WO2022122034A1 - 一种抗ctla-4抗体及融合蛋白的制剂 - Google Patents

一种抗ctla-4抗体及融合蛋白的制剂 Download PDF

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WO2022122034A1
WO2022122034A1 PCT/CN2021/137235 CN2021137235W WO2022122034A1 WO 2022122034 A1 WO2022122034 A1 WO 2022122034A1 CN 2021137235 W CN2021137235 W CN 2021137235W WO 2022122034 A1 WO2022122034 A1 WO 2022122034A1
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composition
ctla
antibody
preparation
monoclonal antibody
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PCT/CN2021/137235
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English (en)
French (fr)
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方国波
蔡则玲
吴珩
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上海赛金生物医药有限公司
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Publication of WO2022122034A1 publication Critical patent/WO2022122034A1/zh

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    • 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
    • A61K9/08—Solutions
    • 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
    • A61K47/02—Inorganic compounds
    • 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
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • 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
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • 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
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/51—Complete heavy chain or Fd fragment, i.e. VH + CH1
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/515—Complete light chain, i.e. VL + CL

Definitions

  • the invention relates to the field of pharmaceutical preparations, in particular to a preparation of an anti-CTLA-4 antibody and a fusion protein.
  • Recombinant anti-CTLA-4 fully human monoclonal antibody is a T cell immune function regulator, which competes with B7-1 molecules on the surface of APC cells to bind to CTLA-4 molecules on the surface of T cells, blocking the immune function regulated by B7-1 molecules. inhibit function.
  • the recombinant anti-CTLA-4 fully human monoclonal antibody has a molecular weight of about 145KD, consists of 1324 amino acids, and is expressed and secreted in CHO cells.
  • the object of the present invention is to provide an anti-CTLA-4 antibody composition, which inhibits the aggregation and degradation of the anti-CTLA-4 antibody, thereby improving the stability of the anti-CTLA-4 antibody.
  • a first aspect of the present invention provides an anti-CTLA-4 antibody composition, characterized in that the composition comprises an anti-CTLA-4 monoclonal antibody, a surfactant, a buffer and a stabilizer.
  • the anti-CTLA-4 antibody has the heavy chain amino acid sequence of SEQ ID NO.: 1 and the light chain amino acid sequence of SEQ ID NO.: 2.
  • the anti-CTLA-4 antibody has the heavy chain amino acid sequence of SEQ ID NO.:3 and the light chain amino acid sequence of SEQ ID NO.:4.
  • the anti-CTLA-4 antibody has the heavy chain amino acid sequence of SEQ ID NO.:5 and the light chain amino acid sequence of SEQ ID NO.:6.
  • the composition is a pharmaceutical composition.
  • the antibody includes a human antibody.
  • the antibody includes a monoclonal antibody.
  • the anti-CTLA-4 monoclonal antibody includes human anti-CTLA-4 monoclonal antibody.
  • the anti-CTLA-4 monoclonal antibody includes an anti-CTLA-4 fully human monoclonal antibody.
  • the anti-CTLA-4 fully human monoclonal antibody includes recombinant anti-CTLA-4 fully human monoclonal antibody.
  • the content of the anti-CTLA-4 monoclonal antibody is 0.5-50 mg/mL, preferably 0.5-40 mg/mL, more preferably 0.5-30 mg/mL, more preferably 0.5-20 mg/mL mL, more preferably 1-15 mg/mL, more preferably 2-10 mg/mL, more preferably 3-8 mg/mL, most preferably 5 mg/mL.
  • the surfactant is selected from the group consisting of positive ionic surfactant, negative ionic surfactant, nonionic surfactant, or a combination thereof.
  • the surfactant is selected from the following group: polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerol fatty acid ester, polysorbate, poloxamer, or a combination thereof.
  • the polysorbate is selected from the following group: polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40, Tween-40) , Polysorbate 60 (PS-60, Tween-60), Polysorbate 80 (PS-80, Tween-80).
  • the poloxamer is selected from the group consisting of poloxamer 188, poloxamer 108, poloxamer 124, or a combination thereof.
  • the surfactant includes polysorbate.
  • the surfactant includes polysorbate 80 (PS-80).
  • the content of the surfactant is 0.001-8wt%, preferably 0.001-3wt%, more preferably 0.001-1wt%, more preferably 0.002-0.5wt%, more preferably 0.002- 0.1wt%, more preferably 0.002-0.06wt%, more preferably 0.005-0.05wt%, more preferably 0.005-0.03wt%, more preferably 0.008-0.02wt%, more preferably 0.008-0.012wt%, most Preferably 0.01 wt%, based on the total weight of the composition.
  • the buffer is selected from the group consisting of citrate-citrate buffer, phosphate-phosphate buffer, or a combination thereof.
  • the medium of the buffer solution is water.
  • the concentration of the buffer is 3-80mM, preferably 5-60mM, more preferably 5-40mM, more preferably 10-30mM, more preferably 15-25mM, more preferably 18 -22mM.
  • the buffer includes phosphate buffer.
  • the concentration of the phosphate buffer is 3-80mM, preferably 5-60mM, more preferably 5-40mM, more preferably 10-30mM, more preferably 15-25mM, more preferably 18-22mM, optimally.
  • the stabilizer is selected from the group consisting of amino acids, sugar alcohols, or a combination thereof.
  • the amino acid is selected from the group consisting of proline, arginine (Arginine), glycine (Glycine), histidine (Histidine), methionine (Methionine), or a combination thereof .
  • the sugar alcohol is selected from the group consisting of sucrose (Sucrose), mannitol (Mannitol), trehalose (Trehalose), maltose (Maltose), sorbitol (Sorbitol), or a combination thereof.
  • the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, or a combination thereof.
  • the stabilizer includes mannitol.
  • the sugar alcohol includes mannitol.
  • the content of the stabilizer is 0.5-50wt%, preferably 0.5-40wt%, more preferably 0.8-30wt%, more preferably 1-20wt%, more preferably 1-15wt% %, more preferably 1-10 wt%, more preferably 1-8 wt%, more preferably 2-7 wt%, most preferably 4 wt% or 5 wt%, based on the total weight of the composition.
  • the composition does not include an osmotic pressure regulator.
  • the composition further includes an osmotic pressure regulator.
  • the osmotic pressure regulator includes inorganic salts.
  • the inorganic salt is selected from the group consisting of sodium salt, potassium salt, calcium salt, magnesium salt, chloride salt, sulfate salt, nitrate salt, or a combination thereof.
  • the inorganic salt is selected from the following group: sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium sulfate, potassium sulfate, calcium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, nitric acid Calcium, magnesium nitrate, or a combination thereof.
  • the inorganic salts include sodium chloride.
  • the content of the osmotic pressure regulator is 0-80 mM, preferably 0-60 mM, more preferably 0-50 mM, more preferably 0-30 mM.
  • the weight ratio of the stabilizer to the surfactant is 350-450:1, preferably 380-420:1, more preferably 390-400:1.
  • the weight ratio of the stabilizer to the surfactant is 450-550:1, preferably 480-520:1, more preferably 490-510:1.
  • the weight ratio of the mannitol to the Tween 80 is 350-450:1, preferably 380-420:1, more preferably 390-400:1.
  • the weight ratio of the mannitol to the Tween 80 is 450-550:1, preferably 480-520:1, more preferably 490-510:1.
  • the content of the osmotic pressure regulator is 0Mm.
  • the content of the osmotic pressure regulator is 10-50 mM, preferably 20-40 mM, more preferably 25-30 mM, and most preferably 0 mM or 30 mM.
  • the composition further includes an antioxidant.
  • the antioxidant includes EDTA (ethylenediaminetetraacetic acid).
  • the pH of the composition is 5.0-7.5, preferably 5.0-7.0, more preferably 5.5-6.5, more preferably 5.8-6.2, and most preferably 6.0.
  • the composition is a liquid preparation.
  • the composition is an oral liquid preparation or an injection liquid preparation.
  • the composition is a pharmaceutical composition.
  • the dosage form of the composition is an oral preparation or an injection preparation.
  • composition includes:
  • Anti-CTLA-4 antibody 1-15mg/mL Phosphate buffer 10-30mM Tween 80 0.005-0.05wt% Mannitol 1-15wt% NaCl 10-50mM.
  • composition includes:
  • Anti-CTLA-4 antibody 2-10mg/mL Phosphate buffer 15-25mM Tween 80 0.005-0.03wt% Mannitol 1-8wt% NaCl 20-40mM.
  • composition includes:
  • Anti-CTLA-4 antibody 1-15mg/mL Phosphate buffer 10-30mM Tween 80 0.005-0.05wt% Mannitol 1-15wt%
  • composition includes:
  • Anti-CTLA-4 antibody 2-10mg/mL Phosphate buffer 15-25mM Tween 80 0.005-0.03wt% Mannitol 1-8wt%
  • freeze-dried preparation is provided, and the freeze-dried preparation is prepared by the following method:
  • composition according to the first aspect of the present invention is freeze-dried to obtain a freeze-dried preparation.
  • the freeze-dried preparation further includes a freeze-dried protective agent.
  • the lyoprotectant is selected from the group consisting of glucose, mannitol, fructose, galactose, or a combination thereof.
