WO2023131213A1 - 一种包含抗FXI/FXIa抗体的药物组合物及其用途 - Google Patents

一种包含抗FXI/FXIa抗体的药物组合物及其用途 Download PDF

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WO2023131213A1
WO2023131213A1 PCT/CN2023/070573 CN2023070573W WO2023131213A1 WO 2023131213 A1 WO2023131213 A1 WO 2023131213A1 CN 2023070573 W CN2023070573 W CN 2023070573W WO 2023131213 A1 WO2023131213 A1 WO 2023131213A1
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pharmaceutical composition
fxi
sucrose
histidine
antigen
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PCT/CN2023/070573
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English (en)
French (fr)
Inventor
沈立君
赵志龙
李磊
汤辰翔
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上海迈晋生物医药科技有限公司
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Priority to CN202380015149.4A priority Critical patent/CN118382643A/zh
Publication of WO2023131213A1 publication Critical patent/WO2023131213A1/zh

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    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C07K16/36—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against blood coagulation factors

Definitions

  • the present disclosure belongs to the field of pharmaceutical preparations, and specifically relates to a pharmaceutical composition comprising an anti-FXI/FXIa antibody and an antigen-binding fragment thereof, and its use as a medicine.
  • the FXI gene is located on human chromosome 4 and encodes a secreted protein consisting of 607 amino acids.
  • the FXI protein molecule contains 4 apple domains and 1 catalytic domain. The form of dimer exists, and the protein FXI that forms a non-covalent complex with HK has a concentration of FXI in human plasma of about 30nM (15-45nM).
  • Human FXI molecules have 88% of the FXI molecules of monkeys and mice , 67% and 58% (two species, three homology data) homology.
  • Therapeutic proteins such as human monoclonal antibodies
  • antibody drugs have a large molecular weight and complex structure, and are prone to degradation, polymerization, or undesired chemical modification, making them unstable.
  • the study of stable formulations of antibody drugs is particularly important.
  • the present disclosure aims to provide a pharmaceutical composition (preparation) comprising an anti-FXI/FXIa antibody that is sufficiently stable and more suitable for administration.
  • the present disclosure provides a pharmaceutical composition comprising a protein (eg, an anti-FXI/FXIa antibody or antigen-binding fragment thereof). And a method for preparing the pharmaceutical composition, and a method for treating or preventing diseases or related pharmaceutical applications thereof are provided.
  • a protein eg, an anti-FXI/FXIa antibody or antigen-binding fragment thereof.
  • a protein in a pharmaceutical composition of the present disclosure has a PI value of about 5.0 to about 6.6, e.g., about 5.2 to about 6.4, about 5.3 to about 6.3, From about 5.4 to about 6.2, from about 5.5 to about 6.1, from about 5.6 to about 6.0, from about 5.7 to about 5.9 (eg, about 5.8), or any range between these points.
  • the PI value is obtained through capillary electrophoresis (ProteinSimple Company, Maurice) detection, and the detection conditions are:
  • Method Prepare and mix the reference substance and the test substance (such as desalted, choose the test substance after desalination), and centrifuge at 13000rpm for 5 minutes. Transfer 80 ⁇ L to Maurice 96-well plate or Maurice iCIEF sample bottle, centrifuge at 13000rpm for 5 minutes (for sample injection vial) or 4000rpm for 5 minutes (for 96-well plate sample injection).
  • a pharmaceutical composition of the present disclosure comprising a protein (e.g., an anti-FXI/FXIa antibody or antigen-binding fragment thereof) has a conductivity of about 100 to about 300 mS/cm, such as about 120 to about 260 mS/cm, about 130 to about 250 mS/cm, about 140 to about 240 mS/cm, about 150 to about 220 mS/cm, about 170 to about 200 mS/cm, about 180 to about 190 mS/cm (eg about 183.7 mS/cm), or these point values any range in between.
  • the conductivity is measured with a conductivity meter (Mettler Corporation; FE38-Standard).
  • compositions of the present disclosure comprising proteins (e.g., anti-FXI/FXIa antibodies or antigen-binding fragments thereof) have an osmolality of from about 250 to about 450 mOsmol/kg, such as from about 270 to about 430 mOsmol/kg, from about 290 to About 410mOsmol/kg, about 300 to about 400mOsmol/kg, about 310 to about 390mOsmol/kg, about 320 to about 380mOsmol/kg, about 330 to about 370mOsmol/kg, about 340 to about 360mOsmol/kg (for example about 350mOsmol/kg ), or any range between these point values.
  • the osmotic pressure is measured by an osmometer (Rozer Company; OM819.C), and the detection method is the freezing point depression method.
  • a protein (eg, an anti-FXI/FXIa antibody or antigen-binding fragment thereof) in a pharmaceutical composition of the present disclosure has a PI value of about 5.7 to about 5.9.
  • a pharmaceutical composition of the present disclosure comprising a protein has a conductivity of about 180 to about 190 mS/cm (e.g., about 183.7 mS/cm), and/or Has an osmolality of about 320 to about 380 mOsmol/kg.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the protein of any one of the above (eg, anti-FXI/FXIa antibody or antigen-binding fragment thereof).
  • the pharmaceutical composition is a self-buffering system.
  • the composition has therapeutic or prophylactic activity.
  • the composition also has advantages such as good stability.
  • the pharmaceutical composition contains basic amino acids such as histidine, arginine, lysine or any combination thereof.
  • the basic amino acid eg, histidine
  • the basic amino acid forms a self-buffering system with a protein (eg, an anti-FXI/FXIa antibody or antigen-binding fragment thereof).
  • the concentration of histidine in any one of the above pharmaceutical compositions is about 1 to about 100 mM, such as about 2 mM to about 80 mM, about 5 mM to about 70 mM, about 10 mM to about 60 mM, about 20 mM to about 50 mM, about 25 mM to about 45 mM, about 30 mM to about 40 mM, about 30 mM to about 100 mM, about 60 mM to about 100 mM, about 30 mM to about 90 mM, or any range between these points.
  • the histidine concentration is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 31 mM, about 32 mM, about 33 mM, about 34 mM, about 35 mM, about 36 mM, about 37 mM , about 38mM, about 39mM, about 40mM, about 41mM, about 42mM, about 43mM, about 44mM, about 45mM, about 46mM, about 47mM, about 48mM, about 49mM, about 50mM, for example about 35mM.
  • the pharmaceutical composition according to any one of the above which comprises a surfactant.
  • the surfactant is a nonionic surfactant.
  • the surfactant is selected from the group consisting of poloxamers (eg, poloxamer 188), polysorbates (eg, polysorbate 20 (ie, PS20), polysorbate 80 (ie, PS80)), Polyhydroxyalkylene, Triton, Sodium Lauryl Sulfonate, Sodium Lauryl Sulfonate, Sodium Octyl Glycoside, Lauryl-Sultaine, Myristyl-Sultaine, Linoleyl-Sultaine Betaine, Stearyl-Sultaine, Lauryl-Sarcosine, Myristyl-Sarcosine, Linoleyl-Sarcosine, Stearyl-Sarcosine, Linoleyl-Betaine, Myristyl-Betaine, Cetyl-Betaine, Laurylamidopropyl
  • the surfactant is a polysorbate or a poloxamer. In some embodiments, the surfactant is polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is poloxamer 188.
  • the concentration of the surfactant is about 0.1 mg/mL to about 10 mg/mL, such as about 0.2 mg/mL to about 8 mg/mL, about 0.5 mg/mL to about 6 mg/mL, about 0.8 mg /mL to about 5mg/mL, about 1.0mg/mL to about 4mg/mL, about 1.2mg/mL to about 3.8mg/mL, about 1.3mg/mL to about 3.6mg/mL, about 1.4mg/mL to about 3.4 mg/mL, about 1.5 mg/mL to about 3.3 mg/mL, about 1.6 mg/mL to about 3.2 mg/mL, about 1.7 mg/mL to about 3 mg/mL, about 1.8 mg/mL to about 2.8 mg/mL mL, about 1.9 mg/mL to about 2.5 mg/mL, about 1.8 mg/mL to about 2.4 mg/mL, about 1.9 mg/mL to about 2.2 mg
  • the pharmaceutical composition according to any one of the above which comprises an osmotic pressure regulator.
  • the osmotic pressure regulator is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), amino acids (including arginine, glycine, semi cystine, histidine, etc.) or salts (sodium chloride, potassium chloride, calcium chloride, etc.).
  • the osmotic pressure regulator is a sugar selected from the group consisting of: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabic Sugar alcohol, xylitol, sorbitol (also known as sorbitol), mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, Maltitol, lactitol and iso-maltulose.
  • the osmotic pressure regulator is one or more selected from the group consisting of sucrose, trehalose, sorbitol, arginine, proline, glycine and sodium chloride.
  • the osmotic pressure regulator is a non-reducing disaccharide; in some embodiments, the osmotic pressure regulator is trehalose or sucrose; in some embodiments, the osmotic pressure regulator is sucrose.
  • the concentration of the osmotic pressure regulator in the pharmaceutical composition according to any one of the above is about 1 mg/mL to about 300 mg/mL, about 1 mg/mL to about 400 mg/mL, for example about 10 mg/mL to about 400 mg/mL, about 5 mg/mL to about 200 mg/mL, about 10 mg/mL to about 150 mg/mL, about 20 mg/mL to about 140 mg/mL, about 30 mg/mL to about 130 mg/mL, about 40 mg/mL to about About 120 mg/mL, 50 mg/mL to about 200 mg/mL, about 50 mg/mL to about 110 mg/mL, about 60 mg/mL to about 100 mg/mL, about 70 mg/mL to about 90 mg/mL (eg, about 80 mg/mL) , or any range between these point values.
  • the concentration of the osmotic pressure regulator is about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 70 mg/mL, about 80mg/mL, about 90mg/mL, about 100mg/mL, about 110mg/mL, about 120mg/mL, about 130mg/mL, about 140mg/mL, about 150mg/mL, about 160mg/mL, about 170mg/mL, about 180mg/mL, about 190mg/mL, about 200mg/mL.
  • the pH value of any one of the pharmaceutical compositions above is about 4.0 to about 8.5, such as about 5.0 to about 8.0, about 6.0 to about 7.5, about 6.5 to about 7.5, about 6.6 to about 7.4 , about 6.7 to about 7.3, about 6.8 to about 7.2, about 6.9 to about 7.1 (eg, about 7.0), or any range between these point values.
  • pH values include about 4.0, about 4.5, about 5.0, about 6.0, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5.
  • the pH of the pharmaceutical composition obtained by exchanging the buffer almost coincides with the pH of the buffer.
  • the pH drift of pharmaceutical preparations is generally small (within the range of ⁇ 0.3). In some embodiments, the pH of the pharmaceutical formulation drifts within ⁇ 0.1.
  • the concentration of the protein (such as an anti-FXI/FXIa antibody or antigen-binding fragment thereof) in the pharmaceutical composition of any of the above is about 0.1 mg/mL to about 1000 mg/mL, such as about 1 mg/mL to about 500 mg/mL, about 1 mg/mL to about 300 mg/mL, about 10 mg/mL to about 400 mg/mL, about 20 mg/mL to about 350 mg/mL, about 30 mg/mL to about 300 mg/mL, about 30 mg/mL to about 200 mg/mL, about 130 mg/mL to about 200 mg/mL, about 40 mg/mL to about 250 mg/mL, about 50 mg/mL to about 200 mg/mL, about 60 mg/mL to about 180 mg/mL, about 50 mg/mL to about 150 mg/mL, about 60 mg/mL to about 150 mg/mL, about 70 mg/mL to about 130 mg/mL, about 80 mg/mL to about 120 mg/mL,
  • the pharmaceutical composition according to any one of the above contains any of a)-g):
  • the pH of the pharmaceutical composition is about 4.0 to about 8.5
  • the pharmaceutical composition may further contain sucrose or trehalose from about 1 mg/mL to about 500 mg/mL, and/or poloxamer 188 or polysorbate from about 0.1 mg/mL to about 10 mg/mL ester.
  • the pharmaceutical composition may further contain about 10 mg/mL to about 400 mg/mL of sucrose or trehalose, and/or about 0.5 mg/mL to about 8.0 mg/mL of poloxamer 188 or poly Sorbitate.
  • protein such as an anti-FXI/FXIa antibody or antigen-binding fragment thereof
  • protein such as an anti-FXI/FXIa antibody or antigen-binding fragment thereof
  • h about 130 mg/mL to about 200 mg/mL of protein (such as an anti-FXI/FXIa antibody or antigen-binding fragment thereof), and about 30 mM to about 100 mM, about 60 mM to about 100 mM, about 60 mM to about 90 mM histidine;
  • protein such as an anti-FXI/FXIa antibody or antigen-binding fragment thereof
  • the pH of the pharmaceutical composition in c) to e), h)-I) is about 6.5 to about 7.5 (eg about 6.7 to about 7.3, eg about 7.0);
  • the pharmaceutical composition in c) to e), h)-I) also contains sucrose or trehalose, and poloxamer 188 or polysorbate (such as polysorbate 80 or polysorbate 20);
  • the pharmaceutical compositions in c) to e), h)-I) contain sucrose at a concentration of about 40 to about 300 mg/mL, about 50 to about 200 mg/mL, about 60 to about 150 mg/mL , about 70 to about 120 mg/mL (eg, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL);
  • the pharmaceutical compositions in c) to e), h)-I) contain poloxamer 188 at a concentration of about 1.5 to about 6 mg/mL, about 1.5 to about 5.0 mg/mL, about 1.8 to about 3.5 mg/mL, about 1.8 to about 2.8 mg/mL (e.g., about 1.8 mg/mL, about 1.9 mg/mL, about 2.0 mg/mL, about 2.1 mg/mL, about 2.2 mg/mL, about 2.3 mg /mL, about 2.4mg/mL, about 2.5mg/mL, about 2.6mg/mL, about 2.7mg/mL, about 2.8mg/mL).
  • poloxamer 188 at a concentration of about 1.5 to about 6 mg/mL, about 1.5 to about 5.0 mg/mL, about 1.8 to about 3.5 mg/mL, about 1.8 to about 2.8 mg/mL (e.g., about 1.8 mg/mL, about 1.9 mg/mL, about 2.0 mg
  • the pharmaceutical composition contains any of the following:
  • the pH of the pharmaceutical composition is about 7.0; or
  • the pH of the pharmaceutical composition is about 6.7;
  • the pH of the pharmaceutical composition is about 7.3; or
  • the pH of the pharmaceutical composition is about 7.0; or
  • the pH of the pharmaceutical composition is about 7.0; or
  • the pH of the pharmaceutical composition is about 7.0; or
  • the pH of the pharmaceutical composition is about 7.0; or
  • the pH of the pharmaceutical composition is about 7.0; or
  • the pH of the pharmaceutical composition is about 7.0; or
  • the pH of the pharmaceutical composition is about 7.0.
  • the present disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising an anti-FXI/FXIa antibody, and a buffer.
  • the buffer is selected from acetate buffer, histidine buffer, Tris-hydrochloride buffer, Tris-citrate buffer, phosphate buffer.
  • the composition has therapeutic or prophylactic activity.
  • the composition also has advantages such as good stability.
  • the pH of the pharmaceutical composition is from about 4.0 to about 8.5, such as from about 5.0 to about 8.0, from about 6.0 to about 7.5, from about 6.5 to about 7.5, from about 6.6 to about 7.4, from about 6.7 to about 7.3, about 6.8 to about 7.2, about 6.9 to about 7.1 (eg, about 7.0), or any range between these points.
  • pH values include about 4.0, about 4.5, about 5.0, about 6.0, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5.
  • the pH of the pharmaceutical composition obtained by exchanging the buffer almost coincides with the pH of the buffer.
  • the pH drift of pharmaceutical preparations is generally small (within the range of ⁇ 0.3). In some embodiments, the pH of the pharmaceutical formulation drifts within ⁇ 0.1.
  • the pharmaceutical composition of any one of the above, wherein the concentration of the anti-FXI/FXIa antibody or antigen-binding fragment thereof is from about 0.1 mg/mL to about 1000 mg/mL, such as from about 1 mg/mL to about 500 mg/mL, about 1 mg/mL to about 300 mg/mL, about 10 mg/mL to about 400 mg/mL, about 20 mg/mL to about 350 mg/mL, about 30 mg/mL to about 300 mg/mL, about 40 mg/mL to about 250 mg/mL, about 50 mg/mL to about 200 mg/mL, about 60 mg/mL to about 180 mg/mL, about 50 mg/mL to about 150 mg/mL, about 60 mg/mL to about 150 mg/mL, about 70 mg/mL to about 150 mg/mL About 130 mg/mL, about 80 mg/mL to about 120 mg/mL, about 90 mg/mL to about 110 mg/mL, about 1 mg/mL to
  • the concentration of the anti-FXI/FXIa antibody is 1 mg/mL to 100 mg/mL. In some embodiments, the concentration of the anti-FXI/FXIa antibody is 40 mg/mL to 100 mg/mL.
  • the concentration of the anti-FXI/FXIa antibody is about 1 mg/mL, about 10 mg/mL, about 20 mg/mL, about 30 mg/mL, about 40 mg/mL, about 45 mg/mL, about 50mg/mL, about 55mg/mL, about 60mg/mL, about 65mg/mL, about 70mg/mL, about 80mg/mL, about 90mg/mL, about 100mg/mL, about 110mg/mL, about 120mg/mL, about 130 mg/mL, about 140 mg/mL, about 150 mg/mL, about 160 mg/mL, about 170 mg/mL, about 180 mg/mL, about 190 mg/mL, or about 200 mg/mL.
  • the surfactant is a nonionic surfactant.
  • the surfactant is selected from the group consisting of poloxamers (e.g., Poloxamer 188), polysorbates (e.g., polysorbate 20, polysorbate 80), polyhydroxyalkylenes, Triton, Sodium Dialkyl Sulfonate, Sodium Lauryl Sulfonate, Sodium Octyl Glycoside, Lauryl-Sultaine, Myristyl-Sultaine, Linoleyl-Sultaine, Stearyl-Sultaine betaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearyl-sarcosine, linoleyl-betaine, myristyl-sarcosine, cetyl Waxyl-Betaine
  • poloxamers e.g.
  • the surfactant is a polysorbate or a poloxamer. In some embodiments, the surfactant is polysorbate 80, polysorbate 20, or poloxamer 188. In some embodiments, the surfactant is poloxamer 188.
  • the surfactant concentration is 0.05 mg/mL, 0.1 mg/mL, 0.2 mg/mL, 0.3 mg/mL, 0.4 mg/mL, 0.5 mg/mL, 0.6 mg/mL, 0.7 mg /mL, 0.8mg/mL, 0.9mg/mL, or 1.0mg/mL, or any range between these point values.
  • the surfactant concentration is 0.4 mg/mL.
  • the surfactant is poloxamer 188 at 0.4 mg/mL.
  • the surfactant is 0.4 mg/mL polysorbate 80.
  • the pharmaceutical composition of any one of the above which includes an osmotic pressure regulator.
  • the osmotic pressure regulator is a sugar (including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars, etc.), amino acids (including arginine, glycine, semi cystine, histidine, etc.) or salts (sodium chloride, potassium chloride, calcium chloride, etc.).
  • the osmotic pressure regulator is a sugar selected from the group consisting of: glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabic Sugar alcohol, xylitol, sorbitol (also known as sorbitol), mannitol, milibiose, melezitose, raffinose, mannotriose, stachyose, maltose, lactulose, maltulose, Maltitol, lactitol and iso-maltulose.
  • the osmotic pressure regulator is one or more selected from the group consisting of sucrose, trehalose, sorbitol, arginine, proline, glycine and sodium chloride.
  • the osmotic pressure regulator is a non-reducing disaccharide; in some embodiments, the osmotic pressure regulator is trehalose or sucrose; in some embodiments, the osmotic pressure regulator is sucrose. In some embodiments, the osmotic pressure regulator is selected from sucrose, proline and sucrose, or glycine and sucrose.
  • the concentration of the osmotic pressure regulator is 1 mg/mL to 300 mg/mL, such as about 5 mg/mL to about 200 mg/mL, about 10 mg/mL to about 150 mg/mL mL, about 20 mg/mL to about 140 mg/mL, about 30 mg/mL to about 130 mg/mL, about 40 mg/mL to about 120 mg/mL, about 50 mg/mL to about 110 mg/mL, about 60 mg/mL to about 100 mg/mL mL, about 75 mg/mL to about 100 mg/mL, about 70 mg/mL to about 90 mg/mL, about 75 mg/mL to about 85 mg/mL, about 75 mg/mL to about 80 mg/mL, about 1 mg/mL to about 100 mg/mL mL, about 1 mg/mL to about 80 mg/mL, about 1 mg/mL to about 40 mg/mL, or any range between these points.
