Compound preparation and application thereof

Abstract

Relates to a compound preparation and application thereof. The compound preparation comprises an anti-VEGF antibody or antigen binding fragment and an integrin GPIIb / IIIa antagonist. According to the compound preparation, the anti-VEGF antibody or antigen binding fragment and the integrin GPIIb / IIIa antagonist in the compound can be kept stable and have good binding activity, and the clinical use safety and effectiveness are improved.

Description

A compound preparation and its application Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a compound preparation and application thereof. Background Art

[0002] Vascular endothelial growth factor (VEGF) is considered to be the most critical factor in physiological and pathological angiogenesis. Vascular endothelial growth factor (VEGF), especially VEGF-A, has been identified as a key factor in inducing tumor angiogenesis. Given the importance of angiogenesis in tumor biology, drug development efforts over the past few decades have been focused on targeting angiogenesis, with VEGF-A as a therapeutic target for inhibiting angiogenesis and normalizing tumor blood vessels, and its effectiveness has been demonstrated in a series of in vivo studies in tumor models, indicating that VEGF monoclonal antibodies inhibiting angiogenesis can inhibit tumor growth (Garcia J, Hurwitz HI, et. All, Bevacizumab In cancer treatment: A review of 15 years of clinical experience and future outlook. Cancer Treat Rev. 2020 Jun;86:102017.). By specifically targeting human vascular endothelial growth factor (VEGF), it inhibits VEGF binding to its receptors, suppressing tumor angiogenesis and reducing the tumor's blood supply, oxygen supply, and other nutrients, thereby inhibiting tumor cell growth. Bevacizumab was approved for marketing by the US Food and Drug Administration in 2004 and was approved for market entry in China in 2010. As one of the key anti-angiogenic drugs, bevacizumab is currently used to treat a variety of malignant tumors.

[0003] However, antibodies, as proteins, are more complex than traditional inorganic and organic drugs. Antibody preparations are prone to degradation during storage due to chemical instability (such as deamidation, racemization, hydrolysis, oxidation, β-elimination, or disulfide exchange) or physical instability (such as denaturation, aggregation, precipitation, or adsorption). The instability of antibody preparations can not only reduce or eliminate drug efficacy but also have adverse effects on patient health.

[0004] Summary of the Invention

[0005] The present invention provides a compound preparation comprising an anti-VEGF antibody or antigen-binding fragment and an integrin GPIIb / IIIa antagonist and application thereof.

[0006] In some embodiments, the present invention provides a formulation comprising an anti-VEGF antibody or antigen-binding fragment and an integrin GPIIb / IIIa antagonist.

[0007] In some embodiments, the pH of the formulation is 3.0-8.0, or 3.0-6.0, or 3.0-5.0, or 3.0-4.5, or 3.8-4.2, or 3.8-4.0. In some embodiments, the pH of the formulation is about 3.0, about 3.5, about 3.8, about 3.9, about 4.0, about 4.2, about 4.5, about 4.8, about 5.0, about 5.2, about 5.4, about 5.5, about 6.0, about 6.5, about 7.0, about 8.0, or a range between any two of these values ​​(including endpoints) or any value therein.

[0008] In some embodiments, the formulation further comprises one or a combination of a buffer, a stabilizer, and a surfactant. In some embodiments, the formulation comprises an anti-VEGF antibody or antigen-binding fragment, an integrin GPIIb / IIIa antagonist, and a buffer. In some embodiments, the formulation further comprises a stabilizer and / or a surfactant.

[0009] In some embodiments, the present invention provides a formulation comprising:

[0010] (1) anti-VEGF antibodies or antigen-binding fragments;

[0011] (2) integrin GPIIb / IIIa antagonists;

[0012] (3) buffer;

[0013] (4) stabilizers; and

[0014] (5) Surfactants

[0015] In some embodiments, the anti-VEGF antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO: 6.

[0016] In some embodiments, the anti-VEGF antibody or antigen-binding fragment comprises a heavy chain variable region and / or a light chain variable region; wherein the heavy chain variable region comprises the sequence shown in SEQ ID NO:7, or a sequence having at least 80% identity to the sequence shown in SEQ ID NO:7, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:7; and / or the light chain variable region comprises the sequence shown in SEQ ID NO:8, or a sequence having at least 80% identity to the sequence shown in SEQ ID NO:8, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:8.

[0017] In some embodiments, the anti-VEGF antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises the sequence shown in SEQ ID NO:7, and the light chain variable region comprises the sequence shown in SEQ ID NO:8.

[0018] In some embodiments, the anti-VEGF antibody comprises a heavy chain and / or a light chain; the heavy chain comprises the sequence shown in SEQ ID NO:9, or a sequence having at least 80% identity to the sequence shown in SEQ ID NO:9, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:9; and / or the light chain comprises the sequence shown in SEQ ID NO:10, or a sequence having at least 80% identity to the sequence shown in SEQ ID NO:10, or an amino acid sequence having one or more conservative amino acid substitutions compared to the sequence shown in SEQ ID NO:10.

[0019] In some embodiments, the anti-VEGF antibody comprises a heavy chain and a light chain; the heavy chain comprises the sequence set forth in SEQ ID NO: 9, and the light chain comprises the sequence set forth in SEQ ID NO: 10. In some embodiments, the anti-VEGF antibody or antigen-binding fragment is bevacizumab, including Avastin or a biosimilar thereof, such as Probexi (BAT1706), Puxinting, Beianting, Hanbetai, Aruito, Boyounuo, Dayoutong, Ankeda, Alymsys, Zirabev, Mvasi, Vegzelma, Onbevzi, Oyavas, Aybintio, and Abevmy.

[0020] Table 1. Amino acid sequence

[0021] In some embodiments, the concentration of the anti-VEGF antibody or antigen-binding fragment is 1-200 mg / mL, or 1-150 mg / mL, or 10-120 mg / mL, or 10-100 mg / mL, or 20-80 mg / mL, or 25-75 mg / mL. In some embodiments, the concentration of the anti-VEGF antibody or antigen-binding fragment is about 1 mg / mL, about 6 mg / mL, about 12 mg / mL, about 20 mg / mL, about 25 mg / mL, about 32.5 mg / mL, about 50 mg / mL, about 60 mg / mL, about 75 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, about 150 mg / mL, about 180 mg / mL, about 200 mg / mL, or a range between any two of these values ​​(including the endpoints) or any value therein.

[0022] In some embodiments, the integrin GPIIb / IIIa antagonist is tirofiban, eptifibatide or batifiban, or a pharmaceutically acceptable salt thereof (e.g., tirofiban hydrochloride). In some embodiments, the integrin GPIIb / IIIa antagonist is batifiban (Compound I disclosed in CN101085809A) or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutically acceptable salt is an inorganic acid salt, and the inorganic acid can be: hypoiodous acid, hypochlorous acid, hypobromous acid, iodic acid, perchloric acid, peroxydisulfuric acid, peroxydicarbonic acid, percarbonic acid, pyrophosphoric acid, pyrosulfuric acid, pyrosulfite, tetrathionic acid, phosphoric acid, thiosulfuric acid, sulfuric acid, chloric acid, metaphosphoric acid, hydroiodic acid, hydroazide, hydrofluoric acid, hydrosulfuric acid, hydrochloric acid, hydrobromic acid, tetraboric acid, carbonic acid, nitric acid, bromic acid, sulfurous acid, phosphorous acid, chlorous acid, hydrochloric acid, nitrous acid, orthophosphoric acid, orthosulfuric acid, orthocarbonic acid and the like. In some embodiments, the pharmaceutically acceptable salt is an organic acid salt, and the organic acid can be: tartaric acid, oxalic acid, malic acid, citric acid (citric acid), ascorbic acid, benzoic acid, salicylic acid, caffeic acid, lactic acid, sorbic acid, fumaric acid, formic acid, acetic acid, benzoic acid, oxalic acid, succinic acid, pyruvic acid, α-ketosuccinic acid, benzenesulfonic acid, or trifluoroacetic acid, maleic acid, tetrasulfonic acid, methanesulfonic acid, fumaric acid, amino acid, etc. In some embodiments, the organic acid is citric acid (citric acid), malic acid, lactic acid, acetic acid, oxalic acid, amino acid. In some embodiments, the pharmaceutically acceptable salt is citrate. In some embodiments, the pharmaceutically acceptable salt is a salt formed by batifiban and a metal ion (e.g., an alkali metal ion (such as sodium or potassium), an alkaline earth metal ion (such as calcium or magnesium) or an aluminum ion) or an organic base (such as diethanolamine, triethanolamine, N-methylglucamine).

