Lyophilized preparation of mRNA adsorbed on lipid nano-emulsion particles

Abstract

Lyophilized preparation of mRNA adsorbed on lipid nano-emulsion particles. The present invention relates to a lyophilized preparation of mRNA adsorbed on lipid nano-emulsion particles. In particular, it provides a method for lyophilizing a liquid preparation of mRNA adsorbed on lipid nano-emulsion particles under conditions that maintain the integrity and pharmaceutical properties of the lyophilized preparation produced for an extended period at a storage temperature of about 5°C.

Description

Technology Field

[0001] The present invention relates to lyophilized formulations of mRNA adsorbed on lipid nano-emulsion particles. In particular, it provides a method for lyophilizing a liquid formulation of mRNA adsorbed on lipid nano-emulsion particles under conditions that maintain the integrity and pharmaceutical properties of the lyophilized formulation produced for an extended period at a storage temperature of about 5°C. Background Technology

[0002] Recently, in many pharmaceutical applications related to therapy, nucleic acids themselves are used for therapeutic purposes. As an example, promising results have been shown in the field of mRNA-based therapy. Here, various types of mRNA molecules are considered important tools not only for gene therapy but also for prophylactic and therapeutic vaccination against many infectious and malignant diseases.

[0003] Both nucleic acid molecules, DNA and mRNA, have been widely used in gene therapy in either naked or complex forms. The use of mRNA is advantageous in modern molecular medicine due to several superior characteristics compared to the use of DNA. As is known, transfection with DNA molecules can lead to serious complications, but this risk does not occur, particularly when using mRNA. The advantages of using mRNA instead of DNA are that virus-derived promoter elements do not need to be administered in vivo, integration into the genome does not occur, and mRNA does not need to translocate to the nucleus for expression.

[0004] Conversely, a major disadvantage of mRNA is that it is unstable and degrades rapidly during the production and formulation stages, as well as before delivery into the cytoplasm of cells. The physicochemical stability of mRNA molecules in solution is very low compared to DNA. mRNA is susceptible to hydrolysis by ribonucleases or divalent cations and typically degrades rapidly within a few hours in solutions at room temperature. To avoid this rapid degradation of mRNA, it is typically stored at -80 to -20°C. However, these storage conditions are costly, particularly for transporting and storing large quantities of mRNA-based therapeutic or vaccine products.

[0005] However, in the field of sensitive biomolecules such as mRNA, lyophilization or freeze-drying methods are selected. Lyophilization typically removes moisture from a frozen sample through sublimation. During lyophilization, the sample is cooled below the freezing point of water and then frozen in a freezing cycle. Subsequently, moisture is removed by sublimation during a drying cycle. However, this freezing / drying of moisture can lead to crystal formation or loss of hydration around the biomolecule, which can further damage the biomolecule through various physicochemical means. Therefore, to prevent such damage, numerous lyoprotectants (also known as cryoprotectants) have been used for lyophilization purposes.

[0006] Preparations using mRNA as an API are inherently unstable in aqueous solutions. The shelf life of these preparations is only a few days at room temperature. To overcome these limitations in such preparations, freeze-drying is used. However, the freeze-drying process is difficult to predict, and if large-scale production of the preparations is required, conditions for each preparation must be determined experimentally. Although freeze-drying of mRNA preparations has been described under laboratory conditions, improved methods are still needed in the field. Specifically, there is a need for methods to industrially apply freeze-drying to complexes, such as mRNA molecules adsorbed onto lipid nano-emulsion particles or nano-carriers in liquids.

[0007] Accordingly, the object of the present invention is to provide a scalable, reproducible, time- and cost-efficient method for freeze-drying mRNA-based complex formulations. A further object of the present invention is to provide a freeze-dried formulation comprising mRNA molecules that is suitable for long-term storage at ambient temperature and, preferably, has increased storage stability compared to prior art formulations, wherein said mRNA molecules have pharmaceutical value.

[0008] explanation

[0009] The present invention relates to lipid nano-emulsion particles or nano-carriers for pharmaceutical applications such as vaccines and therapeutic agents. Adsorbed The present invention provides a method for freeze-drying a liquid formulation of mRNA molecules. Specifically, the present invention relates to lipid nano-emulsion particles or nano-carriers. Adsorbed The present invention relates to a method for freeze-drying mRNA, wherein the method comprises the following stages:

[0010] 1) comprising at least one mRNA and at least one lyophilized protective agent adsorbed on lipid nano-emulsion particles liquid mixtureA step of providing [the substance] to a glass vial, loading the glass vial into a freeze dryer chamber, and pre-cooling it for a desired temperature and time;

[0011] 2) Cool the above mixture to a freezing temperature at a desired cooling rate in the freeze dryer chamber, maintain it for a desired time, and then freeze the mixture at the freezing temperature. Frozen mixture A stage forming;

[0012] 3) A stage for reducing the pressure in the freeze-drying chamber to a pressure below atmospheric pressure in two desired depressurization steps and three desired heating steps, and for primary drying the frozen mixture;

[0013] 4) a step of further treating the frozen mixture in the freeze-drying chamber at a pressure below atmospheric pressure in a desired depressurization step and a desired heating step, and secondarily drying the frozen mixture to form a freeze-dried formulation comprising at least one mRNA adsorbed on lipid nano-emulsion particles and at least one freeze-drying protective agent in the glass vial; and

[0014] 5) A stage for stopping the glass vial, then equilibrating the freeze-drying chamber to atmospheric pressure and temperature under nitrogen gas, and removing the glass vial containing the freeze-drying agent for sealing.

[0015] In a preferred embodiment, the lyophilized preparation is stable at a temperature of about 5°C and can generate a functional immune response when injected into a subject. As specified herein, the phrase "at a temperature of about 5°C" means any temperature of 2 to 8°C regardless of the environment of use. Additionally, as specified herein, "liquid mixture" means the lyophilized or liquid preparation thereof of the vaccine prior to lyophilization.

[0016] In a preferred embodiment, the invention stages 1 to 5 as described above are performed sequentially. Furthermore, the stages may include one or more steps, which may be performed simultaneously or overlap.