  • the third aspect of the present invention provides a use of the composition according to the first aspect of the present invention or the freeze-dried preparation according to the second aspect of the present invention for preparing a medicament for (i) prevention and/or treatment of unresectable or metastatic melanoma; (ii) prevention and/or treatment of advanced renal cell carcinoma; (iii) prevention and/or treatment of metastatic colorectal cancer with high microsatellite instability or mismatch repair deficiency and/or (iv) for adjuvant treatment of melanoma.
  • a fourth aspect of the present invention provides a method for (i) preventing and/or treating unresectable or metastatic melanoma; (ii) preventing and/or treating advanced renal cell carcinoma; (iii) preventing and/or treating microsatellite height insensitivity Metastatic colorectal cancer deficient in stability or mismatch repair; and/or (iv) a method for adjuvant treatment of melanoma, the method comprising: administering to a subject in need a composition according to the first aspect of the present invention .
  • the subject is a human or a non-human mammal.
  • Figure 1 Non-reducing electropherogram of preparation samples incubated at 60°C for 24 hours.
  • the inventors have developed a pharmaceutical composition, which can effectively maintain the stability of the anti-CTLA-4 antibody, and the anti-CTLA-4 antibody in the composition can be used in the compression test (high temperature, freeze-thaw) conditions can maintain its stability, therefore, the composition of the present invention can stabilize the quality of the anti-CTLA-4 antibody, prolong the shelf life of the product, and improve the safety of clinical practice.
  • the present invention has been completed.
  • the terms “comprising,” “including,” and “containing” are used interchangeably to include not only closed definitions, but also semi-closed, and open definitions. In other words, the terms include “consisting of”, “consisting essentially of”.
  • the weight content (wt.%) or concentration (eg, mM, mg/mL) of each component is based on the weight or volume of the pharmaceutical composition.
  • Tris As used herein, tris is abbreviated as Tris, ie “tris” and “Tris” are used interchangeably.
  • EDTA is ethylenediaminetetraacetic acid.
  • phosphate buffer is an aqueous phosphate - phosphate buffer solution, wherein the content is in terms of phosphate ions, eg, 20 mM phosphate buffer refers to a concentration of 20 mM converted to PO43- .
  • anti-CTLA-4 antibody consists of two identical light chains (L) and two identical heavy chains (H). At one end of each heavy chain is a variable region (VH) followed by a number of constant regions. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is opposite the first constant domain of the heavy chain, and the variable domain of the light chain is opposite the variable domain of the heavy chain .
  • the anti-CTLA-4 antibody described in the present invention is not particularly limited, and is preferably a human anti-CTLA-4 antibody, such as an anti-CTLA-4 fully human monoclonal antibody.
  • the anti-CTLA-4 antibody of the present invention has the heavy chain amino acid sequence of SEQ ID NO.: 1 and the light chain amino acid sequence of SEQ ID NO.: 2;
  • amino acid sequence of SEQ ID NO.: 1 is as follows:
  • amino acid sequence of SEQ ID NO.: 2 is as follows:
  • the anti-CTLA-4 antibody of the present invention has the heavy chain amino acid sequence of SEQ ID NO.:3 and the light chain amino acid sequence of SEQ ID NO.:4;
  • amino acid sequence of SEQ ID NO.: 3 is as follows:
  • amino acid sequence of SEQ ID NO.:4 is as follows:
  • the anti-CTLA-4 antibody of the present invention has the heavy chain amino acid sequence of SEQ ID NO.:5 and the light chain amino acid sequence of SEQ ID NO.:6;
  • amino acid sequence of SEQ ID NO.:5 is as follows:
  • amino acid sequence of SEQ ID NO.: 6 is as follows:
  • the present invention provides an anti-CTLA-4 antibody composition, which comprises an anti-CTLA-4 monoclonal antibody, a surfactant, a buffer and a stabilizer.
  • composition of the present invention is preferably a pharmaceutical composition.
  • compositions of the present invention are preferably liquid preparations, such as oral liquid preparations or injectable liquid preparations.
  • the content of the anti-CTLA-4 monoclonal antibody is 0.5-50 mg/mL, preferably 0.5-40 mg/mL, more preferably 0.5-30 mg/mL, more preferably 0.5-20 mg/mL, more preferably 1-15 mg/mL, more preferably 2-10 mg/mL, more preferably 3-8 mg/mL, most preferably 5 mg/mL.
  • the surfactant is selected from the group consisting of positive ionic surfactant, negative ionic surfactant, nonionic surfactant, or a combination thereof.
  • the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid ester, polyoxyethylene hydrogenated castor oil, glycerol fatty acid ester, polysorbate, poloxamer, or a combination thereof .
  • the polysorbate is selected from the group consisting of polysorbate 20 (PS-20, Tween-20), polysorbate 40 (PS-40, Tween-40), polysorbate Alcohol ester 60 (PS-60, Tween-60), polysorbate 80 (PS-80, Tween-80).
  • the poloxamer is selected from the group consisting of poloxamer 188, poloxamer 108, poloxamer 124, or a combination thereof.
  • the surfactants include polysorbates.
  • the surfactant includes polysorbate 80 (PS-80).
  • the content of the surfactant can be 0.001-8wt%, preferably 0.001-3wt%, more preferably 0.001-1wt%, more preferably 0.002-0.5wt%, more preferably preferably 0.002-0.1wt%, more preferably 0.002-0.06wt%, more preferably 0.005-0.05wt%, more preferably 0.005-0.03wt%, more preferably 0.008-0.02wt%, more preferably 0.008-0.012 wt%, optimally 0.01 wt%, based on the total weight of the composition.
  • the buffer is selected from the group consisting of citrate-citrate buffer, phosphate-phosphate buffer, or a combination thereof.
  • the medium of the buffer solution is water.
  • the concentration of the buffer can be 3-80mM, preferably 5-60mM, more preferably 5-40mM, more preferably 10-30mM, more preferably 15-25mM, More preferably 18-22mM.
  • the buffer includes a phosphate buffer.
  • the concentration of the phosphate buffer is 3-80mM, preferably 5-60mM, more preferably 5-40mM, more preferably 10-30mM, more preferably 15-25mM, more preferably 18- 22mM, optimally.
  • the stabilizer is selected from the group consisting of amino acids, sugar alcohols, or a combination thereof.
  • the amino acid is selected from the group consisting of Proline, Arginine, Glycine, Histidine, Methionine, or a combination thereof.
  • the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, maltose, sorbitol, or a combination thereof.
  • the sugar alcohol is selected from the group consisting of sucrose, mannitol, trehalose, sorbitol, or a combination thereof.
  • the stabilizer includes mannitol.
  • the sugar alcohol includes mannitol.
  • the content of the stabilizer can be 0.5-50wt%, preferably 0.5-40wt%, more preferably 0.8-30wt%, more preferably 1-20wt%, more preferably Preferably 1-15 wt%, more preferably 1-10 wt%, more preferably 1-8 wt%, more preferably 2-7 wt%, most preferably 4 wt% or 5 wt%, based on the total weight of the composition.
  • the composition does not include an osmotic pressure regulator.
  • the composition further includes an osmotic pressure regulator.
  • the osmotic pressure regulator includes inorganic salts.
  • the inorganic salt is selected from the group consisting of sodium, potassium, calcium, magnesium, chloride, sulfate, nitrate, or combinations thereof.
  • the inorganic salt is selected from the group consisting of sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium sulfate, potassium sulfate, calcium sulfate, magnesium sulfate, sodium nitrate, potassium nitrate, calcium nitrate, nitric acid Magnesium, or a combination thereof.
  • the inorganic salts include sodium chloride.
  • the content of the osmotic pressure regulator is 0-80 mM, preferably 0-60 mM, more preferably 0-50 mM, more preferably 0-30 mM.
  • the weight ratio of the stabilizer to the surfactant is 350-450:1, preferably 380-420:1, more preferably 390-400:1.
  • the weight ratio of the stabilizer to the surfactant is 450-550:1, preferably 480-520:1, more preferably 490-510:1.
  • the weight ratio of the mannitol to the Tween 80 is 350-450:1, preferably 380-420:1, more preferably 390-400:1.
  • the weight ratio of the mannitol to the Tween 80 is 450-550:1, preferably 480-520:1, more preferably 490-510:1.
  • the content of the osmotic pressure regulator is 0Mm.
  • the content of the osmotic pressure regulator is 10-50 mM, preferably 20-40 mM, more preferably 25-30 mM, and most preferably 0 mM or 30 mM.
  • the composition further includes an antioxidant.
  • the antioxidant includes EDTA (ethylenediaminetetraacetic acid).
  • the pH of the composition is 5.0-7.5, preferably 5.0-7.0, more preferably 5.5-6.5, more preferably 5.8-6.2, and most preferably 6.0.
  • the composition comprises:
  • Anti-CTLA-4 antibody 1-15mg/mL Phosphate buffer 10-30mM Tween 80 0.005-0.05wt% Mannitol 1-15wt% NaCl 10-50mM.
  • the composition comprises:
  • Anti-CTLA-4 antibody 2-10mg/mL Phosphate buffer 15-25mM Tween 80 0.005-0.03wt% Mannitol 1-8wt% NaCl 20-40mM.
  • the composition comprises:
  • Anti-CTLA-4 antibody 1-15mg/mL Phosphate buffer 10-30mM Tween 80 0.005-0.05wt% Mannitol 1-15wt%
  • the composition comprises:
  • Anti-CTLA-4 antibody 2-10mg/mL Phosphate buffer 15-25mM Tween 80 0.005-0.03wt% Mannitol 1-8wt%
  • the present invention also provides a freeze-dried preparation, which is prepared by the following method:
  • composition according to the present invention is freeze-dried to obtain a freeze-dried preparation.
