Sustained release formulations using non-aqueous emulsions

Abstract

A non-aqueous emulsion process for producing polymeric microparticles or polymer coated microparticles is provided. A method produces a sustained release particulate composition by combining a protein powder and a polymer into a hydrocarbon solvent to form a non-aqueous first solution; and adding the first solution to a second solution to form a non-aqueous emulsion, where the second solution includes a fluorocarbon liquid and a fluorine-containing surfactant, the non-aqueous emulsion including a plurality of emulsion hydrocarbon droplets in the fluorocarbon liquid. A subsequent particulate hardening process comprises the steps of: removing the hydrocarbon solvent from the formed emulsion droplets, which can be accomplished by vaporizing the hydrocarbon under ambient conditions while stirring; or accelerated hardening by vacuum or by adding a hydrofluoroester as a co-solvent to the fluorocarbon. The fluorocarbon liquid is removed and washed with an additional fluorocarbon liquid to separate the sustained release microparticles, wherein the sustained release microparticles include one or more protein nuclei and a polymer skin layer.

Description

[0001] This case is a divisional application of the patent application with application number 202080082188.2 filed by the applicant on November 25, 2020, entitled "Sustained Release Formulation Using Non-aqueous Emulsion". The entire contents of the parent case are incorporated into this divisional by reference. Technical Field

[0002] Aspects of the present invention generally relate to drug microsphere formulations and methods of preparing the same using a non-aqueous emulsion system. Background Art

[0003] The sustained release delivery of therapeutic proteins to biologically relevant targets is desirable for the treatment of medical conditions such as cancer, cardiovascular disease, vascular pathologies, orthopedic conditions, dental conditions, wounds, autoimmune diseases, gastrointestinal conditions, 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, therapeutic drugs are slowly released as the polymer degrades over time.

[0004] Sustained release may be desirable for patient compliance. Specifically, reducing the number of injections may be beneficial, especially when a doctor is required to perform an injection as in the case of intraocular treatment. The medical needs of sustained release formulations that effectively deliver drugs over time with as little injection as possible have not yet been met. In other diseases, such as cancer and inflammatory diseases, improved implantable sustained release formulations containing stable and effective protein therapeutics are needed.

[0005] Therapeutic macromolecules such as antibodies and receptor Fc fusion proteins must be formulated in a manner that not only makes the molecules suitable for application to patients, but also maintains their stability during storage and at the site of application. For example, unless the solution is correctly formulated, therapeutic proteins (e.g., antibodies and fusion proteins) in aqueous solutions are prone to degradation, aggregation and / or undesirable chemical modifications. The stability of protein therapeutics in liquid formulations depends not only on the type of excipients used in the formulation, and the amount and ratio of those excipients relative to each other, but also on the concentration of soluble proteins. When preparing therapeutic protein formulations, considerations other than stability must also be considered. Examples of such additional considerations include the viscosity of the solution and the concentration of therapeutic proteins that a given formulation can adapt to. When formulating therapeutic proteins for sustained release, great care must be taken to keep the formulation stable over a period of time and at storage and physiological temperatures, contain sufficient concentrations of antibodies, and have other properties that enable the formulation to be conveniently applied to patients.

[0006] Some sustained-release formulations are produced using a variety of encapsulation methods, including: internal phase separation, interfacial polymerization, formation of multiple emulsions, layer-by-layer adsorption of polyelectrolytes, and soft template technology. Water-in-oil-in-water (W / O / W) multiple emulsions are the most common type of multiple emulsions and are capable of encapsulating aqueous / hydrophilic cores directly in aqueous suspensions. Unfortunately, aqueous emulsion systems present specific problems when used to encapsulate bioactive agents into sustained-release formulations. For example, proteins precipitate at aqueous-organic interfaces with a concomitant decrease in their immunoreactivity (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 proteins. After hydrolysis, protein droplets begin to merge and escape into the aqueous environment and aggregate or precipitate. After hardening, voids and water channels appear in the microparticles where the proteins were once located but have escaped into the aqueous environment.

[0007] Non-aqueous emulsions can replace conventional aqueous emulsions in any situation where the presence of water is not desired. However, there are few reports on non-aqueous emulsions in the literature or prior art. 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-immiscible polar solvents (e.g., formamide, acetonitrile) using existing surfactants instead of water. Previously, perfluorinated water-in-oil (W / F) emulsions have been studied and widely used in droplet-based microfluidics for single-cell or single-molecule bioassays. In these studies, PFPE-PEG-PFPE has been used as a fluorosurfactant (FS) for stabilizing water droplets in fluorocarbon solvents.

[0008] Although many immiscible solvent pairs are available, usually one polar and one non-polar, it is challenging to find a suitable pair for synthesizing polymer microspheres. Typical biodegradable polymers, such as poly(lactide-co-glycolide) (PLGA), polylactic acid (PLA), poly(orthoesters) (POE) are mostly soluble in solvents with moderate polarity such as chloroform, dichloromethane, ethyl acetate, etc. This limits the choice of continuous phase. In addition, compatibility with the process, toxicity, safety, and residual solvents are issues with the use of those organic solvents and need to be considered for use as drug products.

[0009] Fluorocarbons can be used as the continuous phase in non-aqueous emulsion systems due to the following general properties:

[0010] 1. Fluorocarbons are neither "hydrophobic" nor "hydrophilic" and are immiscible with most organic (hydrocarbon) solvents, making them ideal continuous phases for hydrocarbon droplet emulsions.

[0011] 2. Fluorocarbons are non-solvents for proteins and other hydrophilic molecules, hydrocarbon-based polymers and organic excipients, ie, these types of molecules are insoluble in fluorocarbons.

[0012] 3. Fluorocarbons have low viscosity.

[0013] 4. Fluorocarbons are chemically inert and relatively less toxic or corrosive than commonly used hydrocarbon solvents.

[0014] 5. Fluorocarbons are volatile and recyclable.

[0015] Previous literature reports have prepared various emulsion systems containing fluorocarbons by microfluidic 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 for synthesizing polymer microspheres. However, all of the emulsions are still water-based emulsion systems using water as dispersed phase or continuous phase.

[0016] It is therefore an object of the present invention to provide non-aqueous emulsion systems for producing pharmaceutical formulations and methods of using the same.

[0017] Another object of the present invention is to provide a sustained release formulation with improved protein stability and stable sustained release. Summary of the invention

[0018] A non-aqueous emulsion method for producing polymer microparticles and polymer-coated microparticles is provided. One embodiment provides a method for producing a sustained-release or controlled-release microparticle composition by combining 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 to form a non-aqueous emulsion, wherein the second solution includes a fluorocarbon liquid and a fluorosurfactant, and the non-aqueous emulsion contains a plurality of emulsion hydrocarbon droplets in the fluorocarbon liquid. In some embodiments, the emulsion is formed from a bulk emulsion. The method further comprises the steps of removing the hydrocarbon solvent and removing the fluorocarbon liquid to separate the sustained-release or controlled-release microparticles, wherein the sustained-release microparticles contain one or more protein powder cores and a biodegradable or bioerodible polymer cortex. 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 at ambient atmospheric pressure or under vacuum. In some embodiments, the fluorocarbon liquid contains a hydrofluoroether (HFE), or additional HFE is added to the non-aqueous emulsion after emulsification to quickly extract the hydrocarbon into the fluorocarbon liquid to accelerate microsphere hardening. In some embodiments, the protein powder is a micronized protein powder. In some embodiments, the microparticles are washed to remove any residual hydrocarbon solvents, fluorocarbon liquids, fluorosurfactants, or combinations thereof remaining on the microparticles. Exemplary fluorocarbon liquids include perfluorinated C5-C18 compounds, including but not limited to FC-40. In some embodiments, the fluorocarbon liquid contains HFE. Exemplary hydrocarbon solvents include but are not limited to dichloromethane, chloroform, ethyl acetate, and combinations thereof. Exemplary fluorosurfactants are perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether (PFPE-PEG-PFPE) triblock copolymers. Exemplary bioerodible polymers are polyorthoesters (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 diameter of the microparticles is 1.0 μm to 100 μm or 1.0 to 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 pharmaceutically acceptable excipient, such as pH buffered saline, or in an oily vehicle such as medium chain triglycerides. The flowable microparticle composition can be administered parenterally, for example, using a syringe with a 27G needle.

