvinylpyrrolidone is one or more of polyvinylpyrrolidone C12, polyvinylpyrrolidone C25, or polyvinylpyrrolidone C30.

[0043] Preferably, the povidone is povidone C12 and / or povidone K12.

[0044] Preferably, the polyvinylpyrrolidone is polyvinylpyrrolidone K12.

[0045] Preferably, the povidone is one or more of povidone K12, povidone K15 or povidone K17.

[0046] Preferably, the povidone is one or more of povidone K12, povidone K15 or povidone K25.

[0047] Preferably, the povidone is one or more of povidone K12, povidone K15 or povidone K29.

[0048] Preferably, the povidone is one or more of povidone K12, povidone K15 or povidone K30.

[0049] Preferably, the povidone is one or more of povidone K12, povidone K15 or povidone K32.

[0050] Preferably, the povidone is one or more of povidone K12, povidone K17 or povidone K25.

[0051] Preferably, the povidone is one or more of povidone K12, povidone K17 or povidone K29.

[0052] Preferably, the povidone is one or more of povidone K12, povidone K17 or povidone K30.

[0053] Preferably, the povidone is one or more of povidone K12, povidone K25 or povidone K32.

[0054] Preferably, the povidone is one or more of povidone K12, povidone K25 or povidone K29.

[0055] Preferably, the povidone is one or more of povidone K12, povidone K25 or povidone K30.

[0056] Preferably, the povidone is one or more of povidone K12, povidone K25 or povidone K32.

[0057] In lyophilization protectants, the types and proportions of components all affect the lyophilization protection of nucleic acid-lipid nanoparticles. Only within a specific range of component types and proportions can the best lyophilization protection be achieved.

[0058] This invention also protects the use of lyophilization protectants in the preparation of lyophilized formulations of nucleic acid-lipid nanoparticles.

[0059] Nucleic acid-lipid nanoparticles are particles encapsulated with nucleic acid drugs (such as mRNA, circRNA, saRNA, siRNA / ASO, etc.) using lipid nanoparticles (LNPs). Each LNP consists of four components: ionizable lipids, neutral lipids, steroids, and polyethylene glycol-modified lipids. The four components in the nucleic acid-lipid nanoparticles of this invention can be made from different specific materials, and the proportions of the components can be varied as long as they can form nanoparticles and encapsulate and deliver nucleic acid drugs.

[0060] Specifically, the ionizable lipid can be any specific ionizable lipid in the art, which can normally form nanoparticles with the other three components and can encapsulate and deliver nucleic acid drugs.

[0061] Specifically, neutral lipids are one or more of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0062] Specifically, steroids are cholesterol.

[0063] Specifically, the polyethylene glycol-modified lipids are DMG-PEG, PEG-DAG, PEG-PE, PEG-S-DAG, PEG-cer, or PEG dialkoxypropyl carbamate.

[0064] More specifically, the PEGylated lipid is DMG-PEG 2000.

[0065] The present invention also protects a nucleic acid-lipid nanoparticle lyophilized composition comprising nucleic acid-lipid nanoparticles and the aforementioned lyophilization protectant.

[0066] Preferably, the composition further comprises a 20-200 mM Tris solution.

[0067] Preferably, the composition further comprises a 20–160 mM Tris solution.

[0068] Preferably, the composition further comprises a 20-130 mM Tris solution. Preferably, the composition further comprises a 20-100 mM Tris solution.

[0069] Preferably, the composition further comprises a 20-80 mM Tris solution.

[0070] The salt concentration in the solution of nucleic acid-lipid nanoparticles and lyophilization protectants also affects the lyophilization protection of nucleic acid-lipid nanoparticles; the lyophilization protection effect improves with increasing salt concentration. However, excessively high salt concentrations can have certain impacts on subsequent drug development. To balance the lyophilization protection of nucleic acid-lipid nanoparticles with the risks of subsequent development, the salt concentration in the solution should be controlled within an appropriate range, such as controlling the Tris solution concentration between 50 and 200 mM.

[0071] Preferably, the composition further comprises a 50-160 mM Tris solution.

[0072] Preferably, the composition further comprises a 50-130 mM Tris solution.

[0073] Preferably, the composition further comprises a 50-100 mM Tris solution.

[0074] Preferably, the composition further comprises a 50-80 mM Tris solution.

[0075] Preferably, the composition further comprises an 80-200 mM Tris solution.

[0076] Preferably, the composition further comprises an 80–160 mM Tris solution.

[0077] Preferably, the composition further comprises an 80–130 mM Tris solution.

[0078] Preferably, the composition further comprises an 80-100 mM Tris solution.

[0079] Preferably, the composition further comprises a 100-200 mM Tris solution.

[0080] Preferably, the composition further comprises a 100-160 mM Tris solution.

[0081] Preferably, the composition further comprises a 100-130 mM Tris solution.

[0082] Preferably, the composition further comprises a 20mM, 40mM, 50mM, 80mM, 100mM, 130mM, 150mM, 160mM or 200mM Tris solution.

[0083] Preferably, the composition further comprises a 40 mM, 50 mM, 80 mM, 100 mM, 130 mM, 160 mM or 200 mM Tris solution.