  • the freeze-dried preparation further includes a freeze-dried protective agent.
  • the lyoprotectant is selected from the group consisting of glucose, mannitol, fructose, galactose, or a combination thereof.
  • the present invention also provides a use of the composition or the freeze-dried preparation of the present invention for preparing a medicament for (i) preventing and/or treating unresectable or metastatic melanoma; (ii) ) prevention and/or treatment of advanced renal cell carcinoma; (iii) prevention and/or treatment of metastatic colorectal cancer with high microsatellite instability or mismatch repair deficiency; and/or (iv) adjuvant therapy for melanoma.
  • the present invention also provides a method of (i) preventing and/or treating unresectable or metastatic melanoma; (ii) preventing and/or treating advanced renal cell carcinoma; (iii) preventing and/or treating microsatellite high instability or mismatch repair deficient metastatic colorectal cancer; and/or (iv) a method for adjuvant therapy of melanoma, the method comprising: administering to a subject in need the composition or the lyophilized preparation of the present invention.
  • the present invention provides a composition containing an anti-CTLA-4 antibody, which can improve the stability of the anti-CTLA-4 antibody.
  • the anti-CTLA-4 antibody composition of the present invention can maintain the stability of the anti-CTLA-4 antibody for at least 18-24 months at 2-8°C.
  • amino acid sequence of the heavy chain of the recombinant anti-CTLA-4 fully human monoclonal antibody in the following Examples 1-3 is as SEQ ID NO.: 1
  • amino acid sequence of the light chain is as SEQ ID NO.: 2
  • amino acid sequence of SEQ ID NO.: 1 is as follows:
  • amino acid sequence of SEQ ID NO.: 2 is as follows:
  • SEC-HPLC is size exclusion chromatography, which is used to analyze the purity of recombinant anti-CTLA-4 fully human monoclonal antibody, specifically, to detect the aggregation and degradation of recombinant anti-CTLA-4 fully human monoclonal antibody.
  • CEX-HPLC is a cation exchange chromatography used to analyze the purity of the recombinant anti-CTLA-4 fully human monoclonal antibody, specifically, to detect the charge heterogeneity of the recombinant anti-CTLA-4 fully human monoclonal antibody.
  • Non-reducing gel electrophoresis (NR SDS-PAGE) was used to separate the denatured samples according to the size of the molecules.
  • the separated protein bands were stained with Coomassie blue.
  • the content of the stained protein bands was analyzed by software analysis of recombinant anti-CTLA-4 whole human.
  • the source monoclonal antibody corresponds to the relative purity of each component protein, and the chemical degradation of the sample is mainly evaluated.
  • DSC differential scanning calorimetry. Specifically, Tm (melting temperature), Tagg (aggregation temperature), Tonset (denaturation temperature) were determined by the fluorescence change of recombinant anti-CTLA-4 fully human monoclonal antibody in different states. threshold)
  • the precipitation status of recombinant anti-CTLA-4 fully human monoclonal antibody was determined by visual observation.
  • thermo stability is the Tm value, Tagg value and Tonset value of recombinant anti-CTLA-4 fully human monoclonal antibody detected by DSC.
  • thermodynamic parameters of the samples were studied in Table 1, and the thermodynamic effects of pH, NaCl, carbohydrates, arginine, Tween 80, EDTA, buffer type and buffer concentration on the recombinant anti-CTLA-4 fully human monoclonal antibody were investigated. impact on stability.
  • the concentration of recombinant anti-CTLA-4 fully human monoclonal antibody in each preparation is 5 mg/mL; "%” is weight percentage (wt%), based on the total weight of the preparation.
  • the term "purity" as used herein refers to the purity of the recombinant anti-CTLA-4 fully human monoclonal antibody detected by SEC-HPLC and CEX-HPLC.
  • the recombinant anti-CTLA-4 fully human monoclonal antibody monomer molecule is affected by the external environment. , may form multimers, undergo degradation, undergo chemical modifications (including deamination, oxidation, pyroglutamidation, N-terminal lysine removal, etc.), analyze monomers, aggregates, small molecule fragments by SEC-HPLC
  • the relative content of the recombinant anti-CTLA-4 fully human monoclonal antibody was analyzed by CEX-HPLC.
  • Example 1 the influence of various factors on the stability of the sample was determined mainly by investigating the change trend of the thermodynamic parameters of the composition of the excipients and preparations. The effect of sample stability.
  • the concentration of recombinant anti-CTLA-4 fully human monoclonal antibody with a concentration of 5 mg/mL was processed into the preparation formula in Table 3.
  • the preparation was placed in a 37 °C incubator, and after 2 weeks of incubation, the recombinant anti-CTLA-4 fully human monoclonal antibody was determined.
  • the purity and charge heterogeneity of the antibody, the formulation of each preparation and the test results of purity are shown in Table 3.
  • the concentration of recombinant anti-CTLA-4 fully human monoclonal antibody in each preparation is 5 mg/mL; the monomer is the purity of recombinant anti-CTLA-4 fully human monoclonal antibody determined by SEC-HPLC, and the main peak is determined by CEX-HPLC Recombinant anti-CTLA-4 fully human monoclonal antibody purity; "%" is weight percent (wt%), based on the total weight of the formulation.
  • the formulation of the recombinant anti-CTLA-4 fully human monoclonal antibody should include phosphate buffer, mannitol, NaCl, Tween 80, and the recombinant anti-CTLA-4 fully human monoclonal
  • concentration of the cloned antibody was 5 mg/mL, and the preparation samples were prepared according to Table 4.
  • the influence of the concentration of excipients and pH on the physicochemical properties of the recombinant anti-CTLA-4 fully human monoclonal antibody was evaluated, and the preparation prescription was confirmed.
  • test conditions were freeze-thaw accelerated test and high temperature accelerated test.
  • the stability of the formulation composition was investigated by freeze-thaw experiments.
  • the test conditions were that the preparations were frozen at -20°C and thawed at room temperature, and the number of cycles was 3 and 5, respectively.
  • the stability of the preparation composition was evaluated by the appearance before and after freezing and thawing, the relative content of the recombinant anti-CTLA-4 fully human monoclonal antibody and the purity of the recombinant anti-CTLA-4 fully human monoclonal antibody determined by SEC-HPLC.
  • the results of the freeze-thaw acceleration test showed that: during the freeze-thaw process, the recombinant anti-CTLA-4 fully human monoclonal antibody was prone to aggregation and even precipitation; increasing the content of mannitol and reducing the content of NaCl was beneficial to inhibit the recombinant anti-CTLA-4 fully human monoclonal antibody.
  • the aggregation and precipitation of the source monoclonal antibody during freezing and thawing are particularly obvious under the conditions of relatively high pH (F3, F6 and F12), presumably because increasing the mannitol content is beneficial to inhibit the precipitation of NaCl in the form of crystals during cryopreservation ;
  • the concentration of EDTA had no obvious effect on the freeze-thaw stability of recombinant anti-CTLA-4 fully human monoclonal antibody.
  • the concentration of recombinant anti-CTLA-4 fully human monoclonal antibody in each preparation is 5 mg/mL; "%” is weight percentage (wt%), based on the total weight of the preparation.
  • test group A F1, F2 and F3
  • test group B F4, F8 and F6
  • test group C F10, F11 and F12
  • the source monoclonal antibody preparation formulation contains 0.01% Tween 80 to maintain the stability of the recombinant anti-CTLA-4 fully human monoclonal antibody.
  • the concentration of recombinant anti-CTLA-4 fully human monoclonal antibody is 5 mg/mL; "%” is weight percentage (wt%), based on the total weight of the preparation.
  • the concentration of recombinant anti-CTLA-4 fully human monoclonal antibody is 5 mg/mL; "%” is weight percentage (wt%), based on the total weight of the preparation.
  • the pH of the preparation buffer system was 6.0, and the preparation composition should include mannitol and optionally NaCl, and the mannitol content was not Below 40mg/ml, keep the appropriate NaCl content, according to the test results, confirm that the appropriate formulation is F4 or F10, respectively 5mg/mL recombinant anti-CTLA-4 fully human monoclonal antibody/20mM phosphate buffer/0.01% Tween 80/4% mannitol/30 mM NaCl (pH 6.0), 5 mg/mL recombinant anti-CTLA-4 fully human monoclonal antibody/20 mM phosphate buffer/0.01% Tween 80/5% mannitol/0 mM NaCl (pH 6.0), the pH of the preparation buffer system can be controlled within the range of 5.8 to 6.2.

Abstract

本发明提供了一种抗CTLA-4抗体组合物，该组合物包括抗CTLA-4单克隆抗体、表面活性剂、缓冲液和稳定剂。本发明的组合物能够提高抗CTLA-4抗体的稳定性。

Description

一种抗CTLA-4抗体及融合蛋白的制剂 技术领域
本发明涉及药物制剂领域，具体地涉及一种抗CTLA-4抗体及融合蛋白的的制剂。
背景技术
重组抗CTLA-4全人源单克隆抗体是一种T细胞免疫功能调节剂，与APC细胞表面的B7-1分子竞争结合T细胞表面的CTLA-4分子，阻断B7-1分子调节的免疫抑制功能.
