Stable high-concentration self-buffering pharmaceutical composition

Abstract

The invention provides a stable high-concentration self-buffering pharmaceutical composition which comprises VEGF (vascular endothelial growth factor) binding molecules, a stabilizer, a surfactant and the like, and the pH value of the composition is 5.0-7.0. The pharmaceutical composition is not additionally added with a buffering agent, has very high stability, is convenient to store and has a wide application prospect.

Description

A stable high-concentration self-buffering pharmaceutical composition

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on September 16, 2022, with application number CN202211134160.6 and invention name “A stable high-concentration self-buffering pharmaceutical composition”, the Chinese patent application filed with the China Patent Office on September 20, 2022, with application number CN202211141413.2 and invention name “A stable high-concentration self-buffering pharmaceutical composition”, and the Chinese patent application filed with the China Patent Office on September 7, 2023, with application number CN202311153498.0 and invention name “A stable high-concentration self-buffering pharmaceutical composition”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present application relates to the technical field of pharmaceutical compositions, and in particular to a stable high-concentration self-buffering pharmaceutical composition. Background Art

[0003] Vascular endothelial growth factor (VEGF) is a known regulator of angiogenesis and neovascularization and has been shown to be a key factor in the development of tumors and eye 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, Regeneron Pharmaceuticals, Inc. is an anti-VEGF drug that binds tightly to VEGF, reducing vascular permeability and further inhibiting angiogenesis. Since August 2014, the U.S. Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved its use via intravitreal injection for the treatment of macular edema secondary to retinal vein occlusion, wet age-related macular degeneration, and diabetic macular edema.

[0004] However, the existing technology still has many shortcomings and areas for improvement. For example, the concentration of therapeutic antibodies in pharmaceutical formulations varies widely depending on the route of administration and other methods. When injection volume is limited, it is often necessary to develop high-concentration antibody formulations. For example, high-concentration formulations are often required for intravitreal injection or subcutaneous administration. At the same time, the formulation of protein drugs itself can pose many challenges to pharmaceutical scientists. Therefore, it is necessary to find a formulation that stabilizes protein drugs and makes them resistant to degradation, aggregation, misfolding, etc. caused by proteolysis and denaturation. In particular, for engineered proteins that differ in substantial aspects from known proteins, finding appropriate stability conditions can be challenging. It is also desirable to have protein drugs in a form that is convenient for patients. Desirable properties include stability at ambient and refrigerated temperatures, suitability for long-term storage, appropriate administration time and dosage, and minimization of discomfort after administration.

[0005] While formulations suitable for intravitreal administration have been discovered in the prior art, there remains a need for stable, high-concentration liquid formulations of VEGF receptor fusion proteins suitable for ophthalmic administration. Formulations containing high-concentration VEGF receptor fusion proteins can reduce administration volume, shorten ocular injection time, increase dosing volume, extend dosing intervals, reduce dosing frequency, and improve manufacturing and storage efficiency. Furthermore, research has revealed that many commonly used buffers for pharmaceutical protein formulations, particularly for ophthalmic applications, have limitations and risks that could impact drug safety.

[0006] Buffers, such as acetate, succinate, citrate, histidine (imidazole), phosphate, and Tris, have been found to have undesirable limitations and disadvantages when used in the formulation of pharmaceutical proteins. Specifically, the additional components in the formulation complicate the formulation process, increase the risk of harmful components, affect the overall stability and shelf life of the drug, and increase the risk of end-user acceptability. It is generally known in the art that although buffer-free systems exist in the prior art, obtaining effective buffer-free pharmaceutical compositions for specific drugs is a challenging task, and the results are often unpredictable. The technical difficulty of high-concentration self-buffering systems is even higher. At the same time, improving the stability of pharmaceutical compositions is very challenging in the industry. From a formulation perspective, high protein concentrations lead to higher protein aggregation rates because closer contact between different components can lead to intermolecular attraction and self-aggregation events. Due to size exclusion and Donnan equilibrium effects, achieving the pH and excipient concentrations required for the UF / DF step is also a challenge. Higher protein concentrations exacerbate this challenge. Therefore, it is necessary to continue to optimize buffer systems and develop stable, high-concentration liquid formulations of VEGF receptor fusion proteins suitable for ophthalmic administration.

[0007] Summary of the Invention

[0008] One of the objectives of this application is to provide an improved, high-concentration pharmaceutical composition of VEGF-binding molecules that does not require an additional buffer. Specifically, this application provides a buffer-free system, a high-concentration protein self-buffering pharmaceutical composition. Through years of exploration and experimentation, the inventors have ultimately developed a high-concentration, self-buffering aqueous pharmaceutical composition of VEGF-binding molecules. Furthermore, the present application surprisingly discovered that this high-concentration, self-buffering system pharmaceutical composition also exhibits high stability, suggesting broad application prospects.

[0009] This application discloses a self-buffering, high-concentration aqueous pharmaceutical composition of a VEGF-binding molecule. High-concentration systems are inherently prone to aggregation events, making the formulation unstable, and self-buffering, high-concentration systems are even more so. Generally speaking, self-buffering, high-concentration systems are difficult to store at room temperature, and aggregation events are particularly prone to occur. The aflibercept pharmaceutical composition disclosed in this application is stable and has a shelf life sufficient for the production, storage, and distribution of the drug. For example, the self-buffering, high-concentration aflibercept pharmaceutical composition disclosed in this application has a shelf life of at least one month, three months, or six months at room temperature (e.g., 25°C).

[0010] In some embodiments, the present application discloses a pharmaceutical composition wherein, when a packaged sample is stored under accelerated conditions (e.g., 25°C ± 2°C), SEC-HPLC purity of ≥95% of the starting amount of aflibercept in the composition remains unchanged after one month. In other embodiments, when a packaged sample is stored under elevated temperature conditions (40°C ± 2°C), the self-buffered high-concentration aflibercept pharmaceutical composition disclosed herein exhibits superior efficacy compared to a high-concentration aflibercept pharmaceutical composition with a buffered system.

