EA041519B1 - COMPOSITIONS AND METHODS FOR OBTAINING PROTEIN MICROPARTICLES (VERSIONS) - Google Patents

Description

Technical field

This invention relates to methods for preparing a prepared pharmaceutical powder and prepared pharmaceutical powders (variants) that are stable for a long period of time and that retain stability and biological activity at ambient and physiological temperatures for a long period of time.

State of the art

Therapeutic macromolecules, such as antibodies and Fc receptor fusion proteins, must be formulated in such a way as to not only make the molecules suitable for administration to patients, but also maintain their stability during storage and at the site of administration. For example, therapeutic proteins (eg, antibodies) in a liquid solution are subject to degradation, aggregation, and/or unwanted chemical modifications if the solution is not prepared correctly. When preparing a therapeutic protein composition, in addition to stability, other aspects should also be taken into account. Examples of such additional aspects include solution viscosity and antibody concentration, which may be provided by the composition. When preparing a therapeutic protein for sustained release, extreme care should be taken to obtain a composition that remains stable over a long period of time, as well as during storage and physiological temperature, has a sufficient concentration of the antibody or other therapeutic biological substance, and has other properties that enable convenient administration of the composition to patients.

Liquid compositions of biological molecules are generally designed to allow long-term stability when frozen or refrigerated, but often do not provide long-term stability at room temperature. One of the solutions known in the art for maintaining the stability and biological/therapeutic activity of a biological molecule is freeze-drying or freeze-drying the molecule. Lyophilization can provide a dry cake that remains relatively stable at ambient temperature for a relatively long period of time. Stability at room temperature can be especially important when storing and distributing biotherapeutics around the world, especially in locations where electricity and refrigeration are unreliable.

Another problem in the field of pharmaceutical compositions is the need to increase concentrations of therapeutic protein to facilitate the delivery of a large amount of drug in a small space. The problem of maximizing the amount of protein drug per unit volume is exacerbated by the inverse need to reduce the amount of excipients that help stabilize the protein. As the molar ratio of protein drug to stabilizer increases, the amount of protein is maximized at the risk of destabilizing the protein.

US Patent Application Publication No. 2016/0176986 A1 describes a highly concentrated (>200 mg/mL) IgG1 formulation in a non-aqueous solvent. This composition contains spray-dried IgG1 particles suspended in a non-aqueous solution formulated for subcutaneous delivery. The spray-dried particles contain trehalose as a stabilizer and an IgG1 molecule in a 1:2 weight ratio.

Shire et al. (J. Pharm. Sci., Vol. 93, No. 6, June 2004, 1390-1402) describes the creation of highly concentrated protein compositions by lyophilization. Shire describes the optimal molar ratio of lyoprotectant to protein as 300:1 or higher. An antibody formulation with a 500:1 molar ratio of lyoprotectant to antibody has been described to have significant room temperature stability, but undesirably hypertonicity. The same antibody formulated with a lower content of lyoprotectant (at a 250:1 molar ratio of lyoprotectant to antibody) showed improved and effective tonicity, but with significantly reduced stability. The 250:1 compromise formulation requires storage at 2-8°C to maintain acceptable stability.

Ajmera and Scherliesz (Int. J. Pharm., vol. 463, 2014, 98-107) describe the use of high nitrogen amino acids, arginine and histidine, to stabilize catalase during spray drying. The stabilizing effect was due to nitrogen-mediated hydrogen bonding. The weight ratio of amino acid to catalase for effective stabilization was 1:1 or 2:1.

The essence of the invention

In one aspect, this invention relates to a method for preparing a formulated pharmaceutical powder. In accordance with this aspect, an aqueous solution is sprayed containing a heat stabilizer selected from the group consisting of mannitol, isoleucine, proline, and combinations thereof, and an antibody or Fc receptor fusion protein at a weight ratio of 1:5-2:5. Then, the atomized aqueous solution is heated to evaporate water from the aerosol droplets and obtain a prepared pharmaceutical powder, where heat is applied to the atomized aqueous solution to evaporate water from the powder, so that the resulting prepared pharmaceutical powder has a moisture content of

- 1 041519 level equal to 3-6%, while the specified prepared pharmaceutical powder is not subjected to secondary drying and, where the powder has particles having a diameter of less than 10 microns. In one embodiment of the invention, the individual particles containing the protein powder are subsequently coated with a biodegradable polymer.

In one aspect, this invention relates to a method for preparing a formulated pharmaceutical powder. In accordance with this aspect, an aqueous solution is sprayed containing a heat stabilizer selected from the group consisting of mannitol, isoleucine, proline, and combinations thereof, and an antibody or Fc receptor fusion protein in a molar ratio of less than 300:1. Then, the nebulized aqueous solution is heated to evaporate the water from the aerosol droplets and obtain the prepared pharmaceutical powder. In one embodiment of the invention, the individual particles containing the formulated pharmaceutical powder are subsequently coated with a biodegradable polymer.

In one aspect, this invention relates to a method for preparing a formulated pharmaceutical powder. In accordance with this aspect, an aqueous solution is sprayed containing i) a heat stabilizer and from 50 to 180 mg/ml of a VEGF trap protein with a mass ratio of 1:5-2:5, where the heat stabilizer is selected from sucrose, trehalose, mannitol, isoleucine, proline, or combinations thereof; and ii) 0.030.1% (w/v) polyoxyethylene sorbitan monolaurate. Then, the nebulized aqueous solution is heated to evaporate the water from the aerosol droplets and obtain the prepared pharmaceutical powder. In one embodiment of the invention, the individual particles containing the protein powder are subsequently coated with a biodegradable polymer.

In one aspect, this invention relates to a formulated pharmaceutical powder (variants) obtained in accordance with one of the above methods, containing about 6097% (wt./wt.) Fc receptor fusion protein and about 3-40% (wt. /wt.) heat stabilizer selected from the group consisting of mannitol, isoleucine, proline and combinations thereof. According to this aspect, the prepared pharmaceutical powder is not completely dry. Said prepared pharmaceutical powder is not subjected to secondary drying and has particles having a diameter of less than 10 µm. In one embodiment, the individual protein particles of the powder are coated with a biodegradable polymer.

figures

In FIG. 1 is a line graph illustrating particle size distribution in equivalent circular diameter (ECD) by volume of spray-dried particles suspended in ethanol as measured by microflow imaging (MFI). The particles were spray dried at various inlet temperatures: 100° C. (dash-dotted line); 110°C (dashed line); 120°C (dotted line); and 130°C (solid line).

In FIG. 2 is a line graph illustrating the size distribution in equivalent circular diameter (ECD) by volume of spray-dried particles suspended in ethanol as measured by microflow imaging (MFI). The pre-spray dried composition contained 0% (dashed line), 0.03% (dashed line) and 0.1% (solid line) w/v. polysorbate 20 (PS20).

In FIG. 3 is a bar graph illustrating the quantitative distribution of spray-dried particles suspended in ethanol by aspect ratio as measured by microflow imaging (MFI). An aspect ratio of 1 represents the spherical particle morphology. The pre-spray dried composition contained 0% (dashed histogram bars), 0.03% (diagonal hatched histogram bars), and 0.1% (solid filled histogram bars) w/v. polysorbate 20 (PS20).

In FIG. 4 is a line graph illustrating the size distribution in equivalent circular diameter (ECD) by volume of spray-dried particles suspended in ethanol as measured by microflow imaging (MFI). The low solute method (depicted by the dashed line) used about 10 times lower solute concentration than the standard method (depicted by the solid line).

In FIG. 5 is a line graph illustrating the size distribution in equivalent circular diameter (ECD) by volume of spray-dried particles (non-polymer coated protein microparticles; dashed line) and spray-coated particles (polymer-coated protein microparticles, solid line) as measured through microflow imaging (MFI).

In FIG. 6, blocks A and B, are dot plots illustrating the formation rate of high molecular weight (HMW) particles as a function of the square root of time at 50° C. as measured by SEC-UPLC. Rates are calculated based on their weight ratio (6A) and molar ratios (6B) of heat stabilizer to protein. Compositions indicated by solid squares contain only sucrose. Compositions containing other heat stabilizers are shown as open circles.

- 2 041519

Detailed description of the invention

While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of this invention, preferred methods and materials are described hereinafter.

The challenge in the biopharmaceutical industry is to provide stable protein compositions at sufficiently high concentrations while providing an effective dose in a small volume. To maintain protein stability, various stabilizers and other excipients are included in the composition. This provides a compromise between protein stability and protein concentration. This invention relates to an improved composition containing a protein that remains stable at high density. The amount of stable protein provided per unit volume is increased without sacrificing protein stability.

In one aspect, the invention relates to a formulated pharmaceutical powder containing about 60-97% (w/w) glycoprotein and about 3-40% (w/w) heat stabilizer. In another aspect, the invention relates to a formulated pharmaceutical powder containing about 85-97% (w/w) glycoprotein without heat stabilizer. In one embodiment of this aspect, the residual water that remains in the powder stabilizes the protein.

In some embodiments, the prepared pharmaceutical powder contains 60-70% (w/w) glycoprotein, 60-70% (w/w) glycoprotein, 65-75% (w/w) glycoprotein, 70- 80% (w/w) glycoprotein, 75-85% (w/w) glycoprotein, 80-90% (w/w) glycoprotein, 85-95% (w/w) glycoprotein, about 60% (w/w) glycoprotein, about 62% (w/w) glycoprotein, about 64% (w/w) glycoprotein, about 66% (w/w) glycoprotein, about 68 % (w/w) glycoprotein, about 70% (w/w) glycoprotein, about 72% (w/w) glycoprotein, about 74% (w/w) glycoprotein, about 76% ( w/w) glycoprotein, about 78% (w/w) glycoprotein, about 80% (w/w) glycoprotein, about 82% (w/w) glycoprotein, about 84% (w/w) /wt.) glycoprotein, about 86% (wt./wt.) glycoprotein, about 88% (wt./wt.) glycoprotein, about 90% (wt./wt.) glycoprotein, about 92% (wt./wt.) .) glycoprotein, about 94% (w/w) glycoprotein, ca about 96% (w/w) glycoprotein or about 98% (w/w) glycoprotein.

In some embodiments, the prepared pharmaceutical powder contains 3-6% (w/w) heat stabilizer, 5-7% (w/w) heat stabilizer, 6-8% (w/w) heat stabilizer, 7-9 % (w/w) heat stabilizer, 8-10% (w/w) heat stabilizer, 9-11% (w/w) heat stabilizer, 10-12% (w/w) heat stabilizer, 11 -13% (w/w) heat stabilizer, 1214% (w/w) heat stabilizer, 13-15% (w/w) heat stabilizer, 14-16% (w/w) heat stabilizer, 15 -17% (w/w) heat stabilizer, 16-18% (w/w) heat stabilizer, 17-19% (w/w) heat stabilizer, 18-20% (w/w) heat stabilizer , 19-21% (w/w) heat stabilizer, 20-22% (w/w) heat stabilizer, 21-23% (w/w) heat stabilizer, 22-24% (w/w) ) heat stabilizer, 23-25% (w/w) heat stabilizer, 24-26% (w/w) heat stabilizer, 25-27% (w/w) heat stabilizer, 22-24% (w/w) wt.) heat stabilizer, 23-25% (wt./wt.) heat stabilizer ator, 24-26% (w/w) heat stabilizer, 25-27% (w/w) heat stabilizer, 22-24% (w/w) heat stabilizer, 23-25% (w/w) .) heat stabilizer, 24-26% (w/w) heat stabilizer, 25-27% (w/w) heat stabilizer, 26-28% (w/w) heat stabilizer, 27-29% (w/w) / wt.) heat stabilizer, 28-30% (wt./wt.) heat stabilizer, 29-31% (wt./wt.) heat stabilizer, 30-32% (wt./wt.) heat stabilizer, 31-33% ( w/w) heat stabilizer, 32-34% (w/w) heat stabilizer, 33-35% (w/w) heat stabilizer, 34-36% (w/w) heat stabilizer, 35-37 % (w/w) heat stabilizer, 36-38% (w/w) heat stabilizer, 37-39% (w/w) heat stabilizer, 38-40% (w/w) heat stabilizer, or 39 -41% (w/w) heat stabilizer.

In some embodiments of the invention, the prepared pharmaceutical powder according to this invention contains a plurality of micron-sized protein particles. The micron-sized protein particles included in the powder may be referred to herein as micronized protein particles, protein particles, protein microparticles, microparticles, multiple micronized protein particles, multiple protein particles, multiple protein microparticles, multiple microparticles, protein powders. microparticles or contained protein microparticles.

In some embodiments of the invention, the claimed prepared powder is free-flowing under normal storage conditions and during pharmaceutical filling operations. In some embodiments, the claimed powder has a Hausner ratio (i.e., the ratio of powder bulk density to powder bulk density) under filling operations or storage conditions of <1.5, <1.45, <1.4, < 1.35, <1.3, <1.25, <1.2, <1.15 or <1.1.

In some embodiments of the invention, the preferred method for determining bulk

- 3 041519 STI powder is a test for adhesion to the walls of the vial. A transparent glass vial is partially filled with the prepared pharmaceutical powder, then the vial is rotated along its vertical axis. The powder, which is able to crumble freely when the vial is rotated, is free-flowing. A powder that has some degree of static charge that causes it to stick to the walls of the vial, or that requires light tapping or removal of the static charge to cause it to crumble as the vial rotates, is considered free-flowing in some embodiments. In some embodiments, the powder is free-flowing if <30%, <25%, <20%, <15%, <10%, or <5% of the inside of the vial is coated with powder after the vial is rotated. In some embodiments of the invention, the powder is free-flowing if its constituent particles that adhere to the walls of the vial after rotation are removed from the walls of the vial by tapping the vial on a hard surface or with a finger on the vial 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times or 10 times. In some embodiments, a powder is free-flowing if its constituent particles that adhere to the walls of the vial after rotation are removed from the walls of the vial by tapping the vial with an aggregate force of <25 micronewtons (µN), about 25 µN, about 20 µN , about 15 µN, about 10 µN, about 9 µN, about 8 µN, about 7 µN, about 6 µN, about 5 µN, about 4 µN, about 3 µN, about 2 µN, about 1 µN, or <1 µN. In some embodiments of the invention, the powder, in which the vast majority of the constituent particles adhere to the walls of the vials under the influence of static forces and do not crumble, is not free-flowing. In some embodiments, the preferred reference powder, which is a free flowing powder, consists of 50-150 µm glass beads (Malvern QA standard, part # CRM0016, from Whitehouse Scientific, Chester, UK).

In some embodiments, powder flow is measured by angle of repose, compressibility (eg, Hausner ratio), roll flow, through-hole volume, shear cell analysis, rheometry, or filling speed. In some embodiments, flowability is measured using a REVOLUTION powder analyzer (Mercury Scientific Inc., Newtown, Conn.), an EVOLUTION powder analyzer (Mercury), a VOLUTION powder flow analyzer (Mercury), or an FT 300 flow analyzer (Sotax AG, Esch, Switzerland). ). Rao et al., European Journal of Pharmaceutics and Biopharmaceutics, Volume 74, Issue 2, February 2010, pp. 388-396 is included in this document to provide information on the determination of the degree of flowability by drum rotation and avalanche.

In some embodiments of the invention, flowability is determined by the volume passing through the opening. In some embodiments of the invention, where the amount of powder is limiting (for example, 1-2 g or less), passing through the hole is carried out by iteratively breaking vaulted structures, then passing the powder through the hole (for example, 3 mm) and measuring the mass of powder that passes through the hole, depending on the time. The degree of flowability is given in milligrams per second. In some embodiments of the invention, the prepared pharmaceutical powder is flowable if the rate of passage through the 3 mm hole is > 10 mg/s, > 15 mg/s, > 20 mg/s, > 25 mg/s, 30 > mg/s, > 35 mg/s, > 40 mg/s, > 45 mg/s, > 50 mg/s, > 55 mg/s, > 60 mg/s, > 65 mg/s, > 70 mg/s, > 75 mg /s, > 80 mg/s, > 85 mg/s, > 90 mg/s, > 95 mg/s, or > 100 mg/s. Seppala et al., AAPS PharmSciTech, Vol. 11, No. 1, March 2010, pp. 402-408 is included herein to provide information on the flow characteristics of a drug mixture containing an excipient based on the through-hole velocity.

