WO2020108497A1 - 一种抗pd-l1抗体制剂 - Google Patents

一种抗pd-l1抗体制剂 Download PDF

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Publication number
WO2020108497A1
WO2020108497A1 PCT/CN2019/121069 CN2019121069W WO2020108497A1 WO 2020108497 A1 WO2020108497 A1 WO 2020108497A1 CN 2019121069 W CN2019121069 W CN 2019121069W WO 2020108497 A1 WO2020108497 A1 WO 2020108497A1
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Prior art keywords
histidine
concentration
antibody
sodium chloride
liquid formulation
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PCT/CN2019/121069
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English (en)
French (fr)
Inventor
肖亮
邓莉萍
黄海玉
刘启刚
刘立平
郭大成
薛彤彤
王晶翼
Original Assignee
和铂医药（香港）有限公司
四川科伦博泰生物医药股份有限公司
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Application filed by 和铂医药（香港）有限公司, 四川科伦博泰生物医药股份有限公司 filed Critical 和铂医药（香港）有限公司
Priority to CA3121288A priority Critical patent/CA3121288A1/en
Priority to JP2021531364A priority patent/JP2022513693A/ja
Priority to AU2019388808A priority patent/AU2019388808A1/en
Priority to KR1020217019952A priority patent/KR20210096640A/ko
Priority to US17/297,719 priority patent/US20230287122A1/en
Priority to EP19890307.2A priority patent/EP3888678A4/en
Publication of WO2020108497A1 publication Critical patent/WO2020108497A1/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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • the present invention relates to the field of therapeutic antibodies. More specifically, the present invention relates to an anti-PD-L1 antibody preparation and its medical use.
  • PD-1 Programmed Death 1
  • PD-L1 Programmed Cell Death-Ligand 1
  • PD-Ligand 1 stands for Programmed Death Receptor-Ligand 1
  • antigen presenting cells APC
  • MHC matrix-containing cell
  • TCR molecules on the surface of T cells specifically recognize the antigen peptide presented by APC, and simultaneously recognize the MHC molecules that form a complex with the antigen peptide.
  • many immunomodulatory molecules on the surface of T cells participate in the reaction and generate costimulatory signals to promote T cell activation. And play an immune effect.
  • PD-1 is expressed on the surface of activated T cells, B cells, NK cells, monocytes, and DC. It interacts with PD-L1, PD-L2, etc. on the surface of APC cells to transmit inhibitory signals and inhibit T The activation of cells maintains the immune homeostasis of the body.
  • PD-L1 On the surface of tumor cells, there is often high expression of PD-L1, which is combined with the high expression of PD-1 in tumor infiltrating T lymphocytes, which leads to the continuous activation of PD-1 pathway, thereby inhibiting T cell activation and achieving tumor immune escape. Therefore, blocking PD-1/PD-L1 binding has become one of the important strategies in the field of tumor immunotherapy.
  • the PD-L1 antibody can block the binding of tumor PD-L1 to T cell PD-1, eliminating this immunosuppressive effect, allowing T cells to be reactivated to recognize and kill tumor cells.
  • WO2017/148424 discloses a new anti-PD-L1 monoclonal antibody, which can effectively block the binding of PD-1 and PD-L1, showing a better effect on tumors or anemia.
  • PD-L1 antibody in clinical treatment needs to solve the problems of physical and chemical properties suitable for administration and stability during storage.
  • Antibody preparation is the final form of antibody in storage, transportation and clinical treatment. It needs to take into account its physical and chemical properties, physiological properties and stability within the validity period, such as protein content, pH, osmotic pressure, insoluble particles, purity, aggregates, binding activity, etc. .
  • a liquid preparation composed of an anti-PD-L1 antibody and a specific prescription has excellent physiological compatibility and stability, and further developed a high-concentration antibody preparation
  • Concentrated protein preparations overcome the problems of poor stability, high viscosity, and easy turbidity. Therefore, the present application discloses a liquid preparation that is particularly advantageous for long-term storage of drugs and clinical use of drugs, thus completing the present invention.