[0011] In some embodiments, the present application discloses a pharmaceutical composition. When insoluble particles of different particle sizes are tested (≥2 μm, ≥5 μm, ≥10 μm and / or ≥25 μm), the self-buffering high-concentration aflibercept pharmaceutical composition disclosed in the present application shows better effects than the high-concentration aflibercept pharmaceutical composition with a buffer system.

[0012] In some embodiments, the present application discloses a self-buffered, high-concentration 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 an antigen-binding fragment thereof, wherein the concentration of the VEGF-binding molecule is at least about 100 mg / mL; ② a stabilizer selected from at least one of sucrose, trehalose, proline, a pharmaceutically acceptable salt of proline, lysine, a pharmaceutically acceptable salt of lysine, or sorbitol; ③ a surfactant selected from at least one of polysorbate 80, polysorbate 20, or a poloxamer; wherein the aqueous pharmaceutical composition does not contain a buffer and has a pH of about 5.0-7.0.

[0013] In some embodiments, the aqueous pharmaceutical composition does not contain a buffer, which buffer refers to a histidine buffer, a phosphate buffer, a citric acid buffer, an acetic acid buffer, a carbonate buffer, a succinic acid buffer, a tartaric acid buffer, and / or a maleic acid buffer.

[0014] In some embodiments, the self-buffered high concentration VEGF-binding molecule aqueous or lyophilized pharmaceutical composition further comprises: ④ at least one of L-arginine, a pharmaceutically acceptable salt of L-arginine, or sodium chloride.

[0015] In some embodiments, the content of L-arginine or a pharmaceutically acceptable salt of L-arginine is about 0-100 mM, or the content of sodium chloride is about 0-40 mM. In preferred embodiments, the content of L-arginine or a pharmaceutically acceptable salt of L-arginine is about 20-100 mM, or the content of sodium chloride is about 40 mM. In more preferred embodiments, the content of L-arginine or a 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.

[0016] 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.

[0017] In some embodiments, the aqueous pharmaceutical composition comprises about 100-180 mg / mL of the VEGF-binding molecule. In preferred embodiments, the aqueous pharmaceutical composition comprises about 100-125 mg / mL of the VEGF-binding molecule.

[0018] In some embodiments, the concentration of the stabilizer in the aqueous pharmaceutical composition is about 5 w / v%-10 w / v%. In some embodiments, the stabilizer is selected from about 5 w / v%-9 w / v% sucrose or about 5.5 w / v%-10 w / v% trehalose. In preferred embodiments, the concentration of sucrose is about 5 w / v%, about 6 w / v%, about 7 w / %, about 8 w / v% or about 9 w / v%, or the concentration of trehalose is about 5.5 w / v%, about 6.6 w / v%, about 7.7 w / v%, about 8.0 w / v% or about 10 w / v%.

[0019] In some embodiments, the surfactant is selected from at least one of polysorbate 20, polysorbate 80, or poloxamer in an amount of about 0.01 w / v% to 0.05 w / v%. In a preferred embodiment, the surfactant is polysorbate 20, and the content of the polysorbate 20 is about 0.01 w / v%, about 0.03 w / v%, about 0.05 w / v%, 0.07 w / v%, about 0.1 w / v%, or about 0.2 w / v%.

[0020] In some embodiments, the pH of the aqueous pharmaceutical composition is about 5.5-6.4; preferably, the pH is about 5.5, about 5.8, about 6.0, about 6.1, about 6.2, or about 6.4.

[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 w / v%-9 w / v% sucrose or about 5.5 w / v%-10 w / v% trehalose, about 0.01 w / v%-0.2 w / v% polysorbate 20, about 0-100 mM L-arginine hydrochloride, or about 0-40 mM sodium chloride; wherein the pH of the aqueous pharmaceutical composition is about 5.0-7.0.

[0022] In some embodiments, the aqueous pharmaceutical composition comprises a VEGF receptor fusion protein at a concentration of about 100-180 mg / mL, about 5 w / v%-7 w / v% sucrose or about 5.5 w / v%-7.7 w / v% trehalose, about 0.01 w / v%-0.2 w / v% polysorbate 20, about 20-100 mM L-arginine hydrochloride, or about 0-40 mM sodium chloride; wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4.

[0023] In some embodiments, the aqueous pharmaceutical composition is selected from any one of the following:

