Pharmaceutical composition containing Anti-VEGF fusion protein

Abstract

A pharmaceutical composition containing an anti-VEGF fusion protein. The pharmaceutical composition contains anti-VEGF fusion protein A and protein fragment B. By means of controlling the content of protein fragment B in the pharmaceutical composition to 0.1%-5.4%, the polymerization rate of the proteins in the composition can be reduced, the stability of the proteins can be improved, and a better biological activity can be maintained.

Description

A pharmaceutical composition containing an anti-VEGF fusion protein

[0001] This application claims priority to Chinese Patent Application No. 2023118409197, filed December 27, 2023. This application incorporates the entirety of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition containing an anti-VEGF fusion protein. Background Art

[0003] Neovascularization is key to the development and spread of many diseases. Numerous ocular diseases involve angiogenesis, including age-related macular degeneration (AMD), retinal vein occlusion (RVO), diabetic retinopathy (DR), and pathological myopia. VEGF is a highly specific vascular endothelial growth factor that promotes increased vascular permeability, extracellular matrix degeneration, endothelial cell migration and proliferation, and angiogenesis. VEGF is widely distributed in multiple tissues in humans and animals. Low levels of VEGF are produced by normal retinal pigment epithelial cells, vascular endothelial cells, and pericytes. Numerous studies have demonstrated that excessive VEGF expression can induce pathological neovascular eye diseases. Given the importance of VEGF signaling in angiogenesis, blocking VEGF or VEGF receptors to inhibit angiogenesis holds important therapeutic potential for angiogenesis-related diseases, including cancer and retinal vascular lesions. Over the past decade, several anti-VEGF drugs have been developed for the treatment of ocular neovascular diseases, including antibodies or fusion proteins such as bevacizumab, ranibizumab, aflibercept, and conbercept.

[0004] Salt bonds, hydrogen bonds, disulfide bonds, and hydrophobic interactions are the forces that maintain protein conformation. Interactions with metal ions, substrates, cofactors, and other low-molecular-weight ligands stabilize protein conformation. Those skilled in the art are well aware that antibodies or proteins are subject to a variety of environmental factors during storage. Temperature, humidity, oxygen, and ultraviolet light, among others, can all cause various physical or chemical changes in fusion proteins, leading to aggregation, decomposition, oxidation, or denaturation. These changes can reduce protein activity, diminish therapeutic efficacy, and cause serious toxic side effects. Summary of the Invention

[0005] One object of the present invention is to provide a pharmaceutical composition containing an anti-VEGF fusion protein with good stability and stable biological activity.

[0006] In certain embodiments, the anti-VEGF fusion protein of the present invention comprises a dimer of two fusion polypeptides, each comprising the extracellular domain 2 of VEGFR-1, the extracellular domains 3 and 4 of VEGFR-2, and human immunoglobulin IgG4. In certain specific embodiments, the anti-VEGF fusion protein of the present invention comprises a dimer of two fusion polypeptides comprising SEQ ID NO: 1 (SEQ ID NO: 1 is shown in FIG1 ), the two fusion polypeptides being non-covalently linked by a disulfide bond and having a molecular weight of 142 kDa. This fusion protein is hereinafter referred to as fusion protein A.

[0007] Fusion protein A is a fusion protein described in the Chinese patent "Application of VEGF receptor fusion protein in the treatment of eye diseases" (Patent No. ZL200610066257.2), specifically the active component of the FP3 fusion protein, which is formed by the fusion of the immunoglobulin-like region 2 in the human vascular endothelial growth factor VEGF receptor 1 and the immunoglobulin-like regions 3 and 4 in the VEGF receptor 2 with the human immunoglobulin Fc fragment, and has the amino acid sequence as described in SEQ ID NO: 1. Therefore, the content of ZL200610066257.2 can be used to further illustrate the present invention.

[0008] The present invention discovered that, in addition to fusion protein A, proteins obtained through cell expression or purification usually contain low-molecular-weight protein fragments B, C, and D. These may be induced by various stress conditions during cell culture, purification, etc., and are usually related to the breakage of protein covalent bonds caused by spontaneous or enzymatic reactions.

[0009] Protein fragment B is a dimer formed by a truncated fusion polypeptide and a second intact fusion polypeptide in fusion protein A, with a molecular weight of 131.2 kDa. Specifically, one truncated fusion polypeptide in protein fragment B has the amino acid sequence set forth in SEQ ID NO:1, 82-526, more specifically, the amino acid sequence set forth in SEQ ID NO:2, while the other fusion polypeptide has the complete amino acid sequence set forth in SEQ ID NO:1. The two fusion polypeptides in protein fragment B are non-covalently linked by a disulfide bond, as shown in Figure 2.

[0010] Protein fragment C is a dimer formed by a truncated fusion polypeptide and a second intact fusion polypeptide in fusion protein A, with a molecular weight of 122.6 kDa. Specifically, one truncated fusion polypeptide in protein fragment C has the amino acid sequence set forth in SEQ ID NO:1 from positions 142 to 526, more specifically, the amino acid sequence set forth in SEQ ID NO:3, while the other fusion polypeptide has the complete amino acid sequence set forth in SEQ ID NO:1. The two fusion polypeptides in protein fragment C are non-covalently linked by a disulfide bond, as shown in Figure 3.

[0011] Protein fragment D is a fusion polypeptide (monomer) in fusion protein A, has a molecular weight of 71 kDa, and has the amino acid sequence as shown in SEQ ID NO: 1.

[0012] The inventors of the present invention unexpectedly discovered that in a pharmaceutical composition comprising fusion protein A, the content of protein fragment B has a significant impact on stability (e.g., the polymerization rate of fusion protein A) and activity, while protein fragments C and D have less impact on stability and activity. By controlling the content of low-molecular-weight protein fragment B in the pharmaceutical composition, the stability of fusion protein A in the composition can be better maintained, as well as its biological activity.

[0013] Therefore, one aspect of the present invention provides a pharmaceutical composition comprising an anti-VEGF fusion protein A and 0.01-7% of protein fragment B. In certain preferred embodiments, the pharmaceutical composition comprises an anti-VEGF fusion protein A and 0.01-5.4% of protein fragment B. In certain preferred embodiments, the pharmaceutical composition comprises an anti-VEGF fusion protein A and 0.01-4.6% of protein fragment B.

