Formulations containing high concentrations of vegr receptor fusion proteins

Abstract

The present invention provides ophthalmic formulations having high concentrations of vascular endothelial growth factor (VEGF) receptor fusion proteins and high stability during storage. Methods of using the high concentration formulations to treat vascular eye diseases are also provided.

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 813,882, filed March 5, 2019; U.S. Provisional Patent Application No. 62 / 769,876, filed November 20, 2018; U.S. Provisional Patent Application No. 62 / 752,127, filed October 29, 2018; and U.S. Provisional Patent Application No. 62 / 669,506, filed May 10, 2018; each of which is incorporated herein by reference in its entirety. Technical Field

[0002] The embodiments described herein generally relate to formulations containing high concentrations of VEGF receptor fusion protein suitable for intraocular application. More specifically, the embodiments described herein provide liquid pharmaceutical formulations for intravitreal application, wherein the formulation contains greater than 40 mg / ml of VEGF receptor fusion protein and exhibits pharmaceutically acceptable potency, stability, viscosity, and pH.

[0003] sequence list

[0004] An official copy of the sequence list, along with the instruction manual, was submitted electronically via EFS-Web in ASCII format as a sequence list file named "10430P1-US_SEQ_LIST_ST25.txt", created on May 9, 2018, and approximately 7KB in size. The sequence list contained in this ASCII document is part of the instruction manual and is incorporated herein by reference in its entirety. Background Technology

[0005] The development of therapeutically useful liquid formulations requires combinations of varying amounts of components to provide a functional and stable delivery vehicle for the target drug. This is especially true when the drug is a protein, and further, an antibody. Antibody formulations have long presented challenges for drug developers and manufacturers because antibody activity and administration typically require pharmaceutically acceptable potency, molality, protein stability, viscosity, and appropriate pH. These challenges are exacerbated when the antibody concentration in the formulation is high (e.g., exceeding the concentrations of most drug-marketed antibodies by 20 to 40 mg / ml).

[0006] Higher concentrations of antibody formulations can shorten injection time, reduce injection volume, decrease antibody administration frequency, and improve production and storage efficiency. However, as mentioned above, the higher the concentration of the antibody or protein, the more difficult it is to maintain the appropriate activity and delivery parameters of the formulation. In particular, high-concentration antibody and protein formulations often compete with increased protein aggregation and viscosity, leading to lower overall antibody or protein potency, lower manufacturing stability, and poorer storage stability. Due to the problems associated with high-concentration protein or antibody formulations, few pharmaceutically acceptable formulations have been developed. There is a need in the art to prepare high-concentration, highly stable antibody and protein formulations that include appropriate potency, stability, viscosity, osmotic concentration, and pH.

[0007] One such protein requiring high-concentration formulations is the vascular endothelial growth factor (VEGF) receptor fusion protein. VEGF receptor fusion proteins are used in many ophthalmic formulations to block VEGF function, for example... (Regeneron Pharmaceuticals, Inc.). Formulations containing high concentrations of VEGF receptor fusion protein can shorten ocular injection time, reduce injection volume, decrease the number of possible injections per administration cycle, and improve manufacturing and storage efficiency.

[0008] The present invention aims to overcome one or more of the problems discussed above. Summary of the Invention

[0009] The various embodiments described herein include formulations containing high concentrations of protein (e.g., suitable for intravitreal administration), particularly formulations containing high concentrations of vascular endothelial growth factor (VEGF) receptor fusion protein. Formulations containing high concentrations of VEGF receptor fusion protein offer numerous therapeutic and economic benefits, including pharmaceutically acceptable potency, long-term manufacturing and storage stability, and viscosity and pH compatibility with intraocular injection. Formulations containing high concentrations of VEGF receptor fusion protein also reduce ocular dosage, a benefit that avoids adverse effects on the limited volume of the eye.

[0010] The embodiments described herein provide formulations comprising a VEGF receptor fusion protein, a buffer solution, a heat stabilizer, a viscosity reducer, and a surfactant. In other embodiments, the formulations do not contain a viscosity reducer. The formulations of the present invention have a pH and viscosity suitable for injection, particularly suitable for therapeutic ocular injection.

[0011] In one embodiment of the invention, a pharmaceutical formulation of the invention is provided having a VEGF receptor fusion protein at a concentration of at least 41 mg / ml or comprising a single dose of the VEGF receptor fusion protein (discussed herein) at a concentration of less than about 100 μL, less than about 50 μL, about 50 μL, about 57 μL, about 60 μL, about 70 μL, or about 75 μL, a buffer solution, optionally a geothermal stabilizer and / or a viscosity reducer, and a surfactant, wherein the pH of the formulation is about 5.0 to about 6.8 (e.g., 5.8).

[0012] In one embodiment of the present invention, the concentration of the VEGF receptor fusion protein is about 30 mg / ml, 60 mg / ml, 114 mg / ml, 120 mg / ml or 140 mg / ml.

[0013] In one embodiment of the invention, the surfactant may be a nonionic surfactant (e.g., about 0.02% to about 0.1%; or about 0.03% (w / v)). In another embodiment of the invention, the surfactant is a nonionic surfactant having a polyoxyethylene moiety, such as polysorbate 20 (PS20), polysorbate 80 (PS80), poloxamer 188, polyethylene glycol 3350, or mixtures thereof.

[0014] In one embodiment of the invention, the buffer is a histidine-based buffer (e.g., 10 mM or 20 mM), such as histidine, histidine hydrochloride, or histidine acetate; a phosphate-based buffer, such as sodium phosphate (e.g., 10 mM); an acetate-based buffer, such as sodium acetate and acetic acid; or a citrate-based buffer, such as sodium citrate or citric acid. In one embodiment of the invention, if the buffer is a phosphate buffer, the pH is about 5.7 to about 8.0, about 5.8 to about 8.0, about 5.7 to about 7.0, about 5.8 to about 7.0, about 5.9 to about 7.0, or about 6.0 to 7.0; if the buffer is a histidine buffer, the pH is about 5.5 to about 6.5; if the buffer is a citrate buffer, the pH is about 3.0 to about 6.2 or about 5.0 to about 6.0; if the buffer is an acetate buffer, the pH is about 3.7 to about 5.6 or about 5.0 to about 6.0.

[0015] In one embodiment of the invention, the heat stabilizer is a sugar, such as sucrose (e.g., about 2.5%, 5%, or 8%, 10%, or 20% (w / v), for example about 2-20%), mannitol, sorbitol, or trehalose; L-proline (e.g., about 2%, 3%, or 4% (w / v)); glycine (e.g., about 50 mM), glycerol, taurine (e.g., about 50 mM), or propanesulfonic acid (e.g., about 50 mM) or any combination thereof.

[0016] In some embodiments of the invention, the pharmaceutical formulations of the invention comprise a viscosity reducer, such as arginine hydrochloride (e.g., L-arginine monohydrochloride) (e.g., 50 mM), lysine, sodium chloride (e.g., 40 mM or 50 mM), or magnesium chloride. Alternatively, in other embodiments, the formulations of the invention explicitly exclude substantially all (if not all) viscosity reducers.

[0017] In one embodiment of the invention, the pharmaceutical formulation of the invention comprises, consists of, or is substantially composed of any of the formulations A-KKKK described herein.

[0018] In this context, the pharmaceutical formulation of the present invention may comprise a VEGF receptor fusion protein (e.g., apracept or conbercept) at concentrations of: about 41 mg / ml to about 275 mg / ml, about 80 mg / ml to about 275 mg / ml, about 140 mg / ml to about 150 mg / ml, about 140 mg / ml to about 159 or 160 mg / ml; including about 60 mg / ml, about 80 mg / ml, about 100 mg / ml, about 113.3 mg / ml, about 114.3 mg / ml, about 120 mg / ml, about 133.3 mg / ml, about 140 mg / ml, about 150 mg / ml, about 200 mg / ml, or about 250 mg / ml. In one embodiment of the invention, the formulation comprises about 40 mg / ml of the VEGF receptor fusion protein.

[0019] Some aspects of the pharmaceutical formulation of the present invention include a heat stabilizer at a concentration of about 2% (w / v) to about 9% (w / v) or about 4% (w / v) to about 9% (w / v), provided that when the heat stabilizer is taurine or propanesulfonic acid, the concentration of the stabilizer is about 25 mM to about 100 mM, the concentration of the surfactant is about 0.02% (w / v) to about 0.1% (w / v) (e.g., about 0.03%), and the buffer solution is about 5 mM to about 15 mM.

[0020] VEGF receptor fusion proteins can be, for example:

[0021] • Encoded by the nucleic acid sequence of SEQ ID NO: 1 or nucleotides 79-1374 or 79-1371 of SEQ ID NO: 1;

[0022] • Contains amino acid SEQ ID NO: 2 or amino acid 27-457 or 27-458 of SEQ ID NO: 2;

[0023] ·include:

[0024] (1) VEGFR1 component, which contains amino acids 27 to 129 of SEQ ID NO: 2;

[0025] (2) VEGFR2 component, which contains amino acids 130 to 231 of SEQ ID NO: 2;

[0026] (3) A polymeric component (“FcΔC1(a)”) comprising amino acids 232 to 457 or 458 of SEQ ID NO: 2 (the C-terminal amino acid K458 of SEQ ID NO: 2 may or may not be included in the VEGF receptor fusion protein). Note that amino acids 1 to 26 of SEQ ID NO: 2 are the signal sequence;

[0027] ● Contains an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor (e.g., VEGFR1) and an Ig domain 3 of a second VEGF receptor (e.g., VEGFR2), and optionally also contains an Ig domain 4 of a second VEGF receptor (e.g., VEGFR2) and a polymeric component (e.g., the Fc domain of IgG).

[0028] • For comparupip; or

[0029] • For apasys.

[0030] VEGF Trap

[0031] atggtcagctactgggacaccggggtcctgctgtgcgcgctgctcagctgtctgcttctc

[0032] acaggatctagttccggaagtgataccggtagacctttcgtagagatgtacagtgaaatc

[0033] cccgaaattatacacatgactgaaggaagggagctcgtcattccctgccgggttacgtca

[0034] cctaacatcactgttatactttaaaaaagtttccacttgacactttgatccctgatggaaaa

[0035] cgcataatctggggacagtagaaagggcttcatcatatcaaatgcaacgtacaaagaaata

[0036] gggcttctgacctgtgaagcaacagtcaatgggcatttgtataagacaaactatctcaca

[0037] catcgacaaaccaatacaatcatagatgtggttctgagtccgtctcatggaattgaacta

[0038] tctgttggagaaaagcttgtcttaaattgtacagcaagaactgaactaaatgtggggatt

[0039] gacttcaactgggaatacccttcttcgaagcatcagcataagaaacttgtaaaccgagac

[0040] ctaaaaacccagtctgggagtgagatgaagaaatttttgagcaccttaactatagatggt

[0041] gtaacccggagtgaccaaggattgtacacctgtgcagcatccagtgggctgatgaccaag

[0042] aagaacagcacatttgtcagggtccatgaaaaggacaaaactcacacatgcccaccgtgc

[0043] ccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggac

[0044] accctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaa

[0045] gaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagaca

[0046] aagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctg

[0047] caccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctccca

[0048] gcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtac

[0049] accctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtc

[0050] aaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaac

[0051] aactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaag

[0052] ctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcat

[0053] gaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatga

[0054] (SEQ ID NO:1)

[0055] MVSYWDTGVLLCALLSCLLLTGSSSGSDTGRPFVEMYSEIPEIIHMTEGRELVI

[0056] PCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATV

[0057] NGHLYKTNYLTHRQTNTIIDVVLSPSHGIELSVGEKLVLNCTARTELNVGIDF

[0058] NWEYPSSKHQHKKLVNRDLKTQSGSEMKKFLSTLTIDGVTRSDQGLYTCAA

[0059] SSGLMTKKNSTFVRVHEKDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR

[0060] TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV

[0061] LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE

[0062] LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK

[0063] LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0064] (SEQ ID NO:2)

[0065] AAs 1-26 = Signal sequence AAs 27-129 = Flt1-D2 (VEGFR1-D2)

[0066] AAs 130-231 = Flk1-D3 (VEGFR2-D3)

[0067] AAs 232-458 = FcΔC1

[0068] In another embodiment of the invention, a container is provided comprising an aqueous solution of about 5 mM to about 25 mM pharmaceutically acceptable buffer, about 4% (w / v) to about 9% (w / v) pharmaceutically acceptable heat stabilizer, about 0.02% (w / v) to about 0.1% (w / v) pharmaceutically acceptable surfactant, and about 41 mg / ml to about 275 mg / ml of VEGF receptor fusion protein. When the heat stabilizer comprises taurine or propanesulfonic acid, the concentration of taurine or propanesulfonic acid is about 25 mM to about 100 mM. The pH of the aqueous solution is about 5.0 to about 6.8, and may be about 5.5 to about 6.2 (e.g., 5.8). In some aspects, the container is a vial or syringe. In other aspects, the aqueous solution does not contain inorganic salts. When the heat stabilizer is a sugar, it can be sucrose, mannitol, sorbitol, or trehalose, and can be present in an amount between about 4% (w / v) and 9% (w / v); the surfactant can be a nonionic surfactant and can contain a polyoxyethylene moiety, such as polysorbate 20, polysorbate 80, poloxamer 188, or polyethylene glycol 3350, at a concentration of about 0.02% to 0.1% by weight (w / v) per volume, more typically 0.02% to 0.04% (w / v). In one embodiment of the invention, the VEGF receptor fusion protein is encoded by the nucleic acid sequence of SEQ ID NO: 1 or nucleotides 79-1374 or 79-1371 of SEQ ID NO: 1; contains amino acids of SEQ ID NO: 2 or amino acids 27-457 or 27-458 of SEQ ID NO: 2; comprises (1) a VEGFR1 component containing amino acids 27 to 129 of SEQ ID NO: 2; (2) a VEGFR2 component containing amino acids 130 to 231 of SEQ ID NO: 2; and (3) a polymeric component (“FcΔC1(a)”) containing amino acids 232-457 of SEQ ID NO: 2 (SEQ ID NO: 1). NO:2's C-terminal amino acid, K458, may or may not be included in the VEGF receptor fusion protein; comprising an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor (e.g., VEGFR1) and an Ig domain 3 of a second VEGF receptor (e.g., VEGFR2), optionally further comprising an Ig domain 4 of a second VEGF receptor (e.g., VEGFR2) and a polymeric component (e.g., the Fc domain of IgG); for conbercept or apracept.

[0069] In other embodiments, a pharmaceutical formulation of the present invention is provided having a VEGF receptor fusion protein in an aqueous carrier, wherein the viscosity of said aqueous carrier is about 10 cP to about 15 cP at 20°C, more typically about 10 cP to 13 cP at 20°C, and most typically about 11 cP to about 12 cP at 20°C (e.g., about 6.0, 7.3, 11.5, or 12.0 cP at 20°C). In one embodiment of the invention, the viscosity is about 12 cP to about 15 cP at 20°C. The pH of the formulation can be about 5.8 to about 6.5 (e.g., about 5.8). In some cases, the formulation does not contain a viscosity reducer, for example, it does not contain arginine hydrochloride, lysine, sodium chloride, or magnesium chloride. In other cases, the formulation comprises 10 mM histidine hydrochloride or 10 mM histidine acetate. The sugar may be sucrose, mannitol, sorbitol, or trehalose, and may be present in an amount between about 4% (w / v) and 9% (w / v) (e.g., about 5%). The surfactant may be a nonionic surfactant and may contain a polyoxyethylene moiety, such as polysorbate 20, polysorbate 80, poloxamer 188, or polyethylene glycol 3350, at a concentration of about 0.02% to 0.1% by weight (w / v) per volume, more typically 0.02% to 0.04% (w / v) (e.g., 0.03%). Additionally, the formulation may further contain a heat stabilizer selected from taurine or propanesulfonic acid, at a concentration of about 25 mM to about 100 mM, more typically about 50 mM to about 70 mM. In one embodiment of the invention, the formulation comprises a VEGF receptor fusion protein, such as apracept (e.g., at concentrations of about 41-275 mg / ml, about 50 mg / ml, about 100 mg / ml, about 115 mg / ml, about 125 mg / ml, about 150 mg / ml, about 200 mg / ml, or any “high” concentration discussed herein), about 10 mM of histidine-based buffer, about 5% (w / v) sucrose, about 0.03% (w / v) nonionic surfactant, such as polysorbate, such as polysorbate 20, and about 50 mM of arginine, L-arginine, or L-arginine hydrochloride, at a pH of about 5.8.In one embodiment of the invention, the VEGF receptor fusion protein is encoded by the nucleic acid sequence of SEQ ID NO: 1 or nucleotides 79-1374 or 79-1371 of SEQ ID NO: 1; contains amino acids of SEQ ID NO: 2 or amino acids 27-457 or 27-458 of SEQ ID NO: 2; comprises (1) a VEGFR1 component containing amino acids 27 to 129 of SEQ ID NO: 2; (2) a VEGFR2 component containing amino acids 130 to 231 of SEQ ID NO: 2; and (3) a polymeric component (“FcΔC1(a)”) containing amino acids 232-457 of SEQ ID NO: 2 (SEQ ID NO: 1). NO:2's C-terminal amino acid, K458, may or may not be included in the VEGF receptor fusion protein; comprising an immunoglobulin-like (Ig) domain 2 of a first VEGF receptor (e.g., VEGFR1) and an Ig domain 3 of a second VEGF receptor (e.g., VEGFR2), optionally further comprising an Ig domain 4 of a second VEGF receptor (e.g., VEGFR2) and a polymeric component (e.g., the Fc domain of IgG); for conbercept or apracept.

[0070] In yet another embodiment, a pharmaceutical formulation of the present invention is provided, comprising a VEGF receptor fusion protein at a concentration of about 80 mg / ml to about 275 mg / ml in a pharmaceutically acceptable buffer. In some cases, the stable pharmaceutical formulation may also contain taurine or propanesulfonic acid. The formulation is stable at a temperature of about 2°C to about 8°C for about 24-36 months. In some cases, when stored at these temperatures and for these time periods, the VEGF receptor fusion protein exhibits an increase of less than about 5% in the high molecular weight species, and more generally, an increase of less than about 4.5%, less than about 4.0%, less than about 3.5%, less than about 3.0%, less than about 2.5%, less than about 2.0%, and / or less than about 1.5%. In other cases, when the formulation is stored at these temperatures and for these time periods, the VEGF receptor fusion protein exhibits an increase of less than 2.0% to 3.0% in the high molecular weight species.

