Lyophilized RNA composition

Abstract

The present invention relates to the field of RNA formulations, particularly to the lyophilization of RNA. More specifically, the present invention relates to a lyophilized composition and a method for obtaining a lyophilized composition. Furthermore, the present invention provides for the use of the reconstituted composition according to the present invention in human and / or veterinary medicine.

Description

Technical Field

[0001] The present invention relates to the field of RNA formulations, particularly to the lyophilization of RNA. More specifically, the present invention relates to lyophilized compositions and methods for obtaining lyophilized compositions. Furthermore, the present invention provides for the use of the reconstituted compositions according to the invention in human and / or veterinary medicine.

Background Art

[0002] mRNA-based vaccines are a new and promising platform that can be prepared relatively quickly. mRNA vaccines have the advantages of not delivering the viral nucleic acid to the host nucleus (avoiding integration into the host DNA) and having a relatively short half-life in vivo for the injected mRNA. Despite these advantages of efficacy and safety, the instability of mRNA vaccines and the need for storage at ultra-low temperatures remain major limitations. Due to the requirement for storage at ultra-low temperatures and the short shelf life, the distribution of vaccines is mainly delayed in regions with limited resources.

[0003] During production, transportation, and the required storage conditions on the end-user side are regarded as important characteristics of mRNA vaccine pharmaceuticals. The stability of mRNA formulations can be affected to some extent by multiple factors such as excipients, pH, temperature, optimization of the mRNA sequence, selection of an appropriate vector, encapsulation of mRNA into lipid nanoparticles (LNPs), and lyophilization. Freeze-drying or lyophilization is a technique that improves the stability of liquid vaccine formulations by first freezing the formulation and removing the aqueous solvent through a sublimation and desorption process (Patent Document 1).

[0004] In a series of CureVac patent documents, it is claimed in the claims that by using cryoprotectants such as sugars, the activity of mRNA formulations (naked mRNA with or without protamine) is maintained during lyophilization and storage (Patent Document 2, Patent Document 3, Patent Document 4). It is claimed that all quality characteristics analyzed during the experimental period (up to 36 months) meet the stability specifications of a stable and safe RNA pharmaceutical (i.e., appearance, RNA integrity, RNA content, pH value, and osmotic pressure). In 2020, Zhao et al. published the results of a study comparing the performance of lyophilized (luciferase-encoding) LNP-mRNA using various cryoprotectants with the performance of fresh LNP. These authors demonstrated that the reconstituted material maintains mRNA expression efficiency in mice as observed in in vivo bioluminescence imaging studies (Non-Patent Document 1).

[0005] In a recent study by BioNTech / Pfizer (Non-Patent Document 2), it was demonstrated that nucleoside-modified mRNA LNPs can be lyophilized and the physicochemical properties of the lyophilized material do not change significantly for at least 12 weeks after storage at room temperature and at least 24 weeks after storage at 4°C. Importantly, the authors observed that the immunogenicity of the lyophilized influenza mRNA-LNP vaccine does not decrease after storage at 4°C for 24 weeks in mice.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0008] Although several lyophilization protocols for mRNA-LNP formulations have been disclosed, there is still a need for an improvement in the lyophilizable composition that can realize a stable product capable of long-term storage without significantly or at all affecting the biological activity or physicochemical properties of the mRNA vaccine, such as the appearance of the cake, the presence of visible particles after reconstitution, mRNA content, encapsulation efficiency, particle size, polydispersity index (PDI), pH, and osmotic pressure.

[0009] The present invention is intended to solve the above problems, and the object of the present invention is to obtain a composition that is particularly suitable for providing a stable product by lyophilization, maintains its physiochemical properties in the lyophilized state, and ultimately maintains the properties of the mRNA-LNP vaccine. This object is solved by the subject matter of the claims. In particular, the underlying object of the present invention is a composition comprising one or more RNA molecules, one or more lipid nanoparticles, one or more cryoprotectants, and tris(hydroxymethyl)methylamine (TRIS), wherein the composition has been lyophilized and the concentration of TRIS in the composition before lyophilization is 3 mM or less. In particular, this composition can be used in human and / or veterinary medicine.

Means for Solving the Problems

[0010] The present invention further provides a method for lyophilization, comprising mixing an RNA molecule, one or more lipid nanoparticles, a cryoprotectant, and TRIS to form the composition of the present invention, and then lyophilizing the composition by a freezing step, a primary drying step, and a secondary drying step, wherein the primary drying step is carried out at a temperature below the glass transition temperature (Tg’) of the maximally freeze-concentrated solution.

[0011] The inventors have surprisingly discovered that the freeze-drying composition of the present invention is particularly suitable for the lyophilization process. After the lyophilization process is completed, the lyophilized composition is characterized by (i) high mRNA recovery rate after lyophilization and reconstitution, (ii) excellent RNA integrity, and (iii) minimal increase in LNP particle size. Furthermore, with a specific composition, the PDI value of the liquid formulation is improved and an appropriate buffering capacity is ensured. The integrity of the process and the functionality of the formulation are maintained even after lyophilization. In particular, the storage stability is improved, especially with respect to long-term storage and storage under non-cooled conditions. The storage period in the frozen state is up to 36 months at -20°C, up to 24 months at 4°C in the refrigerated state (2 - 8°C), and up to 24 hours at 25°C at room temperature. The method of the present invention is a reproducible and cost-effective method and can be used to produce a composition containing RNA having the above characteristics. The lyophilized composition containing RNA according to the present invention can be advantageously stored, transported, and applied in, for example, the pharmaceutical field (e.g., as a vaccine) without a cold chain while maintaining very high integrity and biological activity of the RNA in the composition.

[0012] In a first aspect, the present invention relates to a composition comprising one or more RNA molecules, one or more lipid nanoparticles, one or more cryoprotectants, and tris(hydroxymethyl)methylamine (TRIS), wherein the composition is lyophilized and the concentration of TRIS in the composition before lyophilization is 3 mM or less.

[0013] In certain embodiments of the present invention, the cryoprotectant is selected from the list comprising trehalose, maltose, sucrose, or combinations thereof, and is particularly sucrose.