  • non-limiting examples of osmolarity regulator concentrations include about 7.5 mg/mL, about 11.5 mg/mL, about 40 mg/mL, about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 75 mg /mL, about 80mg/mL, about 85mg/mL, about 90mg/mL, about 95mg/mL, about 100mg/mL.
  • the aforementioned pharmaceutical composition is an isotonic formulation.
  • the osmolarity regulator is 80 mg/mL sucrose.
  • the osmolarity regulator is 40 mg/mL sucrose.
  • the osmolarity adjusting agent is about 10 mM to about 300 mM proline and about 1 mg/mL to about 200 mg/mL sucrose. In some embodiments, the osmolarity regulator is about 10 mM to about 200 mM proline and about 10 mg/mL to about 100 mg/mL sucrose. In some embodiments, the osmolarity adjusting agent is about 50 mM to about 150 mM proline and about 10 mg/mL to about 80 mg/mL sucrose. In some embodiments, the osmolarity adjusting agent is about 80 mM to about 120 mM proline and about 40 mg/mL to about 80 mg/mL sucrose.
  • the osmolarity regulator is about 100 mM proline and about 40 mg/mL sucrose. In some embodiments, the osmolarity regulator is about 10 mM to about 300 mM glycine and about 1 mg/mL to about 200 mg/mL sucrose. In some embodiments, the osmolarity regulator is about 10 mM to about 200 mM glycine and about 10 mg/mL to about 100 mg/mL sucrose. In some embodiments, the osmolarity regulator is about 50 mM to about 150 mM glycine and about 10 mg/mL to about 80 mg/mL sucrose.
  • the osmolarity regulator is about 80 mM to about 120 mM glycine and about 40 mg/mL to about 80 mg/mL sucrose. In some embodiments, the osmolarity regulator is about 100 mM glycine and about 40 mg/mL sucrose.
  • the pharmaceutical composition of any one of the above is about 5 mM to about 100 mM. In some embodiments, the concentration of the buffer is from about 10 mM to about 30 mM. In some embodiments, the concentration of the buffer is from about 5 mM to about 15 mM. In some embodiments, the concentration of the buffer is about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 40 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100mM, and any range between these point values.
  • the buffer is about 10 mM histidine-HCl. In some embodiments, the buffer is about 10 mM monosodium phosphate-disodium phosphate. In some embodiments, the buffer is about 10 mM Tris-HCl.
  • the pharmaceutical composition as described in any one of the above contains any of a)-i):
  • protein such as an anti-FXI/FXIa antibody
  • protein such as an anti-FXI/FXIa antibody
  • the pH of the pharmaceutical composition in a) to c) is about 4.0 to about 8.5 (eg about 6.5 to about 7.5, eg about 7.0);
  • the pharmaceutical composition in a) to c) also contains sucrose, or trehalose, or glycine and sucrose, or proline and sucrose, and poloxamer 188 or polysorbate (such as polysorbate 80 or Polysorbate 20);
  • the pharmaceutical composition in a) to c) also contains sucrose or trehalose, for example, at a concentration of about 1 mg/mL to about 300 mg/mL, about 10 mg/mL to about 150 mg/mL, about 75 to about 100 mg/mL (eg, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL);
  • sucrose or trehalose for example, at a concentration of about 1 mg/mL to about 300 mg/mL, about 10 mg/mL to about 150 mg/mL, about 75 to about 100 mg/mL (eg, about 70 mg/mL, about 80 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL, about 120 mg/mL);
  • the pharmaceutical composition in a) to c) also contains glycine and sucrose, such as glycine at a concentration of about 10 mM to about 300 mM and about 1 mg/mL to about 200 mg/mL sucrose, about 10 mM to about 200 mM glycine and about 10 mg/mL to about 100 mg/mL sucrose, about 80 mM to about 120 mM glycine, and about 40 mg/mL to about 80 mg/mL sucrose (e.g., about 100 mM glycine and about 40 mg/mL sucrose, about 90 mM glycine and about 50 mg/mL sucrose, about 110 mM glycine and about 30 mg/mL sucrose);
  • sucrose such as glycine at a concentration of about 10 mM to about 300 mM and about 1 mg/mL to about 200 mg/mL sucrose, about 10 mM to about 200 mM glycine and about 10 mg/mL to about
  • the pharmaceutical composition in a) to c) also contains proline and sucrose, such as proline at a concentration of about 10 mM to about 300 mM and about 1 mg/mL to about 200 mg/mL sucrose, about 10 mM to About 200 mM proline and about 10 mg/mL to about 100 mg/mL sucrose, about 80 mM to about 120 mM proline and about 40 mg/mL to about 80 mg/mL sucrose (such as about 100 mM proline and about 40 mg/mL sucrose, about 90 mM proline and about 50 mg/mL sucrose, about 110 mM proline and about 30 mg/mL sucrose);
  • proline and sucrose such as proline at a concentration of about 10 mM to about 300 mM and about 1 mg/mL to about 200 mg/mL sucrose, about 10 mM to About 200 mM proline and about 10 mg/mL to about 100 mg/mL sucrose, about 80 mM to about 120 mM
  • the pharmaceutical composition in a) to c) also contains poloxamer 188 or polysorbate (such as polysorbate 80 or polysorbate 20) at a concentration of about 0.1 to about 10 mg/mL, About 1.5 to about 4.0 mg/mL, about 1.8 to about 3.5 mg/mL, about 1.8 to about 2.8 mg/mL (e.g. about 1.8 mg/mL, about 1.9 mg/mL, about 2.0 mg/mL, about 2.1 mg/mL mL, about 2.2 mg/mL, about 2.3 mg/mL, about 2.4 mg/mL, about 2.5 mg/mL, about 2.6 mg/mL, about 2.7 mg/mL, about 2.8 mg/mL).
  • poloxamer 188 or polysorbate such as polysorbate 80 or polysorbate 20
  • the pH of the pharmaceutical composition is about 6.5 to about 7.5; optionally, the pharmaceutical composition may further contain about 80 mg/mL of sucrose, and about 0.1 mg/mL to about 10 mg/mL of poloxamer 188 or polysorbate.
  • the pH of the pharmaceutical composition is about 6.5 to about 7.5;
  • the pharmaceutical composition may further contain glycine or proline of about 100 mM, sucrose of about 40 mg/mL, and about 0.1 mg/mL to Approximately 10 mg/mL of poloxamer 188 or polysorbate.
  • the pH of the pharmaceutical composition is about 4.0 to about 8.5;
  • the pharmaceutical composition can further contain sucrose from about 10 mg/mL to about 150 mg/mL, and about 0.1 mg/mL to about Poloxamer 188 or polysorbate at 10 mg/mL.
  • the pH of the pharmaceutical composition is about 4.0 to about 8.5; optionally, the pharmaceutical composition may further contain glycine or proline from about 10 mM to about 200 mM, about 10 mg/mL to about 100 mg /mL of sucrose, and about 0.1 mg/mL to about 10 mg/mL of poloxamer 188 or polysorbate.
  • the pH of the pharmaceutical composition is about 6.5 to about 7.5;
  • the pharmaceutical composition can further contain about 80 mg/mL of sucrose, and about 1.5 mg/mL to about 4 mg/mL of porine Loxamer 188 or polysorbate.
  • the pH of the pharmaceutical composition is about 6.5 to about 7.5; optionally, the pharmaceutical composition may further contain glycine or proline of about 100 mM, sucrose of about 40 mg/mL, and about 1.5 mg /mL to about 4 mg/mL of poloxamer 188 or polysorbate.
  • the pharmaceutical compositions of the present disclosure also comprise a solvent.
  • the solvent in the pharmaceutical composition is selected from but not limited to non-toxic physiologically acceptable liquid carriers, such as physiological saline, water for injection, glucose solution (such as 5% glucose injection, glucose sodium chloride injection) and the like.
  • the present disclosure also provides a pharmaceutical composition obtained by diluting 0.9% normal saline or 5% glucose solution, or containing the concentration of the above pharmaceutical composition diluted with 0.9% normal saline or 5% glucose solution which can be used as an intravenous injection Anti-FXI/FXIa antibody or its antigen-binding fragment, histidine, sucrose, poloxamer 188.
  • the concentration of the anti-FXI/FXIa antibody or antigen-binding fragment thereof in the pharmaceutical composition is from about 0.01 mg/mL to about 50 mg/mL.
  • the concentration of the anti-FXI/FXIa antibody or antigen-binding fragment thereof in the pharmaceutical composition is from about 0.1 mg/mL to about 30 mg/mL. In some embodiments, the concentration of the anti-FXI/FXIa antibody or antigen-binding fragment thereof in the pharmaceutical composition is from about 0.20 mg/mL to about 13.30 mg/mL.
  • the protein in the pharmaceutical composition of the present disclosure is an antibody, antibody fragment, or Fc fusion protein.
  • the antigen targeted by the antibody, antibody fragment or Fc fusion protein is selected from the group consisting of: Factor VIIIC, Factor IX, Factor XI/Factor XIa (FXI/FXIa), Tissue Factor, Vascular Endothelial Growth Factor (VEGF) , renin, growth hormone, growth hormone releasing factor, parathyroid hormone, thyrotropin, lipoprotein, alpha-1-antitrypsin, bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), epidermal growth factor (EGF), transforming growth factor (TGF), interferon, M-CSF, GM-CSF, G-CSF, IL-1 to IL-10, HER2, HER3 or HER4 receptors, CTLA-4
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof in the pharmaceutical composition above comprises a heavy chain variable region and a light chain variable region, wherein:
  • the heavy chain variable region comprises HCDR1, HCDR2, HCDR3 shown in SEQ ID NO: 7, 8, 9 respectively, and the light chain variable region comprises respectively shown in SEQ ID NO: 10, 11, 12 LCDR1, LCDR2, LCDR3.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof in the pharmaceutical composition may be selected from murine antibodies, chimeric antibodies, humanized antibodies, such as humanized antibodies.
  • the light chain and heavy chain FR sequences on the light chain and heavy chain variable regions of the humanized anti-FXI/FXIa antibody described in the pharmaceutical composition are derived from human germline light chain and heavy chain, respectively FR or its mutant sequence.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises an antibody heavy chain variable region and a light chain variable region, wherein:
  • a heavy chain variable region sequence such as SEQ ID NO: 5 or a sequence having at least 80%, at least 85%, at least 90% identity thereto, a light chain variable region sequence such as SEQ ID NO: 6 or a sequence having at least 80% identity thereto %, at least 85%, at least 90% identical sequences;
  • a heavy chain variable region sequence such as SEQ ID NO: 13 or a sequence having at least 80%, at least 85%, at least 90% identity thereto, a light chain variable region sequence such as SEQ ID NO: 14 or a sequence having at least 80% identity thereto %, at least 85%, at least 90% identical sequences;
  • a heavy chain variable region sequence such as SEQ ID NO: 15 or a sequence having at least 80%, at least 85%, at least 90% identity thereto, a light chain variable region sequence such as SEQ ID NO: 16 or a sequence having at least 80% identity thereto %, at least 85%, at least 90% identical sequences;
  • a heavy chain variable region sequence such as SEQ ID NO: 17 or a sequence having at least 80%, at least 85%, at least 90% identity thereto, a light chain variable region sequence such as SEQ ID NO: 16 or a sequence having at least 80% identity thereto %, at least 85%, at least 90% identical sequences;
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof in the pharmaceutical composition comprises an antibody heavy chain variable region and a light chain variable region, wherein: the heavy chain variable region sequence is as SEQ ID Shown in NO: 17, and the sequence of the light chain variable region is shown in SEQ ID NO: 16.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof further comprises a heavy chain constant region and a light chain constant region.
  • the heavy chain constant region is selected from the constant regions of human IgG1, IgG2, IgG3, IgG4, IgG4P (i.e. the S241P mutant of IgG4), and the light chain constant region is selected from the human kappa and lambda chains constant region.
  • the sequence of the IgG4P Fc (that is, the IgG4Fc containing S241P) is shown in, for example, SEQ ID NO: 19.
  • sequence of the heavy chain CH1 is as shown in SEQ ID NO: 18 or a sequence having at least 80%, at least 85%, at least 90% identity thereto, and/or the light chain constant region The sequence shown in SEQ ID NO: 20 or a sequence having at least 80%, at least 85%, at least 90% identity thereto.
  • the anti-FXI/FXIa antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, wherein: the heavy chain has a sequence shown in SEQ ID NO: 21 or at least 80%, at least 85%, at least 90% identical sequence, and said light chain has the sequence shown in SEQ ID NO: 22 or a sequence having at least 80%, at least 85%, at least 90% identical thereto.
  • the "at least 90% identity” encompasses at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity.
  • the "at least 90% identity” covers at least 80%, at least 85%, at least 87%, at least 88%, at least 89%, at least 90%, at least 95%, at least 96%, at least 97%, At least 98%, at least 99% identity.
  • the anti-FXI/FXIa antibody in the pharmaceutical composition comprises an antibody heavy chain and a light chain, wherein: the heavy chain sequence is as shown in SEQ ID NO: 21, and the light chain sequence is as shown in SEQ ID NO: 21 ID NO: 22.
  • the antigen-binding fragment of the anti-FXI/FXIa antibody is a Fab, Fv, sFv, Fab', F(ab')2, linear antibody, single chain antibody, scFv, sdAb, sdFv, Nanobody, Peptibodies peptibodies, domain antibodies and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv), eg specifically scFv, Fv, Fab or Fab' fragments.
  • the heavy chain variable region and/or the light chain variable region of the anti-FXI/FXIa antibody comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid changes .
  • the amino acid changes may be conservative substitutions of amino acid residues in the variable region.
  • the above-mentioned anti-FXI/FXIa antibody may have any or more of the following properties:
  • the method for measuring the above-mentioned affinity is, for example, BIACORE TM .
  • the pharmaceutical composition of any one of the foregoing can be intravenous injection, subcutaneous injection, intraperitoneal injection or intramuscular injection.
  • the present disclosure provides a method for preparing any one of the aforementioned pharmaceutical compositions, the method comprising the step of replacing the stock solution of the anti-FXI/FXIa antibody or antigen-binding fragment thereof with a buffer.
  • the present disclosure also provides a liquid formulation comprising the pharmaceutical composition described in any one of the foregoing.
  • the pharmaceutical composition described in the present disclosure has sufficient pharmaceutical stability and can be placed stably for a long time.
  • the present disclosure also provides a method for preparing a freeze-dried preparation of a pharmaceutical composition containing an anti-FXI/FXIa antibody, the method comprising the step of freeze-drying the aforementioned pharmaceutical composition.
  • the present disclosure also provides a lyophilized formulation of a pharmaceutical composition comprising an anti-FXI/FXIa antibody, the lyophilized formulation is obtained by lyophilizing any of the aforementioned pharmaceutical compositions.
  • the lyophilized formulation is stored at 2-8° C. in the dark and is stable for at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months.
  • the lyophilized formulation is stable at 25°C for at least 1 month, at least 3 months, at least 6 months, at least 12 months.
  • the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, or at least 30 days.
  • the present disclosure also provides a reconstitution solution comprising an anti-FXI/FXIa antibody pharmaceutical composition, the reconstitution solution is prepared by reconstitution of any one of the aforementioned lyophilized preparations.
  • the present disclosure further provides a product, which includes a container filled with the pharmaceutical composition, liquid preparation, freeze-dried preparation or reconstitution solution as described in any one of the above containers.
  • the container may be, but is not limited to, a neutral borosilicate glass tubing injection bottle.
  • the present disclosure also provides the pharmaceutical composition according to any of the above, the liquid preparation according to any of the above, the freeze-dried preparation according to any of the above, the reconstitution solution according to any of the above, or the product according to any of the above in Use in the preparation of medicaments for treating diseases.
  • the present disclosure also provides a method for treating or preventing diseases, comprising administering to a subject in need an effective amount of the pharmaceutical composition as described in any one of the above, the liquid preparation as described in any one of the above, or the lyophilized product as described in any one of the above A preparation, a reconstitution solution as in any of the above, or a product as in any of the above.
  • the present disclosure also provides the pharmaceutical composition according to any of the above, the liquid preparation according to any of the above, the freeze-dried preparation according to any of the above, the reconstitution solution according to any of the above, or the product according to any of the above, It is used for the treatment of diseases.
  • the disease of any one of the above which is a thrombotic or thromboembolic disease and/or a thrombotic or thromboembolic complication, or arrhythmia, cardiogenic thromboembolism, diffuse vascular Internal coagulation.
  • said thrombotic or thromboembolic disease is selected from but not limited to: cardiac coronary artery disease, such as acute coronary syndrome (ACS), ST-segment elevation Myocardial infarction (STEMI) and myocardial infarction without ST-segment elevation (non-STEMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention, and peripheral arterial occlusive disease, pulmonary embolism , venous thromboembolism, venous thrombosis, transient ischemic attack, and thrombotic or thromboembolic disease in other vessels in thrombotic stroke and thromboembolic stroke, caused by chronic thromboembolism (CTEPH) Lung disease and pulmonary hypertension, or a combination thereof.
  • CTEPH chronic thromboembolism
  • the present disclosure provides a method for treating or preventing a disease, comprising administering to a subject in need a therapeutically or preventively effective amount of the pharmaceutical composition described in any of the above, the liquid preparation of any of the above, the liquid preparation of any of the above The lyophilized preparation, the reconstitution solution of any of the above, or the product of any of the above, wherein: the disease is a thrombotic or thromboembolic disease and/or a thrombotic or thromboembolic complication, Or arrhythmia, cardiogenic thromboembolism, disseminated intravascular coagulation.
  • the thrombotic or thromboembolic disease or its complications are selected from, but not limited to: coronary artery disease of the heart, such as acute coronary syndrome (ACS), myocardial infarction with ST-segment elevation ( STEMI) and myocardial infarction without ST-segment elevation (non-STEMI), stable angina, unstable angina, reocclusion and restenosis after coronary intervention, and peripheral arterial occlusive disease, pulmonary embolism, venous thromboembolism , venous thrombosis, transient ischemic attack, and thrombotic or thromboembolic disease in other vessels in thrombotic stroke and thromboembolic stroke, lung disease and pulmonary hypertension caused by chronic thromboembolism (CTEPH) or a combination thereof.
  • the administration is by subcutaneous or intravenous injection.
  • FIG 1 In vitro FXIIa-mediated inhibition of FXI activating enzyme activity by anti-FXI/FXIa antibodies.
  • Figure 2 Detection of aPTT/PT anticoagulant activity in human blood, wherein Figures 2A and 2B are the results of aPTT and PT tests of anti-FXI/FXIa antibodies on human blood, respectively. 1209 is another anti-FXI/FXIa antibody obtained through screening in this application.
  • Figure 3 Detection of aPTT/PT anticoagulant activity in monkey blood, wherein Figures 3A and 3B are the results of aPTT and PT tests of anti-FXI/FXIa antibodies on monkey blood, respectively.
  • Figure 4 Animal experiment detection of anti-FXI/FXIa antibody on cynomolgus monkeys, wherein, Figure 4A is the change curve of aPTT, plasma drug concentration, FXI:C% and free FXI of antibody 3882 in cynomolgus monkeys, Figure 4B Figure 4C and Figure 4D show the safety test of antibody 3882 in cynomolgus monkeys, the bleeding time measurement and PT detection results respectively.
  • Figure 5 Anti-FXI/FXIa antibody in vivo pharmacodynamics (PD) test results on cynomolgus monkeys, wherein, Figure 5A is the APTT (s) test results, Figure 5B is the FXI:C (%) test results, 3882 (1mg /kg) for intravenous administration and subcutaneous administration, and the control BAY1213790 (1mg/kg) for intravenous administration.
  • PD pharmacodynamics
  • Figure 6 Screening of the pH value of the buffer system for anti-FXI/FXIa antibody preparations, where Figures 6A, 6B, and 6C show the changing trends of SEC, NRCE, and iCIEF at different pH/buffer 40°C, respectively.
  • Figure 7 Fine screening of pH, excipients and surfactant concentrations in anti-FXI/FXIa antibody preparations, where Figures 7A, 7B, and 7C show the trends of SEC, NRCE, and CEX under different pH/buffer 40°C conditions, respectively.
  • Figure 8 Screening of anti-FXI/FXIa antibody preparation buffer system and surfactant types, in which Figures 8A and 8B show the trends of SEC and CEX of different surfactants at 40°C, respectively.
  • Buffering agent refers to a buffering agent that is resistant to pH changes through the action of its acid-base conjugated component.
  • buffers to control pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, Glycylglycine and other organic acid buffers.
  • a “histidine buffer” is a buffer comprising histidine ions.
  • histidine buffers include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate and other buffers, preferably histidine-hydrochloride buffer agent.
  • Histidine-HCl buffer is formulated with histidine and hydrochloric acid or histidine and histidine hydrochloride.