The constituent protein microparticle of the formulated pharmaceutical powder may be approximately spherical in shape. Some protein microparticles are roughly spherical in shape, while other protein microparticles are more irregular in shape. The shape of protein microparticles can be determined, in particular, by static light scattering (DLS), microflow imaging (MFI) or laser diffraction. DLS is based on measuring the intensity of scattered light at several different angles resulting from intense light directed at a suspension of particles. The Fraunhofer equation is then applied to the scattering data to determine the size and shape of the protein microparticles. US Pat. No. 5,104,221 A is incorporated herein by reference to provide information on the application of the Fraunhofer equation to determine the size and shape of micron sized particles. DLS is typically applied to objects 1 µm or smaller. MFI is based on a series of bright field particle images obtained from a sample stream passing through a flow cell. The image data is analyzed to determine particle size and roundness or aspect ratio. Roundness refers to how round or spherical a protein microparticle is and is expressed on a scale of 0-1, where 1 means absolute sphericity. The term aspect ratio, which generally means the length over the width of an object, is used interchangeably with the term roundness. The aspect ratio can be expressed as the ratio of the length of the minor axis to the length of the major axis. So the more spherical parts

- 4 041519 ca has an aspect ratio close to 1 (for example, 0.98).

Laser diffraction is another method based on Fraunhofer diffraction used to determine the shape and size of particles in the range of 1-100 µm. The laser beam passes through the particle suspension and an intermediate lens focuses the diffracted light onto the sensor. Publication of De Boer et al., Int. J. Pharmaceutics, 249 (1-2): 219-231 (2002) is incorporated herein to provide particle size information by laser diffraction. In one embodiment, the size distribution of the protein microparticles contained in the powder is determined by laser diffraction using a MASTERSIZER 3000 laser particle size analyzer from Malvern (Malvern, UK).

In some embodiments of the invention, the shape of the protein microparticle is nearly spherical. In one embodiment of the invention, the aspect ratio of the protein microparticle is >0.80. In another embodiment, the aspect ratio of the protein microparticle is from about 0.90 to about 0.98. The shape of a protein microparticle can be determined, in particular, by microflow imaging (MFI).

In the context of this document, the term microparticle diameter includes the meaning of any of the following: (a) the diameter of a sphere that describes the microparticle, (b) the diameter of the largest sphere that fits within the boundaries of the microparticle or protein core, (c) any measurement between the circumscribed sphere ( a) and the inscribed sphere (b), including the average between them, (d) the length of the longest axis of the microparticle, (e) the length of the shortest axis of the microparticle, (f) any measurement between the length of the long axis (d) and the length of the short axis ( e) including the average between the two and/or (g) the equivalent circular diameter (ECD), as determined by MFI, light blocking particle studies such as DLS or the like. MFI and DLS are generally described in Sharma et al., Micro-flow imaging: flow microscopy applied to subvisible particulate analysis in protein formulations, 12(3) AAPS J. 455-64 (2010); and B. J. Frisken, Revisiting the Method of Cumulants for the Analysis of Dynamic Light-Scattering Data, 40(24) Applied Optics 4087-91 (2001), which are incorporated herein to provide information on microflow imaging and dynamic light scattering . Diameter is usually expressed in micrometers (μm or micron).

The protein microparticle of the claimed formulated pharmaceutical powder may be nearly spherical in shape and have a diameter in the range of 2 to about 45 microns. In one embodiment of the invention, the majority of the protein microparticles of the powder have a diameter of less than 10 microns, as determined by MFI. In some embodiments of the invention, the size range of protein microparticles of the claimed prepared pharmaceutical powder is 1-10 microns, 2-10 microns, 3-10 microns, 4-10 microns, 5-10 microns, 6-10 microns, 7-10 microns, 8 -10 µm, 9-10 µm, 1-9 µm, 1-8 µm, 1-7 µm, 1-6 µm, 1-5 µm, 1-4 µm, 1-3 µm, 1-2 µm, 2 -9 µm, 2-8 µm, 2-7 µm, 2-6 µm, about 1 µm, about 1.5 µm, about 2 µm, about 2.5 µm, about 3 µm, about 3.5 µm, about 4 µm, approx. 4.5 µm, approx. 5 µm, approx. 5.5 µm, approx. 6 µm, approx. 6.5 µm, approx. 7 µm, approx. 9 µm, about 9.5 or about 10 µm.

In some embodiments of the invention, the diameter of the protein microparticle is <50 microns. In one embodiment of the invention, the diameter of the protein microparticle is <12 microns. In another embodiment of the invention, the diameter of the protein microparticle is < 10 µm. In yet another embodiment of the invention, the diameter of the protein microparticle is from about 0.5 microns to about 7.0 microns. In one specific embodiment of the invention, the diameter of the protein microparticle is about 5.0 microns. In another specific embodiment of the invention, the diameter of the protein microparticle is about 2.5 microns. The diameter of protein microparticles can be determined, in particular, by MFI or static light scattering.

The diameter of the protein microparticle positively correlates with the volume of the protein microparticle. For example, a 10 µm ideal sphere is about ^5x10'4 nL, and a 5 µm ideal sphere is about 7x10'5 nL. In some embodiments of the invention ^, the range of volumes of protein microparticles of the claimed prepared pharmaceutical powder is 5x10'^7'5x10'4 nl, 10'6'5x10'4 nl, 5x10'6'5x10'4 nl, 10'5'5x10'4 nl, 5x10'5'5x10'4 nl, 10'4'5x10' ⁴ nl, about 5x10'7 nl, about 6x10'7 nl, about 7x10'7 ^nl , about 8x10'7 nl, about 9x10'7 nl, about 10 '6 nl, about 2x10'6 nl or about 3x10'6 nl, about 4x10'6 nl, about 5x10'6 nl or about 6x10'6 nl, about 7x10'6 nl, about 8x10'6 nl or about 9x10'6 nl, about 10'5 nl, about 2x10'5 nl or about 3x10'5 nl, about 4x10'5 nl, about 5x10'5 nl or about 6x10'5 nl, about 7x10'5 nl, about 8x10'5 nl or approx ^. 9x10'5 nl, approx. 10' ⁴ nl, approx ^{. 2x10'4 nl, or approx. 3x10 '4 nl , approx} . ^'4 nl or about 9x10'4 nl or 10' ³ nl.

In one embodiment of the invention, the proposed prepared pharmaceutical powder is absolutely dry. A powder that is completely dry may contain up to 3% (w/w) of water. In some embodiments, the completely dry powder contains <3% (w/w) water, <2.9% (w/w) water, <2.8% (w/w) water, < 2.7% (w/w) water, < 2.6% (w/w) water, < 2.5% (w/w) water, < 2.4% (w/w) wt.) water, < 2.3% (wt./wt.) water, < 2.2%

- 5 041519 (w/w) water, < 2.1% (w/w) water, < 2.0% (w/w) water, < 1.9% (w/w) .) water, < 1.8% (w/w) water, < 1.7% (w/w) water, < 1.6% (w/w) water, < 1.5 % (w/w) water, < 1.4% (w/w) water, < 1.3% (w/w) water, < 1.2% (w/w) water, < 1.1% (w/w) water, < 1.0% (w/w) water, < 0.9% (w/w) water, < 0.8% ( w/w) water, < 0.7% (w/w) water, < 0.6% (w/w) water, < 0.5% (w/w) water, < 0.4% (w/w) water, < 0.3% (w/w) water, < 0.2% (w/w) water or < 0.1% (w/w) /wt.) water. In some embodiments, the completely dry powder contains about 3% (w/w) water, about 2.5% (w/w) water, about 2% (w/w) water, about 1. 5% (w/w) water, about 1% (w/w) water, about 0.5% (w/w) water, or about 0% (w/w) water. In some embodiments of the invention, absolutely dry powder contains 03% (wt./wt.) water, 0.05-3% (wt./wt.) water, 0.5-3% (wt./wt.) water, 1-3% (w/w) water, 2-3% (w/w) water, 0-2% (w/w) water, 0.05-2% (w/w) .) water, 0.5-2% (w/w) water, 1-2% (w/w) water, 0-1% (w/w) water, 0.05-1 % (w/w) water or 0.5-1% (w/w) water.

In another embodiment of the invention, the proposed pharmaceutical powder is not completely dry. In some embodiments of the invention, the proposed prepared pharmaceutical powder, which is not absolutely dry, contains no more than 10% (wt./wt.) water. In some embodiments of the invention, the proposed prepared pharmaceutical powder, which is not completely dry, contains more than 3% (wt./wt.) Water and not more than 10% (wt./wt.) Water, 3.5-10% (wt. ./wt.) water, 4-10% (wt./wt.) water, 4.5-10% (wt./wt.) water, 5-10% (wt./wt.) water, 5, 5-10% (w/w) water, 6-10% (w/w) water, 6.5-10% (w/w) water, 7-10% (w/w) .) water, 7.5-10% (w/w) water, 8-10% (w/w) water, 8.5-10% (w/w) water, 910% ( w/w) water, 9.5-10% (w/w) water, >3-9% (w/w) water, >3-9% (w/w) water , >3-8.5% (w/w) water, >3-8% (w/w) water, >3-7.5% (w/w) water, >3- 7% (w/w) water, >3-6.5% (w/w) water, >3-6% (w/w) water, >3-5.5% (w/w) ./wt.) water, >3-5% (wt./wt.) water, >3-4.5% (wt./wt.) water, >3-4% (wt./wt.) water , >3-3.5% (w/w) water, about 3.1% (w/w) water, about 3.5% (w/w) water, about 4% (w/w) ./wt.) water, about 4.5% (wt./wt.) water, about 5% (wt./wt.) water, about 5.5% (w/w) water, about 6% (w/w) water, about 6.5% (w/w) water, about 7% (w/w) water , about 7.5% (w/w) water, about 8% (w/w) water, about 8.5% (w/w) water, about 9% (w/w) ) water, about 9.5% (w/w) water, or about 10% (w/w) water.

In some embodiments of the invention, the proposed prepared pharmaceutical powder may contain up to 10% (w/w) of water. In some embodiments, the powder contains < 10% (w/w) water, < 9.5% (w/w) water, < 9% (w/w) water, < 8.5% (w/w) water, < 8% (w/w) water, < 7.5% (w/w) water, < 7% (w/w) water, < 6, 5% (w/w) water, < 6% (w/w) water, < 5.5% (w/w) water, < 5% (w/w) water, < 4.5% (w/w) water, < 4% (w/w) water, < 3.5% (w/w) water, < 3% (w/w) water , < 2.5% (w/w) water, < 2% (w/w) water, < 1.5% (w/w) water, < 1% (w/w) ) water or < 0.5% (w/w) water. In some embodiments, the powder contains about 10% (w/w) water, about 9.5% (w/w) water, about 9% (w/w) water, about 8.5% (w/w) water, about 8% (w/w) water, about 7.5% (w/w) water, about 7% (w/w) water, about 6, 5% (w/w) water, about 6% (w/w) water, about 5.5% (w/w) water, about 5% (w/w) water, about 4.5% (w/w) water, about 4% (w/w) water, about 3.5% (w/w) water, about 3% (w/w) water , about 2.5% (w/w) water, about 2% (w/w) water, about 1.5% (w/w) water, about 1% (w/w) ) water, about 0.5% (w/w) water, or about 0% (w/w) water. In some embodiments, the powder contains 0.01-10% (w/w) water, 0.01-3% (w/w) water, 0.5-4% (w/w) water, 3-10% (w/w) water, 0-3% (w/w) water, 0.5-3.5% (w/w) water, 1-4% ( w/w) water, 1.5-4.5% (w/w) water, 2-5% (w/w) water, 2.5-5.5% (w/w) wt.) water, 3-6% (wt./wt.) water, 3.5-6.5% (wt./wt.) water, 4-7% (wt./wt.) water, 4, 5-7.5% (w/w) water, 5-8% (w/w) water, 5.5-8.5% (w/w) water, 6-9% ( w/w) water, 6.5-9.5% (w/w) water or 7-10% (w/w) water.

The water content of the microparticles can be determined using any one or more methods known in the art. These methods include gravimetric methods, including thermogravimetry, gas chromatography, near-infrared spectroscopy, coulometry, and the Karl Fischer method. These methods are discussed in J. K. Townes, Moisture content in proteins: its effects and measurement, 705 J. Chromatography A 115-127, 1995, and references therein. For example, you can use the (gravimetric) loss on drying (LOD) method, in which the microparticles are weighed, subjected to heat to distill off water and other volatiles, and then weighed again. The weight loss is due to the water (and other volatiles) contained in the starting material. Near-infrared spectroscopy measures the reflection of waves in the range of 1100 to 2500 nm through a glass vial (glass surface) containing a protein, determining the moisture content without destroying the sample. See United States Pharmacopeia, XXIII Revision, USP Convention, Rockville, MD 1995, pp. 1801-1802; and Savage et. al., Determination of Adequate Moisture Content for Efficient Dry-Heat Viral Inactivation in Lyophilized Factor VIII by Loss on Drying and by Near Infrared Spectroscopy, 26 Biologicals 119-124, 1998. Preferred

- 6 041519 method for determining the water content is the Karl Fischer method (volume or coulometric), which determines the amount of H2O by measuring the oxidation of SO2 with I2, where one mole of I2 is consumed per mole of H2O.

Water can stabilize or otherwise contribute to the stability of the protein of the claimed formulated pharmaceutical powder, and in some embodiments of the invention, replace the heat stabilizer.

In one embodiment, the glycoprotein contained in the formulated pharmaceutical powder is stable. The term stable or stability refers to the maintenance of an acceptable degree of physical and chemical structure or biological function of a glycoprotein after storage under certain conditions or after introduction into a physiologically significant environment. A protein may be stable even if it does not retain 100% of its chemical structure or biological function after being stored or administered for a certain period of time. Under certain circumstances, retention of about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of a protein's structure or function after storage or administration over a period of time can be considered stable. .

Stability can be measured, in particular, by determining the percentage of native molecule that remains in the composition after storage or administration to a patient for a certain period of time at a certain temperature. The percentage of a protein that retains its natural conformation can be determined, in particular, using size exclusion chromatography (eg size exclusion high performance liquid chromatography [SE-HPLC]). To determine the stability of a protein, in one embodiment of the invention, the formulated pharmaceutical powder is solubilized and then the protein is subjected to testing. A native protein includes a protein that is non-aggregated and non-degraded. In some embodiments, at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96%, 97%, 98%, 99%, or 100% of the native form of the protein can be detected in the inventive formulated pharmaceutical powder after storage for a certain period of time at a certain temperature or under physiological conditions after administration to a patient (eg, implantation).

The aggregated protein can be detected as particles transferred as high molecular weight particles onto a gel or chromatographic sieve. The term high molecular weight or HMW particle or protein may be used interchangeably with the term aggregate, aggregates, or aggregated protein. The stable protein of the claimed formulated pharmaceutical powder undergoes an increase in the rate of formation of HMW particles (also called the rate of aggregation) of < 10% per month ^L 1 ^/2 , < 9% per month ^{L 1/2} , < 8% per month ^{L 1/2} , < 7 % per month ^1/2 , < 6% per month ^1/2 , < 5% per month ^1/2 , < 4% per month ^1/2 , < 3% per month ^{1/2 ,} < ² % per month ^{1/2 2} or < 1% per ^{month 1/2} . In a preferred embodiment of the invention ^, the rate of aggregation of the protein contained in the prepared pharmaceutical powder is < 5% per month ^1/2 .

