Method for freeze-drying lipid nanoparticles

Abstract

A method for preparing a lyophilized lipid nanoparticle-nucleic acid composition is provided. The method includes preparing a suspension of lipid nanoparticles using a simple sugar and one or more excipients selected from thiosulfate, potassium sorbate, sodium benzoate, and iodixanol. Freeze-dried lipid nanoparticle-nucleic acid compositions and methods for reconstituting and administering the same are also provided.

Description

[Technical Field]

[0001] This disclosure relates to the field of pharmaceutical manufacturing and products. More specifically, this disclosure relates to methods for preparing lipid nanoparticle-encapsulated nucleic acid compositions and lyophilized lipid nanoparticle nucleic acid products. [Background technology]

[0002] Therapies based on intracellular delivery of nucleic acids to target cells target both extracellular and intracellular barriers. In fact, systemic administration of bare nucleic acid substances is difficult due to all the characteristics that hinder their clinical development: their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, tendency to be taken up by phagocytic cells and trigger immune responses. When exogenous nucleic acid substances enter the human biological system, they are recognized as foreign pathogens by the reticuloendothelial system (RES) and cleared from the bloodstream before they have a chance to encounter target cells inside or outside the vascular system. The half-life of bare nucleic acids in the bloodstream has been reported to be about a few minutes (Kawabata K, Takakura Y, Hashida MPharm Res. 1995 Jun;12(6):825-30). Chemical modification and appropriate delivery methods can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, thereby improving the stability and efficacy of nucleic acid-based therapies. Furthermore, RNA or DNA are anionic hydrophilic polymers, which are unfavorable for cellular uptake, and they are anionic even on their surface. Therefore, the success of nucleic acid-based therapies largely depends on the development of vehicles or vectors that can efficiently and effectively deliver genetic material to target cells and achieve sufficient in vivo expression levels with minimal toxicity.

[0003] While some gene therapies have successfully utilized viral delivery vectors (e.g., AAV), lipid-based formulations are increasingly recognized as one of the most promising delivery systems for RNA and other nucleic acid compounds due to their biocompatibility and ease of large-scale production. One of the most significant advances in lipid-based nucleic acid therapy occurred in August 2018, when Patisiran (ALN-TTR02) became the first siRNA therapeutic to be approved by the Food and Drug Administration (FDA) and the European Commission (EC). ALN-TTR02 is an siRNA formulation based on so-called stable nucleic acid lipid particle (SNALP) transfection technology. Despite the success of Patisiran, the delivery of nucleic acid therapeutics via lipid nanoparticles remains under development.

[0004] Several lipid-based delivery vehicles recognized in the art for nucleic acid therapeutics include polymer-based carriers, e.g., polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, polyvesicular liposomes, proteoliposomes, exosomes of both natural and synthetic origin, natural, synthetic, and semi-synthetic lamellar bodies, nanoparticles, micelles, and emulsions. Among these, lipid nanoparticles have shown high potential as a delivery vehicle for RNA therapeutics. However, the use of lipid nanoparticle delivery technology faces stability issues, requiring in some cases that the lipid nanoparticle suspension must be stored at impractical low temperatures of about -70°C and thawed only immediately before intended administration. Such requirements may limit the development of pharmaceuticals that can be used by patients in their own homes, as well as in the transport and storage of lipid nanoparticle therapeutics in remote and underdeveloped regions of the world (all of which lack adequate equipment for storing lipid nanoparticle suspensions).

[0005] One solution to improve the storability of lipid nanoparticle formulations is to produce them as lyophilized products that can be reconstituted later before administration. Lyophilized lipid nanoparticle compositions can be stored at more practical temperatures, allowing for more convenient modes of distribution and administration.

[0006] Although freeze-drying technology has existed for decades, its application to lipid nanoparticle formulations has not been fully adapted, and conventional freeze-drying methods result in the loss of some of the desired properties, including low polydispersity, small particle size, high encapsulation ratio, and in vivo efficacy, when reconstituted. Therefore, a new solution is needed to provide freeze-dried lipid nanoparticle compositions that exhibit maintained wholeness and efficacy when reconstituted. [Overview of the project]

[0007] This disclosure provides a lyophilization method that results in the preservation of the wholeness of lipid nanoparticles, the wholeness of encapsulated nucleic acids, an acceptable particle size of lipid nanoparticles within the pre-lyophilized particle size, and good polydispersibility of the nanoparticles. The method stems from the discovery that special excipients can be added to a pre-treated suspension of nanoparticles before subjecting the suspension to the lyophilization process. Furthermore, lyophilization parameters are used in combination with these excipients to achieve high-quality lyophilized lipid nanoparticle products. The lyophilized products are readily reconstituted and readily administered as pharmaceutical preparations.

[0008] In some embodiments, a method is provided for lyophilizing a composition comprising RNA-encapsulating lipid nanoparticles, comprising the steps of: providing a suspension of lipid nanoparticles in a liquid medium; preparing the liquid medium to form a pre-treated suspension comprising at least one excipient selected from potassium sorbate, thiosulfate, sodium benzoate, and iodixanol; and subjecting the pre-treated suspension to a lyophilization process.

[0009] In another embodiment, a lyophilized composition is provided comprising lipid nanoparticles encapsulating nucleic acids and one or more excipients selected from potassium sorbate, thiosulfate, sodium benzoate, and iodixanol.

[0010] In another embodiment, a method for storing a lyophilized composition of the Disclosure is provided, comprising storing the lyophilized product at a temperature of about -20°C to about 8°C. In some embodiments, a method for storing a lyophilized composition of the Disclosure is provided, comprising storing the lyophilized product at a temperature of about -20°C to about 25°C. In some embodiments, the lyophilized product is stored at about -20°C. In some embodiments, the lyophilized product is stored at about 2°C to about 8°C. In some embodiments, the lyophilized product is stored at about 20°C to about 25°C.

[0011] Another embodiment provides a method for reconstituting a lyophilized composition of the present disclosure, comprising adding a liquid medium to the lyophilized composition.

[0012] In another embodiment, a method is provided for treating a disease or disorder in a subject, comprising administering a lyophilized composition of the present disclosure, reconstituted in a liquid medium, to the subject.

[0013] Further features and advantages of the subject art are described below, some of which will be evident from the description or acquired through the implementation of the subject art. The advantages of the subject art will be realized and achieved through the description and its embodiments.

[0014] The general explanation above and the detailed explanation below should be understood as illustrative examples intended to provide further explanation of the subject technology. [Brief explanation of the drawing]

[0015] [Figure 1]The characteristics of reconstituted lipid nanoparticles prepared according to the experiment described in Example 3 for a formulation prepared at a concentration of 1 mg RNA / mL are shown (particle size, polydispersity (PDI), and encapsulation rate (Encap (%)). [Figure 2A] The particle size measurement results (bar chart) and encapsulation rate (circles) for formulations prepared using P188 poloxamer added to the suspension before freeze-drying, as described in Example 3, are shown. [Figure 2B] The particle size measurement results (bar chart) and encapsulation rate (circles) for formulations prepared using P188 poloxamer added to the freeze-dried formulation as described in Example 3 are shown. [Figure 3] The concentration dependence of the particle size (bar chart) and encapsulation rate (circles) for lipid nanoparticle concentrations (0.25, 0.5, and 1.0 mg RNA / mL) for P188 formulations treated after lyophilization at different concentrations of P188, as described in Example 3, is shown. [Figure 4] As described in Example 12, the human erythropoietin (hEPO) expression levels of selected reconstituted formulations compared to freeze-thaw controls and PBS-negative controls are shown. [Modes for carrying out the invention]

[0016] It is understood that various configurations of the subject art will be readily apparent to those skilled in the art from this disclosure, and these various configurations of the subject art are illustrated and described as examples. As will be understood, the subject art is capable of other and different configurations, and some of its details are modifiable in various other respects without all departing from the scope of the subject art. Accordingly, the abstract, drawings and detailed description should be considered as examples in nature and not as limitations.

[0017] The detailed descriptions provided below are intended to describe various configurations of the subject art and not to show only configurations in which the subject art can be practiced. The detailed descriptions include specific details to provide a complete understanding of the subject art. However, it will be apparent to those skilled in the art that the subject art can be practiced without these specific details.

[0018] In some embodiments, a method is provided for lyophilizing a composition comprising lipid nanoparticles for encapsulating RNA, the method comprising: a) providing a suspension of lipid nanoparticles in a liquid medium, the liquid medium comprising about 4% w / v to about 22% w / v of saccharide; and b) preparing the liquid medium, thereby forming a pre-treated suspension comprising at least one excipient selected from potassium sorbate, thiosulfate, sodium benzoate, and iodixanol.

[0019] In some embodiments, the method further includes step (c): subjecting the pre-treated suspension to a freeze-drying process comprising i.) an initial freezing step carried out at a temperature of -48±8°C and atmospheric pressure; ii.) a primary drying step carried out at a temperature in the range of -20±2°C to -48±2°C and a pressure in the range of about 25 mTorr to about 100 mTorr; and iii.) a secondary drying step carried out at a temperature in the range of 5±2°C to 30±2°C and a pressure in the range of about 30 mTorr to about 300 mTorr.

[0020] In some embodiments, the method further includes: step (c): subjecting the pre-treated suspension to a freeze-drying process comprising: i.) an initial freezing step carried out at a temperature of -48±8°C and atmospheric pressure; ii.) a primary drying step carried out at a pressure of about 0.03 to about 0.08 mbar, starting at a temperature of -48±8°C and gradually decreasing to a temperature of 0±2°C over a period ranging from about 40 to about 75 hours; and iii.) a secondary drying step carried out at a pressure of about 0.03 to about 0.08 mbar, starting at a temperature of 0±2°C and gradually decreasing to a temperature of about 25±3°C over a period ranging from about 30 to about 50 hours.

[0021] In some embodiments, the liquid medium is an aqueous medium.

[0022] In some embodiments, the RNA in the suspension has a concentration in the range of about 0.05 mg / mL to about 2.0 mg / mL. In some embodiments, the RNA in the suspension has a concentration in the range of about 0.075 mg / mL to about 0.3 mg / mL. In some embodiments, the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 1.5 mg / mL. In some embodiments, the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 1.0 mg / mL. In some embodiments, the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 0.5 mg / mL.

[0023] In some embodiments, the weight ratio of total lipids to RNA in the suspension is approximately 50:1 to approximately 10:1. In some embodiments, the weight ratio of total lipids to RNA in the suspension is approximately 40:1 to approximately 20:1. In some embodiments, the weight ratio of total lipids to RNA in the suspension is approximately 35:1 to approximately 25:1.

[0024] In some embodiments, the pre-treated suspension contains thiosulfate. In some embodiments, the thiosulfate is sodium thiosulfate or potassium thiosulfate. In some embodiments, the thiosulfate has a concentration of about 0.025% w / v to about 1.0% w / v. In some embodiments, the thiosulfate has a concentration of about 0.025% w / v to about 0.75% w / v. In some embodiments, the thiosulfate has a concentration of about 0.025% w / v to about 0.5% w / v. In some embodiments, the thiosulfate has a concentration of about 0.05% w / v to about 0.3% w / v. In some embodiments, the thiosulfate has a concentration of about 0.05% w / v to about 0.25% w / v.

[0025] In some embodiments, the pre-treated suspension contains potassium sorbate. In some embodiments, the potassium sorbate has a concentration of about 0.01 M to about 0.5 M. In some embodiments, the potassium sorbate has a concentration of about 0.02 M to about 0.4 M. In some embodiments, the potassium sorbate has a concentration of about 0.025 M to about 0.3 M. In some embodiments, the potassium sorbate has a concentration of about 0.03 M to about 0.2 M. In some embodiments, the potassium sorbate has a concentration of about 0.035 M to about 0.1 M. In some embodiments, the potassium sorbate has a concentration of about 0.04 M to about 0.08 M. In some embodiments, the potassium sorbate has a concentration of about 0.01 M to about 0.05 M. In some embodiments, the potassium sorbate has a concentration of about 0.02 M to about 0.04 M.

[0026] In some embodiments, the pre-treated suspension contains iodixanol. In some embodiments, the iodixanol has a concentration of about 5% w / v to about 15% w / v. In some embodiments, the iodixanol has a concentration of about 6% w / v to about 13% w / v. In some embodiments, the iodixanol has a concentration of about 7% w / v to about 11% w / v. In some embodiments, the iodixanol has a concentration of about 8% w / v to about 10% w / v.

[0027] In some embodiments, the pre-treated suspension contains sodium benzoate. In some embodiments, the sodium benzoate has a concentration of about 0.01 M to about 0.6 M. In some embodiments, the sodium benzoate has a concentration of about 0.02 M to about 0.5 M. In some embodiments, the sodium benzoate has a concentration of about 0.03 M to about 0.4 M. In some embodiments, the sodium benzoate has a concentration of about 0.04 M to about 0.3 M. In some embodiments, the sodium benzoate has a concentration of about 0.05 M to about 0.2 M.

[0028] In some aspects of any of the above embodiments, the pre-treated suspension further comprises polyvinyl alcohol (PVA). In some embodiments, the PVA has a concentration of about 0.01% w / v to about 0.75% w / v.

[0029] In some aspects of any of the embodiments described above, the pre-treated suspension further comprises NaCl. In some embodiments, the NaCl has a concentration of about 0.005 M to about 0.5 M. In some embodiments, the NaCl has a concentration of about 0.01 M to about 0.4 M. In some embodiments, the NaCl has a concentration of about 0.015 M to about 0.3 M. In some embodiments, the NaCl has a concentration of about 0.015 M to about 0.2 M. In some embodiments, the NaCl has a concentration of about 0.015 M to about 0.1 M. In some embodiments, the NaCl has a concentration of about 0.02 M to about 0.05 M. In some embodiments, the NaCl has a concentration of about 0.03 M to about 0.07 M.

[0030] In some aspects of any of the embodiments described above, the saccharide is sucrose. In some embodiments, the saccharide has a concentration of about 8% w / v to about 20% w / v. In some embodiments, the saccharide has a concentration of about 7% w / v to about 11% w / v. In some embodiments, the saccharide has a concentration of about 8% w / v to about 10% w / v. In some embodiments, the saccharide has a concentration of about 16% w / v to about 20% w / v.

[0031] In some aspects of any of the above embodiments, the liquid medium or pre-treated suspension contains a buffer. In some embodiments, the buffer is selected from MOPS, HEPES, Tris, MES, citrate, and phosphate-buffered saline (PBS). In some embodiments, the buffer is Tris. In some embodiments, the buffer has a concentration of about 10 mM to about 100 mM. In some embodiments, the buffer has a concentration of about 15 mM to about 75 mM. In some embodiments, the buffer has a concentration of about 10 mM to about 40 mM.

[0032] In some aspects of any of the above embodiments, the liquid medium or pre-treated suspension has a pH of about 7.0 to about 8.5.

[0033] In some aspects of any of the embodiments described above, the method further includes, after step (b), dividing the pre-treated suspension into individual containers in predetermined lyophilized volumes. In some embodiments, the predetermined lyophilized volume is in the range of about 0.5 mL to about 10.0 mL. In some embodiments, the predetermined lyophilized volume is in the range of about 1.0 mL to about 3.0 mL.

[0034] In some aspects of any of the above embodiments, the pre-treated suspension further comprises poloxamer. In some embodiments, the poloxamer is poloxamer 188. In some embodiments, the poloxamer is concentrated at a concentration of about 0.01% w / v to about 0.10% w / v. In some embodiments, the poloxamer is concentrated at a concentration of about 0.02% w / v to about 0.8% w / v. In some embodiments, the poloxamer is concentrated at a concentration of about 0.03% w / v to about 0.7% w / v. In some embodiments, the poloxamer is concentrated at a concentration of about 0.04% w / v to about 0.06% w / v.

[0035] In some embodiments, products prepared by the processes described herein are provided.

[0036] In some embodiments, a lyophilized composition is provided comprising lipid nanoparticles encapsulating nucleic acids, monosaccharides, and one or more excipients selected from potassium sorbate, thiosulfate, sodium benzoate, and iodixanol.

[0037] In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is self-replicating RNA. In some embodiments, the RNA is mRNA. In some embodiments, the nucleic acid is approximately 20 to 13,000 nucleotides in length.

[0038] In some embodiments, the weight ratio of total lipids to nucleic acids in the lyophilized composition is about 50:1 to about 10:1. In some embodiments, the weight ratio of total lipids to RNA in the lyophilized composition is about 40:1 to about 20:1. In some embodiments, the weight ratio of total lipids to RNA in the lyophilized composition is about 35:1 to about 25:1.

[0039] In some embodiments of the above embodiments of the lyophilized composition, the lyophilized composition contains potassium sorbate in a weight ratio of about 30:1 to about 250:1 of potassium sorbate to RNA. In some embodiments, the lyophilized composition contains potassium sorbate in a weight ratio of about 40:1 to about 200:1 of potassium sorbate to RNA. In some embodiments, the lyophilized composition contains potassium sorbate in a weight ratio of about 50:1 to about 175:1 of potassium sorbate to RNA.

[0040] In some embodiments of the above embodiments of the lyophilized composition, the lyophilized composition contains sodium thiosulfate in a weight ratio of sodium thiosulfate to RNA of about 0.25:1 to about 40:1. In some embodiments, the lyophilized composition contains sodium thiosulfate in a weight ratio of sodium thiosulfate to RNA of about 2:1 to about 10:1. In some embodiments, the lyophilized composition contains sodium thiosulfate in a weight ratio of sodium thiosulfate to RNA of about 3:1 to about 8:1.

[0041] In some embodiments of the above embodiments of the freeze-dried composition, the freeze-dried composition contains sodium benzoate in a weight ratio of about 1:1 to about 12:1 of sodium benzoate to RNA. In some embodiments, the freeze-dried composition contains sodium benzoate in a weight ratio of about 2:1 to about 10:1 of sodium benzoate to RNA. In some embodiments, the freeze-dried composition contains sodium benzoate in a weight ratio of about 3:1 to about 9:1 of sodium benzoate to RNA.

[0042] In some embodiments of the above embodiments of the lyophilized composition, the lyophilized composition contains iodixanol in a weight ratio of iodixanol to RNA of about 100:1 to about 800:1. In some embodiments, the lyophilized composition contains iodixanol in a weight ratio of iodixanol to RNA of about 150:1 to about 750:1. In some embodiments, the lyophilized composition contains iodixanol in a weight ratio of iodixanol to RNA of about 200:1 to about 700:1. In some embodiments, the lyophilized composition contains iodixanol in a weight ratio of iodixanol to RNA of about 250:1 to about 650:1.