  • Self-buffering means that a substance such as a pharmaceutical protein (e.g., an antibody or antigen-binding fragment thereof; in some embodiments, an anti-FXI/FXIa antibody, such as the 3882 antibody of the present disclosure) is sufficient in the absence of other buffering agents.
  • a pharmaceutical protein e.g., an antibody or antigen-binding fragment thereof; in some embodiments, an anti-FXI/FXIa antibody, such as the 3882 antibody of the present disclosure
  • Tris-citrate buffer is a buffer comprising citrate ions.
  • Tris-citrate buffer include Tris-hydrochloride, Tris-acetate, Tris-phosphate, Tris-sulfate, Tris-citrate and the like, preferably Tris-citrate.
  • Tris-HCl buffer is a buffer that includes hydrochloride ions.
  • Tris-hydrochloride buffer include Tris-hydrochloride, Tris-acetate, Tris-phosphate, Tris-sulfate, Tris-citrate and the like buffers and the like.
  • a preferred citrate buffer is Tris-HCl buffer.
  • a “phosphate buffer” is a buffer that includes phosphate ions.
  • the phosphate buffer include disodium phosphate-sodium dihydrogenphosphate, disodium phosphate-potassium dihydrogenphosphate, and the like.
  • a preferred phosphate buffer is disodium phosphate-sodium dihydrogen phosphate buffer.
  • Acetate buffer is a buffer that includes acetate ions.
  • acetate buffers include acetic acid-sodium acetate, acetic acid-histidine salt, acetic acid-potassium acetate, acetic acid-calcium acetate, acetic acid-magnesium acetate, and the like.
  • a preferred acetate buffer is acetic acid-sodium acetate buffer.
  • “Pharmaceutical composition” means a mixture containing one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, and other chemical components such as physiologically/pharmaceutically acceptable Carriers and Excipients.
  • the purpose of the pharmaceutical composition is to promote the administration to the organism, facilitate the absorption of the active ingredient and thus exert biological activity.
  • “pharmaceutical composition” and “formulation” are not mutually exclusive.
  • the solvent therein is water.
  • the term “about” means that a value is within an acceptable error range for the particular value as determined by one of ordinary skill in the art, depending in part on how it is measured or determined (ie, the limits of the measurement system). For example, “about” can mean within 1 or more than 1 standard deviation every practice in the art. Alternatively, “about” or “comprising essentially” can mean a range of up to 20%. Furthermore, particularly with respect to biological systems or processes, the term can mean up to an order of magnitude or up to 5 times a value. Unless otherwise stated, when a specific value appears in the application and claims, the meaning of "about” or “comprising essentially” should be assumed to be within an acceptable error range for the specific value.
  • the pharmaceutical composition of the present disclosure can achieve a stable effect: the antibody therein substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably, the pharmaceutical composition Afterwards, its physical and chemical stability and its biological activity are substantially retained.
  • the shelf life is generally selected based on the intended shelf life of the pharmaceutical composition.
  • a stable pharmaceutical antibody formulation is one in which no significant changes are observed when stored at refrigerated temperatures (2-8° C.) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years.
  • stable liquid formulations include liquid formulations that exhibit the desired characteristics after a period that includes storage at 25°C including 1 month, 3 months, 6 months, or 1 month at 40°C.
  • Typical acceptable criteria for stability are as follows: Usually no more than about 10%, preferably no more than about 5%, of the antibody monomer is degraded as measured by SEC-HPLC. By visual analysis, drug antibody preparations were colorless or yellow, clear to micro-opalescent. The concentration, pH and osmolality of the formulations have no more than ⁇ 10% variation. Typically not more than about 10%, preferably not more than about 5% truncation is observed, and typically not more than about 10%, preferably not more than about 5%, aggregates are formed.
  • the antibody does not show significant increased aggregation, precipitation and/or denaturation after visual inspection for color and/or clarity, or as measured by UV light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS) , then the antibody "retains its physical stability" in the pharmaceutical formulation. Changes in protein conformation can be assessed by fluorescence spectroscopy (which determines protein tertiary structure) and by FTIR spectroscopy (which determines protein secondary structure).
  • An antibody "retains its chemical stability" in a pharmaceutical formulation if the antibody exhibits no significant chemical changes. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Degradation processes that often alter protein chemical structure include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by methods such as peptide mapping combined with mass spectrometry or MALDI/TOF/MS, etc. method), deamidation (evaluated by methods such as ion-exchange chromatography, capillary isoelectric focusing, peptide mapping, isoaspartic acid measurement, etc.) and isomerization (by measuring isoaspartic acid content, Peptide mapping, etc. to evaluate).
  • An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of biological activity exhibited at the time the pharmaceutical formulation was prepared.
  • the biological activity of an antibody can be determined, for example, by an antigen binding assay.
  • Fractor XI also referred to herein as “coagulation factor XI,” “FXI,” or “fXI,” is a two-chain glycoprotein with a molecular weight of about 160 kilodaltons (kD). The two chains can be the same disulfide-linked polypeptide having a molecular weight of about 80,000 Daltons. FXI contains 4 "apple domains" (A1-A4 from the N-terminus, heavy chain) and a C-terminal catalytic domain (light chain).
  • the four apple domains contain FXI binding sites for other proteins, such as Al for thrombin; A2 for HK, A3 for Factor IX (FIX), GPIb and heparin, A4 for FXIIa.
  • FXI can be converted to its active form, coagulation factor XIa (FXIa), by Factor XIIa (FXIIa).
  • the serine protease FXIa converts factor IX to IXa, which then activates factor X (Xa).
  • Xa can then mediate factor II/thrombin activation.
  • Antibody or “immunoglobulin” is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, traditional antibodies (composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds) Tetrapeptide chain structure antibody), and Fab, Fv, sFv, F(ab')2, linear antibody, single chain antibody, scFv, sdAb, sdFv, nanobody, peptibody, structure Domain antibodies (heavy chain (VH) antibodies, light chain (VL) antibodies) and multispecific antibodies (bispecific antibodies, diabody, triabody and tetrabody, tandem di-scFv, tandem tri-scFv).
  • antibody as used in this disclosure includes full-length antibodies, individual chains thereof, and any portion, domain or fragment thereof that has antigen-binding activity (i.e., antigen-binding fragments), as well as individual chains thereof and any antigen-binding antibody. Active parts, domains or fragments of multispecific antibodies (including but not limited to antigen binding domains or fragments such as VHH domains or VH/VL domains, respectively).
  • Traditional antibodies or immunoglobulins usually have a tetrapeptide chain structure consisting of two identical heavy chains and two identical light chains connected by interchain disulfide bonds. The amino acid composition and arrangement order of the constant region of the heavy chain are different.
  • immunoglobulins can be divided into five classes, or isotypes of immunoglobulins, namely IgM, IgD, IgG, IgA and IgE, and their corresponding Heavy chains are mu chains, delta chains, gamma chains, alpha chains, and epsilon chains, respectively.
  • the same class of Ig can be divided into different subclasses according to the amino acid composition of its hinge region and the number and position of heavy chain disulfide bonds.
  • IgG can be divided into IgG1, IgG2, IgG3, and IgG4.
  • Light chains are classified as either kappa chains or lambda chains by difference in the constant region.
  • Each of the five Ig classes can have either a kappa (kappa) chain or a lambda (lambda) chain.
  • an antibody of the disclosure specifically or substantially specifically binds to human FXI/FXIa.
  • variable region The sequence of about 110 amino acids near the N-terminal of the antibody heavy chain and light chain varies greatly, which is the variable region (Fv region); the rest of the amino acid sequence near the C-terminal is relatively stable, which is the constant region.
  • the variable region includes 3 hypervariable regions (HVR) and 4 framework regions (FR) with relatively conserved sequences. Three hypervariable regions determine the specificity of antibodies, also known as complementarity determining regions (CDR).
  • CDR complementarity determining regions
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of 3 CDR regions and 4 FR regions, and the sequence from the amino terminal to the carboxyl terminal is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the 3 CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the number and position of the CDR amino acid residues in the LCVR region and HCVR region of the antibody or antigen-binding fragment of the present disclosure conform to the known Kabat numbering rules (LCDR1-3, HCDR2-3), or conform to the numbering rules of kabat and chothia (HCDR1).
  • Antibodies of the present disclosure include murine antibodies, chimeric antibodies, humanized antibodies, preferably humanized antibodies.
  • chimeric antibody is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by the murine antibody.
  • To establish a chimeric antibody it is necessary to first establish a hybridoma that secretes a mouse-derived specific monoclonal antibody, then clone the variable region gene from the mouse hybridoma cell, and then clone the constant region gene of the human antibody as required, and then clone the variable region gene of the mouse
  • the gene is connected with the human constant region gene to form a chimeric gene and then inserted into a human vector, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the FXI chimeric antibody further comprises a light chain constant region of a human ⁇ , ⁇ chain or a variant thereof.
  • the antibody heavy chain of the FXI chimeric antibody further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • humanized antibody also known as CDR-grafted antibody (CDR-grafted antibody) refers to the antibody variable region framework grafted with mouse CDR sequences to humans, that is, different types of human germline Antibodies generated from antibody framework sequences. It can overcome the strong antibody variable antibody response induced by chimeric antibodies due to carrying a large number of mouse protein components.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • the germline DNA sequences of the human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase ), as well as in Kabat, EA et al.
  • the humanized antibody of the present disclosure also includes the humanized antibody after affinity maturation of CDR by phage display.
  • mutation in the mutated sequence described in the present disclosure includes, but is not limited to, "back mutation", “conservative modification” or “conservative substitution or substitution”.
  • Consservative modification or “conservative substitution or substitution” in this disclosure refers to other amino acid substitutions in proteins with similar characteristics (such as charge, side chain size, hydrophobicity/hydrophilicity, main chain conformation and rigidity, etc.) Amino acids allow frequent changes without altering the biological activity of the protein. Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al.
  • the "mutated sequence” mentioned in the present disclosure refers to the nucleotide sequence and/or Amino acid sequence Nucleotide and/or amino acid sequences having varying degrees of percent sequence identity.
  • the sequence identity described in this disclosure may be at least 85%, 90% or 95%, for example at least 95%.
  • Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% , 100%.
  • Sequence comparison and determination of percent identity between two sequences can be performed by default settings of the BLASTN/BLASTP algorithm available on the website of the National Center For Biotechnology Institute.
  • binding to FXI/FXIa refers to being capable of interacting with FXI/FXIa or its epitope, which may be of human origin.
  • antigen-binding site in the present disclosure refers to a discrete three-dimensional site on an antigen that is recognized by an antibody or antigen-binding fragment of the present disclosure.
  • the term “specifically binds” refers to as by techniques available in the art, e.g. competition ELISA, measure or Measure what is measured.
  • the term also applies when, for example, the antigen-binding domain of an antibody of the disclosure is specific for a particular epitope carried by many antigens, in which case the antibody carrying the antigen-binding domain is capable of specifically binding multiple antigens carrying that epitope .
  • Constant modification refers to the replacement of an amino acid in a protein with another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.) such that frequent Changes are made without altering the biological activity of the protein.
  • similar characteristics e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.
  • Single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., p. 224, (4th edition)).
  • substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • Amino acid sequence "identity” refers to the sequence similarity between two proteins or polypeptides. When a position in both compared sequences is occupied by the same amino acid residue, eg, if a position is occupied by the same amino acid residue in both polypeptides, then the molecules are identical at that position.
  • Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST2.0 algorithms described in Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. .(1977) Nucleic Acids Res. 25:3389-3402. Software for performing BLAST analyzes is publicly available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • one or more human FR regions are added to the non-human CDR region by genetic engineering methods.
  • the human FR germline sequence can be obtained from the website http://imgt.cines.fr of ImMunoGeneTics (IMGT) by comparing the IMGT human antibody variable region germline gene database and MOE software, or from Immunoglobulin Journal, 2001ISBN012441351 get.
  • Antibodies or antigen-binding fragments engineered in the present disclosure can be prepared and purified using conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems lead to glycosylation of antibodies, especially at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human FXI. Positive clones are expanded in serum-free medium in bioreactors for antibody production.
  • the culture fluid from which the antibody has been secreted can be purified by conventional techniques.
  • administering and “treating” when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid refer to the interaction of an exogenous drug, therapeutic agent, diagnostic agent, or composition with an animal, human, or subject. contact with subjects, cells, tissues, organs or biological fluids.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research and experimental methods.
  • Treatment of cells includes contacting the reagents with the cells, and contacting the reagents with a fluid, wherein the fluid contacts the cells.
  • administeristering” and “treating” also mean in vitro and ex vivo treatment of, for example, a cell by a reagent, diagnostic, binding composition or by another cell.
  • Treatment when applied to human, veterinary or research subjects means therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treating means administering a therapeutic agent, internally or externally, such as a composition comprising any of the conjugated compounds of the present disclosure, to a patient having one or more symptoms of a disease for which the therapeutic agent is known to have an effect.
  • a therapeutic agent is administered in a patient or population to be treated in an amount effective to alleviate one or more symptoms of a disease, to induce regression of such symptoms or to inhibit the progression of such symptoms to any clinically measurable extent.
  • the amount of a therapeutic agent effective to alleviate the symptoms of any particular disease (also referred to as a "therapeutically effective amount”) will vary depending on factors such as the patient's disease state, age and weight, and the ability of the drug to produce the desired effect in the patient.
  • Whether disease symptoms have been alleviated can be assessed by any of the clinical tests commonly used by a physician or other professional health care practitioner to assess the severity or progression of such symptoms.
  • embodiments of the present disclosure e.g., methods of treatment or articles of manufacture
  • any statistical test known in the art such as Student's t-test, chi-square test, Mann and Whitney's The U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test and Wilcoxon test determined that it should reduce the target disease symptoms in a statistically significant number of patients.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease.
  • An effective amount also means an amount sufficient to permit or facilitate diagnosis.
  • Effective amounts for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the general health of the patient, the route and dosage of administration and the severity of side effects.
  • An effective amount may be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Tm value refers to the thermal denaturation temperature of the protein, that is, the temperature at which half of the protein is unfolded. At this time, the spatial structure of the protein is destroyed, so the higher the Tm value, the higher the thermal stability of the protein.
  • Human FXI/FXIa protein (Uniprot Acc No.P03951), as the antigen and detection protein involved in the present disclosure, was purchased from Enzyme research laboratories (FXI:Cat.HFXI1111; FXIa:HFXIa 1111a). The following FXI/FXIa antigens unless otherwise specified refer to human FXI/FXIa.
  • ProteinG filler For the purification of mouse hybridoma supernatant, ProteinG filler (KANEKA: Cat.KanCapTMG) is the first choice for affinity chromatography.
  • the cultured hybridoma is centrifuged to get the supernatant, and a gravity column equipped with ProteinG filler is used for purification.
  • 6M hydrochloric acid Guanidine (Simga: Cat.G3272-1KG) was used to regenerate 3-5 times the column volume, and then use pure water to wash 3-5 times the column volume; then use 1 ⁇ PBS (pH7.4) (Sangon Biotech (Shanghai) co.
  • solution replacement can be performed by methods well known to those skilled in the art, such as using ultrafiltration tubes for ultrafiltration concentration and solution replacement to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required buffer system.
  • the cell culture supernatant expressing the Fc fusion protein or antibody is subjected to high-speed centrifugation, and the supernatant is collected.
  • ProteinA GE, Cat: 17-5474-99
  • affinity column was regenerated with 0.1M NaOH (Sigma: Cat: 71687-500g) for 5 times column volume, and then 1 ⁇ PBS (pH7.4) (Sangon Biotech (Shanghai) co., Ltd.: Cat.E607016-0500) to wash and equilibrate for 5 times the column volume.
  • the cell supernatant was loaded and bound at a low flow rate, and the flow rate was controlled so that the retention time was about 1 min or longer.
  • the chromatographic column was washed with 1 ⁇ PBS (pH7.4) for 5 times the column volume until the UV absorption dropped back to the baseline.
  • 0.1M Glycine (pH3.0) Sigma:Cat.410225-250G
  • elution product utilizes 1M Tris-HCl (pH8.0) (Vetec, Cat. .V900483) quickly adjust the pH to 7-8, temporarily store.
  • solution replacement can be performed by methods well known to those skilled in the art, such as using ultrafiltration tubes for ultrafiltration concentration and solution replacement to the required buffer system, or using molecular exclusion such as G-25 desalting to replace the required buffer system.
  • the optimized PEG-mediated fusion step was used to fuse spleen lymphocytes and myeloma cells Sp2/0-Ag14 cells to obtain hybridoma cells.
  • the hybridoma culture supernatant was purified, cell binding experiment and cell blocking experiment were carried out by combining ELISA method, and the positive well cells were expanded, frozen, preserved and sequenced in time.
  • Hybridoma clones are obtained after further screening of hybridoma cells by ELISA method, and then antibodies are prepared and purified.
  • the positive clone 3807 was obtained, and the corresponding antibody variable region amino acid sequence is as follows:
  • the selected and screened antibodies have high homology
  • the heavy chain variable region germline gene of the heavy chain is used as a template to graft the CDRs of the mouse-derived monoclonal antibody into the corresponding human-derived modules.
  • the three-dimensional structure of the transplanted antibody was simulated and analyzed again, and the specific site in the FR region that affected the structure and shape of the CDR region was restored.
  • the amino acid residues are identified and annotated by the Kabat numbering system.
  • the light chain template of the hybridoma clone 3807 humanized framework is IGKV3-11*01
  • the heavy chain template is IGHV1-69-2*01
  • the sequence of the humanized variable region is as follows (the CDRs are underlined):
  • the VH of antibody 3871 is 3807-VH1 and the VL is 3807-VL3.
  • 3871 is genetically modified to make the antibody more stable and less immunogenic. Only the FR region of the heavy chain variable region was modified, and the light chain variable region remained unchanged.
  • the sequence of the heavy chain variable region of the humanized antibody 3871 gene modification is as follows (the light chain remains unchanged):
  • the VH of antibody 3882 is 3807-VH1 (GTFS), and the VL is 3807-VL3.
  • the above heavy chain variable regions were fused with the corresponding human antibody heavy chain CH1 (SEQ ID NO: 18) and human antibody IgG4 Fc (including S241P mutation) (SEQ ID NO: 19), and the light chain variable regions were fused with Human kappa (SEQ ID NO: 20) was fused to form a recombinant chimeric antibody for subsequent detection.
  • the full-length sequence of the antibody is exemplified by 3882.
  • Antibody expression vectors were prepared by conventional methods, transfected into CHO cells, separated, purified, detected, and the target antibodies were obtained.
  • the Biacore method is used.
  • Use Protein A biosensor chip (Cat.#29127556, GE) to affinity capture a certain amount of antibody to be tested, and then flow through the surface of the chip with human FXI/FXIa under a series of concentration gradients, and use Biacore instrument (Biacore T200, GE ) Real-time detection of reaction signals to obtain association and dissociation curves.
  • Biacore T200, GE Biacore instrument
  • the biochip was washed and regenerated with the regeneration solution configured in the human antibody capture kit or the glycine-hydrochloric acid regeneration solution (Cat.#BR-1003-54, GE) at pH 1.5.
  • the buffer used in the experiment is HBS-EP+10 ⁇ buffer solution (Cat.#BR-1006-69, GE) diluted to 1 ⁇ (pH 7.4) with D.I.Water
  • Human blood/monkey blood was freshly collected in a sodium citrate tube, centrifuged at 3000rpm for 15min, and the upper plasma was taken.
  • the activated partial thromboplastin time (aPTT) in the presence of different concentrations of candidate antibodies (diluted in PBS) was measured with a Sysmex kit, and the candidate antibody to be tested was incubated with plasma at 37°C for 3 minutes, and then added by adding 25mM chloride Calcium reagent initiates coagulation and measures the time when coagulation occurs.
  • the concentration (EC50) of the candidate antibody that prolongs the aPTT by 50% was determined.
  • Bleeding time was observed in the blank control group at 15 minutes (min) and 3 hours (h) after administration; plasma was collected at 15 minutes, 3h, 6h and 1 day (d) after administration, respectively, and aPTT and PT were observed according to the above method.
  • the bleeding time of the administration group was observed at 15min, 3h, 2d, 4d, 1 week (w), 2w and 3w after administration; , 4w, 5w and 6w collected plasma according to the above method and observed aPTT, PT, plasma drug concentration, plasma FXI:C% and plasma free FXI concentration.
  • the animals in both groups were subjected to AV-shunt thrombectomy 1 day after administration for 10 minutes, and the net weight of the thrombus was weighed after thrombus establishment.
  • aPTT, PT and plasma FXI:C% were measured by coagulation instrument and corresponding kits, plasma free FXI concentration and plasma drug concentration were measured by ELISA method.
  • the thrombus weight was compared with the administration group and the control group, and the t test was used for statistical analysis.
