Particles, reagent, pharmaceutical composition, powder inhalant, and method for producing particles

Abstract

To provide novel particles containing VHH antibodies, to provide pharmaceutical compositions and powder inhalants containing such particles, and to provide a method for producing novel particles containing VHH antibodies.SOLUTION: Provided is a particle having a particulate substrate and a VHH antibody dispersed in the substrate and being substantially free of VHH antibody aggregates.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to particles, reagents, pharmaceutical compositions, powder inhalants and methods for producing particles. [Background technology]

[0002] In recent years, the use of antibodies as reagents and pharmaceuticals has been investigated. In particular, antibodies have advantages over small molecule drugs, such as higher efficacy, fewer side effects, and a wider range of applicable diseases.

[0003] VHH antibodies, which are single-domain antibodies, are known as one type of antibody. VHH antibodies have the advantages of being able to be produced at low cost using microorganisms and of being easily modified by protein engineering, such as to produce multivalent or multispecific antibodies, or to modify drugs or compounds. Taking advantage of these advantages, pharmaceuticals using VHH antibodies are being developed (see, for example, Patent Document 1).

[0004] Furthermore, VHH antibodies have been developed as reagents for regenerative medicine that can serve as alternatives to FGF2 (see, for example, Patent Document 2).

[0005] There are concerns that antibody aggregates may reduce the accuracy and functionality of commercially available antibody reagents and antibody diagnostic drugs, including IgG antibodies. It has also been pointed out that antibody aggregates may cause immunogenicity in commercially available antibody drugs, including IgG antibodies.

[0006] Similarly, VHH antibodies may form aggregates in pharmaceuticals, raising concerns about immunogenicity. However, there has been insufficient research into how to suppress VHH antibody aggregate formation, and there is room for further consideration in the development of reagents and pharmaceuticals. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention has been made in view of the above circumstances, and aims to provide novel particles comprising VHH antibodies. It is also a further object of the present invention to provide reagents, pharmaceutical compositions, and powder inhalants comprising such particles. It is also a further object of the present invention to provide a method for producing novel particles comprising VHH antibodies. [Means for solving the problem]

[0008] In order to solve the above-mentioned problems, one aspect of the present invention includes particles having a substrate shaped into a particulate form and a VHH antibody dispersed in the substrate, and which are substantially free of aggregates of the VHH antibody.

[0009] According to the present invention, novel particles containing VHH antibodies can be provided. It is also possible to provide reagents, pharmaceutical compositions, and powder inhalants containing such particles. Furthermore, it is also possible to provide methods for producing novel particles containing VHH antibodies. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a schematic diagram showing one particle (unit particle 1) constituting the particle of this embodiment. [Figure 2] FIG. 2 shows an example of a particle manufacturing apparatus. [Figure 3] FIG. 3 is a schematic cross-sectional view showing an example of a droplet discharge means used in the manufacturing apparatus 100. As shown in FIG. [Figure 4] FIG. 4 is an electron microscope photograph of particle A. [Figure 5] FIG. 5 shows the SEC chromatogram of the raw material VHH antibody. [Figure 6] FIG. 6 is an SEC chromatogram of a mannitol solution. [Figure 7] FIG. 7 is an SEC chromatogram measured for particle A. [Figure 8] FIG. 8 is an SEC chromatogram measured for particle B. [Figure 9] FIG. 9 is an SEC chromatogram measured for particle C. [Figure 10] FIG. 10 shows the results of measuring the binding ability of the VHH antibody used as the starting material. [Figure 11] FIG. 11 shows the results of measuring the binding ability of the VHH antibody contained in particle A. [Figure 12] FIG. 12 shows the results of measuring the binding ability of the VHH antibody contained in particle B. [Figure 13] FIG. 13 shows the results of measuring the binding ability of the VHH antibody contained in particle C. [Figure 14] FIG. 14 is a graph showing the measurement results of the inhalation characteristics. DETAILED DESCRIPTION OF THE INVENTION

[0011] [particle] The particles of this embodiment have a substrate and a VHH antibody (variable domain of heavy chain antibody).

[0012] In this embodiment, the term "particles" refers to a population of particulate compositions comprising a substrate and a VHH antibody, unless otherwise specified.

[0013] The particles are typically functional particles that exhibit a desired function. By appropriately selecting the base material contained therein, the particles can be designed to become functional particles with a desired function. Examples of functional particles include particles that deliver VHH antibody 3 to a target site to exert a desired physiological effect, i.e., particles used in drug delivery systems (DDS particles), and sustained-release particles that continuously release drugs over a long period of time.

[0014] In this embodiment, the term "physiological effect" refers to an effect resulting from the physiological activity of a VHH antibody, such as quantitative and / or qualitative changes or effects on living organisms, tissues, cells, proteins, DNA, RNA, etc.

[0015] In this embodiment, "biological activity" means that a VHH antibody acts on a site (e.g., a target tissue as a target site) to change or affect it. The target site is preferably, for example, a receptor present on the cell surface or inside the cell. When the target site is such a receptor, the biological activity of the VHH antibody binding to the specific receptor transmits a signal to the cell, resulting in a physiological effect. The VHH antibody may be a substance produced by an organism (human or non-human) or an artificially synthesized substance.

[0016] 1 is a schematic diagram showing one particle (unit particle 1) constituting the particle of this embodiment. Unit particle 1 has base particle 2, which is a base material formed into a particulate shape, and VHH antibody 3 dispersed in base particle 2.

[0017] Furthermore, the unit particle 1 is substantially free of VHH antibody aggregates.

[0018] In this embodiment, the term "VHH antibody aggregate" refers to a general multimer in which multiple VHH antibodies of the same type are bound, regardless of the degree to which the three-dimensional structure is maintained.

[0019] Furthermore, "substantially free of aggregates" means that the amount of VHH antibody aggregates is such that they do not adversely affect the pharmacological action.

[0020] Furthermore, "adverse effects" refers to side effects such as immunogenicity and toxicity. Although the acceptable amount differs for each VHH antibody, those skilled in the art can easily estimate the "amount of VHH antibody aggregates that does not cause side effects in a living organism, for example, a human."

[0021] Whether aggregates have the above-mentioned adverse effects can be evaluated by cell assays, functional assays, etc. If aggregates are present, a decrease or abnormality in biological activity may be observed. Furthermore, toxicity tests using animals can also be used to evaluate the toxicity and immunogenicity of aggregates.

[0022] In the unit particle 1, the VHH antibody 3 is dispersed and encapsulated in the base particle 2. The term "encapsulation" is not particularly limited as long as the VHH antibody 3 is temporarily or continuously retained in the base particle 2. Furthermore, not all of the VHH antibody dispersed and encapsulated in the base particle 2 needs to be encapsulated within the base particle, and the VHH antibody may be present on the particle surface.

[0023] In the unit particle 1, the VHH antibody 3 may be uniformly dispersed in the base particle 2, or may be unevenly distributed in the base particle 2. When the particles are used as reagents or pharmaceuticals, it is preferable that the VHH antibody 3 be uniformly dispersed in the base particle 2 from the viewpoint of controlling the release of the VHH antibody.

[0024] When a unit particle 1 contains multiple types of base particles 2, and one of the base particles 2 is unevenly distributed in a specific location within the unit particle 1, the dispersion state of the VHH antibody 3 may differ depending on the type of base particle 2.

[0025] <Base material> The substrate is a substance that serves as the basis for forming the shape of the unit particle 1. The substrate constituting the base particle 2 may be of one type only, or may be of two or more types in combination. The substrate may be a composition combining any of the substrates described below.

[0026] In the unit particle 1, the substrate is formed into a particulate substrate particle 2.

[0027] The substrate is preferably solid at room temperature. There are no particular limitations on the substrate, as long as it is not a substance that inhibits the function of VHH antibody 3. The substrate may be a low molecular weight substance (hereinafter referred to as a low molecular weight substrate) or a high molecular weight substance (hereinafter referred to as a high molecular weight substrate). When the particles according to this embodiment are used for medical purposes, if the substrate is a substance that is not toxic to living organisms, the resulting unit particles 1 (particles) can be suitably applied to living organisms.

