The invention relates to the manufacture, composition, and to an extended release protein therapeutic. Specifically, the invention relates to the manufacture, composition, and use of a plurality of polymer coated protein microspheres for the extended and uniform release of protein in an aqueous-based or physiological environment over time.

The extended release of a therapeutic protein administered toward a biological target, such as e.g., the retina or a tumor, or administered parenterally is desirable for the treatment of many different conditions, including cancers, cardiovascular diseases, vascular conditions, orthopedic disorders, dental disorders, wounds, autoimmune diseases, gastrointestinal disorders, and ocular diseases. Biocompatible and biodegradable polymers for the controlled and extended delivery of drugs have been in use for decades. As the polymer degrades over time, the therapeutic drug is slowly released.

In the case of intraocular therapeutics, there is a significant unmet medical need for extended release formulations to deliver protein therapeutics effectively over time with as few intraocular injections as possible. In the case of other diseases, such as cancer, diseases of inflammation, and other diseases, there is a need for improved implantable extended release formulations containing protein therapeutics.

Applicants have discovered and herein disclose and claim methods of manufacturing and using microparticles containing a biodegradable polymer and a therapeutic protein, which is capable of releasing a therapeutically effective amount of the therapeutic protein uniformly over an extended period of time.

The following documents are also referred to:

• US2011/0104151 A1 which is concerned with microparticle compositions and methods for treating age-related macular degeneration.
• US2008/0305115 A1 which is concerned with reduced-mass, long-acting dosage forms.
• WO03/092665 A2 which is concerned with ocular drug delivery systems and uses thereof.
• KIM B S ET AL,BIOMATERIALS, vol. 30 (5), 2009, p. 902-909 which is concerned with Bovine Serum Albumin-loaded PLGA microcapsules prepared using a mono axial nozzle ultrasonic atomiser, for use in in vivo delivery.

In one aspect, the invention provides a method of manufacturing a microparticle, which comprises a protein core and a polymer cortex. Hence, the present invention provides a method of manufacturing an extended release pharmaceutical composition comprising a protein particle coated with a biodegradable polymer, the method comprising the steps of:

1. (a) subjecting a solution of the therapeutic protein to dispersion and drying by spray-drying to form micronized protein particles, wherein the inlet temperature of the spray dryer is set at a temperature above the boiling point of water, and the outlet temperature is set at a temperature below the boiling point of water and above ambient temperature, and wherein the solution of the therapeutic protein is pumped into the spray dryer at a rate of 2 mL/min to 15 mL/min;
2. (b) suspending the micronized protein particles in an organic solution comprising a biodegradable polymer and an organic solvent and
   , wherein the polymer is polyorthoester (POE) or ethyl cellulose (EC)
3. (c) spray-drying the suspension of (b) to form a population of protein polymer microparticles,

In one embodiment, the manufactured microparticle has a diameter of about two microns to about 70 microns, or a median diameter of about 15 microns to about 30 microns.

The protein particle of step (a) is a micronized protein particle, which is obtained by spray drying a solution comprising the protein. In some embodiments, the protein solution is spray dried via dual-nozzle sonication, single-nozzle sonication, or electrospray. In some embodiments, the resultant micronized protein particle, which forms the core of the manufactured microparticle, has a diameter of from about two microns to about 30 microns, with a median diameter of about 10 microns to about 12 microns.

In one embodiment, the solvent is removed at step (c) by creating a dispersion of the protein-polymer-solvent mixture of step (b) and allowing the solvent to evaporate from the droplets created by the dispersion. The dispersion is created by spray-drying, which may be performed by dual-nozzle sonication, single-nozzle sonication, or electrospray. In one embodiment, the solvent is removed from the droplets by applying heat or air, or by chemical extraction. In one embodiment, the polymer is ethyl cellulose In one embodiment, the polymer is polyorthoester.

In one aspect, the method provided of manufacturing a microparticle comprises the steps of (1) forming a micronized protein particle having a diameter of from about two microns to about 30 microns, with a median diameter of from about 10 microns to 12 microns, by spray-drying a solution containing a protein, wherein the protein is an antigen-binding protein. In some embodiments, the VEGF-Trap protein has the sequence of SEQ ID NO:1; (2) suspending the micronized protein particle in a solution comprising the polymer and a solvent, wherein the polymer is as recited in the claims; and (3) removing the solvent by spray-drying micronized protein particle-polymer-solvent suspension and driving off the solvent by applying heat or air, or by extracting the solvent, wherein a microparticle is formed having a diameter of about two microns to about 70 microns, with a median diameter of from about 15 microns to about 30 microns, and comprising a protein core and a polymer cortex.

In some embodiments, the spray-drying of step (1) or step (3) is performed via dual-nozzle sonication, single-nozzle sonication, or electrospray.

In one embodiment, the method of manufacturing the microparticle comprises the steps of (1) forming a micronized VEGF-Trap particle having a diameter of from about 10 microns to 12 microns by spray-drying a solution containing a VEGF Trap protein; (2) suspending the micronized VEGF Trap particle in a solution comprising polyorthoester incorporating a latent acid and a compatible solvent, or ehtylcellulose and a compatible solvent; and (3) removing the solvent by (a) spray-drying the micronized VEGF Trap particle-polyorthoester-latent acid-solvent suspension or the micronized VEGF Trap particle-ethyl cellulose-solvent suspension and (b) driving off the solvent by applying heat or air, or by extracting the solvent, wherein a microparticle is formed having a diameter of a bout 15 microns to about 30 microns, and comprising a VEGF-Trap core and a polymer cortex of polyorthoester, and copolymers or derivatives thereof.

