Sustained release formulations using non-aqueous emulsions

Abstract

A non-aqueous emulsion method for producing polymeric or polymer-coated microparticles is provided. One method produces a sustained-release microparticle composition by mixing a protein powder and a polymer in a hydrocarbon solvent to form a non-aqueous first solution, and then adding the first solution to a second solution containing a fluorocarbon liquid and a fluorosurfactant to form a non-aqueous emulsion containing multiple emulsion hydrocarbon droplets in the fluorocarbon liquid. The subsequent microparticle hardening process involves removing the hydrocarbon solvent from the formed emulsion droplets, which can be achieved by evaporating the hydrocarbon under ambient conditions with stirring, or by accelerated hardening via vacuum or by adding a hydrofluoroester as a cosolvent to the fluorocarbon. The fluorocarbon liquid is removed and washed with additional fluorocarbon liquid to isolate sustained-release microparticles, which contain one or more protein cores and a polymer frustule.

Description

[Technical field]

[0001] Aspects of the present invention relate generally to drug microsphere formulations and methods for making them using non-aqueous emulsion systems. [Background technology]

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

[0003] Extended release may be desirable for patient compliance. In particular, reducing the number of injections may be beneficial, especially when a physician is required to perform the injection, such as the example of intraocular therapeutics. There is an unmet medical need for extended release formulations to effectively deliver drugs over time with as few injections as possible. For other diseases, such as cancer and inflammatory diseases, there is a need for improved implantable extended release formulations that contain stable and effective protein therapeutics.

[0004] Therapeutic macromolecules such as antibodies and receptor Fc fusion proteins must be formulated in a manner that not only renders the molecules suitable for administration to patients, but also maintains their stability during storage and at the site of administration. For example, therapeutic proteins (e.g., antibodies and fusion proteins) in aqueous solutions are prone to degradation, aggregation, and / or undesirable chemical modification if the solution is not properly formulated. The stability of protein therapeutics in liquid formulations depends not only on the types of excipients used in the formulation and the amounts and ratios of those excipients relative to each other, but also on the concentration of soluble protein. When preparing therapeutic protein formulations, considerations other than stability must also be taken into account. Examples of such additional considerations include the viscosity of the solution and the concentration of therapeutic protein that a given formulation can accommodate. When formulating therapeutic proteins for extended release, great care must be taken to arrive at a formulation that remains stable over time and during storage and at physiological temperatures, contains sufficient antibody concentration, and has other properties that allow it to be conveniently administered to patients.

[0005] Some extended release formulations are produced using various encapsulation methodologies, including internal phase separation, interfacial polymerization, formation of multiple emulsions, layer-by-layer adsorption of polyelectrolytes, and soft template techniques. Water-in-oil-in-water (W / O / W) multiple emulsions are the most common type of multiple emulsions, allowing for the direct encapsulation of an aqueous / hydrophilic core in an aqueous suspension. Unfortunately, aqueous emulsion systems have certain problems when used to encapsulate bioactive agents in extended release formulations. For example, precipitation of proteins occurs at the aqueous-organic interface with a concomitant reduction in their immunoreactivity (see Raghuvanshi, R., et al. Pharm, Dev Technol, 3(2):269-76 (1998)). In some aqueous emulsion systems, water can diffuse into the organic phase and hydrolyze the proteins. After hydrolysis, the protein droplets dissolve and escape into the aqueous environment and begin to aggregate or precipitate. After hardening, voids and water channels appear in the microparticles where proteins once resided but have escaped into the aqueous environment.

[0006] Non-aqueous emulsions can replace normal aqueous emulsions whenever the presence of water is undesirable. However, there are few reports in the literature or prior art regarding non-aqueous emulsions. Two types of hydrocarbon-based non-aqueous emulsion systems are known: (1) two immiscible organic solvents (e.g., hexane / dimethylformamide) stabilized by block copolymers, and (2) oil and an immiscible polar solvent (e.g., formamide, acetonitrile) replacing water using existing surfactants. Previously, perfluorinated water-in-oil (W / F) emulsions have been studied and widely applied in droplet-based microfluidics for single-cell or single-molecule biological assays. In these studies, PFPE-PEG-PFPE is used as a fluorosurfactant (FS) to stabilize water droplets in fluorocarbon solvents.

[0007] Many immiscible solvent pairs are available, usually one polar and the other non-polar, but the challenge is to find a pair that is suitable for the synthesis of polymer microspheres. Most typical biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), poly(orthoester) (POE), are soluble in solvents with moderate polarity, such as chloroform, dichloromethane, and ethyl acetate. This limits the options for the continuous phase. In addition, process compatibility, toxicity, safety, and residual solvents are concerns with using these organic solvents and need to be considered for pharmaceutical use.

[0008] Fluorocarbons can be used as the continuous phase in non-aqueous emulsion systems due to the following general properties: 1. Fluorocarbons are neither "hydrophobic" nor "hydrophilic" and are immiscible with most organic (hydrocarbon) solvents, making them ideal as the continuous phase in hydrocarbon droplet emulsions. 2. Fluorocarbons are non-solvents for proteins and other hydrophilic molecules, hydrocarbon-based polymers, and organic excipients, i.e., these types of molecules are not soluble in fluorocarbons. 3. Fluorocarbons have low viscosity. 4. Fluorocarbons are chemically inert and can be relatively less toxic or corrosive compared to commonly used hydrocarbon solvents. 5. Fluorocarbons are volatile and recyclable.

[0009] Previous literature has reported that various types of emulsion systems containing fluorocarbons have been fabricated through microfluidics methods, such as water-in-fluorocarbon (W / F), water-in-fluorocarbon-in-water (W / F / W) double emulsions, water / fluorocarbon / oil / water (W / F / O / W) triple emulsions, fluorocarbon / hydrocarbon / water (F / H / W) double emulsions, and hydrocarbon / fluorocarbon / water (H / F / W) double emulsions. Some of these emulsions have been used in the synthesis of polymer microspheres. However, they are all aqueous-based emulsion systems that use water as the dispersed or continuous phase.

[0010] It is therefore an object of the present invention to provide non-aqueous emulsion systems for the manufacture of pharmaceutical formulations and methods of their use.

[0011] Another object of the present invention is to provide an extended release formulation with improved protein stability and stable extended release. Summary of the Invention

[0012] A non-aqueous emulsion method for producing polymer microparticles and polymer-coated microparticles is provided. One embodiment provides a method for producing sustained or controlled release microparticle compositions by mixing a protein powder and a biodegradable or bioerodible polymer in a hydrocarbon solvent to form a non-aqueous first solution, and adding the first solution to a second solution, where the second solution includes a fluorocarbon liquid and a fluorosurfactant to form a non-aqueous emulsion containing a plurality of emulsion hydrocarbon droplets in the fluorocarbon liquid. In some embodiments, the emulsion is formed by bulk emulsion. The method further includes removing the hydrocarbon solvent and removing the fluorocarbon liquid to isolate sustained or controlled release microparticles, the sustained release microparticles containing one or more cores of protein powder and a shell of biodegradable or bioerodible polymer. The fluorocarbon liquid and the hydrocarbon liquid can be removed while stirring the non-aqueous emulsion and evaporating the fluorocarbon liquid and the hydrocarbon liquid under ambient atmospheric pressure or under vacuum. In some embodiments, the fluorocarbon fluid contains a hydrofluoroether (HFE) or additional HFE was added to the non-aqueous emulsion after emulsification to quickly extract the hydrocarbon into the fluorocarbon fluid and accelerate microsphere hardening. In some embodiments, the protein powder is a micronized protein powder. In some embodiments, the microparticles are washed to remove residual hydrocarbon solvent, fluorocarbon fluid, fluorosurfactant, or combinations thereof remaining on the microparticles. Exemplary fluorocarbon fluids include perfluoro C5-C18 compounds, including but not limited to FC-40. In some embodiments, the fluorocarbon fluid contains an HFE. Exemplary hydrocarbon solvents include but are not limited to dichloromethane, chloroform, ethyl acetate, and combinations thereof. An exemplary fluorosurfactant is a perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether (PFPE-PEG-PFPE) tri-block copolymer. An exemplary bioerodible polymer is polyorthoester (POE).In some embodiments, 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 have a diameter of 1.0-100 μm or 1.0-200 μm. In one embodiment, the microparticles formed by the disclosed non-aqueous emulsion method are flowable microparticle compositions. The disclosed flowable microparticle compositions can be suspended in a pharma- ceutically acceptable excipient, e.g., pH-buffered saline, or can be suspended in an oil vehicle, such as a medium chain triglyceride. The flowable microparticle composition can be administered parenterally, e.g., using a syringe with a 27G needle.

[0013] Another embodiment provides a method for producing a population of polymer-coated microspheres by emulsifying a dispersed phase comprising 1.0-30.0% w / v spray-dried protein suspended in a hydrocarbon solution, where the hydrocarbon solution comprises 5.0-40% w / v POE, in a continuous phase to form emulsion droplets of the dispersed phase, where the continuous phase comprises 0.1-5.0% w / v fluorosurfactant and optionally HFE. The method further comprises hardening the emulsion droplets by removing the hydrocarbon liquid while stirring the emulsion to form a population of polymer-coated microparticles, and optionally washing the microparticles to remove the hydrocarbon solution, fluorocarbon solution, fluorosurfactant, or combinations thereof. In one embodiment, the hydrocarbon solution and fluorocarbon solution are removed by evaporation at ambient atmospheric pressure or under vacuum.

