WO2025140553A1 - Pharmaceutical composition containing anti-vegf fusion protein - Google Patents

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 anti-VEGF fusion protein

This application claims the priority of Chinese patent application No. 2023118409197 filed on December 27, 2023. This application cites the entire text of the above Chinese patent application.

Technical Field

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

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 pathological myopia. VEGF is a highly specific vascular endothelial growth factor that promotes vascular permeability, extracellular matrix degeneration, endothelial cell migration, proliferation and angiogenesis. VEGF is widely distributed in many tissues in humans and animals. Normal retinal pigment epithelial cells, endothelial cells and pericytes can produce low levels of VEGF. Many studies have confirmed that excessive expression of VEGF can induce pathological neovascular eye diseases. Due to the importance of VEGF signaling to angiogenesis, blocking VEGF or VEGF receptors to inhibit angiogenesis has an important therapeutic effect on diseases related to angiogenesis, including cancer, retinal vascular lesions, etc. In the past decade, some anti-VEGF drugs for the treatment of ocular neovascular eye diseases have been developed, such as bevacizumab, ranibizumab, aflibercept, conbercept and other antibodies or fusion protein drugs.

Salt bonds, hydrogen bonds, disulfide bonds and hydrophobic interactions are forces that maintain protein conformation stability. The interaction between metal ions, substrates, cofactors and other low molecular weight ligands stabilizes protein conformation. It is well known to those skilled in the art that antibodies or proteins are affected by a variety of environmental factors during storage, such as temperature, humidity, oxygen, ultraviolet rays, etc., which can cause a variety of physical or chemical changes in fusion proteins, resulting in protein aggregation, decomposition, oxidation or denaturation. These changes can reduce the activity of the protein, reduce the therapeutic effect and cause serious toxic side effects.

Summary of the invention

On the one hand, the purpose of the present invention is to provide a pharmaceutical composition containing an anti-VEGF fusion protein with good stability and stable biological activity.

In certain embodiments, the anti-VEGF fusion protein of the present invention comprises a dimer of two fusion polypeptides, each polypeptide 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 is a dimer comprising two fusion polypeptides containing SEQ ID NO: 1 (SEQ ID NO: 1 sequence as shown in Figure 1), the two fusion polypeptides are non-covalently linked by disulfide bonds, and the molecular weight is 142kDa. The fusion protein is hereinafter referred to as fusion protein A.

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 number ZL200610066257.2), specifically the active component in 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.

The present invention finds that in addition to the fusion protein A, the protein obtained by cell expression or purification usually contains low molecular weight protein fragments B, C, and D, which 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.

Protein fragment B is a dimer formed by a truncated fusion polypeptide in fusion protein A and another complete fusion polypeptide, with a molecular weight of 131.2 kDa. Specifically, a truncated fusion polypeptide in protein fragment B has an amino acid sequence as described in SEQ ID NO: 1 at positions 82-526, more specifically, an amino acid sequence as described in SEQ ID NO: 2, and the other fusion polypeptide has a 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.

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

Protein fragment D is a fusion polypeptide (monomer) in fusion protein A, with a molecular weight of 71 kDa and an amino acid sequence as described in SEQ ID NO:1.

The inventors of the present invention unexpectedly discovered that in a pharmaceutical composition comprising fusion protein A, the content of protein fragment B has an important influence on stability (e.g., 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.

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

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%.

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%.

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%.

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%.

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%.

In certain embodiments, the content of 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 (fourth volume) of the Chinese Pharmacopoeia, and gel electrophoresis is used to warm the protein containing the loading buffer at 70±2°C for 10 minutes, and load 4 μg of the sample in a 4-15% prefabricated polyacrylamide gel for electrophoresis imaging. The prefabricated gel is added with trichloride fluorescent dye, which is combined with tryptophan in the protein under ultraviolet light at about 300nm 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 value of the main component protein and the impurity protein.

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) by conventional biological methods in the art (such as methods in CN200510073595.4, etc.). The control of fusion protein A, protein fragments B, C, and D can be carried out by conventional purification methods in the art, such as the conventional purification methods described in "Protein Purification and Analysis Technology" published in 2005 (edited by Lu Jian, Beijing Industrial Press).