[0023] In some embodiments, the concentration of the integrin GPIIb / IIIa antagonist is 1-100 mg / mL, or 1-90 mg / mL, or 1-60 mg / mL, or 10-60 mg / mL, or 1-30 mg / mL, or 10-30 mg / mL, or 2-20 mg / mL. In some embodiments, the concentration of the integrin GPIIb / IIIa antagonist is about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 5 mg / mL, about 8 mg / mL, about 10 mg / mL, about 12 mg / mL, about 15 mg / mL, about 18 mg / mL, about 20 mg / mL, about 30 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, or a range between any two of these values ​​(including the endpoints) or any value therein.

[0024] In some embodiments, the weight ratio of the anti-VEGF antibody or antigen-binding fragment to the integrin GPIIb / IIIa antagonist is 1-10: 1, or 1-8: 1, or 1-4: 1. In some embodiments, the weight ratio of the anti-VEGF antibody or antigen-binding fragment to the integrin GPIIb / IIIa antagonist is 1: 1, 5:4, 2: 1, 5:2, 3: 1, 15:4, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, or 10: 1.

[0025] In some embodiments, the buffer is selected from acetic acid, citric acid, phosphate, histidine, histidine salt, citrate, citric acid, citrate, acetate, succinate, glutamate, or a combination thereof. In some embodiments, the buffer is selected from acetate buffer, citric acid buffer, phosphate buffer, or histidine buffer. In some embodiments, the buffer is acetate buffer. In some embodiments, the concentration of the buffer is 1 to 50 mM, or 1 to 40 mM, or 1 to 30 mM, or 5 to 25 mM, or 10 to 20 mM. In some embodiments, the concentration of the buffer is about 1 mM, about 5 mM, about 8 mM, about 10 mM, 13 mM, about 15 mM, about 18 mM, about 20 mM, about 22 mM, about 25 mM, about 27 mM, about 30 mM, about 35 mM, about 40 mM, about 50 mM, or a range (including endpoints) between any two of these values ​​or any value therein. In some embodiments, the buffer is an acetate buffer at about 10-20 mM.

[0026] In some embodiments, the stabilizer is selected from polyols, sugars, amino acids, salts. In some embodiments, the stabilizer is selected from one or more of sodium chloride, arginine or its salts, polyethylene glycol, sorbitol, mannitol, monosaccharides, oligosaccharides, polysaccharides, cyclodextrins or their derivatives. In some embodiments, the stabilizer is selected from one or more of cyclodextrins or their derivatives, sucrose and trehalose. In some embodiments, the cyclodextrins or their derivatives are selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, their hydroxypropylated derivatives (such as hydroxypropyl beta-cyclodextrin), hydroxyethylated derivatives, ethylated derivatives and methylated derivatives, sulfobutyl ether beta-cyclodextrin, branched cyclodextrin, cyclodextrin polymers, or combinations thereof. In some embodiments, the stabilizer is selected from one or more of hydroxypropyl beta-cyclodextrin, sucrose and trehalose. In some embodiments, the stabilizer is a combination of cyclodextrins or their derivatives and oligosaccharides. In a solid state, trehalose is usually present as trehalose dihydrate, so in some embodiments, trehalose dihydrate can be used when preparing the preparation, or other forms of trehalose (such as anhydrous trehalose) can be used. In some embodiments, the concentration of the stabilizer is 1-200 mg / mL, or 1-150 mg / mL, or 10-150 mg / mL, or 20-150 mg / mL, or 50-150 mg / mL, or 70-130 mg / mL, 80-130 mg / mL or 100-120 mg / mL. In some embodiments, the concentration of the stabilizer is about 1 mg / mL, about 3 mg / mL, about 5 mg / mL, about 8 mg / mL, about 10 mg / mL, about 12 mg / mL, about 15 mg / mL, about 18 mg / mL, about 20 mg / mL, about 30 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, about 130 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, or a range between any two of these values ​​(including the endpoints), or any value therein.

[0027] In some embodiments, the stabilizer is a combination of hydroxypropyl beta-cyclodextrin and trehalose. In some embodiments, the concentration of the hydroxypropyl beta-cyclodextrin is 1 to 200 mg / mL, or 1 to 150 mg / mL, or 10 to 150 mg / mL, or 20 to 100 mg / mL, or 20 to 80 mg / mL, or 30 to 70 mg / mL, or 40 to 60 mg / mL. In some embodiments, the concentration of the trehalose is 1 to 200 mg / mL, or 1 to 150 mg / mL, or 10 to 150 mg / mL, or 20 to 120 mg / mL, or 20 to 100 mg / mL, or 20 to 80 mg / mL, or 30 to 70 mg / mL, or 50 to 65 mg / mL. In some embodiments, the concentration of hydroxypropyl beta-cyclodextrin is about 1 mg / mL, about 3 mg / mL, about 5 mg / mL, about 8 mg / mL, about 10 mg / mL, about 12 mg / mL, about 15 mg / mL, about 18 mg / mL, about 20 mg / mL, about 30 mg / mL, about 40 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, or a range between any two of these values ​​(including the endpoints), or any value therein. In some embodiments, the concentration of trehalose is about 1 mg / mL, about 3 mg / mL, about 5 mg / mL, about 8 mg / mL, about 10 mg / mL, about 12 mg / mL, about 15 mg / mL, about 18 mg / mL, about 20 mg / mL, about 30 mg / mL, about 50 mg / mL, about 54.3 mg / mL (about 60 mg / mL of trehalose dihydrate), about 57.9 mg / mL (about 64 mg / mL In some embodiments, the stabilizer is about 40 to 60 mg / mL of hydroxypropyl beta-cyclodextrin and about 30 to 70 mg / mL of trehalose dihydrate. In some embodiments, the stabilizer is about 40 to 60 mg / mL of hydroxypropyl beta-cyclodextrin and about 50 to 65 mg / mL of trehalose.In some embodiments, the stabilizer is about 50 mg / mL of hydroxypropyl beta-cyclodextrin and about 57.9 mg / mL of trehalose (such as about 64 mg / mL of trehalose dihydrate). In some embodiments, the stabilizer is about 50 mg / mL of hydroxypropyl beta-cyclodextrin and about 27.1 mg / mL of trehalose (such as about 30 mg / mL of trehalose dihydrate). In some embodiments, the stabilizer is about 40-60 mg / mL of hydroxypropyl beta-cyclodextrin and about 54.3 mg / mL of trehalose (such as about 60 mg / mL of trehalose dihydrate).

[0028] In some embodiments, the stabilizer is a combination of hydroxypropyl beta-cyclodextrin and sucrose. In some embodiments, the concentration of the sucrose is about 1 mg / mL, about 3 mg / mL, about 5 mg / mL, about 8 mg / mL, about 10 mg / mL, about 12 mg / mL, about 15 mg / mL, about 18 mg / mL, about 20 mg / mL, about 30 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 120 mg / mL, about 130 mg / mL, about 150 mg / mL, about 160 mg / mL, about 170 mg / mL, about 180 mg / mL, about 190 mg / mL, about 200 mg / mL, or the range (including endpoints) between any two values ​​in these numerical values ​​or any value therein. In some embodiments, the stabilizer is about 40-60 mg / mL of hydroxypropyl beta-cyclodextrin and about 30-70 mg / mL of sucrose. In some embodiments, the stabilizer is about 40-60 mg / mL of hydroxypropyl beta-cyclodextrin and about 50-65 mg / mL of sucrose. In some embodiments, the stabilizer is about 50 mg / mL of hydroxypropyl beta-cyclodextrin and about 60 mg / mL of sucrose.

