Stable, highly concentrated, self-buffering pharmaceutical compositions

Abstract

This is a stable, highly concentrated, self-buffering pharmaceutical composition containing a VEGF-binding molecule, a stabilizer, and a surfactant. The pH of the composition is 5.0 to 7.0. The pharmaceutical composition does not contain any additional buffering agents, has very high stability, is convenient to store, and has a wide range of applications.

Description

[Technical Field]

[0001] This application claims priority to the following Chinese patent applications filed with the Patent Office of the People's Republic of China on September 16, 2022, bearing application number CN202211134160.6 and entitled "Stable High-Concentration Self-Buffering Pharmaceutical Composition," the following Chinese patent applications filed with the Patent Office of the People's Republic of China on September 20, 2022, bearing application number CN202211141413.2 and entitled "Stable High-Concentration Self-Buffering Pharmaceutical Composition," and the following Chinese patent applications filed with the Patent Office of the People's Republic of China on September 7, 2023, bearing application number CN202311153498.0 and entitled "Stable High-Concentration Self-Buffering Pharmaceutical Composition," the entire contents of which are incorporated herein by reference.

[0002] <Technical field> The present invention relates to the technical field of pharmaceutical compositions, and in particular to stable, highly concentrated, self-buffering pharmaceutical compositions. [Background technology]

[0003] Vascular endothelial growth factor (VEGF) is a known regulator of angiogenesis and neovascularization and has been shown to be a key factor associated with tumors and ocular diseases or disorders. Many VEGF-binding molecules, including VEGF receptor fusion proteins or VEGF antibodies, have been approved for therapeutic use in humans and other mammals. For example, the anti-VEGF drug EYLEA® (Regeneron Pharmaceuticals, Inc.) tightly binds to VEGF and further inhibits neovascularization by reducing vascular permeability. Since August 2014, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have sequentially approved EYLEA® for the treatment of macular edema secondary to retinal vein occlusion, wet age-related macular degeneration, and diabetic macular edema via intravitreal injection.

[0004] However, the prior art has many drawbacks and areas for improvement. For example, the concentration of therapeutic antibodies in pharmaceutical formulations varies widely depending on the route of administration. When injection volume is limited, it is usually necessary to develop a highly concentrated antibody formulation. For example, for intravitreal injection or subcutaneous administration, it is usually necessary to develop a highly concentrated formulation. At the same time, the preparation of protein drugs itself can present many challenges to pharmaceutical scientists. Therefore, it is necessary to find a formulation that stabilizes protein drugs and resists degradation due to proteolysis, denaturation, aggregation, misfolding, and the like. In particular, it can be difficult to find suitable stability conditions for engineered proteins that are substantially different from known proteins. Protein drugs in a form that is convenient for patients are also desirable. Desirable properties include stability at ambient and refrigerated temperatures, suitability for long-term storage, appropriate administration time and dosage, and minimal discomfort after administration.

[0005] Although the prior art has found formulations suitable for intravitreal administration, there is still a need for a stable liquid formulation of high-concentration VEGF receptor fusion protein suitable for ocular administration.A formulation containing high-concentration VEGF receptor fusion protein can reduce the administration volume, shorten the ocular injection time, increase the dosage, extend the administration interval, reduce the administration frequency, and improve the efficiency of production and storage.At the same time, research has found that many buffers commonly used to prepare drug proteins have certain limitations and risks, especially for ophthalmic drugs, and may affect the safety of the drug.

[0006] Buffers such as acetate, succinate, citrate, histidine (imidazole), phosphate, and Tris have been found to have undesirable limitations and drawbacks when used in the preparation of pharmaceutical proteins. Specifically, additional components in the formulation complicate the formulation process, increase harmful components, affect the overall stability and shelf life of the drug, and simultaneously increase the risk of end-user acceptability. While buffer-salt-free systems exist in the prior art, achieving an effective buffer-salt-free pharmaceutical composition for a specific drug is a challenging task, with results often unpredictable. It is generally known in the art that high-concentration self-buffering systems pose even greater technical challenges. At the same time, increasing the stability of pharmaceutical compositions is extremely difficult industrially. From a formulation perspective, high protein concentrations result in a higher rate of protein aggregation, as closer contact between different components leads to intermolecular attraction and self-aggregation events. Due to size exclusion and Donnan equilibrium effects, achieving a pH and auxiliary material concentration that meets the requirements of the UF / DF process is also a challenge, and higher protein concentrations make this task even more difficult. Therefore, there is a need to further optimize the buffer system and develop stable liquid formulations of high-concentration VEGF receptor fusion proteins suitable for ocular administration. Summary of the Invention [Problem to be solved by the invention]

[0007] One of the objectives of the present invention is to provide an improved high-concentration pharmaceutical composition of a VEGF-binding molecule that has a high concentration and does not require an additional buffering agent. That is, the present invention provides a buffer salt-free, high-concentration protein self-buffering pharmaceutical composition. After many years of research and testing, the inventors have finally prepared a high-concentration self-buffering aqueous pharmaceutical composition of a VEGF-binding molecule. Furthermore, the present inventors have surprisingly found that the high-concentration self-buffering pharmaceutical composition has very high stability and a wide range of applications. [Means for solving the problem]

[0008] The present invention discloses a self-buffering, highly concentrated VEGF-binding molecule aqueous pharmaceutical composition.High-concentration systems are prone to aggregation events that make formulations unstable, and this is particularly true for self-buffering, highly concentrated systems.Generally, self-buffering, highly concentrated systems are difficult to store under room temperature conditions, and are more prone to aggregation.The aflibercept pharmaceutical composition disclosed in the present invention is stable and has a shelf life sufficient for the manufacture, storage, and distribution of drugs.For example, the self-buffering, highly concentrated aflibercept pharmaceutical composition disclosed in the present invention has a shelf life of at least 1 month, 3 months, or 6 months under room temperature storage conditions (e.g., 25°C).

[0009] In some embodiments, the present invention discloses a pharmaceutical composition, wherein the initial amount of aflibercept in the composition with a SEC-HPLC purity of ≥ 95% remains unchanged even after one month when packaged samples are stored under accelerated conditions (e.g., 25°C ± 2°C). In other embodiments, when packaged samples are stored under elevated temperature conditions (40°C ± 2°C), the self-buffered high-concentration aflibercept pharmaceutical composition disclosed in the present invention exhibits superior efficacy to high-concentration aflibercept pharmaceutical compositions with a buffer system.

