Atomization system, atomization method, inhalation solution, liquid medicine bin and combined product

Abstract

The invention relates to an atomization system, an atomization method, an inhalation solution, a liquid medicine bin and a combined product. The atomization system comprises an atomization assembly and a power assembly. The atomization assembly is used for atomizing inhalation solution and comprises an atomization core, the thickness of the atomization core is 50-200 microns, the number of micropores is 2-1000 / cm < 2 >, the average pore diameter of the micropores is 2-50 microns, and the surface roughness is smaller than or equal to 20 microns; the power assembly enables the inhaled solution and the atomization assembly to be extruded relatively to form aerosol particles. According to the atomization system, by optimizing the core parameters of gas atomization, the viscosity loss caused by atomization shearing is reduced, the atomization effect of the shear sensitive substance can be improved, meanwhile, the structural integrity of the shear sensitive substance is considered, and the use effect of the shear sensitive substance is ensured.

Description

Technical Field

[0001] The present application relates to the field of atomization inhalation of shear-sensitive substances and mucosal immunization technology, and in particular to an atomization system, an atomization method, an inhalation solution, a drug solution tank and a combination product. Background Art

[0002] As a new drug delivery system, inhalation preparations can achieve non-invasive drug administration, directly deliver drugs to the lung lesion area, form local high concentrations, without gastrointestinal damage and first-pass effect, achieve high bioavailability and reduce systemic toxic side effects, and effectively improve patient compliance. Therefore, the use of inhalation administration for respiratory disease drug delivery has extremely broad prospects and application potential.

[0003] As a widely used inhalation preparation, inhalation solution needs to be jointly developed with the nebulization system. The compatibility verification of the inhalation drug-device combination is a key factor affecting the drug lung delivery efficiency and therapeutic effect. Compared with conventional inhalation preparations, respiratory disease drugs often contain shear-sensitive substances. It is necessary to develop a safe and effective nebulization method and nebulization system that can meet the aerosol transmission characteristics and shear-sensitive substances. Summary of the invention

[0004] Based on this, some embodiments of the present application provide a nebulization system that can be used for the nebulization of shear-sensitive substances, reduce the viscosity loss of nebulization shear, take into account the deep lung delivery efficiency and biological activity of shear-sensitive substances, and ensure the use effect of shear-sensitive substances after inhalation.

[0005] In addition, other embodiments of the present application also provide a nebulization method, an inhalation solution, a drug solution tank, and a nebulization drug-device combination product.

[0006] An atomization system, comprising:

[0007] An atomizer assembly, the atomizer assembly is used for atomizing an inhalation solution, the atomizer assembly comprises an atomizer core, the atomizer core has a thickness of 50 μm to 200 μm, a number of micropores of 2 / cm2 to 1000 / cm2, an average pore size of the micropores of 2 μm to 50 μm, and a surface roughness of ≤20 μm;

[0008] A power component is used to cause the inhalation solution and the atomization component to be relatively squeezed to form aerosol mist particles.

[0009] In some embodiments, the atomizer core satisfies at least one of the following conditions:

[0010] (1) The number of micropores of the atomizer core is ≥ 100 per square centimeter;

[0011] (2) The average pore size of the micropores of the atomizer core is 2 μm to 15 μm;

[0012] (3) The surface roughness of the atomizer core is ≤5 μm.

[0013] In some embodiments, the power assembly satisfies at least one of the following conditions:

[0014] (1) The mechanical thrust speed of the power component directly acting on the inhaled solution is 20 mm / s to 500 mm / s;

[0015] (2) The power assembly can drive the atomizer core to vibrate at a frequency of 80 kHz to 350 kHz.

[0016] In some embodiments, the inhalation solution includes a shear sensitive substance.

[0017] In some embodiments, the following conditions are met at the same time:

[0018] (1) The aerodynamic mass median diameter of the aerosol particles generated by the inhalation solution after atomization by the atomization system is 2 μm to 8 μm;

[0019] (2) After the inhalation solution is atomized by the atomization system, the percentage of particles smaller than 5 μm in the aerosol mist is 40% to 75%;

[0020] (3) The inhalation solution includes protein particles, which are virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles. The recovery rate of the recombinant protein antigen in the aerosol mist particles generated after the inhalation solution is atomized by the atomization system is 55% to 95%.

[0021] In some embodiments, in the inhalation solution, the concentration of the recombinant protein antigen is 100 μg / mL to 1 mg / mL, the average pore size of the micropores of the atomization core is 2 μm to 15 μm; the aerodynamic mass median diameter of the aerosol mist particles generated after the inhalation solution is atomized by the atomization system is 3.3 μm to 5.4 μm, and the recovery rate of the recombinant protein antigen in the aerosol mist particles is 82% to 87%; or,

[0022] In the inhalation solution, the concentration of the recombinant protein antigen is 1 mg / mL to 2 mg / mL, and the average pore size of the micropores of the atomization core is 10 μm to 15 μm; the aerodynamic mass median diameter of the aerosol mist particles generated after the inhalation solution is atomized by the atomization system is 3.6 μm to 5.9 μm, and the recovery rate of the recombinant protein antigen in the aerosol mist particles is 77% to 89%.

[0023] In some embodiments, the material of the atomizer core is selected from one or more of plastic materials, metal materials and inorganic non-metallic materials.

[0024] In some embodiments, the material of the atomizer core includes one or more of polypropylene, stainless steel and silicon nitride.

[0025] In some embodiments, the atomization system can achieve a single quantitative atomization inhalation solution volume of 15 μL to 200 μL.

[0026] A method for atomizing an inhalation solution comprises the following steps:

[0027] Obtaining the inhalation solution and the above-mentioned atomization system;

[0028] The power component of the atomization system is used to cause relative squeezing of the inhalation solution and the atomization component to form aerosol mist particles.

[0029] In some of the embodiments, the step of using the power component of the atomization system to cause the inhalation solution and the atomization component to be relatively squeezed to form aerosol mist particles includes: using the power component to drive the atomization core of the atomization component to move, the movement causes the atomization core to generate pressure on the inhalation solution, and the inhalation solution is relatively squeezed with the atomization core in the process of passing through the micropores of the atomization core, thereby being converted into the aerosol mist particles.

[0030] In some embodiments, the atomization method satisfies at least one of the following conditions:

[0031] (1) During the atomization process, the mechanical thrust speed of the power component of the atomization system is 20 mm / s to 500 mm / s;

[0032] (2) During the atomization process, the atomization core of the atomization system vibrates at a frequency of 80 kHz to 350 kHz.

[0033] In some embodiments, the atomization method satisfies at least one of the following conditions:

[0034] (1) During the atomization process, the mechanical thrust speed of the power component of the atomization system is 200 mm / s to 500 mm / s;

[0035] (2) During the atomization process, the atomization core of the atomization system vibrates at a frequency of 200 kHz to 350 kHz.

[0036] In some embodiments, at least one of the following conditions is met:

[0037] (1) The aerodynamic mass median diameter of the aerosol mist particles after atomization is 2.7 μm to 5.5 μm;

[0038] (2) The recovery rate of the protein in the aerosol mist particles after atomization is 60% to 90%;

[0039] (3) The effective atomization rate is 0.08 g / min to 0.35 g / min;

[0040] (4) The inhalation coordination between the aerosol delivery process and the user's inhalation process is ≥ 90%;

[0041] (5) Atomization residue ≤ 5%.

[0042] In some embodiments, during the atomization process, the mechanical thrust speed of the power assembly is 200 mm / s to 500 mm / s, the operating pressure of the atomization core with an average pore size of 2 μm subjected to the inhaled solution is 66.67 bar to 166.67 bar, the aerodynamic mass median diameter of the aerosol mist particles after atomization is 2.7 μm to 4.3 μm, and the recovery rate of the protein in the aerosol mist particles after atomization is 63.15% to 87.39%; or,

[0043] During the atomization process, the mechanical thrust speed of the power assembly is 200 mm / s to 500 mm / s, the operating pressure of the atomization core with an average pore size of 15 μm subjected to the inhaled solution is 13.33 bar to 33.33 bar, the aerodynamic mass median diameter of the aerosol mist particles after atomization is 3.6 μm to 5.5 μm, and the recovery rate of the protein in the aerosol mist particles after atomization is 66.89% to 86.26%; or,

[0044] The inhalation solution includes protein particles, which are virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles. During the atomization process, the atomization core of the atomization system vibrates at a frequency of 80kHz to 350kHz, the concentration of the recombinant protein antigen is 100μg / mL to 1mg / mL, the atomization rate of the inhalation solution is 0.356±0.021g / min to 0.709±0.063g / min, the aerodynamic mass median diameter of the aerosol mist particles generated by the inhalation solution after atomization by the atomization system is 3.4μm to 5.1μm, the percentage of particles smaller than 5μm in the aerosol mist particles is 54% to 72%, and the recovery rate of the recombinant protein antigen in the aerosol mist particles is 64% to 86%; or,

[0045] During the nebulization process, the atomization core of the atomization system vibrates at a frequency of 80kHz to 350kHz, the concentration of the recombinant protein antigen is 1mg / mL to 2mg / mL, the atomization rate of the inhalation solution is 0.243±0.028g / min to 0.484±0.076g / min, the aerodynamic mass median diameter of the aerosol particles generated after the inhalation solution is atomized by the atomization system is 4.2μm to 5.3μm, the percentage of particles smaller than 5μm in the aerosol particles is 49% to 66%, and the recovery rate of the recombinant protein antigen in the aerosol particles is 52% to 76%.

[0046] An inhalation solution for implementing the atomization method as described above;

[0047] The inhalation solution includes protein particles, which include virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles; the average particle size of the protein particles is 5nm to 2000nm, and the concentration of the recombinant protein antigen in the inhalation solution is 20μg / mL to 2mg / mL.

[0048] In some embodiments, the protein particle includes the virus-like particle, and the virus-like particle satisfies at least one of the following conditions:

[0049] (1) The average particle size of the virus-like particles is 20 nm to 200 nm;

[0050] (2) the concentration of the recombinant protein antigen in the inhalation solution is 20 μg / mL to 1000 μg / mL;

[0051] (3) The particle size polydispersity coefficient of the virus-like particles is 0.2 to 0.4.

[0052] In some embodiments, the protein particles include the complex particles, and the complex particles meet at least one of the following conditions:

[0053] (1) The average particle size of the composite particles is 5 nm to 2000 nm;

[0054] (2) The concentration of the recombinant protein antigen in the inhalation solution is 0.1 mg / mL to 2 mg / mL.

[0055] In some of the embodiments, at 25°C and standard atmospheric pressure, the surface tension of the inhalation solution is 40mN / m to 70mN / m; and / or, at 25°C and standard atmospheric pressure, the viscosity of the inhalation solution is 0.9mPa.S to 3mPa.S.

