Pharmaceutical composition containing anti-VEGF fusion protein

Abstract

The invention provides a pharmaceutical composition containing an anti-VEGF (vascular endothelial growth factor) fusion protein. The pharmaceutical composition contains a protein of an anti-VEGF fusion protein A and a protein fragment B, by controlling the content of the protein fragment B in the pharmaceutical composition to be 0.01-7%, the polymerization rate of the protein in the composition can be reduced, the stability of the protein can be improved, good biological activity can be maintained, and the pharmaceutical composition has important significance for developing stable protein pharmaceutical preparations.

Description

[0001] This application claims the priority of Chinese Patent Application No. 2023118409197 with an application date of December 27, 2023. This application incorporates the entire text of the above-mentioned Chinese patent application by reference. Technical Field

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

[0003] The formation of new blood vessels is the key to the development and spread of many diseases. Many eye diseases involve angiogenesis, including age-related macular degeneration (AMD), retinal vein occlusion (RVO), diabetic retinopathy (DR), and pathologic myopia, etc. VEGF is a highly specific vascular endothelial growth factor, which has the effects of promoting increased vascular permeability, extracellular matrix degeneration, vascular endothelial cell migration, proliferation, and blood vessel formation. VEGF is widely distributed in many tissues of humans and animals. Normal retinal pigment epithelial cells, vascular endothelial cells, and pericytes in the eye can produce low levels of VEGF. Many studies have confirmed that if VEGF is overexpressed, it can induce pathologic neovascular eye diseases. Due to the importance of VEGF signal transduction in angiogenesis, blocking VEGF or VEGF receptors to inhibit angiogenesis has an important therapeutic effect on diseases related to angiogenesis, including cancer, retinal vascular diseases, etc. In the past decade or so, some anti-VEGF drugs for treating ocular neovascular eye diseases have emerged, such as antibody or fusion protein drugs like bevacizumab, ranibizumab, aflibercept, and conbercept.

[0004] Salt bonds, hydrogen bonds, disulfide bonds, and hydrophobic interactions are the forces that maintain the conformational stability of proteins. The interaction of metal ions, substrates, cofactors, and other low-molecular-weight ligands stabilizes the protein conformation. Those skilled in the art are well aware that antibodies or proteins are affected by various environmental factors during storage. For example, temperature, humidity, oxygen, ultraviolet light, etc. can cause various physical or chemical changes in the fusion protein, resulting in protein aggregation, decomposition, oxidation, or denaturation, etc. These changes can reduce the activity of the protein, decrease the therapeutic effect, and cause serious toxic and 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 polypeptide comprising extracellular domain 2 of VEGFR-1, 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 is a dimer comprising two fusion polypeptides containing the fusion polypeptide described in SEQ ID NO:1 (the SEQ ID NO:1 sequence is as shown in Figure 1 ). The two fusion polypeptides are non-covalently linked by disulfide bonds and have a molecular weight of 142 kDa. Hereinafter, this fusion protein is referred to as fusion protein A.

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

[0008] The present invention discovers that among the proteins expressed or purified in cells, in addition to fusion protein A, there are usually also low-molecular-weight protein fragments B, C, and D, which may be induced to form under various stress conditions during cell culture, purification, etc., and are usually related to the cleavage of protein covalent bonds caused by spontaneous or enzymatic reactions.

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

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

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

[0012] The inventors of the present invention unexpectedly found that in a pharmaceutical composition containing fusion protein A, the content of protein fragment B has an important influence on stability (such as the polymerization rate of fusion protein A) and activity, while protein fragments C and D have less influence 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, on the one hand, the present invention provides a pharmaceutical composition, which contains anti-VEGF fusion protein A and 0.01 - 7% of protein fragment B. In certain preferred embodiments, the pharmaceutical composition contains anti-VEGF fusion protein A and 0.01 - 5.4% of protein fragment B. In certain preferred embodiments, the pharmaceutical composition contains anti-VEGF fusion protein A and 0.01 - 4.6% of protein fragment B.

[0014] In certain embodiments, the content of protein fragment B in the present invention is 0.1 - 7%. In certain preferred embodiments, the content of protein fragment B in the present invention is 0.1 - 5.4%. In certain preferred embodiments, the content of protein fragment B in the present invention is 0.1 - 4.9%. In certain preferred embodiments, the content of protein fragment B in the present invention is 0.1 - 4.4%. In certain preferred embodiments, the content of protein fragment B in the present invention is 0.1 - 4.6%. In certain preferred embodiments, the content of protein fragment B in the present invention is 0.1 - 3.7%. In certain preferred embodiments, the content of protein fragment B in 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 to 7%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6 to 5.4%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6 to 4.9%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6 to 4.6%. In certain preferred embodiments, the content of protein fragment B of the present invention is 1.3 to 5.4%. In certain preferred embodiments, the content of protein fragment B of the present invention is 1.3 to 4.6%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6 to 4.1%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6 to 3.7%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6 to 3.1%.