[0084] Preferably, the composition further comprises a 50 mM, 80 mM, 100 mM, 130 mM, 160 mM or 200 mM Tris solution.

[0085] Preferably, the composition further comprises an 80 mM, 100 mM, 130 mM, 160 mM or 200 mM Tris solution.

[0086] Preferably, the composition further comprises an 80 mM, 100 mM, 130 mM or 160 mM Tris solution.

[0087] Preferably, the composition further comprises an 80 mM, 100 mM or 130 mM solution.

[0088] Preferably, the composition further comprises an 80 mM, 100 mM or 160 mM Tris solution.

[0089] Preferably, the composition further comprises a 100 mM, 130 mM or 160 mM Tris solution.

[0090] Preferably, the composition further comprises 100 mM or 130 mM.

[0091] Preferably, the composition further comprises a 100 mM or 160 mM Tris solution.

[0092] Preferably, the composition further comprises a 50 mM Tris solution.

[0093] Preferably, the composition further comprises an 80 mM Tris solution.

[0094] Preferably, the composition further comprises a 100 mM Tris solution.

[0095] Preferably, the composition further comprises a 130 mM Tris solution.

[0096] Preferably, the composition further comprises a 160 mM Tris solution.

[0097] Preferably, the composition further comprises a 200 mM Tris solution.

[0098] Preferably, the pH value of the Tris solution is 6.5 to 8.0.

[0099] Preferably, the pH value of the Tris solution is 7.0 to 8.0.

[0100] Preferably, the pH of the Tris solution is 7.5.

[0101] Preferably, the pH value of the Tris solution is 6.8 to 8.0.

[0102] Preferably, the pH value of the Tris solution is 6.8 to 7.5.

[0103] Preferably, the pH value of the Tris solution is 6.8 to 7.2.

[0104] Preferably, the pH value of the Tris solution is 6.8 to 7.0.

[0105] Preferably, the pH of the Tris solution is 6.8 ± 0.1.

[0106] Preferably, the pH value of the Tris solution is 7.0 ± 0.3.

[0107] Preferably, the pH value of the Tris solution is 7.0 ± 0.2.

[0108] Preferably, the pH value of the Tris solution is 7.0 ± 0.1.

[0109] Preferably, the pH value of the Tris solution is 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5.

[0110] Preferably, the pH value of the Tris solution is 6.8, 6.9, 7.0, 7.1, 7.2 or 7.3.

[0111] Preferably, the pH value of the Tris solution is 6.8, 6.9, 7.0, 7.1 or 7.2.

[0112] Preferably, the pH of the Tris solution is 6.8.

[0113] Preferably, the pH of the Tris solution is 6.9.

[0114] Preferably, the pH of the Tris solution is 7.0.

[0115] Preferably, the pH of the Tris solution is 7.1.

[0116] Preferably, the pH value of the Tris solution is 7.2.

[0117] Preferably, the pH of the Tris solution is 7.3.

[0118] Preferably, the pH of the Tris solution is 7.4.

[0119] Preferably, in the nucleic acid-lipid nanoparticle freeze-dried composition, the mass ratio of sucrose or trehalose is 5% to 20%.

[0120] Preferably, in the nucleic acid-lipid nanoparticle freeze-dried composition, the mass ratio of sucrose or trehalose is 10% to 20%.

[0121] Preferably, in the nucleic acid-lipid nanoparticle freeze-dried composition, the mass ratio of sucrose or trehalose is 10% to 15%.

[0122] This invention also protects a method for preparing a lyophilized nucleic acid-lipid nanoparticle formulation, which involves preparing a mixture containing nucleic acid-lipid nanoparticles and the aforementioned lyophilization protectant, pre-freezing it at a lower temperature, drying it, and controlling the moisture content to be below 5.0% to obtain a lyophilized nucleic acid-lipid nanoparticle formulation.

[0123] This invention also protects a method for preparing a lyophilized nucleic acid-lipid nanoparticle formulation, wherein the aforementioned lyophilized nucleic acid-lipid nanoparticle composition is pre-frozen at a lower temperature, dried, and the moisture content is controlled to be below 5.0% to obtain a lyophilized nucleic acid-lipid nanoparticle formulation.

[0124] Preferably, the moisture content is controlled to be below 3.0%.

[0125] Preferably, the moisture content is controlled to be below 2.0%.

[0126] Preferably, the mixture is prepared using a Tris solution, wherein the concentration of Tris is 20–100 mM.

[0127] Preferably, the concentration of Tris in the mixture is 50–100 mM.

[0128] Preferably, the concentration of Tris in the mixture is 80–100 mM.

[0129] Preferably, the concentration of Tris in the mixture is 50–200 mM.

[0130] Preferably, the concentration of Tris in the mixture is 50–160 mM.

[0131] Preferably, the concentration of Tris in the mixture is 50–130 mM.

[0132] Preferably, the concentration of Tris in the mixture is 50–80 mM. More preferably, the concentration of Tris in the mixture is 80–200 mM.

[0133] Preferably, the concentration of Tris in the mixture is 80–160 mM.

[0134] Preferably, the concentration of Tris in the mixture is 80–130 mM.

[0135] Preferably, the concentration of Tris in the mixture is 80–100 mM.