  • the antibody test results are shown in Fig. 4A-Fig. 4D, wherein the negative control (NC) is no administration.
  • NC negative control
  • the results showed that 5mpk well inhibited thrombus formation and prolonged the endogenous coagulation time, but had no significant effect on animal bleeding time and exogenous coagulation time.
  • the PK results showed that the half-life was about 20 days.
  • Intravenous administration group animals before administration and 5min after administration 1h, 1d, 2d, 3d, 5d, 1w, 2w, 3w, 4w, subcutaneous administration group before administration and 1h, 1d, 2d after administration , 3d, 5d, 1w, 2w, 3w, 4w, blood samples were collected in sodium citrate blood collection tubes, aPTT and plasma FXI:C% were measured with a coagulation analyzer and corresponding kits.
  • the anti-FXI/FXIa antibodies in Examples 4 to 9 all have a heavy chain sequence as shown in SEQ ID NO: 21, and a light chain sequence as 3882 shown in SEQ ID NO: 22.
  • the buffer in Table 5 was prepared to prepare an antibody preparation with an anti-FXI/FXIa antibody concentration of 120mg/mL, and samples were taken for high temperature 40°C, shaking and freeze-thaw stability studies.
  • Appearance results show that no granules are produced in all formulations by shaking 5D; granules are produced in all formulations by freeze-thawing 5cycle, including 10mM acetic acid-sodium acetate pH4.5 and pH5.0 systems, 10mM histidine-histidine hydrochloride pH 6.5 System and 10mM sodium dihydrogen phosphate-disodium hydrogen phosphate pH 7.0 system produced less particles; 15D at 40°C all formulations produced particles, 10mM histidine-histidine hydrochloride pH 6.5 and 10mM sodium dihydrogen phosphate-phosphoric acid The disodium hydrogen pH 7.0 system produced fewer particles than the other two systems.
  • NRCE The results of NRCE (Fig. 6B) showed that the purity of NRCE decreased at 40°C-15D; in the 10mM Tris-hydrochloric acid system, with the increase of pH (pH 7.5-8.5), the purity of NRCE decreased significantly, and in the range of pH4.5-7.5 In other buffer systems, there was no significant difference in NRCE purity.
  • the buffer system with pH 6.5-7.5 (histidine-histidine hydrochloride, sodium dihydrogen phosphate-disodium hydrogen phosphate and Tris-hydrochloric acid system) is more suitable.
  • the purity inspection items have changed slightly, which are all within the acceptable range.
  • the 10mM histidine-histidine hydrochloride pH 7.0 system is superior to the 10mM histidine-histidine hydrochloride pH 6.5 and 10mM Tris-hydrochloride systems.
  • the antibody has better stability in the pH range of 6.5-7.5, and the histidine-histidine hydrochloride system is better than sodium dihydrogen phosphate-disodium hydrogen phosphate, Tris- Hydrochloric acid, acetic acid-sodium acetate and Tris-citric acid systems, and the 3882 antibody is more sensitive to ionic strength.
  • Appearance results show that no particles are produced in all formulations at 40°C 2W and 40°C 4W.
  • the pH/buffer is determined to be pH 7.0 of the histidine-antibody self-buffer system, and the surfactant is preferably poloxamer 188.
  • Appearance results show: Shaking 3D formulations have no particles; freeze-thaw 5cycle, F1 (35mM His, 0.1% Poloxamer 188), F2 (35mM His, 0.15% Poloxamer 188) have a small amount of particles; At 40°C and 2W, no particles were produced in all formulations; at 40°C and 4W, a small amount of particles were produced in F1 and F2.
  • NRCE results showed that: under freeze-thaw and shaking conditions, there was no significant difference between NRCE and T0; after high temperature, NRCE of different prescriptions decreased compared with T0, and there was no significant difference between the prescriptions.
  • the pH/buffer was determined to be histidine-antibody self-buffering system pH7.0, and the surfactant was 0.2% poloxamer 188.
  • NRCE results showed that: under freeze-thaw and shaking conditions, there was no significant difference between NRCE and T0; after high temperature, NRCE of different prescriptions decreased compared with T0, and there was no significant difference between the prescriptions.
  • the prescription was initially determined as 35mM histidine, 80mg/mL sucrose, 2mg/mL poloxamer 188, pH 7.0, and anti-FXI/FXIa antibody concentration 80mg/mL.
  • histidine-protein self-buffering system uses histidine concentration, protein concentration and poloxamer 188 concentration as variables to carry out DoE design
  • the factor level is histidine concentration 30-40mM
  • protein concentration 70-130mg/mL is histidine concentration 1.8-2.8g/L
  • Minitab software was used to conduct DoE experiments, and a series of prescriptions were obtained.
  • the samples were placed at -35°C/5 rounds of freezing and thawing at room temperature, 40°C-2W, 40°C -26D, shake for 3 days (25°C, 300rpm), check the stability:
  • NRCE results showed that: under freeze-thaw and shaking conditions, there was no significant difference between NRCE and T0; after high temperature, NRCE of different prescriptions decreased compared with T0, and there was no significant difference between the prescriptions.
  • the diluted medicinal solution has good compatibility with the infusion system, and can be used within 24h at 2-8°C + 6h at room temperature after dilution in the infusion system.

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Abstract

一种包含FXI/FXIa抗体的药物组合物及其用途，具体涉及的药物组合物包含抗FXI/FXIa抗体或其抗原结合片段以及缓冲剂。

Description

一种包含抗FXI/FXIa抗体的药物组合物及其用途
本公开要求如下专利申请的优先权：申请日为2022年01月05日，申请号为202210006206.X和申请日为2022年01月18日，申请号为202210053441.2，发明名称为“一种包含抗FXI/FXIa抗体的药物组合物及其用途”的中国专利申请；上述专利申请的全部内容通过引用结合在本公开中。
技术领域
本公开属于药物制剂领域，具体涉及一种包含抗FXI/FXIa抗体及其抗原结合片段的药物组合物，以及其作为药物的用途。
背景技术
FXI基因位于人类第4号染色体，编码由607个氨基酸组成的分泌蛋白，FXI蛋白分子包含4个苹果结构域(apple domains)和1个催化结构域(catalytic domain)，在人血液内是以同二聚体的形式存在，与HK形成非共价复合物的蛋白FXI循环在人血浆里的FXI浓度为约30nM(15-45nM)，人FXI分子与猴和小鼠的FXI分子分别具有88％、67％和58％(两个物种，三个同源性数据)的同源性。
目前已经有多篇文献和临床试验表明，抑制FXI及其转化成的FXIa的活性能够有效的降低血栓的形成，并且不会造成可见的出血风险。从目前披露的临床结果来看，相对于现有的口服抗凝剂(NOAC)，抗FXI/FXIa抗体有着相似或更好的抗血栓效果的同时出血风险更低，患者能够得到更安全的治疗。
治疗性蛋白例如人单克隆抗体，由于其特性，通常作为液体药物制剂来注射给药。但是，抗体药物其分子量大、结构复杂，容易降解、聚合或发生不希望发生的化学修饰等而变得不稳定。为了使抗体适合于给药，并且在储存及随后使用过程中能保持稳定性，发挥更好的效果，抗体药物的稳定制剂研究显得尤为重要。本公开旨在提供一种足够稳定且更适于给药的包含抗FXI/FXIa抗体的药物组合物(制剂)。
发明内容
本公开提供一种药物组合物，其包含蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)。以及提供制备所述药物组合物的方法，及其治疗或预防疾病的方法或相关制药用途。
一些实施方案中，本公开的药物组合物中的蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)具有约5.0至约6.6的PI值，例如约5.2至约6.4、约5.3至约6.3、约5.4至约6.2、约5.5至约6.1、约5.6至约6.0、约5.7至约5.9(例如约5.8)，或者为这些点值之间的任意范围。一些实施方案中，所述PI值是经毛细管电泳仪(ProteinSimple公司，Maurice)检测获得，检测条件为：
实验温度：15℃；聚焦时间：1500V 1min；3000V 6min
方法：将对照品及供试品(如脱盐，选用脱盐后供试品)配制混匀，13000rpm离心5分钟。取80μL转移到Maurice 96孔板或者Maurice iCIEF进样瓶中，13000rpm离心5分钟 (针对进样瓶进样)或者4000rpm离心5分钟(针对96孔板进样)。
一些实施方案中，本公开的含有蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)药物组合物具有约100至约300mS/cm的电导率，例如约120至约260mS/cm、约130至约250mS/cm、约140至约240mS/cm、约150至约220mS/cm、约170至约200mS/cm、约180至约190mS/cm(例如约183.7mS/cm)，或者为这些点值之间的任意范围。一些实施方案中，所述电导率是经电导率仪(梅特勒公司；FE38-Standard)测定。
一些实施方案中，本公开的含有蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)药物组合物具有约250至约450mOsmol/kg的渗透压，例如约270至约430mOsmol/kg、约290至约410mOsmol/kg、约300至约400mOsmol/kg、约310至约390mOsmol/kg、约320至约380mOsmol/kg、约330至约370mOsmol/kg、约340至约360mOsmol/kg(例如约350mOsmol/kg)，或者为这些点值之间的任意范围。一些实施方案中，所述渗透压是经渗透压仪(罗泽公司；OM819.C)测定，检测方法为冰点下降法。
一些实施方案中，本公开的药物组合物中的蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)具有约5.7至约5.9的PI值。一些实施方案中，本公开的含有蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)的药物组合物具有约180至约190mS/cm(例如约183.7mS/cm)的电导率，和/或具有约320至约380mOsmol/kg的渗透压。
自缓冲体系
本公开提供一种药物组合物，其包含如上任一项所述的蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)。所述药物组合物为自缓冲体系。该组合物具有治疗或预防活性。此外，该组合物还可具有稳定性好等优势。
一些实施方案中，所述药物组合物中含有碱性氨基酸，例如组氨酸、精氨酸、赖氨酸或其任意组合。一些实施方案中，所述碱性氨基酸(例如组氨酸)与蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)形成自缓冲体系。
一些实施方案中，如上任一项所述的药物组合物中组氨酸浓度为约1至约100mM，例如约2mM至约80mM、约5mM至约70mM、约10mM至约60mM、约20mM至约50mM、约25mM至约45mM、约30mM至约40mM，约30mM至约100mM，约60mM至约100mM，约30mM至约90mM，或者为这些点值之间的任意范围。一些实施方案中，所述组氨酸浓度为约5mM、约10mM、约15mM、约20mM、约25mM、约30mM、约31mM、约32mM、约33mM、约34mM、约35mM、约36mM、约37mM、约38mM、约39mM、约40mM、约41mM、约42mM、约43mM、约44mM、约45mM、约46mM、约47mM、约48mM、约49mM、约50mM，例如约35mM。
一些实施方案中，如上任一项所述的药物组合物，其包含表面活性剂。在一些实施方案中，所述表面活性剂是非离子表面活性剂。在一些实施方案中，所述表面活性剂选自泊洛沙姆(例如泊洛沙姆188)、聚山梨酯(例如聚山梨酯20(即PS20)、聚山梨酯80(即PS80))、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基- 肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等，或其任意组合。在一些实施方案中，所述表面活性剂为聚山梨酯或泊洛沙姆。在一些实施方案中，所述表面活性剂为聚山梨酯80、聚山梨酯20或泊洛沙姆188。在一些实施方案中，所述表面活性剂为泊洛沙姆188。
一些实施方案中，所述表面活性剂的浓度为约0.1mg/mL至约10mg/mL，例如约0.2mg/mL至约8mg/mL、约0.5mg/mL至约6mg/mL、约0.8mg/mL至约5mg/mL、约1.0mg/mL至约4mg/mL、约1.2mg/mL至约3.8mg/mL、约1.3mg/mL至约3.6mg/mL、约1.4mg/mL至约3.4mg/mL、约1.5mg/mL至约3.3mg/mL、约1.6mg/mL至约3.2mg/mL、约1.7mg/mL至约3mg/mL、约1.8mg/mL至约2.8mg/mL、约1.9mg/mL至约2.5mg/mL、约1.8mg/mL至约2.4mg/mL、约1.9mg/mL至约2.2mg/mL、约1.8mg/mL至约2.2mg/mL、约1.9mg/mL至约2.1mg/mL，或者为这些点值之间的任意范围。例如约0.1mg/mL、约0.2mg/mL、约0.3mg/mL、约0.4mg/mL、约0.5mg/mL、约0.6mg/mL、约0.7mg/mL、约0.8mg/mL、约0.9mg/mL、约1.0mg/mL、约1.1mg/mL、约1.2mg/mL、约1.3mg/mL、约1.4mg/mL、约1.5mg/mL、约1.6mg/mL、约1.7mg/mL、约1.8mg/mL、约1.9mg/mL、约2.0mg/mL、约2.1mg/mL、约2.2mg/mL、约2.3mg/mL、约2.4mg/mL、约2.5mg/mL、约2.6mg/mL、约2.7mg/mL、约2.8mg/mL、约2.9mg/mL、约3.0mg/mL、约3.1mg/mL、约3.2mg/mL、约3.3mg/mL、约3.4mg/mL、约3.5mg/mL、约3.6mg/mL、约3.7mg/mL、约3.8mg/mL、约3.9mg/mL、约4.0mg/mL、约4.5mg/mL、约5.0mg/mL、约5.5mg/mL、约6.0mg/mL、约6.5mg/mL、约7.0mg/mL、约7.5mg/mL、约8.0mg/mL、约8.5mg/mL、约9.0mg/mL、约9.5mg/mL、约10.0mg/mL，例如为约2mg/mL。
一些实施方案中，如上任一项所述的药物组合物，其包含渗透压调节剂。在一些实施方案中，渗透压调节剂为糖(包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等)、氨基酸(包括精氨酸、甘氨酸、半胱氨酸、组氨酸等等)或盐类(氯化钠、氯化钾、氯化钙等)。在一些实施方案中，所述渗透压调节剂为糖，所述糖选自：葡萄糖，蔗糖，海藻糖，乳糖，果糖，麦芽糖，右旋糖苷，甘油，赤藻糖醇，丙三醇，阿拉伯糖醇，木糖醇，山梨糖醇(也称山梨醇)，甘露醇，密里二糖，松三糖，蜜三糖，甘露三糖，水苏糖，麦芽糖，乳果糖，麦芽酮糖，麦芽糖醇，乳糖醇和异-麦芽酮糖。在一些实施方案中，所述渗透压调节剂选自由蔗糖、海藻糖、山梨糖醇、精氨酸、脯氨酸、甘氨酸和氯化钠组成的组中的一种或多种。在一些实施方案中，所述渗透压调节剂为非还原性二糖；在一些实施方案中，所述渗透压调节剂为海藻糖或蔗糖；在一些实施方案中，所述渗透压调节剂为蔗糖。
一些实施方案中，如上任一项所述的药物组合物中所述渗透压调节剂的浓度约1mg/mL至约300mg/mL，约1mg/mL至约400mg/mL，例如约10mg/mL至约400mg/mL、约5mg/mL至约200mg/mL、约10mg/mL至约150mg/mL、约20mg/mL至约140mg/mL、约30mg/mL至约130mg/mL、约40mg/mL至约120mg/mL、50mg/mL至约200mg/mL、约50mg/mL至约 110mg/mL、约60mg/mL至约100mg/mL、约70mg/mL至约90mg/mL(例如约80mg/mL)，或者为这些点值之间的任意范围。一些实施方案中，所述渗透压调节剂的浓度为约10mg/mL、约20mg/mL、约30mg/mL、约40mg/mL、约50mg/mL、约60mg/mL、约70mg/mL、约80mg/mL、约90mg/mL、约100mg/mL、约110mg/mL、约120mg/mL、约130mg/mL、约140mg/mL、约150mg/mL、约160mg/mL、约170mg/mL、约180mg/mL、约190mg/mL、约200mg/mL。
一些实施方案中，如上任一项所述的药物组合物的pH值为约4.0至约8.5，例如为约5.0至约8.0、约6.0至约7.5、约6.5至约7.5、约6.6至约7.4、约6.7至约7.3、约6.8至约7.2、约6.9至约7.1(例如为约7.0)，或者为这些点值之间的任意范围。一些pH值的非限制性实施例包括约4.0、约4.5、约5.0、约6.0、约6.4、约6.5、约6.6、约6.7、约6.8、约6.9、约7.0、约7.1、约7.2、约7.3、约7.4、约7.5、约7.6、约7.7、约7.8、约7.9、约8.0、约8.1、约8.2、约8.3、约8.4、约8.5。