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 different molecular weights is added to a chromatographic column filled with gel particles, these substances move with the flow of the eluent, and there are two modes of movement in the column: vertical downward movement caused by gravity and irregular diffusion. Due to their large diameter, large molecular proteins cannot enter the micropores in the gel particles and can only flow in the gaps between the particles, so they move down faster during elution and are washed out of the gel column first; small molecular proteins can not only diffuse in the gaps between the gel particles, but also penetrate into the pores in the gel. During the downward movement, they continuously reciprocate between the gel and the gaps between the particles to make their travel longer, and finally are eluted out of the column; the time for medium molecular proteins to flow out is between large and small molecules, and the larger the molecule, the earlier it flows out, and finally separates proteins of different molecular sizes in the sample from each other. 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 the process parameters (such as filler, column length, sample loading, 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 fragments of different molecular weights. 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 (such as referring to Lu Jian, "Protein Purification and Analysis Technology", Beijing: Beijing Industrial Press, 2005, pages 40-52). For example, in a specific exemplary embodiment, a chromatographic column is filled with a gel filler suitable for separating 10-400 kDa proteins, and the chromatographic column is equilibrated with a chromatographic equilibration liquid, and the sample volume is controlled to be ≤10%CV and the sample flow rate is controlled to be ≤20 cm/h for 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.

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 commonly used purification method in protein purification technology. The 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 the charged molecule and the filler phase is reversible. When the pH is changed or the buffer with gradually increasing ionic strength is eluted, the substance bound to the filler can be exchanged with the ions in the eluent and eluted into the solution. Due to the different charges of aggregates (HMWs), monomers, low molecular weight fragments (LMWs), host cell proteins (HCP), etc., their binding abilities with the filler are also different, so the order of being eluted into the solution is also different, so they are separated. The present invention can control the content of low molecular weight fragments and other impurities in fusion protein A according to the different charge properties of low molecular weight fragments and other impurities and protein A. The separation principle and process of cation exchange 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. 64-119). For example, in a specific exemplary embodiment, a chromatographic column is filled with a strong cation exchange chromatography filler, and the sample is adjusted to be weakly acidic and the conductivity is less than 30 mS/cm before loading, balancing, intermediate washing, and eluting the target protein. According to the above method, sample proteins with different fragment contents can be obtained according to different peak collection parameters.

For example, in certain embodiments, in the purification process 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 functional ligands and combines two or more interaction modes. Generally speaking, there are two main types of mixed mode chromatography for protein biological products, one is cationic + hydrophobic mode, and the other is anionic + hydrophobic mode. The ligand binds to the target molecule through various effects, such as ionic, hydrophobic and hydrogen bonding, and removes various impurities such as aggregates, low molecular weight fragments, Protein A, HCP, DNA and viruses through 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 routinely adjusted according to the actual separation effect. For example, in a specific exemplary embodiment, a chromatographic column is filled with anionic and hydrophobic mixed mode chromatography fillers, the sample is adjusted to weak alkalinity and the conductivity to between 40 and 60 mS/cm, and the flow-through liquid 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.

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.

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.

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

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.

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 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 lauryl sulfate, sodium oleyl sulfate), polyoxyethylene sodium lauryl sulfate, sodium lauryl sulfosuccinate, propylene glycol, dimethyl sulfoxide, etc.; and natural surfactants, such as lecithin, glycerophospholipids, phosphate sphingolipids, 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.

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 controlled by a buffer or a pH adjuster. 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.

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 generally 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 expected excipients that can be used for pharmaceutical compositions of the present invention include, for example, antioxidants, antimicrobial agents, etc.

Another aspect of the present invention provides a pharmaceutical composition, which 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.

In certain more specific embodiments, the pharmaceutical composition comprises:

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, histidine, 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.

In certain more specific embodiments, the pharmaceutical composition comprises:

10mg/mL to 150mg/mL of fusion protein A;

5-300 mM buffer;

100-300 mM amino acids;

0-20% 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, histidine, 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.