[0071] Embodiments herein may also include pharmaceutical formulations comprising apracept, a pH buffer solution, sugar, and a surfactant, wherein the concentration of apracept is from 41 mg / ml to about 275 mg / ml. Alternatively, embodiments may be stable liquid pharmaceutical formulations comprising conbercept, a pH buffer solution, sugar, and a surfactant, wherein the concentration of conbercept is from 41 mg / ml to about 275 mg / ml. Stable liquid pharmaceutical formulations may also contain taurine or propanesulfonic acid. In some aspects, the concentration of apracept or conbercept is 80 mg / ml or 150 mg / ml. In other aspects, the formulations do not contain inorganic salts, such as sodium chloride.

[0072] Some embodiments involve sterile syringes pre-filled with an aqueous solution containing 80 mg / ml apracept, conbercept, 10 mM histidine hydrochloride, histidine acetate, or sodium phosphate, 5% (w / v) sucrose, mannitol, sorbitol, or trehalose, 0.03% (w / v) polysorbate 20, polysorbate 80, poloxamer 188, or polyethylene glycol 3350, and 40 mM sodium chloride. Aspects described herein include the further addition of approximately 25 mM to approximately 100 mM, and more typically approximately 50 mM to approximately 70 mM, taurine or propanesulfonic acid. In some aspects, the syringe is prefilled with an aqueous solution containing 80 mg / ml apracept, conbercept, 10 mM histidine hydrochloride, histidine acetate, or sodium phosphate, 8% (w / v) sucrose, mannitol, sorbitol, or trehalose, and 0.03% (w / v) polysorbate 20, polysorbate 80, poloxamer 188, or polyethylene glycol 3350. Aspects herein include the further addition of about 25 mM to about 100 mM, more typically about 50 mM to about 70 mM, taurine or propanesulfonic acid. In other respects, the syringe is prefilled with an aqueous solution containing 150 mg / ml apracept, conbercept, 10 mM histidine hydrochloride, histidine acetate, or sodium phosphate, 5% (w / v) sucrose, mannitol, sorbitol, or trehalose, 0.03% (w / v) polysorbate 20, polysorbate 80, poloxamer 188, or polyethylene glycol 3350, and 40 mM sodium chloride, at a pH of approximately 6.2. Further additions of taurine or propanesulfonic acid may be permitted. In other respects, the syringe is prefilled with an aqueous solution containing 150 mg / ml apracept, conbercept, 10 mM histidine hydrochloride, histidine acetate, or sodium phosphate, 8% (w / v) sucrose, mannitol, sorbitol, or trehalose, and 0.03% (w / v) polysorbate 20, polysorbate 80, poloxamer 188, or polyethylene glycol 3350, with a pH of approximately 6.2, and the aqueous solution does not contain inorganic salts. The solution may further include approximately 25 mM to approximately 100 mM, and more typically approximately 50 mM to approximately 70 mM, taurine or propanesulfonic acid. In other respects, the syringe is prefilled with an aqueous solution containing 150 mg / ml apraceptor or conbercept, 10 mM sodium acetate or acetic acid, 5% (w / v) glycerol, and 0.03% (w / v) polysorbate 20, polysorbate 80, poloxamer 188 or polyethylene glycol 3350 and 40 mM sodium chloride, with a pH of approximately 6.2. In other respects, the syringe is prefilled with an aqueous solution containing 150 mg / ml apraceptor or conbercept, 10 mM sodium acetate or acetic acid, 8% (w / v) glycerol, and 0.03% (w / v) polysorbate 20, polysorbate 80, poloxamer 188 or polyethylene glycol 3350 and 40 mM sodium chloride, with a pH of approximately 6.2.

[0073] This invention also provides methods for preparing any of the formulations described herein, including the step of combining each component of the formulation into a single composition. Such methods may include the step of adding the resulting formulation to a vial or injection device. Any composition that is a product of such methods also constitutes part of this invention. For example, embodiments herein also include methods for preparing a formulation by combining a histidine-based, citrate-based, acetate-based, or phosphate-based buffer with sucrose, polysorbate 20, and a VEGF receptor fusion protein; and optionally, one or more additional components, such as those described herein. In some cases, the formulation is prepared to also contain taurine or propanesulfonic acid, but in the absence of taurine or propanesulfonic acid, it may also be free of inorganic salts. In aspects herein, the sucrose content is from about 4% to about 10% per volume weight, the polysorbate 20 content is from about 0.02% to about 0.1% per volume weight, and the concentration of the receptor fusion protein is from about 41 mg / ml to about 275 mg / ml. When the formulation contains taurine or propanesulfonic acid, it is present, for example, in amounts from about 25 mM to about 100 mM, but may also be included in amounts from 50 mM to 70 mM. The method may include loading a predetermined volume of the preparation into a sterile syringe such that the volume contains a dose of 0.1 mg to 10 mg of VEGF receptor fusion protein.

[0074] The present invention also provides a method for administering the formulation of the present invention to a subject (e.g., a human), including intraocular injection (e.g., intravitreal injection) of the formulation into the eye of the subject. The present invention also provides a method for administering the formulation of the present invention to a subject (e.g., a human), including implanting an intravitreal implant comprising the formulation of the present invention into the vitreous body of the subject.

[0075] The embodiments described herein also include methods for treating vascular eye diseases in subjects of need, such as age-related macular degeneration (wet), macular edema following retinal vein occlusion, retinal vein occlusion (RVO), central retinal vein occlusion (CRVO), branch retinal vein occlusion (BRVO), diabetic macular edema (DME), choroidal neovascularization (CNV), iris neovascularization, neovascular glaucoma, postoperative fibrosis of glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, corneal opacity, pterygium, vascular retinopathy, or diabetic retinopathy (e.g., nonproliferative diabetic retinopathy and / or proliferative diabetic retinopathy), by intraocular injection of at least about 2 mg (e.g., 4 mg, 6 mg, or 8 mg) of VEGF receptor fusion protein (e.g., apracept or conbercept), such as any formulation described herein, into the eye of a subject of need. In one embodiment of the invention, the injection volume is 100 μL or less (e.g., 100 μL, 50 μL, or 57 μL). The method comprises intravitreal injection of a premixed aqueous solution having a concentration of VEGF receptor fusion protein of about 41 mg / ml to about 275 mg / ml, pharmaceutically acceptable sugars, pharmaceutically acceptable buffers, and pharmaceutically acceptable surfactants. Treatment methods using premixed aqueous solutions do not require dilution. In aspects herein, the premixed aqueous solution has a pH of about 6.0 to about 6.5 and a viscosity of about 10 cP to 13 cP.

[0076] In terms of treatment methods, the VEGF receptor fusion protein comprises VEGFR1R2-FcΔC1(a) encoded by the nucleic acid sequence of SEQ ID NO: 1, amino acids 27 to 129 of SEQ ID NO: 2, a VEGFR2 component comprising amino acids 130 to 231 of SEQ ID NO: 2, or a polymerized component comprising amino acids 232 to 457 of SEQ ID NO: 2, or any combination thereof. In one embodiment of the invention, the VEGF receptor fusion protein is apracept or conbercept.

[0077] Other implementations based on the disclosure herein will become apparent from the following detailed reading. Attached Figure Description

[0078] Referring to the accompanying drawings, the above and other aspects, features and advantages of the embodiments disclosed herein will become more apparent to those skilled in the art.

[0079] Figure 1AThis is a graph determining the stability of the VEGF receptor fusion protein (apasip) in four different formulations (B, H, D, and G) over a 36-month period at 2°C to 8°C. The stability of the VEGF receptor fusion protein was tested using size exclusion ultra-high performance liquid chromatography (SE-UPLC) to identify the formation of high molecular weight (HMW) species (signs of protein degradation).

[0080] Figure 1B This graph also shows the stability of the VEGF receptor fusion protein (apasip) in four formulations (B, H, D, and G) over a 36-month period at 2°C to 8°C. The stability of the VEGF receptor fusion protein was tested using SE-UPLC to identify the percentage of the predominant species in each formulation.

[0081] Figure 1C The bar graph shows the formation of VEGF receptor fusion protein charge heterogeneity in four formulations (B, H, D, and G) over a period of 36 months at 2°C to 8°C. The levels of acidic substances were tested at manufacturing (0.0), 12 months, 24 months, and 36 months using imaging capillary isoelectric focusing (iCIEF).

[0082] Figure 1D The bar graph shows the formation of VEGF receptor fusion protein charge heterogeneity in four formulations (B, H, D, and G) over a 36-month period at 2°C to 8°C. Major class components were tested using iCIEF at manufacturing (0.0), 12 months, 24 months, and 36 months.

[0083] Figure 2A This graph shows the stability of 150 mg / ml VEGF receptor fusion protein (apasicept) in a formulation of 10 mM sodium phosphate, 8% (w / v) sucrose, and 0.03% polysorbate 20 (w / v) at pH 6.2 (with and without L-arginine hydrochloride) over 28 days at 37°C. The stability of the VEGF receptor fusion protein was tested using SE-UPLC to identify HMW species formation.

[0084] Figure 2B This graph shows the stability of 150 mg / ml VEGF receptor fusion protein in formulations of 10 mM sodium phosphate, 8% (w / v) sucrose, and 0.03% polysorbate 20 (w / v) at pH 6.2 (with and without L-arginine hydrochloride) over a 28-day period at 37°C. The stability of the VEGF receptor fusion protein was tested using SE-UPLC to identify the percentage of the predominant species in each formulation.

[0085] Figure 3AThis is a bar graph showing the viscosity of a 10 mM sodium phosphate buffer formulation containing 155 mg / ml VEGF receptor fusion protein, salt-free, and with 100 mM arginine, 200 mM arginine, 50 mM lysine, 200 mM lysine, 50 mM sodium chloride, or 100 mM sodium chloride. Viscosity is measured in cP at 20°C.

[0086] Figure 3B This is a bar graph showing the viscosity of a 10mM histidine buffer formulation containing 155 mg / ml VEGF receptor fusion protein, without salt, and with 100 mM arginine, 200 mM arginine, 50 mM lysine, 200 mM lysine, 50 mM sodium chloride, or 100 mM sodium chloride. Viscosity is measured in cP at 20°C.

[0087] Figure 4A This graph shows the stability of a 150 mg / ml VEGF receptor fusion protein at 5°C for 12 months in the presence and absence of 50 mM L-arginine monophosphate, in 10 mM sodium phosphate, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 6.2. The VEGF receptor fusion protein concentration was 150 mg / ml. The stability of the VEGF receptor fusion protein was tested using SE-UPLC to identify HMW species formation.

[0088] Figure 4B This graph shows the stability of 150 mg / ml VEGF receptor fusion protein at 5°C for 12 months in 10 mM sodium phosphate, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 6.2 (with and without 50 mM L-arginine hydrochloride). The VEGF receptor fusion protein concentration was 150 mg / ml. The stability of the VEGF receptor fusion protein was tested using SE-UPLC to identify the percentage of the predominant species in each formulation.

[0089] Figure 5 This graph shows the viscosity of the formulation under two buffer conditions (10 mM phosphate buffer or 10 mM histidine buffer) at 20°C. The VEGF receptor fusion protein concentration varied between 10 mg / ml and 170 mg / ml. Viscosity is expressed in mPa·s.

[0090] Figure 6 Dynamic light scattering screening was demonstrated under various formulation conditions, each with protein concentrations ranging from 2 to 10 mg / ml. Diffusion interaction parameters indicated that taurine and PSA (propanesulfonic acid) improved Kd.

[0091] Figure 7This is a graph showing the change in the percentage of high molecular weight (HMW) species in formulations F1-F9 (formulation WW-EEE) over time, measured by size exclusion ultra-high performance liquid chromatography (SE-UPLC) after 6 months of storage at 5°C. Formulations F1-F9 are listed in Table 7-1 of this article.

[0092] Figure 8(AB) shows the percentage of high molecular weight species measured by SE-UPLC after storage at 5°C for 3 months (A) or incubation at 37°C for 28 days (B). Formulations F1-F4 (EEE, SSS, CCC (140 mg / ml) and TTT) are listed in Table 8-1 below.

[0093] Figure 9 The viscosity (cp) of formulations F1-F12 at 20°C at the initial time point is shown. Formulations F1-F12 are listed in Table A below.

[0094] Table A. Formulations F1-F12.

[0095]

[0096]

[0097] Figure 10 The weight molar osmotic concentrations (mmol / Kg) of formulations F1-F12 are shown (listed in Table A of this document).

[0098] Figure 11(AB) Percentage of high molecular weight species in formulations F1-F6 (formulations GGG, HHH, III, JJJ, LLL and KKK) over time, measured by SE-UPLC after storage at 37°C for 28 days (A) or at 5°C for 3 months (B).

[0099] Figure 12 The dynamic light scattering (diffusion coefficient (cm²)) of the formulations shown in Table 9-3 (GGG-RRR) at the initial time points is illustrated. 2 ( / second), radius (nm), and %Pd).

[0100] Figure 13 shows baseline FA images and OCT (30-degree lens) images (OD = oculus dexter (right eye); OS = oculus sinister (left eye)) of two different rabbits (326-OS and 329-OD) before apracept administration. (A) FA image of the left eye of rabbit 326; (B) OCT image of the left eye of rabbit 326; (c) FA image of the right eye of rabbit 329; (D) OCT image of the right eye of rabbit 329.

[0101] Figure 14(AF) shows rabbit FA images on day 1 (A), day 7 (B), and day 14 (C) of a rabbit (326-OS) administered histidine buffer preparation (30-degree lens) (7 mg / eye), as well as the time course of OCT on day 1 (D), day 7 (E), and day 14 (F).

[0102] Figure 15 (AH) shows rabbit FA images of a rabbit (326-OS) administered histidine buffer preparation (55-degree lens) (7 mg / eye) at 3 weeks (A), 4 weeks (B), 7 weeks (C), and 8 weeks (D); and time courses of OCT at 3 weeks (E), 4 weeks (F), 7 weeks (G), and 8 weeks (H).

[0103] Figure 16(AF) shows the rabbit FA images on day 1 (A), week 1 (B), and week 2 (C) and the time course of OCT on day 1 (D), week 1 (E), and week 2 (F) of a rabbit (329-OS) administered phosphate buffer preparation (30-degree lens) (7 mg / eye).

[0104] Figure 17 (AH) shows the rabbit FA images at weeks 3 (A), 4 (B), 7 (C), and 8 (D) and the time course of OCT at weeks 3 (E), 4 (F), 7 (G), and 8 (H) of a rabbit (329-OS) administered phosphate-buffered saline (55-degree lens) (7 mg / eye).

[0105] Figure 18(AB) shows the purity (percentage of natural species) (A) and percentage of high molecular weight (HMW) species (B) in the formulation UUU-BBBB over time at 37°C (up to one month) (see Table 11-1), as analyzed by SE-UPLC.

[0106] Figure 19(AE) shows the stability and purity analysis of the formulation, which contains 114.3 mg / mL VEGF Trap (apasicept) formulated in 10 mM histidine, pH 5.8, 5% sucrose, 0.03% polysorbate 20, and 50 mM arginine hydrochloride. The analysis was performed by size exclusion chromatography to determine the following percentages: (i) high molecular weight species (HMW) after incubation at 5°C or 37°C for 2 months (A) and (ii) major species (main peak) after incubation at 5°C or 37°C for 2 months (B); and the presence of particulate matter visible under a microscope after incubation at 37°C for 28 days as determined by microfluidic imaging (C), the presence of particulate matter visible under a microscope after incubation at 37°C for 28 days as determined by particle light-masking analysis (D), and particulate matter visible under a microscope after incubation at 37°C for 28 days as determined by microscopy (E).

[0107] Figure 20 shows the stability and purity analysis of formulations of apracept at 80, 100, 120, or 140 mg / ml, determined by SE-UPLC of the percentage of high molecular weight species (HMW) after incubation at 2–8°C for 6 months (A) or at 37°C for 28 days (B).

[0108] Figure 21 The percentage of rabbit eyes over time as of administration of 500 mcg or 2 mg apacept was shown by the complete leakage inhibition test (Gehan-Breslow-Wilcoxon test (P 0.0453)). Detailed Implementation

[0109] This invention provides formulations with high concentrations of VEGF receptor fusion proteins (e.g., apracept) that, despite significant technical hurdles, exhibit excellent functional and storage properties. For example, a common method for identifying suitable excipients for formulations containing peptide drugs (e.g., VEGF receptor fusion proteins) is by assessing peptide stability under accelerated stress conditions, such as high temperatures (e.g., 37°C). Excipients unsuitable under non-stress conditions (e.g., low temperatures, such as 5°C) often cause undesirable effects (e.g., protein aggregation) under stress within a short period. This approach is common in the biotechnology and pharmaceutical industries because it accelerates the elimination of excipients unlikely to stabilize drug products. See, for example, Magari, Assessing Shelf Life Using Real-Time and Accelerated Stability Tests, Biopharm Intl. 16(11):36-48 (2003). In some cases, product release can be based on accelerated stability data, but this must be performed simultaneously with real-time shelf-life analysis (non-accelerated). Magari (2003) and the FDA, Guidelines for Submitting Documentation for the Stability of Human Drugs and Biologics, Rockville, MD (1987). However, here, at 5°C, the presence of arginine in the histidine formulation is stable, although arginine appears to tend to decrease in stability under temperature and pressure (37°C). See Figure 8 (AB). This property of the formulation described herein presents technical difficulties that would guide practitioners against selecting arginine; therefore, practitioners are unlikely to choose arginine as an excipient. However, the formulation presented herein achieves high stability by overcoming such technical obstacles. The formulation of the VEGF trap in the histidine buffer also results in a beneficial reduction in viscosity relative to the viscosity observed in the phosphate buffer formulation. A relatively low viscosity is desirable because small needle holes are preferred for intravitreal injection (due to reduced patient discomfort and ocular trauma). Lower viscosity formulations require less force to push the formulation through the needle, making it easier for the therapist to inject the formulation via the needle. In addition, rabbits showed good tolerance to preparations containing histidine and arginine.