[0019] Another embodiment provides a method for producing a population of polymer-coated microspheres by emulsifying a dispersed phase comprising 1.0% w / v to 30.0% w / v spray-dried protein suspended in a hydrocarbon solution into a continuous phase to form emulsion droplets of the dispersed phase, wherein the hydrocarbon solution comprises 5.0% w / v to 40% w / v POE, wherein the continuous phase comprises a fluorocarbon solution comprising 0.1% w / v to 5.0% w / v fluorosurfactant and optionally HFE. The method further comprises hardening the emulsion droplets to form the population of polymer-coated microspheres by removing the hydrocarbon liquid while stirring the emulsion, and optionally washing the microparticles to remove any hydrocarbon solution, fluorocarbon solution, fluorosurfactant, or a combination thereof. In one embodiment, the hydrocarbon solution and the fluorocarbon solution are removed by evaporation at ambient atmospheric pressure or under vacuum.

[0020] Yet another embodiment provides a method for producing polymer-coated microparticles by combining a hydrocarbon solution containing a dissolved polymer with a spray-dried protein powder to produce a dispersed phase; and combining the dispersed phase with a continuous phase to produce emulsion droplets of the dispersed phase in the continuous phase, wherein the continuous phase comprises a fluorocarbon liquid and 0.2% w / v to 5.0% w / v FS and optionally HFE. The method comprises removing the hydrocarbon solution and the fluorocarbon solution by agitating the emulsion under vacuum to harden the microparticles and then harvesting the polymer-coated microparticles. The method also comprises an optional step of washing the harvested microparticles.

[0021] Still another embodiment provides a method for producing microparticles by combining a first solution containing a polymer in a hydrocarbon solvent with a second solution containing a fluorocarbon solvent and a fluorosurfactant and stirring the combined solution to produce an emulsion. The method includes the steps of removing the hydrocarbon solvent under vacuum while stirring the combined solution to harden the microparticles and harvesting the microparticles. The method includes optionally washing the microparticles and drying the microparticles.

[0022] 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 on the polymer surface or in the internal matrix of the microparticles.

[0023] Still another embodiment provides a pharmaceutical composition comprising polymer-coated microparticles produced using the non-aqueous emulsion method disclosed herein.

[0024] In some embodiments, the size of the microparticles can be tuned to a desired diameter or size by varying the formulation composition and process parameters. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1A is a diagram showing the process of producing blank POE microspheres by H / F based bulk emulsion - Scheme 1. Figure 1B The chemical structure of FC-40 is shown. Figure 1C The fluorinated surfactant PFPE-PEG-PFPE (Pico-Surf TM 1), Chemical structure of a perfluoropolyether / poly(ethylene glycol) triblock copolymer. Pico-Surf TM 1 is commercially available, for example 5% (w / w) in FC-40.

[0026] Figure 2A is a micrograph of blank POE microspheres formed by H / F emulsion. Figure 2B is a micrograph showing POE aggregation found at low FS content.

[0027] 3A, 3B, and 3C are micrographs of blank POE microspheres formed by H / F emulsions at low, medium, and high homogenization speeds.

[0028] Figure 4 (Scheme 2) is a diagram showing the process of encapsulating SDP in POE microspheres via S / H / F based bulk emulsion.

[0029] Figure 5 (Scheme 3) is a diagram showing a fluorocarbon-in-hydrocarbon emulsion system for encapsulating protein SDP.

[0030] Figures 6A and 6B are fluorescence images of ethyl acetate droplets containing POE and fluorescently labeled spray-dried protein (F-SDP) dispersed in FC-40. Note that F-SDP retains its original size and morphology within the droplet.

[0031] Figure 7A is a bright field micrograph of VEGF Trap F-SDP encapsulated microspheres. Figure 7B is a fluorescent image of VEGF Trap F-SDP encapsulated microspheres (bar = 20 μm). Figure 7C is a fluorescent image of VEGF Trap F-SDP encapsulated microspheres (bar = 10 μm).

[0032] Figures 8A-8D are fluorescent images of VEGF Trap F-SDP encapsulated POE microspheres placed in an aqueous environment. Note that the F-SDP retains its original size and morphology within the droplets.

[0033] Fig. 9is a line plot of volume density (%) versus size (μm) for microparticles produced in a non-aqueous emulsion process using either dichloromethane (DCM) or ethyl acetate (EtAc).

[0034] Figures 10A and 10B are micrographs of microparticles loaded with 10% w / w and 30% w / w VEGF Trap SDP, respectively.

[0035] Figures 11A and 11B Representative fluorescence images of VEGF Trap F-SDP encapsulated POE microspheres loaded with 10% w / w and 30% w / w SDP, respectively. Note that F-SDP retains its original size and morphology within the droplets.

[0036] 12A-12C are scanning electron microscope (SEM) images of microparticles loaded with 5% w / w, 10% w / w, and 30% w / w SDP, showing an increase in protein on the microparticle surface with increasing SDP loading.

[0037] Figures 13A and 13B are SEM images of the spray-dried proteins with Dv50 of 2.18 μm and 5.63 μm.

[0038] Fig.14 A, 14B, and 14C are bright field, fluorescence, and SEM images of VEGF-Trap F-SDP encapsulated in PLA microspheres.

[0039] Fig.15 A and 15B are bright field and fluorescence images of VEGF-Trap F-SDP encapsulated in PLGA microspheres. DETAILED DESCRIPTION

[0040] I. Definitions

[0041] It should be understood that the present disclosure is not limited to the compositions and methods described herein and the experimental conditions described, because the compositions and methods and experimental conditions can vary. It should also be understood that the terms used herein are only for the purpose of describing certain embodiments and are not intended to be limiting, because the scope of the present disclosure will only be limited by the appended claims.

[0042] Unless otherwise defined, all technical terms and scientific terms used have the same meaning as those commonly understood by those of ordinary skill in the art to which the present disclosure belongs. However, any compositions, methods and materials similar or equivalent to the compositions, methods and materials described herein can be used to practice or test the present invention. All publications mentioned are incorporated herein by reference in their entirety.

[0043] The use of the terms "a", "an", "the" and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0044] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.

[0045] The use of the term "about" is intended to describe values ​​that are within a range of approximately + / -10% above or below the stated value; in other embodiments, these values ​​may be within a range of values ​​that are within a range of approximately + / -5% above or below the stated value; in other embodiments, these values ​​may be within a range of values ​​that are within a range of approximately + / -2% above or below the stated value; in other embodiments, these values ​​may be within a range of values ​​that are within a range of approximately + / -1% above or below the stated value. The above ranges are intended to be determined according to the context, and no further limitations are implied. Unless otherwise indicated herein or otherwise clearly contradicted by the context, all methods described herein may be performed in any suitable order. Unless otherwise required, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended only to better illustrate the present invention and does not limit the scope of the present invention. No language in this specification should be interpreted as indicating that any unclaimed element is necessary to practice the present invention.

[0046] The term "protein" refers to a molecule comprising two or more amino acid residues connected to each other by a peptide bond. Proteins include polypeptides and peptides and may also include modifications such as glycosylation, lipid linkage, sulfation, γ-carboxylation, alkylation, hydroxylation and ADP-ribosylation of glutamic acid residues. Proteins may have scientific or commercial interests, include protein-based drugs, and, among others, proteins also include enzymes, ligands, receptors, antibodies and chimeric or fusion proteins. Proteins are produced by various types of recombinant cells using well-known cell culture methods, and are usually introduced into cells by genetic engineering techniques (e.g., sequences encoding chimeric proteins, or codon-optimized sequences, intron-free sequences, etc.), wherein they may exist as episomes or be integrated into the genome of cells.

[0047] "Antibody" refers to an immunoglobulin molecule consisting of four polypeptide chains, i.e., 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 region and the VL region can be further subdivided into hypervariable regions called complementary determining regions (CDRs), which are interspersed with more conservative regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The term "antibody" includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass. The term "antibody" includes antibody molecules that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from host cells transfected to express the antibody. The term antibody also includes bispecific antibodies, which include heterotetrameric immunoglobulins that can bind to more than one different epitope. Bispecific antibodies are generally described in U.S. Patent No. 8,586,713, which is incorporated by reference into this application.

[0048] "Fc fusion proteins" include part or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, and part or all of the two or more proteins are not found together in nature. The preparation of fusion proteins including certain heterologous polypeptides fused to different parts of a polypeptide derived from an antibody (including an Fc domain) has been described below: for example, 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", Current Protocols in Immunology, Supplement 4, pp. 10.19.1-10.19.11, 1992. A "receptor Fc fusion protein" includes one or more extracellular domains of a receptor coupled to an Fc portion, which in some embodiments includes a hinge region and subsequent CH2 and CH3 domains of an immunoglobulin. In some embodiments, the Fc fusion protein includes 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.