重组抗CTLA-4全人源单克隆抗体分子量约为145KD，由1324个氨基酸组成，在CHO细胞中表达和分泌。
由于其具有复杂的空间结构，在生产、储存过程中容易受到外部环境因素(如pH、温度、光照、冻融、有机溶剂或微生物污染等)的影响，发生形态上的变化，导致产生聚集或其他的化学修饰产物如降解、脱胺、氧化等，影响产品的有效性及安全性。蛋白稳定性易受到缓冲溶液的类型及浓度、pH、盐的种类及浓度、表面活性剂、氨基酸及糖类保护剂、蛋白浓度等的影响，结合产品的理化特性，开发适合重组抗CTLA-4全人源单克隆抗体稳定性优异的制剂处方对临床用药非常重要。
因此，本领域需要开发一种具有稳定性高的重组抗CTLA-4全人源单克隆抗体制剂，提高重组抗CTLA-4全人源单克隆抗体的稳定性。
发明内容
本发明的目的是提供一种抗CTLA-4抗体组合物，所述的组合物抑制抗CTLA-4抗体的聚集和降解，从而提高抗CTLA-4抗体的稳定性。
本发明第一方面，提供一种抗CTLA-4抗体组合物，其特征在于，所述的组合物包括抗CTLA-4单克隆抗体、表面活性剂、缓冲液和稳定剂。
优选地，所述的抗CTLA-4抗体具有如SEQ ID NO.：1的重链氨基酸序列和SEQ ID NO.：2轻链氨基酸序列。
优选地，所述的抗CTLA-4抗体具有如SEQ ID NO.：3的重链氨基酸序列和SEQ ID NO.：4轻链氨基酸序列。
优选地，所述的抗CTLA-4抗体具有如SEQ ID NO.：5的重链氨基酸序列和SEQ ID NO.：6轻链氨基酸序列。
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述的抗体包括人源抗体。
在另一优选例中，所述的抗体包括单克隆抗体。
在另一优选例中，所述的抗CTLA-4单克隆抗体包括人源抗CTLA-4单克隆抗体。
在另一优选例中，所述的抗CTLA-4单克隆抗体包括抗CTLA-4全人源单克隆抗体。
在另一优选例中，所述的抗CTLA-4全人源单克隆抗体包括重组抗CTLA-4全人源单克隆抗体。
在另一优选例中，所述抗CTLA-4单克隆抗体的含量为0.5-50mg/mL，较佳地0.5-40mg/mL，更佳地0.5-30mg/mL，更佳地0.5-20mg/mL，更佳地1-15mg/mL，更佳地2-10mg/mL，更佳地3-8mg/mL，最佳地5mg/mL。
在另一优选例中，所述的表面活性剂选自下组：正离子型表面活性剂、负离子型表面活性剂、非离子型表面活性剂，或其组合。
在另一优选例中，所述的表面活性剂选自下组：聚氧乙烯山梨醇酐脂肪酸酯、聚氧乙烯氢化蓖麻油、甘油脂肪酸酯、聚山梨醇酯、泊洛沙姆，或其组合。
在另一优选例中，所述的聚山梨醇酯选自下组：聚山梨醇酯20(PS–20，吐温-20)、聚山梨醇酯40(PS-40，吐温-40)、聚山梨醇酯60(PS–60，吐温-60)、聚山梨醇酯80(PS-80，吐温-80)。
在另一优选例中，所述的泊洛沙姆选自下组：泊洛沙姆188、泊洛沙姆108、泊洛沙姆124，或其组合。
在另一优选例中，所述的表面活性剂包括聚山梨醇酯。
在另一优选例中，所述的表面活性剂包括聚山梨醇酯80(PS-80)。
在另一优选例中，所述表面活性剂的含量为0.001-8wt％，较佳地0.001-3wt％，更佳地0.001-1wt％，更佳地0.002-0.5wt％，更佳地0.002-0.1wt％，更佳地0.002-0.06wt％，更佳地0.005-0.05wt％，更佳地0.005-0.03wt％，更佳地0.008-0.02wt％，更佳地0.008-0.012wt％，最佳地0.01wt％，以组合物的总重量计。
在另一优选例中，所述的缓冲液选自下组：柠檬酸盐-柠檬酸缓冲液、磷酸盐-磷酸缓冲液，或其组合。
在另一优选例中，所述的缓冲液的介质为水。
在另一优选例中，所述缓冲液的浓度为3-80mM，较佳地5-60mM，更佳地5-40mM，更佳地10-30mM，更佳地15-25mM，更佳地18-22mM。
在另一优选例中，所述的缓冲剂包括磷酸缓冲液。
在另一优选例中，所述的磷酸缓冲液的浓度为3-80mM，较佳地5-60mM，更佳地5-40mM，更佳地10-30mM，更佳地15-25mM，更佳地18-22mM，最佳地。
在另一优选例中，所述的稳定剂选自下组：氨基酸、糖醇，或其组合。
在另一优选例中，所述的氨基酸选自下组：脯氨酸、精氨酸(Arginine)、甘氨酸(Glycine)、组氨酸(Histidine)、甲硫氨酸(Methionine)，或其组合。
在另一优选例中，所述的糖醇选自下组：蔗糖(Sucrose)、甘露醇(Mannitol)、海藻糖(Trehalose)、麦芽糖(Maltose)、山梨醇(Sorbitol)，或其组合。
在另一优选例中，所述的糖醇选自下组：蔗糖、甘露醇、海藻糖、山梨醇，或其组合。
在另一优选例中，所述的稳定剂包括甘露醇。
在另一优选例中，所述的糖醇包括甘露醇。
在另一优选例中，所述的稳定剂的含量为0.5-50wt％，较佳地0.5-40wt％，更佳地0.8-30wt％，更佳地1-20wt％，更佳地1-15wt％，更佳地1-10wt％，更佳地1-8wt％，更佳地2-7wt％，最佳地4wt％或5wt％，以组合物的总重量计。
在另一优选例中，所述的组合物不包括渗透压调节剂。
在另一优选例中，所述的组合物还包括渗透压调节剂。
在另一优选例中，所述的渗透压调节剂包括无机盐。
在另一优选例中，所述的无机盐选自下组：钠盐、钾盐、钙盐、镁盐、氯盐、硫酸盐、硝酸盐，或其组合。
在另一优选例中，所述的无机盐选自下组：氯化钠、氯化钾、氯化钙、氯化镁、硫酸钠、硫酸钾、硫酸钙、硫酸镁、硝酸钠、硝酸钾、硝酸钙、硝酸镁，或其组合。
在另一优选例中，所述的无机盐选包括氯化钠。
在另一优选例中，所述的渗透压调节剂的含量为0-80mM，较佳地0-60mM，更佳地0-50mM，更佳地0-30mM。
在另一优选例中，所述稳定剂与所述表面活性剂的重量比为350-450：1，较佳地380-420：1，更佳地390-400：1。
在另一优选例中，所述稳定剂与所述表面活性剂的重量比为450-550：1，较佳地480-520：1，更佳地490-510：1。
在另一优选例中，所述甘露醇与所述吐温80的重量比为350-450：1，较佳地380-420：1，更佳地390-400：1。
在另一优选例中，所述甘露醇与所述吐温80的重量比为450-550：1，较佳地480-520：1，更佳地490-510：1。
在另一优选例中，所述的渗透压调节剂的含量为0Mm。
在另一优选例中，所述的渗透压调节剂的含量为10-50mM，较佳地20-40mM，更佳地25-30mM，最佳地0mM或30mM。
在另一优选例中，所述的组合物还包括抗氧化剂。
在另一优选例中，所述的抗氧化剂包括EDTA(乙二胺四乙酸)。
在另一优选例中，所述的组合物的pH为5.0-7.5，较佳地5.0-7.0，更佳地5.5-6.5，更佳地5.8-6.2，最佳地6.0。
在另一优选例中，所述的组合物为液体制剂。
在另一优选例中，所述的组合物为口服液体制剂或注射液体制剂。
在另一优选例中，所述的组合物为药物组合物。
在另一优选例中，所述的组合物的剂型为口服制剂或注射制剂。
在另一优选例中，所述的组合物包括：
抗CTLA-4抗体 1-15mg/mL
磷酸缓冲液 10-30mM
吐温80 0.005-0.05wt％
甘露醇 1-15wt％
NaCl 10-50mM。
在另一优选例中，所述的组合物包括：
抗CTLA-4抗体 2-10mg/mL
磷酸缓冲液 15-25mM
吐温80 0.005-0.03wt％
甘露醇 1-8wt％
NaCl 20-40mM。
在另一优选例中，所述的组合物包括：
抗CTLA-4抗体 1-15mg/mL
磷酸缓冲液 10-30mM
吐温80 0.005-0.05wt％
甘露醇 1-15wt％
在另一优选例中，所述的组合物包括：
抗CTLA-4抗体 2-10mg/mL
磷酸缓冲液 15-25mM
吐温80 0.005-0.03wt％
甘露醇 1-8wt％
本发明第二方面，提供一种冻干制剂，所述的冻干制剂通过以下方法制备：
将如本发明第一方面所述的组合物进行冷冻干燥，得到冻干制剂。
在另一优选例中，所述的冻干制剂还包括冻干保护剂。
在另一优选例中，所述的冻干保护剂选自下组：葡萄糖、甘露醇、果糖、半乳糖，或其组合。
本发明第三方面，提供一种如本发明第一方面所述的组合物或如本发明第二方面所述的冻干制剂的用途，用于制备药物，所述药物用于(i)预防和/或治疗无法切除或转移性黑色素瘤；(ii)预防和/或治疗晚期肾细胞癌；(iii)预防和/或治疗微卫星高度不稳定性或错配修复缺陷的转移性结直肠癌；和/或(iv)用于黑色素瘤辅助治疗。