Also disclosed is a microparticle comprising a protein coated with a polymer. In one instance, the microparticle has a diameter of from about 2 microns to about 70 microns. In one instance, the microparticle has a diameter of about 15 microns.

In one instance, the VEGF-Trap protein comprises an amino acid sequence set forth in SEQ ID NO:1.

The polymer is a biodegradable polymer. In one embodiment, the polymer is ethyl cellulose. In one instance, the polymer is polyorthoester.

In one instance, the microparticle comprises a micronized protein core of less that ten microns and a polymer cortex. In one instance, the micronized protein core is at least 50% coated with polymer, which means that no more than 50% of the surface of the micronized protein core is exposed. In one instance, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the surface of the micronized protein core is coated with polymer.

In one instance, the microparticle of greater than 10 microns in size comprises (a) a micronized protein core of less that 10 microns, wherein the protein is a trap-type VEGF-Trap; and (b) a polymer coat, whrein the polymer is as recited in the claims.

In one instance, the microparticle of an average diameter of about 15 microns to about 30 microns comprises (a) a micronized protein core of about 10 to about 12 microns, wherein the protein is a VEGF-Trap protein; and (b) a polymer coat, wherein the polymer is ethyl cellulose or polyorthoester.

Also disclosed is a plurality of microparticles, which range in size from about two microns to about 70 microns, and which comprise a micronized protein core of about two microns to about 30 microns, and a polymer cortex.

In a specific instance, the VEGF-Trap protein comprises the amino acid sequence set forth in SEQ ID NO:1.

In one instance, the polymer is a biocompatible polymer. In one embodiment, the polymer is a bioerodible polymer. In one instance, the polymer is a biodegradable polymer. In one instance, the polymer is ethyl cellulose. In one instance, the polymer is a polyorthoester.

In one instance, the micronized protein core of most microparticles of the plurality of microparticles is at least 50% coated with polymer, which means that no more than 50% of the surface of the micronized protein core is exposed. In one instance, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or 100% of the surface of the micronized protein core is coated with polymer.

In one instance, the plurality of microparticles, which range in size from about two microns to about 70 microns, comprise (a) a micronized protein core of from about two microns to about 30 microns, wherein the protein is a VEGF-Trap protein; and (b) a polymer cortex, wherein the polymer is as recited in the claims.

In one instance, the plurality of microparticles, which range in size from about two microns to about 70 microns, with a median size of from about 15 microns to about 30 microns, comprise (a) a micronized protein core of from about two microns to about 30 microns, with a median size of about 10 microns to about 12 microns, wherein the protein is a VEGF-Trap protein; and (b) a polymer cortex, wherein the polymer is ethyl cellulose or polyorthoester.

Also disclosed is an extended release formulation of a therapeutic protein for the release or delivery of a steady level of the therapeutic protein over time. The extended release formulation comprises a plurality of microparticles, which range in size from about two microns to about 70 microns, each of which comprises a micronized protein core of about two microns to about 30 microns, and a polymer cortex.

In one instance, the VEGF-Trap protein has a primary structure of SEQ ID NO:1. The therapeutic protein comprises an Fc domain.

The polymer cortex comprises a biodegradable polymer. The polymer cortex comprises a polymer selected from ethyl cellulose, and polyorthoester,

In one embodiment, plurality of microparticles comprises a collection of microparticles having a range of thicknesses of the polymercortex, such that individual microparticles of the collection of microparticles degrades at a different rate, which allows for a uniform rate of release of the therapeutic protein.

In one embodiment, the plurality of microparticles comprises a mixture of uncoated micronized protein particles and microparticles having a range of thicknesses of the polymer cortex, which allows for the release of therapeutic protein at periodic intervals based on cortex thickness.

In one embodiment, the plurality of microparticles comprises a mixture of microparticles having polymercortices of varying levels of hydrophobicity to control the timing or duration of degradation and subsequent release. In one embodiment, the microparticles each comprise an inner polymer layer and an outer polymer layer, wherein the outer polymer layer limits the hydration of the inner polymer layer to control release of the therapeutic protein.

In one embodiment, the therapeutic protein is released from the plurality of microparticles at a rate of from about 0.01 mg/week to about 0.30 mg/week for a duration of at least 60 days, when the microparticles are in an aqueous environment. In one embodiment, the aqueous environment is in vitro buffer. In one embodiment, the aqueous environment is in vivo. In one embodiment, the aqueous environment is ex vivo. In one embodiment, the aqueous environment is a vitreous humor.

In one embodiment, the extended release formulation comprises a plurality of microparticles, which range in size from about two microns to about 70 microns and which comprise (a) a core of micronized therapeutic protein of from about two microns to about 30 microns, wherein the therapeutic protein is VEGF-Trap protein; and (b) a polymer cortex of a range of thicknesses, wherein the polymer is as recited in the claims wherein the microparticles release or deliver a steady level of the therapeutic protein at a rate of from about 0.01 mg/week to about 0.30 mg/week for at least 60 days.

In one embodiment, the extended release formulation comprises a plurality of microparticles, which range in size from about two microns to about 70 microns, with a median size of from about 15 microns to about 30 microns, and which comprise (a) a micronized protein core of from about two microns to about 30 microns, with a median size of about 10 microns to about 12 microns, wherein the protein is a VEGF-Trap protein; and (b) a polymer cortex of a range of thicknesses, wherein the polymer is ethyl cellulose, or polyorthoester, such that in an aqueous environment the microparticles release or deliver a steady level of VEGF Trap at a rate of about 0.06 ± 0.02 mg/week for at least 60 days.