[0014] Yet another embodiment provides a method for producing polymer-coated microparticles by mixing a hydrocarbon solution containing a dissolved polymer with a spray-dried protein powder to produce a dispersed phase, and mixing the dispersed phase with a continuous phase to produce emulsion droplets of the dispersed phase in the continuous phase, where the continuous phase comprises a fluorocarbon liquid and 0.2-5.0% w / v FS, and optionally HFE. The method includes removing the hydrocarbon solution and the fluorocarbon solution by stirring the emulsion under vacuum to harden the microparticles, and then collecting the polymer-coated microparticles. The method also includes an optional step of washing the collected microparticles.

[0015] Yet another embodiment provides a method for producing microparticles by mixing a first solution containing a polymer in a hydrocarbon solvent with a second solution containing a fluorocarbon solvent and a fluorosurfactant, and stirring the mixed solution to produce an emulsion. The method includes removing the hydrocarbon solvent under vacuum while stirring the mixed solution to harden the microparticles, and collecting the microparticles. The method optionally includes washing the microparticles and drying the microparticles.

[0016] Another embodiment provides polymer-coated microparticles produced by the non-aqueous emulsion methods described herein. In some embodiments, the microparticles have few or no pores or channels in the polymer surface or interior matrix of the microparticle.

[0017] Yet another embodiment provides a pharmaceutical composition containing the polymer-coated microparticles produced using the non-aqueous emulsion methods disclosed herein.

[0018] In some embodiments, the size of the microparticles can be adjusted to a desired diameter or size by varying the formulation composition and process parameters. [Brief description of the drawings]

[0019]

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[0020] I. Definition It is to be understood that the present disclosure is not limited to the compositions and methods described herein, and the experimental conditions described, and therefore may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure is limited only by the appended claims.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Any compositions, methods, and materials similar or equivalent to those described herein can be used to carry out or test the present invention.All publications mentioned are incorporated herein in their entirety by reference.

[0022] In the context of describing the invention claimed in this application (particularly in the context of the claims), use of the terms "a," "an," "the," and similar referents should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0023] The recitation of ranges of values ​​herein, unless otherwise stated herein, is intended to serve as a shorthand method of referring individually to each separate value falling within that range, and each separate value is incorporated herein as if it were individually recited herein.

[0024] The use of the term "about" is intended to describe any value that is approximately + / -10% above or below the stated value. In other embodiments, the value may be approximately + / -5% above or below the stated value, in other embodiments, the value may be approximately + / -2% above or below the stated value, and in other embodiments, the value may be approximately + / -1% above or below the stated value. The foregoing ranges are intended to be made clear by the context, and no further limitations are implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to better clarify the invention, and do not limit the scope of the invention unless specifically claimed otherwise. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0025] "Protein" refers to a molecule that contains two or more amino acid residues linked together by peptide bonds. Proteins include polypeptides and peptides, and may also include modifications such as glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, alkylation, hydroxylation, and ADP-ribosylation. Proteins may be of scientific or commercial interest, including protein-based drugs, and include enzymes, ligands, receptors, antibodies, and chimeric or fusion proteins, among others. Proteins are produced by various types of recombinant cells using well-known cell culture methods, and are generally introduced into cells by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intron-less sequences, etc.), which may be present as episomes or integrated into the genome of the cell.

[0026] An "antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain has a heavy chain variable region (HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain has a light chain variable region and a light chain constant region. The light chain constant region consists of one domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, which are arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules prepared, expressed, created, or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody. The term antibody also includes bispecific antibodies, which include heterotetrameric immunoglobulins capable of binding to multiple different epitopes. Bispecific antibodies are generally described in U.S. Patent No. 8,586,713, which is incorporated herein by reference.

[0027] An "Fc fusion protein" comprises parts or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, that are not found together in nature. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) has been reported, for example, by Ashkenazi et al., Proc. Natl. Acad. ScL USA 88:10535, 1991; Byrn et al., Nature 344:677, 1990, and Hollenbaugh et al., "Construction of Immunoglobulin Fusion Proteins", pages 10.19.1-10.19.11, 1992 in Current Protocols in Immunology, Suppl. 4. A "receptor Fc fusion protein" comprises one or more extracellular domains of a receptor coupled to an Fc portion, which in some embodiments comprises the hinge region of an immunoglobulin followed by the CH2 and CH3 domains. In some embodiments, the Fc fusion protein comprises two or more different receptor chains that bind to one or more ligands, for example, the Fc fusion protein is a trap, such as an IL-1 trap or a VEGF trap.

[0028] "Micronized protein particle" or "protein particle" refers to a particle containing multiple protein molecules with low, very low, or near-zero amounts of water (e.g., <3% water by weight). As used herein, micronized protein particles are generally spherical in shape and have an ECD ranging from 2 microns to about 35 microns. Micronized protein particles are not limited to a particular protein entity and are suitable for the preparation and delivery of therapeutic proteins. Common therapeutic proteins include, among others, antigen-binding proteins, such as soluble receptor fragments, antibodies (including IgG) and antibody derivatives or fragments, other Fc-containing proteins, including Fc fusion proteins, and trap-type proteins such as VEGF trap (Huang, C., Curr. Opin. Biotechnol. 20:692-99 (2009)).

[0029] II. Preparation of Microsphere Formulations Using Hydrocarbon-Fluorocarbon Emulsions Systems and methods are provided for formulating pharmaceutical compositions using anhydrous emulsion systems. The disclosed anhydrous emulsion methods overcome some of the problems of existing aqueous emulsion systems. For example, comparative studies provided herein between the disclosed anhydrous emulsion systems and existing aqueous emulsion systems show that formulations made using aqueous emulsion systems leak drugs (e.g., protein drugs) from the emulsion droplets into the aqueous continuous phase during manufacturing. This leakage of drugs from the emulsion droplets results in low encapsulation efficacy. The disclosed non-aqueous-based emulsion methods described herein encapsulate drug molecules, including but not limited to hydrophilic drugs such as proteins, with increased encapsulation efficacy compared to aqueous emulsion systems, maintain the original protein particle structure, or a combination thereof. The disclosed anhydrous emulsion systems and methods can produce encapsulated drug formulations by bulk methods (i.e., agitation, homogenization, sonication) and other conventional methods. The systems and methods can also be applied to a wide range of polymeric materials, solid-state payloads, and emulsification methods. Table 1 shows the comparative results of the different emulsions and demonstrates that the non-aqueous emulsion system is a significant improvement in microparticle encapsulation compared to the aqueous emulsion system. [Table 1-1]

[0030] A. Solid-in-Hydrocarbon-in-Fluorocarbon (S / H / F) Emulsions An exemplary non-aqueous S / H / F emulsion method includes mixing a dry protein powder and a biodegradable and / or bioerodible polymer in a hydrocarbon solvent to form a non-aqueous first solution, and adding the first solution to a second solution composed of a fluorocarbon liquid and a fluorosurfactant. The first and second solutions are mixed, for example, by stirring, sonication, cavitation, homogenization, or vortexing, in a manner to form a non-aqueous emulsion containing a plurality of emulsion hydrocarbon droplets in the fluorocarbon liquid. The method includes removing the hydrocarbon solvent and removing the fluorocarbon liquid to isolate microparticles having one or more cores of microparticulated protein and a frustule of biodegradable polymer. In one embodiment, the emulsion is stirred and the hydrocarbon liquid and the fluorocarbon liquid are evaporated under vacuum. The resulting microparticles can be optionally washed to remove the hydrocarbon solvent, the fluorocarbon liquid, the fluorosurfactant, or a combination thereof. The emulsion can be formed using bulk emulsion techniques.