[0136] Preferably, the concentration of Tris in the mixture is 100–200 mM.

[0137] Preferably, the concentration of Tris in the mixture is 100–160 mM.

[0138] Preferably, the concentration of Tris in the mixture is 100–130 mM.

[0139] Preferably, the concentration of Tris in the mixture is 20mM, 40mM, 50mM, 80mM, 100mM, 130mM, 160mM or 200mM.

[0140] Preferably, the concentration of Tris in the mixture is 40mM, 50mM, 80mM, 100mM, 130mM, 160mM or 200mM.

[0141] Preferably, the concentration of Tris in the mixture is 50 mM, 80 mM, 100 mM, 130 mM, 160 mM or 200 mM.

[0142] Preferably, the concentration of Tris in the mixture is 80 mM, 100 mM, 130 mM, 160 mM or 200 mM.

[0143] Preferably, the concentration of Tris in the mixture is 80 mM, 100 mM, 130 mM or 160 mM.

[0144] Preferably, the concentration of Tris in the mixture is 80 mM, 100 mM or 130 mM.

[0145] Preferably, the concentration of Tris in the mixture is 80 mM, 100 mM or 130 mM.

[0146] Preferably, the concentration of Tris in the mixture is 80 mM, 100 mM or 160 mM.

[0147] Preferably, the concentration of Tris in the mixture is 100 mM, 130 mM or 160 mM.

[0148] Preferably, the concentration of Tris in the mixture is 100 mM or 130 mM.

[0149] Preferably, the concentration of Tris in the mixture is 100 mM or 160 mM.

[0150] Preferably, the concentration of Tris in the mixture is 50 mM.

[0151] Preferably, the concentration of Tris in the mixture is 80 mM.

[0152] Preferably, the concentration of Tris in the mixture is 100 mM.

[0153] Preferably, the concentration of Tris in the mixture is 130 mM.

[0154] Preferably, the concentration of Tris in the mixture is 160 mM.

[0155] Preferably, the concentration of Tris in the mixture is 200 mM.

[0156] Preferably, the mass ratio of the aforementioned freeze-drying protectant in the mixture is 5% to 20%.

[0157] Preferably, the mass ratio of the aforementioned freeze-drying protectant in the mixture is 10% to 20%.

[0158] Preferably, the mass ratio of the aforementioned freeze-drying protectant in the mixture is 10% to 15%.

[0159] Preferably, the cooling and pre-freezing process includes pre-cooling, pre-freezing, sublimation drying, and desorption drying;

[0160] The pre-cooling temperature is -10 to room temperature;

[0161] The pre-freezing temperature is -40 to -80℃, and the cooling rate is 0.1 to 6.0℃ / minute;

[0162] Sublimation drying is performed under vacuum conditions with programmed temperature increase at a vacuum level of 0.01–1.0 mbar, at a rate of 0.0–6.0 °C / min, maintaining the temperature at -30–-65 °C for at least 2 hours; at -10–-30 °C for at least 2 hours; and at -10–-0 °C for at least 2 hours.

[0163] The desorption drying was carried out under vacuum conditions, with a vacuum degree of 0.01 to 1.0 mbar, and the temperature was increased to 0 to 25°C at a rate of 0.5 to 2.0°C / min and maintained for at least 2 hours.

[0164] Preferably, the pre-cooling temperature is -10~10℃, -10~5℃, -5~5℃, or 0~5℃.

[0165] Preferably, the pre-freezing temperature is -40 to -80°C, and the cooling rate is 0.1 to 6.0°C / minute.

[0166] Preferably, the pre-freezing temperature is -40 to -60°C, and the cooling rate is 0.1 to 6.0°C / minute.

[0167] Preferably, the pre-freezing temperature is -40 to -60°C, and the cooling rate is 0.1 to 4.0°C / minute.

[0168] Preferably, the pre-freezing temperature is -40 to -60°C, and the cooling rate is 0.1 to 2.0°C / minute.

[0169] Preferably, the temperature rise rate during sublimation drying is 0.1–4.0 °C / min or 0.1–2.0 °C / min.

[0170] Preferably, the cooling and pre-freezing process includes pre-cooling, pre-freezing, sublimation drying, and desorption drying;

[0171] The pre-cooling temperature is 0–5℃;

[0172] The pre-freezing temperature is -40 to -60℃, and the cooling rate is 0.5 to 2.0℃ / minute;

[0173] Sublimation drying is performed under vacuum conditions with a temperature program, a vacuum level of 0.1 to 1 mbar, and a temperature increase of 0.5 to 2.0 °C / min. After each 10 °C increase, the temperature is maintained for at least 2 hours before continuing the temperature program to decrease until the temperature reaches 0 to 5 °C, which is then maintained for at least 2 hours.

[0174] The desorption drying was carried out under vacuum conditions, with a vacuum degree of 0.1 to 1 mbar, and the temperature was increased to 5 to 10°C at a rate of 0.5 to 2.0°C / min and maintained for at least 2 hours.

[0175] This invention also protects the lyophilized formulations prepared by the above-described method for preparing lyophilized nucleic acid-lipid nanoparticle formulations.