[0049] "Micronized protein particles" or "protein particles" means particles containing multiple protein molecules and low, very low or close to zero water content (e.g., <3% by weight of water). As used herein, micronized protein particles are generally spherical in shape and have an ECD in the range of 2 microns to about 35 microns. Micronized protein particles are not limited to any particular protein entity and are suitable for the preparation and delivery of therapeutic proteins. Common therapeutic proteins include, among others, antigen binding proteins, e.g., soluble receptor fragments, antibodies (including IgG) and derivatives or fragments of antibodies, other Fc-containing proteins including Fc fusion proteins, and receptor-Fc fusion proteins including trap-type proteins such as VEGF Trap (Huang, C., Curr. Opin. Biotechnol. 20: 692-99 (2009)).

[0050] II. Production of Microsphere Formulations Using Hydrocarbon-Fluorocarbon Emulsions

[0051] Provided are systems and methods for preparing pharmaceutical compositions using an anhydrous emulsion system. The disclosed anhydrous emulsion method overcomes several problems of existing aqueous emulsion systems. For example, a comparative study between the disclosed anhydrous emulsion system and the existing aqueous emulsion system provided herein shows that formulations produced using aqueous emulsion systems leak drugs, such as protein drugs, from emulsion droplets into the aqueous continuous phase during production. This drug leakage from the emulsion droplets results in low encapsulation efficacy. The disclosed non-aqueous-based emulsion method described herein encapsulates drug molecules including, but not limited to, hydrophilic drugs such as proteins with increased encapsulation efficacy relative to aqueous emulsion systems, retaining the original protein particle structure or a combination thereof. The disclosed anhydrous emulsion system and method can produce encapsulated drug formulations by bulk methods (i.e., stirring, homogenization, ultrasonic treatment) and other conventional methods. The system and method can also be applied to a wide range of polymer materials, solid-state payloads and emulsification methods. Table 1 shows the comparison results of different emulsions, which demonstrates that the non-aqueous emulsion system has significant improvement in microparticle encapsulation compared to the aqueous emulsion system.

[0052] Table 1: Summary of methods and main results

[0053]

[0054] A. Fluorocarbon-in-hydrocarbon (S / H / F) emulsion

[0055] Exemplary non-aqueous S / H / F emulsion methods include combining dried protein powder and biodegradable and / or bioerodible polymers into hydrocarbon solvents to form a non-aqueous first solution and adding the first solution to a second solution made of fluorocarbon liquid and fluorosurfactant. The combination of the first solution and the second solution is in a manner for forming a non-aqueous emulsion containing a plurality of emulsion hydrocarbon droplets in a fluorocarbon liquid, such as by stirring, ultrasonic treatment, cavitation, homogenization or vortex. The method includes removing hydrocarbon solvents and removing fluorocarbon liquids to separate the microparticles with one or more micronized protein cores and biodegradable polymer cortexes. In one embodiment, the emulsion is stirred and hydrocarbons and fluorocarbon liquids are evaporated under vacuum. The resulting microparticles can be optionally washed to remove hydrocarbon solvents, fluorocarbon liquids, fluorosurfactants or their combinations. The emulsion can be formed using bulk emulsion technology.

[0056] One embodiment provides a method of producing a sustained release microparticle composition by combining 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 to form a non-aqueous emulsion, wherein the second solution contains a fluorocarbon liquid, a fluorosurfactant, and optionally HFE, the non-aqueous emulsion containing a plurality of emulsion hydrocarbon droplets in the fluorocarbon liquid, the plurality of emulsion hydrocarbon droplets containing the protein powder. Homogenization, vortexing, ultrasonic treatment, cavitation, stirring, or a combination thereof can be used to form the emulsion. The method further comprises the step of removing the hydrocarbon solvent and the fluorocarbon liquid while stirring the emulsion. The hydrocarbon liquid and the fluorocarbon liquid can optionally be removed by evaporation under vacuum. In other embodiments, the microparticles can be harvested by filtration. Removal of the hydrocarbon liquid and the fluorocarbon liquid hardens the microparticles, which can then be harvested. In some embodiments, HFE can be added to the fluorocarbon to help extract the hydrocarbon from the dispersed phase into the fluorocarbon continuous phase to achieve a faster hardening process. HFE is miscible with fluorocarbons and hydrocarbons, and can therefore act as a co-solvent to increase the solubility of hydrocarbons in the fluorocarbon phase. The sustained release microparticles produced by the non-aqueous emulsion method contain proteins encapsulated in a biodegradable or bioerodible polymer matrix. In some embodiments, the microparticles have a single core-shell structure. In other embodiments, the microparticles have multiple cores dispersed in the polymer. In still other embodiments, the microparticle population includes microparticles having a single core structure encapsulated by a polymer cortex and microparticles having a multi-core structure in the polymer cortex. The fluorocarbon liquid can be a perfluorinated C5-C18 compound, including but not limited to FC-40, and the hydrocarbon solution is selected from the following group: ethyl acetate, chloroform, toluene, ethyl acetate, tetrahydrofuran and dichloromethane or a combination thereof. In one embodiment, the fluorinated surfactant can be Pico-SurfTM 1 Commercially available perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether. 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 acid-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 a truncated version thereof. Typically, the protein is micronized, for example, by spray drying, electrospray drying, reversible precipitation, spray freezing, microtemplating or a combination thereof. In one embodiment, the protein is VEGFTrap protein or a truncated form thereof. Other proteins that can be used in the disclosed method are described below. The microparticles produced by the disclosed method have a polymer cortex without pores or channels. The polymer cortex is not perforated. In some embodiments, the microparticles have a diameter of 1 to 200 μm.

[0057] Another embodiment provides a method for producing polymer-coated microspheres by combining (1) a dispersed phase having 1.0% w / v to 30.0% w / v spray-dried protein suspended in a hydrocarbon solution, wherein the hydrocarbon solution includes 5.0% w / v to 30% w / v POE, into (2) a continuous phase to form emulsion droplets of the dispersed phase, wherein the continuous phase contains a fluorocarbon solution including 0.1% w / v to 5.0% w / v fluorosurfactant. The method comprises hardening the emulsion droplets by removing the hydrocarbon solution to form hardened polymer-coated microspheres. In one embodiment, the fluorocarbon solution can be a perfluorinated C5-C18 compound, including but not limited to FC-40, and the hydrocarbon solution is selected from the group consisting of ethyl acetate, chloroform, toluene, ethyl acetate, tetrahydrofuran, and dichloromethane, or a combination thereof. In one embodiment, the fluorosurfactant can be Pico-Surf TM 1 Commercially available perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether. The method comprises the step of stirring the emulsion under vacuum to remove hydrocarbon and fluorocarbon solutions.

[0058] Still another embodiment provides a method for producing polymer-coated microparticles by combining a hydrocarbon solution containing a dissolved polymer and a spray-dried protein powder to produce a dispersed phase. The method comprises combining the dispersed phase with a continuous phase to produce emulsion droplets of the dispersed phase in the continuous phase, wherein the continuous phase contains a fluorocarbon liquid and 0.1% w / v to 5.0% w / v of a fluorosurfactant, and harvesting the polymer-coated microparticles. The hydrocarbon solution can be selected from the group consisting of ethyl acetate, methylene chloride, chloroform, or a combination thereof. In one embodiment, the fluorocarbon solution contains FC-40, and the surfactant is a Pico-Surf TM1 Commercially available perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether.

[0059] 1. Hydrocarbon solvents

[0060] In some embodiments, the hydrocarbon solvent (also referred to as a hydrocarbon liquid) is selected so that the polymeric material, such as 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 may contain acetonitrile, dimethylformamide, dimethyl sulfoxide, acetone, ethanol, methanol, pentane, propanol, hexane, or a combination thereof.

[0061] 2. Fluoride-containing liquid

[0062] Exemplary fluorine-containing liquids are fluorocarbon liquids including, but not limited to, Flourinert TM 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)butyl-1-amine ( Figure 1B )、Fluorinert TM FC-70 (average MW = 821 g / mol) or a combination thereof. In some embodiments, the fluorocarbon liquid is or contains a hydrofluoroether (HFE). Exemplary HFEs include but are not limited to NOVEC TM 7000(1-methoxyheptafluoropropane), NOVEC TM 7100(Methoxy-nonafluorobutane), NOVEC TM 7200(ethoxy-nonafluorobutane), NOVEC TM 7500 (2-(trifluoromethyl)-3-ethoxydodecafluorohexane). In still other embodiments, the fluorocarbon liquid contains FC-40, FC-70, Novec TM 7500、Novec TM 7100、Novec TM 7000 or a combination thereof. In certain embodiments, the second solution contains a fluorinated surfactant (FS) in addition to the fluorinated liquid. An exemplary FS is a perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether (PFPE-PEG-PFPE) triblock copolymer, which can be Pico-Surf TM 1 is commercially available. In one embodiment, the fluorocarbon liquid or second solution contains FC-40 and Pico-Surf TM 1.

