Lyophilized RNA composition

Abstract

The present invention is in the field of RNA formulations, in particular freeze-drying of RNA. More particularly, the present invention relates to a lyophilized composition and a method of obtaining a lyophilized composition. Furthermore, the invention provides the use of the reconstitution composition according to the invention in human and / or veterinary medicine.

Description

Field of the Invention

[0001] The present invention belongs to the field of RNA preparations, in particular the freeze-drying of RNA. More specifically, the present invention relates to a freeze-dried composition and a method for obtaining a freeze-dried composition. In addition, the present invention provides the use of the reconstituted composition according to the present invention in human and / or veterinary medicine. Background of the Invention

[0003] Vaccines based on mRNA are new and promising platforms that can be prepared in a relatively short period of time. mRNA vaccines have the advantage that they do not deliver viral nucleic acid to the host cell nucleus - avoiding integration with host DNA - and the injected mRNA also has a relatively short in vivo half-life. Despite these advantages of efficacy and safety, the instability and ultra-cold storage requirements of mRNA vaccines remain major limitations. Ultra-cold storage requirements and short shelf life slow down the distribution of vaccines, mainly in resource-limited areas of the world.

[0004] The storage conditions required during preparation, transportation and at the end-user site are considered to be important features of mRNA vaccine drug products. The stability of mRNA formulations can be affected to some extent by a variety of factors, such as excipients, pH, temperature and optimization of the mRNA sequence; appropriate carrier selection; encapsulation of mRNA in lipid nanoparticles (LNPs), and freeze drying. Freeze drying or lyophilization is a technique to improve the stability of liquid vaccine formulations by first freezing the formulation and removing the aqueous solvent through a sublimation and desorption process [WO2012098358].

[0005] In a series of patents from CureVac, it is claimed that the activity of mRNA formulations (naked mRNA with and without protamine) during freeze-drying and storage is maintained by using cryoprotectants such as sugars [WO2016 / 165831; WO2011 / 069528; US9616084]. It is claimed that all quality attributes analyzed during the experimental period (up to 36 months) meet the stability specifications of stable and safe RNA drugs (i.e., appearance, RNA integrity, RNA content, pH and weight molar osmotic pressure concentration (osmolalit y )). In 2020, Zhao et al. published their findings on the performance of freeze-dried LNP-mRNA (encoding luciferase) with different cryoprotectants compared to fresh LNP. These authors demonstrated that the reconstituted material maintained mRNA expression efficiency in mice as observed by in vivo bioluminescence imaging studies [Zhao et al. 2020].

[0006] In a recent study from BioNTech / Pfizer [Muramatsu et al. 2022], it was demonstrated that nucleoside-modified mRNA LNPs could be lyophilized and that the physicochemical properties of the lyophilized material did not change significantly after storage for 12 weeks at room temperature and for at least 24 weeks at 4°C. Importantly, the authors observed no reduction in the immunogenicity of the lyophilized influenza mRNA-LNP vaccine in mice after storage for 24 weeks at 4°C.

[0007] Although some lyophilization protocols for mRNA-LNP formulations have been described, there is still a need for improved lyophilizable compositions to achieve stable products with long shelf life and little or no effect on the biological activity or physicochemical properties of the mRNA vaccines such as cake appearance, presence of visible particles after reconstitution, mRNA content, encapsulation efficiency, particle size and polydispersity index (PDI), pH and weight molar osmolality.

[0008] The present invention aims to solve the above problems, and the object of the present invention is to obtain a composition particularly suitable for lyophilization to provide a stable product and - in the lyophilized state - maintain its physicochemical properties and ultimately maintain the physicochemical properties of the mRNA-LNP vaccine. This purpose is solved by the subject matter of the claims. In particular, based on the purpose of the present invention, it is solved according to the following: 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 wherein the concentration of TRIS in the composition before lyophilization is 3mM or less. In particular, the composition can be used in humans and / or veterinary medicine.

[0009] The present invention further provides a lyophilization method comprising mixing one or more RNA molecules, one or more lipid nanoparticles, a cryoprotectant and TRIS to form a composition according to the present invention; and thereafter lyophilizing the composition by means of 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 maximum freeze-concentrated solution.

[0010] The inventors unexpectedly found that the freeze-dried composition according to the present invention is particularly suitable for freeze-drying process. After the freeze-drying process is completed, the freeze-dried composition is characterized by: (i) high mRNA recovery after freeze drying and reconstruction, (ii) excellent RNA integrity, and (iii) minimal increase in LNP particle size. In addition, the specific composition enables the liquid formulation to have a more favorable PDI value and ensures sufficient buffering capacity. The process integrity and functionality of the formulation are still retained after freeze drying. In particular, storage stability is increased, especially for extended time periods and / or storage under non-cooling conditions. In a frozen state of -20°C, the shelf life is up to 36 months; in a cold state of 4°C (2-8°C), the shelf life is up to 24 months; and at room temperature of 25°C, the shelf life is up to 24 hours. The method according to the present invention can be used to prepare a composition with the above-mentioned characteristics comprising RNA in a reproducible and cost-effective manner. The lyophilized composition comprising RNA according to the present invention can advantageously be stored, transported and used without a cold chain, for example in the medical field (eg as a vaccine), while the integrity and biological activity of the RNA in the composition remain very high. SUMMARY OF THE INVENTION

[0012] In a first aspect, the present invention relates to a composition comprising: one / or more / a RNA molecule, one / or more / a lipid nanoparticle, one or more cryoprotectants 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.

[0013] In a specific embodiment of the invention, the cryoprotectant is selected from the list comprising: trehalose, maltose or sucrose, or a combination thereof; in particular 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 wherein the concentration of TRIS in the composition prior to lyophilization is 3 mM or less.

[0015] In another specific embodiment, the concentration of the cryoprotectant in the composition prior to 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 specific embodiments, the concentration of sucrose in the composition prior to 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 specific embodiments, the concentration of sucrose prior to lyophilization is about 5% to about 20% (w / v) and between 5% to about 20% (w / v), particularly about 5% to about 15% (w / v) and between 5% to about 15% (w / v), more particularly about 8% to about 15% (w / v) and between 8% to about 15% (w / v), even more particularly about 10% to about 15% (w / v) and between 10% to about 15% (w / v).