[0110] Reference will now be made in detail to representative embodiments. It should be understood that the following description is not intended to limit the embodiments to a single preferred embodiment. Rather, they are intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the embodiments described as defined in the appended claims.

[0111] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. As used herein, when referring to specifically enumerated numerical values, the term “about” means that the value may differ from the referenced value by no more than 1%. Thus, for example, as used herein, the expression “about 100” includes 99 and 101 and all values ​​between them (e.g., 99.1%, 99.2%, 99.3%, 99.4%, etc.).

[0112] In one embodiment of the invention, the pharmaceutical formulation of the invention conforms to the USP for small volume injection (SVP) of ophthalmic solutions. <789> For example, each ml contains less than about 50 diameter cells. > 10 μm particles; each ml contains fewer than approximately 5 particles with a diameter of > Particles with a diameter of 25 μm; or containing fewer than approximately 2 particles per ml. > 50□m particles.

[0113] Note that for the purposes of this article, "intravitreal injection" refers to injection into the vitreous humor of the eye (near the retina at the back of the eye). The statements "suitable for intravitreal administration" or "suitable for intravitreal injection" indicate that the formulation under discussion can be safely injected into the vitreous humor of a subject's eye without causing any known adverse reactions associated with intravitreal injection of EYLEA.

[0114] The term "pharmaceutical formulation" in this article refers to a formulation that includes a pharmaceutically acceptable carrier, for example, for administering a VEGF receptor fusion protein (e.g., apracept or conbercept) to a subject for therapeutic / medical use.

[0115] The term "pharmaceutically acceptable" refers to a formulation that is suitable for application to the eyes within the bounds of reasonable medical judgment.

[0116] The term “subject” in this article means any mammalian (e.g., rabbit, mouse, rat, or monkey) subject, especially a human, for example, for whom they expect to be diagnosed, prognosed, or treated with the formulations described herein.

[0117] The term "aqueous" formulation refers to a formulation that contains water.

[0118] The term "treat" or "treatment" refers to therapeutic measures that reverse, stabilize, or eliminate an undesirable disease or condition (such as vascular eye disease or cancer), for example, by reducing, stabilizing, or eliminating any clinically measurable degree of one or more symptoms or indications causing such disease or condition (e.g., in the case of vascular eye disease); by reducing or maintaining a diabetic retinopathy severity score (DRSS); by improving or maintaining visual acuity (e.g., best-corrected visual acuity, for example, by increasing ETDRS letter measurements); by increasing or maintaining visual field and / or decreasing or maintaining central retinal thickness; and, in the case of cancer, by stopping or reversing the growth, survival, and / or metastasis of cancer cells in a subject. Typically, a treatment involves administering one or more therapeutically effective doses of VEGF receptor fusion protein to a subject suffering from the disease or condition.

[0119] "Prevention" or "prevention" refers to preventive measures taken to stop the development of an unwanted disease or condition (e.g., vascular eye disease).

[0120] SE-UPLC can be used in this invention to quantify the presence of high molecular weight species in formulations. SE stands for size exclusion chromatography. UPLC stands for ultra-high performance liquid chromatography. Suitable SE columns of this type of HMW that can be used in UPLC systems to characterize VEGF receptor fusion proteins (e.g., apracept or conbercept) in formulations can resolve molecules with molecular weights ranging from approximately 10,000 to 450,000 Daltons. See, for example, ACQUITY UPLC Protein BEH SEC. Chromatographic column. In one embodiment of the invention, two such columns are connected in series when quantifying the HMW species in a formulation. Compared to HPLC (High Performance Liquid Chromatography), UPLC offers improved sensitivity and resolution. UPLC instruments operate under high pressure and use finer particles (typically less than 2.5 μm) than those used in HPLC. Furthermore, UPLC flows at higher linear velocities compared to HPLC.

[0121] The embodiments described herein include formulations containing high concentrations (e.g., about 60 mg / ml, about 80 mg / ml, about 90 mg / ml, about 100 mg / ml, about 113.3 mg / ml, about 114.3 mg / ml, about 120 mg / ml, about 133.3 mg / ml, about 140 mg / ml, about 150 mg / ml, about 200 mg / ml, or about 250 mg / ml) of VEGF receptor fusion protein (e.g., apracept or conbercept). Suitable formulations included herein contain high concentrations of VEGF receptor fusion protein, buffer solutions, heat stabilizers, and surfactants. In some aspects, suitable formulations also include viscosity reducers. In other aspects, suitable formulations substantially do not include all viscosity reducers. Typical formulations have a pH of about 5.0 to about 6.8 (e.g., 5.8), but may include any pH suitable for administering the VEGF receptor fusion protein to a subject's eye.

[0122] This invention includes formulations comprising a VEGF receptor fusion protein (e.g., apracept or conbercept) bound to one or more other therapeutic agents (e.g., an Ang-2 inhibitor (e.g., an anti-ANG2 antibody or its antigen-binding fragment) or nesvacumab), a Tie-2 receptor activator, an anti-PDGF, PDGF receptor, or PDGF receptor β antibody or its antigen-binding fragment, and / or other VEGF antagonists, such as bevacizumab, ranibizumab, pegattatanib, or a soluble form of human vascular endothelial growth factor receptor 3 (VEGFR-3) comprising extracellular domains 1-3, indicated as an Fc fusion protein), and methods of prevention or treatment comprising administration of such formulations described herein. In one embodiment of the invention, the formulation of the invention comprises a VEGF receptor fusion protein, such as apracept, but does not comprise any other therapeutic agent (e.g., an antibody or its antigen-binding fragment).

[0123] The term "in combination with" indicates that the formulation and other therapeutic agents can be formulated into a single composition, for example, for simultaneous delivery, or formulated separately into two or more compositions (e.g., a kit). The other therapeutic agents themselves can be formulated as their own pharmaceutical preparations. Each can be administered to the subject at the same time as others or at different times; for example, each administration can be given non-simultaneously (e.g., separately or sequentially) within a given time interval. Furthermore, the formulation and other therapeutic agents can be administered to the subject via the same or different routes.

[0124] In one embodiment of the invention, the formulation comprises any one or more of the following: sodium sulfate (e.g., 50 mM); sodium thiocyanate (e.g., 50 mM); sodium citrate (e.g., 40 mM); glycine (e.g., 50 mM); sodium chloride (e.g., 50 mM); lysine (e.g., 50 mM); sodium aspartate (e.g., 50 mM); and / or monosodium glutamate (e.g., 50 mM). For example, in one embodiment, the formulation comprises a combination of: sodium citrate (e.g., 50 mM) and arginine hydrochloride (e.g., 50 mM); glycine (e.g., 50 mM) and arginine hydrochloride (e.g., 50 mM); sodium aspartate (e.g., 50 mM) and arginine hydrochloride (e.g., 50 mM); or monosodium glutamate (e.g., 50 mM) and arginine hydrochloride (e.g., 50 mM).

[0125] VEGF receptor fusion protein and other VEGF inhibitors

[0126] For the purposes of this document, a “VEGF receptor fusion protein” refers to a molecule comprising one or more VEGF receptors or their domains fused to another polypeptide, which interferes with the interaction between VEGF and the native VEGF receptor, for example, by two such fusion polypeptides binding to form a homodimer or other multimer. Such VEGF receptor fusion proteins may be referred to as “VEGF-Trap” or “VEGF Trap”. VEGF receptor fusion proteins falling within this definition and within the scope of this disclosure include chimeric polypeptides that comprise two or more immunoglobulin (Ig)-like domains of the VEGF receptor, such as VEGFR1 (also known as Flt1) and / or VEGFR2 (also known as Flk1 or KDR), and may also comprise multimeric domains (e.g., Fc domains).

[0127] An exemplary VEGF receptor fusion protein is a molecule called VEGF1R2-FcΔC1(a), which is encoded by the nucleic acid sequence of SEQ ID NO: 1 or its nucleotides 79-1374 or 79-1371.

[0128] VEGF1R2-FcΔC1(a) contains three components:

[0129] (1) VEGFR1 component, which contains amino acids 27 to 129 of SEQ ID NO: 2;

[0130] (2) The VEGFR2 component, comprising amino acids 130 to 231 of SEQ ID NO: 2; and

[0131] (3) The polymeric component (“FcΔC1(a)”) contains amino acids 232 to 457 of SEQ ID NO: 2 (the C-terminal amino acid K458 of SEQ ID NO: 2 may or may not be included in the VEGF receptor fusion protein, see U.S. Patent Nos. 7,396,664 or 7,354,579, which are incorporated herein by reference for all purposes). Note that amino acids 1 to 26 of SEQ ID NO: 2 are the signal sequence.

[0132] In one embodiment of the present invention, the VEGF receptor fusion protein comprises amino acids 27-458 or 27-457 of SEQ ID NO: 2.

[0133] In one embodiment of the present invention, the VEGF receptor fusion protein includes...

[0134] (1) Immunoglobulin-like (Ig) domain 2 of the first VEGF receptor (e.g., VEGFR1), and

[0135] (2) Ig domain 3 of the second VEGF receptor (e.g., VEGFR2),

[0136] (3) and optionally, also includes the Ig domain 4 of a second VEGF receptor (e.g., VEGFR2) and

[0137] (4) Polymer components (e.g., the Fc domain of IgG).

[0138] For example, in one embodiment of the invention, the VEGF receptor fusion protein has the following arrangement of the domains:

[0139] ●[VEGFR1 Ig domain 2]-[VEGFR2 Ig domain 3]-[MC] (e.g., its homodimer) or

[0140] ●[VEGFR1 Ig domain 2]-[VEGFR2 Ig domain 3]-[VEGFR2 Ig domain 4]-[MC] (e.g., its homodimer).

[0141] In one embodiment of the invention, the VEGF receptor fusion protein is a VEGF microtrap, which is a VEGF Trap molecule having a truncated multimeric component (e.g., Fc), wherein the microtrap still includes the Fc hinge region. See, for example, WO2005 / 00895 or U.S. Patent No. 7,396,664.

[0142] Note that this disclosure also includes, within its scope, a high-concentration formulation that, instead of the VEGF receptor fusion protein, comprises a VEGF-binding molecule and an anti-VEGF antibody and its antigen-binding fragment.

[0143] ● Bevacizumab (e.g., at a concentration of approximately 80-90 or 88 mg / ml),

[0144] Ranibizumab (e.g., at a concentration of approximately 20-40 mg / ml, such as 21-35, 21 or 35 mg / ml),

[0145] ● Anti-VEGF aptamers, such as piperatanib (e.g., piperatanib sodium),

[0146] • Single chain (e.g., V) L -V H Anti-VEGF antibodies, such as brolucizumab (e.g., concentrations of approximately 200-400 mg / ml, or 200, 210, 400, or 420 mg / ml),

[0147] • Anti-VEGF DARPin, such as Abicipar Pegol DARPin (e.g., concentrations of approximately 70-140, 70, or 140 mg / ml), or

[0148] ● Bispecific anti-VEGF antibodies, for example, which can also bind to ANG2, such as RG7716 (e.g., at concentrations of approximately 100-400, 100, 105, 400, or 420 mg / ml).

[0149] To minimize the reproducibility of the embodiments discussed herein, the scope of the invention is considered to include embodiments in which any formulation discussed herein comprises an anti-VEGF antibody or antibody fragment at any concentration discussed herein, or other VEGF-binding molecules discussed herein (e.g., replaced by anti-VEGF DARPin), replacing the VEGF receptor fusion protein. For example, the invention includes formulations containing 35 or 80 mg / ml ranibizumab, buffer, heat stabilizer, viscosity reducer, and surfactant.

[0150] DARPins are designed ankylosing protein repeats. DARPins typically consist of three to four tightly packed repeat sequences of approximately 33 amino acid residues, each repeat containing a β-turn and two antiparallel α-helices. This rigid framework provides protein stability while enabling the presentation of variable regions, typically six amino acid residues per repeat, for target recognition.

[0151] "Anti-VEGF" antibodies or antibody antigen-binding fragments refer to antibodies or fragments that specifically bind to VEGF.

[0152] Exemplary VEGF receptor fusion proteins include apacept ( Regeneron Pharmaceuticals, Inc.) or Conbercept (commercially sold by Chengdu Kanghong Biotechnology Co., Ltd.). See International Patent Application Publications WO2005 / 121176 or WO2007 / 112675. The terms “apracept” and “conbercept” include their biosimilar forms. References to a biosimilar form of a product (e.g., apracept) generally refer to products containing the same amino acid sequence, but include products that are biosimilar under the USBiologics Price Competition and Innovation Act.

[0153] The pharmaceutical formulation of the present invention is a "high concentration". The high-concentration pharmaceutical formulation of the present invention comprises a VEGF receptor fusion protein at a concentration of at least 41 mg / ml, at least 80 mg / ml, at least 100 mg / ml, at least 125 mg / ml, at least 140 mg / ml, at least 150 mg / ml, at least 175 mg / ml, at least 200 mg / ml, at least 225 mg / ml, at least 250 mg / ml, or at least 275 mg / ml. Alternatively, "high concentration" may refer to the concentration of the VEGF receptor fusion protein included in the formulation being about 140 mg / ml to about 160 mg / ml, at least about 140 mg / ml but less than 160 mg / ml, about 41 mg / ml to about 275 mg / ml, about 70 mg / ml to about 75 mg / ml, or about 80 mg / ml to about 250 mg / ml. In some aspects, the concentration of the VEGF receptor fusion protein in the formulation is any one of the following concentrations: 41 mg / ml; 42 mg / ml; 43 mg / ml; 44 mg / ml; 45 mg / ml; 46 mg / ml; 47 mg / ml; 48 mg / ml; 49 mg / ml; 50 mg / ml; 51 mg / ml; 52 mg / ml; 53 mg / ml; 54 mg / ml; 55 mg / ml; 56 mg / ml; 57 mg / ml; 58 mg / ml; 59 mg / ml; 60 mg / ml; 61 mg / ml; 62 mg / ml; 63 mg / ml; 64 mg / ml; 65 mg / ml; 66 mg / ml; 67 mg / ml; 68 mg / ml; 69 mg / ml; 70 mg / ml; 71 mg / ml; 72 mg / ml; 73 mg / ml; 74 mg / ml; 75 mg / ml; 76 mg / ml; 77 mg / ml; 78 mg / ml; 78 mg / ml; 78 mg / ml; 79 mg / ml; 70 mg / ml; 71 mg / ml; 72 mg / ml; 73 mg / ml; 74 mg / ml; 75 mg / ml; 76 mg / ml; 7 ... mg / ml; 79 mg / ml; 80 mg / ml; 81 mg / ml; 82 mg / ml; 83 mg / ml; 84 mg / ml; 85 mg / ml; 86 mg / ml; 87 mg / ml; 88 mg / ml; 89 mg / ml; mg / ml; 98 mg / ml; 99 mg / ml; 100 mg / ml; 101 mg / ml; 102 mg / ml; 103 mg / ml; 104 mg / ml; 105 mg / ml; 106 mg / ml; 107 mg / ml; 108 mg / ml; mg / ml; 113.3 mg / ml; 114 mg / ml;114.1mg / ml;114.2 mg / ml;114.3 mg / ml;114.4 mg / ml;114.5 mg / ml;114.6 mg / ml,114.7 mg / ml,114.8mg / ml;114.9 mg / ml;115 mg / ml;116 mg / ml;117 mg / ml;118 mg / ml;119mg / ml;120 mg / ml;121 mg / ml;122 mg / ml;123 mg / ml;124 mg / ml;125mg / ml;126 mg / ml;127mg / ml;128 mg / ml;129 mg / ml;130 mg / ml;131mg / ml;132 mg / ml;133 mg / ml;133.3 mg / ml;133.4 mg / ml,134 mg / ml;135mg / ml;136 mg / ml;137 mg / ml;138 mg / ml;139 mg / ml;140mg / ml;141mg / ml;142 mg / ml;143 mg / ml;144 mg / ml;145 mg / ml;146 mg / ml;147mg / ml;148mg / ml;149 mg / ml;150 mg / ml;151 mg / ml;152 mg / ml;153mg / ml;154mg / ml;155 mg / ml;156mg / ml;157mg / ml;158 mg / ml;159 mg / ml;160 mg / ml;161 mg / ml;162 mg / ml;163 mg / ml;164 mg / ml;165 mg / ml;166mg / ml;167 mg / ml;168 mg / ml;169 mg / ml;170 mg / ml;171 mg / ml;172mg / ml;173 mg / ml;174 mg / ml;175 mg / ml;176 mg / ml;177 mg / ml;178mg / ml;179mg / ml;180 mg / ml;181 mg / ml;182 mg / ml;183 mg / ml;184mg / ml;185 mg / ml;186 mg / ml;187 mg / ml;188 mg / ml;189 mg / ml;190mg / ml;191mg / ml;192mg / ml;193mg / ml;194mg / ml;195mg / ml;196mg / ml;197mg / ml;198mg / ml;199mg / ml;200mg / ml;201mg / ml;202mg / ml;203mg / ml;204mg / ml;205mg / ml; 206mg / ml; 207mg / ml; 208mg / ml; 209mg / ml; 210mg / ml; 211mg / ml; 212mg / ml; 213mg / ml ;214mg / ml; 215mg / ml; 216mg / ml; 217mg / ml; 218mg / ml; 219mg / ml; 220mg / ml; 221mg / ml; 222mg / m l; 223mg / ml; 224mg / ml; 225mg / ml; 226mg / ml; 227mg / ml; 228mg / ml; 229mg / ml; 230mg / ml; 231mg / ml; 232mg / ml; 233mg / ml; 234mg / ml; 235mg / ml; 236mg / ml; 237mg / ml; 238mg / ml; 239mg / ml; 240mg / ml; 241mg / ml; 242mg / ml; 243mg / ml; 244mg / ml; 245mg / ml; 246mg / ml; 247mg / ml; 248mg / ml; 249m g / ml; 250mg / ml; 251mg / ml; 252mg / ml; 253mg / ml; 254mg / ml; 255mg / ml; 256mg / ml; 257mg / ml; 258 mg / ml; 259 mg / ml; 260 mg / ml; 261 mg / ml; 262 mg / ml; 263 mg / ml; 264 mg / ml; 265 mg / ml; 266 mg / ml; 267 mg / ml; 268 mg / ml; 269 mg / ml; 270 mg / ml; 271 mg / ml; 272 mg / ml; 273 mg / ml; 274 mg / ml; or 275 mg / ml. Other VEGF receptor fusion protein concentrations are considered, provided that the concentration is effective according to the embodiments described herein.