[0010] In some embodiments, the present invention discloses a pharmaceutical composition, and insoluble particulates of different particle sizes (≧2 μm, ≧5 μm, ≧10 μm, and / or ≧25 μm) were detected, and the self-buffered high-concentration aflibercept pharmaceutical composition disclosed in the present invention showed superior efficacy to high-concentration aflibercept pharmaceutical compositions with a buffer system.

[0011] In some embodiments, the present invention discloses a self-buffering, highly concentrated aqueous or lyophilized pharmaceutical composition of a VEGF-binding molecule, comprising: (1) a VEGF-binding molecule selected from a VEGF receptor fusion protein or an anti-VEGF antibody or antigen-binding fragment thereof, having a concentration of at least about 100 mg / mL; (2) at least one stabilizer selected from sucrose, trehalose, proline, a pharmaceutically acceptable salt of proline, lysine, a pharmaceutically acceptable salt of lysine, and sorbitol; and (3) at least one surfactant selected from polysorbate 80, polysorbate 20, and poloxamer, wherein the aqueous pharmaceutical composition does not contain a buffering agent and has a pH of about 5.0 to 7.0.

[0012] In some embodiments, the aqueous pharmaceutical composition does not comprise a buffer, which refers to a histidine buffer, a phosphate buffer, a citrate buffer, an acetate buffer, a carbonate buffer, a succinate buffer, a tartrate buffer, and / or a maleate buffer.

[0013] In some embodiments, the self-buffering highly concentrated aqueous or lyophilized pharmaceutical composition of a VEGF binding molecule further comprises (4) at least one of L-arginine, a pharmaceutically acceptable salt of L-arginine, and sodium chloride.

[0014] In some embodiments, the content of the L-arginine or the pharmaceutically acceptable salt of L-arginine is about 0 to 100 mM, or the content of the sodium chloride is about 0 to 40 mM. In preferred embodiments, the content of the L-arginine or the pharmaceutically acceptable salt of L-arginine is about 20 to 100 mM, or the content of sodium chloride is about 40 mM. In more preferred embodiments, the content of the L-arginine or the pharmaceutically acceptable salt of L-arginine is about 20 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, or about 100 mM.

[0015] In some embodiments, the concentration of the VEGF binding molecule in the aqueous pharmaceutical composition is at least about 100 mg / mL, at least about 114.3 mg / mL, at least about 125 mg / mL, or at least about 180 mg / mL.

[0016] In some embodiments, the aqueous pharmaceutical composition contains about 100-180 mg / mL of the VEGF-binding molecule. In a preferred embodiment, the aqueous pharmaceutical composition contains about 100-125 mg / mL of the VEGF-binding molecule.

[0017] In some embodiments, the concentration of the stabilizer in the aqueous pharmaceutical composition is about 5% to 10% w / v. In some embodiments, the stabilizer is selected from about 5% to 9% w / v sucrose or about 5.5% to 10% w / v trehalose, and in preferred embodiments, the sucrose concentration is about 5%, 6%, 7%, 8%, or 9% w / v, or the trehalose concentration is about 5.5%, 6.6%, 7.7%, 8.0%, or 10% w / v.

[0018] In some embodiments, the surfactant is about 0.01% to 0.05% w / v of at least one selected from polysorbate 20, polysorbate 80, and poloxamer. In preferred embodiments, the surfactant is polysorbate 20, and the content of polysorbate 20 is about 0.01%, about 0.03%, about 0.05%, 0.07%, about 0.1%, or about 0.2% w / v.

[0019] In some embodiments, the aqueous pharmaceutical composition has a pH of about 5.5 to 6.4. Preferably, the pH is about 5.5, about 5.8, about 6.0, about 6.1, about 6.2, or about 6.4.

[0020] In some embodiments, the aqueous pharmaceutical composition comprises a VEGF receptor fusion protein at a concentration of about 100-180 mg / mL, about 5-9 w / v% sucrose or about 5.5-10 w / v% trehalose, about 0.01-0.2 w / v% polysorbate 20, and about 0-100 mM L-arginine hydrochloride or about 0-40 mM sodium chloride, and has a pH of about 5.0-7.0.

[0021] In some embodiments, the aqueous pharmaceutical composition comprises a VEGF receptor fusion protein at a concentration of about 100-180 mg / mL, about 5-7 w / v% sucrose or about 5.5-7.7 w / v% trehalose, about 0.01-0.2 w / v% polysorbate 20, and about 20-100 mM L-arginine hydrochloride or about 0-40 mM sodium chloride, and has a pH of about 5.5-6.4.

[0022] In some embodiments, the aqueous pharmaceutical composition is any selected from the following:

[0023] Aqueous pharmaceutical composition A contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0024] Aqueous pharmaceutical composition B contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0025] Aqueous pharmaceutical composition C contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0026] Aqueous pharmaceutical composition D contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0027] Aqueous pharmaceutical composition E contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0028] Aqueous pharmaceutical composition F contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0029] Aqueous pharmaceutical composition G contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0030] Aqueous pharmaceutical composition H contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0031] Aqueous pharmaceutical composition I contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0032] Aqueous pharmaceutical composition J contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0033] Aqueous pharmaceutical composition K contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0034] Aqueous pharmaceutical composition L contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0035] Aqueous pharmaceutical composition M contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0036] Aqueous pharmaceutical composition N contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0037] Aqueous pharmaceutical composition O contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0038] Aqueous pharmaceutical composition P contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0039] Aqueous pharmaceutical composition Q contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0040] Aqueous pharmaceutical composition R contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0041] Aqueous pharmaceutical composition S contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0042] Aqueous pharmaceutical composition T contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0043] Aqueous pharmaceutical composition U contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0044] Aqueous pharmaceutical composition V contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0045] Aqueous pharmaceutical composition W contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0046] Aqueous pharmaceutical composition X contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0047] Aqueous pharmaceutical composition Y contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5 w / v % sucrose, approximately 0.01 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0048] Aqueous pharmaceutical composition Z contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 5 w / v % sucrose, approximately 0.05 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0049] Aqueous pharmaceutical composition ZA contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0050] Aqueous pharmaceutical composition ZB contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0051] Aqueous pharmaceutical composition ZC contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0052] Aqueous pharmaceutical composition ZD contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0053] Aqueous pharmaceutical composition ZE contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0054] The aqueous pharmaceutical composition ZF contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0055] Aqueous pharmaceutical composition ZG contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0056] Aqueous pharmaceutical composition ZH contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0057] Aqueous pharmaceutical composition ZI contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0058] Aqueous pharmaceutical composition ZJ contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0059] Aqueous pharmaceutical composition ZK contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0060] Aqueous pharmaceutical composition ZL contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0061] Aqueous pharmaceutical composition ZM contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0062] Aqueous pharmaceutical composition ZN contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0063] The aqueous pharmaceutical composition ZO contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0064] Aqueous pharmaceutical composition ZP contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0065] Aqueous pharmaceutical composition ZQ contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0066] Aqueous pharmaceutical composition ZR contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0067] Aqueous pharmaceutical composition ZS contains a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 5 w / v % sucrose, about 0.03 w / v % polysorbate 20, and about 80 mM L-arginine hydrochloride, and has a pH of about 5.5 to 6.4.