Also disclosed is a pharmaceutical formulation for use in a method for modulating the release of a protein. In one instance, the method comprises the step of making a plurality of microparticles as described in the previous aspect, followed by the step of placing the microparticles into a solvent. The solvent in some instances is aqueous. The solvent can be in vitro, such as in a phosphate buffered solution. The solvent can be in vivo, such as e.g. vitreous humour.

• Figure 1 depicts the relative amount (% volume) of protein particles without a polymer cortex of a given diameter (ECD (µm)) in a population of protein particles manufactured from 50 mg/mL of VEGF Trap protein, 25 mg/mL of VEGF Trap protein, and 25 mg/mL of VEGF Trap protein plus 0.1% polysorbate 80.
• Figure 2 depicts the relative amount (% volume determined by MFI) of microparticles of a given diameter (ECD (µm)) in a population of micoparticles manufactured from 50 mg/mL of VEGF Trap protein plus 50 mg/mL POE, 250 mg/mL POE, and 50 mg/mL EC.
• Figure 3 depicts the amount of VEGF Trap protein in milligrams released from microparticles manufactured from 50 mg/mL POE, 250 mg/mL POE, or 50 mg/mL EC over approximately 60 days.

The micro particle and protein core particle in the subject invention are roughly spherical in shape. Some microparticles and protein cores will approach sphericity, while others 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 which circumscribes the microparticle or protein core, (b) the diameter of the largest sphere that fits within the confines of the microparticle or the protein core, (c) any measure between the circumscribed sphere of (a) and the confined sphere of (b), including the mean 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 measure between the length of the long axis (d) and the length of the short axis (e), including the mean between the two, and/or (g) equivalent circular diameter ("ECD"), as determined by micro-flow imaging (MFI), nanoparticle tracking analysis (NTA), or light obscuration methods such as dynamic light scattering (DLS). See generally Sharma et al., Micro-flow imaging: flow microscopy applied to subvisible particulate analysis in protein formulations, AAPS J. 2010 Sep; 12(3): 455-64. Diameter is generally expressed in micrometers (µm or micron). Diameter can be determined by optical measurement

"Micronized protein particle" or "protein particle" means a particle containing multiple molecules of protein with low, very low, or close to zero amounts of water (e.g., <3% water by weight). As used herein, the micronized protein particle is generally spherical in shape and has an ECD ranging from 2 microns to about 35 microns. The micronized protein particle is suited to the preparation and delivery of a therapeutic protein. Common therapeutic proteins include trap-type proteins (Huang, C., Curr. Opin. Biotechnol. 20: 692-99 (2009)) such as e.g. VEGF-Trap.

A micronized protein particle can be made by any method known in the art for making micron-sized protein particles. For example, the protein particle may be made by inter alia spray-drying (infra), lyophilization, jet milling, hanging drop crystallization (Ruth et al., Acta Crystallographica D56: 524-28 (2000)), gradual precipitation (US 7,998,477 (2011)), lyophilyzation of a protein-PEG (polyethylene glycol) aqueous mixture (Morita et al., Pharma. Res. 17: 1367-73 (2000)), 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)). The present invention employs spray drying.

As used herein, the term "protein" refers to a molecule comprising two or more amino acid residues joined to each other by peptide bonds. Peptides, polypeptides and proteins are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamMa-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Polypeptides can be of scientific or comMercial interest, including protein-based drugs. Polypeptides include, among other things, antibodies and chimeric or fusion proteins. Polypeptides are produced by recombinant animal cell lines using cell culture methods.

An "antibody" is intended to refer to immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected 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 of 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, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to 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" is inclusive of, but not limited to, those that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody. An IgG comprises a subset of antibodies.

"Fc fusion proteins" comprise part or all of two or more proteins, one of which is an Fc portion of an immunoglobulin molecule, that are not fused in their natural state. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., 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", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 - 10.19.11, 1992. "Receptor Fc fusion proteins" comprise one or more of one or more extracellular domain(s) of a receptor coupled to an Fc moiety, which in some embodiments comprises a hinge region followed by a CH2 and CH3 domain of an immunoglobulin. In some embodiments, the Fc-fusion protein contains two or more distinct receptor chains that bind to a single or more than one ligand(s). For example, an Fc-fusion protein is a trap, such as for example an IL-1 trap (e.g., Rilonacept, which contains the IL-1RAcP ligand binding region fused to the IL-1R1 extracellular region fused to Fc of hlgG1; see U.S. Pat. No. 6,927,004). or a VEGF Trap (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 Fc of hlgG1; e.g., SEQ ID NO:1; see U.S. Pat. Nos. 7,087,411 and 7,279,159, which are herein incorporated by reference in their entirety).

As used herein, the term "polymer" refers to a macromolecule comprising repeating monomers connected by covalent chemical bonds. Polymers used in the practice of this invention are biodegradable. Synthetic biodegradable polymers include ethyl cellulose, and polyorthoester.

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

Polyorthoester (POE) is a bioerodible polymer designed for drug delivery. It is generally a polymer of a ketene acetal, preferably a cyclic diketene acetal, such as e.g., 3,9-dimethylene-2,4,8,10-tetraoxa spiro[5.5]-undecane, which is polymerized via glycol condensation to form the orthoester linkages. A description of polyorthoester sysnthesis and various types can be found e.g. in US 4,304,767. Polyorthoesters can be modified to control their drug release profile and degradation rates by swapping in or out various hydrophobic diols and polyols, such as e.g., replacing a hexanetriol with a decanetriol.; as well as adding latent acids, such as e.g., octanedioic acid or the like, to the backbone to increase pH sensitivity. Other modifications to the polyorthoester include the integration of an amine to increase functionality. The formation, description, and use of polyorthoesters are described in US 5,968,543; US 4,764,364; Heller and Barr, Biomacromolecules, 5(5): 1625-32 (2004); and Heller, Adv. Drug. Deliv. Rev., 57: 2053-62 (2005).

