[go: up one dir, main page]

WO2025140553A1 - Composition pharmaceutique contenant une protéine de fusion anti-vegf - Google Patents

Composition pharmaceutique contenant une protéine de fusion anti-vegf Download PDF

Info

Publication number
WO2025140553A1
WO2025140553A1 PCT/CN2024/143188 CN2024143188W WO2025140553A1 WO 2025140553 A1 WO2025140553 A1 WO 2025140553A1 CN 2024143188 W CN2024143188 W CN 2024143188W WO 2025140553 A1 WO2025140553 A1 WO 2025140553A1
Authority
WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
fusion protein
protein
arginine
tween
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/CN2024/143188
Other languages
English (en)
Chinese (zh)
Inventor
周茂
罗祖秀
邓星
黄倩
柯潇
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chengdu Kanghong Biotechnologies Co Ltd
Original Assignee
Chengdu Kanghong Biotechnologies Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chengdu Kanghong Biotechnologies Co Ltd filed Critical Chengdu Kanghong Biotechnologies Co Ltd
Publication of WO2025140553A1 publication Critical patent/WO2025140553A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/179—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0048—Eye, e.g. artificial tears
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the present invention relates to the field of pharmaceutical preparations, and in particular to a pharmaceutical composition containing an anti-VEGF fusion protein.
  • 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.
  • VEGF signaling 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • stability e.g., polymerization rate of fusion protein A
  • protein fragments C and D have less influence on stability and activity.
  • the present invention provides a pharmaceutical composition, which contains an anti-VEGF fusion protein A and 0.01-7% of protein fragment B.
  • the pharmaceutical composition contains an anti-VEGF fusion protein A and 0.01-5.4% of protein fragment B.
  • the pharmaceutical composition contains an anti-VEGF fusion protein A and 0.01-4.6% of protein fragment B.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • the content of fusion protein A and protein fragments described in the present invention is determined by gel electrophoresis (SDS-PAGE).
  • 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 ⁇ g 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).
  • protein fragments B, C, and D can be removed or their content can be controlled by gel chromatography.
  • 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.
  • 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.
  • 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.
  • sample proteins with different fragment contents can be obtained according to different peak collection parameters.
  • 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.
  • 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.
  • 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 s
  • 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.
  • 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.
  • 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.
  • composition which contains:
  • 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 comprises:
  • 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.
  • the pharmaceutical composition comprises:
  • 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.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition contains:
  • the pH is 7.5-8.7.
  • the pH is 7.5-8.7.
  • 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.
  • 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 ⁇ l inoculated in each well.
  • 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 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%.
  • composition of the preparation samples is as follows:
  • Proteins of different purities 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.
  • composition of the preparation samples is as follows:
  • Proteins of different purities 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%.
  • composition of the preparation samples is as follows:
  • the sample formulation is as follows:
  • the sample formulation is as follows:
  • 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%.
  • the sample formulation is as follows:
  • 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.
  • the sample formulation is as follows:
  • 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.
  • 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.
  • 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.
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • General Health & Medical Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Epidemiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Ophthalmology & Optometry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Immunology (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biochemistry (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Vascular Medicine (AREA)
  • Dermatology (AREA)
  • Cell Biology (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Medicinal Preparation (AREA)

Abstract

L'invention concerne une composition pharmaceutique contenant une protéine de fusion anti-VEGF. La composition pharmaceutique contient une protéine de fusion A anti-VEGF et un fragment de protéine B. En réglant la teneur en fragment de protéine B dans la composition pharmaceutique à une valeur comprise entre 0,1 % et 5,4 %, le taux de polymérisation des protéines dans la composition peut être réduit, la stabilité des protéines peut être améliorée, et une meilleure activité biologique peut être maintenue.
PCT/CN2024/143188 2023-12-27 2024-12-27 Composition pharmaceutique contenant une protéine de fusion anti-vegf Pending WO2025140553A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202311840919 2023-12-27
CN202311840919.7 2023-12-27

Publications (1)

Publication Number Publication Date
WO2025140553A1 true WO2025140553A1 (fr) 2025-07-03

Family

ID=96114167

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2024/143188 Pending WO2025140553A1 (fr) 2023-12-27 2024-12-27 Composition pharmaceutique contenant une protéine de fusion anti-vegf

Country Status (2)

