POLYMER PROTEIN MICROPARTICLES

Abstract

Microparticles containing a core of therapeutic protein and a cortex of a biocompatible and biodegradable polymer, and methods of making and using the microparticles are provided. The extended release of a therapeutic protein from the microparticles in a physiological solution is demonstrated over an extended period of time.

Claims

1-25. (canceled)

26. A method of manufacturing an extended release pharmaceutical composition comprising a therapeutic protein particle coated with a biodegradable polymer, the method comprising: a. spray drying an aqueous therapeutic protein solution to form therapeutic protein microparticles wherein the inlet temperature of the spray dryer is set at a temperature greater than the boiling point of water and the outlet temperature that is above ambient and below the boiling point of water to form a powder comprising a population of therapeutic protein microparticles wherein the aqueous solution is pumped into the spray dryer at a rate of 2 mL/min to 15 mL/min, b. suspending the powder comprising the population of therapeutic protein microparticles in a solution comprising polyorthoester (POE) and an organic solvent to form a suspension; and c. spray drying the suspension to form the extended release pharmaceutical composition comprising a population of POE-coated therapeutic protein microparticles, wherein the extended release pharmaceutical composition comprises about 1 to about 500 mg/mL of therapeutic protein and releases the therapeutic protein for at least 60 days at a steady rate of release, wherein extended release pharmaceutical composition can treat conditions selected from the group consisting of cancers, cardiovascular diseases, vascular conditions, orthopedic disorders, dental disorders, wounds, autoimmune diseases, gastrointestinal disorders and ocular diseases.

27. The method of claim 26, wherein the outlet temperature is about 54 to 55° C.

28. The method of claim 27, wherein each therapeutic protein particle of the population of therapeutic protein particles comprises less than 3% (w/w) water.

29. The method of claim 26, wherein the organic solvent is ethyl acetate.

30. The method of claim 26, wherein the spray drying the suspension (c) comprises atomizing the suspension and then applying heat to the atomized suspension at a temperature greater than the flash point of the organic solvent to evaporate the organic solvent to form the population of POE-coated therapeutic protein microparticles.

31. The method of claim 30, wherein the median diameter of the population of POE-coated therapeutic protein microparticles is about 15 microns to about 30 microns.

32. The method of claim 26, wherein each therapeutic protein microparticle of the population of therapeutic protein microparticles has a diameter of about 2 microns to about 14 microns.

33. The method of claim 26, wherein the condition is cancer.

34. The method of claim 26, wherein the condition is cardiovascular disease.

35. The method of claim 26, wherein the condition is a vascular disorder.

36. The method of claim 26, wherein the condition is a wound.

37. The method of claim 26, wherein the condition is a vascular disorder.

38. The method of claim 26, wherein the condition is an autoimmune disease.

39. The method of claim 26, wherein the condition is an orthopedic disorder.

40. The method of claim 26, wherein the condition is a gastrointestinal disorder.

41. The method of claim 26, wherein the condition is an ocular disease.

42. A method of providing an extended release coating for a therapeutic protein comprising: a. atomizing an aqueous solution comprising a therapeutic protein; b. spray drying the atomized aqueous solution at a temperature greater than the boiling point of water to remove water in the solution and form a population of therapeutic protein microparticles; c. coating each therapeutic protein microparticle of the population therapeutic protein microparticles with a biodegradable polymer to form a population of polymer-coated therapeutic protein microparticles, wherein the biodegradable polymer is polyorthoester, wherein the extended release pharmaceutical composition releases the therapeutic protein for at least 60 days at a steady rate of release, wherein extended release pharmaceutical composition can treat conditions selected from the group consisting of cancers, cardiovascular diseases, vascular conditions, orthopedic disorders, dental disorders, wounds, autoimmune diseases, gastrointestinal disorders and ocular diseases.