As used herein, the phrase "spray-dry" means a method of producing a dry powder comprising micron-sized particles from a slurry or suspension by using a spray-dryer. Spray dryers employ an atomizer or spray nozzle to disperse the suspension or slurry into a controlled drop size spray. Drop sizes from 10 to 500 µm can be generated by spray-drying. As the solvent (water or organic solvent) dries, the protein substance dries into a micron-sized particle, forming a powder-like substance; or in the case of a protein-polymer suspension, during drying, the polymer hardened shell around the protein load.

The microparticles in the invention comprise a protein core surrounded by a polymer cortex or coat. Briefly, a micronized protein particle is formed, which is then dispersed in a polymer solution (polymer dissolved in solvent) to form a protein-polymer suspension. The protein-polymer suspension is then dispersed into micronized (atomized) droplets, and the solvent is driven-off to form the microparticle.

In one embodiment, the micronized protein particle is formed by making a solution of the protein and then subjecting that protein solution to dispersion and heatto form a dry powder comprising the protein. In the method of the invention the micronized protein particles are formed by spray-drying. In one embodiment, the protein is a therapeutic protein that is formulated to include buffers, stabilizers and other pharmaceutically acceptable excipients to make a pharmaceutical formulation of the therapeutic protein. Exemplary pharmaceutical formulations are described in US 7,365,165, US 7,572,893, US 7,608,261, US 7,655,758, US 7,807,164, US 2010-0279933, US 2011-0171241, and PCT/US11/54856.

The amount of therapeutic protein contained within the pharmaceutical formulations produced by the present invention may vary depending on the specific properties desired of the formulations, as well as the particular circumstances and purposes for which the formulations are intended to be used. In certain embodiments, the pharmaceutical formulations may contain about 1 mg/mL to about 500 mg/mL of protein; about 5 mg/mL to about 400 mg/mL of protein; about 5 mg/mL to about 200 mg/mL of protein; about 25 mg/mL to about 180 mg/mL of protein; about 25 mg/mL to about 150 mg/mL of protein; or about 50 mg/mL to about 180 mg/mL of protein. For example, the formulations produced by the present invention may comprise about 1 mg/mL; about 2 mg/mL; about 5 mg/mL; 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 86 mg/mL; about 87 mg/mL; about 88 mg/mL; about 89 mg/mL; about 90 mg/mL; about 95 mg/mL; about 100 mg/mL; about 105 mg/mL; about 110 mg/mL; about 115 mg/mL; about 120 mg/mL; about 125 mg/mL; about 130 mg/mL; about 131 mg/mL; about 132 mg/mL; about 133 mg/mL; about 134 mg/mL; about 135 mg/mL; about 140 mg/mL; about 145 mg/mL; about 150 mg/mL; about 155 mg/mL; about 160 mg/mL; about 165 mg/mL; about 170 mg/mL; about 175 mg/mL; about 180 mg/mL; about 185 mg/mL; about 190 mg/mL; about 195 mg/mL; about 200 mg/mL; about 205 mg/mL; about 210 mg/mL; about 215 mg/mL; about 220 mg/mL; about 225 mg/mL; about 230 mg/mL; about 235 mg/mL; about 240 mg/mL; about 245 mg/mL; about 250 mg/mL; about 255 mg/mL; about 260 mg/mL; about 265 mg/mL; about 270 mg/mL; about 275 mg/mL; about 280 mg/mL; about 285 mg/mL; about 200 mg/mL; about 200 mg/mL; or about 300 mg/mL of therapeutic protein.

The pharmaceutical formulations produced by the present invention comprise one or more excipients. The term "excipient," as used herein, means any non-therapeutic agent added to the formulation to provide a desired consistency, viscosity or stabilizing effect.

The pharmaceutical formulations produced by the present invention may also comprise one or more carbohydrate, e.g., one or more sugar. The sugar can be a reducing sugar or a non-reducing sugar. "Reducing sugars" include, e.g., sugars with a ketone or aldehyde group and contain a reactive hemiacetal group, which allows the sugar to act as a reducing agent. Specific examples of reducing sugars include fructose, glucose, glyceraldehyde, lactose, arabinose, mannose, xylose, ribose, rhamnose, galactose and maltose. Non-reducing sugars can comprise an anomeric carbon that is an acetal and is not substantially reactive with amino acids or polypeptides to initiate a Maillard reaction. Specific examples of non-reducing sugars include sucrose, trehalose, sorbose, sucralose, melezitose and raffinose. Sugar acids include, for example, saccharic acids, gluconate and other polyhydroxy sugars and salts thereof.

The amount of sugar contained within the pharmaceutical formulations produced by the present invention will vary depending on the specific circumstances and intended purposes for which the formulations are used. In certain embodiments, the formulations may contain about 0.1% to about 20% sugar; about 0.5% to about 20% sugar; about 1% to about 20% sugar; about 2% to about 15% sugar; about 3% to about 10% sugar; about 4% to about 10% sugar; or about 5% to about 10% sugar. For example, the pharmaceutical formulations of the present invention may comprise about 0.5%; about 1.0%; about 1.5%; about 2.0%; about 2.5%; about 3.0%; about 3.5%; about 4.0%; about 4.5%; about 5.0%; about 5.5%; about 6.0%; 6.5%; about 7.0%; about 7.5%; about 8.0%; about 8.5%; about 9.0%; about 9.5%; about 10.0%; about 10.5%; about 11.0%; about 11.5%; about 12.0%; about 12.5%; about 13.0%; about 13.5%; about 14.0%; about 14.5%; about 15.0%; about 15.5%; about 16.0%; 16.5%; about 17.0%; about 17.5%; about 18.0%; about 18.5%; about 19.0%; about 19.5%; or about 20.0% sugar (e.g., sucrose).