[0014] In another aspect, the present invention relates to a composition comprising one or more RNA molecules, one or more lipid nanoparticles, sucrose, and tris(hydroxymethyl)methylamine (TRIS), wherein the composition is lyophilized and the concentration of TRIS in the composition before lyophilization is 3 mM or less.

[0015] In another specific embodiment, the concentration of the cryoprotectant in the composition before lyophilization is at least about 10% (w / v), particularly at least about 15% (w / v), more particularly at least about 20% (w / v), preferably at least about 15% (w / v).

[0016] In certain embodiments, the concentration of sucrose in the composition before lyophilization is at least about 5% (w / v), particularly at least about 8% (w / v), more particularly at least about 10% (w / v).

[0017] In certain embodiments, the sucrose concentration before lyophilization is from about 5% to about 20% (w / v), particularly from about 5% to about 15% (w / v), more specifically from about 8% to about 15% (w / v), and even more specifically from about 10% to about 15% (w / v).

[0018] In a further embodiment of the present invention, the concentration of TRIS in the composition before lyophilization is from about 0.01 mM to about 3 mM, preferably from about 0.1 mM to about 2 mM, more preferably from about 0.5 mM to 1.5 mM, and most preferably about 1 mM.

[0019] In a further embodiment of the present invention, the concentration of TRIS in the composition before lyophilization is from about 0.01 mM to about 3 mM, preferably from about 0.1 mM to about 3 mM, more preferably from about 0.5 mM to 3 mM, and most preferably from about 1 mM to about 3 mM. Alternatively, it is from about 2 mM to about 3 mM.

[0020] In certain embodiments of the present invention, the lipid nanoparticles comprise a PEG lipid, particularly a PEG2000 lipid, more specifically DMG-PEG2000.

[0021] In certain embodiments, the lipid nanoparticles in the composition before lyophilization comprise about 40 mol% to 60 mol% ionizable lipid, about 5 mol% to 15 mol% phospholipid, about 20 mol% to 40 mol% sterol, and at least 0.5 mol% PEG lipid.

[0022] In yet another embodiment, the composition before lyophilization further comprises water or water for injection (WFI).

[0023] In the following embodiments of the present invention, the one or more RNA molecules are linear or circular RNA molecules.

[0024] In yet another embodiment, the one or more RNA molecules are mRNA molecules.

[0025] In a further aspect, the present invention provides a composition for use in human and / or veterinary medicine.

[0026] In one preferred embodiment, the composition according to the present invention is reconstituted before administration to a subject in need thereof.

[0027] In certain embodiments, reconstitution is carried out using water, particularly water for injection (WFI) or an aqueous solution of a salt, preferably TBS, more preferably TBS containing 20 mM TRIS, most preferably TBS containing 20 mM TRIS and 0.9% NaCl.

[0028] In another specific embodiment, the pH of the reconstituted composition is from about 6 to about 8.

[0029] In yet another further aspect, the present invention provides a method for lyophilizing a composition comprising an RNA molecule and a lipid nanoparticle, the method comprising mixing an RNA molecule, one or more lipid nanoparticles, a cryoprotectant, and TRIS at a concentration of 3 mM or less in water or water for injection to form the composition of the present invention, and lyophilizing the composition by a freezing step, a primary drying step, and a secondary drying step, wherein the primary drying step is carried out at a temperature below the glass transition temperature (Tg’) of the maximally freeze-concentrated solution.

[0030] In a further embodiment of the method of the present invention, the primary drying step is carried out at a temperature of about -30 °C to about -50 °C, preferably at a temperature of about -35 °C to about -45 °C, and particularly at about -40 °C.

Brief Description of the Drawings

[0031] Here, particularly when referring to the drawings, it is emphasized that the details shown are illustrative and for the purpose of exemplarily explaining various embodiments of the present invention only. These are presented to provide what is considered to be the most useful and easily explained for the principles and conceptual aspects of the present invention. In this regard, no attempt has been made to show the details of the structure of the present invention beyond what is necessary for a basic understanding of the present invention. By describing in conjunction with the drawings, it will become apparent to those skilled in the art how some forms of the present invention can actually be implemented.

[0032]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0033] The present invention will be further described below. In the following text, various aspects of the present invention are defined in more detail. Each aspect thus defined can be combined with other aspects unless explicitly indicated to the contrary. In particular, features indicated as preferred or advantageous can be combined with other features indicated as preferred or advantageous.

[0034] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. By way of example, "compound" means one compound or a plurality of compounds.

[0035] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. These terms also include "consisting of" and "consisting essentially of."

[0036] As used herein, the term "about" or "approximately" when used to refer to a measurable value such as a parameter, amount, duration, etc. means that the variation is inclusive of the specified value within + / - 10% or less, preferably + / - 5% or less, more preferably + / - 1% or less, and still more preferably + / - 0.1% or less, as long as such variation is appropriate for the practice of the disclosed invention. It is understood that the value itself to which the modifier "about" or "approximately" refers is also specifically and preferably disclosed.

[0037] In a first aspect, the present invention relates to a composition comprising one or more RNA molecules, one or more lipid nanoparticles, one or more cryoprotectants, and tris(hydroxymethyl)methylamine (TRIS), wherein the concentration of TRIS in the composition before lyophilization is 3 mM or less.

[0038] As used herein, the term "composition" may refer to any mixture of two or more products or compounds for the purpose of long-term stabilization, such as preventing denaturation or aggregation over the expected shelf life. With respect to the present invention, the term "lyophilized composition" in the present invention refers to the final product in the form of a lyophilized powder or solid after the lyophilization process. On the other hand, the term "reconstituted composition" is used for a lyophilized composition in aqueous form after reconstitution, for example, with water, and is used in a state ready to be administered to a subject in need thereof. Finally, the present invention also relates to a "freezedrying composition" which is a composition before the lyophilization process and may be in the form of a solution, suspension, liquid, or aqueous formulation. Concentrations, amounts, ratios, proportions, etc. of, for example, TRIS, cryoprotectants, RNA molecules, LNP used herein relate to the composition after lyophilization (i.e., the lyophilized composition), unless otherwise specified. More specifically, in those cases, the present application refers to the concentrations, amounts, ratios, proportions, etc. of the composition before lyophilization (i.e., the freezedrying composition).