[0016] In certain embodiments, the content of protein fragment B of the present invention is 0.01 to 7%, the content of protein fragment C is 0 to 12.0%, and the content of protein fragment D is 0 to 6.1%. In certain embodiments, the content of protein fragment B of the present invention is 0.1 to 7%, the content of protein fragment C is 0 to 12.0%, and the content of protein fragment D is 0 to 6.1%. In certain embodiments, the content of protein fragment B of the present invention is 0.6 to 7%, the content of protein fragment C is 0 to 12.0%, and the content of protein fragment D is 0 to 6.1%. In certain preferred embodiments, the content of protein fragment B of the present invention is 0.6 to 5.4%, the content of protein fragment C is 0 to 12.0%, and the content of protein fragment D is 0 to 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 contents of the fusion protein A and protein fragments of the present invention are determined by gel electrophoresis (SDS-PAGE). In some specific embodiments, the SDS-PAGE of the present invention is carried out according to the general rule "0541 Electrophoresis Method, Fifth Method SDS-Polyacrylamide Gel Electrophoresis Method" in the fourth part of the Chinese Pharmacopoeia 2020 edition. Gel electrophoresis is used. The protein containing the loading buffer is incubated at 70±2°C for 10 min, and 4 μg is loaded and electrophoresed and imaged in a 4-15% precast polyacrylamide gel. Trichloride fluorescent dye is added to this precast gel, and it covalently binds to tryptophan in the protein under ultraviolet light at about 300 nm to emit fluorescence, making the protein in the gel visible. The protein purity or impurity content is calculated according to the response signal values of the main component protein and the impurity protein.

[0020] The preparation of the fusion protein in the pharmaceutical composition of the present invention is obtained by constructing an expression vector and expressing it in cells (such as CHO cells) through conventional biological methods in the art (such as the methods described in CN200510073595.4, etc.). The control of the fusion protein A and protein fragments B, C, and D can be achieved by conventional purification methods in the art, such as the conventional purification methods described in "Protein Purification and Analysis Techniques" published in 2005 (compiled by Lu Jian, Beijing Industry Press).

[0021] For example, in some embodiments, during the purification of fusion protein A, protein fragments B, C, and D can be removed or their content can be controlled by gel chromatography. During gel chromatography, when a mixed protein sample containing proteins of different molecular weights is added to a chromatography column packed with gel particles, these substances move along with the flow of the eluent, and there are two modes of movement in the column: a vertical downward movement caused by gravity and an irregular diffusion. Since macromolecular proteins have a larger diameter and cannot enter the micropores inside the gel particles, they can only flow in the voids between the particles. Therefore, during elution, they move downward faster and are eluted from the gel column first. Small molecular proteins can not only diffuse in the voids between the gel particles but also penetrate into the internal pores of the gel. During the downward movement, they continuously reciprocate between the inside of the gel and the particle gaps, resulting in a longer travel distance and being eluted from the column last. Medium molecular proteins elute at a time between that of large and small molecules. The larger the molecule, the earlier it elutes, ultimately separating proteins of different molecular sizes 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. Among them, 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 the process parameters (such as packing material, column length, sample loading amount, 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 fragments with different molecular weights. The separation principle and process of gel chromatography are well-known to those skilled in the art and can be adjusted conventionally according to the actual separation effect (see, for example, Lu Jian, "Protein Purification and Analysis Techniques", Beijing: Beijing Industry Press, 2005, pp. 40-52). For example, in a specific exemplary embodiment, a chromatography column is packed with a gel packing material suitable for separating proteins with molecular weights of 10-400 kDa, the chromatography column is equilibrated with a chromatography equilibration solution, the sample loading volume is controlled to be ≤10% CV, and the sample loading flow rate is ≤20 cm / h for sample loading, equilibration, and collection of the target protein. According to the above method, sample proteins with different fragment contents can be obtained according to different peak collection parameters.

[0022] For example, in some embodiments, during the purification process of fusion protein A, the contents of protein A and protein fragments B, C, and D can be controlled by cation exchange chromatography. Ion exchange chromatography is a commonly used purification method in protein purification technology. Its principle is that the charge carried by the substance to be separated can bind to the opposite charge carried by the packing material. The binding between such charged molecules and the packing material is reversible. When the pH is changed or eluted with a buffer solution with gradually increasing ionic strength, the substances bound to the packing material can exchange with the ions in the eluent and be eluted into the solution. Since the charges of aggregates (HMWs), monomers, low molecular weight fragments (LMWs), host cell proteins (HCP), etc. are different, their binding abilities to the packing material are also different, so the order of being eluted into the solution is also different, and thus they are separated. According to the present invention, the contents of low molecular weight fragments and other impurities in fusion protein A can be controlled based on the different charge properties of the low molecular weight fragments and other impurities from protein A. The separation principle and process of cation exchange chromatography are well-known to those skilled in the art and can be adjusted routinely according to the actual separation effect (see, for example, Lu Jian, "Protein Purification and Analysis Techniques", Beijing: Beijing Industry Press, 2005, pp. 64-119). For example, in a specific exemplary embodiment, a strong cation exchange chromatography packing material is used to fill the chromatography column. After adjusting the sample to be loaded to weakly acidic and the conductivity < 30 mS / cm, the sample is loaded, equilibrated, washed in the middle, and the target protein is eluted. According to the above method, sample proteins with different fragment contents can be obtained according to different peak collection parameters.