43. The method of claim 42, wherein the population of therapeutic protein microparticles is dry.

44. The method of claim 42, wherein the population of therapeutic protein microparticles comprises less than 3% water.

45. The method of claim 42, wherein the aqueous solution comprises about 10 mg/mL to 100 mg/mL of the therapeutic protein.

46. The method of claim 42, wherein the coating step (c) further comprises: combining: (1) an organic solvent with the biodegradable polymer to a concentration of between about 10 mg/mL and 300 mg/mL; and (2) the population of therapeutic protein microparticles to a concentration of between about 10 mg/mL and 100 mg/mL to form a slurry; and spray drying the slurry to form the plurality of polymer-coated therapeutic protein microparticles at step (c).

Description

DRAWINGS

[0036] FIG. 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.

[0037] FIG. 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.

[0038] FIG. 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.

DETAILED DESCRIPTION

[0039] The micro particle and protein core particle of 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 September; 12(3): 455-64. Diameter is generally expressed in micrometers (μm or micron). Diameter can be determined by optical measurement

[0040] “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 not limited to any particular protein entity, and is suited to the preparation and delivery of a therapeutic protein. Common therapeutic proteins include inter alia antigen-binding proteins, such as e.g., soluble receptor fragments, antibodies (including IgGs) and derivatives or fragments of antibodies, other Fc containing proteins, including Fc fusion proteins, and receptor-Fc fusion proteins, including the trap-type proteins (Huang, C., Curr. Opin. Biotechnol. 20: 692-99 (2009)) such as e.g. VEGF-Trap.

[0041] The micronized protein particle of the invention 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 (U.S. Pat. No. 7,998,477 (2011)), lyophilization of a protein-PEG (polyethylene glycol) aqueous mixture (Morita et al., Pharma. Res. 17: 1367-73 (2000)), supercritical fluid precipitation (U.S. Pat. No. 6,063,910 (2000)), or high pressure carbon dioxide induced particle formation (Bustami et al., Pharma. Res. 17: 1360-66 (2000)).

[0042] 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.

[0043] 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.

[0044] “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. Sci. 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 hIgG1; see U.S. Pat. No. 6,927,004, which is herein incorporated by reference in its entirety), 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 hIgG1; 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).

[0045] 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 biocompatible and biodegradable. A biocompatible and biodegradable polymer can be natural or synthetic. Natural polymers include polynucleotides, polypeptides, such as naturally occurring proteins, recombinant proteins, gelatin, collagens, fibrins, fibroin, polyaspartates, polyglutamates, polylysine, leucine-glutamate co-polymers; and polysaccharides, such as cellulose alginates, dextran and dextran hydrogel polymers, amylose, inulin, pectin and guar gum, chitosan, chitin, heparin, and hyaluronic acid. Synthetic biocompatible or biodegradable polymers include polylactic acid (PLA), polyglycolic acid (PGA), polylactic-polyglycolic copolymer (PLGA), poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate, PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol 1000 (PLGA-TGPS), polyanhydride poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly (lactic acid) copolymer (PEG-PLA), poly-ε-caprolactone (PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate (PEC), polyisobutyl cyanoacrylate, poly-N-(2-hydroxypropyl)methacrylamide (poly(HPMA)), poly-β-R-hydroxy butyrate (PHB), poly-β-R-hydroxy alkanoate (PHA), poly-β-R-malic acid, phospholipid-cholesterol polymers, 2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethyleneglycol-distearoylphosphatidylehtanolamine (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 torn control rates of degradation, and inter alia poly(ethylene glycol)/poly(butylene terephthalate) copolymers.

[0046] 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 U.S. Pat. No. 4,210,529 (1980) and references therein for a detailed description of ethyl cellulose and methods of making derivatives of ethyl cellulose.