[0018] In another embodiment of the invention, the concentration of TRIS in the composition prior to lyophilization is about 0.01 mM to about 3 mM and between 0.01 mM to about 3 mM, preferably about 0.1 mM to about 2 mM and between 0.1 mM to about 2 mM, more preferably about 0.5 mM and 1.5 mM and between 0.5 mM and 1.5 mM, and most preferably about 1 mM.

[0019] In another embodiment of the invention, the concentration of TRIS in the composition prior to lyophilization is about 0.01 mM to about 3 mM and between 0.01 mM and about 3 mM, preferably about 0.1 mM to about 3 mM and between 0.1 mM and about 3 mM, more preferably about 0.5 mM and 3 mM and between 0.5 mM and 3 mM, most preferably about 1 mM to about 3 mM and between 1 mM and about 3 mM. Alternatively, about 2 mM to about 3 mM and between 2 mM and about 3 mM.

[0020] In a specific embodiment of the present invention, the one or more lipid nanoparticles comprise PEG lipids, particularly PEG2000 lipids, more particularly DMG-PEG2000.

[0021] In a specific embodiment, prior to lyophilization, the lipid nanoparticles in the composition comprise between about 40 mol% and 60 mol% and between 40 mol% and 60 mol% ionizable lipids, between about 5 mol% and 15 mol% and between 5 mol% and 15 mol% phospholipids, between about 20 mol% and 40 mol% and between 20 mol% and 40 mol% sterols, and at least 0.5 mol% PEG lipids.

[0022] In yet further embodiments, prior to lyophilization, the composition further comprises water or water for injection (WFI).

[0023] In the following embodiments of the invention, the one / one or more / one RNA molecule is one / one or more / one linear or circular RNA molecule.

[0024] In yet other embodiments, the one or more RNA molecules are one or more mRNA molecules.

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

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

[0027] In a specific embodiment, reconstitution is performed with water, in particular water for injection (WFI) or an aqueous solution of saline, preferably TBS, more preferably TBS with 20 mM TRIS, most preferably TBS with 20 mM TRIS and 0.9% NaCl.

[0028] In yet another specific embodiment, the reconstituted composition has a pH of about 6 to about 8.

[0029] In yet another aspect, the present invention provides a method for lyophilizing a composition comprising RNA molecules and lipid nanoparticles, the method comprising mixing one or more RNA molecules, 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 a composition according to the present invention; and lyophilizing the composition by means of 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 maximum freeze concentrated solution.

[0030] In a further embodiment of the process of the invention, the primary drying step is carried out at a temperature of about -30°C to about -50°C and between -30°C to about -50°C, preferably at about -35°C and about -45°C and between -35°C to about -45°C, in particular at about -40°C. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] With specific reference now to the accompanying drawings, it is emphasized that the details shown are by way of example and are only for the purpose of illustrative discussion of different embodiments of the present invention. They are provided in order to provide what are considered to be the most useful and easiest descriptions of the principles and concepts of the present invention. In this regard, no attempt is made to illustrate the structural details of the present invention in more detail than is necessary for a basic understanding of the present invention. The description in conjunction with the accompanying drawings makes it apparent to those skilled in the art how several forms of the present invention can be embodied in practice.

[0033] Figure 1: Physicochemical properties of lyophilized compositions containing 3 mM Tris and different amounts of sucrose before lyophilization. Results are presented for freeze-dried cakes reconstituted in WFI (FD cakes) compared to -80°C liquid controls. (A) RNA content ( μ g / ml). (B) Weight molar osmotic pressure concentration (mOsm / kg). (C) Encapsulation efficiency (%).

[0034] Figure 2 : Antibody response after Sars-Cov2 Omicron spike. Before reconstitution, the freeze-dried composition was stored at different temperatures (i.e. 2-8°C, 25°C or 30°C) for 0 months (T0), 3 months (T3) or 5 months (T5). The liquid control was stored at -80°C or at 2-8°C. In order to monitor the development of the immune response in these mice, submandibular blood sampling was performed at D20 and D35. The concentration of anti-Omicron IgG in serum was determined by ELISA.

[0035] Figure 3 : Luciferase expression at 4h, 24h and 48h after LNP injection. Injections were performed with TBS or liquid -80°C samples (control) or freeze-dried compositions that were stored at different temperatures (i.e. 2-8°C, 25°C or 30°C) for 0 months (T0) (Panel A) or 5 months (T5) (Panel B) before reconstitution. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present invention will now be further described. In the following paragraphs, different aspects of the present invention are defined in more detail. Unless clearly indicated to the contrary, each aspect so defined can be combined with any other one or more aspects. In particular, any feature indicated as preferred or advantageous can be combined with any other one or more features indicated as preferred or advantageous.

[0038] As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a compound" means one compound or more than one compound.

[0039] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including," "includes," or "containing," "contains," and 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."

[0040] As used herein, the term "about" or "approximately" when referring to a measurable value such as a parameter, amount, duration, etc., is intended to encompass the specified value as well as variations of + / -10% or less, preferably + / -5% or less, more preferably + / -1% or less, and still more preferably + / -0.1% or less from the specified value, as long as such variations are suitable for performing the disclosed invention. It is to be understood that the value to which the modifier "about" or "approximately" refers itself is also specifically and preferably disclosed.

[0041] In a first aspect, the present invention is directed to a composition comprising: one or more RNA molecules, one or more lipid nanoparticles, one or more cryoprotectants and tris(hydroxymethyl)methylamine (TRIS); and wherein the concentration of TRIS in the composition prior to lyophilization is 3 mM or less.