[0068] Aqueous pharmaceutical composition ZT contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0069] Aqueous pharmaceutical composition ZU contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0070] Aqueous pharmaceutical composition ZV contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0071] Aqueous pharmaceutical composition ZW contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0072] Aqueous pharmaceutical composition ZX contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0073] Aqueous pharmaceutical composition ZY contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5 w / v % sucrose, approximately 0.01 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0074] Aqueous pharmaceutical composition ZZ contains a VEGF receptor fusion protein at a concentration of approximately 125 mg / mL, approximately 5 w / v % sucrose, approximately 0.05 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0075] Aqueous pharmaceutical composition AA contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0076] Aqueous pharmaceutical composition AB contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0077] Aqueous pharmaceutical composition AC contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0078] The aqueous pharmaceutical composition AD contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0079] The aqueous pharmaceutical composition AE contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0080] Aqueous pharmaceutical composition AF contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0081] Aqueous pharmaceutical composition AG contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0082] Aqueous pharmaceutical composition AH contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0083] Aqueous pharmaceutical composition AI contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0084] Aqueous pharmaceutical composition AJ contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0085] Aqueous pharmaceutical composition AK contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0086] Aqueous pharmaceutical composition AL contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 60 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0087] The aqueous pharmaceutical composition AM contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0088] Aqueous pharmaceutical composition AN contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0089] The aqueous pharmaceutical composition AO contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0090] The aqueous pharmaceutical composition AP contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0091] Aqueous pharmaceutical composition AQ contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0092] The aqueous pharmaceutical composition AR contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 70 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0093] The aqueous pharmaceutical composition AS contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0094] The aqueous pharmaceutical composition AT contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 6 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0095] The aqueous pharmaceutical composition AU contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 7 w / v % sucrose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0096] The aqueous pharmaceutical composition AV contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5.5 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0097] The aqueous pharmaceutical composition AW contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 6.6 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0098] Aqueous pharmaceutical composition AX contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 7.7 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 80 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0099] Aqueous pharmaceutical composition AY contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5 w / v % sucrose, approximately 0.01 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0100] Aqueous pharmaceutical composition AZ contains a VEGF receptor fusion protein at a concentration of approximately 180 mg / mL, approximately 5 w / v % sucrose, approximately 0.05 w / v % polysorbate 20, and approximately 50 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0101] Aqueous pharmaceutical composition BA contains a VEGF receptor fusion protein at a concentration of approximately 114.3 mg / mL, approximately 8 w / v % trehalose, approximately 0.03 w / v % polysorbate 20, and approximately 20 mM L-arginine hydrochloride, and has a pH of approximately 5.5 to 6.4.

[0102] Preferably, the VEGF receptor fusion protein is aflibercept.

[0103] In some embodiments, the VEGF-binding molecule comprises two polypeptides, each polypeptide comprising the immunoglobulin-like (Ig) domain 2 of VEGFR1 and the immunoglobulin-like (Ig) domain 3 of VEGFR2. In preferred embodiments, the VEGF-binding molecule is selected from bevacizumab, ranibizumab, aflibercept, or conbercept. In more preferred embodiments, the VEGF-binding molecule is aflibercept.

[0104] In some embodiments, the pharmaceutical composition is stable for at least 6 months, 12 months, or 24 months at 2-8° C. Alternatively, the pharmaceutical composition is stable for at least 1 month, 3 months, or 6 months at 25° C.±2° C.

[0105] In some embodiments, the present invention provides a lyophilized formulation obtained by lyophilizing an aqueous pharmaceutical composition according to the present invention, or by reconstituting the lyophilized formulation to obtain an aqueous pharmaceutical composition according to the present invention.

[0106] In some embodiments, the present invention provides a delivery device comprising the aqueous pharmaceutical composition or the reconstituted lyophilized formulation of the present invention. In one preferred embodiment, the delivery device is a pre-filled syringe.

[0107] In some embodiments, the present invention provides a method for treating an eye disease or disorder mediated by VEGF, comprising applying to a subject an aqueous pharmaceutical composition or a delivery device disclosed herein. In a preferred embodiment, the application site is intravitreal.

[0108] In some embodiments, the present invention provides use of an aqueous pharmaceutical composition or delivery device of the present invention in preparing a pharmaceutical composition for treating an ocular disease or disorder mediated by VEGF, said treatment comprising applying to a subject the aqueous pharmaceutical composition or delivery device of the present invention. In one preferred embodiment, the site of application is intravitreal.

[0109] In some embodiments, the ocular disease or disorder comprises a neovascular or vasculogenic ocular disease.

[0110] In some embodiments, the ocular disease or disorder comprises neovascular (wet) age-related macular degeneration (AMD), age-related macular degeneration (wet), macular edema, macular edema secondary to 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, fibrosis after glaucoma surgery, proliferative vitreoretinopathy (PVR), optic nerve head neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy (DR), non-proliferative diabetic retinopathy, and / or proliferative diabetic retinopathy.

[0111] In some embodiments, a method of delivering a VEGF-binding molecule to a subject is disclosed, comprising applying to the subject an aqueous pharmaceutical composition or a delivery device according to the present invention. In a preferred embodiment, the site of application is intravitreal.