[0043] In some of the above embodiments of the freeze-dried composition, the freeze-dried composition further comprises polyvinyl alcohol (PVA) in a weight ratio of PVA to RNA of about 1:1 to about 12:1.

[0044] In some aspects of any of the above embodiments of the freeze-dried composition, the saccharide further comprises sucrose in a weight ratio of sucrose to RNA of about 100:1 to about 800:1.

[0045] In some aspects of any of the above embodiments of the lyophilized composition, the lyophilized composition further comprises a buffer selected from HEPES, MOPS, Tris, MERS, citrate, and phosphate in a weight ratio of buffer to RNA of about 3:1 to about 150:1.

[0046] In another embodiment, a lyophilized composition is provided comprising lipid nanoparticles for encapsulating RNA, poloxamer, potassium sorbate, and sugar. In some embodiments, the poloxamer is poloxamer 188. In some embodiments, the lyophilized composition contains about 0.001 to about 1.0% w / w of RNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is self-replicating RNA. In some embodiments, the lyophilized composition contains about 0.005 to about 0.8% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.01 to about 0.5% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.02 to about 0.4% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.03 to about 0.3% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.04 to about 0.2% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.5 to about 5.0% w / w of lipids. In some embodiments, the lyophilized composition contains about 1.0 to about 4.0% w / w of lipids. In some embodiments, the lyophilized composition contains about 1.25 to about 3.0% w / w of lipids. In some embodiments, the lyophilized composition contains about 0.5 to about 2.5% w / w of Tris buffer. In some embodiments, the lyophilized composition contains about 0.75 to about 2.25% w / w of Tris buffer. In some embodiments, the lyophilized composition contains about 1.0 to about 2.0% w / w of Tris buffer. In some embodiments, the lyophilized composition contains about 0.75 to about 2.75% w / w of NaCl. In some embodiments, the lyophilized composition contains about 1.0 to about 2.5% w / w of NaCl. In some embodiments, the lyophilized composition contains about 1.25 to about 1.80% w / w of NaCl. In some embodiments, the lyophilized composition contains about 85 to about 96% w / w of sugars. In some embodiments, the freeze-dried composition contains about 88 to about 95% w / w of sugar. In some embodiments, the freeze-dried composition contains about 90 to about 95% w / w of sugar. In some embodiments, the sugar is sucrose.In some embodiments, the freeze-dried composition contains about 0.01 to about 1.0% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.02 to about 0.8% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.03 to about 0.7% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.04 to about 0.6% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.05 to about 0.5% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.06 to about 0.4% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.07 to about 0.3% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.09 to about 0.2% w / w of poloxamer. In some embodiments, the poloxamer is poloxamer 188. In some embodiments, the freeze-dried composition contains about 0.5 to about 5.0% w / w of potassium sorbate. In some embodiments, the freeze-dried composition contains about 0.75 to about 4.0% w / w of potassium sorbate. In some embodiments, the freeze-dried composition contains about 1.0 to about 3.0% w / w of potassium sorbate. In some embodiments, the freeze-dried composition contains about 1.25 to about 2.75% w / w of potassium sorbate.

[0047] In some embodiments, a method for storing a lyophilized composition is provided, comprising storing the lyophilized product described herein at a temperature of about 2°C to about 8°C. In some embodiments, the method comprises storing the lyophilized product at a temperature of about -20°C.

[0048] In some embodiments, a method is provided for reconstituting a lyophilized composition, comprising adding a liquid medium to the lyophilized composition described herein. In some embodiments, the liquid medium is an aqueous medium. In some embodiments, the liquid medium comprises a poloxamer. In some embodiments, the poloxamer is P-188. In some embodiments, the liquid medium further comprises a buffer having a pH of about 7.0 to about 8.5.

[0049] In some embodiments, a method is provided for treating a disease or disorder in a subject, comprising administering to the subject a lyophilized composition described herein, reconstituted in a liquid medium. In some embodiments, the reconstituted lyophilized composition is administered intravenously. In some embodiments, the reconstituted lyophilized composition is administered intramuscularly. In some embodiments, the reconstituted lyophilized composition is administered by inhalation. In some embodiments, the reconstituted lyophilized composition is administered mucous membranely. In some embodiments, the reconstituted lyophilized composition is administered subcutaneously.

[0050] Freeze drying The technique of freeze-drying, also known as cryodesiccation, is based on the physical principle of sublimation, a process in which a solid substance directly transitions to a gaseous state. Therefore, freeze-drying and the principle of sublimation on which it works are in stark contrast to the more common drying technique of direct evaporation, which transitions a liquid substance to a gas. The fundamental processes and techniques used in freeze-drying are well understood in the art. (See Rey, Louis, ed. Freeze-drying / lyophilization of pharmaceutical and biological products. CRC Press, 2016; and Nireesha, GR, et al. Int.j. novel trends in pharm.sci.3.4(2013):87-98). A brief overview is provided below.

[0051] Freeze-drying is a multi-stage operation in which each step is critical. The key parameters affecting the outcome of this process can be highly specific to the type of substance being freeze-dried, and therefore, strict control may be required to obtain a high-quality product. Some of the parameters that must be considered include the substance, e.g., the substance being freeze-dried that must maintain its desired properties and activity; the surrounding medium and its components, e.g., fillers, stabilizers, emulsifiers, antioxidants, antifreeze agents, freeze-drying protectants, and moisture buffers; the equipment used, the process which must be adapted according to the specific requirements and low-temperature behavior of different products under treatment; and the freeze-drying cycle.

[0052] Freeze-drying cycle Regarding the freeze-drying cycle, it is well established that freeze-drying operations include i) preparation of the substance; ii) freezing; iii) sublimation or primary drying; and iv) desorption or secondary drying. After these steps, the freeze-dried product typically undergoes further processing to prepare it for storage.

[0053] Preparation / pretreatment of substances The preparation of the substance to be processed (solid, liquid, paste, emulsion) involves adjusting, as necessary, the matrix it encounters in solution or suspension, the liquid medium in which it exists, pH, tension, and the addition of other excipients, while ensuring that the fundamental properties of the substance are not impaired. The pre-processed substance is then divided into predetermined volumes for optimized lyophilization. Aliquots can be dispensed into individual containers such as vials.

[0054] In some embodiments of the lyophilization methods provided herein, the method further includes dividing a pre-treated suspension into individual containers of a predetermined lyophilization volume prior to step (c). In some embodiments, the individual containers are vials. In some embodiments, the predetermined lyophilization volume is in the range of about 0.5 mL to about 5.0 mL. In some embodiments, the predetermined lyophilization volume is in the range of about 1.0 mL to about 4.0 mL.

[0055] Freezing process In the freezing process, a substance hardens by being exposed to low temperatures. During this critical period, all fluids present become solid, either crystalline, amorphous, or glassy. In the case of water, this typically results in a complex network of ice, but it can also remain embedded in a glassy structure or more or less tightly bound within the interstitial structure. Other liquids and solvents have specific freezing properties. Solutes may concentrate and even crystallize. Simultaneously, the volume expansion of the system as water freezes can induce strong mechanical stress, combined with an osmotic shock resulting from an increase in the concentration of the interstitial fluid.

[0056] Sublimation stage / Primary drying Next, the sublimation stage, or primary drying, takes place when the frozen material, placed under vacuum, is gradually heated to deliver enough energy for the ice to sublimate. During this critical period, a precise balance must be maintained between heat input (heat transfer) and water sublimation (mass transfer) so that drying can proceed without inducing harmful reactions in the frozen material, such as re-melting, swelling, or collapse. Subsequently, continuous and precise adjustment of the operating pressure is required to link the heat input to the evaporation potential of the frozen material.

[0057] Desorption stage / Secondary drying The desorption step, or secondary drying, begins when the ice has been removed by distillation, and a higher vacuum allows for the progressive extraction of bound water above zero degrees. This must be done because over-drying is just as bad as under-drying and can result in undesirable dried structures, denatured products, or products unsuitable for reconstitution. For each product, an appropriate amount of residual moisture must be reached under a given temperature and pressure.

[0058] Freeze-drying of lipid nanoparticle formulations In some embodiments, a method is provided for freeze-drying a composition comprising lipid nanoparticles for encapsulating RNA, the method comprising a.) providing a suspension of lipid nanoparticles in a liquid medium; and b.) preparing the liquid medium, thereby forming a pre-treated suspension comprising at least one excipient selected from potassium sorbate, thiosulfate, sodium benzoate, and iodixanol. In some embodiments, the method further comprises c.) subjecting the pre-treated suspension to a freeze-drying process comprising i.) an initial freezing step carried out at a temperature of -52±6°C and atmospheric pressure; ii.) a primary drying step carried out at a temperature in the range of -25±2°C to -48±2°C and a pressure in the range of about 25 mTorr to about 75 mTorr; and iii.) a secondary drying step carried out at a temperature in the range of 5±2°C to 10±2°C and a pressure in the range of about 85 mTorr to about 200 mTorr.

[0059] In some embodiments, the method further includes subjecting the pre-treated suspension to a freeze-drying process comprising: c) an initial freezing step carried out at a temperature of -48±8°C and atmospheric pressure; ii) a primary drying step carried out at a pressure of about 0.03 to about 0.08 mbar, starting at a temperature of -48±8°C and gradually decreasing to a temperature of 0±2°C over a period ranging from about 40 to about 75 hours; and iii) a secondary drying step carried out at a pressure of about 0.03 to about 0.08 mbar, starting at a temperature of 0±2°C and gradually decreasing to a temperature of about 25±3°C over a period ranging from about 30 to about 50 hours.

[0060] In some embodiments, the freeze-drying cycle shown below is followed: [Table 1]

[0061] Lipid nanoparticles Several lipid-based drug delivery vehicles are used in the technology of delivering nucleic acid drugs, including liposomes, cationic liposomes, and lipid nanoparticles. Conventional liposomes are vesicles composed of at least one bilayer and an internal aqueous compartment. The bilayer membrane of liposomes is usually formed of amphiphilic molecules, such as synthetic or naturally occurring lipids containing spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16:307-321, 1998). The bilayer membrane of liposomes can also be formed of amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). They generally exist as spherical vesicles, and their size can range from 20 nm to several microns.

[0062] Liposomes can be composed of cationic, anionic, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are those made entirely or partially from positively charged lipids, or more specifically, lipids containing both cationic groups and lipophilic moieties. In addition to the general properties of liposomes outlined above, the positively charged moieties of cationic lipids used in cationic liposomes offer several advantages and some unique structural features. For example, the lipophilic moiety of a cationic lipid is hydrophobic and will therefore move itself away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species. Conversely, the cationic moiety can associate with the aqueous medium and, more importantly, with polar molecules and species, and together with them, complex within the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are favored by electrostatic interactions with charged nucleic acids and are therefore being increasingly investigated for use in gene therapy, resulting in complexes that offer biocompatibility, low toxicity, and the potential for large-scale production required for in vivo clinical applications. Cationic lipids suitable for use in cationic liposomes are listed below herein.

[0063] In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNPs) have a structure that includes a single monolayer or bilayer of lipids that encapsulates a compound in a solid phase. Therefore, unlike liposomes, lipid nanoparticles do not have an aqueous or other liquid phase internally; rather, the lipids in the bilayer or monolayer shell directly complex with the internal compound, thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles with relatively uniform dispersion of shape and size. While the source varies depending on the size at which lipid particles are considered nanoparticles, there is some overlap in that lipid nanoparticles can have diameters ranging from 10 nm to 1000 nm. More generally, however, they are considered smaller than 120 nm or even 100 nm.

[0064] In the case of lipid nanoparticle nucleic acid delivery systems, the lipid shell can be formulated to contain an ionizable cationic lipid that can complex and associate with the load electrical backbone of the nucleic acid core. Ionizable cationic lipids with an apparent pKa value below approximately 7 have the advantage of complexing the cationic lipid with the load electrical backbone of the nucleic acid and loading the lipid nanoparticles at a pH value below the pKa of the ionizable lipid when positively charged. Subsequently, at physiological pH values, lipid nanoparticles can adopt a relatively neutral form that can significantly increase the circulating half-life of the particles after IV administration. In relation to nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems, including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutic agents, and low levels of cytotoxicity and immunogenicity.

[0065] Methods for producing suitable lipid components and lipid nanoparticles are well known in the art and are described, for example, in PCT / US2020 / 023442, US 8,058,069, US 8,822,668, US 9,738,593, US 9,139,554, PCT / US2014 / 066242, PCT / US2015 / 030218, PCT / 2017 / 015886, and PCT / US2017 / 067756, the contents of which are incorporated by reference.

[0066] Cationic lipids Lipid nanoparticles preferably contain cationic lipids suitable for forming cationic liposomes or lipid nanoparticles. Cationic lipids are widely studied for nucleic acid delivery because they can bind to negatively charged membranes and induce uptake. Generally, cationic lipids are amphiphilic substances containing a positively hydrophilic head group, two (or more) lipophilic tails, or a steroid moiety, and a ligator between these two domains. Preferably, cationic lipids carry a net positive charge at approximately physiological pH. Cationic liposomes are traditionally the most commonly used nonviral delivery system for oligonucleotides, such as plasmid DNA, antisense oligonucleotides, and siRNA / small hairpin RNA-shRNA. Cationic lipids, such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate), can form complexes or lipoplexes with negatively charged nucleic acids through electrostatic interactions, providing high in vitro transfection efficiency. In some embodiments, the lipid nanoparticles comprise a combination of two or more cationic lipids. The lipid nanoparticles may further comprise lipidoid and / or polymeric components.

[0067] In the lipid nanoparticles of this disclosure, cationic lipids include, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 1,2-dioleoylmethylammonium propane chloride (DOTAP) (also known as N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride and l,2-dioleyloxy-3-trimethylaminopropane chloride salt), N-(l-(2,3-dioleyloxy) Propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), l,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ-DLenDMA), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), l,2- Dilinoleoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), l,2-Dilinoleoxy-3-morpholinopropane (DLin-MA), l,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), l-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), l,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l, 2-Dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), l,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-l,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-l,2-propanediol (DOAP), l,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[l,3]-Dioxolane (DLin-K-DMA) or its analog, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][l,3]dioxol-5-amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraene-19- Il-4-(dimethylamino)butanoate (MC3), l,l'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-l-yl)ethylazanediyl)didodecane-2-ol (C12-200), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxy It may be solan (DLin-K-C2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-M-C3-DMA), 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropane-l-amine (MC3 ether), 4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylbutane-1-amine (MC4 ether), or any combination thereof. Other cationic lipids include N,N-distearyl-N,N-dimethylammonium bromide (DDAB), 3P-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Choi), N-(l-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), dioctadecylamideglycylcarboxyspermine (DOGS), l,2-dioleoyl-sn-3-phosphoethanolamine (DOPE), l,2-dioleoyl-3-dimethylammoniumpropane (DODAP), N-(l,2-dimyristyloxyprop-3-yl)-N,This includes, but is not limited to, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE) and 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (XTC). Furthermore, commercially available preparations of cationic lipids, such as LIPOFECTIN (including DOTMA and DOPE, available from GIBCO / BRL) and Lipofectamine (including DOSPA and DOPE, available from GIBCO / BRL), can be used.

[0068] Other suitable cationic lipids are disclosed in International Publications WO09 / 086558, WO09 / 127060, WO10 / 048536, WO10 / 054406, WO10 / 088537, WO10 / 129709, and WO2011 / 153493; U.S. Patent Publications 2011 / 0256175, 2012 / 0128760, and 2012 / 0027803; U.S. Patent No. 8,158,601; and Love et al., PNAS, 107(5), 1864-69, 2010, the contents of which are incorporated herein by reference.

[0069] Other suitable cationic lipids include those having alternative fatty acid groups and other dialkylamino groups, including those with different alkyl substituents (e.g., N-ethyl-N-methylamino- and N-propyl-N-ethylamino-). These lipids are part of a subcategory of cationic lipids called aminolipids. In some embodiments of the lipid nanoparticles described herein, the cationic lipids are aminolipids. Generally, aminolipids with fewer saturated acyl chains are easier to size, especially when the complex must be sized to less than about 0.3 microns for filtration sterilization purposes. Aminolipids containing unsaturated fatty acids having carbon chain lengths in the range of C14 to C22 may be used. Other scaffolds may also be used to separate the amino group from the fatty acid or aliphatic alkyl portion of the aminolipid.

[0070] In some embodiments, the lipid nanoparticles comprise a cationic lipid of formula I according to patent application PCT / EP2017 / 064066. In this regard, the disclosure of PCT / EP2017 / 064066 is also incorporated herein by reference.

[0071] In some embodiments, the amino lipids or cationic lipids of the present disclosure are ionizable and have at least one protonatable or deprotonatable group such that the lipid is positively charged at a pH below physiological pH (e.g., pH 7.4) and neutral at a second pH, preferably above physiological pH. Of course, the addition or removal of protons as a function of pH is an equilibrium process, and the reference to charged or neutral lipids refers to the nature of the predominant species, and it will be understood that not all of the lipids need to be present in the charged or neutral form. Lipids having multiple protonatable or deprotonatable groups, or lipids that are zwitterions, are not excluded from use in the present disclosure. In certain embodiments, the protonatable lipid has a pKa of protonatable groups in the range of about 4 to about 11. In some embodiments, the ionizable cationic lipid has a pKa of about 5 to about 7. In some embodiments, the pKa of the ionizable cationic lipid is about 6 to about 7.

[0072] In some embodiments, the lipid nanoparticles comprise an ionizable cationic lipid of formula I:

Chemical Formula

[0073] In some embodiments, X 7 S is.

[0074] In some embodiments, X 5 This is -C(O)O-, and thus -C(O)OR 6 X is formed, 6 This is -C(O)O-, and thus -C(O)OR 5 A formation is created.

[0075] In some embodiments, R 7 and R 8 Each of these is independently selected from the group consisting of methyl, ethyl, and isopropyl.