[0028] On the other hand, when the particles according to this embodiment are used in in vitro diagnostic agents (in vitro diagnostic medicines) or reagents, it is possible to use a substance whose base material is toxic to living organisms, as long as the purpose of the in vitro diagnostic agent or reagent is not hindered. Note that an in vitro diagnostic agent means "a pharmaceutical agent that is intended to be used exclusively for the diagnosis of disease and is not to be used directly on the human or animal body" (defined by the "Act on Ensuring Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc.").

[0029] -Low molecule base material- In this embodiment, the low-molecular-weight base material is a compound having a weight-average molecular weight of less than 15,000.

[0030] The low molecular weight substrate is not particularly limited and can be appropriately selected depending on the purpose. For example, examples of low molecular weight substrates include lipids, sugars, cyclodextrins, amino acids, and organic acids. Only one low molecular weight substrate may be used, or two or more low molecular weight substrates may be used in combination. Furthermore, each of the materials described in detail below as low molecular weight substrates may be used alone, or two or more low molecular weight substrates may be used in combination.

[0031] --Lipids-- The lipids are not particularly limited and can be appropriately selected depending on the purpose. Examples of lipids include medium- or long-chain monoglycerides, medium- or long-chain diglycerides, medium- or long-chain triglycerides, phospholipids, vegetable oils, fish oils, seasoning oils, water-insoluble vitamins, fatty acids, mixtures thereof, and derivatives thereof. Examples of vegetable oils include soybean oil, avocado oil, squalene oil, sesame oil, olive oil, corn oil, rapeseed oil, safflower oil, sunflower oil, etc.

[0032] --Sugars-- The sugars are not particularly limited and can be appropriately selected depending on the purpose. Examples of sugars include monosaccharides and polysaccharides such as glucose, mannose, idose, galactose, fucose, ribose, xylose, lactose, sucrose, maltose, trehalose, turanose, raffinose, maltotriose, acarbose, cyclodextrins, amylose (starch), and cellulose, as well as sugar alcohols (polyols) such as glycerin, sorbitol, lactitol, maltitol, mannitol, xylitol, and erythritol, and derivatives thereof.

[0033] --Cyclodextrins-- The cyclodextrins are not particularly limited and can be appropriately selected depending on the purpose. Examples of cyclodextrins include hydroxypropyl-β-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, α-cyclodextrin, and cyclodextrin derivatives.

[0034] --Amino acids-- The amino acids are not particularly limited and can be appropriately selected depending on the purpose. Examples of amino acids include valine, lysine, leucine, threonine, isoleucine, asparagine, glutamine, phenylalanine, aspartic acid, serine, glutamic acid, methionine, arginine, glycine, alanine, tyrosine, proline, histidine, cysteine, tryptophan, and derivatives thereof.

[0035] --Organic acids-- The organic acids are not particularly limited and can be appropriately selected depending on the purpose. Examples of organic acids include adipic acid, ascorbic acid, citric acid, fumaric acid, gallic acid, glutaric acid, lactic acid, malic acid, maleic acid, succinic acid, tartaric acid, and derivatives thereof.

[0036] -Polymer base material- In this embodiment, the polymer-based material is a compound having a weight-average molecular weight of 15,000 or more.

[0037] The high-molecular-weight base material is not particularly limited and can be appropriately selected depending on the purpose. Examples thereof include water-soluble cellulose, polyalkylene glycol, poly(meth)acrylamide, poly(meth)acrylic acid, poly(meth)acrylic acid ester, polyallylamine, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl acetate, biodegradable polyester, polyglycolic acid, polyamino acid, gelatin, proteins such as fibrin, polysaccharides, and derivatives thereof.

[0038] These polymer-based materials may be used alone or in combination of two or more.Furthermore, each of the materials described in detail below as the polymer-based material may be used alone or in combination of two or more.

[0039] --Water-soluble cellulose-- The water-soluble cellulose is not particularly limited and can be appropriately selected depending on the purpose. Examples of the water-soluble cellulose include alkyl celluloses such as methyl cellulose and ethyl cellulose; Hydroxyalkyl celluloses such as hydroxyethyl cellulose and hydroxypropyl cellulose; and Hydroxyalkyl alkyl celluloses such as hydroxyethyl methyl cellulose and hydroxypropyl methyl cellulose Examples include:

[0040] --Polyalkylene glycol-- The polyalkylene glycol is not particularly limited and can be appropriately selected depending on the purpose. Examples of the polyalkylene glycol include polyethylene glycol (PEG), polypropylene glycol, polybutylene glycol, and copolymers thereof.

[0041] --Poly(meth)acrylamide-- The poly(meth)acrylamide is not particularly limited and can be appropriately selected depending on the purpose. Examples of poly(meth)acrylamides include polymers of monomers such as N-methyl(meth)acrylamide, N-ethyl(meth)acrylamide, N-propyl(meth)acrylamide, N-butyl(meth)acrylamide, N-benzyl(meth)acrylamide, N-hydroxyethyl(meth)acrylamide, N-phenyl(meth)acrylamide, N-tolyl(meth)acrylamide, N-(hydroxyphenyl)(meth)acrylamide, N-(sulfamoylphenyl)(meth)acrylamide, N-(phenylsulfonyl)(meth)acrylamide, N-(tolylsulfonyl)(meth)acrylamide, N,N-dimethyl(meth)acrylamide, N-methyl-N-phenyl(meth)acrylamide, and N-hydroxyethyl-N-methyl(meth)acrylamide. These monomers may be polymerized singly or in combination of two or more.

[0042] --Poly(meth)acrylic acid-- The poly(meth)acrylic acid is not particularly limited and can be appropriately selected depending on the purpose. Examples of the poly(meth)acrylic acid include homopolymers such as polyacrylic acid and polymethacrylic acid, and copolymers such as acrylic acid-methacrylic acid copolymer.

[0043] --Poly(meth)acrylic acid ester-- The poly(meth)acrylic acid ester is not particularly limited and can be appropriately selected depending on the purpose. Examples of the poly(meth)acrylic acid ester include polymers of monomers such as ethylene glycol di(meth)acrylate, diethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, glycerol poly(meth)acrylate, polyethylene glycol (meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, and 1,3-butylene glycol di(meth)acrylate. These monomers may be polymerized alone or in combination of two or more.

[0044] --Polyallylamine-- The polyallylamine is not particularly limited and can be appropriately selected depending on the purpose. Examples of polyallylamine include polymers of diallylamine and triallylamine. These monomers may be polymerized singly or in combination of two or more.

[0045] --Polyvinylpyrrolidone-- Commercially available polyvinylpyrrolidone can be used. There are no particular limitations on the commercially available polyvinylpyrrolidone, and it can be appropriately selected depending on the purpose. Examples of commercially available polyvinylpyrrolidone include Plasdone C-15 (manufactured by ISP TECHNOLOGIES), Kollidon VA64, Kollidon K-30, Kollidon CL-M (all manufactured by Kawarlal), and Kollicoat IR (manufactured by BASF).

[0046] --Polyvinyl alcohol-- The polyvinyl alcohol is not particularly limited and can be appropriately selected depending on the purpose. Examples of polyvinyl alcohol include silanol-modified polyvinyl alcohol, carboxyl-modified polyvinyl alcohol, and acetoacetyl-modified polyvinyl alcohol.

[0047] --Polyvinyl acetate-- The polyvinyl acetate is not particularly limited and can be appropriately selected depending on the purpose. Examples of polyvinyl acetate include vinyl acetate-crotonic acid copolymer and vinyl acetate-itaconic acid copolymer.

[0048] --Biodegradable polyester-- The biodegradable polyester is not particularly limited and can be appropriately selected depending on the purpose. Examples of the biodegradable polyester include polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymer, polylactic acid; poly-ε-caprolactone; succinate polymers such as polyethylene succinate, polybutylene succinate, and polybutylene succinate adipate; polyhydroxyalkanoates such as polyhydroxypropionate, polyhydroxybutyrate, and polyhydroxyparate; and polyglycolic acid.

[0049] --Polyamino acids-- The polyamino acid is not particularly limited and can be appropriately selected depending on the purpose. The polyamino acid may be a polymer of any combination of the amino acids exemplified in the above amino acid section, but is preferably a polymer of a single amino acid. Preferred polyamino acids include, for example, amino acid homopolymers such as poly-α-glutamic acid, poly-γ-glutamic acid, polyaspartic acid, polylysine, polyarginine, polyornithine, and polyserine, and copolymers thereof.