[0112] The pharmaceutical composition provided by the present invention does not require the addition of additional buffering agents, has very high stability, is convenient to store, and has a wide range of application prospects. The drawings further illustrate the novel features disclosed in the present invention. The features and advantages disclosed in the present invention will be better understood with reference to the drawings, but it should be understood that these drawings are used only to illustrate specific embodiments illustrating the principles disclosed in the present invention and are not intended to limit the scope of the appended claims. [Brief explanation of the drawings]

[0113] [Figure 1] FIG. 1 is a trend graph of the change in purity of the main peak of SEC of formulations A2 and A3 at 40°C ± 2°C. [Figure 2] FIG. 2 is a trend graph showing the change in purity of the SEC polymer of formulations A2 and A3 under different storage conditions. [Figure 3] FIG. 3 is a trend graph of the change in purity of the main peak of SEC of formulations B1, B2, B3, B4, and B5 under different storage conditions. [Figure 4]FIG. 4 shows viscosity curves corresponding to formulations B1, B2, B3, B4, and B5. [Figure 5] FIG. 5 is a trend graph of the change in purity of the main peak of the SEC of preparations C1 to C10 at 37° C.±2° C. [Figure 6] FIG. 6 is a trend graph of the change in SEC purity of formulations F5 and F6 at 2 to 8°C and 37°C±2°C. DETAILED DESCRIPTION OF THE INVENTION

[0114] The term "about" includes and describes the value or parameter itself. For example, "about x" includes and describes "x" itself. As used herein, the term "about," when used in connection with a measurement or to modify a value, unit, constant, or range of values, includes the value or parameter itself as well as a variation of ±1% to 10%. In some examples, the term "about," when used in connection with a measurement or to modify a value, unit, constant, or range of values, refers to a variation of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%.

[0115] The term "aqueous" pharmaceutical composition refers to a pharmaceutical composition that contains water.

[0116] The term "self-buffering" is meant to exclude components traditionally used to introduce buffering capacity into a formulation, such as, but not limited to, histidine buffer, phosphate buffer, citrate buffer, acetate buffer, carbonate buffer, succinate buffer, tartrate buffer, maleate buffer, or mixtures thereof. Proteins are known to provide buffering capacity to formulations in addition to any buffering capacity present in a formulation that does not contain a buffering component. Thus, these terms do not imply that a formulation must not have buffering capacity.

[0117] The term "high concentration" means that the formulation contains at least 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, 109 mg / mL, 110 mg / mL, 111 mg / mL, 112 mg / mL, 113 mg / mL, 114 mg / mL, 114.1 mg / 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, 114.8 mg / mL, 114.9 mg / mL, 114.10 mg / mL, 114.11 mg / mL, 114.12 mg / mL, 114.13 mg / mL, 114.14 mg / mL, 114.15 mg / mL, 114.16 mg / mL, 114.17 mg / mL, 114.18 mg / mL, 114.19 mg / mL, 114.20 mg / mL, 114.21 mg / mL, 114.22 mg / mL, 114.23 mg / mL, 114.24 mg / mL, 114.25 mg / mL, 114.26 mg / mL, 114.27 mg / mL, 114.28 mg / mL, 114.29 ... mL, 114.7mg / mL, 114.8mg / mL, 114.9mg / mL, 115mg / mL, 116mg / mL, 117mg / mL, 118mg / mL, 119mg / mL, 120mg / mL, 121mg / mL, 122mg / mL, 1 23mg / mL, 124mg / mL, 125mg / mL, 126mg / mL, 127mg / mL, 128mg / mL, 129mg / mL, 130mg / mL, 131mg / mL, 132mg / mL, 133mg / mL, 134mg / mL, 135 mg / mL, 136mg / mL, 137mg / mL, 138mg / mL, 139mg / mL, 140mg / mL, 141mg / mL, 142mg / mL, 143mg / mL, 144mg / mL, 145mg / mL, 146mg / mL, 147m g / mL, 148mg / mL, 149mg / mL, 150mg / mL, 151mg / mL, 152mg / mL, 153mg / mL, 154mg / mL, 155mg / mL, 156mg / mL, 157mg / mL, 158mg / mL, 159mg VEGF receptor fusion protein at concentrations of 160mg / mL, 161mg / mL, 162mg / mL, 163mg / mL, 164mg / mL, 165mg / mL, 166mg / mL, 167mg / mL, 168mg / mL, 169mg / mL, 170mg / mL, 171mg / mL, 172mg / mL, 173mg / mL, 174mg / mL, 175mg / mL, 176mg / mL, 177mg / mL, 178mg / mL, 179mg / mL, or 180mg / mL.

[0118] The term "VEGF-binding molecule" refers to a VEGF receptor fusion protein, an anti-VEGF antibody or antigen-binding fragment, or an antibody or fragment that specifically binds to VEGF. An exemplary VEGF receptor fusion protein includes aflibercept (Regeneron Pharmaceuticals, Inc.), see International Patent Application Publication No. WO2005 / 121176 or WO2007 / 112675.

[0119] The term "VEGF receptor fusion protein" refers to a molecule comprising one or more VEGF receptors or domains thereof fused to another polypeptide, thereby disrupting the interaction between VEGF and a native VEGF receptor. For example, two such fusion polypeptides may combine to form a homodimer or other multimer. Such a VEGF receptor fusion protein is referred to as a "VEGF-Trap" or "VEGF Trap." VEGF receptor fusion proteins within the scope of the present invention and falling within this definition include chimeric polypeptides comprising two or more immunoglobulin (Ig)-like domains of a VEGF receptor, such as VEGFR1 (also known as Flt1) and / or VEGFR2 (also known as Flk1 or KDR), and may include a multimerization domain (e.g., an Fc domain).

[0120] The term "aflibercept" includes its biosimilar forms, and the biosimilar forms of aflibercept generally refer to products that contain the same amino acid sequence.The amino acid sequence and nucleic acid sequence of exemplary aflibercept are described in the literature, see WO2000075319A1.

[0121] The term "treatment," as used herein, refers to a therapeutic approach according to the present invention, wherein the method of "treatment" involves administering an antibody of the present invention to a subject in need of such treatment (e.g., a subject having a VEGF-mediated ocular disorder or a subject who may ultimately acquire such a disorder) in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of, the disorder or recurring disorder, or prolong the survival of the subject beyond that expected in the absence of such treatment.

[0122] The term "subject" or "patient" refers to human and non-human mammals, including, but not limited to, primates, rabbits, pigs, horses, dogs, cats, sheep, and cows.

[0123] Protein content was measured using a Lunatic microspectrophotometer. Blank subtraction was performed with purified water, and the UV absorbance at 280 nm was corrected for light scattering at 330 nm. Protein concentration was calculated according to Equation 1, where the extinction coefficient ε is 1.5 (L / g cm). -1 )

number

[0124] Size Exclusion Chromatography (SEC): Polymers, monomers, and fragments are quantified by size exclusion chromatography. The measurements are performed on a Waters e2695-2489 HPLC system using a TSKgel G3000SWXL 7.8x300mm 5µm 250Å column. The mobile phase is potassium phosphate buffer. Samples are diluted to 1mg / mL with the mobile phase, and the injection volume is 25µL. Proteins are eluted isocratically for 30 minutes at a flow rate of 0.5mL / min, and the absorbance of the eluate is measured at 215nm. Integration is performed using Empower3 software.