[0039] In connection with the present invention, the term "lyophilisation" (also called freeze drying) is used to maintain or improve the shelf life of a product and to make storage, distribution, and transportation more convenient. The lyophilization process typically consists of the following three consecutive steps: i) a freezing step in which the solvent (e.g., water) crystallizes into ice, ii) a primary drying step in which the solvent is removed by sublimation under vacuum, and iii) a secondary drying step in which most of the unfrozen solvent is removed by diffusion and desorption.

[0040] In connection with the present invention, "TRIS" or tris(hydroxymethyl)aminomethane, or what is known as tromethamine or THAM in pharmaceutical applications, has the chemical formula (HOCH 2 ) 3CNH 2 It is understood as an organic compound represented by this. This is routinely used as a component of buffer solutions such as TAE buffer and TBE buffer, especially for nucleic acid solutions. TRIS is frequently used to enhance the permeability of cell membranes or as a compound of metal ions in solutions. As used herein, TRIS functions as a buffer to maintain the pH of the solution within an acceptable range before lyophilization. Many other buffers covering a wide pH range, such as acetate, citrate, glycine, histidine, phosphoric acid (sodium or potassium), and diethanolamine, can also be selected in formulation and may thus be equally suitable for the present invention. Any pH adjuster can be used, but the use of tris(hydroxymethyl)aminomethane hydrochloride is recommended.

[0041] In some embodiments, the concentration of TRIS in the composition before lyophilization can be about 3 mM or less, such as about 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01 mM.

[0042] In certain embodiments, the concentration of TRIS in the composition before lyophilization is preferably about 0.5 mM, about 1 mM or about 1.5 mM.

[0043] In a further embodiment of the present invention, the concentration of TRIS in the composition before lyophilization is about 0.01 to about 3 mM, preferably about 0.1 to about 2 mM, more preferably about 0.5 to 1.5 mM, and most preferably about 1 mM.

[0044] In another embodiment, the concentration of TRIS in the composition before lyophilization is between about 0.01 mM and about 3 mM, preferably between about 0.1 mM and about 3 mM, more preferably between about 0.5 mM and about 2 mM, and most preferably between about 1 mM and about 3 mM. Alternatively, it is between about 2 mM and about 3 mM.

[0045] In certain embodiments, the concentration of TRIS in the composition before lyophilization is about 1 mM, about 2 mM, or about 3 mM.

[0046] According to the present invention, the composition to be lyophilized also contains at least one cryoprotectant. As used herein, unless otherwise specified, the term "cryoprotectant" refers to an excipient and is understood as a permeable or non-permeable substance used to protect the composition and its related compounds from damage caused by freezing, for example, by ice formation. Conventional cryoprotectants are generally glycols (alcohols containing at least two hydroxyl groups) and sugars such as ethylene glycol, propylene glycol, glycerol, and trehalose. Generally, the sugars preferred in this context have high water substitution activity and high glass transition temperature. In particular, sucrose and trehalose are non-reducing sugars and maintain the structural integrity of cells in a non-toxic manner during freezing and thawing.

[0047] In certain embodiments, the cryoprotectant can be selected from the list comprising sodium citrate, sodium chloride, sorbitol, polysorbate, trehalose, mannose, mannitol, maltose, sucrose, glucose, fructose, lactose, histidine, arginine, lysine, dextran, maltodextrin, cyclodextrin, polyvinylpyrrolidone (PVP), glycine, glycerol, polyethylene glycol (PEG), propylene glycol, and / or mixtures thereof.

[0048] In certain embodiments of the present invention, the cryoprotectant is selected from the list comprising trehalose, maltose, sucrose, or combinations thereof, particularly sucrose.

[0049] In certain embodiments, the cryoprotectant is sucrose.

[0050] Accordingly, the present invention relates to a composition comprising one or more RNA molecules, one or more lipid nanoparticles, sucrose, and tris(hydroxymethyl)methylamine (TRIS), wherein the concentration of TRIS in the composition before lyophilization is 3 mM or less.

[0051] In certain embodiments, the composition of the present invention before lyophilization comprises at least about 0.01% (w / v), at least about 0.1% (w / v), at least about 0.5% (w / v), at least about 1% (w / v), at least about 2.5% (w / v), at least about 5% (w / v), at least about 10% (w / v), at least about 15% (w / v), at least about 20% (w / v), at least about 25% (w / v), at least about 30% (w / v), particularly at least about 15% (w / v) cryoprotectant.

[0052] In another specific embodiment, the concentration of the cryoprotectant in the composition before lyophilization is from about 0.01% to about 30% (w / v), preferably from about 10% to about 20% (w / v), and most preferably about 15% (w / v).

[0053] In another embodiment, the sucrose concentration before lyophilization is from about 5% to about 20% (w / v) sucrose, such as about 5%, 6%, 8%, 9%, 10% to about 15%, 16%, 17%, 18%, 19%, 20% (w / v) sucrose.

[0054] In another embodiment, the sucrose concentration before lyophilization is from about 5% to about 20% (w / v), particularly from about 5% to about 15% (w / v), more specifically from about 8% to about 15% (w / v), and even more specifically from about 10% to about 15% (w / v). In one embodiment, the concentration of the cryoprotectant in the composition before lyophilization is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% (w / v).

[0055] In one embodiment, the concentration of the cryoprotectant in the composition before lyophilization is 15% (w / v).

[0056] In another embodiment, the sucrose concentration in the composition before lyophilization is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% (w / v).

[0057] In a specific embodiment, the sucrose concentration in the composition before lyophilization is 15% (w / v).

[0058] In a further embodiment, the composition before lyophilization of the present invention contains a concentration of 1 mM TRIS and 15% sucrose (w / v).

[0059] In yet another embodiment, the composition before lyophilization of the present invention contains TRIS at a concentration of 3 mM or less and sucrose from about 5% to about 20% (w / v), such as about 5%, 6%, 8%, 9%, 10% to about 15%, 16%, 17%, 18%, 19%, 20% (w / v) of sucrose.