[0050] --gelatin-- The gelatin is not particularly limited and can be appropriately selected depending on the purpose. Examples of gelatin include lime-processed gelatin, acid-processed gelatin, gelatin hydrolysate, and gelatin enzyme dispersion.

[0051] Further, as the gelatin, a gelatin derivative obtained by covalently bonding a hydrophobic group to the above gelatin may be used. The hydrophobic group is not particularly limited and can be appropriately selected according to the purpose. For example, polyesters such as polylactic acid, polyglycolic acid, and poly-ε-caprolactone; lipids such as cholesterol and phosphatidylethanolamine; an alkyl group, an aromatic group containing a benzene ring; a heteroaromatic group, or a mixture thereof, etc. can be mentioned.

[0052] Among the above materials, the substrate is preferably a substance having no biotoxicity, particularly a biodegradable substance such as a biodegradable polymer. By adopting a biodegradable substance as the substrate, the unit particle 1 containing the substrate can be suitably contained in pharmaceutical preparations, functional foods, functional cosmetics, etc.

[0053] Among the various materials described above, saccharides are preferable as the substrate, and further, either one or both of lactose and mannitol are preferable. The unit particle 1 using a saccharide as the substrate is excellent in stability and can suitably protect the VHH antibody 3 dispersed therein. In addition, the unit particle 1 using a saccharide as the substrate can be used as a formulation for pulmonary administration, which is preferable.

[0054] <VHH antibody> The VHH antibody 3 is a low molecular weight single domain antibody using the variable region of a heavy chain antibody. The VHH antibody 3 may be a natural VHH antibody derived from camelids or one artificially designed and synthesized in animal cells, cell-free translation systems, etc. Further, the VHH antibody 3 may be one obtained by chemically modifying an existing VHH antibody, or may be a multivalent VHH antibody.

[0055] Multivalent VHH antibodies refer to the binding of multiple heterologous VHH antibodies and are distinguished from multimers formed by the binding of multiple homologous VHH antibodies, i.e., VHH antibody aggregates. In this case, the VHH antibody species may be either non-multivalent or multivalent. Multivalent VHH antibodies and multimerized VHH antibodies can be distinguished, for example, by using HPLC or titer measurement. Multivalent VHH antibodies and VHH antibody aggregates can be distinguished by known HPLC or titer measurement.

[0056] -Aggregation rate- As described above, the unit particle 1 does not substantially contain aggregates of the VHH antibody 3. Hereinafter, aggregates of the VHH antibody 3 will be simply referred to as "aggregates." This makes it possible to eliminate concerns about reduced accuracy or function when the unit particle 1 is used as a diagnostic agent or reagent, and to easily maintain the pharmacological effect upon administration and suppress side effects when used as a pharmaceutical.

[0057] The proportion of aggregates present in unit particle 1 can be determined by size exclusion chromatography (SEC). Specifically, a solution is prepared by dissolving particles, which are a group of unit particle 1, and the aggregates contained in the resulting solution are analyzed by SEC, and the proportion is determined from the peak area ratio of the measurement results (chromatogram). A solution containing dissolved particles is obtained by dissolving 10 mg of particles in 100 μL of phosphate-buffered saline (PBS, pH 7.4) and filtering it as a standard procedure before SEC. In SEC, relatively larger molecules have shorter retention times, and smaller molecules have longer retention times. Therefore, the VHH antibody peak has a longer retention time than the aggregate peak.

[0058] Furthermore, unless VHH antibodies are actively aggregated, most of the VHH antibodies contained in the particles are expected to be monodispersed in the matrix, and there will be more non-aggregated VHH antibodies than aggregates. Therefore, when a solution containing dissolved particles is analyzed by SEC, the VHH antibody peak is expected to be larger than the aggregate peak.

[0059] From the above, in the SEC analysis results of a solution in which particles are dissolved, it can be determined that peaks having a shorter retention time than the main peak are peaks derived from aggregates. In this embodiment, the proportion of aggregates in the particles is expressed by the "aggregate ratio" (%) defined by the following formula (A): Aggregate rate (%) = Peak area A / Peak area B × 100 ... (A) Peak area A: Area of ​​the peak derived from aggregates Peak area B: Area of ​​all peaks derived from VHH antibodies

[0060] The particle agglomeration rate is preferably 5% or less, more preferably 3% or less, and theoretically preferably 0%.

[0061] -Binding ability- In the following explanation, the strength of the binding between a VHH antibody 3 and an antigen, in other words, the affinity between a VHH antibody 3 and an antigen, is referred to as “binding ability.” A VHH antibody 3 with high binding ability binds strongly to an antigen.

[0062] The binding ability of VHH antibody 3 in the particles can be measured by any method using a measuring device that utilizes surface plasmon resonance (SPR) or biolayer interferometry (BLI), such as a biomolecular interaction analysis system (Octet system, manufactured by SARTORIUS).

[0063] The binding capacity of VHH antibody 3 in the particles is determined by the dissociation constant (K D It has been confirmed that the binding ability of VHH antibody 3 contained in the particles is no different from that of the VHH antibody used as the starting material.

[0064] <Particle properties> The particles may have the following physical properties:

[0065] -Particle size distribution- The particle size distribution of the particles is preferably narrow. Examples of indices that represent the particle size distribution of the particles include the relative span factor (RSF) and the volume average particle size (Dv) / number average particle size (Dn).

[0066] --Relative Span Factor (RSF)-- RSF is defined as (D90-D10) / D50. When measuring the volumetric particle size distribution of particles, D90 represents the cumulative 90% by volume from the small particle side of the cumulative particle size distribution. Similarly, D50 represents the cumulative 50% by volume from the small particle side of the cumulative particle size distribution, and D10 represents the cumulative 10% by volume from the small particle side of the cumulative particle size distribution.

[0067] The RSF of the particles is preferably greater than 0 and less than or equal to 1.2 (0<[RSF]≦1.2), more preferably greater than 0 and less than or equal to 1.0 (0<[RSF]≦1.0), even more preferably greater than 0 and less than or equal to 0.8 (0<[RSF]≦0.8), and even more preferably greater than 0 and less than or equal to 0.6 (0<[RSF]≦0.6).

[0068] Examples of methods for measuring RSF include a method using a concentrated system analyzer (model number: FPAR-1000, manufactured by Otsuka Electronics Co., Ltd.) that employs dynamic light scattering.

[0069] --Volume average particle size (Dv) / Number average particle size (Dn)-- Dv / Dn is the value obtained by dividing Dv by Dn, and is preferably 1.00 or more and 1.50 or less, and more preferably 1.00 or more and 1.20 or less.

[0070] An example of a method for measuring Dv and Dn is a method using a laser diffraction / scattering particle size distribution analyzer (device name: Microtrac MT3000II, manufactured by Microtrac Bell Co., Ltd.).

[0071] Particles with a particle size distribution (RSF, Dv / Dn) within the above range have a narrow distribution width of the particle size distribution, and the proportion of coarse particles relative to the center of the particle size distribution is reduced. As a result, even when the particles are to be contained in a pharmaceutical composition and must be sterilized by filtration before use, they are less likely to clog a sterilization filter, allowing for simple and efficient sterilization by filtration.

[0072] Furthermore, by having the particle size distribution (RSF, Dv / Dn) within the above ranges, the size of the unit particles 1 becomes uniform, and the content of VHH antibody 3 and base material (base material particle 2) in the unit particles 1 and the surface area of ​​the unit particles 1 become uniform. This results in a uniform amount of VHH antibody eluted from each unit particle 1. Therefore, when exposed to different environments (environments A and B), the amount of VHH antibody eluted from the unit particles 1 in environment A becomes uniform to the amount of VHH antibody eluted from the unit particles 1 in environment B, making it possible to provide particles that enable highly controlled release of VHH antibodies.

[0073] -Particle size- The volume average particle size (Dv) of the particles is preferably 0.5 μm or more and 100 μm or less. The Dv of the particles can be appropriately selected depending on the purpose. In particular, when the particles are used as a powder inhalant, the Dv is more preferably 0.5 μm or more and 10 μm or less. When used as a powder inhalant, particles with a volume average particle size of 0.5 μm or more and 10 μm or less are easily delivered to the respiratory tract efficiently.

[0074] -In vitro inhalation properties- When the particles of this embodiment are used as an inhalation formulation, it is advisable to also measure their in vitro inhalation characteristics, which are important indicators for estimating the inhalation efficiency of the particles in vivo.