  • the present invention provides a liquid formulation comprising:
  • Anti-PD-L1 antibody with a concentration of about 10 mg/ml to about 150 mg/ml; SEQ ID NOs: light chain CDRs shown in 4-6;
  • the liquid preparation has a pH of about 5.0 to about 7.0; and, the liquid preparation is free of added sugar and/or sugar alcohol;
  • liquid preparation has an osmotic pressure equivalent to that of human blood.
  • the anti-PD-L1 antibody liquid formulation disclosed herein is characterized by the absence of added sugar and/or sugar alcohol, and therefore may also be referred to herein as a sugar-free antibody formulation.
  • the liquid formulation does not contain sugars and/or sugar alcohols selected from the group consisting of sucrose, glucose, trehalose, mannitol and sorbitol.
  • the anti-PD-L1 antibody has a heavy chain variable region shown in SEQ ID NO: 7 and a light chain variable region shown in SEQ ID NO: 8. In certain preferred embodiments, the anti-PD-L1 antibody has a heavy chain shown in SEQ ID NO: 9 and a light chain shown in SEQ ID NO: 10.
  • the concentration of the anti-PD-L1 antibody is 10 mg/ml to 30 mg/ml.
  • the concentration of the anti-PD-L1 antibody is 10 mg/ml to 120 mg/ml, preferably 30 mg/ml to 70 mg/ml.
  • the concentration of the anti-PD-L1 antibody is about 15 mg/ml to about 25 mg/ml. In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is about 20 mg/ml.
  • the concentration of the anti-PD-L1 antibody is about 50 mg/ml to about 70 mg/ml. In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is about 60 mg/ml.
  • the concentration of the anti-PD-L1 antibody is about 80 mg/ml to about 150 mg/ml. In certain preferred embodiments, the concentration of the anti-PD-L1 antibody is about 90 mg/ml, about 100 mg/ml, about 110 mg/ml, or about 120 mg/ml.
  • the sodium chloride concentration is from about 100 mM to about 160 mM. In certain preferred embodiments, the sodium chloride concentration is about 105 mM, about 110 mM, about 115 mM, about 120 mM, about 130 mM, about 135 mM, about 140 mM, about 145 mM, about 150 mM, or about 155 mM. In certain more preferred embodiments, the sodium chloride concentration is about 140 mM.
  • the buffer is selected from: citric acid-citrate buffer, histidine buffer or acetate-acetate buffer.
  • the histidine in the histidine buffer is present alone or in the form of histidine hydrochloride or histidine acetate.
  • the buffer is a histidine buffer.
  • the histidine in the histidine buffer is present alone or in the form of histidine-hydrochloride or histidine-acetate.
  • the concentration of the buffer is about 10 mM to about 20 mM. In certain preferred embodiments, the concentration of the buffer is about 20 mM. In certain preferred embodiments, the liquid formulation contains a histidine buffer at a concentration of about 20 mM, preferably the histidine in the histidine buffer is present alone or as histidine hydrochloride or Histidine acetate exists.
  • the buffer solution is 20 mM histidine-histidine hydrochloride.
  • the pH of the liquid preparation is adjusted with a buffer solution, and the buffer solution may further be adjusted with a pH adjusting agent (for example, hydrochloric acid, acetic acid, phosphoric acid, etc.).
  • a pH adjusting agent for example, hydrochloric acid, acetic acid, phosphoric acid, etc.
  • the liquid formulation has a pH of about 5.2 to about 6.8.
  • the liquid formulation has a pH of about 5.6 to about 6.8.
  • the liquid formulation has a pH of about 5.6 to about 6.0.
  • the pH of the liquid formulation is about 5.0, about 5.2, about 5.6, about 5.8, about 6.0, about 6.4, about 6.8, or about 7.0.
  • the pH of the liquid formulation is 5.8 ⁇ 0.1.
  • the pH of the liquid formulation is about 5.8.
  • the liquid formulation has an osmolality of about 260 mOsmol/kg to about 380 mOsmol/kg. In certain preferred embodiments, the liquid formulation has an osmolality of about 280 mOsm/kg to about 380 mOsmol/kg (eg, 280 mOsmol/kg to about 360 mOsmol/kg). In certain preferred embodiments, the liquid formulation has an osmolality of about 280 mOsm/kg to about 330 mOsmol/kg. In certain preferred embodiments, the liquid formulation has substantially the same osmotic pressure as human blood.