[0047] Poly-D,L-lactide-co-glycolide (PLGA) is also a well-known Food and Drug Administration (FDA) approved biocompatible and biodegradable polymer used in tissue engineering and pharmaceutical delivery systems. PLGA is a polyester comprising glycolic acid and lactic acid monomers. For a description of the synthesis of PLGA and manufacture of PLGA nanoparticles, see Astete and Sabliov, Biomater. Sci. Polym. Ed., 17(3): 247-89 (2006) and references therein.

[0048] 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, which hydrolyses rapidly in the body to form a non-toxic or low toxicity hydroxycarboxylic acid. 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 and use of PCL-based microspheres and nanospheres as delivery systems, see Sinha et al., Int. J. Pharm., 278(1): 1-23 (2004) and references therein.

[0049] 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 synthesis and various types can be found e.g. in U.S. Pat. No. 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 U.S. Pat. Nos. 5,968,543; 4,764,364; Heller and Barr, Biomacromolecules, 5(5): 1625-32 (2004); and Heller, Adv. Drug. Deliv. Rev., 57: 2053-62 (2005).

[0050] 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.

[0051] The microparticles of 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.

[0052] 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 heat to form a dry powder comprising the protein. One method to form the micronized protein particles is 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 U.S. Pat. Nos. 7,365,165, 7,572,893, 7,608,261, 7,655,758, 7,807,164, US 2010-0279933, US 2011-0171241, and PCT/US11/54856.

[0053] The amount of therapeutic protein contained within the pharmaceutical formulations of 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 of 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.

[0054] The pharmaceutical formulations of 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.

[0055] The pharmaceutical formulations of 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.

[0056] The amount of sugar contained within the pharmaceutical formulations of 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).

[0057] The pharmaceutical formulations of 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 poly(ethylene 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 of 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.

[0058] The amount of surfactant contained within the pharmaceutical formulations of 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 of the present invention 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).

[0059] The pharmaceutical formulations of 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 to 11 mM±1.65 mM; 10 mM±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.

[0060] The pharmaceutical formulations of 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.

[0061] In one particular embodiment, 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.

[0062] In some embodiments, the therapeutic protein solution contains VEGF Trap protein at about 25 mg/mL or about 50 mg/mL in 10 mM phosphate, pH 6.2, 2% sucrose, and optionally 0.1% polysorbate.

[0063] The therapeutic protein formulation is then subjected to dispersion and drying to form micronized protein particles. One method of making the micronized protein particles is to subject 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 (Büchi 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 (e.g., VEGF Trap solution or IgG 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 m.sup.3/h and the spray gas at 530 L/h.

[0064] 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.

[0065] 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 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.

[0066] In some embodiments, the polymer can be ethyl cellulose (“EC”), poly(lactic acid) (“PLA”), polyorthoester (“POE”), poly-D,L-lactide-co-glycolide (“PLGA”), or poly-ε-caprolactone (“PCL”). 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.

[0067] 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).

[0068] In one embodiment, the protein particle-polymer solution suspension is subjected the 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 m.sup.3/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 (e.g., 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 m.sup.3/h and the spray gas at about 530 L/h.

[0069] 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.

[0070] The microparticles of 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.

[0071] The microparticles of 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.

EXAMPLES

[0072] 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, and are not intended to limit the scope of what the inventors regard as their 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.

[0073] 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.

Example 1: Micronized Proteins

[0074] 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, Büchi 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 m.sup.3/h and spray gas at 530 L/h. The VGT solution was pumped at about 7 mL/min.

[0075] The size of the resultant VGT particles was measured by micro-flow imaging (MFI) and dynamic light imaging (DLS). FIG. 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.

[0076] 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).

TABLE-US-00001 TABLE 1 Median protein particle sizes (μm) as determined by MFI and DLS Median Median Formulation size by MFI (μm) size by DLS (μm) 50 mg/mL VEGF Trap 7 7.6 25 mg/mL VEGF Trap 6 5.9 25 mg/mL VEGF Trap, 6 7.1 0.1% polysorbate 80

Example 2: Micronized Protein Suspensions in Organic Polymer Solutions

[0077] 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”).