In certain more specific embodiments, the pharmaceutical composition comprises:

10-150 mg/mL 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.

In certain more specific embodiments, the pharmaceutical composition comprises:

10-120 mg/mL fusion protein A;

10 mM citrate buffer;

100 mM arginine;

5% sucrose; and,

0.05% Tween 20;

The pH is 7.5-8.7.

In some specific embodiments, 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 to 8.7, and the system pH is preferably adjusted to 7.7±0.2 using hydrochloric acid.

In some specific embodiments, 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;

The pH is 7.5-8.7.

In some specific embodiments, 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;

The pH is 7.5-8.7.

In some specific embodiments, 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;

The pH is 7.5-8.7.

In some specific embodiments, the pharmaceutical composition contains:

10mg/mL to 120mg/mL of fusion protein A;

10 mM tromethamine;

100 mM arginine; and,

5% sucrose;

The pH is 7.5-8.7.

In some specific embodiments, the pharmaceutical composition contains:

10mg/mL to 120mg/mL of fusion protein A;

290 mM glutamate;

290 mM arginine; and,

78mM NaOH;

The pH is 7.5-8.7.

In some specific embodiments, the pharmaceutical composition contains:

10mg/mL to 120mg/mL of fusion protein A;

5 mM phosphate; preferably 5 mM sodium dihydrogen phosphate;

100 mM arginine;

10% trehalose; and,

0.01% P188;

The pH is 7.5-8.7.

Another object of the present invention is to provide a kind of container or delivery device comprising the pharmaceutical composition described in the present invention purpose one.In some specific embodiments, the example of the container includes but is not limited to bottle, syringe, ampoule, bottle, cartridge and pouch.The syringe can be used by standard syringe and needle, automatic syringe device and micro-infusion device.In some preferred embodiments, delivery device of the present invention is a pre-filled injection device, and a sufficient amount of the pharmaceutical composition prepared according to the disclosure is included in the pre-filled container, which helps to distribute protein preparations for parenteral administration (injection or infusion).In some embodiments, container or pre-filled container include at least one pharmaceutical unit dosage form, which 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, 3.0 mL, 4.0 mL, 5.0 mL, 6.0 mL, 7.0 mL, 8.0 mL, 9.0 mL, 10.0 mL, 11.0 mL, 12.0 mL, 13.0 mL, 14.0 mL, 15.0 mL, 16.0 mL, 17.0 mL, 18.0 mL, 19.0 mL, 20.0 mL, 21.0 mL, 22.0 mL, 23.0 mL, 24.0 mL, 25.0 mL, 26.0 mL, 27.0 mL, 28.0 mL, 29.0 mL, 30.0 mL, 31.0 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 volume of the pharmaceutical composition of the present invention.

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. Lyophilization methods are well known to those of ordinary skill in the art, and include, for example, sublimation of water from a frozen preparation under controlled conditions. The lyophilizable preparation can be redissolved into a solution, suspension, emulsion, or any other form suitable for administration or use. Reconstitution can typically be performed by adding an aqueous solution to dissolve the lyophilized preparation.

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 eye disease. Or provide the pharmaceutical composition described in the first object of the present invention for treating an eye disease. The method of "treatment" adopts 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 eye disorder or a subject who may eventually acquire such a disorder) to prevent, cure, delay the disorder or recurring disorder, reduce the severity of the disorder or recurring disorder, or improve one or more symptoms of the disorder or recurring disorder.

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 retinal branch 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.

The dosage for treatment can be easily determined by a physician with ordinary skills 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 mode of administration. Typically, the therapeutically effective composition is administered at a dosage of from 10 mg/mL to about 200 mg/mL per dose. Preferably, the dosage 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 dosage of the anti-VEGF fusion protein used in the method of the present invention is 10 mg/mL. In other preferred embodiments, the dosage of the anti-VEGF fusion protein used in the method 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 affected ocular tissue to affect the eye of the mammal. In certain embodiments, the dosage of each eye is at least about 0.5 mg up to 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.