The pharmaceutical formulations produced by the present invention may also comprise one or more surfactant. As used herein, the term "surfactant" means a substance which reduces the surface tension of a fluid in which it is dissolved and/or reduces the interfacial tension between oil and water. Surfactants can be ionic or non-ionic. Exemplary non-ionic surfactants that can be included in the formulations of the present invention include, e.g., alkyl polyethylene oxide), alkyl polyglucosides (e.g., octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Specific non-ionic surfactants that can be included in the formulations produced by the present invention include, e.g., polysorbates such as polysorbate 20, polysorbate 28, polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, polysorbate 81, and polysorbate 85; poloxamers such as poloxamer 188, poloxamer 407; polyethylene-polypropylene glycol; or polyethylene glycol (PEG). Polysorbate 20 is also known as TWEEN 20, sorbitan monolaurate and polyoxyethylenesorbitan monolaurate.

The amount of surfactant contained within the pharmaceutical formulations produced by the present invention may vary depending on the specific properties desired of the formulations, as well as the particular circumstances and purposes for which the formulations are intended to be used. In certain embodiments, the formulations may contain about 0.05% to about 5% surfactant; or about 0.1% to about 0.2% surfactant. For example, the formulations may comprise about 0.05%; about 0.06%; about 0.07%; about 0.08%; about 0.09%; about 0.10%; about 0.11%; about 0.12%; about 0.13%; about 0.14%; about 0.15%; about 0.16%; about 0.17%; about 0.18%; about 0.19%; about 0.20%; about 0.21%; about 0.22%; about 0.23%; about 0.24%; about 0.25%; about 0.26%; about 0.27%; about 0.28%; about 0.29%; or about 0.30% surfactant (e.g., polysorbate 20).

The pharmaceutical formulations produced by the present invention may also comprise one or more buffers. In some embodiments, the buffer has a buffering range that overlaps fully or in part the range of pH 5.5 - 7.4. In one embodiment, the buffer has a pKa of about 6.0 ± 0.5. In certain embodiments, the buffer comprises a phosphate buffer. In certain embodiments, the phosphate is present at a concentration of 5 mM ± 0.75 mM to 15 mM ± 2.25 mM; 6 mM ± 0.9 mM to 14 mM ± 2.1 mM; 7 mM ± 1.05 mM to 13 mM ± 1.95 mM; 8 mM ± 1.2 mM to 12 mM ± 1.8 mM; 9 mM ± 1.35 mM to11 mM ± 1.65 mM; 10mM ± 1.5 mM; or about 10 mM. In certain embodiments, the buffer system comprises histidine at 10 mM ± 1.5 mM, at a pH of 6.0 ± 0.5.

The pharmaceutical formulations produced by the present invention may have a pH of from about 5.0 to about 8.0. For example, the formulations of the present invention may have a pH of about 5.0; about 5.2; about 5.4; about 5.6; about 5.8; about 6.0; about 6.2; about 6.4; about 6.6; about 6.8; about 7.0; about 7.2; about 7.4; about 7.6; about 7.8; or about 8.0.

The therapeutic protein is a VEGF Trap protein. Pharmaceutical formulations for the formation of micronized VEGF Trap protein particles may contain from about 10 mg/mL to about 100 mg/mL VEGF Trap 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. Solutions may contain one or more buffers of from about 5 mM to about 50 mM. In one embodiment, the buffer is about 10 mM phosphate at a pH of about 6 ± 0.5. Solutions may also contain sucrose at a concentration of from about 1% to about 10%. In one embodiment, the solution contains sucrose at about 2% w/w.

In some embodiments, the therapeutic protein solution contains VEGF Trap protein at about25 mg/mL or about 50 mg/mL in 10 mM phosphate, pH 6.2, 2% sucrose, and optionally 0.1% polysorbate.

The therapeutic protein formulation is then subjected to dispersion and drying to form micronized protein particles. The micronized protein particles are made by subjecting the protein solution to spray-drying. Spray-drying is generally known in the art and may be performed on equipment such as e.g., a BÜCHI Mini Spray Dryer B-290 (Buchi Labortechnik AG, Flawil, CH). In one particular embodiment, the protein solution (e.g., but not limited to any one of the VEGF Trap formulations described above) is pumped into the spray dryer at a rate of about 2 mL/min to about 15 mL/min, or about 7 mL/min. The inlet temperature of the spray dryer is set at a temperature above the boiling point of water, such as e.g., at about 130°C. The outlet temperature at a temperature below the boiling point of water and above ambient temperature, such as e.g., 55°C. In one specific embodiment, a protein solution (VEGF Trap solution) is pumped into a BÜCHI Mini Spray Dryer B-290 at about 7 mL/min, with an inlet temperature of about 130°C and an outlet temperature of about 55°C, with the aspirator set at 33 m³/h and the spray gas at 530 L/h.

The resulting micronized protein particles range in size from about 1 µm to about 100 µm in diameter, depending upon the particular formulation and concentration of protein and excipients. In some embodiments, the micronized protein particles have a diameter of from about 1 µm to about 100 µm, from about 1 µm to about 40 µm, from about 2 µm to about 15 µm, from about 2.5 µm to about 13 µm, from about 3 µm to about 10 µm, about 5 µm, about 6 µm, about 7 µm, about 8 µm, about 9 µm, about 10 µm, about 11 µm, or about 12 µm.