[0023] For example, in some embodiments, during the purification process of fusion protein A, the contents of protein A and protein fragments B, C, and D can be controlled by mixed-mode chromatography. The structure of the functional ligand is optimized in mixed-mode chromatography, combining two or more interaction modes. Generally speaking, there are mainly two types of mixed-mode chromatography used for protein-based biological products, one is the cation + hydrophobic mode, and the other is the anion + hydrophobic mode. The ligand binds to the target molecule through multiple interactions, such as ionic interaction, hydrophobic interaction, and hydrogen bond interaction. Various impurities such as aggregates, low molecular weight fragments, Protein A, HCP, DNA, and viruses are removed by the flow-through mode. The separation principle and process of mixed-mode chromatography are well-known to those skilled in the art and can be adjusted routinely according to the actual separation effect. For example, in a specific exemplary embodiment, a chromatography column is filled with an anion and hydrophobic mixed-mode chromatography packing material. The sample to be loaded is adjusted to weakly alkaline and the conductivity is between 40 and 60 mS / cm, and the flow-through fraction is collected to obtain the target protein. According to the above method, sample proteins with different fragment contents can be obtained according to different peak collection parameters.

[0024] In certain embodiments, the concentration of the anti-VEGF fusion protein A of the present invention is from 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 from 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 the pH is from 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, etc.

[0027] Suitable osmotic pressure regulators for use with the present invention include, but are not limited to, one or more of sugars, glycerol and propylene glycol. As sugars, 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. 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 can 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, sorbitan fatty acid esters, sorbitan trioleate, glycerol fatty acid esters, polyglycerol fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene glycerol fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene hydrogenated castor oils (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 dodecyl sulfate, sodium oleyl sulfate), polyoxyethylene sodium dodecyl sulfate, sodium dodecyl sulfosuccinate, propylene glycol, dimethyl sulfoxide, etc.; and natural surfactants such as lecithin, glycerophospholipid, phosphosphingolipid, etc. Preferred surfactants are polyoxyethylene sorbitan fatty acid esters such as polysorbates 20, 40, 60, 80, or poloxamer 188. More preferred surfactants are polysorbate 20 or 80.

[0029] For the pharmaceutical composition of the present invention, considering the stability of the fusion protein of the present invention, the pH is preferably 6.8 to 8.7. This pH range can be achieved by a buffer or a pH regulator for pH control. 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 contains histidine, the composition can act both as a buffer and as a free amino acid, but when using a histidine buffer, usually non-histidine free amino acids are included, e.g., a histidine buffer and lysine / arginine are included. The amino acids can exist in the form of any suitable salt, such as hydrochloride salts, e.g., arginine-HCl. Other intended excipients that can be used in the pharmaceutical composition of the present invention can also include, for example, antioxidants, antibacterial agents, etc.

[0031] On the other hand, the present invention provides a pharmaceutical composition, which contains:

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

[0033] 5 - 300 mM buffer;

[0034] 10 - 500 mM amino acid;

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

[0036] 0 - 0.1% surfactant;

[0037] pH is 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 some more specific embodiments, the pharmaceutical composition contains:

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

[0044] 5 - 300 mM buffer;

[0045] 10 - 300 mM amino acid;

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

[0047] 0 - 0.1% surfactant;

[0048] pH is 6.8 - 8.7;

[0049] The buffer is selected from one or more of citric acid, phosphate, histidine, glutamic acid and tromethamine; the osmotic pressure regulator is selected from one or more of sucrose, trehalose, mannitol and sorbitol;

[0050] The surfactant is selected from one or more of Tween 20, Tween 80 and P188;

[0051] The amino acid is selected from one or more of glutamic acid, arginine and histidine.

[0052] In some more specific embodiments, the pharmaceutical composition contains:

[0053] 10 mg / mL to 150 mg / mL of fusion protein A;

[0054] 5 - 300 mM buffer;

[0055] 100 - 300 mM amino acid;

[0056] 0 - 20% osmotic pressure regulator; and,

[0057] 0 - 0.1% surfactant;

[0058] pH is 6.8 - 8.7;

[0059] The buffer is selected from one or more of citric acid, phosphate, histidine, glutamic acid and tromethamine; the osmotic pressure regulator is selected from one or more of sucrose, trehalose, mannitol and sorbitol;

[0060] The surfactant is selected from one or more of Tween 20, Tween 80 and P188;

[0061] The amino acid is selected from one or more of glutamic acid, arginine and histidine.