  • the osmolarity corresponding to human blood is osmolality of about 280 mOsmol/kg to about 400 mOsmol/kg, preferably osmolality of 290 mOsmol/kg to about 320 mOsmol/kg It is more preferably osmolality from 295 mOsmol/kg to about 315 mOsmol/kg.
  • the liquid formulation further includes a surfactant.
  • the surfactant is polysorbate 80/polysorbate 20 or poloxamer 188. In certain preferred embodiments, the surfactant is polysorbate 80.
  • the concentration of the surfactant is from about 0.01% w/v to about 0.02% w/v. In certain preferred embodiments, the concentration of the surfactant is about 0.02% w/v. In certain preferred embodiments, the liquid formulation comprises polysorbate 80 at a concentration of about 0.02% w/v.
  • the liquid formulation comprises:
  • the anti-PD-L1 antibody at a concentration of about 20 mg/ml, about 60 mg/ml, about 90 mg/ml, or about 120 mg/ml;
  • a buffer with a concentration of about 20 mM is selected from the group consisting of histidine buffer, citric acid-citrate buffer, and acetate-acetate buffer;
  • the liquid preparation has a pH of about 5.2 to about 6.8 (for example, a pH of about 5.6 to about 6.8, for example a pH of about 5.6 to about 6.0, for example a pH of about 5.8); and, the liquid preparation does not contain sugar And/or sugar alcohol.
  • the liquid formulations of the present invention can be stable at 5°C ⁇ 3°C and can be stored for at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least About 6 months, at least about 9 months, at least about 1 year or at least about 2 years.
  • the liquid formulations of the present invention are stable at about 40°C and can be stored for at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least about 6 Month, at least about 9 months, at least about 1 year or at least about 2 years.
  • the liquid formulations of the present invention are stable at room temperature (eg, about 25°C) and can be stored for at least about 2 weeks, at least about 1 month, at least about 2 months, at least about 3 months, at least About 6 months, at least about 9 months, at least about 1 year or at least about 2 years.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-hydrochloride buffer at a concentration of 20 mM , A concentration of 0.02% w/v polysorbate 80; and the pH of the liquid formulation is 5.8.
  • the liquid formulation is stable at 40°C for at least 28 days, and stable at 5°C ⁇ 3°C for at least 12 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-hydrochloride buffer at a concentration of 20 mM , A concentration of 0.02% w/v polysorbate 80; and, the pH of the liquid formulation is 5.0.
  • the liquid formulation is stable at 40°C for at least 28 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-hydrochloride buffer at a concentration of 20 mM , A concentration of 0.02% w/v polysorbate 80; and the pH of the liquid formulation is 7.0.
  • the liquid formulation is stable at 40°C for at least 28 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-acetate buffer at a concentration of 20 mM , A concentration of 0.02% w/v polysorbate 80; and the pH of the liquid formulation is 5.8.
  • the liquid formulation is stable at 40°C for at least 28 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 140 mM, and an acetate-sodium acetate buffer at a concentration of 20 mM at a concentration of 0.02% w/v polysorbate 80; and the pH of the liquid formulation is 5.8.
  • the liquid formulation is stable at 40°C for at least 28 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 140 mM, and citric acid-sodium citrate buffer at a concentration of 20 mM, Polysorbate 80 at a concentration of 0.02% w/v; and the pH of the liquid formulation is 5.8.
  • the liquid formulation is stable at 40°C for at least 28 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 140 mM, and a group in the form of histidine alone at a concentration of 20 mM Acid buffer, polysorbate 80 with a concentration of 0.02% w/v; and the pH of the liquid preparation is 5.8.
  • the liquid formulation is stable at 40°C for at least 28 days, 25°C for at least 6 months, and 5°C ⁇ 3°C for at least 36 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-histidine hydrochloride buffer at a concentration of 20 mM Liquid, polysorbate 80 with a concentration of 0.02% w/v; and the pH of the liquid preparation is 5.8.
  • the liquid formulation is stable at 40°C for at least 28 days, 25°C for at least 6 months, and 5°C ⁇ 3°C for at least 36 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 60 mg/ml, sodium chloride at a concentration of 140 mM, and a group in the form of histidine alone at a concentration of 10 mM Acid buffer, polysorbate 80 with a concentration of 0.02% w/v; and the pH of the liquid preparation is 5.7.