[0056] In some embodiments, at least one of the following conditions is met:

[0057] (1) The recombinant protein antigens include influenza virus subtype-specific hemagglutinin and neuraminidase, parainfluenza virus F protein and HN protein subtypes, norovirus capsid different subtypes, novel coronavirus spike protein or respiratory syncytial virus F protein and G protein;

[0058] (2) The recombinant protein antigen is a dimer or trimer of a single antigen, or the recombinant protein antigen is a heteropolymer;

[0059] (3) The virus-like particles are non-covalently self-assembled into a 12- to 15-mer high-order structure based on the oligomeric subunits of the recombinant protein antigen;

[0060] (4) The complex particles are assembled into lipid-protein nanoparticles, polymer-protein nanoparticles or inorganic-protein nanoparticles based on the oligomeric subunits of the recombinant protein antigen by nanotechnology;

[0061] (5) The purity of the recombinant protein antigen is 95% to 98%.

[0062] In some of the embodiments, at least one of emulsification, heat induction, pH induction, solvent distillation, fluid control, and material design methods is used to cause the oligomeric subunits of the recombinant protein antigen to self-assemble to form the virus-like particles, or the oligomeric subunits of the recombinant protein antigen are assembled with lipids, polymers, or inorganic particles to form the complex particles.

[0063] In some embodiments, the lipid comprises one or more of phospholipids, cholesterol and medium-chain glycerides, wherein the number of carbon atoms of the medium-chain glycerides is 6 to 12; and / or,

[0064] The polymer includes one or more of PLGA and PEI; and / or,

[0065] The inorganic particles include one or more of gold, iron oxide and silicon dioxide.

[0066] In some embodiments, the inhalation solution is atomized and then inhaled through the nasal cavity or oral cavity for respiratory mucosal immunization.

[0067] A medicine liquid tank is used to store the above-mentioned inhalation solution, and the medicine liquid tank is used to be detachably connected to the above-mentioned atomization system.

[0068] In some of the embodiments, the drug liquid tank can achieve a single quantitative atomization inhalation solution volume of 15 μL to 200 μL.

[0069] A nebulizer drug-device combination product comprises the above-mentioned nebulizer system, the above-mentioned inhalation solution and the above-mentioned drug solution tank.

[0070] The present application has found that shear-sensitive substances are affected by physical and chemical factors during the atomization process. For example, the average pore size, thickness, surface roughness, and number of micropores of the atomization core in the atomization system will affect the shear force received during the atomization process of the inhaled solution. This will in turn affect the atomization efficiency, aerosol mist particle distribution, and biological activity of the aerosol-state inhaled solution, thereby affecting the pulmonary delivery efficiency and use effect of the shear-sensitive substance. Based on this, some embodiments of the present application provide an atomization system, which includes an atomization component and a power component. By optimizing the core parameters of aerosolization, it can be used for the atomization of shear-sensitive substances, reduce the viscosity loss of atomization shear, and make the shear force received during the atomization process of the inhaled solution suitable. While ensuring the structural integrity and activity retention (high recovery rate) of the aerosol-state shear-sensitive substance, it can reduce the particle size of the aerosol mist particles after atomization, increase the deposition rate of target sites such as the bronchial tubes of the lungs, and improve the use effect of the shear-sensitive substance. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0072] Figure 1 A schematic diagram of the structure of an atomization system in some embodiments of the present application;

[0073] Figure 2 A schematic diagram of a process flow of an atomization method according to some embodiments of the present application;

[0074] Figure 3 The graph is a result of atomization rate of inhalation solutions of different concentrations;

[0075] Figure 4 This is a comparison chart of the atomization rate of the inhaled solution under different numbers of micropores;

[0076] Figure 5 This is a comparison chart of the atomization rate of the inhaled solution under different surface roughness;

[0077] Figure 6 This is a comparison chart of the atomization rate of the inhaled solution at different atomizer core thicknesses;

[0078] Figure 7 The figure is a comparison of the recovery rate of the recombinant protein antigen in the aspirated solution under different micropore numbers;

[0079] Figure 8 This is a comparison chart of the recovery rate of recombinant protein antigen in the aspirated solution under different surface roughness;

[0080] Fig. 9 This is a comparison chart of the recovery rate of recombinant protein antigen in the inhaled solution under different atomizer core thicknesses;

[0081] Fig.10 The results of protein immunoblotting of the inhaled solution under different micropore numbers, surface roughness and atomizer core thickness;

[0082] Fig.11 The graph shows the atomization rate of the inhaled solution and the recovery rate of the recombinant protein antigen at different atomization core vibration frequencies;

[0083] Fig.12 The figure is the aerodynamic evaluation result of the aerosol mist particles after the inhalation solution is atomized at different atomizer core vibration frequencies;

[0084] Fig.13 The graph shows the atomization rate of the inhaled solution and the recovery rate of the recombinant protein under the average pore diameter of the micropores of different atomizer cores;

[0085] Fig.14 The figure is the aerodynamic evaluation result of the aerosol mist particles after the inhalation solution is atomized under the average pore size of the micropores of different atomizer cores;

[0086] Fig.15 This is the relationship between aerosol particle distribution and recovery rate under different atomization processes;

[0087] Fig.16 This is the result diagram of the inhalation coordination of the atomization system;

[0088] Fig.17 The graph shows the antibody titer of different tissue samples at different times. DETAILED DESCRIPTION

[0089] In order to facilitate the understanding of the present application, the present application will be described more comprehensively below in conjunction with the specific embodiments. The preferred embodiments of the present application are provided in the specific embodiments. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0090] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present application belongs. The terms used herein in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0091] Unless otherwise specified or there is a contradiction, the terms and phrases used in this application have the following meanings:

[0092] In this application, "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include at least one of the features.

[0093] In the description of the present application, “plurality” means at least two, for example, two, three, etc., unless otherwise clearly and specifically defined.

[0094] In this application, "one or several" refers to any one, any two or more of the listed items. Among them, "several" refers to any two or more of the listed items.

[0095] In this application, the percentage concentration involved, unless otherwise specified, refers to the final concentration. The final concentration refers to the proportion of the added component in the system after the addition of the component.

[0096] In this document, "further", "further", "particularly", "for example", "such as", "example", "for example", etc. are used for descriptive purposes, indicating that the previous and subsequent technical solutions are related in terms of the content covered, but should not be understood as a limitation on the previous technical solution, nor can it be understood as a limitation on the protection scope of this document. In this document, unless otherwise specified, A (such as B) means that B is a non-limiting example of A, and it can be understood that A is not limited to B.

[0097] Herein, "optionally", "optional", and "optional" mean optional, that is, any one of the two parallel schemes of "yes" or "no". If multiple "optional" items appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" item is independent of each other. In this application, descriptions such as "optionally contain", "optionally include", etc. mean "contain or not contain". "Optional component X" means that component X exists or does not exist, or means that component X is contained or not contained.

[0098] When a numerical range is disclosed in this application, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed in this application should be understood to include any and all subranges included therein.

[0099] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0100] The terms "including" and "having" and any variations thereof in the embodiments of the present application are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or device that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units that are not listed, or may optionally include other steps or components that are inherent to these processes, methods, products, or devices.

[0101] Reference to "embodiments" in this application means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described in this application may be combined with other embodiments.

[0102] In the flowchart of the present application, it should be understood that although the various steps in the flowchart are displayed in sequence as indicated by the arrows, these steps are not necessarily performed in the order indicated by the arrows. Unless otherwise clearly stated in the text, there is no strict order restriction on the execution of these steps. They can be performed in other orders, and at least part of the steps in the figure may include multiple sub-steps or multiple stages. These sub-steps or stages are not necessarily performed at the same time, but can be performed at different times. The order of execution is not necessarily sequential, but can be performed in turn or alternating with other steps or at least part of other sub-steps or stages.

[0103] Vaccines come in different forms, including inactivated split vaccines, influenza subunit vaccines, and live attenuated influenza vaccines. Among them, inactivated split vaccines are derived from chicken embryos. The vaccine ingredients produced by chicken embryos are complex, and the purity of purification is directly related to immunogenicity and safety. It is easy to cause allergic reactions and cannot be used for people who are allergic to egg protein. The production process is heavily dependent on the supply of chicken embryos. The source of vaccine strains is limited by the attenuation, reassortment, and distribution of the strains. The production process uses live viruses, which has certain biosafety requirements for GMP (cleanse wall) workshops and is costly.

[0104] The influenza subunit vaccine is further purified on the basis of the chicken embryo lysate vaccine, removing the internal protein of the virus, enriching the HA (hemagglutinin) and NA (neuraminidase) components, and enhancing the theoretical safety. This technology also has the following problems: on the one hand, it leads to reduced immunogenicity of the subunit vaccine and dependence on adjuvants. On the other hand, the production cost of subunit vaccines is also relatively high, which is not conducive to popularization and application in large-scale vaccination. The attenuated vaccine is prepared from attenuated strains obtained by genetic engineering or natural selection, which more effectively simulates natural infection and stimulates a stronger immune response. However, it still has the following problems: first, there is a risk of mutation, toxicity and transmission, which is especially not suitable for people with low immune function and immunodeficiency. Secondly, the production process is complicated and the production cycle is long, and more control steps are required to ensure the stability and consistency of the virus strain.

[0105] At present, most vaccines are mainly administered by intramuscular injection, which is often accompanied by adverse reactions such as muscle soreness and swelling, and local discomfort is greater, and patient compliance is poor. Intramuscular injection is mainly based on humoral immunity and relies on enhancers such as adjuvants, which may cause local or systemic side effects. In addition, intramuscular injection also has the risk of cross-contamination, high vaccination requirements, and cumbersome procedures, which affect vaccination efficiency.

[0106] Therefore, some researchers have developed non-injectable, low-adjuvant-dependent mucosal immunization vaccines, most of which are live attenuated vaccines, such as oral polio vaccine (OPV) and nasal influenza vaccine (LAIV) products. The mucosal vaccination route can simulate the natural infection process, and the immunization process is simpler and more economical. It has been confirmed in the oral enteral immunization route. It not only provides individual protection, but also can establish a herd immunity barrier in the community. At present, the vaccination route for local respiratory tract immunization is the nasal cavity, which is more suitable for sensitive populations and specific influenza strains, and fails to cover the lower respiratory tract and lungs. The lower respiratory tract and lungs are more in line with the immunization sites of natural respiratory infection. In addition, live attenuated vaccines have infection risks and production process impurity risks.

[0107] As research deepens, inhalation preparations as a strategy for delivering vaccines for respiratory diseases have shown extremely broad prospects and application potential. The world's first inhalation vaccine uses a recombinant new coronavirus vector as an antigen. Without changing the injection formula, it uses a nebulizer administration technology. The vaccine is atomized into tiny particles and inhaled into the lungs through the mouth to complete the vaccination. However, this method still has the following problems: Compared with conventional inhalation preparations, protein vaccines are sensitive to antigen structure and activity. During the atomization process, the integrity of their spatial conformation is easily affected by physical and chemical factors, resulting in the integrity of their protein structure and biological activity being affected. In addition, the size and deposition distribution of aerosol mist particles and the stability of their components need to meet the requirements for entry into the lungs, which places high demands on atomization technology. Equipment that can simultaneously meet the atomization performance requirements and maintain the activity of the vaccine is expensive, not portable, and depends on the facility configuration and management process of the vaccination site.

[0108] Therefore, there is a lack of systematic combination products for inhalable vaccines, and there is an urgent need to improve the preparation technology and atomization methods of inhalable vaccines. This atomization method can also be used for the atomization of other shear-sensitive substances.