[0062] In some more specific embodiments, the pharmaceutical composition contains:

[0063] 10 - 150 mg / mL of fusion protein A;

[0064] 10 - 250 mM citric acid buffer;

[0065] 100 - 250 mM arginine or histidine;

[0066] 5 - 20% sucrose or trehalose; and,

[0067] 0 - 0.1% Tween 20, Tween 80 or P188;

[0068] pH is 7.5 - 8.7.

[0069] In some more specific embodiments, the pharmaceutical composition contains:

[0070] 10 - 120 mg / mL of fusion protein A;

[0071] 10 mM citrate buffer;

[0072] 100 mM arginine;

[0073] 5% sucrose; and,

[0074] 0.05% Tween 20;

[0075] pH 7.5 - 8.7.

[0076] In some specific embodiments, the pharmaceutical composition contains:

[0077] 10 mg / mL - 120 mg / mL fusion protein A;

[0078] 10 mM citrate buffer;

[0079] 100 mM arginine; and,

[0080] 5% trehalose;

[0081] pH 7.5 - 8.7, preferably adjusting the system pH to 7.7 ± 0.2 with hydrochloric acid.

[0082] In some specific embodiments, the pharmaceutical composition contains:

[0083] 10 - 120 mg / mL fusion protein A;

[0084] 100 mM citrate buffer;

[0085] 250 mM arginine;

[0086] 20% sucrose; and,

[0087] 0.1% Tween 20;

[0088] pH 7.5 - 8.7.

[0089] In some specific embodiments, the pharmaceutical composition contains:

[0090] 10 - 120 mg / mL fusion protein A;

[0091] 250 mM citrate buffer;

[0092] 100 mM histidine;

[0093] 8% sucrose; and,

[0094] 0.1% Tween 20;

[0095] pH 7.5 - 8.7.

[0096] In some specific embodiments, the pharmaceutical composition contains:

[0097] 10 - 120 mg / mL of fusion protein A;

[0098] 10 mM of citric acid buffer;

[0099] 250 mM of arginine;

[0100] 20% of sucrose; and,

[0101] 0.1% of Tween 20;

[0102] The pH is 7.5 - 8.7.

[0103] In some specific embodiments, the pharmaceutical composition contains:

[0104] 10 mg / mL - 120 mg / mL of fusion protein A;

[0105] 10 mM of tromethamine;

[0106] 100 mM of arginine; and,

[0107] 5% of sucrose;

[0108] The pH is 7.5 - 8.7.

[0109] In some specific embodiments, the pharmaceutical composition contains:

[0110] 10 mg / mL - 120 mg / mL of fusion protein A;

[0111] 290 mM of glutamic acid;

[0112] 290 mM of arginine; and,

[0113] 78 mM of NaOH;

[0114] The pH is 7.5 - 8.7.

[0115] In some specific embodiments, the pharmaceutical composition contains:

[0116] 10 mg / mL - 120 mg / mL of fusion protein A;

[0117] 5 mM of phosphate; preferably 5 mM of sodium dihydrogen phosphate;

[0118] 100 mM of arginine;

[0119] 10% of trehalose; and,

[0120] 0.01% of P188;

[0121] The pH is 7.5 to 8.7.

[0122] Another object of the present invention is to provide a container or delivery device comprising the pharmaceutical composition of the first object of the present invention. In some specific embodiments, examples of the container include, but are not limited to, vials, syringes, ampoules, bottles, cartridges, and sachets. The syringe can be administered by a standard syringe and needle, an autoinjector device, and a microinfusion device. In some preferred embodiments, the delivery device of the present invention is a prefilled injection device, and a sufficient amount of the pharmaceutical composition formulated according to the present disclosure is contained in the prefilled container, which helps to dispense the protein preparation for parenteral administration (injection or infusion). In some embodiments, the container or prefilled container contains at least one pharmaceutical unit dosage form, which may be particularly suitable for self-administration. For example, the unit dose per vial, cartridge, or prefilled container (e.g., prefilled syringe or disposable pen) may 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 a larger volume of the pharmaceutical composition of the present invention.

[0123] Another object of the present invention is to provide a lyophilized preparation prepared by lyophilizing the pharmaceutical composition of the first object of the present invention. The lyophilization method is well known to those of ordinary skill in the art and includes, for example, the sublimation of water from the frozen preparation under controlled conditions. The lyophilizable preparation can be redissolved into a solution, suspension, emulsion, or any other suitable form for administration or use. Typically, the lyophilized preparation can be reconstituted by adding an aqueous solution.