[0125] Reduced capillary electrophoresis (rCE-SDS): % (main peak 1 + main peak 2) purity was measured by reduced capillary electrophoresis (rCE-SDS). This measurement was performed on a SCIEX PA800plus capillary electrophoresis system using a 50 μm ID uncoated quartz capillary with an effective separation length of 20 cm, a total separation length of 30.2 cm, a PDA 220 nm bandwidth of 10 nm, and integration processing using 32 Karat software.

[0126] Non-reducing SDS-PAGE (nrSDS-PAGE): The percent main peak purity was measured by non-reducing SDS-PAGE electrophoresis. This measurement was performed using polyacrylamide gel electrophoresis, with a separating gel concentration of 8% and a stacking gel concentration of 4.5%, with electrophoresis detection performed under a constant current of 10 mA per block gel. The gel was stained with Coomassie Brilliant Blue R250 and destained with a destaining solution, after which scanning imaging was performed using a gel imaging system to analyze the purity of the target band.

[0127] Imaging capillary isoelectric focusing (iCIEF): Imaging capillary isoelectric focusing (iCIEF) was used to measure charge heterogeneity and perform identification experiments. These measurements were performed using an imaging capillary electrophoresis system, the Maurice C system, with a capillary effective separation length of 5 cm and a focusing time of 6 min. The focusing program was 1.5 kV for 1 min and 3 kV for 5 min for detection. Integration was performed using Empower3 software.

[0128] MFI Microflow Imaging Particle Analysis: The number of particles is measured by the MFI Microflow Imaging Particle Analysis System. This measurement is performed using the MFI particle analysis system MFI5200, the test sample volume is 1 mL, the total effective volume is 0.7 mL, and the analysis volume is 0.51 mL, and the analysis is performed using the MVSS analysis software.

[0129] Viscosity measurement using m-VROC type viscometer: e-VROC built with Rheosense technology TM is an excellent tool for simultaneously measuring extensional and shear viscosity. The viscometer uses MEMS pressure sensors to measure the pressure of upstream and downstream constrictions and records the change in flow rate. The fluid undergoes a nearly constant elongation due to the contraction / expansion, allowing the apparent extensional viscosity to be calculated.

[0130] Determining osmolality by freezing point depression: The freezing point depression of a solution is used to indirectly measure its osmolality. For an ideal dilute solution, the freezing point depression satisfies the relationship △Tf = Kf m, where △Tf is the freezing point depression, Kf is the freezing point depression constant (1.86 when water is the solvent), and m is the molality. The osmolality satisfies the relationship Po = ko m, where Po is the osmotic pressure, ko is the osmotic pressure constant, and m is the molality of the solution. Since the concentrations in both equations are equal, the osmolality of a solution can be measured by freezing point depression.

[0131] Measurement of binding activity by ELISA: The binding activity of recombinant human vascular endothelial growth factor receptor-antibody fusion protein injection to VEGF165 was measured using the ELISA method. First, human VEGF165 protein was coated onto a 96-well plate, then gradient diluted versions of this product were added, followed by secondary antibody, coloring solution, and stop solution. The absorbance was read and the binding ability of this product to the human VEGF165 antigen was calculated. The test data was subjected to a four-parameter fitting analysis using Soft Max Pro or other similar software, and the results were automatically analyzed using the following formula: The logarithm of the reference product (self-prepared) concentration is on the X axis, and the absorbance value is on the Y axis. The software calculates the median effective concentration (EC 50 ) and create an "S" shaped curve.

number

[0132] The concentrations of the VEGF-binding molecule, stabilizer, surfactant, L-arginine, pharmaceutically acceptable salt of L-arginine, and sodium chloride described in the present invention refer to the concentrations of the VEGF-binding molecule, stabilizer, surfactant, L-arginine, pharmaceutically acceptable salt of L-arginine, and sodium chloride, respectively, in the aqueous pharmaceutical composition.

[0133] The experimental conditions in the following examples are as follows: shaking at room temperature (25±2°C) for one week (25°C 1W), freezing and thawing three times (one cycle consists of freezing at -40°C for 16 hours and thawing at 25°C for 8 hours), leaving at 2-8°C for one month (2-8°C 1M), leaving at 2-8°C for six months (2-8°C 6M), leaving at 25°C±2°C for one month (25°C 1M), leaving at 40°C±2°C for one week (40°C 1W), leaving at 40°C±2°C for two weeks (40°C 2W), or leaving at 40°C±2°C for one month (40°C 1M).

[0134] Example 1: Preparation of Self-Buffering Formulation Samples

[0135] The long-term efficacy, physical stability, and charge isoform formation of four different formulations containing VEGF receptor fusion protein (aflibercept) at approximately 40 mg / mL, 114.3 mg / mL, and 180 mg / mL were measured. The component contents of the four formulations are listed in Table 1.1. To prepare the formulation solutions, buffer exchange was performed using the desired auxiliary material solution, and the solution was concentrated to the required antibody concentration by ultrafiltration. After ultrafiltration concentration was completed, the auxiliary material was added to the antibody solution as a mother liquor. All formulations were sterile filtered through a 0.22 μm low-protein-binding filter, filled into sterile 2 mL vials under aseptic conditions, and sealed using film-coated rubber stoppers and aluminum-plastic combination caps. The presence (%) of high molecular weight species (HMWS, LMWS) and the main peak (Monomer / MAIN) were determined by SEC-HPLC, non-reversible SDS-PAGE, and rapid cell chromatography-SDS to evaluate the stability of aflibercept during storage under different conditions for each formulation.

[0136] Below are the different formulations and their stability results.

[0137] [Table 1-1]

[0138] [Table 1-2]

[0139] [Table 1-3]

[0140] [Table 1-4]

[0141] [Table 1-5]

[0142] [Table 1-6]

[0143] Test Examples A1 and A2 were buffered, while Test Examples A3 and A4 were self-buffered. As can be seen from the experimental results, after shaking at room temperature (25±2°C) for one week, freezing and thawing three times, and leaving at 2-8°C for six months, the samples of Test Examples A1, A2, and A3 were relatively stable, with no obvious changes in SEC-HPLC purity. After leaving at 25°C for one month, Test Examples A1, A2, A3, and A4 showed no obvious changes in purity, except for a significant decrease in nr-SDS-PAGE purity.