[0075] A cascade impactor is commonly used as a means for measuring inhalation characteristics, and examples thereof include, but are not limited to, an Andersen Non-Bubbly Sampler (manufactured by Tokyo Dylec Co., Ltd.).

[0076] The inhalation characteristics can be calculated as the fine particle fraction (FPF) value from the amount of particles deposited in stages 2 to 7 of the cascade impactor. Particles with higher FPF values ​​are expected to have better inhalation efficiency in vivo.

[0077] [Pharmaceutical composition] The pharmaceutical composition contains the above-described particles. The pharmaceutical composition also contains additives, such as those used for formulation, as needed. The additives are not particularly limited and can be appropriately selected depending on the purpose. Examples of additives include excipients, flavoring agents, disintegrants, fluidizing agents, adsorbents, lubricants, odorants, surfactants, flavorings, colorants, antioxidants, masking agents, antistatic agents, and wetting agents. In the pharmaceutical composition, these additives may be contained in the particles, i.e., in the base particles. The pharmaceutical composition may also be a mixture of the above-described particles and these additives.

[0078] In this specification, the term "pharmaceutical composition" refers to a composition used as a drug or quasi-drug. Both "drugs" and "quasi-drugs" refer to those defined in Article 2 of the Act on Ensuring Quality, Efficacy and Safety of Pharmaceuticals, Medical Devices, etc.

[0079] The additive substance may be used alone or in combination of two or more kinds. Furthermore, with regard to each material described in detail below as the additive substance, it may be used alone or in combination of two or more kinds.

[0080] --Excipients-- The excipient is not particularly limited and can be appropriately selected depending on the purpose. Examples of the excipient include lactose, sucrose, mannitol, glucose, fructose, maltose, erythritol, maltitol, xylitol, palatinose, trehalose, sorbitol, crystalline cellulose, talc, anhydrous silicic acid, anhydrous calcium phosphate, precipitated calcium carbonate, calcium silicate, etc.

[0081] --Flavoring agent-- The flavoring agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of flavoring agents include L-menthol, sucrose, D-sorbitol, xylitol, citric acid, ascorbic acid, tartaric acid, malic acid, aspartame, acesulfame potassium, thaumatin, sodium saccharin, dipotassium glycyrrhizinate, sodium glutamate, 5'-sodium inosinate, and 5'-sodium guanylate.

[0082] --Disintegrant-- The disintegrant is not particularly limited and can be appropriately selected depending on the purpose. Examples of the disintegrant include low-substituted hydroxypropyl cellulose, carmellose, carmellose calcium, carboxymethyl starch sodium, croscarmellose sodium, crospovidone, hydroxypropyl starch, and corn starch.

[0083] --Superplasticizer-- The fluidizing agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of the fluidizing agent include light anhydrous silicic acid, hydrous silicon dioxide, and talc.

[0084] As the light anhydrous silicic acid, commercially available products can be used. There are no particular limitations on the commercially available light anhydrous silicic acid, and it can be appropriately selected depending on the purpose. As the light anhydrous silicic acid, for example, Adsolider 101 (manufactured by Freund Corporation: average pore diameter: 21 nm) can be mentioned.

[0085] --Adsorbent-- Commercially available products can be used as the adsorbent. There are no particular limitations on the commercially available adsorbent, and it can be appropriately selected depending on the purpose. Examples of adsorbents include trade name: Carplex (component name: synthetic silica, registered trademark of DSL. Japan Co., Ltd.), trade name: Aerosil (registered trademark of Nippon Aerosil Co., Ltd.) 200 (component name: hydrophilic fumed silica), trade name: Sylysia (component name: amorphous silicon dioxide, registered trademark of Fuji Silysia Chemical Co., Ltd.), and trade name: Alkamac (component name: synthetic hydrotalcite, registered trademark of Kyowa Chemical Co., Ltd.).

[0086] --lubricant-- The lubricant is not particularly limited and can be appropriately selected depending on the purpose. Examples of the lubricant include magnesium stearate, calcium stearate, sucrose fatty acid ester, sodium stearyl fumarate, stearic acid, polyethylene glycol, and talc.

[0087] --Odor masking agent-- The flavoring agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of the flavoring agent include trehalose, malic acid, maltose, potassium gluconate, anise essential oil, vanilla essential oil, and cardamom essential oil.

[0088] --Surfactants-- The surfactant is not particularly limited and can be appropriately selected depending on the purpose. Examples of surfactants include polysorbates such as polysorbate 80, polyoxyethylene-polyoxypropylene copolymers, and sodium lauryl sulfate.

[0089] --Fragrance-- The flavoring agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of the flavoring agent include lemon oil, orange oil, and peppermint oil.

[0090] --Colorant-- The coloring agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of the coloring agent include titanium oxide, food yellow No. 5, food blue No. 2, red ferric oxide, and yellow ferric oxide.

[0091] --Antioxidants-- The antioxidant is not particularly limited and can be appropriately selected depending on the purpose. Examples of the antioxidant include sodium ascorbate, L-cysteine, sodium sulfite, vitamin E, etc.

[0092] --Concealing agent-- The masking agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of the masking agent include titanium oxide.

[0093] --Anti-static agent-- The antistatic agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of the antistatic agent include talc and titanium oxide.

[0094] --Wetting agent-- The wetting agent is not particularly limited and can be appropriately selected depending on the purpose. Examples of wetting agents include polysorbate 80, sodium lauryl sulfate, sucrose fatty acid esters, macrogol, and hydroxypropyl cellulose (HPC).

[0095] The pharmaceutical composition formulation is not particularly limited and can be appropriately selected depending on the purpose. Examples of the formulation include a large intestine delivery formulation, a lipid microsphere formulation, a dry emulsion formulation, a self-emulsifying formulation, a dry syrup, a powder formulation for nasal administration, a powder formulation for pulmonary administration, a wax matrix formulation, a hydrogel formulation, a polymeric micelle formulation, a mucoadhesive formulation, a gastric floating formulation, a liposome formulation, and a solid dispersion formulation.

[0096] Dosage forms of pharmaceutical compositions include, for example, tablets, capsules, suppositories, other solid dosage forms, etc.; aerosols for intranasal or pulmonary administration, etc.; liquid preparations such as injectables, intraocular preparations, intraotic preparations, and oral preparations.

[0097] The route of administration of the pharmaceutical composition is not particularly limited and can be appropriately selected depending on the purpose. Examples of the route of administration include oral administration, nasal administration, rectal administration, vaginal administration, subcutaneous administration, intravenous administration, and pulmonary administration. Among these, pulmonary administration is preferred.

[0098] The pharmaceutical composition of this embodiment is preferably a powder inhalant in the form of an aerosol. The particles described above have uniform unit particle shape and size, and can reach the deep lungs by riding on air currents. The ability of the particles to reach the deep lungs can be confirmed by testing using a known pulmonary cascade impactor (Andersen-type cascade impactor). Tests using a pulmonary cascade impactor have confirmed that the particles (pharmaceutical composition) of this embodiment can successfully reach stage 7, suggesting their suitability as a powder inhalant.

[0099] The particles may be functional particles depending on various applications. The functional fine particles are not particularly limited and can be appropriately selected depending on the purpose. Examples include immediate-release particles, sustained-release particles, pH-dependent release particles, pH-independent release particles, enteric-coated particles, and controlled-release coated particles.

[0100] [reagent] The reagent contains the above-described particles and may contain additives such as stabilizers in addition to a dispersion medium, as needed. Such a reagent may be, for example, a research reagent, a reagent for regenerative medicine, or a reagent to be added to a culture medium for cultured meat. Furthermore, the reagent may be used in cosmetics, etc.

[0101] [Particle manufacturing method] The particle manufacturing method of this embodiment includes a step of ejecting a raw material liquid containing a substrate, a VHH antibody, and a solvent from an ejection hole into the gas phase to form droplets of the raw material liquid, and a step of removing the solvent from the droplets in the gas phase to obtain particles containing the substrate and the VHH antibody.

[0102] The particle production device of this embodiment comprises a substrate, a droplet ejection means for ejecting droplets of a raw material liquid containing a VHH antibody and a solvent, and a granulation means for removing the solvent from the droplets to obtain particles.

[0103] In this application, "removal" means that the solvent contained in the droplets is removed from the droplets. Removal is not limited to removal of all the solvent contained in the droplets, and the solvent contained in the droplets may remain in the particles as long as the particles are obtained.