[0124] Another object of the present invention is to provide the use of the pharmaceutical composition of the first object of the present invention in the preparation of a medicament for treating eye diseases. Alternatively, it provides the pharmaceutical composition of the first object of the present invention for treating eye diseases. The method of "treatment" involves 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 ultimately develop such a disorder) to prevent, cure, delay the disorder or a recurrent disorder, reduce the severity of the disorder or a recurrent disorder, or improve one or more symptoms of the disorder or a recurrent disorder.

[0125] In some specific embodiments, the ocular disease is an ocular neovascular disease. In some more specific embodiments, the ocular neovascular disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization, or corneal neovascularization ocular diseases. 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 some other preferred embodiments, the disease is selected from age-related macular degeneration, diabetic macular edema, or diabetic retinopathy.

[0126] The dosage for treatment can be readily determined by a physician having 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 composition of the present invention is determined by considering the required dosage volume and the mode of administration. Generally, the therapeutically effective composition is administered at a dose of from 10 mg / mL to about 200 mg / mL per dose. Preferably, the dose used in the method of the present invention is 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 dose of the anti-VEGF fusion protein used in the method of the present invention is 10 mg / mL. In some other preferred embodiments, the dose of the anti-VEGF fusion protein used in the method of the present invention is 120 mg / mL. Delivery of the pharmaceutical composition to the subject can be effected by, for example, intravitreal injection, subretinal injection, choroidal injection (e.g., suprachoroidal injection), or topical administration (e.g., eye drops) or by injection into the affected ocular tissue to affect the eye of a mammal. In certain embodiments, the dose per eye is at least about 0.5 mg to up to about 10 mg. Preferred doses per eye include 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 dose can be administered in various volumes suitable for ocular administration.

[0127] The beneficial effects of the present invention are as follows:

[0128] Provided is a pharmaceutical composition comprising an anti-VEGF fusion protein A and a protein fragment B. By controlling the content of protein fragment B in the pharmaceutical composition, the polymerization of protein A in the pharmaceutical composition can be reduced, and the stability can be improved; and the pharmaceutical composition can maintain good biological activity, and even after undergoing high-temperature treatment, the biological activity can still be maintained well.

[0129] The polymers of the fusion protein A described in the present invention mainly refer to polymers formed by intermolecular forces such as covalent bonds or hydrogen bonds between protein molecules, and are complexes composed of two or more proteins, such as dimers, tetramers, and hexamers. Brief Description of the Drawings

[0130] Figure 1 Fusion protein A SEQ ID NO:1 sequence.

[0131] Figure 2 Schematic diagram of the structure of protein fragment B.

[0132] Figure 3 Schematic diagram of the structure of protein fragment C. Detailed Description of the Invention

[0133] The SEC-HPLC described in the present invention is carried out according to the general rule "0514 Molecular Exclusion Chromatography" in the fourth part of the Chinese Pharmacopoeia 2020 edition. Using a hydrophilic silica gel size exclusion chromatography column TSK G3000 SWXL, the sample loading amount is 50-200 μg, the mobile phase is 20 mM disodium hydrogen phosphate, 150 mM sodium chloride, 200 mM arginine, pH 7.2, the flow rate is 0.5 ml / min, the detection wavelength is 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 this SEC-HPLC method.

[0134] The SEC-UPLC described in the present invention is carried out according to the general rule "0514 Molecular Exclusion Chromatography" in the fourth part of the Chinese Pharmacopoeia 2020 edition. Using an ultra-high performance liquid chromatography 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), controlling the sample loading amount 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.

[0135] The SDS-PAGE stain-free method described in the present invention is carried out according to the "Fifth Method: SDS-Polyacrylamide Gel Electrophoresis" in the General Rules (Part IV) of the Chinese Pharmacopoeia 2020 Edition. Using gel electrophoresis, the protein containing the loading buffer is incubated at 70 ± 2 °C for 10 min, and 4 μg of the sample is electrophoresed and imaged in a 4-15% precast polyacrylamide gel. A trichloride fluorescent dye is added to the precast gel, which covalently binds to tryptophan in the protein under ultraviolet light at about 300 nm to emit fluorescence, making the protein in the gel visible. The protein purity or impurity content is calculated according to the response signal values of the main component protein and the impurity protein. The non-reducing purity (%) of the protein in the examples of the present invention was detected by this SDS-PAGE method.