[0024] An aqueous pharmaceutical composition A comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0025] An aqueous pharmaceutical composition B comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0026] An aqueous pharmaceutical composition C comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0027] An aqueous pharmaceutical composition D comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0028] An aqueous pharmaceutical composition E comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0029] An aqueous pharmaceutical composition F comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0030] An aqueous pharmaceutical composition G comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0031] An aqueous pharmaceutical composition H comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0032] An aqueous pharmaceutical composition I comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0033] An aqueous pharmaceutical composition J comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0034] An aqueous pharmaceutical composition K comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0035] An aqueous pharmaceutical composition L comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0036] An aqueous pharmaceutical composition M comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0037] An aqueous pharmaceutical composition N comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0038] An aqueous pharmaceutical composition O comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0039] An aqueous pharmaceutical composition P comprising: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0040] An aqueous pharmaceutical composition Q comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0041] An aqueous pharmaceutical composition R comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0042] An aqueous pharmaceutical composition S comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0043] An aqueous pharmaceutical composition T comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0044] An aqueous pharmaceutical composition U comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0045] An aqueous pharmaceutical composition V comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0046] An aqueous pharmaceutical composition W comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0047] An aqueous pharmaceutical composition X comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0048] An aqueous pharmaceutical composition Y comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5 w / v% sucrose, about 0.01 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0049] An aqueous pharmaceutical composition Z comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 5 w / v% sucrose, about 0.05 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0050] An aqueous pharmaceutical composition ZA comprises: 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 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0051] The aqueous pharmaceutical composition ZB comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0052] An aqueous pharmaceutical composition ZC comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0053] An aqueous pharmaceutical composition ZD comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0054] An aqueous pharmaceutical composition ZE comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0055] The aqueous pharmaceutical composition ZF comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0056] The aqueous pharmaceutical composition ZG comprises: 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 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0057] An aqueous pharmaceutical composition ZH comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0058] An aqueous pharmaceutical composition ZI comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0059] An aqueous pharmaceutical composition ZJ comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0060] The aqueous pharmaceutical composition ZK comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0061] An aqueous pharmaceutical composition ZL comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0062] An aqueous pharmaceutical composition ZM comprises: 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 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0063] An aqueous pharmaceutical composition ZN comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0064] The aqueous pharmaceutical composition ZO comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0065] The aqueous pharmaceutical composition ZP comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0066] An aqueous pharmaceutical composition ZQ comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0067] An aqueous pharmaceutical composition ZR comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0068] The aqueous pharmaceutical composition ZS comprises: 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, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0069] An aqueous pharmaceutical composition ZT comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0070] An aqueous pharmaceutical composition ZU comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0071] An aqueous pharmaceutical composition ZV comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0072] An aqueous pharmaceutical composition ZW comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0073] An aqueous pharmaceutical composition ZX comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0074] An aqueous pharmaceutical composition ZY comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 5 w / v% sucrose, about 0.01 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0075] An aqueous pharmaceutical composition ZZ comprises: a VEGF receptor fusion protein at a concentration of about 125 mg / mL, about 5 w / v% sucrose, about 0.05 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0076] The aqueous pharmaceutical composition AA comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0077] An aqueous pharmaceutical composition AB comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0078] An aqueous pharmaceutical composition AC comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0079] An aqueous pharmaceutical composition AD comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0080] An aqueous pharmaceutical composition AE comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0081] Aqueous pharmaceutical composition AF comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0082] The aqueous pharmaceutical composition AG comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0083] An aqueous pharmaceutical composition AH comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0084] The aqueous pharmaceutical composition AI comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0085] Aqueous pharmaceutical composition AJ comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0086] The aqueous pharmaceutical composition AK comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0087] An aqueous pharmaceutical composition AL comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 60 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0088] The aqueous pharmaceutical composition AM comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0089] An aqueous pharmaceutical composition AN comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0090] The aqueous pharmaceutical composition AO comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0091] The aqueous pharmaceutical composition AP comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0092] The aqueous pharmaceutical composition AQ comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0093] The aqueous pharmaceutical composition AR comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 70 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0094] The aqueous pharmaceutical composition AS comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0095] The aqueous pharmaceutical composition AT comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 6 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0096] The aqueous pharmaceutical composition AU comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 7 w / v% sucrose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0097] The aqueous pharmaceutical composition AV comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5.5 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0098] The aqueous pharmaceutical composition AW comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 6.6 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0099] The aqueous pharmaceutical composition AX comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 7.7 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 80 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0100] An aqueous pharmaceutical composition AY comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5 w / v% sucrose, about 0.01 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0101] The aqueous pharmaceutical composition AZ comprises: a VEGF receptor fusion protein at a concentration of about 180 mg / mL, about 5 w / v% sucrose, about 0.05 w / v% polysorbate 20, and about 50 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0102] An aqueous pharmaceutical composition BA comprises: a VEGF receptor fusion protein at a concentration of about 114.3 mg / mL, about 8 w / v% trehalose, about 0.03 w / v% polysorbate 20, and about 20 mM L-arginine hydrochloride, wherein the pH of the aqueous pharmaceutical composition is about 5.5-6.4;

[0103] Preferably, the VEGF receptor fusion protein is aflibercept.

[0104] 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 a more preferred embodiment, the VEGF-binding molecule is aflibercept.

[0105] In some embodiments, the pharmaceutical composition is stable at 2-8°C for at least 6 months, 12 months, or 24 months; or the pharmaceutical composition is stable at 25°C ± 2°C for at least 1 month, 3 months, or 6 months.

[0106] In some embodiments, the present application provides a lyophilized preparation, which is obtained by lyophilizing the aqueous pharmaceutical composition according to the present application, or the aqueous pharmaceutical composition of the present application is obtained by reconstitution of the lyophilized preparation.

[0107] In some embodiments, the present application provides a delivery device comprising the aqueous pharmaceutical composition or the reconstituted lyophilized formulation of the present application. In a preferred embodiment, the delivery device is a pre-filled syringe.

[0108] In some embodiments, the present application provides a method for treating an eye disease or disorder mediated by VEGF, the method comprising administering to a subject an aqueous pharmaceutical composition or delivery device disclosed herein. In a preferred embodiment, the site of administration is intravitreal.

[0109] In some embodiments, the present application provides the use of the aqueous pharmaceutical composition or delivery device of the present application in the preparation of a pharmaceutical composition for treating an ocular disease or disorder mediated by VEGF, the treatment comprising administering the aqueous pharmaceutical composition or delivery device of the present application to a subject. In a preferred embodiment, the site of administration is within the vitreous body.

[0110] In some embodiments, the ocular disease or disorder comprises an ocular neovascular disease or an angiogenic eye disease.

[0111] 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 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, post-operative fibrosis of glaucoma, proliferative vitreoretinopathy (PVR), optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy (DR), non-proliferative diabetic retinopathy and / or proliferative diabetic retinopathy.

[0112] In some embodiments, a method of delivering a VEGF-binding molecule to a subject is disclosed, comprising administering to the subject an aqueous pharmaceutical composition or a delivery device of the present application. In a preferred embodiment, the site of administration is intravitreal.

[0113] The pharmaceutical composition provided in this application does not require the addition of additional buffers, has high stability, is convenient for storage, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The accompanying drawings further illustrate the novel features disclosed in this application. The features and advantages disclosed in this application can be better understood with reference to these drawings, but it should be understood that these drawings are only used to illustrate specific implementations of the principles disclosed in this application and are not intended to limit the scope of the appended claims.

[0115] Figure 1 is a graph showing the purity change trend of the main SEC peak of formulations A2 and A3 at 40°C ± 2°C.

[0116] Figure 2 is a graph showing the SEC aggregate purity change trends of preparations A2 and A3 under different storage conditions.