[0076] In some embodiments, L 5 and L 6 Each is independent of C1-C 10 It is alkyl. In some embodiments, L 5 It is a C1-C3 alkyl group, L 6 is a C1-C5 alkyl group. In some embodiments, L 6 is a C1-C2 alkyl group. In some embodiments, L 5 and L 6Each of these is a linear C7 alkyl group. In some embodiments, L 5 and L 6 Each of these is a linear C9 alkyl group.

[0077] In some embodiments, R 5 and R 6 Each of these is an alkenyl independently. In some embodiments, R 6 is an alkenil. In some embodiments, R 6 is a C2-C9 alkenyl. In some embodiments, the alkenyl contains a single double bond. In some embodiments, R 5 and R 6 Each of them is an alkyl group. In some embodiments, R 5 is a branched alkyl group. In some embodiments, R 5 and R 6 Each is independently selected from the group consisting of C9 alkyl, C9 alkenyl, and C9 alkynyl. In some embodiments, R 5 and R 6 Each is independent of C 11 Alkyl, C 11 Alkenyl and C 11 Selected from the group consisting of alkynnyls. In some embodiments, R 5 and R 6 Each is independently selected from the group consisting of C7 alkyl, C7 alkenyl, and C7 alkynyl. In some embodiments, R 5 is -CH((CH2) p CH3)2 or -CH((CH2) p CH3)((CH2) p-1 CH3) [wherein p is 4 to 8.] In some embodiments, p is 5 and L 5 is a C1-C3 alkyl group. In some embodiments, p is 6 and L 5 C3 is. In some embodiments, p is 7. In some embodiments, p is 8 and L 5 is a C1-C3 alkyl group. In some embodiments, R 5 is -CH((CH2)p CH3)((CH2) p-1 CH3)[where p is 7 or 8.]

[0078] In some embodiments, R 4 is ethylene or propylene. In some embodiments, R 4 is n-propylene or isobutylene.

[0079] In some embodiments, L 7 is absent, R 4 is ethylene, X 7 is S, R 7 and R 8 are each methyl. In some embodiments, L 7 is absent, R 4 is n-propylene, X 7 is S, R 7 and R 8 are each methyl. In some embodiments, L 7 is absent, R 4 is ethylene, X 7 is S, R 7 and R 8 are each ethyl.

[0080] In some embodiments, X 7 is S, X 5 is -C(O)O-, thereby forming -C(O)O-R 6 , X 6 is -C(O)O-, thereby forming -C(O)O-R 5 , L 5 and L 6 are each independently a linear C3-C7 alkyl, L 7 is absent, R 5 is -CH((CH2) p CH3)2, R 6 is C7-C 12 alkenyl. In some further embodiments, p is 6 and R 6 is C9 alkenyl.

[0081] Helper lipids and sterols The RNA lipid nanoparticles of this disclosure may contain helper lipids, which can be called neutral helper lipids, noncationic lipids, noncationic helper lipids, anionic lipids, anionic helper lipids, or neutral lipids. Lipid formulations, particularly cationic liposomes and lipid nanoparticles, have been shown to undergo increased uptake into cells when helper lipids are present in the formulation (Curr. Drug Metab. 2014;15(9):882-92). For example, several studies have shown that neutral and zwitterionic lipids, such as 1,2-dioleoylsn-glycero-3-phosphatidylcholine (DOPC), dioleoyl-phosphatidyl-ethanolalamine (DOPE), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), are more conjugate (i.e., promote fusion) than cationic lipids, but can affect the pleomorphism of lipid-nucleic acid complexes, which promotes the transition from the lamellar phase to the hexagonal phase, thereby inducing cell membrane fusion and disruption (Nanomedicine (Lond). 2014 Jan;9(1):105-20). Furthermore, the use of helper lipids may help reduce any potential adverse effects of using many common cationic lipids, such as toxicity and immunogenicity.

[0082] Non-limiting examples of non-cationic lipids suitable for the lipid nanoparticles of this disclosure include phospholipids, such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl Examples include oil-phosphatidylethanolamine (POPE), palmitoyloleoyl-phosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), monomethyl-phosphatidylethanolamine, dimethyl-phosphatidylethanolamine, dierydoyl-phosphatidylethanolamine (DEPE), stearoyloleoyl-phosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof. Other diacylphosphatidylcholines and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having a carbon chain of C10 to C24, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0083] Further examples of noncationic lipids include sterols such as cholesterol and their derivatives. One study concluded that, as a helper lipid, cholesterol broadens the charge spacing of the lipid layer interacting with nucleic acids, allowing the charge distribution to more closely match that of the nucleic acids (JRSoc.Interface.2012 Mar 7;9(68):548-561). Non-limiting examples of cholesterol derivatives include polar analogs such as 5α-cholestanol, 5α-coprostanol, cholesteryl-(2'-hydroxy)-ethyl ether, cholesteryl-(4'-hydroxy)-butyl ether, and 6-ketocholestanol; non-polar analogs such as 5α-cholestanol, cholestenone, 5α-cholestanone, 5α-cholestanone, and cholesteryl decanoate; and mixtures thereof. In preferred embodiments, the cholesterol derivative is a polar analog such as cholesteryl-(4'-hydroxy)-butyl ether.

[0084] In some embodiments, the helper lipids present in the lipid nanoparticles consist of or comprise a mixture of one or more phospholipids and cholesterol or a derivative thereof. In other embodiments, the neutral lipids present in the lipid nanoparticles consist of or comprise one or more phospholipids, for example, cholesterol-free lipid nanoparticles. In yet another embodiment, the neutral lipids present in the lipid nanoparticles consist of or comprise cholesterol or a derivative thereof, for example, phospholipid-free lipid nanoparticles.

[0085] Other examples of helper lipids include phosphorus-free products containing lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine lauryl sulfate, alkyl-aryl sulfate polyethyl oxy-fatty acid amides, dioctadecyldimethylammonium bromide, ceramides, and sphingomyelin.

[0086] In some embodiments, the helper lipids constitute about 2 mol% to about 20 mol%, about 3 mol% to about 18 mol%, about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, about 6 mol% to about 12 mol%, about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or a range within these).

[0087] Cholesterol or cholesterol derivatives in lipid nanoparticles may account for up to approximately 40 mol%, 45 mol%, 50 mol%, 55 mol%, or 60 mol% of the total lipids present in the lipid nanoparticles. In some embodiments, cholesterol or cholesterol derivatives account for approximately 15 mol% to 45 mol%, 20 mol% to 40 mol%, 25 mol% to 35 mol%, or 28 mol% to 35 mol%, or approximately 25 mol%, 26 mol%, 27 mol%, 28 mol%, 29 mol%, 30 mol%, 31 mol%, 32 mol%, 33 mol%, 34 mol%, 35 mol%, 36 mol%, or 37 mol% of the total lipids present in the lipid nanoparticles.

[0088] In some embodiments, the phospholipid component in the mixture may represent about 2 mol% to about 20 mol%, about 3 mol% to about 18 mol%, about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, about 6 mol% to about 12 mol%, about 5 mol% to about 10 mol%, about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or a range within these).

[0089] The percentage of helper lipids present in lipid nanoparticles is the target amount, and the actual amount of helper lipids present in the formulation may vary, for example, by ±5 mol%.

[0090] Lipid nanoparticles containing cationic lipid compounds or ionizable cationic lipid compounds (or a combination of two cationic lipids) may consist, on a molar basis, of approximately 30–70% cationic lipid compounds(or more), approximately 25–40% cholesterol, approximately 2–15% helper lipids, and approximately 0.5–5% polyethylene glycol (PEG) lipids, relative to the total lipids present in the formulation. In some embodiments, the composition consists of approximately 40–65% cationic lipid compounds, approximately 25–35% cholesterol, approximately 3–9% helper lipids, and approximately 0.5–3% PEG lipids, relative to the total lipids present in the formulation.

[0091] The formulation may be a lipid particle formulation containing, for example, 8-30% nucleic acids, 5-30% helper lipids, and 0-20% cholesterol; 4-25% cationic lipids, 4-25% helper lipids, 2-25% cholesterol, 10-35% cholesterol-PEG, and 5% cholesterol-amine; or 2-30% cationic lipids, 2-30% helper lipids, 1-15% cholesterol, 2-35% cholesterol-PEG, and 1-20% cholesterol-amine; or up to 90% cationic lipids and 2-10% helper lipids, or even 100% cationic lipids.

[0092] Lipid conjugate The lipid nanoparticles described herein may further comprise lipid conjugates. Conjugated lipids are useful in preventing particle aggregation. Suitable conjugated lipids include, but are not limited to, PEG-lipid conjugates, cationic polymer-lipid conjugates, and mixtures thereof. Furthermore, lipid delivery vehicles can be used for specific targeting by attaching ligands (e.g., antibodies, peptides, and carbohydrates) to their surface or to the ends of the conjugated PEG chains (Front Pharmacol. 2015 Dec 1;6:286).

[0093] In preferred embodiments, the lipid conjugate is a PEG lipid. When polyethylene glycol (PEG) is included in lipid nanoparticles as a coating or surface ligand, a technique called PEGylation helps protect the nanoparticles from the immune system and avoid uptake by RES (Nanomedicine (Lond). 2011 Jun;6(4):715-28). PEGylation is widely used to stabilize lipid nanoparticles and their payloads through physical, chemical, and biological mechanisms. Surfactant-like PEG lipids (e.g., PEG-DSPE) can penetrate lipid nanoparticles to form a hydrate layer and a steric barrier on the surface. Based on the degree of PEGylation, the surface layer can generally be divided into two types: brush-like and mushroom-like layers. In formulations stabilized with PEG-DSPE, PEG will take on a mushroom structure at low levels of PEGylation (typically less than 5 mol%) and will shift to a brush structure as the PEG-DSPE content increases beyond a certain level (Journal of Nanomaterials. 2011;2011:12). Increased PEGylation has been shown to significantly increase the circulating half-life of lipid nanoparticles (Annu. Rev. Biome Eng. 2011 Aug 15;13():507-30; J. Control Release. 2010 Aug 3;145(3):178-81).

[0094] Suitable examples of PEG lipids include, but are not limited to, PEG bonded to dialkyloxypropyl (PEG-DAA), PEG bonded to diacylglycerol (PEG-DAG), PEG bonded to phospholipids such as phosphatidylethanolamine (PEG-PE), PEG compounded with ceramide, PEG compounded with cholesterol, or derivatives thereof, and mixtures thereof.

[0095] PEG is a linear, water-soluble polymer of ethylene PEG repeat units having two terminal hydroxyl groups. PEGs are classified by their molecular weight and include: monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S), monomethoxypolyethylene glycol succinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol torecylate (MePEG-TRES), monomethoxypolyethylene glycol imidazolyl carbonyl (MePEG-IM), and compounds containing terminal hydroxyl groups instead of terminal methoxy groups (e.g., HO-PEG-S, HO-PEG-S-NHS, HO-PEG-NH2).

[0096] The PEG portion of the PEG-lipid conjugates described herein may contain an average molecular weight in the range of about 550 daltons to about 10,000 daltons. In specific cases, the PEG portion has an average molecular weight of about 750 daltons to about 5,000 daltons (e.g., about 1,000 daltons to about 5,000 daltons, about 1,500 daltons to about 3,000 daltons, about 750 daltons to about 3,000 daltons, about 750 daltons to about 2,000 daltons). In preferred embodiments, the PEG portion has an average molecular weight of about 2,000 daltons or about 750 daltons. The average molecular weight may be any value or a partial value within the enumerated range, including the endpoints.

[0097] In certain cases, PEG may be substituted with alkyl, alkoxy, acyl, or aryl groups, as desired. PEG may be compounded directly with lipids or linked to lipids via a linker moiety. For example, any linker moiety suitable for linking PEG to lipids may be used, including non-ester-containing and ester-containing linker moieties. In preferred embodiments, the linker moiety is a non-ester-containing linker moiety. Suitable non-ester-containing linker moieties include, but are not limited to, amides (-C(O)NH-), aminos (-NR-), carbonyls (-C(O)-), carbamates (-NHC(O)O-), ureas (-NHC(O)NH-), disulfides (-SS-), ethers (-O-), succinyls (-(O)CCH2CH2C(O)-), succinamidyls (-NHC(O)CH2CH2C(O)NH-), ethers, and combinations thereof (such as linkers containing both carbamate and amide linker moieties). In preferred embodiments, a carbamate linker is used to bind PEG to lipids.

[0098] In other embodiments, the ester-containing linker moiety is used to bind PEG to lipids. Suitable ester-containing linker moieties include, for example, carbonates (-OC(O)O-), succinoyl, phosphate esters (-O-(O)POH-O-), and combinations thereof.

[0099] Phosphatidylethanolamine having various acyl chain groups with various chain lengths and degrees of saturation can be complexed with PEG to form a lipid complex. Such phosphatidylethanolamine is commercially available or can be isolated or synthesized using conventional techniques known to those skilled in the art. Phosphatidylethanolamine containing saturated or unsaturated fatty acids having a carbon chain length in the range of C10 - C20 is preferred. Phosphatidylethanolamine containing mono- or di-unsaturated fatty acids, as well as mixtures of saturated and unsaturated fatty acids, can also be used. Suitable phosphatidylethanolamines include, but are not limited to, dimyristoyl-phosphatidylethanolamine (DMPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dioleoyl-phosphatidylethanolamine (DOPE), and distearoyl-phosphatidylethanolamine (DSPE).

[0100] In some embodiments, the PEG-DAA complex is a PEG-didecyloxypropyl (C 10 ) complex, a PEG-dilauryl-oxypropyl (C 12 ) complex, a PEG-dimyristyloxypropyl (C 14 ) complex, a PEG-dipalmityloxypropyl (C 16 ) complex, or a PEG-distearyloxypropyl (C 18 ) complex. In these embodiments, PEG preferably has an average molecular weight of 750 or 2,000 daltons. In certain embodiments, the terminal hydroxyl group of PEG is substituted with a methyl group.

[0101] In addition to the foregoing, other hydrophilic polymers can be used instead of PEG. Suitable polymers that can be used instead of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl, methacrylamide, polymethacrylamide, and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized cellulose such as hydroxymethylcellulose or hydroxyethylcellulose.

[0102] In some embodiments, the lipid complex (e.g., PEG lipid) accounts for about 0.1 mol% to about 2 mol%, about 0.5 mol% to about 2 mol%, about 1 mol% to about 2 mol%, about 0.6 mol% to about 1.9 mol%, about 0.7 mol% to about 1.8 mol%, about 0.8 mol% to about 1.7 mol%, about 0.9 mol% to about 1.6 mol%, about 0.9 mol% to about 1.8 mol%, about 1 mol% to about 1.8 mol%, about 1 mol% to about 1.7 mol%, about 1.2 mol% to about 1.8 mol%, about 1.2 mol% to about 1.7 mol%, about 1.3 mol% to about 1.6 mol%, or about 1.4 mol% to about 1.6 mol% (or any fraction of these or a range within these) of the total lipids present in the lipid nanoparticles. In other embodiments, the lipid conjugate (e.g., PEG-lipid) accounts for approximately 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, or 5% (or any percentage or range thereof) of the total lipids present in the lipid nanoparticles. The amount may be any value or a partial value within the enumerated range, including the endpoints.

[0103] The proportion of lipid conjugates (e.g., PEG-lipids) present in the lipid nanoparticles of this disclosure is a target amount, and the actual amount of lipid conjugates present in the formulation may vary, for example, by ±0.5 mol%. Those skilled in the art will understand that the concentration of lipid conjugates may change depending on the rate at which the lipid conjugates and lipid nanoparticles used become fused.

[0104] Lipid nanoparticles - nucleic acid preparations In connection with this disclosure, lipid nanoparticle delivery vehicles typically help transport nucleic acids (e.g., RNA) to target cells or tissues. Exemplary nucleic acids include both DNA and RNA. In preferred embodiments, the lipid nanoparticles comprise RNA, cationic lipids (e.g., one or more cationic lipids or salts thereof), phospholipids, and conjugated lipids that inhibit particle aggregation (e.g., one or more PEG-lipid conjugates). The lipid nanoparticles may also contain cholesterol.

[0105] In some embodiments, the RNA is completely encapsulated within the lipid portion of lipid nanoparticles so that the RNA is resistant to nuclease degradation in aqueous solution.

[0106] The term "RNA" means a molecule containing at least one ribonucleotide residue. "Ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribofuranose moiety. This term includes isolated RNA such as double-stranded RNA, single-stranded RNA, and partially purified RNA; essentially pure RNA; synthetic RNA; recombinant RNA; and modified RNA, which differs from naturally occurring RNA due to the addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations include the addition of non-nucleotide substances to or within interfering RNA, such as at the terminus(s) of an interfering RNA, or at one or more nucleotides of the RNA. The nucleotides in the RNA molecules of this disclosure may also include non-standard nucleotides, such as nucleotides that do not occur naturally, or chemically synthesized nucleotides or deoxyribonucleotides. These modified RNAs may be called analogs, or analogs of naturally occurring RNA. As used herein, the terms “ribonucleic acid” and “RNA” mean molecules containing at least one ribonucleotide residue, including siRNA, antisense RNA, single-stranded RNA, microRNA, mRNA, non-coding RNA, and polyvalent RNA.

[0107] In some embodiments, RNA is self-replicating RNA. In some embodiments, RNA is mRNA. In some embodiments, RNA is siRNA. In some embodiments, nucleic acid is approximately 1,000 to 13,000 nucleotides in length.

[0108] The lipid nanoparticles of this disclosure also typically have a total lipid:RNA ratio (mass / mass ratio) of about 1:1 to about 100:1, about 1:1 to about 50:1, about 2:1 to about 45:1, about 3:1 to about 40:1, about 5:1 to about 38:1, or about 6:1 to about 40:1, or about 7:1 to about 35:1, or about 8:1 to about 30:1; or about 10:1 to about 25:1; or about 8:1 to about 12:1; or about 13:1 to about 17:1; or about 18:1 to about 24:1; or about 20:1 to about 30:1. In some preferred embodiments, the total lipid:RNA ratio (mass / mass ratio) is about 10:1 to about 25:1. In some embodiments, the weight ratio of total lipid to RNA in the suspension is about 50:1 to about 10:1. In some embodiments, the weight ratio of total lipids to RNA in the suspension is approximately 40:1 to approximately 20:1. In some embodiments, the weight ratio of total lipids to RNA in the suspension is approximately 35:1 to approximately 25:1. The ratio may be any value or a secondary value within the described range, including the endpoints.