[0017] The present invention discloses a method for producing a stable formulation by freeze-drying an mRNA formulation adsorbed onto lipid nano-emulsion particles in a liquid in the presence of a freeze-drying protective agent, preferably a carbohydrate freeze-drying protective agent selected from the group of carbohydrates consisting of mannitol, sucrose, glucose, mannose, or trehalose, under desired freezing and drying conditions, wherein the produced stable formulation possesses excellent integrity of mRNA after the completion of the freeze-drying process, and specifically, storage reliability is increased with respect to storage under long-term and non-cooling conditions. Furthermore, the invention is suitable for use on an industrial scale.

[0018] In the context of the present invention, freeze-drying mRNA adsorbed onto lipid nano-emulsion particles in a liquid in the presence of a freeze-drying protective agent was previously difficult and unreliable; however, the method disclosed herein induces these exceptional properties of the formulation according to the present invention. Furthermore, it is reproducible on an industrial scale and cost-effective. Additionally, the prior art has not proposed a method for freeze-drying adsorbed mRNA complexes or molecules under the freeze-drying conditions disclosed herein.

[0019] In the context of the present invention, stage 1) Provided by liquid mixtureThe formulation comprises at least one mRNA adsorbed on the surface of lipid nano-emulsion particles or nano-carriers in liquid together with a sugar that is a freeze-drying protective agent. Such mRNA complexes are produced by the method disclosed herein and comprise nano-emulsion particles containing cationic lipids in water prepared under controlled conditions. The surface of such particles is suitable for the adsorption of negatively charged single-strand mRNA molecules, and said particles are also referred to as nano-carriers.

[0020] In a preferred embodiment, the liquid mixture comprises a nano-carrier, the particles comprise or consist of at least one cationic lipid compound, and the complex preferably exists as a nanoparticle as defined herein. The nano-carrier optionally further comprises, together with one or more TLR4 agonist adjuvants, other components such as squalene and one or more surfactants such as polysorbate-80 or sorbitan monostearate in a buffered stabilized solution.

[0021] In a preferred embodiment, the liquid mixture provided in step 1) comprises at least one mRNA adsorbed onto lipid nano-emulsion particles, and the size of the nanoparticles, preferably the average size, is in the range of 50 to 500 nm, more preferably 50 to 300 nm. In a particularly preferred embodiment, the size of the nanoparticles is 50 to 150 nm.

[0022] In a preferred embodiment, the liquid mixture provided in step 1) of the method of the present invention comprises water as a solvent, which elutes additional components, such as a lyophilizing protective agent or a buffer and a desired excipient. In this invention, the water is preferably pyrogen-free water or water for injection (WFI).

[0023] The liquid provided in step 1) of the method of the present invention may include a buffer, for example, a buffer containing citric acid or a similar buffer. However, any other suitable buffering system may be used for a similar effect.

[0024] In a preferred embodiment, the liquid mixture provided in step 1) comprises at least one lyophilizing agent, said lyophilizing agent is selected from the group of carbohydrates. Such carbohydrates are suitable for the manufacture of pharmaceutical formulations and include, but are not limited to, disaccharides such as sucrose, trehalose, etc. Additionally, said sugar, such as trehalose, preferably has a low tendency to crystallize. Among the liquid mixture provided in step 1) of the method of the present invention, the lyophilizing agent is preferably selected from the group consisting of mannitol, sucrose, glucose, mannose, or trehalose.

[0025] The weight ratio of particles to freeze-drying protective agent, preferably carbohydrates, more preferably sugars, even more preferably sucrose, in the liquid mixture provided in stage 1) above is preferably in the range of 10 to 40%, more preferably 10 to 20%.

[0026] In one embodiment, the liquid mixture provided in step 1) of the method of the present invention contains at least one mRNA at a concentration of at least 300 μg / mL, preferably at least 10 μg / mL.

[0027] The liquid mixture provided in step 1) of the method of the present invention is preferably a liquid or semi-liquid formulation and comprises at least one mRNA and at least one lyophilized protective agent as defined herein. The at least one mRNA and at least one lyophilized protective agent are preferably dissolved in the liquid mixture provided in step 1), which comprises step 1A). In a preferred embodiment, the liquid mixture is an aqueous solution of at least one mRNA and at least one lyophilized protective agent, preferably comprising a solvent as defined herein. The liquid mixture used herein may also be a viscous solution, an emulsion, a dispersion, a suspension, etc. In step 1A), a vial containing a desired amount of the liquid mixture is loaded into the freeze-drying chamber of a freeze-dryer. The vial is then pre-cooled, preferably from about 25°C to about 5°C.

[0028] According to the method of the present invention, the liquid mixture provided in step 1) is introduced into a freeze dryer or the freeze-drying chamber of the freeze dryer. In this invention, the term 'freeze dryer' refers to a device for freeze-drying a liquid or semi-liquid formulation. Preferably, the freeze dryer used herein may be controlled for parameters characterizing the freeze-drying process, such as the temperature and pressure in the freeze-drying chamber containing the liquid to be freeze-dried. Such control is preferably performed in a semi-automatic or automatic manner, for example, by programming the device before the freeze-drying process begins, so that the device performs the desired freeze-drying process by applying, for example, specific predetermined steps (e.g., freeze, dry), and preferably transitions from one such step to another under predetermined temperature and pressure. Additionally, the freeze dryer preferably includes a freeze-drying chamber, wherein the atmosphere may be controlled, preferably by flooding the chamber with nitrogen.

[0029] According to a preferred embodiment, after step 1), the liquid mixture is applied to step 2) of the method in a freeze-drying chamber, wherein step 2A) comprises freezing the liquid mixture to a temperature below 5°C, preferably in the range of -40 to -60°C, most preferably in the range of -50 to -60°C, at a cooling rate of about 0.8 to 1.0°C / min. Next, the liquid mixture is maintained at a temperature in the range of -50 to -60°C for about 500 minutes, more preferably about 300 minutes. Additionally, steps 2A) and 2B) comprise introducing the liquid mixture into a freeze-drying chamber, wherein the pressure in the freeze-drying chamber is approximately equal to the pressure according to standard atmosphere (about 760 mTorr).