[0014] In certain embodiments, the content of protein fragment B of the present invention is 0.1-7%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.1-5.4%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.1-4.9%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.1-4.4%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.1-4.6%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.1-3.7%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.1-3.1%.

[0015] In certain embodiments, the content of protein fragment B of the present invention is 0.6-7%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6-5.4%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6-4.9%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6-4.6%. In certain preferred embodiments, the content of protein fragment B of the present invention is 1.3-5.4%. In certain preferred embodiments, the content of protein fragment B of the present invention is 1.3-4.6%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6-4.1%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6-3.7%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6-3.1%.

[0016] In certain embodiments, the content of protein fragment B of the present invention is 0.01-7%, the content of protein fragment C is 0-12.0%, and the content of protein fragment D is 0-6.1%. In certain embodiments, the content of protein fragment B of the present invention is 0.1-7%, the content of protein fragment C is 0-12.0%, and the content of protein fragment D is 0-6.1%. In certain embodiments, the content of protein fragment B of the present invention is 0.6-7%, the content of protein fragment C is 0-12.0%, and the content of protein fragment D is 0-6.1%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6-5.4%, the content of protein fragment C is 0-12.0%, and the content of protein fragment D is 0-6.1%.

[0017] In certain embodiments, the purity of the anti-VEGF fusion protein A of the present invention is greater than 77%. In certain preferred embodiments, the purity of the anti-VEGF fusion protein A of the present invention is greater than 78%, preferably greater than 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%.

[0018] In certain embodiments, the purity of the anti-VEGF fusion protein A of the present invention is 77-98%. In certain preferred embodiments, the purity of the anti-VEGF fusion protein A of the present invention is 77-96%. In certain preferred embodiments, the purity of the anti-VEGF fusion protein A of the present invention is 77-87%. In certain embodiments, the purity of the anti-VEGF fusion protein A of the present invention is 80-98%. In certain embodiments, the purity of the anti-VEGF fusion protein A of the present invention is 80-96%. In certain preferred embodiments, the purity of the anti-VEGF fusion protein A of the present invention is 80-87%.

[0019] In certain embodiments, the content of the fusion protein A and protein fragments described in the present invention is determined by gel electrophoresis (SDS-PAGE). In some specific embodiments, the SDS-PAGE described in the present invention is in accordance with the general rule "0541 Electrophoresis Method Fifth Method SDS-polyacrylamide gel electrophoresis method" of the 2020 edition (Part IV) of the Chinese Pharmacopoeia. Gel electrophoresis is used to warm the protein containing the loading buffer at 70±2°C for 10 minutes, and 4 μg of the sample is electrophoresed in a 4-15% precast polyacrylamide gel for imaging. Trichloride fluorescent dye is added to the precast gel, which combines with the tryptophan in the protein under ultraviolet light at about 300 nm to emit fluorescence, so that the protein in the gel is visualized, and the protein purity or impurity content is calculated based on the response signal values ​​of the main component protein and the miscellaneous protein.

[0020] The fusion protein in the pharmaceutical composition of the present invention is prepared by constructing an expression vector and expressing it in cells (e.g., CHO cells) using conventional biological methods in the art (e.g., methods described in CN200510073595.4). Fusion protein A and protein fragments B, C, and D can be purified using conventional purification methods in the art, such as those described in "Protein Purification and Analysis Techniques" (edited by Lu Jian, Beijing Industrial Press), published in 2005.

[0021] For example, in certain embodiments, during the purification of fusion protein A, protein fragments B, C, and D can be removed or their content controlled by gel chromatography. During gel chromatography, when a mixed protein sample containing different molecular weights is added to a chromatographic column packed with gel particles, these substances migrate with the flow of the eluent. Within the column, there are two modes of movement: vertical downward movement due to gravity and irregular diffusion. Larger proteins, due to their larger diameter, cannot enter the micropores within the gel particles and can only flow through the spaces between the particles. Therefore, they migrate downward faster during elution and are the first to be washed out of the gel column. Smaller proteins, in addition to diffusing in the spaces between the gel particles, can also penetrate into the pores of the gel. During downward migration, they continuously move back and forth between the gel and the interparticle spaces, resulting in a longer travel distance and ultimately being eluted out of the column. Medium-molecular-weight proteins elute at a time between large and small molecules; larger molecules elute earlier, ultimately separating proteins of different molecular sizes from one another in the sample. The molecular weight of the fusion protein A of the present invention is about 142 kDa, while the molecular weights of the low molecular weight fragments B, C, and D are between 71 and 131 kDa, wherein the molecular weight of fragment B is about 131.2 kDa, the molecular weight of fragment C is about 122.6 kDa, and the molecular weight of fragment D is about 71 kDa. Therefore, according to the different molecular weights of the low molecular weight fragments and the fusion protein A, gel chromatography can be used and process parameters (such as filler, column length, sample load, flow rate, etc.) can be adjusted according to the conventional methods in the art to separate the target protein from the low molecular weight fragments, thereby controlling the content of the different molecular weight fragments. The separation principle and process of gel chromatography are well known to those skilled in the art and can be routinely adjusted according to the actual separation effect (e.g., see Lu Jian, ed., Protein Purification and Analysis Technology, Beijing: Beijing Industrial Press, 2005, pp. 40-52). For example, in a specific exemplary embodiment, a chromatography column is packed with a gel filler suitable for separating proteins between 10 and 400 kDa. The column is equilibrated with a chromatography equilibration solution, and the sample volume is controlled to be ≤10% CV and the sample flow rate is controlled to be ≤20 cm / h. The sample is then loaded, equilibrated, and the target protein is collected. According to the above method, sample proteins with different fragment contents can be obtained using different peak collection parameters.