The micronized protein particles are then coated with a biocompatible and biodegradable polymer. This is can be accomplished by suspending the micronized protein particles in a polymer solution. A polymer solution is essentially a polymer dissolved in a solvent. For example, the biocompatible and biodegradable polymer may be dissolved in inter alia methylene chloride, tetrahydrofuran, ethyl acetate, or or some other useful solvent. Ethyl acetate is widely known as a safe solvent and is often used in the preparation of drugs, implants and foodstuffs.

The polymer can be ethyl cellulose ("EC"), or polyorthoester ("POE"). The polymer can be dissolved in the solvent (e.g., ethyl acetate) at a concentration of from about 10 mg/mL to about 300 mg/mL, from about 15 mg/mL to about 295 mg/mL, from about 20 mg/mL to about 290 mg/mL, from about 25 mg/mL to about 280 mg/mL, from about 30 mg/mL to about 270 mg/mL, from about 35 mg/mL to about 265 mg/mL, from about 40 mg/mL to about 260 mg/mL, from about 45 mg/mL to about 260 mg/mL, from about 50 mg/mL to about 255 mg/mL, from about 55 mg/mL to about 250 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 75 mg/mL, about 100 mg/mL, about 125 mg/mL, about 150 mg/mL, about 175 mg/mL, about 200 mg/mL, about 225 mg/mL, or about 250 mg/mL.

The micronized protein particles are added to the polymer solution at about 10 mg/mL to about 100 mg/mL, about 15 mg/mL to about 95 mg/mL, about 20 mg/mL to about 90 mg/mL, about 25 mg/mL to about 85 mg/mL, about 30 mg/mL to about 80 mg/mL, about 35 mg/mL to about 75 mg/mL, about 40 mg/mL to about 70 mg/mL, about 45 mg/mL to about 65 mg/mL, about 50 mg/mL to about 60 mg/mL, at about 25 mg/mL, at about 30 mg/mL, at about 35 mg/mL, at about 40 mg/mL, at about 45 mg/mL, or at about 50 mg/mL. The particles are mixed to form a slurry or suspension, which is then subjected to dispersion and drying to form the polymer coated protein particle (i.e., microparticle).

The protein particle-polymer solution suspension is subjected to spray-drying, which is performed in a manner similar to the method for manufacturing the micronized protein particles, but with a reduced intake temperature to protect against igniting the organic solvent or polymer. Briefly, the protein particle-polymer solution suspension is pumped into the spray dryer at a rate of about 5 mL/min to about 20 mL/min, or about 12.5 mL/min. The suspension was pumped at 12.5 mL/min into the spray dryer with an aspirator air and spray gas flow rate of about 530 L/h and 35 m³/h (mm), respectively. The inlet temperature was set at 90° and the outlet temperature was set at about 54°C. The inlet temperature of the spray dryer is set at a temperature above the flash point of the solvent, such as e.g., at about 90°C. The outlet temperature at a temperature below the intake temperature and above ambient temperature, such as e.g., about 54°C. In one particular embodiment, a suspension containing about 50 mg/mL of protein particle (VEGF Trap) in about 50 mg/mL to about 250 mg/mL polymer/ethyl acetate solution is pumped into a BÜCHI Mini Spray Dryer B-290 at about 12.5 mL/min, with an inlet temperature of about 90°C and an outlet temperature of about 54°C, with the aspirator set at about 35 m³/h and the spray gas at about 530 L/h.

The resulting microparticles, which contain a protein particle core within a polymer cortex, have a range of diameters of 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 20 µm to about 50 µm, about 15 µm, about 20 µm, about 25 µm, or about 30 µm. The size variation in large part reflects the thickness of the polymer cortex, although the diameter of the protein core could contribute to size variation to some extent. Manipulating the starting concentration of the polymer solution, and/or the polymer itself can control the diameter of the microparticle. For example, those microparticles which were manufactured using 50 mg/mL polymer have a median size of about 15 µm to 20 µm, whereas those microparticles which were manufactured using 250 mg/mL polymer had a median size of about 30 µm.

The microparticles produced by the instant invention are useful in the time-release or extended release of protein therapeutics. For example, it is envisioned that the VEGF Trap microparticles are useful in the extended release of VEGF Trap therapeutic protein in, for example, the vitreous for the treatment of vascular eye disorders, or subcutaneous implantation for the extended release of VEGF Trap to treat cancer or other disorders.

The microparticles produced by the instant invention release protein in a physiological aqueous environment at about 37°C at a relatively constant rate over an extended period of time, to at least 60 days. In general, those microparticles manufactured with a higher concentration of polymer (e.g., 250 mg/mL) tended to show a relatively linear protein release profile; whereas those microparticles manufactured with a lower concentration of polymer (e.g., 50 mg/mL) tended to show an initial burst followed by an onset of a delayed burst release. Furthermore, microparticles formed from a higher concentration of polymer showed a slower rate of release of protein than those formed from a lower concentration of particles. The quality of protein released from the microparticles over time was consistent with the quality of the stating protein material. Little to no protein degradation occurred.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, sizes, etc.) but some experimental errors and deviations should be accounted for.

In the following examples, VEGF-Trap protein ("VGT"), which is a dimer of the polypeptide comprising the amino acid sequence SEQ ID NO:1, serves as an exemplar receptor-Fc-fusion protein.