Country Link
CN (1) CN120204358A (fr)
WO (1) WO2025140553A1 (fr)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1706867A (zh) * 2004-06-08 2005-12-14 成都康弘科技实业（集团）有限公司 抑制血管新生的融合蛋白质及其用途
CN1793179A (zh) * 2005-11-04 2006-06-28 余波 包含vegf受体片段的优化融合蛋白质及其医疗应用
US20060217311A1 (en) * 2005-03-25 2006-09-28 Daniel Dix VEGF antagonist formulations
CN1915427A (zh) * 2006-03-31 2007-02-21 成都康弘生物科技有限公司 Vegf受体融合蛋白在治疗眼睛疾病中的应用
CN103212075A (zh) * 2012-01-19 2013-07-24 成都康弘生物科技有限公司 一种含有vegf拮抗剂的滴眼液
CN110618229A (zh) * 2018-06-20 2019-12-27 成都康弘生物科技有限公司 一种蛋白的非还原肽图分析方法
CN110618202A (zh) * 2018-06-20 2019-12-27 成都康弘生物科技有限公司 一种检测蛋白纯度的方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1706867A (zh) * 2004-06-08 2005-12-14 成都康弘科技实业（集团）有限公司 抑制血管新生的融合蛋白质及其用途
US20060217311A1 (en) * 2005-03-25 2006-09-28 Daniel Dix VEGF antagonist formulations
CN1793179A (zh) * 2005-11-04 2006-06-28 余波 包含vegf受体片段的优化融合蛋白质及其医疗应用
CN1915427A (zh) * 2006-03-31 2007-02-21 成都康弘生物科技有限公司 Vegf受体融合蛋白在治疗眼睛疾病中的应用
CN103212075A (zh) * 2012-01-19 2013-07-24 成都康弘生物科技有限公司 一种含有vegf拮抗剂的滴眼液
CN110618229A (zh) * 2018-06-20 2019-12-27 成都康弘生物科技有限公司 一种蛋白的非还原肽图分析方法
CN110618202A (zh) * 2018-06-20 2019-12-27 成都康弘生物科技有限公司 一种检测蛋白纯度的方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG TAO, ZHANG DA-PENG, YANG YANG, XU JING, SHI JING-PING, QIAN WEI-ZHU, LI BO-HUA, WEI YU-QUAN, WANG HAO: " Characterization of a novel vascular endothelial growth factor receptor-immunoglobulin fusion protein variant", CURRENT IMMUNOLOGY., no. 5, 30 September 2010 (2010-09-30), pages 361 - 364, XP093330039 *

Also Published As

Publication number Publication date
CN120204358A (zh) 2025-06-27

Similar Documents

Publication Publication Date Title
Zhang et al. The pharmacology study of a new recombinant human VEGF receptor-fc fusion protein on experimental choroidal neovascularization
CN118948760A (zh) 含有高浓度vegf受体融合蛋白的制剂
AU2012326084B2 (en) Etanercept formulations stabilized with combinations of sugars and polyols
KR102177437B1 (ko) 리포좀 제제
TWI675844B (zh) 多靶向融合蛋白及其應用
CN103533950A (zh) Vegf拮抗剂在治疗血管生成眼病中的用途
JP6450364B2 (ja) 眼送達のためのキメラサイトカイン製剤
TW201711697A (zh) 抗－ｃｇｒｐ抗體調配物
RS63703B1 (sr) Upotreba antagonista vegf za lečenje angiogenih očnih poremećaja
WO2012111762A1 (fr) Préparation pharmaceutique d'anticorps anti-cd40 très concentrée
KR20120130752A (ko) 혈관신생-관련된 안질환의 치료를 위한 조성물 및 방법
US20150196645A1 (en) Stable Aqueous Formulations of Etanercept
US20160130321A1 (en) Use of a vegf antagonist in treating macular edema
TW201838646A (zh) 眼用藥用組合物
WO2019020777A1 (fr) Formulation liquide d'un antagoniste du vegf
Foy et al. Local tolerance and systemic safety of pegaptanib sodium in the dog and rabbit
CN103816115B (zh) 一种含有抑制血管增生的融合蛋白的药物组合物及用途
JP2024533245A (ja) Ｖｅｇｆ受容体融合タンパク質の製剤
WO2025140553A1 (fr) Composition pharmaceutique contenant une protéine de fusion anti-vegf
WO2022242527A1 (fr) Formulation de protéine hybride anticorps-cytokine monoclonale
WO2024056058A1 (fr) Composition pharmaceutique stable à haute concentration à tamponnage spontané
EP4438629A1 (fr) Molécule de liaison de ligand multispécifique et son utilisation
WO2023281367A1 (fr) Formulations liquides stables d'aflibercept
RU2817350C2 (ru) Липосомальные препараты
WO2024236190A1 (fr) Annexine a5 monomère destinée à être utilisée dans le traitement d'un oedème maculaire ou d'une occlusion veineuse rétinienne

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24911472

Country of ref document: EP

Kind code of ref document: A1