[0104] A known dry granulation method for obtaining particles in a gas phase is, for example, the spray drying method. In the spray drying method, a liquid containing particulate material is sprayed into the air and dried to obtain small atomized particles. There are various spraying methods, including a pressure nozzle method in which the liquid is pressurized and sprayed from a nozzle, and a disk method in which the liquid is sent to a rapidly rotating disk and scattered by centrifugal force.

[0105] While the spray-drying method can produce particles with a high percentage of VHH antibodies retained within the particle (VHH antibody retention rate) after the particle production process, it is generally difficult to produce particles with a small diameter. When the spraying method is a disk type, it may be possible to produce small particles, but this requires large-scale equipment. Furthermore, the droplets tend to coalesce in the air, making it difficult to produce particles with a narrow particle size distribution. To prevent droplets from coalescing in the air, the droplets must be heated after spraying so that they dry quickly. However, this method is not suitable for particle materials such as VHH antibodies, which have the risk of their physiological activity being altered by heating.

[0106] In contrast, the method employed in this embodiment allows particles to be produced more easily and stably than the spray drying method.

[0107] - Droplet formation process - In the particle production method of this embodiment, first, a raw material liquid containing a substrate, a VHH antibody, and a solvent is discharged into the gas phase to form droplets of the raw material liquid (droplet formation step).

[0108] In this step, various methods can be used as long as they can eject the raw material liquid from the ejection holes to form droplets. Among these, a method in which vibration is imparted to the raw material liquid and the raw material liquid is ejected from the ejection holes is preferred.

[0109] The method for vibrating and discharging the raw material liquid is not particularly limited, and examples thereof include the following methods (a) to (c). All of methods (a) to (c) are methods for discharging the raw material liquid from a liquid storage part that stores the raw material liquid and has a discharge hole.

[0110] (a) A method in which the volume changer that changes the volume of the liquid storage section is vibrated and the volume is changed according to the vibration period. (b) A method of discharging the raw material liquid from the discharge hole while vibrating the entire liquid storage unit in the direction of discharging the raw material liquid. (c) A method of vibrating a thin film having discharge holes formed therein

[0111] The volume-changing means in method (a) is not particularly limited as long as it can change the volume of the liquid storage section, and can be selected appropriately depending on the purpose. For example, a piezoelectric element that expands and contracts when a voltage is applied can be used.

[0112] Method (b) includes, for example, a means using the technology described in JP-A-2007-199463.

[0113] Method (c) includes, for example, a means using the technology described in JP-A-2008-292976.

[0114] When the raw material liquid is vibrated and ejected, a so-called inkjet nozzle can be used as the ejection means. The ejection mechanism of the inkjet nozzle can be, for example, a liquid column resonance method, a membrane vibration method, a liquid vibration method, a Rayleigh fragmentation method, or the like.

[0115] --solvent-- The solvent is a liquid that dissolves the substrate, and preferably also dissolves the VHH antibody.

[0116] Examples of the solvent include water, aliphatic halogenated hydrocarbons (e.g., dichloromethane, dichloroethane, chloroform, etc.), alcohols (e.g., methanol, ethanol, propanol, etc.), ketones (e.g., acetone, methyl ethyl ketone, etc.), ethers (e.g., diethyl ether, dibutyl ether, 1,4-dioxane, etc.), aliphatic hydrocarbons (e.g., n-hexane, cyclohexane, n-heptane, etc.), aromatic hydrocarbons (e.g., benzene, toluene, xylene, etc.), organic acids (e.g., acetic acid, propionic acid, etc.), esters (e.g., ethyl acetate, etc.), amides (e.g., dimethylformamide, dimethylacetamide, etc.), and mixed solvents of two or more of these solvents.

[0117] Among these, from the viewpoint of solubility and the risk of denaturation of VHH antibodies, water, saline solutions containing water as the main component, and buffer solutions such as phosphate buffer, TRIS buffer, acetate buffer, heptane buffer, and HEPES buffer are preferred, with water and phosphate buffer being more preferred.

[0118] The solvent content is preferably 70% by mass or more and 99.5% by mass or less, and more preferably 90% by mass or more and 99% by mass or less, based on the mass of the raw material liquid. When the solvent content is 70% by mass or more and 99.5% by mass or less, production stability is improved in terms of the solubility of the particulate material and the liquid viscosity.

[0119] --Raw material liquid-- The viscosity of the raw material liquid is not particularly limited and can be appropriately selected depending on the purpose. The viscosity of the raw material liquid is, for example, preferably 0.5 mPa·s or more and 15.0 mPa·s or less, and more preferably 0.5 mPa·s or more and 10.0 mPa·s or less.

[0120] The viscosity of the raw material liquid is measured, for example, using a viscoelasticity measuring device (device name: MCR rheometer, manufactured by Anton Paar) at 25°C and a shear rate of 10 s -1 It is preferable that the viscosity of the raw material liquid is 0.5 mPa·s or more and 15.0 mPa·s or less, because this allows suitable ejection in the above-mentioned means for ejecting droplets.

[0121] The surface tension of the raw material liquid is not particularly limited and can be appropriately selected depending on the purpose. The surface tension of the raw material liquid is preferably 10 mN / m or more and 60 mN / m or less, and more preferably 20 mN / m or more and 50 mN / m or less.

[0122] The surface tension of the raw material liquid can be measured, for example, using a handy surface tensiometer (device name: PocketDyne, manufactured by KRUSS) by the maximum bubble pressure method under conditions of 25° C. and a lifetime of 1,000 ms.

[0123] When the surface tension of the raw material liquid is 0.5 mPa·s or more and 15.0 mPa·s or less, the droplets can be suitably discharged by the above-mentioned means for discharging the droplets.

[0124] -Process for obtaining particles- Next, the solvent is evaporated from the droplets in the gas phase to remove the solvent, thereby obtaining particles (particle obtaining step). The solvent is removed while the droplets ejected into the gas phase fly in the gas phase.

[0125] In this manufacturing method of the present embodiment, the temperature of the gas phase is controlled to be equal to or lower than the aggregation temperature (Tagg) of the VHH antibody. Furthermore, the temperature of the gas phase is preferably controlled to be lower than the aggregation temperature of the VHH antibody. The "temperature of the gas phase" is preferably controlled to be the "maximum temperature of the airflow that can come into contact with the droplets" in the device used to manufacture the particles.

[0126] For example, the aggregation temperature of the VHH antibody used in the Examples described below is approximately 49° C. In this case, the gas phase temperature is preferably, for example, 20° C. or higher and 49° C. or lower, more preferably 20° C. or higher and 45° C. or lower, and even more preferably 20° C. or higher and 35° C. or lower.

[0127] The aggregation temperature of VHH antibodies varies depending on the type of VHH antibody. Therefore, prior to carrying out the droplet formation step, it is advisable to carry out a step of measuring the aggregation temperature of the VHH antibody to be used, and then set the gas phase temperature to a temperature equal to or lower than the aggregation temperature of the VHH antibody based on the measurement results.

[0128] The inventors' studies have confirmed that VHH antibodies aggregate (form aggregates) when heated. Therefore, by controlling the gas phase temperature to a temperature equal to or lower than the aggregation temperature of VHH antibodies, preferably lower than the aggregation temperature, it is possible to prevent VHH antibodies from aggregating and forming aggregates during granulation.

[0129] Depending on the manufacturing equipment used for granulation, it may be difficult to directly measure and control the temperature of the gas phase. In such cases, a preliminary experiment may be conducted to determine the relationship between the temperature at the inlet of the airflow introduced into the granulation space (airflow inlet temperature) and the aggregation rate of the resulting particles, and the gas phase temperature may be roughly calculated from the airflow inlet temperature. When the above relationship is determined by gradually increasing the airflow inlet temperature under constant ejection conditions for the raw material liquid, the temperature at which the aggregation rate begins to increase sharply can be roughly calculated to correspond to the temperature at which the gas phase temperature exceeds the aggregation temperature of the VHH antibody.

[0130] In this process, droplets are ejected into a transport air current (gas phase) to vaporize the solvent from the droplets, thereby forming granules. A preferred method for vaporizing the solvent from the droplets is, for example, to make the transport direction of the transport air current approximately perpendicular to the ejection direction of the droplets.

[0131] It is also preferable to appropriately adjust the temperature, vapor pressure, type of gas, etc. of the transport airflow.