[0117] FIG3 is a graph showing the SEC main peak purity change trends of preparations B1, B2, B3, B4, and B5 under different storage conditions.

[0118] FIG4 is a viscosity curve diagram corresponding to formulations B1, B2, B3, B4, and B5.

[0119] FIG5 is a graph showing the purity conversion trend of the main SEC peak of formulations C1-C10 at 37°C±2°C.

[0120] FIG6 is a graph showing the SEC purity conversion trends of formulations F5 and F6 at 2-8°C and 37°C±2°C. DETAILED DESCRIPTION

[0121] The term "about" includes and describes the value or parameter itself. For example, "about x" includes and describes "x" itself. As used herein, when used in conjunction with a measurement or to modify a value, unit, constant, or a range of values, the term "about" includes, in addition to the value or parameter itself, a variation of ±1%-10%. In some embodiments, when used in conjunction with a measurement or to modify a value, unit, constant, or a range of values, the term "about" refers to a variation of ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%.

[0122] The term "aqueous" pharmaceutical composition refers to a pharmaceutical composition that includes water.

[0123] The term "self-buffering" refers to the exclusion of components traditionally used to introduce buffering capacity into a formulation, such as buffers including, but not limited to, histidine buffers, phosphate buffers, citrate buffers, acetate buffers, carbonate buffers, succinate buffers, tartaric acid buffers, maleate buffers, or mixtures thereof, and the like. Proteins are known to provide buffering capacity to a formulation in addition to any buffering capacity present in a formulation without a buffer component. Therefore, these terms do not imply that a formulation cannot have any buffering capacity.

[0124] The term "high concentration" formulation contains a VEGF receptor fusion protein concentration of 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, 115 mg / mL, 116 mg / mL, 117 mg / mL, 118 mg / mL, 119 mg / mL, 120 mg / mL, 121 mg / mL, 122 mg / mL, 123 mg / mL, 124 mg / mL, 125 mg / mL, 126 mg / mL, 127 mg / mL, 128 mg / mL, 129 mg / mL, 130 mg / mL, 131 mg / mL, 132 mg / mL, 133 mg / mL, 134 mg / mL, 135 mg / mL, 136 mg / mL, 137 mg / mL, 138 mg / mL, 139 mg / mL, 140 mg / mL, 141 mg / mL, 142 mg / mL, 143 mg / mL, 144 mg / mL, 145 mg / mL, 146 mg / mL, 147 mg / mL, 148 mg / mL, 149 mg / mL, 150 mg / mL, 151 mg / mL, 152 mg / mL, 153 mg / mL, 154 mg / mL, 155 mg / mL, 156 mg / mL, 157 mg / 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, 166 mg / mL, 167 mg / mL, 168 mg / mL, 169 mg / mL, 170 mg / mL, 171 mg / mL, 172 mg / mL, 173 mg / mL, 174 mg / mL, 175 mg / mL, 176 mg / mL, 177 mg / mL, 178 mg / mL, 179 mg / mL, 180 mg / mL.

[0125] 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. Exemplary VEGF receptor fusion proteins include aflibercept (Regeneron Pharmaceuticals, Inc.), see International Patent Application Publication Nos. WO 2005 / 121176 or WO 2007 / 112675.

[0126] The term "VEGF receptor fusion protein" refers to a molecule comprising one or more VEGF receptors or domains thereof fused to another polypeptide that interferes with the interaction between VEGF and a native VEGF receptor, e.g., where two such fusion polypeptides bind 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 within the scope of the present application include chimeric polypeptides comprising two or more immunoglobulin (Ig)-like domains of a VEGF receptor, e.g., VEGFR1 (also known as Flt1) and / or VEGFR2 (also known as Flk1 or KDR), and may also comprise a multimerization domain (e.g., an Fc domain).

[0127] The term "aflibercept" includes biosimilar forms thereof, which generally refer to products comprising the same amino acid sequence. For example, the amino acid sequence and nucleic acid sequence of aflibercept have been described in the literature, such as those shown in WO2000075319A1.

[0128] The term "treatment" as used herein refers to the treatment methods described herein. The method of "treatment" employs administering the antibodies of the present application to a subject in need of such treatment (e.g., a subject suffering from a VEGF-mediated ocular disorder or a subject who may eventually acquire such a disorder) to prevent, cure, delay the onset of the disorder or recurring disorder, lessen the severity of the disorder or recurring disorder, or ameliorate one or more symptoms of the disorder or recurring disorder, or to prolong the survival of the subject beyond the expected survival period in the absence of such treatment.

[0129] The term "subject" or "patient" refers to humans and non-human mammals, including but not limited to primates, rabbits, pigs, horses, dogs, cats, sheep, and cows.

[0130] Protein content determination: Protein content was determined using a Lunatic microspectrophotometer. Purified water was used for blank subtraction, and the UV absorption at 280 nm was corrected by light scattering at 330 nm. Protein concentration was calculated according to Formula 1, where the extinction coefficient ε is 1.5 (L / g·cm -1 ).

[0131] Size Exclusion Chromatography (SEC): Size exclusion chromatography is used to quantify aggregates, monomers, and fragments. This assay utilizes a TSKgel G3000SWXL 7.8x300mm 5μm The column was run on a Waters E2695-2489 HPLC system. The mobile phase was potassium phosphate buffer. The sample was diluted to 1 mg / mL with the mobile phase, and the injection volume was 25 μL. The protein was eluted isocratically at a flow rate of 0.5 mL / min for 30 min, and the absorbance of the eluate was monitored at 215 nm. Integration was performed using Empower 3 software.

[0132] Reduced Capillary Electrophoresis (rCE-SDS): The % (main peak 1 + main peak 2) purity was determined by reduced capillary electrophoresis (rCE-SDS). This determination was performed on a SCIEX PA800plus capillary electrophoresis system using 50 μm ID non-coated quartz capillaries, an effective capillary separation length of 20 cm, a total separation length of 30.2 cm, a PDA 220 nm bandwidth of 10 nm for detection, and integration using 32Karat software.