The specified amount of time after which stability is measured can be at least 14 days, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months , at least 24 months or more. The temperature at which the microparticles can be in the stability evaluation can be any temperature from about -80 to about 50°C, for example, stored at about -80°C, about -30°C, about -20°C, about 0 °C, about 4-8°C, about 5°C, about 25°C or other ambient temperatures, about 35°C, about 37°C or other physiological temperatures, about 45 or about 50°C.

Stability can be measured by determining the percentage of protein that forms an aggregate (ie high molecular weight particles) after a certain amount of time at a certain temperature, the stability being inversely proportional to the percentage of high molecular weight (HMW) particles that form. The percentage of HMW protein particles can be determined by size exclusion chromatography after solubilization as described above. A protein microparticle can also be considered stable if less than about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1% protein. Preferably, <10%, <5%, or <2% of the protein contained in the formulated pharmaceutical powder is present as HMW particles.

Stability can be measured by determining the percentage of protein that degrades or is otherwise detected as small molecular weight (LMW) particles in the microparticle after a certain amount of time at a certain temperature, the stability being inversely proportional to the percentage of LMW particles that are detected in the solubilized microparticle. The percentage of LMW protein particles can be determined using size exclusion chromatography as

- 7 041519 described above. A protein microparticle can also be considered stable if, after three months at ambient temperature, less than about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or 0.1% protein.

In some embodiments of the invention, the claimed formulated pharmaceutical powder contains a buffer. Buffers are well known in the art. In general, a buffer is included in the aqueous protein solution starting material used to prepare the inventive powder. In some embodiments of the invention, the buffer is included in the starting material at a concentration of from 1 mm to 100 mm. In some specific embodiments of the invention, the buffer is included in the source material at a concentration of about 10 mm. In some embodiments, the buffer is present in the starting material at a concentration of 5 mM ± 0.75 mM to 15 mM ± 2.25 mM; from 6 mM ± 0.9 mM to 14 mM ± 2.1 mM; from 7 mM ± 1.05 mM to 13 mM ± 1.95 mM; from 8 mM ± 1.2 mM to 12 mM ± 1.8 mM; from 9 mM ± 1.35 mM to 11 mM ± 1.65 mM; 10 mM ± 1.5 mM; or about 10 mm. In some embodiments, the starting material buffer system contains histidine, phosphate, and/or acetate at a concentration of 10 mM ± 1.5 mM.

In some embodiments, the buffer is present in the claimed formulated pharmaceutical powder at a concentration of < 10% (w/w), < 9.5% (w/w), < 9% (w/w) , < 8.5% (w/w), < 8% (w/w), < 7.5% (w/w), < 7% (w/w), < 6.5% (w/w), < 6% (w/w), 5.5% (w/w), < 5% (w/w), < 4.5 % (w/w), < 4% (w/w), < 3.5% (w/w), < 3% (w/w), < 2.5% ( w/w), < 2% (w/w), < 1.5% (w/w), < 1% (w/w) or < 0.5% (w/w) /wt.). In some embodiments of the invention, the buffer is present in the claimed prepared pharmaceutical powder at a concentration of 0.1-0.5% (w/w), 0.5-1% (w/w), 0.5- 1.5% (w/w), 1-2% (w/w), 1.5-2.5% (w/w), 2-3% (w/w) ), 2.5-3.5% (w/w), 3-4% (w/w), 3.5-4.5% (w/w), 4-5% (w/w), 4.5-5.5% (w/w), 5-6% (w/w), 5.5-6.5% (w/w) ), 6-7% (w/w), 6.5-7.5% (w/w), 8-9% (w/w), 8.5-9.5% (w/w) or 910% (w/w).

In some embodiments, the buffer is selected from buffers that are included in the pH range of about 3 to about 9, or in the pH range of about 3.7 to about 8.0. For example, the protein-containing aqueous solution starting material may have a pH of about 3.4, about 3.6, about 3.8, about 4.0, about 4.2, about 4.4, about 4.6, about 4.8, 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 buffer may be a combination of separate buffers, such as, for example, a combination of histidine and acetate (histidine-acetate buffer). In one embodiment, the buffer has a buffering range of about 3.5 to about 6, or about 3.7 to about 5.6, such as the acetate buffered range. In one embodiment, the buffer has a buffering range of about 5.5 to about 8.5, or about 5.8 to about 8.0, such as the phosphate buffered range. In one embodiment, the buffer has a buffering range of about 5.0 to about 8.0, or about 5.5 to about 7.4, such as a histidine buffered range.

In one embodiment of the invention, the claimed formulated pharmaceutical powder does not contain additional buffer. Wherein, any buffering capacity of the powder or liquid aqueous source material is provided by the inclusion of protein, a heat stabilizer, if present, or water.

Surfactant (one or more) can also be included in the original water-containing protein-containing material to obtain microparticles. In the context of this document, the term surfactant means a substance that reduces the surface tension of the liquid in which it is dissolved and/or reduces the interfacial tension between oil and water. Surfactants may be ionic or non-ionic. Typical non-ionic surfactants that can be included in the starting material (and subsequently in the prepared pharmaceutical powder) include, for example, alkyl poly(ethylene oxide), alkyl polyglucosides (for example, octyl glucoside and decyl maltoside), fatty alcohols such as cetyl alcohol and oleyl alcohol, MEA cocamide, DEA cocamide and TEA cocamide. Particular nonionic surfactants that may be included in the starting material include, for example, polyoxyethylene sorbitan esters (also referred to as 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 polyoxyethylene sorbitan monolaurate.

The amount of surfactant contained in the starting material solution may vary depending on the specific properties and purposes required of the powder. The bulk properties of the powder, such as flowability, can be affected by controlling the surface content.

- 8 041519 active substance. Without wishing to be bound by theory, it is believed that the surfactant acts on the interface between the air and the surface of the aqueous protein droplets (the starting material for the inventive powder) by reducing the surface tension. Higher concentrations of surfactant result in rounder and smoother protein microparticles, which can improve flowability. Lower concentrations of surfactant result in less round and more bumpy protein microparticles.

In some embodiments of the invention, the initial aqueous solution containing the claimed protein may contain from about 0.015% (w/v) to about 0.1% (w/v) of a surfactant (for example, polysorbate 20 or polysorbate 80). For example, the starting material may contain about 0.015%; about 0.016%; about 0.017%; about 0.018%; about 0.019%; about 0.02%; about 0.021%; about 0.022%; about 0.023%; about 0.024%; about 0.025%; about 0.026%; about 0.027%; about 0.028%; about 0.029%; about 0.03%; about 0.031%; about 0.032%; about 0.033%; about 0.034%; about 0.035%; about 0.036%; about 0.037%; about 0.038%; about 0.039%; about 0.04%; about 0.041%; about 0.042%; about 0.043%; about 0.044%; about 0.045%; about 0.046%; about 0.047%; about 0.048%; about 0.049%; about 0.05%; about 0.051%; about 0.052%; about 0.053%; about 0.054%; about 0.055%; about 0.056%; about 0.057%; about 0.058%; about 0.059%; about 0.06%; about 0.061%; about 0.062%; about 0.063%; about 0.064%; about 0.065%; about 0.066%; about 0.067%; about 0.068%; about 0.069%; about 0.07%; about 0.071%; about 0.072%; about 0.073%; about 0.074%; about 0.075%; about 0.076%; about 0.077%; about 0.078%; about 0.079%; about 0.08%; about 0.081%; about 0.082%; about 0.083%; about 0.084%; about 0.085%; about 0.086%; about 0.087%; about 0.088%; about 0.089%; about 0.09%; about 0.091%; about 0.092%; about 0.093%; about 0.094%; about 0.095%; about 0.096%; about 0.097%; about 0.098%; about 0.099% or about 0.10% surfactant (eg, polysorbate 20 or polysorbate 80).

In one embodiment of the invention, the formulated pharmaceutical powder contains an amphipathic non-ionic surfactant such as a fatty acid ester of polyoxyethylene sorbitan. In one embodiment, the surfactant is polysorbate 20 or polysorbate 80. In one embodiment, the weight ratio of polysorbate to protein in the claimed formulated pharmaceutical powder is 0.003:1-1:5, 0.03:10, 0.3 :50, 0.3:25, 1:50, 0.3:10, 3:50, 3:25 or 1:5.

Heat stabilizers may be included in the formulated pharmaceutical powder and hence the aqueous solution to inhibit or reduce the formation of aggregates and other thermal stress degradation products. Heat stabilizers can be amino acids, preferably amino acids with hydrophobic side chains, carbohydrates, sugar alcohols, polymers, copolymers and block copolymers, polypeptides, surfactants, and the like. Examples of suitable heat stabilizers include Pluronic F68, arginine, lysine, amino acids with hydrophobic side chains including glycine, proline, valine, leucine and isoleucine, sorbitol, mannitol, glycerin, trehalose, dextrose, sucrose and other carbohydrates, various cyclodextrins, sodium chloride and their combinations. See Goldberg et al., Formulation development of therapeutic monoclonal antibodies using high-throughput fluorescence and static light scattering techniques: role of conformational and colloidal stability, 100(4) J. Pharm. sci. 1306-1315, 2011 and Bhambhani et al., Formulation design and highthroughput excipient selection based on structural integrity and conformational stability of dilute and highly concentrated IgG1 monoclonal antibody solutions, 101(3) J. Pharm. sci. 1120-1135, 2012.

The carbohydrate may be a reducing sugar or a non-reducing sugar. Reducing sugars include, for example, sugars with a ketone or aldehyde group and contain a reactive hemiacetal group which enables the sugar to function 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 may contain an anomeric carbon that is acetal and does not substantially react with amino acids or polypeptides to initiate a Maillard reaction. Specific examples of reducing sugars include sucrose, trehalose, sorbose, sucralose, melesitose, and raffinose. Sugar acids include, for example, sugar acids, gluconate and other polyhydroxy sugars and their salts.

In some embodiments of the invention, heat stabilizers are included based on the mass or molar ratio to the protein. Without wishing to be bound by theory, it is believed that heat stabilizers partially replace the water that surrounds the protein, allowing the protein to remain stable (water displacement), thereby allowing water to be removed while preserving the structure of the protein. In accordance with one aspect of the present invention, the ratio of protein to stabilizer is maximized to provide a composition containing higher amounts of protein per unit volume while maintaining the proper protein structure. In one embodiment, for every 5 parts by weight of protein in the formulated pharmaceutical powder, there is < 2 parts by weight of heat stabilizer. For example, if the initial aqueous solution for obtaining particles contains 5

- 9 041519 mg / ml of protein, then as a result, the heat stabilizer will be included in an amount of < 2 mg / ml, while maintaining the ratio of 5 parts of protein to < 2 parts of heat stabilizer in the claimed prepared pharmaceutical powder. In one embodiment, for every 5 parts by weight of protein in the formulated pharmaceutical powder, there is < 1 part of heat stabilizer by weight. For example, if the initial aqueous solution contains 5 mg/mL of protein, then eventually the heat stabilizer will be included in an amount of < 1 mg/mL, maintaining a ratio of 5 parts of protein by weight to < 1 part of heat stabilizer by weight in the claimed formulated pharmaceutical powder. In some embodiments of the invention, the weight ratio of protein to heat stabilizer is 5:2-100:1, 5:2-10:3, 20:7-4:1, 10:3-5:1, 4:1-20:3 , 5:1-10:1, 20:3-20:1, 10:1-40:1, 20:1-50:1 or 40:1-100:1.

In another embodiment of the invention, for each mol of protein, the prepared pharmaceutical powder contains < 300 mol of heat stabilizer. For example, if the starting material of the initial aqueous solution contains 1 nM of protein, then in the end the heat stabilizer will be included in an amount of < 300 nM, while maintaining the molar ratio of heat stabilizer to protein at < 300: 1 in the claimed prepared pharmaceutical powder. In some embodiments of the invention, the molar ratio of heat stabilizer to protein is 350:1-1:1, 350:1-300:1, 325:1275:1, 300:1-250:1, 275:1-225:1, 250 :1-200:1, 225:1-175:1, 200:1-150:1, 175:1-125:1, 150:1-100:1, 125:1-75:1, 100:1 -50:1, 75:1-25:1 or 50:1 - < 1:1.

The heat stabilizer component may contain more than one molecular particle. For example, the heat stabilizer may be composed of one or more of sucrose, trehalose, mannitol, arginine (Arg), and the hydrophobic amino acids glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline ( Pro), phenylalanine (Phe), methionine (Met) and tryptophan (Trp) in various relative amounts added to bring to the total cumulative amount of heat stabilizer. In table. 1 and 2 are examples of the ratios of the heat stabilizer and protein used in specific aqueous solutions of the starting material, and in table. 4 shows examples of the ratios of heat stabilizer and protein used in specific pharmaceutical powders prepared, as well as the stability of the protein in the powder obtained from these starting material compositions. 3.

In some embodiments of the invention, the declared heat stabilizer may be a macromolecule (> 200 g per mol). Macromolecules that are used as a heat stabilizer include disaccharides such as sucrose, trehalose, lactose, maltose, cellobiose, and the like. Sucrose and trehalose are the preferred macromolecular stabilizers.

Table 1 Pharmaceutical Powder Starting Material Containing Heat Stabilizers

(O^TE DROTT Protein Heat stabilizer (mg/ml) Iraoist (mg/ml) sucrose Trehalose Mannitol Isoleucine 1 50 10 - — - 2 50 20 - - - 3 50 10 - 5 5 4 50 - 10 - - 5 50 - 20 - - 6 50 - 10 5 5 7 50 - - 10 10 8 50 - - - - 9 50 - 10 — - 10 50 - 20 - - eleven 50 - 10 - - 12 5 1 - — - 13 5 2 - - - 14 5 1 - 0.5 0.5 15 5 - 1 — - 16 5 - 2 - - 17 5 - 1 0.5 0.5 18 5 - - 1 1

- 10 041519

Table 2. Pharmaceutical Powder Starting Material Containing Heat Stabilizers, Buffer and Surfactant

composition PROTEIN PHOSPH (MG/M AT L) (NM) SUCKAROSE (% W/V) THERMOSTA) i TREHALOSE A(% W/V) ZILIZATO MANNITE (% W/V) R ISOLEUCI N(% W/V) POLYSORB AT 20 (% W/V) 19 50 10 2 0.015 20 50 10 1 0.015 21 50 10 1 0.5 0.5 0.015 22 50 10 2 0.015 23 50 10 1 0.5 0.5 0.015 24 50 10 1 1 0.015 25 50 10 2 0.03 26 50 10 1 0.03 27 50 10 1 0.5 0.5 0.03 28 50 10 2 0.03 29 50 10 1 0.5 0.5 0.03 thirty 50 10 1 1 0.03 31 50 10 2 0.06 32 50 10 1 0.06 33 50 10 1 0.5 0.5 0.06 34 50 10 2 0.06 35 50 10 1 0.5 0.5 0.06 36 50 10 1 1 0.06 37 50 10 2 0.1 38 50 10 1 0.1 39 50 10 1 0.5 0.5 0.1 40 50 10 2 0.1 41 50 10 1 0.5 0.5 0.1 42 50 10 1 1 0.1 43 5 1 0.2 0.015 44 5 1 0.1 0.015 45 5 1 0.1 0.05 0.05 0.015 46 5 1 0.2 0.015 47 5 1 0.1 0.05 0.05 0.015 48 5 1 0.1 0.1 0.015 49 5 1 0.2 0.03 50 5 1 0.1 0.03 51 5 1 0.1 0.05 0.05 0.03 52 5 1 0.2 0.03 53 5 1 0.1 0.05 0.05 0.03 54 5 1 0.1 0.1 0.03 55 5 1 0.2 0.06 56 5 1 0.1 0.06 57 5 1 0.1 0.05 0.05 0.06 58 5 1 0.2 0.06 59 5 1 0.1 0.05 0.05 0.06 60 5 1 0.1 0.1 0.06 61 5 1 0.2 0.1 62 5 1 0.1 0.1 63 5 1 0.1 0.05 0.05 0.1 64 5 1 0.2 0.1 65 5 1 0.1 0.05 0.05 0.1 66 5 1 0.1 0.1 0.1