[0063] In some embodiments, FS is

[0064] in

[0065] Where: n ~ 37, x + z ~ 6.0, y ~ 12.5. Or where n = 3.7, x + z ~ 3.6, y ~ 9.0. (Lee, M. et al., Lab Chip., 7: 14(3): 509-13 (2014)).

[0066] In one embodiment, HFE has the following chemical structure:

[0067]

[0068] 2-(Trifluoromethyl)-3-ethoxydodecafluorohexane.

[0069] Other HFEs suitable for use in the process are a class of molecules in which all of the hydrogen atoms are located on carbons that are not fluorine-substituted and are separated from the fluorinated carbon by ether oxygens (i.e., RfORh). The HFE has a molecular structure that can be linear, branched, or cyclic or a combination thereof (e.g., alkylcycloaliphatic), and preferably contains no olefinic unsaturation, having a total of about 4 to about 20 carbon atoms. Such HFEs are known and readily available as substantially pure compounds or mixtures. Due to the lipophilicity and fluorinophilicity of HFE, it is miscible with fluorocarbons and hydrocarbons. When added to a hydrocarbon / fluorocarbon emulsion, the HFE can act as a co-solvent to extract hydrocarbons into the fluorocarbon phase and accelerate the hardening process.

[0070] In some embodiments, the hydrocarbon solvent, fluorocarbon, or both are removed by evaporation while stirring the emulsion, optionally under vacuum. In some embodiments, the microparticles are harvested by filtration, optionally under vacuum.

[0071] The percentage of HFE in the fluorocarbon phase can be 0-20% v / v, with increasing the percentage of HFE increasing the hydrocarbon extraction rate. However, the percentage of HFE cannot be too high because the size and morphology of the microspheres may become more difficult to control.

[0072] 3. Erodible or biodegradable polymers

[0073] In one embodiment, the polymer is a biodegradable or bioerodible polymer. In some embodiments, the polymer is selected from the group consisting of branched or linear polyethylene glycol (PEG), polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-polyglycolic acid copolymer (PLGA), poly-D,L-lactide-co-glycolide (PLGA), PLGA-fumaric acid ethylene oxide, PLGA-α-tocopheryl succinate esterified to polyethylene glycol 1000 (PLGA-TGPS), polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxybutyric acid-co-hydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly(lactic acid) copolymer (PEG-PLA), poly-ε-caprolactone (PCL), polyalkyl cyanoacrylate (PAC), poly(ethyl) cyanoacrylate (PEC), polyisobutyl cyanoacrylate, poly-N-(2-hydroxypropyl) methacrylamide (poly(HPMA)), poly-β-R-hydroxybutyric acid (PHA), polyisobutyl cyanoacrylate (PEC), polyisobutyl cyanoacrylate (PHA ... ester (PHB), poly-β-R-hydroxyalkanoate (PHA), poly-β-R-malic acid, phospholipid-cholesterol polymer, 2-dioleoyl-sn-glyceryl-3-phosphatidylcholine / polyethylene glycol-distearoylphosphatidylethanolamine (DOPC / PEG-DSPE) / cholesterol, polysaccharides, cellulose, ethylcellulose, methylcellulose, alginate, dextran and dextran hydrogel polymer, amylose, inulin, pectin and guar gum, shell Polysaccharides, chitin, heparin, hyaluronic acid, polyrotaxanes and polypseudorotaxanes based on cyclodextrin (CD), polyaspartate, polyglutamate, polyleucine, leucine-glutamate copolymers, polybutylene succinate, 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.

[0074] As used herein, the term "polymer" refers to a macromolecule comprising repeating monomers connected by covalent chemical bonds. Polymers are biocompatible and biodegradable, erodible. Biocompatible and biodegradable polymers can be natural or synthetic. Natural polymers include polynucleotides, polypeptides, such as naturally occurring proteins, recombinant proteins, gelatin, collagen, fibrin, fibroin, polyaspartate, polyglutamate, polylysine, leucine-glutamate copolymers; and polysaccharides, such as cellulose alginate, dextran and dextran hydrogel polymers, amylose, inulin, pectin and guar gum, chitosan, chitin, heparin and hyaluronic acid. The synthetic biocompatible or biodegradable polymers include polylactic acid (PLA), polyglycolic acid (PGA), polylactic acid-polyglycolic acid copolymer (PLGA), poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate, PLGA-α-tocopheryl succinate esterified to polyethylene glycol 1000 (PLGA-TGPS), polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxybutyric acid-co-hydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly(lactic acid) copolymer (PEG-PLA), poly-ε-caprolactone (PCL), polyalkyl cyanoacrylate (PAC), poly(ethyl) cyanoacrylate (PEC), polycyanoacrylate isobutyl acrylate, poly-N-(2-hydroxypropyl) methacrylamide (poly(HPMA)), poly-β-R-hydroxybutyrate (PHB), poly-β-R-hydroxyalkanoate (PHA), poly-β-R-malic acid, phospholipid-cholesterol polymer, 2-dioleoyl-sn-glyceryl-3-phosphatidylcholine / polyethylene glycol-distearoylphosphatidylethanolamine (DOPC / PEG-DSPE) / cholesterol, ethyl cellulose, cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes, polybutylene succinate (PBS), polyorthoesters, polyorthoester-polyamidine copolymers, polyorthoester-diamine copolymers, polyorthoesters incorporating latent acids to control degradation rates, especially poly(ethylene glycol) / poly(butylene terephthalate) copolymers.

[0075] Ethylcellulose (EC) is a well-known and readily available biomaterial used in pharmaceutical and food science. Ethylcellulose is a cellulose derivative in which some of the glucose hydroxyls are replaced by ether. See Martinac et al., J. Microencapsulation, 22(5):549-561 (2005) and references therein, which describe methods for using ethylcellulose as a biocompatible polymer for making microspheres. See also US 4,210,529 (1980) and references therein for a detailed description of ethylcellulose and methods for preparing ethylcellulose derivatives.

[0076] Poly-D, L-lactide-co-glycolide (PLGA) is also a well-known biocompatible and biodegradable polymer approved by the Food and Drug Administration (FDA) for tissue engineering and drug delivery systems. PLGA is a polyester comprising glycolic acid and lactic acid monomers. For a description of the synthesis of PLGA and the manufacture of PLGA nanoparticles, see Astete and Sabliov, Biomater. Sci. Polym. Ed., 17(3): 247-89 (2006) and references therein.

[0077] 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 vivo to form non-toxic or low-toxic hydroxycarboxylic acids. For a description of the manufacture of PCL, see Labet and Thielemans, Chemical Society Reviews 38:3484–3504 (2009) and references therein. For a description of the manufacture of PCL-based microspheres and nanospheres and their use as delivery systems, see Sinha et al., Int. J. Pharm., 278(1):1-23 (2004) and references therein.

[0078] Polyorthoester (POE) is a bioerodible polymer designed for drug delivery. The polyorthoester is typically a ketene acetal, preferably a cyclic diene ketene acetal, for example, a polymer of 3,9-dimethyl-2,4,8,10-tetraoxaspiro[5.5]-undecane, which is polymerized by condensation of ethylene glycol to form an orthoester bond. For example, descriptions of polyorthoester synthesis and various types can be found in US 4,304,767. Polyorthoesters can be modified to control their drug release profile and degradation rate by: swapping in or out various hydrophobic diols and polyols, such as replacing hexanetriol with decantriol; and adding latent acids to the main chain, such as glycolide, suberic acid, etc. to increase pH sensitivity. Customized forms of POE can include glycolic acid in the POE main chain to regulate mass loss and drug release. Other modifications to polyorthoesters include integrated amines to increase functionality. The formation, description, and use of polyorthoesters are described in: US 5,968,543; US 4,764,364; Heller and Barr, Biomacromolecule, 5(5): 1625-32 (2004); and Heller, Adv. Drug. Deliv. Rev., 57: 2053-62 (2005).