[0133] Non-reducing SDS-PAGE (nrSDS-PAGE): The % main peak purity was determined by non-reducing SDS-PAGE electrophoresis using polyacrylamide gel electrophoresis with an 8% separating gel concentration and a 4.5% stacking gel concentration. Electrophoresis was performed at a constant current of 10 mA / gel. After staining with Coomassie Brilliant Blue R250 and destaining with a destaining solution, the gel was scanned and imaged using a gel imaging system to analyze the purity of the target band.

[0134] Imaging capillary isoelectric focusing (iCIEF): Charge heterogeneity and identification experiments were performed using imaging capillary isoelectric focusing (iCIEF). This determination was performed on an imaging capillary electrophoresis instrument, a Maurice C system, with an effective capillary length of 5 cm, a focusing time of 6 minutes, a focusing program of 1.5 kV to 1 minute, and detection at 3 kV to 5 minutes. Integration was performed using Empower 3 software.

[0135] MFI Microfluidic Imaging Particle Analysis: Particle counts were determined using the MFI Microfluidic Imaging Particle Analysis System. This determination was performed on an MFI Particle Analysis System MFI 5200 with a sample volume of 1 mL, a total active volume of 0.7 mL, and an analysis volume of 0.51 mL. Analysis was performed using MVSS analysis software.

[0136] Viscosity measurement by m-VROC viscometer: e-VROC technology builtTM The viscometer is an excellent tool for measuring both extensional and shear viscosities simultaneously. It uses MEMS pressure sensors to measure pressure upstream and downstream of a contraction and record changes in flow rate. The fluid experiences nearly constant extension through contraction / expansion, enabling the calculation of apparent extensional viscosity.

[0137] Freezing point depression method for determining osmolality: Measuring the freezing point depression of a solution indirectly determines its osmolality. In an ideal dilute solution, freezing point depression follows the relationship ΔTf = Kf·m, where ΔTf is the freezing point depression, Kf is the freezing point depression constant (1.86 when water is the solvent), and m is the molarity. Osmotic pressure, on the other hand, follows the relationship Po = k·m, where Po is the osmotic pressure, k is the osmotic pressure constant, and m is the molarity of the solution. Since the concentrations in both equations are equivalent, the freezing point depression method can be used to determine the osmolality of a solution.

[0138] ELISA determination of binding activity: The binding activity of recombinant human vascular endothelial growth factor receptor-antibody fusion protein injection to VEGF165 was determined by ELISA method. First, human VEGF165 protein was coated on a 96-well plate, and then gradient dilutions of this product were added. Then, secondary antibody, colorimetric solution, and stop solution were added in sequence. The absorbance value was read and the binding ability of this product to human VEGF165 antigen was calculated. The experimental data were subjected to four-parameter fitting analysis using Soft Max Pro or other similar software, and the results were automatically analyzed according to the following formula: the logarithm of the reference sample (self-made) concentration was on the X-axis, and the absorbance value was on the Y-axis. The software will give the half effective concentration (EC50) of the test sample and the reference sample. 50 ) and draw an “S” curve.

[0139] The concentration of VEGF binding molecules, the concentration of stabilizers, the concentration of surfactants, the concentration of L-arginine, the concentration of pharmaceutically acceptable salts of L-arginine, and the concentration of sodium chloride described in this application refer to the concentrations of VEGF binding molecules, stabilizers, surfactants, L-arginine, the concentrations of pharmaceutically acceptable salts of L-arginine, and sodium chloride in the aqueous pharmaceutical composition, respectively.

[0140] The experimental conditions in the following examples are: shaking at room temperature (25±2°C) for one week (25°C 1W), freeze-thaw cycles 3 times (freezing at -40°C for 16 hours and thawing at 25°C for 8 hours as one cycle), placing at 2-8°C for 1 month (2-8°C 1M), placing at 2-8°C for 6 months (2-8°C 6M), placing at 25°C±2°C for 1 month (25°C 1M), placing at 40°C±2°C for one week (40°C 1W), placing at 40°C±2°C for two weeks (40°C 2W) or placing at 40°C±2°C for 1 month (40°C 1M).

[0141] Example 1: Preparation of self-buffered system formulation samples

[0142] Four different formulations of the VEGF receptor fusion protein (aflibercept) at approximately 40 mg / mL, approximately 114.3 mg / mL, and approximately 180 mg / mL were tested for long-term potency, physical stability, and charge variant formation. The component contents of the four formulations are shown in Table 1.1. To prepare the formulation solutions, the desired excipient solutions were buffer exchanged and concentrated by ultrafiltration to the desired antibody concentration. After ultrafiltration and concentration, the excipients to be added were added to the antibody solution as a stock solution. All formulations were sterile filtered through a 0.22 μm low protein binding filter and aseptically filled into sterile 2 mL vials, sealed with film-coated rubber stoppers and aluminum-plastic combination caps. The stability of aflibercept during storage under different conditions was evaluated for each formulation using SEC-HPLC, nr-SDS-PAGE, and rCE-SDS to determine the presence (%) of high molecular weight species (HMWS, LMWS) and the main peak (monomer / main).

[0143] The following are different formulations and their stability results:

[0144] Table 1.1 Composition of preparations containing aflibercept

[0145] Note: “-” means not added.

[0146] Table 1.2 Stability data of formulation A1

[0147] Note: “ / ” means not detected.

[0148] Table 1.3 Stability data of formulation A2

[0149] Note: “ / ” means not detected.

[0150] Table 1.4 Stability data of formulation A3

[0151] Note: “ / ” means not detected.

[0152] Table 1.5 Stability data of formulation A4

[0153] Note: “ / ” means not detected.