通常，通过置换缓冲剂获得的药物组合物的pH与缓冲剂pH几乎一致。同时，本领域技术人员公知，在药物制剂的过程中，有时可能会存在pH飘移，但药物制剂的pH的飘移一般很小(±0.3范围内)。在一些实施方案中，药物制剂的pH的飘移在±0.1范围内。
一些实施方案中，如上任一项所述的药物组合物中的蛋白(例如抗FXI/FXIa抗体或其抗原结合片段)的浓度为约0.1mg/mL至约1000mg/mL，例如约1mg/mL至约500mg/mL、约1mg/mL至约300mg/mL、约10mg/mL至约400mg/mL、约20mg/mL至约350mg/mL、约30mg/mL至约300mg/mL、约30mg/mL至约200mg/mL、约130mg/mL至约200mg/mL、约40mg/mL至约250mg/mL、约50mg/mL至约200mg/mL、约60mg/mL至约180mg/mL、约50mg/mL至约150mg/mL、约60mg/mL至约150mg/mL、约70mg/mL至约130mg/mL、约80mg/mL至约120mg/mL、约90mg/mL至约110mg/mL，或者为这些点值之间的任意范围。例如，约1mg/mL、约5mg/mL、约10mg/mL、约15mg/mL、约20mg/mL、约25mg/mL、约30mg/mL、约35mg/mL、约40mg/mL、约45mg/mL、约50mg/mL、约55mg/mL、约60mg/mL、约61mg/mL、约62mg/mL、约63mg/mL、约64mg/mL、约65mg/mL、约66mg/mL、约67mg/mL、约68mg/mL、约69mg/mL、约70mg/mL、约71mg/mL、约72mg/mL、约73mg/mL、约74mg/mL、约75mg/mL、约76mg/mL、约77mg/mL、约78mg/mL、约79mg/mL、约80mg/mL、约81mg/mL、约82mg/mL、约83mg/mL、约84mg/mL、约85mg/mL、约86mg/mL、约87mg/mL、约88mg/mL、约89mg/mL、约90mg/mL、约91mg/mL、约92mg/mL、约93mg/mL、约94mg/mL、约95mg/mL、约96mg/mL、约97mg/mL、约98mg/mL、约99mg/mL、约100mg/mL、约101mg/mL、约102mg/mL、约103mg/mL、约104mg/mL、约105mg/mL、约106mg/mL、约107mg/mL、约108mg/mL、约109mg/mL、约110mg/mL、约111mg/mL、约112mg/mL、约113mg/mL、约114mg/mL、约115mg/mL、约116mg/mL、约117mg/mL、约118mg/mL、约119mg/mL、约120mg/mL、约121mg/mL、约122mg/mL、约123mg/mL、约124mg/mL、约125mg/mL、约126mg/mL、约127mg/mL、约128mg/mL、约129mg/mL、约130mg/mL、约131mg/mL、约132mg/mL、约133mg/mL、约134mg/mL、约135mg/mL、约140mg/mL、约145mg/mL、约150mg/mL、约155mg/mL、约160mg/mL、约165mg/mL、约170mg/mL、约175mg/mL、约180mg/mL、约185mg/mL、约190mg/mL、约195mg/mL、约200mg/mL。
一些实施方案中，如上任一项所述的药物组合物含有a)-g)中任一：
a)约1mg/mL至约500mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，约1mM至约100mM的组氨酸，所述药物组合物的pH为约4.0至约8.5；可选地，所述药物组合物可进一步含有约1mg/mL至约500mg/mL的蔗糖或海藻糖，和/或约0.1mg/mL至约10mg/mL的泊洛沙姆188或聚山梨酯。
b)约10mg/mL至约300mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，约5mM至约80mM的组氨酸，所述药物组合物的pH为约6.0至约8.0；可选地，所述药物组合物可进一步含有约10mg/mL至约400mg/mL的蔗糖或海藻糖，和/或约0.5mg/mL至约8.0mg/mL的泊洛沙姆188或聚山梨酯。
c)约30mg/mL至约200mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，和约10mM至约60mM、约20mM至约50mM、约30mM至约40mM(例如约35mM)的组氨酸；
d)约50mg/mL至约150mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，和约10mM至约60mM、约20mM至约50mM、约30mM至约40mM(例如约35mM)的组氨酸；
e)约70mg/mL至约130mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，和约10mM至约60mM、约20mM至约50mM、约30mM至约40mM(例如约35mM)的组氨酸；
h)约130mg/mL至约200mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，和约30mM至约100mM、约60mM至约100mM、约60mM至约90mM的组氨酸；
I)约30mg/mL至约200mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，和约30mM至约100mM、约60mM至约100mM、约60mM至约90mM的组氨酸；
其中，c)至e)、h)-I)中的药物组合物的pH为约6.5至约7.5(例如约6.7至约7.3，例如约7.0)；
可选地，c)至e)、h)-I)中的药物组合物还含有蔗糖或海藻糖，和泊洛沙姆188或聚山梨酯(例如聚山梨酯80或聚山梨酯20)；
一些实施方案中，c)至e)、h)-I)中的药物组合物中含有蔗糖，浓度为约40至约300mg/mL、约50至约200mg/mL、约60至约150mg/mL、约70至约120mg/mL(例如约70mg/mL、约80mg/mL、约90mg/mL、约100mg/mL、约110mg/mL、约120mg/mL)；
一些实施方案中，c)至e)、h)-I)中的药物组合物中含有泊洛沙姆188，浓度为约1.5至约6mg/mL、约1.5至约5.0mg/mL、约1.8至约3.5mg/mL、约1.8至约2.8mg/mL(例如约1.8mg/mL、约1.9mg/mL、约2.0mg/mL、约2.1mg/mL、约2.2mg/mL、约2.3mg/mL、约2.4mg/mL、约2.5mg/mL约2.6mg/mL、约2.7mg/mL、约2.8mg/mL)。
f)约70mg/mL至约130mg/mL的蛋白(如抗FXI/FXIa抗体)，约30mM至约40mM(例如约35mM)的组氨酸，约60至约200mg/mL蔗糖，和约1.8至约2.8mg/mL的泊洛沙姆188；
g)约70mg/mL至约130mg/mL的蛋白(如抗FXI/FXIa抗体)，约30mM至约40mM(例如约35mM)的组氨酸，约80mg/mL蔗糖，和约2.0mg/mL的泊洛沙姆188。
一些实施方案中，药物组合物含有如下中的任一：
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约35mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约2mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.0；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约35mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约2mg/mL的泊洛沙姆188，
所述药物组合物的pH为约6.7；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约35mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约2mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.3；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约30mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约1.8mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.0；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约30mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约2.8mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.0；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约40mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约1.8mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.0；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约40mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约2.3mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.0；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约40mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约2.8mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.0；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约35mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约2.3mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.0；或
(a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
(b)约35mM的组氨酸，
(c)约80mg/mL的蔗糖，
(d)约1.8mg/mL的泊洛沙姆188，
所述药物组合物的pH为约7.0。
其他缓冲剂体系
本公开提供一种药物组合物，其包含抗FXI/FXIa抗体，以及缓冲剂。一些实施方案中，所述缓冲剂选自醋酸盐缓冲剂、组氨酸盐缓冲剂、Tris-盐酸盐缓冲剂、Tris-柠檬酸盐缓冲剂、磷酸盐缓冲剂。该组合物具有治疗或预防活性。此外，该组合物还可具有稳定性好等优势。
一些实施方案中，所述药物组合物的pH值为约4.0至约8.5，例如为约5.0至约8.0、约6.0至约7.5、约6.5至约7.5、约6.6至约7.4、约6.7至约7.3、约6.8至约7.2、约6.9至约7.1(例如为约7.0)，或者为这些点值之间的任意范围。一些pH值的非限制性实施例包括约4.0、约4.5、约5.0、约6.0、约6.4、约6.5、约6.6、约6.7、约6.8、约6.9、约7.0、约7.1、约7.2、约7.3、约7.4、约7.5、约7.6、约7.7、约7.8、约7.9、约8.0、约8.1、约8.2、约8.3、约8.4、约8.5。
通常，通过置换缓冲剂获得的药物组合物的pH与缓冲剂pH几乎一致。同时，本领域技术人员公知，在药物制剂的过程中，有时可能会存在pH飘移，但药物制剂的pH的飘移一般很小(±0.3范围内)。在一些实施方案中，药物制剂的pH的飘移在±0.1范围内。
在一些实施方案中，如上任一项所述的药物组合物，其中所述抗FXI/FXIa抗体或其抗原结合片段的浓度为约0.1mg/mL至约1000mg/mL，例如约1mg/mL至约500mg/mL、约1mg/mL至约300mg/mL、约10mg/mL至约400mg/mL、约20mg/mL至约350mg/mL、约30mg/mL至约300mg/mL、约40mg/mL至约250mg/mL、约50mg/mL至约200mg/mL、约60mg/mL至约180mg/mL、约50mg/mL至约150mg/mL、约60mg/mL至约150mg/mL、约70mg/mL至约130mg/mL、约80mg/mL至约120mg/mL、约90mg/mL至约110mg/mL、约1mg/mL至约300mg/mL、约1mg/mL至约200mg/mL，或者为这些点值之间的任意范围。在一些实施方案中，所述抗FXI/FXIa抗体的浓度为1mg/mL至100mg/mL。在一些实施方案中，所述抗FXI/FXIa抗体的浓度为40mg/mL至100mg/mL。在一些非限制性实施方案中，所述抗FXI/FXIa抗体的浓度为约1mg/mL、约10mg/mL、约20mg/mL、约30mg/mL、约40mg/mL、约45mg/mL、约50mg/mL、约55mg/mL、约60mg/mL、约65mg/mL、约70mg/mL、约80mg/mL、约90mg/mL、约100mg/mL、约110mg/mL、约120mg/mL、约130mg/mL、 约140mg/mL、约150mg/mL、约160mg/mL、约170mg/mL、约180mg/mL、约190mg/mL或约200mg/mL。
在一些实施方案中，如上任一项所述的药物组合物，其中所述药物组合物包含表面活性剂。在一些实施方案中，所述表面活性剂是非离子表面活性剂。在一些实施方案中，所述表面活性剂选自泊洛沙姆(例如泊洛沙姆188)、聚山梨酯(例如聚山梨酯20、聚山梨酯80)、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-磺基甜菜碱、肉豆蔻基-磺基甜菜碱、亚油基-磺基甜菜碱、硬脂基-磺基甜菜碱、月桂基-肌氨酸、肉豆蔻基-肌氨酸、亚油基-肌氨酸、硬脂基-肌氨酸、亚油基-甜菜碱、肉豆蔻基-甜菜碱、鲸蜡基-甜菜碱、月桂酰胺基丙基-甜菜碱、柯卡酰胺基丙基-甜菜碱、亚油酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-甜菜碱、棕榈酰胺基丙基-甜菜碱、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-二甲基胺、棕榈酰胺基丙基-二甲基胺、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等。在一些实施方案中，所述表面活性剂为聚山梨酯或泊洛沙姆。在一些实施方案中，所述表面活性剂为聚山梨酯80、聚山梨酯20或泊洛沙姆188。在一些实施方案中，所述表面活性剂为泊洛沙姆188。
在一些实施方案中，如上任一项所述的药物组合物，其中所述表面活性剂浓度为0.05-mg/mL至10mg/mL，例如0.1mg/mL-10mg/mL、0.05mg/mL至1.0mg/mL、0.1mg/mL至1.0mg/mL、0.2mg/mL至0.8mg/mL。在一些实施方案中，所述表面活性剂浓度为0.2mg/mL至0.6mg/mL。在一些实施方案中，所述表面活性剂浓度为0.05mg/mL、0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL或1.0mg/mL，或者为这些点值之间的任意范围。在一些实施方案中，所述表面活性剂浓度为0.4mg/mL。在一些实施方案中，所述表面活性剂为0.4mg/mL的泊洛沙姆188。在一些实施方案中，所述表面活性剂为0.4mg/mL的聚山梨酯80。
在一些实施方案中，如上任一项所述的药物组合物，其包括渗透压调节剂。在一些实施方案中，渗透压调节剂为糖(包括单糖，二糖，三糖，多糖，糖醇，还原性糖，非还原性糖等等)、氨基酸(包括精氨酸、甘氨酸、半胱氨酸、组氨酸等等)或盐类(氯化钠、氯化钾、氯化钙等)。在一些实施方案中，所述渗透压调节剂为糖，所述糖选自：葡萄糖，蔗糖，海藻糖，乳糖，果糖，麦芽糖，右旋糖苷，甘油，赤藻糖醇，丙三醇，阿拉伯糖醇，木糖醇，山梨糖醇(也称山梨醇)，甘露醇，密里二糖，松三糖，蜜三糖，甘露三糖，水苏糖，麦芽糖，乳果糖，麦芽酮糖，麦芽糖醇，乳糖醇和异-麦芽酮糖。在一些实施方案中，所述渗透压调节剂选自由蔗糖、海藻糖、山梨糖醇、精氨酸、脯氨酸、甘氨酸和氯化钠组成的组中的一种或多种。在一些实施方案中，所述渗透压调节剂为非还原性二糖；在一些实施方案中，所述渗透压调节剂为海藻糖或蔗糖；在一些实施方案中，所述渗透压调节剂为蔗糖。在一些实施方案中，所述渗透压调节剂选自蔗糖，脯氨酸和蔗糖，或甘氨酸和蔗糖。
在一些实施方案中，如上任一项所述的药物组合物，渗透压调节剂浓度为1mg/mL至300mg/mL，例如约5mg/mL至约200mg/mL、约10mg/mL至约150mg/mL、约20mg/mL至 约140mg/mL、约30mg/mL至约130mg/mL、约40mg/mL至约120mg/mL、约50mg/mL至约110mg/mL、约60mg/mL至约100mg/mL、约75mg/mL至约100mg/mL、约70mg/mL至约90mg/mL、约75mg/mL至约85mg/mL、约75mg/mL至约80mg/mL、约1mg/mL至约100mg/mL、约1mg/mL至约80mg/mL、约1mg/mL至约40mg/mL，或者为这些点值之间的任意范围。在一些实施方案中，渗透压调节剂浓度非限制性实施包括约7.5mg/mL、约11.5mg/mL、约40mg/mL、约60mg/mL、约65mg/mL、约70mg/mL、约75mg/mL、约80mg/mL、约85mg/mL、约90mg/mL、约95mg/mL、约100mg/mL。在一些实施方案中，前述药物组合物为等渗制剂。在一些实施方案中，所述渗透压调节剂为80mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为40mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约10mM至约300mM的脯氨酸和约1mg/mL至约200mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约10mM至约200mM的脯氨酸和约10mg/mL至约100mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约50mM至约150mM的脯氨酸和约10mg/mL至约80mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约80mM至约120mM的脯氨酸和约40mg/mL至约80mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约100mM的脯氨酸和约40mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约10mM至约300mM的甘氨酸和约1mg/mL至约200mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约10mM至约200mM的甘氨酸和约10mg/mL至约100mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约50mM至约150mM的甘氨酸和约10mg/mL至约80mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约80mM至约120mM的甘氨酸和约40mg/mL至约80mg/mL蔗糖。在一些实施方案中，所述渗透压调节剂为约100mM的甘氨酸和约40mg/mL蔗糖。
在一些实施方案中，如上任一项所述的药物组合物，所述缓冲剂的浓度为约5mM至约100mM。在一些实施方案中，所述缓冲剂的浓度为约10mM至约30mM。在一些实施方案中，所述缓冲剂的浓度为约5mM至约15mM。在一些实施方案中，所述缓冲剂的浓度为约5mM、约10mM、约15mM、约20mM、约25mM、约30mM、约40mM、约50mM、约60mM、约70mM、约80mM、约90mM或约100mM，以及这些点值之间的任一范围。在一些实施方案中，所述缓冲剂为约10mM组氨酸-盐酸盐。在一些实施方案中，所述缓冲剂为约10mM磷酸二氢钠-磷酸氢二钠。在一些实施方案中，所述缓冲剂为约10mM Tris-盐酸。
一些实施方案中，如上任一项所述的药物组合物含有a)-i)中任一：
a)约1mg/mL至约300mg/mL的蛋白(如抗FXI/FXIa抗体)，和约5mM至约100mM、约10mM至约30mM、约5mM至约15mM(例如约10mM)的组氨酸-盐酸盐或磷酸二氢钠-磷酸氢二钠或Tris-盐酸；
b)约50mg/mL至约150mg/mL的蛋白(如抗FXI/FXIa抗体)，和约5mM至约100mM、约10mM至约30mM、约5mM至约15mM(例如约10mM)的组氨酸-盐酸盐或磷酸二氢钠-磷酸氢二钠或Tris-盐酸；
c)约70mg/mL至约130mg/mL的蛋白(如抗FXI/FXIa抗体)，和约5mM至约100mM、约10mM至约30mM、约5mM至约15mM(例如约10mM)的组氨酸-盐酸盐或磷酸二氢钠 -磷酸氢二钠或Tris-盐酸；
其中，a)至c)中的药物组合物的pH为约4.0至约8.5(例如约6.5至约7.5，例如约7.0)；
可选地，a)至c)中的药物组合物还含有蔗糖、或海藻糖、或甘氨酸和蔗糖、或脯氨酸和蔗糖，和泊洛沙姆188或聚山梨酯(例如聚山梨酯80或聚山梨酯20)；
一些实施方案中，a)至c)中的药物组合物中还含有蔗糖或海藻糖，例如浓度为约1mg/mL至约300mg/mL、约10mg/mL至约150mg/mL、约75至约100mg/mL(例如约70mg/mL、约80mg/mL、约90mg/mL、约100mg/mL、约110mg/mL、约120mg/mL)；
一些实施方案中，a)至c)中的药物组合物中还含有甘氨酸和蔗糖，例如浓度为约10mM至约300mM的甘氨酸和约1mg/mL至约200mg/mL蔗糖、约10mM至约200mM的甘氨酸和约10mg/mL至约100mg/mL蔗糖、约80mM至约120mM的甘氨酸和约40mg/mL至约80mg/mL蔗糖(例如约100mM的甘氨酸和约40mg/mL蔗糖、约90mM的甘氨酸和约50mg/mL蔗糖、约110mM的甘氨酸和约30mg/mL蔗糖)；
一些实施方案中，a)至c)中的药物组合物中还含有脯氨酸和蔗糖，例如浓度为约10mM至约300mM的脯氨酸和约1mg/mL至约200mg/mL蔗糖、约10mM至约200mM的脯氨酸和约10mg/mL至约100mg/mL蔗糖、约80mM至约120mM的脯氨酸和约40mg/mL至约80mg/mL蔗糖(例如约100mM的脯氨酸和约40mg/mL蔗糖、约90mM的脯氨酸和约50mg/mL蔗糖、约110mM的脯氨酸和约30mg/mL蔗糖)；
一些实施方案中，a)至c)中的药物组合物中还含有泊洛沙姆188或聚山梨酯(例如聚山梨酯80或聚山梨酯20)，浓度为约0.1至约10mg/mL、约1.5至约4.0mg/mL、约1.8至约3.5mg/mL、约1.8至约2.8mg/mL(例如约1.8mg/mL、约1.9mg/mL、约2.0mg/mL、约2.1mg/mL、约2.2mg/mL、约2.3mg/mL、约2.4mg/mL、约2.5mg/mL约2.6mg/mL、约2.7mg/mL、约2.8mg/mL)。