[0030] The method of the present invention further comprises a step 2) of cooling the liquid mixture to a freezing temperature, wherein the cooling is performed at a defined cooling rate [step 2A)] and a step [step 2B) of freezing the liquid mixture at a freezing temperature to obtain a frozen mixture.

[0031] In a preferred embodiment, the freezing temperature is a predetermined temperature. Regarding the quality of the freeze-dried product, it is important to select an appropriate freezing temperature. Specifically, the temperature of the frozen mixture containing one mRNA and a freeze-drying protective agent must be maintained below the collapse temperature of the liquid mixture. According to a preferred embodiment, in the method of the present invention, the freezing temperature is lower than the collapse temperature of the liquid mixture provided in stage 1) or the frozen mixture obtained in stage 2), respectively.

[0032] In a more preferred embodiment, the freezing temperature is equal to or lower than the glass transition temperature of the liquid mixture provided in stage 1) or the frozen mixture obtained in stage 2).

[0033] In relation to the method of the present invention, the temperatures defined herein typically refer to each temperature in a freeze-drying chamber. The temperature in the freeze-drying chamber is preferably determined by determining the temperature of the shelf temperature, the temperature of the liquid mixture provided in Stage 1), or the temperature of the frozen mixture obtained in Stage 2), each, or a part thereof. As used herein, the term 'shelf temperature' typically relates to a temperature measured through at least one probe, which is preferably located on the surface of the shelf. The temperature of the liquid mixture provided in Stage 1) and the temperature of the frozen mixture obtained in Stage 2), or a part thereof, preferably correspond to the respective shelf temperature.

[0034] In a preferred embodiment, the freeze-drying is controlled using a storage temperature as a parameter. For example, the freeze-drying process is typically programmed using the temperature defined herein as the storage temperature and exemplified in the table of the Examples section. Optionally, the actual temperature of the liquid mixture provided in Stage 1) and / or the frozen mixture obtained in Stage 2), or a portion thereof, may be measured directly, for example, while the production process is being established, in addition to the storage temperature.

[0035] According to a preferred embodiment, step 2) of the method of the present invention comprises the step of cooling the liquid mixture provided in step 1) to a freezing temperature, wherein the cooling rate is a defined rate. Preferably, the cooling rate in step 2) is less than 1.5 °C / min. Alternatively, the cooling rate in step 2) may be in the range of 0.05 to 1.0 °C / min.

[0036] In a specific embodiment of the method of the present invention, the freezing temperature is maintained for at least 200 minutes, more preferably for at least 300 minutes, and most preferably for at least 500 minutes.

[0037] The method of the present invention further comprises a step 3) of primary drying, wherein step 3) comprises a step of reducing the pressure in the freeze-drying chamber to a pressure below atmospheric pressure, approximately 100 mTorr [Step 3A)], and maintaining it at said pressure for a desired time [Step 3B)], followed by a step of increasing the temperature from about -60 to about -40°C at a rate of 0.05 to 0.09°C / min [Step 3C)], and maintaining it at said temperature for a desired time [Step 3D)]. In an additional subsequent step [Step 3E)], the pressure is reduced to about 75 mTorr, and the temperature is increased to about -20°C at a rate of 0.05 to 0.09°C / min (e.g., 0.07°C / min) and maintained at this position for a desired time [Step 3F)]. In the next step [step 3G)], the temperature is increased from about -20 to about 5°C at a rate of 0.06 to 0.1°C / min (e.g., 0.08°C / min) and maintained at the said temperature for a desired time [step 3H)]. According to a preferred embodiment, step 3) includes the step of reducing the pressure in the freeze-drying chamber to a pressure in the range of about 120 to about 70 mTorr.

[0038] Preferably, after freezing the liquid mixture provided in Stage 1), the pressure in the freeze-drying chamber is further reduced to a pressure below atmospheric pressure. In a preferred embodiment, the pressure in the freeze-drying chamber is reduced to a pressure below atmospheric pressure before or at the start of the drying process. Most preferably, the pressure is reduced before increasing the temperature.

[0039] The drying temperature is preferably lower than the decay temperature of the frozen mixture obtained in stage 2). More preferably, the drying temperature in stage 3) is lower than the glass transition temperature of the frozen mixture obtained in stage 2). In a specific embodiment, the drying temperature is in the range of about -70 to about 10°C, preferably about -60 to about 5°C. The overall drying rate from the freezing temperature to the final drying temperature is preferably in the range of 0.05 to 1.2°C / min.

[0040] The method of the present invention further comprises a secondary drying step 4), said step 4) comprising a step [step 4A)] of further reducing the pressure in a freeze-drying chamber to a pressure of about 35 mTorr, which is less than atmospheric pressure, while simultaneously increasing the temperature from 5 to 25°C at a rate of 0.05 to 0.09°C / min. Then maintaining it for a desired time [step 4B)]. According to a preferred embodiment, said step 4) comprises a step of further reducing the pressure in the freeze-drying chamber to a pressure in the range of about 50 to about 30 mTorr.

[0041] The method of the present invention further comprises a step 4), wherein step 4) further comprises the step of further reducing the pressure in the freeze-drying chamber to a pressure below atmospheric pressure and increasing the temperature to room temperature. Preferably, the pressure in the freeze-drying chamber is further reduced to a pressure below atmospheric pressure after drying the frozen mixture provided in step 3). In a preferred embodiment, the pressure in the freeze-drying chamber is further reduced to a pressure below atmospheric pressure prior to or at the start of a secondary drying process. Most preferably, the pressure is reduced before increasing the temperature.

[0042] The method of the present invention further comprises step 5), wherein the glass vial closure and vacuum break by nitrogen are achieved at room temperature, and the vial is recovered.

[0043] Changes in pressure as well as cooling and heating rates in stages 2), 3), and 4) affect the quality of the lyophilized formulation and are the core of the present invention disclosed herein for maintaining the integrity and / or biological activity of the mRNA complex formulation during lyophilization. According to a preferred embodiment, the heating rate in stages 2), 3), and 4) of the method of the present invention is therefore preferably in the range of 0.05 to 1.2 °C / min, more preferably in the range of 0.06 to 1.0 °C / min.