Table 3 Pharmaceutical Powder Starting Material Containing Heat Stabilizers, Buffer and Surfactant

Components of liquid starting material (mg/ml) Sample Protein sucrose Trehalose Mannitol Isoleucine Proline Phosphate Polysorbate 67 50 0 0 0 0 0 1.5 0 68 5 0.5 0.25 0 0.25 0 0.15 0.15 69 5 0.5 0 0.25 0.25 0 0.15 0.15 70 5 0 0.5 0.25 0.25 0 0.15 0.15 71 5 0.5 0 0 0.5 0 0.15 0.15 72 5 0.5 0 0 0 0.5 0.15 0.15 73 5 0.5 0 0.5 0 0 0.15 0.15 74 5 1 0 0 0 0 0.15 0.15 75 5 2 0 0 0 0 0.15 0.2 76 5 2 0 0 0 0 0.15 0 77 5 0 2 0 0 0 0.15 0 78 5 0 1 0.5 0.5 0 0.15 0 79 50 5 0 0 0 0 1.5 0 80 50 10 0 0 0 0 1.5 0 81 50 5 0 2.5 2.5 0 1.5 0 82 50 20 0 0 0 0 1.5 0 83 50.6 9 0 4.5 4.5 0 1.35 0 84 50 10 0 10 0 0 1.5 0 85 50 10 0 0 10 0 1.5 0 86 50 10 0 5 5 0 1.5 0 87 50 0 0 10 10 0 1.5 0 88 50 0 0 20 0 0 1.5 0 89 50 0 0 0 20 0 1.5 0

- 11 041519

Table 4. Pharmaceutical Powder Component Ratios and Aggregation Degree

Degree °/mae/mae Weight ratio Molar ratio of aggregation at o . . heat stabilizer / heat stabilizer / 50°C

Glycoprotein sample in ^g g g g

powder Glycoprotein glycoprotein (%HMW/month L) 67 97.5 0.0 0 20.0 68 82.0 0.2 93 10.3 69 82.0 0.2 109 9.4 70 82.0 0.2 106 9.5 71 82.0 0.2 121 8.6 72 82.0 0.2 133 8.4 73 82.0 0.2 97 9.3 74 82.0 0.2 67 I,8 75 71.6 0.4 134 7.6 76 73.4 0.4 134 8.2 77 73.4 0.4 122 8.5 78 73.4 0.4 212 5.7 79 90.1 0.1 34 14.8 80 83.8 0.2 67 12.5 81 83.8 0.2 109 12.2 82 73.4 0.4 134 8.2 83 75.6 0.4 194 7.5 84 73.4 0.4 193 6.2 85 73.4 0.4 243 6.0 86 73.4 0.4 218 5.4 87 73.4 0.4 302 4.9 88 73.4 0.4 253 6.8 89 73.4 0.4 351 7.8

In some embodiments of the invention, the claimed heat stabilizer contains one or more small molecules (<200 grams per mol) without a macromolecular stabilizer. At a given weight ratio, the inclusion of low molecular weight heat stabilizers can maximize the ratio of heat stabilizer to protein per mole, thereby providing a stability advantage. Small molecules that are used as a heat stabilizer include monosaccharides such as ribose, deoxyribose, glucose, fructose, galactose, and the like, sugar alcohols, and other polyols such as erythritol, glycerol, threitol, arabitol, xylitol, ribitol, mannitol. , sorbitol, galactitol, fucit, iditol, inositol and the like, and amino acids such as arginine and the hydrophobic amino acids glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine and tryptophan and the like. Hydrophobic amino acids have aliphatic side chains with very small dipole moments and a (polar) carboxylic acid head. Therefore, free hydrophobic amino acids are amphipathic. Without wishing to be bound by theory, it is believed that the amphipathic heat stabilizer may provide some protection to the protein from denaturation of the water-air interface. Preferred low molecular weight heat stabilizers include mannitol, isoleucine and proline.

In some embodiments, the heat stabilizer contains only sucrose, only trehalose, only isoleucine, only mannitol, or only proline. In some embodiments of the invention, the heat stabilizer contains a combination of sucrose and trehalose. In some embodiments, the heat stabilizer contains a combination of sucrose and trehalose in a weight ratio of 3:1-1:3, about 2:1, about 4:3, about 1:1, about 3:4, or about 1:2.

In some embodiments of the invention, the heat stabilizer contains a combination of sucrose, mannitol and isoleucine. In some embodiments, the heat stabilizer contains a combination of sucrose, mannitol, and isoleucine in a weight ratio of 4:3:1, 2:1:1, 4:1:3, 2:3:1, 1:1:1, or 2: 1:3.

In some embodiments of the invention, the prepared pharmaceutical powders containing sucrose have a higher degree of aggregation than powders that use mannitol, isoleucine, proline, or combinations thereof (Fig. 6).

In some embodiments of the invention, the heat stabilizer contains a combination of trehalose, mannitol and isoleucine. In some embodiments, the heat stabilizer contains a combination of trehalose, mannitol, and isoleucine in a weight ratio of 4:3:1, 2:1:1, 4:1:3, 2:3:1, 1:1:1, or 2: 1:3.

In some embodiments of the invention, the heat stabilizer contains a combination of sucrose and isoleucine. In some embodiments, the heat stabilizer contains a combination of sucrose and trehalose in a weight ratio of 3:1-1:3, about 2:1, about 4:3, about 1:1, about 3:4, or about 1:2.

In some embodiments of the invention, the heat stabilizer contains a combination of sucrose and proline. In some embodiments, the heat stabilizer contains a combination of sucrose and proline in a weight ratio of 3:1-1:3, about 2:1, about 4:3, about 1:1, about 3:4, or about 1:2.

- 12 041519

In some embodiments of the invention, the heat stabilizer contains a combination of sucrose and mannitol. In some embodiments, the heat stabilizer contains a combination of sucrose and mannitol in a weight ratio of 3:1-1:3, about 2:1, about 4:3, about 1:1, about 3:4, or about 1:2.

In some embodiments of the invention, the heat stabilizer contains a combination of mannitol and isoleucine. In some embodiments, the heat stabilizer contains a combination of mannitol and isoleucine in a weight ratio of 3:1-1:3, about 2:1, about 4:3, about 1:1, about 3:4, or about 1:2.

In one embodiment, the heat stabilizer is sucrose present in the claimed formulated pharmaceutical powder in a weight ratio to protein of about 1:5 to 2:5. In another embodiment, the pharmaceutical powder prepared contains sucrose, mannitol, isoleucine and protein in a weight ratio of about 2:1:1:10.

In one embodiment, the heat stabilizer is trehalose present in the claimed formulated pharmaceutical powder in a weight ratio to protein of about 1:5 to 2:5. In another embodiment, the pharmaceutical powder prepared contains trehalose, mannitol, isoleucine and protein in a weight ratio of about 2:1:1:10.

In one embodiment, the formulated pharmaceutical powder contains about 71-75% (w/w) glycoprotein, 14-15% (w/w) mannitol, 14-15% (w/w) isoleucine or proline , 2-2.5% (w/w) buffer and 3-8% (w/w) water.

In some embodiments of the invention, the prepared pharmaceutical powder is obtained from an aqueous solution containing the claimed protein and other excipients. This initial aqueous solution is also referred to as the starting material. In some embodiments of the invention, the source material may contain from about 0.005 to about 5% of the heat stabilizer; about 0.01% to about 4% heat stabilizer; about 0.02% to about 3% heat stabilizer, or about 0.05% to about 2% heat stabilizer, in a weight/volume (w/v) ratio. In some embodiments of the invention, the prepared pharmaceutical powder according to this invention may contain (w/v) about 0.01%; 0.02%; 0.03%; 0.04%; 0.05%; 0.06%; 0.07%; 0.08%; 0.09%;

0.10%; 0.11%; 0.12%; 0.13%; 0.14%; 0.15% 0.16%; 0.17%; 0.18%; 0.19%; 0.20%; 0.21%; 0.22%; 0.23% 0.24%; 0.25%; 0.26%; 0.27%; 0.28%; 0.29%; 0.30%; 0.31%; 0.32%; 0.33%; 0.34%; 0.35%; 0.36%; 0.37% 0.38%; 0.39%; 0.40%; 0.41%; 0.42%; 0.43%; 0.44%; 0.45%; 0.46%; 0.47%; 0.48%; 0.49%; 0.50%; 0.51% 0.52%; 0.53%; 0.54%; 0.55%; 0.56%; 0.57%; 0.58%; 0.59%; 0.60%; 0.61%; 0.62%; 0.63%; 0.64%; 0.65% 0.66%; 0.67%; 0.68%; 0.69%; 0.70%; 0.71% 0.72%; 0.73%; 0.74%; 0.75%; 0.76%; 0.77%; 0.78%; 0.79% 0.80%; 0.81%; 0.82%; 0.83%; 0.84%; 0.85% 0.86%; 0.87%; 0.88%; 0.89%; 0.90%; 0.91%; 0.92%; 0.93% 0.94%; 0.95%; 0.96%; 0.97%; 0.98%; 0.99%; 1.0%; 1.1%; 1.2%; 1.3% 1.4%; 1.5%; 1.6%; 1.7%; 1.8%; 1.9%

2.0%; 2.1%; 2.2%; 2.3%; 2.4%; 2.5%; 2.6%; 2.7%; 2.8%; 2.9%; 3.0%; 3.1%; 3.2%; 3.3%; 3.4%; 3.5%; 3.6%; 3.7%; 3.8%; 3.9%; 4.0%; 4.1%; 4.2%; 4.3%; 4.4%; 4.5%; 4.6%; 4.7%; 4.8%; 4.9% or about 5.0% heat stabilizers.

In an aspect of the present invention in which no heat stabilizer is present in the powder, the residual water serves to stabilize the protein.

The protein contained in the claimed formulated pharmaceutical powder is not limited to any particular protein compound. As used herein, the term protein includes therapeutic proteins, recombinant proteins used in research or therapy, decoys and other Fc receptor fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, human antibodies, bispecific antibodies, antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. Proteins can be produced using recombinant cell based production systems such as insect baculovirus systems, yeast systems (eg Pichia sp.), mammalian systems (eg CHO cells and CHO derivatives such as CHO-Ki cells). Publication of Ghaderi et al., Production platforms for biotherapeutic glycoproteins: Occurrence, impact, and challenges of non-human sialylation, 28 Biotechnol Genet Eng Rev. 147-75 (2012) is incorporated herein by reference to refer to therapeutic proteins and their preparation.

In some embodiments, the protein is a therapeutic protein. Therapeutic proteins can be antigen-binding proteins such as, for example, soluble receptor fragments, antibodies (including IgG) and antibody derivatives or fragments, other Fc-containing proteins, including Fc-containing fusion proteins, and receptor-Fc-fusion proteins, including decoy-type proteins such as, for example, aflibercept (VEGF decoy molecule), riloncept (IL1 decoy molecule), and etanercept (TNF decoy molecule). Publication Huang, C, Curr. Opin. Biotechnol. 20: 692-99 (2009) is incorporated herein to refer to trap-type molecules. US Pat. US Patent No. 6927044 B2 is included in this document to provide information

- 13 041519 information about rilonacept. US Pat. No. 8,063,182 B2 is included herein to provide information about etanercept.

The term protein includes any amino acid polymer containing more than about 50 amino acids covalently linked through amide bonds. Proteins contain one or more amino acid polymer chains, commonly known in the art as polypeptides. A protein may contain one or more polypeptides that form a single functioning biomolecule. Polypeptides typically contain more than 50 amino acids, while peptides typically contain 50 amino acids or less.

Proteins can contain various covalent and non-covalent modifications. Some proteins may have disulfide bridges (i.e., bridges between cysteine residues that form cystine). These covalent bonds may be within one polypeptide chain or between two separate polypeptide chains. For example, disulfide bridges are required to ensure the correct structure and function of insulin, immunoglobulins, protamine, and the like. For a recent review on disulfide bond formation, see Oka and Bulleid, Forming disulfides in the endoplasmic reticulum, 1833(11) Biochim Biophys Acta 2425-9 (2013).

In addition to the formation of disulfide bonds, proteins can undergo other post-translational modifications. These modifications include lipidation (eg, myristiolation, palmitoylation, farnesylation, geranylgeranylation, and glycosylphosphatidylinositol (GPI) anchoring), alkylation (eg, methylation), acylation, amidation, glycosylation (eg, addition of glycosyl groups to arginine, asparagine, cysteine, hydroxylysine, serine , threonine, tyrosine and/or tryptophan) and phosphorylation (i.e. addition of a phosphate group to serine, threonine, tyrosine and/or histidine). For a recent review on post-translational modification of proteins produced in eukaryotes, see Mowen and David, Unconventional post-translational modifications in immunological signaling, 15(6) Nat Immunol 512-20 (2014) and Blixt and Westerlind, Arraying the post-translational glycoproteome ( PTG), 18 Curr Opin Chem Biol. 62-9 (2014).

In one embodiment, the protein contained in the formulated pharmaceutical powder is a glycoprotein that encompasses any protein containing a glycosyl group. Antibodies and receptor Fc fusion proteins (also known as decoy proteins or decoy molecules) are examples of glycoproteins. Antibodies produced in heterologous mammalian systems are also glycosylated at different residues (e.g., asparagine) with different polysaccharides and may differ from species to species, which may affect the antigenicity of therapeutic antibodies (see Butler and Spearman, The choice of mammalian). cell host and possibilities for glycosylation engineering, 30 Curr Opin Biotech 107-112 (2014)). The N-glycans of IgG molecules typically include mannosylated N-acetylglucosamine, which in some cases may include additional galactose and/or fucose groups. Antibodies typically have a mass of about 150-170 kDa (kg/mol), with glycosylation providing about 2-3% of the mass. See Plomp et al., Recent Advances in Clinical Glycoproteomics of Immunoglobulins (Igs), Mol Cell Proteomics. 2016 Jul; 15(7):2217-28.

Trap molecules are N-glycosylated in a similar manner, as they contain the Fc domain of an immunoglobulin. Trap molecules have a larger molecular weight range from about 50 kD for etanercept, about 100 kD for aflibercept, to about 250 kD for riloncept. Given the smaller size of the aflibercept polypeptide chain, the relative level of the contribution of glycosylation to the mass of the aflibercept molecule is greater than that of the antibody, i.e. about 5-16% of the total mass or more. Without wishing to be bound by theory, it is believed that differences in relative glycosylation of different proteins may influence the optimal ratio of heat stabilizer to protein in the claimed formulated pharmaceutical powder.

For example, in one specific embodiment where the protein is aflibercept, the formulated pharmaceutical particle comprises about 62% polypeptide, about 12% glycan, about 25% sucrose, and about 2% phosphate by weight. In another specific embodiment where the protein is aflibercept, the formulated pharmaceutical particle comprises about 71% polypeptide, about 13% glycan, about 2% phosphate, and about 14.1% sucrose by weight.

For example, in a particular embodiment where the protein is an IgG1 molecule, the formulated pharmaceutical particle comprises about 81% protein, about 1.5% histidine, about 16% sucrose, and about 0.2% polysorbate 80 by weight.

For example, in a particular embodiment where the protein is an IgG4 molecule, the formulated pharmaceutical particle comprises about 45% protein, about 0.4% acetate, about 56% sucrose, and about 0.4% polysorbate 20 by weight.

Immunoglobulins (also known as antibodies) are examples of proteins having multiple polypeptide chains and significant post-translational modifications. The canonical immunoglobulin protein (eg, IgG) contains four polypeptide chains - two light chains and two heavy chains. Each light chain is linked to one heavy chain through a cystine disulfide bond, and

- 14 041519 two heavy chains are linked to each other via two cystine disulfide bonds. Immunoglobulins produced in mammalian systems are also glycosylated at various residues (eg, asparagine residues) with various polysaccharides and may differ from species to species, which may affect the antigenicity of therapeutic antibodies. Butler and Spearman, The choice of mammalian cell host and possibilities for glycosylation engineering, 30 Curr Opin Biotech 107-112 (2014) is incorporated herein by reference to provide information on heterologous glycoprotein production in mammalian cell systems.