  • the liquid formulation is stable at 40°C for at least 14 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 60 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-histidine hydrochloride buffer at a concentration of 10 mM Liquid, polysorbate 80 with a concentration of 0.02% w/v; and the pH of the liquid preparation is 5.7.
  • the liquid formulation is stable at 40°C for at least 14 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 60 mg/ml, sodium chloride at a concentration of 140 mM, and a group in the form of histidine alone at a concentration of 20 mM Acid buffer, polysorbate 80 with a concentration of 0.02% w/v; and the pH of the liquid preparation is 5.8.
  • the liquid formulation is stable at 5°C for at least 6 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 60 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-histidine hydrochloride buffer at a concentration of 20 mM Liquid, polysorbate 80 with a concentration of 0.02% w/v; and the pH of the liquid preparation is 5.8.
  • the liquid formulation is stable at 5°C for at least 6 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 90 mg/ml, sodium chloride at a concentration of 140 mM, and a group in the form of histidine alone at a concentration of 20 mM Lysine buffer with a concentration of 0.02% w/v polysorbate 80; and the pH of the liquid formulation is 5.8.
  • the liquid formulation is stable at 40°C for at least 13 days and 25°C ⁇ 2°C for at least 2 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 90 mg/ml, sodium chloride at a concentration of 140 mM, and a group in the form of histidine alone at a concentration of 20 mM Lysine buffer with a concentration of 0.02% w/v polysorbate 80; and the pH of the liquid formulation is 5.8.
  • the liquid formulation is stable at 40°C for at least 13 days and 25°C ⁇ 2°C for at least 2 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 120 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-hydrochloride buffer at a concentration of 20 mM , The concentration is 0.02% w/v polysorbate 80; and, the pH of the liquid formulation is 5.8.
  • the liquid formulation is stable at 40°C for at least 13 days and 25°C ⁇ 2°C for at least 2 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 120 mg/ml, sodium chloride at a concentration of 140 mM, and histidine-hydrochloride buffer at a concentration of 20 mM , The concentration is 0.02% w/v polysorbate 80; and, the pH of the liquid formulation is 5.8.
  • the liquid formulation is stable at 40°C for at least 13 days and 25°C ⁇ 2°C for at least 2 months.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 100 mM, and citric acid-sodium citrate buffer at a concentration of 20 mM,
  • the pH of the liquid preparation is 5.6.
  • the Tm value of the liquid formulation sample is greater than 65°C and is stable at 40°C for at least 3 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 100 mM, and citric acid-sodium citrate buffer at a concentration of 20 mM, Also, the pH of the liquid preparation is 6.0. In such embodiments, the Tm value of the liquid formulation sample is greater than 65°C and is stable at 40°C for at least 3 days.
  • the liquid formulation comprises: the anti-PD-L1 antibody at a concentration of 20 mg/ml, sodium chloride at a concentration of 100 mM, and citric acid-sodium citrate buffer at a concentration of 20 mM, Also, the pH of the liquid preparation is 6.8. In such embodiments, the Tm value of the liquid formulation sample is greater than 65°C and is stable at 40°C for at least 3 days.
  • the specification of the liquid preparation is 150 mg (10 ml)/bottle to 2000 mg (10 ml)/bottle.
  • the specifications of the liquid preparation are selected from the group consisting of 200 mg (10 ml)/bottle, 600 mg (10 ml)/bottle, 900 mg (10 ml)/bottle, 1200 mg (10 ml)/bottle, or 1500 mg (10 ml) /bottle.
  • the anti-PD-L1 antibody liquid preparation of the present invention can be prepared by combining various components at a predetermined concentration using a method known in the art.
  • the anti-PD-L1 antibody can be dialyzed into a solution containing other components in the liquid preparation and adjusted to the desired concentration.
  • the preparation was filtered and sterilized using a 0.22 ⁇ m pore size filter.
  • the prepared preparation is packaged for convenient use.
  • the packaging material may be a glass bottle (eg, vial), a metal alloy container, a pre-filled syringe, or a pen-type syringe.
  • the anti-PD-L1 antibody liquid preparation of the present invention may be contained in any container suitable for storing drugs and other pharmaceutical compositions.