[0176] Compared with the prior art, the present invention has the following beneficial effects:

[0177] The freeze-drying protectant of this invention is composed of sucrose or trehalose and povidone in a specific ratio. It has a good freeze-drying protection effect on nucleic acid-lipid nanoparticles and can ensure that there are no significant differences in particle size, uniformity, encapsulation efficiency, nucleic acid integrity and expression performance before and after freeze-drying.

[0178] Meanwhile, by adjusting the salt concentration in the mixture of nucleic acid-lipid nanoparticles and lyophilization protectant, the lyophilization protection effect on nucleic acid-lipid nanoparticles was further enhanced, making the performance of the lyophilized nucleic acid-lipid nanoparticles closer to that before lyophilization. Attached Figure Description

[0179] Figure 1 shows the expression performance of Fluc-LNPs in HeLa cells before and after freeze-drying in Example 4.

[0180] Figure 2 shows the expression performance of Fluc-LNPs in 293T cells before and after freeze-drying in Example 5. Detailed Implementation

[0181] The present invention will be further described in detail below with reference to specific embodiments. These embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0182] The model mRNA used in the following examples is the mRNA encoding firefly luciferase (Fluc).

[0183] The LNP used consists of ionizable lipids (SM102), DSPC, cholesterol, and DMG-PEG 2000 in a molar ratio of 48.5:11.1:38.9:1.5.

[0184] Preparation of mRNA-LNPs (lipid nanoparticles encapsulating mRNA): Ionizable lipids (SM102), DSPC, cholesterol, and DMG-PEG 2000 were dissolved in anhydrous ethanol at a molar ratio of 48.5:11.1:38.9:1.5 to prepare a lipid-ethanol phase with a total lipid concentration of 25 mM; model mRNA was dissolved in acetate-sodium acetate buffer (200 mM) at pH 5.0 to prepare an mRNA-buffer phase with a concentration of 266.8 μg / mL. The lipid-ethanol phase and mRNA-buffer phase were rapidly mixed at a flow rate ratio of 1:3 in a microfluidic device (injection pump model: SPM, manufacturer: Duko Industrial Technology; mixing chip manufacturer: FluidicLab, model: LNP-B0). The mixed sample was placed in a dialysis bag (30kDa) and dialyzed overnight at 4°C in Tris buffer (pH 7.5) of different concentrations (e.g., 20mM, 100mM) to obtain Flux-LNP model samples with different buffers.

[0185] Povidone is available in various polymers, among which the commonly used ones are povidone C12, povidone C15, povidone C17, povidone C25, povidone C30, povidone K12, povidone K15, povidone K17, povidone K25, povidone K29, povidone K30, or povidone K32. All of the listed povidone products can be used in this invention. For ease of experimentation and understanding, povidone C12 or povidone K12 was used in the following examples.

[0186] Example 1: Screening of Lyophilization Protectant Components

[0187] The lyophilization protectant consists of one or two of sucrose, trehalose, and mannitol. The required amounts are weighed out and dissolved in 20 mM Tris salt buffer (pH 7.5) to prepare a lyophilization protectant solution with a sugar concentration (sucrose, trehalose, or mannitol used in this application) of 20% (w / w).

[0188] The composition to be lyophilized (Fluc-LNPs to be lyophilized): The model Fluc-LNPs sample (20mM Tris buffer) was mixed with an equal volume of lyophilization protectant solution to obtain the composition to be lyophilized (Fluc-LNPs to be lyophilized). The concentration of sugars (sucrose, trehalose, or mannose) in the Fluc-LNPs sample to be lyophilized was 10% (w / w), and the concentration of Tris salt was 20mM.

[0189] The lyophilized Fluc-LNPs samples were aliquoted into vials, 300 μL per vial, and placed in a lyophilizer. The lyophilization program was set up for lyophilization to obtain the lyophilized Fluc-LNPs formulation. Different experimental groups were set up according to the different lyophilization protectant solutions used, with 3 samples in each group. The test data are the average of the test data of the 3 samples.

[0190] Table 1 Freeze-drying procedure for Fluc-LNPs

[0191] Detection of lyophilized samples: The lyophilized Fluc-LNPs samples were reconstituted with water for injection, and the particle size, polydispersity index (DLS, Malvern), and encapsulation efficiency (Ribogreen) were measured to evaluate the performance of the lyophilized samples.

[0192] The performance test results of different test group samples after freeze-drying are shown in Table 2.

[0193] Table 2. Performance test results of samples from different test groups before and after freeze-drying.

[0194] "NT" in the table indicates that it was not detected.

[0195] Whether sucrose, trehalose, or mannitol is used as a single lyophilization protectant, or a combination of sucrose / mannitol or trehalose / mannitol is used, the particle size of Fluc-LNPs increases significantly after lyophilization, resulting in poorer uniformity (significantly increased polydispersity index) and a significant decrease in encapsulation efficiency. Therefore, the performance of lyophilized Fluc-LNPs is significantly reduced and does not meet requirements. Thus, neither sucrose, trehalose, nor mannitol as a single lyophilization protectant, nor a combination of sucrose / mannitol or trehalose / mannitol as a lyophilization protectant, can effectively protect the performance of lyophilized Fluc-LNPs.