Solutions containing 25 mg/mL VEGF Trap protein ("VGT"), 25 mg/mL VGT plus 0.1% polysorbate 80, and 50 mg/mL VGT in 10 mM phosphate, 2% sucrose, pH 6.2 were each independently atomized in a spray dry micronizer (BÜCHI Mini Spray Dryer B-290, Buchi Labortechnik AG, Flawil, CH) to form droplets containing VEGF Trap. Heat was applied to evaporate the water from the droplets, resulting in a powder containing VEGF Trap. The inlet temperature was set at 130°C and outlet temperature at about 55°C. The aspirator was set at 33 m³/h and spray gas at 530 L/h. The VGT solution was pumped at about 7 mL/min.

The size of the resultant VGT particles was measured by micro-flow imaging (MFI) and dynamic light imaging (DLS). Figure 1 depicts the particle size distribution as determined by MFI for the VGT particles derived from each of the 25 mg/mL VGT, 25 mg/mL VGT plus 0.1% polysorbate 80, and 50 mg/mL VGT concentrations. For all concentrations, the equivalent circular diameter (ECD) of VGT particles ranged from about 1 µm to about 39 µm, with the majority of particles ranging in size of from about 2 µm to about 14 µm. For the 25 mg/mL VGT solution, the particles clustered in the range of about 2.5 µm to about 8.8 µm, with a mode of about 6 µm. For the 25 mg/mL VGT plus 0.1% polysorbate 80 solution, the particles clustered in the range of about 2.5 µm to about 9.7 µm, with a mode of about 6 µm. For the 50 mg/mL VGT solution, the particles clustered in the range of about 2.7 µm to about 12.8 µm, with a mode of about 7 µm. Median diameters for each formulation, as determined by both MFI and DLS methods, are described in Table 1.

VGT particles were reconstituted in water for injection and examined via size exclusion, i.e., size exclusion - ultra performance liquid chromatography (SE-UPLC) to determine protein purity. No change in purity was noted after micronization relative to starting material (see Table 3). Formulation Median size by MFI (µm) Median size by DLS (µm) 50mg/mL VEGF Trap 7 7.6 25mg/mL VEGF Trap 6 5.9 25mg/mL VEGF Trap, 0.1% polysorbate 80 6 7.1

Various polymers were used or are contemplated for use in the manufacture of the polymer cortex of the microparticles. Those polymers include inter alia ethyl cellulose ("EC"), polyorthoester ("POE"), poly-D,L-lactide-co-glycolide ("PLGA"), and poly-ε-caprolactone ("PCL").

Micronized VEGF Trap particles were suspended in a solution of 50 mg/mL ethyl cellulose in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-50-EC suspension".

Micronized VEGF Trap particles were suspended in a solution of 100 mg/mL ethyl cellulose in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-100-EC suspension".

Micronized VEGF Trap particles are suspended in a solution of 250 mg/mL ethyl cellulose in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-250-EC suspension".

Micronized VEGF Trap particles were suspended in a solution of 50 mg/mL polyorthoester containing about 5% latent acid in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-50-POE suspension".

Micronized VEGF Trap particles were suspended in a solution of 250 mg/mL polyorthoester containing about 5% latent acid in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-250-POE suspension".

Micronized VEGF Trap particles were suspended in a solution of 50 mg/mL PLGA in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-50-PLGA suspension".

Micronized VEGF Trap particles were suspended in a solution of 200 mg/mL PLGA in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-200-PLGA suspension".

Micronized VEGF Trap particles were suspended in a solution of 250 mg/mL PLGA in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-250-PLGA suspension".

Micronized VEGF Trap particles are suspended in a solution of 50 mg/mL PCL in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-50-PCL suspension".

Micronized VEGF Trap particles are suspended in a solution of 250 mg/mL PCL in ethyl acetate at a concentration of about 50 mg/mL VGT; herein designated "VGT-250-PCL suspension".

PCL has a low Tg and may not be suitable for heat-drying as described below, but can be used for solvent extraction in an aqueous bath with polyvinyl alcohol (PVA), for example.

Each VGT polymer suspension, which was made according to Example 2 (supra), was subjected to spray drying using a BÜCHI Mini Spray Dryer B-290 (Buchi Labortechnik AG, Flawil, CH). Briefly, each suspension was atomized to form microdroplets, which were subsequently heat dried to remove the solvent and form the polymer-coated protein microparticles. The suspension was pumped at 12.5 mL/min into the spray dryer with an aspirator air and spray gas flow rate of about 530 L/h and 35 m³/h, respectively. The inlet temperature was set at 90° and the outlet temperature was set at about 54°C.

Spray dried polymer coated protein particles manufactured according to the exemplified process generate a plurality of microparticles having a range of equivalent circular diameters of from about 2.5 µm to about 65 µm (Figure 2). The size variation in large part reflects the thickness of the polymer cortex, although the diameter of the protein core could contribute to size variation to some extent.

The diameter of the microparticle correlates with the starting concentration of the polymer solution (Table 2,

Figure 2). Those microparticles which were manufactured using 50 mg/mL polymer had a median size of about 17 µm ± 2.8 µm. Those microparticles which were manufactured using 250 mg/mL polymer had a median size of about 29 µm.

The stability of the VEGF-Trap protein was assessed using quantitative size exclusion chromatography (SE-UPLC), which allows for the quantification of smaller degradation products and larger aggregation products relative to the intact monomer. The results are described in Table 3. Essentially, the protein remained stable throughout the spray drying and spray coating processes.