  • the liquid preparation of the present invention may be contained in a sealed and sterilized plastic container, metal alloy container, or glass container having a volume, such as an ampoule, vial, or syringe. Therefore, in certain preferred embodiments, the liquid formulations of the present invention are contained in glass vials (eg, ampoules or vials), syringes, or micro-infusion sets. In certain exemplary embodiments, the liquid formulations of the present invention are contained in glass vials (eg, ampoules or vials).
  • the invention also provides an article comprising a container containing the liquid formulation of the invention.
  • suitable containers include, for example, plastic containers, metal alloy containers, or glass containers, such as ampoules, vials, or syringes.
  • the article of the invention comprises a container containing the liquid formulation of the invention.
  • a suitable container is a glass bottle, a metal alloy container or a pre-flush syringe.
  • the article may further include a package insert printed with instructions for use.
  • the anti-PD-L1 antibody liquid preparation of the present invention can be administered to a subject by a parenteral route, such as injection (for example, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or percutaneous, mucosal, nasal, respiratory, and/ Or oral administration.
  • a parenteral route such as injection (for example, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or percutaneous, mucosal, nasal, respiratory, and/ Or oral administration.
  • the liquid formulation is administered to a subject (eg, human) by intravenous injection or subcutaneous injection.
  • the anti-PD-L1 antibody liquid preparation antibody of the present invention can be used to prevent and/or treat tumors or anemia by inhibiting and/or blocking intracellular signaling mediated by PD-1 binding to PD-L1.
  • the invention also relates to the use of the liquid formulation of the invention in the preparation of a medicament for:
  • the liquid formulations of the present invention are administered separately, in combination, simultaneously, or sequentially with additional pharmaceutically active agents for the prevention and/or treatment and/or adjuvant treatment of tumors or anemia.
  • the additional pharmaceutically active agent is, for example, a chemotherapeutic drug.
  • the tumor is selected from breast cancer, lung cancer such as non-small cell lung cancer and lung squamous cell carcinoma, liver cancer, gastric cancer, intestinal cancer such as colon cancer or rectal cancer, esophageal cancer, ovarian cancer, cervical cancer , Kidney cancer, prostate cancer, bladder cancer, pancreatic cancer, Merkel cell carcinoma, cholangiocarcinoma, nasopharyngeal carcinoma, and head and neck squamous cell carcinoma, glioma, melanoma leukemia, and lymphoma.
  • lung cancer such as non-small cell lung cancer and lung squamous cell carcinoma
  • liver cancer gastric cancer
  • intestinal cancer such as colon cancer or rectal cancer
  • esophageal cancer ovarian cancer
  • cervical cancer Kidney cancer
  • prostate cancer bladder cancer
  • pancreatic cancer Merkel cell carcinoma
  • cholangiocarcinoma cholangiocarcinoma
  • nasopharyngeal carcinoma and head and neck squamous cell carcinoma
  • the lymphoma is Hodgkin's lymphoma or non-Hodgkin's lymphoma.
  • the non-Hodgkin lymphoma is one of peripheral T cell lymphoma, hemangioimmunoblast T cell lymphoma, NK/T cell lymphoma, and B cell non-Hodgkin lymphoma. One or more.
  • the term "antibody” shall include, but is not limited to, immunity that specifically binds to an antigen and includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds globulin.
  • Each heavy chain contains a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region contains three constant domains CH1, CH2 and CH3.
  • Each light chain contains a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the constant region of the light chain contains a constant domain CL.
  • VH and VL regions can also be subdivided into regions with high denaturation (called complementarity determining regions (CDR)), with more conserved regions (called framework regions (FR)) interspersed therebetween.
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of 3 CDRs and 4 FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 arranged from amino terminus to carboxy terminus.
  • the variable regions (VH and VL) of each heavy/light chain pair respectively form an antigen binding site.
  • the heavy chain can be classified as ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ , and the antibody isotypes are defined as IgM, IgD, IgG, IgA and IgE, "light chain” is divided into two strict types: kappa ( ⁇ ) and lambda ( ⁇ ).
  • antibody includes, for example, naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies. Non-human antibodies can be humanized by recombinant methods to reduce their immunogenicity in humans. Unless specifically stated otherwise, "antibody” as used herein should be understood to include intact antibody molecules, both immunoglobulin IgG.
