Sustained release formulations using non-aqueous emulsions

Abstract

To provide a system for producing encapsulated protein microparticles.SOLUTION: The present invention provides a system capable of carrying out the steps of: combining protein powder and a polymer into a hydrocarbon solvent to form a non-aqueous first solution; adding the first solution to a second solution, wherein the second solution comprises a fluorocarbon liquid and a fluorosurfactant; agitating the combined solutions to form a non-aqueous emulsion comprising multiple emulsion hydrocarbon droplets in the fluorocarbon liquid; removing the hydrocarbon solvent; and removing the fluorocarbon liquid to isolate the microparticles, wherein the microparticles comprise protein encapsulated within a matrix of polymer.SELECTED DRAWING: Figure 1A

Description

[Technical Field]

[0001] Aspects of the present invention generally relate to pharmaceutical microsphere formulations and non-aqueous emulsions. and methods for producing them using the system. [Background technology]

[0002] Extended release delivery of therapeutic proteins to biologically relevant targets is a promising treatment for cancer, cardiovascular disease, and other conditions. diseases, vascular conditions, orthopedic disorders, dental disorders, wounds, autoimmune diseases, gastrointestinal disorders, and eye diseases Biocompatible and biodegradable polymers are desirable for the treatment of medical conditions such as Dozens of other implantable delivery devices for controlled and extended delivery of drugs are available. For example, in some polymer-based delivery devices, the polymer As it degrades over time, the therapeutic agent is slowly released.

[0003] Extended release may be desirable for patient compliance, particularly reducing the number of injections. This is especially true when a doctor is required to administer the injection, such as with intraocular medications. Extended-release drugs can be beneficial for effective drug delivery over time with as few injections as possible. There is an unmet medical need for formulations of other diseases, e.g., cancer and inflammation. For infectious diseases, improved implantable extension drugs containing stable and effective protein therapeutics are There is a need for extended release formulations.

[0004] Therapeutic macromolecules such as antibodies and receptor Fc fusion proteins are used to administer the molecules to patients. and maintaining their stability during storage and at the site of administration. For example, therapeutic proteins (e.g., antibodies) in aqueous solutions must be formulated in a and fusion proteins) may degrade, aggregate, and / or The stability of protein therapeutics in liquid formulations is Only the types of excipients used in the formulation and the amounts and ratios of those excipients to each other When preparing therapeutic protein formulations, Considerations other than stability must also be taken into account. Examples of such additional considerations include: These include the viscosity of the solution and the concentration of therapeutic protein that a given formulation can accommodate. When formulating a therapeutic protein for remain stable at room temperature, contain sufficient antibody concentrations, and allow for convenient administration to patients Great care must be taken to arrive at a formulation that has other properties that contribute to the

[0005] Some extended-release formulations are based on internal phase separation, interfacial polymerization, the formation of multiple emulsions, polymer Using various encapsulation methodologies, including layer-by-layer adsorption of electrolytes and soft templating techniques, Water-in-oil-in-water (W / O / W) multiple emulsions are the most common type of multiple emulsion. Heavy emulsion, allowing for direct encapsulation of aqueous / hydrophilic cores in aqueous suspension Unfortunately, aqueous emulsion systems are difficult to use to encapsulate bioactive agents into extended-release formulations. For example, precipitation of proteins can lead to their immunoreactivity. occurs at the aqueous-organic interface with a concomitant decrease in reactivity (Raghuvanshi, R. ,et al.Pharm,Dev Technol,3(2):269-76(199 8) In some aqueous emulsion systems, water diffuses into the organic phase, forming a After hydrolysis, the protein droplets dissolve and escape into the aqueous environment. After hardening, the proteins that were once present in the aqueous environment begin to aggregate or precipitate. The escaped particles have voids and water channels.

[0006] Whenever the presence of water is undesirable, non-aqueous emulsions are preferred over conventional aqueous emulsions. However, there is no literature or prior art information on non-aqueous emulsions. There are few reports in the field of non-aqueous emulsions based on two types of hydrocarbons. Systems are known, namely: (1) two immiscible polymers stabilized by block copolymers; (2) compatible organic solvents (e.g., hexane / dimethylformamide), and (3) existing surfactants. The use of a solvent to displace oil and water with an immiscible polar solvent (e.g., formamide, acetone) Previously, perfluorinated water-in-oil (W / F) emulsions were investigated. It has been developed as a droplet-based microfluidic device for single-cell or single-molecule biological assays. These studies have shown that PFPE-PEG-P FPE acts as a fluorosurfactant (FS) to stabilize water droplets in fluorocarbon solvents. It is used in

[0007] Many immiscible solvent pairs are available, usually one polar and the other non-polar; The challenge is to find a pair that is suitable for the synthesis of polymer microspheres. Biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA), poly Lactic acid (PLA), poly(orthoester) (POE) are mostly It is soluble in solvents with moderate polarity such as methyl ether and ethyl acetate. In addition, process compatibility, toxicity, safety, and residual solvents are issues that must be addressed. These organic solvents are of concern and should not be investigated for pharmaceutical use. This requires further investigation.

[0008] Fluorocarbons are well suited as the continuous phase in non-aqueous emulsion systems due to the following general properties: It can be used as such. 1. Fluorocarbons are neither "hydrophobic" nor "hydrophilic" and are similar to most organic (hydrocarbon) It is immiscible with the solvent, which allows the fluorocarbon to act as the continuous phase of the hydrocarbon droplet emulsion. This made it ideal. 2. Fluorocarbons are highly soluble in proteins and other hydrophilic molecules, hydrocarbon-based polymers, and It is a non-solvent for organic excipients, i.e., these types of molecules are soluble in fluorocarbons. It's not about sex. 3. Fluorocarbons have low viscosity. 4. Fluorocarbons are chemically inert and less reactive than commonly used hydrocarbon solvents. It can be relatively less toxic or corrosive. 5. Fluorocarbons are volatile and recyclable.

[0009] Previous literature has demonstrated the use of microfluidics to synthesize water-in-fluorocarbon (W / F) and water Water-in-fluorocarbon (W / F / W) double emulsion, water / fluorocarbon / oil / water (W / F / O / W) triple emulsion, fluorocarbon / hydrocarbon / water (F / H / W) double emulsion , and hydrocarbon / fluorocarbon / water (H / F / W) double emulsions. It has been reported that various types of emulsion systems containing these emulsions have been prepared. Some of these compounds have been used to synthesize polymer microspheres. However, they are all aqueous-based emulsions that use water as the dispersed or continuous phase. It is of the N type.

[0010] Therefore, the object of the present invention is to provide a non-aqueous emulsion system for the preparation of pharmaceutical formulations and its The present invention provides methods for using these.

[0011] Another object of the present invention is to provide a method for the preparation of a prolonged release drug having improved protein stability and stable extended release. The object is to provide a long-release formulation. Summary of the Invention

[0012] Nonaqueous emulsion for producing polymer microparticles and polymer-coated microparticles One embodiment provides a method for producing a protein powder and a biodegradable or bioerodible material. mixing a polymer in a hydrocarbon solvent to form a non-aqueous first solution; Adding the solution to a second solution produces a sustained- or controlled-release particulate composition. wherein the second solution comprises a fluorocarbon liquid and a fluorosurfactant. A non-aqueous emulsion containing a plurality of emulsion hydrocarbon droplets in a fluorocarbon liquid, In some embodiments, the emulsion is a bulk emulsion. The method involves removing the hydrocarbon solvent, removing the fluorocarbon liquid, and then forming a sustained release The method further comprises isolating the sustained release or controlled release microparticles, wherein the sustained release microparticles are The present invention relates to a pharmaceutical composition comprising one or more cores of a porous powder and a shell of a biodegradable or bioerodible polymer. The fluorocarbon liquid and the hydrocarbon liquid are added to the non-aqueous emulsion while stirring. The hydrocarbon liquid can be removed by evaporation under ambient atmospheric pressure or under vacuum. In some embodiments, the fluorocarbon liquid includes a hydrofluoroether (HFE). or after emulsification, additional HFE is added to the non-aqueous emulsion to form a hydrocarbon The microspheres were quickly extracted into a fluorocarbon liquid to accelerate hardening. In some embodiments, the protein powder is a micronized protein powder. In some embodiments, the particulates are washed to remove any residual hydrocarbon solvent, fluorine, or other organic solvent remaining on the particulates. Fluorocarbon liquids, fluorosurfactants, or combinations thereof. Carbon dioxide liquids include perfluoro C5-C18 compounds, including but not limited to FC-40. In some embodiments, the fluorocarbon liquid contains HFE. Typical hydrocarbon solvents include dichloromethane, chloroform, ethyl acetate, and combinations thereof. Exemplary fluorosurfactants include, but are not limited to, perfluorosurfactants, perfluorosiloxanes, and combinations thereof. Perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether (P PFPE-PEG-PFPE tri-block copolymer. In some embodiments, the protein is a polyorthoester (POE). The protein is an antibody or antigen-binding fragment thereof, a fusion protein, or a recombinant protein. In one embodiment, the protein is a spray-dried VEGF trap protein. In some embodiments, the microparticles are 1.0 to 100 μm or 1.0 to 200 μm. In one embodiment, the non-aqueous emulsion formed by the disclosed non-aqueous emulsion method has a diameter of 1.5 mm. The disclosed flowable particulate compositions are pharmaceutically acceptable carriers. It may be suspended in an acceptable vehicle, such as pH buffered saline, or medium chain triglycerides. The flowable particulate composition can be suspended in an oily vehicle such as cereals containing ... It can be administered parenterally using a syringe.