[0109] Based on this, some embodiments of the present application provide an inhalation solution and a nebulization system for realizing efficient nebulization inhalation thereof, which can reduce the particle size of aerosol mist particles, increase the lung deposition rate, and enhance the therapeutic effect of shear-sensitive substances while ensuring a high recovery rate of aerosol shear-sensitive substances (structural integrity and activity retention).

[0110] Specifically, the first aspect of the present application provides an inhalation solution, including protein particles, the protein particles including virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles. The average particle size of the protein particles is 5nm to 2000nm. In the inhalation solution, the concentration of the recombinant protein antigen is 20μg / mL to 2mg / mL.

[0111] Some embodiments of the present application use genetically engineered yeast to solve the production disadvantages of recombinant protein antigens, and use virus-like self-aggregation systems of different subtypes of influenza antigens to solve the weak immune response of nanoantigens, thereby providing a safer and more effective technical route for airway mucosal immunity by inhalation vaccination. In addition, the protein particles of some embodiments of the present application have specific immunogenicity and can simulate the natural infection process without relying on adjuvants and other enhancers to stimulate specific immune responses.

[0112] In some embodiments, the genetically engineered cell host of the recombinant protein antigen includes at least one of yeast cells (such as Saccharomyces cerevisiae), insect cells (such as Spodoptera frugiperda Sf9 cells), mammalian cells (such as CHO cells), or plant cells.

[0113] Specifically, the recombinant protein antigen is a protein of a specific part of a pathogen produced by a genetically engineered cell host, and the post-translational glycosylation modification is intact.

[0114] In some embodiments, the recombinant protein antigen includes any one of influenza virus subtype-specific hemagglutinin and neuraminidase, parainfluenza virus F protein and HN protein subtypes, norovirus capsid different subtypes, novel coronavirus spike protein, or respiratory syncytial virus F protein and G protein. In the inhalation solution, the above-mentioned recombinant protein antigen is used so that the inhalation solution can be used for the treatment and prevention of different respiratory diseases.

[0115] In some embodiments, the recombinant protein antigen may include a dimer or trimer of a single antigen. Alternatively, the recombinant protein antigen may include a heteromultimer.

[0116] In some of the embodiments, the purity of the recombinant protein antigen is 95% to 98%.

[0117] Specifically, the virus-like particles can be non-covalently self-assembled into a 12-15-mer high-order structure based on the oligomeric subunits of the recombinant protein antigen. The complex particles are assembled into lipid-protein nanoparticles, polymer-protein nanoparticles or inorganic-protein nanoparticles through nanotechnology based on the oligomeric subunits of the recombinant protein antigen.

[0118] It is understood that oligomers refer to complexes formed by two or more protein monomers through non-covalent and / or covalent interactions, including dimers, trimers, tetramers, polymers, etc. Taking virus-like particles as an example, when the recombinant protein antigen includes a dimer or trimer of a single antigen, the virus-like particles are obtained by non-covalent self-assembly based on the subunits of the dimer or trimer of the recombinant protein antigen. When the recombinant protein antigen includes a heteropolymer, the virus-like particles are obtained by non-covalent self-assembly based on the subunits of the heteropolymer of the recombinant protein antigen.

[0119] The above-mentioned recombinant protein antigen is used to form virus-like particles through self-assembly or to assemble with other components to form complex particles, which can be used in the treatment and prevention of different respiratory diseases.

[0120] In some of the embodiments, at least one of emulsification, heat induction, pH induction, solvent distillation, fluid control, and material design methods is used to cause the oligomeric subunits of the recombinant protein antigen to self-assemble into virus-like particles, or the oligomeric subunits of the recombinant protein antigen to assemble with lipids, polymers, or inorganic particles to form complex particles.

[0121] By adopting the above method, the oligomeric subunits of the recombinant protein antigen can be self-assembled to form virus-like particles or assembled with other substances to form complex particles.

[0122] It can be understood that the above only provides a few ways to form virus-like particles or complex particles, but is not limited thereto.

[0123] Specifically, the lipid may include one or more of phospholipids, cholesterol and medium chain glycerides. The carbon number of medium chain glycerides is 6 to 12. The polymer includes one or more of polylactic acid-co-glycolic acid (PLGA) and polyethyleneimine (PEI). The inorganic particles include one or more of gold, iron oxide and silicon dioxide.

[0124] The above substances can be assembled with oligomeric subunits of recombinant protein antigens to form different complex particles.

[0125] In some embodiments, the average particle size of the protein particles is 5nm to 2000nm, and the concentration of the recombinant protein antigen in the inhalation solution is 20μg / mL to 2mg / mL. For example, the average particle size of the protein particles can be, but is not limited to, 5nm, 10nm, 50nm, 80nm, 100nm, 150nm, 200nm, 400nm, 800nm, 1000nm, 1500nm, 2000nm, or a range consisting of any two of these values. In the inhalation solution, the concentration of the recombinant protein antigen can be, but is not limited to, 20μg / mL, 50μg / mL, 100μg / mL, 200μg / mL, 500μg / mL, 800μg / mL, 1000μg / mL, 1500μg / mL, 1800μg / mL, 2000μg / mL, or a range consisting of any two of these values.

[0126] In some of the embodiments, the average particle size of the virus-like particles is 20nm to 200nm. The average particle size of the virus-like particles is within the above range, the particle size is small, and in the actual atomization process, it is not easily affected by the shear of the micropores of the atomization core. Optionally, the average particle size of the virus-like particles is 20nm to 100nm. Furthermore, the particle size polydispersity coefficient of the virus-like particles is 0.2 to 0.4. The particle size polydispersity coefficient of the virus-like particles is small, and the particle size distribution is relatively uniform.

[0127] Further, the protein particles include virus-like particles, and the concentration of the recombinant protein antigen in the inhalation solution is 20μg / mL to 1000μg / mL. The concentration of the recombinant protein antigen is within the above range, has a suitable surface tension and viscosity, and the shear force required to form tiny droplets during the atomization process is suitable, which can ensure the integrity of the protein activity and structure. Further, the protein particles include virus-like particles, and the concentration of the recombinant protein antigen in the inhalation solution is 100μg / mL to 1000μg / mL.

[0128] In some other embodiments, the average particle size of the composite particles is 5 nm to 2000 nm.

[0129] Furthermore, the protein particles include complex particles, and the concentration of the recombinant protein antigen in the inhalation solution is 0.1 mg / mL to 2 mg / mL.

[0130] The protein particles include complex particles. The concentration of the recombinant protein antigen is within the above range. The particles have suitable surface tension and viscosity. The shear force required to form tiny droplets during atomization is suitable, and the activity and structural integrity of the protein can be ensured.

[0131] In some embodiments, the surface tension of the inhaled solution is 40 mN / m to 70 mN / m at 25° C. and standard atmospheric pressure. For example, the surface tension of the inhaled solution may be, but is not limited to, 40 mN / m, 45 mN / m, 50 mN / m, 55 mN / m, 60 mN / m, 65 mN / m, 70 mN / m, or a range consisting of any two of these values.

[0132] In some embodiments, at 25°C and standard atmospheric pressure, the viscosity of the inhalation solution is 0.9mPa.S to 3mPa.S. For example, the viscosity of the inhalation solution may be, but is not limited to, 0.9mPa.S, 1mPa.S, 1.2mPa.S, 1.4mPa.S, 1.6mPa.S, 1.8mPa.S, 2mPa.S, 2.2mPa.S, 2.4mPa.S, 2.6mPa.S, 2.8mPa.S, 3mPa.S or a range consisting of any two of these values. Optionally, at 25°C and standard atmospheric pressure, the viscosity of the inhalation solution is 1mPa.S to 2mPa.S.

[0133] The above-mentioned inhalation solution has suitable surface tension and viscosity, and the shear force required to form tiny droplets during the atomization process is suitable, which can ensure the integrity of protein activity and structure.

[0134] In some embodiments, the inhalation solution can be inhaled through the nose or mouth after being aerosolized for respiratory mucosal immunization.

[0135] The above-mentioned inhalation solution includes virus-like particles self-assembled by recombinant protein antigens or complex particles assembled by recombinant protein antigens and one or more of lipids, polymers and inorganic particles. Compared with attenuated live vaccines, it has high safety and high immunogenicity. In addition, by controlling the particle size of protein particles and the concentration of the inhalation solution, it has good stability, and can be atomized to obtain suitable aerosol mist particles for delivery to target sites such as the lungs and bronchi, simulating the natural infection process and enhancing mucosal immunity and protective efficacy. And the shear force required to form tiny droplets during the atomization process is appropriate, which can ensure the integrity of protein activity and structure.

[0136] The second aspect of the present application provides an atomization system. Figure 1 , including an atomization component 120 and a power component 130.

[0137] The atomizer assembly 120 is used for atomizing the inhaled solution. The atomizer assembly 120 may include an atomizer core having a thickness ranging from 50 μm to 200 μm, a number of micropores ranging from 2 to 1000 per square centimeter, an average pore size of 2 to 50 μm, and a surface roughness of ≤20 μm.

[0138] The power assembly 130 can cause the inhalation solution and the atomization assembly 120 to be squeezed relative to each other to form aerosol mist particles.

[0139] It can be understood that in the present application, the atomizer core refers to a mesh used to atomize the inhalation solution, which is atomized by vibration rather than heating through the action of a power component. In addition, by optimizing the structure of the atomizer core, the shear force to which the inhalation solution is subjected during the atomization process is appropriate to ensure the activity and structural integrity of shear-sensitive substances.

[0140] Specifically, the power component can be a component that can cause the inhalation solution and the atomization component to be relatively squeezed to form aerosol mist particles, for example, the power component includes a vibration motor or a vibrator, which atomizes the inhalation solution through the atomization core through vibration to form fine and uniform aerosol mist particles.

[0141] In one example, the thickness of the atomizer core may be, but is not limited to, 50 μm, 60 μm, 80 μm, 100 μm, 120 μm, 140 μm, 150 μm, 160 μm, 180 μm, 200 μm, or a range consisting of any two of these values.

[0142] In one example, the number of micropores of the atomization core may be, but is not limited to, 2, 10, 20, 50, 100, 200, 300, 350, 400, 450, 500, 600, 800, 1000, or a range consisting of any two of these values.

[0143] Optionally, the number of micropores of the atomization core is ≥ 100 / cm2. That is, the number of micropores of the atomization core is 100 / cm2 to 1000 / cm2. The number of micropores of the atomization core within the above range is conducive to improving the atomization rate while ensuring the integrity of the protein structure.

[0144] In one example, the average pore size of the micropores of the atomizer core may be, but is not limited to, 2 μm, 4 μm, 5 μm, 6 μm, 8 μm, 10 μm, 12 μm, 15 μm, 20 μm, 25 μm, 30 μm, 40 μm, 50 μm, or a range consisting of any two of these values.

[0145] Optionally, the average pore size of the micropores of the atomizer core is 2 μm to 15 μm. The average pore size of the micropores of the atomizer core within the above range is conducive to improving the atomization rate and recovery rate. At the same time, the aerodynamic particle size of the aerosol mist particles generated after atomization is smaller, which is more conducive to lung delivery.