[0079] 4. Protein drugs

[0080] In some embodiments, the microparticle formulation produced by the disclosed anhydrous emulsion method and system comprises 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, for example, as described in U.S. Patents Nos. 7,087,411, 7,279,159 and 8144840, which are incorporated herein by reference in their entirety). In some embodiments, the VEGF Trap protein is a truncated form of VEGF Trap, as described in U.S. Patent No. 7,396,664, which is incorporated by reference in its entirety.

[0081] 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 includes a chimeric hinge. In still other embodiments, the antibody includes 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.

[0082] In some embodiments, the antibody is selected from the group consisting of: anti-programmed cell death 1 antibody (e.g., an anti-PD1 antibody as described in U.S. Pat. No. 9,987,500), anti-programmed cell death ligand-1 (e.g., an anti-PD-L1 antibody as described in U.S. Pat. No. 9,938,345), anti-D114 antibody, anti-angiopoietin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Pat. No. 9,402,898), anti-angiopoietin-like 3 antibody (e.g., an anti-AngPtl3 antibody as described in U.S. Pat. No. 9,018,356), anti-platelet-derived growth factor receptor antibody ( For example, anti-PDGFR antibodies as described in U.S. Pat. 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- 9 antibody (e.g., an anti-PCSK9 antibody as described in U.S. Pat. No. 8,062,640 or U.S. Pat. No. 9,540,449), an anti-growth and differentiation factor-8 antibody (e.g., an anti-GDF8 antibody, also known as an anti-myostatin antibody, as described in U.S. Pat. No. 8,871,209 or 9,260,515), an anti-glucagon receptor (e.g., an anti-GCGR antibody as described in U.S. Pat. No. 9,587,029 or 9,657,099), an anti-VEGF antibody, an anti-IL1R antibody, an interleukin 4 receptor antibody (e.g., as described in U.S. Patent Application Publication No. US201 No. 4 / 0271681A1 (abandoned) or U.S. Pat. No. 8,735,095 or 8,945,559), anti-interleukin 6 receptor antibody (e.g., anti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173,880), anti-IL1 antibody, anti-IL2 antibody, anti-IL3 antibody, anti-IL4 antibody, anti-IL5 antibody, anti-IL6 antibody, anti-IL7 antibody, anti-interleukin 33 (e.g., anti-IL33 antibody as described in 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. US2019 / 0031741A1), anti-cluster of differentiation 3 (e.g., anti-CD3 antibodies as described in U.S. Pat. No. 9,657,102), anti-cluster of differentiation 20 (e.g., anti-CD20 antibodies as described in U.S. Pat. No. 9,657,102 and US20150266966A1 and U.S. Pat. No. 7,879,984), anti-CD19 antibodies, anti-CD28 antibodies, anti-cluster of differentiation-48 (e.g., anti-CD48 antibodies as described in U.S. Pat. No. 9,228,014), anti-Fel d1 antibody (e.g., as described in U.S. Patent No. 9,079,948), anti-Middle East Respiratory Syndrome virus (e.g., anti-MERS antibody as described in U.S. Patent No. 9718872), anti-Ebola virus antibody (e.g., as described in U.S. Patent No. 9,771,414), anti-Zika virus antibody, anti-lymphocyte activation gene 3 antibody (e.g., anti-LAG3 antibody or anti-CD223 antibody), anti-nerve growth factor antibody (e.g., anti-NGF antibody as described in U.S. Patent Application Publication No. US2016 / 0017029 (abandoned) and U.S. Patent Nos. 8,309,088 and 9,353,176) and anti-protein Y antibody. In some embodiments, the bispecific antibody is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Pat. Nos. 9,657,102 and US20150266966A1), an anti-CD3 x anti-Mucin 16 bispecific antibody (e.g., anti-CD3 x anti-Muc16 bispecific antibody), and an anti-CD3 x anti-prostate specific membrane antigen bispecific antibody (e.g., anti-CD3 x anti-PSMA bispecific antibody). 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 pegol), cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtansinealirocumab, evinacumab, evolocumab, fasinumab, golimumab, guselkumab, ibritumomab tiuxetan), idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab umab), ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab, raxibacumab, reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab,tocilizumab, tocilizumab, trastuzumab, trevogrumab, ustekinumab, and vedolizumab.

[0083] In some embodiments, the protein in the complex is a recombinant protein (e.g., an Fc fusion protein) containing an Fc portion and another domain. In some embodiments, the Fc fusion protein is a receptor Fc fusion protein containing one or more extracellular domains of a receptor coupled to the Fc portion. In some embodiments, the Fc portion includes the hinge region of IgG and subsequent CH2 and CH3 domains. In some embodiments, the receptor Fc fusion protein contains two or more different receptor chains that bind to a single ligand or multiple ligands. For example, the Fc fusion protein is a TRAP protein, such as IL-1trap (e.g., rilonacept, which contains the IL-1RAcP ligand binding region fused to the Il-1R1 extracellular region fused to the Fc of hIgG1; see U.S. Pat. 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 a ScFv-Fc fusion protein, which contains one or more of the one or more antigen binding domains of an antibody (such as a variable heavy chain fragment and a variable light chain fragment) coupled to the Fc portion.

[0084] In certain embodiments, the initial protein is in the form of a dry powder, such as a micronized dry powder. In certain embodiments, the protein is a spray-dried powder (SDP). Using spray-dried protein instead of a protein solution has the advantages of higher protein loading in microparticles and better protein stability in the encapsulation process. In certain embodiments, the dry protein molecule remains solid during the entire encapsulation process and storage conditions and is surrounded by a stabilizer. In certain embodiments, the spray-dried protein of the encapsulation shows high recovery and low aggregates, which may be due to minimized surface interactions, because only a small portion of surface proteins are exposed to the interface. In certain embodiments, the protein is micronized before encapsulation.

[0085] B. Microparticles

[0086] One embodiment provides a pharmaceutical composition produced using the disclosed non-aqueous emulsion system. In some embodiments, the pharmaceutical composition contains microparticles having a polymer cortex and a micronized protein core. In some embodiments, the microparticles are approximately spherical in shape. Some microparticles and protein cores will be nearly spherical, while other microparticles and protein cores will be more irregular in shape. Thus, as used herein, the term "diameter" means each and any of the following: (a) the diameter of a sphere circumscribing a microparticle or protein core; (b) the diameter of the largest sphere that fits within the confines of a microparticle or protein core; (c) any measurement between the circumscribing sphere of (a) and the confining sphere of (b), including the average between the two; (d) the length of the longest axis of a microparticle or protein core; (e) the length of the shortest axis of a microparticle or protein core; (f) any measurement between the major axis length (d) and the minor axis length (e), including the average between the two, and / or (g) an equivalent circular diameter ("ECD") determined by microfluidics imaging (MFI), nanoparticle tracking analysis (NTA), or by a light scattering method such as static light scattering (SLS), dynamic light scattering (DLS), or laser diffraction analysis, etc. Diameters are typically expressed in micrometers (μm or micrometers). Diameters can be determined by optical measurements or scanning electron microscopy measurements.

[0087] Microparticles produced by the disclosed non-aqueous emulsion process have a plurality of protein molecules with low, very low or near zero amounts of water (e.g., <3 wt % water). As used herein, micronized protein particles have an ECD in the range of 2 microns to about 35 microns, or 2.0 μm to 50 μm, or 5.0 μm to 15.0 μm, or about 10 μm. The micronized protein particles are not limited to any particular protein entity, and are suitable for the preparation and delivery of therapeutic proteins comprising the proteins described above.

[0088] For example, protein particles can be micronized by spray drying, freeze drying and grinding, jet milling, reversible precipitation in a non-solvent, granulation, gradual 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" means a method of producing a dry powder including micron-sized particles from a slurry or suspension by using a spray dryer. The spray dryer uses an atomizer or nozzle to disperse the suspension or slurry into a controlled droplet size spray. Droplet sizes of 10 μm to 500 μm can be produced by spray drying. As the solvent (water or organic solvent) dries, the protein material dries into micron-sized particles, thereby forming a powdery material; or in the case of a protein-polymer suspension, during drying, the polymer hardens the shell around the protein load.

[0089] In some embodiments, the micronized protein is a VEGF Trap protein. The pharmaceutical formulation used to form the micronized VEGF Trap protein particles can contain about 10 mg / mL to about 100 mg / mL VEGF Trap protein, about 1.0 mg / mL to about 50 mg / mL protein, about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 55 mg / mL, about 60 mg / mL, about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 85 mg / mL, about 90 mg / mL, about 95 mg / mL, or about 100 mg / mL VEGF Trap protein.