[0031] One embodiment provides a method for producing a sustained release microparticle composition by mixing a protein powder and a biodegradable or bioerodible polymer in a hydrocarbon solvent to form a non-aqueous first solution, and adding the first solution to a second solution containing a fluorocarbon liquid, a fluorosurfactant, and optionally an HFE to form a non-aqueous emulsion containing a plurality of emulsion hydrocarbon droplets containing the protein powder in the fluorocarbon liquid. The emulsion can be formed using homogenization, vortexing, sonication, cavitation, stirring, or a combination thereof. The method further includes removing the hydrocarbon solvent and the fluorocarbon liquid while stirring the emulsion. The hydrocarbon and fluorocarbon liquid can be removed by evaporation, optionally under vacuum. In other embodiments, the microparticles can be collected by filtration. Removing the hydrocarbon liquid and the fluorocarbon liquid hardens the microparticles, which can then be collected. In some embodiments, for a faster hardening process, an HFE can be added to the fluorocarbon to facilitate extraction of the hydrocarbon from the dispersed phase into the fluorocarbon continuous phase. HFE is miscible with both fluorocarbons and hydrocarbons and can therefore act as a co-solvent to enhance the solubility of the hydrocarbons in the fluorocarbon phase. The sustained release microparticles produced by the non-aqueous emulsion method contain proteins encapsulated within a matrix of biodegradable or bioerodible polymers. In some embodiments, the microparticles have a single core-shell structure. In other embodiments, the microparticles have multiple cores dispersed within the polymer. In still other embodiments, the population of microparticles includes microparticles having a single core structure encapsulated by a polymer frustule and microparticles having multiple core structures within a polymer frustule. The fluorocarbon liquid can be a perfluoro C5-C18 compound, including but not limited to FC-40, and the hydrocarbon solution is selected from the group of ethyl acetate, chloroform, toluene, ethyl acetate, tetrahydrofuran, and dichloromethane, or combinations thereof.In one embodiment, the fluorosurfactant is perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether, commercially available as Pico-Surf™ 1. In some embodiments, the bioerodible polymer is POE. In other embodiments, the polymer is selected from the group consisting of polylactic acid and poly(lactic-co-glycolic acid). In general, the protein is an antibody or an antigen-binding fragment thereof, a fusion protein, a recombinant protein, or a fragment or truncated version thereof. Typically, the protein is microparticleized by, for example, spray drying, electrospray drying, reversible precipitation, spray freezing, microtemplating, or a combination thereof. In one embodiment, the protein is a VEGF trap protein or a truncated form thereof. Other proteins that can be used in the disclosed methods are described below. The microparticles produced by the disclosed methods have a polymer frustule that lacks pores or channels. The polymer frustule is not poreized. In some embodiments, the microparticles have a diameter of 1-200 μm.

[0032] Another embodiment provides a method for producing polymer-coated microspheres by mixing (1) a dispersed phase having 1.0-30.0% w / v spray-dried protein suspended in a hydrocarbon solution, the hydrocarbon solution comprising 5.0-30% w / v POE, into (2) a continuous phase to form emulsion droplets of the dispersed phase, the continuous phase comprising a fluorocarbon solution comprising 0.1-5.0% w / v fluorosurfactant. The method includes hardening the emulsion droplets by removing the hydrocarbon solution to form hardened polymer-coated microspheres. In one embodiment, the fluorocarbon solution can be a perfluoro C5-C18 compound, including but not limited to FC-40, and the hydrocarbon solution is selected from the group of ethyl acetate, chloroform, toluene, ethyl acetate, tetrahydrofuran, and dichloromethane, or combinations thereof. In one embodiment, the fluorosurfactant is perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether, commercially available as Pico-Surf™ 1. The method includes agitating the emulsion under vacuum to remove the hydrocarbon solution and the fluorocarbon solution.

[0033] Yet another embodiment provides a method for producing polymer-coated microparticles by mixing a hydrocarbon solution containing a dissolved polymer and a spray-dried protein powder to produce a dispersed phase. The method includes mixing the dispersed phase with a continuous phase to produce emulsion droplets of the dispersed phase in the continuous phase, where the continuous phase contains a fluorocarbon liquid and 0.1-5.0% w / v fluorosurfactant, and collecting the polymer-coated microparticles. The hydrocarbon solution can be selected from the group consisting of ethyl acetate, dichloromethane, chloroform, or combinations thereof. In one embodiment, the fluorocarbon solution contains FC-40 and the surfactant is perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether, commercially available as Pico-Surf™ 1.

[0034] 1. Hydrocarbon solvents In some embodiments, the hydrocarbon solvent (also referred to as a hydrocarbon liquid) is selected so that the polymeric material, for example, a biodegradable or bioerodible polymer, is soluble in the hydrocarbon. In some embodiments, the hydrocarbon solvent is selected from the group consisting of dichloromethane, chloroform, toluene, ethyl acetate, tetrahydrofuran, or a combination thereof. In some embodiments, the hydrocarbon solvent can contain acetonitrile, dimethylformamide, dimethylsulfoxide, acetone, ethanol, methanol, pentane, propanol, hexane, or a combination thereof.

[0035] 2. Fluorine solution Exemplary fluoro fluids are fluorocarbon fluids, including, but not limited to, Flourinert™ 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 (FIG. 1B), Fluorinert™ FC-70 (average MW=821 g / mol), or combinations thereof. In some embodiments, the fluorocarbon fluid is or contains a hydrofluoroether (HFE). Exemplary HFEs include, but are not limited to, NOVEC™ 7000 (1-methoxyheptafluoropropane), NOVEC™ 7100 (methoxy-nonafluorobutane), NOVEC™ 7200 (ethoxy-nonafluorobutane), and NOVEC™ 7500 (2-(trifluoromethyl)-3-ethoxydodecafluorohexane). In yet other embodiments, the fluorocarbon fluid contains FC-40, FC-70, Novec™ 7500, Novec™ 7100, Novec™ 7000, or combinations thereof. In certain embodiments, the second solution contains a fluorosurfactant (FS) in addition to the fluoro fluid. An exemplary FS is a perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether (PFPE-PEG-PFPE) triblock copolymer commercially available as Pico-Surf™ 1. In one embodiment, the fluorocarbon liquid or second solution contains FC-40 and Pico-Surf™ 1.

[0036] In some embodiments, the FS is [ka] where n=about 37, x+z=about 6.0, and y=about 12.5, or n=3.7, x+z=about 3.6, and y=about 9.0 (Lee, M. et al. Lab Chip., 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 are a class of molecules in which all hydrogen atoms reside on carbons that have no fluorine substitution and are separated from the fluorinated carbon by ether oxygen, i.e., RfORh. HFEs have molecular structures that can be linear, branched, cyclic, or combinations thereof (e.g., alkylcycloaliphatic), and preferably are free of ethylenic unsaturation and have a total of about 4 to about 20 carbon atoms. Such HFEs are known and readily available as either essentially pure compounds or mixtures. Due to the lipophilic and fluorophilic nature of HFEs, they are miscible with both fluorocarbons and hydrocarbons. When added to a hydrocarbon / fluorocarbon emulsion, they can act as co-solvents to extract the hydrocarbon into the fluorocarbon phase and accelerate the curing process.

[0039] In some embodiments, the hydrocarbon solvent, the fluorocarbon, or both are removed by evaporation, optionally under vacuum, while stirring the emulsion, hi some embodiments, the microparticles are collected by filtration, optionally under vacuum.

[0040] The percentage of HFE in the fluorocarbon phase can be 0-20% v / v, but increasing the percentage of HFE increases the hydrocarbon extraction rate, however, the percentage of HFE cannot be too high because it may become more difficult to control the size and morphology of the microspheres.

[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-polyglycolic acid copolymer (PLGA), poly-d,l-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate, PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol 1000 (PLGA-TGPS), polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly Poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-PVA), polyethylene glycol-poly(lactic acid) copolymer (PEG-PLA), poly-ε-caprolactone (PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl) cyanoacrylate (PEC), polyisobutyl cyanoacrylate, poly-N-(2-hydroxypropyl) methacrylamide (poly(HPMA)), poly-β-R-hydroxybutyrate (PHB), poly-β-R-hydroxyalkanoate (PHA), poly-β-R-phosphoric acid ... gonolic acid, phospholipid-cholesterol polymers, 2-dioleoyl-sn-glycero-3-phosphatidylcholine / polyethylene glycol-distearoylphosphatidylethanolamine (DOPC / PEG-DSPE) / cholesterol, polysaccharides, cellulose, ethylcellulose, methylcellulose, alginates, dextran and dextran hydrogel polymers, amylose, inulin, pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid, cyclodextrin (CD)-based polysaccharides, Selected from the group consisting of taxanes and polypseudorotaxanes, polyaspartates, polyglutamates, polylucines, leucine-glutamic acid copolymers, polybutylene succinates, gelatin, collagen, fibrin, fibroin, polyorthoesters, polyorthoester-polyamidine copolymers, polyorthoester-diamine copolymers, polyorthoesters incorporating latent acids, poly(ethylene glycol) / poly(butylene terephthalate) copolymers, and combinations and copolymers thereof.In one embodiment, the polymer is poly-ε-caprolactone (PCL) or a derivative or copolymer thereof. In one embodiment, the polymer is PLGA or a derivative or copolymer thereof. In one embodiment, the polymer is ethylcellulose or a derivative or copolymer thereof. In one embodiment, the polymer is a polyorthoester or a derivative or copolymer thereof. In one embodiment, the polymer is a polyesteramide.