[0132] Furthermore, as long as the collected particles remain in a solid state, the solvent may remain in the collected particles, and a drying step may be additionally provided as a separate step.

[0133] The resulting particles can be suitably collected by a particle collecting means. The particle collecting means is not particularly limited and can be appropriately selected depending on the purpose. Examples of the particle collecting means include cyclone collection and back filters.

[0134] [Particle manufacturing equipment] 2 is a schematic diagram showing a manufacturing apparatus 100, which is an example of a particle manufacturing apparatus. The manufacturing apparatus 100 is used to implement the above-described particle manufacturing method. As shown in FIG. 2, the manufacturing apparatus 100 has a droplet discharge unit 10 and a granulation unit 20.

[0135] -Droplet discharge part- The droplet discharge unit 10 discharges the above-mentioned raw material liquid into a gas phase to form droplets. The droplet discharge unit 10 includes a droplet discharge means 11, a container 12, a liquid supply pipe 13, and a pump 15.

[0136] --Droplet discharge means-- 3 is a schematic cross-sectional view showing an example of droplet discharge means used in the manufacturing apparatus 100. The droplet discharge means 11 vibrates the raw material liquid L and discharges it into a gas phase to form droplets D. The droplet discharge means 11 employs the above-mentioned method (a) as a method for vibrating and discharging the raw material liquid.

[0137] 3 is a schematic cross-sectional view showing an example of the droplet discharge means 11. As shown in FIG.

[0138] The main body 110 has a storage section 110S that stores the raw material liquid L. The main body 110 also has a plurality of discharge holes 110a that communicate with the storage section 110S. The main body 110 receives the raw material liquid L from a storage container 12 (not shown), and stores the raw material liquid L in the storage section 110S.

[0139] The discharge holes 110a are not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include openings provided in a nozzle plate or the like.

[0140] The cross-sectional shape of the discharge hole 110a is not particularly limited and can be appropriately selected depending on the purpose. For example, (1) A tapered shape in which the opening diameter becomes smaller from the inside (the storage section S side) to the outside (the side from which the liquid is discharged) (2) A rounded shape with an opening that narrows from the inside (the storage section S side) to the outside (the side from which the liquid is dispensed) (3) A shape in which the opening diameter narrows at a certain nozzle angle from the inside (the side of the storage section S) to the outside (the side from which the liquid is discharged) (4) Combination of the shape of (1) and the shape of (2) Among these, the shape (3) is preferable because it maximizes the pressure applied to the raw material liquid L at the discharge holes 110a.

[0141] The "nozzle angle" refers to the angle between the ejection direction of droplets ejected from the ejection hole 110a (direction 1) and the direction of the airflow near the nozzle (direction 2). When direction 1 and direction 2 are the same, the nozzle angle is 0°, and when direction 1 and direction 2 are perpendicular, the nozzle angle is 90°.

[0142] The nozzle angle in the shape of (3) is not particularly limited and can be appropriately selected depending on the purpose, but is preferably between 60° and 90°. When the nozzle angle is between 60° and 90°, droplet ejection can be stabilized.

[0143] The size (inner diameter) of the ejection hole 110a is not particularly limited and can be selected appropriately depending on the purpose. The "size of the ejection hole 110a" refers to the opening diameter (inner diameter) of the ejection port 110a that opens into the outer surface of the droplet ejection means 11.

[0144] The size (inner diameter) of the discharge hole 110a is, for example, preferably 1000 μm or less, more preferably 1.0 μm or more and 1000 μm or less, even more preferably 1.0 μm or more and 500 μm or less, and particularly preferably 1.0 μm or more and 50 μm or less.

[0145] If the shape of the discharge hole 110a is not a perfect circle, the diameter (circle equivalent diameter) of a perfect circle having an area equivalent to the area of ​​the discharge hole 110a is used.

[0146] The elastic plate 111 forms part of the wall surface of the storage section 110S and is deformed by receiving stress. When the elastic plate 111 is deformed to become convex toward the storage section 110S, the volume of the storage section 110S changes (decreases).

[0147] The elastic plate 111 can be manufactured using various materials as long as they do not adversely affect the base material or VHH antibodies contained in the raw material liquid L and do not impair the effects of the invention. For example, the elastic plate 111 can be made of metal or resin.

[0148] The volume changing means 112 is in contact with the elastic plate 111 and is provided on the opposite side of the elastic plate 111 from the storage section 110S. The volume changing means 112 deforms the elastic plate 111 by applying a drive voltage, thereby changing (reducing) the volume of the storage section 110S. As the volume of the storage section 110S decreases, the surplus raw material liquid L that was contained in the storage section 110S and that can no longer be contained in the storage section 110S is discharged from the discharge hole 110a, and droplets D are formed.

[0149] Furthermore, by applying a drive voltage of a predetermined frequency to the volume changing means 112, the volume changing means 112 vibrates the elastic plate 111 in accordance with the frequency of the drive voltage, which imparts vibration to the raw material liquid L, causing the raw material liquid L to be discharged from the discharge hole 110a.

[0150] The frequency of the driving voltage is not particularly limited and can be appropriately selected depending on the purpose. For example, the frequency is preferably 1 kHz or higher, more preferably 150 kHz or higher, and even more preferably 300 kHz to 500 kHz.

[0151] When the frequency of the driving voltage, i.e., the vibration imparted to the raw material liquid L via the elastic plate 111, is 1 kHz or higher, the droplets D can be ejected from the ejection holes 110a with good reproducibility. Furthermore, when the frequency of the driving voltage is 150 kHz or higher, production efficiency can be improved.

[0152] There are no particular limitations on the volume change means 112, as long as it can deform the elastic plate 111 and change the volume of the storage section 110S, and it can be selected appropriately depending on the purpose. An example of the volume change means 112 is a piezoelectric element (piezo element) that expands and contracts when a voltage is applied. There are no particular limitations on the piezoelectric element, and its shape, size, and material can be selected appropriately.

[0153] The material of the piezoelectric element is not particularly limited and can be appropriately selected depending on the purpose, and examples thereof include piezoelectric ceramics such as lead zirconate titanate (PZT), piezoelectric polymers such as polyvinylidene fluoride (PVDF), and single crystals such as quartz, LiNbO3, LiTaO3, and KNbO3.

[0154] --Containment Container-- The storage container 12 is a container that contains a raw material solution L containing a substrate, a VHH antibody, and a solvent. The storage container 12 may be flexible or inflexible.

[0155] The material of the storage container 12 is not particularly limited and can be appropriately selected depending on the purpose. The material of the storage container 12 may be made of, for example, resin or metal. Furthermore, the storage container 12 may have a sealed structure or a non-sealed structure.

[0156] --Liquid supply pipe, liquid return pipe, pump-- The liquid supply pipe 13 connects the droplet discharge means 11 and the container 12. The liquid supply pipe 13 supplies the raw material liquid L from the container 12 to the droplet discharge means 11.

[0157] The pump 15 supplies the raw material liquid L contained in the container 12 to the droplet discharge means 11 .

[0158] A pressure gauge P1 may be provided in the path of the liquid supply pipe 13. The pressure gauge P1 indicates the pressure of the raw material liquid L in the liquid supply pipe 13 and in the container 110S. The liquid sending pressure to the droplet discharge means 11 is managed by the pressure gauge P1.

[0159] -Granulation section- The granulating section 20 has a chamber 21 and a collecting means 22 .

[0160] --Chamber-- The chamber 21 is a cylindrical member having an internal space 21S in which the droplet discharge means 11 is disposed. In the chamber 21, the solvent is removed from the droplets D discharged by the droplet discharge means 11, and particles P are formed.

[0161] The chamber 21 has an airflow inlet 211 provided at the top end and an airflow outlet 212 provided at the bottom end.

[0162] Gas whose temperature is controlled to be equal to or lower than the aggregation temperature of VHH antibodies is introduced through the air flow inlet 211. There are no particular limitations on the type of gas, and for example, air or a non-flammable gas such as nitrogen may be used.

[0163] The introduced gas forms a downward air current in the internal space 21S. As a result, the droplets D discharged from the droplet discharge means 11 are carried downward by the downward air current in addition to gravity. The speed of the downward air current is preferably equal to or greater than the discharge speed of the droplets D. The downward air current may be a laminar flow, a swirling flow, or a turbulent flow.