[0060] In another embodiment, the composition before lyophilization of the present invention contains TRIS at a concentration of 3 mM or less and sucrose from about 5% to about 20% (w / v), such as about 8% to about 20% (w / v), about 8% to about 15% (w / v), about 10% to about 15% (w / v) of sucrose.

[0061] Regarding the present invention, the composition according to the present invention comprises at least one or more RNA molecules delivered in one or more media comprising lipids, liposomes, lipid nanoparticles, polymers, or polymer-based nanoparticles.

[0062] As described in the specification, lipids refer to any component belonging to the categories of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols, glycolipids, and polyketides.

[0063] Furthermore, regarding the present invention, a liposome is a spherical vesicular structure having at least one lipid bilayer that forms a hollow spherical shape surrounding an aqueous phase. Therefore, target cargos such as nucleic acid-containing formulations, pharmaceuticals, proteins / peptides can be encapsulated within either the aqueous compartment (in the case of water-soluble / hydrophilic) or the lipid bilayer (in the case of lipid-soluble / lipophilic) of the liposome.

[0064] Regarding the present invention, the term "lipid nanoparticle" or LNP refers to nano-sized particles composed of one or more lipids, for example, a combination of different lipids that are particularly useful for encapsulating various nucleic acids (RNA and DNA) as drugs or vaccines within a non-aqueous core. More specifically, lipid nanoparticles are generally spherical and composed of a solid lipid core stabilized by surfactants. The core lipids may be fatty acids, acylglycerols, waxes, and mixtures thereof. Biomembrane lipids such as phospholipids, sphingomyelin, bile salts (sodium taurocholate), and sterols (cholesterol) can be used as stabilizers. Lipids used in LNPs may include, for example, but are not limited to, at least one phospholipid, at least one modified lipid (such as PEG lipid) (e.g., PEG2000 lipid), at least one ionizable lipid, and at least one sterol. The lipid nanoparticles and their compositions of the present invention are generally known in the art.

[0065] With respect to the present invention, the term "PEG lipid" or "PEGylated lipid" means any suitable lipid modified with a PEG (polyethylene glycol) group. The PEG lipid particularly suitable for the present invention is characterized by being a diC14-PEG lipid. When the term C14-PEG lipid is used with respect to the present invention, this means a diC14-PEG lipid, i.e., a lipid having two C14 lipid tails. The C14-PEG lipid includes a polyethylene glycol moiety that defines the molecular weight of the lipid and a fatty acid tail containing 14 C atoms. For example, the PEG lipid according to the present invention may be, for example, DMG-PEG, more specifically DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), or DMPE-PEG, more specifically DMPE-PEG2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]) and the like, which are diC14-PEG lipids. Alternatively, longer-chain PEG lipids such as diC16 lipids and diC18 lipids can also be suitably used. In certain embodiments, the diC18-PEG2000 lipid is selected from the list including (distearoyl-based)-PEG2000 lipids such as DSG-PEG2000 lipid (2-distearoyl-rac-glycero-3-methoxypolyethylene glycol-2000), or DSPE-PEG2000 lipid (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]); or (dioleoyl-based) PEG2000 lipids such as DOG-PEG2000 lipid (1,2-dioleoyl-rac-glycerol) or DOPE-PEG2000 lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) or PEG5000 lipid.

[0066] In certain embodiments, the PEG2000 lipid contains at least three oxygen atoms at the fatty acid terminus of the lipid. Examples of such PEG lipids include DMG-PEG2000, DSPE-PEG2000, and the like.

[0067] In certain embodiments of the present invention, the PEG2000 lipid is DMG-PEG2000.

[0068] As used herein, the term "ionizable" (or cationic) in the context of a compound or lipid means that the compound or lipid contains a neutral group that has the ability to generate and dissociate ions (usually H+ ions), thereby becoming positively charged itself. Alternatively, the neutral group in the compound or lipid can generate electrons and thereby become negatively charged. Any type of ionizable lipid can be suitably used as used herein. For example, suitable ionizable lipids are ionizable amino lipids containing two identical or different termini linked via an S-S bond.

[0069] As used herein, the term "phospholipid" means a lipid molecule consisting of a hydrophilic "head" composed of two hydrophobic fatty acid "tails" and a phosphate group. Since the two components are most often linked by a glycerol molecule, the phospholipids of the present invention are preferably glycerol phospholipids. Further, the phosphate group is often modified with simple organic molecules such as choline (i.e., becoming phosphocholine) or ethanolamine (i.e., becoming phosphoethanolamine).

[0070] As used herein, the term "sterol" is also called steroid alcohol and is a subgroup of steroids that occur naturally in plants, animals, and fungi or are produced by some bacteria. Any suitable sterol can be used in the present invention, and suitable sterols are selected from the list including, for example, cholesterol, ergosterol, campesterol, oxysterol, androsterol, desmosterol, nikasterol, sitosterol, and stigmasterol, preferably cholesterol.

[0071] In certain embodiments, the LNP contains from about 10 mol% to about 60 mol% of ionizable lipid, preferably in the range of about 40 mol% to 60 mol%.

[0072] In another specific embodiment, the LNP contains from about 15 mol% to 50 mol% of sterol, preferably contains from about 20 mol% to 40 mol% of sterol.

[0073] In a further embodiment, the LNP contains from about 0.5 mol% to 10 mol% of the PEG2000 lipid, preferably in the range of about 0.5 mol% to 5 mol%.

[0074] In another embodiment, the LNP contains at least 0.5 mol%, such as at least 1 mol%, such as at least 1.5 mol%, such as at least 2 mol%, such as at least 2.5 mol%, such as at least 3 mol% of the PEG2000 lipid.

[0075] In another specific embodiment, the LNP contains from about 5 mol% to 40 mol% of the phospholipid, preferably contains from about 5 mol% to 15 mol%.

[0076] In certain embodiments, the lipid nanoparticles in the composition before lyophilization contain from about 40 mol% to 60 mol% of ionizable lipid, from about 5 mol% to 15 mol% of phospholipid, from about 20 mol% to 40 mol% of sterol, and at least 0.5 mol% of the PEG2000 lipid.

[0077] In yet a further embodiment, the composition before lyophilization further contains water or water for injection (WFI).