[0090] In some embodiments, microparticles produced using the disclosed non-aqueous emulsion systems contain a protein particle core within a polymer cortex having a diameter 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 μm to 15 μm, about 20 μm, about 25 μm, or about 30 μm. The size variation largely reflects the thickness of the polymer cortex, although the diameter of the protein core may account for the size variation to some extent.

[0091] 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 pharmaceutically acceptable excipient, such as pH buffered saline. The flowable microparticle compositions can be administered parenterally, for example, using a syringe such as a syringe with a 27G needle.

[0092] Microparticles can be used for delayed release or sustained release of protein therapeutics. In some embodiments, microsphere formulations are injected intravitreally, suprachoroidally or subcutaneously. For example, it is envisioned that VEGF Trap microparticles can be used to release VEGF Trap therapeutic proteins in, for example, the vitreous body for the treatment of vascular ophthalmic disorders, or can be used for subcutaneous implantation to release VEGF Trap to treat other disorders.

[0093] The microparticles of the invention release protein at a relatively constant rate in a physiological aqueous environment at about 37°C over an extended period of time up to at least 60 days, 90 days, 120 days or 150 days.

[0094] One embodiment provides a microsphere composition produced using the non-aqueous emulsion method disclosed herein, wherein the composition contains>100 mg of spray-dried protein. In one embodiment, the non-aqueous emulsion method has a yield of>90% and produces microparticles with>99% purity and>10% w / w loading and <10% burst for a 50-100 μL injection volume.

[0095] Examples

[0096] Example 1: Blank microspheres were synthesized by H / F based bulk emulsion.

[0097] Materials and methods

[0098] Emulsion systems based on oil and water are often used for polymer microparticles or nanoparticle synthesis, wherein the hydrophobic polymer material is dissolved in the organic phase and dispersed in the aqueous continuous phase. However, for water-soluble polymers, for example, PEG, carboxymethyl cellulose (CMC) and polymers that are easily hydrolyzed in the presence of water, including polyanhydrides, aliphatic polyesters (such as polylactic acid) with short midblocks and some poly (amino acids) (such as poly (glutamic acid)), conventional emulsion systems based on water are not ideal. The following examples demonstrate the effectiveness of the disclosed H / F emulsion system for producing the above-mentioned water-soluble or water-degradable polymer microparticles. In certain embodiments, first those polymers are dissolved in a hydrocarbon solvent, and the hydrocarbon solvent comprises a polar solvent, such as acetonitrile, tetrahydrofuran, and a weakly polar solvent, such as DCM, chloroform. Then this polymer solution is added to a continuous phase, i.e. a fluorocarbon liquid, such as in the FC-40 with FS (e.g., Picosurf 1). Prepare the emulsion by stirring, vortexing or other emulsification methods. The emulsion droplets eventually harden into polymer spheres by evaporation or extraction of the hydrocarbon solvent.

[0099] In a specific embodiment, for blank POE microspheres synthesized by H / F bulk emulsion, as in Scheme 1 ( Figure 1A ) showed that 200 μL of DCM containing about 10% w / v, 20% w / v, 30% w / v and 40% w / v POE was added to a 0.5% w / w FS Pico-Surf TM 1 (Sphere Fluidics) in 2mL FC-40. Emulsification is achieved by vortexing. The emulsion droplets are lighter than FC-40 and float on the top of the solution. Take an aliquot and drop it on a slide for microscope imaging. Stirring under vacuum for 3 hours makes the microspheres harden. First, the hardened polymer spheres in FC-40 are vacuum filtered through a 0.22 micron PES membrane. FC-40 passes through the filter and the microspheres are retained. Then the microspheres are washed with additional FC-40 and the microspheres are completely dried under vacuum. In another example with the same process, DCM containing about 30% w / v POE is used in the hydrocarbon phase, and FC40 containing about 0.01%, 0.1% and 0.5% FS is used in the fluorocarbon phase to evaluate the impact of FS concentration.

[0100] result

[0101] In the presence of FS, a mixture of hydrocarbons and fluorocarbons can form H / F emulsions. In one example, DCM is dispersed in FC-40 (see Figure 1B ) as H / F emulsion, and PFPE-PEG-PFPE was used as FS (see Figure 1CStructure of FS in FC-40). Increasing concentrations of FS were added to the FC-40 fluorocarbon phase. Testing showed that 0.1-5% w / w FS was required to prevent coalescence of the DCM droplets (Figure 2A). If less than 0.1% w / w SF was added, a wider size distribution was observed. If no SF was used, the DCM droplets were unstable. The dispersed DCM droplets would quickly merge together and the two phases would separate quickly. The results showed that a sufficient amount of FS must be used to produce a stable H / F emulsion and that stirring must be continued during the hardening process to successfully produce polymer microspheres. (Figure 2B).

[0102] DCM containing POE is added and vortexed in FC-40 to form droplets containing POE. DCM is evaporated in an open container under ambient conditions or under vacuum to harden the droplets into POE microspheres (Figures 2A and 2B). The size of the microspheres is related to the droplet size and the POE content in the organic phase. Higher POE concentrations produce larger microsphere sizes (Table 1).

[0103] Table 1: Microsphere size of POE spheres produced using DCM containing different concentrations of POE.

[0104] diameter 10%w / v POE 20%w / v POE 30%w / v POE 40%w / v POE Dv(10)(μm) 0.9 1.3 3.1 7.1 Dv(50)(μm) 2.7 7.2 17 34.8 Dv(90)(μm) 6.5 13.4 30.1 67.4

[0105] Example 2: Effect of homogenization speed.

[0106] Materials and methods

[0107] One (1) mL of DCM containing 30% w / v or 40% w / v POE was added to 9 mL of FC-40 with 0.5% (w / w) FS FC-40 and emulsified with a VWR handheld homogenizer 200 with a VWR 7 mm×95 mm sawtooth generator probe at one of three homogenization speeds, i.e., low (about 50% of full power), medium (about 60% of full power), and high (about 70% of full power). The formed emulsion was stirred under vacuum. The formed microspheres were washed and dried under vacuum.

[0108] result

[0109] As shown in Figure 3, for 30% POE, low homogenization speed produced larger microsphere size, while high homogenization speed produced smaller size (Table 2). 40% POE showed the same trend. These results show that adjusting the homogenization speed can control the microsphere size.

[0110] Table 2: Microsphere size of POE spheres produced at different homogenization speeds.

[0111] diameter Low speed Medium high speed Dv(10)(μm) 2.8 2.0 1.1 Dv(50)(μm) 16.1 13.5 5.4 Dv(90)(μm) 31.5 31.6 12.0

[0112] Example 3: General procedure for encapsulation of protein SDP in POE microspheres via S / H / F-based bulk emulsion method.

[0113] Materials and methods

[0114] As in Figure 4 As shown in the previous article, bulk emulsion synthesis can be divided into three steps: preparation, emulsification, and hardening. Due to the different parameters used in these three steps, the properties of the products will be different. The general procedure is described as follows:

[0115] For formulation, 10%-30% w / w of total solid weight of VEGF Trap SDP (or fluorescently labeled SDP (F-SDP) for fluorescent imaging) was dispersed in 500 μL of ethyl acetate containing 10-35% w / v POE by vortexing and then sonicating for 5 minutes. These suspensions were then added to 9.5 mL of FC-40 with 0.1-0.5% w / w FS. Emulsification can be achieved by stirring, vortexing or homogenizing 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 hardened on the slide by evaporation under ambient conditions. For hardening the microspheres, one of the following three methods was applied: (a) stirring the solution overnight under ambient conditions in an open container and allowing the ethyl acetate to evaporate; (b) stirring the solution under vacuum for at least 2 hours to accelerate solvent evaporation; (c) adding NOVEC 7500 or a mixture of FC-40 and NOVEC 7500 to the emulsion under stirring. HFE acts as a co-solvent to help extract ethyl acetate from the hydrocarbon phase into the fluorocarbon phase and achieves a rapid hardening process (usually within a few minutes).

[0116] Finally, the hardened 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 completely dried under vacuum.

[0117] The size of the microspheres was measured by laser diffraction analysis using a Malvern Mastersizer 3000 and by liquid sampling by dispersing the product powder in a 0.01% w / v PVA solution. The morphology of the product was measured using scanning electron microscopy (SEM).