[0109] The lipid nanoparticles disclosed herein typically have wavelengths of approximately 30nm to 150nm, 40nm to 150nm, 50nm to 150nm, 60nm to 130nm, 70nm to 110nm, 70nm to 100nm, 80nm to 100nm, 90nm to 100nm, 70nm to 90nm, 80nm to 90nm, 70nm to 80nm, or approximately 30nm, 35nm, and 4nm. It has an average diameter of 0 nm, approximately 45 nm, approximately 50 nm, approximately 55 nm, approximately 60 nm, approximately 65 nm, approximately 70 nm, approximately 75 nm, approximately 80 nm, approximately 85 nm, approximately 90 nm, approximately 95 nm, approximately 100 nm, approximately 105 nm, approximately 110 nm, approximately 115 nm, approximately 120 nm, approximately 125 nm, approximately 130 nm, approximately 135 nm, approximately 140 nm, approximately 145 nm, or approximately 150 nm, and is substantially nontoxic. The diameter may be any value or a partial value within the enumerated range, including the endpoints.

[0110] In relation to nucleic acids, complete encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that enhances fluorescence when associated with nucleic acids. Encapsulation is determined by adding the dye to lipid nanoparticles, measuring the resulting fluorescence, and comparing it to the fluorescence observed when a small amount of nonionic surfactant is added. The surfactant-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may also be calculated as E=(I0-I) / I0, where I and I0 refer to the fluorescence intensity before and after the addition of the surfactant.

[0111] In some embodiments, lipid nanoparticles make up approximately 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 30% to 95%, 40% to 95%, 50% to 95%, 60% to 95%, 70% to 95%, 80% to 95%, 85% to 95%, 90% to 95%, 30% to 90%, 40% to 90%, 50% to 90% Approximately 60% to 90%, 70% to 90%, 80% to 90%, or at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% (or any portion or range thereof) of the RNA is completely encapsulated within the lipid portion of the formulation so that the RNA is contained within it. The amount may be any value or partial value within the enumerated range, including the endpoints.

[0112] Suspensions and liquid media A suspension is a heterogeneous mixture in which solute particles do not dissolve but are suspended throughout the bulk of the solvent and remain free to float in the medium. The internal phase (solid) is dispersed throughout the external phase (fluid), which can be facilitated by the use of specific excipients or suspending agents. The liquid medium used to suspend lipid nanoparticles may include any suitable liquid medium known in the art. Suitable liquids used in pharmaceutical suspensions include alcohols, glycerin, polyethylene glycol, and polypropylene glycol. The mechanism by which these liquids provide penetration is that they are miscible with water and reduce the liquid-air interface tension. The liquid penetrates into the individual particles, facilitating penetration. In some embodiments, the liquid medium is an aqueous medium.

[0113] In this disclosure, the concentration of lipid nanoparticles in a suspension is disclosed as the concentration of encapsulated RNA per 1 mL of suspension. In some embodiments, the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 2.0 mg / mL. In some embodiments, the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 1.5 mg / mL. In some embodiments, the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 1.0 mg / mL. In some embodiments, the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 0.5 mg / mL.

[0114] Excipients, freeze-drying protective agents, antifreeze agents, and buffer solutions Lipid nanoparticle-RNA formulations may be pre-treated to facilitate lyophilization and reconstitution. Typically, a buffered suspension of the lipid nanoparticle-RNA formulation is combined with special excipients, some of which function as lyophilization protectants and / or antifreeze agents. As used herein, the term “lyophilization protectant” refers to a substance, compound, or excipient added to a composition to protect the active ingredient during the drying step of lyophilization, to assist in the storage or stabilization of the lyophilized product, and / or to assist in the easier reconstitution of the lyophilized product. Lyophilization protectants may also be used as bulking agents. As used herein, “antifreeze agent” refers to a substance, compound, or excipient added to a biological or pharmaceutical composition to protect it from freeze damage.

[0115] In some embodiments, the pre-treated suspension contains a freeze-drying protective agent. In some embodiments, the pre-treated suspension contains an antifreeze agent.

[0116] Suitable examples of excipients used in the freeze-drying process as either freeze-drying protective agents or antifreeze agents include saccharide compounds (e.g., monosaccharides, disaccharides, etc.). Examples of protective sugar compounds include monosaccharides, e.g., C 5-6Aldoses and ketoses, as well as disaccharides, such as sucrose, lactose, maltose, trehalose, cellobiose, kordibiose, sakebiose, isomaltose, sophorose, laminaribiose, genthiobiose, turanose, maltulose, isomaltulose, genthiobiulose, mannobiose, melibiose, melibiulose, and xylobiose.

[0117] In some embodiments, the pre-treated suspension contains thiosulfate. Thiosulfate can be any suitable thiosulfate salt for in vivo administration. In some embodiments, thiosulfate is sodium thiosulfate or potassium thiosulfate. In some embodiments, thiosulfate is sodium thiosulfate. In some embodiments, the pre-treated suspension has a thiosulfate concentration of about 0.025% w / v to about 1.0% w / v. In some embodiments, thiosulfate is sodium thiosulfate. In some embodiments, the pre-treated suspension has a thiosulfate concentration of about 0.025% w / v to about 0.75% w / v. In some embodiments, thiosulfate is sodium thiosulfate. In some embodiments, the pre-treated suspension has a thiosulfate concentration of about 0.025% w / v to about 0.5% w / v. In some embodiments, thiosulfate is sodium thiosulfate. In some embodiments, the pre-treated suspension has a thiosulfate concentration of about 0.05% w / v to about 0.3% w / v. In some embodiments, the thiosulfate is sodium thiosulfate. In some embodiments, the pre-treated suspension has a thiosulfate concentration of about 0.05% w / v to about 0.25% w / v.

[0118] In some embodiments, the pre-treated suspension contains potassium sorbate. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of about 0.01 M to about 0.5 M. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of about 0.02 M to about 0.4 M. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of about 0.025 M to about 0.3 M. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of about 0.03 M to about 0.2 M. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of about 0.035 M to about 0.1 M. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of about 0.04 M to about 0.08 M. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of about 0.015 M to about 0.06 M. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of about 0.02 M to about 0.04 M. In some embodiments, the pre-treated suspension has a potassium sorbate concentration of approximately 0.005, 0.006, 0.007, 0.008, 0.009, 0.010, 0.011, 0.012, 0.013, 0.014, 0.015, 0.016, 0.017, 0.018, 0.019, 0.020, 0.021, 0.022, 0.023, 0.024, 0.025, 0.026, 0.027, 0.028, 0.029, 0.030, 0.035, 0.040, 0.045, 0.050, 0.055, 0.060, 0.070, 0.080, 0.090, or 0.10 M.

[0119] In some embodiments, the pre-treated suspension contains iodixanol. In some embodiments, the pre-treated suspension has an iodixanol concentration of about 5% w / v to about 15% w / v. In some embodiments, the pre-treated suspension has an iodixanol concentration of about 6% w / v to about 13% w / v. In some embodiments, the pre-treated suspension has an iodixanol concentration of about 7% w / v to about 11% w / v. In some embodiments, the pre-treated suspension has an iodixanol concentration of about 8% w / v to about 10% w / v.

[0120] In some embodiments, the pre-treated suspension contains sodium benzoate. In some embodiments, the pre-treated suspension has a sodium benzoate concentration of about 0.01 M to about 0.6 M. In some embodiments, the pre-treated suspension has a sodium benzoate concentration of about 0.02 M to about 0.5 M. In some embodiments, the pre-treated suspension has a sodium benzoate concentration of about 0.03 M to about 0.4 M. In some embodiments, the pre-treated suspension has a sodium benzoate concentration of about 0.04 M to about 0.3 M. In some embodiments, the pre-treated suspension has a sodium benzoate concentration of about 0.05 M to about 0.2 M.

[0121] In some embodiments, the pre-treated suspension contains a combination of excipients selected from thiosulfate, potassium sorbate, iodixanol, and sodium benzoate at concentrations as specified herein.

[0122] In some embodiments, a pre-treated solution comprising thiosulfate, potassium sorbate, iodixanol, and / or sodium benzoate further comprises polyvinyl alcohol (PVA). PVA is a water-soluble synthetic polymer having the ideal formula [CH2CH(OH)]n. Any suitable PVA may be used in the pre-treated suspension of this disclosure. Types of PVA are known in the art and are commercially available from several sources (Sigma-Aldrich, TCI, Alfa Aesar, VWR). In some embodiments, the PVA has an average molecular weight of about 9 kDa to about 186 kDa. In some embodiments, the PVA is PVA3 as described herein, having a molecular weight of about 27 kDa. In some embodiments, the PVA is PVA10 as described herein, having a molecular weight of about 13 kDa to about 23 kDa. In some embodiments, the pre-treated suspension has a PVA concentration of about 0.01% w / v to about 0.75% w / v.

[0123] In some embodiments, a pre-treated suspension comprising thiosulfate, potassium sorbate, iodixanol, and / or sodium benzoate further comprises NaCl. In some embodiments, the pre-treated suspension has an NaCl concentration of about 0.005 M to about 0.5 M. In some embodiments, the pre-treated suspension has an NaCl concentration of about 0.01 M to about 0.4 M. In some embodiments, the pre-treated suspension has an NaCl concentration of about 0.015 M to about 0.3 M. In some embodiments, the pre-treated suspension has an NaCl concentration of about 0.015 M to about 0.2 M. In some embodiments, the pre-treated suspension has an NaCl concentration of about 0.015 M to about 0.1 M. In some embodiments, the pre-treated suspension has an NaCl concentration of about 0.02 M to about 0.05 M. In some embodiments, the pre-treated suspend has an NaCl concentration of approximately 1 mM to 500 mM, 2 mM to 475 mM, 3 mM to 450 mM, 4 mM to 425 mM, 5 mM to 400 mM, 6 mM to 375 mM, 7 mM to 350 mM, 8 mM to 325 mM, 9 mM to 300 mM, 10 mM to 275 mM, 15 mM to 250 mM, 20 mM to 200 mM, 25 mM to 150 mM, 30 mM to 100 mM, 35 mM to 75 mM, 40 mM to 60 mM, 45 mM to 55 mM, or 25 mM to 75 mM.

[0124] In some embodiments, a pre-treated suspension comprising thiosulfate, potassium sorbate, iodixanol, and / or sodium benzoate further comprises sucrose. In some embodiments, the pre-treated suspension has a sucrose concentration of about 5% w / v to about 15% w / v. In some embodiments, the pre-treated suspension has a sucrose concentration of about 7% w / v to about 11% w / v. In some embodiments, the pre-treated suspension has a sucrose concentration of about 8% w / v to about 10% w / v.

[0125] In some embodiments, a liquid medium or pre-treated suspension containing thiosulfate, potassium sorbate, iodixanol, and / or sodium benzoate further comprises a buffer. In some embodiments, the buffer is selected from MOPS, HEPES, Tris, MES, citrate, and phosphate-buffered saline (PBS). In some embodiments, the buffer has a concentration of about 7 mg / mL to about 15 mg / mL. In some embodiments, the liquid medium or pre-treated suspension has a pH of about 7.4. In some embodiments, the liquid medium or pre-treated suspension has a pH of about 7.0 to about 8.0.

[0126] In one embodiment, a liquid medium is provided comprising encapsulated RNA at a concentration of about 0.005 mg / mL to about 2.0 mg / mL, potassium sorbate at a concentration of about 0.005 M to about 0.5 M, poloxamer at a concentration of about 0.005 to about 0.5% w / v, sugar at a concentration of about 4% to about 22% w / v, NaCl at a concentration of about 5 mM to about 500 mM, and a buffer having a pH of about 7.4 to about 8.0 at a concentration of about 1 mM to about 300 mM. In some embodiments, the poloxamer is poloxamer 188 (also known as P188). In some embodiments, the sugar is sucrose. In some embodiments, the RNA is at a concentration of about 0.010 to about 1.5 mg / mL. In some embodiments, the RNA is at a concentration of about 0.050 to about 0.8 mg / mL. In some embodiments, the potassium sorbate is at a concentration of about 0.010 M to about 0.3 M. In some embodiments, the potassium sorbate is at a concentration of about 0.015 M to about 0.1 M. In some embodiments, the poloxamer is at a concentration of about 0.10 to about 0.40% w / v. In some embodiments, the poloxamer is at a concentration of about 0.015 to about 0.30% w / v. In some embodiments, the poloxamer is at a concentration of about 0.020 to about 0.20% w / v. In some embodiments, the poloxamer is at a concentration of about 0.030 to about 0.10% w / v. In some embodiments, the sugar is at a concentration of about 8 to about 20% w / v. In some embodiments, the sugar is at a concentration of about 12 to about 20% w / v. In some embodiments, the sugar is at a concentration of about 16 to about 20% w / v. In some embodiments, the buffer is Tris. In some embodiments, the buffer is at a concentration of about 2 mM to about 250 mM. In some embodiments, the buffer solution has a concentration of approximately 3 mM to approximately 200 mM. In some embodiments, the buffer solution has a concentration of approximately 4 mM to approximately 150 mM. In some embodiments, the buffer solution has a concentration of approximately 5 mM to approximately 100 mM. In some embodiments, the buffer solution has a concentration of approximately 8 mM to approximately 50 mM. In some embodiments, the buffer solution has a concentration of approximately 10 mM to approximately 40 mM. In some embodiments, the buffer solution has a concentration of approximately 12 mM to approximately 30 mM. In some embodiments, the buffer solution has a concentration of approximately 15 mM to approximately 25 mM.

[0127] Freeze-dried composition In another embodiment, a lyophilized composition is provided comprising lipid nanoparticles encapsulating nucleic acids and one or more excipients selected from potassium sorbate, thiosulfate, sodium benzoate, and iodixanol. In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is mRNA. In some embodiments, the RNA is siRNA. In some embodiments, the RNA is self-replicating RNA.

[0128] In some embodiments, a lyophilized composition comprising potassium sorbate, thiosulfate, sodium benzoate, and / or iodixanol has a total lipid-to-nucleic acid weight ratio of about 50:1 to about 10:1 in the lyophilized composition. In some embodiments, the lyophilized composition has a total lipid-to-nucleic acid weight ratio of about 40:1 to about 20:1 in the lyophilized composition. In some embodiments, the lyophilized composition has a total lipid-to-nucleic acid weight ratio of about 35:1 to about 25:1 in the lyophilized composition. In some embodiments, the lyophilized composition has a total lipid-to-nucleic acid weight ratio of about 45:1 to about 30:1 in the lyophilized composition.

[0129] Potassium sorbate, thiosulfate, sodium benzoate, and / or iodixanol may be present in weight ratio to nucleic acid (e.g., RNA) of the selected excipients described below herein.

[0130] In some embodiments, the freeze-dried composition contains potassium sorbate in a weight ratio of about 30:1 to about 250:1 of potassium sorbate to nucleic acid. In some embodiments, the freeze-dried composition contains potassium sorbate in a weight ratio of about 40:1 to about 200:1 of potassium sorbate to nucleic acid. In some embodiments, the freeze-dried composition contains potassium sorbate in a weight ratio of about 50:1 to about 175:1 of potassium sorbate to nucleic acid. In some embodiments, the freeze-dried composition contains potassium sorbate in a weight ratio of about 5:1 to about 150:1 of potassium sorbate to nucleic acid. In some embodiments, the freeze-dried composition contains potassium sorbate in a weight ratio of about 10:1 to about 125:1 of potassium sorbate to nucleic acid. In some embodiments, the freeze-dried composition contains potassium sorbate in a weight ratio of about 15:1 to about 100:1 of potassium sorbate to nucleic acid. In some embodiments, the lyophilized composition contains potassium sorbate in a weight ratio of about 20:1 to about 80:1 of potassium sorbate to nucleic acid. In some embodiments, the lyophilized composition contains potassium sorbate in a weight ratio of about 25:1 to about 60:1 of potassium sorbate to nucleic acid. In some embodiments, the lyophilized composition contains potassium sorbate in a weight ratio of about 30:1 to about 50:1 of potassium sorbate to nucleic acid.

[0131] In some embodiments, the lyophilized composition contains sodium thiosulfate in a weight ratio of about 1:1 to about 12:1 of sodium thiosulfate to nucleic acid. In some embodiments, the lyophilized composition contains sodium thiosulfate in a weight ratio of about 2:1 to about 10:1 of sodium thiosulfate to nucleic acid. In some embodiments, the lyophilized composition contains sodium thiosulfate in a weight ratio of about 3:1 to about 8:1 of sodium thiosulfate to nucleic acid.

[0132] In some embodiments, the freeze-dried composition contains sodium benzoate in a weight ratio of about 1:1 to about 12:1 of sodium benzoate to nucleic acid. In some embodiments, the freeze-dried composition contains sodium benzoate in a weight ratio of about 2:1 to about 10:1 of sodium benzoate to nucleic acid. In some embodiments, the freeze-dried composition contains sodium benzoate in a weight ratio of about 3:1 to about 9:1 of sodium benzoate to nucleic acid.

[0133] In some embodiments, the lyophilized composition contains iodixanol in a weight ratio of about 100:1 to about 800:1 of iodixanol to nucleic acid. In some embodiments, the lyophilized composition contains iodixanol in a weight ratio of about 150:1 to about 750:1 of iodixanol to nucleic acid. In some embodiments, the lyophilized composition contains iodixanol in a weight ratio of about 200:1 to about 700:1 of iodixanol to nucleic acid. In some embodiments, the lyophilized composition contains iodixanol in a weight ratio of about 250:1 to about 650:1 of iodixanol to nucleic acid.

[0134] In some embodiments, a lyophilized composition comprising potassium sorbate, thiosulfate, sodium benzoate, and / or iodixanol further comprises polyvinyl alcohol (PVA) in a weight ratio of PVA to nucleic acid of about 1:1 to about 12:1.

[0135] In some embodiments, a freeze-dried composition comprising potassium sorbate, thiosulfate, sodium benzoate, and / or iodixanol further comprises sucrose in a weight ratio of sucrose to nucleic acid of about 100:1 to about 800:1.

[0136] In some embodiments, a lyophilized composition comprising potassium sorbate, thiosulfate, sodium benzoate, and / or iodixanol further comprises a buffer selected from HEPES, MOPS, Tris, MERS, citrate, and phosphate, in a weight ratio of nucleic acid to buffer of about 3:1 to about 150:1. In some embodiments, the buffer is Tris.