[0078] For the purposes of the present invention, the term "water for injection" is understood as a preparation of sterile, pyrogen-free, solute-free distilled water, purified by distillation or reverse osmosis, having a pH of about 5.0 to 7.0. This is used only as a sterile solvent or diluent medium for drugs or solutions suitable for parenteral administration and is intended for use in the manufacture or injection of pharmaceuticals.

[0079] In the following embodiments of the present invention, the one or more RNA molecules are linear or circular RNA molecules.

[0080] As used herein, the term "RNA" refers to a molecule containing ribonucleotide residues, preferably a molecule consisting entirely or substantially of ribonucleotide residues. "Ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. In particular, this term refers to single-stranded RNA, but also to isolated RNA such as double-stranded RNA, partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from natural RNA by addition, deletion, substitution, and / or alteration of one or more nucleotides. Such alterations include the addition of non-nucleotide substances to the ends or within the RNA, for example to one or more nucleotides of the RNA. Nucleotides within an RNA molecule may also include non-natural nucleotides, non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may sometimes be referred to as analogs or analogues of naturally occurring RNA.

[0081] In yet another embodiment, the one or more RNA molecules are mRNA molecules.

[0082] According to the present invention, the term "RNA" includes "mRNA" which means "messenger RNA", preferably related thereto and generated using DNA as a template, and related to a "transcript" encoding a peptide or protein. mRNA usually includes a 5' untranslated region (5'-UTR), a coding region for a protein or peptide, and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is generated by in vitro transcription using a DNA template. In one embodiment of the present invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to those skilled in the art. For example, there are various commercially available in vitro transcription kits.

[0083] To avoid misunderstanding, the composition according to the present invention includes an LNP further comprising one or more mRNA molecules, or the composition according to the present invention can include a combination of a number of mRNA molecules, such as one or more mRNA molecules encoding an immunomodulatory protein and / or one or more mRNA molecules encoding an antigen-specific protein and / or a disease-specific protein.

[0084] In a very particular embodiment, the composition according to the present invention includes one or more mRNA molecules encoding at least one immunostimulatory protein selected from the list including CD40L, CD70, and caTLR4.

[0085] That is, the present invention provides a lyophilized composition comprising an mRNA molecule encoding CD40L, an mRNA molecule encoding CD70, and an mRNA molecule encoding caTLR4, wherein the mRNA molecules are formulated in lipid nanoparticles, and the composition further comprises one or more cryoprotectants and TRIS at a concentration of 3 mM or less before lyophilization. In a further aspect, the present invention provides a composition for use in human medicine and / or veterinary medicine.

[0086] In a preferred embodiment, the lyophilized composition according to the present invention is reconstituted before being administered to a subject in need thereof. The composition in this reconstituted form is further referred to as the "reconstituted composition". In certain embodiments, the reconstituted composition according to the present invention is a pharmaceutical composition comprising the lyophilized composition and at least one or more pharmaceutically acceptable agents such as excipients, carriers, diluents, etc.

[0087] With respect to the present invention, the term "pharmaceutical composition" refers to a composition having pharmaceutical properties such as a vaccine (or vaccine composition). In other words, it refers to a composition that produces a pharmacological and / or physiological effect. In some embodiments, pharmaceutically acceptable agents include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents for achieving a composition that can be used as an administration form. Additional suitable pharmaceutical carriers and diluents, as well as the pharmaceuticals necessary for their use, are described in "Remington's Pharmaceutical Sciences". After lyophilization, the resulting dry composition can be reconstituted using any suitable medium / buffer such as water, water for injection, TRIS buffered saline (TBS), and / or phosphate buffered saline (PBS), but is not limited thereto.

[0088] In certain embodiments, reconstitution is carried out using water or an aqueous solution of salt, preferably TBS, more preferably TBS containing 20 mM TRIS, and most preferably TBS containing 20 mM TRIS and 0.9% NaCl.

[0089] In certain embodiments, reconstitution is carried out using water for injection (WFI). In more specific embodiments, the pH of the reconstituted composition is from about 6 to about 8.

[0090] In certain embodiments, the reconstituted composition has a pH of about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, preferably a pH of about 7.0, and preferably a pH of about 7.4.

[0091] The present invention provides a method for preventing and treating human and veterinary diseases by administering a composition or pharmaceutical composition to a subject in need thereof.

[0092] For the purposes of this application, the terms "treatment", "treating", "treat" and the like refer to obtaining the desired pharmacological and / or physiological effect. This effect may be prophylactic in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely stabilizing or curing a disease and / or the adverse effects resulting from the disease. "Treatment" includes all treatments of diseases in mammals, particularly humans, and includes the following: (a) preventing the occurrence of a disease or symptom in a subject who may be susceptible to the disease or symptom but has not yet been diagnosed, (b) suppressing the symptoms of a disease, i.e., stopping the progression of the disease, or (c) alleviating the symptoms of a disease, i.e., causing regression of the disease or symptom.

[0093] In certain embodiments, the present invention provides a pharmaceutical composition as defined herein, which is suitable for use in parenteral administration, more specifically intravenous, intratumoral, intradermal, intraperitoneal, intramuscular or intra-articular administration, preferably intramuscular administration, after reconstitution.

[0094] In certain embodiments, the present invention provides a reconstituted composition or pharmaceutical composition for use in the treatment or prevention of a pathogen in a subject, particularly a viral pathogen, more specifically the SARS-CoV-2 virus.

[0095] In another embodiment, the reconstituted composition or pharmaceutical composition as defined herein is provided for use in the prevention and / or treatment of cell proliferative diseases.

[0096] In yet another aspect, the present invention provides a freeze-drying method comprising: a) mixing an RNA molecule, one or more lipid nanoparticles, a cryoprotectant, and TRIS to thereby form a composition before lyophilization according to the present invention; and b) lyophilizing the composition by a freezing step, a primary drying step, and a secondary drying step, wherein the primary drying step is carried out at a temperature below the glass transition temperature (Tg’) of the maximally freeze-concentrated solution.