[0042] As used herein, the term "composition" can be any mixture of two or more products or compounds that are intended for long-term stabilization, such as preventing denaturation or aggregation within the expected shelf life. In the context of the present invention, the term "lyophilized composition" of the present invention refers to the final product in the form of a lyophilized powder or solid form after the lyophilization process. On the other hand, the term "reconstructed composition" is used for a lyophilized composition that is in aqueous form and is ready to be given to a subject in need after reconstruction in, for example, water. Finally, the present invention also relates to a "lyophilized composition", which is a composition before the lyophilization process, which can be in the form of a solution, suspension, liquid or aqueous preparation. Unless otherwise specified, the concentration, amount, ratio, ratio, etc. of, for example, TRIS, one or more cryoprotectants, one or more RNA molecules, one or more LNPs as used herein, relates to the composition (i.e., lyophilized composition) after lyophilization. More specifically, in those cases, the application will refer to the concentration, amount, ratio, ratio, etc. of the composition (i.e., lyophilized composition) before lyophilization.

[0043] In the context of the present invention, the term "freeze drying" (also known as freeze drying) is used to maintain or improve the shelf life of a product, making it more convenient for storage, distribution and transportation. The freeze drying process generally comprises three consecutive steps: i) a freezing step, in which a solvent (such as, for example, water) crystallizes into ice, ii) a primary drying step, in which the solvent is removed by sublimation under vacuum, iii) a secondary drying step, in which most of the unfrozen solvent is removed by diffusion and desorption.

[0044] In the context of the present invention, the term "TRIS" or tris (hydroxymethyl) aminomethane, or tromethamine or THAM as it is referred to during medical use, is understood to be an organic compound with the chemical formula (HOCH2) 3CNH2. It is conventionally used as a component in buffer solutions such as TAE and TBE buffers, especially for solutions of nucleic acids. Tris is commonly used to increase the permeability of cell membranes or as a complex with metal ions in solution. As used herein, TRIS serves as a buffer to maintain the pH of the solution within an acceptable range before lyophilization. Many other buffers covering a wide pH range can be used to select in a formulation, for example, acetate, citrate, glycine, histidine, phosphate (sodium or potassium) and diethanolamine, and therefore may also be suitable in the context of the present invention. Although any pH adjusting agent can be used, it is preferred to use tris (hydroxymethyl) aminomethane hydrochloride.

[0045] In some embodiments, the concentration of TRIS in the composition prior to 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.

[0046] In a specific embodiment, prior to lyophilization, the concentration of TRIS in the composition is preferably about 0.5 m M, about 1 m M or about 1.5 m M.

[0047] In another embodiment of the invention, the concentration of TRIS in the composition prior to lyophilization is about 0.01 to about 3 mM and between 0.01 to about 3 mM, preferably about 0.1 mM to about 2 mM and between 0.1 mM to about 2 mM, more preferably about 0.5 mM and 1.5 mM and between 0.5 mM and 1.5 mM, and most preferably about 1 mM.

[0048] In another embodiment, the concentration of TRIS in the composition prior to lyophilization is about 0.01 to about 3 mM and between 0.01 to about 3 mM, preferably about 0.1 mM to about 3 mM and between 0.1 mM to about 3 mM, more preferably about 0.5 mM and 2 mM and between 0.5 mM to 2 mM, most preferably about 1 mM to about 3 mM and between 1 mM to about 3 mM. Alternatively, about 2 mM to about 3 mM and between 2 mM to about 3 m Between M.

[0049] In a specific embodiment, prior to lyophilization, the concentration of TRIS in the composition is about 1 m M, about 2 m M or about 3 m M.

[0050] According to the present invention, freeze-dried compositions also include at least one cryoprotectant. As used herein and unless otherwise indicated, the term "cryoprotectant" refers to an excipient and should be construed as a permeability or non-permeability material for protecting compositions and their related compounds from the freezing damage caused by, for example, ice formation. Conventional cryoprotectants are typically glycols (alcohol containing at least two hydroxyl groups) and sugars, such as ethylene glycol, propylene glycol, glycerol and trehalose. Typically, preferred sugars have high water displacement activity and high glass transition temperature in this context. Especially, sucrose and trehalose are non-reducing sugars, which keep the structural integrity of cells in a non-toxic manner during freezing and thawing.

[0051] In a specific embodiment, 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.

[0052] In a specific embodiment of the invention, the cryoprotectant is selected from the list comprising: trehalose, maltose or sucrose, or a combination thereof; in particular sucrose.

[0053] In specific embodiments, the cryoprotectant is sucrose.

[0054] 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); and wherein the concentration of TRIS in the composition prior to lyophilization is 3 mM or less.

[0055] In specific embodiments, prior to lyophilization, the composition of the invention 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), and in particular at least about 15% (w / v) of cryoprotectant.

[0056] In another specific embodiment, the concentration of the cryoprotectant in the composition prior to lyophilization is about 0.01% to about 30% (w / v) and between 0.01% to about 30% (w / v), preferably about 10% to about 20% (w / v) and between 10% to about 20% (w / v), and most preferably about 15% (w / v).

[0057] In another embodiment, the concentration of sucrose prior to lyophilization is about 5% to about 20% (w / v) and between 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.

[0058] In another embodiment, before lyophilization, the concentration of sucrose is about 5% to about 20% (w / v) and between 5% to about 20% (w / v), particularly about 5% to about 15% (w / v) and between 5% to about 15% (w / v), more particularly about 8% to about 15% (w / v) and between 8% to about 15% (w / v), even more particularly about 10% to about 15% (w / v) and between 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).

[0059] In one embodiment, the concentration of the cryoprotectant in the composition prior to lyophilization is 15% (w / v).

[0060] In another embodiment, the concentration of sucrose in the composition prior to 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).

[0061] In a specific embodiment, the concentration of sucrose in the composition prior to lyophilization is 15% (w / v).

[0062] In a further embodiment, the composition of the invention prior to lyophilization comprises TRIS at a concentration of 1 mM and 15% sucrose (w / v).

[0063] In yet other embodiments, the compositions of the invention prior to lyophilization comprise TRIS at a concentration of 3 mM or less and about 5% to about 20% (w / v) and sucrose between 5% to about 20% (w / v), such as about 5%, 6%, 8%, 9%, 10% to about 15%, 16%, 17%, 18%, 19%, 20% (w / v).