[0137] In some embodiments, the freeze-dried composition comprising potassium sorbate, thiosulfate, sodium benzoate, and / or iodixanol further comprises NaCl. In some embodiments, the freeze-dried composition comprises about 0.5% w / w to about 5.0% w / w of NaCl. In some embodiments, the freeze-dried composition comprises about 0.6% w / w to about 4.5% w / w of NaCl. In some embodiments, the freeze-dried composition comprises about 0.7% w / w to about 4.0% w / w of NaCl. In some embodiments, the freeze-dried composition comprises about 0.8% w / w to about 3.5% w / w of NaCl. In some embodiments, the freeze-dried composition comprises about 0.9% w / w to about 3.0% w / w of NaCl. In some embodiments, the freeze-dried composition comprises about 1.0% w / w to about 2.5% w / w of NaCl. In some embodiments, the freeze-dried composition comprises about 1.1% w / w to about 2.4% w / w of NaCl. In some embodiments, the freeze-dried composition contains about 1.2% w / w to about 2.3% w / w of NaCl. In some embodiments, the freeze-dried composition contains about 1.3% w / w to about 2.1% w / w of NaCl. In some embodiments, the freeze-dried composition contains about 1.4% w / w to about 2.0% w / w of NaCl. In some embodiments, the freeze-dried composition contains about 1.4% w / w to about 1.6% w / w of NaCl. In some embodiments, the freeze-dried composition contains about 0.75% w / w to about 2.25% w / w of NaCl. In some embodiments, the freeze-dried composition contains about 1.0% w / w to about 2.0% w / w of NaCl.

[0138] In some embodiments, the freeze-dried composition contains about 85 to about 96% w / w sugar. In some embodiments, the sugar is sucrose. In some embodiments, the freeze-dried composition contains about 88 to about 95% w / w sugar. In some embodiments, the freeze-dried composition contains about 90 to about 95% w / w sugar. In some embodiments, the freeze-dried composition contains about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% w / w sugar. The term "about" in the aforementioned sugar percentages means ±0.5%.

[0139] In some embodiments, the freeze-dried composition contains about 0.01 to about 1.0% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.02 to about 0.8% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.03 to about 0.7% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.04 to about 0.6% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.05 to about 0.5% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.06 to about 0.4% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.07 to about 0.3% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.09 to about 0.2% w / w of poloxamer. In some embodiments, the poloxamer is poloxamer 188.

[0140] In one embodiment, a lyophilized composition is provided comprising lipid nanoparticles for encapsulating RNA, poloxamer, potassium sorbate, and sugar. In some embodiments, the poloxamer is poloxamer 188. In some embodiments, the lyophilized composition contains about 0.001 to about 1.0% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.005 to about 0.8% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.01 to about 0.5% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.02 to about 0.4% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.03 to about 0.3% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.04 to about 0.2% w / w of RNA. In some embodiments, the lyophilized composition contains about 0.5 to about 5.0% w / w of lipid. In some embodiments, the lyophilized composition contains about 1.0 to about 4.0% w / w of lipid. In some embodiments, the lyophilized composition contains about 1.25 to about 3.0% w / w of lipids. In some embodiments, the lyophilized composition contains about 0.5 to about 2.5% w / w of Tris buffer. In some embodiments, the lyophilized composition contains about 0.75 to about 2.25% w / w of Tris buffer. In some embodiments, the lyophilized composition contains about 1.0 to about 2.0% w / w of Tris buffer. In some embodiments, the lyophilized composition contains about 0.75 to about 2.75% w / w of NaCl. In some embodiments, the lyophilized composition contains about 1.0 to about 2.5% w / w of NaCl. In some embodiments, the lyophilized composition contains about 1.25 to about 1.80% w / w of NaCl. In some embodiments, the lyophilized composition contains about 85 to about 96% w / w of sugars. In some embodiments, the lyophilized composition contains about 88 to about 95% w / w of sugars. In some embodiments, the freeze-dried composition contains about 90 to about 95% w / w sugar. In some embodiments, the sugar is sucrose. In some embodiments, the freeze-dried composition contains about 0.01 to about 1.0% w / w poloxamer.In some embodiments, the freeze-dried composition contains about 0.02 to about 0.8% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.03 to about 0.7% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.04 to about 0.6% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.05 to about 0.5% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.06 to about 0.4% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.07 to about 0.3% w / w of poloxamer. In some embodiments, the freeze-dried composition contains about 0.09 to about 0.2% w / w of poloxamer. In some embodiments, the poloxamer is poloxamer 188. In some embodiments, the freeze-dried composition contains about 0.5 to about 5.0% w / w of potassium sorbate. In some embodiments, the freeze-dried composition contains about 0.75 to about 4.0% w / w of potassium sorbate. In some embodiments, the freeze-dried composition contains about 1.0 to about 3.0% w / w of potassium sorbate. In some embodiments, the freeze-dried composition contains about 1.25 to about 2.75% w / w of potassium sorbate.

[0141] Storage, reconstitution, and administration of lyophilized compositions Lyophilized compositions prepared by the processes described herein or the lyophilized compositions described herein may be stored stably at higher temperatures than lipid nanoparticle suspensions. Typically, lipid nanoparticle suspensions are stored at -70°C, which is not a suitable temperature for transport and storage for facilities lacking equipment capable of achieving and maintaining this temperature. Lyophilized compositions may be stored stably at temperatures above 70°C. In some embodiments, methods for storing lyophilized compositions are provided herein, comprising storing the lyophilized product of the present disclosure at temperatures between about -20°C and about 8°C. In some embodiments, the methods include storing the lyophilized product of the present disclosure at temperatures of about -20, -19, -18, -17, -16, -15, -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, or 8°C. In some embodiments, the method includes storing the lyophilized product of the present disclosure at a temperature of about -20°C to about 8°C.

[0142] In some embodiments, a method is provided for reconstituting a lyophilized composition of the present disclosure, comprising adding a liquid medium to the lyophilized composition.

[0143] In some embodiments, a method is provided for treating a disease or disorder in a subject, comprising administering a lyophilized composition of the present disclosure, reconstituted in a liquid medium, to the subject. In some embodiments, the reconstituted lyophilized composition is administered intravenously. In some embodiments, the reconstituted lyophilized composition is administered intramuscularly. In some embodiments, the reconstituted lyophilized composition is administered by inhalation. Methods for intravenous, intramuscular, and inhalation administration are known in the art and are readily adaptable to the reconstituted formulations described herein.

[0144] definition Throughout this specification, substituents of the compounds of the disclosure are disclosed in groups or ranges. It is specifically intended that this disclosure includes each individual partial combination of members of such groups and ranges. For example, the term "C 1-6 Specifically, "alkyl" is intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.

[0145] The term "anionic lipid" refers to lipids that are negatively charged at physiological pH. Examples of these lipids include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups bound to neutral lipids.

[0146] The phrase "at least one of" preceding a sequence of items, along with the terms "and" or "or" used to separate them into any of the items, modifies the list as a whole, rather than each element (i.e., each item). The phrase "at least one of" does not require the selection of at least one of each listed item. Rather, it allows for meanings that include at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. For example, the phrase "at least one of A, B, and C," or "at least one of A, B, or C," means A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C, respectively.

[0147] The terms “include,” “have,” etc., are used in the specification or claims and are intended to be comprehensive in the same manner as the term “comprise” is interpreted when used in a transitional clause of the claims.

[0148] The term "cationic lipid" refers to amphiphilic lipids and their salts having a positive hydrophilic tip group; one, two, three or more hydrophobic fatty acids or aliphatic alkyl chains; and a connector between these two domains. Ionizable or protonizable cationic lipids usually have a pK a It is protonated (i.e., becomes positively charged) at a pH below pK a At pH levels above this, it is substantially neutral. Preferred ionizable cationic lipids are those with a pKa below the physiological pH, which is typically 7.4. Cationic lipids of this disclosure may also be referred to as titrable cationic lipids. Cationic lipids can be “aminolipids” having a protonable tertiary amine (e.g., pH titrable) head group. Some exemplary aminolipids are C 18 These can contain alkyl chains, each alkyl chain independently having 0 to 3 (e.g., 0, 1, 2, or 3) double bonds; and having an ether, ester, or ketal bond between the tip group and the alkyl chain. Examples of such cationic lipids include, but are not limited to, DSDMA, DODMA, DLinDMA, DLenDMA, γ-DLenDMA, DLin-K-DMA, DLin-K-C2-DMA (also known as DLin-C2K-DMA, XTC2, and C2K), DLin-K-C3-DMA A, DLin-K-C4-DMA, DLen-C2K-DMA, y-DLen-C2K-DMA, DLin-M-C2-DMA (also known as MC2), DLin-M-C3-DMA (also known as MC3), and (DLin-MP-DMA) (also known as 1-Bl 1).

[0149] The term “contains” is intended to be open and allow for the inclusion of additional elements or processes, but does not require such inclusion. Where the term “contains” is used herein, the term “consistes of” is therefore included and disclosed.

[0150] The term "composition" means any product containing a specific amount of a specific component, and any product obtained directly or indirectly from a specific combination of specific components.

[0151] The term "commercially available chemicals" and the chemicals used in the examples described herein can be obtained from standard commercial suppliers, such as, for example, Acros Organics (Pittsburgh, Pa.), Sigma-Adrich Chemical (Milwaukee, Wis.), Avocado Research (Lancashire, UK), Bionet (Cornwall, UK), Boron Molecular (Research Triangle Park, NC), Combi-Blocks (San Diego, Calif.), Eastman Organic Chemicals, Eastman Kodak Company (Rochester, NY), Fisher Scientific Co. (Pittsburgh, Pa.), Frontier Scientific (Logan, Utah), ICN Biomedicals, Inc. (Costa Mesa, Calif.), Lancaster Synthesis (Windham, NH), Maybridge Chemical Co. (Cornwall, UK), Pierce Chemical Co. (Rockford, Ill.), and Riedel de Examples include Haen (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland, Oregon), and Wako Chemicals USA, Inc. (Richmond, Va.).

[0152] The phrase "compounds described in chemical literature" can be identified through reference books and databases relating to chemical compounds and chemical reactions, as is well known to those skilled in the art. Suitable reference books and articles that detail the synthesis of reactants useful in the preparation of the compounds disclosed herein, or that refer to articles establishing the preparation of the compounds disclosed herein, include, for example, “Synthetic Organic Chemistry”, John Wiley and Sons, Inc. New York; SRSandler et al, “Organic Functional Group Preparations”, 2nd Ed., Academic Press, New York, 1983; HOHouse, “Modern Synthetic Reactions”, 2nd Ed., WABenjamin, Inc. Menlo Park, Calif., 1972; TLGlichrist, “Heterocyclic Chemistry”, 2nd Ed. John Wiley and Sons, New York, 1992; J. March, “Advanced Organic Chemistry: reactions, mechanisms and structure”, 5th Ed., Wiley Interscience, New York, 2001 is cited as an example, and preferred and similar reactants can also be identified by the Chemical Abstract Service of the American Chemical Society, which is available in most public and university libraries, as well as by indexes of known chemicals prepared by online databases (for further details, contact the American Chemical Society, Washington, DC). Chemicals that are known but not commercially available in catalogs can be prepared by custom chemical synthesis companies, and many standard chemical suppliers (such as those mentioned above) offer custom synthesis services.

[0153] As used herein, the term “effective dose” of a drug means an amount sufficient to produce a beneficial or desired outcome, such as a clinical outcome, and therefore, “effective dose” depends on the context in which the term is applied. For example, in the context of administering a drug to treat cancer, an effective dose of the drug is an amount sufficient to achieve the treatment of cancer as defined herein, compared to, for example, a response that would be obtained without the administration of the drug.

[0154] The term "fully encapsulated" means that the nucleic acids (e.g., mRNA) within the nucleic acid-lipid particles are not significantly degraded after exposure to serum or nuclease assays that significantly degrade free RNA. When fully encapsulated, in a process that typically degrades 100% of free nucleic acids, preferably less than 25% of the nucleic acids within the particles are degraded, more preferably less than 10%, and most preferably less than 5%. "Fully encapsulated" also means that the nucleic acid-lipid particles do not rapidly degrade into their constituent parts upon in vivo administration.

[0155] The term "nucleic acid" refers to deoxyribonucleotides or ribonucleotides, and polymers thereof in single-stranded or double-stranded forms. This term encompasses nucleic acids, whether synthetic, natural, or unnatural, that contain known nucleotide analogs or modified skeletal residues or linkages, and that possess similar binding properties to the reference nucleic acid and are metabolized in a similar manner to the reference nucleic acid. Examples of such analogs, but not limited to, include phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0156] The term "compound" includes all stereoisomers, geometric isomers, tautomers, and isotopes of the structure described.

[0157] The term "delivery" means the act or manner of delivering a compound, substance, element, part, cargo, or payload.

[0158] The term “delivery agent” or “delivery vehicle” means, at least in part, any substance that facilitates the in vivo delivery of polynucleotides to targeted cells.

[0159] "Expression" of a nucleic acid sequence means one or more of the following: (1) creation of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and / or 3' end processing); (3) translation of RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein.

[0160] "Features" refer to performance, properties, or inherent elements.

[0161] The term "hydrophobic lipid" non-limitingly refers to compounds having nonpolar groups, including but not limited to saturated and unsaturated aliphatic hydrocarbon groups, which may be substituted with one or more aromatic, alicyclic, or heterocyclic groups. Preferred examples include, but are not limited to, diacylglycerol, dialkylglycerol, NN-dialkylamino, 1,2-diacyloxy-3-aminopropane, and 1,2-dialkyl-3-aminopropane.

[0162] The term "lipid" refers to organic compounds containing fatty acid esters, characterized by being insoluble in water but soluble in many organic solvents. Lipids are generally classified into at least three classes: (1) "simple lipids," which include fats, oils, and waxes; (2) "complex lipids," which include phospholipids and glycolipids; and (3) "derived lipids," such as steroids.

[0163] The term "lipid delivery vehicle" refers to a lipid formulation that can be used to deliver therapeutic nucleic acids (e.g., mRNA) to a target site (e.g., cells, tissues, organs, etc.). Lipid delivery vehicles can be nucleic acid-lipid particles, which can be formed from cationic lipids, non-cationic lipids (e.g., phospholipids), complex lipids to prevent particle aggregation (e.g., PEG lipids), and optionally cholesterol. Typically, therapeutic nucleic acids (e.g., mRNA) can be protected from enzymatic degradation by encapsulating them in the lipid portion of the particles.

[0164] The term "lipid-encapsulated" refers to nucleic acids, such as mRNA, that are fully encapsulated, partially encapsulated, or both, within a lipid formulation. In preferred embodiments, the nucleic acid (e.g., mRNA) is fully encapsulated within the lipid particles.

[0165] The term "lipid complex" refers to a complex lipid that inhibits the aggregation of lipid particles. Examples of such lipid complexes include, but are not limited to, PEG-lipid complexes such as PEG bonded to dialkyloxypropyl (e.g., PEG-DAA complex), PEG bonded to diacylglycerol (e.g., PEG-DAG complex), PEG bonded to cholesterol, PEG bonded to phosphatidylethanolamine, and PEG-lipid complexes such as PEG bonded to ceramide, cationic PEG lipids, polyoxazoline (POZ)-lipid complexes, and PEG bonded to polyamide oligomers, as well as mixtures thereof. PEG or POZ may be directly bonded to lipids or linked to lipids via a linker moiety. Any linker moiety suitable for linking PEG or POZ to lipids can be used, for example, containing ester-free and ester-containing linker moieties. In certain preferred embodiments, ester-free linker moieties such as amides or carbamates are used.

[0166] The term "amphipathic lipid" refers to a lipid substance in which the hydrophobic portion faces the hydrophobic phase, while the hydrophilic portion faces the aqueous phase. The hydrophilic characteristic stems from the presence of polar or charged groups such as carbohydrates, phosphoric acid, carboxyl, sulfat, amino, sulfhydryl, nitro, hydroxyl, and other equivalent groups. Hydrophobicity can be conferred by the inclusion of nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups, and one or more aromatic, alicyclic, or heterocyclic groups. Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0167] The term "messenger RNA" (mRNA) refers to any polynucleotide that codes for a target protein or polypeptide and, when translated, can produce the encoded target protein or polypeptide in vitro, in vivo, in situ, or ex vivo.

[0168] The term “nucleotide” means nucleotides that have a natural base (standard) or a modified base, which are well known in the art. Such a base is usually located at the 1' position of the nucleotide sugar moiety. Nucleotides typically consist of a base, a sugar, and a phosphate group. Nucleotides can be unmodified or modified with a sugar, phosphate, and / or base moiety (similarly referred to as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides, and others; see, for example, Usman and McSwiggen (above), all incorporated herein by reference; Eckstein, et al. International PCT Publication WO92 / 07065; Usman, et al. International PCT Publication WO 93 / 15187; Uhlman & Peyman (above)). There are several examples of modified nucleic acid bases known in the art, as summarized in Limbach, et al., Nucleic Acids Res. 22:2183, 1994. Some non-limiting examples of base modifications that can be incorporated into nucleic acid molecules include inosine, purine, pyridine-4-one, pyridine-2-one, phenyl, pseudouracil, 2,4,6-trimethoxybenzene, 3-methyluracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidine (e.g., 5-methylcytidine), 5-alkyluridine (e.g., ribothymidine, 5-halolyzine (e.g., 5-bromouridine), or 6-azapyrimidine or 6-alkylpyrimidine (e.g., 6-methyluridine)), propyne, and others (Burgin, et al., Biochemistry 35:14090, 1996; Uhlman & Peyman, cited above). In this embodiment, “modified base” means a nucleotide base other than adenine, guanine, cytosine, and uracil at the 1' position, or their equivalents.

[0169] The term "patient" means a person who may be seeking treatment, may need treatment, needs treatment, is receiving treatment, will receive treatment, or is receiving care from a professional trained in a particular disease or condition.

[0170] The term "pharmaceutically acceptable" is used herein to mean a compound, substance, composition, and / or dosage form that is suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, within reasonable limits of medical judgment, and that is commensurate with a reasonable benefit / risk ratio.