[0118] To measure the protein content of the microspheres, a predetermined amount of microspheres was first dissolved in 200 μL of ethyl acetate and then extracted with 1% pure water, the aqueous phase was collected and centrifuged to remove the turbid suspension. Protein purity and concentration were measured by SEC-UPLC.

[0119] To measure burst release, a predetermined amount of microspheres was incubated in 1 mL of PBS for 1 hour at 37° C. The mixture was centrifuged and the supernatant was subjected to SEC-UPLC to obtain protein concentration.

[0120] result

[0121] The above results show that a stable H / F emulsion is formed in the presence of sufficient SF. This non-aqueous emulsion can successfully produce blank POE spheres. This anhydrous method is used again to incorporate SDP into POE microspheres. In one example, 10% w / w total solid weight of VEGF Trap F-SDP is introduced into ethyl acetate (containing 20% ​​w / v POE) as a suspension, and this suspension in FC-40 (containing 0.5% w / w FS) is emulsified by stirring and vortexing. Immediately after emulsification, an aliquot is transferred to a slide for microscopy imaging. As shown in Figures 6A and 6B, ethyl acetate is dispersed in droplets in FC-40, and SDP particles are clearly confined within the ethyl acetate droplets. In contrast to the S / O / W system (data not shown), there is no sign of protein leakage into the fluorocarbon continuous phase. Importantly, in this H / F system, the SDP particles in the droplets retain their original pit shape in the powder state. Since there is no water in the H / F system to reconstitute the SDP, the SDP retains its original solid particle form. After hardening, POE microspheres containing single or multiple SDP particles can be clearly observed by bright field and fluorescence microscopy images (Figures 7A, 7B, and 7C). After evaporating the hydrocarbon and fluorocarbon solvents on a glass slide, water was added to test the burst release and encapsulation quality. As shown in Figure 8A-D It is shown in Figure 2 that the SDP-encapsulated POE microspheres retain their integrity after the microsphere product is placed in water. No immediate release of the protein is observed, and the shape of the SDP particles remains unchanged, indicating that the SDP particles are well protected by the polymer matrix and isolated from the aqueous environment. These results show that H / F emulsions are effective solutions for encapsulating proteins and other hydrophilic drugs into polymer matrices and have the potential to achieve high encapsulation efficiency, high yield, while minimizing burst release - all of which are major challenges when using water-based W / O / W or S / O / W methods.

[0122] The procedure disclosed herein is an example of using a S / H / F non-aqueous based bulk emulsion method for encapsulating protein SDP in POE microspheres. The method is repeatable, scalable, and adjustable. By changing the parameters in the formulation and process, the product properties can be adjusted and controlled. The effects of some of those parameters are disclosed in Example 4.

[0123] Example 4: Effect of hydrocarbon solvents

[0124] Materials and methods

[0125] Microparticles were produced using dichloromethane or ethyl acetate as hydrocarbons as described in Example 2. DCM containing 35% w / v POE and ethyl acetate containing 35% w / v POE were prepared. Ten percent (10%) w / w total solid weight of protein powder was suspended in 0.5 mL of a solution of POE 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 produce an emulsion and stirred under house vacuum for 1.5 hours. The formed microspheres were isolated by filtration, washed with FC-40, and dried under vacuum.

[0126] result

[0127] Fig. 9 The size distribution of microparticles produced using the same formulation and process conditions is shown, except for the type of hydrocarbon solvent (dichloromethane or ethyl acetate). Microparticles produced using either hydrocarbon show encapsulation of the spray-dried protein. Using dichloromethane produces larger microparticles. See Table 2 below. The results show that under the same formulation and process conditions, using different hydrocarbon solvents will result in different microsphere sizes. The size of the microspheres produced by DCM is larger than the size of the microspheres produced by ethyl acetate. Therefore, the hydrocarbon solvent can be intentionally selected to control the size of the microspheres.

[0128] Table 3: Particle size of SDP loaded POE spheres produced with DCM or ethyl acetate.

[0129] diameter DCM EtA Dv(10)(μm) 5.2 3.2 Dv(50)(μm) 30.7 21.2 Dv(90)(μm) 64.9 42.3

[0130] Example 5: Effect of protein loading

[0131] Materials and methods

[0132] Microparticles were prepared as described in Example 2 with only a change in protein loading. DCM containing thirty-five percent (35%) w / v POE was prepared. Protein powders of 5% w / w, 10% w / w and 30% w / w total solid weight were suspended in a solution of 0.5 mL of POE 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 produce an emulsion, and then a 1:1 v:v mixture of 6 mL of Novec7500 and FC-40 was added to the emulsion within one minute. Then after stirring for another minute, the formed microspheres were separated by filtration, washed with FC-40, and dried under vacuum.

[0133] result

[0134] As shown in Table 3, increasing the amount of protein powder in the formulation produced larger POE microparticle size as measured by laser diffraction analysis, and the protein loading in the resulting final POE microsphere product also increased as observed by protein extraction experiments, bright field and confocal fluorescence microscopy. The bright field image of the 30% w / w protein powder loading showed darker and less transparent microspheres than the 10% w / w protein powder, indicating that more drug was encapsulated in the microsphere product ( Figures 10A and 10B Representative confocal images confirmed that SDP was encapsulated in the POE matrix in its original form from the cross-sectional view of the microspheres ( Figures 11A and 11B ). More SDP particles were observed in the 30% w / w loaded microspheres. Likewise, the encapsulated SDP retained its original pit shape, indicating that it was intact during the entire manufacturing process. Additionally, SEM images demonstrated that increasing the protein powder loading resulted in more protein particles being adsorbed to the POE microparticle surface (Figures 12A-12C). Therefore, the results demonstrated that up to 30% w / w protein powder can be effectively encapsulated in microspheres. Due to the lack of physical space in the microspheres in this formulation, protein particles may be adsorbed to the surface of the microspheres in the case of >30% w / w protein powder loading. If such effects are desired for therapeutic efficacy, surface-adsorbed proteins may produce burst release of the drug when in contact with water. Proteins will not be lost to the continuous phase as expected in a water-based emulsion system.

[0135] Table 4: Particle size of SDP loaded POE spheres produced at different SDP loadings.

[0136] diameter 5％ 10％ 30％ Dv(10)(μm) 4.9 5.2 17.1 Dv(50)(μm) 20.6 30.7 40.7 Dv(90)(μm) 43.0 64.9 81.3

[0137] Example 6: Design of Experiments (DOE) for encapsulation of SDP into POE microspheres using H / F bulk emulsification.

[0138] Materials and methods

[0139] A DOE study was conducted to evaluate the impact of the key factors of synthesis in the design space on the properties of the final product. Ten runs were performed in the designed experiment according to the general procedure described in Example 2. Protein powder loading, protein powder particle size (Dv (50) size was 2.2um and 5.6um, see SEM images in Figure 13), polymer concentration and HFE concentration were changed, while the following formulations and process conditions remained unchanged, for example, the volume of hydrocarbon and fluorocarbon phases, homogenization speed, FS concentration (Table 4). The measured responses included microsphere size (Dv50, laser diffraction span), encapsulation efficiency, burst release at 37°C for 1 hour, and SEM images.

[0140] result

[0141] The results of the DOE are summarized in Table 5.

[0142] Table 5: Experimental design and measured responses for SDP encapsulation DOE study.

[0143]

[0144] *Microspheres were dissolved in ethyl acetate and proteins were extracted using water and quantified using SEC-UPLC **Microspheres were incubated in PBS at 37°C for 1 hour. Released proteins were quantified using SEC-UPLC.

[0145] A custom designed DOE fit on microsphere size (where R 2 =0.76) revealed the main effects of protein powder loading and POE concentration (where p value < 0.05, see the relevant results in Table 6). In addition, the fitting of the burst release (R 2 =0.92) showed that only protein powder loading significantly affected burst release (where p value <0.05, see related results in Table 7). The results indicate that increasing the amount of protein powder in the formulation will result in a higher effective load in the final product, but will also increase the burst release percentage. The burst release may be caused by surface adsorbed protein particles. The maximum amount of protein powder internalized in the polymer microspheres is determined by the physical space of a given microsphere size. Simply increasing the protein powder concentration in the formulation suspension will not increase drug encapsulation beyond a certain threshold, which in this example is about 30% w / w.

[0146] Table 6: Correlation of factors with microsphere size.

[0147] project Valuation Standard error t ratio Prob>|t| VIF intercept 23.03 0.924421 24.91 <.0001* . SDP load (%) (5,25) 3.4875 1.033534 3.37 0.0118* 1 [Polymer] (%; w / v) (25,35) 2.99 0.924421 3.23 0.0144* 1

[0148] Table 7: Positive correlation of SDP loading and burst release.