[0064] In another embodiment, the pre-lyophilized composition of the invention comprises TRIS at a concentration of 3 mM or less and about 5% to about 20% (w / v) and between 5% to about 20% (w / v) sucrose, such as about 8% to about 20% (w / v) and between 8% to about 20% (w / v), about 8% to about 15% (w / v) and between 8% to about 15% (w / v), about 10% to about 15% (w / v) and between 10% to about 15% (w / v) sucrose.

[0065] In the context of the present invention, the composition according to the present invention comprises at least one or more RNA molecules delivered in one or more vehicles comprising lipids, liposomes, lipid nanoparticles, polymers or polymer-based nanoparticles.

[0066] As used herein, lipid refers to any component from the classes of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, sterols, prenols, glycolipids, polyketides.

[0067] Furthermore, in the context of the present invention, a liposome is a spherical vesicle structure having at least one lipid bilayer formed in the shape of a hollow sphere surrounding an aqueous phase. Thus, any cargo, such as a nucleic acid-containing formulation, a drug, a protein / peptide, can be encapsulated in the aqueous compartment within the liposome (if it is water-soluble / hydrophilic) or within the lipid bilayer (if it is fat-soluble / lipophilic).

[0068] In the context of the present invention, the term "lipid nanoparticle" or LNP refers to a nanoparticle composed of one or more lipids, for example, a combination of different lipids that are particularly useful for encapsulating a variety of nucleic acids (RNA and DNA) as drugs or vaccines in a non-aqueous core. More specifically, the shape of the lipid nanoparticle is generally spherical and is composed of a solid lipid core stabilized by a surfactant. The core lipid can be a fatty acid, an acylglycerol, a wax, and a mixture thereof. Biomembrane lipids such as phospholipids, sphingomyelin, bile salts (sodium taurocholate) and sterols (cholesterol) can be used as stabilizers. The possible lipid used in the LNP can be, for example, but not limited to, at least one phospholipid, at least one modified lipid, such as a PEG lipid (e.g., a PEG2000 lipid), at least one ionizable lipid, and at least one sterol. The lipid nanoparticle of the present disclosure and compositions thereof are generally known in the art.

[0069] In the context of the present invention, the term "PEG lipid" or alternatively "PEGylated lipid" refers to any suitable lipid modified with a PEG (polyethylene glycol) group. In the context of the present invention, particularly suitable PEG lipids are characterized as di-C14-PEG lipids. Wherein in the context of the present invention, the term C14-PEG lipid is used, this refers to a di-C14-PEG lipid, i.e. a lipid with 2 C14 lipid tails. The C14-PEG lipid contains a polyethylene glycol moiety that defines the molecular weight of the lipid, and a fatty acid tail comprising 14 C-atoms. For example, in the context of the present invention, the PEG lipid can be a diC14-PEG lipid, such as, for example, DMG-PEG, more particularly DMG-PEG2000 (1,2-dimyristoyl-rac-glyceryl-3-methoxypolyethylene glycol-2000), or DMPE-PEG, more particularly DMPE-PEG2000 (1,2-dimyristoyl-sn-glyceryl-3-phosphoethanolamine-N-[methoxy (polyethylene glycol) -2000). Alternatively, longer chain PEG lipids, such as diC16-lipids or diC18-lipids, can also be suitably used. In a specific embodiment, the di-C18-PEG2000 lipid is selected from the list comprising: (distearoyl-based)-PEG2000 lipids, such as DSG-PEG2000 lipids (2-distearoyl-rac-glyceryl-3-methoxypolyethylene glycol-2000) or DSPE-PEG2000 lipids (1,2-distearoyl-sn-glyceryl-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]); or (dioleoyl-based)-PEG2000 lipids, such as DOG-PEG2000 lipids (1,2-dioleoyl-rac-glycerol) or DOPE-PEG2000 lipids (1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000]) or PEG5000 lipids.

[0070] In a specific embodiment, the PEG2000 lipid comprises at least 3 oxygen atoms in the fatty acid tail of the lipid. Examples of such PEG lipids are DMG-PEG2000, DSPE-PEG2000.

[0071] In a specific embodiment of the present invention, the PEG2000 lipid is DMG-PEG2000.

[0072] In the context of the present invention, in the context of a compound or lipid, the term "ionizable" (or alternatively cationic) refers to the presence of any uncharged group in the compound or lipid that can dissociate by producing ions (usually H+ ions) and thus itself becomes positively charged. Alternatively, any uncharged group in the compound or lipid can produce electrons and thus become negatively charged. As used herein, any type of ionizable lipid can be suitably used. For example, a suitable ionizable lipid is an ionizable amino lipid that comprises 2 identical or different tails connected via SS bonds.

[0073] In the context of the present invention, the term "phospholipid" refers to a lipid molecule consisting of two hydrophobic fatty acid "tails" and a hydrophilic "head" consisting of phosphate groups.

[0074] These two components are most commonly linked together by a glycerol molecule, and therefore the phospholipids of the present invention are preferably glycero-phospholipids. In addition, the phosphate group is usually modified with simple organic molecules such as choline (i.e. to provide phosphocholine) or ethanolamine (i.e. to provide phosphoethanolamine).

[0075] In the context of the present invention, the term "sterol", also referred to as sterol (steroid alcohol), is a subgroup of steroids naturally occurring in plants, animals and fungi or that can be produced by some bacteria. In the context of the present invention, any suitable sterol can be used, such as selected from the list comprising: cholesterol, ergosterol, campesterol, oxysterol, antrosterol, desmosterol, nicasterol (nicasterol), sitosterol and stigmasterol; preferably cholesterol.

[0076] In specific embodiments, the LNP comprises about 10 mol% and 60 mol% and between 10 mol% and 60 mol% of the ionizable lipid; preferably about 40 mol% and 60 mol% and between 40 mol% and 60 mol%.

[0077] In yet another specific embodiment, the LNP comprises about 15 mol% and between 50 mol% and 50 mol% sterol; preferably about 20 mol% and between 20 mol% and 40 mol%.