Antibodies are often used as therapeutic biomolecules. In the context of this document, the term antibody includes immunoglobulin molecules consisting of four polypeptide chains, two heavy (H) chains and two light (L) chains linked by disulfide bonds. Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) that alternate with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the amino to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3, CDR light chains may be abbreviated as LCDR1, LCDR2 and LCDR3 The term high affinity antibody refers to antibodies having a binding affinity for their target of at least 10-9 M, at least 10-1 M, at least 10-11 M; or at least 10-12 M, when measured by surface plasmon resonance, for example, BIACORE™, or when determining the affinity in solution by ELISA.

The term "bispecific antibody" includes an antibody capable of selectively binding two or more epitopes. Bispecific antibodies typically contain two different heavy chains, with each heavy chain specifically binding to a different epitope, either on two different molecules (for example, antigens) or on the same molecule (for example, on the same antigen). ). If the bispecific antibody is able to selectively bind two different epitopes (a first epitope and a second epitope), then the affinity of the first heavy chain for the first epitope will typically be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain. for the second epitope, and vice versa. The epitopes recognized by a bispecific antibody may be on one or another target (eg, on the same or a different protein). Bispecific antibodies can be obtained, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleotide sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen can be fused to nucleotide sequences encoding different heavy chain constant regions, and such sequences can be expressed in a cell that expresses an immunoglobulin light chain. A typical bispecific antibody has two heavy chains, each containing three heavy chain CDRs, followed (from the N-terminus to the C-terminus) by a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain, which does not confer antigen-binding specificity but can bind to each heavy chain, or which can bind to each heavy chain and which can bind one or more epitopes linked by antigen-binding regions of the heavy chain, or which can bind to each heavy chain and bind one or both heavy chains with one or both epitopes.

The expression heavy chain or heavy chain of an immunoglobulin includes the sequence of the constant region of the heavy chain of an immunoglobulin from any organism and, unless otherwise indicated, includes the variable domain of the heavy chain. Heavy chain variable domains contain three heavy chain CDRs and four FR regions unless otherwise noted. Heavy chain fragments contain CDRs, CDRs and FRs, and combinations thereof. A typical heavy chain has, following the variable domain (from the N-terminus to the C-terminus), a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain. A functional heavy chain fragment includes a fragment that is capable of specifically recognizing an antigen (eg, recognizing an antigen with a KD value in the micromolar, nanomolar, or picomolar range), that is capable of being expressed and secreted from a cell, and that contains at least one CDR.

The term light chain includes the immunoglobulin light chain constant region sequence from any organism, and unless otherwise indicated, includes human kappa and lambda light chains. Light chain variable domains (VLs) typically contain three light chain CDRs and four framework (FR) regions, unless otherwise noted. Typically, a full-length light chain contains, in the direction from the amino-terminus to the carboxyl-terminus, a VL domain that contains FR1-CDR1-FR2-CDR2

- 15 041519

FR3-CDR3-FR4, and a light chain constant domain. Light chains that can be used in accordance with this invention include chains that, for example, do not selectively bind either a first or a second antigen selectively bound by an antigen-binding protein. Suitable light chains include chains that can be identified by screening for the most commonly used light chains in existing antibody libraries (libraries in liquids or computer simulations) where the light chains do not inherently interfere with the affinity and/or selectivity of the antigen-binding domains of the antigen-binding proteins. Suitable light chains include chains that can bind one or both epitopes that are linked by antigen-binding regions of an antigen-binding protein.

The variable domain expression includes an immunoglobulin light or heavy chain amino acid sequence (modified as desired) that contains the following amino acid regions in sequence from N-terminus to C-terminus (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4. The expression "variable domain" includes an amino acid sequence capable of folding into a canonical domain (VH or VL) having a double beta-sheet structure, where the beta-sheets are connected by a disulfide bond between the remainder of the first beta-sheet and the second beta-sheet.

The phrase complementarity determining region or term CDR includes an amino acid sequence encoded by the nucleotide sequence of an organism's immunoglobulin genes that is normally (i.e., in a wild-type animal) present between two framework regions in the light or heavy chain variable region of an immunoglobulin molecule (e.g., antibody or receptor T cells). The CDR may be encoded by, for example, a germline sequence or a rearranged or non-rearranged sequence and, for example, a naive or mature B cell or T cell. In some cases (for example, in the case of CDR3), CDRs may be encoded by two or more sequences (for example, germline sequences) that are not contiguous (for example, in the unrearranged nucleotide sequence), but are contiguous in the nucleotide sequence of B cells, for example, by splicing or joining sequences (eg, V-D-J recombination to form heavy chain CDR3).

The expression Fc-containing protein includes antibodies, bispecific antibodies, immunoadhesins, and other binding proteins that contain at least a functional portion of the CH2 and CH3 region of an immunoglobulin. A functional region refers to a region of CH2 and CH3 that can bind an Fc receptor (eg, FcyR or FcRn, ie neonatal Fc receptor) and/or that can be involved in complement activation. If the CH2 and CH3 region contains deletions, substitutions and/or insertions, or other modifications that render it unable to bind any Fc receptor and unable to activate complement, the CH2 and CH3 region is non-functional.

Fc-containing proteins may contain modifications in immunoglobulin domains, including if these modifications affect one or more effector functions of the binding protein (for example, modifications that affect FcyR binding, FcRn binding, and hence the half-life and/or CDC activity ). Such modifications include, but are not limited to, the following modifications and combinations thereof, with reference to the immunoglobulin constant region EU numbering: 238, 239, 248, 249, 250, 252, 254, 255, 256, 258, 265, 267, 269, 270, 272, 276, 278, 280, 283, 285, 286,

289, 290, 292, 293, 294, 295, 296, 297, 298, 301, 303, 305, 307, 308, 309, 311, 312, 315, 318, 320, 322, 324,

326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 340, 342, 344, 356, 358, 359, 360, 361, 362,

373, 375, 376, 378, 380, 382, 383, 384, 386, 388, 389, 398, 414, 416, 419, 428, 430, 433, 434, 435, 437, 438 and

439.

For example, and not as a limitation, a binding protein is an Fc-containing protein and exhibits an increased serum half-life (compared to the same Fc-containing protein without the listed modifications) and has a modification at position 250 (eg, E or Q); 250 and 428 (for example, L or F); 252 (eg L/Y/F/W or T), 254 (eg S or T) and 256 (eg S/R/Q/E/D or T); or a modification at position 428 and/or 433 (eg L/R/SI/P/Q or K) and/or 434 (eg H/F or Y); or a modification at position 250 and/or 428; or a modification at position 307 or 308 (eg, 308F, V308F) and 434. In another example, the modification may include modification 428L (eg, M428L) and 434S (eg, N434S); modification 428L, 259I (for example, V259I) and 308F (for example, V308F); modification 433K (for example, H433K) and 434 (for example, 434Y); modification 252, 254 and 256 (for example, 252Y, 254T and 256E); modification 250Q and 428L (for example, T250Q and M428L); modification 307 and/or 308 (for example, 308F or 308P).

Some recombinant Fc-containing proteins contain receptors or receptor fragments, ligands or ligand fragments that have related binding partners in biological systems. Receptor Fc fusion proteins refer to recombinant molecules that contain a soluble receptor fused to the Fc domain of an immunoglobulin. Some receptor Fc fusion proteins may contain the ligand-binding domains of several different receptors that affect this one. These Fc receptor fusion proteins are known as decoys or decoy molecules. Rilonacept and

- 16 041519 aflibercept are examples of commercially available decoys that target IL1R (see US Pat. No. 7,927,583) and VEGF (see US Pat. No. 7,087,411), respectively. Other recombinant Fc-containing proteins include recombinant proteins containing a peptide fused to an Fc domain, such as Centocor's MIMETIBODY™ technology. Recombinant Fc-containing proteins are described in C. Huang, Receptor-Fc fusion therapeutics, traps, and MIMETIBODY technology, 20(6) Curr. Opin. Biotechnol. 692-9 (2009).

Fc fusion proteins include some or all of two or more proteins, one of which is the Fc portion of an immunoglobulin molecule, that are not fused in their natural state. The production of fusion proteins containing certain heterologous polypeptides fused to different regions of antibody-derived polypeptides (including the Fc domain) is described, for example, in 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 contain one or more extracellular domains of a receptor coupled to an Fc fragment, which in some embodiments comprises a hinge region followed by an immunoglobulin CH2 and CH3 domain. . In some embodiments, the Fc fusion protein contains two or more different receptor chains that bind to one or more ligands. For example, an Fc fusion protein is a decoy, such as, for example, an IL-1 decoy (e.g., rilonacept, which contains an IL-1RAcP ligand-binding region fused to an IL-1R1 extracellular region fused to an Fc in hIgG1, see US Pat. 6927004, which is incorporated herein by reference in its entirety) or a VEGF decoy (e.g., aflibercept, which contains the Ig domain 2 of the VEGF Flt1 receptor fused to the Ig domain 3 of the VEGF Flk1 receptor fused to the Fc in hIgG1, e.g., SEQ ID no: 1, see US Pat. Nos. 7,087,411 and 7,279,159, which are incorporated herein by reference in their entirety).

A VEGF antagonist refers to any drug, drug, or other molecule that antagonizes or otherwise reduces the activity of vascular endothelial growth factor (VEGF). VEGF is a member of the PDGF family of growth factors. It acts predominantly on endothelial cells, stimulating mitogenesis and cell migration, and thereby stimulating vasculogenesis and angiogenesis. VEGF can be used ectopically to treat ischemia, in particular coronary heart disease. Conversely, VEGF antagonists can be used to inhibit angiogenesis. Anti-angiogenic agents are used in the treatment of cancer, because tumors require neovascularization to continue growing and metastasizing, and in the treatment of choroidal neovascularization, which leads to the wet form of age-related macular degeneration. For a review of VEGF and drugs used to stimulate or inhibit VEGF activity, see Wu et al., A systems biology perspective on sVEGFR1: its biological function, pathogenic role and therapeutic use, 14(3) J. Cell Mol. Med. 528-52 (2010); Yadav et al., Tumor Angiogenesis and Angiogenic Inhibitors: A Review, 9(6) J. Clin. Diagn. Res. XE01-XE05 (2015); and I. Zachary, VEGF signaling: integration and multi-tasking in endothelial cell biology, 31 (Pt 6) Biochem. soc. Trans. 1171-7 (2003).

VEGF inhibitors include small molecules that inhibit VEGF-stimulated tyrosine kinases, including, for example, lapatinib, sunitinib, sorafenib, axitinib, and paxopanib (Yadav, 2015). Macromolecular inhibitors of VEGF include the monoclonal antibody bevacizumab, the Fab fragment ranibizumab, the aflibercept trap, and the PEGylated aptamer pegatinib (Id).

Other growth factors involved in angiogenesis and serving as therapeutic targets for neovascularization include, in particular, angiopoietin-2 (Ang2) and platelet-derived growth factor (PDGF). Ang2 is a constituent element in the angiopoietin-Tie (AT) pathway and is therefore involved in vascular remodeling. The AT pathway, in which angiopoietin-1 binds and acts as an agonist of the cognate Tie2 receptor, promotes endothelial cell survival and the formation, maturation, and maintenance of blood vessels. Ang2 is also involved in lymphangiogenesis. Ang2 also binds to and modulates Tie2 signaling, and appears to act as a Tie2 antagonist in the presence of Ang1. See Thurston and Daly, The Complex Role of Angiopoietin-2 in the Angiopoietin - Tie Signaling Pathway, 2(9) Cold Spring Harb. perspective. Med. a006650 (2012) and Chintharlapalli et al., Angiopoietin-2: an attractive target for improved antiangiogenic tumor therapy, 73(6) Cancer Res. 1649-57 (2013).

Ang2 causes cancer and inflammation. It has an increased level of expression in various carcinomas, cytomas and sarcomas, as well as in sepsis and inflammatory diseases. Ang2 plays a role in leukocyte recruitment. Blocking Ang2 leads to partial inhibition of the growth of human tumor xenografts (Thurston, 2012). Anti-Ang2 antibodies and other peptide inhibitors that bind to Ang2 are under development for the treatment of diseases and conditions caused or exacerbated by angiogenesis. Antibodies to Ang2 are described, for example, in US patent No. 6166185; 7521053; 7205275; and publications of US Patent Application No. 2006/0018909; 2006/0246071; 2006/068953; 2007/0154482 and 2011/0027286.

The PDGF system contains a family of dimeric growth factor ligands and receptor tyrosines

- 17 041519 Az. It contains five (5) ligand isoforms: PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD, and PDGF-AB; and two (2) receptors: PDGFRA (alpha receptor) and PDGFRB (beta receptor). PDGF is involved in embryonic cell division and tissue remodeling, as well as angiogenesis at a later stage of development. Kinase inhibitors such as sorafenib, nilotinib, dasatinib, sunitinib, nintedanib and imatinib are PDGF inhibitors used to treat cancer. Some anti-PDGFR antibodies are also under development to counteract PDGF signaling for the treatment of cancer and other angiogenesis-related diseases. See Kono et al., Adding to the mix: fibroblast growth factor and platelet-derived growth factor receptor pathways as targets in non-small cell lung cancer, 12(2) Curr. Cancer Drug Targets 107-23 (2012); Bauman et al., Antagonism of Platelet-Derived Growth Factor Receptor in Non-Small Cell Lung Cancer: Rationale and Investigations, 13 Clin. Cancer Res. 4632s (2007) and Stock et al., Platelet-derived growth factor receptor-α: a novel therapeutic target in human hepatocellular cancer, 6 Mol. Cancer Ther. 1932-41 (2007); US patents No. 5468468; 7740850; 8425911 and 8574578; and U.S. Patent Application Publication No. 2009/0053241; 2011/0177074; 2012/0009199; 2012/0027767 and 2014/0193402.

The claimed prepared pharmaceutical powder can be made from the original aqueous solution (precursor). Therefore, the proposed protein can be included in the source material at certain concentrations. The protein concentration as well as the total solute concentration in the starting material can affect the size of the resulting protein microparticle in the powder, therefore the size of the microparticles can be controlled by adjusting the protein concentration or the concentration of other solutes in the starting material. Without wishing to be bound by theory, it is believed that lower concentrations of protein or other solutes in the starting material may result in a finer powder containing smaller protein microparticles. Higher concentrations of protein or other solutes in the starting material may result in a coarser powder containing larger protein microparticles.

The protein concentration in the initial aqueous solution can vary from as little as 1 mg/mL or less to a practical amount, such as about 175-200 mg/mL or more. In some embodiments of the invention, the initial aqueous solution contains the claimed protein at a concentration of from about 1 to about 500 mg/ml of protein; about 5 to about 400 mg/ml protein; about 5 to about 200 mg/ml protein; about 25 to about 180 mg/ml of protein; about 25 to about 150 mg/ml of protein; or about 50 to about 180 mg/ml of protein. In some embodiments of the invention, the initial aqueous solution contains the claimed protein at a concentration of 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 2 35 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.

As described above, the therapeutic protein can be an antigen binding protein such as an antibody, antibody fragment, decoy molecule or other Fc receptor fusion protein, soluble receptors, and the like. In one particular embodiment, the therapeutic protein is aflibercept, a VEGF antagonist decoy molecule. The starting material solutions for the formation of aflibercept-containing microparticles may contain from about 1 to about 100 mg/ml of aflibercept, about 2 mg/ml, about 3 mg/ml, about 4 mg/ml, about 5 mg/ml, about 6 mg/ml, about 7 mg/ml, about 8 mg/ml, about 9 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 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 at a concentration of from about 5 to about 50 mm. In one embodiment, the buffer contains about 10 mM phosphate at a pH of about 6 ± 0.5.