[0154] In one embodiment of the invention, the concentration of the pharmaceutical formulation of the invention is such that it comprises about 4, 6, 8, 10, 12, 14, 16, 18, or 20 mg of VEGF receptor fusion protein (e.g., apracept), or an amount of such protein at any acceptable dose discussed herein, in about 100 μL or less, about 75 μL or less, or about 70 μL or less, for example about 50 μL; 51 μL; 52 μL; 53 μL; 54 μL; 5 ...

[0155] □l; 56□l; 57□l; 58□l; 59□l; 60□l; 61□l; 62□l; 63□l; 64□l; 65□l; 66□l; 67□l; 68□l; 69□l; 70□l; 71□l; 72□l; 73□l; 74□l; 75□l; 76□l; 77□l; 78□l; 79□l; 80□l; 81□l; 82□l; 83□l; 84□l; 85□l; 86□l; 87□l; 88□l; 89□l; 90□l; 91□l; 92□l; 93□l; 94□l; 95□l; 96□l; 97□l; 98□l; 99□l; or 100□l.

[0156] This invention includes any formulation listed under “Illustrative Formulations” herein, but wherein the concentration of VEGF receptor fusion protein (e.g., apracept) is replaced by the concentration presented in this section (“VEGF Receptor Fusion Proteins and Other VEGF Inhibitors”).

[0157] buffer solution

[0158] The buffer solution used herein refers to a solution that resists pH changes by using an acid-base conjugate. The buffer solution is capable of maintaining a pH in the range of about 5.0 to about 6.8, and more generally, in the range of about 5.8 to about 6.5, and most typically, in the range of about 6.0 to about 6.5. In some cases, the pH of the formulations of the present invention is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, or about 6.8. Examples of buffer solutions used in the formulations herein include histidine-based buffer solutions, such as histidine, histidine hydrochloride, and histidine acetate. The buffer solution included in the formulations herein may alternatively be a phosphate-based buffer solution, such as sodium phosphate, an acetate-based buffer solution, such as sodium acetate or acetic acid, or may be a citrate-based buffer solution, such as sodium citrate or citric acid. It is also recognized that the buffer solution can be a mixture of the above-described components, provided that the buffer solution can buffer the formulation within the pH range described above. In some cases, the buffer solution is about 5 mM to about 25 mM, or more typically about 5 mM to about 15 mM. The buffer solution can be about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, or about 25 mM.

[0159] In one embodiment of the invention, histidine and histidine hydrochloride are used to prepare a histidine-based buffer solution.

[0160] surfactants

[0161] The surfactants used herein refer to components that protect high concentrations of VEGF receptor fusion proteins from various surface and interface-induced stresses. Thus, surfactants can be used to limit or minimize the aggregation of VEGF receptor fusion proteins and promote protein solubility. Suitable surfactants have been shown to be nonionic and may include surfactants having a polyoxyethylene moiety. Exemplary surfactants of this class include: polysorbate 20, polysorbate 80, poloxamer 188, polyethylene glycol 3350, and mixtures thereof. Surfactants in formulations may be present at about 0.02% to about 0.1% (w / v) per volume weight, and more typically about 0.02% to about 0.04% (w / v). In some cases, the surfactant is about 0.02% (w / v), about 0.03% (w / v), about 0.04% (w / v), about 0.05% (w / v), about 0.06% (w / v), about 0.07% (w / v), about 0.08% (w / v), about 0.09% (w / v), or about 0.1% (w / v).

[0162] Heat stabilizer

[0163] The heat stabilizers used in this article refer to components that provide thermal stability against the thermal denaturation of VEGF receptor fusion proteins and prevent loss of efficacy or activity of the VEGF receptor fusion proteins. Suitable heat stabilizers include sugars, and may be sucrose, trehalose, sorbitol, or mannitol, or may be amino acids, such as L-proline, L-arginine (e.g., L-arginine hydrochloride), or taurine. Additionally, heat stabilizers may include substituted acrylamides or propane sulfonic acids, or may be compounds such as glycerol.

[0164] In some cases, the formulations described herein include sugars and taurine, sugars and amino acids, sugars and propane sulfonic acid, sugars and taurine, glycerol and taurine, glycerol and propane sulfonic acid, amino acids and taurine, or amino acids and propane sulfonic acid. Additionally, the formulations may include sugars, taurine and propane sulfonic acid, glycerol, taurine and propane sulfonic acid, and L-proline, taurine and propane sulfonic acid.

[0165] The embodiments described herein typically contain heat stabilizers present individually, either independently or in a combination of the following total concentrations: about 2% (w / v) to about 10% (w / v), or about 4% (w / v) to about 10% (w / v), or about 4% (w / v) to about 9% (w / v), or about 5% (w / v) to about 8% (w / v). The concentration of the heat stabilizer in the formulation may be about 2% (w / v), about 2.5% (w / v), about 3% (w / v), about 4% (w / v), about 5% (w / v), about 6% (w / v), about 7% (w / v), about 8% (w / v), about 9% (w / v), about 10% (w / v), or about 20% (w / v).

[0166] Regarding taurine and propanesulfonic acid, in one embodiment of the invention, the amount of these heat stabilizers present in the formulation may be from about 25 mM to about 100 mM, more typically from about 50 mM to about 75 mM (compared to other heat stabilizers).

[0167] viscosity reducer

[0168] Viscosity reducers are commonly used to reduce or prevent protein aggregation. Viscosity reducers used herein include sodium chloride, magnesium chloride, D- or L-arginine (e.g., L-arginine monohydrochloride), lysine, or mixtures thereof. When present herein, viscosity reducers may be present in amounts from about 10 mM to about 100 mM, and more typically from about 30 mM to about 75 mM, and even more typically from about 40 mM to about 70 mM. In some cases, viscosity reducers are present in amounts of about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM, or about 100 mM.

[0169] formulation viscosity

[0170] Formulations according to embodiments of this invention may also have pharmaceutically acceptable viscosities for intraocular administration, such as intravitreal injection. Viscosity is generally a measure of the resistance of a fluid to deformation under shear or tensile stress (typically measured using techniques known in the art, such as a viscometer or rheometer). Typical viscosities of formulations according to embodiments of this invention are about 5.0 cP (centipoise) to about 15 cP, about 11 cP to about 14 cP, about 12 cP to about 15 cP, or about 11 cP to about 12 cP. Therefore, the formulation viscosity described herein can be approximately 5.0 cP, approximately 6.0, approximately 7.1 cP, approximately 7.2 cP, approximately 7.3 cP, approximately 7.4 cP, approximately 7.5 cP, approximately 7.6 cP, approximately 10 cP, approximately 10.5 cP, approximately 11.0 cP, approximately 11.5 cP, approximately 12.0 cP, approximately 12.5 cP, approximately 13.0 cP, approximately 13.5 cP, approximately 14.0 cP, approximately 14.5 cP, or approximately 15.0 cP (e.g., measured at 20°C).

[0171] The various embodiments described herein do not require the inclusion of inorganic salts or other viscosity reducers to maintain these highly useful viscosities. Typically, high-concentration protein solutions require viscosity reducers to prevent protein aggregation and high viscosity, which makes the formulation difficult to inject intravitreal and reduces the potency of VEGF receptor fusion proteins. Thus, the embodiments described herein comprise formulations with virtually no or no added sodium chloride (NaCl), magnesium chloride (MgCl2), D- or L-arginine hydrochloride, lysine, or other viscosity reducers.

[0172] Formulation weight molar permeation concentration

[0173] Weight molar osmotic concentration is a key attribute of injectable formulations. Products that match physiological osmotic conditions are desirable. Furthermore, weight molar osmotic concentration can identify the soluble contents in the solution. In one embodiment of the invention, the weight molar osmotic concentration of the formulation is less than or equal to about 506 mmol / Kg or about 250 to about 506 mmol / Kg, for example, about 250, 260, 270, 280, 290, 299, 300, 310, 314, 315, 316, 324, 343, 346, 349, 369, 384, 403, 426, 430, or 506 mmol / Kg. In another embodiment of the invention, the weight molar osmotic concentration is less than about 250 mmol / Kg.

[0174] Purity and stability of the formulation

[0175] The formulations containing high concentrations of VEGF receptor fusion protein described herein are stable during manufacturing and storage. The term "stable" as used herein means that the formulation containing the VEGF receptor protein maintains chemical and physical stability during manufacturing and storage; for example, it retains its integrity and exhibits minimal degradation, denaturation, or unfolding. The stability of the VEGF receptor protein can be determined using analytical techniques available in the art at different temperatures and time periods. In particular, various bioassays (e.g., VEGF assays used to determine the VEGF receptor fusion protein of this article) can be used. 165 The chemical stability (potency) of the VEGF receptor was determined using the combined BAF / 3VEGFR1 / EPOR cell line, and its physical stability was determined by size exclusion (SE) chromatography, UPLC (ultra-high performance liquid chromatography) size exclusion (SE) chromatography, visual appearance, OD, pH, charge variant formation, and high molecular weight (HMW) species formation rate. Stable VEGF receptor fusion proteins exhibited limited variation in OD, pH, charge variant formation, and HMW species formation.

[0176] As used herein, the term "high molecular weight" (HMW) in a formulation containing a given VEGF Trap (e.g., apracept) refers to any class of peptides or peptide complexes eluted from the formulation by a size exclusion column (e.g., SE-UPLC) before the VEGF Trap peptide and / or its homodimer (e.g., having a higher molecular weight). The percentage of HMW class refers to the percentage of this class relative to the total amount of peptides in the formulation, as analyzed, for example by SE-UPLC.

[0177] In one embodiment of the invention, as discussed more fully in the following examples, formulations that are stable over periods of up to 12 months, up to 24 months, and / or up to 36 months when stored at about 2°C to about 8°C exhibit significant VEGF receptor fusion protein potency and physical stability.

[0178] In embodiments of the present invention, the formulation of the present invention is the following:

[0179] • After approximately 28 days at about 37°C, the high molecular weight derivatives showed approximately 3%, 5%, 10%, and 11% [of something unspecified].

[0180] 12%, 13%, 14%, 15%, 16%, 17%, 18%, 20%, 30%, 35% or 37% (or

[0181] An increase of 10-15% or 15-20% (or 10-20%) (e.g., measured by SE-UPLC or SEC).

[0182] ● After approximately 28 days at about 37°C, the main species showed approximately 5%, 6%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, or 22% (or

[0183] A reduction of 5-20%, 5-10%, 10-15%, or 15-20% (e.g., through SE-UPLC or SEC)

[0184] Measurement)

[0185] ●After approximately 28 days at approximately 37°C, the results showed approximately 80%, 81%, 82%, 83%, 84%, and 85%...

[0186] 86% or 87% (or about 80-85%) or more of the protein as the dominant species (e.g., by SE-

[0187] UPLC or SEC measurement)

[0188] ● After approximately 28 days at approximately 37°C, the low molecular weight species increased by approximately 1, 1.5, or 2% (or 1-

[0189] 2% (e.g., measured by SE-UPLC or SEC)

[0190] • After approximately one month at approximately 37°C, the high molecular weight species increased by approximately 16%; and / or the main species decreased by approximately 17% and / or the low molecular weight species increased by approximately 0.5% or <1%; and / or after approximately two months at approximately 5°C, the main species decreased by approximately 1%, and / or the high molecular weight species increased by approximately 1%, and / or

[0191] Or low molecular weight (LMW) species with no significant or detectable amount; and / or with approximately 97% of the major species after approximately 2 months at approximately 5°C (e.g., measured by SE-UPLC or SEC).

[0192] ● After approximately 12 months at approximately 2-8°C, the number of high molecular weight species increased by approximately 3-3.5% (e.g., through...).

[0193] SE-UPLC or SEC measurement)

[0194] • After approximately 12 months at approximately 2–8°C, the major species reduction is less than approximately 1% or 1%, 2%, 3% or 4% (e.g., 1–4% or 3–4%) (as measured by SE-UPLC or SEC).

[0195] ● After approximately 12 months at approximately 2–8°C, approximately 94% or 95% or more of the protein is the dominant species (e.g., as measured by SE-UPLC or SEC).

[0196] ●After about 3 months at approximately 2-8°C, the number of high molecular weight species increased by approximately 1% or 2% (e.g., as measured by SE-UPLC or SEC).

[0197] ● After approximately 6 months at approximately 2–8°C, the high molecular weight species increased by approximately <1%, 1%, or 2% (e.g., as measured by SE-UPLC or SEC).

[0198] At approximately 5°C, approximately 5 1 / 2 or 6 months later, the total high molecular weight species have about 2.5, 3.0 or 3.5 (or about 2.5-3.5%) (e.g., as measured by SE-UPLC or SEC).

[0199] • After approximately 6 months at approximately 2-8°C, the major species decreased by less than approximately 1% or 1% or 2% (e.g., 0.5-2% or 1-2%) (as measured by SE-UPLC or SEC).

[0200] • After about 6 months at about 2-8°C, approximately 96% or 97% or 98% (or 96-98%) or more of protein is the dominant species (e.g., as measured by SE-UPLC or SEC).

[0201] • After approximately 24 or 36 months at approximately 2–8°C, the increase in high molecular weight species is less than approximately 5, 6, or 7% (e.g., approximately 1.5, 2, 3, 4, or 5%) (or 1.5–5% or 1.5–2.5%); and / or the total high molecular weight species comprise approximately 3.0, 3.25, 3.5, 4.0, 4.5, or 5%; and / or the major species decrease by approximately 2% or 3% (or 2–3%); and / or the total amount of the major species is approximately 95% or 96% or more (or 95–96%) (e.g., measured by SE-UPLC or SEC).

[0202] • After approximately one month at approximately 37°C, apacept in the formulation with approximately 97, 98, 99, or 100% (or 97-100%) of the apacept can be recovered by RP-HPLC.

[0203] • Immediately after production and purification, it contains less than about 1.5%, 2%, 2.5%, 3.0%, or 3.5% of high molecular weight species (e.g., as measured by SE-UPLC or SEC).

[0204] ● After about 6 months at about 37°C, at least about 70% or 75% (e.g., 70-75%) of apacilpeptide is used as the dominant species / peak (e.g., measured by capillary isoelectric focusing (cIEF) or imaging capillary isoelectric focusing (non-acidic and non-basic species)).

[0205] ● After about 36 months at about 2-8°C, the acidity species increase by about 1 or 2% (or 1-2%) (e.g., by capillary isoelectric focusing (cIEF) or imaging capillary isoelectric focusing measurement).

[0206] ● After about 36 months at about 2-8°C, the major species / peak decreases by about 1% or less (e.g., by capillary isoelectric focusing (cIEF) or capillary isoelectric focusing measurement by imaging).

[0207] ● After about 36 months at about 2-8°C, the main species / peaks are about 78-79% (e.g., by capillary isoelectric focusing (cIEF) or capillary isoelectric focusing measurement by imaging).

[0208] and / or

[0209] • When administered intravitreal to mammals (e.g., humans, rabbits, or mice) (e.g., those with vascular eye diseases such as wet AMD), it does not cause any adverse reactions associated with EYLEA (e.g., when...).

[0210] Different clinically distinct adverse events were observed with EYLEA administered intravitreally at doses of 0.5 or 2.0 mg.

[0211] It may not cause clinically obvious inflammation in the eye, prolonged increase in intraocular pressure (IOP), long-term increase or decrease in blood pressure and / or retinal detachment.

[0212] Furthermore, in embodiments of the invention, high concentrations of VEGF receptor fusion proteins are stable because they form little or no acidic charge heterogenes during manufacturing and storage, for example, by means of electro-focusing tests such as imaging capillary.

[0213] In embodiments of the present invention, the formulations of the present invention exhibit less than or equal to about 8% of low molecular weight (LMW) species.

[0214] In embodiments of the present invention, the formulation of the present invention has an endotoxin content of less than about 0.2, 0.4 or 0.5 EU (endotoxin units) / ml;

[0215] In embodiments of the present invention, the formulations of the present invention are substantially free of particulate matter or particulate matter with a size of about 1, 2, 5, 10, 25 or 50 micrometers (or larger).

[0216] In an embodiment of the present invention, when the formulation of the present invention is analyzed by non-reducing CE-SDS (SDS capillary gel electrophoresis), at least 97% of the total peak area is the main peak.

[0217] In an embodiment of the present invention, when the formulation of the present invention is analyzed by size exclusion UPLC (SE-UPLC), at least 93, 94 or 95% of the total peak area is the main peak and less than or equal to 3.5, 4, 5 or 6% is aggregates.

[0218] In embodiments of the present invention, the formulations of the present invention are prepared at approximately 2°C, 3°C, 4°C, 5°C, 6°C, 7°C or 8°C, 2-8°C (e.g., an average temperature of 5°C), 23°C, 25°C, 30°C or 37°C.

[0219] Instructional formulations

[0220] Illustrative high-concentration VEGF receptor fusion protein formulations include the following:

[0221] Formulation A: 80 mg / ml apracept, 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0222] Formulation B: 80 mg / ml apracept, 10 mM phosphate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0223] Formulation C: 80 mg / ml apracept, 10 mM citrate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0224] Formulation D: 80 mg / ml apracept, 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 6.2.

[0225] Formulation E: 80 mg / ml apracept, 10 mM phosphate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0226] Formulation F: 80 mg / ml apracept, 10 mM citrate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0227] Formulation G: 80 mg / ml apracept, 10 mM histidine-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, in particular, excluding viscosity reducers.

[0228] Formulation H: 80 mg / ml apracept, 10 mM phosphate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, in particular, excluding viscosity reducers.

[0229] Formulation I: 80 mg / ml apracept, 10 mM citrate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0230] Formulation J: 80 mg / ml apracept, 10 mM histidine-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, in particular, excluding viscosity reducers.