[0144] The test results after one month of storage at 40°C ± 2°C are shown in Tables 1.2-1.4. Figure 1 shows the trend graph of SEC main peak purity change for Formulations A2 and A3 at 40°C ± 2°C, and Figure 2 shows the trend graph of SEC polymer purity change for Formulations A2 and A3 under different storage conditions. As can be seen from the above results, the SEC-HPLC purity of Test Example A3 was significantly improved compared to Test Example A2. Specifically, Test Example A3 had a 5.9% improvement in SEC-HPLC main peak purity and a 5.9% reduction in aggregates compared to Test Example A2. In industrial applications, improving protein stability is challenging, and a 1-2% improvement in purity is highly significant, with a 5.9% improvement being a significant advance. Furthermore, preparing a self-buffering, high-concentration formulation during the preparation process significantly reduces the cost of auxiliary materials. The test results of Test Examples A1-A4 demonstrate that the self-buffering formulations of the present invention, free of histidine and phosphate buffers, exhibited excellent stability throughout the study.

[0145] [Table 1-7]

[0146] [Table 1-8]

[0147] Tables 1.7 and 1.8 show the MFI detection results for insoluble particles. For intravitreal injection products, reducing aggregates is expected to reduce a range of adverse effects, such as immunogenicity, in patients. Test Example A3 showed improved nr-SDS-PAGE and rCE-SDS purity compared to A1 and A2. At the same time, Test Example A3 also showed improved insoluble particle concentration compared to A1 and A2. In all analyses of insoluble particle sizes ≥2 μm, ≥5 μm, ≥10 μm, and ≥25 μm, the A3 formulation had a lower concentration of insoluble particle concentration. This is a surprising result, as improved insoluble particle concentration can reduce the incidence of adverse effects in patients due to injection in ophthalmic administration. Therefore, these test results demonstrate the excellent potential of the self-buffering formulation of the present invention for ophthalmic administration.

[0148] Example 2: Comparison of the stability of self-buffering formulations containing different concentrations of L-arginine hydrochloride

[0149] Test Examples B1 to B7 are self-buffering formulations containing different concentrations of L-arginine hydrochloride, and the contents of each component are shown in Table 2.1. The aflibercept concentration in B1 to B7 was approximately 114.3 mg / mL. In Example 2, the effects of adding 20 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, and 100 mM of L-arginine hydrochloride on the stability of the pharmaceutical composition were examined.

[0150] [Table 2-1]

[0151] [Table 2-2]

[0152] [Table 2-3]

[0153] [Table 2-4]

[0154] [Table 2-5]

[0155] [Table 2-6]

[0156] [Table 2-7]

[0157] [Table 2-8]

[0158] The physical stability of L-arginine hydrochloride self-buffering formulations with different concentrations was evaluated at 2-8°C, 25°C ± 2°C, and 37°C ± 2°C using SEC-HPLC, nr-SDS-PAGE, and rCE-SDS. The experimental results are shown in Tables 2.2-2.8 and Figures 3-4. Figure 3 shows the purity trend graph of the main peak of SEC for formulations B1, B2, B3, B4, and B5 under different storage conditions. Figure 4 shows the viscosity curves for formulations B1, B2, B3, B4, and B5.

[0159] As can be seen from the above test results, the stability of the pharmaceutical compositions containing 20 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, and 100 mM L-arginine hydrochloride under the conditions of 2-8°C 3M and 25°C 1M was all very good, and their purity did not change significantly compared to the initial value (time 0). At the same time, different concentrations of L-arginine hydrochloride did not significantly affect the viscosity of the formulation.

[0160] Example 3: Comparison of the stability of self-buffering formulations containing different types and concentrations of stabilizers

[0161] Test Examples C1 to C10 are self-buffering formulations containing stabilizers of different types and concentrations, each containing approximately 114.3 mg / mL of aflibercept. The contents of each component are shown in Table 3.1. In Example 3, the effects of stabilizers of different types and concentrations on the stability of the pharmaceutical compositions were examined.

[0162] [Table 3-1]

[0163] [Table 3-2]

[0164]

Table 3-3

[0165]

Table 3-4

[0166]

Table 3-5

[0167]

Table 3-6

[0168]

Table 3-7

[0169]

Table 3-8

[0170]

Table 3-9

[0171]

Table 3-10

[0172]

Table 3-11

[0173] Tables 3.2-3.11 show the percentages of Monomer / MAIN (main peak), HMWS (polymer), and fragments (LMWS) for formulations C1-C10 measured by SEC-HPLC, rCE-SDS, and nr-SDS-PAGE at 0 h, 37°C ± 2°C, 2-8°C, and 25°C ± 2°C. Figure 5 shows the change in main peak purity by SEC for formulations C1-C10 at 37°C ± 2°C. The following observations can be made from the addition of auxiliary materials such as sucrose, trehalose, sorbitol, proline, and lysine hydrochloride to prepare 10 highly pure self-buffered, high-concentration formulations C1-C10. The 0 h SEC-HPLC main peak purity was 98.7% or higher. The results at 37°C 1M showed that the SEC-HPLC main peak purities of formulations C1 to C3 were between 86.1% and 88.8%, those of formulations C4 to C6 were between 88.7% and 91.0%, and those of formulations C7, C8, and C9 were 78.4%, 67.3%, and 85.1%, respectively, and no obvious differences were observed in the purity changes of each formulation by rCE-SDS and nr-SDS-PAGE.

[0174] The above test results indicate that sucrose and trehalose have significant protective effects against aflibercept, and when the content ranges of sucrose and trehalose are examined, it is found that both pharmaceutical compositions have excellent stability when the sucrose concentration is in the range of 5 w / v% to 7 w / v% and the trehalose concentration is in the range of 5.5 w / v% to 7.7 w / v%.

[0175] Example 4: Comparison of the stability of different self-buffering formulations

[0176] The pH, protein content, and polysorbate 20 content of different self-buffering formulations were adjusted, and the components are shown in Table 4.1. The stability of aflibercept formulations was measured under the following conditions: sucrose was 6 w / v%, L-arginine hydrochloride was 50 mM, pH was 5.5, 5.8, and 6.1, respectively, protein contents were approximately 100 mg / mL, approximately 114.3 mg / mL, and approximately 125 mg / mL, respectively, and polysorbate 20 was 0.01 w / v%, 0.03 w / v%, and 0.05 w / v%, respectively.