d)约1mg/mL至约300mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，约10mM的组氨酸-盐酸盐或磷酸二氢钠-磷酸氢二钠或Tris-盐酸，所述药物组合物的pH为约6.5至约7.5；可选地，所述药物组合物可进一步含有约80mg/mL的蔗糖，和约0.1mg/mL至约10mg/mL的泊洛沙姆188或聚山梨酯。
e)约1mg/mL至约300mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，约10mM的组氨酸-盐酸盐或磷酸二氢钠-磷酸氢二钠或Tris-盐酸，所述药物组合物的pH为约6.5至约7.5；可选地，所述药物组合物可进一步含有约100mM的甘氨酸或脯氨酸，约40mg/mL的蔗糖，和约0.1mg/mL至约10mg/mL的泊洛沙姆188或聚山梨酯。
f)约50mg/mL至约150mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，约5mM至约100mM的组氨酸-盐酸盐或磷酸二氢钠-磷酸氢二钠或Tris-盐酸，所述药物组合物的pH为约4.0至约8.5；可选地，所述药物组合物可进一步含有约10mg/mL至约150mg/mL的蔗糖，和约0.1mg/mL至约10mg/mL的泊洛沙姆188或聚山梨酯。
g)约50mg/mL至约150mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，约5mM至约100mM的组氨酸-盐酸盐或磷酸二氢钠-磷酸氢二钠或Tris-盐酸，所述药物组合物的pH为约4.0至约8.5；可选地，所述药物组合物可进一步含有约10mM至约200mM的甘氨酸或 脯氨酸，约10mg/mL至约100mg/mL的蔗糖，和约0.1mg/mL至约10mg/mL的泊洛沙姆188或聚山梨酯。
h)约70mg/mL至约130mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，约5mM至约15mM的组氨酸-盐酸盐或磷酸二氢钠-磷酸氢二钠或Tris-盐酸，所述药物组合物的pH为约6.5至约7.5；可选地，所述药物组合物可进一步含有约80mg/mL的蔗糖，和约1.5mg/mL至约4mg/mL的泊洛沙姆188或聚山梨酯。
i)约70mg/mL至约130mg/mL的蛋白(如抗FXI/FXIa抗体或其抗原结合片段)，约5mM至约15mM的组氨酸-盐酸盐或磷酸二氢钠-磷酸氢二钠或Tris-盐酸，所述药物组合物的pH为约6.5至约7.5；可选地，所述药物组合物可进一步含有约100mM的甘氨酸或脯氨酸，约40mg/mL的蔗糖，和约1.5mg/mL至约4mg/mL的泊洛沙姆188或聚山梨酯。
本公开的药物组合物还包含溶剂。药物组合物中溶剂选自但不限于无毒性的生理学可接受的液体载体，如生理盐水、注射用水、葡萄糖溶液(如5％葡萄糖注射液、葡萄糖氯化钠注射液)等。
本公开还提供一种药物组合物，其是经0.9％生理盐水或5％葡萄糖溶液稀释获得的，或含有上述药物组合物经0.9％生理盐水或5％葡萄糖溶液稀释后的可作为静脉注射剂浓度的抗FXI/FXIa抗体或其抗原结合片段、组氨酸、蔗糖、泊洛沙姆188。在一些实施方案中，所述药物组合物中抗FXI/FXIa抗体或其抗原结合片段的浓度为约0.01mg/mL至约50mg/mL。在一些实施方案中，所述药物组合物中抗FXI/FXIa抗体或其抗原结合片段的浓度为约0.1mg/mL至约30mg/mL。在一些实施方案中，所述药物组合物中抗FXI/FXIa抗体或其抗原结合片段的浓度为约0.20mg/mL至约13.30mg/mL。
在一些实施方案中，本公开药物组合物中的蛋白是抗体、抗体片段或Fc融合蛋白。在一些实施方案中，所述抗体、抗体片段或Fc融合蛋白靶向的抗原选自：因子VIIIC、因子IX、因子XI/因子XIa(FXI/FXIa)、组织因子、血管内皮生长因子(VEGF)、肾素、生长激素、生长激素释放因子、甲状旁腺激素、促甲状腺激素、脂蛋白、α-1-抗胰蛋白酶、骨衍生神经营养因子(BDNF)、神经营养蛋白-3、-4、-5、或-6(NT-3、NT-4、NT-5、或NT-6)、表皮生长因子(EGF)、转化生长因子(TGF)、干扰素、M-CSF、GM-CSF、G-CSF、IL-1至IL-10、HER2、HER3或HER4受体、CTLA-4、PD-1、PD-L1、PD-L2、TIM3、41BB、41BBL、OX40、OX40L、GITRL、GITR、CD25、CD27、CD70、CD80、TNFRSF25、CD40L、CD40、B7H3、B7H4、VISTA、TMIGD2、HHLA2-TMIGD2、嗜乳脂蛋白(包括BTNL2)、Siglec家族、TIGIT及PVR家族成员、KIRs、ILTs及LIRs、CD79、GARP、GITR、PSMA、神经纤毛蛋白(Neuropilin)、CD160、CD30及CD155；和任何以上多肽的片段。在一些实施方案中，所述药物组合物中的蛋白是抗FXI/FXIa抗体或其抗原结合片段。
在一些实施方案中，上述药物组合物中所述抗FXI/FXIa抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：
所述重链可变区包含分别如SEQ ID NO：7、8、9所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：10、11、12所示的LCDR1、LCDR2、LCDR3。
在一些实施方案中，药物组合物中所述抗FXI/FXIa抗体或其抗原结合片段可选自鼠源 抗体、嵌合抗体、人源化抗体，例如人源化抗体。
在可选实施方案中，药物组合物中所述的人源化抗FXI/FXIa抗体轻链和重链可变区上的轻链和重链FR序列分别来源于人种系轻链和重链的FR或其突变序列。
在一些实施方案中，药物组合物中所述抗FXI/FXIa抗体或其抗原结合片段包含抗体重链可变区和轻链可变区，其中：
重链可变区序列如SEQ ID NO：5或与之具有至少80％、至少85％、至少90％同一性的序列，轻链可变区序列如SEQ ID NO：6或与之具有至少80％、至少85％、至少90％同一性的序列；
重链可变区序列如SEQ ID NO：13或与之具有至少80％、至少85％、至少90％同一性的序列，轻链可变区序列如SEQ ID NO：14或与之具有至少80％、至少85％、至少90％同一性的序列；
重链可变区序列如SEQ ID NO：15或与之具有至少80％、至少85％、至少90％同一性的序列，轻链可变区序列如SEQ ID NO：16或与之具有至少80％、至少85％、至少90％同一性的序列；
重链可变区序列如SEQ ID NO：17或与之具有至少80％、至少85％、至少90％同一性的序列，轻链可变区序列如SEQ ID NO：16或与之具有至少80％、至少85％、至少90％同一性的序列；
在一些具体实施方案中，药物组合物中所述抗FXI/FXIa抗体或其抗原结合片段包含抗体重链可变区和轻链可变区，其中：所述重链可变区序列如SEQ ID NO：17所示，和所述轻链可变区序列如SEQ ID NO：16所示。
在一些实施方案中，所述抗FXI/FXIa抗体或其抗原结合片段进一步包含重链恒定区和轻链恒定区。在可选的实施方案中，所述重链恒定区选自人IgG1、IgG2、IgG3、IgG4、IgG4P(即IgG4的S241P突变体)恒定区，所述轻链恒定区选自人κ和λ链恒定区。所述IgG4P Fc(即包含S241P的IgG4Fc)的序列例如SEQ ID NO：19所示。在一些实施方案中，所述重链CH1的序列如SEQ ID NO：18所示或与之具有至少80％、至少85％、至少90％同一性的序列，和/或所述轻链恒定区的序列如SEQ ID NO：20所示或与之具有至少80％、至少85％、至少90％同一性的序列。
在一些实施方案中，所述抗FXI/FXIa抗体或其抗原结合片段包含重链和轻链，其中：所述重链具有SEQ ID NO：21所示的序列或与之具有至少80％、至少85％、至少90％同一性的序列，和所述轻链具有SEQ ID NO：22所示的序列或与之具有至少80％、至少85％、至少90％同一性的序列。
本公开中，所述“至少90％同一性”涵盖至少90％、至少95％、至少96％、至少97％、至少98％、至少99％同一性。
本公开中，所述“至少90％同一性”涵盖至少80％、至少85％、至少87％、至少88％、至少89％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％同一性。
在一些具体实施方案中，药物组合物中所述抗FXI/FXIa抗体包含抗体重链和轻链，其中：所述重链序列如SEQ ID NO：21所示，和所述轻链序列如SEQ ID NO：22所示。
在一些实施方案中，所述抗FXI/FXIa抗体的抗原结合片段为Fab、Fv、sFv、Fab’、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)，例如具体为scFv、Fv、Fab或Fab’片段。
在一些实施方案中，所述抗FXI/FXIa抗体的重链可变区和/或轻链可变区包含1、2、3、4、5、6、7、8、9或10个氨基酸变化。所述氨基酸变化可以是可变区中的氨基酸残基保守性置换。
在一些实施方案中，上述抗FXI/FXIa抗体可以具有以下任一或任几项的特性：
防止内在凝血途径或共同凝血途径的活化；
阻断FXI和/或FXIa结合至凝血因子IX、凝血因子XIIa或凝血酶中的一个或多个及凝血途径的其他组分；
阻断FIX、FXI及FXIa中的一个或多个结合至血小板受体；
具有以≤10 -9KD结合至人FXI和/或FXIa蛋白；
结合至FXI/FXIa时阻止FXI/FXIa催化结构域呈现活性构象；
可用于皮下给药或静脉给药。
上述亲和力的测定方式例如是BIACORE TM。
一些实施方案中，前述任一项的药物组合物，可以是静脉注射剂、皮下注射剂、腹腔注射剂或肌肉注射剂。
在一些实施方案中，本公开提供一种制备前述任一项药物组合物的方法，所述方法包括将抗FXI/FXIa抗体或其抗原结合片段原液经缓冲剂置换的步骤。
本公开还提供一种液体制剂，其包含前述任一项所述的药物组合物。
本公开所述药物组合物已具有足够成药稳定性，可以长期稳定放置。
本公开还提供一种制备包含抗FXI/FXIa抗体的药物组合物的冻干制剂的方法，所述方法包括将前述药物组合物经冷冻干燥的步骤。
本公开还提供一种包含抗FXI/FXIa抗体的药物组合物的冻干制剂，所述冻干制剂通过将前面任一所述的药物组合物经冷冻干燥获得。
在一些实施方案中，冻干制剂于2-8℃避光保存，稳定至少1个月，至少3个月，至少6个月，至少12个月，至少18个月，至少24个月，至少30个月。
在一些实施方案中，所述冻干制剂于25℃稳定至少1个月，至少3个月，至少6个月，至少12个月。
在一些实施方案中，所述冻干制剂于40℃稳定至少7天，至少14天或至少30天。
本公开还提供一种包含抗FXI/FXIa抗体药物组合物的复溶溶液，所述复溶溶液是通过将前述任一项的冻干制剂经复溶制备获得。
本公开进一步提供一种制品，其包括容器，该容器中装有如上任一项所述的药物组合物、液体制剂、冻干制剂或复溶溶液。在一些实施方案中，所述容器可以为但不限于中性硼硅玻璃管制注射剂瓶。
本公开还提供如上任一项所述的药物组合物、如上任一项的液体制剂、如上任一项的冻 干制剂、如上任一项的复溶溶液或如上任一项所述的制品在制备用于治疗疾病的药物中的用途。
本公开还提供一种治疗或预防疾病的方法，包括给予有需要的受试者有效量的如上任一项所述的药物组合物、如上任一项的液体制剂、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的制品。
本公开还提供如上任一项所述的药物组合物、如上任一项的液体制剂、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的制品，其用作治疗疾病的用途。
在一些实施方案中，如上任一项所述疾病，其为血栓形成性或血栓栓塞性疾病和/或血栓形成性或血栓栓塞性并发症，或心律失常、心源性血栓栓塞、弥漫性血管内凝血。
在一些实施方案中，如上任一项所述疾病，所述血栓形成性或血栓栓塞性疾病选自但不限于：心脏冠状动脉疾病，诸如急性冠状动脉综合征(ACS)、有ST段升高的心肌梗死(STEMI)和无ST段升高的心肌梗死(非STEMI)、稳定型心绞痛、不稳定型心绞痛、冠状动脉介入后的再闭塞和再狭窄，以及导致外周动脉闭塞性疾病、肺栓塞、静脉血栓栓塞、静脉血栓形成、短暂性脑缺血发作以及血栓形成性脑卒中和血栓栓塞性脑卒中的其它血管中的血栓形成性或血栓栓塞性疾病，由慢性血栓栓塞(CTEPH)引起的肺病和肺动脉高压或其组合。
本公开提供一种治疗或预防疾病的方法，包括向有需要的受试者施用治疗或预防有效量的如上任一项所述的药物组合物、如上任一项的液体制剂、如上任一项的冻干制剂、如上任一项的复溶溶液或如上任一项所述的制品，其中：所述疾病为血栓形成性或血栓栓塞性疾病和/或血栓形成性或血栓栓塞性并发症，或心律失常、心源性血栓栓塞、弥漫性血管内凝血。在一些实施方案中，所述血栓形成性或血栓栓塞性疾病或其并发症选自但不限于：心脏冠状动脉疾病，诸如急性冠状动脉综合征(ACS)、有ST段升高的心肌梗死(STEMI)和无ST段升高的心肌梗死(非STEMI)、稳定型心绞痛、不稳定型心绞痛、冠状动脉介入后的再闭塞和再狭窄，以及导致外周动脉闭塞性疾病、肺栓塞、静脉血栓栓塞、静脉血栓形成、短暂性脑缺血发作以及血栓形成性脑卒中和血栓栓塞性脑卒中的其它血管中的血栓形成性或血栓栓塞性疾病，由慢性血栓栓塞(CTEPH)引起的肺病和肺动脉高压或其组合。在一些实施方案中，所述施用的方式为皮下或静脉注射。
附图说明
图1：抗FXI/FXIa抗体的体外FXIIa介导的FXI活化酶活抑制试验。
图2：人血的aPTT/PT抗凝血活性检测，其中，图2A、2B分别为抗FXI/FXIa抗体在人血上的aPTT和PT试验结果。1209为本申请筛选获得的另一个抗FXI/FXIa抗体。
图3：猴血的aPTT/PT抗凝血活性检测，其中，图3A、3B分别为抗FXI/FXIa抗体在猴血上的aPTT和PT试验结果。
图4：抗FXI/FXIa抗体在食蟹猴上的动物实验检测，其中，图4A为抗体3882在食蟹猴体内的aPTT、血浆药物浓度、FXI:C％和游离FXI的变化曲线，图4B为抗体3882在食蟹猴体内的血栓生成抑制作用，图4C和图4D为抗体3882在食蟹猴体内的安全性测试，分别出 血时间测定和PT检测结果。
图5：抗FXI/FXIa抗体在食蟹猴上体内药效学(PD)试验结果，其中，图5A为APTT(s)检测结果，图5B为FXI：C(％)检测结果，3882(1mg/kg)为静脉给药和皮下给药两种方式，对照BAY1213790(1mg/kg)为静脉给药方式。
图6：抗FXI/FXIa抗体制剂缓冲体系pH值的筛选，其中，图6A、6B、6C分别为不同pH/buffer 40℃条件下SEC、NRCE、iCIEF变化趋势。
图7：抗FXI/FXIa抗体制剂中pH、辅料和表面活性剂浓度的细筛，其中，图7A、7B、7C分别为不同pH/buffer 40℃条件下SEC、NRCE、CEX变化趋势。
图8：抗FXI/FXIa抗体制剂缓冲体系和表面活性剂种类的筛选，其中，图8A、8B分别为不同表面活性剂在40℃条件下SEC、CEX变化趋势。
具体实施方式
术语
为了更容易理解本公开，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本公开所属领域的一般技术人员通常理解的含义。
“缓冲剂”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲剂。将pH控制在适当范围中的缓冲剂的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸、草酸盐、乳酸盐、磷酸盐、柠檬酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲剂。
“组氨酸缓冲剂”是包含组氨酸离子的缓冲剂。组氨酸缓冲剂的实例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲剂，优选组氨酸-盐酸盐缓冲剂。组氨酸-盐酸盐缓冲剂是组氨酸与盐酸或组氨酸与组氨酸盐酸盐配制而成。
“自缓冲的”是指在没有其它缓冲剂的情况下，物质如药物蛋白(例如抗体或其抗原结合片段；一些实施方案中，为抗FXI/FXIa抗体，例如为本公开的3882抗体)足以抵制某个应用的pH变化的能力。
“Tris-柠檬酸盐缓冲剂”是包括柠檬酸根离子的缓冲剂。Tris-柠檬酸盐缓冲剂的实例包括组Tris-盐酸盐，Tris-醋酸盐，Tris-磷酸盐，Tris-硫酸盐，Tris-柠檬酸盐等缓冲剂等，优选Tris-柠檬酸盐。
“Tris-盐酸盐缓冲剂”是包括盐酸根离子的缓冲剂。Tris-盐酸盐缓冲剂的实例包括Tris-盐酸盐，Tris-醋酸盐，Tris-磷酸盐，Tris-硫酸盐，Tris-柠檬酸盐等缓冲剂等。优选的柠檬酸盐缓冲剂为Tris-盐酸盐缓冲剂。
“磷酸盐缓冲剂”是包括磷酸离子的缓冲剂。磷酸盐缓冲剂的实例包括磷酸氢二钠酸-磷酸二氢钠、磷酸氢二钠酸-磷酸二氢钾等。优选的磷酸盐缓冲剂为磷酸氢二钠酸-磷酸二氢钠缓冲剂。
“醋酸盐缓冲剂”是包括醋酸根离子的缓冲剂。醋酸盐缓冲剂的实例包括醋酸-醋酸钠、醋酸-组氨酸盐、醋酸-醋酸钾、醋酸-醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲剂为醋酸-醋酸钠缓冲剂。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。本文中，“药物组合物”和“制剂”并不互相排斥。
本公开中所述药物组合物的溶液形式，若无特殊说明，其中的溶剂均为水。
本文所用术语“约”是指数值在由本领域一般技术人员所测定的具体值的可接受误差范围内，所述数值部分取决于怎样测量或测定(即测量体系的限度)。例如，在本领域每一次实行中“约”可意味着在1内或超过1的标准差。或者，“约”或“基本上包含”可意味着至多20％的范围。此外，特别对于生物学系统或过程而言，该术语可意味着至多一个数量级或数值的至多5倍。除非另外说明，否则当具体值在本申请和权利要求中出现时，“约”或“基本上包含”的含义应该假定为在该具体值的可接受误差范围内。
本公开所述的药物组合物能够达到一种稳定的效果：其中的抗体在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性，优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的药物抗体制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，且甚至更优选地最多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃保存包括1个月、3个月、6个月或在40℃保存1个月在内的时段后表现出期望的特征。稳定性的典型的可接受的标准如下：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗体单体发生降解。通过视觉分析，药物抗体制剂是无色或黄色、澄清至微乳光。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的截短，通常形成不超过约10％、优选不超过约5％的聚集。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和SDS-PAGE等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗体在药物制剂中“保留它的生物活性”。抗体的生物活性可以例如通过抗原结合测定来确定。
“因子XI”在本文中也称为“凝血因子XI”、“FXI”或“fXI”，其是分子量为约160 千道尔顿(kD)的双链糖蛋白。两条链可为相同的经二硫键连接的分子量为约80,000道尔顿的多肽。FXI含有4个“苹果结构域(apple domains)”(来自N-末端的A1-A4，重链)和C-末端催化结构域(轻链)。不希望受限于特定理论，认为四个苹果结构域包含对其它蛋白的FXI结合位点，诸如A1对凝血酶；A2对HK，A3对因子IX(FIX)、GPIb和肝素，A4对FXIIa。FXI可通过因子XIIa(FXIIa)转化为其活性形式，凝血因子XIa(FXIa)。丝氨酸蛋白酶FXIa转化凝血因子IX为IXa，后者随后激活凝血因子X(Xa)。Xa随后可介导凝血因子II/凝血酶激活。
本公开所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
“抗体”或“免疫球蛋白”以最广义使用，并且涵盖各种抗体结构，包括但不限于传统的抗体(由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构抗体)，以及具有抗原结合活性的Fab、Fv、sFv、F(ab’)2、线性抗体、单链抗体、scFv、sdAb、sdFv、纳米抗体、肽抗体peptibody、结构域抗体(重链(VH)抗体、轻链(VL)抗体)和多特异性抗体(双特异性抗体、diabody、triabody和tetrabody、串联二-scFv、串联三-scFv)。本公开中使用的术语“抗体”包括全长抗体、其单个的链及其任意具有抗原结合活性的部分(也即抗原结合片段)、结构域或片段以及包含其单个的链及任意具有抗原结合活性的部分、结构域或片段的多特异性抗体(包括但不限于抗原结合结构域或片段，分别例如VHH结构域或VH/VL结构域)。传统的抗体或免疫球蛋白通常是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。重链恒定区的氨基酸组成和排列顺序不同，据此，可将免疫球蛋白分为五类，或称为免疫球蛋白的同种型，即IgM、IgD、IgG、IgA和IgE，其相应的重链分别为μ链、δ链、γ链、α链、和ε链。同一类Ig根据其铰链区氨基酸组成和重链二硫键的数目和位置的差别，又可分为不同的亚类，如IgG可分为IgG1、IgG2、IgG3、IgG4。轻链通过恒定区的不同分为κ链或λ链。五类Ig中每类Ig都可以有κ(kappa)链或λ(lambda)链。在一些实施例中，本披露的抗体特异性地或基本上特异性地结合到人FXI/FXIa。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1，CDR1，FR2，CDR2，FR3，CDR3，FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本公开所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDR2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
本公开的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体 的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后插入人载体中，最后在真核工业系统或原核工业系统中表达嵌合抗体分子。在本公开一个优选的实施方案中，所述的FXI嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的FXI嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、IgG4或其变体的重链恒定区。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网 www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本公开的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
本公开中所述的突变序列中的“突变”包括但不限于“回复突变”、“保守修饰”或“保守置换或取代”。本公开中所述的“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等
Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