The beneficial effects of the present invention are:

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 and the stability can be improved; and the pharmaceutical composition can maintain good biological activity, and the biological activity can still be well maintained even after high-temperature treatment.

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, a tetramer, or a hexamer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Fusion protein A SEQ ID NO:1 sequence.

Figure 2 Schematic diagram of the structure of protein fragment B.

Figure 3 Schematic diagram of the structure of protein fragment C.

DETAILED DESCRIPTION

The SEC-HPLC described in the present invention is in accordance with the general rule "0514 Molecular Exclusion Chromatography" of the 2020 edition (Part 4) of the Chinese Pharmacopoeia, using a hydrophilic silica size exclusion chromatography column TSK G3000 SWXL, 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 embodiments of the present invention is detected by the SEC-HPLC method.

The SEC-UPLC described in the present invention is in accordance with the general rule "0514 Molecular Exclusion Chromatography" of the 2020 edition (Volume 4) 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, 1.7μm, 4.6mm*150mm), the sample amount is controlled to be 4-12μg, the mobile phase is 20mM disodium hydrogen phosphate, 150mM sodium chloride, 200mM arginine, pH7.2, the flow rate is 0.3ml/min, the detection wavelength is 280nm, and the polymer (%) is calculated by the area normalization method.

The SDS-PAGE stain-free method 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 4) of the Chinese Pharmacopoeia. The protein containing the loading buffer is warmed at 70±2°C for 10 minutes, and 4 μg is loaded for electrophoresis imaging in a 4-15% precast polyacrylamide gel. The precast gel is added with trichloride fluorescent dye, which combines with tryptophan in the protein under ultraviolet light at about 300nm to emit fluorescence, so that the protein in the gel can be visualized, and 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-reduced purity (%) of the protein in the embodiment of the present invention is detected by the SDS-PAGE method.

The biological activity luciferase reporter gene method described in the present invention is detected according to 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 vascular endothelial growth factor (VEGF)-stimulated cell luciferase is different by blocking different concentrations of protein to determine the biological activity of the protein. In the experiment, the protein standard/test sample was gradient diluted to 30000 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 a cell suspension of 5×10 ⁵ cells/mL with DMEM test medium and then inoculated into a 96-well cell culture plate, with 80 μl inoculated in each well. Different concentrations of standard/test sample mixed solutions were added, 20 μl per well, and cultured at 37°C±1°C and 5% carbon dioxide for 5.8-6 hours. After equilibration at room temperature for 10 to 15 minutes, 100 μl of color substrate was added to each well, and after being placed at room temperature for 3 to 5 minutes, the microplate reader was immediately placed, and the fluorescence response value of each well was measured using a chemiluminescence module. The rhVEGF165 working solution was added to the cell well as a positive control, and the DMEM test medium was added to the cell well as a negative control. The results were measured in the same way and recorded. The four-parameter curve was drawn with the concentration of the test sample and the standard sample as the horizontal coordinate and the average fluorescence response as the vertical coordinate to calculate the half effective concentration (EC50) of the test sample and the standard sample using a computer program or a four-parameter regression calculation method. 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 sample ÷ EC50 of the test sample × 100%. The biological activity (%) of the protein in the examples of the present invention was evaluated by this method. The biological activity of the preparation samples in the examples of the present invention after preparation was tested to be higher than 85%.

The data tested in all the embodiments of the present invention are the average values of 6 parallel samples (n=6).

Example 1

The composition of the preparation samples is as follows:

Use hydrochloric acid to adjust the system pH to 7.7 ± 0.2

Proteins of different purities (see Table 1, the protein includes fusion protein A, protein fragments B, C, and D, and the content of specific components is shown in the "Protein non-reducing purity (%)" in the table, the same below) were taken 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 is basically consistent 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 the two groups of samples was greater than 80%, and the activity was well maintained.