[0013] Another embodiment is a spray-dried 1.0-30.0% w / v suspension in a hydrocarbon solution. The dispersed phase containing the protein (where the hydrocarbon solution contains 5.0-40% w / v POE) The polymer coating is prepared by emulsifying the polymer in a continuous phase to form emulsion droplets of the dispersed phase. The present invention provides a method for producing a population of crystalline microspheres, wherein the continuous phase is 0. The method includes the step of preparing an emulsion containing 1 to 5.0% w / v of a fluorosurfactant and optionally an HFE. The emulsion droplets are hardened by removing the hydrocarbon liquid while stirring the mixture. forming a population of polymer-coated microparticles and optionally washing the microparticles to form a carbon Remove hydrogen chloride solutions, fluorocarbon solutions, fluorosurfactants, or combinations thereof. In one embodiment, the hydrocarbon solution and the fluorocarbon solution are It is removed by evaporation under atmospheric pressure or under vacuum.

[0014] Yet another embodiment involves spray drying a hydrocarbon solution containing a dissolved polymer. mixing the dispersed phase with a continuous phase to form a dispersed phase; and mixing the dispersed phase with a continuous phase to form a dispersed phase. Polymer-coated microparticles are produced by generating emulsion droplets of the dispersed phase in The present invention provides a method for producing a fluorocarbon polymer, wherein the continuous phase comprises a fluorocarbon liquid and 0.2 to 5.0% w / v. The method comprises stirring the emulsion under vacuum. The hydrocarbon solution and the fluorocarbon solution are removed by the addition of a polymer. and collecting the coated microparticles. The method also includes washing the collected microparticles. Optional steps included.

[0015] Yet another embodiment is a method for preparing a first solution containing a polymer in a hydrocarbon solvent by dissolving a fluorinated carbon dioxide in the first solution. mixing the mixture with a second solution containing a solvent and a fluorosurfactant; and agitating the resulting solution to form an emulsion. The method comprises removing the hydrocarbon solvent under vacuum while stirring the mixed solution to obtain a fine hardening the particles and collecting the particulates. Washing the microparticles and drying the microparticles.

[0016] Another embodiment is a polymer produced by the non-aqueous emulsion process described herein. In some embodiments, the microparticles are provided with a polymeric coating of the microparticles. Polymers with few or no pores or channels on the surface or in the internal matrix stomach.

[0017] Yet another embodiment is a non-aqueous emulsion prepared using the non-aqueous emulsion method disclosed herein. The present invention provides a pharmaceutical composition containing the polymer-coated microparticles.

[0018] In some embodiments, the size of the microparticles is determined by the formulation composition and process parameters. By varying the diameter or size, the desired diameter or size can be adjusted. [Brief explanation of the drawings]

[0019] [Figure 1]Figure 1A shows the process scheme 1 for blank POE microsphere production via H / F-based bulk emulsion. Figure 1B shows the chemical structure for FC-40. Figure 1C shows the chemical structure of the fluorosurfactant PFPE-PEG-PFPE (Pico-Surf™ 1), a perfluoropolyether / poly(ethylene glycol) triblock copolymer. Pico-Surf™ 1 is commercially available, for example, as 5% (w / w) in FC-40. [Figure 2] Figure 2A is a photomicrograph of blank POE microspheres formed via H / F emulsion, and Figure 2B is a photomicrograph showing POE aggregation observed at low FS content. [Figure 3] Figures 3A, 3B, and 3C are photomicrographs of blank POE microspheres formed via H / F emulsions at low, medium, and high homogenization speeds. [Figure 4] (Scheme 2) Illustrates the process of SDP encapsulation within POE microspheres via S / H / F-based bulk emulsion. [Figure 5] (Scheme 3) Hydrocarbon-in-fluorocarbon emulsion system for encapsulation of protein SDP. [Figure 6] Figures 6A and 6B are fluorescence images of ethyl acetate droplets containing POE and fluorescently labeled spray-dried protein (F-SDP) dispersed in FC-40. Note that the F-SDP retained its original size and morphology within the droplets. [Figure 7] Figure 7A is a bright-field micrograph of VEGF-trapped F-SDP-encapsulated microspheres. Figure 7B is a fluorescent image of VEGF-trapped F-SDP-encapsulated microspheres (bar = 20 μm). Figure 7C is a fluorescent image of VEGF-trapped F-SDP-encapsulated microspheres (bar = 10 μm). [Figure 8] 8A-8D are fluorescence images of VEGF-trapped F-SDP-encapsulated POE microspheres placed in an aqueous environment. Note that F-SDP retained its original size and morphology within the droplets. [Figure 9] 1 is a line graph of volume density (%) versus size (μm) for microparticles produced using dichloromethane (DCM) or ethyl acetate (EtAc) in a non-aqueous emulsion method. [Figure 10] 10A and 10B are photomicrographs of microparticles loaded with 10% and 30% w / w VEGF-trapping SDP, respectively. [Figure 11] Figures 11A and 11B are representative fluorescence images of VEGF-trapped F-SDP-encapsulated POE microspheres loaded with 10% and 30% w / w SDP, respectively. Note that F-SDP retained its original size and morphology within the droplets. [Figure 12] 12A-12C are scanning electron microscope (SEM) images of microparticles loaded with 5%, 10%, and 30% w / w SDP, showing that increasing SDP loading increases the protein on the surface of the microparticles. [Figure 13] Figures 13A and 13B are SEM images of spray-dried proteins with Dv50 of 2.18 μm and 5.63 μm. [Figure 14] 14A, 14B, and 14C are bright field, fluorescence, and SEM images of VEGF-trapped F-SDP encapsulated in PLA microspheres. [Figure 15] 15A and 15B are bright field and fluorescent images of VEGF-trapped F-SDP encapsulated in PLGA microspheres. DETAILED DESCRIPTION OF THE INVENTION

[0020] I. Definition The present disclosure is not limited to the compositions and methods described herein, and the experimental conditions described. It should be understood that the scope of the present disclosure is limited only by the appended claims. The terminology used herein is intended to describe only particular embodiments. It should be understood that this is for illustrative purposes only and is not intended to be limiting.

[0021] Unless otherwise defined, all technical and scientific terms used herein are defined by the It has the same meaning as commonly understood by a person skilled in the art to which the disclosure belongs. Any compositions, methods, and materials similar or equivalent to those described herein are included within the scope of the present invention. All publications mentioned are hereby incorporated by reference. is incorporated herein in its entirety.

[0022] In the context of describing the invention claimed in this application (especially in the context of the claims), In this specification, the use of the terms "a," "an," "the," and similar referents Unless otherwise indicated or clearly contradicted by context, the singular and plural It should be interpreted to cover both the number forms.

[0023] The recitation of ranges of values ​​herein simply refers to the range, unless otherwise indicated herein. is intended to serve as a shorthand for individually referring to each separate value contained within, Each separate value is incorporated herein as if it were individually recited herein. can be.

[0024] Use of the term "about" means that the stated value is exceeded by approximately + / - 10%. In other embodiments, the term "value" is intended to describe values ​​that are either above or below the The values ​​may vary above or below the stated value within approximately + / - 5%. In other embodiments, the value may be within a range of values, and in other embodiments, the value may be approximately the recited value. It may be within a range of + / - 2% above or below any other value. In some cases, the values ​​exceed or are within the stated range of approximately + / - 1%. The range may be any value less than or equal to the value of the formula. All methods described herein are intended to be within the scope of the present invention and no further limitations are implied. Unless otherwise indicated in the specification or clearly contradicted by context, any preferred Any and all examples or exemplary methods provided herein may be used in any suitable order. The use of such language (e.g., "etc.") is merely intended to better clarify the invention. and does not limit the scope of the invention unless specifically claimed otherwise. No language in the document shall be construed as requiring any non-claimed element to be essential to the practice of the invention. should not be construed as indicating

[0025] A "protein" is a molecule made up of two or more amino acid residues linked together by peptide bonds. Proteins include polypeptides and peptides, and also refer to molecules containing glycosyl groups. hydroxylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation Proteins may include modifications such as carboxylation, and ADP-ribosylation. may be of scientific or commercial interest, including protein-based drugs; These include, inter alia, enzymes, ligands, receptors, antibodies, and chimeric or fusion proteins. Proteins can be grown in various types of recombinant cells using well-known cell culture methods. are produced using genetic engineering techniques (e.g., sequences encoding chimeric proteins, or or codon-optimized sequences, intron-less sequences, etc.) are introduced into cells, which It may exist as a plasmid or may be integrated into the genome of the cell.

[0026] An "antibody" is a protein that contains two heavy (H) chains and two nucleotides interconnected by disulfide bonds. An immunoglobulin molecule consists of four polypeptide chains: one light (L) chain; It has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region is C Each light chain contains three domains: H1, CH2, and CH3. The light chain constant region consists of one domain (CL). The VL and VL regions are interspersed with more conserved regions called framework regions (FR). They can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs). Each VH and VL consists of three CDRs and four FRs, which are amino-terminal From the carboxy terminus to the CDR1, FR2, CDR2, FR3, CDR3, The term "antibody" refers to any isotype or subclass of antibody. "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins. The term "antibody" refers to a cell isolated from a host cell transfected to express the antibody. antibody molecules prepared, expressed, created, or isolated by recombinant means, such as antibodies The term antibody also includes bispecific antibodies, which are antibodies that bind to multiple different epitopes. Bispecific antibodies include heterotetrameric immunoglobulins that can bind to antibodies of the same or different origin. No. 8,586,713, which is incorporated herein by reference. Be absorbed.

[0027] An "Fc fusion protein" is a protein that contains part or all of two or more proteins, among which One of them is the Fc portion of an immunoglobulin molecule, and they are not found together in nature. Certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) Preparation of fusion proteins containing peptides can be carried out, for example, as described in Ashkenazi et al., Proc.Natl.Acad.ScL USA 88:10535,1991;Byr n et al., Nature 344:677, 1990, and Hollenba ugh et al.,Current Protocols in Immunolo gy, Suppl. 4, "Construction of Immunoglo bulin Fusion Proteins”, pages 10.19.1-10.19. 11, 1992. "Receptor-Fc fusion protein" is a protein that contains an Fc portion. The extracellular domains of one or more coupled receptors are included, which in some implementations In the form of an immunoglobulin hinge region followed by CH2 and CH3 domains In some embodiments, the Fc fusion protein is linked to one or more ligands. For example, Fc fusion proteins are traps that contain two or more different receptor chains that bind to each other. , such as IL-1 trap or VEGF trap.