[0164] The temperature of the gas phase in the internal space 21S is controlled to be equal to or lower than the aggregation temperature of the VHH antibody. The droplets D ejected into the internal space 21S are heated in the internal space 21S and exposed to a downward air current. This removes the solvent from the droplets D, forming particles P.

[0165] The chamber 21 may have a pressure gauge P2 that measures the pressure in the internal space 21S. The pressure in the internal space 21S is managed by the pressure gauge P2.

[0166] If the measurement value of the pressure gauge P1 of the droplet discharge unit 10 is greater than the measurement value of the pressure gauge P2, the raw material liquid L may seep out from the discharge hole 110a. On the other hand, if the measurement value of the pressure gauge P1 is smaller than the measurement value of the pressure gauge P2, gas from the internal space 21S may enter the droplet discharge means 11, causing the discharge of droplets D to stop. Therefore, it is preferable that the measurement value of the pressure gauge P1 and the measurement value of the pressure gauge P2 are approximately the same.

[0167] --Collection means-- The formed particles P pass through the airflow outlet 212 and are collected by the collecting means 22. There are no particular limitations on the collecting means 22 and it can be appropriately selected depending on the purpose. Examples of the collecting means 22 include cyclone collection and a back filter.

[0168] The particles P collected by the collecting means 22 may be further stored in a storage section 23.

[0169] Furthermore, if the particles P collected by the collecting means 22 contain a large amount of solvent, it is preferable to perform secondary drying as appropriate. As the secondary drying, a commonly known drying means such as fluidized bed drying or vacuum drying can be used.

[0170] In such a manufacturing apparatus 100, it is preferable to suppress coalescence of the droplets D before drying by a known method, thereby making it possible to obtain particles P with a narrow particle size distribution.

[0171] The above-described production method makes it possible to produce particles in which VHH antibodies are dispersed in a base material.

[0172] The particles produced are produced using an ejection means that forms droplets by vibration, etc., which reduces the risk of the VHH antibody's higher-order structure changing due to external stress as a particle material, and as a result, can suppress the progression of VHH antibody aggregation and the decrease in binding ability.

[0173] Furthermore, the particles produced do not undergo the process of particle production, where the drug migrates to another phase, as occurs with, for example, the emulsion method, and therefore the above-described production method can produce particles with a high proportion of VHH antibodies retained in the particles (VHH antibody retention rate).

[0174] The above-described particle 1 can provide novel particles that contain a VHH antibody and are substantially free of VHH antibody aggregates. Furthermore, pharmaceutical compositions, reagents, and powder inhalants that contain such particle 1 can be provided.

[0175] Furthermore, the above-described particle production method makes it possible to easily produce particles that are substantially free of VHH antibody aggregates.

[0176] In particular, when particle 1 is used as a powder inhalant, the following effects can be expected.

[0177] First, biopharmaceuticals, including antibody-based drugs, have grown to account for more than half of the pharmaceutical market, but most formulations are injectable. This limits the indications for these drugs. On the other hand, for respiratory and pulmonary diseases, pulmonary formulations, which can deliver drugs directly to the lesion, are suitable, and it is expected that biopharmaceuticals will be utilized in this field.

[0178] Since pulmonary preparations must be in powder form, biopharmaceuticals must also be provided as powders. When providing biopharmaceuticals as powders, one possible approach is to mechanically grind freeze-dried biopharmaceuticals. However, the powders obtained by crushing freeze-dried products have non-uniform particle sizes. Therefore, it is known that most of the drug in the powdered biopharmaceuticals obtained in this way is deposited in the bronchi and does not reach the deep lungs (alveoli).

[0179] In contrast, the particles of the present embodiment have uniform unit particle shapes and sizes, and can be carried by air currents to reach the deep lungs. The ability of the particles to reach the deep lungs can be confirmed by a test using a cascade impactor.

[0180] Furthermore, it has been confirmed that the binding ability of the VHH antibodies contained in the particles is no different from that of the raw VHH antibodies. In other words, the VHH antibodies contained in the particles can be evaluated as being equivalent to the VHH antibodies that were the raw material before being made into particles, and are expected to exert the desired effect on lesions.

[0181] As a result, the particles of this embodiment can be used as an effective pulmonary formulation, and are expected to be effective medicines for infectious diseases such as COVID-19 and RS virus infection, as well as respiratory diseases such as lung cancer and COPD.

[0182] While the preferred embodiments of the present invention have been described above with reference to the accompanying drawings, the present invention is not limited to these examples. The shapes and combinations of the components shown in the above examples are merely examples, and various modifications can be made based on design requirements, etc., without departing from the spirit of the present invention. [Example]

[0183] The present invention will be described below with reference to examples, but the present invention is not limited to these examples.

[0184] [Example 1] (Preparation of formulation containing VHH antibody) Mannitol (Tokyo Chemical Industry Co., Ltd.), anti-human serum albumin (HSA)-VHH antibody (Tagg: 49°C), and Dulbecco's PBS (DPBS) were mixed in the mass ratio shown in Table 1 and stirred at 600 rpm for 1 hour using a stirrer, thereby obtaining an aqueous solution in which mannitol was completely dissolved (Formulation A).

[0185] [Table 1]

[0186] (Preparation of VHH antibody-containing particles) Production of particle A Using a manufacturing apparatus configured as shown in Fig. 2, the obtained prescription liquid A was discharged using a droplet discharge means (manufactured by Ricoh Co., Ltd.) having a piezoelectric (PZT) element. Water was removed from the obtained droplets to obtain particles A of Example 1. The manufacturing conditions for particles A were as follows.

[0187] -Particle manufacturing conditions- Discharge hole shape: perfect circle Discharge hole diameter: 8μm PZT drive frequency: 310KHz PZT drive voltage: 10V Dry air flow rate: 50L / min dry nitrogen Dry air temperature (air inlet temperature): 35℃

[0188] Figure 4 is an electron microscope photograph (magnification: 5000x, accelerating voltage: 5 kV) of particle A. From Figure 4, it can be seen that the obtained particle A has a diameter of 2 to 3 μm. It can also be seen that particle A does not contain coarse particles or fine particles, and the particle size is uniform.

[0189] [Example 2] Particles B of Example 2 were obtained in the same manner as in Example 1, except that the temperature of the dry air stream was changed to 50°C.

[0190] [Comparative Example 1] Particles C of Comparative Example 1 were obtained in the same manner as in Example 1, except that the temperature of the dry air stream was changed to 65°C.

[0191] [Particle evaluation] (measuring the particle size of microparticles) The average particle size (Dv) and volume cumulative particle size (D10, D50, D90) of each particle were measured using a Microtrac MT3000II. From the measurement results, the RSF was calculated based on the following formula. RSF=(D90-D10) / D50

[0192] It should be noted that D10, D50, and D90 are the volume particle sizes when the volume cumulative frequency is 10%, 50%, and 90%, respectively.

[0193] The measurement results are shown in Table 2.

[0194] [Table 2]

[0195] From Table 2, it can be seen that particles A, B, and C all have similar sizes and particle size distributions.

[0196] (VHH antibody aggregate rate) The aggregation rate of each particle was measured by size exclusion chromatography (SEC) using an ACQUITY UPLC H-Class PLUS Bio (Waters Corporation) under the following measurement conditions using a solution prepared under the following conditions.

[0197] ·solution 10 mg of particles were dissolved in 100 μL of phosphate buffered saline (PBS, pH 7.4), and the solution was filtered through a filter (Millipore Ultrafree MC UFC30LH00, manufactured by Merck) to prepare a sample solution for SEC analysis. Measurement conditions Column: ACQUITY Premier Protein SEC Column, 250Å, 1.7μm, 4.6×150mm Mobile phase: sodium phosphate, pH7.4 Flow rate: 0.3ml / min Column temperature: 25℃ Injection volume: 5μL Total measurement time: 12 min Detection light: UV280nm

[0198] Figure 5 is an SEC chromatogram of the raw material VHH antibody. Figure 6 is an SEC chromatogram of the mannitol solution. From Figures 5 and 6, peaks with retention times shorter than approximately 5.28 minutes can be considered to be VHH antibody aggregates.

[0199] Figure 7 is an SEC chromatogram measured for particle A. Figure 8 is an SEC chromatogram measured for particle B. Figure 9 is an SEC chromatogram measured for particle C. Figures 7 to 9 show that the amount of VHH antibody aggregates increases as the temperature of the dry air stream during granulation increases.