[0171] As used herein, the term “pharmaceutically acceptable excipients” means any component other than the compounds described herein (e.g., vehicles capable of suspending or dissolving active compounds) that is substantially non-toxic and non-inflammatory to the patient. Examples of excipients include anti-adhesives, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavorings, fragrances, lubricants (flow enhancers), preservatives, printing inks, adsorbents, suspending agents or dispersants, sweeteners, and hydration water. Examples of excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0172] The term "pharmaceutically acceptable salt" refers to a derivative of the disclosed compound in which the parent compound has been modified by converting an existing acidic or base site to its salt form (for example, by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic salts of basic residues (e.g., amines) and alkali or organic salts of acidic residues (e.g., carboxylic acids). Representative acid addition salts include acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxyethanesulfone. Examples include salts, lactobionates, lactates, laurates, lauryl sulfates, malates, maleates, malons, methanesulfons, 2-naphthalenesulfons, nicotinates, nitrates, oleates, oxalates, palmitates, pamoates, pectins, persulfates, 3-phenylpropionates, phosphates, picrates, pivalates, propions, stearates, succinates, sulfates, tartrates, thiocyans, toluenesulfons, undecanoates, and valersates. Typical alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations (including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine). Examples of pharmaceutically acceptable salts of this disclosure include conventional non-toxic salts of parent compounds formed from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of this disclosure can be synthesized from a parent compound containing a basic or acidic moiety by conventional chemical methods.Typically, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of a suitable base or acid in water, an organic solvent, or a mixture thereof; non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, and acetonitrile are usually preferred. A list of suitable salts can be found in Remington's Pharmaceutical Sciences, 17. th These findings are found in *Pharmaceutical Salts: Properties, Selection, and Use*, PHStahl and CGWermuth (eds.), Wiley-VCH, 2008, and in *Journal of Pharmaceutical Science*, 66, 1-19 (1977), the entirety of each of these works is incorporated herein by reference.

[0173] The terms “purify,” “refined,” and “clean” mean to make substantially pure or clean by removing unwanted components, contaminants, mixtures, or impurities from a substance.

[0174] The term "significant" or "remarkably" is used interchangeably with the term "substantially."

[0175] The term "stable" means a compound that is robust enough to survive isolation from a reaction mixture to a useful purity, and preferably, can be formulated into an effective therapeutic agent.

[0176] The terms "to stabilize," "stable," and "stable region" all mean to make something stable or to become stable.

[0177] The term "substantially" means a qualitative state that fully or nearly fully describes the characteristics or properties of an object. Those skilled in the art in the field of biology will understand that biological and chemical phenomena rarely, if any, reach completion and / or move toward completion, or achieve or avoid absolute results. Therefore, as used herein, the term "substantially" expresses a potential lack of completeness, which is characteristic of many biological and chemical phenomena.

[0178] The term “to treat” means to partially or completely alleviate, reduce, improve, cure, delay the onset, inhibit the progression, reduce the severity, and / or reduce the occurrence of one or more symptoms or characteristics of a particular infection, disease, disorder, and / or condition. For example, “to treat” cancer may mean inhibiting the survival, growth, and / or expansion of the tumor. Treatment may be administered to subjects who show no signs of the disease, disorder, and / or condition, and / or subjects who show only early signs of the disease, disorder, and / or condition, in order to reduce the risk of developing a condition associated with the disease, disorder, and / or condition.

[0179] The term "in vitro" refers to events that occur in an artificial environment, such as in a test tube or reaction vessel, during cell culture, or in a petri dish, rather than within a living organism (e.g., an animal, plant, or microorganism).

[0180] The term "in vivo" refers to events that occur within living organisms (for example, animals, plants, or microorganisms, or their cells or tissues).

[0181] The term "neutral lipid" refers to lipid species that exist in either an uncharged or neutral zwitterionic form at a selected pH. Examples of such lipids at physiological pH include diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebroside, and diacylglycerol.

[0182] The term "noncationic lipid" refers to amphiphilic lipids, neutral lipids, or anionic lipids as described herein.

[0183] The term “oligomer” can be used interchangeably with “polynucleotide” and refers to a molecule containing at least two monomers, including oligonucleotides such as DNA and RNA. In the case of oligomers containing RNA monomers and / or unlocked nucleic acid (UNA) monomers, the oligomers of this disclosure may contain sequences in addition to the coding sequence (CDS). These additional sequences may be untranslated sequences, i.e., sequences that have not been converted into proteins by the host cell. These untranslated sequences may include a 5' cap, a 5' untranslated region (5'UTR), a 3' untranslated region (3'UTR), and a tail region, for example, a poly-A tail region. As will be described in more detail herein, any of these untranslated sequences may contain one or more UNA monomers—these UNA monomers cannot be translated by the host cell's mechanisms. In the context of this disclosure, “mRNA sequence,” “mRNA sequence,” “translatable polynucleotide,” or “translatable compound” means a sequence containing an RNA coding region (e.g., the coding sequence of human CFTR or a codon-optimized version thereof) that can be converted to a region, for example, a protein or a fragment thereof, for example, a human CFTR protein or a fragment thereof.

[0184] The term “subject” means any living organism to which a composition according to this disclosure can be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and / or plants. [Examples]

[0185] Additional embodiments of this disclosure are described in further detail in the following embodiments, which are not intended to limit the scope of the claims in any way.

[0186] Example 1: General substances and methods The experiments performed in the examples described herein were carried out using lipid nanoparticle compositions produced according to a well-known process, for example, the one described in U.S. Patent Application No. 16 / 823,212, and this content is incorporated by reference for the specific purpose of teaching the lipid nanoparticle production process. The lipid nanoparticle compositions and lyophilized products were characterized for several properties. The materials and methods for these characterization processes, as well as a general method for producing the lipid nanoparticle compositions used in the lyophilization experiments, are provided in these examples.

[0187] Manufacturing of lipid nanoparticles The lipid nanoparticle formulations used in this example were prepared by mixing lipids (cationic lipids: helper lipids: cholesterol: PEG-lipids) with RNA dissolved in citrate buffer in ethanol. The mixture was instantaneously diluted with phosphate buffer. The ethanol was removed by dialysis to phosphate buffer using a regenerated cellulose membrane (100 kD MWCO) or by tangential flow filtration (TFF) using a modified polyethersulfone (mPES) hollow fiber membrane (100 kD MWCO). Once the ethanol was completely removed, the buffer was replaced with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer containing 10-300 (e.g., 40-60) mM NaCl and 5-15% sucrose, pH 7.3. The formulation was concentrated and then filtered through a 0.2 μm PES filter. Next, the RNA concentration in the formulation was measured using a Ribogreen fluorescence assay, and the concentration was adjusted to the desired final concentration by diluting with HEPES buffer (pH 7.2-8.5) containing 10-100 (e.g., 40-60) mM NaCl and 0-15% sucrose, along with glycerol. If not immediately used for further research, the final formulation was filtered through a 0.2 μm filter, filled into glass vials, stoppered, capped, and allowed to stand at -70±5°C. The lipid nanoparticle formulations were characterized for pH and osmotic pressure. Lipid and RNA content were measured by high-performance liquid chromatography (HPLC), and mRNA integrity was measured using a fragment analyzer.

[0188] Dynamic light scattering (DLS) The average particle size (z) and polydispersity index (PDI) of the lipid nanoparticle formulations used in the examples were measured by dynamic light scattering using a Malvern Zetasizer Nano ZS (UK).

[0189] RiboGreen Assay The encapsulation efficiency of lipid nanoparticle formulations was characterized using the RiboGreen fluorescence assay. RiboGreen is a proprietary fluorescent dye (Molecular Probes / Invitrogen, a division of Life Technologies, now part of Thermo Fisher Scientific, Eugene, Oregon, USA) used for the detection and quantification of nucleic acids, including both RNA and DNA. In its free form, RiboGreen exhibits almost no fluorescence and has very slight absorption properties. When bound to nucleic acids, the dye fluoresces with an intensity several orders of magnitude greater than in its unbound state. The fluorescence can then be detected by a sensor (fluorescent photometer), and the nucleic acid can be quantified.

[0190] Western blot The in vivo efficacy of lipid nanoparticle formulations was tested by measuring applicable protein expression or knockdown activity using a Western blot assay. In the assay, cells transfected with the applicable lipid nanoparticle formulation at an appropriate density were seeded in Dulbecco's Modified Eagle Medium (DMEM) / Fetal Bovine Serum (FBS) medium using a 96-well collagen plate. At optimal confluence, the cells were transfected with the lipid nanoparticle formulation and diluted in a transfection reagent mix (MessengerMax and Opti-MEM). The cells were then placed in a CO2 incubator and grown. At the desired time, the medium was removed and the cells were fixed in 4% fresh paraformaldehyde (PFA) for 20 minutes. Subsequently, the fixative was removed and the cells were permeabilized several times in Tris-buffered saline (TBST) with TWEEN for 5 minutes each time. After permeabilization washing was complete, the cells were incubated in blocking buffer (ODYSSEY® Blocking Buffer (PBS) (Li-Cor, Lincoln, NE)) for 45 minutes. Next, the primary antibody was added and incubated at room temperature for 1 hour. Then, the cells were washed several times in TBST, diluted in blocking buffer, and incubated with the secondary antibody containing the CellTag700 strain for 1 hour. Finally, the cells were washed several times in TBST and then given a final wash in Tris-buffered saline (TBS). The plates were imaged using a Licor (Lincoln, Nebraska USA) detection system, and the data were normalized to the total number of cells labeled with CellTag700.

[0191] Example 2: Evaluation of various excipients (freeze-drying protective agents) in pre-treated suspensions Experiments were conducted to evaluate the effects of various excipients on the quality of lyophilized products of lipid nanoparticle formulations prepared as described in Example 1. The quality of the lyophilized lipid nanoparticle formulations was evaluated by analyzing the formulations after lyophilization and comparing them to the lipid nanoparticle formulations before lyophilization and after a conventional freeze / thaw cycle (i.e., freezing at approximately -70°C and then thawing at room temperature).

[0192] Analysis of lipid nanoparticle formulations included analysis of particle size, polydispersity (PDI), and encapsulation efficiency (%Encap). The particle size after lyophilization was compared to the particle size before lyophilization, and the difference was reported as delta (δ). Various compositions tested were screened for meeting characteristic thresholds, including minimal particle size increase (δ<10nm), maintenance of PDI (<0.2), and maintenance of high encapsulation efficiency (>85%).

[0193] Lyophilized lipid nanoparticle formulations were prepared by first pre-treating a suspension of lipid nanoparticle formulations prepared according to Example 1 after filtration by adding the excipients specified below to achieve the listed concentrations. A lyophilization cycle was applied with a slow freezing gradient with primary drying at -20°C followed by secondary drying at 25°C. Lyophilization cycles were performed using a Millrock Revo Freeze Dryer (model number RV85S4) with 2.0 mL aliquots of the suspension having a lipid nanoparticle concentration of 0.25 mg RNA / mL. After lyophilization, the lyophilized product was reconstituted in 2.0 mL of water and analyzed as described above.

[0194] The excipients investigated in the research disclosed herein are listed in Table 1 below. [Table 2]

[0195] Two of the above excipients, namely human albumin (HA) and polyvinyl alcohol 1 (PVA1), were used for initial evaluation using the parameters and results listed in Table 2 below. Comparative formulations without excipients or containing glycerol were also studied. [Table 3]

[0196] As shown in Table 2, the tested lyophilization cycles and excipients did not produce lipid nanoparticle formulations with suitable properties. For HA, concentrations of 1.0% w / v and 0.05% w / v PVA1 provided the best results.

[0197] Example 3: Evaluation of the effect of lipid nanoparticle concentration Additional experiments were conducted to study the effects of lipid nanoparticle concentration (measured as RNA concentration in suspension), buffer concentration, salt concentration, antifreeze concentration, and lyophilization protective agent (poloxamer) concentration on the quality and properties of the lyophilized product.

[0198] These parameters were studied in nine different lyophilization experiments outlined in Table 3 below. Lyophilized lipid nanoparticle formulations were prepared by first pre-treating a suspension of lipid nanoparticle formulations containing approximately 21 nucleotides of siRNA, prepared according to Example 1 after filtration, to include the excipients and conditions listed in Table 3. After pre-treatment, the pre-treated formulations were lyophilized under conditions similar to those described in Example 2. Each of Experiments 1-9 listed in Table 3 was performed at lipid nanoparticle concentrations of 0.25 mg / mL, 0.5 mg / mL, 1.0 mg / mL, and 2.0 mg / mL. The resulting lyophilized compositions were then reconstituted and characterized for particle size, encapsulation rate, and PDI. [Table 4]

[0199] The results of the study at 1 mg lipid nanoparticles / mL are shown in Figure 1. Regarding particle size parameters, A3, B2, C1 / C3, and D2 showed the smallest particle size, and A3 showed the best particle size overall. Regarding encapsulation efficiency, parameters A3, B2, C1, and D1 showed the best results for their respective excipient groups, and D1 showed the best encapsulation efficiency overall. Finally, regarding polydispersity (PDI), parameters A3, B2, C3, and D1 showed the best PDI for their respective excipient groups, and C3 showed the best PDI overall. Therefore, these experiments determined that a composition containing A3 (15% glycerol), B2 (10 mM buffer), C1 (0 mM salt), and D1 / D2 (0%~0.1% poloxamer) would provide the best results for a given excipient. This same analysis was also performed for the concentrations of the tested lipid nanoparticles, and the optimal conditions found for each concentration are shown in Table 4. [Table 5]

[0200] Further studies were conducted to determine the effect of adding poloxamer (a freeze-drying protective agent) at concentrations of 0.1% and 0.2% P188 before and after freeze-drying for conditions 1-4. Figure 2A shows the results for the pre-freeze-drying experiment, and Figure 2B shows the results for the post-freeze-drying experiment. It can be seen that adding poloxamer after freeze-drying (as part of the reconstruction) was found to be the best way to maintain the inclusion rate (shown as circles in the chart).

[0201] Finally, Figure 3 shows the results for characterizing lipid nanoparticle formulations treated with poloxamer after lyophilization under different concentrations of lipid nanoparticles. It can be seen that a particle size of approximately 85 nm was observed at a concentration of approximately 0.25 mg / mL, while larger particle sizes were observed at higher concentrations. The encapsulation rate is shown as a bar curve in this figure, indicating good encapsulation for each formulation.

[0202] Example 4: Evaluation of two additional PVA excipients After obtaining results from Examples 2 and 3, further studies were conducted to compare the effects of other PVA excipients on the freeze-dried product. The two PVA excipients were PVA2 and PVA3, listed in Table 1 above. The experimental conditions and results are provided in Table 5 below. In these experiments, the freeze-drying cycle of Example 2 was repeated, except that the primary drying temperature was changed to -25°C.

Table 6

[0203] Generally, none of the formulations showed acceptable values for both δ and encapsulation efficiency, but groups 13 and 15 showed excellent values for δ. Further studies were conducted with different lyophilization cycles, and also the effect of adding sucrose was evaluated.

[0204] Example 5: Further study on PVA excipients using different lyophilization cycles The experiment of Example 4 was extended to study the effect of the lyophilization cycle and further the effect of adding sucrose. In this study, the lyophilization cycles of Examples 2 and 4 were changed to load the suspension in vials and freeze at a primary drying temperature of -48°C, -35°C and a secondary drying process at 10°C, and the volume of the lyophilized suspension was reduced to 1.5 mL. The conditions and results of this study are provided in Table 6 below. <e

Table 7

[0205] In this further study, lower drying temperatures resulted in higher quality in the lipid nanoparticle formulations, which was most readily observed when compared to the formulation with 1.0% HA (Group 4). It was also found that high sucrose improved the particle size but decreased the encapsulation efficiency.

[0206] Example 6: Evaluation of Various PVA Excipients A further study was designed and conducted to compare the effects of different PVA excipients at various concentrations. The PVA excipients used in this study were PVA1, PVA2, PVA3, PVA4, PVA5, PVA6, and PVA7. The conditions and results of the lyophilization experiments are provided in Table 7 below. The lyophilization cycle of Example 5 was used in this experiment. [Table 8]

[0207] The lowest δ value in combination with acceptable values for encapsulation efficiency and PDI was observed for the PVA3 formulation. The results of Group 14 also suggest that PVA3 in combination with human albumin (HA) has a better effect than when using only PVA3 (Groups 9 - 12).

[0208] Example 7: Additional Round of Evaluation of PVA Excipients Using the findings of Example 6, further studies were conducted to compare them with other PVA excipients, particularly PVA8, PVA9, and PVA10. Lipid nanoparticle formulations were prepared as described in the previous examples, and in these experiments, the lyophilization cycle of Example 5 was applied except that a primary drying temperature of -25°C was applied. The specific conditions and results are shown in Table 8. [Table 9-1] [Table 9-2]

[0209] The results suggest that PVA10 showed excellent results in groups 48, 49, and 51, exhibiting good inclusion and delta values. PVA3 also showed excellent values ​​in groups 12-13 and 16-19.

[0210] Example 8: Direct comparative study of PVA3 and PVA10 Based on the findings of Example 7, further experiments were designed to directly compare formulations using PVA3 or PVA10. In these experiments, lipid nanoparticle formulations were prepared as described in Example 1, and the lyophilization cycle of Example 5 was applied. The conditions and results for these experiments are provided in Table 9. [Table 10-1] [Table 10-2] [Table 10-3]

[0211] The delta values ​​from these studies were large for some groups, with those showing delta values ​​less than 5 having low inclusion efficiencies. Conversely, other groups showed good inclusion efficiencies of over 90%, but also exhibited higher delta values. PVA3 and PVA10 were equivalent under the tested conditions, and the optimal conditions from this experiment appear to be those of groups 33 and 63.

[0212] Example 9: Research using potassium sorbate Next, an experiment was designed to evaluate the effect of potassium sorbate (KS) on lyophilized lipid nanoparticles that may contain PVA. Lipid nanoparticle formulations were prepared as described in Example 1, and the lyophilization cycle of Example 5 was applied. Specific conditions and results are shown in Table 10. [Table 11]

[0213] The group receiving a combination of human albumin and PVA did not show acceptable results. Groups 12 and 13 showed good delta values ​​and good inclusion efficiency. The conditions of these groups were selected for further study.