  • anti-PD-L1 antibody refers to an antibody capable of specifically binding to PD-L1.
  • specifically binds means that the antibody and antigen form a complex that is relatively stable under physiological conditions. Specific binding can be characterized by an equilibrium dissociation constant (K D ) of about 1 ⁇ 10 ⁇ 9 M or less. Methods for determining whether two molecules specifically bind are well known in the art, such as bioluminescence interferometry or surface plasmon resonance technology (eg Biacore).
  • Biacore surface plasmon resonance technology
  • the anti-PD-L1 antibodies described herein comprise the heavy chain CDRs shown in SEQ ID NOs: 1-3, respectively, and the light chain shown in SEQ ID NOs: 4-6, respectively CDR.
  • the anti-PD-L1 antibodies described herein have a heavy chain variable region shown in SEQ ID NO: 7 and a light chain variable region shown in SEQ ID NO: 8.
  • the anti-PD-L1 antibodies described herein have a heavy chain as shown in SEQ ID NO: 9 and a light chain as shown in SEQ ID NO: 10. Lys at the end of the heavy chain is easily deleted, but this deletion does not affect biological activity, see Dick, LW et al., Biotechnol. Bioeng., 100:1132-1143.
  • the term "programmed death-1 (PD-1)” refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on previously activated T cells in vivo and binds two ligands PD-L1 and PD-L2. The complete hPD-1 sequence can be found under GenBank accession number U64863.
  • the term "programmed death ligand-1 (PD-L1)” is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which binds to PD-1 Down-regulate T cell activation and cytokine secretion.
  • PD-L1 as used herein includes human PD-L1 (hPD-L1), variants, isoforms and species homologues of PD-L1, as well as having at least one common table with hPD-L1 Analogs.
  • the complete PD-L1 sequence can be found under GenBank accession number Q9NZQ7.
  • liquid formulation means a combination of at least one active ingredient (eg, an antibody, which is capable of exerting biological activity in a subject) and at least one inactive ingredient, the inactive ingredient should be combined with When the active ingredients are combined, they are suitable for administration to a subject.
  • active ingredients eg, an antibody, which is capable of exerting biological activity in a subject
  • inactive ingredient should be combined with When the active ingredients are combined, they are suitable for administration to a subject.
  • liquid formulations do not contain ingredients that are unacceptably toxic to subjects who will administer the formulation.
  • the subject may include a mammal, preferably a human.
  • the expression "stability" of an antibody means that an antibody substantially retains its physical stability and/or chemical stability and/or biological activity after storage.
  • the storage period is generally selected based on the predetermined shelf life of the preparation.
  • Analytical techniques for measuring antibody stability are well known in the art, and stability can be determined at a selected temperature and for a selected period of time (for example, storage at 40°C for at least 28 days and antibody stability during detection, or Store at 5°C ⁇ 3°C for at least 12 months and test the antibody stability during the test).
  • Chemical changes may involve size changes (eg, shearing), which can be evaluated, for example, by using size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser analytical ionization/time-of-flight mass spectrometry (MALDI/TOF MS) .
  • size changes eg, shearing
  • SDS-PAGE size exclusion chromatography
  • MALDI/TOF MS matrix-assisted laser analytical ionization/time-of-flight mass spectrometry
  • Other types of chemical changes include changes in charge (for example, changes due to deamidation), which can be evaluated by ion exchange chromatography, for example.
  • an antibody in a preparation has the biological activity for its intended use, the antibody retains its biological activity in the preparation.
  • the biological activity of the antibody in the preparation is within about 70% to 130% (for example, 80% to 130%) of the biological activity exhibited when preparing the preparation, it is considered to maintain its Biological activity (eg, determined by antigen binding assay).
  • buffer refers to a buffer solution that resists changes in pH by the action of its acid-base conjugate component.
  • buffers include acetate, succinate, gluconate, histidine, citrate, glycylglycine, and other organic acid buffers.
  • buffers suitable for use in the liquid formulations disclosed herein are selected from citric acid-citrate buffers, histidine buffers, and acetate-acetate buffers.
  • histidine buffer refers to a buffer containing histidine, and the histidine in the buffer may exist alone or in the following form: for example, histidine hydrochloride, histidine ethyl Acid salt, histidine phosphate or histidine sulfate.