本发明第四方面，提供一种(i)预防和/或治疗无法切除或转移性黑色素瘤；(ii)预防和/或治疗晚期肾细胞癌；(iii)预防和/或治疗微卫星高度不稳定性或错配修复缺陷的转移性结直肠癌；和/或(iv)用于黑色素瘤辅助治疗的方法，所述的方法包括：给予所需对象如本发明第一方面所述的组合物。
在另一优选例中，所述的对象为人或非人哺乳动物。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图
图1制剂样品60℃孵育24小时非还原电泳图谱。
图2制剂样品60℃孵育24小时非还原电泳图谱。
具体实施方式
本发明人通过广泛而深入的研究，研发出一种药物组合物，所述的药物组合物能够有效保持抗CTLA-4抗体的稳定性，组合物中的抗CTLA-4抗体能够在加压试验(高温、冻融)条件下均能保持其稳定性，因此，本发明所述的组合物能够稳定抗CTLA-4抗体的质量，延长产品的货架期，提高临床实用的安全性。在此基础上，完成了本发明。
术语
除非另有定义,否则本文中所用的所有技术和科学术语的含义与本发明所属领域普通技术人员普遍理解的含义相同。
如本文所用，术语“包含”、“包括”、“含有”可互换使用，不仅包括封闭式定义，还包括半封闭、和开放式的定义。换言之，所述术语包括了“由……构成”、“基本上 由……构成”。
如本文所用，“mM”为mmol/L单位，例如，1mM＝1mmol/L。
在本发明中在所述的药物组合物中，各个组分的重量含量(wt.％)或浓度(如mM、mg/mL)均以药物组合物的重量或体积计。
如本文所用，三羟甲基氨基甲烷简称为Tris，即“三羟甲基氨基甲烷”与“Tris”可互换使用。
如本文所用，术语“EDTA”为乙二胺四乙酸。
如本文所用，术语“磷酸缓冲液”为磷酸-磷酸盐缓冲水溶液，其中，含量以磷酸根离子计，例如20mM磷酸缓冲液是指换算成PO 4 3-的浓度为20mM。
抗CTLA-4抗体
如本文所用，术语“抗CTLA-4抗体”由两个相同的轻链(L)和两个相同的重链(H)组成。每条重链的一端有可变区(VH)，其后是多个恒定区。每条轻链的一端有可变区(VL)，另一端有恒定区；轻链的恒定区与重链的第一个恒定区相对，轻链的可变区与重链的可变区相对。
本发明所述的抗CTLA-4抗体并没有特别的限定，优选为人抗CTLA-4抗体，例如抗CTLA-4全人源单克隆抗体。
优选地，本发明所述的抗CTLA-4抗体具有SEQ ID NO.：1的重链氨基酸序列和SEQ ID NO.：2轻链氨基酸序列；
SEQ ID NO.：1的氨基酸序列如下：
Figure PCTCN2021137235-appb-000001
SEQ ID NO.：2的氨基酸序列如下：
Figure PCTCN2021137235-appb-000002
Figure PCTCN2021137235-appb-000003
优选地，本发明所述的抗CTLA-4抗体具有SEQ ID NO.：3的重链氨基酸序列和SEQ ID NO.：4的轻链氨基酸序列；
SEQ ID NO.：3的氨基酸序列如下：
Figure PCTCN2021137235-appb-000004
SEQ ID NO.：4的氨基酸序列如下：
Figure PCTCN2021137235-appb-000005
优选地，本发明所述的抗CTLA-4抗体具有SEQ ID NO.：5的重链氨基酸序列和SEQ ID NO.：6的轻链氨基酸序列；
SEQ ID NO.：5的氨基酸序列如下：
Figure PCTCN2021137235-appb-000006
SEQ ID NO.：6的氨基酸序列如下：
Figure PCTCN2021137235-appb-000007
组合物
本发明提供一种抗CTLA-4抗体组合物，所述的组合物包括抗CTLA-4单克隆抗体、表面活性剂、缓冲液和稳定剂。
本发明所述的组合物优选为药物组合物。
本发明所述的组合物优选为液体制剂，例如口服液体制剂或注射液体制剂。
在本发明的一个优选例中，所述所述抗CTLA-4单克隆抗体的含量为0.5-50mg/mL，较佳地0.5-40mg/mL，更佳地0.5-30mg/mL，更佳地0.5-20mg/mL，更佳地1-15mg/mL，更佳地2-10mg/mL，更佳地3-8mg/mL，最佳地5mg/mL。
在本发明另一优选例中，所述的表面活性剂选自下组：正离子型表面活性剂、负离子型表面活性剂、非离子型表面活性剂，或其组合。
代表性地，所述的表面活性剂选自下组：聚氧乙烯山梨醇酐脂肪酸酯、聚氧乙烯氢化蓖麻油、甘油脂肪酸酯、聚山梨醇酯、泊洛沙姆，或其组合。
代表性地，所述的聚山梨醇酯选自下组：聚山梨醇酯20(PS–20，吐温-20)、聚山梨醇酯40(PS-40，吐温-40)、聚山梨醇酯60(PS–60，吐温-60)、聚山梨醇酯80(PS-80，吐温-80)。
代表性地，所述的泊洛沙姆选自下组：泊洛沙姆188、泊洛沙姆108、泊洛沙姆124，或其组合。
典型地，所述的表面活性剂包括聚山梨醇酯。
典型地，所述的表面活性剂包括聚山梨醇酯80(PS-80)。
在本发明所述的组合物中，所述表面活性剂的含量可以为0.001-8wt％，较佳地0.001-3wt％，更佳地0.001-1wt％，更佳地0.002-0.5wt％，更佳地0.002-0.1wt％，更佳地0.002-0.06wt％，更佳地0.005-0.05wt％，更佳地0.005-0.03wt％，更佳地0.008-0.02wt％，更佳地0.008-0.012wt％，最佳地0.01wt％，以组合物的总重量计。
在本发明的一个优选例中，所述的缓冲液选自下组：柠檬酸盐-柠檬酸缓冲液、磷酸盐-磷酸缓冲液，或其组合。
在另一优选例中，所述的缓冲液的介质为水。
在本发明所述的组合物中，所述缓冲液的浓度可以为3-80mM，较佳地5-60mM，更佳地5-40mM，更佳地10-30mM，更佳地15-25mM，更佳地18-22mM。
代表性地，所述的缓冲剂包括磷酸缓冲液。
代表性地，所述的磷酸缓冲液的浓度为3-80mM，较佳地5-60mM，更佳地5-40mM，更佳地10-30mM，更佳地15-25mM，更佳地18-22mM，最佳地。
在本发明的一个优选例中，所述的稳定剂选自下组：氨基酸、糖醇，或其组合。
代表性地，所述的氨基酸选自下组：脯氨酸、精氨酸(Arginine)、甘氨酸(Glycine)、组氨酸(Histidine)、甲硫氨酸(Methionine)，或其组合。
代表性地，所述的糖醇选自下组：蔗糖(Sucrose)、甘露醇(Mannitol)、海藻糖(Trehalose)、麦芽糖(Maltose)、山梨醇(Sorbitol)，或其组合。
代表性地，所述的糖醇选自下组：蔗糖、甘露醇、海藻糖、山梨醇，或其组合。
典型地，所述的稳定剂包括甘露醇。
典型地，所述的糖醇包括甘露醇。
在本发明所述的组合物中，所述的稳定剂的含量可以为0.5-50wt％，较佳地0.5-40wt％，更佳地0.8-30wt％，更佳地1-20wt％，更佳地1-15wt％，更佳地1-10wt％，更佳地1-8wt％，更佳地2-7wt％，最佳地4wt％或5wt％，以组合物的总重量计。
在本发明的一个优选例中，所述的组合物不包括渗透压调节剂。
在本发明的一个优选例中，所述的组合物还包括渗透压调节剂。
在另一优选例中，所述的渗透压调节剂包括无机盐。
代表性地，所述的无机盐选自下组：钠盐、钾盐、钙盐、镁盐、氯盐、硫酸盐、硝酸盐，或其组合。
代表性地，所述的无机盐选自下组：氯化钠、氯化钾、氯化钙、氯化镁、硫酸钠、硫酸钾、硫酸钙、硫酸镁、硝酸钠、硝酸钾、硝酸钙、硝酸镁，或其组合。
典型地，所述的无机盐选包括氯化钠。
在本发明的一个优选例中，所述的渗透压调节剂的含量为0-80mM，较佳地0-60mM，更佳地0-50mM，更佳地0-30mM。
在本发明的一个优选例中，所述稳定剂与所述表面活性剂的重量比为350-450：1，较佳地380-420：1，更佳地390-400：1。
在本发明的一个优选例中，所述稳定剂与所述表面活性剂的重量比为450-550：1，较佳地480-520：1，更佳地490-510：1。
在本发明的一个优选例中，所述甘露醇与所述吐温80的重量比为350-450：1，较佳地380-420：1，更佳地390-400：1。
在本发明的一个优选例中，所述甘露醇与所述吐温80的重量比为450-550：1，较佳地480-520：1，更佳地490-510：1。
在本发明的一个优选例中，所述的渗透压调节剂的含量为0Mm。