[0154] Table 1.6 Viscosity and osmotic pressure data for formulations A2-A4

[0155] Test Example A1 and Test Example A2 are schemes with a buffer system, and Test Example A3 and Test Example A4 are self-buffer system schemes. The experimental results show that: under the conditions of shaking at room temperature (25±2℃) for one week, freeze-thaw cycles 3 times, and storage at 2-8℃ for 6 months, the samples of Test Example A1, Test Example A2, and Test Example A3 are relatively stable, and there is no significant change in SEC-HPLC purity. Under the condition of storage at 25℃ for 1 month, except for a significant decrease in nr-SDS-PAGE purity, there is no significant change in other purities of Test Example A1, Test Example A2, Test Example A3, and Test Example A4.

[0156] Under the condition of being placed at 40℃±2℃ for 1 month, the test results are shown in Tables 1.2-1.4. The SEC main peak purity transformation trend diagram of preparations A2 and A3 at 40℃±2℃ is shown in Figure 1, and the SEC aggregate purity transformation trend diagram of preparations A2 and A3 under different storage conditions is shown in Figure 2. From the above results, it can be seen that the SEC-HPLC purity of Test Example A3 has been greatly improved compared with Test Example A2. Specifically, compared with Test Example A2, the SEC-HPLC main peak purity of Test Example A3 increased by 5.9% and the aggregates decreased by 5.9%. In industry, improving protein stability is very difficult. An increase of 1-2% in purity is of great significance. An increase of 5.9% in purity is a very significant breakthrough. In addition, the preparation of high-concentration preparations of the self-buffering system saves more excipient costs during the preparation process. In summary, the test results of Test Examples A1-A4 show that the self-buffering preparations of the present application that do not contain histidine and phosphate buffers have shown excellent stability throughout the research process.

[0157] Table 1.7 Insoluble particles—MFI results

[0158] Table 1.8 Insoluble particles—MFI results (morphological analysis)

[0159] Tables 1.7 and 1.8 show the MFI test results of insoluble particles. For ophthalmic intravitreal injection products, the reduction of aggregates is expected to reduce a series of adverse reactions such as immunogenicity in patients. Compared with A1 and A2, the purity of test example A3 was improved in nr-SDS-PAGE and rCE-SDS. At the same time, compared with A1 and A2, the insoluble particles in test example A3 were improved. In the analysis of insoluble particles ≥2μm, ≥5μm, ≥10μm and ≥25μm, the A3 preparation had a lower concentration of insoluble particles. This is a surprising result. For ophthalmic administration, the improvement of insoluble particles can reduce the rate of adverse reactions in patients due to injection. Therefore, the results of this test show that the self-buffering preparation of the present application has excellent prospects in ophthalmic administration.

[0160] Example 2: Comparison of the Stability of Self-Buffering System Formulations Containing Different L-Arginine Hydrochloride Concentrations

[0161] Test Examples B1-B7 are self-buffered formulations containing varying concentrations of L-arginine hydrochloride. The contents of the individual components are shown in Table 2.1. The concentration of aflibercept in B1-B7 was approximately 114.3 mg / mL. Example 2 examined the effects of adding 20 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, and 100 mM L-arginine hydrochloride, respectively, on the stability of the pharmaceutical compositions.

[0162] Table 2.1 Preparations containing different concentrations of L-arginine hydrochloride

[0163] Table 2.2 Stability data of formulation B1

[0164] Note: “ / ” means not detected.

[0165] Table 2.3 Stability data of formulation B2

[0166] Note: “ / ” means not detected.

[0167] Table 2.4 Stability data of formulation B3

[0168] Note: “ / ” means not detected.

[0169] Table 2.5 Stability data of formulation B4

[0170] Note: “ / ” means not detected.

[0171] Table 2.6 Stability data of formulation B5

[0172] Note: “ / ” means not detected.

[0173] Table 2.7 Stability data of formulation B6

[0174] Note: “ / ” means not detected.

[0175] Table 2.8 Stability data of formulation B7

[0176] Note: “ / ” means not detected.

[0177] The physical stability of self-buffered preparations of L-arginine hydrochloride at different concentrations at 2-8°C, 25°C±2°C, and 37°C±2°C was evaluated by 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 is a trend chart of the purity change of the SEC main peak of preparations B1, B2, B3, B4, and B5 under different storage conditions, and Figure 4 is a viscosity curve corresponding to preparations B1, B2, B3, B4, and B5.

[0178] The above test results show that under the conditions of 2-8°C 3M and 25°C 1M, the stability of the pharmaceutical compositions containing 20mM, 40mM, 50mM, 60mM, 70mM, 80mM, and 100mM L-arginine hydrochloride is very excellent, and their various purities do not change significantly compared with the initial stage (0 time). At the same time, different concentrations of L-arginine hydrochloride have no obvious effect on the viscosity of the preparation.

[0179] Example 3: Comparison of the stability of self-buffered system formulations containing different types and concentrations of stabilizers

[0180] Test Examples C1-C10 are self-buffered formulations containing different types and concentrations of stabilizers, each containing approximately 114.3 mg / mL of aflibercept. The contents of the various components are shown in Table 3.1. Example 3 examined the effects of different stabilizer types and concentrations on the stability of the pharmaceutical composition.

[0181] Table 3.1 Preparations containing aflibercept

[0182] Note: “-” means not added.

[0183] Table 3.2 Stability data of formulation C1

[0184] Note: “ / ” means not detected.

[0185] Table 3.3 Stability data of formulation C2

[0186] Note: “ / ” means not detected.

[0187] Table 3.4 Stability data of formulation C3

[0188] Note: “ / ” means not detected.

[0189] Table 3.5 Stability data of formulation C4

[0190] Note: “ / ” means not detected.

[0191] Table 3.6 Stability data of formulation C5

[0192] Note: “ / ” means not detected.

[0193] Table 3.7 Stability data of formulation C6

[0194] Note: “ / ” means not detected.

[0195] Table 3.8 Stability data of formulation C7

[0196] Note: “ / ” means not detected.

[0197] Table 3.9 Stability data of formulation C8

[0198] Note: “ / ” means not detected.

[0199] Table 3.10 Stability data of formulation C9

[0200] Note: “ / ” means not detected.

[0201] Table 3.11 Stability data of formulation C10

[0202] Note: “ / ” means not detected.