[0146] In one example, the surface roughness of the atomizer core can be, but is not limited to, 2 μm, 4 μm, 5 μm, 6 μm, 8 μm, 10 μm, 12 μm, 15 μm, 20 μm, or a range consisting of any two of these values. Optionally, the surface roughness of the atomizer core is ≤ 5 μm. The above arrangement is conducive to improving the recovery rate while ensuring the structural integrity of the shear-sensitive substance.

[0147] In some embodiments, the material of the atomizer core is selected from one or more of a plastic material, a metal material, and an inorganic non-metal material. For example, the plastic material may be, but is not limited to, a polyolefin, such as polypropylene. The metal material may be, but is not limited to, stainless steel, and the inorganic non-metal material may be, but is not limited to, silicon nitride.

[0148] Specifically, the material of the atomizer core is selected from one or more of polypropylene, stainless steel and silicon nitride. The material of the atomizer core has certain strength and good safety.

[0149] In some embodiments, the mechanical thrust speed of the power assembly directly acting on the inhaled solution is 20 mm / s to 500 mm / s. For example, the mechanical thrust speed can be, but is not limited to, 20 mm / s, 50 mm / s, 100 mm / s, 150 mm / s, 200 mm / s, 250 mm / s, 30 mm / s, 350 mm / s, 400 mm / s, 450 mm / s, 500 mm / s, or a range consisting of any two of these values.

[0150] Optionally, the mechanical thrust speed of the power component directly acting on the inhaled solution is 200 mm / s to 500 mm / s.

[0151] The mechanical thrust speed refers to the rate of change of the relative compression between the inhaled solution and the atomization component over time. Specifically, when the inhaled solution is compressed and becomes aerosol mist particles through the micropores, it interacts with the atomization core due to the Bernoulli principle (such as shear force, turbulence, etc.), which will generate reverse thrust. The speed of the fluid or particle movement caused by this thrust is the mechanical thrust speed.

[0152] In some embodiments, the power assembly can drive the atomization core to vibrate at a frequency of 80kHz to 350kHz. For example, the vibration frequency can be, but is not limited to, 100kHz, 120kHz, 140kHz, 160kHz, 180kHz, 200kHz, 220kHz, 240kHz, 260kHz, 280kHz, 300kHz, 350kHz, or a range consisting of any two of these values. Optionally, the power assembly can drive the atomization core to vibrate at a frequency of 200kHz to 350kHz. With the above arrangement, the inhaled solution has a higher atomization rate and a suitable recovery rate. At the same time, the aerodynamic particle size of the aerosol mist particles produced after atomization is smaller, which is conducive to lung delivery.

[0153] Specifically, the power component can be a component that can cause the inhalation solution and the nebulizer component to be relatively squeezed to form aerosol mist particles. For example, the power component includes different mechanical energy and electrical energy to provide the shear force required to generate aerosol mist particles, so that the inhalation solution passes through the micropores of the nebulizer core to form fine and uniform aerosol mist particles.

[0154] In some embodiments, the volume of inhaled solution atomized in a single quantitative atomization that can be achieved by the atomization system is 15 μL to 200 μL. Single atomization refers to the volume of inhaled solution atomized in a single use by a patient, and has different volumes according to different specifications.

[0155] In some embodiments, the atomization system may further include a liquid medicine tank, which is used to store the inhalation solution. Specifically, the atomization system includes a liquid medicine tank, an atomization component, and a power component.

[0156] The medicine liquid tank can be used to store the inhalation solution. Specifically, the medicine liquid tank can be a multi-specification, single-dose, replaceable module.

[0157] In some embodiments, the drug liquid tank is detachably connected to the atomization assembly.

[0158] In some embodiments, the volume of inhaled solution for a single quantitative atomization that can be achieved by the liquid medicine tank is 15 μL to 200 μL. For example, the liquid medicine tank can be a single-dose, replaceable module with a volume of 15 μL to 200 μL. The volume of the liquid medicine tank 110 can be, but is not limited to, any volume specification in the range of 15 μL to 200 μL.

[0159] It can be understood that the volume of inhalation solution that can be achieved by the medicine liquid warehouse for a single quantitative atomization refers to the overall volume of the medicine liquid warehouse that meets the single inhalation vaccination dose of different populations. During the atomization process, the medicine liquid warehouse is filled with the inhalation solution, and the volume of the medicine liquid warehouse corresponds to the volume of the inhalation solution. Therefore, in actual use, a medicine liquid warehouse of appropriate volume can be selected according to the amount of inhalation solution to be atomized. The use of a medicine liquid warehouse with the above-mentioned inhalation solution volume can achieve quantitative atomization, and through the optimization of the atomization system, it has good atomization stability, and the residual amount after atomization is ≤5%, which can achieve low-residue and efficient atomization. It can be understood that atomization stability means that during the atomization process, precise micro-atomization can be achieved, and the standard deviation (SD) of atomization is small.

[0160] It is understood that in other embodiments, the atomization system may not contain a liquid medicine tank, and during atomization, the atomization system can be connected to the liquid medicine tank. When the liquid medicine tank is connected to the atomization system, the liquid medicine tank and the atomization component can be connected, and the atomization core can be immersed in the inhalation solution in the liquid medicine tank.

[0161] In some embodiments, the mass median aerodynamic diameter of the aerosol particles generated by the inhalation solution after atomization by the atomization system is 2 μm to 8 μm. Mass median aerodynamic diameter (MMAD) is a parameter used to measure particle size in the field of aerodynamics.

[0162] Specifically, MMAD refers to the particle size at which the mass of particles larger and smaller than the particle size each accounts for 50% of the total mass of the particles when the particles are sorted by aerodynamic particle size. That is, in a particle size distribution, 50% of the mass is distributed below this particle size, and 50% of the mass is distributed above this particle size. The smaller the aerodynamic mass median diameter of the aerosol mist particles generated after the inhaled solution is atomized by the atomization system, the easier it is for the aerosol mist particles to be transported to the distal branches of the airway, and the better the lung deposition. In this embodiment, the MMAD of the aerosol mist particles generated after the inhaled solution is atomized by the atomization system is smaller, which is more conducive to delivery to the lungs.

[0163] In some embodiments, the percentage of particles smaller than 5 μm in the aerosol mist generated by the inhalation solution after atomization by the atomization system (FPF) is 40% to 75%. FPF is the effective site deposition amount, which specifically refers to the percentage of aerosol mist particles of the inhalation solution deposited in the lungs. The higher the FPF, the higher the delivery efficiency of the inhalation solution and the higher the lung deposition amount. FPF within the above range is conducive to ensuring the use effect of the inhalation solution.

[0164] In some embodiments, the inhalation solution includes a shear-sensitive substance. For example, in some of these embodiments, the inhalation solution can be an inhalation solution of a recombinant protein vaccine. Specifically, the inhalation solution includes protein particles, which are virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles. Specifically, the inhalation solution is as described in the first aspect above, and will not be repeated here.

[0165] In some embodiments, the recovery rate of the recombinant protein antigen in the aerosol mist generated by the inhalation solution after atomization by the atomization system is 55% to 95%. For example, the recovery rate of the recombinant protein antigen can be, but is not limited to, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or a range consisting of any two of these values.

[0166] In some of the embodiments, in the inhalation solution, the concentration of the recombinant protein antigen is 100 μg / mL to 1 mg / mL, and the average pore size of the micropores of the atomization core is 2 μm to 15 μm; the aerodynamic mass median diameter of the aerosol particles generated after the inhalation solution is atomized by the atomization system is 3.3 μm to 5.4 μm, and the recovery rate of the recombinant protein antigen in the aerosol particles is 82% to 87%.

[0167] In other embodiments, in the inhalation solution, the concentration of the recombinant protein antigen is 1 mg / mL to 2 mg / mL, and the average pore size of the micropores of the atomization core is 10 μm to 15 μm; the aerodynamic mass median diameter of the aerosol particles generated after the inhalation solution is atomized by the atomization system is 3.6 μm to 5.9 μm, and the recovery rate of the recombinant protein antigen in the aerosol particles is 77% to 89%.

[0168] The research in this application found that by adjusting the preferred average pore size of the micropores of the atomization core for inhalation solutions of different concentrations of recombinant protein antigens, the aerosol mist particle size and protein activity of the aerosol vaccine can be taken into account, which is beneficial to further improve the immune effect of the inhalation solution.

[0169] It can be understood that in other embodiments, the inhalation solution may also be other solutions, such as other protein solutions with specific conformations that need to be used by nebulization, or solutions containing shear-sensitive therapeutic drugs that need to be used by nebulization, etc.

[0170] New protein inhalation preparations are in the ascendant and need to be developed and verified in combination with a nebulization system. The nebulization drug-device combination parameters of protein solutions require both lung deposition depth and delivery efficiency, as well as dual screening of protein activity and efficacy. Furthermore, there is a lack of research on the adaptation mechanism of the nebulization drug-device combination parameters for inhaled vaccines, which limits its widespread application in the prevention and treatment of respiratory diseases.

[0171] In some embodiments of the present application, represented by recombinant protein antigens that are highly sensitive to shear force, the parameters of the atomization system are adjusted to protect the vaccine from physical and chemical damage during the atomization process, ensure that it retains biological activity, and is fully delivered to the target site of the lungs, thereby improving the inhalation efficiency and immune effect of the recombinant protein vaccine. This can not only significantly improve the efficacy of the drug, reduce the dosage of the drug, and reduce side effects, but also is expected to provide a safer and more effective treatment for respiratory diseases and even other suitable diseases. In addition, the study of the adjustment of atomization parameters will also promote the innovation of inhalation drug delivery technology, promote the development of personalized medicine, and open up new ways for the precise delivery and disease management of nanoprotein vaccines.

[0172] In addition, traditional conventional atomization technology and corresponding equipment only solve the aerosol generation and pulmonary delivery of small molecules of chemical drugs, and are not yet compatible, let alone developed system parameters suitable for efficient atomization of shear-sensitive, tertiary-structured immunogenic active ingredients. However, the present application found that the average pore size, vibration frequency, operating pressure, etc. of the micropores of the atomization core in the atomization system will affect the pressure on the fluid, and thus affect the atomization efficiency, aerosol mist particle distribution, and protein structure stability of the aerosol antigen protein, thereby affecting the pulmonary delivery efficiency and inhalation immune effect. Based on this, for the aerosol generation of influenza nanoantigen self-assembled virus-like particles with strict requirements on spatial conformation and structural integrity, a pharmaceutical and medical device product composed of a recombinant protein vaccine inhalation solution and an atomization system is provided, which can also be used to solve the atomization inhalation and pulmonary delivery of other protein solutions with complex conformations, filling the gap in the research on efficient atomization inhalation of biological macromolecules.

[0173] The third aspect of the present application provides a method for atomizing an inhaled solution, which can be used for efficient and highly active atomization of recombinant protein vaccines. Figure 2 , including the following steps:

[0174] Step S210: obtaining the above-mentioned inhalation solution and the above-mentioned atomization system.

[0175] Step S230: using the power component of the atomization system to cause the inhalation solution and the atomization component to be squeezed relative to each other to form aerosol mist particles.