[0196] Example 2: Screening of Lyophilization Protectant Components

[0197] The lyophilization protectant consists of any one of sucrose, trehalose, or mannitol, combined with either PEG4000 or povidone C12. Weigh out the required amount and dissolve each component in 20 mM Tris buffer (pH 7.5) to prepare a lyophilization protectant solution with a sugar concentration of 20% (w / w). When the lyophilization protectant is composed solely of povidone C12, prepare a 12% (w / w) lyophilization protectant solution.

[0198] The freeze-drying of Fluc-LNPs, sample aliquoting, freeze-drying procedure, and post-freeze-drying sample testing were all the same as in Example 1.

[0199] The performance test results of different test group samples after freeze-drying are shown in Table 3.

[0200] Table 3. Performance test results of samples from different test groups before and after freeze-drying

[0201] "NT" in the table indicates that it was not detected.

[0202] As shown in experimental groups 9-11, the particle size of the Fluc-LNPs samples increased significantly after freeze-drying, the uniformity deteriorated, and the encapsulation rate decreased significantly. This indicates that the two-component freeze-drying protectants of sucrose / PEG4000, trehalose / PEG4000, or mannitol / PEG4000 cannot effectively protect the performance of LNPs after freeze-drying.

[0203] When povidone C12 was used alone as a lyophilization protectant, the particle size of the freeze-dried Fluc-LNPs samples increased significantly, and the uniformity deteriorated. When povidone C12 was combined with sucrose or trehalose as a lyophilization protectant, the changes in particle size and uniformity of the freeze-dried Fluc-LNPs samples were significantly reduced, and the encapsulation efficiency was better. In particular, when povidone C12 was combined with sucrose as a lyophilization protectant, the changes in particle size and uniformity of the freeze-dried Fluc-LNPs samples were the lowest, superior to the same proportion of povidone C12 and trehalose.

[0204] As can be seen from experimental groups 13-25, when povidone C12 is combined with any one of the components of sucrose, trehalose or mannitol as a freeze-drying protectant, the ratio of the two components also affects the performance of the Fluc-LNPs samples after freeze-drying.

[0205] As shown in experimental groups 13-17, when the sucrose / povidone C12 mass ratio was 10:1 or 1:1, the particle size variation of Fluc-LNPs after freeze-drying was relatively large; when the mass ratio was 7:1 to 3:1, the changes in particle size, uniformity, and encapsulation efficiency of Fluc-LNPs after freeze-drying were relatively small. This indicates that when sucrose / povidone C12 is used as a freeze-drying protectant at a mass ratio of 7:1 to 3:1, the freeze-drying protection effect on Fluc-LNPs samples is optimal.

[0206] As can be seen from experimental groups 18-22, when trehalose and povidone C12 are combined as freeze-drying protectants, only when the mass ratio is 5:1 (experimental group 20) are the particle size and uniformity of the freeze-dried Fluc-LNPs samples comparable to those of experimental groups 14-16, but the encapsulation rate decreases significantly.

[0207] As can be seen from experimental groups 23-25, when mannitol and povidone C12 are combined as freeze-drying protectants, the particle size of Fluc-LNPs changes significantly after freeze-drying, and the freeze-drying protection effect on Fluc-LNPs samples is not good.

[0208] Example 3 Effect of Buffer Solution

[0209] The freeze-drying protectant is composed of any one of sucrose, trehalose, or mannitol, combined with povidone C12. The mass ratio of sucrose / povidone C12 and trehalose / povidone C12 is 5:1, while the mass ratio of mannitol / povidone C12 is 5:1 or 1:1. Alternatively, the mass ratio of sucrose / povidone C12 may be 3:1.

[0210] The lyophilization protectant solutions were prepared using different buffer solutions, namely 20mM Tris buffer, 20mM Tris + 50mM sodium chloride buffer, 50mM Tris buffer, 80mM Tris buffer, 100mM Tris buffer, 130mM Tris buffer, or 160mM Tris buffer, to investigate the effect of different buffer solutions on the lyophilization protection of Fluc-LNPs samples.

[0211] When preparing the Fluc-LNP model, the buffer solution used was the same as that used for the lyophilization protectant.

[0212] Fluc-LNPs to be lyophilized: The model Fluc-LNPs sample (using the same buffer as the lyophilization protectant buffer) was mixed with an equal volume of the lyophilization protectant solution to obtain the composition to be lyophilized (Fluc-LNPs to be lyophilized). The sugar concentration in the lyophilization protectant of the Fluc-LNPs sample to be lyophilized was 10% (w / w).

[0213] In experimental groups 26-34, the LNP consisted of SM102, DSPC, cholesterol, and DMG-PEG 2000 in a molar ratio of 48.5:11.1:38.9:1.5. In experimental groups 35-39, the LNP consisted of compound 1, DSPC, cholesterol, and DMG-PEG 2000 in a molar ratio of 48.5:11.1:38.9:1.5; the structure of compound 1 is as follows:

[0214] The dispensing and freezing procedures for the freeze-dried Fluc-LNPs were the same as in Example 1.

[0215] The performance results of Fluc-LNPs samples before and after lyophilization with different buffer solutions are shown in Table 4.

[0216] Table 4. Performance test results of samples with different buffer solutions before and after freeze-drying

[0217] "NT" in the table indicates that it was not detected.