[0203] Tables 3.2-3.11 show the monomer / MAIN (main peak), HMWS (polymer), and fragment (LMWS) percentage results of SEC-HPLC, rCE-SDS, and nr-SDS-PAGE of C1-C10 preparations at 0h, 37℃±2℃, 2-8℃, and 25℃±2℃. Figure 5 shows the purity change trend of the SEC main peak of preparations C1-C10 at 37℃±2℃. From the above results, it can be seen that ten self-buffered high-concentration preparations C1-C10 with high purity were prepared by adding excipients such as sucrose, trehalose, sorbitol, proline, and lysine hydrochloride, and the 0h SEC-HPLC main peak purity was above 98.7%; the results at 37℃1M showed that the SEC-HPLC main peak purity of C1-C3 preparations was between 86.1% and 88.8%; the SEC-HPLC main peak purity of C4-C6 preparations was between 88.7% and 91.0%; the SEC-HPLC main peak purity of C7, C8, and C9 preparations was 78.4%, 67.3%, and 85.1%, respectively; no significant difference was found in the purity changes of each preparation by rCE-SDS and nr-SDS-PAGE.

[0204] The above test results show that sucrose and trehalose have a significant protective effect on aflibercept. Further exploration of the content range of sucrose and trehalose found that when the sucrose concentration is within the range of 5w / v%-7w / v% and the trehalose concentration is within the range of 5w / v%-7.7w / v%, the pharmaceutical composition has excellent stability.

[0205] Example 4: Comparison of the stability of different self-buffering system preparations

[0206] The pH, protein content, and polysorbate 20 content of different self-buffered formulations were adjusted, as shown in Table 4.1. The stability of aflibercept formulations was tested under the following conditions: 6 w / v% sucrose, 50 mM L-arginine hydrochloride, pH values ​​of 5.5, 5.8, and 6.1, protein contents of approximately 100 mg / mL, approximately 114.3 mg / mL, and approximately 125 mg / mL, respectively, and polysorbate 20 of 0.01 w / v%, 0.03 w / v%, and 0.05 w / v%, respectively.

[0207] Table 4.1 Preparations containing aflibercept

[0208] Table 4.2 Stability data of formulation D1

[0209] Note: “ / ” means not detected.

[0210] Table 4.3 Stability data of formulation D2

[0211] Note: “ / ” means not detected.

[0212] Table 4.4 Stability data of formulation D3

[0213] Note: “ / ” means not detected.

[0214] Table 4.5 Stability data of formulation D4

[0215] Note: “ / ” means not detected.

[0216] Table 4.6 Stability data of formulation D5

[0217] Note: “ / ” means not detected.

[0218] Table 4.7 Stability data of formulation D6

[0219] Note: “ / ” means not detected.

[0220] Table 4.8 Stability data of formulation D7

[0221] Note: “ / ” means not detected.

[0222] Table 4.9 Stability data of preparation D8

[0223] Note: “ / ” means not detected.

[0224] Table 4.10 Stability data of formulation D9

[0225] Note: “ / ” means not detected.

[0226] Table 4.11 Stability data of formulation D10

[0227] Note: “ / ” means not detected.

[0228] Table 4.12 Viscosity and osmotic pressure data of formulations D1-D10

[0229] The test results are shown in Tables 4.2-4.12. These results demonstrate that the SEC-HPLC main peak purity of formulations D1-D10 at 0 h was above 99.0%. The SEC-HPLC main peak purity of formulations D1-D10 at 2-8°C (1M) and 25°C (1M) ranged from 97.7% to 98.9%. The viscosity of the formulations ranged from 5.6 to 10.1 cP, and the osmotic pressure was 342 to 387 mOsmol / kg. These results demonstrate that the stability of the high-concentration self-buffering formulations remains excellent within the protein content range of 100-125 mg / mL, pH 5.5-6.1, and polysorbate 20 content of 0.01-0.05 w / v%.

[0230] Example 5: Formulation Dilution Test

[0231] 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 information of the two formulations. To prepare the formulation solutions, the intended excipient solutions were used for dilution. The excipients to be added were added to the antibody solution as stock solutions. All formulations were sterile-filtered through a 0.22 μm low protein-binding filter and aseptically filled into sterile 2 mL vials, sealed with film-coated rubber stoppers and aluminum-plastic combination caps. SEC-HPLC was used to determine the presence (%) of high molecular weight species (HMWS, LMWS) and the main peak (monomer / main). The stability of aflibercept in both formulations during storage at 37°C was evaluated to characterize the intravitreal stability of the drug product.

[0232] Table 5.1 Preparations containing aflibercept

[0233] Table 5.2 Stability data of formulation E1

[0234] Table 5.3 Stability data of formulation E2

[0235] Note: “ / ” means not detected.

[0236] The test results are shown in Tables 5.2-5.3. From the above results, it can be found that after the stability test at 37°C for 3M, the SEC-HPLC main peak purity of Formulation 1 and Formulation 2 did not change significantly, indicating that the simulated diluted aflibercept has good stability in the vitreous body.

[0237] Example 6: Lyophilization and pH testing of formulations

[0238] To further confirm the pH range and investigate the stability of the lyophilized formulations, the stability of the formulations was investigated at pH 5.8, 6.0, 6.2, and 6.4, and lyophilized formulation samples were prepared. All formulations were sterile filtered through a 0.22 μm low protein-binding filter and aseptically filled into sterile 2 mL vials, sealed with a film-coated rubber stopper and an aluminum-plastic combination cap. These formulation solutions were then stored at 37°C for 1 mL and 2-8°C for 3 mL to investigate the stability of the samples. Table 6.1 shows information for the different formulations, including aflibercept at approximately 114.3 mg / mL. Tables 6.2-6.7 present the stability results for the different formulations.

[0239] Table 6.1 Preparations containing aflibercept

[0240] Note: “-” means not added.

[0241] Table 6.2 Stability data of formulation F1

[0242] Note: “ / ” means not detected.

[0243] Table 6.3 Stability data of formulation F2

[0244] Note: “ / ” means not detected.