[0022] For example, in certain embodiments, the content of protein A and protein fragments B, C, and D can be controlled by cation exchange chromatography during the purification process of fusion protein A. Ion exchange chromatography is a purification method commonly used in protein purification technology. Its principle is that the charge carried by the separated substance can be combined with the opposite charge carried by the filler. The binding effect between this charged molecule and the filler phase is reversible. When the pH is changed or the buffer solution with gradually increasing ionic strength is used for elution, the substance bound to the filler can be exchanged with the ions in the eluent and eluted into the solution. Since the charges of aggregates (HMWs), monomers, low molecular weight fragments (LMWs), host cell proteins (HCPs), etc. are different, their binding abilities to the fillers are also different, so the order in which they are eluted into the solution is also different, thereby being separated. The present invention can control the content of low molecular weight fragments and other impurities in fusion protein A based on the different charge properties of low molecular weight fragments and other impurities from protein A. The separation principles and procedures of cation exchange chromatography are well known to those skilled in the art and can be routinely adjusted based on the actual separation effect (e.g., see Lu Jian, ed., Protein Purification and Analysis Technology, Beijing: Beijing Industrial Press, 2005, pp. 64-119). For example, in a specific exemplary embodiment, a chromatography column is packed with a strong cation exchange chromatography filler, and the sample is adjusted to a weakly acidic state with a conductivity of <30 mS / cm before loading, equilibration, intermediate washing, and elution of the target protein. According to the above method, sample proteins with different fragment contents can be obtained according to different peak collection parameters.

[0023] For example, in certain embodiments, during the purification of fusion protein A, mixed-mode chromatography can be used to control the content of protein A and protein fragments B, C, and D. Mixed-mode chromatography optimizes the structure of the functional ligand and combines two or more interaction modes. Generally speaking, there are two main types of mixed-mode chromatography used for protein biopharmaceuticals: cationic + hydrophobic mode and anionic + hydrophobic mode. The ligand binds to the target molecule through various interactions, such as ionic, hydrophobic, and hydrogen bonding. The flow-through mode removes various impurities, including aggregates, low molecular weight fragments, protein A, HCPs, DNA, and viruses. The separation principles and processes of mixed-mode chromatography are well known to those skilled in the art and can be routinely adjusted based on the actual separation effect. For example, in a specific exemplary embodiment, a chromatography column is packed with anionic and hydrophobic mixed-mode chromatography filler. The sample is adjusted to a weakly alkaline state and a conductivity between 40 and 60 mS / cm. The flow-through is collected to obtain the target protein. According to the above method, sample proteins with different fragment contents can be obtained by varying peak collection parameters.

[0024] In certain embodiments, the concentration of the anti-VEGF fusion protein A of the present invention is 1 mg / mL to 200 mg / mL. In certain preferred embodiments, the concentration of the anti-VEGF fusion protein A of the present invention is 10 mg / mL to 150 mg / mL, 10 mg / mL to 140 mg / mL, 10 mg / mL to 130 mg / mL, or 10 mg / mL to 120 mg / mL. In some specific embodiments, the concentration of the anti-VEGF fusion protein A of the present invention is 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, or 120 mg / mL.

[0025] In certain embodiments, the pharmaceutical composition of the present invention further comprises: a buffer; an osmotic pressure regulator, and / or a surfactant; an amino acid; and a pH of 6.8 to 8.7.

[0026] Suitable buffers for use with the present invention include, but are not limited to, organic acid salts, such as Tris-HCl, citric acid, phosphate, histidine, succinate or acetate buffers, and the like.

[0027] Suitable osmotic pressure regulators for use with the present invention include, but are not limited to, one or more of sugar, glycerol, and propylene glycol. As sugar, it may include, but is not limited to, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, or sorbose, etc.; disaccharides such as lactose, sucrose, trehalose, or cellobiose, etc.; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, or starch, etc.; and sugar alcohols such as mannitol, xylitol, maltitol, lactitol, xylitol, or sorbitol (glucitol), etc. As preferably, the osmotic pressure regulator is selected from one or more of sucrose, trehalose, mannitol, and sorbitol. More preferably, the osmotic pressure regulator is selected from sucrose or trehalose. In some embodiments, the osmotic pressure regulator of the present invention may also act as a stabilizer, such as sugar.

[0028] Suitable surfactants for use with the present invention include, but are not limited to, nonionic surfactants, ionic surfactants, and zwitterionic surfactants. Typical surfactants for use in the present invention include, but are not limited to, sorbitol fatty acid esters, sorbitol trioleate, glycerol fatty acid esters, polyglycerol fatty acid esters, polyoxyethylene sorbitol fatty acid esters, polyoxyethylene glycerol fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene hydrogenated castor oil (e.g., polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil), polyoxyethylene beeswax derivatives, polyoxyethylene lanolin derivatives, and polyoxyethylene fatty acid amides; C10-C18 alkyl sulfates (e.g., sodium hexadecyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate), polyoxyethylene sodium lauryl sulfate, sodium lauryl sulfosuccinate, propylene glycol, dimethyl sulfoxide, etc.; and natural surfactants, such as lecithin, glycerophospholipids, sphingolipid phosphates, etc. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters such as polysorbate 20, 40, 60, 80, or poloxamer 188. More preferred surfactants are polysorbate 20 or 80.

[0029] The pharmaceutical composition of the present invention, based on the stability of the fusion protein of the present invention, preferably has a pH of 6.8 to 8.7. This pH range can be achieved by a buffer or a pH adjuster to control the pH. In certain embodiments, the pH of the pharmaceutical composition of the present invention is between 7.0 and 8.7. In certain embodiments, the pH of the pharmaceutical composition of the present invention is between 7.5 and 8.7. In certain embodiments, the pH of the pharmaceutical composition of the present invention is between 7.7 and 8.7. In one embodiment, the pH of the pharmaceutical composition of the present invention is about 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6 or 8.7. In a preferred embodiment, the pH of the pharmaceutical composition is about 7.7±0.2.

[0030] Suitable free amino acids for use in the present invention include, but are not limited to, arginine, lysine, histidine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid or aspartic acid. Preferably, basic amino acids are included, i.e., arginine, lysine and / or histidine. If the composition includes histidine, the composition can serve as both a buffer and a free amino acid, but when a histidine buffer is used, non-histidine free amino acids are typically included, such as a histidine buffer and lysine / arginine. The amino acid can exist in the form of any suitable salt, such as a hydrochloride, such as arginine-HCl. Other excipients that may be included in pharmaceutical compositions of the present invention include, for example, antioxidants, antimicrobial agents, etc.