[0044] In a preferred embodiment, the disclosed method comprises at least two drying steps, namely a primary drying stage 3) and a secondary drying stage 4). In the primary drying stage 3), free, i.e., unbound water surrounding the mRNA forming a complex with the nano-carrier and other components is typically removed from the solution. Subsequently, in the secondary drying stage 4), thermal energy is applied to remove water bound at the molecular level to the mRNA complex. In both cases, the hydration sphere surrounding the mRNA complex is lost. Preferably, the primary drying stage 3) includes a step of heating the frozen mixture to a primary drying temperature, which is preferably lower than the secondary drying temperature at which the frozen mixture is heated in the secondary drying stage 4). More preferably, the pressure in the primary drying stage 3) ('primary drying pressure') is higher than the pressure in the secondary drying stage 4) ('secondary drying pressure').

[0045] According to an additional embodiment, step 3) includes a step of reducing the pressure in the freeze-drying chamber to a primary drying pressure, which is applied before or simultaneously with heating from the freezing temperature to the primary drying temperature, and is maintained or reduced during the primary drying stage 3); subsequently, the step of reducing the pressure in the freeze-drying chamber to a secondary drying pressure, which is applied before or simultaneously with heating from the primary drying temperature to the secondary drying temperature, and is maintained during the secondary drying stage 4).

[0046] The above first drying step 3) can be performed at a normal pressure of about 760 mTorr, or by lowering the pressure to the first drying pressure. Preferably, the first drying pressure is in the range of about 70 to 760 mTorr. In this first drying stage, the pressure is typically controlled by the application of a partial vacuum. Using a vacuum increases the sublimation rate, which is useful for a careful drying process. This stage is performed slowly to avoid applying a lot of heat and to avoid possible changes or damage to the composite structure in the frozen mixture.

[0047] In a preferred embodiment, the primary drying stage includes a step of adjusting the temperature to a primary drying temperature, which is preferably in the range of about -70 to about 10°C. As an additional alternative, the primary drying stage 3) is performed in several steps at the defined primary drying temperature and primary drying pressure.

[0048] Preferably, the temperature is reduced from the freezing temperature to the primary drying temperature at a defined cooling rate. More preferably, the temperature is increased in two primary drying stages [stages 3C) and 3E)], preferably from the freezing temperature to the primary drying temperature, at a cooling rate in the range of 0.04 to 0.09 °C / min, more preferably about 0.07 °C / min. The pressure is reduced from about 760 to about 100 mTorr in two primary drying stages [stages 3A) and 3E)], and then reduced from about 100 to about 35 mTorr. One embodiment of the temperature and pressure maintenance, as well as the cooling and heating cycles, is presented as an example in Table 2.

[0049] In the second drying stage 4), non-frozen water molecules bound to the mRNA complex are typically removed, as ice is generally removed in the first drying stage 3). In this second drying stage 4), the temperature is typically raised to about 25°C, which is higher than in the first drying stage 3), to destroy the physicochemical interactions formed between the water molecules and the frozen mixture. Additionally, the pressure may be lowered in this stage to facilitate desorption.

[0050] In a preferred embodiment, the secondary drying stage 4) includes the step of adjusting the temperature to a secondary drying temperature and / or the step of adjusting the pressure to a secondary drying pressure. In a specific embodiment, the secondary drying temperature is higher than the primary drying temperature and / or the secondary drying pressure is lower than the primary drying pressure. More preferably, the secondary drying temperature is in the range of about 5 to about 25°C. The pressure is preferably adjusted to a secondary drying pressure. The secondary drying pressure is preferably in the range of about 30 to about 50 mTorr.

[0051] After the completion of stage 4) of the method of the present invention, a lyophilized formulation is typically obtained comprising at least one mRNA and at least one lyophilized protective agent adsorbed on lipid nano-emulsion particles or nano-carriers.

[0052] In a preferred embodiment, after step 4), the glass vial is closed with a rubber stopper, and the freeze-drying chamber is filled with nitrogen or carbon dioxide gas. The glass vial containing the freeze-drying agent obtained according to the method of the present invention is closed before opening the freeze-drying chamber by equilibrating it to atmospheric pressure.

[0053] The lyophilized formulation obtained by the method of the present invention is stable for several months, particularly at about 5°C. The storage stability of the mRNA is typically determined by determining the relative (structural) integrity and biological activity after a given storage period.

[0054] The storage stability of the lyophilized formulations is described in Tables 5, 6, and 7. Briefly, lyophilized formulations containing 10%, 20%, or 40% sucrose were designated as formulations A, B, and C, respectively, and various bioanalytical tests were performed at five time points during storage of these formulations at approximately 5°C from day 0 to a maximum of 9 months. The physicochemical properties of mRNA formulations adsorbed onto lipid nano-emulsion particles or nano-carriers were tested as shown in Tables 5, 6, and 7; and Figures 1, 2, and 3. Four important parameters of the formulations—particle size, dispersion, mRNA content, and immunogenicity (IgG titer in mouse tests)—were compared. Lyophilized formulations containing approximately 20% sucrose as a lyophilized protective agent demonstrated the desired results along with the stability of various parameters over a period of approximately 9 months. The differences between these parameters were negligible when compared to storage at 0 days and 9 months at approximately 5°C. The integrity of the mRNA molecules of the above preparations was not impaired, and they exhibited excellent immunogenicity when tested in rat and hamster models with the production of sufficient IgG antibodies against the SARS-CoV-2 spike protein. Tables 5, 6, and 7 and Figures 1, 2, and 3 describe the characteristics of various lyophilized preparations prepared by the method of the present invention disclosed herein. Figures 4 and 5 describe the immunogenicity characteristics of the lyophilized preparations.

[0055] The lyophilized formulation obtainable by the method of the present invention can be stored at a temperature of about 5°C for a significantly longer period than the corresponding mRNA molecule in WFI or other injectable solutions. Specifically, the lyophilized formulation obtained by the method of the present invention can be stored at room temperature, thereby simplifying the transport and storage processes.

[0056] Preferably, the relative integrity of mRNA in the lyophilized preparation obtained by the method of the present invention is at least 70%, more preferably at least 90%, after storage at a temperature of about 5°C for at least 3 months, more preferably for at least 6 months, most preferably for at least 1 year.