[0097] As described herein, the term “glass transition temperature” (Tg) is understood as the temperature at which a frozen substance changes from a glassy (brittle) state to a rubbery (soft or flexible) state. Generally, in the spray freeze-drying method of amorphous materials, two different glass transition temperatures are considered: (i) the glass transition temperature (Tg’) of the maximally freeze-concentrated solution, which is related to the state of the frozen solution; (ii) the glass transition temperature (Tg) of the dried solid phase, which is maintained after the start of primary drying. In particular, in order to determine the shelf temperature during primary drying to avoid collapse, it is more appropriate to consider Tg’ rather than Tg. The shelf life of the lyophilized protein may be affected by the product temperature during the primary drying step. By carrying out primary drying below Tg’, it is possible to maintain the physical stability (cake shape) of the lyophilized product.

[0098] In certain embodiments, the primary drying can also be carried out at a temperature below the collapse temperature (Tc).

[0099] As used herein, the term "collapse temperature" (Tc) is understood as the temperature at which a substance softens until it can no longer support its structure. The collapse phenomenon negatively affects the properties of the final lyophilized product, causing loss of volatiles during storage, deterioration of reconstitution behavior, non-uniform moisture distribution, and extensive caking. Typically, the Tc of a material tends to be different from the temperature applied during drying. In some embodiments, the product temperature during primary drying is maintained 2 - 5 °C lower than the Tc to prevent collapse and maintain an elegant cake structure. For example, in a lyophilization process where the product temperature is -25 °C and the Tc is -20 °C, the appearance is improved. Generally, the value of the collapse temperature is 1 - 3 °C higher than the Tg’ value. Generally, the target product temperature in the primary drying stage of an optimized lyophilization process is several degrees lower than the critical threshold corresponding to the glass transition temperature (Tg’) of the frozen concentrated phase. The temperatures defined herein with respect to the method of the present invention typically refer to the respective temperatures within the lyophilization chamber. Depending on the type of equipment, the temperature within the lyophilization chamber may be measured in various ways.

[0100] In certain embodiments, the primary drying is carried out at a temperature about 0.5 °C, about 1 °C, about 2 °C, about 3 °C, about 4 °C, about 5 °C, about 6 °C, about 7 °C, about 8 °C, about 9 °C, about 10 °C lower than the collapse temperature (Tc). As an example, for the product described in the present application, the collapse temperature of the composition is -33 °C, the Tg’ is about -35 °C, and the primary drying temperature is -40 °C.

[0101] In certain embodiments of the method of the present invention, the primary drying step is carried out at about -30 °C to about -50 °C, preferably about -35 °C to about -45 °C, particularly about -40 °C.

[0102] In a preferred embodiment of the present invention, the primary drying temperature is at least 0.5 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, or 15 °C lower than the Tg’ of a given composition.

[0103] Preferably, the primary drying temperature in step b) of the method of the present invention is less than -25°C, more preferably less than -30°C, and most preferably less than -35°C. Even more preferably, the primary drying temperature in step b) of the method of the present invention is in the range of -55°C to -25°C, preferably in the range of -50°C to -30°C, more preferably in the range of -45°C to -35°C, and most preferably in the range of -43°C to -37°C. In a particularly preferred embodiment, the primary drying temperature is about -40°C.

[0104] In certain embodiments, the temperature of the primary drying step rises to the temperature of the secondary drying step. In some embodiments, the primary drying temperature begins at about -30°C to about -50°C, preferably about -35°C to about -45°C, particularly about -40°C, and rises up to about 10°C, after which the secondary drying step is initiated.

[0105] Tg is typically determined empirically. Methods for determining the glass transition temperature of a substance or composition are known in the art and include, for example, using freeze-drying microscopy, differential thermal analyzers (e.g., differential scanning calorimetry), or electrical impedance analyzers (dielectric resistance analysis). The freezing temperature is preferably pre-determined by selecting a temperature below the Tg of the given composition.

[0106] In a preferred embodiment of the present invention, the freezing temperature is at least 0.5°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C lower than the Tg of the given composition. For example, if the primary drying step is carried out at -40°C, which is lower than the Tg of the given composition, the freeze-drying step can be carried out at, for example, -50°C.

[0107] Preferably, the freezing temperature in step b) of the method of the present invention is less than -30°C, more preferably less than -35°C, and most preferably less than -40°C. Further preferably, the freezing temperature in step b) of the method of the present invention is in the range of -65°C to -35°C, preferably in the range of -60°C to -40°C, more preferably in the range of -55°C to -45°C, and most preferably in the range of -53°C to -47°C. In a particularly preferred embodiment, the freezing temperature is about -50°C.

Examples

[0108] The present invention will be described by the following examples. It will be understood by those skilled in the art that various changes and modifications can be easily made without departing from the technical idea and essential features of the present invention. Therefore, it is understood that the examples described below are illustrative in all respects and do not limit the scope of the present invention in any way.

[0109] Materials and Methods:

[0110] Raw Materials:

[0111] DSCP, cholesterol, and DMG-PEG2000 were purchased from Avanti Polar Lipids Inc. (Birmingham, Alabama, USA). The unique ionizable substance was synthesized by eTheRNA Imminotherapies or an external contractor. eGFP mRNA was produced at eTheRNA Imminotherapies in Niel, Belgium.

[0112] Preparation of the composition to be lyophilized

[0113] Lipid nanoparticles (LNPs) encapsulating EGFP mRNA were produced using a mixing device that mixes a solution containing EGFP mRNA in sodium acetate buffer (100 mM, pH 4) with a lipid solution containing ionizable lipid (50 mol %), helper lipid (10 mol %), cholesterol (38.5 mol %), and DMG-PEG2000 at a pre-defined flow rate ratio. The bulk after mixing was diluted with water for injection (WFI) and processed by tangential flow filtration (TFF) in three sub-steps: concentration, diafiltration, and final concentration. The bulk obtained from the TFF process was diluted with cryoprotectant to obtain a final TRIS buffer concentration of 1 mM in 15% sucrose. Next, the solution was sterile filtered using a 0.22 μm polyethersulfone (PBS) filter.