[0214] Example 10: Further research on potassium sorbate and sodium benzoate In this study, the experimental conditions of Example 9 were further investigated for RNA concentration at 1.0 mg / mL. Studies including PVA11 and polysorbate 20 (PS20) were also conducted. Lipid nanoparticle formulations were prepared as described in Example 1 and lyophilized as in Example 5. The conditions and results are provided in Table 11 below (Note: F / T indicates freeze-thaw formulations frozen at -70°C, then thawed and characterized). [Table 12-1] [Table 12-2] [Table 12-3]

[0215] The results show that the selected formulation from Example 9 (0.05% PVA + 0.05M NaCl + 0.1M KS) is reproducible and provides good results in terms of particle size preservation (delta), PDI, and encapsulation rate, even at higher RNA concentrations (see groups 41, 42, and 43). Under some conditions, sodium benzoate (NaB) also showed good results (groups 44 and 55). Polysorbate 20 was also shown to be a good antifreeze agent, maintaining the integrity of lipid particles even at 0.05% w / v (group 36), although not as a lyophilization protectant. Other salts and excipients tested did not show comparable efficacy to potassium sorbate.

[0216] Example 11: Research on PVA-free formulations In this experiment, prior knowledge was applied to determine whether lipid nanoparticles could be lyophilized without the use of PVA, but instead by using a hydrophobic salt in combination with human albumin. The formulations were prepared as described in Example 1, and the lyophilization cycle of Example 5 was applied. Specific conditions and results are provided in Table 12 below. [Table 13-1] [Table 13-2] [Table 13-3]

[0217] The results indicate that replacing PVA with a hydrophobic salt combined with human albumin (HA) does not produce a suitable lyophilized lipid nanoparticle formulation.

[0218] Example 12: Research on alternative excipients This study was designed to test whether potassium sorbate could be replaced by other excipients. The study also evaluated how these excipients function at reduced RNA concentrations, performed at a concentration of 0.25 mg RNA / mL. The excipients tested included iodixanol and L-proline (Pro). Lipid nanoparticle formulations were prepared as described in Example 1 and subjected to the lyophilization cycle of Example 5. Specific conditions and corresponding results for the pre-treated suspensions compared to the freeze-thaw formulations are provided in Table 13. [Table 14-1] [Table 14-2] [Table 14-3]

[0219] The results of this study indicate that the concentration of excipients used in pre-treated formulations can be reduced as long as the same ratio of excipient to RNA is used (see groups 1-3). Sodium benzoate (NaB) showed results at the same level as potassium sorbate (KS) at 0.25 mg RNA / mL. Experiments with L-proline showed that it was not an effective alternative to potassium sorbate. In contrast, iodixanol was shown to be a potent lyophilization protectant, further improving formulations that contained PVA and proline (groups 22-26), but could also be used in formulations that did not contain PVA (groups 47 and 48).

[0220] Example 12: In vivo study of the reconstituted formulation Selected formulations from previous studies were tested for in vivo efficacy. Lipid nanoparticles containing 0.25 mg / mL mRNA encoding human EPO protein (hEPO) were used for general testing of the ability to successfully transfect samples and measure the efficiency of mRNA translation in vivo. The formulations were lyophilized using the lyophilization cycle of Example 5 in calculated amounts to achieve a final volume of 3.0 mL. The experiment also included measurements of a control freeze-thaw formulation in 5% glycerol and a negative PBS control. The formulation conditions are provided in Table 14, which also shows the characterization of these formulations prior to in vivo studies. [Table 15]

[0221] The results of the study are shown in Figure 4. All reconstituted formulations showed good hEPO expression, and compositions 3 and 4 showed expression close to that of the freeze-thaw control.

[0222] Example 13: Testing of different formulations with large mRNA levels The lyophilized compositions developed in the prior examples were tested to determine whether they could be applied to mRNA lipid nanoparticle formulations containing large mRNA sizes. Two mRNAs were tested; mRNA1 had a size of approximately 1332 nucleotides, and mRNA2 had a size of approximately 4868 nucleotides. The formulations were prepared as described in Example 1, and the lyophilization cycle of Example 5 was applied. The conditions and results for the lyophilization study are provided in Table 15. [Table 16-1] [Table 16-2]

[0223] The results indicate that the lyophilized formulations are effective for large mRNA constructs. In particular, formulations from groups 25, 26, and 27 showed excellent results when reconstituted.

[0224] Example 14: Additional Formulation This experiment tested the effects of P188 and other combinations on lyophilized lipid nanoparticle formulations using mRNA2. Lipid nanoparticle formulations were prepared as described in Example 1, and the lyophilization cycle of Example 5 was applied. Results for specific formulation conditions and for the lyophilized and reconstituted formulations are provided in Table 16. [Table 17]

[0225] The results showed that all tested groups exhibited encapsulation efficiencies exceeding 99%, indicating that formulations other than those containing iodixanol can be used. Furthermore, groups 1 and 7 showed similar δ values ​​to group 17, which used iodixanol.

[0226] Example 15: Freeze-drying of self-replicating RNA Self-replicating RNA (also known as replicon RNA) is typically larger than average mRNA, and the study was designed to determine whether self-replicating RNA lipid nanoparticle formulations can be efficiently lyophilized.

[0227] Formulations were prepared as described in Example 1 using self-replicating RNA concentrations in the range of 0.10 to 2.0 mg / mL. The following lyophilization cycle was used: Initial freezing (shelf temperature): -52℃, 30 minutes Freeze for an additional 5 minutes, vacuum set point 300 mTorr Primary drying: 1.-48℃, maintained for 30 minutes, vacuum set point 50mTorr 2.15-60 minutes at -40°C, vacuum setpoint 50 mTorr 3. Maintain at -40°C, vacuum setpoint 50 mTorr, until a pressure difference of 4 mTorr is reached. (The pressure difference indicates the change in relative humidity in the freeze-drying chamber). Secondary drying at 5°C, vacuum setpoint 100 mTorr, for 1200 minutes.

[0228] Each of the following formulation conditions shown in Table 17 was tested: [Table 18]

[0229] The results under the above conditions showed that reconstitution yielded lyophilized lipid nanoparticle formulations with appropriate size, polydispersity, and delta value.

[0230] Example 15: Lyophilization of self-replicating RNA lipid nanoparticle formulation The process carried out in this embodiment is performed using a lipid nanoparticle composition produced according to a well-known process, for example, the one described in U.S. Patent Application No. 16 / 823,212, and this content is incorporated by reference for the specific purpose of teaching the lipid nanoparticle production process. The lipid nanoparticle composition and the lyophilized product were characterized for several properties. The materials and methods for these characterization processes, as well as a general method for producing the lipid nanoparticle composition used in the lyophilization experiment, are provided in this embodiment.

[0231] Lipid nanoparticle manufacturing The lipid nanoparticle formulations used in this embodiment were prepared by mixing lipids (ionizable cationic lipids (ATX-126): helper lipids: cholesterol: PEG-lipids) with RNA dissolved in citrate buffer in ethanol. The mixture was instantaneously diluted with phosphate buffer. The ethanol was removed by dialysis to phosphate buffer using a regenerated cellulose membrane (100 kD MWCO) or by tangential flow filtration (TFF) using a modified polyethersulfone (mPES) hollow fiber membrane (100 kD MWCO). Once the ethanol was completely removed, the buffer was replaced with HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer containing 10-300 (e.g., 40-60) mM NaCl and 5-15% sucrose, pH 7.3. The formulation was concentrated and then filtered through a 0.2 μm filter using a PES filter. Next, the RNA concentration in the formulation was measured using a RiboGreen fluorescence assay, and the concentration was adjusted to the desired final concentration by diluting with HEPES buffer (pH 7.2-8.5) containing 10-100 (e.g., 40-60) mM NaCl and 0-15% sucrose, along with glycerol. If not immediately used for further research, the final formulation was filtered through a 0.2 μm filter, filled into glass vials, stoppered, capped, and allowed to stand at -70±5°C. The lipid nanoparticle formulations were characterized for pH and osmotic pressure. Lipid and RNA content were measured by high-performance liquid chromatography (HPLC), and mRNA integrity was measured using a fragment analyzer.

[0232] freeze-drying process Self-replicating RNA (also known as replicon RNA) is typically larger than average mRNA, and this study was designed to determine whether self-replicating RNA lipid nanoparticle formulations can be efficiently lyophilized. The quality of the lyophilized lipid nanoparticle formulations was assayed by analyzing the lyophilized formulations and comparing them to the lipid nanoparticle formulations before lyophilization and after conventional freeze / thaw cycles (i.e., frozen at approximately -70°C and then thawed at room temperature).

[0233] Analysis of lipid nanoparticle formulations included analysis of particle size, polydispersity (PDI), and encapsulation efficiency (%Encap). The particle size after lyophilization was compared to the particle size before lyophilization, and the difference could be reported as delta (δ). Various compositions tested were screened for meeting characteristic thresholds, including minimal particle size increase (δ<10nm), maintenance of PDI (<0.2), and maintenance of high encapsulation efficiency (>85%).

[0234] Lipid nanoparticle formulations were prepared as described above using self-replicating RNA with a length exceeding 11,000 nucleotides. Next, the obtained lipid nanoparticle formulations were treated with buffer exchange and then treated with a pre-lyophilization suspension containing self-replicating RNA at concentrations of 0.05–2.0 mg / mL, 0.01–0.05 M potassium sorbate, 0.01–0.10% w / v poloxamer 188 (Kolliphor®), 14–18% w / v sucrose, 25–75 mM NaCl, and 15–25 mM pH 8.0 Tris buffer. Next, the pre-lyophilization formulations were lyophilized using a Millrock Revo Freeze Dryer (model number RV85S4) with aliquots of 2.0 mL of the suspension and the lyophilization cycles provided in Table 18 below. [Table 19]

[0235] Lyophilized particles prepared according to the method described above were reconstituted with 2 mL of water and characterized using DLS and RiboGreen. The results provided in Table 19 below show that the lyophilized composition was found to produce a lyophilized lipid nanoparticle formulation with appropriate size, polydispersity, and delta value (approximately 5.3 nm) upon reconstitution. [Table 20]

[0236] Further consideration The above-mentioned explanations enable those skilled in the art to implement the various configurations described herein. While the subject art is described with reference to various figures and configurations, it should be understood that these are for illustrative purposes only and should not be construed as limiting the scope of the subject art.

[0237] Many other ways of implementing the subject art may exist. Various functions and elements described herein can be separated from those shown without departing from the scope of the subject art. Various modifications to these configurations will be readily apparent to those skilled in the art, and the general definitions defined herein are applicable to other configurations. Therefore, many changes and modifications can be made to the subject art by those skilled in the art without departing from the scope of the subject art.

[0238] It is understood that any particular order or sequence of steps in the disclosed process is illustrative and describes an exemplary approach. It is understood that any particular order or sequence of steps in the process may be rearranged based on design priorities. Some steps may be performed simultaneously. The claims for the attached method present elements of various steps in a sample order and are not intended to limit the present order or sequence.

[0239] When used herein, the phrase “at least one of” preceding a sequence of items, along with the terms “and” or “or” used to separate them into any of the items, modifies the list as a whole, rather than each element (i.e., each item). The phrase “at least one of” does not require the selection of at least one of each listed item. Rather, it allows for meanings including at least one of any one of the items, and / or at least one of any combination of the items, and / or at least one of each of the items. For example, the phrase “at least one of A, B, and C” or “at least one of A, B, or C” means A only, B only, or C only; any combination of A, B, and C; and / or at least one of each of A, B, and C, respectively.

[0240] Furthermore, to the extent that terms such as “include” and “have” are used in the specification or claims, such terms are intended to be comprehensive in the same manner as the term “comprise” is interpreted when used in a transitional clause of the claims.

[0241] In one or more aspects, the terms “about,” “substantially,” and “approximately” may provide industrially acceptable tolerances and / or relatives between items for their corresponding terms, e.g., less than 1 percent to 5 percent.

[0242] References to elements in the singular form are intended to mean "one or more" unless otherwise specified, and not "one and unique." The masculine pronoun (e.g., "his") includes the feminine and neuter genders (e.g., "her" and "it"), and vice versa. The term "several" refers to one or more. Underlined and / or italicized headings and subheadings are for convenience only and are not intended to limit the subject art, nor to be referenced in connection with the interpretation of the description of the subject art. Any structural and functional equivalents of elements of the various configurations described through this disclosure, whether known or hereafter known to those skilled in the art, are expressly incorporated herein by reference and are intended to be encompassed by the subject art. Furthermore, nothing disclosed herein is intended to be publicly displayed, whether or not such disclosure is expressly enumerated above.