[0029] In some embodiments, the mass ratio of the hydroxypropyl beta-cyclodextrin and the VEGF antagonist is 0.5-4.0. In some embodiments, when the pH is 4.0, the mass ratio of the hydroxypropyl beta-cyclodextrin and the VEGF antagonist is 1.0-2.5. In some embodiments, when the pH is 4.0, the mass ratio of the hydroxypropyl beta-cyclodextrin and the VEGF antagonist is 1.2-2.4. In some embodiments, when the pH is 3.8, the mass ratio of the hydroxypropyl beta-cyclodextrin and the VEGF antagonist is 0.6-2.5. In some embodiments, when the pH is 3.8, the mass ratio of the hydroxypropyl beta-cyclodextrin and the VEGF antagonist is 0.8-2.4.

[0030] In some embodiments, the surfactant is a nonionic surfactant. In some embodiments, the surfactant is selected from poloxamer (e.g., poloxamer 188), tween (e.g., tween 20 or tween 80), or a combination thereof. In some embodiments, the concentration of the surfactant is 0.05-10 mg / mL, or 0.1-10 mg / mL, or 0.1-5 mg / mL, or 0.2-1 mg / mL, or 0.2-0.6 mg / mL. In some embodiments, the concentration of the surfactant is about 0.05 mg / mL, about 0.08 mg / mL, about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.8 mg / mL, about 1 mg / mL, about 3 mg / mL, about 6 mg / mL, about 8 mg / mL, about 10 mg / mL, or the range (including endpoints) between any two values ​​in these numerical values ​​or any value therein. In some embodiments, the surfactant is about 0.4 mg / mL of Tween 20 or Tween 80.

[0031] In some embodiments, the formulation comprises:

[0032] (1) Anti-VEGF antibody or antigen-binding fragment: 1-200 mg / mL;

[0033] (2) Batifiban: 1–100 mg / mL;

[0034] (3) Buffer: 1-50 mM;

[0035] (4) Stabilizer: 1-200 mg / mL; and

[0036] (5) Tween: 0.05-10 mg / mL;

[0037] The pH of the preparation is 3.0-8.0, preferably 3.0-6.0, preferably 3.0-5.0, preferably 3.5-4.5, preferably 3.5-4.0, preferably 3.8-4.2, or preferably 3.8-4.0, for example, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1 or about 4.2.

[0038] In some embodiments, the stabilizer is a combination of cyclodextrin or its derivatives and disaccharides.

[0039] In some embodiments, the formulation comprises:

[0040] (1) Anti-VEGF antibody or antigen-binding fragment: 1-200 mg / mL;

[0041] (2) Batifiban: 1–100 mg / mL;

[0042] (3) Acetate buffer, histidine buffer or citric acid buffer: 1-50 mM;

[0043] (4) Hydroxypropyl beta-cyclodextrin: 1-200 mg / mL;

[0044] (5) Trehalose or sucrose: 1-200 mg / mL; and

[0045] (6) Tween 20 or Tween 80: 0.05-10 mg / mL;

[0046] The pH of the preparation is 3.0 to 8.0, preferably 3.0 to 6.0, preferably 3.0 to 5.0, preferably 3.5 to 4.5, preferably 3.5 to 4.0, preferably 3.8 to 4.2, or preferably 3.8 to 4.0.

[0047] In some embodiments, the formulation comprises:

[0048] (1) Anti-VEGF antibody or antigen-binding fragment: 1-150 mg / mL;

[0049] (2) Batifiban: 1-90 mg / mL;

[0050] (3) Acetate buffer: 1-40 mM;

[0051] (4) Hydroxypropyl beta-cyclodextrin: 1-150 mg / mL;

[0052] (5) Trehalose or sucrose: 10-150 mg / mL; and

[0053] (6) Tween 20 or Tween 80: 0.1-10 mg / mL;

[0054] The pH of the preparation is 3.0 to 8.0, preferably 3.0 to 6.0, preferably 3.0 to 5.0, preferably 3.5 to 4.5, preferably 3.5 to 4.0, and preferably 3.8 to 4.2.

[0055] In some embodiments, the formulation comprises:

[0056] (1) Anti-VEGF antibody or antigen-binding fragment: 10-120 mg / mL;

[0057] (2) Batifiban: 1-60 mg / mL;

[0058] (3) Acetate buffer: 1-30 mM;

[0059] (4) Hydroxypropyl beta-cyclodextrin: 20-100 mg / mL;

[0060] (5) Trehalose or sucrose: 20-100 mg / mL; and

[0061] (6) Tween 20 or Tween 80: 0.1-10 mg / mL;

[0062] The pH of the preparation is 3.0 to 8.0, preferably 3.0 to 6.0, preferably 3.0 to 5.0, preferably 3.5 to 4.5, preferably 3.5 to 4.0, preferably 3.8 to 4.2, or preferably 3.8 to 4.0.

[0063] In some embodiments, the formulation comprises:

[0064] (1) Anti-VEGF antibody or antigen-binding fragment: 10-100 mg / mL;

[0065] (2) Batifiban: 10-60 mg / mL;

[0066] (3) Acetate buffer: 1-30 mM;

[0067] (4) Hydroxypropyl beta-cyclodextrin: 20-80 mg / mL;

[0068] (5) Trehalose or sucrose: 20-80 mg / mL; and

[0069] (6) Tween 20 or Tween 80: 0.1-5 mg / mL;

[0070] The pH of the preparation is 3.0 to 8.0, preferably 3.0 to 6.0, preferably 3.0 to 5.0, preferably 3.5 to 4.5, preferably 3.5 to 4.0, preferably 3.8 to 4.2, or preferably 3.8 to 4.0.

[0071] In some embodiments, the formulation comprises:

[0072] (1) Anti-VEGF antibody or antigen-binding fragment: 20-80 mg / mL;

[0073] (2) Batifiban: 10-30 mg / mL;

[0074] (3) Acetate buffer: 5-25 mM;

[0075] (4) Hydroxypropyl beta-cyclodextrin: 30-70 mg / mL;

[0076] (5) Trehalose or sucrose: 30-70 mg / mL; and

[0077] (6) Tween 20 or Tween 80: 0.2-1 mg / mL;

[0078] The pH of the preparation is 3.0 to 8.0, preferably 3.0 to 6.0, preferably 3.0 to 5.0, preferably 3.5 to 4.5, preferably 3.5 to 4.0, preferably 3.8 to 4.2, or preferably 3.8 to 4.0.

[0079] In some embodiments, the formulation comprises the following components:

[0080] (1) Anti-VEGF antibody or antigen-binding fragment: 20-80 mg / mL;

[0081] (2) Batifiban: 10-30 mg / mL;

[0082] (3) Acetate buffer: 5-25 mM;

[0083] (4) Hydroxypropyl beta-cyclodextrin: 40-60 mg / mL;

[0084] (5) Trehalose or sucrose: 50-65 mg / mL; and

[0085] (6) Tween 20 or Tween 80: 0.2-1 mg / mL;

[0086] The pH of the preparation is 3.0 to 8.0, preferably 3.0 to 6.0, preferably 3.0 to 5.0, preferably 3.5 to 4.5, preferably 3.5 to 4.0, preferably 3.8 to 4.2, or preferably 3.8 to 4.0.

[0087] In some embodiments, the formulation comprises:

[0088] (1) Anti-VEGF antibody or antigen-binding fragment: 32.5 mg / mL;

[0089] (2) Batifiban: 20 mg / mL;

[0090] (3) Acetate buffer: 10-20 mM;

[0091] (4) Hydroxypropyl beta-cyclodextrin: 40-60 mg / mL, preferably 50 mg / mL;

[0092] (5) Trehalose dihydrate: 50-65 mg / mL, preferably 60 mg / mL; and

[0093] (6) Tween 80: 0.2-1 mg / mL, preferably 0.4 mg / mL;

[0094] The pH of the preparation is 3.5 to 4.2, or preferably 3.8 to 4.0.

[0095] In some embodiments, the formulation comprises:

[0096] (1) Anti-VEGF antibody or antigen-binding fragment: 25 mg / mL;

[0097] (2) Batifiban: 10-20 mg / mL;

[0098] (3) Acetate buffer: 10-20 mM;

[0099] (4) Hydroxypropyl beta-cyclodextrin: 40-60 mg / mL, for example, 40, 50 or 60 mg / mL;

[0100] (5) sucrose: 60 mg / mL; and

[0101] (6) Tween 20 or Tween 80: 0.4 mg / mL;

[0102] The pH of the preparation is 3.5 to 4.2, or preferably 3.8 to 4.0.