在本发明的一个优选例中，所述的渗透压调节剂的含量为10-50mM，较佳地20-40mM，更佳地25-30mM，最佳地0mM或30mM。
在本发明的一个优选例中，所述的组合物还包括抗氧化剂。
在另一优选例中，所述的抗氧化剂包括EDTA(乙二胺四乙酸)。
在本发明的一个优选例中，所述的组合物的pH为5.0-7.5，较佳地5.0-7.0，更佳地5.5-6.5，更佳地5.8-6.2，最佳地6.0。
在本发明的一个优选例中，所述的组合物包括：
抗CTLA-4抗体 1-15mg/mL
磷酸缓冲液 10-30mM
吐温80 0.005-0.05wt％
甘露醇 1-15wt％
NaCl 10-50mM。
在本发明的一个优选例中，所述的组合物包括：
抗CTLA-4抗体 2-10mg/mL
磷酸缓冲液 15-25mM
吐温80 0.005-0.03wt％
甘露醇 1-8wt％
NaCl 20-40mM。
在本发明的一个优选例中，所述的组合物包括：
抗CTLA-4抗体 1-15mg/mL
磷酸缓冲液 10-30mM
吐温80 0.005-0.05wt％
甘露醇 1-15wt％
在本发明的一个优选例中，所述的组合物包括：
抗CTLA-4抗体 2-10mg/mL
磷酸缓冲液 15-25mM
吐温80 0.005-0.03wt％
甘露醇 1-8wt％
冻干制剂
本发明还提供一种冻干制剂，所述的冻干制剂通过以下方法制备：
将如本发明所述的组合物进行冷冻干燥，得到冻干制剂。
在另一优选例中，所述的冻干制剂还包括冻干保护剂。
在另一优选例中，所述的冻干保护剂选自下组：葡萄糖、甘露醇、果糖、半乳糖， 或其组合。
用途
本发明还提供一种如本发明所述组合物或所述冻干制剂的用途，用于制备药物，所述药物用于(i)预防和/或治疗无法切除或转移性黑色素瘤；(ii)预防和/或治疗晚期肾细胞癌；(iii)预防和/或治疗微卫星高度不稳定性或错配修复缺陷的转移性结直肠癌；和/或(iv)用于黑色素瘤辅助治疗。
本发明还提供一种(i)预防和/或治疗无法切除或转移性黑色素瘤；(ii)预防和/或治疗晚期肾细胞癌；(iii)预防和/或治疗微卫星高度不稳定性或错配修复缺陷的转移性结直肠癌；和/或(iv)用于黑色素瘤辅助治疗的方法，所述的方法包括：给予所需对象如本发明所述组合物或所述冻干制剂。
本发明的主要优点包括：
1、本发明提供一种含有抗CTLA-4抗体的组合物，所述的组合物能够提高抗CTLA-4抗体的稳定性。
2、本发明所述的抗CTLA-4抗体组合物够保持抗CTLA-4抗体在2～8℃至少18～24个月的稳定性。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
实施例
以下实施例1-3中的重组抗CTLA-4全人源单克隆抗体的重链的氨基酸序列为如SEQ ID NO.：1，轻链的氨基酸序列为如SEQ ID NO.：2，
SEQ ID NO.：1的氨基酸序列如下：
Figure PCTCN2021137235-appb-000008
Figure PCTCN2021137235-appb-000009
SEQ ID NO.：2的氨基酸序列如下：
Figure PCTCN2021137235-appb-000010
通用测定方法
SEC-HPLC为体积排阻色谱法，用于分析重组抗CTLA-4全人源单克隆抗体的纯度，具体地，检测重组抗CTLA-4全人源单克隆抗体的聚集及降解。
CEX-HPLC为阳离子交换色谱，用于分析重组抗CTLA-4全人源单克隆抗体的纯度，具体地，检测重组抗CTLA-4全人源单克隆抗体的电荷异质性。
非还原凝胶电泳(NR SDS-PAGE)根据变性后的样品分子大小进行分离，分离的蛋白条带采用考马斯蓝染色，染色的蛋白条带含量应用分析软件分析重组抗CTLA-4全人源单克隆抗体对应各组分蛋白相对纯度，主要评估样品的化学降解。
DSC为差示扫描量热法，具体地，通过重组抗CTLA-4全人源单克隆抗体在不同状态下的荧光的变化来测定Tm(熔解温度)、Tagg(聚集温度)、Tonset(变性温度阈值)
目测法观察来确定重组抗CTLA-4全人源单克隆抗体的沉淀状况。
实施例1单因素制剂组成的热稳定性
本文所用的术语“热稳定性”是通过DSC检测重组抗CTLA-4全人源单克隆抗体Tm值、Tagg值及Tonset值。
考察pH、盐离子、糖、氨基酸、表面活性剂及抗氧化剂等对重组抗CTLA-4全人源单克隆抗体热稳定性影响，制剂配方及热稳定性测定结果见表1。
表1制剂处方及热稳定性测定结果
Figure PCTCN2021137235-appb-000011
Figure PCTCN2021137235-appb-000012
注：除A18重组抗CTLA-4全人源单克隆抗体浓度为28mg/ml外，其余制剂处方重组抗CTLA-4全人源单克隆抗体浓度均为5mg/ml；“％”为重量百分比(wt％)，以制剂的总重量计。
表1对样品的热力学参数进行研究，考察了pH、NaCl、糖类、精氨酸、吐温80、EDTA、缓冲液类型及缓冲液浓度对重组抗CTLA-4全人源单克隆抗体的热稳定性的影响。
1)pH的影响考察了重组抗CTLA-4全人源单克隆抗体在pH5.0～pH7.5时的热稳定性(A5～A9)，结果显示：当pH＜6.0时，制剂样品热稳定性值Tonset、Tm及Tagg值相对偏低，在pH6.0～7.5范围内的制剂样品热稳定性值Tonset、Tm相对稳定，且pH6.0的样品有最高Tagg值。
2)NaCl浓度的影响考察了NaCl浓度范围在0～120mM内的制剂样品(A1～A4)热稳定性，测定的热稳定性值没有明显差异，结果表明NaCl浓度对样品的热稳定性无明显的影响。
3)糖的影响考察了甘露醇含量在0～5％范围内制剂样品的热稳定性(A3，A10， A11)，海藻糖浓度范围为0～10％(A3，A12，A13)的制剂样品的热稳定性，蔗糖浓度范围为0～10％(A3，A14，A15)的制剂样品的热稳定性，结果显示随着糖的浓度增加，热稳定性值都有增加的趋势，但甘露醇、海藻糖、蔗糖种类对制剂样品的热稳定性没有明显的影响。
4)精氨酸浓度的影响：考察了精氨酸浓度在0～60mM范围内的制剂样品(A3，A16，A17)的热稳定性，含有精氨酸的制剂样品热稳定性没有差异，相对于不含有精氨酸的制剂样品测定的热稳定性值略有下降，结果表明精氨酸浓度对重组抗CTLA-4全人源单克隆抗体的热稳定性并无明显影响。
5)重组抗CTLA-4全人源单克隆抗体浓度的影响考察了制剂体系重组抗CTLA-4全人源单克隆抗体浓度(A3，A18)对制剂热稳定性的影响，当重组抗CTLA-4全人源单克隆抗体浓度从增加至28mg/ml时，Tagg明显下降。
6)吐温80浓度的影响考察了聚山梨酯80浓度在0～0.1％范围内的制剂样品(S3，S19，S20)的热稳定性，测定的热稳定性值没有明显差异，表明吐温80的浓度对重组抗CTLA-4全人源单克隆抗体的热稳定性并无明显影响。
7)EDTA浓度的影响考察了EDTA浓度在0～0.01％范围内的制剂样品(A3，A21，A22)的热稳定性，热稳定性值没有明显差异，结果表明EDTA浓度对重组抗CTLA-4全人源单克隆抗体的热稳定性无明显影响。
8)缓冲溶液的影响考察了三种缓冲溶液制剂体系下样品(A3，A23，A24)热稳定性，测定的热稳性值没有明显差异，结果表明磷酸缓冲液、柠檬酸缓冲液和柠檬酸钠-磷酸缓冲液均适用于重组抗CTLA-4全人源单克隆抗体制剂。
上述结果表明：制剂体系pH、重组抗CTLA-4全人源单克隆抗体浓度、糖的含量对重组抗CTLA-4全人源单克隆抗体热稳定性有明显的影响。根据表1的结果，结合拟定注射剂量，选择重组抗CTLA-4全人源单克隆抗体浓度为5mg/mL，溶液缓冲溶液为pH6.0，含有多种辅料组成的制剂，在室温条件下储存约12个月后考察重组抗CTLA-4全人源单克隆抗体的热稳定性，制剂配方及热稳定性测定结果见表2，结果显示：
9)缓冲溶液类型及浓度的影响考察了3种缓冲体系包括磷酸缓冲液、磷酸—柠檬酸缓冲溶液、柠檬酸缓冲溶液在不同浓度条件下的制剂样品稳定性，磷酸缓冲液浓度分别为20mM、40mM、60mM(B7，B8，B9)，磷酸-柠檬酸缓冲溶液浓度分别为13mM、26mM、40mM(B4，B5，B6)，柠檬酸缓冲溶液浓度分别为10mM、20mM、30mM(B1，B2，B3)，结果显示：随着缓冲溶液浓度的变化，测定热稳定性值Tm1没有发生明显的变化，而柠檬酸缓冲溶液、磷酸-柠檬酸缓冲溶液测定的Tagg值随溶液浓度的增加略有下降趋势；