Table 1

Example 2

The composition of the preparation samples is as follows:

Use hydrochloric acid to adjust the system pH to 7.7 ± 0.2

Proteins of different purities (see Table 2) were taken and prepared into samples 3 and 4 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 2. As can be seen from Table 2, as the content of protein fragment B in the formulation samples increased, the content of polymer in the samples increased more significantly after placement, and the polymerization rate of the polymer was faster. At the same time, the biological activity of the protein after being placed at 35°C for 15 days was determined. The biological activity of the protein in sample 3 was greater than 75%, and the biological activity of the protein in sample 4 was less than 65%.

Table 2

Example 3

The composition of the preparation samples is as follows:

Use hydrochloric acid to adjust the system pH to 7.7 ± 0.2

Proteins of different purities (see Table 3) were taken and prepared into samples 5 and 6 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 3. From the results in Table 3, it can be seen that when the content of protein fragment B is basically the same, when the content of other components (such as protein fragment C) is significantly different, the polymer content of the two groups of formulation samples after being placed at 25°C is basically the same. At the same time, the biological activity of the proteins of the two groups of samples after being placed at 35°C for 15 days was determined, and the results showed that the biological activity of the proteins of the two groups of samples was greater than 80%.

Table 3

Example 4

The composition of the preparation samples is as follows:

Use hydrochloric acid to adjust the system pH to 7.7 ± 0.2

Proteins of different purities (see Table 4) were taken and prepared into 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 remains basically the same, and when the content of other components (protein A, C, D, etc.) is significantly different, the polymer content in the two groups of formulation samples after placement is basically the same. At the same time, the biological activity of the proteins of the two groups of samples after being placed at 35°C for 15 days was determined, and the results showed that the biological activity of the proteins of the two groups of samples was greater than 80%.

Table 4

Example 5

The composition of the preparation samples is as follows:

Use hydrochloric acid to adjust the system pH to 7.7 ± 0.2

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 increases, the content of polymer in the formulation sample increases significantly after placement, and the polymerization rate is faster. At the same time, the biological activity of the protein in the sample was determined 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%.

Table 5

Example 6

The composition of the preparation samples is as follows:

Use hydrochloric acid to adjust the system pH to 7.7 ± 0.2

Proteins of different purities (see Table 6) were taken and prepared into samples 16-23 according to the above formulation composition. The biological activities of the proteins of the samples were measured after being placed at 35° C. for 15 days, as shown in Table 6.

Table 6

Example 7

The sample formulation is as follows:

Use hydrochloric acid to adjust the system pH to 7.7 ± 0.2

The proteins described in Table 7 were taken and prepared into samples 24-25 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 7. At the same time, the biological activity of the protein in the samples was determined after being placed at 35°C for 15 days. The results showed that the biological activity of the protein in the two groups of samples was greater than 80%.

Table 7

Example 8

The sample formulation is as follows:

Adjust the system pH to 7.7±0.2

The proteins described in Table 8 were prepared into samples 26-27 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 8. At the same time, the biological activity of the protein in the samples was determined after being placed at 35°C for 15 days, and the results showed that the biological activity of the protein in the two groups of samples was greater than 80%.

Table 8

Example 9

The sample formulation is as follows:

Adjust the system pH to 7.7±0.2

The proteins described in Table 9 were prepared into samples 28-29 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 9. At the same time, the biological activity of the protein in the samples was determined after being placed at 35°C for 15 days, and the results showed that the biological activity of the protein in the two groups of samples was greater than 80%.

Table 9

Example 10

The sample formulation is as follows:

Use hydrochloric acid to adjust the system pH to 7.7 ± 0.2

The proteins described in Table 10 were prepared into samples 30-31 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 10. At the same time, the biological activity of the protein in the samples was determined after being placed at 35°C for 15 days, and the results showed that the biological activity of the protein in the two groups of samples was greater than 80%.

Table 10

Embodiment 11

The sample formulation is as follows:

Adjust the system pH to 7.7±0.2

The proteins described in Table 11 were prepared into samples 32-33 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 11. At the same time, the biological activity of the protein in the samples was determined after being placed at 35°C for 15 days, and the results showed that the biological activity of the protein in the two groups of samples was greater than 80%.