[0136] The biological activity luciferase reporter gene method described in the present invention is detected according to the "Method for Determining the Biological Activity of Conbercept" in the General Rules (Part IV) of the Chinese Pharmacopoeia 2020 Edition. 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. By blocking the expression of luciferase in cells by vascular endothelial growth factor (VEGF) with different concentrations of protein, the biological activity of the protein is determined. In the experiment, the protein standard / product is serially diluted to 30,000 ng / mL and then further diluted to 1.21 ng / mL, with a total of 11 concentration gradients. The 11 gradient standards / samples are respectively mixed with the rhVEGF165 working solution in equal volume and incubated at 37 °C ± 1 °C and 5% carbon dioxide for 20-40 minutes, with 2 replicates for each gradient. Take HEK293 cells and prepare them into 5×10 5After inoculating the cell suspension at cells / mL into a 96-well cell culture plate, 80 μL was inoculated into each well. Different concentrations of standard product / test sample mixed solutions were added, 20 μL to each well, and cultured at 37°C ± 1°C and 5% carbon dioxide for 5.8 - 6 hours. After equilibrating at room temperature for 10 - 15 minutes, 100 μL of chromogenic substrate was added to each well. After standing at room temperature for 3 - 5 minutes, it was immediately placed in an enzyme-linked immunosorbent assay (ELISA) reader, and the fluorescence response value of each well was measured using the chemiluminescence module. Adding the rhVEGF165 working solution to the cell well served as the positive control, and adding the DMEM test medium to the cell well served as the negative control. The experiment was measured in the same way, and the experimental results were recorded. It was processed using a computer program or the four-parameter regression calculation method. With the concentrations of the test sample and the standard product as the abscissa and the average fluorescence response as the ordinate, a four-parameter curve was plotted, and the half-maximal effective concentration (EC50) of the test sample and the standard product was calculated. The relative biological potency of the test sample was calculated according to the following formula. Relative biological potency of the test sample (%) = EC50 of the standard product ÷ EC50 of the test sample × 100%. The protein biological activity (%) in the examples of the present invention was evaluated by this method. The biological activities of the formulated samples in the examples of the present invention were all higher than 85% after detection.

[0137] All the test data in the examples of the present invention are the averages of 6 parallel samples (n = 6).

[0138] Example 1

[0139] The composition of the formulated sample is as follows:

[0140]

[0141] Adjust the pH of the system to 7.7 ± 0.2 with hydrochloric acid

[0142] Take proteins with different purities (see Table 1, this protein contains fusion protein A, protein fragments B, C, and D, and for the specific component contents, refer to "Protein non-reducing purity (%)" in the table, the same below), and prepare Samples 1 and 2 according to the above formulation composition. The samples were placed at 25°C for 2 weeks and 4 weeks. After that, the polymer content in the samples was determined by size exclusion chromatography-high performance liquid chromatography (SEC-HPLC), and the results are shown in Table 1. It can be seen from the results in Table 1 that when the content of protein fragment B is basically the same, in the case of significant differences in the contents of other components (fusion protein A or protein fragments C, D, etc.), after standing at 25°C for 2 weeks and 4 weeks, the polymer contents of the two groups of samples are basically the same. At the same time, the biological activity of the protein after standing at 35°C for 15 days was measured, and the results showed that the protein biological activities of the two groups of samples were both greater than 80%, and the activity was well maintained.

[0143] Table 1

[0144]

[0145] Example 2

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

[0147]

[0148]

[0149] Adjust the pH of the system to 7.7 ± 0.2 with hydrochloric acid

[0150] Take proteins of different purities (see Table 2), and prepare Samples 3 and 4 according to the above preparation composition. After the samples are placed at 25°C for 2 and 4 weeks, the polymer content in the samples is determined by SEC-HPLC, and the results are shown in Table 2. It can be seen from Table 2 that as the content of protein fragment B in the preparation samples increases, after storage, the polymer content in the samples increases more significantly, and the polymerization rate of the polymer is faster. At the same time, the biological activity of the protein after being placed at 35°C for 15 days is measured. The biological activity of the protein in Sample 3 is greater than 75%, and the biological activity of the protein in Sample 4 is less than 65%.

[0151] Table 2

[0152]

[0153] Example 3

[0154] The composition of the preparation samples is as follows:

[0155]

[0156] Adjust the pH of the system to 7.7 ± 0.2 with hydrochloric acid

[0157] Take proteins of different purities (see Table 3), and prepare Samples 5 and 6 according to the above preparation composition. After the samples are placed at 25°C for 2 and 4 weeks, the polymer content in the samples is determined by SEC-HPLC, and the results are shown in Table 3. It can be known from the results in Table 3 that when the content of protein fragment B is basically the same, in the case of significant differences in the content of other components (such as protein fragment C), the polymer content of the two groups of preparation samples remains basically the same after being placed at 25°C. At the same time, the biological activity of the proteins in the two groups of samples after being placed at 35°C for 15 days is measured, and the results show that the biological activities of the proteins in the two groups of samples are both greater than 80%.

[0158] Table 3

[0159]

[0160] Example 4

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

[0162]

[0163] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0164] Take proteins of different purities (see Table 4) and prepare Samples 7 and 8 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, 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 basically the same and the contents of other components (protein A, C, D, etc.) differed significantly, the polymer contents in the two groups of formulated samples were basically the same after storage. At the same time, the biological activities of the proteins in the two groups of samples were determined after being placed at 35 °C for 15 days, and the results showed that the biological activities of the proteins in both groups of samples were greater than 80%.