[0031] Another aspect of the present invention provides a pharmaceutical composition comprising:

[0032] 1mg / mL to 200mg / mL fusion protein A;

[0033] 5-300 mM buffer;

[0034] 10-500 mM amino acids;

[0035] 0-30% osmotic pressure regulator; and,

[0036] 0-0.1% surfactant;

[0037] pH 6.8-8.7;

[0038] The buffer is selected from one or more of Tris-HCl, citric acid, phosphate, histidine, glutamic acid, succinate, tromethamine and acetate buffer;

[0039] The osmotic pressure regulator is selected from one or more of sucrose, trehalose, mannitol, glycerol, propylene glycol and sorbitol;

[0040] The surfactant is selected from one or more of polyethylene glycol, Tween 20, Tween 80, P188, propylene glycol and dimethyl sulfoxide;

[0041] The amino acid is selected from one or more of lysine, arginine, histidine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid and aspartic acid.

[0042] In certain more specific embodiments, the pharmaceutical composition comprises:

[0043] 1mg / mL to 200mg / mL fusion protein A;

[0044] 5-300 mM buffer;

[0045] 10-300 mM amino acids;

[0046] 0-30% osmotic pressure regulator; and,

[0047] 0-0.1% surfactant;

[0048] pH 6.8-8.7;

[0049] The buffer is selected from one or more of citric acid, phosphate, histidine, glutamic acid and tromethamine;

[0050] The osmotic pressure regulator is selected from one or more of sucrose, trehalose, mannitol and sorbitol;

[0051] The surfactant is selected from one or more of Tween 20, Tween 80 and P188;

[0052] The amino acid is selected from one or more of glutamic acid, arginine and histidine.

[0053] In certain more specific embodiments, the pharmaceutical composition comprises:

[0054] 10mg / mL to 150mg / mL fusion protein A;

[0055] 5-300 mM buffer;

[0056] 100-300 mM amino acids;

[0057] 0-20% osmotic pressure regulator; and,

[0058] 0-0.1% surfactant;

[0059] pH 6.8-8.7;

[0060] The buffer is selected from one or more of citric acid, phosphate, histidine, glutamic acid and tromethamine;

[0061] The osmotic pressure regulator is selected from one or more of sucrose, trehalose, mannitol and sorbitol;

[0062] The surfactant is selected from one or more of Tween 20, Tween 80 and P188;

[0063] The amino acid is selected from one or more of glutamic acid, arginine and histidine.

[0064] In certain more specific embodiments, the pharmaceutical composition comprises:

[0065] 10-150 mg / mL fusion protein A;

[0066] 10-250 mM citrate buffer;

[0067] 100-250 mM arginine or histidine;

[0068] 5-20% sucrose or trehalose; and,

[0069] 0-0.1% Tween 20, Tween 80, or P188;

[0070] The pH is 7.5-8.7.

[0071] In certain more specific embodiments, the pharmaceutical composition comprises:

[0072] 10-120 mg / mL fusion protein A;

[0073] 10 mM citrate buffer;

[0074] 100 mM arginine;

[0075] 5% sucrose; and

[0076] 0.05% Tween 20;

[0077] The pH is 7.5-8.7.

[0078] In some specific embodiments, the pharmaceutical composition contains:

[0079] 10mg / mL to 120mg / mL fusion protein A;

[0080] 10 mM citrate buffer;

[0081] 100 mM arginine; and,

[0082] 5% trehalose;

[0083] The pH is 7.5 to 8.7, and hydrochloric acid is preferably used to adjust the system pH to 7.7±0.2.

[0084] In some specific embodiments, the pharmaceutical composition contains:

[0085] 10-120 mg / mL fusion protein A;

[0086] 100 mM citrate buffer;

[0087] 250 mM arginine;

[0088] 20% sucrose; and

[0089] 0.1% Tween 20;

[0090] The pH is 7.5-8.7.

[0091] In some specific embodiments, the pharmaceutical composition contains:

[0092] 10-120 mg / mL fusion protein A;

[0093] 250 mM citrate buffer;

[0094] 100 mM histidine;

[0095] 8% sucrose; and

[0096] 0.1% Tween 20;

[0097] The pH is 7.5-8.7.

[0098] In some specific embodiments, the pharmaceutical composition contains:

[0099] 10-120 mg / mL fusion protein A;

[0100] 10 mM citrate buffer;

[0101] 250 mM arginine;

[0102] 20% sucrose; and

[0103] 0.1% Tween 20;

[0104] The pH is 7.5-8.7.

[0105] In some specific embodiments, the pharmaceutical composition contains:

[0106] 10mg / mL to 120mg / mL fusion protein A;

[0107] 10 mM tromethamine;

[0108] 100 mM arginine; and,

[0109] 5% sucrose;

[0110] The pH is 7.5-8.7.

[0111] In some specific embodiments, the pharmaceutical composition contains:

[0112] 10mg / mL to 120mg / mL fusion protein A;

[0113] 290 mM glutamate;

[0114] 290 mM arginine; and,

[0115] 78 mM NaOH;

[0116] The pH is 7.5-8.7.

[0117] In some specific embodiments, the pharmaceutical composition contains:

[0118] 10mg / mL to 120mg / mL fusion protein A;

[0119] 5 mM phosphate; preferably 5 mM sodium dihydrogen phosphate;

[0120] 100 mM arginine;

[0121] 10% trehalose; and

[0122] 0.01% P188;

[0123] The pH is 7.5-8.7.

[0124] Another object of the present invention is to provide a kind of container or the delivery device that comprises the pharmaceutical composition described in purpose of the present invention one.In some specific embodiments, the example of described container includes but is not limited to bottle, syringe, ampoule, bottle, cartridge and pouch.Described syringe can be used by standard syringe and needle, automatic syringe device and microinfusion device.In some preferred embodiments, delivery device of the present invention is pre-filled injection device, and enough pharmaceutical composition prepared according to the disclosure is included in pre-filled container, and it helps to distribute protein preparation for parenteral administration (injection or infusion).In some embodiments, container or pre-filled container comprise at least one pharmaceutical unit dosage form, and it can be particularly suitable for self-administration. For example, a unit dose per vial, cartridge, or prefilled container (e.g., a prefilled syringe or disposable pen) can contain about 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, 5.0 mL, 5.5 mL, 6.0 mL, 6.5 mL, 7.0 mL, 7.5 mL, 8.0 mL, 8.5 mL, 9.0 mL, 9.5 mL or about 10.0 mL or more of the pharmaceutical composition of the present invention.