[0057] More preferably, after storage at a temperature of about 5°C, the biological activity of the mRNA of the lyophilized preparation is preferably at least 70%, more preferably at least 90%, of the biological activity of the newly prepared mRNA, as defined above in relation to the relative integrity of the mRNA. The biological activity is preferably determined by analyzing the amount of protein expressed from each of the reconstituted mRNA and the newly prepared mRNA complexed on the nano-carrier, for example, after transfection into a mammalian cell line or a subject. Alternatively, the biological activity may be determined by measuring the induction of an immune response in the subject.

[0058] In a preferred embodiment, the lipid nano-emulsion particle or nano-carrier comprises a cationic lipid such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), a liquid lipid such as squalene, a hydrophobic surfactant such as sorbitan monostearate, a hydrophilic surfactant such as polysorbate-80, and a self-replicating mRNA molecule capable of expressing a SARS-CoV2 spike protein mutant or variant.

[0059] In an additional aspect, the present invention further provides the use of the method of the present invention in the manufacture of pharmaceutical preparations, or said lyophilized preparations can be used directly as pharmaceuticals.

[0060] Additionally, the lyophilized formulations of the present invention disclosed herein may comprise a pharmaceutically acceptable carrier and / or vehicle. In the context of the present invention, the pharmaceutically acceptable carrier typically comprises a liquid or non-liquid base of the pharmaceutical lyophilized formulation of the present invention. Brief explanation of the drawing

[0061] The drawings shown below are merely exemplary and will explain the invention in further ways. These drawings should not be interpreted as limiting the invention thereto. Fig. 1:Electrophoresis images show the integrity of mRNA after adsorption onto lipid nano-emulsion particles or nano-carriers present in the lyophilized formulation on day 0 after the lyophilization process. Lane 1 is an RNA molecular weight marker with a single-stranded RNA transcript, where the first band is 9 kb, the sixth band is 3 kb, and the tenth band is 0.5 kb. Lane 2 contains a purified self-replicating SARS-CoV-2 S-protein expression mRNA molecule of approximately 12 kb. Lane 5 is a liquid formulation containing 10% sucrose, where the RNA is mRNA bound to a nano-carrier immobilized in the well. Lane 6 is the same liquid formulation containing 20% ​​sucrose. Lane 7 is the same liquid formulation containing 40% sucrose. The samples in lanes 5 through 7 were maintained at approximately 25°C. Lane 9 is a lyophilized preparation containing 10% sucrose, where RNA is mRNA bound to a nano-carrier immobilized in a well. Lane 10 is the same lyophilized preparation containing 20% ​​sucrose. Lane 11 is the same lyophilized preparation containing 40% sucrose. Lane 13 is the mRNA solvent extracted from the lyophilized preparation containing 10% sucrose, where RNA is mRNA that has migrated to approximately 12 kb in the well. Lane 14 is the same extract preparation containing 20% ​​sucrose. Lane 15 is the same extract preparation containing 40% sucrose. The samples in lanes 9 through 11 and lanes 13 through 15 were stored at approximately 5°C prior to use. Lane 17 is a lyophilized preparation containing 10% sucrose, where RNA is mRNA bound to a nano-carrier immobilized in a well. Lane 18 is the same lyophilized preparation containing 20% ​​sucrose. Lane 19 is the same lyophilized preparation containing 40% sucrose. Lane 21 is the mRNA solvent extracted from the lyophilized preparation containing 10% sucrose, where the RNA is mRNA that has moved to approximately 12 kb in the well. Lane 22 is the same extract preparation containing 20% ​​sucrose.Lane 23 is the same extract preparation containing 40% sucrose. The samples in lanes 17 to 19 and lanes 21 to 23 were stored at approximately 25°C before use. Lane 25 is an extract liquid preparation containing 10% sucrose as a control. In all preparations, the nano-carrier used is a GNP type having self-replicating SARS-CoV-2 S-protein coding mRNA expressed in vitro from a plasmid template. Fig. 2:Electrophoresis images show the integrity of mRNA after adsorption onto lipid nano-emulsion particles or nano-carriers present in the lyophilized preparation on day 90 after the lyophilization process. Lane 1 is an RNA molecular weight marker with a single-stranded RNA transcript, a first band of 9 kb, a sixth band of 3 kb, and a tenth band of 0.5 kb. Lane 3 is a lyophilized preparation containing 10% sucrose, where the RNA is mRNA bound to a nano-carrier immobilized in the well. Lane 4 is the same lyophilized preparation containing 20% ​​sucrose. Lane 5 is the same lyophilized preparation containing 40% sucrose. Lane 6 is the mRNA solvent extracted from the lyophilized preparation containing 10% sucrose, where the RNA is mRNA that has migrated to a position of approximately 12 kb in the well. Lane 7 is the same extract containing 20% ​​sucrose. Lane 8 is the same extract containing 40% sucrose. The samples in lanes 3 through 8 were stored at approximately 25°C for 90 days prior to use. Lane 10 is a lyophilized preparation containing 10% sucrose, where the RNA is mRNA bound to a nano-carrier immobilized in the well. Lane 11 is the same lyophilized preparation containing 20% ​​sucrose. Lane 12 is the same lyophilized preparation containing 40% sucrose. Lane 13 is the mRNA solvent extracted from the lyophilized preparation containing 10% sucrose, where the RNA is mRNA that has migrated to approximately 12 kb in the well. Lane 14 is the same extract containing 20% ​​sucrose. Lane 15 is the same extract containing 40% sucrose. The samples in lanes 10 through 15 were stored at approximately 5°C for 90 days prior to use. In all formulations, the nano-carrier used is a GNP type containing self-replicating SARS-CoV-2 S-protein coding mRNA expressed in vitro from a plasmid template. Fig. 3:Electrophoresis images show the integrity of mRNA in lyophilized preparations treated with RNAse at the end of storage for 9 months at approximately 5°C. Lane 1 is an RNA molecular weight marker with a single-stranded RNA transcript, where the first band is 9 kb, the sixth band is 3 kb, and the tenth band is 0.5 kb. Lane 2 contains a purified, self-replicating SARS-CoV-2 S-protein expression mRNA molecule of approximately 12 kb that is untreated with RNase. Lane 3 contains the same mRNA molecule that has been degraded by treatment with RNase. Lane 4 is the untreated preparation A. Lane 5 is the treated preparation A. Lane 6 is mRNA extracted from the untreated preparation A. Lane 7 is mRNA extracted from the treated preparation A. Lane 8 is the untreated preparation B. Lane 9 is the treated preparation B. Lane 10 is mRNA extracted from the untreated preparation B. Lane 11 is mRNA extracted from the treated preparation B. Lane 12 is the untreated preparation C. Lane 13 is the treated preparation C. Lane 14 is mRNA extracted from the untreated preparation C. Lane 15 is mRNA extracted from the treated preparation C. Fig. 4: This shows the immune response generated by vaccine formulation B comprising the mRNA molecule and nano-carrier of the present invention disclosed herein. The SARS-CoV-2 spike protein-generating self-replicating mRNA construct was adsorbed onto a nano-carrier called GNP and injected into rats and hamsters to generate a potent IgG response. Fig. 5: The present invention demonstrates immune responses generated by various vaccine formulations comprising mRNA molecules and nano-carriers. The SARS-CoV-2 spike protein-generating self-replicating mRNA construct was adsorbed onto a nano-carrier called GNP and injected into mice to produce a potent IgG response as well as surrogate virus-neutralizing antibodies. Specific details for implementing the invention