[0028] "Microparticulated protein particles" or "protein particles" are those that have low, very low, or or near-zero amounts of water (e.g., <3% water by weight). As used herein, micronized protein particles generally refer to particles containing The micronized particles are spherical in shape and have an ECD in the range of 2 microns to about 35 microns. Protein particles are not limited to specific protein entities and are useful in the preparation and delivery of therapeutic proteins. Common therapeutic proteins include, among others, antigen-binding proteins. These include, for example, soluble receptor fragments, antibodies (including IgG) and antibody derivatives or fragments, Other Fc-containing proteins, including Fc fusion proteins, as well as traps such as VEGF traps Wrapped proteins (Huang, C., Curr. Opin. Biotechnol. 20:692-99(2009)).

[0029] II. Preparation of Microsphere Formulations Using Hydrocarbon-Fluorocarbon Emulsions A system and method for formulating pharmaceutical compositions using an anhydrous emulsion system is provided. The disclosed waterless emulsion method provides a solution to some of the existing water-based emulsion systems. For example, the disclosed waterless emulsion system and the existing A comparative test between the aqueous emulsion system and the product manufactured using the aqueous emulsion system was carried out. The agent transports the drug (e.g., protein drug) from the emulsion droplets into the aqueous continuous phase during production. This leakage of the drug from the emulsion droplets also leads to low encapsulation efficiency. The non-aqueous based emulsion method disclosed herein can be used to Drug molecules, including but not limited to any hydrophilic drug, are compared to aqueous emulsion systems. encapsulation with increased encapsulation efficiency, maintaining the original protein particle structure or their assembly. The disclosed anhydrous emulsion systems and methods are bulk processes (i.e., stirring). by agitation, homogenization, sonication and other conventional methods. The system and method can also be used to prepare encapsulated drug formulations using a wide range of polymeric materials. Table 1 shows the different emulsions that can be applied to different materials, solid-state payloads, and emulsification methods. The results of the comparison of the non-aqueous emulsion system and the aqueous emulsion system were shown. This demonstrates a significant improvement in prostate encapsulation. [Table 1-1]

[0030] A. Solid-in-hydrocarbon-in-fluorocarbon (S / H / F) emulsion An exemplary non-aqueous S / H / F emulsion method is described for dry protein powders as well as biodegradable a non-aqueous first solution by mixing a non-aqueous and / or bioerodible polymer in a hydrocarbon solvent; forming a second solution comprising a fluorocarbon liquid and a fluorosurfactant; and adding the first solution to the liquid. The mixing of the first and second solutions can be performed, for example, by by stirring, sonication, cavitation, homogenization, or vortexing. A non-aqueous emulsion is formed containing multiple emulsion hydrocarbon droplets in a fluorocarbon liquid. The method includes the steps of removing the hydrocarbon solvent and removing the fluorocarbon liquid. and microparticles having one or more cores of microparticulated proteins and a shell of biodegradable polymer. and isolating the particles. In one embodiment, the emulsion is agitated to The hydrogen chloride and fluorocarbon liquids are evaporated under vacuum. The resulting microparticles are optionally washed and , hydrocarbon solvents, fluorocarbon liquids, fluorosurfactants, or combinations thereof The emulsion can be formed using bulk emulsion techniques. can.

[0031] In one embodiment, the protein powder and the biodegradable or bioerodible polymer are mixed in a hydrocarbon mixing a fluorocarbon liquid, a fluorine-based surfactant, and a solvent to form a non-aqueous first solution; The first solution is added to a second solution containing a fluorocarbon, a fluorine-containing agent, and optionally HFE. A non-aqueous emulsion containing a plurality of emulsion hydrocarbon droplets containing protein powder in a liquid. and forming a dispersion of the sustained release microparticle composition. Emulsions can be prepared by homogenization, vortexing, sonication, cavitation, stirring, or a combination thereof. The method further includes removing the hydrocarbon solvent and the fluorocarbon liquid while stirring. The hydrogen and fluorocarbon liquid can be removed by evaporation, optionally under vacuum. In other embodiments, the particulates can be collected by filtration. Removal of the fluorocarbon liquid hardens the microparticles, which can then be collected. In some embodiments, HFE is added to the fluorocarbon for a faster curing process. This can facilitate extraction of the hydrocarbon from the dispersed phase into the fluorocarbon continuous phase. HFE is miscible with both fluorocarbons and hydrocarbons, and therefore the fluorocarbon phase It can act as a co-solvent to enhance the solubility of hydrocarbons in non-aqueous emulsions. The sustained release microparticles produced by the method are composed of a matrix of biodegradable or bioerodible polymers. In some embodiments, the microparticles contain proteins encapsulated within the microparticles. In another embodiment, the microparticles are dispersed within a polymer. In yet another embodiment, the population of microparticles has a plurality of cores coated with a polymer. Microparticles with a single core structure encapsulated by a shell and microparticles with multiple core structures within a polymer furrow The fluorocarbon liquid includes, but is not limited to, FC-40. The hydrocarbon solution may be a C5 to C18 fluoro compound, and the hydrocarbon solution may be ethyl acetate, chloroform, or the like. Form, toluene, ethyl acetate, tetrahydrofuran, and dichloromethane, or In one embodiment, the fluorosurfactant is selected from the group consisting of P Perfluoropolyether-b-polyether, commercially available as ico-Surf™ 1 In some embodiments, the preferred embodiment is polyethylene glycol-b-perfluoropolyether. In another embodiment, the bioerodible polymer is POE. Selected from the group consisting of lactic acid and poly(lactic-co-glycolic acid). The protein may be an antibody or antigen-binding fragment thereof, a fusion protein, a recombinant protein, or the like. Typically, the protein is a fragment or truncated version of the For example, spray drying, electrospray drying, reversible precipitation, spray freezing, microtemplates In one embodiment, the protein is micronized by a method such as micronization, micronization, or a combination thereof. The protein is a VEGF trap protein or a truncated form thereof. Other proteins that can be used in the methods disclosed are described below. The microparticles produced by the method have a polymeric shell that lacks pores or channels. The polymer shell is not porous. In some embodiments, the microparticles have a particle size of 1 to 200 microns. It has a diameter of 1 μm.

[0032] Another embodiment is (1) a 1.0 to 30.0% w / v spray-dried hydroxybenzoate suspended in a hydrocarbon solution. The dispersed phase having dry protein, the hydrocarbon solution containing 5.0 to 30% w / v POE (2) mixing a dispersed phase, including the dispersed phase, into a continuous phase to form emulsion droplets of the dispersed phase; and a method for producing polymer-coated microspheres, wherein the method comprises: The phase contains a fluorocarbon solution containing 0.1 to 5.0% w / v of a fluorosurfactant. The method involves hardening the emulsion droplets by removing the hydrocarbon solution to form a hardened polymer. In one embodiment, the fluorocarbon solution is , and perfluoro C5 to C18 compounds, including but not limited to FC-40. The hydrocarbon solution can be ethyl acetate, chloroform, toluene, ethyl acetate, tetrahydrofuran, The solvent is selected from the group consisting of toluene, toluene, toluene dichloromethane, and dichloromethane, or a combination thereof. In an embodiment, the fluorosurfactant is commercially available as Pico-Surf™ 1. Perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether The method involves stirring the emulsion under vacuum to separate the hydrocarbon solution and the fluorine. Removing the carbon fluoride solution.

[0033] Yet another embodiment involves the use of a hydrocarbon solution containing a dissolved polymer and a spray drying tank. The polymer-coated microparticles are prepared by mixing the polymer powder with the dispersed phase. The present invention provides a method for producing a dispersed phase by mixing a dispersed phase with a continuous phase. To generate emulsion droplets of the fluorocarbon liquid and 0.1-5 (containing 0.0% w / v fluorosurfactant) and polymer-coated microparticles were collected. The hydrocarbon solution may be ethyl acetate, dichloromethane, chloroform, or or a combination thereof. The fluorocarbon solution contained FC-40, and the surfactants were Pico-Surf™ 1 and Perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether is commercially available as It is a fluoropolyether.

[0034] 1. Hydrocarbon solvents In some embodiments, the hydrocarbon solvent (also referred to as a hydrocarbon liquid) is The material, e.g., a biodegradable or bioerodible polymer, is soluble in the hydrocarbon. In some embodiments, the hydrocarbon solvent is selected from dichloromethane, chloroform, toluene, ethyl acetate, tetrahydrofuran, or a combination thereof In some embodiments, the hydrocarbon solvent is selected from the group consisting of acetonitrile, dichloromethane, ethanol, methylparaben ... Methylformamide, dimethyl sulfoxide, acetone, ethanol, methanol, pentaerythritol, The solvent may contain ethanol, propanol, hexane, or a combination thereof.

[0035] 2. Fluorosol An exemplary fluoro fluid is a fluorocarbon fluid, such as Flourinert™ FC-4 0 (average MW = 650 g / mol) 1,1,2,2,3,3,4,4,4-nonafluoro -N,N-bis(1,1,2,2,3,3,4,4,4-nonafluorobutyl)butane- 1-Amine (Figure 1B), Fluorinert™ FC-70 (average MW = 821 g / mol), or combinations thereof. In an embodiment, the fluorocarbon liquid is a hydrofluoroether (HFE) or or containing it. Exemplary HFEs include NOVEC™ 7000 (1-methoxy NOVEC 7100 (Methoxy-Nonafluoropropane), NOVEC™ 7200 (ethoxy-nonafluorobutane), NOVEC™ Trademark) 7500 (2-(trifluoromethyl)-3-ethoxydodecafluorohexane) In yet another embodiment, the fluorocarbon liquid includes, but is not limited to: FC-40, FC-70, Novec (trademark) 7500, Novec (trademark) 7100, Novec™ 7000, or combinations thereof. In this case, the second solution contains a fluorosurfactant (FS) in addition to the fluoro liquid. An exemplary FS is a perfluoropolyether ester commercially available as Pico-Surf™ 1. Perfluoro-b-polyethylene glycol-b-perfluoropolyether (PFPE-PE In one embodiment, the fluorocarbon liquid or The second solution contains FC-40 and Pico-Surf™ 1.