Table 11

Example 12

The sample formulation is as follows:

Adjust the system pH to 7.7±0.2

The proteins described in Table 12 were prepared into samples 34-35 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 12. At the same time, the biological activity of the protein in the samples was determined after being placed at 35°C for 15 days, and the results showed that the biological activity of the protein in the two groups of samples was greater than 80%.

Table 12

Example 13

The sample formulation is as follows:

Use hydrochloric acid to adjust the system pH to 7.9 ± 0.2

The proteins described in Table 13 were prepared into samples 36-37 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 13. At the same time, the biological activity of the protein in the samples was determined after being placed at 35°C for 15 days, and the results showed that the biological activity of the protein in the two groups of samples was greater than 80%.

Table 13

Embodiment 14

The sample formulation is as follows:

Use hydrochloric acid to adjust the system pH to 7.7-8.7

Proteins of different purities as described in Table 14 were taken and prepared into samples 38-40 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 14.

Table 14

Embodiment 15

The sample formulation is as follows:

Use hydrochloric acid to adjust the system pH to 5 ± 0.5

Proteins of different purities as described in Table 15 were taken and 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.

Table 15

Example 16 Protein Purification

1. Gel chromatography

The column was filled with gel filler suitable for separating 10-400 kDa proteins, and the column was balanced with chromatography balance solution, and the sample volume was controlled to be ≤10%CV and the sample flow rate was ≤20 cm/h for loading, balancing, and collecting the target protein. The proteins in samples 1-4, 9-23, and 41-44 were obtained according to different peak collection parameters.

2. Cation exchange chromatography

The chromatography column was filled 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.

3. Mixed Mode Chromatography

The column was filled with anionic and hydrophobic mixed mode chromatography fillers, the sample was adjusted to weak alkalinity and the conductivity to between 40 and 60 mS/cm, and the flow-through was collected to obtain the target protein. The proteins in samples 7-8 and 24-37 were obtained according to different peak collection parameters.

Claims (20)

A pharmaceutical composition, characterized in that it 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%. The pharmaceutical composition according to claim 1, characterized in that the content of protein fragment B in the pharmaceutical composition 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%. The pharmaceutical composition according to claim 1, characterized in that the content of fusion protein A in the pharmaceutical composition is 77-98%, preferably 80-98%. The pharmaceutical composition according to claim 3, characterized in that the content of fusion protein A in the pharmaceutical composition is 77-87%, preferably 80-87%. 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. 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, and more preferably 7.7±0.2. 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. 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. 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. The pharmaceutical composition according to claim 8, characterized in that the pharmaceutical composition contains: 10-150 mg/mL 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. The pharmaceutical composition according to claim 1, characterized in that the pharmaceutical composition contains: 10-120 mg/mL 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: 10mg/mL to 120mg/mL of fusion protein A; 10 mM tromethamine; 100 mM arginine; and, 5% sucrose; pH 7.5-8.7; Alternatively, the pharmaceutical composition contains: 10mg/mL to 120mg/mL of fusion protein A; 290 mM glutamate; 290 mM arginine; and, 78mM NaOH; pH 7.5-8.7; Alternatively, the pharmaceutical composition contains: 10mg/mL to 120mg/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: 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. 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). A container or delivery device comprising the pharmaceutical composition of any one of claims 1 to 12. The container or delivery device of claim 13, wherein the container is a vial or a syringe. The container or delivery device of claim 13, wherein the delivery device is a prefilled injection device. A lyophilized preparation prepared by lyophilizing the pharmaceutical composition according to any one of claims 1 to 12. Use of the pharmaceutical composition according to any one of claims 1 to 12 in the preparation of a medicament for treating eye diseases. A method for treating an ocular disease, comprising administering the pharmaceutical composition of any one of claims 1 to 12 to a subject. The use according to claim 17 or the method according to claim 18, characterized in that the eye disease is an ocular neovascular disease; preferably, the ocular neovascular disease is selected from retinal neovascularization, choroidal neovascularization, iris neovascularization or corneal neovascularization eye disease. 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.