[0103] In some embodiments, the formulation comprises:

[0104] (1) Anti-VEGF antibody or antigen-binding fragment: 25-75 mg / mL, e.g., 25, 50, or 75 mg / mL;

[0105] (2) Batifiban: 20 mg / mL;

[0106] (3) Acetate buffer: 20 mM;

[0107] (4) Hydroxypropyl beta-cyclodextrin: 40-60 mg / mL, for example, 40, 50 or 60 mg / mL;

[0108] (5) Trehalose dihydrate: 60 mg / mL; and

[0109] (6) Tween 20 or Tween 80: 0.4 mg / mL;

[0110] The pH of the preparation is 3.5 to 4.2, or preferably 3.8 to 4.0.

[0111] In some embodiments, the formulation comprises:

[0112] (1) Anti-VEGF antibody or antigen-binding fragment: 50 mg / mL;

[0113] (2) Batifiban: 20 mg / mL;

[0114] (3) Acetate buffer: 20 mM;

[0115] (4) Hydroxypropyl beta-cyclodextrin: 40-60 mg / mL, for example, 40, 50 or 60 mg / mL;

[0116] (5) Trehalose dihydrate: 60 mg / mL; and

[0117] (6) Tween 80: 0.4 mg / mL;

[0118] The pH of the preparation is 3.5 to 4.2, or preferably 3.8 to 4.0.

[0119] In some embodiments, the formulation comprises:

[0120] (1) Anti-VEGF antibody or antigen-binding fragment: 75 mg / mL;

[0121] (2) Batifiban: 20 mg / mL;

[0122] (3) Acetate buffer: 20 mM;

[0123] (4) Hydroxypropyl beta-cyclodextrin: 40-60 mg / mL, for example, 40, 50 or 60 mg / mL;

[0124] (5) Trehalose dihydrate: 60 mg / mL; and

[0125] (6) Tween 80: 0.4 mg / mL;

[0126] The pH of the preparation is 3.5 to 4.2, or preferably 3.8 to 4.0.

[0127] In some embodiments, the pH of the formulation is 3.0-8.0, preferably 3.0-6.0, preferably 3.0-5.0, preferably 3.5-4.5, preferably 3.5-4.0, preferably 3.8-4.2, or preferably 3.8-4.0, for example, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4.0, about 4.1 or about 4.2.

[0128] In some embodiments, the formulation comprises:

[0129] (1) Anti-VEGF antibody or antigen-binding fragment: 25 mg / mL;

[0130] (2) Batifiban: 20 mg / mL;

[0131] (3) Histidine buffer: 10 mM;

[0132] (4) Hydroxypropyl beta-cyclodextrin: 50 mg / mL;

[0133] (5) Trehalose dihydrate: 64 mg / mL; and

[0134] (6) Tween 80: 0.4 mg / mL;

[0135] The pH of the preparation is 5.0 to 5.85, such as about 5.00, about 5.25, or about 5.50.

[0136] In some embodiments, the preparation is a liquid preparation. In some embodiments, the solvent of the liquid preparation is water, such as water for injection or purified water.

[0137] In some embodiments, the liquid preparation is administered intravenously, subcutaneously, intraocularly, intramuscularly, or the like.

[0138] In some embodiments, the present invention also provides a solid preparation, which is lyophilized from the preparation described herein.

[0139] In some embodiments, the present invention further provides a reconstituted preparation obtained by reconstituting the solid preparation described herein with a pharmaceutically acceptable solvent (such as water for injection or purified water).

[0140] In some embodiments, the liquid formulation of the present invention can be prepared using methods known to those skilled in the art.

[0141] In some embodiments, the present invention provides a method for preparing the liquid preparation described herein, comprising: taking the prescribed amount of each component, adding water to dissolve and mix, and adjusting the pH value to obtain a liquid preparation.

[0142] In some embodiments, the present invention provides a method for preparing the liquid preparation described herein, comprising: preparing a buffer solution, ultrafiltration of the anti-VEGF antibody or antigen-binding fragment into the buffer solution, adding an integrin GPIIb / IIIa antagonist and excipients, and diluting the anti-VEGF antibody or antigen-binding fragment to a specified concentration to obtain a liquid preparation.

[0143] In some embodiments, the present invention also provides use of the formulation described herein in the preparation of a medicament for treating a VEGF-related disease.

[0144] In some embodiments, the present invention also provides for use of the formulations described herein in treating VEGF-related diseases.

[0145] In some embodiments, the present invention also provides a method for treating a VEGF-related disease, comprising administering a therapeutically effective amount of a formulation described herein to a patient in need thereof. In some embodiments, the patient is a mammal. In some embodiments, the patient is a human.

[0146] In some embodiments, the VEGF-related disease is a cancer or tumor. In some embodiments, the cancer is selected from colorectal cancer, lung cancer, glioblastoma, hepatocellular carcinoma, epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, renal cell carcinoma, glioblastoma, glioma, glioma, tumor metastasis, neuromyelitis optica, B-cell chronic lymphocytic leukemia, sarcoma, liver cancer, multiple myeloma, brain cancer.

[0147] In some embodiments, the VEGF-related disease is an eye disease in which VEGF is overexpressed. In some embodiments, the eye disease in which VEGF is overexpressed is an eye fundus lesion in which VEGF is overexpressed. In some embodiments, the VEGF-related disease is selected from macular degeneration, age-related macular degeneration (AMD), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, choroidal neovascularization secondary to pathological myopia, iris neovascularization, intraocular neovascularization, ocular neovascularization, neovascular glaucoma, macular edema, diabetic macular edema (DME, including localized, non-central DME and diffuse, including central DME), cystoid macular edema (CME), retinopathy, diabetic retinopathy (DR, including proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinal diseases, retinopathy of prematurity (ROP), family Familial exudative vitreoretinopathy (FEVR), hypertensive retinopathy, retinal vein occlusion (RVO) (including branch retinal vein occlusion and central retinal vein occlusion), retinal myeloid nerve fiber (CNV (including myopic CNV)), pathological myopia, Hippel-Lindau disease, Coronz disease, Norrie disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, retinitis pigmentosa (RP), retinal angiomatous proliferation, macular telangiectasia, retinal degeneration, vasculitis, optic disc edema, retinitis, conjunctivitis (including infectious and non-infectious (e.g., allergic) conjunctivitis), Leber congenital amaurosis, uveitis (including infectious and non-infectious uveitis), choroiditis, ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury, and Sjögren's disease.

[0148] In some embodiments, the present invention provides a container comprising a formulation described herein. In some embodiments, the container can be any container conventionally used in the art for storing drugs, such as a prefilled syringe, injection pen, vial (such as a vial), ampoule, pouch, etc.

[0149] The compound preparation of the present invention has good stability, can make the anti-VEGF antibody or antigen-binding fragment and integrin GPIIb / IIIa antagonist in the compound remain stable and have good binding activity, and improve the safety and effectiveness of clinical use. BRIEF DESCRIPTION OF THE DRAWINGS

[0150] Figure 1 shows the SEC test results of compound preparations with different pH values ​​at high temperature for 7 days;

[0151] Figure 2 shows the SEC test results of compound preparations with different pH values ​​after 7 days of illumination;

[0152] FIG3 shows the RP-HPLC test results of compound preparations with different pH values ​​at high temperature for 7 days;

[0153] FIG4 shows the RP-HPLC test results of compound preparations with different pH values ​​after irradiation for 7 days;

[0154] Figure 5 shows the pH variation trends of bevacizumab-SEC monomers (left) and aggregates (right) in compound preparations with different bevacizumab concentrations; from left to right, the bevacizumab concentrations are 25, 50, and 75 mg / mL; the pH values ​​of each group are 3.8, 3.9, 4.0, 4.1, and 4.2, respectively;

[0155] Figure 6 shows the pH variation trend of batifiban-SEC monomer (left) and aggregate (right) in compound preparations with different bevacizumab concentrations; from left to right, the bevacizumab concentrations are 25, 50, and 75 mg / mL; and the pH of each group from left to right is 3.8, 3.9, 4.0, 4.1, and 4.2, respectively.