[0176] Specifically, the inhalation solution and the atomization system are respectively the same as those described above and will not be repeated here.

[0177] Optionally, step S220 may be included between step S210 and step S230: adding the inhalation solution to the atomization system. It is understandable that in some embodiments, if the atomization system includes a liquid medicine tank, the atomization method includes step S220, adding the inhalation solution to the liquid medicine tank of the atomization system. In other embodiments, the atomization system may not include a liquid medicine tank. In this case, the atomization method does not include step S220, and during atomization, the atomization system is connected to the liquid medicine tank. Specifically, the liquid medicine tank may be docked with the atomization component of the atomization system so that the atomization core can be immersed in the inhalation solution in the liquid medicine tank.

[0178] In some embodiments, the step of using a power component of the nebulizer system to relatively squeeze the inhaled solution and the nebulizer component to form aerosol mist particles includes: using the power component to drive the atomizer core of the nebulizer component to move, and the movement can cause the atomizer core to generate pressure on the inhaled solution, so that the inhaled solution is relatively squeezed with the atomizer core in the process of passing through the micropores of the atomizer core, thereby converting into a state of aerosol mist particles.

[0179] In some embodiments, during the atomization process, the mechanical thrust speed of the power component of the atomization system is 20 mm / s to 500 mm / s.

[0180] Optionally, during the atomization process, the mechanical thrust speed of the power component of the atomization system is 200 mm / s to 500 mm / s.

[0181] In some embodiments, during the atomization process, the atomization core of the atomization system vibrates at a frequency of 80 kHz to 350 kHz.

[0182] Optionally, during the atomization process, the atomization core of the atomization system vibrates at a frequency of 200kHz to 350kHz.

[0183] Specifically, in some embodiments, during the atomization process, the inhaled solution is subjected to an operating pressure of 0.4 bar to 200 bar for the atomizer core having an average pore size of 2 μm to 15 μm. At this time, the median particle size D of the aerosol mist particles after atomization is 50 In the range of 2μm~50μm.

[0184] Optionally, the average pore size of the micropores of the atomizer core is 2μm to 15μm, and the operating pressure of the atomizer core to which the inhaled solution is subjected may be but is not limited to 1.33bar, 6.67bar, 13.33bar, 33.33bar, 66.67bar, 166.67bar or a range consisting of any two close to these values. The median particle size of the aerosol mist particles of the aerosol vaccine generated is between 2.75μm and 6.01μm, which meets the particle size requirements for inhaled lung deposition.

[0185] The operating pressure of the atomizer core on the inhaled solution is the pressure applied by the atomizer assembly to the inhaled solution under normal working conditions, and is a key factor in promoting the inhaled solution to pass through the micropores of the atomizer core to form fine droplets. The magnitude of the operating pressure directly affects the atomization effect, the distribution of aerosol mist particles, the uniformity of aerosol mist particles, and the structure and activity of the recombinant protein antigen in the aerosol mist particles.

[0186] In some embodiments, when the aerodynamic mass median diameter of the aerosol mist particles after atomization is 2 μm to 8 μm, the recovery rate of the protein in the aerosol mist particles is 55% to 95%.

[0187] Optionally, the aerodynamic mass median diameter of the aerosol mist particles after atomization is 2.7 μm to 5.5 μm. Optionally, the recovery rate of the protein in the aerosol mist particles after atomization is 60% to 90%.

[0188] In some embodiments, when the average pore size of the micropores of the atomization core is 2 μm and the mechanical thrust speed during the atomization process is 200 mm / s to 500 mm / s, or the average pore size of the micropores of the atomization core is 15 μm and the mechanical thrust speed during the atomization process is 200 mm / s to 500 mm / s, the aerodynamic mass median particle size of the aerosol mist particles after atomization is 2.7 μm to 5.5 μm, and the average recovery rate of protein in the aerosol mist particles is 63% to 88%.

[0189] Specifically, in some of the embodiments, during the atomization process, the mechanical thrust speed of the power component of the atomization system is 200 mm / s to 500 mm / s, the operating pressure of the atomization core with an average pore size of 2 μm for the inhaled solution is 66.67 bar to 166.67 bar, the corresponding aerodynamic mass median diameter of the aerosol particles after atomization is 2.7 μm to 4.3 μm, and the recovery rate of protein in the aerosol particles after atomization is 63.15% to 87.39%.

[0190] In other embodiments, during the atomization process, the mechanical thrust speed of the power component of the atomization system is 200 mm / s to 500 mm / s, the operating pressure of the atomization core with an average pore size of 15 μm for the inhaled solution is 13.33 bar to 33.33 bar, the aerodynamic mass median diameter of the aerosol particles after atomization is 3.6 μm to 5.5 μm, and the recovery rate of protein in the aerosol particles after atomization is 66.89% to 86.26%.

[0191] In other embodiments, the inhalation solution includes protein particles, which are virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles. During the atomization process, the atomization core of the atomization system vibrates at a frequency of 80kHz to 350kHz, the concentration of the recombinant protein antigen is 100μg / mL to 1mg / mL, the atomization rate of the inhalation solution is 0.356±0.021g / min to 0.709±0.063g / min, and the aerodynamic mass median diameter of the aerosol particles generated after the inhalation solution is atomized by the atomization system is 3.4μm to 5.1μm, the percentage of particles smaller than 5μm in the aerosol particles is 54% to 72%, and the recovery rate of the recombinant protein antigen in the aerosol particles is 64% to 86%.

[0192] In other embodiments, during the nebulization process, the atomization core of the nebulization system vibrates at a frequency of 80kHz to 350kHz, the concentration of the recombinant protein antigen is 1mg / mL to 2mg / mL, the atomization rate of the inhalation solution is 0.243±0.028g / min to 0.484±0.076g / min, the aerodynamic mass median diameter of the aerosol particles generated after the inhalation solution is atomized by the nebulization system is 4.2μm to 5.3μm, the percentage of particles smaller than 5μm in the aerosol particles is 49% to 66%, and the recovery rate of the recombinant protein antigen in the aerosol particles is 52% to 76%.

[0193] In some embodiments, the effective atomization rate is 0.08 g / min to 0.35 g / min. Effective atomization rate = atomization rate × FPF × recovery rate. FPF here refers to the fine particle fraction, that is, the percentage of particles smaller than 5 μm in the aerosol mist generated after the inhaled solution is atomized by the atomization system, and the recovery rate refers to the recovery rate of the recombinant antigen protein.

[0194] The atomization rate is obtained by the following test method: adjust the air compressor pump's inhalation flow rate to 7L / min with a flow meter, take a Cambridge filter, weigh and record its weight. Add the inhalation solution to the atomization system's drug tank, take out the filter after atomization for 1 minute and weigh it to obtain the atomization rate. Repeat the test 3 times and take the average value as the atomization rate.

[0195] In some embodiments, the concentration of the recombinant protein antigen is 100 μg / mL to 1 mg / mL, and the effective atomization rate is 0.271±0.027 g / min when the average pore size of the atomization core micropores is 5 μm to 15 μm. In other embodiments, the concentration of the recombinant protein antigen is 1 mg / mL to 2 mg / mL, and the effective atomization rate is 0.295 g / min to 0.307 g / min when the average pore size of the atomization core micropores is 10 μm to 15 μm.

[0196] In some embodiments, the inhalation coordination between the aerosol delivery process and the user's inhalation process is ≥ 90%, wherein the inhalation coordination refers to the percentage of the aerosol recovery amount to the release amount during a single inhalation in a standard breathing mode.

[0197] In some embodiments, the atomization residual amount is ≤5%, wherein the atomization residual amount refers to the percentage of the mass of the remaining drug liquid after atomization to the mass of the total drug liquid before atomization.

[0198] In some embodiments, the aerosolized particles are administered to mice through the airway, and the IgG (immunoglobulin G) antibody titer that produces an immune response in the respiratory tract three weeks after the secondary immunization can reach 5.9log10. Antibody titer is used to measure the minimum concentration (i.e., maximum dilution) required for a certain antibody to recognize a specific antigen epitope. It is generally expressed as: the maximum dilution that can still produce a positive result. Antibody titer is usually detected by ELISA (enzyme-linked immunosorbent assay) method.

[0199] The above-mentioned nebulization method enables the recombinant protein vaccine to be used for nebulization as an inhalation solution for airway mucosal immunization, and has good aerosol mist particle characteristics and immune effects.

[0200] A fourth aspect of the present application provides a medicine liquid tank for storing the above-mentioned inhalation solution, and the medicine liquid tank is used for detachably docking with the above-mentioned atomization system.

[0201] In some of the embodiments, the volume of the inhaled solution that can be achieved by the drug liquid tank for single quantitative atomization is 15 μL to 200 μL.

[0202] The specific medicine liquid tank is the same as described above and will not be repeated here.

[0203] The fifth aspect of the present application provides a nebulized drug-device combination product, comprising the above-mentioned nebulization system, the above-mentioned inhalation solution and the above-mentioned drug solution tank.

[0204] The atomized drug-device combination product of this application can meet the safety and activity requirements of the ingredients of inhalable vaccines, realize the integrated design of drug-device combination products, and conform to the development direction of portable use and self-vaccination of vaccines. In addition, the above-mentioned atomized drug-device combination product can realize one-time micro-atomized inhalation and mucosal inoculation of highly active vaccines, thereby standardizing the vaccination operation and simplifying the inhalation immunization procedures, and providing a scalable solution to meet the needs of seasonal and community outbreak epidemic prevention.

[0205] In order to make the purpose and advantages of the present application clearer, the atomization system, atomization method and its effects of the present application are further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and shall not be used to limit the present application. The following embodiments do not include other components except inevitable impurities unless otherwise specified. The drugs and instruments used in the embodiments are conventionally selected in the art unless otherwise specified. The experimental methods for which specific conditions are not specified in the embodiments are implemented according to conventional conditions, such as the conditions described in the literature and books or the methods recommended by the manufacturer.

[0206] Example 1

[0207] This example provides a method for preparing an inhalation solution of a recombinant protein vaccine with different concentrations to evaluate the stability of a virus-like particle antigen, comprising the following steps:

[0208] A glycoengineered yeast strain that highly expresses influenza virus hemagglutinin antigen HA recombinant protein was constructed to express HA protein, and purification schemes such as anions and cations, molecular sieves, etc. were combined to prepare hollow protein particles (HA-VLPs) composed of single or multiple subtypes of virus-like assembly, and different concentrations of inhalation solutions were prepared by dilution with physiological saline. Among them, the concentration of the inhalation solution refers to the concentration of the recombinant protein antigen.

[0209] The average particle size and polydispersity index (PDI) of the protein particles in the prepared inhalation solutions of different concentrations were tested. Specifically, the average particle size and PDI of the nanoparticles were measured by dynamic light scattering (DLS). The test results are shown in Table 1 below.