[0042] As used herein, the term "polymer" refers to a macromolecule that contains repeating monomers linked by covalent chemical bonds. The polymer is biocompatible and biodegradable erodible. Biocompatible and biodegradable polymers can be natural or synthetic. Natural polymers include polynucleotides, naturally occurring proteins that are polypeptides, recombinant proteins, gelatin, collagen, fibrin, fibroin, polyaspartates, polyglutamates, polylysine, leucine-glutamate copolymers, and the like, and polysaccharides such as cellulose alginate, dextran and dextran hydrogel polymers, amylose, inulin, pectin and guar gum, chitosan, chitin, heparin, and hyaluronic acid.Synthetic biocompatible or biodegradable polymers include polylactic acid (PLA), polyglycolic acid (PGA), polylactic-polyglycolic acid copolymer (PLGA), poly-d,l-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate, PLGA-alpha-co-copheryl succinate esterified to polyethylene glycol 1000 (PLGA-TGPS), polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxybutyrate-co-hydroxyvalerate) (PHB-PVA), polyethylene glycol-poly(lactic acid) copolymer (PEG-PLA), poly-ε-caprolactone (PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl) cyanoacrylate (PEC), polyisobutyl cyanoacrylate, poly-N-(2-hydroxypropyl) These include methacrylamide (poly(HPMA)), poly-β-R-hydroxybutyrate (PHB), poly-β-R-hydroxyalkanoates (PHA), poly-β-R-malic acid, phospholipid-cholesterol polymers, 2-dioleoyl-sn-glycero-3-phosphatidylcholine / polyethylene glycol-distearoylphosphatidylethanolamine (DOPC / PEG-DSPE) / cholesterol, ethylcellulose, cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes, polybutylene succinate (PBS), polyorthoesters, polyorthoester-polyamidine copolymers, polyorthoester-diamine copolymers, polyorthoesters incorporating latent acids to control degradation rate, and poly(ethylene glycol) / poly(butylene terephthalate) copolymers, among others.

[0043] Ethylcellulose (EC) is a well-known and readily available biomaterial used in pharmaceutical and food sciences. It is a cellulose derivative in which some of the glucose hydroxyl groups are replaced with ethyl ethers. See Martinac et al., J. Microencapsulation, 22(5):549-561 (2005) and references therein, which describe the use of ethylcellulose as a biocompatible polymer in the manufacture of microspheres. See also US 4,210,529 (1980) and references therein, for a detailed description of ethylcellulose and methods for making derivatives of ethylcellulose.

[0044] Poly-d,l-lactide-co-glycolide (PLGA) is also a well-known U.S. Food and Drug Administration (FDA) approved biocompatible and biodegradable polymer used in tissue engineering and drug delivery systems. PLGA is a polyester containing glycolic acid and lactic acid monomers. See Astete and Sabliov, Biomater. Sci. Polym. Ed., 17(3):247-89 (2006) and references therein for a description of the synthesis of PLGA and the manufacture of PLGA nanoparticles.

[0045] Poly-ε-caprolactone (PCL) is another biocompatible and biodegradable polymer approved by the FDA for use in humans as a drug delivery device. PCL is a polyester of ε-caprolactone that rapidly hydrolyzes in the body to form non-toxic or low-toxicity hydroxycarboxylic acids. For a description of how to manufacture PCL, see Labet and Thielemans, Chemical Society Reviews 38:3484-3504 (2009) and references therein. For a description of the manufacture and use of PCL-based microspheres and nanospheres as delivery systems, see Sinha et al., Int. J. Pharm., 278(1):1-23 (2004) and references therein.

[0046] Polyorthoesters (POEs) are biodegradable polymers designed for drug delivery. They are generally polymers of ketene acetals, preferably cyclic diketene acetals (e.g., 3,9-dimethylidene-2,4,8,10-tetraoxaspiro[5.5]undecane, etc.), and are polymerized via glycol condensation to form orthoester linkages. The synthesis of polyorthoesters and descriptions of various types can be found, for example, in US4,304,767. Polyorthoesters can be modified to control their drug release profiles and degradation rates by incorporating and removing various hydrophobic diols and polyols, such as by replacing hexanetriol with decanetriol, and by adding latent acids, such as glycolide, octanedioic acid, etc., to the backbone to increase pH sensitivity. Custom forms of POE can include glycolic acid in the POE backbone to adjust mass loss and drug release. Other modifications to polyorthoesters include incorporating amines to enhance functionality. The formation, description, and use of polyorthoesters are described in US5,968,543, US4,764,364, Heller and Barr, Biomacromolecules, 5(5):1625-32(2004), and Heller, Adv. Drug. Deliv. Rev., 57:2053-62(2005).

[0047] 4. Protein Drugs In some embodiments, the microparticle formulations produced by the disclosed water-free emulsion method and system contain a drug. Exemplary drugs include, but are not limited to, proteins, fusion proteins and fragments thereof, antibodies and antigen-binding fragments thereof. In one embodiment, the protein is a VEGF trap protein (e.g., Aflibercept, which contains the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG1), as described, for example, in U.S. Patent Nos. 7,087,411, 7,279,159, and 8,144,840, which are incorporated herein by reference in their entirety. In some embodiments, the VEGF trap protein is a truncated form of the VEGF trap, as described in U.S. Patent No. 7,396,664, which is incorporated herein by reference in its entirety.

[0048] In some embodiments, the protein in the microparticle formulation is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen-binding antibody fragment, a single chain antibody, a diabody, a triabody, or a tetrabody, a bispecific tetravalent immunoglobulin G-like molecule called a dual variable domain immunoglobulin (DVD-IG), an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgG1 antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In another embodiment, the antibody comprises a chimeric hinge. In yet another 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 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgG1 / IgG4 antibody.

[0049] In some embodiments, the antibody includes an anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Pat. No. 9,987,500, an anti-programmed cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Pat. No. 9,938,345), an anti-Dll4 antibody, an anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Pat. No. 9,402,898), an anti-angiopoietin-like 3 antibody (e.g., an anti-AngPtl3 antibody as described in U.S. Pat. No. 9,018,356), an anti-platelet derived growth factor receptor antibody ...018,356), an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-Dll4 antibody, an anti-D No. 9,265,827), anti-Erb3 antibodies, anti-prolactin receptor antibodies (e.g., anti-PRLR antibodies as described in U.S. Pat. No. 9,302,015), anti-complement 5 antibodies (e.g., anti-C5 antibodies as described in U.S. Pat. No. 9,795,121), anti-TNF antibodies, anti-epidermal growth factor receptor antibodies (e.g., anti-EGFR antibodies as described in U.S. Pat. No. 9,132,192 or anti-EGFRvIII antibodies as described in U.S. Pat. No. 9,475,875), anti-proprotein convertase subtilisin kexin- No. 8,062,640 or U.S. Pat. No. 9,540,449), anti-growth and differentiation factor-8 antibodies (e.g., anti-GDF8 antibodies, also known as anti-myostatin antibodies and described in U.S. Pat. No. 8,871,209 or 9,260,515), anti-glucagon receptor (e.g., anti-GCGR antibodies as described in U.S. Pat. No. 9,587,029 or 9,657,099), anti-VEGF antibodies, anti-IL1R antibodies, interleukin 4 receptor antibodies (e.g., U.S. Pat. App. No. 6,223,611, and U.S. Pat ... No. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibodies (e.g., anti-IL6R antibodies as described in U.S. Pat. Nos. 7,582,298, 8,043,617, or 9,173,880), anti-IL1 antibodies, anti-IL2 antibodies, anti-IL3 antibodies, anti-IL4 antibodies, anti-IL5 antibodies, anti-IL6 antibodies, anti-IL7 antibodies, anti-interleukin 33 (e.g., U.S. Pat. Nos. 9,453,072 or 9,637,535), anti-respiratory syncytial virus antibodies (e.g., anti-RSV antibodies as described in U.S. Pat. Nos. 9,447,173 and 10,125,188, and U.S. Patent Application Publication No. 2019 / 0031741A1), anti-group 3 (e.g., anti-CD3 antibodies as described in U.S. Pat. No. 9,657,102), anti-group 20 (e.g., anti-CD20 antibodies as described in U.S. Pat. Nos. 9,657,102 and US2015 / 0266966A1, and U.S. Pat. No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-group 48 (e.g., anti-CD48 antibodies as described in U.S. Pat. No. 9,228,014), anti-Fel d1 antibodies (e.g., as described in U.S. Pat. No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibodies as described in U.S. Pat. No. 9,718,872), anti-Ebola virus 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., anti-NGF antibodies as described in U.S. Patent Application Publication No. 2016 / 0017029 (now abandoned), and U.S. Pat. Nos. 8,309,088 and 9,353,176), and anti-protein Y antibodies. In some embodiments, the bispecific antibody is an anti-CD3 x anti-CD20 bispecific antibody (e.g., as described in U.S. Pat. No. 9,657,102 and US2015 / 0266966A1), anti-CD3 x anti-mucin 16 bispecific antibodies (e.g., anti-CD3 x anti-Muc16 bispecific antibodies), and anti-CD3 x anti-prostate specific membrane antigen bispecific antibodies (e.g., anti-CD3 x anti-PSMA bispecific antibodies). In some embodiments, the protein of interest is selected from the group consisting of abciximab, adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab, atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodalumab, brolucizumab, canakinumab, capromab pendetide, certolizumab pegol, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtansine alirocumab, evinacumab, evolocumab, fasinumab, golimumab, veloc ... , guselkumab, ibritumomab tiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, The agent is selected from the group consisting of omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, linucumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, trevogrumab, ustekinumab, and vedolizumab.