[0078] In further embodiments, the LNP comprises about 0.5 mol% and 10 mol% and between 0.5 mol% and 10 mol% of the PEG2000 lipid; preferably about 0.5 mol% and 5 mol% and between 0.5 mol% and 5 mol%.

[0079] In another embodiment, the LNP comprises 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.

[0080] In another specific embodiment, said LNP comprises about 5 mol% and 40 mol% and between 5 mol% and 40 mol% of said phospholipid; preferably about 5 mol% and 15 mol% and between 5 mol% and 15 mol%.

[0081] In a specific embodiment, prior to lyophilization, the lipid nanoparticles in the composition comprise about 40 mol% and 60 mol% and between 40 mol% and 60 mol% ionizable lipids, about 5 mol% and 15 mol% and between 5 mol% and 15 mol% phospholipids, about 20 mol% and 40 mol% and between 20 mol% and 40 mol% sterols, and at least 0.5 mol% of said PEG2000 lipid.

[0082] In yet further embodiments, prior to lyophilization, the composition further comprises water or water for injection (WFI).

[0083] In the context of the present invention, the term "water for injection" is understood to be a sterile, pyrogen-free, solute-free preparation of distilled water having a pH of about 5.0 to 7.0 purified by distillation or reverse osmosis. It is used only as a sterile solvent or diluent vehicle for drugs or solutions suitable for parenteral administration and is intended for use in the preparation of drug products or for injection.

[0084] In the following embodiments of the invention, the one or more RNA molecules are one or more linear or circular RNA molecules.

[0085] In the context of the present invention, the term "RNA" refers to a molecule comprising ribonucleotide residues and preferably completely or substantially composed of ribonucleotide residues." Ribonucleotide " refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. In particular, the term refers to single-stranded RNA, but may also refer to double-stranded RNA, isolated RNA (such as partially purified RNA), substantially pure RNA, synthetic RNA, recombinant RNA, and RNA modified by adding, deleting, replacing and / or changing one or more nucleotides that are different from naturally occurring RNA. Such a change may include adding non-nucleotide materials, such as being added to one or more ends or interior of RNA, for example, at one or more nucleotides of RNA. The nucleotides in the RNA molecule may also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. The RNAs of these changes may be referred to as analogs or analogs of naturally occurring RNA.

[0086] In yet further embodiments, the one or more RNA molecules are one or more mRNA molecules.

[0087] According to the present invention, the term "RNA" includes and preferably relates to "mRNA", which refers to "messenger RNA" and refers to a "transcript" that can be produced using DNA as a template and encodes a peptide or protein. mRNA generally comprises a 5' untranslated region (5'-UTR), a protein or peptide coding region and a 3' untranslated region (3'-UTR). mRNA has a limited half-life in cells and in vitro. Preferably, mRNA is produced by in vitro transcription using a DNA template. In one embodiment of the invention, RNA is obtained by in vitro transcription or chemical synthesis. In vitro transcription methods are known to technicians. For example, there are a variety of commercially available in vitro transcription kits.

[0088] To avoid any misunderstanding, the compositions according to the present invention comprise LNPs further comprising one or more mRNA molecules, or they may comprise a combination of multiple mRNA molecules, such as one or more mRNA molecules encoding immunomodulatory proteins and / or one or more mRNA molecules encoding antigen-specific proteins and / or disease-specific proteins.

[0089] In a very specific embodiment, the composition comprises one or more mRNA molecules encoding at least one immunostimulatory protein selected from the list comprising CD40L, CD70 and caTLR4.

[0090] Thus, the present invention provides a lyophilized composition comprising an mRNA molecule encoding CD40L, an mRNA molecule encoding CD70, an mRNA molecule encoding caTLR4, wherein the mRNA molecule is formulated in a lipid nanoparticle, and wherein the composition further comprises one or more cryoprotectants, and TRIS at a concentration of 3 mM or less prior to lyophilization. In a further aspect, the present invention provides a composition for use in human and / or veterinary medicine.

[0091] In a preferred embodiment, the lyophilized composition according to the present invention is reconstituted before administration to a subject in need thereof. This reconstituted form of the composition is further referred to as a "reconstituted composition". In a specific embodiment, the reconstituted composition according to the present invention is a pharmaceutical composition comprising a lyophilized composition and at least one or more pharmaceutically acceptable agents such as excipients, carriers, diluents.

[0092] In the context of 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 provides a pharmacological and / or physiological effect. In some embodiments, pharmaceutically acceptable agents include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve compositions that can be used as dosage forms. Other suitable pharmaceutical carriers and diluents and pharmaceutical excipients for their use are described in Remington's Pharmaceutical Sciences. After freeze drying, any suitable medium / buffer, such as, but not limited to, water, water for injection, Tris-buffered saline (TBS) and / or phosphate-buffered saline (PBS) can be used to reconstitute the resulting dried composition.

[0093] In a specific embodiment, reconstitution is performed using water or an aqueous solution of saline, preferably TBS, more preferably TBS with 20 mM TRIS, most preferably TBS with 20 mM TRIS and 0.9% NaCl.

[0094] In a specific embodiment, reconstitution is performed using water for injection (WFI).In yet another specific embodiment, the reconstituted composition has a pH of about 6 to about 8.

[0095] In specific 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 about 7.0, preferably about 7.4.

[0096] The present invention provides methods for preventing and treating human and veterinary disorders by administering a composition or pharmaceutical composition to a subject in need thereof.

[0097] In the context of this application, the terms "treatment", "treating", "treat" and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely stabilizing or curing a disease and / or adverse reactions attributable to the disease. "Treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (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 as having the disease or symptom; (b) inhibiting the symptoms of a disease, i.e., arresting its development; or (c) alleviating the symptoms of a disease, i.e., causing regression of the disease or symptom.

[0098] In a specific embodiment, the invention provides a pharmaceutical composition as defined herein which, after reconstitution, is suitable for parenteral administration; more particularly for intravenous, intratumoral, intradermal, intraperitoneal, intramuscular or intranodal administration, preferably intramuscular administration.