[0036] In some embodiments, the FS is [ka] wherein n=about 37, x+z=about 6.0, and y=about 12.5, or n= 3.7, x + z = approximately 3.6, y = approximately 9.0 (Lee, M. et al. Lab C hip.,7:14(3):509-13(2014)).

[0037] In one embodiment, the HFE has the following chemical structure: [ka]

[0038] Other HFEs suitable for use in the process have all hydrogen atoms attached to carbon atoms that have no fluorine substitution. A class of molecules in which atoms are separated from fluorinated carbon by ether oxygen, The HFE may be linear, branched, cyclic, or a combination thereof. (e.g., alkylcycloaliphatic), and preferably Such HFEs are free of ethylene unsaturation and have a total of about 4 to about 20 carbon atoms. are well known and readily available as either essentially pure compounds or mixtures. Due to the lipophilic and fluorophilic nature of HFEs, they interact with both fluorocarbons and hydrocarbons. When added to hydrocarbon / fluorocarbon emulsions, they act as co-solvents. can act as a catalyst to extract hydrocarbons into the fluorocarbon phase and accelerate the hardening process. .

[0039] In some embodiments, the hydrocarbon solvent, the fluorocarbon, or both, is The emulsion is removed by evaporation, optionally under vacuum, with stirring. In this embodiment, the microparticles are collected by filtration, optionally under vacuum.

[0040] The proportion of HFE in the fluorocarbon phase can be 0-20% v / v, but the proportion of HFE Increasing the ratio increases the hydrocarbon extraction rate. Do not use too high a proportion of HFE as it may be more difficult to control the size and morphology. It is not possible.

[0041] 3.Erodible or biodegradable polymers In one embodiment, the polymer is a biodegradable or bioerodible polymer. In some embodiments, the polymer is a branched or linear polyethylene glycol ( PEG), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-polyglycol Poly(d,l-lactide-co-glycolide) (PLGA), PLGA-ethylene oxide fumarate, esterified to polyethylene glycol 1000 PLGA-alpha-tocopheryl succinate (PLGA-TGPS), poly- Poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxybenzoate) Hydroxybutyrate-co-hydroxyvalerate (PHB-PVA), polyethylene glycol-poly Poly(lactic acid) copolymer (PEG-PLA), poly-ε-caprolactone (PCL), poly -Alkyl-cyano-acrylate (PAC), poly(ethyl) cyanoacrylate (P EC), Polyisobutyl cyanoacrylate, Poly-N-(2-hydroxypropyl)methyl Poly(HPMA) and poly-β-R-hydroxybutyrate (PHB) , poly-β-R-hydroxyalkanoate (PHA), poly-β-R-malic acid, phosphorus Lipid-cholesterol polymer, 2-dioleoyl-sn-glycero-3-phosphatidylinositol Alcohol / Polyethylene glycol-distearoylphosphatidylethanolamine ( DOPC / PEG-DSPE) / Cholesterol, polysaccharide, cellulose, ethyl cellulose, methylcellulose, alginate, dextran and dextran hydrochloride ologel polymers, amylose, inulin, pectin and guar gum, chitosan, chitin , heparin, hyaluronic acid, cyclodextrin (CD)-based polyrotaxanes and Polypseudorotaxane, polyaspartate, polyglutamate, polyrusin, leucine Polyglutamic acid copolymer, polybutylene succinate, gelatin, collagen, fibrils fibroin, polyorthoester, polyorthoester-polyamidine copoly mer, polyorthoester-diamine copolymer, polyorthoester incorporating latent acid poly(ethylene glycol) / poly(butylene terephthalate) copolymers, and and combinations and copolymers thereof. The polymer is poly-ε-caprolactone (PCL) or its derivatives or copolymers. In one embodiment, the polymer is PLGA or a derivative or copoly In one embodiment, the polymer is ethyl cellulose or a derivative thereof. In one embodiment, the polymer is a polyorthoester or copolymer thereof. In one embodiment, the polymer is a polyester a derivative or copolymer of It's Mido.

[0042] As used herein, the term "polymer" refers to a group of molecules linked together by covalent chemical bonds. Polymers are large molecules containing repeating monomers that are biocompatible, biodegradable, and erodible. Biocompatible and biodegradable polymers can be natural or synthetic. Natural polymers include polynucleotides, naturally occurring proteins, and polypeptides. , recombinant protein, gelatin, collagen, fibrin, fibroin, polyasparagine phosphate, polyglutamate, polylysine, leucine-glutamate copolymer, etc. , and the polysaccharides cellulose alginate, dextran and dextrose. Ran hydrogel polymer, amylose, inulin, pectin and guar gum, chitosan , chitin, heparin, and hyaluronic acid. Degradable polymers include polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-poly Polyglycolic acid copolymer (PLGA), poly-d,l-lactide-co-glycolide (P LGA), PLGA-ethylene oxide fumarate, polyethylene glycol 1000 Esterified PLGA-alpha-co-copheryl succinate (PLGA-TGPS ), polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH ), poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-PVA), polyethylene PEG-PLA, poly-ε-caprolactone (P CL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl) cyanoacrylate acrylate (PEC), polyisobutyl cyanoacrylate, poly-N-(2-hydroxy Propyl) methacrylamide (poly(HPMA)), poly-β-R-hydroxybutyrate Poly-β-R-hydroxyalkanoate (PHB), poly-β-R-hydroxyalkanoate (PHA), poly-β-R-hydroxyalkanoate (PHB), poly-β-R-hydroxyalkanoate (PHA ...B), poly-β-R-hydroxyalkanoate (PHB), poly-β Glycolic acid, phospholipid-cholesterol polymer, 2-dioleoyl-sn-glycero-3- Phosphatidylcholine / Polyethylene Glycol-Distearoylphosphatidylethanolamine Cholesterol (DOPC / PEG-DSPE) / Cholesterol, Ethylcellulose, Cyclopentasiloxane CD-based polyrotaxanes and polypseudorotaxanes, polybromo Styrene succinate (PBS), polyorthoester, polyorthoester-polyamidite copolymer, polyorthoester-diamine copolymer, and latent acid to control degradation rate. Incorporated polyorthoesters, as well as poly(ethylene glycol) / poly (butylene terephthalate) copolymers.

[0043] Ethylcellulose (EC) is a well-known and readily available polymer used in pharmaceutical and food science. It is a biomaterial that can be obtained by converting some of the glucose hydroxyl groups to ethyl ether. It is a cellulose derivative substituted with methylcellulose. Martinac et al., J. Mic roencapsulation,22(5):549-561(2005) and References are given to the use of biocompatible polymers in the manufacture of microspheres. A method using ethyl cellulose is described. and methods for preparing derivatives of ethyl cellulose are described in US Pat. No. 4,210,529 (198 0) and the references therein.

[0044] Poly-d,l-lactide-co-glycolide (PLGA) has also been used in tissue engineering and pharmaceuticals. Well-known biocompatible and FDA-approved compounds used in the delivery system PLGA is a biodegradable polymer. It is a polyethylene glycol (PEG) copolymer containing glycolic acid and lactic acid monomers. Aste is a representative for the synthesis of PLGA and the description of the fabrication of PLGA nanoparticles. te and Sabliov,Biomater.Sci.Polym.Ed.,17 (3):247-89 (2006) and references therein.

[0045] Poly-ε-caprolactone (PCL) is a promising candidate for human use as a drug delivery device. It is another biocompatible and biodegradable polymer approved by the FDA for use in CL is a polyester of ε-caprolactone that is rapidly hydrolyzed in the body and is non-toxic. or to form a hydroxycarboxylic acid with low toxicity. For a description of the method for producing PCL, see L abet and Thielemans,Chemical Society Rev See Jews 38:3484-3504 (2009) and references therein. Fabrication and characterization of PCL-based microspheres and nanospheres as delivery systems For instructions on use, see Sinha et al., Int. J. Pharm., 278( 1):1-23 (2004) and references therein.

[0046] Polyorthoesters (POEs) are bioerodible polymers designed for drug delivery. It is generally a ketene acetal, preferably a cyclic diketene acetal (e.g., 3,9-dimethylene-2,4,8,10-tetraoxaspiro[5.5]undecane It is a polymer of ethylene glycol, etc., which is polymerized via glycol condensation to form orthoester bonds. A description of polyorthoester synthesis and various types is given in, for example, US Pat. ,767. Polyorthoesters can be found, for example, in the form of hexanetriol. Various hydrophobic diols and polyols can be used, such as by replacing decanetriol with by incorporating or excluding methyl methyl ethers, as well as by incorporating or excluding methyl ethers such as glycolide, octane, By adding latent acids such as diacids to the backbone to increase pH sensitivity, these drugs It can be modified to control the release profile and degradation rate. The tamform contains glycolic acid in the POE backbone to modulate mass loss and drug release. Other modifications to polyorthoesters include the addition of amino groups to increase functionality. The formation, description, and use of polyorthoesters are described in US5 ,968,543,US4,764,364,Heller and Barr,Bio macromolecules,5(5):1625-32(2004), and Hel ler,Adv.Drug.Deliv.Rev.,57:2053-62(2005) is described in.