[0231] Formulation K: 80 mg / ml apracept, 10 mM phosphate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, in particular, excluding viscosity reducers.

[0232] Formulation L: 80 mg / ml apracept, 10 mM citrate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0233] Formulation M: 150 mg / ml apracept, 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0234] Formulation N: 150 mg / ml apracept, 10 mM phosphate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0235] Formulation O: 150 mg / ml apracept, 10 mM citrate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0236] Formulation P: 150 mg / ml apracept, 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 6.2.

[0237] Formulation Q: 150 mg / ml apracept, 10 mM phosphate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0238] Formulation R: 150 mg / ml apracept, 10 mM citrate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0239] Formulation S: 150 mg / ml apracept, 10 mM histidine-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, in particular, excluding viscosity reducers.

[0240] Formulation T: 150 mg / ml apracept, 10 mM phosphate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2 (e.g. 6.2), and optionally, specifically excluding viscosity reducers.

[0241] Formulation U: 150 mg / ml apracept, 10 mM citrate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0242] Formulation V: 150 mg / ml apracept, 10 mM histidine-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0243] Formulation W: 150 mg / ml apracept, 10 mM phosphate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, in particular, excluding viscosity reducers.

[0244] Formulation X: 150 mg / ml apracept, 10 mM citrate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0245] Formulation Y: 80 mg / ml conbercept, 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0246] Formulation Z: 80 mg / ml conbercept, 10 mM phosphate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0247] Formulation AA: 80 mg / ml conbercept, 10 mM citrate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0248] Formulation BB: 80 mg / ml Conbercept, 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 6.2.

[0249] Formulation CC: 80 mg / ml conbercept, 10 mM phosphate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0250] Formulation DD: 80 mg / ml Conbercept, 10 mM citrate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0251] Formulation EE: 80 mg / ml conbercept, 10 mM histidine-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0252] Formulation FF: 80 mg / ml conbercept, 10 mM phosphate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0253] Formulation GG: 80 mg / ml conbercept, 10 mM citrate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0254] Formulation HH: 80 mg / ml conbercept, 10 mM histidine-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0255] Formulation II: 80 mg / ml conbercept, 10 mM phosphate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0256] Formulation JJ: 80 mg / ml conbercept, 10 mM citrate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0257] Formulation KK: 150 mg / ml conbercept, 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0258] Formulation LL: 150 mg / ml Conbercept, 10 mM phosphate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0259] Formulation MM: 150 mg / ml Conbercept, 10 mM citrate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0260] Formulation NN: 150 mg / ml Conbercept, 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 6.2.

[0261] Formulation OO: 150 mg / ml Conbercept, 10 mM phosphate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0262] Formulation PP: 150 mg / ml Conbercept, 10 mM citrate-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 80 and 40 mM sodium chloride, pH 5.8 to 6.2.

[0263] Formulation QQ: 150 mg / ml Conbercept, 10 mM histidine-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0264] Formulation RR: 150 mg / ml conbercept, 10 mM phosphate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0265] Formulation SS: 150 mg / ml conbercept, 10 mM citrate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 20, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0266] Formulation TT: 150 mg / ml conbercept, 10 mM histidine-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0267] Formulation UU: 150 mg / ml conbercept, 10 mM phosphate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0268] Formulation VV: 150 mg / ml conbercept, 10 mM citrate-based buffer, 8% (w / v) sucrose, and 0.03% (w / v) polysorbate 80, pH 5.8 to 6.2, and optionally, specifically excluding viscosity reducers.

[0269] Formulation WW: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 10 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 50 mM taurine, pH 5.8.

[0270] Formulation XX: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 20 mM histidine-based buffer, 4% (w / v) proline, 0.03% (w / v) polysorbate 20 and 50 mM arginine hydrochloride, pH 5.8.

[0271] Formulation YY: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 20 mM histidine-based buffer, 2.5% (w / v) sucrose, 2.0% (w / v) proline, 0.03% (w / v) polysorbate 20 and 50 mM taurine, pH 5.8.

[0272] Formulation ZZ: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 10 mM histidine-based buffer, 2.5% (w / v) sucrose, 2.0% (w / v) proline, 0.03% (w / v) polysorbate 20 and 50 mM arginine hydrochloride, pH 5.8.

[0273] Formulation AAA: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 20 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 50 mM PSA, pH 5.8.

[0274] Formulation BBB: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 20 mM histidine-based buffer, 2.5% (w / v) sucrose, 2.0% (w / v) proline, 0.03% (w / v) polysorbate 20 and 50 mM PSA, pH 5.8.

[0275] Formulation CCC: 80, 100, 120 or 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 20 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 50 mM arginine hydrochloride, pH 5.8.

[0276] Formulation DDD: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 10 mM histidine-based buffer, 4% (w / v) proline, 0.03% (w / v) polysorbate 20 and 50 mM PSA, pH 5.8.

[0277] Formulation EEE: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 20 mM histidine-based buffer, 5% (w / v) sucrose and 0.03% (w / v) polysorbate 20, and optionally without heat stabilizers, pH 5.8.

[0278] Formulation FFF: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 10 mM sodium phosphate, 5% (w / v) sucrose and 0.03% polysorbate 20, pH 6.2.

[0279] Formulation GGG: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM sodium sulfate

[0280] Formulation HHH: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM sodium thiocyanate

[0281] Formulation III: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 40 mM sodium citrate

[0282] Formulation JJJ: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose, 0.03% polysorbate 20; 50 mM glycine

[0283] Formulation KKK: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose, 0.03% polysorbate 20; 50 mM sodium chloride

[0284] Formulation LLL: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM lysine

[0285] Formulation MMM: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM sodium aspartate

[0286] Formulation NNN: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM sodium glutamate

[0287] Formulation OOO: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM sodium citrate; 50 mM arginine hydrochloride

[0288] Formulation PPP: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM glycine; 50 mM arginine hydrochloride

[0289] Formulation QQQ: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM sodium aspartate; 50 mM arginine hydrochloride

[0290] Formulation RRR: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM histidine, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 50 mM sodium glutamate; 50 mM arginine hydrochloride

[0291] Formulation SSS: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM His, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 10 mM L-arginine hydrochloride

[0292] Formulation TTT: 140 mg / ml VEGF receptor fusion protein (e.g., apracept); 20 mM His, pH 5.8; 5% sucrose; 0.03% polysorbate 20; 100 mM L-arginine hydrochloride

[0293] Formulation UUU: 30 mg / ml VEGF receptor fusion protein (e.g., apracept), 10% sucrose, 10 mM phosphate, 0.03% polysorbate 20, pH 6.2

[0294] Formulation VVV: 30 mg / ml VEGF receptor fusion protein (e.g., apracept), 20% sucrose, 10 mM phosphate, 0.03% polysorbate 20, pH 6.2

[0295] Formulation WWW: 60 mg / ml VEGF receptor fusion protein (e.g., apracept), 10% sucrose, 10 mM phosphate, 0.03% polysorbate 20, pH 6.2

[0296] Formulation XXX: 60 mg / ml VEGF receptor fusion protein (e.g., apracept), 20% sucrose, 10 mM phosphate, 0.03% polysorbate 20, pH 6.2

[0297] Formulation YYY: 120 mg / ml VEGF receptor fusion protein (e.g., apracept), 10% sucrose, 10 mM phosphate, 0.03% polysorbate 20, pH 6.2

[0298] Formulation ZZZ: 120 mg / ml VEGF receptor fusion protein (e.g., apracept), 20% sucrose, 10 mM phosphate, 0.03% polysorbate 20, pH 6.2

[0299] Formulation AAAA: 120 mg / ml VEGF receptor fusion protein (e.g., apracept), 10% sucrose, 10 mM phosphate, 0.03% polysorbate 20, 50 mM NaCl, pH 6.2

[0300] Formulation BBBB: 120 mg / ml VEGF receptor fusion protein (e.g., apracept), 20% sucrose, 10 mM phosphate, 0.03% polysorbate 20, 50 mM NaCl, pH 6.2

[0301] Formulation CCCC: 140 mg / ml VEGF receptor fusion protein (e.g., apracept), 10 mM sodium phosphate, 5% sucrose, 40 mM sodium chloride, 0.03% PS20, pH 6.2

[0302] Formulation DDDD: 80 mg / ml VEGF receptor fusion protein (e.g., apracept), 20 mM histidine-based buffer, 5% (w / v) sucrose, 0.03% (w / v) polysorbate 20 and 50 mM L-arginine hydrochloride, pH 5.8.

[0303] Formulation EEEE: 120.0 mg / ml VEGF receptor fusion protein (e.g., apracept) (e.g., ±12 mg / ml), 20 mM histidine-based buffer (e.g., ±2 mM), 5% (w / v) sucrose (e.g., ±0.5%), 0.03% (w / v) polysorbate 20 (e.g., 0.02-0.04%), and 50 mM L-arginine hydrochloride (e.g., ±5 mM), pH 5.8 (e.g., 5.6-6.0 or 5.5-6.1).

[0304] Formulation FFFF: 113.3 mg / ml VEGF receptor fusion protein (e.g., apracept) (e.g., 102-125 mg / ml), 20 mM histidine-based buffer (e.g., ±2 mM), 5% (w / v) sucrose (e.g., ±0.5%), 0.03% (w / v) polysorbate 20 (e.g., 0.02-0.04%), and 50 mM L-arginine hydrochloride (e.g., ±5 mM), pH 5.8 (e.g., 5.6-6.0 or 5.5-6.1).

[0305] Formulation GGGG: 114.3 mg / ml VEGF receptor fusion protein (e.g., apracept) (e.g., 103-126 mg / ml), 10 mM histidine-based buffer (e.g., ±1 mM), 5% (w / v) sucrose (e.g., ±0.5%), 0.03% (w / v) polysorbate 20 (e.g., 0.02-0.04%), and 50 mM L-arginine hydrochloride (e.g., ±5 mM), pH 5.8 (e.g., 5.6-6.0 or 5.5-6.1).

[0306] Formulation HHHH: 100.0 mg / ml VEGF receptor fusion protein (e.g., apracept) (e.g., ±10 mg / ml), 20 mM histidine-based buffer (e.g., ±2 mM), 5% (w / v) sucrose (e.g., ±0.5%), 0.03% (w / v) polysorbate 20 (e.g., 0.02-0.04%), and 50 mM L-arginine hydrochloride (e.g., ±5 mM), pH 5.8 (e.g., 5.6-6.0 or 5.5-6.1).

[0307] Formulation IIII: 133.3 mg / ml VEGF receptor fusion protein (e.g., apracept) (e.g., ±13 mg / ml), 20 mM histidine-based buffer (e.g., ±2 mM), 5% (w / v) sucrose (e.g., ±0.5%), 0.03% (w / v) polysorbate 20 (e.g., 0.02-0.04%), and 50 mM L-arginine hydrochloride (e.g., ±5 mM), pH 5.8 (e.g., 5.6-6.0 or 5.5-6.1).

[0308] Formulation JJJJ: 150 mg / ml apracept (e.g., apracept) (e.g., ±15 mg / ml), 10 mM sodium phosphate, 8% (w / v) sucrose (e.g., ±0.8%), 0.03% (w / v) polysorbate 20 (e.g., 0.02-0.04%) and 50 mM L-arginine hydrochloride, pH 6.2 (e.g., 6.0-6.4 or 5.9-6.5).

[0309] Formulation KKKK: 114.3 mg / ml VEGF receptor fusion protein (e.g., apracept) (e.g., ±14 mg / ml), 20 mM histidine-based buffer (e.g., ±2 mM), 5% (w / v) sucrose (e.g., ±0.5%), 0.03% (w / v) polysorbate 20 (e.g., 0.02-0.04%), and 50 mM L-arginine hydrochloride (e.g., ±5 mM), pH 5.8 (e.g., 5.6-6.0 or 5.5-6.1);

[0310] Or any formulation described herein.

[0311] In embodiments of the invention, the concentration of any formulation component listed above or discussed herein (e.g., all components) (e.g., any one of formulations A-KKKK) is ± about 3%, 5%, or about 10% of the concentration specifically mentioned.

[0312] Manufacturing method

[0313] Embodiments herein include a method of manufacturing a pharmaceutical formulation of the present invention comprising a VEGF receptor fusion protein (e.g., any of formulations A-KKKK described herein), the method comprising combining the components of the formulation into a single composition and optionally introducing the formulation into a container or device (e.g., a vial) or delivery device (e.g., a pre-filled syringe). The formulation, vial, or device that is a product of this method is part of the present invention.

[0314] In embodiments of the present invention, a method for preparing a pharmaceutical formulation containing a VEGF receptor fusion protein (e.g., any of formulations A-KKKK described herein) comprises the steps of: culturing host cells (e.g., Chinese hamster ovary cells) containing one or more polynucleotides encoding a VEGF receptor fusion protein (e.g., apracept) in a culture medium and under conditions expressing the protein; and purifying the protein from the host cells and / or the culture medium, and combining a portion of the protein with an excipient of the pharmaceutical formulation described herein. Again, formulations, vials, or devices that are products of this method are part of the present invention.

[0315] In embodiments of the invention, the amount and type of VEGF receptor fusion protein required for achieving high concentrations and end uses in the formulation are determined. The same determination is made regarding the amount and type of buffer solution, the amount and type of surfactant, the amount and type of heat stabilizer, and the inclusion or specific exclusion of viscosity reducers. These components are combined and mixed to ensure that the pH of the formulation is at a desired value, for example, between about 5.0 and about 6.8 (e.g., 5.8), and / or the viscosity is at a desired value, for example, about 6.0, 7.3, 11.5, or 12.0 cP at 20°C. In embodiments of the invention, the formulation containing a high concentration of VEGF receptor fusion protein can be sterilized and stored under stable conditions, such as 2°C to 8°C (e.g., 5°C), for up to 24 or 36 months.

[0316] For example, Hardman et al. (2001), *Goodman and Gilman's The Pharmacological Basis of Therapeutics*, McGraw-Hill, New York, NY; Gennaro (2000), *The Science and Practice of Pharmacy*, Lippincott, Williams, and Wilkins, New York, NY; and Avis et al. (eds.)

[0317] (Author), (1993), Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, New York; Lieberman et al. (eds.), (1990), Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, New York; Lieberman et al. (eds.), (1990), Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, New York;

[0318] Weiner and Kotkoskie, (2000), Excipient Toxicity and Safety, Marcel Decker, New York.

[0319] Vascular eye diseases and cancer

[0320] The pharmaceutical formulations of the present invention containing a VEGF receptor fusion protein (e.g., any of pharmaceutical formulations A-KKKK) can be used to treat or prevent any vascular eye disease by administering a therapeutically effective amount of the VEGF receptor fusion protein in the formulation of the present invention to a subject in need, for example by intravitreal injection. Vascular eye disease, as used herein, refers to any eye disease caused by or associated with the growth or proliferation of blood vessels and / or leakage of blood vessels. Non-limiting examples of vascular eye diseases that can be treated or prevented using the formulations and methods described herein include:

[0321] Age-related macular degeneration (wet type),

[0322] ● Macular edema,

[0323] • Macular edema following retinal vein occlusion

[0324] ●Retinal vein occlusion (RVO)

[0325] Central retinal vein occlusion (CRVO),

[0326] ● Branch retinal vein occlusion (BRVO)

[0327] ● Diabetic macular edema (DME)

[0328] • Choroidal neovascularization (CNV)

[0329] • Iris neovascularization,

[0330] ● Neovascular glaucoma

[0331] Fibrosis after glaucoma surgery

[0332] ●Proliferative vitreoretinopathy (PVR)

[0333] ● New blood vessel formation in the optic nerve head

[0334] ● Corneal neovascularization,

[0335] • Retinal neovascularization

[0336] • Vitreous neovascularization

[0337] • Corneal opacity,

[0338] ·Pterygium,

[0339] Vascular retinopathy

[0340] • Diabetic retinopathy (e.g., nonproliferative diabetic retinopathy (e.g., characterized by a diabetic retinopathy severity grade (DRSS) of approximately 47 or 53) or proliferative diabetic retinopathy; e.g., in subjects without DME) and

[0341] ● Diabetic retinopathy in patients with diabetic macular edema (DME).

[0342] The pharmaceutical formulations of the present invention containing a VEGF receptor fusion protein (e.g., any of formulations A-KKKK) can be used to treat or prevent any cancer by administering a therapeutically effective amount of the VEGF receptor fusion protein in the formulation of the present invention to a subject in need, for example by intramuscular, intratumoral, subcutaneous, or intravenous injection. Cancers include those whose growth, proliferation, survival, and / or metastasis depend to some extent on angiogenesis. In embodiments of the present invention, said cancer is colorectal cancer, lung cancer, skin cancer, breast cancer, brain cancer, stomach cancer, kidney cancer, prostate cancer, liver cancer, or pancreatic cancer.

[0343] Therefore, the present invention provides a method for treating or preventing vascular eye diseases in subjects with such need, the method comprising intraocular administration of a therapeutically effective amount of VEGF receptor fusion protein (e.g., apacept) (e.g., about 4, 6, or 8.0, 8.1, 8.4, or 8.5 mg), for example in a pharmaceutical formulation according to the invention, into the vitreous body of the subject's eye. In embodiments of the invention, the VEGF receptor fusion protein is administered to both eyes. In embodiments of the invention, a therapeutically effective dose of the VEGF receptor fusion protein is administered approximately every 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks. In embodiments of the invention, this method of treatment or prevention is performed in the absence of a significant increase in blood pressure (systolic and / or diastolic) and / or the absence of hypertension (e.g., grade 1, 2, or 3) and / or abnormally high intraocular pressure in the subject. In embodiments of the invention, the method includes the following steps: after the administration, monitoring for a significant increase in the subject's blood pressure (systolic and / or diastolic) and / or the development of hypertension (e.g., grade 1, 2, or 3) and / or abnormally high intraocular pressure.