[0062] Examples

[0063] The embodiments presented below are for illustrative purposes only and are intended to further explain the invention and should not be interpreted as limiting it.

[0064] Example 1: Preparation of mRNA adsorbed onto lipid nano-emulsion particles in a liquid

[0065] Methods for preparing self-replicating mRNA constructs and mRNA capable of expressing SARS-CoV-2 spike protein antigen [COVID-19 S protein antigen] and other similar antigens have been previously disclosed by the inventors. Similarly, methods for preparing lipid nano-emulsion particles or nano-carriers [also referred to herein as GNP] have been previously disclosed by the inventors. Briefly, the mRNA adsorbed on the nano-carrier in a liquid comprises a cationic lipid, such as DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), a liquid lipid, such as squalene, a hydrophobic surfactant, such as sorbitan monostearate, a hydrophilic surfactant, such as polysorbate-80, and a self-replicating mRNA molecule of interest [e.g., SARS-CoV-2 spike protein mutant expression] in a buffer, such as a citrate buffer, which forms a liquid formulation used in the freeze-drying method disclosed herein. The above nano-carrier or GNP optionally also contains an immunostimulator compound, such as a toll-like receptor 4 antagonist adjuvant, such as monophosphoryl lipid-A [MPL] or glucopyranosyl lipid-A [GLA] compound.

[0066] Example 2: Preparation and Characteristics of Liquid Formulation

[0067] The preparation of the nano-carrier or GNP was achieved through a three-part process. In the first part, an oil phase was prepared using all hydrophobic materials forming part of the carrier. Here, to prepare about 5 mL of the oil phase, about 3 g of DOTAP, about 3.7 g of sorbitan monostearate, and about 3.75 g of squalene were mixed in a glass container. The mixture was heated to about 65°C until all components were well mixed with uniform consistency. In the second part, about 3.7 g of polysorbate-80 was mixed with 90 mL of 10 mM sodium citrate, pH 6.0 buffer solution and kept warm at 65°C. In the third part, both the oil phase and the aqueous phase were mixed for about 15 minutes in a high-shear mixer operating at about 5000 RPM. Next, this mixture was passed through a high-pressure homogenizer about 10 times at about 30,000 psi and primed with the remaining aqueous phase to provide about 100 mL of a nano-carrier solution. The nano-carrier solution optionally contained an immunostimulator, such as MPL or GLA, in an amount of about 0.5 μg / mL if desired. While the nano-carrier GNP did not contain MPL or GLA, GNP-M contained an MPL adjuvant and GNP-G contained a GLA adjuvant [see Table 1].

[0068] The adsorption of mRNA molecules onto the nano-carrier was carried out through a very meticulous and precise process of mixing the nano-carrier solution with the mRNA solution to form a stable complex. Here, the ratio of nitrogen [present on the DOTAP molecule] to phosphate [present on the RNA molecule] [N:P ratio] was obtained by measuring the association of the mRNA molecules with the nano-carrier particles, as the mRNA molecules carry a negative charge and the DOTAP molecules carry a positive charge, thereby inducing the adsorption of the mRNA molecules onto the nano-carrier. To achieve a stable complex of the nano-carrier and the mRNA molecules, various N:P ratios of DOTAP to mRNA amount ranging from 1 to 150 were attempted, while the mRNA amount was kept constant. This led to an ideal N:P ratio of 5 to 15 for obtaining a stable complex of mRNA adsorbed onto the nano-carrier. Therefore, to prepare the above complex, the aforementioned nano-carrier solution was diluted to about 6 mg / mL of DOTAP using a 10 mM sodium citrate, pH 6.0 solution containing about 200 mg / mL of sucrose. Then, about 50 mL of this diluted nano-carrier solution was placed in a 1000 mL container and placed in a shaker rotating at about 180 RPM. Then, about 50 mL of the mRNA solution prepared in Example 3 was slowly added over about 10 minutes using a syringe pump under constant stirring conditions at a temperature of about 5 °C. Then, the mixture was allowed to form a complex at 5 °C for about 30 minutes, and then the complex solution was filtered through 0.45 μm and 0.22 μm membrane filters to obtain a sterile vaccine solution. To determine the amount of RNA molecules adsorbed on the nano-carrier particles and the changes in the characteristics of the nano-carriers, the average particle size and particle size distribution parameters were measured by dynamic light scattering using a Zetasizer Nano ZS system [Malvern Panalytical].Table 1 provides the changes observed in the above parameters of the nano-carrier upon adsorption of mRNA molecules.