[0218] As shown in experimental groups 26-31 and 35-39, the effect of buffer salt concentration on the particle size of lyophilized Fluc-LNPs, in descending order of particle size increase, is: 20mM Tris buffer > 20mM Tris, 50mM sodium chloride buffer > 80mM Tris buffer. When the buffer is greater than 50mM Tris buffer, the increase in particle size after lyophilization is not significant, and the uniformity and encapsulation efficiency of the lyophilized Fluc-LNPs samples do not change significantly.

[0219] As shown in experimental groups 32-34, the particle size of Fluc-LNPs was significantly increased after freeze-drying with the freeze-drying protectant composed of mannitol / povidone C12 (mass ratios of 5:1 and 1:1, respectively), indicating that the freeze-drying protection effect on Fluc-LNPs was poor.

[0220] The results above show that the salt concentration of the Fluc-LNPs buffer has a significant impact on the particle size of LNPs after lyophilization. The higher the salt concentration, the smaller the particle size change of the LNPs after lyophilization. Furthermore, a higher salt concentration is more advantageous in maintaining the uniformity of the lyophilized Fluc-LNPs samples.

[0221] Example 4: Detection of expression performance in cells

[0222] Fluc-LNPs samples from experimental groups 26-39 after lyophilization were selected to test their expression in cells.

[0223] Test method:

[0224] 1. Cell plating

[0225] 1.1 Remove 293T cells or HeLa cells from the CO2 incubator and transfer them to a biosafety cabinet that has been sterilized with UV light for 30 minutes.

[0226] 1.2 Discard the old culture medium and wash once with PBS. Digest with 2 mL of trypsin for 2 min, then add 4 mL of DMEM complete culture medium to stop the digestion. Transfer all cell suspension to a 15 mL centrifuge tube and centrifuge at 500 × g for 5 min (25 °C).

[0227] 1.3 Discard the supernatant, resuspend the sample in an appropriate amount of DMEM complete medium, and add 10 μL of the resuspended sample to 70 μL of trypan blue for staining; add 10 μL of the sample to a hemocytometer and count the cells using a fluorescence microscope at a count density of 510 × 10⁻⁶. 4 .

[0228] 1.4 Take 196 μL of cell suspension and add 9804 μL of DMEM complete culture medium to adjust the cell density to 1.0 × 10⁻⁴. 5 per mL.

[0229] 1.5 Add 0.125 mL of cell culture medium (adjusted to density) to a 96-well plate, resulting in a cell volume of 1.25 × 10⁻⁶ cells. 4 Cells were placed in a 37°C, 5% CO2 incubator for 18 hours.

[0230] 2. Cell transfection

[0231] Remove the 96-well plate and add 0.5 μg of mRNA-LNPs sample solution to each well of cell culture. Gently shake the wells to distribute the sample evenly and incubate in a CO2 incubator for 24 hours. Prepare three replicates for each mRNA-LNPs sample.

[0232] 3. Detection of luciferase expression levels

[0233] 3.1 After the culture is completed, add 90 μL of detection reagent to each well sample and incubate at room temperature for 5 min.

[0234] 3.2 Transfer 110 μL to the corresponding black plate and detect the fluorescence value in the Luminescence mode of the microplate reader.

[0235] The cells used in experimental groups 35-39 were HeLa cells, and the expression results of the cells before and after freeze-drying are shown in Figure 1.

[0236] The results showed that when the salt concentration of the Fluc-LNPs buffer was >50mM, the expression of luciferase in Fluc-LNPs samples in HeLa cells was less different before and after lyophilization. In particular, when the salt concentration of the buffer was >100mM, there was no significant difference in the expression of Fluc-LNPs before and after lyophilization.

[0237] Example 5: Stability Study of Lyophilized Samples

[0238] The components of the lyophilization protectant are sucrose / povidone C12 and trehalose / povidone C12, with mass ratios of 7:1, 5:1 and 3:1, respectively. Referring to Example 3, different concentrations of Tris buffer were used to prepare the lyophilization protectant solution.

[0239] The Fluc-LNPs to be lyophilized were obtained by mixing equal volumes of model Fluc-LNPs and lyophilization protectant solution. The buffer solutions for the model Fluc-LNPs and the lyophilization protectant were the same. The concentration of sucrose or trehalose in the Fluc-LNPs sample to be lyophilized was 10% (w / w).

[0240] The dispensing of the freeze-dried Fluc-LNPs samples was the same as in Example 1.

[0241] The freeze-drying process is as follows:

[0242] Pre-cooling: Set the partition temperature to 4℃ and maintain it for 30 minutes.

[0243] Pre-freezing: The temperature of the partition is reduced to -50℃ at a rate of 1℃ / min and maintained for 2 hours.

[0244] Sublimation drying: The temperature of the partition is increased to -30°C at a rate of 1°C / min and maintained for 18 hours, with a vacuum degree of 0 to 0.3 mbar;

[0245] The temperature of the partition was increased to -20°C at a rate of 1°C / minute and maintained for 10 hours, with a vacuum degree of 0 to 0.6 mbar.

[0246] The temperature of the partition was increased to -10°C at a rate of 1°C / minute and maintained for 4 hours, with a vacuum degree of 0 to 0.2 mbar.