[0156] the term

[0157] Unless otherwise stated, each of the following terms shall have the meaning set forth below.

[0158] definition

[0159] It should be noted that the term "a" entity refers to one or more of that entity, for example, "a preparation" should be understood as one or more preparations, and therefore, the terms "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein.

[0160] As used herein, the terms "comprising" or "including" mean that compositions and methods, etc. include the listed elements, such as components or steps, but do not exclude others. "Essentially consisting of" means that compositions and methods exclude other elements that have a fundamental effect on the characteristics of the combination, but do not exclude elements that have no essential effect on the composition or method. "Consisting of" means excluding elements not specifically listed. Embodiments defined by each of these transition terms are within the scope of the present invention. For example, when a composition is described as including ingredients A, B, and C, a composition consisting essentially of A, B, and C and a composition consisting of A, B, and C are independently within the scope of the present invention.

[0161] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue that has a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions are unlikely to substantially alter the functional properties of a protein. Examples of amino acid classes with chemically similar side chains include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; and 6) acidic side chains: aspartic acid and glutamic acid.

[0162] "Homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing positions in each sequence that can be aligned. When a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. "At least 80% identity" means about 80% identity, about 81% identity, about 82% identity, about 83% identity, about 85% identity, about 86% identity, about 87% identity, about 88% identity, about 90% identity, about 91% identity, about 92% identity, about 94% identity, about 95% identity, about 98% identity, about 99% identity, or a range (including endpoints) between any two of these values ​​or any value therein.

[0163] "Antibody" and "antigen-binding fragment" refer to polypeptides or polypeptide complexes that specifically recognize and bind to an antigen. An antibody can be a complete antibody, any antigen-binding fragment thereof, or a single chain thereof. Therefore, the term "antibody" includes any protein or peptide that contains at least a portion of an immunoglobulin molecule with biological activity that binds to an antigen. Antibodies and antigen-binding fragments include, but are not limited to, the complementarity determining regions (CDRs) of a heavy chain or light chain or its ligand-binding portion, the heavy chain variable region (VH), the light chain variable region (VL), the heavy chain constant region (CH), the light chain constant region (CL), the framework regions (FRs), or any portion thereof, or at least a portion of a binding protein. CDR regions include the CDR regions (LCDR1-3) of the light chain and the CDR regions (HCDR1-3) of the heavy chain. Antibodies and antigen-binding fragments can specifically recognize and bind to polypeptides or polypeptide complexes of one or more (e.g., two) antigens. Antibodies or antigen-binding fragments that specifically recognize and bind to multiple (e.g., two) antigens can be referred to as multispecific (e.g., bispecific) antibodies or antigen-binding fragments.

[0164] The term "antibody fragment" or "antigen-binding fragment" refers to a portion of an antibody. Antibody fragments of the present invention may be structured in forms similar to monospecific antibody fragments such as F(ab')2, F(ab)2, Fab', Fab, Fv, and scFv. Regardless of their structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes aptamers, Spiegelmers, and diabodies. The term "antigen-binding fragment" also includes any synthetic or genetically engineered protein that functions as an antibody by binding to a specific antigen to form a complex.

[0165] The antibodies and antigen-binding fragments disclosed in the present invention include, but are not limited to, polyclonal, monoclonal, multispecific, fully human, humanized, primatized, chimeric antibodies, single-chain antibodies, epitope-binding fragments (e.g., Fab, Fab' and F(ab')2, single-chain Fv (scFv)).

[0166] The antibodies disclosed herein can be derived from any animal, including birds and mammals. Preferably, the antibodies are human, mouse, donkey, rabbit, goat, camel, llama, condricthoid (e.g., shark), horse, or chicken antibodies.

[0167] Glycoprotein IIb / IIIa (GPIIb / IIIa), also known as integrin αIIbβ3, belongs to a large family of cell surface receptors known as integrins. GPIIb / IIIa is the most abundant receptor on the platelet surface (approximately 80,000 copies per platelet), accounting for 1% to 2% of all platelet proteins. The GPIIb / IIIa receptor consists of two subunits: GPIIb (or αIIb), with a molecular weight of approximately 140 kDa, and GPIIIa (or β3), with a molecular weight of approximately 105 kDa. GPIIb / IIIa antagonists are also known as integrin GPIIb / IIIa antagonists. In some embodiments, the GPIIb / IIIa antagonist is selected from tirofiban or a salt thereof (e.g., tirofiban hydrochloride), Eptifibatide (trade name Integrilin), Abciximab, Batifiban (patent ZL200610083865.4 and patent ZL201210319865.5), etc. Batifiban has a high affinity for the platelet glycoprotein receptor GPIIb / IIIa, which means it can inhibit platelet aggregation and also inhibit vascular smooth muscle growth by inhibiting the vitronectin receptor.

[0168] "About" or "approximately" refers to the conventional error range of the corresponding numerical value that is readily known to those skilled in the relevant art. In some embodiments, "about" or "approximately" mentioned herein refers to the described numerical value and its ±10%, ±5% or ±1% range.

[0169] The term "buffer," also referred to as a buffer system or buffer system, includes but is not limited to organic acid salts such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, or phthalic acid and their salts; Tris, tromethamine hydrochloride, or phosphate buffer. In addition, amino acids and their salts can also be used as buffers. Such amino acid components include but are not limited to glycine, histidine, arginine, lysine, ornithine, isoleucine, leucine, alanine, glutamic acid, or aspartic acid. In the present invention, when it comes to a preparation containing a buffer or a buffer system, it also means that the preparation contains a buffer, and the buffer is prepared by the buffer. In some embodiments, the buffer is a histidine buffer, and the histidine buffer forms a histidine buffer in a solvent (e.g., water for injection or purified water) or a liquid preparation; for example, containing 10 mM histidine buffer in a liquid preparation also means containing 10 mM histidine buffer in the liquid preparation.

[0170] The amount of a buffer as used herein refers to the total amount of the buffer pair in the buffer system comprising the buffer. In some embodiments, the amount of a buffer is expressed in molar concentration, where the value refers to the molar concentration of the buffer pair in the buffer system comprising the buffer. For example, when a histidine buffer composed of L-histidine and L-histidine hydrochloride is used as the buffer, a given concentration of the histidine buffer (e.g., 10 mM) is the combined concentration of L-histidine and L-histidine hydrochloride (e.g., 5 mM L-histidine and 5 mM L-histidine hydrochloride).

[0171] The term "stabilizer" refers to a pharmaceutical excipient that is used to protect the active pharmaceutical ingredient and / or formulation from chemical and / or physical degradation during production, storage and application. Stabilizers include, but are not limited to, polyols such as mannitol, sorbitol, glycerol, arabinol, propylene glycol, polyethylene glycol, etc.; sugars such as monosaccharides (e.g., fructose, mannose, galactose, glucose, sorbose, etc.), oligosaccharides (e.g., disaccharides such as lactose, sucrose, trehalose, cellobiose, maltose, etc.; trisaccharides such as raffinose, gentiotriose, melezitose, etc.; cyclodextrin or its derivatives such as α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, its hydroxypropylated derivatives (e.g., hydroxypropyl beta-cyclodextrin (also known as hydroxypropyl-β-cyclodextrin, abbreviated as HP-β-CD)), hydroxyethylated derivatives, Ethylated derivatives and methylated derivatives, sulfobutyl ether β-cyclodextrin, branched cyclodextrin, cyclodextrin polymers, etc.), polysaccharides (such as maltodextrin, dextran, starch, glycogen, cellulose, chitin, etc.), sugar derivatives (such as sugar alcohols (such as mannitol, sorbitol, xylitol, maltitol, lactitol, glucitol, etc.), sugar acids (such as gluconic acid, glucuric acid, etc.), sugar amines, glycosides (such as methyl glucoside, etc.), etc.); ion stabilizers, which include salts such as sodium chloride (NaCl), etc.; or amino acids such as arginine, arginine-HCl, proline, glycine, methionine, etc. The stabilizer in the preparation of the present invention has the function of regulating osmotic pressure, so the stabilizer can also be called an osmotic pressure regulator.