[0125] Another object of the present invention is to provide a lyophilized formulation prepared by lyophilizing the pharmaceutical composition described in the first object of the present invention. Lyophilization methods are well known to those skilled in the art and include, for example, sublimation of water from a frozen formulation under controlled conditions. The lyophilizable formulation can be reconstituted into a solution, suspension, emulsion, or any other form suitable for administration or use. Reconstitution can typically be achieved by dissolving the lyophilized formulation in an aqueous solution.

[0126] Another object of the present invention is to provide the use of the pharmaceutical composition described in the first object of the present invention in the preparation of a medicament for treating an ocular disease. Alternatively, the pharmaceutical composition described in the first object of the present invention is provided for use in treating an ocular disease. The method of "treating" comprises administering the pharmaceutical composition of the present invention 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 develop such a disorder) to prevent, cure, delay the onset of the disorder or recurring disorder, reduce the severity of the disorder or recurring disorder, or ameliorate one or more symptoms of the disorder or recurring disorder.

[0127] In some specific embodiments, the eye disease is an eye neovascular disease. In some more specific embodiments, the eye neovascular disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization or corneal neovascularization eye disease. In some preferred embodiments, the disease is selected from age-related macular degeneration, macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, macular edema caused by central retinal vein occlusion, macular edema caused by branch retinal vein occlusion, diabetic macular edema, diabetic retinopathy, polypoidal choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia or retinopathy of prematurity. In other preferred embodiments, the disease is selected from age-related macular degeneration, diabetic macular edema or diabetic retinopathy.

[0128] The therapeutic dosage can be readily determined by a physician with ordinary skill in treating the disease or condition using known dosage adjustment techniques. For example, the therapeutically effective amount of the anti-VEGF fusion protein used in the pharmaceutical compositions of the present invention is determined by considering the desired dose volume and mode of administration. Typically, therapeutically effective compositions are administered at a dose of from 10 mg / mL to about 200 mg / mL per dose. Preferably, the dosage used in the methods of the present invention is from about 100 mg / mL to about 120 mg / mL (i.e., about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 mg / mL). In some preferred embodiments, the dosage of the anti-VEGF fusion protein used in the methods of the present invention is 10 mg / mL. In other preferred embodiments, the dosage of the anti-VEGF fusion protein used in the methods of the present invention is 120 mg / mL. The pharmaceutical composition can be delivered to the subject by, for example, intravitreal injection, subretinal injection, choroidal injection (e.g., suprachoroidal injection) or topical application (e.g., eye drops) or by injection into the eye tissue to affect the eye of a mammal. In certain embodiments, the dosage of each eye is at least about 0.5 mg to as high as about 10 mg. The preferred dosage of each eye includes about 0.5 mg, 0.6 mg, 0.7 mg, 0.8 mg, 0.9 mg, 1.0 mg, 1.2 mg, 1.4 mg, 1.6 mg, 1.8 mg, 2.0 mg, 2.5 mg, 3.0 mg, 3.5 mg, 4.0 mg, 4.5 mg, 5.0 mg, 5.5 mg, 6.0 mg, 6.5 mg, 7 mg, 7.5 mg, 8 mg, 8.5 mg, 9 mg, 9.5 mg, or 10 mg. The dosage can be delivered in various volumes suitable for eye administration.

[0129] The beneficial effects of the present invention are:

[0130] Provided is a pharmaceutical composition comprising an anti-VEGF fusion protein A and a protein fragment B. By controlling the content of the protein fragment B in the pharmaceutical composition, the aggregation of the protein A in the pharmaceutical composition can be reduced, thereby improving the stability; and the pharmaceutical composition can maintain good biological activity, and the biological activity can still be well maintained even after undergoing high-temperature treatment.

[0131] The polymer of fusion protein A described in the present invention mainly refers to a polymer formed by intermolecular forces such as covalent bonds or hydrogen bonds between protein molecules, and is a complex composed of two or more proteins, such as a dimer, tetramer, or hexamer. BRIEF DESCRIPTION OF THE DRAWINGS

[0132] Figure 1. Fusion protein A SEQ ID NO: 1 sequence.

[0133] Figure 2 Schematic diagram of the structure of protein fragment B.

[0134] Figure 3 Schematic diagram of the structure of protein fragment C. DETAILED DESCRIPTION

[0135] The SEC-HPLC described in the present invention is in accordance with the general rule "0514 Molecular Exclusion Chromatography" of the 2020 edition (Part IV) of the Chinese Pharmacopoeia. A hydrophilic silica size exclusion chromatography column TSK G3000 SWXL is used, with a sample load of 50 to 200 μg, a mobile phase of 20 mM disodium hydrogen phosphate, 150 mM sodium chloride, 200 mM arginine, pH 7.2, a flow rate of 0.5 ml / min, a detection wavelength of 280 nm, and the polymer content is calculated by the area normalization method. Unless otherwise specified, the polymer content (%) in the examples of the present invention is detected by the SEC-HPLC method.

[0136] The SEC-UPLC described in the present invention is in accordance with the general rule "0514 Molecular Exclusion Chromatography" of the 2020 edition (Volume IV) of the "Chinese Pharmacopoeia", using an ultra-high liquid phase and a size exclusion chromatography column based on ethylene bridge hybrid (BEH) particle technology (ACQUITY UPLC Protein BEH SEC Column, 200A, 1.7 μm, 4.6 mm*150 mm), the sample amount is controlled to be 4-12 μg, the mobile phase is 20 mM disodium hydrogen phosphate, 150 mM sodium chloride, 200 mM arginine, pH 7.2, the flow rate is 0.3 ml / min, the detection wavelength is 280 nm, and the polymer (%) is calculated by the area normalization method.