[0099] In a specific embodiment, the present invention provides a reconstituted composition or a pharmaceutical composition for treating or preventing a pathogen in a subject; particularly a viral pathogen, more particularly the SARS-CoV-2 virus.

[0100] In another embodiment, there is provided a reconstituted composition or a pharmaceutical composition as defined herein for use in the prevention and / or treatment of a cell proliferative disorder.

[0101] In yet another aspect, the present invention provides a method of lyophilization, comprising: a) mixing one or more RNA molecules, one or more lipid nanoparticles, a cryoprotectant and TRIS to form a composition before lyophilization according to the present invention; and b) lyophilizing the composition by means of 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 maximum freeze-concentrated solution.

[0102] As used herein, the term "glass transition temperature" (Tg) is understood to be the temperature at which a frozen material changes from a glassy state (brittle) to a rubbery state (pliable or soft). Typically, in spray freeze drying processes for amorphous materials, two different types of glass transition temperatures are considered: (i) the glass transition temperature (Tg') of the maximum frozen concentrated solution associated with being in a frozen solution state; (ii) the glass transition temperature (Tg) of a well-maintained dry solid phase after the start of primary drying. In particular, in order to determine the storage temperature during primary drying to avoid collapse, it is more relevant to consider Tg' than Tg. The shelf life of freeze-dried proteins may be affected by the product temperature during the primary drying step. Primary drying below Tg' will make it possible to maintain the physical stability (i.e., the shape of the block) of the freeze-dried product.

[0103] In a specific embodiment, the primary drying may also be carried out at a temperature below the collapse temperature (Tc).

[0104] As used herein, the term "collapse temperature" (Tc) is understood to be the temperature at which the material softens to the point at which it cannot support its structure. The collapse phenomenon has a deleterious effect on the properties of the final freeze-dried product, resulting in volatile losses during storage, poor reconstitution behavior, uneven moisture distribution, and a large amount of caking. Typically, the Tc of the material tends to be different from the temperature applied during drying. In some embodiments, the product temperature during primary drying is maintained at 2°C-5°C below its Tc to avoid collapse and maintain an aesthetically pleasing block structure. For example, a freeze-drying process in which the product temperature is -25°C and Tc is -20°C produces a good appearance. Typically, the value of the collapse temperature is 1-3°C higher than the Tg' value. In general, the target product temperature during the primary drying stage of the optimized freeze-drying process is several degrees lower than the critical threshold of the glass transition temperature (Tg') corresponding to the freeze-concentrated phase. The temperature defined herein for the method according to the present invention generally refers to the corresponding temperature in the freeze-drying chamber. Depending on the type of instrument, the temperature in the freeze-drying chamber can be determined in different ways.

[0105] In a specific embodiment, the primary drying is carried out at a temperature below the collapse temperature (Tc) by 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. As an example, for the product described in this application, the collapse temperature of the composition is -33°C, Tg' is about -35°C, and the primary drying temperature is carried out at -40°C.

[0106] In a specific embodiment of the process of the present invention, the primary drying step is carried out at about -30°C to about -50°C and between -30°C to about -50°C, preferably at about -35°C and about -45°C and between -35°C to about -45°C, in particular at about -40°C.

[0107] In a preferred embodiment of the present invention, the primary drying temperature is equal to or 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.

[0108] Preferably, the primary drying temperature in step b) of the process according to the invention is below -25°C, more preferably below -30°C and most preferably below -35°C. Further preferably, the primary drying temperature in step b) of the process according to the invention is in the range of -55°C to -25°C, preferably -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.

[0109] In specific embodiments, the primary drying step temperature is ramped to the secondary drying temperature. In some embodiments, the primary drying temperature starts at about -30°C to about -50°C and between -30°C to about -50°C, preferably about -35°C and about -45°C and between -35°C to about -45°C, especially about -40°C, and ramped to about 10°C, wherein the secondary drying step is then started.

[0110] Tg is usually determined empirically. Methods for determining the glass transition temperature of a substance or composition are known in the art, and include, for example, by using a freeze-drying microscope, a differential thermal analyzer (e.g., differential scanning calorimetry) or an electrical impedance analyzer (dielectric resistance analysis). The freezing temperature is preferably predetermined by selecting a temperature equal to or lower than the Tg of a given composition.

[0111] In a preferred embodiment of the invention, the freezing temperature is equal to or 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 below the Tg of the given composition. For example, if the primary drying step is carried out at -40°C (which is below the Tg of the given composition), the freeze drying step can be carried out at, for example, -50°C.

[0112] Preferably, the freezing temperature in step b) of the process according to the invention is below -30°C, more preferably below -35°C and most preferably below -40°C. Further preferably, the freezing temperature in step b) of the process according to the invention is in the range of -65°C to -35°C, preferably -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. Example

[0113] The present invention will now be described by means of the following examples. It will be appreciated by those skilled in the art that various changes and modifications may be easily made without departing from the technical spirit or essential features of the present invention. Therefore, it should be understood that the embodiments described below are illustrative in all respects and do not limit the scope of the present invention in any way.

[0114] Materials and methods:

[0115] Raw materials:

[0116] DSPC, cholesterol and DMG-PEG2000 were purchased from Avanti Polar Lipids Inc. (Birmingham, Alabama, USA). Proprietary ionizable species were synthesized at eTheRNA Immunotherapies or by external parties. eGFP mRNA was produced at eTheRNA Immunotherapies in Niel, Belgium.

[0117] Preparation of freeze-dried compositions

[0118] Lipid nanoparticles (LNPs) encapsulating EGFP mRNA were prepared using a mixing device, wherein a solution containing EGFP mRNA in sodium acetate buffer (100 mM, pH 4) was mixed with a lipid solution containing ionizable lipids (50% mol%), auxiliary lipids (10% mol%), cholesterol (38.5% mol%) and DMG-PEG2000 at a predefined flow rate ratio. The mixed bulk was diluted in water for injection (WFI) and processed in three substeps via tangential flow filtration (TFF): concentration, diafiltration and final concentration. The material obtained by the TFF process was diluted with a cryoprotectant to obtain a final Tris buffer concentration of 1 mM in 15% sucrose. The solution was then sterile filtered using a 0.22 μm polyethersulfone (PES) filter T.