[0050] In some embodiments, the protein in the complex is a recombinant protein (e.g., an Fc fusion protein) that includes an Fc portion and another domain. In some embodiments, the Fc fusion protein is a receptor Fc fusion protein, and includes one or more extracellular domains of a receptor coupled to an Fc portion. In some embodiments, the Fc portion includes the CH2 and CH3 domains following the hinge region of an IgG. In some embodiments, the receptor Fc fusion protein includes two or more different receptor chains that bind either a single ligand or multiple ligands. For example, the Fc fusion protein is a trap protein, such as an IL-1 trap (e.g., rilonacept, which includes the IL-1RAcP ligand binding domain fused to the IL-1R1 extracellular domain fused to the Fc of hIgG1, see U.S. Patent No. 6,927,004, which is incorporated herein by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which includes the Ig domain 2 of the VEGF receptor Flt1 fused to the Ig domain 3 of the VEGF receptor Flk1 fused to the Fc of hIgG10). In other embodiments, the Fc fusion protein is an ScFv-Fc fusion protein, which comprises one or more antigen-binding domains, such as a variable heavy and variable light fragment of an antibody coupled to an Fc portion.

[0051] In some embodiments, the initial protein is in the form of a dry powder, for example, a micronized dry powder. In some embodiments, the protein is a spray-dried powder (SDP). The use of spray-dried protein instead of a solution of protein has the advantage of higher protein loading in the microparticles and better protein stability during the encapsulation process. In some embodiments, the dry protein molecules remain in a solid state and surrounded by a stabilizing material throughout the entire encapsulation process and storage conditions. In some embodiments, the encapsulated spray-dried protein shows high recovery and low aggregation, which may be due to the fact that only a small percentage of surface protein is exposed to the interface, thereby minimizing surface interactions. In some embodiments, the protein is micronized before encapsulation.

[0052] B. Microparticles One embodiment provides a pharmaceutical composition manufactured using the disclosed non-aqueous emulsion system. In some embodiments, the pharmaceutical composition contains microparticles having a polymeric shell and a micronized protein core. In some embodiments, the microparticles are substantially spherical in shape. Some microparticles and protein cores will approach true spheres, while others will be more irregular in shape. Thus, as used herein, the term "diameter" refers to (a) the diameter of a sphere defining the microparticle or protein core, (b) the diameter of the largest sphere that fits within the boundaries of the microparticle or protein core, (c) any measurement between the defined sphere of (a) and the sphere within the boundaries of (b) (including the average value between the two), (d) the length of the longest axis of the microparticle or protein core, (e) the length of the shortest axis of the microparticle or protein core, (f) any measurement between the length of the long axis (d) and the length of the short axis (e) (including the average value between the two), and / or (g) the equivalent circular diameter ("ECD"), as determined by microflow imaging (MFI), nanoparticle tracking analysis (NTA), or the volume average diameter or number average diameter by light scattering methods such as static light scattering (SLS), dynamic light scattering (DLS), or laser diffraction analysis. The diameter is generally expressed in micrometers (μm or micron). The diameter can be determined by optical measurement or scanning electron microscope measurement.

[0053] Microparticles manufactured by the disclosed non-aqueous emulsion method contain multiple molecules of protein with a low, very low, or near-zero amount of water (e.g., <3% water by weight). As used herein, the micronized protein particles have an ECD in the range of 2 microns to about 35 microns, or 2.0 to 50 μm, or 5.0 to 15.0 μm, or about 10 μm. The micronized protein particles are not limited to a specific protein entity and are suitable for the preparation and delivery of therapeutic proteins including the above proteins.

[0054] For example, protein particles can be micronized by spray drying, freeze drying and milling, jet milling, reversible precipitation in a non-solvent, granulation, progressive precipitation (US 7,998,477 (2011)), supercritical fluid precipitation (US 6,063,910 (2000)), or high pressure carbon dioxide induced particle formation (Bustami et al., Pharma. Res. 17:1360-66 (2000)). As used herein, the phrase "spray drying" refers to a method of producing a dry powder containing micron-sized particles from a slurry or suspension by using a spray dryer. A spray dryer uses an atomizer or atomizing nozzle to disperse a suspension or slurry into a controlled droplet size spray. Droplet sizes of 10 to 500 μm can be produced by spray drying. When the solvent (water or organic solvent) dries, the protein material either dries to micron size, forming a powder-like material, or, in the case of protein-polymer suspensions, forms a polymeric hardening shell around the protein payload upon drying.

[0055] In some embodiments, the microparticulated protein is a VEGF trap protein. The pharmaceutical preparation for forming the microparticulated VEGF trap protein particles may contain about 10mg / mL to about 100mg / mL of VEGF trap protein, about 1.0 to about 50mg / mL of protein, about 10mg / mL, about 15mg / mL, about 20mg / mL, about 25mg / mL, about 30mg / mL, about 35mg / mL, about 40mg / mL, about 45mg / mL, about 50mg / mL, about 55mg / mL, about 60mg / mL, about 65mg / mL, about 70mg / mL, about 75mg / mL, about 80mg / mL, about 85mg / mL, about 90mg / mL, about 95mg / mL, or about 100mg / mL of VEGF trap protein.

[0056] In some embodiments, microparticles produced using the disclosed non-aqueous emulsion systems contain a protein particle core within a polymer furrow and have diameters ranging from about 2 μm to about 70 μm, about 5 μm to about 65 μm, about 10 μm to about 60 μm, about 15 μm to about 55 μm, about 10 μm to about 50 μm, about 1.0 to 15 μm, about 20 μm, about 25 μm, or about 30 μm. Size variation largely reflects the thickness of the polymer furrow, although the diameter of the protein core may also contribute to size variation to some extent.

[0057] In one embodiment, the microparticles formed by the disclosed non-aqueous emulsion method are flowable microparticle compositions.The disclosed flowable microparticle compositions can be suspended in a pharma- ceutically acceptable excipient, for example, pH-buffered saline.The flowable microparticle compositions can be administered parenterally, for example, by using a syringe, such as a 27G needle syringe.

[0058] Microparticles are useful for the periodic or sustained release of protein therapeutics.In some embodiments, microparticle formulations are injected into the vitreous, into the choroid, or subcutaneously.For example, it is envisioned that VEGF trap microparticles are useful for the extended release of VEGF trap therapeutic protein in the vitreous, for example for the treatment of vascular eye disorders, or for the extended release of VEGF trap for the treatment of other disorders by subcutaneous implantation.

[0059] The microparticles of the invention release protein at a relatively constant rate over extended periods of at least 60, 90, 120, or up to 150 days in a physiological aqueous environment at about 37°C.

[0060] One embodiment provides a composition of microspheres produced using the non-aqueous emulsion method disclosed herein, the composition containing >100 mg of spray-dried protein. In one embodiment, the non-aqueous emulsion method produces microparticles with >10% w / w loading and <10% burst in an injection volume of 50-100 μL, with a yield of >90%, and a purity of >99%. EXAMPLES

[0061] Example 1: Blank microsphere synthesis via H / F-based bulk emulsion. material and method Oil and water-based emulsion systems are frequently used for the synthesis of polymer microparticles or nanoparticles, where hydrophobic polymeric materials are dissolved in an organic phase and dispersed in an aqueous continuous phase. However, for water-soluble polymers, such as PEG, carboxymethylcellulose (CMC), and polymers that readily hydrolyze in the presence of water, including polyanhydrides, aliphatic polyesters with short midblocks such as polylactic acid, and certain poly(amino acids) such as poly(glutamic acid), traditional water-based emulsion systems are not ideal. The following examples demonstrate the utility of the disclosed H / F emulsion system for producing the above water-soluble or water-degradable polymer microparticles. In some embodiments, the polymers are first dissolved in a hydrocarbon solvent, including polar solvents (e.g., acetonitrile, tetrahydrofuran), and less polar solvents (e.g., DCM, chloroform). This polymer solution is then added to the continuous phase, which is a fluorocarbon liquid (e.g., FC-40) with FS (e.g., Picosurf1). The emulsion is created through stirring, vortexing, or other emulsification methods. The emulsion droplets are eventually hardened into polymer spheres by evaporating or extracting the hydrocarbon solvent.

[0062] In certain embodiments, as shown in Scheme 1 (Figure 1A), for the synthesis of blank POE microspheres via an H / F bulk emulsion, 200 μL of POE at approximately 10%, 20%, 30%, and 40% w / v in DCM was added to 2 mL of FC-40 containing 0.5% w / w FS Pico-Surf™ 1 (Sphere Fluidics). Emulsification was achieved by vortex stirring. The emulsion droplets were lighter than FC-40 and floated on top of the solution. Aliquots were taken and dropped onto a slide glass for microscopy imaging. The microspheres were cured with stirring under vacuum for 3 hours. The cured polymer spheres in FC-40 were first vacuum filtered through a 0.22 micron PES membrane. FC-40 passed through the filter and the microspheres were retained. The microspheres were then washed with additional FC-40 and dried completely under vacuum. In another example by the same process, approximately 30% w / v POE in DCM was used in the hydrocarbon phase and approximately 0.01%, 0.1%, and 0.5% FS in FC40 were used in the fluorocarbon phase to evaluate the effect of FS concentration.