[0047] 4. Protein drugs In some embodiments, the water-free emulsions produced by the disclosed methods and systems The microparticle formulations used include drugs. Exemplary drugs include proteins, fusion proteins, and the like. These include, but are not limited to, proteins and fragments thereof, antibodies and antigen-binding fragments thereof. In one embodiment, the protein is prepared using a method described, for example, in U.S. Pat. No. 7,087,411, ,279,159, and 8,144,840, which are incorporated herein by reference in their entireties. VEGF trap proteins (e.g., hIgG1) as described in the incorporated herein. VEGF receptor fused to Fc of Flk1. VEGF receptor fused to Ig domain 3 of Flk1. The antibody, Aflibercept, contains the Ig domain 2 of Flt1. In embodiments, the VEGF trap protein is A truncated form of VEGF trap as described in U.S. Pat. No. 7,396,664. .

[0048] In some embodiments, the protein in the microparticle formulation is an antibody, a human antibody, a humanized antibody, or a Antibodies, chimeric antibodies, monoclonal antibodies, multispecific antibodies, bispecific antibodies, antigen-binding antibodies antibody fragments, single-chain antibodies, diabodies, triabodies, or tetrabodies, dual variable domain antibodies Bispecific tetravalent immunoglobulin G-like molecules called DVD-IG , IgD antibody, IgE antibody, IgM antibody, IgG antibody, IgG1 antibody, IgG2 antibody, I In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In another embodiment, the antibody comprises a chimeric hinge. In one embodiment, the antibody comprises a chimeric Fc. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody. is.

[0049] In some embodiments, the antibody includes an anti-programmed cell death 1 antibody (e.g., U.S. Pat. No. 6,413,999). Anti-PD1 antibodies, anti-programmed cell death ligands, such as those described in U.S. Pat. No. 9,987,500, 1 (e.g., anti-PD-L1 antibodies such as those described in U.S. Patent No. 9,938,345), anti-D ll4 antibody, anti-angiopoietin-2 antibody (see, e.g., U.S. Pat. No. 9,402,898 anti-ANG2 antibodies, such as those described in U.S. Pat. No. 9,529,492; anti-angiopoietin-like 3 antibodies, such as those described in U.S. Pat. No. 9,529,492; anti-AngPtl3 antibodies, such as those described in US Pat. No. 5,018,356; anti-platelet-derived growth factor receptor antibodies (e.g., anti-PDGFR antibodies as described in U.S. Pat. No. 9,265,827), anti Erb3 antibodies, anti-prolactin receptor antibodies (e.g., those described in U.S. Pat. No. 9,302,015) anti-PRLR antibodies as described in U.S. Pat. No. 9,795,121); anti-complement 5 antibodies (e.g., anti-C5 antibodies, such as those described in US Pat. No. 6,449,462; anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., US Pat. No. 6,449,462); Anti-EGFR antibodies such as those described in U.S. Patent No. 9,132,192 or U.S. Patent No. 9,477 anti-EGFRvIII antibodies as described in US Pat. No. 5,875, anti-proprotein convertase subunits, Antibodies to thiazin-kexin-9 (see, e.g., U.S. Pat. No. 8,062,640 or U.S. Pat. No. 6,062,640) anti-PCSK9 antibodies, such as those described in US Pat. No. 9,540,449; anti-growth and differentiation factor-8 antibodies, Antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies, U.S. Patent No. 8,449,949; ... ,871,209 or 9,260,515), antiglucagon receptors The compounds (e.g., those described in U.S. Pat. Nos. 9,587,029 or 9,657,099) Anti-GCGR antibodies, anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor Antibodies (e.g., U.S. Patent Application Publication No. US2014 / 0271681A1 (abandoned) or as described in U.S. Pat. Nos. 8,735,095 or 8,945,559 anti-IL4R antibody), anti-interleukin 6 receptor antibody (e.g., U.S. Pat. No. 7,582, 298, 8,043,617, or 9,173,880; L6R antibody), anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 (e.g., U.S. Patent Nos. 9,449,462, ... anti-IL33 antibodies, such as those described in US Pat. Nos. 53,072 or 9,637,535; anti-respiratory Syncytial virus antibodies (e.g., U.S. Patent Nos. 9,447,173 and 10,125 ,188, and U.S. Patent Application Publication No. 2019 / 0031741A1. anti-RSV antibodies), anti-cluster 3 (e.g., as described in U.S. Pat. No. 9,657,102), anti-CD3 antibodies, such as anti-Cluster of Differentiation 20 antibodies (e.g., U.S. Pat. No. 9,657,102 and and US2015 / 0266966A1, and U.S. Patent No. 7,879,984. anti-CD20 antibodies, such as those listed above), anti-CD19 antibodies, anti-CD28 antibodies, and anti-cluster of differentiation 48 antibodies (e.g., anti-CD48 antibodies, such as those described in U.S. Pat. No. 9,228,014; anti-Fel d antibodies, 1 antibody (e.g., as described in U.S. Pat. No. 9,079,948), anti-Middle East Respiratory Syndrome virus anti-MERS antibodies, such as those described in U.S. Pat. No. 9,718,872; anti-ebol Ebola antibodies (e.g., anti-Ebola as described in U.S. Pat. No. 9,771,414); anti-Zika virus antibodies, anti-lymphocyte activation gene 3 antibodies (e.g., anti-LAG3 antibodies, or anti-CD223 antibodies), anti-nerve growth factor antibodies (e.g., U.S. Patent Application Publication No. 2016 / 00 17029 (now abandoned), and U.S. Patents 8,309,088 and 9,3 53,176), and anti-protein Y antibodies. In some embodiments, the bispecific antibody is an anti-CD3 x anti-CD20 bispecific antibody. (described in U.S. Patent No. 9,657,102 and US2015 / 0266966A1) , anti-CD3 × anti-mucin 16 bispecific antibodies (e.g., anti-CD3 × anti-Muc16 bispecific antibody), and anti-CD3 x anti-prostate specific membrane antigen bispecific antibodies (e.g., anti-CD3 x anti In some embodiments, the antibody is selected from the group consisting of a PSMA bispecific antibody. Cardiac target proteins include abciximab, adalimumab, adalimumab-atto, and a do-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, Basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, bri Natumomab, brentuximab vedotin, brodalumab, brolucizumab, canakinumab , capromab pendetide, certolizumab pegol, cemiplimab, cetuximab, denos Mab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, Emicizumab-kxwh, emtansine alirocumab, evinacumab, evolocumab, Acinumab, golimumab, guselkumab, ibritumomab tiuxetan, idarucizumab , infliximab, infliximab-abda, infliximab-dyyb, ipi Limumab, ixekizumab, mepolizumab, necitumumab, nesbacumab, nivolumab, Oviltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab ib, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, lambda Nibizumab, raxibacumab, reslizumab, linucumab, rituximab, sarilumab, Secukinumab, siltuximab, tocilizumab, trastuzumab, trevo The compound is selected from the group consisting of guromulab, ustekinumab, and vedolizumab.

[0050] In some embodiments, the protein in the complex comprises an Fc portion and another domain. In some embodiments, the recombinant protein (e.g., an Fc fusion protein) comprises: In the present invention, the Fc fusion protein is a receptor-Fc fusion protein, and is coupled to the Fc portion. In some embodiments, the F The c portion contains the CH2 and CH3 domains following the hinge region of IgG. In some embodiments, the receptor-Fc fusion protein binds to a single ligand or multiple ligands. For example, an Fc fusion protein may contain two or more different receptor chains that bind to either: A trap protein, for example, an IL-1 trap (e.g., fused to the Fc of hIgG1) Contains the IL-1RAcP ligand binding domain fused to the IL-1R1 extracellular domain. Rilonacept, U.S. Patent No. 6,927,027, which is incorporated herein by reference in its entirety. See issue 04), or VEGF trap (e.g., fused to the Fc of hIgG10 VEGF receptor Flt1 fused to Ig domain 3 of VEGF receptor Flk1 Ig domain 2-containing aflibercept or ziv-aflibercept) In another embodiment, the Fc fusion protein is an ScFv-Fc fusion protein. These include variable heavy and variable light chain fragments of an antibody coupled to an Fc portion. The antibody comprises one or more antigen-binding domains of

[0051] In some embodiments, the initial protein is a dry powder, e.g., a micronized powder. In some embodiments, the protein is in the form of a spray-dried powder. The use of spray-dried proteins instead of solutions of proteins results in microparticles. Higher protein loading capacity in the encapsulation process and better protein stability during the encapsulation process In some embodiments, the dry protein molecules have the advantage of being completely encapsulated. It remains in a solid state and surrounded by a stabilizing material during processing and storage. In some embodiments, the encapsulated spray-dried protein exhibits high recovery and low aggregation. This is manifested by only a small proportion of surface proteins being exposed at the interface. This may be due to the minimization of surface interactions. The protein is micronized before encapsulation.

[0052] B. Microparticles One embodiment provides pharmaceutical compositions prepared using the disclosed non-aqueous emulsion systems. In some embodiments, the pharmaceutical composition comprises a polymer shell and a micronized protein. In some embodiments, the microparticles are in the form of Some microparticles and protein cores will approach perfect spheres, while others will The diameter of the sphere will be more irregular in shape. The terms are Microflow Imaging (MFI), Nanoparticle Tracking Analysis (NTA) or determined by static light scattering (SLS), dynamic light scattering (DLS), or laser diffraction (a) Determined as the volume average diameter or number average diameter by light scattering methods such as diametral analysis; (b) the diameter of the sphere defining the microparticle or protein core; (c) the diameter of the largest sphere that fits within the boundary of (a) and (b) (d) any measurement between a particle or a sphere within the particle, including the average value between the two; (e) the length of the longest axis of the microparticle or protein core, (f ) Any measurement between the major axis length (d) and the minor axis length (e) (the average between the two) (g) Equivalent Circular Diameter ("ECD"), and / or (g) The diameter is generally expressed in micrometers (μm or microns). , can be determined by optical measurements or scanning electron microscopy measurements.