[0119] Freeze drying process

[0120] Contains 800 μA vial of the above lipid composition encapsulating eGFP mRNA in 1 mM Tris and 15% sucrose was subjected to a freeze-drying cycle using the following as described:

[0121] 1. Temperature calibration at 20℃

[0122] 2. Freeze to -50°C for 100 minutes at a rate of (0.7°C / min) to achieve a temperature below Tg'

[0123] 3. Maintain -50℃ temperature for 60 minutes

[0124] 4. Primary Drying Sub-Step 1: Maintain temperature at -50°C for 15 minutes and turn on vacuum at pressure setting: 0.0720 mbar

[0125] 5. Primary drying sub-step 2: Maintain temperature at -50°C for 3 hours. Vacuum setting: 0.0720 mbar

[0126] 6. Primary Drying Sub-Step 3: Raise the temperature to -40°C at a rate of 0.08°C / min over 2 hours while maintaining the vacuum set point constant: 0.0720 mbar

[0127] 7. Primary Drying Sub-Step 4: Maintain temperature at -40°C for 70 hours and use the same vacuum setting of -0.0720 mbar

[0128] 8. Primary Drying Sub-Step 5: Raise the temperature to 10°C at a rate of 0.08°C / min over 10 hours. Maintain the vacuum setting at 0.0720 mbar.

[0129] 9. Secondary Drying Sub-Step 1: Maintain the temperature at 10°C for 15 minutes while reducing the vacuum setting to 0.0100 mbar

[0130] 10. Secondary Drying Sub-Step 2: Maintain the temperature at 10°C for 5 hours and 50 minutes, using a vacuum setting of 0.0100 mbar.

[0131] result

[0132] Example 1 - Freeze Drying Composition Selection - Stage 1

[0133] A freeze-dried composition screen was performed to select the best cryoprotectant formulation that produced the best cake appearance and quality attributes after reconstitution. TBS and WFI were tested in combination with trehalose and / or sucrose. Notably, samples freeze-dried in WFI were reconstituted with TBS, while samples freeze-dried in TBS were reconstituted in WFI. The data in Table 1 show that sucrose is superior to trehalose as a cryoprotectant, while no improvement in cake appearance and particle quality attributes was observed using 20% ​​or 25% cryoprotectant. Therefore, as shown in Example 2, further formulation optimization was performed using 15% sucrose.

[0134] Table 1. Summary table of selection criteria used for freeze-dried composition selection.

[0135]

[0136] A: Uniform, coherent mass without collapse or melting.

[0137] B: Poor lump, melting present.

[0138] X: Translucent, homogeneous solution with no visible particles.

[0139] Y: Translucent, foamy solution with no visible particles.

[0140] Z: Translucent, foamy solution with visible particles present.

[0141] Example 2 - Freeze Drying Composition Selection - Stage 2

[0142] A freeze-dried composition containing 15% sucrose was further optimized to improve mRNA encapsulation efficiency and recovery, reduce PDI values ​​and obtain better particle size control after reconstitution.

[0143] Several formulations without or with increasing TRIS concentrations were tested in the presence of 15% sucrose and subjected to freeze drying as detailed above and reconstitution with TBS under the lyophilization process. Of the four conditions tested, 1 mM Tris with 15% sucrose produced the best particle quality attributes: particle size was maintained and mRNA encapsulation efficiency remained high.

[0144] Table 2. Summary table of selection criteria used for formulation selection.

[0145]

[0146] A: Uniform, coherent mass without collapse or melting.

[0147] X: Translucent, homogeneous solution with no visible particles.

[0148] Example 3 - Freeze Drying Composition Selection - Stage 3

[0149] In order to evaluate whether the Tris concentration lower than 5mM (such as 3mM) is also suitable as a freeze-dried composition, further optimization experiments were carried out. Before freeze drying, different amounts of sucrose (5%, 8%, 10%, 15% and 20% w / v) (Table 3) were tested in the composition comprising 3mMTris. The results of freeze-dried blocks (FD blocks) reconstructed in WFI compared with -80°C liquid controls are provided.

[0150] Table 3. Summary table of selection criteria used for freeze-dried composition selection.

[0151]

[0152]

[0153] A: Uniform, coherent mass without collapse or melting.

[0154] B: There is some shrinkage at the bottom.

[0155] C: Poor lump, melting present.

[0156] The size of the various LNPs remained in the range of 107-123 nm. The PDI remained very low, meaning a value below 0.2. By increasing the amount of sucrose, the mRNA content decreased ( Figure 1 A) and the osmolality increases ( Figure 1 B), while the pH was kept constant at pH 7.4 (data not shown).

[0157] Encapsulation efficiency was lower compared to the -80°C liquid control but showed consistency between formulations with 8-15% w / v sucrose ( Figure 1 C). In addition, these formulations showed similar encapsulation efficiency to the formulation with 1 mM Tris, 15% sucrose (Table 2). As is apparent from Tables 2 and 3, formulations containing 1 mM Tris or 3 mM Tris with varying amounts of sucrose performed equally well and were also superior to formulations containing 20 mM Tris or 5 mM Tris and 15% sucrose.

[0158] Example 4 - Stability Study of 1 mM Tris Composition

[0159] To investigate the stability of the lyophilized compositions, the selected lyophilized compositions (1 mM Tris, 15% sucrose) were prepared, freeze dried, and placed under two different storage conditions: (1) 2-8°C and (2) 25°C for 3 months. A control sample of liquid (non-lyophilized) material containing the drug product was used in this study. At each time point, after reconstitution with TBS (20 mM Tris, 0.9% NaCl), the different quality attributes of the particles were tested. As highlighted in Tables 4 and 5, for the blocks stored at 2-8°C, all the quality attributes tested remained stable over time. Among them were the key quality attributes of particle size, encapsulation efficiency, and mRNA content. For the blocks stored at 25°C (accelerated storage conditions), except for a slight decrease in mRNA content, the quality attributes remained stable over time. It is also worth mentioning that the mRNA integrity and in vitro functionality of the reconstituted blocks were retained over time compared to T0. This indicates that the mRNA remains intact and is not unstable during the freeze drying process and when stored at 2-8°C in lyophilized form. Furthermore, it can be inferred that the lipid nanoparticles are not unstable.