[0344] Application mode

[0345] The pharmaceutical formulations of the present invention, comprising a VEGF receptor fusion protein, can be administered according to known medically approved delivery systems. In embodiments herein, these delivery systems may include administration of the formulation to a patient via ocular, intraocular, intrachoroidal, intravitreal, or subconjunctival injection. Alternatively, the pharmaceutical formulations of the present invention may also be administered to a patient via topical routes such as eye drops, ocular gels, ointments, etc. Other possible routes of delivery for the formulations herein include intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral administration.

[0346] In an embodiment of the invention, intravitreal injection of the pharmaceutical formulation of the invention comprises the following steps: piercing the eye with a syringe and needle (e.g., a 30-gauge needle) containing the formulation, and injecting the formulation (e.g., less than or equal to about 100 μL; about 40, 50, 55, 56, 57, 57.1, 58, 60, 70, or 75 μL) into the vitreous humor of the eye (e.g., having a sufficient volume to deliver a therapeutically effective amount of VEGF receptor fusion protein, such as about 4, 5, 6, 7, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 8.9, 9, 10, 12, 14, 16, 18, or 20 mg of VEGF receptor fusion protein). Optionally, the method includes the step of administering a local anesthetic (e.g., procaine, lidocaine, or tetracaine), an antibiotic (e.g., a fluoroquinolone), a preservative (e.g., povidone-iodine), and / or a pupillary dilator to the eye to be injected. In embodiments of the invention, a sterile area is established around the eye to be injected prior to injection. In embodiments of the invention, after intraocular injection, the subject's intraocular pressure and / or blood pressure are monitored for elevation. In embodiments of the invention, the other eye is also injected using the same procedure.

[0347] Dosage of VEGF receptor fusion protein

[0348] Each dose of high-concentration VEGF receptor fusion protein administered to the subject during treatment may contain the same or substantially the same amount of fusion protein. Alternatively, the amount of any dose may be different or variable during treatment.

[0349] An effective or therapeutically effective amount of VEGF receptor fusion protein for the treatment or prevention of cancer (e.g., at least partially mediated by angiogenesis) or vascular eye disease refers to an amount of VEGF receptor fusion protein sufficient to cause regression, stabilization, or elimination of cancer or vascular eye disease, for example by regressing, stabilizing, or eliminating one or more symptoms or indications of cancer or vascular eye disease to any clinically measurable extent (e.g., in the case of vascular eye disease), by reducing or maintaining the severity score of diabetic retinopathy (DRSS), by improving or maintaining visual acuity (e.g., best-corrected visual acuity, for example, by increasing ETDRS letter measurements), increasing or maintaining visual field and / or reducing or maintaining central retinal thickness, and, in the case of cancer, by stopping or reversing the growth, survival, and / or metastasis of cancer cells in a subject. In embodiments of the present invention, the effective or therapeutically effective amount of VEGF receptor fusion protein for treating or preventing vascular ophthalmopathy is from about 0.5 mg to about 10 mg or from 0.5 mg to about 20 mg per dose, including: about 0.5 mg or more; or about 2 mg or more, for example, about 2.1 mg; 2.2 mg; 2.3 mg; 2.4 mg; 2.5 mg; 2.6 mg; 2.7 mg; 2.8 mg; 2.9 mg; 3.0 mg; 3.1 mg; 3.2 mg; 3.3 mg; 3.4 mg; g; 3.5mg; 3.6mg; 3.7mg; 3.8mg; 3.9mg; 4.0mg; 4.1mg; 4.2mg; 4.3mg; 4.4mg; 4.5mg; 4.6mg; 4.7mg; 4.8mg; 4.9m g; 5.0mg; 5.1mg; 5.2mg; 5.3mg; 5.4mg; 5.5mg; 5.6mg; 5.7mg; 5.8mg; 5.9mg; 6.0mg; 6.1mg; 6.2mg; 6.3mg; 6.4mg ; 6.5mg; 6.6mg; 6.7mg; 6.8mg; 6.9mg; 7.0mg; 7.1mg; 7.2mg; 7.3mg; 7.4mg; 7.5mg; 7.6mg; 7.7mg; 7.8mg; 7.9mg 8.0mg; 8.1mg; 8.2mg; 8.3mg; 8.4mg; 8.5mg; 8.6mg; 8.7mg; 8.8mg; 8.9mg; 9mg; 9.1mg; 9.2mg; 9.3mg; 9.4mg; 9. 5mg; 9.6mg; 9.7mg; 9.8mg; 9.9mg, 10.0mg, 10.1mg; 10.2mg; 10.3mg; 10.4mg; 10.5mg; 10.6mg; 10.7mg; 10.8mg ; 10.9mg; 11mg; 11.1mg; 11.2mg; 11.3mg; 11.4mg; 11.5mg; 11.6mg; 11.7mg; 11.8mg; 11.9mg; 12mg; 12.1mg; 12.2mg; 12.3mg; 12.4mg; 12.5mg; 12.6mg; 12.7mg; 12.8mg; 12.9mg; 13mg; 13.1mg; 13.2mg; 13.3mg; 13.4mg; 13.5mg; 13.6mg; 13.7mg; 13.8mg; 13.9mg; 14mg; 14.1mg; 14.2mg; 14.3mg; 14.4mg; 14.5mg; 14.6mg; 14.7mg; 14.8mg; 14.9mg; 15mg; 15.1mg; 15.2mg; 15.3mg; 15.4mg; 15.5mg; 15.6mg; 15.7mg; 15.8mg; 15.9mg; 16mg; 16.1m g; 16.2mg; 16.3mg; 16.4mg; 16.5mg; 16.6mg; 16.7mg; 16.8mg; 16.9mg; 17mg; 17.1mg; 17.2mg; 17.3mg; 17.4mg; 17.5mg; 17.6mg; 17.7mg; 17.8mg; 17.9mg; 18mg; 18. 1mg; 18.2mg; 18.3mg; 18.4mg; 18.5mg; 18.6mg; 18.7mg; 18.8mg; 18.9mg; 19mg; 19.1mg; 19.2mg; 19.3mg; 19.4mg; 19.5mg; 19.6mg; 19.7mg; 19.8mg; 19.9mg; or 20mg. In embodiments of the invention, an effective amount or therapeutically effective amount of VEGF receptor fusion protein for treating or preventing cancer is approximately 4 mg / kg (e.g., intravenous injection). This dose may be administered, for example, once every two weeks.

[0350] In embodiments of the invention, the VEGF receptor fusion protein is administered in a volume sufficient to deliver, for example, the desired dose of the fusion protein as described above. In embodiments of the invention, the delivery volume (e.g., for the treatment or prevention of vascular ophthalmopathy, e.g., via intravitreal injection) is less than or equal to about 100 μL (e.g., about any of the following volumes: 25 μL; 26 μL; 27 μL; 28 μL; 29 μL; 30 μL; 31 μL; 32 μL; 33 μL; 34 μL; 35 μL; 36 μL; 37 μL; 38 μL; 39 μL; 40 μL; 41 μL; 42 μL; 43 μL; 44 μL; 45 μL; 46 μL; 47 μL; 48 μL). 49 microliters; 50 microliters; 51 microliters; 52 microliters; 53 microliters; 54 microliters; 55 microliters; 56 microliters; 57 microliters; 58 microliters; 59 microliters; 60 microliters; 61 microliters; 62 microliters; 63 microliters; 64 microliters; 65 microliters; 66 microliters; 67 microliters; 68 microliters; 69 microliters; 70 microliters; 71 microliters; 72 microliters; 73 microliters; 74 microliters; 75 microliters; 76 microliters; 77 microliters; 78 microliters; 79 microliters; 80 microliters; 81 microliters; 82 microliters; 83 microliters; 84 microliters; 85 microliters;

[0351] 86 μL; 87 μL; 88 μL; 89 μL; 90 μL; 91 μL; 92 μL; 93 μL;

[0352] 94 microliters; 95 microliters; 96 microliters; 97 microliters; 98 microliters or 99 microliters).

[0353] In embodiments of the invention, the formulation is administered in a volume of about 60 μL or less, about 70 μL or less, about 75 μL or less, or about 100 μL or less (e.g., by intravitreal injection for the treatment or prevention of vascular eye diseases). For example, in embodiments of the invention, about 2, 4, 6, 8.0, 8.1, 8.0-8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, or 8.9, 9.0, or 10 mg of VEGF receptor fusion protein is administered in about 50, 60, 70, or 75 μL.

[0354] The scope of the invention also includes methods for delivering 0.5 or 2 mg of VEGF receptor fusion protein in a low volume, such as less than 50 μL (e.g., about 1, 5, 10, 17, 17.5, 18, 18.5, 20, 30, 40 or 45 μL) in the pharmaceutical formulation of the invention (e.g., for the treatment or prevention of vascular eye diseases, such as by intravitreal injection).

[0355] The present invention also includes a composition comprising, or consisting of, or substantially comprising a “single-dose volume” of the pharmaceutical formulation of the present invention, i.e., a volume (e.g., about 50 μL or 60 μL, 70 μL or 75 μL, or any volume containing about 4, 6, 8 or 10 mg of VEGF receptor fusion protein) in the pharmaceutical formulation of the present invention. As described below, containers (e.g., vials or injection devices) of formulations comprising a single-dose volume, optionally including a small excess fill volume, are also part of the present invention.

[0356] Containers and injection devices

[0357] High-concentration VEGF receptor fusion protein formulations (e.g., any of formulations A-KKKK) according to embodiments herein can be pre-packaged or pre-loaded in a variety of useful containers and injection devices. Therefore, the present invention includes containers and injection devices containing such formulations. In embodiments herein, the container is a vial that can be sterile. In another embodiment herein, the container is a test tube that can be sterile. In embodiments of the invention, the injection device (which can be sterile) is a syringe (e.g., a pre-filled syringe or an auto-injector). In one embodiment of the invention, the injection device is an intravitreal implant, such as a refillable intravitreal implant.

[0358] A "pre-filled" syringe is a syringe that is filled with the formulation of the present invention before it is sold or used by a physician or patient.

[0359] In this article, "sterile" means free of pathogens or free of substantially all or all living microorganisms and their spores.

[0360] The syringes used herein include a cylinder, which is made of, for example, glass or a polymer such as the cycloolefin described in U.S. Patent Publication 2017 / 0232199 (which is incorporated herein for all purposes), a plunger, and a needle.

[0361] Containers and injection devices may be coated with silicone (e.g., silicone oil) or baked silicone (e.g., silicone oil). < 40□g or < 100□g).

[0362] In embodiments of the invention, the container or injection device is substantially metal-free, substantially tungsten-free, or low in tungsten.

[0363] In one embodiment of the invention, the syringe includes one or more dose line graduations and / or a dose measurement system.

[0364] The container according to the embodiments described herein can contain high concentrations of VEGF receptor fusion protein formulations. In some aspects, the container or injection device may include a label stating instructions for use. In some cases, the container or injection device described herein may include a packaging insert with instructions for use as described throughout this specification.

[0365] In other embodiments, a volume containing a single or multiple doses (e.g., 2 or more) of a high concentration of VEGF receptor fusion protein (e.g., VEGF receptor fusion protein in doses of 2 mg, 4 mg, 6 mg, 8 mg, or 10 mg) (as described above) may be pre-packaged into a container or injection device, such as a sterile syringe, for storage until use. In one example, the volume in the container comprises a single dose of VEGF receptor fusion protein, optionally also including a small amount of excess fill volume. The sterile pre-filled syringe can be stored under storage conditions (e.g., 2°C–8°C) for, for example, up to 12 months, 24 months, or 36 months. Excess fill refers to an excess volume sufficient to allow for withdrawal and / or administration of an appropriate volume. In one embodiment of the invention, the container has a single dose volume or multiple dose volumes and approximately 5%–10% excess fill volume.

[0366] Syringe sizes can be, for example, 0.3cc, 0.5cc, or 1cc. Sterile syringes typically include needles suitable for intravitreal injections, usually about 1 / 2 inch in length, or 12.5mm to 16mm, and can be 29, 30, 31, 32, or 33 gauge, depending on the patient's and healthcare professional's preference. Other needle lengths and gauges can be used as long as the needle effectively completes the intravitreal injection.

[0367] Although the invention has been specifically shown and described with reference to various embodiments, those skilled in the art will understand that changes in form and detail may be made to the various embodiments disclosed herein without departing from the spirit and scope of the invention, and that the various embodiments disclosed herein are not intended to be limiting of the scope of the claims.

[0368] Other formulations

[0369] This invention includes formulations containing greater than 40 mg / ml of VEGF receptor fusion protein (e.g., apracept or conbercept) and:

[0370] (a) A buffer solution containing a histidine salt (e.g., histidine-HCl or histidine-acetate, e.g., 10 mM to 50 mM) and a pH of 5.7 to 6.2; a sugar (e.g., greater than 6% but not greater than 10%), such as sucrose, trehalose, mannitol or glucose; and a surfactant selected from polysorbate 20 and polysorbate 80 (e.g., 0% to 0.1%).

[0371] (b) A buffer containing histidine, such as L-histidine / histidine hydrochloride (e.g., 10 mM); a nonionic surfactant, such as polysorbate 20 (e.g., 0.03%); an inorganic salt, such as NaCl (e.g., 40 mM); and a carbohydrate, such as sucrose (e.g., 5%), for example, at pH 6.0-6.5 (e.g., 6.2 or 6.5);

[0372] (c) Citric acid (e.g., 5 mM, 10 mM, 15 mM, 20 mM, 25 mM or 30 mM), sucrose (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%), arginine (e.g., 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM or 50 mM or 100 mM), polysorbate 20 (e.g., 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09% or 0.10%);

[0373] (d) Buffer solutions, such as phosphates, histidines, acetates, succinates, citrates, glutamates, and / or lactates (e.g., 5-20 or 5-50 mM); nonionic surfactants, such as polysorbates (e.g., PS20 or PS80), polyethylene glycol dodecyl ether, poloxamer, 4-(1,1,3,3-tetramethylbutyl)phenyl-polyethylene glycol, alkyl sugars, or alkyl glycosides; tonicating agents, such as polyols or amino acids, such as sucrose, trehalose, sorbitol, mannitol, glycerol, proline, arginine, methionine, glycine, or lysine, wherein the final tonicotinic concentration of the formulation is approximately 300 mOsm / kg, and wherein the concentration of chloride anions is less than approximately 10 mM; pH 5.0-6.5; or

[0374] (e) 10 mM sodium phosphate, 40 mM sodium chloride, 0.03% polysorbate 20 and 5% sucrose, pH 6.2.

[0375] Example

[0376] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. Efforts have been made to ensure the accuracy of the figures used, but some experimental errors and biases should be taken into account. Any formulations listed in these examples are part of this invention.

[0377] In these embodiments, when an experiment is conducted at 2-8°C, the temperature target is 5°C, with a tolerance of ±3°C variation.

[0378] Example 1The 80 mg / ml VEGF receptor fusion protein formulation maintained its potency, physical stability, and charge over a period of 36 months.

[0379] The long-term potency, physical stability, and charge heteromorph formation of four different formulations containing 80 mg / ml VEGF receptor fusion protein (apasicept) were tested. The ingredient list for each of the four formulations is shown in Table 1-1. The formation of VEGF receptor fusion protein HMW species and the percentage of major species were assessed for each formulation during 36 months of storage at 2°C to 8°C by SE-UPLC. Each formulation containing VEGF receptor fusion protein was also evaluated using imaging capillary isoelectric focusing at the same time course and temperature. Finally, the potency of each formulation was tested by bioassay at the same time course and temperature.

[0380] Table 1-1. Formulations containing VEGF receptor fusion protein

[0381]

[0382] Table 1-2. %HMW Types

[0383]

[0384] Table 1-3. % Main Peak

[0385]

[0386]

[0387] Table 1-4. % of Acid Types

[0388]

[0389] Table 1-5. % Main Categories

[0390]

[0391]

[0392] Data shown Figure 1A , 1B 1C and 1D. Figure 1A The percentage of HMW species formed for each of the four formulations is shown. Compared to sodium phosphate, the rate of HMW species formation in VEGF TRAP is slightly slower when the formulation is histidine-based. Figure 1A As shown, after 36 months of storage at 2°C to 8°C, the %HMW class increased by 2.6% in sodium phosphate formulations (formulations 1 and 2), compared to an increase of 1.6% to 1.9% in histidine buffer (formulations 3 and 4). Figure 1B As shown, the data were confirmed, with SE-UPLC used to identify the percentage of major VEGF receptor fusion protein species present within the same time and temperature range. As a point of comparison, the current concentration of VEGF receptor fusion proteins with 10 mM sodium phosphate was used. The formulation (40 mg / ml) was tested, which showed that the %HMW class increased by 1.2% (not shown) after storage at 2°C to 8°C for 36 months. In addition, the potency of each of the four 80 mg / ml VEGF TRAP formulations was tested and compared with... Activities were compared (not shown). This was achieved through all four formulations and... The bioassay method maintains potency. These data indicate that the formulation described in this paper can maintain efficacy with... The VEGF receptor fusion protein exhibits considerable stability.

[0393] Reference Figure 1C and 1D The percentage of VEGF TRAP charge heterogenes formed after storage at 2°C to 8°C for 36 months was also tested. Figure 1C The results showed that VEGF TRAP in formulations 1-4 did not exhibit a significant change in the percentage of acidic species over a 36-month period. Figure 1D The same findings regarding the percentage of major species were shown over a 36-month period. The finding that VEGFTRAP retains its charge heteromorph species during storage indicates that the quality of VEGF TRAP is maintained and that processes such as deamination, N-terminal pyroglutamic acid formation, isomerization, and aggregation did not occur during storage.

[0394] The data in Example 1 show that, Formulations with twice the VEGF TRAP concentration maintained physical stability, quality, and potency over a 36-month period at 2°C to 8°C. Furthermore, while all four formulations showed similar potency during this period, the histidine-containing formulation resulted in a slightly smaller increase in HMW species formation (a sign of protein degradation), suggesting that histidine may be the buffer of choice in certain situations. However, the histidine- and phosphate-based buffers exhibited excellent stability throughout the study.

[0395] Example 2 Stability of VEGF TRAP at 150 mg / ml in sodium phosphate buffer / sucrose formulation.

[0396] The physical stability of two sodium phosphate formulations of 150 mg / ml VEGF Trap (apasip) was tested at 37°C for 28 days. The ingredient list for each formulation is shown in Table 2-1. The formation of high molecular weight wheat (HMW) species and the percentage of major species were assessed for each formulation during 28 days of storage at 37°C using SE-UPLC.