In some embodiments of the invention, the protein microparticles of the claimed formulated pharmaceutical powder are coated with a polymer. The term polymer includes macromolecules containing repeating monomers linked by covalent chemical bonds. The polymers used for therapeutic microparticles are biocompatible and biodegradable. Bioso

- 18 041519 inclusive or biodegradable polymer may be natural or synthetic. Natural polymers include polynucleotides, polypeptides such as natural proteins, recombinant proteins, gelatin, collagens, fibrins, fibroin, polyaspartates, polyglutamates, polyleucine, copolymers of leucine and glutamate; 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 acid polyglycolic acid copolymer (PLGA), poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate, PLGA-alpha- tocopheryl succinate esterified to polyethylene glycol 1000 (PLGA-TGPS), poly[1,6-bis(p-carboxyphenoxy)hexane] (pCPH) polyanhydride, poly(hydroxybutyric acid-cohydroxyvaleric acid) (PHBPVA), polyethylene glycol-poly(lactic acid) copolymer ) (PEG-PLA), poly-ε-caprolactone (PCL), polyalkyl cyanoacrylate (PAC), poly(ethyl) cyanoacrylate (PEC), polyisobutyl cyanoacrylate, poly-N-(2hydroxypropyl)methacrylamide (poly(HPMA)), poly-β -R-hydroxybutyrate (PHB), poly-v-N-hydroxyalkanoate (PHA), poly-v-N-malic acid, phospholipid-cholesterol polymers, 2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethylene glycol-distearoylphosphatidylethanolamine (DOPC/PEGDSPE )/cholesterol, ethylcellulose, polyrotaxanes and polypseudorothaxanes based on cyclodexter ine (CD), polybutylene succinate (PBS), polyorthoesters, polyorthoester-polyamidine copolymers, polyorthoester-diamine copolymers, polyorthoesters containing latent acids to control the degradation rate, and in particular poly(ethylene glycol)/poly(butylene terephthalate) copolymers ).

Ethylcellulose (EC) is a well known and readily available biomaterial used in the pharmaceutical and food industries. It is a derivative of cellulose in which some of the hydroxyl groups of glucose have been replaced by ethyl ether. Martinac et al., 22(5) J. Microencapsulation 549-561 (2005) is incorporated herein to provide information on the use of ethyl cellulose as a biocompatible polymer in the manufacture of microspheres. US Pat. No. 4,210,529 is incorporated herein to provide information on ethylcellulose and ethylcellulose derivatives.

Poly-D,L-lactide-coglycolide (PLGA) is also a well known FDA approved biocompatible and biodegradable polymer for use in tissue engineering and pharmaceutical delivery systems. PLGA is a polyester containing glycolic acid and lactic acid monomers. Publication Astete and Sabliov, 17(3) Biomater. sci. Polym. Ed. 247-89 (2006) is incorporated herein to provide information on the synthesis of PLGA and the production of nanoparticles from PLGA.

Poly-ε-caprolactone (PCL) is another biocompatible and biodegradable polymer approved by the FDA for human use as a drug delivery device. PCL is a polyester of s-caprolactone that is rapidly hydrolyzed in the body to form a non-toxic or low-toxic hydroxycarboxylic acid. Labet and Thielemans, 38 Chemical Society Reviews 3484-3504 (2009) is included in this document to provide information on obtaining PCL. Publication Sinha et al., 278(1) Int. J Pharm. 1-23 (2004)) is included herein to provide information on the production of microspheres and nanospheres based on PCL.

Polyorthoester (POE) is a biodegradable polymer designed for drug delivery. Typically, it is a polymer of ketene acetal, preferably cyclic diketene acetal, such as, for example, 3,9-dimethylene-2,4,8,10-tetraoxapiro[5,5]-undecane, which polymerizes by condensation of glycol to form orthoester linkages . Polyorthoesters can be modified to control their drug release profile and degradation rate by adding or removing various hydrophobic diols and polyols, such as replacing hexanetriol with decanetriol, as well as adding hidden acids such as octanedioic acid or the like to the base for increasing sensitivity to changes in pH. Other modifications of the polyorthoester include the integration of an amine to increase functionality. US Patent No. 5968543; US patent No. 4764364; US patent No. 4304767; Heller and Barr publications, 5(5) Biomacromolecules 1625-32 (2004); and Heller, 57 Adv. drug. Deliv. Rev. 2053-62 (2005) are incorporated herein to provide information on polyorthoesters.

Polyethylene glycol (PEG) can be crosslinked to form a gel in which protein microparticles are incorporated. Gibas and Janik, Review: synthetic polymer hydrogels for biomedical applications, 4(4) Chemistry & Chemical Technology 297-304 (2010) is incorporated herein to provide information on cross-linked PEG gels.

The polymers that are preferred for applications that involve spray drying heat have high glass transition temperatures. In some embodiments, the high glass transition temperature is >10°C, >15°C, >20°C, >25°C, >30°C, >

- 19 041519

35°C, > 45°C or > 30°C.

In one aspect, this invention relates to a method for obtaining the claimed formulated pharmaceutical powder by spray drying the initial aqueous solution containing the claimed protein and any additional excipients (also called starting material, described in this document). The term spray drying means a process for preparing a micron-sized powder from a solution, slurry or suspension using a spray dryer. Spray dryers use an atomizer or spray nozzle to disperse the slurry or slurry into a spray with controlled droplets. By spray drying, droplets ranging in size from 10 to 500 µm can be obtained. As the solvent (water or organic solvent) dries, the proteinaceous substance dries up to become a micron-sized particle (i.e., a proteinaceous microparticle) to form a powdery substance; or in the case of a polymer suspension with protein microparticles, with the formation of a hardened polymer shell around the protein content. Spray drying can be carried out on equipment such as, for example, the BUCHI Mini Spray Dryer B-290 (Buchi Labortechnik AG, Flaville, Switzerland). Elversson & MillqvistFureby, Aqueous two-phase systems as a formulation concept for spray-dried protein, 294(1,2) International Journal of Pharmaceutics 73-87 (2005) is incorporated herein by reference to provide information on spray drying.

In one particular embodiment, the feedstock is pumped into the spray dryer at a rate of about 2 to about 15 ml/min, or about 7 ml/min. In one embodiment, the nozzle pressure is from about 35 psi to about 100 psi. In one embodiment, the nozzle pressure is about 75 psi. The inlet temperature of the spray dryer is set at or above the boiling point of water, for example, from about 100 to 130°C. The outlet temperature is set at a level below the boiling point of water and above ambient temperature, for example, about 55°C. In one particular embodiment, the protein solution (e.g., VEGF Trap protein solution or IgG solution) is pumped into a BUCHI Mini Spray Dryer B290 at a rate of about 7 ml/min at an inlet temperature of about 100, 110, 120 or 130° C. and an outlet temperature of about 55° C., with the aspirator set at 33 m ³ /h and spraying gas at 530 l/h.

In one embodiment, a formulated pharmaceutical powder is obtained by subjecting the claimed starting material to atomization to form a mist of atomized droplets, and then applying heat to the mist of atomized droplets to form a formulated pharmaceutical powder containing protein. In some embodiments of the invention, the resulting prepared pharmaceutical powder is subjected to additional drying (also known as secondary drying). Post-drying includes hot drying, lyophilization and nitrogen purge. In other embodiments of the invention, the resulting prepared pharmaceutical powder is not subjected to additional drying. In some embodiments of the invention, the resulting prepared pharmaceutical powder is not subjected to subsequent hot drying. In some embodiments of the invention, the resulting prepared pharmaceutical powder is not subjected to subsequent lyophilization. In some embodiments of the invention, the resulting prepared pharmaceutical powder is not subjected to subsequent drying by blowing with nitrogen.

In one embodiment of the invention, the starting material contains a heat stabilizer and a glycoprotein in a weight ratio of about or in the range of 1:50-2:5. In one embodiment, for every 5 parts by weight of protein, the starting material contains <2 parts by weight of heat stabilizer. For example, if the starting material contains 5 mg/mL of protein, then the total heat stabilizer will be included in an amount of < 2 mg/mL, maintaining a ratio of 5 parts of protein to < 2 parts of heat stabilizer in the resulting claimed formulated pharmaceutical powder, as described above in this document. In one embodiment, for every 5 parts by weight of protein, the starting material contains < 1 part of heat stabilizer by weight. For example, if the starting material contains 5 mg/mL of protein, then the total heat stabilizer will be included in an amount of < 1 mg/mL, maintaining a ratio of 5 parts of protein by weight to 1 part of heat stabilizer by weight in the claimed formulated pharmaceutical powder as described above in this document. In some embodiments of the invention, the weight ratio of protein to heat stabilizer is 5:2-100:1, 5:2-10:3, 20:7-4:1, 10:3-5:1, 4:1-20:3 , 5:1-10:1, 20:3-20:1, 10:140:1, 20:1-50:1 or 40:1-100:1.

In another embodiment of the invention, for each mol of protein, the starting material contains < 300 mol of heat stabilizer. For example, if the starting material contains 1 nM of protein, then the final amount of heat stabilizer will be included in the amount of < 300 nM, maintaining the molar ratio of heat stabilizer to protein at < 300:1 in the resulting claimed cooked headlight.

- 20 041519 matceutical powder, as described above in this document. In some embodiments of the invention, the molar ratio of heat stabilizer to protein is 350:1-1:1, 350:1-300:1, 325:1275:1, 300:1-250:1, 275:1-225:1, 250 :1-200:1, 225:1-175:1, 200:1-150:1, 175:1-125:1, 150:1-100:1, 125:1-75:1, 100:1 -50:1, 75:1-25:1 or 50:1 - < 1:1.

In yet another embodiment of the invention, the starting material contains a protein without a heat stabilizer.

In one embodiment of the invention, the resulting prepared pharmaceutical powder is absolutely dry, as described above in this document.

In another embodiment of the invention, the resulting prepared pharmaceutical powder is not completely dry, as described above in this document. In some embodiments of the invention, the prepared pharmaceutical powder, which is not completely dry, contains from about 3% to about 10% (wt./wt.) water, 3.5-10% (wt./wt.) water, 4- 10% (w/w) water, 4.5-10% (w/w) water, 5-10% (w/w) water, 5.5-10% (w/w) .) water, 6-10% (w/w) water, 6.5-10% (w/w) water, 7-10% (w/w) water, 7.5-10 % (w/w) water, 8-10% (w/w) water, 8.5-10% (w/w) water, 9-10% (w/w) water , 9.5-10% (w/w) water, >3-9% (w/w) water, >3-9% (w/w) water, >3-8.5 % (w/w) water, >3-8% (w/w) water, >3-7.5% (w/w) water, >3-7% (w/w) .) water, >3-6.5% (w/w) water, >3-6% (w/w) water, >3-5.5% (w/w) water, >3-5% (w/w) water, >3-4.5% (w/w) water, >3-4% (w/w) water, >3-3.5 % (w/w) water, about 3.1% (w/w) water, about 3.5% (w/w) water, about 4% (w/w) water, about 4.5% (w/w) water, about 5% (w/w) water, about 5.5% (w/w) water, about 6% (w/w) water, about 6.5% (w/w) water, about 7% (w/w) water, about 7.5% (w/w) water , about 8% (w/w) water, about 8.5% (w/w) water, about 9% (w/w) water, about 9.5% (w/w) ) water or about 10% (w/w) water.

In some embodiments of the invention, the heat stabilizer contained in the claimed source material contains sucrose or trehalose, or a combination of trehalose and sucrose.

In other embodiments of the invention, the heat stabilizer contained in the claimed source material does not contain a molecule with a molecular weight of more than 200 g/mol. In some embodiments of the invention, the heat stabilizer contained in the claimed starting material is selected from the group consisting of mannitol, isoleucine, proline, and combinations thereof.

In other embodiments of the invention, the heat stabilizer contained in the claimed source material contains trehalose or sucrose in combination with mannitol, isoleucine or proline.

In one embodiment of the invention, the claimed starting material also contains a buffer. Preferred buffers include phosphate, histidine and acetate. In one embodiment, the starting material contains 0.5-10 mM buffer.

In one embodiment of the invention, the claimed source material does not contain a buffer. In this case, the starting material is buffered with the claimed protein as such and with water.

In one embodiment of the invention, the claimed starting material also contains a non-ionic detergent. Nonionic detergents include polysorbates such as polysorbate 20 and polysorbate 80. In one embodiment, the nonionic surfactant is present in the starting material at a concentration of 0.01-0.2% (w/v).

In some embodiments of the invention, the claimed starting material contains the claimed protein, one or more (i) buffers, (ii) heat stabilizers, (iii) and/or surfactants. In one embodiment, the aqueous solution contains 2-200 mg/ml of protein, 0.5-2% (w/v) heat stabilizer(s), 1-10 mM buffer(s), and 0.005-0.3% ( w/v) surfactant(s). In a specific embodiment of the invention, the aqueous solution contains 50 mg/ml protein, 2% wt./about. one or more heat stabilizers, 10 mm buffer and from 0.015% to 0.1% wt./about. one or more non-ionic surfactants. In another embodiment of the invention, the aqueous solution contains 5 mg/ml protein, 0.2% wt./about. one or more heat stabilizers, 1 mm buffer and from 0.015 to 0.1% wt./about. one or more non-ionic surfactants.

In another embodiment of the invention, the protein microparticles included in the powder, formed from the claimed starting material, are subsequently coated with a biodegradable polymer. In some embodiments of the invention, the claimed formulated pharmaceutical powder is suspended in a polymer solution. In one embodiment, the polymer solution is prepared by dissolving the polymer in an organic solvent such as methylene chloride, tetrahydrofuran, ethyl acetate, dichloromethane, ethanol, or some other suitable solvent. Ethyl acetate is widely known as a safe solvent and is often used in the preparation of medicines, implants, and foods.

In some embodiments, the polymer may be ethylcellulose (EC), poly(lactic acid) (PLA), polyorthoester (POE), poly-D,L-lactide-co

- 21 041519 glycolide (PLGA) or poly-8-caprolactone (PCL). In some embodiments, the polymer is polylactic acid (PLA), polyglycolic acid (PGA), cross-linked polyethylene glycol (PEG), poly-D,L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate, PLGA alpha-tocopheryl succinate, esterified to polyethylene glycol 1000 (PLGA-TGPS), poly[1,6-bis(p-carboxyphenoxy)hexane] polyanhydride (pCPH), poly(hydroxybutyric acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly(lactic acid) copolymer (PEGPLA), poly-ε-caprolactone (PCL), polyalkyl cyanoacrylate (PAC), poly(ethyl) cyanoacrylate (PEC), polyisobutyl cyanoacrylate, poly-K-(2-hydroxypropyl) methacrylamide (poly (HPMA)), poly-v- Rhydroxybutyrate (PHB), poly-β-R-hydroxyalkanoate (PHA), poly-β-R-malic acid, phospholipidcholesterol polymers, 2-dioleoyl-sn-glycero-3-phosphatidylcholine/polyethylene glycol distearoylphosphatidylethanolamine (DOPC/PEG-DSPE)/cholesterol , polysaccharides, cellulose, ethylcellulose, methylcellulose, alginates, dextran and dextran e hydrogel polymers, amylose, inulin, pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid, polyrotaxanes and polypseudorothaxanes based on cyclodextrin (CD), polyaspartates, polyglutamates, polyleucine, copolymers of leucine and glutamate, polybutylene succinate (PBS), gelatin, collagens, fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine copolymer, polyorthoester-diamine copolymers, hidden acid-containing polyorthoesters, poly(ethylene glycol)/poly(butylene terephthalate) copolymer, and combinations and copolymers thereof.

The polymer can be dissolved in a solvent (eg, ethyl acetate) at a concentration of from about 10 mg/mL to about 300 mg/mL (i.e., 1-30% [w/v]), from about 15 to about 295 mg/mL, about 20 to about 290 mg/mL, about 25 to about 280 mg/mL, about 30 to about 270 mg/mL, about 35 to about 265 mg/mL, about 40 to about 260 mg/mL, about 45 to about 260 mg/mL, about 50 to about 255 mg/mL, about 55 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. In one embodiment, the polymer concentration in the internal feed of the spray dryer is < 10% (w/v) and the polymer concentration in the external feed is < 25% (w/v).