The average ratio of protein to polymer by weight was also determined for the manufactured microparticles. A collection of microparticles manufactured with varying polymers and polymer concentration was extracted and subjected to quantitative reverse phase chromatography (RP-HPLC). The results are presented in Table 3. The data may be interpreted to support the theory that a higher starting concentration of polymer yields a thicker polymer cortex on the microparticle. Material Range (µm) Median (µm) Mode (µm) VEGF-Trap ("VGT") (50 mg/mL) 2.5 - 29.4 10 - 12 8.3 VGT (50 mg/mL) + POE (50 mg/mL) 2.5 - 64.5 15 9.4 VGT (50 mg/mL) + POE (250 mg/mL) 2.5 - 49.4 29 28.5 VGT (50 mg/mL) + EC (50 mg/mL) 2.5 - 49.6 19 16.5 Material VGT starting material VGT Extracted from Coated Polymers¹ % Native % Native² % w/w VGT/polymer³ VGT starting material 97.7 - - Reconstituted VGT 97.6 - - VGT (50 mg/mL) + POE (50 mg/mL) - 96.3 14.6 VGT (50 mg/mL) + POE (250 mg/mL) - 97.7 1.8 VGT (50 mg/mL) + EC (50 mg/mL) - 97.1 6.1 ¹ Based on extracted VEGF Trap after 1 hour reconstitution to remove uncoated VEGF Trap. ²Average of percent native by SE-UPLC (n=4). ³Average of percent weight to weight loading of VGT to polymer by RP-HPLC (n=4).

The release of protein from microparticles was determined by suspending various batches of microparticles in buffer (10 mM phosphate, 0.03% polysorbate 20, pH 7.0) and measuring the amount and quality of protein released into solution over time while incubated at 37°C. At 1-2 week intervals, the microparticles were pelleted by mild centrifugation and 80% of the supernatant containing released protein was collected for subsequent analysis. An equivalent amount of fresh buffer was replaced and the microparticles were resuspended by mild vortexing and returned to the 37°C incubation chamber. Protein amount and quality in the supernatant was assessed by size exclusion chromatography.

In general, those microparticles manufactured with a higher concentration of polymer (e.g., 250 mg/mL) tended to show a relatively linear protein release profile; whereas those microparticles manufactured with a lower concentration of polymer (e.g., 50 mg/mL) tended to show an initial burst followed by an onset of a delayed burst release. The data showing the extended release of protein, which remained stable, for up to about 60 days is depicted in Figure 3 (release data). Table 4 summarizes the linear rate-of-release data. Material VEGF Trap protein release (mg VGT/week) VGT (50 mg/mL) + POE (50 mg/mL) 0.14 ± 0.16 VGT (50 mg/mL) + POE (250 mg/mL) 0.06 ± 0.02 VGT (50 mg/mL) + EC (50 mg/mL) 0.031 ± 0.02

Particle size distributions were controlled by polymer concentration and atomization spray gas flow. Increased polymer concentration shifted the distribution towards larger particles (200 mg/mL PLGA at 45 mm spray gas flow v. 100 mg/mL PLGA at 45 mm spray gas flow; see Table 5). Similarly, a lower atomization spray gas flow resulted in larger droplets and thus, larger particles (100 mg/mL PLGA at 25 mm spray gas flow v. 100 mg/mL PLGA at 45 mm spray gas flow; see Table 5). [PLGA] (mg/mL) Gas Flow Rate (m³/h) Particle size range (microns) Mode of particle size (microns) Percent total volume of particles with 15 micron particle size Protein alone NA 2.5-25 3.5 1.5% 100 25 2.5-40 9.4 3.7% 100 45 2.5-30 9.4 3.7% 200 45 2.5-30 10.2-15.4 5.4%

VEGF Trap or IgG was spray coated with low molecular weight (202S) poly(lactic acid) (PLA-LMW), high molecular weight (203S) poly(lactic acid) (PLA-HMW), polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric acid-cohydroxyvaleric acid) (PHB-PVA), PEG-poly(lactic acid) block copolymer (PEG-PLA), and poly-D,L-lactide-co-glycolide (PLGA). 25 mg/mL of spray-dried protein was combined with 50-100 mg/mL polymer. In vitro release assays were performed in 10 mM phosphate buffer, pH7.2 at 37°C. The results are depicted in Table 6. Polymer Protein Relative number of particles at 15 microns Time to 100% protein release PLA-LMW VEGF Trap 0.8 x 10² 3 days PLA-HMW VEGF Trap 0.8 x 10² 3 days pCPH VEGF Trap 1 x 10² 3 days PHB-PVA VEGF Trap 5 x 10² 1 days PEG-PLA VEGF Trap 0.6 x 10² 6 hours PLGA IgG 1 x 10² 8 days

VEGF Trap and IgG were extracted from their respective polymer coats and measured for purity by SE-UPLC. The results are summarized in Table 7. The proteins generally were compatible with the spray coating process for the polymers tested. Protein remained stable for at least 14 days for those polymers that continued to release protein. Protein Polymer % Purity by Size Exclusion Chromatography After spray coating 1 day in vitro release (IVR) 3 days IVR 14 days IVR VEGF Trap POE (AP141) 97.7 98.3 98.2 96.7 VEGF Trap PLA-LMW 97.0 97.4 92.8 - VEGF Trap PLA-HMW 93.9 97.3 95.4 - VEGF Trap PEG-PLA 89.9 91.2 - - VEGF Trap pCPH 89.2 94.2 84.8 - VEGF Trap PHB-PVA 97.4 96.2 - - VEGF Trap PLGA 96.6 97.8 - 93.6 IgG PLGA 99.2 98.0 - 92.0

• <110> Regeneron Pharmaceuticals, Inc.
• <120> Polymer Protein Microparticles
• <130> 1110A
• <140> Not available
  <141> 2012-11-18
• <150> US 61/561,525
  <151> 2011-11-18
• <160> 1
• <170> Patentln version 3.5
• <210> 1
  <211> 415
  <212> PRT
  <213> Artificial Sequence
• <220>
  <223> synthetic
• <400> 1