[0243] Although the detailed descriptions include many specific examples, these should not be interpreted as limiting the scope of the subject art, but merely as illustrating different embodiments and aspects of the subject art. It should be understood that the scope of the subject art includes other embodiments not discussed in detail above. Various other modifications, changes, and variations can be made in the arrangement, operation, and details of the methods and apparatus of the subject art disclosed herein without departing from the scope of this disclosure. Unless otherwise expressed, references to elements in the singular are not intended to mean "one and only one," but rather "one or more." In addition, an apparatus or method does not need to solve all problems that can be solved (or have all the advantages achievable) by different embodiments of this disclosure in order to be included within the scope of this disclosure. In this specification, "can" and its derivatives should be understood as "occasionally" or "optionally," as opposed to a definitive possibility. The present invention includes the following embodiments. [Item 1] A method for freeze-drying a composition containing lipid nanoparticles for encapsulating RNA, wherein the method is: a. A step of providing a suspension of lipid nanoparticles in a liquid medium, wherein the liquid medium contains a saccharide in an amount of about 4% w / v to about 22% w / v; and b. The step of preparing the liquid medium, thereby forming a pre-treated suspension containing at least one excipient selected from potassium sorbate, thiosulfate, sodium benzoate, and iodixanol. The method, including the method described above. [Section 2] Step (c): c. The pre-treated suspension i. Initial freezing process carried out at a temperature of -48±8℃ and atmospheric pressure; ii. A primary drying process carried out at a temperature in the range of -20±2℃ to -48±2℃ and a pressure in the range of approximately 25 mTorr to approximately 100 mTorr; and iii. A secondary drying process carried out at a temperature in the range of 5±2℃ to 30±2℃ and a pressure in the range of approximately 30mTorr to approximately 300mTorr. Subject to a freeze-thaw process that includes The method described in item 1, further comprising: [Section 3] Step (c): c. The pre-treated suspension i. Initial freezing process carried out at a temperature of -48±8℃ and atmospheric pressure; ii. A primary drying process carried out at a pressure of approximately 0.03 to approximately 0.08 mbar, starting at a temperature of -48 ± 8°C and gradually decreasing to a temperature of 0 ± 2°C over a period of approximately 40 to approximately 75 hours; and iii. A secondary drying process performed at a pressure of approximately 0.03 to 0.08 mbar, starting at a temperature of 0 ± 2°C and gradually increasing to a temperature of approximately 25 ± 3°C over a period of approximately 30 to 50 hours. Subject to a freeze-thaw process that includes The method described in item 1, further comprising: [Clause 4] The method according to any one of Clauses 1 to 3, wherein the liquid medium is an aqueous medium. [Item 5] The method according to any of the preceding items, wherein the RNA in the suspension has a concentration in the range of about 0.05 mg / mL to about 2.0 mg / mL. [Clause 6] The method according to Clause 5, wherein the RNA in the suspension has a concentration in the range of about 0.075 mg / mL to about 0.3 mg / mL. [Clause 7] The method according to Clause 5, wherein the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 1.5 mg / mL. [Clause 8] The method according to Clause 5, wherein the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 1.0 mg / mL. [Clause 9] The method according to Clause 5, wherein the RNA in the suspension has a concentration in the range of about 0.1 mg / mL to about 0.5 mg / mL. [Item 10] The method according to any one of the preceding items, wherein the weight ratio of total lipids to RNA in the suspension is approximately 50:1 to approximately 10:1. [Clause 11] The method according to Clause 10, wherein the weight ratio of total lipids to RNA in the suspension is about 40:1 to about 20:1. [Clause 12] The method according to Clause 10, wherein the weight ratio of total lipids to RNA in the suspension is about 35:1 to about 25:1. [Clause 13] The pre-treated suspension comprising thiosulfate, according to the method of any one of the preceding clauses. [Clause 14] The method according to Claim 13, wherein the thiosulfate is sodium thiosulfate or potassium thiosulfate. [Item 15] The method according to item 13, wherein the thiosulfate has a concentration of about 0.025% w / v to about 1.0% w / v. [Item 16] The method according to item 13, wherein the thiosulfate has a concentration of about 0.025% w / v to about 0.75% w / v. [Item 17] The method according to item 13, wherein the thiosulfate has a concentration of about 0.025% w / v to about 0.5% w / v. [Item 18] The method according to item 13, wherein the thiosulfate has a concentration of about 0.05% w / v to about 0.3% w / v. [Item 19] The method according to item 13, wherein the thiosulfate has a concentration of about 0.05% w / v to about 0.25% w / v. [Clause 20] The method according to any one of the preceding clauses, wherein the pretreated suspension comprises potassium sorbate. [Clause 21] The method according to Clause 20, wherein the potassium sorbate has a concentration of about 0.01 M to about 0.5 M. [Clause 22] The method according to Clause 20, wherein the potassium sorbate has a concentration of about 0.02 M to about 0.4 M. [Item 23] The method according to item 20, wherein the potassium sorbate has a concentration of about 0.025 M to about 0.3 M. [Clause 24] The method according to Clause 20, wherein the potassium sorbate has a concentration of about 0.03 M to about 0.2 M. [Clause 25] The method according to Clause 20, wherein the potassium sorbate has a concentration of about 0.035 M to about 0.1 M. [Clause 26] The method according to Clause 20, wherein the potassium sorbate has a concentration of about 0.04 M to about 0.08 M. [Clause 27] The method according to Clause 20, wherein the potassium sorbate has a concentration of about 0.01 M to about 0.05 M. [Clause 28] The method according to Clause 20, wherein the potassium sorbate has a concentration of about 0.02 M to about 0.04 M. [Clause 29] The method according to any one of the preceding clauses, comprising the pre-treated suspension, comprising iodixanol. [Clause 30] The method according to Clause 29, wherein the iodixanol has a concentration of about 5% w / v to about 15% w / v. [Clause 31] The method according to Clause 29, wherein the iodixanol has a concentration of about 6% w / v to about 13% w / v. [Clause 32] The method according to Clause 29, wherein the iodixanol has a concentration of about 7% w / v to about 11% w / v. [Clause 33] The method according to Clause 29, wherein the iodixanol has a concentration of about 8% w / v to about 10% w / v. [Clause 34] The pre-treated suspension comprises sodium benzoate, as described in any one of the preceding clauses. [Clause 35] The method according to Clause 34, wherein the sodium benzoate has a concentration of about 0.01 M to about 0.6 M. [Clause 36] The method according to Clause 34, wherein the sodium benzoate has a concentration of about 0.02 M to about 0.5 M. [Clause 37] The method according to Clause 34, wherein the sodium benzoate has a concentration of about 0.03 M to about 0.4 M. [Clause 38] The method according to Clause 34, wherein the sodium benzoate has a concentration of about 0.04 M to about 0.3 M. [Clause 39] The method according to Clause 34, wherein the sodium benzoate has a concentration of about 0.05 M to about 0.2 M. [Clause 40] The method according to any one of the preceding clauses, wherein the pretreated suspension further comprises polyvinyl alcohol (PVA). [Clause 41] The method according to Clause 40, wherein the PVA has a concentration of about 0.01% w / v to about 0.75% w / v. [Clause 42] The method according to any one of the preceding clauses, wherein the pretreated suspension further comprises NaCl. [Clause 43] The method according to Clause 42, wherein the NaCl has a concentration of about 0.005 M to about 0.5 M. [Clause 44] The method according to Clause 42, wherein the NaCl has a concentration of about 0.01 M to about 0.4 M. [Clause 45] The method according to Clause 42, wherein the NaCl has a concentration of about 0.015 M to about 0.3 M. [Clause 46] The method according to Clause 42, wherein the NaCl has a concentration of about 0.015 M to about 0.2 M. [Clause 47] The method according to Clause 42, wherein the NaCl has a concentration of about 0.015 M to about 0.1 M. [Clause 48] The method according to Clause 42, wherein the NaCl has a concentration of about 0.02 M to about 0.05 M. [Clause 49] The method according to Clause 42, wherein the NaCl has a concentration of about 0.03 M to about 0.07 M. [Clause 50] The method according to any one of the preceding clauses, wherein the saccharide is sucrose. [Clause 51] The method according to Clause 50, wherein the sucrose has a concentration of about 8% w / v to about 20% w / v. [Clause 52] The method according to Clause 50, wherein the sucrose has a concentration of about 7% w / v to about 11% w / v. [Clause 53] The method according to Clause 50, wherein the sucrose has a concentration of about 8% w / v to about 10% w / v. [Clause 54] The method according to Clause 50, wherein the sucrose has a concentration of about 16% w / v to about 20% w / v. [Clause 55] The method according to any one of the preceding clauses, wherein the liquid medium or pre-treated suspension comprises a buffer. [Clause 56] The method according to Clause 55, wherein the buffer is selected from MOPS, HEPES, Tris, MES, citrate, and phosphate-buffered saline (PBS). [Clause 57] The method according to Clause 56, wherein the buffer is Tris. [Clause 58] The buffer solution is in a concentration of approximately 10 mM to approximately 100 mM, as described in any one of Clauses 55 to 57. [Clause 59] The method according to Clause 58, wherein the buffer solution has a concentration of about 15 mM to about 75 mM. [Clause 60] The method according to Clause 58, wherein the buffer solution has a concentration of about 10 mM to about 40 mM. [Clause 61] The method according to any one of the preceding clauses, wherein the liquid medium or the pre-treated suspension has a pH of about 7.0 to about 8.5. [Clause 62] The method according to any one of the preceding clauses, further comprising, after step (b), dividing the pre-treated suspension into individual containers in predetermined lyophilized volumes. [Clause 63] The method according to Clause 62, wherein the predetermined lyophilized volume is in the range of about 0.5 mL to about 10.0 mL. [Clause 64] The method according to Clause 62, wherein the predetermined lyophilized volume is in the range of about 1.0 mL to about 3.0 mL. [Clause 65] The method according to any one of the preceding clauses, further comprising the pretreated suspension poloxamer. [Clause 66] The method according to Claim 65, wherein the poloxamer is poloxamer 188. [Clause 67] The method according to Clause 65 or Clause 66, wherein the poloxamer is present in a concentration of about 0.01% w / v to about 0.10% w / v. [Clause 68] The method according to Clause 65 or Clause 66, wherein the poloxamer is present in a concentration of about 0.02% w / v to about 0.8% w / v. [Clause 69] The method according to Clause 65 or Clause 66, wherein the poloxamer is present in a concentration of about 0.03% w / v to about 0.7% w / v. [Clause 70] The method according to Clause 65 or Clause 66, wherein the poloxamer is present in a concentration of about 0.04% w / v to about 0.06% w / v. [Clause 71] A product prepared by the process described in any one of Clauses 1 to 70. [Item 72] A lyophilized composition comprising lipid nanoparticles for encapsulating nucleic acids, monosaccharides, and one or more excipients selected from potassium sorbate, thiosulfate, sodium benzoate, and iodixanol. [Clause 73] The freeze-dried composition according to Clause 72, wherein the nucleic acid is RNA. [Clause 74] The freeze-dried composition according to Clause 73, wherein the RNA is self-replicating RNA. [Clause 75] The freeze-dried composition according to Clause 73, wherein the RNA is mRNA. [Clause 76] The nucleic acid is a lyophilized composition according to any one of Clauses 73 to 75, wherein the nucleic acid has a length of about 20 nucleotides to about 13,000 nucleotides. [Clause 77] The freeze-dried composition according to any one of Clauses 72 to 76, wherein the weight ratio of total lipids to nucleic acids in the freeze-dried composition is about 50:1 to about 10:1. [Clause 78] The freeze-dried composition according to Clause 77, wherein the weight ratio of total lipids to RNA in the freeze-dried composition is about 40:1 to about 20:1. [Clause 79] The freeze-dried composition according to Clause 77, wherein the weight ratio of total lipids to RNA in the freeze-dried composition is about 35:1 to about 25:1. [Clause 80] The freeze-dried composition according to any one of Clauses 72 to 79, wherein the freeze-dried composition contains potassium sorbate in a weight ratio of potassium sorbate to RNA of about 30:1 to about 250:1. [Clause 81] The freeze-dried composition according to any one of Clauses 72 to 79, wherein the freeze-dried composition contains potassium sorbate in a weight ratio of about 40:1 to about 200:1 of potassium sorbate to RNA. [Clause 82] The freeze-dried composition according to any one of Clauses 72 to 79, wherein the freeze-dried composition contains potassium sorbate in a weight ratio of potassium sorbate to RNA of about 50:1 to about 175:1. [Clause 83] The freeze-dried composition according to any one of Clauses 72 to 82, wherein the freeze-dried composition comprises sodium thiosulfate in a weight ratio of sodium thiosulfate to RNA of about 0.25:1 to about 40:1. [Clause 84] The freeze-dried composition according to any one of Clauses 72 to 82, wherein the freeze-dried composition comprises sodium thiosulfate in a weight ratio of sodium thiosulfate to RNA of about 2:1 to about 10:1. [Clause 85] The freeze-dried composition according to any one of Clauses 72 to 82, wherein the freeze-dried composition comprises sodium thiosulfate in a weight ratio of sodium thiosulfate to RNA of about 3:1 to about 8:1. [Clause 86] The freeze-dried composition according to any one of Clauses 72 to 85, wherein the freeze-dried composition comprises sodium benzoate in a weight ratio of about 1:1 to about 12:1 of sodium benzoate to RNA. [Clause 87] The freeze-dried composition according to any one of Clauses 72 to 85, wherein the freeze-dried composition comprises sodium benzoate in a weight ratio of about 2:1 to about 10:1 of sodium benzoate to RNA. [Clause 88] The freeze-dried composition according to any one of Clauses 72 to 85, wherein the freeze-dried composition comprises sodium benzoate in a weight ratio of about 3:1 to about 9:1 of sodium benzoate to RNA. [Clause 89] The freeze-dried composition according to any one of Clauses 72 to 88, wherein the freeze-dried composition contains iodixanol in a weight ratio of iodixanol to RNA of about 100:1 to about 800:1. [Clause 90] The freeze-dried composition according to any one of Clauses 72 to 88, wherein the freeze-dried composition comprises iodixanol in a weight ratio of iodixanol to RNA of about 150:1 to about 750:1. [Clause 91] The freeze-dried composition according to any one of Clauses 72 to 88, wherein the freeze-dried composition comprises iodixanol in a weight ratio of iodixanol to RNA of about 200:1 to about 700:1. [Clause 92] The freeze-dried composition according to any one of Clauses 72 to 88, wherein the freeze-dried composition comprises iodixanol in a weight ratio of iodixanol to RNA of about 250:1 to about 650:1. [Clause 93] The lyophilized composition according to any one of Clauses 72 to 92, further comprising polyvinyl alcohol (PVA) in a weight ratio of PVA to RNA of about 1:1 to about 12:1. [Clause 94] The lyophilized composition according to any one of Clauses 72 to 93, wherein the saccharide is sucrose in a weight ratio of sucrose to RNA of about 100:1 to about 800:1. [Clause 95] The lyophilized composition according to any one of Clauses 72 to 94, further comprising a buffer selected from HEPES, MOPS, Tris, MERS, citrate, and phosphate in a weight ratio of buffer to RNA of about 3:1 to about 150:1. [Item 96] A lyophilized composition comprising lipid nanoparticles for encapsulating RNA, poloxamer, potassium sorbate, and sugar. [Clause 97] The composition according to Claim 96, wherein the poloxamer is poloxamer 188. [Clause 98] The freeze-dried composition according to clause 96 or 97, comprising about 0.001 to about 1.0% w / w of the RNA. [Clause 99] The composition according to Clause 98, wherein the freeze-dried composition contains about 0.005 to about 0.8% w / w of the RNA. [Clause 100] The composition according to Clause 98, wherein the freeze-dried composition contains about 0.01 to about 0.5% w / w of the RNA. [Clause 101] The composition according to Clause 98, wherein the freeze-dried composition contains about 0.02 to about 0.4% w / w of the RNA. [Clause 102] The composition according to Clause 98, wherein the freeze-dried composition contains about 0.03 to about 0.3% w / w of the RNA. [Clause 103] The composition according to Clause 98, wherein the freeze-dried composition contains about 0.04 to about 0.2% w / w of the RNA. [Item 104] The freeze-dried composition is the composition according to any one of items 96 to 103, wherein the freeze-dried composition contains about 0.5 to about 5.0% w / w of lipids. [Item 105] The composition according to item 104, wherein the freeze-dried composition contains about 1.0 to about 4.0% w / w of lipids. [Item 106] The composition according to item 104, wherein the freeze-dried composition contains about 1.25 to about 3.0% w / w of lipids. [Clause 107] The lyophilized composition comprises about 0.5 to about 2.5% w / w of Tris buffer, as described in any one of Clauses 96 to 106. [Clause 108] The composition according to Clause 107, wherein the lyophilized composition contains about 0.75 to about 2.25% w / w of Tris buffer. [Clause 109] The composition according to Clause 107, wherein the lyophilized composition contains about 1.0 to about 2.0% w / w of Tris buffer. [Clause 110] The freeze-dried composition is the composition according to any one of Clauses 96 to 109, wherein the freeze-dried composition contains about 0.75 to about 2.75% w / w of NaCl. [Item 111] The composition according to item 110, wherein the freeze-dried composition contains about 1.0 to about 2.5% w / w of NaCl. [Item 112] The composition according to item 110, wherein the freeze-dried composition contains about 1.25 to about 1.80% w / w of NaCl. [Item 113] The freeze-dried composition is the composition according to any one of items 96 to 112, wherein the freeze-dried composition contains about 85 to about 96% w / w of the sugar. [Clause 114] The composition according to Clause 113, wherein the freeze-dried composition contains about 88 to about 95% w / w of the sugar. [Item 115] The composition according to item 113, wherein the freeze-dried composition contains about 90 to about 95% w / w of the sugar. [Clause 116] The composition according to any one of claims 96 to 115, wherein the sugar is sucrose. [Clause 117] The freeze-dried composition comprises about 0.01 to about 1.0% w / w of the poloxamer, as described in any one of Clauses 96 to 116. [Clause 118] The composition according to Clause 117, wherein the freeze-dried composition contains about 0.02 to about 0.8% w / w of the poloxamer. [Clause 119] The composition according to Clause 117, wherein the freeze-dried composition contains about 0.03 to about 0.7% w / w of the poloxamer. [Clause 120] The composition according to Clause 117, wherein the freeze-dried composition contains about 0.04 to about 0.6% w / w of the poloxamer. [Clause 121] The composition according to Clause 117, wherein the freeze-dried composition contains about 0.05 to about 0.5% w / w of the poloxamer. [Clause 122] The composition according to Clause 117, wherein the freeze-dried composition contains about 0.06 to about 0.4% w / w of the poloxamer. [Clause 123] The composition according to Clause 117, wherein the freeze-dried composition contains about 0.07 to about 0.3% w / w of the poloxamer. [Clause 124] The composition according to Clause 117, wherein the freeze-dried composition contains about 0.09 to about 0.2% w / w of the poloxamer. [Clause 125] The composition according to any one of Clauses 96 to 124, wherein the poloxamer is poloxamer 188. [Clause 126] The freeze-dried composition comprises about 0.5 to about 5.0% w / w of potassium sorbate, as described in any one of Clauses 96 to 125. [Clause 127] The composition according to Clause 126, wherein the freeze-dried composition contains about 0.75 to about 4.0% w / w of potassium sorbate. [Clause 128] The composition according to Clause 126, wherein the freeze-dried composition contains about 1.0 to about 3.0% w / w of potassium sorbate. [Clause 129] The composition according to Clause 126, wherein the freeze-dried composition contains about 1.25 to about 2.75% w / w of potassium sorbate. [Item 130] A method for storing a freeze-dried composition according to any one of items 72 to 129, comprising storing the freeze-dried product at a temperature of about 2°C to about 8°C. [Item 131] A method for storing a freeze-dried composition according to any one of items 72 to 129, comprising storing the freeze-dried product at a temperature of about -20°C. [Item 132] A method for reconstituting a freeze-dried composition described in any one of items 72 to 129, comprising adding a liquid medium to the freeze-dried composition. [Clause 133] The method according to Clause 132, wherein the liquid medium is an aqueous medium. [Clause 134] The method according to Clause 132 or Clause 133, wherein the liquid medium comprises a poloxamer. [Clause 135] The method described in Clause 134, wherein the poloxamer is P-188. [Clause 136] The method according to any one of Clauses 132 to 135, wherein the liquid medium further comprises a buffer having a pH of about 7.0 to about 8.5. [Clause 137] A method for treating a disease or disorder in a subject, comprising administering to the subject a lyophilized composition according to any one of Clauses 72 to 129, which has been reconstituted in a liquid medium. [Clause 138] The method according to Clause 137, wherein the reconstituted lyophilized composition is administered intravenously. [Clause 139] The method according to Clause 137, wherein the reconstituted lyophilized composition is administered intramuscularly. [Clause 140] The method according to Clause 137, wherein the reconstituted lyophilized composition is administered by inhalation. [Clause 141] The method according to Clause 137, wherein the reconstituted lyophilized composition is administered to a mucous membrane. [Clause 142] The reconstituted lyophilized composition is administered subcutaneously, according to the method of Clause 137.

Claims

1. A pharmaceutical composition comprising a solid lyophilized composition, wherein the solid lyophilized composition is i. Lipid nanoparticles encapsulating 0.001% w / w to 1.0% w / w of ribonucleic acid (RNA) relative to the weight of the solid freeze-dried composition, ii. 0.01% w / w to 1.0% w / w of poloxamer based on the weight of the solid freeze-dried composition, and 0.5% w / w to 5.0% w / w of potassium sorbate based on the weight of the solid freeze-dried composition, iii. 85% w / w to 96% w / w of sugar relative to the weight of the solid freeze-dried composition. A pharmaceutical composition containing the above.

2. A pharmaceutical composition comprising a solid lyophilized composition, wherein the solid lyophilized composition is i. Lipid nanoparticles encapsulating 0.001 to 1.0% w / w of RNA relative to the weight of the solid, freeze-dried composition, ii. 0.01% w / w to 1.0% w / w of poloxamer and 0.5% w / w to 5.0% w / w of potassium sorbate relative to the weight of the solid freeze-dried composition. iii. A salt in an amount of 0.75% w / w to 2.75% w / w relative to the weight of the solid freeze-dried composition, and iv. 85% w / w to 96% w / w of sugar relative to the weight of the solid freeze-dried composition. Includes, Here, when the solid freeze-dried composition is reconstituted in a liquid medium, the lipid nanoparticles have a change in average diameter of less than 15 nm compared to the average diameter before freeze-drying. Pharmaceutical composition.

3. The pharmaceutical composition according to claim 1 or 2, wherein the poloxamer is poloxamer 188.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the solid freeze-dried composition contains 0.5 to 5.0% w / w of lipids.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the solid lyophilized composition comprises 0.5 to 2.5% w / w Tris buffer.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the solid freeze-dried composition contains 0.75 to 2.75% w / w of NaCl.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the sugar is sucrose.

8. A method for storing a solid freeze-dried composition according to any one of claims 1 to 7, comprising storing the solid freeze-dried product at a temperature of 2°C to 8°C.

9. A method for storing a solid freeze-dried composition according to any one of claims 1 to 7, comprising storing the solid freeze-dried product at a temperature of -20°C.

10. A method for reconstituting a solid freeze-dried composition according to any one of claims 1 to 7, comprising adding a liquid medium to the solid freeze-dried composition.

11. The method according to claim 10, wherein the liquid medium is an aqueous medium.

12. The method according to claim 10 or 11, wherein the liquid medium further comprises a buffer having a pH of 7.0 to 8.

5.

13. A pharmaceutical composition for treating a disease or disorder in a subject, comprising a solid lyophilized composition according to any one of claims 1 to 7, wherein the solid lyophilized composition, reconstituted in a liquid medium, is administered to the subject.

14. The pharmaceutical composition according to claim 13, wherein the reconstituted solid lyophilized composition is administered intravenously, mucous membrane, or subcutaneously.

15. The pharmaceutical composition according to claim 13, wherein the reconstituted solid freeze-dried composition is administered intramuscularly.

16. The pharmaceutical composition according to claim 13, wherein the reconstituted solid lyophilized composition is administered by inhalation.

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