The inventive formulated protein powder is then added to the polymer solution at a concentration of about 10 to about 100 mg/mL, about 15 to about 95 mg/mL, about 20 to about 90 mg/mL, about 25 to about 85 mg. /ml, about 30 to about 80 mg/ml, about 35 to about 75 mg/ml, about 40 to about 70 mg/ml, about 45 to about 65 mg/ml, about 50 to about 60 mg /ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml or about 50 mg/ml. The powder and polymer solution are mixed to form a slurry or suspension, which is then dispersed and dried to form a polymer-coated pharmaceutical powder. The preferred polymer solution contains the polymer dissolved in a solvent which does not dissolve the suspended protein particles or their excipients. For example, in those embodiments in which the powder contains sucrose as an excipient, ethanol is not preferred because sucrose can dissolve in ethanol and leave behind protein in a particle that is insoluble.

In one embodiment of the invention, the suspension or suspension of powder and polymer is subjected to spray drying, which is carried out in a manner similar to the method for obtaining the claimed formulated pharmaceutical powder, but with a decrease in inlet temperature (Tin) to protect against ignition of the organic solvent or polymer. For example, if the organic solvent is dichloromethane, Tin may be 40°C; if the solvent is ethyl acetate, Tin may be 77°C and if the solvent is ethanol, Tin may be 78°C. Briefly, a slurry or suspension of powder and polymer is pumped into a spray dryer at a rate of about 0.5 to about 20 ml/min, about 1.2 ml/min, about 2.6 ml/min, or about 12.5 ml /min In one embodiment of the invention, a spray dryer with a dual feed nozzle is used to spray the slurry. In one embodiment of the invention, the viscosity of the polymer solution is <20 centipoise at 20°C. In one embodiment, the internal polymer concentration is < 10% (w/v) and the external polymer concentration is < 25%. In one embodiment, the microparticles are suspended in a polymer solution at <10% (w/v). In one embodiment, Tin is solvent dependent, eg 40°C for dichloromethane, 77°C for ethyl acetate and 78°C for ethanol. In one embodiment, T _out is less than the glass transition temperature of the claimed formulated pharmaceutical powder and depends on Tin, feed rate, solvent, and aspirator. In one embodiment of the invention T _max nozzle is < 150°C. In one embodiment of the invention, the maximum delivery rate from the outer channel is about 1.2 ml/min and the maximum delivery rate from the inner channel is about 2.6 ml/min. In one embodiment of the invention, the power supplied to the ultrasonic disperser of the spray dryer is about 0.5-2 W, and the aspirator is set to about 80%.

- 22 041519

The resulting formulated polymer coated pharmaceutical powder contains the constituent protein microparticles surrounded by a polymer shell. Such polymer-coated protein microparticles may take any of a variety of shapes. In one embodiment, certain polymer coated protein microparticles comprise 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5 protein microparticles contained within a single protein microparticle with polymer coating. In some embodiments of the invention, a single polymer-coated protein microparticle contains 1, 2, 3, 4, 5, b, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 protein microparticles. In some embodiments of the invention, the prepared pharmaceutical powder contains a polymer-coated protein microparticle containing 1-2, 2-3, or 3-4 protein microparticles.

In some embodiments, the polymer coated protein microparticles have a diameter range of about 2 to about 70 microns, about 5 to about 65 microns, about 10 to about 60 microns, about 15 to about 55 microns, about 20 up to about 50 µm, about 15 µm, about 20 µm, about 25 µm, or about 30 µm. The change in size largely reflects the thickness of the polymer shell, although the diameter of the protein shell may contribute to some extent to the change in size.

Controlling the initial concentration of the polymer solution, or the polymer itself, can be used to control the diameter of the final micron-sized particle.

In some embodiments of the invention, the resulting prepared polymer-coated pharmaceutical powder is subjected to additional drying (also known as secondary drying). Post-drying includes hot drying, lyophilization and nitrogen purge. In other embodiments of the invention, the resulting polymer-coated pharmaceutical powder is not further dried. In some embodiments of the invention, the resulting polymer-coated pharmaceutical powder is not post-dried. In some embodiments of the invention, the resulting prepared polymer-coated pharmaceutical powder is not subjected to subsequent lyophilization. In some embodiments of the invention, the resulting polymer-coated pharmaceutical powder is not post-dried with a nitrogen blast.

The polymer-coated microparticles of this invention are used in sustained release or sustained release protein therapeutics. For example, aflibercept microparticles are contemplated for use in the vitreous sustained release of aflibercept for the treatment of ocular vascular disorders, or for subcutaneous implantation for sustained release of a VEGF trap in the treatment of cancer or other diseases.

U.S. Published Application No. US 2013/0129830 A1, which was published May 23, 2013, and U.S. Provisional Application No. 62/405610, which was filed October 7, 2016, are incorporated herein by reference in their entirety. .

Examples

The following examples are provided so as to provide those skilled in the art with a description of how to make and use the methods and compositions of this invention and are not intended to limit the scope of what the inventors consider their invention.

Example 1 Spray Drying Parameters

SUBSTANCE: proposed is a method for obtaining a prepared pharmaceutical powder containing a protein by applying a spray drying step to an aqueous solution of a starting material containing a protein. A solution of the source material containing 50 mg/ml aflibercept, 10 mm phosphate (pH 6.2) and 2% wt./about. sucrose were spray dried using a BUCHI B290 mini spray dryer (Flavile, Switzerland). Here, the inlet temperature was varied from 100 to 130° C. in increments of ten degrees, and the ECD of the resulting particles was determined by MFI. As shown in FIG. 1, the temperature of the drying gas at the inlet does not affect the size distribution or morphology of the protein microparticles.

At each inlet temperature, less than one percent of the resulting particles were larger than 10 microns. Brief exposure to high temperatures and evaporative cooling prevented degradation of the aflibercept protein during spray drying.

Example 2 Protein Integrity and Stability

The molecular integrity of aflibercept in the formulated pharmaceutical powder obtained after spray drying was assessed. The reconstituted aflibercept powder was compared with the liquid formulation of aflibercept for ophthalmic administration and with the original formulation containing aflibercept. The ophthalmic composition contained 40 mg/ml aflibercept, 5% (w/v) sucrose, 0.03% (w/v) polysorbate 20, 40 mM NaCl and 10 mM phosphate, pH 6.2. Preliminary

- 23 041519 Heavily spray dried starting material and reconstituted spray dried compositions contained 50 mg/ml aflibercept, 2% (w/v) sucrose and 10 mM phosphate, pH 6.2. Aflibercept in the prepared pharmaceutical powder obtained after spray drying retained its nativeness and biological activity (see Table 5).

The stability of aflibercept in the spray dried powder was evaluated by measuring the formation of high molecular weight particles. After 3, 6 and 12 months using SE-UPLC recovered protein was determined by the formation of high molecular weight (HMW) particles at 37°C. The spray-dried aflibercept powder was incubated at 37° C. under dry conditions and then reconstituted to 50 mg/ml aflibercept for analysis. The rate of formation of HMW particles was determined by linear regression of the square root of time for time points collected after 3, 6, or 12 months at 37°C.

The results, which are shown in table. 6 indicate that the aggregation rate of spray-dried aflibercept is within pharmaceutically acceptable parameters.

Table 5. Integrity of Spray-Dried Aflibercept

Ophthalmologists- Pre Restore- weighty subjected lazy composition spray dried aflibercept drying aflibercept spraying aflibercept % Recovery (RP-HPLC) 99 100 96 Purity % Native 99.3 99.1 99.0 (SE-UPLC) Analysis of options, different charges % Basic 79.5 79.9 79.8 (iCIEF) Purity according to SDS-PAGE (electrophoresis in SDS- PAAG) % Basic 97.0 97.5 96.9 (Unreconstituted) Purity by SDS-PAGE % Basic 99.0 99.0 99.0 (Refurbished) mDSC t _t 1 (°C) 67.6 - 67.3 T _t 2 (°C) 85.3 - 85.6 FTIR % os-Spiral 8.3 - 8.3 % β-Sheet 39.2 - 39.5 % Relative activity (bioassay) 84 109 103

Table 6. Stability of Prepared Spray-Dried Aflibercept Powders

Size Base formulation 37°C particles (50 mg/mL aflibercept, 10 mM phosphate, pH _{(%n0 SE_upLC )} µm 1% w/v Sucrose 5.7% µm 2% w/v Sucrose 3, 7%

Example 3. Heat stabilizers

It has been shown that the addition of excipients to the starting material improves the thermal stability of the protein microparticles. Freeze-dried and spray-dried aflibercept (ie, 2.5 µm particles prepared from 5 mg/ml aflibercept starting material) was incubated at 50° C. under dry conditions and reconstituted to 5 mg/ml aflibercept for analysis. The results are given in Table 1. 7. The flowability of the powder in the compositions containing mannitol and isoleucine appeared to be similar to the flowability of the compositions containing only sucrose or trehalose. The inclusion of mannitol and isoleucine improved the thermal stability of spray-dried compositions containing less, i. 2.5 µm, particles.

Table 7. Thermal stability of 2.5 µm protein microparticles Thermal stabilizers + Formation rate

Basic composition

HMW Particle Size Particles (5 mg/mL aflibercept at aflibercept, 50°C (% HMW/L/mo.

1 mM phosphate, pH 6.2) by SE-UPLC) Spray dried 0.2% w/v 2.5 µm sucrose 8.5% Spray dried _ 0.2% w/v 2.5 µm trehaloses 0.1% w/v sucrose 8.8% Spray dried 2.5 µm 0.05% w/v mannitol 5.9%

0.05% May . /about . Not

- 24 041519

0.1% w/v trehaloses

Spray dried

2.5 µm 0.05% w/v

mannitol

5.7%

0.05% w/v Not

0.1% w/v

dried mannitol

2.5 µm 7.0% spray

0.1% May. /about . Not

Example 4 Particle Size and Shape

Nozzle inlet temperature, surfactant incorporation, dissolved aflibercept concentration, and incorporation of thermal stabilizers were evaluated for their effect on particle size or shape. Protein concentration and surfactant incorporation in each case resulted in smaller, and in the case of surfactant, rounder microparticles. These results are summarized in Table. 8.

Table 8. Size and shape of particles

Spray parameter Impact on size Effect on morphology Effect on protein stability drying particles particles Inlet temperature No No No (110-130°C) + Polysorbate 20 (<0.1% w/v) | Concentration Decrease Higher sphericity No Increase in HMW particles (-0.3%) after spray drying Decrease in the rate of formation solute Decrease No + Heat stabilizer No No HMW in dry thermal stress conditions. The value depends on the amount and type of heat stabilizer.

As described in Example 1, the inlet temperature was varied from 100 to 130° C. in increments of ten degrees, and the ECD of the resulting particles was determined by MFI. As shown in FIG. 1, the temperature of the drying gas at the inlet does not affect the particle size distribution or morphology. At each inlet temperature, less than one percent of the resulting particles were larger than 10 microns.

Adding 0.03-0.1% wt./about. polysorbate 20 to starting material resulted in finer particles upon spray drying compared to starting material solutions that did not contain polysorbate 20 (FIG. 2). Powder from compositions containing more than 0.1% wt./about. polysorbate 20 showed more agglomeration and was less free-flowing (i.e., more sticky). Powders with low flowability are difficult to process in subsequent processes. Therefore, compositions containing less than 0.1% wt./about. polysorbate 20 are preferred. It was also found that the addition of 0.03% wt./about. polysorbate 20 to a solution of starting material leads to the formation of more spherical particles (ie, a higher aspect ratio [minor axis/major axis]) when spray dried (see Fig. 3).

The lower solute concentration in the starting material solution resulted in a reduction in the size of the spray-dried aflibercept particles to about 2.5 µm (compared to 5 µm). A spray drying method using 50 ml/ml aflibercept, 10 mM phosphate (pH 6.2), 2% sucrose (w/v) as the starting material solution resulted in particles of -5 μm in diameter. Decreasing the concentration of the solute by a factor of 10 in the starting material solution (i.e. 5 ml/ml aflibercept, 1 mM phosphate [pH 6.2], 0.2% sucrose [w/v]) resulted in a decrease in the size of the protein microparticles by half to -2.5 µm (see Fig. 4). This means an 8-fold decrease in the particle volume (2 ³ ). Spray drying a more dilute starting material requires 10 times more time (-1 hour/g) to obtain the same amount of powder as when using compositions with a higher concentration. The higher surface to volume ratio of smaller protein microparticles, combined with longer exposure to exit temperature during spray drying, can subject the protein to thermal stress and can reduce stability to some extent.

Example 5 Polymer Coating

Protein microparticles according to this invention can be coated with a polymer. This process may be called sputtering. Briefly, the polyorthoester was dissolved in dichloromethane, ethyl acetate or ethanol to bring the viscosity to about < 20 centipoise at 20°C. When using a dual spray nozzle (BUCHI B-290 mini spray dryer (Flavile, Switzerland)) the polymer concentration was < 10% (w/v) internal and < 25% (w/v) external submission. The microparticles were suspended in a polymer solution at less than 10% w/v. Tin was 40°C when

- 25 041519 the solvent was dichloromethane, 77°C when the solvent was ethyl acetate and 78°C when the solvent was ethanol. T _out was maintained below the glass transition temperature (Tg) using a heat jacket filled with circulating ice water to cool the cyclone. T _out depended on Tin, feed rate, type of solvent and aspirator. The T _max of the nozzle was kept below 150°C. The maximum delivery rates from the outer channel and the inner channel of the dual delivery nozzle were 1.2 and 2.6 ml/min, respectively. The power supplied to the ultrasonic disperser ranged from 0.5 to 2 W; and the aspirator was set to 80%.

By MFI (see FIG. 5), a significant change in size distribution towards larger particles was observed after spray coating of polyorthoester (POE) onto the protein microparticles.

Example 6 Comparison of Protein Characteristics

Protein (aflibercept) from reconstituted protein microparticles (Recon DP) was compared to pre-spray dried protein (Pre-SD FDS) and aflibercept formulated as a liquid formulation (EYLEA® DP) to determine any adverse effects caused by spray drying. In one experiment, each of these three compositions was subjected to rapid sedimentation - analytical ultracentrifugation (SV-AUC). This method is used to detect and quantify protein aggregates (see Arthus et al., Detection of protein aggregates by sedimentation velocity analytical ultracentrifugation (SV-AUC): sources of variability and their relative importance, 98(10) J. Pharm. Sci. 3522-39, 2009). SV-AUC demonstrated that all lots are comparable in terms of HMW particles (Table 9). EYLEA® DP may have a somewhat lower aggregate content, while reconstituted spray-dried aflibercept may contain aggregates with a change in particle size. But given the variability in repeated samples, none of these trends are clearly significant, and overall these results provide no clear or strong evidence that the three samples are not comparable.

Table 9. Protein aggregation as determined by SV-AUC

Monomer sedimentation coefficient, S (average value ± SD)

EYLEA® DP 5.161 ± 0.004

Pre-SD FDS 5.181 ± 0.014

Recon DP 5.174 + 0.002% Estimated Dimer (7.5-8.0 S) (Mean ± SD) % Total Aggregates (Mean ± SD)

1.3±0.7 1.7±1.2

1.5±0.5 2.2±1.2

1.5 + 0.5 2.3 +1.2

In another experiment, each of these three compositions was subjected to size exclusion chromatography multi-angle laser light scattering (SEC-MALLS). This method is also used to detect and quantify protein aggregates as well as misfolded proteins (see P. J. Wyatt, Light scattering and the absolute characterization of macromolecules, 272 Anal. Chim. Acta 1-40, 1993; Odaka et al., Ligand-Binding Enhances the Affinity of Dimerization of the Extracellular Domain of the Epidermal Growth Factor Receptorl, 122 J. Biochem.116-121, 1997; J. S. Philo, Is any measurement method optimal for all aggregate sizes and types?8(3) AAPS J. E564-71, 2006). SEC-MALLS demonstrated that all lots were comparable in terms of peak area percentage and calculated mass for each particle type (see Table 10).