[0137] The SDS-PAGE stain-free method described in the present invention is in accordance with the general rule "0541 Electrophoresis Method, Method 5 SDS-polyacrylamide gel electrophoresis" of the 2020 edition (Part IV) of the Chinese Pharmacopoeia. Gel electrophoresis is used to warm the protein containing the loading buffer to 70±2°C for 10 minutes, and 4 μg is loaded for electrophoresis imaging in a 4-15% precast polyacrylamide gel. Trichloride fluorescent dye is added to the precast gel, which combines with the tryptophan in the protein under ultraviolet light of about 300 nm to emit fluorescence, so that the protein in the gel is visualized, and the protein purity or impurity content is calculated according to the response signal values ​​of the main component protein and the miscellaneous protein. The non-reducing purity (%) of the protein in the embodiment of the present invention is detected by the SDS-PAGE method.

[0138] The biological activity luciferase reporter gene method described in the present invention is detected in accordance with the general rule "3535 Conbercept Biological Activity Assay" of the 2020 edition (Part IV) of the "Chinese Pharmacopoeia". This method uses human embryonic kidney cells (HEK293) stably transfected with the vascular endothelial growth factor receptor 2 (VEGFR2) gene and the luciferase reporter gene luc2P. The expression of luciferase in the cells is stimulated by blocking the vascular endothelial growth factor (VEGF) at different concentrations of protein to determine the biological activity of the protein. In the experiment, the protein standard / test sample was gradiently diluted to 30,000 ng / mL and then diluted down to 1.21 ng / mL, for a total of 11 concentration gradients. The 11 gradient standards / samples were mixed with equal volumes of rhVEGF165 working solution, incubated at 37°C ± 1°C and 5% carbon dioxide for 20 to 40 minutes, and 2 replicates were made for each gradient. HEK293 cells were taken and prepared into 5×10 5 Cells were plated in a 96-well cell culture plate with a cell suspension of 100 μl / mL. A standard / test sample mixture of varying concentrations was added at 20 μl per well and incubated at 37°C ± 1°C, 5% CO2 for 5.8–6 hours. After equilibration at room temperature for 10–15 minutes, 100 μl of chromogenic substrate was added to each well. After 3–5 minutes at room temperature, the cells were immediately placed in a microplate reader and the fluorescence response of each well was measured using a chemiluminescence module. The rhVEGF165 working solution was added to the wells as a positive control, and DMEM test medium was added to the wells as a negative control. The assay was performed using the same method and the results were recorded. Four-parameter curves were plotted using a computer program or four-parameter regression method, with the test sample and standard concentrations as the horizontal axis and the average fluorescence response as the vertical axis. The median effective concentration (EC50) of the test sample and standard was calculated. The relative biological potency of the test sample was calculated using the following formula: Relative biological potency (%) of the test sample = EC50 of the standard sample ÷ EC50 of the test sample × 100%. The biological activity (%) of the proteins in the examples of the present invention was evaluated using this method. The biological activity of the formulation samples in the examples of the present invention after preparation was tested to be higher than 85%.

[0139] The data tested in all the examples of the present invention are the average values ​​of 6 parallel samples (n=6).

[0140] Example 1

[0141] The composition of the preparation sample is as follows:

[0142] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0143] Proteins of different purities were taken (see Table 1, the protein includes fusion protein A, protein fragments B, C, and D, and the content of specific components can be found in the "Protein non-reducing purity (%)" in the table, the same below), and prepared into sample 1 and sample 2 according to the above-mentioned formulation composition. The samples were placed at 25°C for 2 weeks. After 4 weeks, the polymer content in the samples was determined by SEC-HPLC, and the results are shown in Table 1. From the results in Table 1, it can be seen that when the content of protein fragment B is basically the same, when the content of other components (fusion protein A or protein fragments C, D, etc.) is significantly different, the polymer content of the two groups of samples remains basically the same after 2 weeks and 4 weeks at 25°C. At the same time, the biological activity of the protein was determined after being placed at 35°C for 15 days. The results showed that the biological activity of the protein in both groups of samples was greater than 80%, and the activity was well maintained.

[0144] Table 1

[0145] Example 2

[0146] The composition of the preparation sample is as follows:

[0147] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0148] Proteins of varying purity (see Table 2) were prepared into Samples 3 and 4 according to the above formulation composition. After the samples were stored at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC. The results are shown in Table 2. As can be seen from Table 2, as the content of protein fragment B in the formulation samples increased, the polymer content in the samples increased more significantly after storage, and the polymer polymerization rate was faster. The biological activity of the proteins was also measured after storage at 35°C for 15 days. The biological activity of the protein in Sample 3 was greater than 75%, while that of the protein in Sample 4 was less than 65%.

[0149] Table 2

[0150] Example 3

[0151] The composition of the preparation sample is as follows:

[0152] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0153] Proteins of varying purity (see Table 3) were prepared into Samples 5 and 6 according to the above formulation composition. After the samples were stored at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC, as shown in Table 3. As shown in Table 3, when the protein fragment B content was essentially the same, and when the content of other components (such as protein fragment C) differed significantly, the polymer content of the two formulation samples remained essentially the same after storage at 25°C. The biological activity of the proteins in both sample groups was also determined after storage at 35°C for 15 days, and the results showed that the biological activity of the proteins in both sample groups was greater than 80%.

[0154] Table 3

[0155] Example 4

[0156] The composition of the preparation sample is as follows:

[0157] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0158] Proteins of varying purity (see Table 4) were prepared into Samples 7 and 8 according to the above formulation composition. After the samples were stored at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC, and the results are shown in Table 4. As can be seen from Table 4, when the content of protein fragment B in the composition remained essentially the same, and when the contents of other components (proteins A, C, D, etc.) differed significantly, the polymer content in the two groups of formulation samples after storage was essentially the same. The biological activity of the proteins in the two groups of samples was also determined after storage at 35°C for 15 days. The results showed that the biological activity of the proteins in both groups of samples was greater than 80%.

[0159] Table 4

[0160] Example 5

[0161] The composition of the preparation sample is as follows:

[0162] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0163] Proteins of different purities (see Table 5) were taken and prepared into samples 9-15 according to the above formulation composition. After the samples were placed at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC. The results are shown in Table 5. As can be seen from Table 5, as the content of protein fragment B in the composition increased, the content of polymer in the formulation sample increased significantly after placement, and the polymerization rate was faster. At the same time, the biological activity of the protein in the sample was measured after being placed at 35°C for 15 days. The biological activity of the protein in samples 9-13 was greater than 80%, the biological activity of the protein in sample 14 was greater than 75%, and the biological activity of the protein in sample 15 was less than 65%.