[0177] [Table 4-1]

[0178] [Table 4-2]

[0179] [Table 4-3]

[0180] [Table 4-4]

[0181] [Table 4-5]

[0182] [Table 4-6]

[0183] [Table 4-7]

[0184] [Table 4-8]

[0185] [Table 4-9]

[0186] [Table 4-10]

[0187] [Table 4-11]

[0188] [Table 4-12]

[0189] The test results are shown in Tables 4.2-4.12. As can be seen from the above results, the 0h SEC-HPLC main peak purity of formulations D1-D10 was 99.0% or higher. As can be seen from the results at 2-8°C 1M and 25°C 1M, the SEC-HPLC main peak purity of formulations D1-D10 ranged from 97.7% to 98.9%, the formulation viscosity was 5.6-10.1 cP, and the osmolality was 342-387 mOsmol / kg. As can be seen from the above results, the stability of the high-concentration self-buffering formulations was maintained at a good level within the protein content range of 100-125 mg / mL, pH 5.5-6.1, and polysorbate 20 content range of 0.01-0.05 w / v%.

[0190] Example 5: Formulation dilution test

[0191] To simulate the intravitreal stability of aflibercept after dilution, the stability of formulations containing 2 mg / mL and 0.1 mg / mL aflibercept was tested. Table 5.1 shows the composition of the two formulations. To prepare the formulation solutions, dilutions were performed using the desired auxiliary material solution. The auxiliary material was added to the antibody solution as a mother solution. All formulations were sterile filtered through a 0.22 μm low-protein-binding filter and filled into sterile 2 mL vials under aseptic conditions. The vials were sealed using film-coated rubber stoppers and aluminum-plastic combination caps. SEC-HPLC was used to identify the presence (%) of high molecular weight species (HMWS, LMWS) and the main peak (Monomer / MAIN). The stability of aflibercept in the two formulations during storage at 37°C was evaluated, and the intravitreal stability of the drug product was characterized.

[0192] [Table 5-1]

[0193] [Table 5-2]

[0194] [Table 5-3]

[0195] The test results are shown in Tables 5.2 to 5.3. As can be seen from the above results, the stability study at 37°C 3M showed no obvious change in the SEC-HPLC main peak purity of Formulation 1 and Formulation 2, indicating that aflibercept after simulated dilution has good stability in the vitreous body.

[0196] Example 6: Lyophilization and pH testing of formulations

[0197] To further confirm the pH range and examine the stability of the lyophilized formulations, the stability of the formulations was examined at pH 5.8, 6.0, 6.2, and 6.4. Simultaneously, lyophilized formulation samples were prepared. All formulations were sterile filtered through a 0.22 μm low-protein-binding filter and filled into sterile 2 mL vials under aseptic conditions. These vials were sealed using film-coated rubber stoppers and aluminum-plastic combination caps. These formulation solutions were stored at 37°C for 1M and 2-8°C for 3M, and the stability of the samples was examined. Table 6.1 shows the information for the different formulations, where the aflibercept content is approximately 114.3 mg / mL. Tables 6.2-6.7 show the stability results for the different formulations.

[0198] [Table 6-1]

[0199] [Table 6-2]

[0200] [Table 6-3]

[0201] [Table 6-4]

[0202] [Table 6-5]

[0203] [Table 6-6]

[0204] [Table 6-7]

[0205] The test results are shown in Tables 6.2-6.7 and Figure 6. Figure 6 shows the SEC purity trend graph for formulations F5 and F6 at 2-8°C and 37°C ± 2°C. As can be seen from the results, after stability studies at 2-8°C 3M and 37°C 1M, there was no significant difference in the SEC-HPLC main peak purity of formulations F1-F4, indicating that the formulations have good stability within the pH range of 5.8-6.4. At 37°C 1M, there was no significant difference in the SEC-HPLC main peak purity of F5 and F6, indicating that the lyophilized formulations of the present invention have excellent stability and feasibility.

[0206] The above-described embodiments of the invention are illustrative only, and those skilled in the art will recognize or be able to ascertain, without undue experimentation, numerous equivalents to the specific compounds, materials, and procedures. All such equivalents are within the scope of the invention and are encompassed by the claims.

Claims

1. 1. An aqueous pharmaceutical composition comprising: (1) a VEGF-binding molecule selected from a VEGF receptor fusion protein, or an anti-VEGF antibody or antigen-binding fragment thereof, at a concentration of at least 100 mg / mL; (2) at least one stabilizer selected from sucrose, trehalose, proline, a pharmaceutically acceptable salt of proline, lysine, a pharmaceutically acceptable salt of lysine, and sorbitol; (3) at least one surfactant selected from polysorbate 80, polysorbate 20, and poloxamer; The aqueous pharmaceutical composition does not contain a buffer and has a pH of 5.0 to 7.

0.

2. The aqueous pharmaceutical composition further comprises (4) at least one of L-arginine, a pharmaceutically acceptable salt of L-arginine, and sodium chloride; 2. The aqueous pharmaceutical composition according to claim 1, wherein the content of the L-arginine or the pharmaceutically acceptable salt of the L-arginine is preferably 0 to 100 mM, or the content of the sodium chloride is preferably 0 to 40 mM.

3. the content of the L-arginine or the pharmaceutically acceptable salt of the L-arginine is 20 to 100 mM, or the content of the sodium chloride is 40 mM; 3. The aqueous pharmaceutical composition according to claim 2, wherein the content of the L-arginine or the pharmaceutically acceptable salt of L-arginine is preferably 20 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, or 100 mM.

4. the concentration of the VEGF-binding molecule in the aqueous pharmaceutical composition is at least 100 mg / mL, at least 114.3 mg / mL, at least 125 mg / mL, or at least 180 mg / mL; The aqueous pharmaceutical composition according to any one of claims 1 to 3, wherein the concentration of the VEGF-binding molecule in the aqueous pharmaceutical composition is preferably 100 to 180 mg / mL, more preferably 100 to 125 mg / mL.

5. the concentration of the stabilizer is 5 w / v % to 10 w / v %; 5. The aqueous pharmaceutical composition according to claim 1, wherein the stabilizer is selected from sucrose or trehalose, the concentration of the sucrose being 5 w / v% to 9 w / v%, and the concentration of the trehalose being 5.5 w / v% to 10 w / v%.