[0063] Results In the presence of FS, the hydrocarbon and fluorocarbon mixture was able to form an H / F emulsion. 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 used as FS (see the structure of FS in Figure 1C). The concentration of FS was increased and added to the FC-40 fluorocarbon phase. The tests showed that 0.1 - 5% w / w FS was required to prevent the adhesion of DCM droplets (Figure 2A). When less than 0.1% w / w SF was added, a wider size distribution was observed. When no SF was used, the DCM droplets were not stable. The dispersed DCM droplets immediately merged together and the two phases quickly separated. The results showed the need to use a sufficient amount of FS to produce a stable H / F emulsion and to stir continuously during the curing process to successfully manufacture polymer microspheres. (Figure 2B).

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

[0065] Example 2: Effect of homogenization speed. material and method One mL of 30% or 40% w / v POE in DCM was added to 9 mL of FC-40 containing 0.5% (w / w) FS FC-40 and emulsified using a VWR Handheld Homogenizer 200 equipped with a VWR 7 mm x 95 mm sawtooth generator probe at one of three homogenization speeds: low (approximately 50% of full power), medium (approximately 60% of full power), and high (approximately 70% of full power). The formed emulsion was stirred under vacuum. The formed microspheres were washed and dried under vacuum.

[0066] result As shown in Figure 3, at 30% POE, low homogenization speeds resulted in larger microsphere sizes, while high homogenization speeds resulted in smaller sizes (Table 2). 40% POE showed the same trend. These results indicate that the microsphere size can be controlled by adjusting the homogenization speed. [Table 2]

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

[0068] For compounding, VEGF-trapping SDP (or fluorescently labeled SDP (F-SDP) for fluorescence imaging) at 10%-30% w / w total solids weight was dispersed in 500 μL of ethyl acetate containing 10-35% w / v POE by vortexing followed by 5 min of sonication. These suspensions were then added to 9.5 mL of FC-40 supplemented with 0.1-0.5% w / w FS. Emulsification can be achieved by stirring, vortexing, or homogenization using a benchtop homogenizer. The structure of the emulsion is shown in Figure 5. Immediately after emulsification, an in-process aliquot was taken and dropped onto a glass slide for microscopic imaging. The droplets were allowed to harden onto the slide through evaporation under ambient conditions. To harden the microspheres, one of three methods was applied: (a) the solution was stirred overnight in an open container under ambient conditions to evaporate the ethyl acetate, (b) the solution was stirred under vacuum for at least 2 hours for faster solvent evaporation, and (c) NOVEC7500, or a mixture of FC-40 and NOVEC7500, was added to the emulsion under stirring. HFE acts as a co-solvent that promotes the extraction of ethyl acetate from the hydrocarbon phase into the fluorocarbon phase, allowing for a fast hardening process (typically within minutes).

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

[0070] Microsphere size was measured using liquid sampling by dispersing product powder in 0.01% w / v PVA solution and laser diffraction analysis using a Malvern Mastersizer 3000. Product morphology was measured using scanning electron microscopy (SEM).

[0071] To measure the protein content of the microspheres, a given amount of microspheres was first dissolved in 200 μL of ethyl acetate and then extracted once with pure water, the aqueous phase was collected and centrifuged to remove the cloudy suspension. Protein purity and concentration were measured by SEC-UPLC.

[0072] To measure burst release, a given amount of microspheres was incubated in 1 mL of PBS for 1 h at 37° C. The mixture was centrifuged and the supernatant was subjected to SEC-UPLC for protein concentration.

[0073] result The above results indicated the formation of a stable H / F emulsion with sufficient SF present. This non-aqueous emulsion can successfully produce blank POE spheres. This anhydrous method was again used to incorporate SDP into POE microspheres. In one example, VEGF-trapped F-SDP at 10% total solids weight (w / w) was introduced as a suspension into ethyl acetate (containing 20% ​​w / v POE) and this suspension in FC-40 (containing 0.5% w / w FS) was emulsified by stirring and vortexing. Immediately after emulsification, an aliquot was transferred onto a glass slide for microscopic imaging. As shown in Figures 6A and 6B, the ethyl acetate dispersed into the FC-40 into droplets, and the SDP particles were clearly trapped within the ethyl acetate droplets. In contrast to the S / O / W system (data not shown), there was no sign of protein leakage into the fluorocarbon continuous phase. Importantly in this H / F system, the SDP particles in the droplets retained the concave shape of their initial powder state. Since there was no water present to reconstitute SDP in the H / F system, the SDP maintained its initial solid microparticle shape. After curing, POE microparticles containing single or multiple SDP particles could be clearly observed through bright field and fluorescence microscopy images (Figures 7A, 7B, and 7C). After evaporation of the hydrocarbon and fluorocarbon solvents on the glass slide, water was added to test the burst release and encapsulation quality. As shown in Figures 8A-D, the SDP-encapsulated POE microspheres retained their integrity after the microsphere preparations were placed in water. No immediate release of protein was observed, and the shape of the SDP particles remained the same, indicating that the SDP particles were well protected by the polymer matrix and shielded from the aqueous environment. These results suggest that H / F emulsions are an effective solution for encapsulating proteins and other hydrophilic drugs in polymer matrices, with the potential to achieve high encapsulation efficiency, high yield while minimizing burst release, all of which are major challenges when using aqueous-based W / O / W or S / O / W methods.

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

[0075] Example 4: Effect of Hydrocarbon Solvent material and method Microparticles were prepared as described in Example 2, using dichloromethane or ethyl acetate as the hydrocarbon. 35% w / v POE in DCM and 35% w / v POE in ethyl acetate were prepared. Protein powder at 10% w / w total solids weight was suspended in 0.5 mL of POE solution in DCM or ethyl acetate. These suspensions were transferred to 9.5 mL of FC-40 containing 0.5% w / w FS in a 20 mL scintillation vial. These mixtures were homogenized to form emulsions and stirred under house vacuum for 1.5 hours. The formed microparticles were isolated by filtration, washed with FC-40, and dried under vacuum.

[0076] result FIG. 9 shows the size distribution of microparticles produced using the same formulation and process conditions, except that the type of hydrocarbon solvent was either dichloromethane or ethyl acetate. Microparticles produced using both hydrocarbons show encapsulation of the spray-dried protein. The use of dichloromethane results in larger microparticles. See Table 2 below. The results suggested that the use of different hydrocarbon solvents under the same formulation and process conditions resulted in microspheres of different sizes. DCM produced larger microsphere sizes than ethyl acetate. Thus, the hydrocarbon solvent can be purposefully selected to control the microsphere size. [Table 3]

[0077] Example 5: Effect of protein loading material and method Microparticles were prepared as described in Example 2, only the protein loading was varied. 35% w / v POE in DCM was prepared. 5%, 10%, and 30% w / w total solid weight protein powders were suspended in 0.5 mL of POE solution in DCM. These suspensions were transferred to 9.5 mL of FC-40 containing 0.5% w / w FS in a 20 mL scintillation vial. These mixtures were homogenized for about 1 minute to generate emulsions, and then 6 mL of a 1:1 v:v mixture of Novec7500 and FC-40 was added to the emulsion within 1 minute. Then, after another minute of stirring, the formed microspheres were 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 resulted in larger POE microparticle size as measured by laser diffraction analysis, and increased protein loading in the final POE microsphere preparation as observed via protein extraction experiments, bright field and confocal fluorescence microscopy. Bright field images at 30% w / w protein powder loading showed darker and less transparent microspheres than 10% w / w protein powder, indicating that more drug was encapsulated in the microsphere preparation (Figures 10A and 10B). Representative confocal images confirmed that the SDP was encapsulated in the POE matrix in its original shape from the cross-sectional images of the microspheres (Figures 11A and 11B). More SDP particles were observed in the 30% w / w loaded microspheres. Again, the encapsulated SDP maintained their original concave shape, indicating that they were intact during the entire manufacturing process. Furthermore, SEM images showed that with increasing protein powder loading, more protein particles were adsorbed on the surface of POE microparticles (Figures 12A-12C). Thus, the results showed that protein powder up to 30% w / w could be efficiently encapsulated within the microspheres. At protein powder loadings >30% w / w, protein particles could become adsorbed on the surface of the microspheres due to the possible lack of physical space within the microspheres of this formulation. The surface-adsorbed protein could result in a burst release of drug upon contact with water, if such an effect is desired for therapeutic efficacy. Protein is not lost to the continuous phase as expected in aqueous-based emulsion systems. [Table 4]

[0079] Example 6: Design of Experiments (DOE) for encapsulating SDP into POE microspheres using H / F bulk emulsification. material and method A DOE study was performed to evaluate the impact of key factors of synthesis in the designed space on the properties of the final product. Ten runs of the designed experiment were performed following the general procedure described in Example 2. Protein powder loading, protein powder particle size (Dv(50) size 2.2um and 5.6um, see SEM images in Figure 13), polymer concentration, and HFE concentration were varied while formulation and process conditions such as volumes of hydrocarbon and fluorocarbon phases, homogenization speed, FS concentration, etc. were kept constant (Table 4). Measured responses included microsphere size (Dv50, spanned by laser diffraction), encapsulation efficiency, burst release at 37°C in 1 hour, and SEM images.