[0164] Table 5

[0165] Example 6

[0166] The composition of the preparation sample is as follows:

[0167] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0168] Proteins of different purities (see Table 6) were prepared into samples 16-23 according to the above formulation composition. The biological activities of the samples were also measured after storage at 35°C for 15 days, as shown in Table 6.

[0169] Table 6

[0170] Example 7

[0171] The sample formulation is as follows:

[0172] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0173] The proteins described in Table 7 were prepared into samples 24 and 25 according to the above formulation composition. After aging the samples at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC, as shown in Table 7. The biological activity of the proteins in the samples was also determined after aging at 35°C for 15 days. The results showed that the biological activity of the proteins in both groups was greater than 80%.

[0174] Table 7

[0175] Example 8

[0176] The sample formulation is as follows:

[0177] Adjust the system pH to 7.7±0.2

[0178] The proteins described in Table 8 were prepared into samples 26 and 27 according to the above formulation composition. After aging the samples at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC, as shown in Table 8. The biological activity of the proteins in the samples was also determined after aging at 35°C for 15 days. The results showed that the biological activity of the proteins in both groups was greater than 80%.

[0179] Table 8

[0180] Example 9

[0181] The sample formulation is as follows:

[0182] Adjust the system pH to 7.7±0.2

[0183] The proteins described in Table 9 were prepared into samples 28 and 29 according to the above formulation composition. After aging the samples at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC, as shown in Table 9. The biological activity of the proteins in the samples was also determined after aging at 35°C for 15 days. The results showed that the biological activity of the proteins in both groups was greater than 80%.

[0184] Table 9

[0185] Example 10

[0186] The sample formulation is as follows:

[0187] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0188] The proteins described in Table 10 were prepared into samples 30 and 31 according to the above formulation composition. After aging the samples at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC. The results are shown in Table 10. The biological activity of the proteins in the samples was also determined after aging at 35°C for 15 days. The results showed that the biological activity of the proteins in both groups was greater than 80%.

[0189] Table 10

[0190] Example 11

[0191] The sample formulation is as follows:

[0192] Adjust the system pH to 7.7±0.2

[0193] The proteins described in Table 11 were prepared into samples 32 and 33 according to the above formulation composition. After aging the samples at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC. The results are shown in Table 11. The biological activity of the proteins in the samples was also determined after aging at 35°C for 15 days. The results showed that the biological activity of the proteins in both groups was greater than 80%.

[0194] Table 11

[0195] Example 12

[0196] The sample formulation is as follows:

[0197] Adjust the system pH to 7.7±0.2

[0198] The proteins described in Table 12 were prepared into samples 34-35 according to the above formulation composition. After aging the samples at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC. The results are shown in Table 12. The biological activity of the proteins in the samples was also determined after aging at 35°C for 15 days. The results showed that the biological activity of the proteins in both groups was greater than 80%.

[0199] Table 12

[0200] Example 13

[0201] The sample formulation is as follows:

[0202] Adjust the system pH to 7.9 ± 0.2 with hydrochloric acid

[0203] The proteins described in Table 13 were prepared into samples 36 and 37 according to the above formulation composition. After aging the samples at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC. The results are shown in Table 13. The biological activity of the proteins in the samples was also determined after aging at 35°C for 15 days. The results showed that the biological activity of the proteins in both groups was greater than 80%.

[0204] Table 13

[0205] Example 14

[0206] The sample formulation is as follows:

[0207] Use hydrochloric acid to adjust the system pH to 7.7-8.7

[0208] Proteins of varying purity as described in Table 14 were prepared into samples 38-40 according to the above formulation composition. After the samples were stored at 25°C for 2 and 4 weeks, the polymer content in the samples was determined by SEC-HPLC. The results are shown in Table 14.

[0209] Table 14

[0210] Example 15

[0211] The sample formulation is as follows:

[0212] Adjust the system pH to 5±0.5 with hydrochloric acid

[0213] Proteins of varying purity as described in Table 15 were prepared into samples 41-44 according to the above formulation composition. The polymer content in the samples was then determined by SEC-UPLC, and the results are shown in Table 15.

[0214] Table 15

[0215] Example 16 Protein Purification

[0216] 1. Gel chromatography

[0217] A chromatography column was packed with a gel filler suitable for separating proteins between 10 and 400 kDa. The column was equilibrated with a chromatography equilibration solution. The sample volume was controlled at ≤10% CV and the flow rate was controlled at ≤20 cm / h. The sample was loaded, equilibrated, and the target protein was collected. Proteins in samples 1-4, 9-23, and 41-44 were obtained using different peak collection parameters.

[0218] 2. Cation exchange chromatography

[0219] The chromatography column was packed with strong cation exchange chromatography filler, and the sample was adjusted to weak acidity and conductivity <30mS / cm before loading, balancing, intermediate washing, and elution of the target protein. The proteins in samples 5-6 and 38-40 were obtained according to different peak collection parameters.

[0220] 3. Mixed-mode chromatography

[0221] The column was packed with anionic and hydrophobic mixed-mode chromatography media. The sample was loaded to a weakly alkaline state and the conductivity was adjusted to between 40 and 60 mS / cm. The flow-through was collected to obtain the target protein. Proteins in samples 7-8 and 24-37 were obtained using different peak collection parameters.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains an anti-VEGF fusion protein A and a protein fragment B. The fusion protein A is a dimer comprising two fusion polypeptides as set forth in SEQ ID NO:1; the protein fragment B is a dimer comprising two fusion polypeptides as set forth in SEQ ID NO:1 and SEQ ID NO:2, and the content of the protein fragment B in the pharmaceutical composition is 0.01-7%, preferably 0.01-5.4%, more preferably 0.01-4.6%.

2. The pharmaceutical composition according to claim 1, wherein In the pharmaceutical composition, the content of the protein fragment B is 0.6-7%, preferably 0.6-5.4%, more preferably 0.6-4.6% or 1.3-5.4%, even more preferably 1.3-4.6%.

3. The pharmaceutical composition according to claim 1, wherein In the pharmaceutical composition, the content of the fusion protein A is 77-98%; preferably 80-98%.