[0053] The fine particles produced by the disclosed non-aqueous emulsion method are low to very low or near-zero amounts of water (e.g., <3% water by weight). As used herein, micronized protein particles contain molecules of 2 microns or less. ~ about 35 microns, or 2.0 to 50 μm, or 5.0 to 15.0 μm, or about 10 μm. Micronized protein particles are particles that contain specific protein entities. Suitable for the preparation and delivery of therapeutic proteins, including but not limited to the proteins listed above. .

[0054] For example, protein particles can be prepared by spray drying, freeze drying and milling, jet milling, Reversible precipitation in non-solvents, granulation, gradual precipitation (US 7,998,477 (2011)), ultra Critical fluid precipitation (US 6,063,910 (2000)), or high-pressure carbon dioxide-induced particles Formation (Bustami et al.,Pharma.Res.17:1360-66( As used herein, the term "spray drying" may be used. The term "spray dryer" refers to the process of converting a slurry or suspension into micron-sized particles by using a spray dryer. A spray dryer is a method for producing a dry powder containing particles of a substance. A nozzle is used to disperse a suspension or slurry into a controlled droplet size spray. Droplet sizes of 0-500 μm can be produced by spray drying. When the organic solvent dries, the protein material dries to a micron-sized, powder-like substance. or in the case of a protein-polymer suspension, upon drying, the protein A polymeric hardening shell is formed around the payload.

[0055] In some embodiments, the microparticulated protein is a VEGF trap protein. The pharmaceutical formulation for forming the micronized VEGF trap protein particles comprises about 10 mg / mL to about 100 mg / mL of VEGF trap protein, about 1.0 to about 50 mg / m L of protein, approximately 10 mg / mL, approximately 15 mg / mL, approximately 20 mg / mL, approximately 25 mg / mL, approx. 30mg / mL, approx. 35mg / mL, approx. 40mg / mL, approx. 45mg / mL, Approx. 50mg / mL, approx. 55mg / mL, approx. 60mg / mL, approx. 65mg / mL, approx. 70m g / mL, approx. 75 mg / mL, approx. 80 mg / mL, approx. 85 mg / mL, approx. 90 mg / mL , about 95 mg / mL, or about 100 mg / mL of VEGF trap protein. obtain.

[0056] In some embodiments, the compositions are prepared using the disclosed non-aqueous emulsion systems. The microparticles contain a protein particle core within a polymer shell, and range in size from about 2 μm to about 70 μm, and about 5 μm to about 70 μm. μm ~ approx. 65 μm, approx. 10 μm ~ approx. 60 μm, approx. 15 μm ~ approx. 55 μm, approx. 10 μm ~ approx. Diameters in the range of 50 μm, about 1.0 to 15 μm, about 20 μm, about 25 μm, or about 30 μm The size variation mostly reflects the thickness of the polymer shell, but the protein core The diameter of the tube may also contribute to some size variation.

[0057] In one embodiment, the microparticles formed by the disclosed non-aqueous emulsion method are The disclosed flowable particulate compositions are prepared by mixing a pharmaceutically acceptable excipient with a The flowable particulate composition may be administered parenterally, for example, by suspension in a vehicle such as pH-buffered saline. can be administered using a syringe, for example, a syringe with a 27G needle.

[0058] Microparticles are useful for the time-release or sustained release of protein therapeutics. In embodiments, the microparticle formulation is injected intravitreally, into the choroid, or subcutaneously. For example, VEGF-trapped microparticles can be used to induce VEGF in the vitreous for the treatment of vascular ophthalmic disorders, for example. EGF traps therapeutic proteins for extended release or to treat other disorders It is envisioned that it will be useful in subcutaneous implantation for extended release of F-trap.

[0059] The microparticles of the present invention can be stored in a physiological aqueous environment at about 37°C for at least 60 days, 90 days, or , 120 days, or 150 days, at a relatively constant rate. Emit quality.

[0060] One embodiment is a maize product made using the non-aqueous emulsion method disclosed herein. A composition of microspheres is provided, the composition comprising >100 mg of spray-dried protein. In one embodiment, the non-aqueous emulsion process has a yield of >90% and 9% purity with >10% w / w loading and <1% ion concentration in 50-100 μL injection volumes Microparticles with a burst of 0% are produced. [Example]

[0061] Example 1: Blank microsphere synthesis via H / F-based bulk emulsion. material and method Oil and water-based emulsion systems contain polymer microparticles or nanoparticles. It is frequently used for the synthesis of polymers, where a hydrophobic polymeric material is dissolved in an organic phase and an aqueous interface is used. However, water-soluble polymers such as PEG, carboxymethylcellulose, CMC, as well as polyanhydrides and polylactic acid, which readily hydrolyze in the presence of water. Aliphatic polyesters with short midblocks such as For polymers containing specific poly(amino acids), such as The following examples are based on the water-soluble or water-degradable polymer microparticles described above. Several experiments demonstrate the utility of the disclosed H / F emulsion system for producing In the form, these polymers are first dissolved in a polar solvent (e.g., acetonitrile, tetrahydrofuran, etc.). dihydrofuran), and less polar solvents (e.g., DCM, chloroform), The polymer solution is then dissolved in a hydrocarbon solvent. Add the continuous phase, which is a fluorocarbon liquid (e.g., FC-40) with rf1. The emulsion is created through mixing, stirring, or other emulsification methods. The polymer is finally cured to form a polypropylene by evaporating or extracting the hydrocarbon solvent. It becomes a mer sphere.

[0062] In certain embodiments, H / F bulk emulsions are prepared as shown in Scheme 1 (FIG. 1A). For blank POE microsphere synthesis via HCl, approximately 10%, 20% in DCM 200 μL of POE, 30%, and 40% w / v, 0.5% w / w FS Pico -Surf™ 1 (Sphere Fluidics) added to 2 mL of FC-40 Emulsification was achieved by vortex mixing. The emulsion droplets were larger than those of FC-40. The cells floated lightly to the top of the solution. An aliquot was taken and placed on a glass slide for microscopic imaging. The microspheres were hardened under vacuum with stirring for 3 hours. The cured polymer spheres in were first vacuum filtered through a 0.22 micron PES membrane. The C-40 passed through the filter, while the microspheres were retained. The wafer was washed with additional FC-40 and thoroughly dried under vacuum. In the examples, about 30% w / v POE in DCM was used in the hydrocarbon phase, and FC40 Approximately 0.01%, 0.1%, and 0.5% FS in the fluorocarbon phase were used to obtain FS concentrations. The impact was assessed.

[0063] result In the presence of FS, hydrocarbon and fluorocarbon mixtures form H / F emulsions In one example, DCM was dispersed in FC-40 as an H / F emulsion ( (See the structure of FC-40 in Figure 1B), and PFPE-PEG-PFPE was treated with FS (See the structure of FS in Figure 1C). The FC-40 fluorocarbon phase was then added. The results showed that 0.1 to 5% w / w of FS was required (Figure 2A). When SF was added, a broader size distribution was observed. The droplets were not stable. The dispersed DCM droplets quickly merged together, and the two phases rapidly separated. The results show that a sufficient amount of FS is used to produce a stable H / F emulsion. and during the curing process to successfully produce polymer microspheres. The need for continuous stirring was demonstrated (Figure 2B).

[0064] Adding POE to DCM and vortexing in FC-40 resulted in a significant improvement in the POE-containing liquid. Evaporation of DCM under ambient conditions or under vacuum in an open vessel resulted in the formation of droplets. , which resulted in the droplets hardening into POE microspheres (Figures 2A and 2B). The size of the microspheres was related to the droplet size and POE content in the organic phase. Higher POE concentrations result in larger microsphere sizes (Table 1). [Table 1-2]

[0065] Example 2: Effect of homogenization speed. material and method 1 mL of 30% or 40% w / v POE in DCM was added to 0.5% (w / w) FS F C-40 was added to 9 mL of FC-40 and injected into a VWR 7 mm x 95 mm sawtooth generator. Using a VWR handheld homogenizer 200 equipped with lobes at three homogenization speeds Low (approximately 50% of full power), medium (approximately 60% of full power), and high (approximately 70% of full power) The emulsion was stirred under vacuum. The spheres were washed and dried under vacuum.

[0066] result As shown in Figure 3, at 30% POE, a lower homogenization speed resulted in larger microspheres. High homogenization speed resulted in smaller particle sizes, whereas high homogenization speed resulted in smaller particle sizes (Table 2). The %POE showed the same trend. These results suggest that the homogenization speed can be adjusted to This demonstrates that the microsphere size can be controlled. [Table 2]

[0067] Example 3: POE microspheres via S / H / F-based bulk emulsion method General procedure for protein SDP encapsulation in . material and method As shown in Figure 4, bulk emulsion synthesis involves three steps: compounding, emulsification, and curing. The properties of the product depend on the different parts used in these three steps. The general procedure is as follows:

[0068] By combining, VEGF trap SDP (or For fluorescence imaging, fluorescently labeled SDP (F-SDP) was added to 10–35% w / v POE. into 500 μL of ethyl acetate containing 100 μL of HCl, vortexed, and then sonicated for 5 min. These suspensions were then dispersed by wave treatment. The emulsion was added to 9.5 mL of FC-40 containing 100% ethanol. This can be achieved by homogenization using a benchtop homogenizer. The structure of the emulsion is shown in Figure 5. An aliquot was taken during the process immediately after emulsification and imaged under a microscope. The droplets were dropped onto a glass slide for solubility. The droplets hardened onto the slide through evaporation under ambient conditions. To harden the microspheres, (a) the solution was left in an open container overnight. (b) stirring the solution under ambient conditions to evaporate the ethyl acetate; (c) evaporating the solution under conditions of faster solvent evaporation; Stir under vacuum for at least 2 hours to remove the residue. (c) NOVEC7500 or FC adding a mixture of PEG-40 and NOVEC 7500 to the emulsion under stirring; One of three methods was applied: HFE, which is the transfer of acetic acid esters from a hydrocarbon phase to a fluorocarbon phase; Co-solvents that facilitate extraction of the chill, allowing for a rapid curing process (typically within minutes) It acts as.