[0210] Table 1 Average particle size and PDI of protein particles at different concentrations

[0211] Concentration (μg / mL) Average particle size (nm) PDI 1000 36.14±1.76 0.33±0.03 500 45.17±9.32 0.29±0.05 100 62.41±17.54 0.37±0.03

[0212] Table 1 shows the average particle size and PDI of protein particles at different concentrations. As can be seen from Table 1, as the concentration of the recombinant protein antigen decreases, the average particle size of the protein particles tends to increase. However, for inhalation solutions with a concentration of 100 to 1000 μg / ml, the average particle size of the protein particles is less than 100 nm, which is much smaller than the pore size of the micropores of the atomizer core, so the possibility of clogging is small. The PDI of the protein particles is between 0.3 and 0.4, and the nanoparticle size distribution is relatively uniform.

[0213] Example 2

[0214] Inhalation solutions of different concentrations were prepared according to the preparation method of Example 1.

[0215] The inhalation solutions of different concentrations were tested as follows:

[0216] The surface tension test was conducted using the platinum plate method of a fully automatic surface tension meter. The viscosity test was conducted using a rotational viscometer. The atomization system was used to measure the atomization rate of inhalation solutions of different concentrations. In the atomization system, the average pore size of the micropores of the atomization core was 5 μm, the vibration frequency of the atomization core was 150 kHz, the number of micropores of the atomization core was 150 / cm2, the surface roughness of the atomization core was 5 μm, and the thickness of the atomization core was 100 μm.

[0217] The specific test method of atomization rate is as follows: Use a flow meter to adjust the air compressor pump's inhalation flow rate to 7L / min, take a Cambridge filter, weigh and record its weight. Add the inhalation solution to the liquid tank of the atomization system, take out the filter after atomization for 1 minute and weigh it to obtain the atomization rate. Repeat the test 3 times and take the average value as the atomization rate of each concentration of the inhalation solution.

[0218] The surface tension and viscosity data of the above-mentioned inhalation solutions of different concentrations at 25° C. and standard atmospheric pressure are shown in Table 2 below.

[0219] Table 2 Surface tension and viscosity of inhalation solutions at different concentrations

[0220] prescription Concentration (μg / mL) Surface tension (mN / m) Viscosity(mPa.S) 1 100 68.21 1.052 2 500 60.43 1.256 3 1000 54.76 1.843 Control group Normal saline 72.75 0.894

[0221] Experimental results: As can be seen from Table 2, as the concentration of the recombinant protein antigen increases, the surface tension of the inhalation solution decreases at 25°C and standard atmospheric pressure, and the surface tension of the inhalation solution of different concentrations is lower than that of normal saline. The viscosity of the inhalation solution increases with the concentration of the inhalation solution, and is greater than the viscosity of normal saline.

[0222] The results of the atomization rate of inhalation solutions of different concentrations are as follows Figure 3 As shown. Figure 3It can be seen that as the concentration of the recombinant protein antigen in the inhaled solution increases, the atomization rate decreases. This is because as the concentration of the recombinant protein antigen increases, its surface tension and viscosity increase, and the required atomization shear force increases, so the atomization rate decreases.

[0223] Example 3

[0224] An inhalation solution with a recombinant protein antigen concentration of 500 μg / mL was prepared according to the method of Example 1. Under the condition of a constant inhalation solution, this example explores the effects of the number of micropores, surface roughness and thickness of the atomization core in the atomization system on the atomization rate of the inhalation solution and the recovery rate of the recombinant protein antigen.

[0225] The inhalation solution with a recombinant protein antigen concentration of 500 μg / mL was atomized using an atomization system with different atomization parameters (as shown in Table 3). The atomization rate test method was the same as in Example 2.

[0226] The recovery rate test method is as follows: refer to the device 1 of the 2020 edition of the "Chinese Pharmacopoeia" General Rules 0951 for the determination of aerodynamic characteristics of fine particles of inhalation preparations. Add the inhalation solution to the nebulization system, start the air pump and start nebulization. After nebulization, collect the recovered liquid in the system, compare the sample content before and after nebulization, that is, use the BCA method (bicinchoninic acid method or bicinchoninic acid method) to quantify the total protein, and use the ELISA method (enzyme-linked immunosorbent assay) to quantify the characteristic protein, and calculate the recovery rate. And use western-blot (protein immunoblotting) to explore whether protein impurities are generated after nebulization.

[0227] Table 3 Atomization parameters of different groups

[0228]

[0229] The experimental results are as follows Figures 4 to 10 shown. Figure 4 This is a comparison chart of the atomization rate of the inhaled solution under different micropore numbers. Figure 5 This is a comparison chart of the atomization rate of the inhaled solution under different surface roughness. Figure 6 The figure is a comparison of the atomization rate of the inhaled solution under different atomizer core thicknesses. Figures 4 to 6 It can be seen that as the number of micropores increases, the atomization rate of the inhaled solution increases. When the number of micropores is 200 / cm2, the atomization rate can reach 0.281±0.039g / min. The change of the surface roughness and thickness of the atomizer core has no obvious effect on the atomization rate.

[0230] Figure 7 The figure is a comparison of the recovery rate of the recombinant protein antigen in the aspirated solution under different numbers of micropores. Figure 8The figure is a comparison of the recovery rate of recombinant protein antigen in the aspirated solution under different surface roughness. Fig. 9 The figure is a comparison of the recovery rate of recombinant protein antigen in the inhaled solution under different atomizer core thicknesses. Figure 7 to Figure 9 It can be seen that the number of micropores in the atomizer core increases, and there is no significant change in the recovery rate of the recombinant protein antigen in the inhaled solution. The BCA recovery rate is about 88%, and the ELISA recovery rate is about 83%. When the surface roughness of the atomizer core is 8μm, the BCA recovery rate and the ELISA recovery rate both decrease to 82.55±0.71% and 78.39±1.61%, respectively. When the thickness of the atomizer core reaches 200μm, the BCA recovery rate and the ELISA recovery rate also decrease to 83.62±0.83% and 76.42±2.42%, respectively. This shows that the increase in the surface roughness and thickness of the atomizer core will lead to a decrease in the recovery rate.

[0231] The integrity of the vaccine structure was further verified by protein gel electrophoresis, and the following results were obtained: Fig.10 The experimental results are shown. Fig.10 The following is the protein immunoblotting result of the inhaled solution under different micropore numbers, surface roughness and atomizer core thickness. Fig.10 It can be seen that none of the parameter groups tested above produced obvious tailing phenomenon, that is, the vaccine structure is relatively complete within the current parameter range, and the atomization parameters can be used for subsequent verification.

[0232] Example 4

[0233] Inhalation solutions with recombinant protein antigen concentrations of 100 μg / mL and 1 mg / mL were prepared according to the method of Example 1. This example investigates the effect of the vibration frequency of the atomizer core in the atomization system on the atomization rate of the inhalation solution, the recovery rate of the recombinant protein antigen, and the aerodynamic properties of the aerosol mist particles after atomization.

[0234] In the atomization system, when the average pore size, number of micropores, surface roughness and thickness of the micropores of the atomization core are constant, the vibration frequency of the atomization core is adjusted to atomize the inhalation solutions of different concentrations prepared above. Among them, the average pore size of the micropores of the atomization core is 5μm, the number of micropores is 150 / cm2, the surface roughness is 5μm and the thickness of the atomization core is 100μm. The vibration frequency of the atomization core is 80kHz, 200kHz and 350kHz.

[0235] The atomization rate test method is the same as in Example 2. The ELISA recovery test method is the same as in Example 2. A new generation particle impactor (NGI) was used to study the aerodynamic properties, referring to the "Chinese Pharmacopoeia" 2020 edition of the four general rules 0951 inhalation preparation fine particle aerodynamic characteristics determination method. The assembled NGI and L-type connecting tube were precooled in a cooling device (5°C) for 90 minutes, the vacuum pump was turned on, the flow meter was connected to the L-type connecting tube, and the flow control valve was adjusted so that the gas flow rate at the inlet of the L-type connecting tube was 15L / min (±5%), and the flow meter was removed. Add the inhalation solution to the atomization system, connect the atomization system to the L-type connecting tube, turn on the driving airflow until the atomization is completed, and turn off the driving airflow. Collect the aerosol mist particles of the L-type connecting tube, each level and the additional filter paper, and then use ELISA to quantify the nanoprotein vaccine. CITAS software is used to calculate the APSD (aerodynamic particle size distribution) evaluation parameters: fine particle ratio (FPF) and aerodynamic mass median diameter (MMAD). According to the atomization rate, FPF and recovery rate, the effective atomization rate is calculated according to the following formula: Effective atomization rate = atomization rate × FPF × recovery rate.

[0236] The results of the atomization rate of the inhaled solution and the recovery rate of the recombinant protein antigen at different atomizer core vibration frequencies are shown in the figure below. Fig.11 As shown. Fig.11 It can be seen that with the increase of vibration frequency, the atomization rate of inhalation solutions of different concentrations showed an upward trend, and the increase in the atomization rate of the low concentration group was greater than that of the high concentration group (such as Fig.11 The ELISA recovery rate decreases with the increase of vibration frequency (as shown in Figure 2A). Fig.11 (As shown in B), the ELISA recovery rate of the high concentration group decreased more significantly. When the vibration frequency of the atomizer core was 350 kHz, the ELISA recovery rate dropped to 52.44±7.55%.

[0237] The aerodynamic evaluation results of the aerosol mist particles after the inhaled solution is atomized at different atomizer core vibration frequencies are shown in the figure below. Fig.12 As shown. Fig.12 It can be seen that when the vibration frequency increases, the FPF of the aerosol mist particles after the inhalation solution of different concentrations is on the rise, and the MMAD becomes smaller. When the vibration frequency is 350kHz, the FPF of the low concentration group is as high as 71.93±2.16%, and the MMAD is 3.37±0.38μm.

[0238] The effective atomization rate was further calculated based on the above atomization rate, FPF and recovery rate results, and the results are shown in Table 4. As can be seen from Table 4, for the low concentration group, the increase in its vibration frequency can effectively increase the atomization rate, and the impact on the recovery rate is limited. Therefore, the higher the vibration frequency within the test range of this embodiment, the higher the effective atomization rate. The high concentration group is more sensitive to changes in vibration frequency, and too high a vibration frequency will lead to a significant decrease in the recovery rate. Therefore, for inhalation solutions of different concentrations, a better effective atomization rate can be achieved by optimizing the vibration frequency. Preferably, at both high and low concentrations, a vibration frequency of 200kHz can be used to achieve a better effective atomization rate.

[0239] Table 4 Results of effective atomization rate of inhaled solution at different atomizer core vibration frequencies

[0240]

[0241] Example 5

[0242] Inhalation solutions with recombinant protein antigen concentrations of 100 μg / mL and 1 mg / mL were prepared according to the method of Example 1. This example explores the effect of the average pore size of the micropores of the atomization core in the atomization system on the atomization rate of the inhalation solution, the recovery rate of the recombinant protein antigen, the proportion of fine particles, the aerodynamic mass median diameter and the delivery rate.

[0243] In the atomization system, when the vibration frequency, number of micropores, surface roughness and thickness of the atomization core are constant, the average pore size of the micropores of the atomization core is adjusted to atomize the inhalation solutions of different concentrations prepared above. Among them, the vibration frequency of the atomization core is 200kHz, the number of micropores is 150 / square centimeter, the surface roughness is 5μm and the thickness of the atomization core is 100μm. The average pore size of the micropores of the atomization core is 2μm, 5μm, 10μm and 15μm.