[0160] It was concluded that a freeze-dried composition consisting of 1 mM Tris, 15% sucrose, pH 7.4 produced a stable cake when stored at 2-8°C.

[0161] Table 4: Summary of three-month stability data at 2-8°C and 25°C as storage conditions

[0162]

[0163] A: Uniform, coherent mass without collapse or melting.

[0164] Table 5: Summary of three-month stability data at 2-8°C and 25°C as storage conditions

[0165]

[0166] Example 5 - In vivo stability test

[0167] In order to study whether the storage conditions of 3mM Tris, 15% w / v sucrose composition after freeze drying at 2-8°C, 25°C or 30°C for 0 months (T0m), 3 months (T3m) or 5 months (T5m) affect the activity of mRNA, an immune study was performed in which BALB / C mice were injected with LNPs containing Sars-Cov2Omicron spike mRNA or Fluc mRN. On the 1st and 21st days of the experiment, mice received a total of 2 intramuscular injections in the biceps of the hind limbs, and the injections were injected with 50 μl of LNPs encapsulating Sars-Cov2Omicron spike mRNA (5 μg) in TBS or buffer (negative control group). In order to monitor the development of immune responses in these mice, submandibular blood sampling was performed at D20 and D35. The concentration of anti-Omicron IgG in serum was determined by ELISA.

[0168] Overall, the lyophilized samples showed comparable IgG titers to the liquid control or the liquid form of the lyophilized material at -80°C ( Figure 2 At D20, some changes were detected, with lower expression in freeze-dried samples at 2-8 °C at T0 and lower antibody titers for freeze-dried samples at 30 °C at T3m. A slight decrease in antibody titers was observed over time, with a total IgG of 10 at T0. 6 -10 7 , at T3m, total IgG is 10 5-6 -10 7 And at T5m, total IgG is 10 5 -10 6 .

[0169] Next, expression levels were detected via bioluminescence (BLI). Mice received an intramuscular injection (50 μl; 2 μg) of Fluc mRNA LNP or TBS buffer (negative control group) in the biceps of the hind limb. At different time points (4h, 24h, 48h) after injection, mice were placed in a bioluminescence scanner and luciferase expression was measured after intraperitoneal injection of luciferin. After 24h, the highest expression of muscle was observed, especially for the 10-fold increase in the expression of Fluc mRNA LNP at T0 (48h). Figure 3 A) and T5m( Figure 3 B) both for the liquid -80°C sample and for the lyophilized sample stored at a temperature of 2-8°C. It can be concluded that the lyophilized samples, especially the 2-8°C lyophilized samples and the liquid -80°C control sample, show comparable expression even after 5 months of storage.

[0170] References

[0171] Muramatsu H,Lam K,Bajusz C,LaczkóD,KarikóK,SchreinerP,Martin A,Lutwyche P,Heyes J,Pardi N,Lyophilization provideslong-term stability for alipid nanoparticle-formulated nucleoside-modified mRNA vaccine,MolecularTherapy(2022).

[0172] Zhao P.,Hou X.,Yan J.Long-term storage of lipid-likenanoparticles formRNA delivery.Bioact Mater.2020;5(2):358-363.

Claims

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 prior to lyophilization, the concentration of TRIS in the composition is 3 mM or less and the concentration of sucrose is less than 20% (w / v).

2. The composition according to claim 1, wherein Prior to lyophilization, the concentration of sucrose in the composition is at least about 5% (w / v), particularly at least about 8% (w / v), more particularly at least about 10% (w / v).

3. The composition according to claim 1, wherein Prior to lyophilization, the concentration of sucrose is about 5% to about 20% (w / v) and between 5% to about 20% (w / v), particularly about 5% to about 15% (w / v) and between 5% to about 15% (w / v), more particularly about 8% to about 15% (w / v) and between 8% to about 15% (w / v), even more particularly about 10% to about 15% (w / v) and between 10% to about 15% (w / v).

4. The composition according to any one of claims 1 or 3, wherein Prior to lyophilization, the concentration of TRIS in the composition is about 0.01 mM to about 3 mM and between 0.01 mM to about 3 mM, preferably about 0.1 mM to about 3 mM and between 0.1 mM to about 3 mM, more preferably about 0.5 mM and 3 mM and between 0.5 mM to 3 mM, most preferably about 1 mM to about 3 mM and between 1 mM to about 3 mM.

5. The composition according to any one of claims 1 or 4, wherein The one or more lipid nanoparticles comprise PEG lipids, particularly PEG2000 lipids, more particularly DMG-PEG2000.

6. The composition according to any one of claims 1 to 5, wherein The one or more lipid nanoparticles comprise between about 40 mol% and 60 mol% ionizable lipids, between about 5 mol% and 15 mol% phospholipids, between about 20 mol% and 40 mol% sterols, and at least 0.5 mol% PEG lipids.

7. The composition according to any one of claims 1 to 6, wherein Prior to lyophilization, the composition further comprises water, in particular water for injection (WFI).

8. The composition according to any one of claims 1 to 7, wherein The one or more RNA molecules are one or more linear or circular RNA molecules.

9. The composition according to any one of claims 1 to 8, wherein The one or more RNA molecules are one or more mRNA molecules.

10. The composition according to any one of claims 1 to 9 for use in human and / or veterinary medicine.

11. The composition according to any one of claims 1 to 10 which is reconstituted prior to administration to a subject in need thereof.

12. The composition according to claim 11, wherein The reconstitution is performed using water or an aqueous solution of saline, preferably TBS, more preferably TBS with 20 mM TRIS, most preferably TBS with 20 mM TRIS and 0.9% NaCl.

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

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

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

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