本公开所述的“突变序列”是指对本公开的核苷酸序列和/或氨基酸序列进行适当的替换、插入或缺失等突变修饰情况下，得到的与本公开的核苷酸序列和/或氨基酸序列具有不同百分比序列同一性程度的核苷酸序列和/或氨基酸序列。本公开中所述的序列同一性可以至少为85％、90％或95％，例如至少为95％。非限制性实施例包括85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，100％。两个序列之间的序列比较和同一性百分比测定可以通过National Center For Biotechnology Institute网站上可得的BLASTN/BLASTP算法的默认设置来进行。
本公开的术语“与FXI/FXIa结合”，指能与FXI/FXIa或其表位相互作用，所述FXI/FXIa或其表位可以是人源的。本公开的术语“抗原结合位点”指抗原上不连续的，由本公开抗体或抗原结合片段识别的三维空间位点。
术语“特异性结合”指如通过本领域可用的技术，例如竞争ELISA、
Figure PCTCN2023070573-appb-000001
测定或
Figure PCTCN2023070573-appb-000002
测定所测定的。该术语还适用于当例如本公开抗体的抗原结合结构域对许多抗原携带的特定表位特异的情况，在该情况下携带抗原结合结构域的抗体能够特异性结合携带该表位的多种抗原。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸 置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
氨基酸序列“同一性”是指两个蛋白或多肽之间的序列相似性。当两个比较序列中的位置均被相同氨基酸残基占据时，例如如果两个多肽的一个位置都被同一个氨基酸残基占据时，那么所述分子在该位置是一致的。适于确定序列同一性百分比和序列相似性百分比的算法的实例是BLAST和BLAST2.0算法，它们分别描述于Altschul et al.(1990)J.Mol.Biol.215:403-410和Altschul et al.(1977)Nucleic Acids Res.25:3389-3402。用于执行BLAST分析的软件可在美国国家生物技术信息中心公开获取(http://www.ncbi.nlm.nih.gov/)。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或者从免疫球蛋白杂志，2001ISBN012441351上获得。
本公开工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达系统会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人FXI特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲剂的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本公开的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽 管本公开的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“Tm值”是指蛋白质热变性温度，即一半蛋白去折叠时的温度，此时蛋白的空间结构被破坏，所以Tm值越高，蛋白热稳定性越高。
实施例
以下结合实施例进一步描述本公开，但这些实施例并非限制着本公开的范围。本公开实施例中未注明具体条件的实验方法，通常按照常规条件，如冷泉港的抗体技术实验手册，分子克隆手册；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例1.抗FXI/FXIa的杂交瘤单克隆抗体的筛选和制备
1、人FXI/FXIa抗原及检测蛋白的制备
人FXI/FXIa蛋白(Uniprot Acc No.P03951)作为本公开涉及的抗原及检测用蛋白，购自Enzyme research laboratories(FXI:Cat.HFXI1111；FXIa:HFXIa 1111a)。以下FXI/FXIa抗原未特殊说明的均指人FXI/FXIa。
>人FXI/FXIa的氨基酸序列：
Figure PCTCN2023070573-appb-000003
同时人工合成以下KLH偶联的FXIa特异性多肽用于小鼠免疫。
KLH-CFYGVESPKILRVYSGIL     (SEQ ID NO：2)
KLH-CGYRKLRDKIQNTLQKAKIPL    (SEQ ID NO：3)
KLH-CGVQEIIIHDQYKMAESGYDI    (SEQ ID NO：4)
2、FXI/FXIa相关重组蛋白的纯化，以及杂交瘤抗体、重组抗体的纯化
(1)鼠杂交瘤上清分离纯化/ProteinG亲和层析：
对于小鼠杂交瘤上清纯化，首选ProteinG填料(KANEKA:Cat.KanCapTMG)进行亲和层析，将培养所得杂交瘤离心取上清，装有ProteinG填料的重力柱用来纯化，首先利用6M盐酸胍(Simga:Cat.G3272-1KG)再生3-5倍柱体积，然后利用纯水清洗3-5倍柱体积；再用1×PBS(pH7.4)(Sangon Biotech(Shanghai)co.，Ltd.:Cat.E607016-0500)缓冲体系作为平衡缓冲剂对层析柱平衡3-5倍柱体积；细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间；利用1×PBS(pH7.4)洗涤层析柱3-5倍柱体积至紫外吸收回落至基线；利用0.1M Glycine(pH3.0)(Sigma:Cat.410225-250G)缓冲剂进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)(Vetec，Cat.V900483)快速调节pH至7-8，暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系。
(2)Protein A亲和层析提取带人Fc标签的融合蛋白或者抗体：
首先，将表达Fc融合蛋白或者抗体的细胞培养上清进行高速离心，收取上清。ProteinA(GE，Cat:17-5474-99)亲和柱用0.1M NaOH(Sigma:Cat:71687-500g)再生5倍柱体积，然后用1×PBS(pH7.4)(Sangon Biotech(Shanghai)co.，Ltd.:Cat.E607016-0500)清洗平衡5倍柱体积。细胞上清利用低流速上样结合，控制流速使保留时间约1min或更长时间，结合完毕后利用1×PBS(pH7.4)冲洗层析柱5倍柱体积至紫外吸收回落至基线。利用0.1M Glycine(pH3.0)(Sigma:Cat.410225-250G)缓冲剂进行样品洗脱，根据紫外检测收集洗脱峰，洗脱产物利用1M Tris-HCl(pH8.0)(Vetec，Cat.V900483)快速调节pH至7-8，暂存。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系。
(3)阴离子层析精纯抗体：
首先，用平衡缓冲剂20mM Tris pH 8.0(Vetec，Cat.V900483)稀释样品至电导小于3ms/cm，Q HP(GE，Cat:17-1014-01)阴离子层析柱用0.5M NaOH(Sigma:Cat:71687-500g)再生5倍柱体积，然后用20mM Tris pH 8.0(Vetec，Cat.V900483)清洗平衡15倍柱体积。样品低流速上样结合，控制流速使保留时间约2min或更长时间，结合完毕后利用20mM Tris pH 8.0(Vetec，Cat.V900483)冲洗层析柱10倍柱体积至紫外吸收回落至基线。用洗脱缓冲剂20mM Tris pH 8.0(Vetec，Cat.V900483)0.5M NaCl(Vetec，Cat.V900058)进行梯度0-100％洗脱，根据SEC-UPLC(Column：Waters ACQUITY
Figure PCTCN2023070573-appb-000004
Protein BEH SEC column
Figure PCTCN2023070573-appb-000005
1.7um，Cat.186005225)纯度检测收集洗脱峰。对于洗脱产物可以利用本领域技术人员熟知的方法进行溶液置换，如利用超滤管进行超滤浓缩及溶液置换至所需的缓冲体系，或者利用分子排阻如G-25脱盐替换成所需的缓冲体系。
3、抗体筛选
取5只SJL白小鼠，5只Balb/c白小鼠，分别用25μg的FXIa抗原和3个KLH偶联的多肽与佐剂混合免疫。时间为第0、14、35天。第0天腹膜内(IP)注射25μg/只的乳化后抗原。第14、35天注射12.5ug/只。于第21、42天取血，用ELISA方法确定小鼠血清中的抗体滴度。在第4-5次免疫以后，选择血清中抗体滴度高并且滴度趋于平台的小鼠进行脾细胞融合。
采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0-Ag14细胞进行融合得到杂交瘤细胞。根据杂交瘤细胞生长密度，用结合ELISA方法对杂交瘤培养上清进行纯 化、细胞结合实验和细胞阻断实验，阳性的孔细胞及时进行扩增冻存保种和测序。ELISA方法对杂交瘤细胞进一步筛选后获得杂交瘤克隆，然后制备并纯化抗体。
获得阳性克隆3807，对应的抗体可变区氨基酸序列如下：
>3807-VH：
Figure PCTCN2023070573-appb-000006
>3807-VL：
Figure PCTCN2023070573-appb-000007
表1.抗体3807的重链及轻链CDR区序列
Figure PCTCN2023070573-appb-000008
实施例2.抗FXI/FXIa杂交瘤抗体的人源化和免疫原性的改造
1、人源化
通过对抗体分子3807进行三维同源建模，结合V-base人种系序列数据库，IMGT人类抗体重链可变区种系基因数据库进行比对的结果，挑选与筛选出来的抗体同源性高的重链可变区种系基因作为模板，将小鼠来源单克隆抗体的CDR移植到相应的人源模块中。对移植后的抗体再次进行三维结构模拟并分析，将FR区中影响CDR区结构形态的特定位点进行恢复突变。其中氨基酸残基由Kabat编号系统确定并注释。
杂交瘤克隆3807人源化架构的轻链模板为IGKV3-11*01，重链模板为IGHV1-69-2*01，人源化可变区序列如下(下划线为CDR)：
>3807VH-CDR graft
Figure PCTCN2023070573-appb-000009
>3807VL-CDR graft
Figure PCTCN2023070573-appb-000010
杂交瘤克隆3807的回复突变位点和突变方式设计如表2。
表2.各重链可变区及轻链可变区序列
Figure PCTCN2023070573-appb-000011
其中3807-VH1和3807-VL3具体序列如下。
>3807-VH1：
Figure PCTCN2023070573-appb-000012
>3807-VL3：
Figure PCTCN2023070573-appb-000013
抗体3871的VH为3807-VH1，VL为3807-VL3。
2、免疫原性改造
对3871进行基因改造，使抗体具有更高的稳定性，更低的免疫原性。仅对重链可变区FR区进行了改造，轻链可变区未变动。
人源化抗体3871基因改造的重链可变区序列如下(轻链不变)：
>3807-VH1(GTFS)
Figure PCTCN2023070573-appb-000014
抗体3882的VH为3807-VH1(GTFS)，VL为3807-VL3。
将以上各重链可变区与对应的人源抗体重链CH1(SEQ ID NO：18)和人抗体IgG4的Fc(含S241P突变)(SEQ ID NO：19)融合，轻链可变区与人源kappa(SEQ ID NO：20)融合，组成重组嵌合抗体进行后续检测。
>人抗体重链CH1：
Figure PCTCN2023070573-appb-000015
>人抗体IgG4PFc(即含S241P突变)：
Figure PCTCN2023070573-appb-000016
Figure PCTCN2023070573-appb-000017
>人抗体轻链Cκ：
Figure PCTCN2023070573-appb-000018
以3882示例抗体全长序列。
>3882-HC：
Figure PCTCN2023070573-appb-000019
>3882-LC：
Figure PCTCN2023070573-appb-000020
经常规方法制备抗体的表达载体，转染CHO细胞，分离，纯化，检测，获得目的抗体。
实施例3.抗FXI/FXIa抗体功能验证
1.亲和力测定
采用Biacore方法。用Protein A生物传感芯片(Cat.#29127556，GE)亲和捕获一定量的待测抗体，然后于芯片表面流经一系列浓度梯度下的人FXI/FXIa，利用Biacore仪器(Biacore T200，GE)实时检测反应信号从而获得结合和解离曲线。在每个循环解离完成后，用人抗体捕获试剂盒里配置的再生溶液或pH1.5的甘氨酸-盐酸再生溶液(Cat.#BR-1003-54，GE)将生物芯片洗净再生。实验中用到的缓冲剂为HBS-EP+10×缓冲溶液(Cat.#BR-1006-69，GE)用D.I.Water稀释至1×(pH 7.4)
实验得到的数据用BIAevaluation version 4.1，GE软件以(1:1)Langmuir模型进行拟合，得出亲和力数值，测试结果见表3。
表3.抗FXI/FXIa抗体的亲和力测定
抗体 抗原 ka(1/Ms) kd(1/s) KD(M)
3871 FXI 1.32E+06 2.66E-05 2.00E-11
3882 FXI 1.973E+6 5.846E-5 2.963E-11
3807 FXIa 1.59E+06 2.35E-04 1.48E-10
3871 FXIa 1.24E+06 7.08E-04 5.70E-10
3882 FXIa 1.06E+06 3.87E-04 3.64E-10
2、体外FXIIa介导的FXI活化酶活抑制试验
检测抗FXI/FXIa抗体与胰蛋白酶原FXI结合阻断FXIIa对FXI酶切的能力。
预先设置SpectraMax M5酶标仪到37℃，冰上预冷384孔板后，加入分别用缓冲剂(20mM HEPES，pH 7.4，150mM NaCl和0.1％BSA)稀释的10ul FXI(12ug/mL)、10ul FXIIa(7.8ug/mL)、10ul的FXI/FXIa待测抗体(300ug/mL)、10ul的Dextran(100ug/mL)，在37℃孵育60min，然后放冰上孵育5min，加入10ul S-2366(8mM)(Chromogenix，S821090)后，酶标仪检测。结果见图1，其中，使用人IgG同种型作为阴性对照(NC)。
3、人血/猴血的aPTT/PT抗凝血活性检测
用枸橼酸钠管新鲜采集人血/猴血，3000rpm离心15min，取上层血浆。
用Sysmex试剂盒测定在不同浓度的候选抗体(PBS稀释)的存在下的活化部分凝血活酶时间(aPTT)，将所要测试的候选抗体用血浆在37℃下孵育3分钟，然后通过加入25mM氯化钙试剂启动凝血，测定发生凝血时的时间。测定使aPTT延长50％的候选抗体的浓度(EC50)，部分抗体测试结果见图2A、图3A和表4。其中，1209为本申请筛选获得的另一个抗FXI/FXIa抗体。
用Sysmex试剂盒测定在不同浓度的候选抗体(PBS稀释)存在下的凝血酶原时间(PT)，将所要测试的候选抗体用血浆在37℃下孵育3分钟，然后通过加入促凝血酶原激酶启动凝血，测定发生凝血时的时间。部分抗体测试结果见图2B和图3B。
表4.抗FXI/FXIa抗体的aPTT/PT测定
抗体 血浆 aPTT1.5(ug/mL) EC 50(ug/mL)
3882 人血 0.91 1.24
3882 猴血 0.98 1.25
4、食蟹猴体内药代动力学(PK)/药效学(PD)试验
正常成年雄性食蟹猴(体重在4.0-4.7kg范围)，按照体重分配入组(体重第1、4、5入一组，体重第2、3、6入另一组)，每组3只，观察14天。
给药前一天以枸橼酸钠采血管采集血样，作为给药前样本，测定aPTT、PT、血浆药物浓度、血浆活性XI因子(Factor XI,FXI)比例(FXI:C％，即有凝血活性的FXI的比例)和血浆游离FXI浓度；同时测定每只动物的出血时间。两组动物中，其中一组空白不给药；另一组给药3882，5毫克/千克体重(mg/kg,mpk)。药物按照5毫克/毫升(mg/mL)浓度溶于磷酸盐缓冲剂(PBS)，静脉推注给药。
空白对照组于给药后15分钟(min)、3小时(h)分别观测出血时间；于给药后15min、3h、6h和1天(d)分别按照上述方法采集血浆并观测aPTT和PT。给药组于给药后15min、3h、2d、4d、1周(w)、2w和3w分别观测出血时间；于给药后15min、3h、6h、1d、2d、4d、1w、2w、3w、4w、5w和6w分别按照上述方法采集血浆并观测aPTT、PT、血浆药物浓度、血浆FXI:C％和血浆游离FXI浓度。两组动物均于给药1d后进行AV-shunt造栓，造栓时间10min，造栓后称取栓净重。
其中aPTT、PT和血浆FXI:C％以凝血仪和相应试剂盒测定，血浆游离FXI浓度和血浆 药物浓度以ELISA方法测定。血栓重量以给药组与对照组进行对比，以t检验进行统计分析。
抗体测试结果参见图4A-图4D，其中，阴性对照(NC)为未给药。结果显示，5mpk良好的抑制了血栓生成，同时，延长了内源性凝血时间，但对动物出血时间和外源性凝血时间没有显著影响，PK结果显示半衰期为20天左右。
5、食蟹猴体内药效学(PD)试验
正常成年雄性食蟹猴(体重在7-9kg范围)6只，随机分为3组，分别为：BAY1213790静脉给药1毫克/千克体重(mg/kg)组；3882静脉给药1毫克/千克体重(mg/kg)组；3882皮下给药1毫克/千克体重(mg/kg)组。静脉给药组动物在给药前及给药后5min,1h、1d、2d、3d、5d、1w、2w、3w、4w，皮下给药组在给药前及给药后1h、1d、2d、3d、5d、1w、2w、3w、4w，以枸橼酸钠采血管采集血样，以凝血仪和相应试剂盒测定aPTT和血浆FXI:C％。
测试结果参见图5A，5B。结果显示，3882皮下给药和静脉给药均能显著延长内源性凝血时间，抑制FXI的活性。在等剂量给药条件下，3882和BAY1213790相比，3882对内源性凝血时间的延长维持时间更长，对FXI的抑制作用更强。
>BAY1213790的重链：
Figure PCTCN2023070573-appb-000021
>BAY1213790的轻链：
Figure PCTCN2023070573-appb-000022
实施例4至实施例9中的抗FXI/FXIa抗体均为重链序列如SEQ ID NO：21所示，轻链序列如SEQ ID NO：22所示的3882。
实施例4.抗FXI/FXIa抗体制剂缓冲体系pH值的筛选
配制表5中缓冲剂，制备抗FXI/FXIa抗体浓度为120mg/mL的抗体制剂，取样品进行高温40℃、振摇和冻融稳定性研究。
表5.抗FXI/FXIa抗体pH/和缓冲剂筛选结果
Figure PCTCN2023070573-appb-000023
Figure PCTCN2023070573-appb-000024
注：C＝无色；CL＝澄明；WO＝微乳光；SO＝强乳光；P+(少量颗粒)；P++(很多颗粒)；P+++(大量颗粒)
外观结果显示：振摇5D所有处方均无颗粒产生；冻融5cycle所有处方均有颗粒产生，其中10mM醋酸-醋酸钠pH4.5和pH5.0体系、10mM组氨酸-盐酸组氨酸pH 6.5体系及10mM磷酸二氢钠-磷酸氢二钠pH 7.0体系产生颗粒较少；40℃15D所有处方的外观均产生颗粒，10mM组氨酸-盐酸组氨酸pH 6.5和10mM磷酸二氢钠-磷酸氢二钠pH 7.0体系产生的颗粒数少于其余两种体系。
SEC(图6A)结果显示：40℃-15D所有处方的聚集体均有所增加；40℃-15D在pH 4.5-6.5范围内，随着pH的升高，SEC聚集体明显降低；40℃-15D当pH从8.0增加至8.5，SEC聚集体明显增加；当pH在6.0-8.0之间时，40℃-15D SEC和T0相比，聚集体增加<2.5％，在可接受范围内。
NRCE(图6B)结果显示：40℃-15D NRCE纯度下降；在10mM Tris-盐酸体系内，随着pH 的升高(pH 7.5-8.5)，NRCE纯度明显下降，在pH4.5-7.5范围的其他buffer体系中，NRCE纯度无明显差异。
iCIEF(图6C)结果显示：在同一buffer体系内，随着pH升高，酸性峰明显增加。
综合外观、SEC、NRCE和iCIEF结果，pH 6.5-7.5的缓冲体系(组氨酸-盐酸组氨酸、磷酸二氢钠-磷酸氢二钠和Tris-盐酸体系)较为适合。
实施例5.抗FXI/FXIa抗体制剂中pH、辅料和表面活性剂浓度的筛选
在pH 6.5-7.5范围内，选择10mM组氨酸-盐酸组氨酸、10mM磷酸二氢钠-磷酸氢二钠、10mM Tris-柠檬酸和10mM Tris-盐酸缓冲体系，制备含120mg/mL抗FXI/FXIa抗体，不同种类辅料和不同种类表面活性剂的制剂。将样品放置于40±2℃、75±5％RH培养箱中，考察稳定性。结果显示，3882抗体对离子强度较敏感，高离子强度下抗体稳定性偏低，且容易出现颗粒。
选择10mM组氨酸-盐酸组氨酸pH6.5、7.0和10mM Tris-盐酸pH7.0、7.5的缓冲体系，制备含60mg/mL抗FXI/FXIa抗体，0.4-0.8mg/mL聚山梨酯80，辅料为80mg/mL蔗糖或者100mM甘氨酸+40mg/mL蔗糖或者100mM脯氨酸+40mg/mL蔗糖的制剂，进行高温40℃、振摇和冻融稳定性研究：
F2)10mM组氨酸-盐酸组氨酸pH6.5，0.4mg/mL聚山梨酯80，80mg/mL蔗糖；
F3)10mM组氨酸-盐酸组氨酸pH6.5，0.6mg/mL聚山梨酯80，80mg/mL蔗糖；
F4)10mM组氨酸-盐酸组氨酸pH6.5，0.8mg/mL聚山梨酯80，80mg/mL蔗糖；
F5)10mM组氨酸-盐酸组氨酸pH6.5，0.4mg/mL聚山梨酯80，100mM甘氨酸+40mg/mL蔗糖；
F6)10mM组氨酸-盐酸组氨酸pH6.5，0.4mg/mL聚山梨酯80，100mM脯氨酸+40mg/mL蔗糖；
F7)10mM组氨酸-盐酸组氨酸pH7.0，0.4mg/mL聚山梨酯80，80mg/mL蔗糖；
F8)10mM组氨酸-盐酸组氨酸pH7.0，0.4mg/mL聚山梨酯80，100mM脯氨酸+40mg/mL蔗糖；
F10)10mMTris-盐酸pH7.0，0.4mg/mL聚山梨酯80，80mg/mL蔗糖；
F11)10mMTris-盐酸pH7.5，0.4mg/mL聚山梨酯80，80mg/mL蔗糖；
表7.抗FXI/FXIa抗体中pH、辅料和表面活性剂浓度细筛结果
Figure PCTCN2023070573-appb-000025
Figure PCTCN2023070573-appb-000026
注：C＝无色；CL＝澄明；WO＝微乳光；SO＝强乳光；P(几个颗粒)；P+(少量颗粒)；P++(很多颗粒)；P+++(大量颗粒)；
外观结果显示：冻融5cycle，10mM组氨酸-盐酸组氨酸pH 6.5体系均产生大量颗粒；其中F4、F6处方在振摇3D后也出现大量颗粒，F2、F3和F5振摇后无颗粒产生；10mM组氨酸-盐酸组氨酸pH 7.0体系(F7、F8)在振摇3D均无颗粒产生，冻融5cycle后，仅F7产生少量颗粒；40℃2W仅F7和F8无颗粒产生，其余处方均产生颗粒，其中，F3(8％蔗糖+0.06％聚山 梨酯80)、F4(8％蔗糖+0.08％聚山梨酯80)、F5(100mM Gly+4％蔗糖+0.04％聚山梨酯80)、F6(100mM Pro+4％蔗糖+0.04％聚山梨酯80)均产生大量颗粒，F2(8％蔗糖+0.04％聚山梨酯80)仅少量颗粒；40℃1M，所有处方均产生颗粒，F7和F8处方仍然表现较好，F7处方少量颗粒，F8几个颗粒，10mM组氨酸-盐酸组氨酸pH 7.0体系明显优于10mM组氨酸-盐酸组氨酸pH 6.5和10mM Tris-盐酸体系；40℃1M，F2-F6均产生大量颗粒，表明加入甘氨酸、脯氨酸以及增加聚山梨酯80浓度对外观无明显改善。