[0244] The test method for the atomization rate of the inhaled solution is the same as in Example 1. The test method for the ELISA recovery rate is the same as in Example 2. The method for studying the aerodynamic properties is the same as in Example 3.

[0245] The results of the atomization rate of the inhaled solution and the recovery rate of the recombinant protein antigen under different average pore diameters of the atomizer core are as follows: Fig.13 As shown. Fig.13 It can be seen that when the average pore size of the micropores of the atomizer core increases, the atomization rate of the inhalation solution of different concentrations and the recovery rate of the recombinant protein antigen show an upward trend. The upward trend of the high concentration group is more obvious. Under the condition of an average pore size of 15μm, the atomization rate of the inhalation solution of different concentrations is higher than 0.7g / min, and the ELISA recovery rate is close to 90%.

[0246] Taking the average pore size of the micropores of the atomizer core as a variable, the aerodynamic evaluation results of the aerosol mist particles after the inhalation solution is as follows: Fig.14 As shown. Fig.14 It can be seen that the larger the average pore size of the micropores of the atomizer core, the smaller the FPF and the larger the MMAD, indicating that the aerosol mist particles generated by the atomization of the atomizer core with a small pore size have a smaller aerodynamic particle size and are more conducive to lung delivery.

[0247] The effective atomization rate was further calculated based on the atomization rate, FPF and recovery rate, and the effective atomization rate = atomization rate × FPF × recovery rate. The results of the effective atomization rate are shown in Table 5. As can be seen from Table 5, the effective atomization rate of the low concentration group at an average pore size of 5μm to 15μm remained relatively stable, at 0.271±0.027g / min; while the high concentration group required a larger average pore size, that is, a better effective atomization rate (0.295g / min~0.307g / min) at 10μm~15μm.

[0248] Table 5 Results of effective atomization rate of inhaled solution at different average pore diameters of atomizer core micropores

[0249]

[0250] Example 6

[0251] An inhalation solution with a recombinant protein antigen concentration of 100 μg / mL was prepared according to the method of Example 1. This example explores the effects of mechanical thrust speed and operating pressure on the aerosol particle distribution after inhalation solution atomization and the recovery rate of the recombinant protein antigen during the atomization process.

[0252] In the atomization system, when the number of micropores, surface roughness and thickness of the atomization core are constant, the average pore size of the micropores of the atomization core is adjusted, and the mechanical thrust speed is used to atomize the inhalation solution prepared above. Among them, the number of micropores of the atomization core is 150 / square centimeter, the surface roughness is 5μm and the thickness of the atomization core is 100μm. The average pore size of the micropores of the atomization core is 2μm, 15μm and 50μm. The mechanical thrust speed is 20mm / s, 200mm / s and 500mm / s.

[0253] The aerosol particle distribution of the inhaled solution after atomization under different mechanical thrust speeds and operating pressures was measured using Synpatek HELOS & SPRAYER, and the ELISA recovery rate test method was the same as in Example 2. The experimental results are shown in Table 6 and Fig.15 Table 6 shows the aerosol particle distribution and protein recovery rate of the inhalation solution after nebulization under different nebulization parameter combinations. Fig. 9 The aerosol particle distribution (D 50) and protein activity (recovery rate).

[0254] It can be seen from Table 6 that the average pore size of the atomizer core micropores is 2 μm. When the mechanical thrust speed (v) is 20 mm / s, 200 mm / s and 500 mm / s, the operating pressure (OP) of the inhaled solution is 6.77 bar, 66.67 bar and 166.67 bar, respectively. The corresponding aerosol particle size D 50 They are 5.22μm, 4.30μm and 2.75μm respectively.

[0255] Furthermore, the average pore size of the atomizer core micropores is 15 μm. When the mechanical thrust speed (v) is 20 mm / s, 200 mm / s, and 500 mm / s, the operating pressure of the inhaled solution is 1.33 bar, 13.33 bar, and 33.33 bar, respectively, corresponding to the aerosol mist particle D 50 The sizes are 6.01μm, 5.44μm and 3.64μm respectively. The average pore size of the atomizer core micropores is 50μm. When the mechanical thrust speed (v) is 20mm / s, 200mm / s and 500mm / s respectively, the operating pressure of the inhaled solution is 0.4bar, 4bar and 10bar respectively. The aerosol mist particle D 50 The sizes are 46.83μm, 20.29μm and 10.56μm respectively.

[0256] On the other hand, when the average pore size of the atomization core micropores is 2μm, the average recovery rates of the atomized protein are 55.03%, 84.34% and 67.53% when the mechanical thrust speed (v) is 20mm / s, 200mm / s and 500mm / s, respectively. For the atomization core with an average pore size of 15μm, the average recovery rates of the recombinant protein vaccine after atomization are basically the same at mechanical thrust speeds of 20mm / s and 200mm / s, which are 68.28% and 69.28%, respectively, while the average recovery rate of the recombinant protein vaccine after atomization is 80.61% when the mechanical thrust speed is 500mm / s. The recovery rates of the recombinant protein vaccine after atomization are basically close at different mechanical thrust speeds for the atomization core with an average pore size of 50μm, which are 69.24%, 65.76% and 69.35%, respectively.

[0257] Therefore, when the power assembly of the atomization system of some embodiments of the present application applies a mechanical thrust speed in the range of 0-500 mm / s, the operating pressure of the inhaled solution during the atomization process does not exceed 170 bar, and the atomization recovery rate of the target protein in the aerosol mist particles is not less than 50%. Further, under the above mechanical thrust speed, the median particle size of the generated aerosol mist particles is between 2.75 μm and 6.01 μm under the condition that the average pore size of the micropores is 2 μm to 15 μm, which meets the particle size requirements for inhaled lung deposition.

[0258] like Fig.15 , based on the dual screening of the median particle size of aerosol mist particles and the protein atomization stability of different combinations of atomization parameters, preferably, the average pore size of the micropores of the atomization core for atomizing the above-mentioned recombinant protein inhalation solution is 2μm, and the average pore size of the micropores of the atomization core is 15μm, and the operating pressure does not exceed 100bar under the condition of a mechanical thrust speed of 200mm / s, and the median particle size of the aerosol mist particles generated is 2.75μm and 5.44μm, respectively, and the average recovery rates of the recombinant protein vaccine are 67.53% and 84.34%, respectively.

[0259] Table 6 Aerosol particle distribution and recovery rate of different atomization parameter combinations

[0260]

[0261] * In the above tests, three biological replicates were used in each group using the biological assay method for specific antigens.

[0262] Example 7

[0263] An inhalation solution with a recombinant protein antigen concentration of 100 μg / mL was prepared according to the method of Example 1. This example verifies the micro-atomization volume range and residual amount.

[0264] The parameters of the atomization system were set as follows: the vibration frequency of the atomization core was 200 kHz, the number of micropores was 150 / cm2, the average pore diameter of the micropores was 2 μm, the surface roughness was 5 μm, and the thickness of the atomization core was 100 μm.

[0265] The weighing method is used to determine the quantitative volume range and residual amount of different volumes of drug solution tanks. First, weigh the weight of the drug solution tank alone (m1) and the total weight after adding the inhalation solution (m2). After nebulization, measure the weight of the drug solution tank and the residual inhalation solution (m3), and calculate the nebulized amount and residual amount.

[0266] The residual amount results under different volumes of liquid medicine tanks (15 μL, 100 μL, 200 μL) are shown in Table 7. The residual amount refers to the percentage of the mass of the remaining inhaled solution after nebulization to the mass of the total inhaled solution before nebulization.

[0267] Table 7 Quantitative volume range and residual volume results of different volumes of liquid medicine tanks

[0268]

[0269] It can be seen from Table 7 that the atomization system and atomization method of the present application can achieve accurate micro-atomization of a volume of 15 μL to 200 μL, with a small SD and good stability; and the residual amount is less than 5%, which can achieve low-residue and efficient atomization.

[0270] Example 8

[0271] An inhalation solution with a recombinant protein antigen concentration of 500 μg / mL was prepared according to the method of Example 1.

[0272] In this embodiment, a breathing simulator is used to measure the inhalation coordination of the atomization system. The parameters of the atomization system are set as follows: the vibration frequency of the atomization core is 200kHz, the number of micropores is 150 / cm2, the average pore size of the micropores is 2μm, the surface roughness is 5μm, and the thickness of the atomization core is 100μm. Connect the breathing simulator and the filter paper device, take 200mg of the inhalation solution and place it in the atomization system. Connect the atomization system nozzle to the filter paper device, and set the breathing simulator to the adult breathing mode (tidal volume 500mL, breathing rate 15BPM, breathing waveform is sinusoidal, and the inhalation-exhalation ratio is 1:1). Turn on the breathing simulator, start the nebulizer at the beginning of the breathing cycle, and end the atomization after running a breathing cycle. Collect the filter paper and weigh it, and calculate the inhalation coordination, that is, the percentage of aerosol recovery and release. The experimental results are as follows Fig.16 shown. Fig.16 is the inhalation coordination of the atomization system. Fig.16 It can be seen that under the standard adult breathing pattern, the inspiratory synergy of the nebulizer system is greater than 90%, with an average value of 94.01±1.63%, which has good inspiratory synergy.

[0273] Example 9

[0274] An inhalation solution with a recombinant protein antigen concentration of 200 μg / mL was prepared according to the method of Example 1.

[0275] This example verifies the immune effect of the inhaled solution in animals through an animal immunization test. The specific experimental grouping is shown in Table 8. The positive control group was administered with a commercially available nasal spray device, and the experimental group was administered with an atomization system. The parameters of the atomization system are as follows: the atomization core is made of stainless steel, the average pore size of the micropores is 2μm, the number of micropores is 1000 / cm2, the surface roughness is 5μm, the thickness is 100μm, and the vibration frequency is 200kHz. The mouse was placed in a mouse holder to fix its head and breathe normally. Use a connector to connect the spray outlet of the atomization system and the head of the holder so that the mouse head is exposed to the aerosol for autonomous inhalation. Samples were collected by orbital blood sampling 3 weeks after the first immunization (Prime) and the second immunization (Boost), and 200μL of blood was taken from each mouse to separate serum (Serum). CO 2 After killing the mice, the bronchoalveolar lavage fluid (BALF) was taken out to detect the specific antibody IgG content in the serum and bronchoalveolar lavage fluid, and the antibody titer was defined as the reciprocal of the highest dilution.

[0276] Table 8 Immunoassay Grouping

[0277]

[0278] Antibody titer test results Fig.17 As shown in the figure, the titers of specific antibodies in the serum of the experimental group after the first and second immunizations were significantly higher than those of the control group. The titers of local mucosal antibodies measured in the bronchoalveolar lavage fluid of the experimental group after the second immunization were also higher than those of the control group. It can be seen that the immune effect of the aerosol recombinant protein antigen produced by the selected nebulization combination system is better than that of the commercially available nasal vaccination device.

[0279] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0280] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as limiting the scope of protection of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several modifications and improvements can be made, which all belong to the scope of protection of the present application. It should be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent of this application shall be based on the contents of the attached claims, and the description and drawings can be used to explain the contents of the claims.