[0165] Table 4

[0166]

[0167] Example 5

[0168] The composition of the formulated samples is as follows:

[0169]

[0170] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0171] Take proteins of different purities (see Table 5) and prepare 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 polymer content in the formulated samples increased significantly after storage, and the polymerization rate was faster. At the same time, the biological activities of the proteins in the samples were determined after being placed at 35 °C for 15 days. The biological activities of the proteins in Samples 9 - 13 were all greater than 80%, the biological activity of the protein in Sample 14 was greater than 75%, and that in Sample 15 was less than 65%.

[0172] Table 5

[0173]

[0174] Example 6

[0175] The composition of the formulated samples is as follows:

[0176]

[0177] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0178] Take proteins of different purities (see Table 6) and prepare Samples 16 - 23 according to the above formulation composition. At the same time, determine the biological activity of the proteins in the samples after being placed at 35°C for 15 days. See Table 6 for details.

[0179] Table 6

[0180]

[0181]

[0182] Example 7

[0183] The prescription of the preparation sample is as follows:

[0184]

[0185] Adjust the pH of the system to 7.7 ± 0.2 with hydrochloric acid

[0186] Take the proteins described in Table 7 and prepare Samples 24 - 25 according to the above formulation composition. After the samples are placed at 25°C for 2 and 4 weeks, determine the polymer content in the samples by SEC - HPLC. The results are shown in Table 7. At the same time, determine the biological activity of the proteins in the samples after being placed at 35°C for 15 days. The results show that the biological activities of the proteins in both groups of samples are greater than 80%.

[0187] Table 7

[0188]

[0189] Example 8

[0190] The prescription of the preparation sample is as follows:

[0191]

[0192] Adjust the pH of the system to 7.7 ± 0.2

[0193] Take the proteins described in Table 8 and prepare Samples 26 - 27 according to the above formulation composition. After the samples are placed at 25°C for 2 and 4 weeks, determine the polymer content in the samples by SEC - HPLC. The results are shown in Table 8. At the same time, determine the biological activity of the proteins in the samples after being placed at 35°C for 15 days. The results show that the biological activities of the proteins in both groups of samples are greater than 80%.

[0194] Table 8

[0195]

[0196] Example 9

[0197] The prescription of the preparation sample is as follows:

[0198]

[0199] Adjust the system pH to 7.7 ± 0.2

[0200] Take the protein described in Table 9 and prepare Samples 28 - 29 according to the above formulation composition. After the samples are placed at 25°C for 2 and 4 weeks, the polymer content in the samples is determined by SEC-HPLC, and the results are shown in Table 9. At the same time, the biological activity of the protein in the samples after being placed at 35°C for 15 days is determined, and the results show that the biological activities of the proteins in both groups of samples are greater than 80%.

[0201] Table 9

[0202]

[0203] Example 10

[0204] The formulation sample prescription is as follows:

[0205]

[0206] Adjust the system pH to 7.7 ± 0.2 with hydrochloric acid

[0207] Take the protein described in Table 10 and prepare Samples 30 - 31 according to the above formulation composition. After the samples are placed at 25°C for 2 and 4 weeks, the polymer content in the samples is determined by SEC-HPLC, and the results are shown in Table 10. At the same time, the biological activity of the protein in the samples after being placed at 35°C for 15 days is determined, and the results show that the biological activities of the proteins in both groups of samples are greater than 80%.

[0208] Table 10

[0209]

[0210] Example 11

[0211] The formulation sample prescription is as follows:

[0212]

[0213] Adjust the system pH to 7.7 ± 0.2

[0214] Take the protein described in Table 11 and prepare Samples 32 - 33 according to the above formulation composition. After the samples are placed at 25°C for 2 and 4 weeks, the polymer content in the samples is determined by SEC-HPLC, and the results are shown in Table 11. At the same time, the biological activity of the protein in the samples after being placed at 35°C for 15 days is determined, and the results show that the biological activities of the proteins in both groups of samples are greater than 80%.

[0215] Table 11

[0216]

[0217] Example 12

[0218] The formulation sample prescription is as follows:

[0219]

[0220]

[0221] Adjust the pH of the system to 7.7 ± 0.2

[0222] Take the protein described in Table 12 and prepare Samples 34 - 35 according to the above formulation composition. After the samples are placed at 25°C for 2 and 4 weeks, the polymer content in the samples is determined by SEC - HPLC, and the results are shown in Table 12. At the same time, the biological activity of the protein in the samples after being placed at 35°C for 15 days is determined, and the results show that the biological activities of the proteins in both groups of samples are greater than 80%.