[0069] Finally, the cured polymer spheres in FC-40 were first passed through a 0.22 μm PES membrane. The mixture was filtered under vacuum. The FC-40 passed through the filter, while the microspheres were retained. The microspheres were then washed with additional FC-40 and thoroughly dried under vacuum.

[0070] The size of the microspheres was determined by dispersing the product powder in a 0.01% w / v PVA solution. Liquid sampling by immersion was performed using the Malvern Mastersizer The morphology of the product was determined by laser diffraction analysis using a scanning electron microscope. The measurements were carried out using a scanning electron microscope (SEM).

[0071] To measure the protein content of the microspheres, a predetermined amount of microspheres was First, dissolve the sample in 200 μL of ethyl acetate, then extract it once with pure water, collect the aqueous phase, and centrifuge. The cloudy suspension was removed by centrifugation. Protein purity and concentration were confirmed by SEC-UPLC. was measured by.

[0072] To measure burst release, a predetermined amount of microspheres was dissolved in 1 mL of PBS for 37 min. The mixture was then centrifuged and the supernatant was analyzed for protein concentration. The mixture was subjected to EC-UPLC.

[0073] result The above results indicated the formation of stable H / F emulsions in the presence of sufficient SF. This non-aqueous emulsion can be used to successfully produce blank POE spheres. The anhydrous method was again used to incorporate SDP into POE microspheres. VEGF Trap F-SDP was added as a suspension in ethyl acetate at a total solid weight of 10% (w / w). (containing 20% ​​w / v POE) and FC-40 (containing 0.5% w / w FS) This suspension in ) was emulsified by stirring and vortexing. Immediately after emulsification, The specimen was transferred onto a glass slide for microscopic imaging. As shown in Figures 6A and 6B The ethyl acetate dispersed in the FC-40 to form droplets, and the SDP particles were clearly visible within the ethyl acetate droplets. In contrast to the S / O / W system (data not shown), the protein There was no sign of leakage of the material into the fluorocarbon continuous phase. The SDP particles in the droplets retained the concave shape of their initial powder state. Since there was no water in the F system to reconstitute the SDP, the SDP took on the initial solid particulate form. After curing, the POE microparticles containing single or multiple SDP particles were observed in bright field and This can be clearly observed through fluorescence microscopy images (Figures 7A, 7B, and 7C). Evaporation of hydrocarbon and fluorocarbon solvents on glass slides followed by burst release with the addition of water The quality of the encapsulation was tested. As shown in Figures 8A-D, the microsphere preparation was immersed in water. After placement in the SDP-encapsulated POE microspheres, their integrity was No immediate release of protein was observed, and the shape of the SDP particles remained the same. This is because the SDP particles are well protected by the polymer matrix and shielded from the aqueous environment. These results show that H / F emulsions are effective in preventing the formation of proteins and other parent substances. It is an effective solution for encapsulating aqueous drugs in a polymer matrix, and provides a burst release This suggests that it is possible to achieve high encapsulation efficiency and high yield while minimizing leakage. These are all based on water-based W / O / W or S / O / W methods. is a major issue.

[0074] The procedure disclosed herein is for protein SDP encapsulation in POE microspheres. This is an example of using the S / H / F non-aqueous based bulk emulsion method for recrystallization. Reproducible, scalable, and adjustable. By varying these parameters, product properties can be adjusted and controlled. The effect of some of the parameters is disclosed in Example 4.

[0075] Example 4: Effect of hydrocarbon solvents material and method Microparticles were prepared as described in Example 2, with the hydrocarbon being dichloromethane or Ethyl acetate was used: 35% w / v POE in DCM and 35% in ethyl acetate. w / v POE was prepared by dissolving 10% w / w of the total solids in DCM or The samples were suspended in 0.5 mL of POE solution in ethyl acetate. In a distillation vial, 9.5 mL of FC-40 containing 0.5% w / w FS was added. These mixtures were homogenized to form an emulsion and then sintered under house vacuum for 1.5 hours. The formed microparticles were isolated by filtration, washed with FC-40, and dried under vacuum. It was dried.

[0076] result Figure 9 shows the results except that the hydrocarbon solvent was either dichloromethane or ethyl acetate. shows the size distribution of microparticles produced using the same formulation and process conditions. Microparticles made using these hydrocarbons also show encapsulation of spray-dried proteins. The use of fluoromethane results in larger particulates. See Table 2 below. The results are Under the same formulation and process conditions, the use of different hydrocarbon solvents can result in different cycles. It was suggested that DCM produces larger microspheres than ethyl acetate. Therefore, the hydrocarbon solvent was purposely chosen to minimize the microsphere size. The sphere size can be controlled. [Table 3]

[0077] Example 5: Effect of protein loading material and method Microparticles were prepared as described in Example 2, with only the protein loading varied. 35% w / v POE in M ​​was prepared. 5%, 10%, and 30% w / w total solids weight A quantity of protein powder was suspended in 0.5 mL of POE solution in DCM. 9.5 mL containing 0.5% w / w FS in a 20 mL scintillation vial The mixture was homogenized for about 1 minute to form an emulsion, and then Add 6 mL of a 1:1 v:v mixture of Novec 7500 and FC-40 within 1 minute of The emulsion was then stirred for another minute, after which the formed microspheres were The product was isolated by filtration, washed with FC-40 and dried under vacuum.

[0078] result As shown in Table 3, increasing the amount of protein powder in the formulation improved the laser diffraction analysis. Larger POE particle sizes, as measured by HPLC, and improved protein extraction Experimentally, the final POE microspheres were observed via bright field and confocal fluorescence microscopy. Increased protein loading in the product was obtained. Brightfield images show microspheres that are darker and less transparent than the 10% w / w protein powder. , indicating that more drug was encapsulated in the microsphere preparation (Fig. 10A and and 10B). Representative confocal images show that SDPs are initially deposited in the POE matrix. The cross-sectional image of the microspheres confirmed that the encapsulation was in the shape of a microsphere (Fig. 11A and and 11B). More SDP particles were observed in the 30% w / w loaded microspheres. Again, the encapsulated SDPs maintained their original concave shape and remained intact throughout the entire manufacturing process. Furthermore, SEM images showed that the protein powder remained intact during the processing period. With increasing loading, more protein particles were adsorbed onto the surface of the POE microparticles. The results thus show that protein powders up to 30% w / w It was shown that >30% w / w of Protein powder loading may result in a lack of physical space within the microspheres of this formulation. Because of this property, protein particles can become adsorbed onto the surface of the microspheres. Surface-adsorbed proteins can be easily absorbed into water if such an effect is desired for therapeutic efficacy. Contact with the drug can result in a burst release of the drug. Protein is not lost to the continuous phase as would be the case. [Table 4]

[0079] Example 6: Encapsulating SDP into POE microspheres using H / F bulk emulsification Design of Experiments (DOE). material and method DOE studies are performed to determine the critical factors of the synthesis in the designed space on the properties of the final product. The effects of the 10 runs in the designed experiment were evaluated according to the general method described in Example 2. Protein powder loading, protein powder particle size (Dv(50) Sizes are 2.2um and 5.6um (see SEM image in Figure 13), polymer concentration, and HFE concentration, while varying the volumes of, e.g., hydrocarbon and fluorocarbon phases, The formulation and process conditions, such as homogenization speed and FS concentration, were kept constant (Table 4). sphere size (Dv50, spanned by laser diffraction), encapsulation efficiency, 1 hour at 37°C Measured response including burst emission at , SEM images.

[0080] result The results of the DOE are summarized in Table 5. [Table 5]

[0081] Fitting of custom-designed DOE for microsphere size (R 2 =0.76) revealed a main effect of protein powder loading and POE concentration ( p-value <0.05, see correlation results in Table 6). Fitting (R 2 = 0.92), only protein powder loading significantly affected burst release. (p value < 0.05, see correlation results in Table 7). Increasing the amount of protein powder in the formulation increases the payload in the final product, The burst release rate is also increased. It is likely caused by protein particles internalized in polymer microspheres. The maximum amount of protein powder that can be accommodated is determined by the physical space available for a given microsphere size. Simply increasing the protein powder concentration in the formulated suspension does not result in a certain threshold ( Drug encapsulation does not increase beyond 100% w / w (which in this example was about 30% w / w). [Table 6] [Table 7-1]

[0082] Example 7. Application of the S / H / F emulsion-based encapsulation method to different proteins. The disclosed H / F-based emulsion systems and processes are suitable for use with different polymers and curing agents. This could be a platform technology applicable to therapeutic proteins. In a particular example of the present invention, Protein powder of recombinant IgG4 (MW = approx. 145 kDa), recombinant IgG1 (MW = approx. Protein powder (146 kDa) or recombinant fusion protein (MW = approximately 64 kDa) The protein powders were each treated with POE microspheres by the same process as in Example 2. The results are summarized in Table 7. Encapsulated protein powder in the microsphere preparation The amount of protein was determined by extraction assay and was in agreement with the target value. After the process, the recombinant fusion protein was maintained for IgG1 and for IgG4. The decrease is slight (less than 2%), indicating good process suitability. [Table 7-2]

[0083] Other biodegradable polymers, such as PLGA and PLA, have also been used in H / F-based emulsions. In a specific example of the present invention, through a process similar to that disclosed in Example 2, The fluorescently labeled VEGF trap F-SDP was then coated onto PLGA (lactide:glycolide = 50:50, Mw 42–65 kDa, Sigma Aldrich) and PLA (Al (Cycloether terminated, Mw 18,000-28,000, Sigma Aldrich) Bright-field and fluorescent microscopic images of the protein powder were taken. was successfully encapsulated inside polymer microspheres (PLA 14A-C, and Fig. 15A-B for PLGA).