Claims

1. An atomization system, characterized in that: include: An atomizer assembly, the atomizer assembly is used for atomizing an inhalation solution, the atomizer assembly comprises an atomizer core, the atomizer core has a thickness of 50 μm to 200 μm, a number of micropores of 2 / cm2 to 1000 / cm2, an average pore size of the micropores of 2 μm to 50 μm, and a surface roughness of ≤20 μm; A power component is used to cause the inhalation solution and the atomization component to be relatively squeezed to form aerosol mist particles.

2. The atomization system according to claim 1, characterized in that: The atomizer core meets at least one of the following conditions: (1) The number of micropores of the atomizer core is ≥ 100 per square centimeter; (2) The average pore size of the micropores of the atomizer core is 2 μm to 15 μm; (3) The surface roughness of the atomizer core is ≤5 μm.

3. The atomization system according to claim 1, characterized in that: The power assembly meets at least one of the following conditions: (1) The mechanical thrust speed of the power component directly acting on the inhaled solution is 20 mm / s to 500 mm / s; (2) The power assembly can drive the atomizer core to vibrate at a frequency of 80 kHz to 350 kHz.

4. The atomization system according to claim 1, characterized in that: The inhalation solution includes a shear sensitive substance.

5. The atomization system according to any one of claims 1 to 4, characterized in that: The following conditions must be met at the same time: (1) The aerodynamic mass median diameter of the aerosol particles generated by the inhalation solution after atomization by the atomization system is 2 μm to 8 μm; (2) After the inhalation solution is atomized by the atomization system, the percentage of particles smaller than 5 μm in the aerosol mist is 40% to 75%; (3) The inhalation solution includes protein particles, which are virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles. The recovery rate of the recombinant protein antigen in the aerosol mist particles generated after the inhalation solution is atomized by the atomization system is 55% to 95%.

6. The atomization system according to claim 5, characterized in that: In the inhalation solution, the concentration of the recombinant protein antigen is 100 μg / mL to 1 mg / mL, the average pore size of the micropores of the atomization core is 2 μm to 15 μm; the aerodynamic mass median diameter of the aerosol mist particles generated after the inhalation solution is atomized by the atomization system is 3.3 μm to 5.4 μm, and the recovery rate of the recombinant protein antigen in the aerosol mist particles is 82% to 87%; or, In the inhalation solution, the concentration of the recombinant protein antigen is 1 mg / mL to 2 mg / mL, and the average pore size of the micropores of the atomization core is 10 μm to 15 μm; the aerodynamic mass median diameter of the aerosol mist particles generated after the inhalation solution is atomized by the atomization system is 3.6 μm to 5.9 μm, and the recovery rate of the recombinant protein antigen in the aerosol mist particles is 77% to 89%.

7. The atomization system according to claim 1 or 2, characterized in that: The material of the atomizer core is selected from one or more of plastic materials, metal materials and inorganic non-metallic materials.

8. The atomization system according to claim 7, characterized in that: The material of the atomizer core is selected from one or more of polypropylene, stainless steel and silicon nitride.

9. The atomization system according to claim 1, characterized in that: The atomization system can achieve a single quantitative atomization inhalation solution volume of 15 μL to 200 μL.

10. A method for atomizing an inhalation solution, characterized in that: The steps include: Obtaining the inhalation solution and the atomization system according to any one of claims 1 to 9; The power component of the atomization system is used to cause the inhalation solution and the atomization component to be relatively squeezed to form aerosol mist particles.

11. The method for atomizing an inhalation solution according to claim 10, characterized in that: The step of using the power component of the atomization system to cause the inhalation solution and the atomization component to be relatively squeezed to form aerosol mist particles comprises: The power assembly is used to drive the atomizing core of the atomizing assembly to move, and the movement causes the atomizing core to generate pressure on the inhalation solution. The inhalation solution is relatively squeezed with the atomizing core in the process of passing through the micropores of the atomizing core, thereby being converted into the aerosol mist particles.

12. The atomization method according to claim 11, characterized in that: The atomization method satisfies at least one of the following conditions: (1) During the atomization process, the mechanical thrust speed of the power component of the atomization system is 20 mm / s to 500 mm / s; (2) During the atomization process, the atomization core of the atomization system vibrates at a frequency of 80 kHz to 350 kHz.

13. The atomization method according to claim 12, characterized in that: The atomization method satisfies at least one of the following conditions: (1) During the atomization process, the mechanical thrust speed of the power component of the atomization system is 200 mm / s to 500 mm / s; (2) During the atomization process, the atomization core of the atomization system vibrates at a frequency of 200 kHz to 350 kHz.

14. The atomization method according to claim 12 or 13, characterized in that: At least one of the following conditions is met: (1) The aerodynamic mass median diameter of the aerosol mist particles after atomization is 2.7 μm to 5.5 μm; (2) The recovery rate of the protein in the aerosol mist particles after atomization is 60% to 90%; (3) The effective atomization rate is 0.08 g / min to 0.35 g / min; (4) The inhalation coordination between the aerosol delivery process and the user's inhalation process is ≥90%; (5) Atomization residue ≤ 5%.

15. The atomization method according to claim 14, characterized in that: During the atomization process, the mechanical thrust speed of the power assembly is 200 mm / s to 500 mm / s, the operating pressure of the atomization core with an average pore size of 2 μm subjected to the inhaled solution is 66.67 bar to 166.67 bar, the aerodynamic mass median diameter of the aerosol mist particles after atomization is 2.7 μm to 4.3 μm, and the recovery rate of the protein in the aerosol mist particles after atomization is 63.15% to 87.39%; or, During the atomization process, the mechanical thrust speed of the power assembly is 200 mm / s to 500 mm / s, the operating pressure of the atomization core with an average pore size of 15 μm subjected to the inhaled solution is 13.33 bar to 33.33 bar, the aerodynamic mass median diameter of the aerosol mist particles after atomization is 3.6 μm to 5.5 μm, and the recovery rate of the protein in the aerosol mist particles after atomization is 66.89% to 86.26%; or, The inhalation solution includes protein particles, which are virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles. During the atomization process, the atomization core of the atomization system vibrates at a frequency of 80kHz to 350kHz, the concentration of the recombinant protein antigen is 100μg / mL to 1mg / mL, the atomization rate of the inhalation solution is 0.356±0.021g / min to 0.709±0.063g / min, the aerodynamic mass median diameter of the aerosol mist particles generated by the inhalation solution after atomization by the atomization system is 3.4μm to 5.1μm, the percentage of particles smaller than 5μm in the aerosol mist particles is 54% to 72%, and the recovery rate of the recombinant protein antigen in the aerosol mist particles is 64% to 86%; or, During the nebulization process, the atomization core of the atomization system vibrates at a frequency of 80kHz to 350kHz, the concentration of the recombinant protein antigen is 1mg / mL to 2mg / mL, the atomization rate of the inhalation solution is 0.243±0.028g / min to 0.484±0.076g / min, the aerodynamic mass median diameter of the aerosol particles generated after the inhalation solution is atomized by the atomization system is 4.2μm to 5.3μm, the percentage of particles smaller than 5μm in the aerosol particles is 49% to 66%, and the recovery rate of the recombinant protein antigen in the aerosol particles is 52% to 76%.

16. An inhalation solution, characterized in that Used to implement the atomization method according to any one of claims 10 to 15; The inhalation solution includes protein particles, which include virus-like particles self-assembled from recombinant protein antigens, or complex particles assembled from recombinant protein antigens and one or more of lipids, polymers and inorganic particles; the average particle size of the protein particles is 5nm to 2000nm, and the concentration of the recombinant protein antigen in the inhalation solution is 20μg / mL to 2mg / mL.

17. The inhalation solution according to claim 16, characterized in that The protein particle includes the virus-like particle, and the virus-like particle satisfies at least one of the following conditions: (1) The average particle size of the virus-like particles is 20 nm to 200 nm; (2) the concentration of the recombinant protein antigen in the inhalation solution is 20 μg / mL to 1000 μg / mL; (3) The particle size polydispersity coefficient of the virus-like particles is 0.2 to 0.

4.

18. The inhalation solution according to claim 16, characterized in that The protein particles include the composite particles, and the composite particles meet at least one of the following conditions: (1) The average particle size of the composite particles is 5 nm to 2000 nm; (2) The concentration of the recombinant protein antigen in the inhalation solution is 0.1 mg / mL to 2 mg / mL.

19. The inhalation solution according to any one of claims 16 to 18, characterized in that At 25° C. and standard atmospheric pressure, the surface tension of the inhalation solution is 40 mN / m to 70 mN / m; and / or, at 25° C. and standard atmospheric pressure, the viscosity of the inhalation solution is 0.9 mPa.S to 3 mPa.S.

20. The inhalation solution according to any one of claims 16 to 18, characterized in that At least one of the following conditions is met: (1) The recombinant protein antigens include influenza virus subtype-specific hemagglutinin and neuraminidase, parainfluenza virus F protein and HN protein subtypes, norovirus capsid different subtypes, novel coronavirus spike protein or respiratory syncytial virus F protein and G protein; (2) The recombinant protein antigen is a dimer or trimer of a single antigen, or the recombinant protein antigen is a heteropolymer; (3) The virus-like particles are non-covalently self-assembled into a 12- to 15-mer high-order structure based on the oligomeric subunits of the recombinant protein antigen; (4) The complex particles are assembled into lipid-protein nanoparticles, polymer-protein nanoparticles or inorganic-protein nanoparticles based on the oligomeric subunits of the recombinant protein antigen by nanotechnology; (5) The purity of the recombinant protein antigen is 95% to 98%.

21. The inhalation solution according to claim 20, characterized in that At least one of emulsification, heat induction, pH induction, solvent distillation, fluid control, and material design methods is used to make the oligomeric subunits of the recombinant protein antigen self-assemble to form the virus-like particles, or the oligomeric subunits of the recombinant protein antigen are assembled with lipids, polymers, or inorganic particles to form the complex particles.

22. The inhalation solution according to claim 21, characterized in that The lipids include one or more of phospholipids, cholesterol and medium-chain glycerides, wherein the number of carbon atoms of the medium-chain glycerides is 6 to 12; and / or, The polymer includes one or more of PLGA and PEI; and / or, The inorganic particles include one or more of gold, iron oxide and silicon dioxide.

23. The inhalation solution according to any one of claims 16 to 18 and 21 to 22, characterized in that The inhalation solution is atomized and inhaled through the nasal cavity or oral cavity for respiratory mucosal immunization.

24. A liquid medicine tank, characterized in that: Used to store the inhalation solution according to any one of claims 16 to 23, the liquid medicine tank is used for detachably docking with the atomization system according to any one of claims 1 to 9.

25. The liquid medicine tank according to claim 24, characterized in that: The volume of the inhaled solution that can be achieved by the drug liquid tank for single quantitative atomization is 15 μL to 200 μL.

26. An atomized drug-device combination product, characterized in that: It comprises the atomization system according to any one of claims 1 to 9, the inhalation solution according to any one of claims 16 to 23, and the drug solution tank according to claim 24 or 25.

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