[0247] The temperature of the partition is increased to 0°C at a rate of 1°C / minute and maintained for 2 hours, with a vacuum degree of 0–0.2 mbar.

[0248] Analysis and drying: The temperature of the partition is increased to 5°C at a rate of 1°C / minute and maintained for 6 hours, with a vacuum degree of 0 to 0.2 mbar.

[0249] Because the number of samples increased in this experiment, the duration of each temperature stage in the sublimation drying process was extended.

[0250] One batch of samples was lyophilized and then rehydrated to test its performance and expression performance in cells. The results are shown in Table 5 and Figure 2 (Figure 2 shows the expression results in 293T cells).

[0251] Table 5. Performance test results of samples before and after freeze-drying.

[0252] The buffer solutions for experimental groups 40-42 were all 100 mM Tris. The LNP consisted of SM102, DSPC, cholesterol, and DMG-PEG 2000 in a molar ratio of 48.5:11.1:38.9:1.5.

[0253] As shown in Table 5 and Figure 2, the particle size, uniformity, encapsulation efficiency, integrity, mRNA concentration, and luciferase expression level of Fluc-LNPs did not change significantly before and after freeze-drying.

[0254] A batch of samples was freeze-dried and then used for stability testing at 2–8°C. The specific method was as follows:

[0255] The freeze-dried Fluc-LNPs were stored at 2–8°C, and samples were taken at 15 days, 1 month, 2 months, 3 months, 6 months, 9 months, and 12 months. After reconstitution with water for injection, the freeze-dried Fluc-LNPs were analyzed for particle size, polydispersity index (PDI), encapsulation efficiency (EE), mRNA integrity, and mRNA concentration.

[0256] Table 6 shows the test data of samples in experimental groups 43-46 (Fluc-LNPs samples to be freeze-dried are the same as those in experimental groups 36-39) before freeze-drying, 0 days after freeze-drying, and 1 month after freeze-drying.

[0257] Table 6. Performance test results of samples before freeze-drying, 0 days after freeze-drying, and 1 minute after freeze-drying.

[0258] The results showed that, within the observation period, when the buffer salt concentration of the freeze-dried Fluc-LNPs was within the range of 80-160 mM, the various parameters of the freeze-dried Fluc-LNPs remained stable.

[0259] Example 6: Detection of the expression performance of nucleic acid-lipid nanoparticles in animals before and after freeze-drying.

[0260] Flux-LNPs to be lyophilized were prepared according to Example 3 (100 mM Tris buffer, pH 6.5, 6.8, or 7.5, respectively), wherein the lyophilization protectant was sucrose:povidone K12 = 3:1 (mass ratio), and the LNP was compound 1:DSPC:Cholesterol:DMG-PEG2K = 48.5:38.9:11.1:1.5. Lyophilization was performed according to the method of Example 1.

[0261] The performance test results of each group of samples before and after freeze-drying are shown in Table 7.

[0262] Table 7 Performance test results before and after freeze-drying

[0263] Flux-LNPs before and after lyophilization were diluted to 20 ng / uL and administered to male SD rats via tail vein injection at a dose of 0.05 mg / Kg. Whole blood samples of 0.3 mL were collected at 1, 2, 4, 6, 8, 10, 24, 32, and 48 h after injection. The samples were anticoagulated with K2 EDTA / heparin sodium and centrifuged within 30 min after an ice bath. Gluc protein was detected using a Gaussian luciferase assay kit.

[0264] The test results are shown in Table 8.

[0265] Table 8. Results of in vivo expression levels in rats before and after freeze-drying for each group of samples.

[0266] The results showed that the pH of the buffer solution had a certain effect on the expression of the samples before and after lyophilization. The expression of the samples was better when the pH was in the range of 6.8-7.5.

[0267] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A lyoprotectant for nucleic acid-lipid nanoparticles, characterized in that, It is composed of sucrose and povidone in a mass ratio of 10 to 1:1, or trehalose and povidone in a mass ratio of 10 to 1:

1. The molecular weight of the povidone is 2000 to 66800.

2. The lyoprotectant according to claim 1, wherein The mass ratio of sucrose to povidone is 7-1:1 or 5-1:1; Preferably, the mass ratio of sucrose to povidone is 7:1, 5:1, or 3:1; Alternatively, the mass ratio of trehalose to povidone is 7–1:1 or 5–1:1; Preferably, the mass ratio of trehalose to povidone is 7:1, 5:1, or 3:

1.

3. The lyoprotectant according to claim 2, wherein The K value of the povidone is 10 to 32; Preferably, the K value of the povidone is 12, 15, 17, 25 or 30; Preferably, the povidone is povidone C12, povidone C15, povidone C17, povidone C25, povidone C30, povidone K12, povidone K15, povidone K17, povidone K25, povidone K29, povidone K30 or povidone K32.

4. Use of the lyophilization protectant according to any one of claims 1 to 3 in the preparation of lyophilized formulations of nucleic acid-lipid nanoparticles.

5. A nucleic acid-lipid nanoparticle lyophilized composition, characterized in that, It comprises nucleic acid-lipid nanoparticles and the lyophilization protectant as described in any one of claims 1 to 3.