4. The pharmaceutical composition according to claim 3, characterized in that, In the pharmaceutical composition, the content of the fusion protein A is 77-87%; preferably 80-87%.

5. The pharmaceutical composition according to claim 1, wherein In the pharmaceutical composition, the concentration of the fusion protein A is 1 mg / mL - 200 mg / mL, preferably 10 mg / mL - 150 mg / mL, more preferably 10 mg / mL - 120 mg / mL.

6. The pharmaceutical composition according to claim 1, wherein The pH value of the pharmaceutical composition is 6.8-8.7, preferably 7.5-8.7, more preferably 7.7±0.

2.

7. The pharmaceutical composition according to any one of claims 1-6, characterized in that, The pharmaceutical composition further comprises: a buffer; an osmotic pressure regulator; a surfactant; and, an amino acid; The pH is 6.8-8.

7.

8. The pharmaceutical composition according to claim 6, characterized in that, The pharmaceutical composition contains: 1 mg / mL - 200 mg / mL of the fusion protein A; 5-300 mM of the buffer; 10-500 mM of the amino acid; 0-30% of the osmotic pressure regulator; and, 0-0.1% of the surfactant; The pH is 6.8-8.7; The buffer is selected from one or more of Tris-HCl, citric acid, phosphate, histidine, glutamic acid, succinate, tromethamine and acetate buffer; The osmotic pressure regulator is selected from one or more of sucrose, trehalose, mannitol, glycerol, propylene glycol and sorbitol; The surfactant is selected from one or more of polyethylene glycol, Tween 20, Tween 80, P188, propylene glycol and dimethyl sulfoxide; The amino acid is selected from one or more of lysine, arginine, histidine, ornithine, isoleucine, leucine, alanine, glycine, glutamic acid and aspartic acid.

9. The pharmaceutical composition according to claim 7, wherein The pharmaceutical composition contains: 1 mg / mL - 200 mg / mL of the fusion protein A; 5-300 mM of the buffer; 10-300 mM of the amino acid; 0-30% of the osmotic pressure regulator; and, 0-0.1% of the surfactant; The pH is 6.8-8.7; The buffer is selected from one or more of citric acid, phosphate, arginine, glutamic acid and tromethamine; The osmotic pressure regulator is selected from one or more of sucrose, trehalose, mannitol and sorbitol; The surfactant is selected from one or more of Tween 20, Tween 80 and P188; The amino acid is selected from one or more of glutamic acid, arginine and histidine.

10. The pharmaceutical composition according to claim 8, wherein, The pharmaceutical composition contains: 10-150 mg / mL of the fusion protein A; 10 - 250 mM citrate buffer; 100 - 250 mM arginine or histidine; 5 - 20% sucrose or trehalose; and, 0 - 0.1% Tween 20, Tween 80 or P188; pH 7.5 - 8.

7.

11. The pharmaceutical composition according to claim 1, wherein The pharmaceutical composition contains: 10 - 120 mg / mL of fusion protein A; 10 mM citrate buffer; 100 mM arginine; 5% sucrose; and, 0.05% Tween 20; pH 7.5 - 8.7; Alternatively, the pharmaceutical composition contains: 10 - 120 mg / mL of fusion protein A; 100 mM citrate buffer; 250 mM arginine; 20% sucrose; and, 0.1% Tween 20; pH 7.5 - 8.7; Alternatively, the pharmaceutical composition contains: 10 - 120 mg / mL of fusion protein A; 250 mM citrate buffer; 100 mM histidine; 8% sucrose; and, 0.1% Tween 20; pH 7.5 - 8.7; Alternatively, the pharmaceutical composition contains: 10 - 120 mg / mL of fusion protein A; 10 mM citrate buffer; 250 mM arginine; 20% sucrose; and, 0.1% Tween 20; pH 7.5 - 8.7; Alternatively, the pharmaceutical composition contains: 10 mg / mL - 120 mg / mL of fusion protein A; 10 mM tromethamine; 100 mM arginine; and, 5% sucrose; pH 7.5 - 8.7; Alternatively, the pharmaceutical composition contains: 10 mg / mL - 120 mg / mL of fusion protein A; 290 mM glutamic acid; 290 mM arginine; and, 78 mM NaOH; pH 7.5 - 8.7; Alternatively, the pharmaceutical composition contains: 10 mg / mL - 120 mg / mL of fusion protein A; 5 mM phosphate; preferably 5 mM sodium dihydrogen phosphate; 100 mM arginine; 10% trehalose; and, 0.01% P188; pH 7.5 - 8.7; Alternatively, the pharmaceutical composition contains: 10 mg / mL - 120 mg / mL of fusion protein A; 10 mM citrate buffer; 100 mM arginine; and, 5% trehalose; pH 7.5 - 8.

7.

12. The pharmaceutical composition according to claim 1, wherein The content of fusion protein A or protein fragment B is determined by gel electrophoresis (SDS - PAGE).

13. A container or delivery device comprising the pharmaceutical composition according to any one of claims 1 - 12.

14. The container or delivery device according to claim 13, characterized in that, The container is a vial or syringe.

15. The container or delivery device according to claim 13, characterized in that, The delivery device is a pre - filled injection device.

16. A lyophilized preparation prepared by lyophilizing the pharmaceutical composition according to any one of claims 1 - 12.

17. Use of the pharmaceutical composition according to any one of claims 1 - 12 in the preparation of a medicament for treating eye diseases.

18. A method for treating eye diseases, comprising administering to a subject the pharmaceutical composition according to any one of claims 1 - 12.

19. The use according to claim 17 or the method according to claim 18, characterized in that, The ocular disease is an ocular neovascular disease; preferably, the ocular neovascular disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization or corneal neovascularization ocular diseases.

20. The use or method according to claim 19, characterized in that, The ocular neovascular disease is selected from age-related macular degeneration, macular edema, macular edema secondary to retinal vein occlusion, retinal vein occlusion, macular edema caused by central retinal vein occlusion, macular edema caused by branch retinal vein occlusion, diabetic macular edema, diabetic retinopathy, polypoidal choroidal vasculopathy, choroidal neovascularization secondary to degenerative myopia or retinopathy of prematurity; Preferably, the ocular neovascular disease is selected from age-related macular degeneration, diabetic macular edema or diabetic retinopathy.