[0397] Table 2-1. Formulation containing VEGF receptor fusion protein (apascept) in Example 2

[0398]

[0399] Table 2-2. %HMW Types

[0400]

[0401]

[0402] Table 2-3. % Main Peak

[0403]

[0404] Figure 2A The results showed no significant difference in the %HMW class formed between the two formulations during 28 days of storage at 37°C, as confirmed by the percentage of major class determined by SE-UPLC. Figure 2B During the 28-day period, there were no changes in appearance, turbidity, or pH of either formulation (not shown).

[0405] Then the results were compared with those containing 40 mg / ml The formulation was compared with formulation 1 of Example 1 (80 mg / ml VEGF TRAP in sodium phosphate buffer). As expected, compared with Compared to other formulations, the 150 mg / ml VEGFTRAP formulation showed a higher percentage of HMW species formation, and a slightly higher percentage of HMW species formation compared to formulation 1 of Example 1.

[0406] Further testing was conducted on formulations containing high concentrations of VEGF TRAP, with histidine-containing formulations showing better protection of these molecules than sodium phosphate-based formulations. Furthermore, incorporation of sodium chloride into each test formulation effectively resulted in instability of VEGF TRAP at 37°C. Data from Example 2 demonstrate that the formulations presented herein provide excellent protection of physical stability even at high concentrations of VEGF receptor fusion protein (150 mg / ml) and relatively high storage temperatures.

[0407] Example 3: For VEGFTRAP at 150 mg / ml, a pharmaceutically acceptable formulation viscosity can be obtained.

[0408] The viscosity properties of numerous formulations containing VEGF Trap (apasip) were tested. Formulations with VEGF Trap concentrations ranging from 10 mg / ml to 160 mg / ml were tested in the presence and absence of various viscosity reducers.

[0409] Figure 3A Formulations containing 155 mg / ml VEGF Trap in 10 mM sodium phosphate buffer, 5% sucrose, and pH 6.2, with no inorganic salts bound, and containing arginine, lysine, sodium chloride, and magnesium chloride, showed almost no difference in viscosity. Viscosity was measured in cP at 20°C in each case. Viscosities were observed to range between approximately 17 cP (without inorganic salts) and 15 cP (100 mM magnesium chloride), with almost no change between the use of viscosity reducers or their complete presence. Figure 3B A series of similar formulations were shown, except that the alkaline buffer was 10 mM histidine and pH 5.8. Here, the absence of any inorganic salts provided a formulation with a viscosity of 11.5 cP, and 50 mM lysine provided a viscosity of 14 cP. The histidine-containing buffer exhibited excellent viscosity, consistent with other low-concentration biological injectables.

[0410] The viscosity of VEGF traps at pH 6.2 in the absence of arginine, with 10 mM sodium phosphate and 5% sucrose, and with 50 mM arginine hydrochloride, was tested at concentrations ranging from 10 mg / ml to 160 mg / ml. For all tested VEGF trap concentrations, the presence of viscosity-reducing agents resulted in similar viscosities. Figure 3A and 3B As shown, even at higher protein concentrations, viscosity reducers have almost no positive effect on formulation viscosity. This is a surprising result, as arginine and other viscosity reducers have previously been shown to have beneficial properties for high protein concentrations.

[0411] Table 3-1. Viscosity-phosphate formulations (cP at 20°C)

[0412] Buffer composition 10mM sodium phosphate, 5% sucrose, pH 6.2 No viscosity reducer 17.3 100mM arginine 16.2 200mM arginine 15.8 50mM lysine 16.2 100mM sodium chloride 16.5 50mM magnesium chloride 16.3 100mM magnesium chloride 15.3

[0413] Table 3-2. Viscosity-histidine preparations (cP at 20℃)

[0414] Buffer composition 10mM histidine, pH 5.8 No viscosity reducer 11.5 100mM arginine 12.0 200mM arginine 12.7 50mM lysine 14.0 200mM lysine 14.1 50mM sodium chloride 13.1 100mM sodium chloride 12.1 50mM magnesium chloride 12.6 100mM magnesium chloride 11.4

[0415] Example 4: Arginine hydrochloride improves the effect of sodium phosphate buffer / sucrose formulation on 150 mg / ml VEGF TRAP stability Qualitative analysis.

[0416] The physical stability of two sodium phosphate formulations of 150 mg / ml VEGF Trap (apasip) was tested over 12 months at 2–8°C. The ingredient list for each of the two formulations is shown in Table 4-1. Formulation 2 also contains 50 mM arginine hydrochloride. The formation of high molecular weight wheat (HMW) species and the percentage of major species were assessed by SE-UPLC during 12 months of storage at 37°C for each formulation.

[0417] Table 4-1. Formulation containing VEGF receptor fusion protein (apasip) in Example 3

[0418]

[0419] Figure 4A The formulation showed that the inclusion of 50 mM arginine hydrochloride improved the physical stability of VEGF TRAP. This stabilizing property of VEGF TRAP... Figure 4B This was confirmed in the presence of 50 mM arginine hydrochloride, where the percentage of major species in VEGF TRAP remained high. This data suggests that, in certain cases, the addition of arginine hydrochloride can provide stabilization for stored VEGF TRAP. Size exclusion chromatography data are listed in Tables 4-2 and 4-3.

[0420] Table 4-2. %HMW Types

[0421]

[0422]

[0423] Table 4-3. % Main Peak

[0424]

[0425] Example 5: Viscosities in histidine and phosphate buffer ranged from 10 mg / ml to 170 mg / ml VEGF TRAP

[0426] Figure 5 The results show that both 10 mM phosphate buffer and 10 mM histidine buffer exhibit very useful viscosity across a range of VEGF Trap (apasip) concentrations. At higher VEGF Trap concentrations, the histidine buffer shows improved viscosity (relative to phosphate). Viscosities were measured at 20 °C.

[0427] Example 6: Taurine and propanesulfonic acid provide improved efficacy for high-concentration VEGFTrap (apascept) formulations. stability.

[0428] Experiments were conducted to determine the effects of various formulation combinations on protein stability. Figure 6Dynamic light scattering plots are shown for formulations with any values ​​ranging from 2 mg / ml protein to 10 mg / ml protein (apascept). Formulations containing 70 mM taurine or 70 mM PSA showed reduced self-interactions at higher concentrations. These data suggest that taurine and PSA may be useful inclusions in high-concentration protein formulations, as discussed in this case.

[0429] Example 7: Long-term study of the stability of various histidine-containing preparations.

[0430] Nine different apracept formulations were tested with varying buffer concentrations, heat stabilizers, and hydrophobic salts. Table 7-1 summarizes the nine formulations, F1-F9.

[0431] Table 7-1. Formulations F1-F9.

[0432]

[0433] VEGFT = VEGF Trap (Apacrip)

[0434] Fourteen (14) mL of each formulation was mixed into a 15 mL Falcon tube. The formulation was then aseptically filtered using a 0.22 μM syringe filter and transferred into sterile 2 mL Type 1 glass vials. In a laminar flow hood, eight (8) vials of each formulation were filled to a volume of 0.4 mL.

[0435] SE-UPLC (molecular weight class) was performed on all samples to determine the chemical stability of each formulation.

[0436] Figure 7 The percentage change in HMW species over time for each formulation F1-F9 is shown in Table 7-2 (see also Table 7-2). Formulations F4 and F7 showed the lowest percentage of HMW species after 4 months at 5°C. The results indicate that the addition of excipients such as arginine and proline significantly reduces the rate of %HMW species formation in VEGF Trap formulations.

[0437] Table 7-2. SE-UPLC analysis (2-8℃)

[0438]

[0439]

[0440] Example 8: Stability study of 140 mg / mL VEGFTrap with different arginine concentrations.

[0441] The effects of different concentrations of arginine hydrochloride on the stability of four 140 mg / mL VEGF Trap (apasicept) ophthalmic drug formulations were investigated. Stability assessments were performed under storage conditions ranging from 2 to 8 °C. The drug (DP) was also incubated under stress (37 °C). The four formulations (F1-F4) evaluated in this stability study are described in Table 8-1 below.

[0442] Table 8-1. Formulations F1-F4

[0443]

[0444] Thaw approximately 145 mL of the 187 mg / mL VEGF Trap histidine formulation. Mix 36 (36) mL of each formulation except F3; mix 39 mL of F3. Each formulation was sterilized by filtration through a 0.22 μm Durapore PVDF sterile filter in an LFH (laminar flow hood) and then filled. Stability studies were conducted using clean, pyrogen-free 2 mL Type I Schott glass vials with 13 mm serum stoppers (S2-F451 4432 / 50B2-40).

[0445] SE-UPLC analysis of the formulations was performed as described above. The HMW measurements of SE-UPLC% over time for each formulation are listed in Figure 8(AB). See also Tables 8-2 and 8-3.

[0446] When stored at 5°C, the %HMW species formation rate of these formulations decreased, proportional to the concentration of arginine hydrochloride. However, this effect was reversed under stress. The formulation showed a significant increase in %HMW species proportional to the concentration of arginine hydrochloride at 37°C. This property makes it highly unlikely that the beneficial effects of arginine on apracept will be found in the presence of histidine buffer. Typically, for formulation development in the biotechnology industry, the effects of various excipients on the drug are first screened under stress (e.g., at high temperatures, such as 37°C) for a short period. The aim of this approach is to rapidly eliminate excipients that may not perform well under prolonged stress-free conditions (e.g., at lower temperatures, such as 5°C). Although arginine has an effect on stability at 37°C, it is still considered a useful excipient. Since formulated pharmaceuticals are typically stored at 4–5°C for several months after production, apracept in arginine and histidine buffer would be a valuable formulation for long-term stable storage of the drug.

[0447] Table 8-2. SE-UPLC analysis (2-8℃)

[0448]

[0449]

[0450] Table 8-3. SE-UPLC analysis (37℃)

[0451]

[0452] Example 9: VEGFTrap stability in the presence of counterions from the Hofmeister series and other excipients Qualitative analysis.

[0453] The effects of various counterions and other excipients on the stability of several formulations were determined.

[0454] A. Counterion screening

[0455] Counterions (e.g., sulfate, thiocyanate, and citrate) were tested in sodium salt form with a high concentration of VEGF Trap (apasicept) formulation (140 mg / ml). Other amino-added excipients (e.g., glycine and lysine) were also tested.

[0456] Thaw approximately 42 ml of 187 mg / ml VEGF Trap ophthalmic medication. Prepare 50 ml of intermediate formulation of the drug substance (155.56 mg / ml), which has a concentration of 110% of the formulation drug substance of 140 mg / ml and the target excipient concentration (excluding anti-counterions, glycine, and lysine).

[0457] The intermediate formulations were further diluted with a 0.5 M excipient stock solution (anti-counterion, glycine, or lysine) to produce the 140 mg / ml formulations listed in Table 9-1 below. Each final formulation was sterilized in a laminar flow hood using a 0.22 μm PVDF syringe filter and then filled into clean, pyrogen-free 2 mL Type I Schott glass vials fitted with a 13 mm serum stopper S2-F451.

[0458] 4432 / 50 82-40 (ELN item number 19700004, flush number 0 000078949) plugged. Sixty (60) vials contain 0.5 ml of each preparation. Six (6) vials contain 1.5 ml.

[0459] Table 9-1. Formulations used for anti-counterion screening

[0460]

[0461] B. Screening for glutamic acid and aspartic acid

[0462] The stability of a high-concentration formulation (140 mg / ml) of VEGF Trap (apasip) in the presence of organic counterions bound to arginine hydrochloride was tested. Furthermore, the compatibility of two novel counterions (glutamate and aspartic acid) with high-concentration VEGF Trap (with and without arginine hydrochloride) was also tested. Table 9-2 below summarizes the tested formulations.

[0463] Thaw approximately 50 ml of VEGF Trap drug at 187 mg / ml. Prepare 60 ml of intermediate formulation drug substance (155.56 mg / ml) having 110% of the formulated drug substance at 140 mg / ml and the target excipient concentration (excluding anti-counterions, citrate, glycine, glutamic acid, and aspartic acid). The intermediate formulation drug substance is further diluted with 1M excipient stock solution (arginine hydrochloride, sodium citrate, glycine, monosodium glutamate, and monosodium aspartate) to produce the 140 mg / ml formulated drug substance formulation listed in the table below. Each final formulation was sterilized in a laminar flow hood using a 0.22 μm PVDF syringe filter and then placed into clean, pyrogen-free 2 mL Type I Schott glass vials, sealed with a 13 mm serum stopper S2-F4514432 / 50B2-40 (ELN item number 19700004, rinse number 0000078949). Sixty (60) vials contained 0.5 mL of each formulation. Six (6) vials contained 1.5 mL.

[0464] Table 9-2. Formulations used for screening glutamic acid and aspartic acid

[0465]

[0466]

[0467] Using RHEOSENSE Viscosity testing is performed using a viscometer. Approximately 0.5 mL of undiluted sample is loaded onto a glass syringe. The sample is equilibrated to the desired temperature and then injected into the chip or measurement cell. Viscosity is calculated by measuring the pressure drop from the inlet to the outlet, which is related to the shear stress on the chip wall. The result is expressed as mPas⁻¹ or cp. See also Figure 9 See Table 9-3.

[0468] The osmotic concentration by weight was measured using a VAPRO vapor pressure osmometer. Approximately 10 μL of undiluted sample was inoculated into a paper tray. The dew point temperature drop, a function of the solution vapor pressure, was measured by a sensitive thermocouple and reported as the osmotic concentration by weight of the solution. Results are expressed as mmol / kg or mOsm. See also Figure 10 See Table 9-3.

[0469] High molecular weight species in certain formulations over time after storage at 37°C or 5°C were also evaluated by SE-UPLC. See Figure 11(AB) and Tables 9-4 and 9-5.

[0470] Dynamic light scattering experiments are conducted using... The analysis was performed using Plate Reader II. Approximately 100 μL of undiluted sample was loaded onto a 96-well plate. 35 acquisitions were performed per well at 25 °C. Autocorrelation analysis using a regularized function was performed on DYNAMICS v7.1 software. Radius (nm) vs. % intensity plots and % mass vs. radius (nm) plots were generated to determine the mean molecular radius and % polydispersity value (%Pd) for each sample. See also... Figure 12 .

[0471] Table 9-3. Viscosity and weight molar permeation concentration of various formulations

[0472]

[0473] Table 9-4. SE-UPLC analysis; %HMW (37℃)

[0474]

[0475]

[0476] Table 9-5. SE-UPLC analysis; %HMW (5℃)

[0477]

[0478] Example 10: Tolerance of IVT delivery of high doses of VEGF trap (140 mg / ml) in normal rabbits.

[0479] Intravitreal injection of anti-VEGF therapy is currently the standard of care for treating neovascular age-related macular degeneration, diabetic macular edema, and retinal vascular occlusive disease. However, monthly or bi-monthly intravitreal injections place a significant burden on patients, caregivers, and physicians. There is an urgent need for more effective and durable therapies in clinical practice. This study aimed to investigate the tolerability of a stable formulation of a high-dose VEGF Trap (140 mg / ml, equivalent to 14 times the clinical dose) in the eyes of normal New Zealand white rabbits.

[0480] Two formulations of high-dose VEGF-Trap (apasip) in histidine-buffered saline or phosphate-buffered saline were tested. Three rabbits in each group received 7 mg VEGF Trap at 50 mg / L via a single bilateral intravitreal injection of each formulation. Ocular signs of inflammation were monitored by slit-lamp scanning, optical coherence tomography (OCT), and fundus angiography on day 1, day 4, week 1, and then weekly until week 12 following intravitreal administration. Intraocular pressure was measured with Tonopen before, 10 minutes after, and 30 minutes after intravitreal injection, and at each follow-up time point. Animals were euthanized at week 12.

[0481] Give the rabbit's eyes any of the following:

[0482] (1) 140 mg / ml apracept, 20 mM histidine, 5% sucrose, 50 mM arginine HCl, 0.03% PS20, pH 5.8;

[0483] or

[0484] (2) 140 mg / ml apracept, 10 mM sodium phosphate, 5% sucrose, 40 mM sodium chloride, 0.03% PS20, pH 6.2.

[0485] Two of the six eyes in the histidine-buffered saline group developed vitreous opacities up to 8 weeks post-IVT, which, confirmed by slit-lamp examination, were due to localized cataracts caused by surgery-related posterior lens injury. One of the six eyes in the phosphate-buffered saline group also developed vitreous opacities up to 8 weeks post-IVT, which, confirmed by slit-lamp examination, were due to localized cataracts caused by (surgery-related) posterior lens injury.

[0486] Within 12 weeks following a single intravitreal administration, the tested high-dose VEGFTrap (140 mg / ml) formulation was well tolerated in normal New Zealand white rabbit eyes. No abnormalities or signs of inflammation were observed in the fundus following a single intravitreal injection of 7 mg VEGF Trap. The vitreous humor was clear, and retinal vascular morphology was normal. No retinal detachment, hemorrhage, or optic nerve head changes were observed. Intraocular pressure remained unchanged compared to baseline. Baseline fluorescein angiography (FA) and OCT images of the rabbit eyes are shown in Figure 13 (AD). Figures 14, 15, 16, and 17 show FA and OCT images of the rabbit eyes over time at 8 weeks for each treatment group. Data from the left eye (OS) or right eye (OD) of rabbits 326 and 329 are shown.

[0487] Example 11: Evaluation of the stability of formulation UUU-BBBB

[0488] This study examined the stability of 60 mg / ml and 120 mg / ml VEGF Trap (apasip) incubated at 37°C for up to 6 months in 10 mM phosphate, 10% or 20% sucrose, 0 or 50 mM NaCl, 0.03% polysorbate 20, and pH 6.2. Table 11-1 below lists the formulations used in this stability study.

[0489] Table 11-1. Formulation UUU-BBBB

[0490]

[0491] Basic formulation: 10 mM phosphate, 0.03% polysorbate 20, pH 6.2 VEGF Trap = apracept

[0492] Before dispensing, the formulation is sterilized by filtration using a PVDF 0.2μm filter in a laminar flow hood.