[0114] Lyophilization process

[0115] Vials containing 800 μL of the above lipid composition encapsulating eGFP mRNA in 1 mM TRIS and 15% sucrose are subjected to a lyophilization cycle using the method shown below:

[0116] 1. Temperature calibration at 20 °C 2. Freeze for 100 minutes to -50 °C at a rate of 0.7 °C / min to a temperature below Tg’ 3. Maintain the temperature at -50 °C for 60 minutes 4. Primary drying sub-step 1: Maintain the temperature at -50 °C for 15 minutes and turn on the vacuum switch. Pressure setpoint: 0.0720 mbar 5. Primary drying sub-step 2: Maintain the temperature at -50 °C for 3 hours. Pressure setpoint: 0.0720 mbar 6. Primary drying sub-step 3: Raise the temperature to -40 °C at a rate of 0.08 °C / min within 2 hours while keeping the vacuum setpoint at 0.0720 mbar constant 7. Primary drying sub-step 4: The temperature is maintained at -40 °C for 70 hours with the same vacuum setpoint of 0.0720 mbar 8. Primary drying sub-step 5: The temperature is raised to 10°C within 10 hours at a rate of 0.08°C / min. The vacuum setpoint is maintained at 0.0720 mbar. 9. Secondary drying sub-step 1: Maintain the temperature at 10°C for 15 minutes and lower the vacuum setpoint to 0.0100 mbar. 10. Secondary drying sub-step 2: Use a vacuum setpoint of 0.0100 mbar and maintain the temperature at 10°C for 5 hours and 50 minutes.

[0117] Results

[0118] Example 1 - Selection of the composition to be lyophilized - Phase 1

[0119] Screening of the composition to be lyophilized was carried out, and the optimal cryoprotectant formulation that brings the best cake characteristics and quality characteristics after reconstitution was selected. TBS and WFI were tested in combination with trehalose and / or sucrose. It should be noted that the samples lyophilized with WFI were reconstituted with TBS, and the samples lyophilized with TBS were reconstituted with WFI. The data in Table 1 show that sucrose is superior to trehalose as a cryoprotectant, but no improvement in the appearance of the cake and the quality characteristics of the particles was observed even when 20% or 25% of the cryoprotectant was used. Therefore, as shown in Example 2, further formulation optimization was carried out using 15% sucrose.

[0120] Table 1 Summary table regarding the selection criteria of the composition to be lyophilized.

Table 1

[0121] Example 2 - Selection of the composition to be lyophilized - Phase 2

[0122] The composition to be lyophilized containing 15% sucrose was further optimized to improve the encapsulation efficiency and recovery rate of mRNA, reduce the PDI value, and improve the control of particle size after reconstitution.

[0123] Several formulations with increased or no TRIS concentration were tested in the presence of 15% sucrose, lyophilized by the above lyophilization process, and reconstituted with TBS. Among the four conditions tested, the combination of 1 mM TRIS and 15% sucrose resulted in the best particle quality characteristics, such as maintaining the particle size and high encapsulation efficiency of mRNA.

[0124] Table 2 Summary table regarding the selection criteria for formulation selection

Table 2

[0125] Example 3 - Selection of the composition to be lyophilized - Phase 3

[0126] To evaluate whether TRIS concentrations less than 5 mM, such as 3 mM, are also suitable as the composition to be lyophilized, further optimization experiments were carried out. Before lyophilization, compositions containing 3 mM TRIS were tested with different amounts of sucrose (5%, 8%, 10%, 15% and 20% w / v) (Table 3). The results of comparing the freeze-dried cake (FD cake) reconstituted with WFI and the liquid control at -80 °C are shown.

[0127] Table 3 Summary table regarding the selection criteria for the composition to be lyophilized.

Table 3

[0128] The sizes of the various LNPs remained in the range of 107 - 123 nm. The PDI maintained a very low value (less than 0.2). Increasing the amount of sucrose decreased the mRNA content (Figure 1A) and increased the osmotic pressure (Figure 1B), but the pH remained constant at pH 7.4 (data not shown).

[0129] Compared with the -80°C liquid control, although the encapsulation efficiency was low, consistency was observed among the formulations containing 8 - 15% w / v sucrose (Figure 1C). Furthermore, these formulations showed an encapsulation efficiency similar to that of the formulation containing 1 mM TRIS and 15% sucrose (Table 2). As is evident from Tables 2 and 3, formulations containing 1 mM TRIS or 3 mM TRIS and various amounts of sucrose functioned equally well and were superior to formulations containing 20 mM TRIS or 5 mM TRIS and 15% sucrose.

[0130] Example 4 - Stability Study of 1 mM TRIS Composition

[0131] To investigate the stability of the lyophilized composition, the selected composition to be lyophilized (1 mM TRIS, 15% sucrose) was prepared, lyophilized, and placed under two different storage conditions: (1) 2 - 8°C and (2) 25°C for 3 months. In this study, control samples of the liquid (non-lyophilized) substance containing the drug product were used. At each time point, after reconstitution with TBS (20 mM TRIS, 0.9% NaCl), various quality characteristics of the particles were tested. As shown in Tables 4 and 5, for the bulk stored at 2 - 8°C, all the quality characteristics tested remained stable as a function of time. These include important quality characteristics such as particle size, encapsulation efficiency, and mRNA content. For the bulk stored at 25°C (accelerated storage conditions), except for a slight decrease in mRNA content, the quality characteristics remained stable over time. It is also worth mentioning that the integrity and in vitro functionality of the mRNA in the reconstituted bulk were maintained over time compared to T0. This suggests that mRNA is not destabilized during the lyophilization process or when stored at 2 - 8°C in the lyophilized state. Furthermore, it can be inferred that the lipid nanoparticles were not destabilized.

[0132] In conclusion, the lyophilized composition composed of 1 mM TRIS, 15% sucrose, and pH 7.4 results in a stable bulk when stored at 2 - 8°C.

[0133] Summary of 3-month stability data at storage conditions of 2-8 °C and 25 °C in Table 4 [Table 4] A: Homogeneous and cohesive mass, not crumbling or melting.