10)糖的影响在磷酸缓冲液组合辅料条件下，考察单糖甘露醇(B12、B13、B14)与二糖海藻糖(B8、B10、B11)的热稳定性，糖的含量对制剂的热稳定性没有表现出明 显的差异，且两者有相近的Tonset、Tm1值，但甘露醇有相对较高的Tagg值。
11)NaCl与甘露醇的影响在磷酸缓冲液体系下，含有甘露醇与NaCl辅料的制剂样品(B15、B16、B17)的热稳定性没有随NaCl含量的变化而表现出明显的差异；在含有NaCl的制剂中，随着甘露醇浓度的提高，样品Tagg值有增加的趋势(B16、B18、B19)。
上述结果表明：缓冲溶液浓度对重组抗CTLA-4全人源单克隆抗体的热稳定性没有明显影响，制剂体系组成中的甘露醇有助于维持重组抗CTLA-4全人源单克隆抗体的稳定性。
表2制剂处方选择及热稳定性测定结果
Figure PCTCN2021137235-appb-000013
Figure PCTCN2021137235-appb-000014
注：各制剂中重组抗CTLA-4全人源单克隆抗体浓度为5mg/mL；“％”为重量百分比(wt％)，以制剂的总重量计。
实施例2组合制剂体系对纯度的影响
本文所用的术语“纯度”是指由SEC-HPLC及CEX-HPLC检测的重组抗CTLA-4全人源单克隆抗体纯度，重组抗CTLA-4全人源单克隆抗体单体分子由于外部环境影响，可能会形成多聚体、产生降解、发生化学修饰(包括脱胺、氧化、焦谷酰胺化、N-末端赖氨酸去除等)，通过SEC-HPLC分析单体、聚集体、小分子片段的相对含量；CEX-HPLC分析重组抗CTLA-4全人源单克隆抗体电荷异质性。
实施例1中主要通过考察辅料及制剂组成热力学参数的变化趋势确定各种因素对样品稳定性的影响，表3通过考察不同制剂组成在加速试验条件下的样品理化性质的变化进一步确认辅料组成对样品稳定性的影响。
将浓度为5mg/mL重组抗CTLA-4全人源单克隆抗体浓度处理成表3制剂处方，制剂置于37℃恒温箱中，孵育2周后，测定重组抗CTLA-4全人源单克隆抗体的纯度及电荷异质性，各制剂配方及纯度检测结果见表3。
制剂组成对重组抗CTLA-4全人源单克隆抗体纯度影响如下：
1)缓冲溶液影响考察了3种缓冲体系包括磷酸缓冲液、磷酸-柠檬酸缓冲溶液、柠檬酸缓冲溶液体系条件下制剂样品的稳定性，在含有海藻糖的制剂样品中，柠檬酸缓冲溶液体系有相对较高的纯度，在含有甘露糖的制剂样品中，缓冲溶液的类型对测定的重组抗CTLA-4全人源单克隆抗体纯度没有影响，但从整体而言，在含有辅料甘露醇制剂样品中测定的重组抗CTLA-4全人源单克隆抗体纯度要高于海藻糖，该结果同甘露醇能提高制剂的Tagg值判断一致；在含有海藻糖与甘露醇的制剂样品中，缓冲溶液的浓度对测定的IEC主峰纯度没有明显影响，但柠檬酸缓冲溶液体系中测定的IEC主峰纯度相对于另外两种缓冲体系偏低。
2)糖的影响改变制剂组成中海藻糖、甘露醇浓度，测定的SEC纯度没有明显的变化，但制剂组成中含有甘露醇，测定的SEC纯度相对于海藻糖较高，在含有NaCl制剂组成中，增加甘露醇含量，测定的SEC纯度没有明显的变化。
3)NaCl的影响：在含有甘露醇制剂组成中，改变NaCl浓度对SEC纯度没有明显的影响，但当NaCl浓度增加至90mM时，测定的SEC纯度有轻微下降趋势。
4)NaCl与甘露醇组合的影响：在含有甘露醇制剂组成中，改变NaCl浓度，测定的IEC主峰纯度没有明显影响；在含有NaCl制剂组成中，增加甘露醇含量，测定的IEC主峰纯度也没有明显影响；但在含有甘露醇与NaCl制剂中，测定的IEC主峰纯度高于不含有NaCl的甘露醇制剂。
结果表明：在制剂体系组成添加甘露醇有助于抑制重组抗CTLA-4全人源单克隆抗体聚集，含有甘露醇与NaCl组合的制剂有助于抑制重组抗CTLA-4全人源单克隆抗体降解。
表3制剂配方及纯度检测结果
Figure PCTCN2021137235-appb-000015
Figure PCTCN2021137235-appb-000016
注：各制剂中重组抗CTLA-4全人源单克隆抗体浓度为5mg/mL；单体为SEC-HPLC测定的重组抗CTLA-4全人源单克隆抗体纯度，主峰为CEX-HPLC测定的重组抗CTLA-4全人源单克隆抗体纯度；“％”为重量百分比(wt％)，以制剂的总重量计。
实施例3制剂组成的确认
根据实施例1和2研究结果，初步确定了重组抗CTLA-4全人源单克隆抗体的制剂组成应包括磷酸缓冲液、甘露醇、NaCl、吐温80，重组抗CTLA-4全人源单克隆抗体浓度为5mg/mL，按照表4进行制备制剂样品，评估辅料浓度、pH对重组抗CTLA-4全人源单克隆抗体理化性质的影响，确认制剂处方。
试验条件分别为冻融加速试验、高温加速试验。
通过冻融实验，考察制剂组成的稳定性。试验条件为制剂在-20℃冷冻，室温 融化，循环次数分别为3次及5次。通过冻融前后的外观、重组抗CTLA-4全人源单克隆抗体相对含量及SEC-HPLC测定重组抗CTLA-4全人源单克隆抗体纯度，评估制剂组成的稳定性。
冻融前后外观及重组抗CTLA-4全人源单克隆抗体含量测定结果见表4，重组抗CTLA-4全人源单克隆抗体纯度结果见表5。
冻融加速试验结果显示：冻融过程中，重组抗CTLA-4全人源单克隆抗体易于发生聚集，甚至产生沉淀；提高甘露醇含量，降低NaCl含量，有利于抑制重组抗CTLA-4全人源单克隆抗体冻融期间的聚集与沉淀，在pH相对较高的条件下现象尤为明显(F3、F6及F12)，推测原因是提高甘露醇含量有利于抑制NaCl在冻存期间以晶体形式析出；EDTA的浓度对重组抗CTLA-4全人源单克隆抗体的冻融稳定性并无明显影响。
表4冻融制剂外观及重组抗CTLA-4全人源单克隆抗体浓度
Figure PCTCN2021137235-appb-000017
Figure PCTCN2021137235-appb-000018
注：“－”表示澄清液体，“+”轻微乳光，“++”表示浑浊，“+++”表示析出沉淀；各制剂中重组抗CTLA-4全人源单克隆抗体浓度为5mg/mL；“％”为重量百分比(wt％)，以制剂的总重量计。
表5冻融制剂重组抗CTLA-4全人源单克隆抗体纯度变化
Figure PCTCN2021137235-appb-000019
注：各制剂中重组抗CTLA-4全人源单克隆抗体浓度为5mg/mL；“％”为重量百分比(wt％)，以制剂的总重量计。
通过高温加速实验，制剂在60℃，24小时孵育，评估制剂稳定性，理化性质 检测方法包括SEC-HPLC、CEX-HPLC、NR SDS-PAGE。检测结果见表6、表7、图1及图2。
结果显示：
1)pH影响
比较三组制剂处方样品，试验组A(F1、F2及F3)、试验组B(F4、F8及F6)、试验组C(F10、F11及F12)，结果显示：随着pH升高，聚集体有增加趋势(见表6)，电荷异质性中的主峰、碱性峰明显下降，酸性峰对应增加。
2)NaCl影响
试验组(F5、F8及F7)中逐渐提高NaCl浓度，观察到重组抗CTLA-4全人源单克隆抗体聚集体逐渐增加，重组抗CTLA-4全人源单克隆抗体纯度呈下降趋势，但电荷异构体主峰相对含量有增加的趋势。
3)甘露醇影响
试验C组中提高甘露醇含量至5％，不添加NaCl，能明显抑制聚集体生成，但电荷异构体主峰呈相对下降趋势。
4)EDTA影响
试验组(F8、F13及F14)中逐渐提高EDTA浓度，对多聚体形成抑制及IEC主峰含量改变没有明显影响。
5)吐温80影响
试验组(F8、F9)中提高吐温80含量时，多聚体增加及IEC主峰含量下降，可能与高温条件下吐温80发生降解相关，结合冻融试验结果，重组抗CTLA-4全人源单克隆抗体制剂处方含有0.01％吐温80能维持重组抗CTLA-4全人源单克隆抗体的稳定性。
表6 60℃高温孵育24小时测定重组抗CTLA-4全人源单克隆抗体纯度(SEC-HPLC)变化
Figure PCTCN2021137235-appb-000020
Figure PCTCN2021137235-appb-000021
注：重组抗CTLA-4全人源单克隆抗体浓度为5mg/mL；“％”为重量百分比(wt％)，以制剂的总重量计。
表7 60℃高温孵育24小时测定重组抗CTLA-4全人源单克隆抗体纯度(CEX-HPLC)变化
Figure PCTCN2021137235-appb-000022
Figure PCTCN2021137235-appb-000023
注：重组抗CTLA-4全人源单克隆抗体浓度为5mg/mL；“％”为重量百分比(wt％)，以制剂的总重量计。