[0069]

[0070] Example 3: Freeze-drying process and parameter optimization

[0071] A general scheme of a freeze-drying cycle for freeze-drying complex drug formulations such as those disclosed herein is presented in Table 2. Briefly, it comprises five stages with various temperature and pressure steps that effectively aid in freeze-drying liquid formulations of mRNA adsorbed on lipid nano-emulsion particles or nano-carriers into stable freeze-dried formulations for at least 6 months at a temperature of about 5°C. In initial experiments, the liquid formulations used herein were freeze-dried using a freeze-drying cycle having a 5-day process cycle. Once the workability of the freeze-drying process was established to obtain the desired results, the process cycle could be further shortened to a 3-day cycle, as disclosed in Tables 3 to 4A.

[0072]

[0073]

[0074]

[0075]

[0076] Example 4: Long-term stability and functional properties of lyophilized mRNA preparations

[0077] To determine the effects of long-term storage on lyophilized preparations of mRNA adsorbed onto lipid nano-emulsion particles or nano-carriers, the lyophilized preparation containing about 10% sucrose [Preparation A, see Table 5], the lyophilized preparation containing about 20% sucrose [Preparation B, see Table 6], or the lyophilized preparation containing about 40% sucrose [Preparation C, see Table 7] were stored at about 5°C for up to 9 months and tested for various physicochemical parameters listed in Tables 5, 6, and 7. Additionally, RNase protection assays were performed on the preparations to evaluate the integrity of the mRNA molecules present in the preparations [see Figures 1 to 3]. This data demonstrates the integrity and stability of the vaccine preparations under the disclosed storage conditions. The vaccine preparations contain mRNA transcripts capable of expressing the full-length SAR-CoV-2 spike protein in amounts of 5 to 300 μg / mL. In addition to other components of the above vaccine formulation, it comprises: 0.2 to 1.0 mg / mL of DOTAP; 0.2 to 1.0 mg / mL of squalene; 0.2 to 1.0 mg / mL of sorbitan monostearate; 0.2 to 1.0 mg / mL of polysorbate-80; and 0.2 to 1.0 mg / mL of citric acid monohydrate.

[0078]

[0079]

[0080]

[0081] Example 5: Immunogenicity Study in Rats and Hamsters

[0082] Immunogenicity studies were performed on the vaccine formulation obtained in Example 3 to determine the immunogenicity characteristics of mRNA molecules adsorbed to the nano-carrier and the immune response formed. Here, the vaccine formulation or the control group was injected into populations of Wistar rats and Syrian hamsters. The study on Wistar rats was conducted on 16 animals, with each group consisting of 8 rats. Group 1 was injected with 100 μL of the reconstituted lyophilized vaccine of Formulation B, with a total mRNA content of 5 μg, on Day 1, followed by an equal amount of booster dose on Day 29. Group 2 was injected with 100 μL of the reconstituted lyophilized vaccine of Formulation B, with a total mRNA content of 10 μg, on Day 1, followed by an equal amount of booster dose on Day 29. Immunogenicity was analyzed at four time points: before blood collection (2 days prior to priming injection), Day 14, Day 28, and Day 43. Immunogenicity analysis was performed using the standard indirect ELISA method. An exponential increase in immunogenic response was observed in both groups injected with doses of 5 μg and 10 μg of the lyophilized vaccine, respectively (see Figure 4A). The study on Syrian hamsters was conducted on 22 animals, with the control group consisting of 4 animals and the three test groups consisting of 6 animals. The control group was injected with 100 μL of GNP (nano-carrier alone) on Day 1, followed by the injection of the same amount of booster dose on Day 29. The first test group was injected with the reconstituted lyophilized mRNA vaccine of Preparation B containing 10 μg of total mRNA on Day 1, followed by the injection of the same amount of booster dose on Day 29. The second test group was injected with the reconstituted lyophilized mRNA vaccine of Preparation B containing 25 μg of total mRNA on Day 1, followed by the injection of the same amount of booster dose on Day 29.The third test group was injected alone with the priming dose of the reconstituted lyophilized vaccine of Preparation B, containing 25 μg of total mRNA, on Day 1, without a booster dose. Immunogenicity was analyzed at five time points: before blood collection (2 days prior to the priming injection), Day 14, Day 28, Day 43, and Day 56. Immunogenicity analysis was performed using the standard indirect ELISA method. In the control group injected with GNP, no significant increase in immune response was observed until Day 56. All three test groups showed a significant increase in immunogenicity on Day 14 compared to titers before blood collection. On Day 28, immunogenicity decreased slightly in the first test group, while the second test group maintained its immune response. The first and second test groups showed a significant increase in immune response on Day 43 and a slight decrease on Day 56. The third test group shows a decrease in titer from day 28 up to day 56. Therefore, it is evident that the above-mentioned lyophilized preparation of the mRNA vaccine is stable and immunogenic in various animal models [see Fig. 4B].

[0083] Example 6: Immunogenicity Study in Mice

[0084] The immunogenicity of the above mRNA vaccine preparation B was further tested in a C57BL / 6 mouse model. The study was conducted in groups of 32 animals, each group consisting of 8 animals. The first test group was injected with 100 μL of reconstituted lyophilized vaccine preparation B with a total mRNA content of 0.5 μg on day 1, followed by the injection of an equal amount of booster dose on day 29. The second test group was injected with 100 μL of reconstituted lyophilized vaccine preparation B with a total mRNA content of 2 μg on day 1, followed by the injection of an equal amount of booster dose on day 29. The third test group was injected with 100 μL of reconstituted lyophilized vaccine preparation B with a total mRNA content of 5 μg on day 1, followed by the injection of an equal amount of booster dose on day 29. In the fourth test group, 100 μL of reconstituted lyophilized vaccine preparation C with a total mRNA content of 10 μg was injected on day 1, followed by the injection of the same amount of booster dose on day 29. Immunogenicity was analyzed at three time points: days 14, 28, and 43. Immunogenicity analysis was performed using the standard indirect ELISA method. The first test group, injected with 0.5 μg of the vaccine, did not show a significant increase in titer from day 14 to day 43. A dose-dependent exponential increase in titer was observed in the test groups injected with doses of 2 μg, 5 μg, and 10 μg from day 14 to day 43 [see Fig. 5A].