6. the concentration of sucrose is 5%, 6%, 7%, 8% or 9% w / v, or 6. The liquid pharmaceutical composition according to claim 5, wherein the concentration of the trehalose is 5.5 w / v%, 6.6 w / v%, 7.7 w / v%, 8.0 w / v%, or 10 w / v%.

7. 7. The aqueous pharmaceutical composition according to claim 1, wherein the concentration of the surfactant is 0.01 w / v % to 0.2 w / v %.

8. the surfactant is selected from polysorbate 20, 8. The aqueous pharmaceutical composition according to claim 7, wherein the concentration of polysorbate 20 is 0.01 w / v%, 0.03 w / v%, 0.05 w / v%, 0.07 w / v%, 0.1 w / v%, or 0.2 w / v%.

9. the pH of the aqueous pharmaceutical composition is 5.5 to 6.4; The aqueous pharmaceutical composition according to any one of claims 1 to 8, wherein the pH is preferably 5.5, 5.8, 6.0, 6.1, 6.2 or 6.

4.

10. a VEGF receptor fusion protein at a concentration of 100-180 mg / mL; 5 w / v% to 9 w / v% sucrose or 5.5 w / v% to 10 w / v% trehalose; 0.01 w / v% to 0.2 w / v% polysorbate 20; 0-100 mM L-arginine hydrochloride or 0-40 mM sodium chloride; The aqueous pharmaceutical composition according to any one of claims 1 to 9, wherein the pH is 5.0 to 7.

0.

11. a VEGF receptor fusion protein at a concentration of 100-180 mg / mL; 5 w / v% to 7 w / v% sucrose or 5.5 w / v% to 7.7 w / v% trehalose; 0.01 w / v% to 0.2 w / v% polysorbate 20; 40-80 mM L-arginine hydrochloride or 0-40 mM sodium chloride; The aqueous pharmaceutical composition according to any one of claims 1 to 9, wherein the pH is 5.5 to 6.

4.

12. An aqueous pharmaceutical composition, comprising: an aqueous pharmaceutical composition A comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition B comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition C comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition D comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition E comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition F comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition G comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition H comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition I comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition J comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition K comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition L comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition M comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition N comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition O comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition P comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition Q comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition R comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition S comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition T comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition U comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition V comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition W comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition X comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition Y comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5 w / v % sucrose, 0.01 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition Z comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 5 w / v % sucrose, 0.05 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZA comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZB comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZC comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZD comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZE comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZF comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZG comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZH comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZI comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZJ comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZK comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZL comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZM comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZN comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZO comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZP comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZQ comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZR comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZS comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZT comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZU comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZV comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZW comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZX comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZY comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5 w / v % sucrose, 0.01 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition ZZ comprising a VEGF receptor fusion protein at a concentration of 125 mg / mL, 5 w / v % sucrose, 0.05 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AA comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AB comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AC comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AD comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AE comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AF comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AG comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AH comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AI comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AJ comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AK comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AL comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 60 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AM comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AN comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AO comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AP comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AQ comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AR comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 70 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AS comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AT comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 6 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AU comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 7 w / v % sucrose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AV comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5.5 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AW comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 6.6 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AX comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 7.7 w / v % trehalose, 0.03 w / v % polysorbate 20, and 80 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AY comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5 w / v % sucrose, 0.01 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition AZ comprising a VEGF receptor fusion protein at a concentration of 180 mg / mL, 5 w / v % sucrose, 0.05 w / v % polysorbate 20, and 50 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; an aqueous pharmaceutical composition BA comprising a VEGF receptor fusion protein at a concentration of 114.3 mg / mL, 8 w / v % trehalose, 0.03 w / v % polysorbate 20, and 20 mM L-arginine hydrochloride, and having a pH of 5.5 to 6.4; is selected from Preferably, the VEGF receptor fusion protein is aflibercept.

13. The VEGF binding molecule comprises two polypeptides: The aqueous pharmaceutical composition according to any one of claims 1 to 9, wherein each polypeptide comprises immunoglobulin-like domain 2 of VEGFR1 and immunoglobulin-like domain 3 of VEGFR2.

14. The aqueous pharmaceutical composition according to any one of claims 1 to 9, wherein the VEGF-binding molecule is selected from bevacizumab, ranibizumab, aflibercept, or conbercept.

15. 15. The aqueous pharmaceutical composition of claim 14, wherein the VEGF-binding molecule is aflibercept.

16. the aqueous pharmaceutical composition is stable at 2-8°C for at least 6 months, 12 months, or 24 months; or 16. The aqueous pharmaceutical composition according to any one of claims 1 to 15, wherein the aqueous pharmaceutical composition is stable at 25°C ± 2°C for at least 1 month, 3 months, or 6 months.

17. A lyophilized formulation comprising: The lyophilized formulation is obtained by lyophilizing the aqueous pharmaceutical composition according to any one of claims 1 to 16, or A freeze-dried formulation, which can be reconstituted to obtain the aqueous pharmaceutical composition according to any one of claims 1 to 16.

18. A delivery device comprising the aqueous pharmaceutical composition of any one of claims 1 to 16 or the lyophilized formulation of claim 17 after reconstitution.

19. 20. The delivery device of claim 18, wherein the delivery device is a pre-filled syringe.

20. 1. A method of treating an ocular disease or disorder mediated by VEGF, comprising: applying to a subject an aqueous pharmaceutical composition according to any one of claims 1 to 16 or a delivery device according to claim 18 or 19; Preferably, the site of application is intravitreal.

21. Use of the aqueous pharmaceutical composition of any one of claims 1 to 16 or the delivery device of claim 18 or 19 in the preparation of a pharmaceutical composition for treating an eye disease or disorder mediated by VEGF.

22. 22. The method of claim 20 or the use of claim 21, wherein the ocular disease or disorder comprises a neovascular or an angiogenic ocular disease.

23. 23. The method or use of claim 22, wherein the ocular disease or disorder comprises neovascular age-related macular degeneration, age-related macular degeneration, macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, choroidal neovascularization, iris neovascularization, neovascular glaucoma, fibrosis after glaucoma surgery, proliferative vitreoretinopathy, optic nerve head neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy, non-proliferative diabetic retinopathy and / or proliferative diabetic retinopathy.

24. 1. A method for delivering a VEGF binding molecule to a subject, comprising: applying to the subject an aqueous pharmaceutical composition according to any one of claims 1 to 16 or a delivery device according to claim 18 or 19; Preferably, the site of application is intravitreal.

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