[0080] result The DOE results 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). In addition, the fitting for burst release (R 2 =0.92), indicating that only protein powder loading significantly affects burst release (p-value <0.05, see correlation results in Table 7). The results suggest that increasing the amount of protein powder in the formulation increases the payload in the final product, but also increases the burst release rate. The burst release is likely caused by protein particles adsorbed on the surface. The maximum amount of protein powder internalized in the polymer microspheres is determined by the physical space for a given microsphere size. Simply increasing the protein powder concentration in the formulation suspension does not increase drug encapsulation beyond a certain threshold, which was about 30% w / w in this example. [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 system and process can be a platform technology applicable to different polymers and therapeutic proteins. In a specific example of the present invention, protein powder of recombinant IgG4 (MW=about 145 kDa), protein powder of recombinant IgG1 (MW=about 146 kDa), or protein powder of recombinant fusion protein (MW=about 64 kDa) were encapsulated in POE microspheres, respectively, by the same process as in Example 2. The results are summarized in Table 7. The amount of encapsulated protein powder in the microsphere preparation was determined by extraction assay and was consistent with the target value. Protein purity was maintained for recombinant fusion protein, IgG1, and slightly decreased (less than 2%) for IgG4 after the encapsulation process, indicating good process compatibility. [Table 7-2]

[0083] Other biodegradable polymers, such as PLGA and PLA, are also used in H / F-based emulsions. In a specific example of the present invention, fluorescently labeled VEGF trap F-SDP was encapsulated in PLGA (lactide:glycolide=50:50, Mw 42-65 kDa, Sigma Aldrich) and PLA (alkyl ether terminated, Mw 18,000-28,000, Sigma Aldrich) microspheres, respectively, through a similar process disclosed in Example 2. Bright field and fluorescent microscopy images showed that the protein powder was successfully encapsulated inside the polymer microspheres (Figures 14A-C for PLA, and Figures 15A-B for PLGA).

[0084] In the foregoing specification, the invention has been described in relation to specific embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is capable of additional embodiments, and that some of the details described herein may be varied considerably without departing from the underlying principles of the invention.

[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 the appended claims, rather than the foregoing specification, that the scope of the invention is indicated. Reference should be made to the claims. <Supplementary Note> Item 1 A method for producing polymer microparticles or polymer-coated microparticles, comprising: mixing a protein powder and a polymer in a hydrocarbon solvent to form a non-aqueous first solution; forming step; adding the first solution to a second solution, the second solution comprising a fluorocarbon liquid and a fluoro surfactant; stirring the mixed solution to form a non-aqueous emulsion containing a plurality of emulsion hydrocarbon droplets in the fluorocarbon liquid; removing the hydrocarbon solvent; removing the fluorocarbon liquid to isolate the microparticles, the microparticles comprising a protein encapsulated within a matrix of the polymer step including, method. Item 2 The method according to Item 1, wherein the microparticles comprise a single core-shell structure. Item 3 The method according to Item 1, wherein at least one of the microparticles comprises a plurality of cores dispersed in the polymer. Item 4 The method according to any one of Items 1 to 3, wherein the microparticles comprise a combination of microparticles having a single core structure encapsulated by a polymer and microparticles having a plurality of core structures encapsulated by a polymer. Item 5 The method according to any one of Items 1 to 4, wherein the fluorocarbon liquid comprises a perfluorinated C5-C18 compound. Item 6 The method according to any one of Items 1 to 5, wherein the hydrocarbon solution is selected from the group consisting of dichloromethane, chloroform, toluene, ethyl acetate, tetra hydrofuran, or a combination thereof. Item 7 The method according to any one of Items 1 to 6, wherein the fluorocarbon solution comprises Flourinert (trademark) 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. Item 8 The method according to Item 1, wherein the hydrocarbon solvent comprises dichloromethane, ethyl acetate, or a combination thereof. Item 9 The method according to any one of Items 1 to 8, wherein the fluorocarbon solution comprises a hydrofluoroether. Item 10 The method according to any one of Items 1 to 9, wherein the fluorosurfactant comprises perfluoropolyether-b-polyethylene glycol -b-perfluoropolyether. Item 11 The method according to any one of Items 1 to 10, wherein the fluorosurfactant comprises perfluoropolyether-b-polyethylene glycol -b-perfluoropolyether. Item 11. The method according to any one of items 1 to 10, wherein the polymer comprises a polyorthoester (POE). Section 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 items 1 to 10, Section 13 The protein is an antibody or an antigen-binding fragment thereof, a fusion protein, a recombinant protein, Item 13. The method of any one of items 1 to 12, wherein the polypeptide is a polypeptide of the present invention or a fragment or truncated form thereof. Section 14 Item 14. The method of item 13, wherein the protein is a VEGF trap protein. Section 15 Item 15. The method of item 14, wherein the protein is a truncated form of a VEGF trap protein. Section 16 Item 16. The method according to any one of items 1 to 15, wherein the microparticles have a diameter of 1 to 200 μm. Section 17 The protein powder contains particles having a diameter of microparticulated protein of 0.5 to 20 μm. Item 17. The method according to any one of Items 1 to 16. Section 18 Protein powders can be prepared by spray drying, electrospray drying, reversible precipitation, spray freezing, microfiltration, and Item 18. The method according to any one of Items 1 to 17, wherein the particles are microparticulated by a clotplate method, or a combination thereof. Section 19 The emulsion may be homogenized, vortexed, sonicated, cavitated, stirred, Item 19. The method according to any one of items 1 to 18, wherein the mixture is formed using a mixture of the above-mentioned components, agitation, or a combination thereof. Section 20 20. The method according to any one of items 1 to 19, wherein the hydrocarbon solvent is removed while stirring the mixed solution. Section 21 21. The method of claim 20, wherein the hydrocarbon solvent is removed under vacuum to harden the microparticles. Section 22 22. The method according to any one of claims 1 to 21, wherein the hydrocarbon solvent is removed by evaporation. Section 23 23. The method of any one of claims 1 to 22, wherein the fluorocarbon liquid is removed by filtration, optionally under vacuum. Section 24 A hydrofluoroether is used as a co-solvent to extract the hydrocarbons. 24. The method according to item 22 or 23. Section 25 24. Microparticles produced by the method according to any one of items 1 to 23, which are sustained release microparticles. Section 26 Item 26. A sustained release composition comprising the microparticles according to item 25. Section 27 The microparticles have few or no pores or channels in the polymer shell. Item 27. The microparticle according to any one of items 1 to 26. Section 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 contains a protein, and the hydrocarbon solution contains 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, a fluorocarbon solution containing 0.1-5.0% w / v of a fluorosurfactant; The emulsion droplets are hardened by removing the hydrocarbon liquid to form a hardened emulsion. Forming polymeric microspheres or polymer-coated microspheres and The method includes: Section 29 The non-aqueous emulsion is stirred and the hydrocarbon solution is heated under ambient atmospheric pressure while stirring, 29. The method of claim 28, wherein the organic solvent is removed by evaporation, or under vacuum. Section 30 The hardened polymer microspheres or polymer-coated microspheres 30. The method of claim 29, wherein the a is collected by vacuum filtration. Section 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. The continuous phase is a mixture of a fluorocarbon liquid and 0.1 to 5.0% w / v fluorine. a surfactant; collecting the polymer-coated microparticles; A method comprising: Section 32 The spray-dried protein may be an antibody, a recombinant protein, a fusion protein, or the like. 32. The method of claim 31, wherein the polypeptide is a fragment of Item 33 The protein is a VEGF trap protein or a truncated VEGF trap protein. 33. The method of claim 32, wherein the quality of the material is high. Section 34 The hydrocarbon solution may be dichloromethane, chloroform, toluene, ethyl acetate, tetrachloroethane, 32. The method of claim 31, wherein the aryl group is selected from the group consisting of aryl, aryloxy ... Section 35 The fluorocarbon liquid is a fluorocarbon liquid having a molecular weight of 1,1,2,2,3,3,4,4, Item 35. The method according to any one of items 31 to 34, comprising 4-nonafluorobutyl)amine. Section 36 The particulates are removed from the hydrocarbon solution by evaporation or under vacuum with stirring. Item 36. The method according to any one of items 31 to 35, wherein the curing is performed by Section 37 37. The method of claim 36, further comprising collecting the hardened microparticles. Section 38 Item 38. Microparticles produced by the method according to any one of Items 31 to 37. Section 39 Item 39. A pharmaceutical composition comprising the microparticles according to Item 38. Section 40 40. The pharmaceutical composition of claim 39, further comprising one or more excipients. Section 41 Item 41. The pharmaceutical composition according to item 40, wherein the pharmaceutical composition is a sustained release composition. Section 42 Item 42. The pharmaceutical composition according to any one of Items 39 to 41, wherein the pharmaceutical composition is formulated for parenteral administration. Section 43 Item 43. The method of any one of items 1 to 42, wherein the pharmaceutical composition comprises more than 100 mg of spray-dried protein. Section 44 The hydrofluoroether is 4-ethoxy-1,1,1,2,2,3,3,4, 10. The method of claim 9, comprising 5,6,6,6-didecafluoro-5-(trifluoromethyl)hexane. Section 45 1. A method for producing a microparticle, comprising: A first solution containing a polymer in a hydrocarbon solvent is mixed with a fluorocarbon solvent and a fluorinated surfactant. mixing the solution with a second solution containing a dispersing agent; 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; and A method comprising: Section 46 The hydrocarbon solvent is dichloromethane, chloroform, toluene, ethyl acetate, tetrachloroethane, Hydrofuran, acetonitrile, ethanol, methanol, propanol, dimethylformamide dimethylsulfoxide, dimethylsulfoxide, or a combination thereof. 46. ​​The method according to claim 45. Section 47 The fluorocarbon solvent is selected from the group consisting of 1,1,2,2,3,3,4,4 47. The method of claim 45 or 46, comprising reacting the compound with 1,4-nonfluorobutylamine. Section 48 The polymer is POE, polylactic acid, poly(lactic-co-glycolic acid), or the like. Item 48. The method according to any one of items 45 to 47, comprising a combination of: Section 49 50. The method of any one of items 45 to 49, wherein the microparticle comprises a polymeric shell and a hollow core. Item 50 The microparticles have few or no pores or channels in the polymer shell. 50. The method of any one of paragraphs 49, wherein the method is not Section 51 The diameter of the microparticles varies depending on the hydrocarbon solvent, the stirring speed, the polymer concentration, or a combination thereof. Item 50 or 51, the method of claim 50 or 51, wherein the desired diameter is adjusted by changing the combination. Section 52 Item 45 to 51. The microparticle according to any one of items 45 to 51.