[0493] Store vials containing each formulation at 37°C for one month.

[0494] Table 11-2. Results of reversed-phase high-performance liquid chromatography (RP-HPLC)

[0495]

[0496]

[0497] VGFT = VEGF Trap, Apacip

[0498] Table 11-3. Size exclusion ultra-high performance liquid chromatography (SE-UPLC) results

[0499]

[0500] VGFT = VEGF Trap, Apacip

[0501] Visual inspection and optical density measurements showed that all formulations were essentially free of particles. After one month at 37°C, RP-HPLC recovery showed no significant loss of protein (Table 11-2), and this trend continued for six months.

[0502] The primary degradation pathway of VEGF traps is aggregation under these conditions (Figure 18(AB); Table 11-3). The stability of VEGF traps depends on protein and sucrose concentrations. Formulations with lower VEGF trap concentrations and higher sucrose concentrations are more stable. Maintaining the same protein:sucrose ratio does not result in the same degradation rate. For example, F1 and F4 have the same protein:sucrose ratio of 3:1, but F4 is less stable than F1 due to its higher protein concentration. Similar trends were observed over 6 months.

[0503] Example 12 Repeated-dose in vivo toxicology studies in monkeys using different apacept formulations.

[0504] In this embodiment, the safety of various formulations in cynomolgus monkeys was evaluated. For groups 1–3, a total of 7 intravitreal doses were administered to both eyes approximately Q4W; for groups 4–9, a total of 3 intravitreal doses were administered to both eyes approximately Q4W. Terminal necropsy was performed approximately one week after the last administration (N = 3 / sex / group), and recovery necropsy was performed approximately 12 weeks after the last administration (2 / sex / group).

[0505] Perform the following assessment:

[0506] Safety assessments were conducted based on clinical signs, weight, vital signs, electrocardiogram data (at the end of administration and at the end of recovery), blood pressure measurements (via tail cannula), and clinical and anatomical pathology.

[0507] • Regular, comprehensive ophthalmological examinations, including slit-lamp microscopy, indirect ophthalmoscopy, and intraocular pressure measurement, should be performed during medication and recovery.

[0508] Fundus photography, fluorescein angiography, and electroretinography were performed before treatment, at week 9 (all groups), at week 26 (groups 1-3 only), and at the end of the recovery period.

[0509] • Blood and hyaline (at termination) are used for bioanalysis, ADA analysis, and toxicological assessment.

[0510] Table 13. Summary of Research Groups*

[0511]

[0512]

[0513] *Each of the formulations described herein is part of the invention.

[0514] The safety profile of the formulations tested in any one or more of groups 1, 2, 3, 4, 5, 6, 7, 8 and / or 9 in cynomolgus monkeys is expected to be comparable to that of EYLEA, as determined in the assessments discussed above.

[0515] Example 13 Stability study of products using VEGF traps.

[0516] Prior to filling, a formulation containing 114.3 mg / mL VEGF Trap (apasip) and 10 mM histidine, pH 5.8, 5% sucrose, 0.03% polysorbate 20, and 50 mM arginine hydrochloride was prepared in an LFH (laminar flow hood) using a 0.22 μm Durapore PVDF sterile filter. Clean, pyrogen-free 3 mL Type I Schott glass vials were filled with 0.3 mL of the drug and sealed with 13 mm serum stoppers (S2-F451 4432 / 50B2-40). After storage at 37°C or 5°C, the purity of the samples was analyzed at different time points by size exclusion chromatography (SEC) to determine the presence (%) of high molecular weight species (HMW), low molecular weight species (LMW), and the major peak (main peak); and the presence of particles of various sizes was determined by microfluidic imaging, HIAC liquid particle counting (with light obstruction), and microscopy.

[0517] Figure 19(AE) shows the stability and purity data after storage at 37°C, 25°C, and 2–8°C. See also Table 13-1-13-4.

[0518] Table 13-1. Purity Analysis by Size Exclusion Chromatography (SEC)

[0519]

[0520]

[0521] Table 13-2: Microfluidic Imaging / Particles Visible Only Under a Microscope

[0522]

[0523] Table 13-3. Microscopically Visible Particulate Matter / Microscopically Visible Particles as Measured by High-Intensity Optical Angle Control (HIAC)

[0524]

[0525] Table 13-4. Microscopic analysis of particles visible only under a microscope

[0526]

[0527]

[0528] Example 14 Stability study of VEGF trap concentration with varying concentrations.

[0529] The effects of different concentrations of VEGF Trap on the stability of four VEGF Trap (apasip) ophthalmic formulations were investigated. Concentrations ranged from 80 to 140 mg / mL. Stability assessments were performed under storage conditions of 2–8 °C. The drug (DP) was also incubated under stress (37 °C). The four formulations evaluated in this stability study (each referred to as “CCC” in this document) are described in Table 14-1 below.

[0530] Prior to filling, approximately 27 mL of each formulation was prepared in an LFH (laminar flow hood) using a 0.22 μm Durapore PVDF sterile filter. Clean, pyrogen-free 2 mL Type I Schott glass vials were filled and sealed with 13 mm serum stoppers (S2-F4514432 / 50B2-40). After storage at 37 °C or 2–8 °C, the purity of the samples was analyzed at different time points by size exclusion ultra-high performance liquid chromatography (SE-UPLC) to determine the presence (%) of high molecular weight species (HMW) and major peaks (main peaks).

[0531] Figure 20(AB) shows the data for the percentage of HMW after 6 months of incubation at 2-8°C or 37°C. See also Tables 14-2 and 14-3. When stored at 37°C and 5°C, these formulations showed a positive correlation between the HMW formation rate and the concentration of VEGF Trap. The 140 mg / mL formulation showed the highest rate of % HMW species formation.

[0532] Table 14-1. Formulations tested.

[0533]

[0534] Table 14-2. SE-UPLC analysis (2-8℃)

[0535]

[0536] Table 14-3. SE-UPLC analysis (37℃)

[0537]

[0538]

[0539] Example 15 In the D,L-AAA model of persistent neovascular leakage, high doses of Eylea had a prolonged duration of action.

[0540] In this embodiment, the effect of a fourfold increase in dose on the duration of apracept's effect in inhibiting chronic retinal vascular leakage in the D,L-AAA model was determined.

[0541] The animals used were New Zealand white rabbits 3 months after DL-AAA (DL-α-aminohexanoic acid) disease induction. The treatment group consisted of:

[0542] ● Placebo (formulation buffer) 50 ml / eye; n = 6 eyes

[0543] ● Apasil 500 mcg / eye (in 50 ml clin); n = 7 eyes

[0544] ● Apacipur 2 mcg / eye (in 50 mlcl); n = 8 eyes

[0545] The formulation used in each treatment group was: 10 mM histidine, 8% sucrose, 0.03% PS20, pH

[0546] 5.8. Ophthalmic examinations performed at baseline and at weeks 1, 2, 4, 6, 7, 9, 10, 11, 13, and 18 included intraocular pressure (IOP), red-light-free (RF) imaging (to determine vascular morphology), fluorescein angiography (FA; to determine vascular leakage), and optical coherence tomography (OCT; to determine vitreous inflammation). Serum (ADA) and plasma (drug levels) were collected at baseline and at weeks 1, 2, 4, 6, and 9.

[0547] FA test data for each group are as follows: Figure 21 As shown in the figures, these data indicate that the duration of inhibition of vascular permeability in the retina of animals receiving higher doses of apacept was longer than that in animals receiving lower doses. At all sampling times up to 18 weeks post-dose, the number of treated eyes with complete inhibition of vascular permeability was significantly increased in the 2 mg dose group.

Claims

1. An aqueous pharmaceutical preparation comprising: A VEGF receptor fusion protein at a concentration of at least 100 mg / ml, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; sucrose; L-arginine; Histidine-based buffers; and Nonionic surfactants selected from polysorbate 20 and / or polysorbate 80; The pH of the preparation is 5.0 to 6.8; The VEGF receptor fusion protein described therein has less than 3.5% high molecular weight species immediately after manufacture and purification and / or less than or equal to 6% high molecular weight species after storage at 2-8°C for 24 months.

2. The formulation according to claim 1 is suitable for intravitreal administration.

3. The formulation according to claim 1 further comprises sodium sulfate, sodium thiocyanate, glycine, NaCl, sodium aspartate, and / or sodium glutamate.

4. The formulation according to claim 2, further comprising sodium sulfate, sodium thiocyanate, glycine, NaCl, sodium aspartate, and / or monosodium glutamate.

5. The formulation according to any one of claims 1-4, wherein the VEGF receptor fusion protein is apacept.

6. The formulation according to any one of claims 1-4, wherein the VEGF receptor fusion protein is a VEGF receptor fusion protein at a concentration of the following: Approximately 100 mg / ml; • Approximately 111.5 mg / ml; • Approximately 112.0 mg / ml; • Approximately 113.3 mg / ml; • Approximately 114.3 mg / ml; • Approximately 115.6 mg / ml; • Approximately 116.3 mg / ml; • Approximately 120 mg / ml; Approximately 133 mg / ml; • Approximately 140 mg / ml; • Approximately 150 mg / ml; • Approximately 200 mg / ml; or • Approximately 250 mg / ml.

7. The formulation according to any one of claims 1 to 4, wherein: (i) a weight molar osmotic concentration of 299 to 506 mmol / Kg; and / or (ii) The viscosity at 20°C is 6-15 cP.

8. The formulation according to any one of claims 1-4, wherein the pH is 5.8 to 6.

5.

9. The formulation according to claim 8, wherein the pH is about 5.

8.

10. The formulation according to any one of claims 1-4, wherein the histidine-based buffer comprises histidine hydrochloride.

11. The formulation according to claim 10, comprising 10 mM to 20 mM histidine hydrochloride.

12. The formulation according to any one of claims 1-4, comprising about 2.5% w / v, about 5% w / v, about 8% w / v, about 10% w / v or about 20% w / v sucrose.

13. The formulation according to any one of claims 1-4, comprising a surfactant at a concentration of about 0.03% w / v.

14. The formulation according to any one of claims 1-4, wherein L-arginine is L-arginine hydrochloride.

15. A formulation comprising a component selected from one of the following: Formulation ZZ: 140 mg / ml VEGF receptor fusion protein, 10 mM histidine-based buffer, 2.5% w / v sucrose, 2.0% w / v proline, 0.03% w / v polysorbate 20 and 50 mM arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein consists of two identical polypeptides composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation CCC: 80, 100, 120 or 140 mg / ml VEGF receptor fusion protein, 20 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20 and 50 mM arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein consists of two identical polypeptides composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation OOO: 140 mg / ml VEGF receptor fusion protein; 20 mM histidine, pH 5.8; 5% w / v sucrose; 0.03% w / v polysorbate 20; 50 mM sodium citrate; 50 mM arginine hydrochloride, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation PPP: 140 mg / ml VEGF receptor fusion protein; 20 mM histidine, pH 5.8; 5% w / v sucrose; 0.03% w / v polysorbate 20; 50 mM glycine; 50 mM arginine hydrochloride, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation QQQ: 140 mg / ml VEGF receptor fusion protein; 20 mM histidine, pH 5.8; 5% w / v sucrose; 0.03% w / v polysorbate 20; 50 mM sodium aspartate; 50 mM arginine hydrochloride, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation RRR: 140 mg / ml VEGF receptor fusion protein; 20 mM histidine, pH 5.8; 5% w / v sucrose; 0.03% w / v polysorbate 20; 50 mM sodium glutamate; 50 mM arginine hydrochloride, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation SSS: 140 mg / ml VEGF receptor fusion protein; 20 mM histidine, pH 5.8; 5% w / v sucrose; 0.03% w / v polysorbate 20; 10 mM L-arginine hydrochloride, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation TTT: 140 mg / ml VEGF receptor fusion protein; 20 mM histidine, pH 5.8; 5% w / v sucrose; 0.03% w / v polysorbate 20; 100 mM L-arginine hydrochloride, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation DDDD: 80.0 mg / ml VEGF receptor fusion protein, 20 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20 and 50 mM L-arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation EEEE: 120.0 mg / ml VEGF receptor fusion protein, 20 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20 and 50 mM L-arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation FFFF: 113.3 mg / ml VEGF receptor fusion protein, 20 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20 and 50 mM L-arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation GGGG: 114.3 mg / ml VEGF receptor fusion protein, 10 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20 and 50 mM L-arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation HHHH: 100.0 mg / ml VEGF receptor fusion protein, 20 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20 and 50 mM L-arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; Formulation IIII: 133.3 mg / ml VEGF receptor fusion protein, 20 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20, and 50 mM L-arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein consists of two identical polypeptides composed of amino acids 27-458 or 27-457 of SEQ ID NO: 2; and Formulation KKKK: 114.3 mg / ml VEGF receptor fusion protein, 20 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20 and 50 mM L-arginine hydrochloride, pH 5.8, wherein the VEGF receptor fusion protein consists of two identical polypeptides composed of amino acids 27-458 or 27-457 of SEQ ID NO:

2.

16. The formulation of claim 15, wherein the VEGF receptor fusion protein is apacept.

17. The formulation of claim 15, wherein the VEGF receptor fusion protein has less than 3.5% of high molecular weight species immediately after manufacture and purification and / or less than or equal to 6% of high molecular weight species after storage at 2-8°C for 24 months.

18. The formulation of claim 16, wherein the VEGF receptor fusion protein has less than 3.5% of high molecular weight species immediately after manufacture and purification and / or less than or equal to 6% of high molecular weight species after storage at 2-8°C for 24 months.

19. A container or injection device comprising the formulation of any one of claims 1-18.

20. The container or injection device according to claim 19, wherein it is a vial or a syringe.

21. The container or injection device according to claim 19, wherein it is a pre-filled syringe.

22. A method for preparing a formulation according to any one of claims 1-18, comprising combining the components of the formulation into a single composition.

23. The method of claim 22, further comprising loading a volume of the formulation into a syringe.

24. A formulation which is the product of the method of claim 22 or 23.

25. Use of the formulation of any one of claims 1-18 and 24 in the preparation of a medicament for intraocular injection into the eye of a subject.

26. Use of an aqueous pharmaceutical formulation of a VEGF receptor fusion protein in the preparation of a medicament for treating vascular ocular diseases in subjects in need, wherein the VEGF receptor fusion protein comprises two identical polypeptides consisting of amino acids 27-458 or 27-457 of SEQ ID NO: 2, wherein the medicament is used for intravitreal injection of at least 8 mg of the VEGF receptor fusion protein into the eye of the subject, wherein the aqueous pharmaceutical formulation comprises the VEGF receptor fusion protein at a concentration of at least 100 mg / ml; sucrose; L-arginine; a histidine-based buffer; and a nonionic surfactant selected from polysorbate 20 and / or polysorbate 80; wherein the pH of the formulation is from 5.0 to 6.8; wherein the VEGF receptor fusion protein has less than 3.5% high molecular weight species immediately after manufacture and purification and / or less than or equal to 6% high molecular weight species after storage at 2-8°C for 24 months.

27. The use according to claim 26, wherein the drug is used to inject about 8 mg of the VEGF receptor fusion protein into the vitreous cavity of the eye of the subject, and wherein the VEGF receptor fusion protein is apacept.

28. Use of the formulation of any one of claims 1-18 or 24 in the preparation of a medicament for treating vascular eye disease in a subject in need, wherein the medicament is used to inject 8 mg of the VEGF receptor fusion protein of the formulation into the eye of the subject intravitreal.

29. The use according to any one of claims 26-28, wherein the vascular eye disease is age-related macular degeneration, macular edema, retinal vein occlusion, choroidal neovascularization, iris neovascularization, neovascular glaucoma, postoperative fibrosis of glaucoma, proliferative vitreoretinopathy, optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, corneal opacity, vascular retinopathy, and / or diabetic retinopathy.

30. The use according to any one of claims 26-28, wherein the vascularized eye disease is wet age-related macular degeneration, macular edema after retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, nonproliferative diabetic retinopathy, and / or proliferative diabetic retinopathy.

31. The use according to any one of claims 26-28, wherein the drug is used for intravitreal injection of the VEGF receptor fusion protein in an amount of about 100 μL, about 90 μL, about 80 μL, about 75 μL, about 70 μL, about 60 μL, or about 50 μL.

32. The use according to any one of claims 26-28, wherein the VEGF receptor fusion protein is contained in a formulation comprising: about 5% w / v sucrose; histidine buffer; and about 0.03% w / v surfactant.

33. The use according to any one of claims 26-28, wherein the drug is administered by injection of the VEGF receptor fusion protein once every 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 weeks.

34. The formulation according to claim 1, comprising 140 mg / ml apracept; 20 mM histidine-based buffer; 5% w / v sucrose; 0.03% w / v polysorbate 20; 10 mM L-arginine; pH 5.

8.

35. The formulation according to claim 1, comprising 103-126 mg / ml VEGF receptor fusion protein, 10±1 mM histidine-based buffer, 5±0.5% w / v sucrose, 0.02-0.04% w / v polysorbate 20 and 50±5 mM L-arginine, pH 5.5-6.1, wherein the VEGF receptor fusion protein is composed of two identical polypeptides, which are composed of amino acids 27-458 or 27-457 of SEQ ID NO:

2.

36. The formulation of claim 35, wherein the VEGF receptor fusion protein is apacept.

37. The formulation according to claim 1, comprising 140 mg / ml apracept, 10 mM histidine-based buffer, 2.5% w / v sucrose, 2.0% w / v proline, 0.03% w / v polysorbate 20 and 50 mM L-arginine, pH 5.

8.

38. The formulation according to claim 1, comprising 114.3 mg / ml VEGF receptor fusion protein, 10 mM histidine-based buffer, 5% w / v sucrose, 0.03% w / v polysorbate 20 and 50 mM L-arginine, pH 5.8, wherein the VEGF receptor fusion protein is composed of two identical polypeptides consisting of amino acids 27-458 or 27-457 of SEQ ID NO:

2.

39. The formulation of claim 38, wherein the VEGF receptor fusion protein is apacept.

40. The formulation according to any one of claims 34-39, wherein L-arginine is L-arginine hydrochloride.

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