6. The nucleic acid-lipid nanoparticle lyophilized composition of claim 5, wherein, The composition also contains a 20–100 mM Tris solution; Preferably, the composition further comprises a 50-100 mM Tris solution; Preferably, the composition further comprises an 80-100 mM Tris solution.

7. The nucleic acid-lipid nanoparticle lyophilized composition of claim 5, wherein, The composition also contains a 20-200 mM Tris solution; Preferably, the composition further comprises a 50-200 mM Tris solution; Preferably, the composition further comprises an 80-200 mM Tris solution; Preferably, the composition further comprises an 80–160 mM Tris solution; Preferably, the composition further comprises an 80-130 mM Tris solution; Preferably, the composition further comprises an 80-100 mM Tris solution; Preferably, the composition further comprises a 100-200 mM Tris solution; Preferably, the composition further comprises a 100-160 mM Tris solution; Preferably, the composition further comprises a 100-130 mM Tris solution.

8. The nucleic acid-lipid nanoparticle lyophilized composition of claim 6 or 7, wherein, The pH value of the Tris solution is 6.5–8.0; Preferably, the pH value of the Tris solution is 7.0 to 8.0; Preferably, the pH value of the Tris solution is 7.5; Preferably, the pH value of the Tris solution is 6.8 to 8.0; Preferably, the pH value of the Tris solution is 6.8 to 7.5; Preferably, the pH value of the Tris solution is 6.8 to 7.2; Preferably, the pH value of the Tris solution is 6.8 to 7.

0.

9. The nucleic acid-lipid nanoparticle lyophilized composition of claim 6 or 7, wherein, In the nucleic acid-lipid nanoparticle freeze-dried composition, the mass ratio of sucrose or trehalose as described in any one of claims 1 to 3 is 5% to 20%; Preferably, in the nucleic acid-lipid nanoparticle freeze-dried composition, the mass ratio of sucrose or trehalose as described in any one of claims 1 to 3 is 10% to 20%; Preferably, in the nucleic acid-lipid nanoparticle freeze-dried composition, the mass ratio of sucrose or trehalose as described in any one of claims 1 to 3 is 10% to 15%.

10. A method of preparing a nucleic acid-lipid nanoparticle lyophilized formulation, characterized in that, A mixture containing nucleic acid-lipid nanoparticles and any one of the lyophilization protectants described in claims 1 to 3 is prepared, pre-frozen at a lower temperature, dried, and the moisture content is controlled to be below 5.0% to obtain a lyophilized formulation of nucleic acid-lipid nanoparticles. Alternatively, the nucleic acid-lipid nanoparticle freeze-dried composition according to any one of claims 5 to 9 is subjected to pre-freezing at a lower temperature, followed by drying to control the moisture content to be below 5.0%, thereby preparing a nucleic acid-lipid nanoparticle freeze-dried formulation.

11. The preparation method according to claim 10, characterized in that, The mixture was prepared using a Tris solution, wherein the concentration of Tris was 20–100 mM. Preferably, the concentration of Tris in the mixture is 50–100 mM; Preferably, the concentration of Tris in the mixture is 80–100 mM.

12. The preparation method according to claim 10, characterized in that, The mixture was prepared using a Tris solution, wherein the concentration of Tris was 20–200 mM. Preferably, the concentration of Tris in the mixture is 50–200 mM; Preferably, the concentration of Tris in the mixture is 80–200 mM; Preferably, the concentration of Tris in the mixture is 80–160 mM; Preferably, the concentration of Tris in the mixture is 80–130 mM; Preferably, the concentration of Tris in the mixture is 80–100 mM; Preferably, the concentration of Tris in the mixture is 100–200 mM; Preferably, the concentration of Tris in the mixture is 100–160 mM; Preferably, the concentration of Tris in the mixture is 100–130 mM.

13. The preparation method according to claim 10, characterized in that, The mass ratio of sucrose or trehalose as described in any one of claims 1 to 3 in the mixture is 5% to 20%; Preferably, the mass ratio of sucrose or trehalose according to any one of claims 1 to 3 in the mixture is 10% to 20%; Preferably, the mass ratio of sucrose or trehalose according to any one of claims 1 to 3 in the mixture is 10% to 15%.

14. The preparation method according to claim 13, characterized in that, The cooling and pre-freezing process includes pre-cooling, pre-freezing, sublimation drying, and desorption drying; The pre-cooling temperature is -10°C to room temperature; The pre-freezing temperature is -40 to -80°C, and the cooling rate is 0.1 to 6.0°C / minute. The sublimation drying is performed under vacuum conditions with programmed temperature increase. The vacuum level is 0.01 to 1.0 mbar, and the temperature is increased at a rate of 0 to 6.0 °C per minute. The temperature is maintained at -30 to -65 °C for at least 2 hours; at -10 to -30 °C for at least 2 hours; and at -10 to -0 °C for at least 2 hours. The analytical drying is carried out under vacuum conditions, with a vacuum degree of 0.01 to 1.0 mbar, and the temperature is increased to 0 to 25°C at a rate of 0.5 to 2.0°C / min and maintained for at least 2 hours.

15. A nucleic acid-lipid nanoparticle lyophilized formulation, characterized in that, The lyophilized formulation prepared by any of the preparation methods described in claims 10 to 14.

Images