Claims

1. 1. A method for producing polymeric or polymer-coated microparticles, comprising the steps of: The protein powder and the 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, the second solution being a mixture of a fluorocarbon liquid and a fluorocarbon. and 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, The process includes encapsulating the protein within a polymer matrix. A method comprising:

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

3. At least one of the microparticles comprises a plurality of cores dispersed within the polymer.

2. The method according to claim 1.

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

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

13. The method according to claim 1.

5. Any of claims 1 to 4, wherein the fluorocarbon liquid comprises a perfluoro C5 to C18 compound.

3. The method according to claim 1 .

6. The hydrocarbon solvent is dichloromethane, chloroform, toluene, ethyl acetate, tetrachloroethane, 6. Any of claims 1 to 5, selected from the group consisting of hydrofuran, hydrochloric acid, ethyl alcohol ... The method according to any one of claims 1 to 5.

7. The fluorocarbon liquid is Flourinert™ 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. Item 7. The method according to any one of items 1 to 6.

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

9. 9. The method according to claim 1, wherein the fluorocarbon liquid comprises a hydrofluoroether. The method according to

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

11. 11. The method according to claim 1, wherein the polymer comprises a polyorthoester (POE). The method according to claim 5.

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 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.

16. The microparticles according to any one of claims 1 to 15, wherein the microparticles have a diameter of 1 to 200 µm. How to.

17. The protein powder comprises 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, microfiltration, and The method according to any one of claims 1 to 1, wherein the microparticles are formed by a clostable template method, or a combination thereof.

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

19. The emulsion is subjected to homogenization, vortexing, sonication, cavitation, stirring, etc.

19. The method of claim 1, wherein the composition is formed using a method comprising the steps of: 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.

2. The method according to any one of claims 1 to 11.

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

22. A process according to any one of the preceding claims, wherein the hydrocarbon solvent is removed by evaporation. How to.

23. 23. The method according to claim 1, wherein the fluorocarbon liquid is removed by filtration. How to.

24. A hydrofluoroether is used as a co-solvent to extract the hydrocarbons. The method according to claim 22 or 23.

25. The method of any one of claims 1 to 23, wherein the microparticles are sustained release microparticles.

26. To prepare a sustained release composition comprising polymeric microparticles or polymer-coated microparticles, 26. A method for producing a microparticle comprising producing the microparticle by the method of claim 25.

27. 27. The method of claim 1, wherein the microparticle lacks pores or channels in the polymer shell. The method according to any one of claims 1 to 5.

28. 1. A method for producing polymer-coated microspheres, comprising: (1) 1.0-30.0% w / w total solids spray-dried protein suspended in a hydrocarbon solution The dispersed phase comprises a substance, and the hydrocarbon solution is 5.0-35% w / v polyorthoether. Includes Stell (POE) , the dispersed phase, (2) mixing the dispersed phase into a continuous phase to form a non-aqueous emulsion containing emulsion droplets of the dispersed phase; The continuous phase is a fluorinated mixture containing 0.1 to 5.0% w / v of a fluorosurfactant. a carbonaceous liquid; The emulsion droplets are hardened by removing the hydrocarbon solvent to form a hardened emulsion. forming a polymer-coated microsphere; The method includes:

29. The non-aqueous emulsion is stirred and the hydrocarbon solvent is added at ambient atmospheric pressure during stirring.

29. The method of claim 28, wherein the organic solvent is removed by evaporation, or under vacuum.

30. The hardened polymer-coated microspheres are collected by vacuum filtration.

30. The method of claim 29 .

31. A method for producing a polymer-coated microparticle, comprising the steps of: The hydrocarbon solvent containing the dissolved polymer is mixed with the spray-dried protein powder to form a dispersion. generating a dispersed phase; The dispersed phase is mixed with a continuous phase to form emulsion droplets of the dispersed phase in the continuous phase. forming a non-aqueous emulsion, the continuous phase being a mixture of a fluorocarbon liquid and 0.1 -5.0% w / v fluorosurfactant; collecting the polymer-coated microparticles; A method comprising:

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

33. The protein is a VEGF trap protein or a truncated VEGF trap protein. The method of claim 32, wherein the

34. The hydrocarbon solvent is dichloromethane, chloroform, toluene, ethyl acetate, tetrachloroethane, 32. The method of claim 31, wherein the aryl group is selected from the group consisting of aryl, aryloxy ... How to.

35. The fluorocarbon liquid is a fluorocarbon liquid having a molecular weight of 1,1,2,2,3,3,4,4, The method of any one of claims 31 to 34, comprising the step of reacting 4-nonafluorobutyl)amine. 。

36. The particulate is agitated to remove the hydrocarbon solvent by evaporation or under vacuum. 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 method for producing a pharmaceutical composition comprising polymer-coated microparticles, the method comprising producing the microparticles by a method according to any one of claims 31 to 37.

39. 39. The method of claim 38, wherein the pharmaceutical composition further comprises one or more excipients.

40. 40. The method of claim 39, wherein the pharmaceutical composition is a sustained release composition.

41. 41. Any of claims 38 to 40, wherein the pharmaceutical composition is formulated for parenteral administration.

3. The method according to claim 1 .

42. 42. Any of claims 38 to 41, wherein the pharmaceutical composition comprises more than 100 mg of spray-dried protein. The method according to any one of claims 1 to 5.

43. The hydrofluoroether is 4-ethoxy-1,1,1,2,2,3,3,4, 5,6,6,6-didecafluoro-5-(trifluoromethyl)hexane.

9. The method according to claim 9.

44. 1. A method for producing a microparticle, comprising: A non-aqueous first solution comprising a polymer in a hydrocarbon solvent is added to a fluorocarbon solvent and a fluorine-containing solvent. mixing the second solution containing the system surfactant; agitating the combined solution to form a non-aqueous 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 drying the microparticles; and Including, The microparticles are sustained release microparticles and the fluorocarbon solvent is a hydrofluoroacetate. A method including a tell.

45. The hydrocarbon solvent is dichloromethane, chloroform, toluene, ethyl acetate, tetrachloroethane, Hydrofuran, acetonitrile, ethanol, methanol, propanol, dimethylformamide dimethylsulfoxide, dimethylsulfoxide, or a combination thereof.

45. The method of claim 44.

46. 1. A method for producing a microparticle, comprising: A first solution containing a polymer in a hydrocarbon solvent is mixed with a fluorocarbon solvent and a fluorinated surfactant. mixing the solution with a second solution containing a dispersing agent; 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 drying the microparticles; and Including, The fluorocarbon solvent is selected from the group consisting of 1,1,2,2,3,3,4,4 ,4-nonafluorobutyl)amine.

47. 1. A method for producing a microparticle, comprising: A first solution containing a polymer in a hydrocarbon solvent is mixed with a fluorocarbon solvent and a fluorinated surfactant. mixing the solution with a second solution containing a dispersing agent; 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 drying the microparticles; and Including, The polymer may be polyorthoester (POE), polylactic acid, poly(lactic acid-co-glycol), carboxylic acid), or a combination thereof.

48. 48. Any one of claims 44 to 47, wherein the microparticle comprises a polymeric shell and a hollow core. The method described above.

49. 49. The method of claim 48, wherein the microparticle lacks pores or channels in the polymeric shell. How to.

50. The diameter of the microparticles varies depending on the hydrocarbon solvent, the stirring speed, the polymer concentration, or a combination thereof.

50. The method of claim 49, wherein the desired diameter is adjusted by varying the combination.

51. 26. The method of claim 25, wherein the microparticle comprises a polymeric shell.

52. The method of any one of claims 1 to 22, wherein the fluorocarbon liquid is removed by vacuum.

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