[0084] In the foregoing specification, the invention has been described with reference to specific embodiments thereof. Although many details are presented for purposes of illustration, the present invention is capable of additional embodiments. and some of the details described herein may be omitted without departing from the basic principles of the invention. It will be apparent to one skilled in the art that the present invention may be varied in various ways.

[0085] All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. and therefore it is to be relied upon in the appended patents, rather than the foregoing specification, as indicating the scope of the invention. Reference should be made to the claims.

Claims

1. 1. A method for producing polymeric or polymer-coated microparticles, comprising: The protein powder and polymer are mixed in a hydrocarbon solvent to form a non-aqueous first solution. forming a The first solution is added to a second solution, and the second solution is a mixture of a fluorocarbon liquid and a fluorocarbon. a surfactant; The mixed solution is stirred to form a plurality of emulsion hydrocarbons in the fluorocarbon liquid. forming a non-aqueous emulsion containing droplets; removing the hydrocarbon solvent; removing the fluorocarbon liquid to isolate the particles, a step of encapsulating a protein within a polymer matrix; A method comprising:

2. The method of claim 1 , wherein the microparticles comprise a single core-shell structure.

3. 10. The method of claim 9, wherein at least one of the microparticles comprises a plurality of cores dispersed within the polymer.

1. The method according to claim 1.

4. The microparticles have a single core structure encapsulated by a polymer and a polymer encapsulated nanoparticle.

4. The method of claim 1, further comprising the step of:

10. The method according to claim 1.

5. 5. The method according to claim 1, wherein the fluorocarbon liquid comprises a perfluoro C5 to C18 compound.

1. The method according to claim 1.

6. The hydrocarbon solution may be dichloromethane, chloroform, toluene, ethyl acetate, tetrachloromethane, 6. Any of claims 1 to 5, selected from the group consisting of: hydrofuran, or a combination thereof. The method according to any one of claims 1 to 4.

7. The fluorocarbon solution is Fluorinert™ FC-40 (average MW=650 g / mol) 1,1,2,2,3,3,4,4,4-nonafluoro-N,N-bis(1, 1,2,2,3,3,4,4,4-nonafluorobutyl)butan-1-amine The method according to any one of claims 1 to 6.

8. The hydrocarbon solvent comprises dichloromethane, ethyl acetate, or a combination thereof. The method of claim 1 .

9. 9. The method according to claim 1, wherein the fluorocarbon solution contains a hydrofluoroether. The method described in paragraph .

10. The fluorosurfactant is perfluoropolyether-b-polyethylene glycol -b-The method according to any one of claims 1 to 9, comprising a perfluoropolyether.

11. 11. The method according to claim 1, wherein the polymer comprises a polyorthoester (POE). The method described in paragraph .

12. The polymer is selected from the group consisting of polylactic acid and poly(lactic-co-glycolic acid). The method according to any one of claims 1 to 10,

13. The protein is an antibody or an antigen-binding fragment thereof, a fusion protein, a recombinant protein, or the like.

13. The method according to claim 1, wherein the hydroxybenzoate is a hydroxybenzoate or a fragment or truncated form thereof. method.

14. The method of claim 13, wherein the protein is a VEGF trap protein.

15. 15. The method of claim 14, wherein the protein is a truncated form of a VEGF trap protein. How to do it.

16. 16. The method according to claim 1, wherein the microparticles have a diameter of 1 to 200 μm. How to do it.

17. The protein powder contains particles having a diameter of microparticulated protein of 0.5 to 20 μm. The method according to any one of claims 1 to 16.

18. Protein powders can be prepared by spray drying, electrospray drying, reversible precipitation, spray freezing, micro-freezing, and 1 to 3, which are microparticulated by a clotplate method, or a combination thereof.

8. The method according to any one of claims 7 to 7.

19. The emulsion may be homogenized, vortexed, sonicated, cavitated, stirred, 19. The method of claim 1, wherein the granules are formed using a granule-forming method, ... The method described.

20. The method according to any one of claims 1 to 19, wherein the hydrocarbon solvent is removed while stirring the mixed solution.

10. The method according to any one of claims 1 to 9.

21. 21. The method of claim 20, wherein the hydrocarbon solvent is removed under vacuum to harden the microparticles. How to post.

22. 22. A method according to any one of claims 1 to 21, wherein the hydrocarbon solvent is removed by evaporation. How to do it.

23. 23. The method of claim 1, wherein the fluorocarbon liquid is removed by filtration, optionally under vacuum. The method according to any one of claims 1 to 4.

24. Hydrofluoroethers are used as co-solvents to extract the hydrocarbons.

24. The method according to claim 22 or 23.

25. A microparticle produced by the method according to any one of claims 1 to 23, Emitted particles, particles.

26. 26. A sustained release composition comprising the microparticles of claim 25.

27. The microparticles have few or no pores or channels in the polymer shell. The microparticle according to any one of claims 1 to 26.

28. To produce polymer microspheres or polymer-coated microspheres A method for (1) 1.0-30.0% w / w total solids spray-dried protein suspended in a hydrocarbon solution The dispersed phase comprises a protein, and the hydrocarbon solution comprises 5.0 to 35% w / v POE. , the dispersed phase, (2) mixing the dispersed phase into a continuous phase to form emulsion droplets of the dispersed phase, and the continuous phase a fluorocarbon solution containing 0.1-5.0% w / v fluorosurfactant; The emulsion droplets are hardened by removing the hydrocarbon liquid to form a hardened emulsion. Forming polymer microspheres or polymer-coated microspheres and A method comprising:

29. The non-aqueous emulsion is stirred, and the hydrocarbon solution is heated under ambient atmospheric pressure during stirring, 29. The method of claim 28, wherein the hydroxyl group is removed by evaporation under vacuum.

30. The hardened polymer microspheres or polymer-coated microspheres 30. The method of claim 29, wherein the a is collected by vacuum filtration.

31. 1. A method for producing polymeric or polymer-coated microparticles, comprising: The hydrocarbon solution containing the dissolved polymer is mixed with the spray-dried protein powder to form a dispersion. generating a dispersed phase; mixing the dispersed phase with a continuous phase to form emulsion droplets of the dispersed phase in the continuous phase; wherein the continuous phase is a mixture of a fluorocarbon liquid and 0.1 to 5.0% w / v fluorine a process including a surfactant; collecting the polymer-coated microparticles; A method comprising:

32. The spray-dried protein may be an antibody, a recombinant protein, a fusion protein, or any of the foregoing. The method of claim 31 , wherein the fragment is a fragment of

33. The protein is a VEGF trap protein or a truncated VEGF trap protein.

33. The method of claim 32, wherein the quality is

34. The hydrocarbon solution may be dichloromethane, chloroform, toluene, ethyl acetate, tetrachloromethane, 32. The method of claim 31 , wherein the carboxylic acid is selected from the group consisting of: methyl methyl ether, ...; How to do it.

35. The fluorocarbon liquid is a fluorocarbon liquid containing 1,1,2,2,3,3,4,4, The method of any one of claims 31 to 34, comprising 4-nonafluorobutyl)amine. 。

36. The particles are removed from the hydrocarbon solution by evaporation or under vacuum with stirring. The method according to any one of claims 31 to 35, wherein the curing is carried out by

37. 37. The method of claim 36, further comprising collecting the hardened particulates.

38. A microparticle produced by the method of any one of claims 31 to 37.

39. 39. A pharmaceutical composition comprising the microparticles of claim 38.

40. 40. The pharmaceutical composition of claim 39, further comprising one or more excipients.

41. 41. The pharmaceutical composition of claim 40, wherein the pharmaceutical composition is a sustained release composition.

42. 42. Any of claims 39 to 41, wherein the pharmaceutical composition is formulated for parenteral administration. The pharmaceutical composition according to any one of claims 1 to 4.

43. 43. The pharmaceutical composition of any one of claims 1 to 42, wherein the pharmaceutical composition comprises more than 100 mg of spray-dried protein. The method according to any one of claims 1 to 5.

44. The hydrofluoroether is 4-ethoxy-1,1,1,2,2,3,3,4, Claims: 5,6,6,6-didecafluoro-5-(trifluoromethyl)hexane 9. The method according to claim 9.

45. 1. A method for producing microparticles, comprising: A first solution containing a polymer in a hydrocarbon solvent is added to a fluorocarbon solvent and a fluorine-based surfactant. mixing the resulting solution with a second solution containing a dispersant; agitating the mixed solution to form an emulsion; The hydrocarbon solvent is removed under vacuum while stirring the mixed solution to form the fine particles. and hardening the collecting the particulates; and Optionally, washing the microparticles; and drying the microparticles; A method comprising:

46. The hydrocarbon solvent is selected from the group consisting of dichloromethane, chloroform, toluene, ethyl acetate, tetrachloromethane ... Hydrofuran, acetonitrile, ethanol, methanol, propanol, dimethylformamide dimethylsulfoxide, dimethylsulfoxide, or a combination thereof.

46. ​​The method of claim 45.

47. The fluorocarbon solvent is selected from the group consisting of (1,1,2,2,3,3,4,4)trifluoromethylbis( ...

47. The method of claim 45 or 46, comprising the step of:

48. The polymer is POE, polylactic acid, poly(lactic acid-co-glycolic acid), or any of these.

48. The method of any one of claims 45 to 47, comprising a combination of:

49. 50. Any one of claims 45 to 49, wherein the microparticle comprises a polymeric shell and a hollow core. The method described below.

50. The microparticles have few or no pores or channels in the polymer shell.

50. The method of any one of claims 49, wherein

51. The diameter of the microparticles may vary depending on the hydrocarbon solvent, the stirring speed, the polymer concentration, or a combination thereof.

52. The method according to claim 50 or 51, wherein the diameter is adjusted to a desired value by changing the combination of the two. How to post.

52. Microparticles according to any one of claims 45 to 51.

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