[0134] Summary of 3-month stability data at storage conditions of 2-8 °C and 25 °C in Table 5 [Table 5]

[0135] Example 5 - In Vivo Stability Test

[0136] To investigate whether the storage conditions affect the activity of mRNA, an immunological study was conducted in which BALB / C mice were injected with LNPs containing Sars-Cov2 Omicron spike mRNA or Fluc mRNA after freeze-drying a 3 mM TRIS, 15% w / v sucrose composition and storing it at 2-8 °C, 25 °C, or 30 °C for 0 months (T0m), 3 months (T3m), or 5 months (T5m). On the 1st and 21st days of the experiment, a total of 2 intramuscular injections of 50 μl of LNP encapsulating 5 μg of Sars-Cov2 Omicron spike mRNA in TBS or buffer (negative control group) were given to the biceps femoris muscle of the hindlimbs of the mice. To monitor the development of the immune response in these mice, submandibular blood sampling was performed on D20 and D35. The anti-Omicron IgG concentration in the serum was measured by ELISA.

[0137] Overall, the freeze-dried samples showed IgG titers equivalent to those of the liquid control at -80 °C or the liquid form of the freeze-dried substance (Figure 2). At D20, some variation was detected, with expression decreasing in the freeze-dried samples at 2-8 °C at T0 and antibody titers decreasing in the freeze-dried samples at 30 °C at T3m. Over time, the antibody titers decreased slightly, with total IgG being 10 6 ~10 7individuals, total IgG was 10 at T3m 5~6 ~10 7 individuals, total IgG was 10 at T5m 5 ~10 6 and it was observed that there were such numbers of individuals.

[0138] Next, the expression levels were detected by bioluminescence (BLI). The biceps femoris muscle of the hindlimb of a mouse was intramuscularly injected once (50 μl, 2 μg) with Fluc mRNA LNP or TBS buffer (negative control group). At various time points after injection (4 hours, 24 hours, 48 hours), luciferin was administered by intraperitoneal injection to the mouse, and then the mouse was placed in a bioluminescence scanner to measure the expression of luciferase. In muscle, the highest expression was observed 24 hours later, both at T0 (Figure 3A) and T5m (Figure 3B), especially in liquid -80°C samples, and also in lyophilized samples stored at a temperature of 2 - 8°C. It can be concluded that lyophilized samples, especially 2 - 8°C samples and liquid -80°C control samples, showed equivalent expression even after 5 months of storage.

[0139] References Muramatsu H, Lam K, Bajusz C, Laczko D, Kariko K, Schreiner P, Martin A, Lutwyche P, Heyes J, Pardi N, Lyophilization provides long - term stability for a lipid nanoparticle - formulated nucleoside - modified mRNA vaccine, Molecular Therapy (2022). Zhao P., Hou X., Yan J. Long - term storage of lipid - like nanoparticles for mRNA delivery. Bioact Mater. 2020;5(2):358 - 363.

[0140] Terms in the drawings RNA content RNA content sucrose sucrose liquid liquid FD cake / WFI FD cake / WFI Osmolality Osmolality Encapsulation Efficiency Encapsulation Efficiency Liquid control Liquid control Freeze-dried Freeze-dried Endpoint titers Total IgG Endpoint titers Total IgG Avg Radiance Average Radiance months months Muscle Muscle Average radiance Average Radiance Liquid Liquid

Claims

**Claim 1** A composition comprising one or more RNA molecules, one or more lipid nanoparticles, sucrose, and tris(hydroxymethyl)methylamine (TRIS), wherein the composition is lyophilized, and wherein the concentration of TRIS in the composition before lyophilization is 3 mM or less, and the concentration of sucrose is less than 20% (w / v). **Claim 2** The composition according to claim 1, wherein the concentration of sucrose in the composition before lyophilization is at least about 5% (w / v), particularly at least about 8% (w / v), more particularly at least about 10% (w / v). **Claim 3** The composition according to claim 1, wherein the concentration of sucrose in the composition before lyophilization is from about 5% to about 20% (w / v), particularly from about 5% to about 15% (w / v), more specifically from about 8% to about 15% (w / v), even more specifically from about 10% to about 15% (w / v). **Claim 4** The composition according to any one of claims 1 to 3, wherein the concentration of TRIS in the composition before lyophilization is from about 0.01 mM to about 3 mM, preferably from about 0.1 mM to about 3 mM, more preferably from about 0.5 mM to 3 mM, and most preferably from about 1 mM to about 3 mM. **Claim 5** The composition according to any one of claims 1 to 4, wherein the lipid nanoparticles comprise a PEG lipid, particularly a PEG2000 lipid, more specifically DMG-PEG2000. **Claim 6** The composition according to any one of claims 1 to 5, wherein the lipid nanoparticles comprise from about 40 mol% to 60 mol% of an ionizable lipid, from about 5 mol% to 15 mol% of a phospholipid, from about 20 mol% to 40 mol% of a sterol, and at least 0.5 mol% of a PEG lipid. **Claim 7** The composition according to any one of claims 1 to 6, wherein the composition before lyophilization further comprises water, particularly water for injection (WFI). **Claim 8** The composition according to any one of claims 1 to 7, wherein the one or more RNA molecules are linear or circular RNA molecules. **Claim 9** The composition according to any one of claims 1 to 8, wherein the one or more RNA molecules are mRNA molecules. **Claim 10** The composition according to any one of claims 1 to 9, which is a composition for use in human and / or veterinary medicine. **Claim 11** The composition according to any one of claims 1 to 10, which is a composition that is reconstituted before administration to a subject in need thereof. **Claim 12** The reconstitution is carried out using water or an aqueous solution of a salt, preferably TBS, more preferably TBS having 20 mM TRIS, and most preferably TBS having 20 mM TRIS and 0.9% NaCl, of the composition according to claim 11.

13. The composition according to claim 11 or 12, having a pH of from about 6 to about 8.

14. A method for lyophilizing a composition comprising an RNA molecule and lipid nanoparticles, the method comprising: a. Mixing an RNA molecule, one or more lipid nanoparticles, a cryoprotectant, and TRIS at a concentration of 3 mM or less in water, particularly water for injection. b. The step of lyophilizing the composition includes a freezing step, a primary drying step, and a secondary drying step, wherein the primary drying step is carried out at a temperature below the glass transition temperature (Tg') of the maximally freeze-concentrated solution. Method.

15. The method according to claim 14, wherein the primary drying step is carried out at a temperature of from about -30 °C to about -50 °C, preferably at a temperature of from about -35 °C to about -45 °C, and particularly at about -40 °C.

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