[0085] Example 7: Lyophilized mRNA vaccine neutralizing antibody reaction

[0086] The SARS-CoV-2 Alternative Virus Neutralization Test (sVNT) was performed using the cPass SARS-CoV-2 Neutralization Antibody Detection Kit [Genscript]. The analysis was performed according to the manufacturer's protocol. Briefly, the samples were diluted 10-fold with dilution buffer. The diluted samples, along with the positive and negative controls provided in the kit, were incubated with an equal volume of the 1000-fold HRP-conjugated RBD supplied in the kit. Subsequently, incubation was performed at 37°C for approximately 30 minutes. Then, approximately 100 µL of all samples and controls were collected from the ACE-2 protein-coated wells provided with the kit. The reaction was carried out in the dark at 37°C for approximately 15 minutes. After 15 minutes, the wells were washed four times before adding 100 µL of the TMB substrate provided with the kit. The samples were allowed to develop color in the dark for 15 minutes before stopping the reaction with the 50 µL HCl solution provided with the kit. The plate was read at 450 nm on a plate reader. The inhibition percentage was calculated as (1 - (OD of sample / OD of negative control)) x 100%.

[0087] The neutralizing antibody response of the above lyophilized mRNA vaccine was tested in a C57BL / 6 mouse model. The study conducted in C57BL / 6 mice was performed in groups of 24 animals, with each group consisting of 6 animals. In the first test group, 100 μL of a liquid vaccine with a total mRNA content of 5 μg was injected on day 1, followed by the injection of an equal amount of booster dose on day 29. In the second group, 100 μL of reconstituted lyophilized vaccine preparation A (containing 10% sucrose) with a total mRNA content of 5 μg was injected on day 1, followed by the injection of an equal amount of booster dose on day 29. In the third test group, 100 μL of reconstituted lyophilized vaccine preparation B (containing 20% ​​sucrose) with a total mRNA content of 5 μg was injected on day 1, followed by the injection of an equal amount of booster dose on day 29. In the fourth test group, 100 μL of a reconstituted lyophilized vaccine formulation (containing 40% sucrose) with a total mRNA content of 5 μg was injected on Day 1, followed by the injection of the same amount of booster dose on Day 29. Neutralizing antibody responses were analyzed at four time points: before blood collection (2 days prior to priming injection), Day 14, Day 28, and Day 43. An increase in neutralizing antibody responses was observed in all test groups on Day 14 and Day 28 compared to the response before blood collection. On Day 43, a significant increase in neutralizing antibody responses was observed not only in the test group injected with the frozen mRNA vaccine, but also in the three test groups injected with lyophilized mRNA vaccine formulations containing 10%, 20%, and 40% sucrose, respectively (see Figure 5B).

Claims

Claim 1 A method for preparing a freeze-dried formulation of mRNA, the method comprising: (a) providing a liquid mixture having mRNA adsorbed on lipid nano-emulsion particles and a lyoprotectant to a glass vial, wherein the lipid nano-emulsion particles comprise at least one cationic lipid compound; (b) pre-cooling the liquid mixture to a temperature of 5 to 25°C in a freeze-dryer chamber; (c) freezing the liquid mixture to a freezing temperature of -70 to -45°C at a cooling rate of 0.07 to 1.2°C / min in the freeze-dryer chamber and maintaining it for a time of 200 to 500 minutes to form a frozen mixture; (d) reducing the pressure in the freeze-dryer chamber to a pressure below atmospheric pressure in two depressurization steps of 760 mTorr to 100 mTorr and 100 mTorr to 35 mTorr, and the temperature 0.05 to 0.05 under three heating steps from -60℃ to -40℃, from -40℃ to -20℃, and from -20℃ to 5℃.A method comprising: (e) a step of first drying the frozen mixture by increasing the heating rate to 9 ℃ / min and maintaining it for a time of 1000 to 1600 minutes; (e) a step of further heating the frozen mixture in the freeze-dryer chamber to a pressure further below atmospheric pressure from 75 mTorr to 35 mTorr and increasing the temperature from 5℃ to 25℃ to secondarily dry the frozen mixture thereby forming a freeze-dried preparation; and (f) a step of stopping the glass vial, then equilibrating the freeze-dryer chamber to atmospheric pressure and temperature under nitrogen gas, and removing the glass vial containing the freeze-dried preparation for pharmaceutical application, wherein the freeze-dried preparation exhibits stability for 30 to 300 days at a temperature of 2 to 8℃ while maintaining mRNA integrity and biological activity. Claim 2 A method according to claim 1, wherein the mRNA is an mRNA capable of expressing a protein molecule. Claim 3 A method according to claim 1, wherein the liquid mixture comprises the mRNA in an amount of 5 to 300 μg / mL. Claim 4 The method of claim 1, wherein the lipid nano-emulsion particles further comprise squalene, polysorbate-80, and sorbitan monostearate. Claim 5 The method of claim 1, wherein the freeze-drying protective agent is selected from the carbohydrate group consisting of mannitol, sucrose, glucose, mannose, or trehalose. Claim 6 A method according to claim 1, wherein the liquid mixture comprises a freeze-drying protective agent in an amount of 10 to 40 weight percent. Claim 7 The method of claim 1, wherein the freezing is performed at a cooling rate of 0.07 to 1.2 ℃ / min. Claim 8 A method according to claim 1, wherein, under the secondary drying step (e), the holding step at 25°C is maintained for a time of 400 to 700 minutes. Claim 9 A method according to claim 1, wherein, under the secondary drying step (e), the temperature increase is performed at a heating rate of 0.05 to 0.9 ℃ / min. Claim 10 The method of claim 1, wherein the freeze-dried formulation is stable for a period of 30 to 300 days at a temperature of 5°C. Claim 11 A method according to claim 1, wherein the lyophilized preparation exhibits immunogenicity in various animal models of mice, rats, and hamsters. Claim 12 The method of claim 1, wherein the lyophilized preparation maintains mRNA integrity in the presence of RNase treatment. Claim 13 A freeze-dried preparation obtained by the method of claim 1, wherein the mRNA forms a stable mRNA complex that maintains its integrity when stored for up to 300 days at a temperature of 5°C. Claim 14 A preparation according to claim 13, wherein the mRNA is a self-replicating replicon mRNA transcript. Claim 15 A method according to claim 6, wherein the liquid mixture comprises a freeze-drying protective agent in an amount of 20 to 40 weight percent. Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete

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