[0149] project Valuation Standard error t ratio Prob>|t| VIF intercept 63.190484 4.008836 15.76 <.0001* . SDP load (%) (5,25) 43.17019 4.482105 9.63 <.0001* 1

[0150] Example 7: Application of S / H / F emulsion-based encapsulation method in different proteins.

[0151] The disclosed H / F-based emulsion system and process can be a platform technology suitable for different polymers and therapeutic proteins. In a specific example of the present invention, protein powder (MW~145kDa) of recombinant IgG4, protein powder (MW~146kDa) of recombinant IgG1 or protein powder (MW~64kDa) of recombinant fusion protein are respectively encapsulated into POE microspheres by the same process as in Example 2. The results are summarized in Table 7. The amount of protein powder encapsulated in the microsphere product is determined by extraction assay and the amount is matched to the target value. After the encapsulation process, the protein purity of the recombinant fusion protein IgG1 remains unchanged or the protein purity of IgG4 decreases slightly (less than 2%), indicating good process compatibility.

[0152] Table 7: SDP results of encapsulating different types of proteins in POE microspheres by S / H / F emulsion.

[0153]

[0154] *Microspheres were dissolved in ethyl acetate and proteins were extracted using water and quantified using SEC-UPLC **Microspheres were incubated in PBS at 37°C for 1 hour. Released proteins were quantified using SEC-UPLC.

[0155] 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 end-capped, Mw 18,000-28,000, Sigma-Aldrich) microspheres, respectively, by a similar process disclosed in Example 2. Bright field and fluorescence microscopy images indicated that the protein powder was successfully encapsulated in the polymer microspheres ( Fig.14 AC targets PLA, and Fig.15 AB for PLGA).

[0156] Although the present invention has been described in the foregoing specification with respect to certain embodiments of the invention and many details have been listed for the purpose of illustration, it will be apparent to those skilled in the art that the present invention is susceptible to other embodiments and that certain details described herein may be significantly modified without departing from the basic principles of the invention.

[0157] All references cited herein are incorporated by reference in their entirety.The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof, and, accordingly, reference should be made to the appended claims rather than to the foregoing specification as indicating the scope of the invention.

Claims

1. A method for producing polymer microparticles or polymer-coated microparticles, the method comprising: combining a protein powder and a polymer in a hydrocarbon solvent to form a non-aqueous first solution; adding the first solution to a second solution, wherein the second solution comprises a fluorocarbon liquid and a fluorosurfactant; agitating the combined solutions to form a non-aqueous emulsion comprising a plurality of emulsion hydrocarbon droplets in the fluorocarbon liquid; removing the hydrocarbon solvent; as well as The fluorocarbon liquid is removed to isolate the microparticles, wherein the microparticles include a protein encapsulated within a polymer matrix.

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

3. The method of claim 1, wherein the at least one microparticle comprises a plurality of cores dispersed within the polymer.

4. The method according to any one of claims 1 to 3, wherein the microparticles include microparticles comprising a combination of a single core structure encapsulated by a polymer and microparticles comprising a multi-core structure encapsulated by a polymer.

5. The method of any one of claims 1 to 4, wherein the fluorocarbon liquid comprises a perfluorinated C5-C18 compound.

6. The method according to any one of claims 1 to 5, wherein the hydrocarbon solution is selected from the group consisting of dichloromethane, chloroform, toluene, ethyl acetate, tetrahydrofuran or a combination thereof.

7. The method according to any one of claims 1 to 6, wherein the fluorocarbon solution comprises Flourinert TM 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.

8. The method of any one of claims 1, wherein the hydrocarbon solvent comprises dichloromethane, ethyl acetate, or a combination thereof.

9. The method of any one of claims 1 to 8, wherein the fluorocarbon solution comprises a hydrofluoroether.

10. The method according to any one of claims 1 to 9, wherein the fluorosurfactant comprises perfluoropolyether-b-polyethylene glycol-b-perfluoropolyether.

11. The method of any one of claims 1 to 10, wherein the polymer comprises a polyorthoester (POE).

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

13. The method of any one of claims 1 to 12, wherein the protein is an antibody or antigen-binding fragment thereof, a fusion protein, a recombinant protein or a fragment or truncated version thereof.

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

15. The method of claim 14, wherein the protein is a truncated form of the VEGF Trap protein.

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

17. The method according to any one of claims 1 to 16, wherein the protein powder comprises particles comprising micronized protein having a diameter of 0.5 μm to 20 μm.

18. The method of any one of claims 1 to 17, wherein the protein powder is micronized by spray drying, electrospray drying, reversible precipitation, spray freezing, microtemplating, or a combination thereof.

19. The method of any one of claims 1 to 18, wherein the emulsion is formed using homogenization, vortexing, sonication, cavitation, stirring, or a combination thereof.

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

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

22. The process of any one of claims 1 to 21, wherein the hydrocarbon solvent is removed by evaporation.

23. The method of any one of claims 1 to 22, wherein the fluorocarbon liquid is removed by filtration, optionally under vacuum.

24. A process according to any one of claims 22 to 23, wherein a hydrofluoroether is used as a co-solvent to extract the hydrocarbon.

25. A microparticle produced by a method according to any one of claims 1 to 23, wherein the microparticle is a sustained release microparticle.

26. A sustained release composition comprising the microparticles according to claim 25.

27. The microparticle of any one of claims 1 to 26, wherein the microparticle has few or no pores or channels in the polymer cortex.

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

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

30. The method of claim 29, wherein the hardened polymer microspheres or polymer-coated microspheres are harvested by vacuum filtration.

31. A method for producing polymer microparticles or polymer-coated microparticles, the method comprising: combining a hydrocarbon solution including a dissolved polymer with a spray dried protein powder to produce a dispersed phase; The dispersed phase is combined with a continuous phase to produce emulsion droplets of the dispersed phase in the continuous phase, wherein The continuous phase comprises a fluorocarbon liquid and 0.1% w / v to 5.0% w / v of a fluorinated surfactant; as well as The polymer-coated microparticles are harvested.

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

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

34. The method of claim 31, wherein the hydrocarbon solution is selected from the group consisting of dichloromethane, chloroform, toluene, ethyl acetate, tetrahydrofuran, or combinations thereof.

35. The method of any one of claims 31 to 34, wherein the fluorocarbon liquid comprises trifluoromethyl)bis(1,1,2,2,3,3,4,4,4-nonafluorobutyl)amine.

36. A method according to any one of claims 31 to 35, wherein the microparticles are hardened by removing the hydrocarbon solution by evaporation or under vacuum while stirring.

37. The method of claim 36, further comprising harvesting the hardened microparticles.

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

39. A pharmaceutical composition comprising the microparticles according to claim 38.

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

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

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

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

44. The method of claim 9, wherein the hydrofluoroether comprises 4-ethoxy-1,1,1,2,2,3,3,4,5,6,6,6-eicosylfluoro-5-(trifluoromethyl)hexane.

45. A method for producing microparticles, the method comprising: combining a first solution comprising a polymer in a hydrocarbon solvent with a second solution comprising a fluorocarbon solvent and a fluorosurfactant; stirring the combined solutions to produce an emulsion; removing the hydrocarbon solvent under vacuum while stirring the combined solutions to harden the microparticles; harvesting the microparticles; optionally washing the microparticles; and drying the microparticles.

46. ​​The method of claim 45, wherein the hydrocarbon solvent is selected from the group consisting of dichloromethane, chloroform, toluene, ethyl acetate, tetrahydrofuran, acetonitrile, ethanol, methanol, propanol, dimethylformamide, dimethyl sulfoxide, or combinations thereof.

47. The method of any one of claims 45 or 46, wherein the fluorocarbon solvent comprises trifluoromethyl)bis(1,1,2,2,3,3,4,4,4-nonafluorobutyl)amine.

48. The method of any one of claims 45 to 47, wherein the polymer comprises POE, polylactic acid, poly(lactic-co-glycolic acid), or a combination thereof.

49. The method of any one of claims 45 to 49, wherein the microparticles comprise a polymeric skin and a hollow core.

50. The method of any one of claims 49, wherein the microparticles have few or no pores or channels in the polymeric cortex.

51. The method of any one of claims 50 to 51, wherein the diameter of the microparticles is adjusted to a desired diameter by varying the hydrocarbon solvent, stirring speed, polymer concentration, or a combination thereof.

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

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