[0223] Table 12

[0224]

[0225] Example 13

[0226] The formulation sample prescription is as follows:

[0227]

[0228] Adjust the pH of the system to 7.9 ± 0.2 with hydrochloric acid

[0229] Take the protein described in Table 13 and prepare Samples 36 - 37 according to the above formulation composition. After the samples are placed at 25°C for 2 and 4 weeks, the polymer content in the samples is determined by SEC - HPLC, and the results are shown in Table 13. At the same time, the biological activity of the protein in the samples after being placed at 35°C for 15 days is determined, and the results show that the biological activities of the proteins in both groups of samples are greater than 80%.

[0230] Table 13

[0231]

[0232] Example 14

[0233] The formulation sample prescription is as follows:

[0234]

[0235]

[0236] Adjust the pH of the system to 7.7 - 8.7 with hydrochloric acid

[0237] Take the proteins with different purities described in Table 14 and prepare samples 38 - 40 according to the above formulation composition. After the samples are placed at 25°C for 2 and 4 weeks, the polymer content in the samples is determined by SEC-HPLC, and the results are shown in Table 14.

[0238] Table 14

[0239]

[0240] Example 15

[0241] The formulation sample prescription is as follows:

[0242]

[0243] Adjust the system pH to 5 ± 0.5 with hydrochloric acid

[0244] Take the proteins with different purities described in Table 15 and prepare samples 41 - 44 according to the above formulation composition. Then the polymer content in the samples is determined by SEC-UPLC, and the results are shown in Table 15.

[0245] Table 15

[0246]

[0247] Protein purification in Example 16

[0248] 1. Gel chromatography

[0249] Pack a chromatography column with a gel filler suitable for separating proteins with a molecular weight of 10 - 400 kDa, equilibrate the chromatography column with a chromatography equilibration solution, control the sample loading volume ≤ 10% CV and the sample loading flow rate ≤ 20 cm / h for sample loading, equilibration, and collect the target protein. Obtain the proteins in samples 1 - 4, 9 - 23, 41 - 44 according to different peak collection parameters.

[0250] 2. Cation exchange chromatography

[0251] Pack a chromatography column with a strong cation exchange chromatography filler, adjust the sample to be loaded to weakly acidic and the conductivity < 30 mS / cm before sample loading, equilibration, intermediate cleaning, and elute the target protein. Obtain the proteins in samples 5 - 6, 38 - 40 according to different peak collection parameters.

[0252] 3. Mixed-mode chromatography

[0253] Pack a chromatography column with an anion and hydrophobic mixed-mode chromatography filler, adjust the sample to be loaded to weakly basic and the conductivity to be between 40 - 60 mS / cm, collect the flow-through fraction, and obtain the target protein. Obtain the proteins in samples 7 - 8, 24 - 37 according to 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, wherein the fusion protein A is a dimer comprising two fusion polypeptides comprising SEQ ID NO: 1; the protein fragment B is a dimer comprising two fusion polypeptides comprising 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%, and more preferably 0.01-4.6%.

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

3. The pharmaceutical composition according to claim 1, characterized in that In the pharmaceutical composition, the content of 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 fusion protein A is 77-87%, preferably 80-87%.

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

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

2.

7. The pharmaceutical composition according to any one of claims 1 to 6, characterized in that The pharmaceutical composition further comprises: Buffer; Osmotic pressure regulators; a surfactant; and, Amino acids; 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 to 200 mg / mL of fusion protein A; 5-300 mM buffer; 10-500 mM amino acids; 0-30% osmotic pressure regulator; and, 0-0.1% surfactant; pH 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, characterized in that The pharmaceutical composition contains: 1 mg / mL to 200 mg / mL of fusion protein A; 5-300 mM buffer; 10-300 mM amino acids; 0-30% osmotic pressure regulator; and, 0-0.1% surfactant; pH 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, characterized in that The pharmaceutical composition contains: 10-150 mg / mL of 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; The pH is 7.5-8.

7.

11. The pharmaceutical composition according to claim 1, characterized in that 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 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 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 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 to 120 mg / mL fusion protein A; 10 mM tromethamine; 100 mM arginine; and, 5% sucrose; pH 7.5-8.7; Alternatively, the pharmaceutical composition contains: 10mg / mL~120mg / mL fusion protein A; 290mM glutamate; 290 mM arginine; and, 78 mM NaOH; pH 7.5-8.7; Alternatively, the pharmaceutical composition contains: 10 mg / mL to 120 mg / mL 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: 10mg / mL to 120mg / mL of fusion protein A; 10 mM citrate buffer; 100 mM arginine; and, 5% trehalose; The pH is 7.5-8.

7.

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

13. A container or delivery device comprising the pharmaceutical composition of 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 a syringe.

15. The container or delivery device of claim 13, wherein: 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 to 12.

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

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

19. The use according to claim 18, 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.

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