Ethyl Cellulose Coating

[0078] 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”.

[0079] 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”.

[0080] 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”.

Polyorthoester Coating

[0081] 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”.

[0082] 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”.

Poly-D,L-Lactide-Co-Glycolide Coating

[0083] 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”.

[0084] 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”.

[0085] 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”.

Poly-ε-Caprolactone Coating

[0086] 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”.

[0087] 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”.

[0088] 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.

Example 3: Dispersion of Protein-Polymer Fine Droplets and Solvent Removal

[0089] 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 (Büchi 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 m.sup.3/h, respectively. The inlet temperature was set at 90° and the outlet temperature was set at about 54° C.

Example 4: Characterization of Protein-Polymer Microparticles

[0090] 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 (FIG. 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.

[0091] The diameter of the microparticle correlates with the starting concentration of the polymer solution (Table 2, FIG. 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.

Example 5: Protein Stability Post Spray Dry

[0092] 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.

[0093] 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.

TABLE-US-00002 TABLE 2 Equivalent circular diameter values Range Median Mode Material (μm) (μm) (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

TABLE-US-00003 TABLE 3 Protein stability and loading VGT VGT Extracted from starting Coated Polymers.sup.1 material % % w/w Material % Native Native.sup.2 VGT/polymer.sup.3 VGT starting material 97.7 — — Reconstituted VGT 97.6 — — VGT (50 mg/mL) + — 96.3 14.6 POE (50 mg/mL) VGT (50 mg/mL) + — 97.7 1.8 POE (250 mg/mL) VGT (50 mg/mL) + — 97.1 6.1 EC (50 mg/mL) .sup.1Based on extracted VEGF Trap after 1 hour reconstitution to remove uncoated VEGF Trap. .sup.2Average of percent native by SE-UPLC (n = 4). .sup.3Average of percent weight to weight loading of VGT to polymer by RP-HPLC (n = 4).

Example 6: Protein Release from Microparticles

[0094] 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.

[0095] 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 FIG. 3 (release data). Table 4 summarizes the linear rate-of-release data.

TABLE-US-00004 TABLE 4 Protein release dynamics VEGF Trap protein release Material (mg VGT/week) VGT (50 mg/mL) + POE (50 mg/mL) 0.14 ± 0.16 VGT (50 mg/mL) + POE (250 0.06 ± 0.02 mg/mL) VGT (50 mg/mL) + EC (50 mg/mL) 0.031 ± 0.02 

Example 7: Particle Size can be Manipulated by Polymer Concentration and Spray Gas Flow

[0096] 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).

TABLE-US-00005 TABLE 5 Particle Size (all metrics are approximate) Particle Mode of Percent total Gas Flow size particle volume of particles [PLGA] Rate range size with 15 micron (mg/mL) (m.sup.3/h) (microns) (microns) 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%

Example 8: Particle Size and Protein Release Across Various Polymers

[0097] 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.

TABLE-US-00006 TABLE 6 Polymer dependent particle size and protein release (all metrics are approximate) Relative number of particles at 15 Time to 100% Polymer Protein microns protein release PLA-LMW VEGF Trap 0.8 × 10.sup.2 3 days PLA-HMW VEGF Trap 0.8 × 10.sup.2 3 days pCPH VEGF Trap   1 × 10.sup.2 3 days PHB-PVA VEGF Trap   5 × 10.sup.2 1 days PEG-PLA VEGF Trap 0.6 × 10.sup.2 6 hours PLGA IgG   1 × 10.sup.2 8 days

Example 9: Protein Stability in Various Polymers

[0098] 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.

TABLE-US-00007 TABLE 7 % Purity by Size Exclusion Chromatography After spray 1 day in vitro 3 days 14 Protein Polymer coating release (IVR) IVR days IVR VEGF Trap POE 97.7 98.3 98.2 96.7 (AP141) 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