Table 10. Protein aggregation as determined by SEC-MALLS

Peak 1 (HMW) Peak 2 (main - clear)

Sample , _{π λ} Peak area , _{π xp}

MM (kDa) _o MM (kDa) Peak area %

EYLEA® DP 264.2 (0.0) 1.0 (0.4) 119.6 (1.0) 99.0(0.0)

Pre-SD FDS 264.9 (3.0) 0.5 (0.0) 119.9 (0.0) 99.5(0.0)

Recon DP 268.1 (6.0) 1.2 (0.0) 120.4 (1.0) 98.8(0.0)

The three aflibercept formulations were also subjected to capillary electrophoresis (CE) oligosaccharide fingerprinting (see Chen et al., Profiling glycoprotein n-linked oligosaccharide by capillary electrophoresis, 19(15) Electrophoresis 2639-44, 1998) and tryptic fragment fingerprinting by ultrahigh performance fluid chromatography-mass spectroscopy (UPLC-MS) (see Sinha et al., Comparison of LC and LC/MS methods for quantifying N-glycosylation in recombinant IgGs, 19(11) J. Am. Soc. Mass. Spectrom. 1643- 54, 2008) to assess glycosylation and other post-translational modifications as well as primary sequence. (CE)-oligosaccharide fingerprinting showed that all lots are comparable (Table 11).

- 26 041519

Table 11. Post-translational modifications

Post-translational modification EYLEA®DP PreSD FDS Recon DP C-terminal Without Lys ⁴³² 96.2% 96.4% 96.5% peptides with and without Lys ⁴³² With Lys ⁴³² 3.8% 3.6% 3.5% Level Asn ⁸⁴ 78.9% 79.5% 79.3% deamidation (isoAsp) in the motif Asn ⁸⁴ -Gly isoAsp ⁸⁴ 21.1% 20.5% 20.7% Level Met ¹⁰ 97.6% 97.5% 97.0% oxidation in Met ¹⁰ Met (O) ¹⁰ 2.4% 2.5% thirty% Level Met ¹⁹² 96.6% 96.6% 96.4% oxidation in Met ¹⁹² Met(O) ¹⁹² 3.4% 3.4% 3.6% Oxidation level in Met ²³⁷ Met ²³⁷ Met (0) ²³⁷ 97.2% 2.8% 97.2% 2.8% 97.2% 2.8% DYLTHR peptide level (associated with NR1 band) ⁹¹ dither ⁹⁶ 0.29% 0.34% 0.33% Glycation of the N-terminal peptide in Agd ⁵ Modified N end 9.2% 9.0% 9.0% The level of non- Glyco Ziliro in Anna 64.3% 63.8% 64.6% glycosylation of Asn ⁶⁸ non-glycosylated 35.7% 36.2% 35.4%

Peptide map chromatograms under reducing and non-reducing conditions were visually comparable among the three aflibercept samples. No unique or significant peaks corresponding to mutant sequences were observed in the spray dried sample. All post-translational modifications were present in all samples: C-terminal lysine removal, asparagine deamidation, methionine oxidation, and asparagine glycation. Site-specific glycosylation profiles at the 5 N-linked glycosylation sites are shown in all three samples. The disulfide-binding peptide profiles in all three samples corresponded to the disulfide-binding profiles expected for aflibercept. No significant levels of randomized or free cysteine-containing peptides were identified in the unreduced peptide maps in the three samples analyzed. The results of peptide mapping analyzes of spray-dried aflibercept were in accordance with the EYLEA® DP reference standard.

Example 7 Effect of Humidity on Protein Stability in Prepared Pharmaceutical Powders

Spray-dried compositions of three different proteins were prepared according to the method described in Example 1. Four prepared powders for each of the three proteins were prepared with different amounts of moisture. The three proteins were IgG1, IgG4 and a decoy molecule (aflibercept). Each aflibercept powder contained 83.8% (w/w) aflibercept, 2.1% (w/w) phosphate and 14.1% (w/w) sucrose, and the water content ranged from < 0.5% to > 6%. Each IgG1 powder contained 80.9% (w/w) IgG1, 1.5% (w/w) histidine, 16.4% (w/w) sucrose, and 0.2% polysorbate 80, and the water content ranged from <0.5% to >6%. Each IgG4 powder contained 44.5% (w/w) IgG1, 0.4% (w/w) acetate, 56.3% (w/w) sucrose, and 0.4% polysorbate 20, and the water content ranged from <0.5% to >6%.

The powders were stored at 50°C for 6 weeks. Samples were received at three weeks, one month and six weeks. Samples were reconstituted and proteins were assessed for changes in high molecular weight particles by SE-UPLC. The aggregation rate was calculated based on the HMW values obtained from the reconstituted samples. The results are presented in table. 12.

- 27 041519

Table 12 Effect of Moisture in Prepared Pharmaceutical Powder on Protein Stability

Molecule Content moisture Aggregation rate at 50 °C (% water, May ./May.) (% HMW/month^) 0.36 1.08 IgG4 0.80 1.08 3.71 1.38 12.94 11, 62 0.01 12, 60 T gg C 1 0.88 11.07 ± y ± 5, 31 8.74 13, 06 20, 84 0.01 13.29 aflibercept 0.92 12.47 5.45 10.99 14.12 27.20

CLAIM

Claims (49)

CLAIM 1. A method for obtaining a prepared pharmaceutical powder, including: a) spray drying an aqueous solution containing: i) at least one heat stabilizer selected from the group consisting of mannitol, isoleucine, proline, and combinations thereof, and an antibody or Fc receptor fusion protein in a weight ratio of 1:52:5; and ii) less than 0.1% (w/v) polysorbate 20; moreover, spray drying occurs at an inlet temperature of approximately 120°C and an outlet temperature of approximately 55°C; And b) applying heat to said atomized aqueous solution to form said formulated pharmaceutical powder, wherein heat is applied to the atomized aqueous solution to evaporate water from the powder so that the resulting formulated pharmaceutical powder has a moisture level of 3-6%, at wherein said prepared pharmaceutical powder is not subjected to secondary drying and wherein the powder has particles having a diameter of less than 10 µm. 2. The method according to claim 1, characterized in that said heat stabilizer does not contain a molecule with a molecular weight of more than 200 g/mol. 3. The method according to claim 1, characterized in that said aqueous solution contains 2-200 mg/ml of said receptor-Re-fusion protein and 0.1-2% (w/v) of said heat stabilizer. 4. The method according to claim 3, characterized in that said heat stabilizer contains (i) mannitol and (ii) isoleucine or proline. 5. The method according to claim 3, characterized in that said aqueous solution does not contain a buffer. 6. Method according to claim 3, characterized in that said aqueous solution contains 0.5-10 mM buffer. 7. The method according to claim 3, characterized in that said aqueous solution contains 0.01-0.2% (w/v) of a non-ionic surfactant. 8. The method according to claim 1, further comprising suspending the specified prepared pharmaceutical powder in an aqueous solution containing an organic solvent such as dichloromethane, ethyl acetate or ethanol and a biodegradable polymer; and spray drying said suspension to form a coated pharmaceutical powder, wherein said pharmaceutical powder is not secondary dried until said coated pharmaceutical powder is formed. 9. A method for obtaining a prepared pharmaceutical powder, including: a) spray drying an aqueous solution containing: i) a heat stabilizer selected from the group consisting of mannitol, isoleucine, proline, and combinations thereof, and an antibody or receptor Re-fusion protein in a molar ratio of less than 300 mol of said heat stabilizer per mole of said antibody or receptor Re-fusion protein ; and ii) less than 0.1% (w/v) polysorbate 20; moreover, spray drying occurs at an inlet temperature of approximately 120°C and an outlet temperature of approximately 55°C; And b) applying heat to said atomized aqueous solution to form said formulated pharmaceutical powder, where heat is applied to the atomized aqueous solution such that the resulting prepared pharmaceutical powder has a moisture content of 3-6%, wherein said prepared pharmaceutical powder not subjected to secondary drying and where the powder has particles having a diameter of less than 10 microns. 10. The method according to claim 9, characterized in that said heat stabilizer does not contain a molecule with a molecular weight of more than 200 g/mol. - 28 041519 11. The method of claim 9, wherein said aqueous solution contains 2-200 mg/ml of said receptor-Fc fusion protein and 0.1-2% (w/v) of said heat stabilizer. 12. The method according to claim 11, characterized in that said heat stabilizer contains (i) mannitol and (ii) isoleucine or proline. 13. The method according to claim 11, characterized in that said aqueous solution does not contain a buffer. 14. The method according to claim 11, characterized in that said aqueous solution contains 0.5-10 mM buffer. 15. The method according to claim 11, characterized in that said aqueous solution contains 0.01-0.2% (w/v) of a non-ionic surfactant. 16. The method according to claim 9, further comprising suspending the specified prepared pharmaceutical powder in an aqueous solution containing an organic solvent such as dichloromethane, ethyl acetate or ethanol and a biodegradable polymer; and spray drying said suspension to form a coated pharmaceutical powder, wherein said pharmaceutical powder is not secondary dried until said coated pharmaceutical powder is formed. 17. Prepared pharmaceutical powder obtained in accordance with the method according to claim 1, containing approximately 60-97% (wt./wt.) receptor-Fc-fused protein and approximately 3-40% (wt./wt.) heat stabilizer, selected from the group consisting of mannitol, isoleucine, proline and combinations thereof, wherein said prepared pharmaceutical powder contains 3-6% water, and said prepared pharmaceutical powder has not been subjected to secondary drying and where the powder has particles having a diameter of less than 10 microns. 18. Prepared pharmaceutical powder according to claim 17, additionally containing 0.5-10 mm buffer. 19. Prepared pharmaceutical powder according to claim 18, characterized in that said buffer is selected from the group consisting of phosphate, histidine and acetate. 20. The prepared pharmaceutical powder according to claim 17, characterized in that said heat stabilizer does not contain a molecule with a molecular weight of more than 200 g/mol. 21. The prepared pharmaceutical powder according to claim 17, characterized in that said heat stabilizer is selected from the group consisting of mannitol, isoleucine, proline, and combinations thereof. 22. The prepared pharmaceutical powder according to claim 17, containing 71-75% (wt./wt.) receptor Fc fusion protein, 14-15% (wt./wt.) mannitol, 14-15% (wt./wt.) ) isoleucine or proline, 2-2.5% (w/w) buffer and 3-8% (w/w) water. 23. The prepared pharmaceutical powder according to claim 17, further comprising a polymer coating. 24. A method for obtaining a prepared pharmaceutical powder, including: a) spraying an aqueous solution containing: i) a heat stabilizer and from 50 to 180 mg/ml VEGF trap protein with a mass ratio of 1:5-2:5, where the heat stabilizer is selected from sucrose, trehalose, mannitol, isoleucine, proline, or a combination thereof; and ii) 0.03-0.1% (w/v) polyoxyethylene sorbitan monolaurate; where spray drying occurs at an inlet temperature of 100 to 130°C and an outlet temperature of 55°C; And b) applying heat to said atomized aqueous solution to form said prepared pharmaceutical powder, where heat is applied to the atomized aqueous solution so that the resulting pharmaceutical powder has a moisture content of 3-6%, where said prepared pharmaceutical powder is not subjected to secondary drying and the powder contains spherical particles with a diameter of less than 10 microns. 25. The method of claim 24, wherein said VEGF decoy protein is aflibercept. 26. The method according to claim 24 or 25, wherein said heat stabilizer does not contain a molecule with a molecular weight greater than 200 g/mol. 27. The method of claim 24 or 25, wherein said aqueous solution contains 0.1-2% (w/v) of said heat stabilizer. 28. The method of claim 27, wherein said aqueous solution does not contain a buffer. 29. The method of claim 27, wherein said aqueous solution contains 0.5-10 mM buffer. 30. The method of claim 27, wherein said aqueous solution contains 0.01-0.2% (w/v) of a nonionic surfactant. 31. The method of claim 24 or 25, further comprising suspending said formulated pharmaceutical powder in an organic solvent such as dichloromethane, ethyl acetate or ethanol containing a biodegradable polymer; and spray drying said suspension to form a coated pharmaceutical powder, wherein said coated pharmaceutical powder is not subjected to secondary drying prior to forming said formulated coated pharmaceutical powder. 32. A method for producing a prepared pharmaceutical powder, including: a) spraying an aqueous solution containing: - 29 041519 i) a heat stabilizer and 50 to 180 mg/ml of a VEGF trap protein at a molar ratio of less than 300 mol of said heat stabilizer per mole of said VEGF trap protein, wherein the heat stabilizer is selected from sucrose, trehalose, mannitol, isoleucine, proline, or a combination thereof; and ii) 0.03-0.1% (w/v) polyoxyethylene sorbitan monolaurate, where spray drying occurs at an inlet temperature of 100 to 130°C and an outlet temperature of 55°C; And b) applying heat to said atomized aqueous solution to form said prepared pharmaceutical powder, where heat is applied to the atomized aqueous solution so that the resulting prepared pharmaceutical powder has a moisture content of 3-6%, where said prepared pharmaceutical powder is not subjected to secondary drying and the powder contains spherical particles with a diameter of less than 10 microns. 33. The method of claim 32, wherein said VEGF decoy protein is aflibercept. 34. The method according to claim 32 or 33, wherein said heat stabilizer does not contain a molecule with a molecular weight of more than 200 g/mol. 35. The method of claim 32 or 33, wherein said heat stabilizer is selected from the group consisting of mannitol, isoleucine, proline, and combinations thereof. 36. The method of claim 32 or 33, wherein said aqueous solution contains 0.1-2% (w/v) of said heat stabilizer. 37. The method according to claim 32 or 33, wherein said aqueous solution does not contain a buffer. 38. The method of claim 32 or 33, wherein said aqueous solution contains 0.5-10 mM buffer. 39. The method of claim 32 or 33, wherein said aqueous solution contains 0.01-0.2% (w/v) of a nonionic surfactant. 40. The method of claim 32 or 33, further comprising suspending said formulated pharmaceutical powder in an organic solvent such as dichloromethane, ethyl acetate or ethanol containing a biodegradable polymer; and spray drying said suspension to form a coated pharmaceutical powder, wherein said formulated coated pharmaceutical powder is not subjected to secondary drying prior to forming said formulated coated pharmaceutical powder. 41. Prepared pharmaceutical powder obtained in accordance with the method according to claim 1, containing 60-97% (wt./wt.) VEGF trap protein and 3-40% (wt./wt.) heat stabilizer, where the heat stabilizer is selected from sucrose, trehalose, mannitol, isoleucine, proline, or combinations thereof, and wherein said prepared pharmaceutical powder contains 3-6% water, wherein said prepared pharmaceutical powder is not subjected to secondary drying. 42. Prepared pharmaceutical powder of claim 41, additionally containing 0.5-10 mM buffer. 43. The formulated pharmaceutical powder of claim 42, wherein said buffer is selected from the group consisting of phosphate, histidine and acetate. 44. The formulated pharmaceutical powder of claim 41, wherein said VEGF decoy protein is aflibercept. 45. Prepared pharmaceutical powder of claim 41 or 44, where the heat stabilizer does not contain a molecule with a molecular weight of more than 200 g/mol. 46. The prepared pharmaceutical powder of claim 41 or 44, wherein the heat stabilizer is selected from the group consisting of mannitol, isoleucine, proline, and combinations thereof. 47. Prepared pharmaceutical powder according to any one of claims 41 or 44, containing 71-75% (wt./wt.) VEGF trap protein, 14-15% (wt./wt.) mannitol, 14-15% (wt.) ./wt.) isoleucine or proline, 2-2.5% (wt./wt.) buffer and 3-8% (wt./wt.) water. 48. The pharmaceutical formulation of claim 41, wherein said VEGF decoy protein is an antibody or Fc receptor fusion protein. 49. The prepared pharmaceutical powder according to claim 41 or 44 further containing a polymer coating.