SEC(图7A)结果显示：冻融、振摇条件下，SEC与T0相比无明显差异；高温后不同处方SEC与T0相比均有所下降；F2-F8(10mM组氨酸-盐酸组氨酸体系)处方间没有明显差异；F10、F11(10mM Tris-盐酸体系)处方SEC纯度略高于10mM组氨酸-盐酸组氨酸体系。
NRCE(图7B)结果显示：冻融、振摇条件下，NRCE与T0相比无明显差异；高温后不同处方NRCE与T0相比均有所下降，处方间无明显差异。
CEX(图7C)结果显示：冻融、振摇条件下，酸性峰与T0相比无明显差异；高温后不同处方酸性峰与T0相比均有所增加，在同一buffer体系内，随着pH升高，酸性峰明显增加。
综合外观、SEC、NRCE和CEX结果，纯度检项略有变化，均在可接受范围内。就外观而言，10mM组氨酸-盐酸组氨酸pH 7.0体系优于10mM组氨酸-盐酸组氨酸pH 6.5和10mM Tris-盐酸体系。根据pH/Buffer筛选和辅料、表面活性剂筛选的结果，抗体在pH 6.5-7.5范围稳定性较好，组氨酸-盐酸组氨酸体系优于磷酸二氢钠-磷酸氢二钠、Tris-盐酸、醋酸-醋酸钠和Tris-柠檬酸等体系，且3882抗体对离子强度较为敏感。我们采用组氨酸-抗体自缓冲体系进一步优化制剂。
实施例6.抗FXI/FXIa抗体制剂缓冲体系和表面活性剂种类筛选
选择组氨酸-抗体自缓冲体系pH7.0，制备含60mg/mL蛋白，辅料为80mg/mL蔗糖，表面活性剂为泊洛沙姆188或聚山梨酯80的抗FXI/FXIa抗体制剂。样品放置40±2℃、75±5％RH培养箱中，考察稳定性：
F1)35mM组氨酸pH7.0，1mg/mL聚山梨酯80，80mg/mL蔗糖；
F2)35mM组氨酸pH7.0，1mg/mL泊洛沙姆188，80mg/mL蔗糖；
F3)35mM组氨酸pH7.0，2mg/mL泊洛沙姆188，80mg/mL蔗糖；
表8.缓冲体系确认和表面活性剂种类筛选
Figure PCTCN2023070573-appb-000027
Figure PCTCN2023070573-appb-000028
注：C＝无色；Y＝微黄色；Y+＝黄色；CL＝澄明
外观结果显示：40℃2W和40℃4W所有处方均无颗粒产生。
SEC(图8A)结果显示：高温后不同处方SEC与T0相比均有所下降，其中F1处方(35mM His+8％蔗糖+0.1％聚山梨酯80)下降最多，明显低于F2(35mM His+8％蔗糖+0.1％泊洛沙姆188)和F3(35mM His+8％蔗糖+0.2％泊洛沙姆188)。
NRCE结果显示：高温后不同处方NRCE与T0相比均有所下降，处方间无明显差异。
CEX(图8B)结果显示：高温后不同处方酸性峰与T0相比均有所增加，其中F1处方增加最多，明显高于F2和F3。
综合外观、SEC、NRCE和CEX结果，当使用组氨酸-抗体自缓冲体系时，pH能维持在7.0左右，所有处方40℃均无颗粒产生，使用表面活性剂聚山梨酯80的处方纯度检项与T0差异显著。因此，确定pH/buffer为组氨酸-抗体自缓冲体系pH7.0，表面活性剂优选为泊洛沙姆188。
实施例7.抗FXI/FXIa抗体制剂缓冲体系确认和表面活性剂浓度筛选
考虑到药物的最终剂量，将抗FXI/FXIa抗体浓度提高，选择组氨酸-抗体自缓冲体系pH7.0，制备含80mg/mL蛋白，辅料为80mg/mL蔗糖，表面活性剂为1-2mg/mL泊洛沙姆188的制剂。样品放置-35℃/室温冻融5轮、40℃-2W、40℃-4W、振摇3天(25℃，300rpm)，考察稳定性：
F1)35mM组氨酸pH7.0，1mg/mL泊洛沙姆188，80mg/mL蔗糖；
F2)35mM组氨酸pH7.0，1.5mg/mL泊洛沙姆188，80mg/mL蔗糖；
F3)35mM组氨酸pH7.0，2mg/mL泊洛沙姆188，80mg/mL蔗糖；
表9.抗FXI/FXIa抗体缓冲体系确认和表面活性剂浓度筛选结果
Figure PCTCN2023070573-appb-000029
注：Y＝微黄色；WO＝微乳光；SO＝强乳光；P+(少量颗粒)
外观结果显示：振摇3D所有处方均无颗粒产生；冻融5cycle，F1(35mM His，0.1％泊洛沙姆188)、F2(35mM His，0.15％泊洛沙姆188)有少量颗粒产生；40℃2W所有处方均无颗粒产生；40℃4W，F1、F2有少量颗粒产生。
SEC结果显示：冻融、振摇条件下，SEC与T0相比无明显差异；高温后不同处方SEC与T0相比均有所下降，处方间无明显差异。
NRCE结果显示：冻融、振摇条件下，NRCE与T0相比无明显差异；高温后不同处方NRCE与T0相比均有所下降，处方间无明显差异。
CEX结果显示：冻融、振摇条件下，酸性峰与T0相比无明显差异；高温后不同处方酸性峰与T0相比均有所增加，处方间无明显差异。
综合外观、SEC、NRCE和CEX结果，确定pH/buffer为组氨酸-抗体自缓冲体系pH7.0，表面活性剂为0.2％泊洛沙姆188。
实施例8.抗FXI/FXIa抗体制剂中抗体浓度的筛选
选择组氨酸-抗体自缓冲体系pH7.0，制备含60mg/mL-100mg/mL抗FXI/FXIa抗体，2mg/mL泊洛沙姆188，辅料为80mg/mL蔗糖的制剂。样品放置-35℃/室温冻融5轮、40℃-2W、40℃-4W、振摇3天(25℃，300rpm)，考察稳定性：：
F1)35mM组氨酸pH7.0，2mg/mL泊洛沙姆188，80mg/mL蔗糖，60mg/mL蛋白；
F2)35mM组氨酸pH7.0，2mg/mL泊洛沙姆188，80mg/mL蔗糖，80mg/mL蛋白；
F3)35mM组氨酸pH7.0，2mg/mL泊洛沙姆188，80mg/mL蔗糖，100mg/mL蛋白；
表10.抗FXI/FXIa抗体制剂蛋白浓度筛选结果
Figure PCTCN2023070573-appb-000030
注：Y＝微黄色；WO＝微乳光
外观结果显示：振摇3D，冻融5cycle，40℃2W及40℃4W所有处方均无颗粒产生；
SEC结果显示：冻融、振摇条件下，SEC与T0相比无明显差异；高温后不同处方SEC与T0相比均有所下降，处方间无明显差异。
NRCE结果显示：冻融、振摇条件下，NRCE与T0相比无明显差异；高温后不同处方NRCE与T0相比均有所下降，处方间无明显差异。
CEX结果显示：冻融、振摇条件下，酸性峰与T0相比无明显差异；高温后不同处方酸性峰与T0相比均有所增加，处方间无明显差异。
综合外观、SEC、NRCE和CEX结果，处方初步确定为35mM组氨酸，80mg/mL蔗糖，2mg/mL泊洛沙姆188，pH 7.0，抗FXI/FXIa抗体浓度80mg/mL。
实施例9.抗FXI/FXIa抗体制剂处方稳健性考察
考虑到生产的可操作性，使用组氨酸-蛋白自缓冲体系，组氨酸浓度、蛋白浓度和泊洛沙姆188浓度为变量进行DoE设计，因子水平为组氨酸浓度30-40mM、蛋白浓度70-130mg/mL、泊洛沙姆188浓度1.8-2.8g/L，应用Minitab软件进行DoE实验，得到一系列处方，样品放置-35℃/室温冻融5轮、40℃-2W、40℃-26D、振摇3天(25℃，300rpm)，考察稳定性：
F1)30mM组氨酸pH7.0，1.8mg/mL泊洛沙姆188，80mg/mL蔗糖，70mg/mL蛋白；
F2)30mM组氨酸pH7.0，1.8mg/mL泊洛沙姆188，80mg/mL蔗糖，130mg/mL蛋白；
F3)30mM组氨酸pH7.0，2.8mg/mL泊洛沙姆188，80mg/mL蔗糖，70mg/mL蛋白；
F4)30mM组氨酸pH7.0，2.8mg/mL泊洛沙姆188，80mg/mL蔗糖，130mg/mL蛋白；
F5)40mM组氨酸pH7.0，1.8mg/mL泊洛沙姆188，80mg/mL蔗糖，70mg/mL蛋白；
F6)40mM组氨酸pH7.0，1.8mg/mL泊洛沙姆188，80mg/mL蔗糖，130mg/mL蛋白；
F7)40mM组氨酸pH7.0，2.8mg/mL泊洛沙姆188，80mg/mL蔗糖，70mg/mL蛋白；
F8)40mM组氨酸pH7.0，2.8mg/mL泊洛沙姆188，80mg/mL蔗糖，130mg/mL蛋白；
F9)35mM组氨酸pH7.0，2.3mg/mL泊洛沙姆188，80mg/mL蔗糖，100mg/mL蛋白；
F10)35mM组氨酸pH7.0，2.3mg/mL泊洛沙姆188，80mg/mL蔗糖，100mg/mL蛋白；
F11)35mM组氨酸pH7.0，2.3mg/mL泊洛沙姆188，80mg/mL蔗糖，100mg/mL蛋白；
F12)35mM组氨酸pH7.0，2mg/mL泊洛沙姆188，80mg/mL蔗糖，120mg/mL蛋白；
表11.抗FXI/FXIa抗体制剂处方稳健性结果
Figure PCTCN2023070573-appb-000031
Figure PCTCN2023070573-appb-000032
Figure PCTCN2023070573-appb-000033
注：C＝无色；Y＝微黄色；CL＝澄明
外观结果显示：振摇3D，冻融5cycle，40℃2W及40℃26D所有处方均无颗粒产生；
SEC结果显示：冻融、振摇条件下，SEC与T0相比无明显差异；高温后不同处方SEC与T0相比均有所下降，处方间无明显差异。
NRCE结果显示：冻融、振摇条件下，NRCE与T0相比无明显差异；高温后不同处方NRCE与T0相比均有所下降，处方间无明显差异。
CEX结果显示：冻融、振摇条件下，酸性峰与T0相比无明显差异；高温后不同处方酸性峰与T0相比均有所增加，处方间无明显差异。
综合外观、SEC、NRCE和CEX结果，即抗FXI/FXIa抗体在70～130mg/mL、组氨酸浓度30-40mM、泊洛沙姆188浓度为1.8-2.8g/L范围之内纯度项差异不显著，制剂稳定性均较好。
实施例10.抗FXI抗体制剂使用中稳定性
取抗FXI抗体制剂成品，分别使用0.9％生理盐水注射液(输液袋材质：聚丙烯)和5％葡萄糖注射液(输液袋材质：聚丙烯)在输液袋中分别稀释至0.20mg/mL、13.30mg/mL，同时考察稀释后药液在一次性输液管路(管路材质：聚丙烯，滤膜材质：聚醚砜)中2～8℃24h+室温光照6h的稳定性。实验结果见表12。
抗FXI抗体制剂使用5％葡萄糖和0.9％生理盐水稀释后(0.20～13.30mg/mL)在2～8℃24h+室温光照6h，所考察检项均在可接受标准范围内。因此，稀释后药液与输液系统相容性良好，可以在输液系统中稀释后于2～8℃24h+室温6h内使用。
Figure PCTCN2023070573-appb-000034

Claims (28)

  1. 一种药物组合物，其包含抗FXI/FXIa抗体或其抗原结合片段，以及缓冲剂，所述缓冲剂选自醋酸盐缓冲剂、组氨酸盐缓冲剂、Tris-盐酸盐缓冲剂、Tris-柠檬酸盐缓冲剂和磷酸盐缓冲剂中的一种或多种；
    优选地，所述缓冲剂为组氨酸盐缓冲剂、Tris-盐酸盐缓冲剂或磷酸盐缓冲剂。
  2. 如权利要求1所述的药物组合物，其中所述缓冲剂或所述药物组合物的pH值为约4.0至约8.5，优选为约6.5至约7.5，更优选为约7.0。
  3. 如权利要求1或2所述的药物组合物，所述缓冲剂的浓度为约5mM至约100mM，优选为约10mM至约30mM，更优选为约5mM至约15mM。
  4. 如权利要求1至3任一项所述的药物组合物，其中所述抗FXI/FXIa抗体或其抗原结合片段的浓度为约1mg/mL至约300mg/mL，优选为约50mg/mL至约150mg/mL。
  5. 如权利要求1至4任一项所述的药物组合物，其中还包括表面活性剂；
    优选地，所述表面活性剂为泊洛沙姆188、聚山梨酯80或聚山梨酯20。
  6. 如权利要求5所述的药物组合物，其中所述表面活性剂的浓度为约0.1mg/mL至约10mg/mL，优选为约1.5mg/mL至约4mg/mL，优选为约0.4mg/mL至约0.8mg/mL。
  7. 如权利要求1至6任一项所述的药物组合物，其中还包括渗透压调节剂；
    优选地，所述渗透压调节剂选自由蔗糖、海藻糖、山梨糖醇、精氨酸、脯氨酸、甘氨酸和氯化钠组成的组中的一种或更多种；
    更优选地，所述渗透压调节剂选自蔗糖，脯氨酸和蔗糖，或甘氨酸和蔗糖。
  8. 如权利要求7所述的药物组合物，其中所述渗透压调节剂选自：
    约10mg/mL至约150mg/mL的蔗糖，
    约10mM至200mM的甘氨酸和约10mg/mL至约100mg/mL的蔗糖，或
    约10mM至200mM的脯氨酸和约10mg/mL至约100mg/mL的蔗糖；
    优选地，所述渗透压调节剂选自：
    约80mg/mL的蔗糖，
    约100mM的甘氨酸和约40mg/mL的蔗糖，或
    约100mM的脯氨酸和约40mg/mL的蔗糖。
  9. 一种药物组合物，其包含抗FXI/FXIa抗体或其抗原结合片段，所述药物组合物为自缓冲体系；优选地，所述药物组合物中包含碱性氨基酸；更优选地，所述碱性氨基酸为组氨 酸。
  10. 如权利要求9所述的药物组合物，其中所述抗FXI/FXIa抗体或其抗原结合片段的浓度为约1mg/mL至约300mg/mL，优选为约30mg/mL至约200mg/mL，更优选为约70mg/mL至约130mg/mL。
  11. 如权利要求9或10所述的药物组合物，所述碱性氨基酸的浓度为约1mM至约100mM，优选10mM至约60mM，更优选为约30mM至约40mM，最优选为约35mM。
  12. 如权利要求9至11任一项所述的药物组合物，其pH值为约4.0至约8.5，优选为约6.5至约7.5，更优选为约6.7至约7.3，最优选为约7.0。
  13. 如权利要求9至12任一项所述的药物组合物，其中还包括渗透压调节剂；
    优选地，所述渗透压调节剂选自由蔗糖、海藻糖、山梨糖醇、精氨酸、脯氨酸、甘氨酸和氯化钠中的一种或多种；
    更优选地，所述渗透压调节剂为蔗糖。
  14. 如权利要求13所述的药物组合物，所述渗透压调节剂的浓度为约10mg/mL至约400mg/mL，优选为约50mg/mL至约200mg/mL，更优选为约80mg/mL。
  15. 如权利要求9至14任一项所述的药物组合物，其中还包括表面活性剂；
    优选地，所述表面活性剂为泊洛沙姆188、聚山梨酯80或聚山梨酯20。
  16. 如权利要求15所述的药物组合物，其中所述表面活性剂的浓度为约0.1mg/mL至约10mg/mL，优选为约1.8mg/mL至约2.8mg/mL，更优选为约2mg/mL。
  17. 一种药物组合物，其包含(a)和(b)，可选地，还包含(c)和/或(d)，其中，
    (a)约1mg/mL至约500mg/mL的抗FXI/FXIa抗体，
    (b)约1mM至约100mM的组氨酸，
    (c)约10mg/mL至约400mg/mL的蔗糖或海藻糖，
    (d)约0.5mg/mL至约80mg/mL的泊洛沙姆、聚山梨酯80或聚山梨酯20，
    所述药物组合物的pH为约4.0至约8.5；
    或者，
    (a)约30mg/mL至约200mg/mL的抗FXI/FXIa抗体，
    (b)约10mM至约60mM的组氨酸，
    (c)约50mg/mL至约200mg/mL的蔗糖，
    (d)约1.5mg/mL至约5.0mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约6.5至约7.5；
    或者，
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体，
    (b)约30mM至约40mM的组氨酸，
    (c)约60mg/mL至约150mg/mL的蔗糖，和
    (d)约1.8mg/mL至约2.8mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约6.7至约7.3；
    或者，
    (a)约70mg/mL至约200mg/mL的抗FXI/FXIa抗体，
    (b)约30mM至约100mM的组氨酸，
    (c)约60mg/mL至约150mg/mL的蔗糖，和
    (d)约1.8mg/mL至约2.8mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约6.7至约7.3。
  18. 根据权利要求17所述的药物组合物，其中，所述药物组合物包含：
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约35mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约2mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.0；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约35mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约2mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约6.7；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约35mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约2mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.3；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约30mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约1.8mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.0；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约30mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约2.8mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.0；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约40mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约1.8mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.0；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约40mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约2.3mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.0；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约40mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约2.8mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.0；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约35mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约2.3mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.0；或
    (a)约70mg/mL至约130mg/mL的抗FXI/FXIa抗体或其抗原结合片段，
    (b)约35mM的组氨酸，
    (c)约80mg/mL的蔗糖，
    (d)约1.8mg/mL的泊洛沙姆188，
    所述药物组合物的pH为约7.0。
  19. 一种药物组合物，其是权利要求17或18的药物组合物经0.9％生理盐水或5％葡萄糖溶液稀释获得的，或含有权利要求16的药物组合物经0.9％生理盐水或5％葡萄糖溶液稀释后的可作为静脉注射剂浓度的抗FXI/FXIa抗体或其抗原结合片段、组氨酸、蔗糖、泊洛沙姆188；
    优选地，其中抗FXI/FXIa抗体或其抗原结合片段的浓度为约0.01mg/mL至约50mg/mL，
    更优选地，其中抗FXI/FXIa抗体或其抗原结合片段的浓度为约0.1mg/mL至约30mg/mL，
    最优选地，其中抗FXI/FXIa抗体或其抗原结合片段的浓度为约0.20mg/mL至约13.30mg/mL。
  20. 如权利要求1至19任一项所述的药物组合物，其中所述抗FXI/FXIa抗体或其抗原结合片段包含重链可变区和轻链可变区，其中：所述重链可变区包含分别如SEQ ID NO：7、8、9所示的HCDR1、HCDR2、HCDR3，和所述轻链可变区包含分别如SEQ ID NO：10、11、12所示的LCDR1、LCDR2、LCDR3。
  21. 如权利要求1至20任一项所述的药物组合物，其中所述抗FXI/FXIa抗体或其抗原结合片段包含重链可变区和轻链可变区，其中，所述重链可变区和轻链可变区选自：
    重链可变区序列如SEQ ID NO：5或与之具有至少90％同一性的序列，轻链可变区序列如SEQ ID NO：6或与之具有至少90％同一性的序列；
    重链可变区序列如SEQ ID NO：13或与之具有至少90％同一性的序列，轻链可变区序列如SEQ ID NO：14或与之具有至少90％同一性的序列；
    重链可变区序列如SEQ ID NO：15或与之具有至少90％同一性的序列，轻链可变区序列如SEQ ID NO：16或与之具有至少90％同一性的序列；或
    重链可变区序列如SEQ ID NO：17或与之具有至少90％同一性的序列，轻链可变区序列如SEQ ID NO：16或与之具有至少90％同一性的序列。
  22. 如权利要求1至21中任一项所述的药物组合物，其中所述抗FXI/FXIa抗体或其抗原结合片段还含有人IgG1 Fc或人IgG4 Fc，所述人IgG4 Fc优选含有S241P突变。
  23. 如权利要求1至22中任一项所述的药物组合物，其中所述抗FXI/FXIa抗体或其抗原结合片段包含重链和轻链，其中：所述重链序列包含SEQ ID NO：21或与之具有至少90％同一性的序列，和所述轻链序列包含SEQ ID NO：22或与之具有至少90％同一性的序列。
  24. 一种冻干制剂，所述冻干制剂通过将权利要求1至23任一项所述的药物组合物经冷冻干燥获得，或所述冻干制剂复溶后可形成权利要求1至23任一项所述的药物组合物。
  25. 一种复溶溶液，其是通过将权利要求24所述的冻干制剂经复溶制备获得。
  26. 根据权利要求1至23任一项所述的药物组合物或权利要求25所述的复溶溶液，其为静脉注射剂、皮下注射剂、腹腔注射剂或肌肉注射剂；优选为静脉或皮下注射剂。
  27. 一种制品，其包括容器，该容器中装有如权利要求1至23中任一项所述的药物组合物、权利要求24所述的冻干制剂或权利要求25所述的复溶溶液。
  28. 一种治疗或预防疾病的方法，包括向有需要的受试者施用治疗或预防有效量的如权利要求1至22中任一项所述的药物组合物、权利要求23所述的冻干制剂、权利要求25 所述的复溶溶液或权利要求27所述的制品，其中：
    所述疾病为血栓形成性或血栓栓塞性疾病和/或血栓形成性或血栓栓塞性并发症，或心律失常、心源性血栓栓塞、弥漫性血管内凝血；
    优选地，所述血栓形成性或血栓栓塞性疾病或其并发症选自：心脏冠状动脉疾病，诸如急性冠状动脉综合征(ACS)、有ST段升高的心肌梗死(STEMI)和无ST段升高的心肌梗死(非STEMI)、稳定型心绞痛、不稳定型心绞痛、冠状动脉介入后的再闭塞和再狭窄，以及导致外周动脉闭塞性疾病、肺栓塞、静脉血栓栓塞、静脉血栓形成、短暂性脑缺血发作以及血栓形成性脑卒中和血栓栓塞性脑卒中的其它血管中的血栓形成性或血栓栓塞性疾病，由慢性血栓栓塞(CTEPH)引起的肺病和肺动脉高压或其组合；
    优选地，所述施用的方式为皮下或静脉注射。
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