[0200] Table 3 shows the aggregate rate calculated from the SEC measurement results for each particle. Table 3 shows that particle C in Comparative Example 1 was unsuitable, with an aggregate rate exceeding 5%. In Comparative Example 1, the temperature of the dry air stream was much higher than the aggregation temperature of VHH antibodies (49°C), and it is highly likely that the temperature of the gas phase was also higher than the aggregation temperature of VHH antibodies, which is thought to have resulted in the formation of large amounts of VHH antibody aggregates.

[0201] [Table 3]

[0202] (Measurement of binding ability) The binding ability of the VHH antibody contained in each particle was measured using the Octet system. Octet RED 384 (SARTORIUS) was used for the measurement. A 96-well black well plate (SARTORIUS) was used for sensor hydration, and a 384-well tilted bottom plate (SARTORIUS) was used for sample measurement.

[0203] Measurement conditions The plate was placed in 200 μL of PBS-T (0.05% Tween 20, pH 7.4) and the Anti-Penta-HIS (HIS1K) biosensor was immersed for 10 minutes. The biosensor was then loaded with a 10 μg / mL His-tagged VHH antibody (10 μg / mL) ligand solution for 120 seconds. After equilibration with PBS-T for 30 seconds, the biosensor was incubated for 100 seconds with various concentrations of human serum albumin (200, 100, 50, 25, 12.5, 6.25, and 3.13 nmol / L).

[0204] The dissociation constant was measured by immersing the sensor chip in a well containing PBS-T for 600 seconds. The data were processed using Octet software version (1.2.1.5) (Molecular Devices), and the K was calculated from the global fitting parameters of a 1:1 binding model. on , K. off Calculate [K off ] / [Kon ] to the dissociation constant K D asked for.

[0205] Figure 10 shows the results of measuring the binding ability of the raw material VHH antibody. Figure 11 shows the results of measuring the binding ability of the VHH antibody contained in particle A. Figure 12 shows the results of measuring the binding ability of the VHH antibody contained in particle B. Figure 13 shows the results of measuring the binding ability of the VHH antibody contained in particle C. In all of Figures 10 to 13, the horizontal axis represents time (seconds) and the vertical axis represents phase difference (nm).

[0206] 10 to 13, symbol A indicates the results using an analyte solution with a concentration of 200 nmol / L, symbol B indicates the results using an analyte solution with a concentration of 100 nmol / L, symbol C indicates the results using an analyte solution with a concentration of 50 nmol / L, symbol D indicates the results using an analyte solution with a concentration of 25 nmol / L, symbol E indicates the results using an analyte solution with a concentration of 12.5 nmol / L, symbol F indicates the results using an analyte solution with a concentration of 6.2 nmol / L, and symbol G indicates the results using an analyte solution with a concentration of 3.1 nmol / L.

[0207] Table 4 shows the dissociation constant K of the VHH antibody contained in each particle. D The evaluation results confirmed that, regardless of the amount of aggregates, the binding ability of the VHH antibody contained in the particles was no different from that of the VHH antibody in the starting material.

[0208] [Table 4]

[0209] (Measurement of in vitro inhalation characteristics) The inhalation characteristics were evaluated using an under-type cascade impactor. Measurements were performed under the following measurement conditions, referring to the method described in USP 2000 "Physical Tests and Determinations / Aerosols" and "Multistage Cascade Impactor Apparatus."

[0210] Measurement conditions Equipment: Andersen sampler (AN-200, manufactured by Tokyo Dylec Co., Ltd.) Pump flow rate: 28.3L / min

[0211] Approximately 30 mg of the prepared powder A was filled into a Japanese Pharmacopoeia No. 2 HPMC capsule and evaluated under an air flow rate of 28.3 L / min. The amount of particles deposited on each stage was calculated by measuring the weight of each stage before and after measurement using a precision balance.

[0212] Figure 14 is a graph showing the measurement results of inhalation characteristics. In Figure 14, C indicates the results using a capsule, and D indicates the results using an inhalation device. The results in Figure 14 show that particles A of this embodiment can reach stage 7, suggesting that they are suitable for use as powder inhalation formulations.

[0213] The above results confirmed that the present invention is useful.

[0214] The present invention includes the following aspects.

[0215] [1] Particles comprising a particulate substrate and a VHH antibody dispersed in the substrate, the particles being substantially free of aggregates of the VHH antibody.

[0216] [2] The particle according to [1], wherein the ratio of the aggregates to the VHH antibody is 3% or less.

[0217] [3] Particles according to [1] or [2], having a volume average particle size of 0.5 μm or more and 100 μm or less.

[0218] [4] The particles according to [3], having a volume average particle size of 0.5 μm or more and 10 μm or less.

[0219] [5] Particles according to any one of [1] to [4], having a RelativeSpanFactor of 1.2 or less.

[0220] [6] The particles according to any one of [1] to [5], wherein the base material is a sugar.

[0221] [7] The particles according to [6], wherein the base material is either lactose or mannitol or both.

[0222] [8] A pharmaceutical composition comprising the particles described in any one of [1] to [7].

[0223] [9] [8] A powder inhalant in which the pharmaceutical composition according to [9] [8] is in the form of an aerosol.

[0224]

[10] A reagent comprising the particles according to any one of [1] to [7].

[0225]

[11] A method for producing particles described in any one of [1] to [7], comprising the steps of: ejecting a raw material liquid containing the substrate, the VHH antibody, and a solvent from an ejection hole into a gas phase to form droplets of the raw material liquid; and removing the solvent from the droplets in the gas phase to obtain particles containing the substrate and the VHH antibody, wherein the temperature of the gas phase is controlled to be below the aggregation temperature of the VHH antibody.

[0226]

[12] The method for producing particles according to

[11] , wherein the inner diameter of the discharge hole is 1000 μm or less.

[0227]

[13] The method for producing particles according to

[11] or

[12] , wherein in the step of forming the droplets, the raw material liquid is vibrated and discharged from the discharge hole. [Explanation of symbols]

[0228] 1...unit particle, 2...base particle, 3...VHH antibody, 10...droplet discharge section, 11...droplet discharge means, 12...storage container, 13...liquid supply pipe, 15...pump, 20...granulation section, 21...chamber, 21S...internal space, 22...collection means, 23...storage section, 100...manufacturing apparatus, 110...main body, 110a...discharge hole, 110S...storage section, 111...elastic plate, 112...volume changing means, 211...air flow inlet, 212...air flow outlet, D...droplet, L...raw material liquid, P...particle, P1, P2...pressure gauge [Prior art documents] [Patent documents]

[0229] [Patent Document 1] Japanese Patent Application Publication No. 2023-156197 [Patent Document 2] International Publication No. 2022 / 270518

Claims

1. A base material formed into particles; and a VHH antibody dispersed in the substrate, Particles that are substantially free of aggregates of the VHH antibody.

2. The particle according to claim 1, wherein the proportion of aggregates relative to the VHH antibody is 3% or less.

3. 3. The particles according to claim 1, wherein the volume average particle size is 0.5 μm or more and 100 μm or less.

4. 4. The particles according to claim 3, having a volume average particle size of 0.5 μm or more and 10 μm or less.

5. 3. The particles according to claim 1, having a relative span factor of 1.2 or less.

6. 3. The particle according to claim 1, wherein the substrate is a sugar.

7. 7. The particle according to claim 6, wherein the substrate is either or both of lactose and mannitol.

8. A pharmaceutical composition comprising the particles of claim 1 or 2.

9. 9. A powder inhalant in which the pharmaceutical composition according to claim 8 is in the form of an aerosol.

10. A reagent comprising the particles according to claim 1 or 2.

11. A method for producing particles according to claim 1 or 2, comprising: a step of ejecting a raw material liquid containing the substrate, the VHH antibody, and a solvent from an ejection hole into a gas phase to form droplets of the raw material liquid; removing the solvent from the droplets in the gas phase to obtain particles comprising the substrate and the VHH antibody; A method for producing particles, wherein the temperature of the gas phase is controlled to be equal to or lower than the aggregation temperature of the VHH antibody.

12. The method for producing particles according to claim 11, wherein the inner diameter of the discharge hole is 1000 μm or less.

13. The method for producing particles according to claim 11, wherein in the step of forming the droplets, the raw material liquid is discharged from the discharge hole while being vibrated.

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