[0172] The term "surfactant" includes, but is not limited to, Tween (e.g., Tween 20 and Tween 80); Poloxamer (e.g., Poloxamer 188); Triton; sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, or stearyl sulfobetaine; lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, or stearyl sarcosine; linoleyl betaine, myristyl betaine Or cetyl betaine; lauroyl propyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine or isostearamidopropyl betaine (e.g., lauroyl propyl); myristamidopropyl dimethylamine, palmitamidopropyl dimethylamine or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol, etc. Tween is also known as polysorbate (e.g., Tween 20 is known as polysorbate 20, and Tween 80 is known as polysorbate 80). In some embodiments, the surfactant is Tween 20.

[0173] The preparations described herein can be formulated using the excipients or their hydrates. For example, histidine hydrochloride, also known as histidine hydrochloride, can be anhydrous histidine hydrochloride or a histidine hydrochloride hydrate, such as histidine hydrochloride monohydrate. For example, "5 mM histidine hydrochloride" refers to 5 mmol of histidine hydrochloride or histidine hydrochloride monohydrate dissolved in a solvent to form a 1 L solution; 1.06 mg of histidine hydrochloride monohydrate includes 1.06 mg of histidine hydrochloride monohydrate or a corresponding amount of histidine hydrochloride. Another example is trehalose, which can be anhydrous trehalose or a trehalose hydrate, such as trehalose dihydrate.

[0174] Purified water as described herein is pharmaceutical water obtained by distillation, ion exchange, reverse osmosis, or other suitable methods from drinking water and does not contain any additives. Water for injection as described herein is water obtained by distillation of purified water.

[0175] "Stability" and "stable" herein refer to the fact that in a liquid formulation comprising polypeptides and proteins (including protein fragments thereof), the polypeptides and proteins (including protein fragments thereof) do not or only rarely aggregate, degrade or fragment under given production, preparation, transportation and storage conditions, and maintain their biological activity. The stability of the polypeptides and proteins (including protein fragments thereof) can be assessed by measuring the degree of aggregation, degradation or fragmentation of the formulation, for example, by techniques such as SEC-HPLC, IEC-HPLC, and CE-SDS.

[0176] As used herein, "in need thereof" means that a patient has been identified as being in need of a particular approach or treatment. In some embodiments, identification can be performed by any diagnostic means.

[0177] "Patient" refers to any mammal in need of diagnosis, prognosis or treatment, including humans, dogs, cats, rabbits, mice, horses, cows, etc.

[0178] "Treatment" refers to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent, slow, ameliorate or halt an undesirable physiological change or disorder, such as the progression of a disease, including but not limited to the following results, whether detectable or undetectable, relief of symptoms, reduction in the severity of the disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement, palliation, alleviation or elimination of the disease state (whether partial or complete), prolongation of life expectancy compared to that expected in the absence of treatment, etc. Patients in need of treatment include those already suffering from the disease or disorder, those susceptible to the disease or disorder, or those in need of prevention of the disease or disorder, and those who can or are expected to benefit from the administration of the antibodies or pharmaceutical compositions disclosed herein for detection, diagnostic procedures and / or treatment.

[0179] An "effective amount" is an amount of an active compound or agent that elicits a biological or medicinal response in a tissue, system, animal, individual, or human.

[0180] The dosage of the preparation of the present invention for human administration will vary with the patient's age, weight, gender, dosage form, health condition and severity of the disease.

[0181] Antibody preparation

[0182] DNA encoding the antibody can be designed and synthesized according to conventional methods based on the antibody amino acid sequence described herein, placed into an expression vector, and then transfected into host cells. The transfected host cells are cultured in culture medium to produce monoclonal antibodies. In some embodiments, the antibody expression vector includes at least one promoter element, the antibody coding sequence, a transcription termination signal, and a polyA tail. Other elements include enhancers, Kozak sequences, and donor and acceptor sites for RNA splicing on both sides of the inserted sequence. Efficient transcription can be achieved using the early and late promoters of SV40, and the early promoters of long terminal repeats from retroviruses such as RSV, HTLV1, HIV1, and cytomegalovirus. Promoters from other cells, such as the actin promoter, can also be used. Suitable expression vectors may include pIRES1neo, pRetro-Off, pRetro-On, PLXSN, or pLNCX, pcDNA3.1 (+ / -), pcDNA / Zeo (+ / -), pcDNA3.1 / Hygro (+ / -), PSVL, PMSG, pRSVcat, pSV2dhfr, pBC12MI and pCS2, etc. Commonly used mammalian cells include HEK293 cells, Cos1 cells, Cos7 cells, CV1 cells, mouse L cells and CHO cells, etc.

[0183] All publications, patents, and patent applications cited herein are incorporated by reference in their entirety for all purposes. DETAILED DESCRIPTION

[0184] The technical solutions of the present invention are further illustrated below by means of specific examples. These examples do not limit the scope of protection of the present invention. Non-essential modifications and adjustments made by others based on the concepts of the present invention remain within the scope of protection of the present invention. Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources or by known preparation methods.

[0185] Example 1: Stability study of batifiban samples at different concentrations

[0186] Batifiban samples with concentrations of 2, 5, 10, 15, and 20 mg / mL were prepared with purified water and placed at 4°C and 40°C overnight before light inspection. The samples of batifiban at different concentrations at 4°C and 40°C were all clear without precipitation, indicating that batifiban had good stability in the concentration range of 2-20 mg / mL.

[0187] Example 2: Effects of different pH and HP-β-CD content on the solubilization and stability of batifiban

[0188] Table 2. Prescribing Information

[0189] Table 3. Batifiban and impurity contents of the formulations after 7 days under different conditions

[0190] As can be seen from Table 3, under 40°C conditions, pH has a greater impact on the impurities of the formulation, and the impurities decrease as the pH decreases from 6.0 to 5.0. Under light conditions (4000 lx, 25°C), the HP-β-CD concentration has a greater impact on the impurities of batifiban, and the impurity content of the formulations with HP-β-CD concentrations of 50 mg / mL and 100 mg / mL is low.

[0191] Example 3: Effects of pH, temperature and light on compound preparations

[0192] Table 4. Prescribing Information

[0193] STD is a standard product of bevacizumab, and its formulation composition is 25 mg / mL bevacizumab + 50 mM phosphate buffer (PB) + 0.4 mg / mL Tween 20, pH 6.1.

[0194] Prescriptions A1-A9 were prepared into transparent glass bottles, and the stability of the samples was tested under high temperature (40°C) and light conditions (4000lx, 25°C light box).

[0195] Table 5. Light inspection results

[0196] According to the light inspection results (Table 5), the yellowing under light was caused by batifiban, and within the pH range of the experimental setting, the higher the pH, the deeper the yellowing. At 40°C, the high-temperature compound samples all showed turbidity. The turbidity of batifiban at high temperature was related to pH. Batifiban alone did not show turbidity at pH = 5.00 (prescription A7). pH has a greater impact on the compound.

[0197] In addition, prescriptions A1 and A7 were prepared into brown EP tubes (brown bottles), and the stability of the samples was tested under 4°C, high temperature (40°C) and light (4000lx, 25°C light box) conditions, respectively.

[0198] Table 6. SEC results of samples of formulations A1 and A7 at 4°C, high temperature (40°C), and light (GZ) conditions

[0199] Table 7. RP-HPLC test results

[0200] As shown in Tables 6 and 7, the compound sample's appearance changed from clear to yellow after 7 days of illumination under standard trans-bottle illumination. Physical and chemical analysis revealed an increase in aggregates. However, the use of brown EP tubing effectively mitigated the adverse effects of illumination. Therefore, brown vials may be used as packaging materials for subsequent compound samples. The RP-HPLC results were consistent with those of the SEC-HPLC.

[0201] Table 8. Capillary isoelectric focusing (CIEF) test results

[0202] From the CIEF test results (Table 8), it is speculated that the pH of the experimental design is between the isoelectric points of the two. Bevacizumab is positively charged and batifiban is negatively charged. During the experiment, the two aggregated and precipitated, so the pH was adjusted and optimized.