[0245] Table 6.4 Stability data of formulation F3

[0246] Note: “ / ” means not detected.

[0247] Table 6.5 Stability data of formulation F4

[0248] Note: “ / ” means not detected.

[0249] Table 6.6 Stability data of formulation F5 lyophilized powder

[0250] Note: “ / ” means not detected.

[0251] Table 6.7 Stability data of formulation F6 lyophilized powder

[0252] Note: “ / ” means not detected.

[0253] The test results are shown in Tables 6.2-6.7 and Figure 6, where Figure 6 is a graph showing the SEC purity trend of formulations F5 and F6 at 2-8°C and 37°C ± 2°C. The results show that after stability studies at 2-8°C for 3 M and 37°C for 1 M, there was no significant difference in the SEC-HPLC main peak purity of formulations F1-F4, indicating that the formulations had good stability within the pH range of 5.8-6.4. At 37°C for 1 M, there was no significant difference in the SEC-HPLC main peak purity of formulations F5 and F6, demonstrating that the lyophilized formulations of the present application have excellent stability and feasibility.

[0254] The embodiments of the present application described above are merely exemplary, and any person skilled in the art will recognize or determine the equivalents of numerous specific compounds, materials, and operations without requiring undue experimentation. All of these equivalents are within the scope of the present application and are encompassed by the claims.

Claims

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

0.

2. The aqueous pharmaceutical composition according to claim 1, wherein The aqueous pharmaceutical composition further comprises: ④ at least one of L-arginine, a pharmaceutically acceptable salt of L-arginine, or sodium chloride; Preferably, the content of the L-arginine or the pharmaceutically acceptable salt of L-arginine is 0-100 mM, or the content of the sodium chloride is 0-40 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 20-100 mM, or the content of the sodium chloride is 40 mM; Preferably, the content of the L-arginine or the pharmaceutically acceptable salt of L-arginine is 20 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM or 100 mM.

4. 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 at least 100 mg / mL, at least 114.3 mg / mL, at least 125 mg / mL, or at least 180 mg / mL; Preferably, the concentration of the VEGF binding molecule in the aqueous pharmaceutical composition is 100-180 mg / mL, preferably 100-125 mg / mL.

5. The aqueous pharmaceutical composition according to any one of claims 1 to 4, wherein The concentration of the stabilizer is 5w / v%-10w / v%; preferably, the stabilizer is selected from sucrose or trehalose, the concentration of the sucrose is 5w / v%-9w / v%, and the concentration of the trehalose is 5.5w / v%-10w / v%.

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

7. The aqueous pharmaceutical composition according to any one of claims 1 to 6, wherein The concentration of the surfactant is 0.01 w / v%-0.2 w / v%.

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

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

4.

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

0.

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

4.

12. An aqueous pharmaceutical composition selected from: The aqueous pharmaceutical composition A comprises: 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, wherein: The pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition B comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition C comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition D comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition E comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition F comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition G comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition H comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition I comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition J comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition K comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition L comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition M comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition N comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition O comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition P comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition Q comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition R comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition S comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition T comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition U comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition V comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition W comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition X comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition Y comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition Z comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZA comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZB comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZC comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZD comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZE comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZF comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZG comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZH comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZI comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition ZJ comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZK comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition ZL comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZM comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZN comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZO comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZP comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZQ comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZR comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZS comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZT comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZU comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZV comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZW comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition ZX comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition ZY comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition ZZ comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AA comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition AB comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition AC comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AD comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AE comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition AF comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AG comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AH comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AI comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Aqueous pharmaceutical composition AJ comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AK comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AL comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AM comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AN comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AO comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AP comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AQ comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AR comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AS comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AT comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AU comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AV comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AW comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AX comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; An aqueous pharmaceutical composition AY comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition AZ comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; The aqueous pharmaceutical composition BA comprises: 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, wherein the pH of the aqueous pharmaceutical composition is 5.5-6.4; Preferably, the VEGF receptor fusion protein is aflibercept.

13. The aqueous pharmaceutical composition according to any one of claims 1 to 9, wherein The VEGF binding molecule comprises two polypeptides, each polypeptide comprising the immunoglobulin-like domain 2 of VEGFR1 and the 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. The aqueous pharmaceutical composition according to claim 14, wherein The VEGF binding molecule is aflibercept.

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

17. A lyophilized preparation, which is obtained by lyophilizing the aqueous pharmaceutical composition according to any one of claims 1 to 16, or the aqueous pharmaceutical composition according to any one of claims 1 to 16 is obtained by reconstitution of the lyophilized preparation.

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

19. The delivery device of claim 18, which is a pre-filled syringe.

20. A method for treating an ocular disease or disorder mediated by VEGF, the method comprising administering to a subject the aqueous pharmaceutical composition of any one of claims 1-16 or the delivery device of claim 18 or 19; preferably, the site of administration is within the vitreous.

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

22. The method of claim 20 or the use of claim 21, wherein The ocular diseases or disorders include ocular neovascular diseases or angiogenic eye diseases.

23. The method or use according to claim 22, wherein The ocular diseases or disorders include neovascular age-related macular degeneration, macular edema, macular edema following retinal vein occlusion, retinal vein occlusion, central retinal vein occlusion, branch retinal vein occlusion, diabetic macular edema, choroidal neovascularization, iris neovascularization, neovascular glaucoma, post-operative fibrosis of glaucoma, proliferative vitreoretinopathy, optic disc neovascularization, corneal neovascularization, retinal neovascularization, vitreous neovascularization, pannus, pterygium, vascular retinopathy, diabetic retinopathy, non-proliferative diabetic retinopathy and / or proliferative diabetic retinopathy.

24. A method of delivering a VEGF-binding molecule to a subject, comprising the step of administering to the subject the aqueous pharmaceutical composition of any one of claims 1-16 or the delivery device of claim 18 or 19; preferably, the site of administration is within the vitreous body.

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