图1及图2所示，60℃孵育24小时，所有制剂降解均有增加，多聚体增加较为明显。制剂缓冲体系pH增加至6.5时，对应制剂为F3、F12、F6，观察到多聚体明显增加，见图1泳道6及泳道9、图2泳道4；制剂中甘露醇含量较低时，多聚体含量明显增加，对应制剂为F1、F2、F3，见图1泳道4及泳道5、6；制剂中添加EDTA没有达到抑制多聚体形成的作用；F4(图2泳道2)、F5(图2泳道3)、F10(图1泳道7)加样孔中样品组分条带颜色相对较浅，表明F4、F5、F10制剂聚集体相对含量较少，该结果和SEC-HPLC测定结果一致；
对制剂组成中的缓冲体系pH、甘露醇、NaCl、EDTA含量进行了确认分析，试验结果表明：制剂缓冲体系适宜pH为6.0，制剂组成中应包含甘露醇和任选地NaCl，其中甘露醇含量不低于40mg/ml，保持适宜的NaCl含量，根据试验结果，确认适宜的制剂处方为F4或F10，分别为5mg/mL重组抗CTLA-4全人源单克隆抗体/20mM磷酸缓冲液/0.01％吐温80/4％甘露醇/30mM NaCl(pH6.0)、5mg/mL重组抗CTLA-4全人源单克隆抗体/20mM磷酸缓冲液/0.01％吐温80/5％甘露醇/0mM NaCl (pH6.0)，制剂缓冲体系pH可控制在5.8～6.2范围内。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种抗CTLA-4抗体组合物，其特征在于，所述的组合物包括抗CTLA-4单克隆抗体、表面活性剂、缓冲液和稳定剂。
  2. 如权利要求1所述的组合物，其特征在于，所述抗CTLA-4单克隆抗体的含量为0.5-50mg/mL，较佳地0.5-40mg/mL，更佳地0.5-30mg/mL，更佳地0.5-20mg/mL，更佳地1-15mg/mL，更佳地2-10mg/mL，更佳地3-8mg/mL，最佳地5mg/mL。
  3. 如权利要求1所述的组合物，其特征在于，所述的表面活性剂选自下组：聚氧乙烯山梨醇酐脂肪酸酯、聚氧乙烯氢化蓖麻油、甘油脂肪酸酯、聚山梨醇酯、泊洛沙姆，或其组合；
    所述的缓冲液选自下组：柠檬酸盐-柠檬酸缓冲液、磷酸盐-磷酸缓冲液，或其组合；和/或
    所述的稳定剂选自下组：氨基酸、糖醇，或其组合。
  4. 如权利要求3所述的组合物，其特征在于，所述的聚山梨醇酯选自下组：聚山梨醇酯20(PS–20，吐温-20)、聚山梨醇酯40(PS-40，吐温-40)、聚山梨醇酯60(PS–60，吐温-60)、聚山梨醇酯80(PS-80，吐温-80)；
    所述的氨基酸选自下组：脯氨酸、精氨酸(Arginine)、甘氨酸(Glycine)、组氨酸(Histidine)、甲硫氨酸(Methionine)，或其组合；和/或
    所述的糖醇选自下组：蔗糖(Sucrose)、甘露醇(Mannitol)、海藻糖(Trehalose)、麦芽糖(Maltose)、山梨醇(Sorbitol)，或其组合。
  5. 如权利要求3所述的组合物，其特征在于，所述的表面活性剂包括聚山梨醇酯80(PS-80)。
  6. 如权利要求1所述的组合物，其特征在于，所述表面活性剂的含量为0.001-8wt％，较佳地0.001-3wt％，更佳地0.001-1wt％，更佳地0.002-0.5wt％，更佳地0.002-0.1wt％，更佳地0.002-0.06wt％，更佳地0.005-0.05wt％，更佳地0.005-0.03wt％，更佳地0.008-0.02wt％，更佳地0.008-0.012wt％，最佳地0.01wt％，以组合物的总重量计；
    所述缓冲液的浓度为3-80mM，较佳地5-60mM，更佳地5-40mM，更佳地10-30mM，更佳地15-25mM，更佳地18-22mM；和/或
    所述的稳定剂的含量为0.5-50wt％，较佳地0.5-40wt％，更佳地0.8-30wt％，更佳地1-20wt％，更佳地1-15wt％，更佳地1-10wt％，更佳地1-8wt％，更佳地2-7wt％，最佳地4wt％或5wt％，以组合物的总重量计。
  7. 如权利要求6所述的组合物，其特征在于，所述的缓冲液包括磷酸缓冲液。
  8. 如权利要求1所述的组合物，其特征在于，所述的组合物还包括渗透压调节剂，所述的渗透压调节剂包括无机盐；
    优选地，所述的渗透压调节剂的含量为0-80mM，较佳地0-60mM，更佳地0-50mM，更佳地0-30mM。
  9. 如权利要求8所述的组合物，其特征在于，所述的无机盐选自下组：氯化钠、氯化钾、氯化钙、氯化镁、硫酸钠、硫酸钾、硫酸钙、硫酸镁、硝酸钠、硝酸钾、硝酸钙、硝酸镁，或其组合。
  10. 如权利要求1所述的组合物，其特征在于，所述的组合物还包括抗氧化剂，所述的抗氧化剂包括EDTA(乙二胺四乙酸)。
  11. 如权利要求1所述的组合物，其特征在于，所述的组合物的pH为5.0-7.5，较佳地5.0-7.0，更佳地5.5-6.5，更佳地5.8-6.2，最佳地6.0。
  12. 一种如权利要求1所述的组合物，其特征在于，所述的抗CTLA-4抗体具有如SEQ ID NO.：1的重链氨基酸序列和SEQ ID NO.：2轻链氨基酸序列；
    所述的抗CTLA-4抗体具有如SEQ ID NO.：3的重链氨基酸序列和SEQ ID NO.：4轻链氨基酸序列；或
    所述的抗CTLA-4抗体具有如SEQ ID NO.：5的重链氨基酸序列和SEQ ID NO.：6轻链氨基酸序列。
  13. 一种冻干制剂，所述的冻干制剂通过以下方法制备：
    将如权利要求1所述的组合物进行冷冻干燥，得到冻干制剂。
  14. 如权利要求1所述的组合物或如权利要求13所述冻干制剂的用途，其特征在于，用于制备药物，所述药物用于(i)预防和/或治疗无法切除或转移性黑色素瘤；(ii)预防和/或治疗晚期肾细胞癌；(iii)预防和/或治疗微卫星高度不稳定性或错配修复缺陷的转移性结直肠癌；和/或(iv)用于黑色素瘤辅助治疗。
  15. 一种(i)预防和/或治疗无法切除或转移性黑色素瘤；(ii)预防和/或治疗晚期肾细胞癌；(iii)预防和/或治疗微卫星高度不稳定性或错配修复缺陷的转移性结直肠癌；和/或(iv)用于黑色素瘤辅助治疗的方法，所述的方法包括：给予所需对象如权利要求1所述的组合物。
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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101325968A (zh) * 2005-03-08 2008-12-17 法玛西雅厄普约翰有限责任公司 抗ctla-4抗体组合物
CN103861102A (zh) * 2005-03-08 2014-06-18 辉瑞产品公司 抗ctla-4 抗体组合物
CN106390115A (zh) * 2015-07-29 2017-02-15 上海君实生物医药科技股份有限公司 一种人源化单克隆抗体的稳定制剂
CN110840830A (zh) * 2019-10-25 2020-02-28 北京东方百泰生物科技有限公司 一种抗ctla-4单克隆抗体的注射制剂
CN111655728A (zh) * 2017-12-20 2020-09-11 和铂医药(上海)有限责任公司 结合ctla-4的抗体及其用途
CN112569183A (zh) * 2020-12-11 2021-03-30 上海赛金生物医药有限公司 一种抗ctla-4抗体及融合蛋白的制剂

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101325968A (zh) * 2005-03-08 2008-12-17 法玛西雅厄普约翰有限责任公司 抗ctla-4抗体组合物
CN103861102A (zh) * 2005-03-08 2014-06-18 辉瑞产品公司 抗ctla-4 抗体组合物
CN106390115A (zh) * 2015-07-29 2017-02-15 上海君实生物医药科技股份有限公司 一种人源化单克隆抗体的稳定制剂
CN111655728A (zh) * 2017-12-20 2020-09-11 和铂医药(上海)有限责任公司 结合ctla-4的抗体及其用途
CN110840830A (zh) * 2019-10-25 2020-02-28 北京东方百泰生物科技有限公司 一种抗ctla-4单克隆抗体的注射制剂
CN112569183A (zh) * 2020-12-11 2021-03-30 上海赛金生物医药有限公司 一种抗ctla-4抗体及融合蛋白的制剂

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