[0203] Example 4: Optimization of pH 3.5-pH 5.0 for Bevacizumab and Batifiban Prescriptions

[0204] Table 9. Prescription design

[0205] The compound preparation samples in Table 9 were dispensed into 2 mL brown vials and placed in a high temperature (40°C) and light (4000 lx, 25°C light box) for two weeks (0-7-14 days). The light inspection results are shown in Table 10.

[0206] Table 10 Light inspection results

[0207] Note: High temperature: GW; light: GZ.

[0208] Table 11. SEC-HPLC test results

[0209] Note: High temperature: GW; Light: GZ

[0210] The results in Figure 1 and Table 11 show that as the pH of the compound samples increased from 4.00 to 5.0, the aggregates and fragments of both bevacizumab and batifiban decreased. Aggregates were less abundant at pH 4.50 and 5.00. At these conditions, opalescence and turbidity appeared in the high-temperature samples inspected under illumination for 7 days, while the brown-bottle samples treated with illumination were all clear. Compared with the compound samples in clear bottles, the brown-bottle packaging significantly reduced the adverse effects of light on the compound samples, demonstrating superior stability under illumination.

[0211] Table 12. RP-HPLC test results

[0212] Note: High temperature: GW; Light: GZ

[0213] As can be seen from Table 12, Figures 3 and 4, the RP-HPLC test results are consistent with those of SEC-HPLC. With the increase of pH, the main peak content of bevacizumab increased significantly, the main peak content of batifiban increased slightly, and the Pre-peak and Post-peak decreased. Under light conditions, the stability of each formulation was good. Combining the results of light inspection and physical and chemical tests, the pH range of 3.8-4.2 was optimal.

[0214] Example 5: Investigation of stabilizers and surfactants for bevacizumab and batifiban compound

[0215] Table 13 Prescription design

[0216] Note: PS20: Tween 20; PS80: Tween 80; Tre: trehalose dihydrate; Suc: sucrose.

[0217] The compound preparation samples in Table 13 were dispensed into 2 mL brown vials and placed in a high temperature (40°C) and light (4000 lx, 25°C light box) test for two weeks.

[0218] Table 14SEC-HPLC detection results

[0219] Note: High temperature: GW; Light: GZ

[0220] The results (Table 14) show that the Tre content significantly affects the bevacizumab content of the compound, with higher Tre content producing less aggregates. There was no significant difference between Tre and Suc as protective agents, and there was no significant difference between PS80 and PS20. However, according to light inspection results, the formulation containing PS20 exhibited a slight opalescence after 14 days of high-temperature storage.

[0221] Example 6: Investigation of Buffer Types in Bevacizumab and Batifiban Compounds

[0222] Table 15 Prescription design

[0223] Note: PS80: Tween 80; Tre: Trehalose dihydrate.

[0224] The compound preparation samples listed in Table 15 were dispensed into 2 mL brown vials and placed in a high-temperature (40°C) and light-exposed (4500 ± 500 lx, 25°C light box) test for two weeks. Formulations D2 and D3, using CB (citric acid buffer) as the buffer, exhibited significant turbidity compared to formulation D1 after 7 days of high-temperature storage. The turbidity increased with increasing bevacizumab concentration.

[0225] Example 7: DOE experimental design further optimizes the prescription

[0226] Table 16 Prescription design

[0227] Note: PS80: Tween 80; PS20: Tween 20; Tre: Trehalose dihydrate.

[0228] The compound preparation samples listed in Table 16 were dispensed into 2 mL brown vials and placed under high temperature (40°C) and light (4500 ± 500 lx, 25°C light box) for two weeks. The light test results are shown in Table 17. Analysis of the mass concentration ratio of bevacizumab to HP-β-CD revealed that at pH 4.2, all formulations became turbid. At pH 4.0, formulations with an HP-β-CD / bevacizumab ratio greater than 1.0 became clear after 14 days at high temperature, while formulations with an HP-β-CD / bevacizumab ratio less than or equal to 1.0 became turbid. At pH 3.8, formulations with an HP-β-CD / bevacizumab ratio less than 0.5 became turbid after 14 days at high temperature, while formulations with an HP-β-CD / bevacizumab ratio greater than 0.5 became clear after 14 days at high temperature. At pH 4.2, formulation E12 became clear after 7 days at high temperature.

[0229] Table 17 Light inspection results

[0230] Note: High temperature: GW; Light: GZ

[0231] Table 18SEC-HPLC test results

[0232] Note: High temperature: GW; Light: GZ

[0233] It can be seen from Table 18, Figures 5 and 6 that the monomer contents of bevacizumab and batifiban in the compound decreased with the increase of bevacizumab concentration, while the aggregates increased. Within the pH range set in the experiment, as the pH increased, the monomer content showed a trend of gradual increase, while the aggregates gradually decreased.

Claims

1. A preparation comprising an anti-VEGF antibody or antigen-binding fragment and an integrin GPIIb / IIIa antagonist, wherein the anti-VEGF antibody or antigen-binding fragment comprises HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 2, HCDR3 shown in SEQ ID NO: 3, LCDR1 shown in SEQ ID NO: 4, LCDR2 shown in SEQ ID NO: 5, and LCDR3 shown in SEQ ID NO:

6.

2. The preparation according to claim 1, characterized in that The pH of the preparation is 3.0 to 8.0, or 3.0 to 6.0, or 3.0 to 5.0, or 3.5 to 4.5, or 3.8 to 4.2, or 3.8 to 4.

0.

3. The preparation according to claim 1 or 2, characterized in that The formulation further comprises one or a combination of a buffer, a stabilizer and a surfactant.

4. The preparation according to any one of claims 1 to 3, comprising: (1) anti-VEGF antibodies or antigen-binding fragments; (2) integrin GPIIb / IIIa antagonists; (3) Buffer; (4) stabilizers; and (5) surfactant; Alternatively, the pH of the formulation is 3.0 to 8.0, or 3.0 to 6.0, or 3.0 to 5.0, or 3.5 to 4.5, or 3.8 to 4.2, or 3.8 to 4.

0.

5. The preparation according to any one of claims 1 to 4, characterized in that The concentration of the anti-VEGF antibody or antigen-binding fragment is 1-200 mg / mL, or 1-150 mg / mL, or 10-120 mg / mL, or 10-100 mg / mL, or 20-80 mg / mL, or 25-75 mg / mL; and, The integrin GPIIb / IIIa antagonist is tirofiban, eptifibatide or batifiban, or a pharmaceutically acceptable salt thereof; or, the concentration of the integrin GPIIb / IIIa antagonist is 1-100 mg / mL, or 1-90 mg / mL, or 1-60 mg / mL, or 10-60 mg / mL, or 1-30 mg / mL, or 2-20 mg / mL, or 10-30 mg / mL, or 20 mg / mL; Alternatively, the mass ratio of the anti-VEGF antibody or antigen-binding fragment to the integrin GPIIb / IIIa antagonist is 1-10:1, or 1-8:1, or 1-4:1; Alternatively, the buffer is selected from acetic acid, acetate, histidine, histidine salt, phosphate, citric acid, citrate, citric acid, citrate, succinate, glutamate, or a combination thereof; Alternatively, the buffer is selected from acetate buffer, citrate buffer, histidine buffer; Alternatively, the concentration of the buffer is 1-50 mM, or 1-40 mM, or 1-30 mM, or 1-25 mM, or 20 mM, or 10 mM; Alternatively, the stabilizer is selected from polyols, sugars, amino acids, salts; Alternatively, the stabilizer is selected from one or more of sodium chloride, arginine or its salts, polyethylene glycol, sorbitol, mannitol, monosaccharides, oligosaccharides, polysaccharides, and sugar derivatives; Alternatively, the stabilizer is selected from one or more of cyclodextrin or its derivatives, sucrose and trehalose; Alternatively, the cyclodextrin or its derivatives are selected from α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxypropylated derivatives, hydroxyethylated derivatives, ethylated derivatives and methylated derivatives thereof, sulfobutyl ether β-cyclodextrin, branched cyclodextrin, cyclodextrin polymers, or combinations thereof; Alternatively, the stabilizer is a combination of hydroxypropyl beta-cyclodextrin and trehalose or sucrose; Alternatively, the concentration of the stabilizer is 1-200 mg / mL, or 10-150 mg / mL, or 50-150 mg / mL, or 70-130 mg / mL, or 80-130 mg / mL; Alternatively, the surfactant is selected from poloxamer and Tween; alternatively, the surfactant is selected from poloxamer 188, Tween 20 and Tween 80, or a combination thereof; alternatively, the concentration of the surfactant is 0.05-10 mg / mL, or 0.1-10 mg / mL, or 0.1-5 mg / mL, or 0.2-1 mg / mL, or 0.2-0.6 mg / mL, or 0.4 mg / mL.

6. The preparation according to any one of claims 1 to 5, comprising: (1) Anti-VEGF antibody or antigen-binding fragment: 1-200 mg / mL; (2) Batifiban: 1-100 mg / mL; (3) Acetate buffer, histidine buffer or citric acid buffer: 1-50 mM; (4) Stabilizer: 1 to 200 mg / mL; and (5) Tween 20 or Tween 80: 0.05-10 mg / mL; in, The pH of the preparation is 3.0-8.0, and the stabilizer is a combination of cyclodextrin or its derivatives and oligosaccharides; or, the stabilizer is a combination of hydroxypropyl beta-cyclodextrin and trehalose; or, the stabilizer is a combination of hydroxypropyl beta-cyclodextrin and sucrose; Alternatively, the formulation comprises: (1) Anti-VEGF antibody or antigen-binding fragment: 1-200 mg / mL; (2) Batifiban: 1-100 mg / mL; (3) Acetate buffer: 1-50 mM; (4) Hydroxypropyl beta-cyclodextrin: 1-200 mg / mL; (5) trehalose or sucrose: 1-200 mg / mL; and (6) Tween 20 or Tween 80: 0.05-10 mg / mL; Wherein, the pH of the preparation is 3.0 to 6.0; Alternatively, the formulation comprises: (1) Anti-VEGF antibody or antigen-binding fragment: 1-150 mg / mL; (2) Batifiban: 1-90 mg / mL; (3) Acetate buffer: 1-40 mM; (4) Hydroxypropyl beta-cyclodextrin: 10-150 mg / mL; (5) trehalose or sucrose: 10-150 mg / mL; and (6) Tween 20 or Tween 80: 0.1-10 mg / mL; Wherein, the pH of the preparation is 3.0 to 6.0; Alternatively, the formulation comprises: (1) Anti-VEGF antibody or antigen-binding fragment: 10-120 mg / mL; (2) Batifiban: 1-60 mg / mL; (3) Acetate buffer: 1-30 mM; (4) Hydroxypropyl beta-cyclodextrin: 20-100 mg / mL; (5) trehalose or sucrose: 20-100 mg / mL; and (6) Tween 20 or Tween 80: 0.1-5 mg / mL; Wherein, the pH of the preparation is 3.0 to 6.0; Alternatively, the formulation comprises: (1) Anti-VEGF antibody or antigen-binding fragment: 10-100 mg / mL; (2) Batifiban: 10-60 mg / mL; (3) Acetate buffer: 1-30 mM; (4) Hydroxypropyl beta-cyclodextrin: 20-80 mg / mL; (5) Trehalose or sucrose: 20-80 mg / mL; and (6) Tween 20 or Tween 80: 0.1-5 mg / mL; Wherein, the pH of the preparation is 3.0 to 6.0; Alternatively, the formulation comprises: (1) Anti-VEGF antibody or antigen-binding fragment: 20-80 mg / mL; (2) Batifiban: 1-30 mg / mL; (3) Acetate buffer: 5-25 mM; (4) Hydroxypropyl beta-cyclodextrin: 20-80 mg / mL; (5) Trehalose or sucrose: 20-80 mg / mL; and (6) Tween 20 or Tween 80: 0.2-1 mg / mL; Wherein, the pH of the preparation is 3.0 to 6.0; Alternatively, the formulation comprises: (1) Anti-VEGF antibody or antigen-binding fragment: 20-80 mg / mL; (2) Batifiban: 10-30 mg / mL; (3) Acetate buffer: 5-25 mM; (4) Hydroxypropyl beta-cyclodextrin: 30-70 mg / mL; (5) Trehalose dihydrate or sucrose: 30-70 mg / mL; and (6) Tween 20 or Tween 80: 0.2-1 mg / mL; Wherein, the pH of the preparation is 3.5 to 4.5; Alternatively, the formulation comprises: (1) Anti-VEGF antibody or antigen-binding fragment: 20-80 mg / mL; (2) Batifiban: 20 mg / mL; (3) Acetate buffer: 5-25 mM; (4) Hydroxypropyl beta-cyclodextrin: 40-60 mg / mL; (5) Trehalose dihydrate or sucrose: 50-65 mg / mL; and (6) Tween 20 or Tween 80: 0.2-1 mg / mL; Wherein, the pH of the preparation is 3.5 to 4.5; Alternatively, the formulation comprises: (1) Anti-VEGF antibody or antigen-binding fragment: 25-75 mg / mL; (2) Batifiban: 20 mg / mL; (3) Acetate buffer: 10-20 mM; (4) Hydroxypropyl beta-cyclodextrin: 40-60 mg / mL; (5) Trehalose dihydrate or sucrose: 50-65 mg / mL; and (6) Tween 20 or Tween 80: 0.2-1 mg / mL; Wherein, the pH of the preparation is 3.5-4.

5.

7. The preparation of any one of claims 1 to 6, wherein the anti-VEGF antibody or antigen-binding fragment comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises the sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the sequence shown in SEQ ID NO:

8.

8. The preparation according to claim 7, characterized in that The anti-VEGF antibody comprises a heavy chain and a light chain; the heavy chain comprises the sequence shown in SEQ ID NO:9, and the light chain comprises the sequence shown in SEQ ID NO:10; Alternatively, the anti-VEGF antibody is bevacizumab.

9. A solid preparation, characterized in that: It is prepared by freeze-drying the preparation according to any one of claims 1 to 8.

10. Use of the preparation according to any one of claims 1 to 9 or the solid preparation according to claim 8 in the preparation of a medicament for treating VEGF-related diseases.

11. Use of the preparation according to any one of claims 1 to 8 or the solid preparation according to claim 9 in treating VEGF-related diseases; or, the VEGF-related disease is cancer or a tumor; for example, the cancer or tumor is selected from colorectal cancer, lung cancer, glioblastoma, hepatocellular carcinoma, epithelial ovarian cancer, fallopian tube cancer, primary peritoneal cancer, cervical cancer, breast cancer, renal cell carcinoma, glioblastoma, glioma, glioma, tumor metastasis, neuromyelitis optica, B-cell chronic lymphocytic leukemia, sarcoma, liver cancer, multiple myeloma, brain cancer; Alternatively, the disease is an eye disease in which VEGF is overexpressed; for example, the VEGF-related disease is selected from macular degeneration, age-related macular degeneration (AMD), corneal neovascularization, diseases associated with corneal neovascularization, retinal neovascularization, diseases associated with retinal / choroidal neovascularization, choroidal neovascularization secondary to pathological myopia, iris neovascularization, intraocular neovascularization, ocular neovascularization, neovascular glaucoma, macular edema, diabetic macular edema (DME, including localized, non-central DME and diffuse, including central DME), cystoid macular edema (CME), retinopathy, diabetic retinopathy (DR, including proliferative DR (PDR), non-proliferative DR (NPDR), and high altitude DR), other ischemia-related retinopathy, retinopathy of prematurity (ROP), familial exudative vitreoretinopathy (FEVR), hypertensive retinopathy, retinal vein occlusion (RVO) (including branch retinal vein occlusion and central retinal vein occlusion), retinal myeloid nerve fiber (CNV (including myopic CNV)), pathological myopia, Hippel-Lindau disease, Coronz disease, Norrie disease, osteoporosis-pseudoglioma syndrome (OPPG), subconjunctival hemorrhage, rubeosis, retinitis pigmentosa (RP), retinal angiomatous proliferation, macular telangiectasia, retinal degeneration, vasculitis, optic disc edema, retinitis, conjunctivitis (including infectious conjunctivitis and non-infectious (e.g., allergic) conjunctivitis), Leber congenital amaurosis, uveitis (including infectious and non-infectious uveitis), choroiditis, ocular histoplasmosis, blepharitis, dry eye, traumatic eye injury and Sjögren's disease.

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