  • a histidine buffer eg histidine hydrochloride buffer
  • L-histidine free base, solid
  • a pH adjusting agent may be added according to actual conditions in the subsequent preparation steps to obtain a liquid formulation with a suitable pH range.
  • the histidine in the histidine buffer suitable for use in the liquid formulations disclosed herein is present alone or in the form of histidine hydrochloride or histidine acetate.
  • citric acid-citrate buffer is a mixture that includes citric acid and citrate.
  • the citric acid-citrate buffer is a citric acid-sodium citrate buffer, that is, a mixture comprising citric acid and sodium citrate.
  • a citric acid-citrate buffer (for example, citric acid-sodium citrate buffer) can be prepared by adding a base (for example, sodium hydroxide) required for salt formation to citric acid.
  • acetic acid-acetate buffer includes a mixture of acetic acid and acetate.
  • the acetate-acetate buffer is an acetate-sodium acetate buffer, that is, a mixture comprising acetic acid and sodium acetate.
  • Acetic acid-acetate buffer (for example, acetic acid-sodium acetate buffer) can be prepared by adding alkali required for salt formation (for example, sodium hydroxide) to acetic acid.
  • sugars include general composition (CH 2 O)n and its derivatives, including monosaccharides, disaccharides, trisaccharides, polysaccharides, reducing sugars, non-reducing sugars, etc.
  • Non-limiting examples herein include glucose, sucrose, seaweed Sugar, lactose, fructose, maltose, dextran, etc.
  • Sugar alcohols can be obtained from corresponding sugars, that is, reducing aldehyde groups or ketone groups on sugar molecules to hydroxyl groups to obtain sugar alcohols; sugar alcohols have the general formula H(CHOH)nH, and non-limiting examples herein include , Erythritol, threitol, arabitol, xylitol, sorbitol, mannitol, maltitol, lactitol, etc.
  • the liquid preparation of the anti-PD-L1 antibody of the present invention does not contain sugar and/or sugar alcohol, so the preparation may also be referred to herein as a sugar-free antibody preparation.
  • the anti-PD-L1 antibody liquid preparation of the present invention does not contain sugars and/or sugar alcohols selected from the group consisting of sucrose, trehalose, glucose, mannitol and sorbitol. It is known in the art that generally, sugar and/or sugar alcohols (eg, sucrose, trehalose, glucose, mannitol, and sorbitol, etc.) need to be added to antibody preparations as antibody stabilizers.
  • sugar and/or sugar alcohols can also be used As an effective osmotic pressure regulator, it can maintain the stability of the preparation (eg, isotonic, that is, has substantially the same osmotic pressure as human blood).
  • osmolality refers to a measure of the osmolality of dissolved solute particles in an aqueous solution.
  • the solute particles include two kinds of ions and non-ionized molecules.
  • Osmolality is expressed as the concentration of osmotically active particles dissolved in 1 kg of solvent (ie water) (ie osmolarity).
  • solvent ie water
  • osmolality is expressed in terms of milli-osmolarity/kg water (mOsmol/kg). It is known to those skilled in the art that a desired level of osmolality can be achieved by adding one or more osmolality adjusting agents, such as sugars and/or sugar alcohols.
  • the term "isotonic" means that the preparation of interest has substantially the same osmotic pressure as human blood.
  • the isotonic formulation will generally have an osmotic pressure of about 260-330 mOsmol/kg.
  • the isotonicity can be measured using, for example, vapor pressure or frozen osmometer.
  • the term "mass to volume ratio” or "w/v” refers to the percentage weight (in grams) of a single component relative to the total volume (in milliliters) of the mixture containing the component. For example, a 20 mg component in a total volume of 100 ml is 0.02% w/v.
  • the term "about” refers to a value or composition within an acceptable error range for a particular value or composition determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, That is the limitation of the measurement system.
  • a measurable value eg, concentration of substance, time, temperature, etc.
  • it means to include a range of ⁇ 5% of the given value, for example, the expression "about 10 mM” means 9.5-10.5mM.
  • the art In the process of preparing antibody preparations, the art generally needs to add sugar and/or sugar alcohol to maintain the stability of the antibody, however, the use of sugar and/or sugar alcohol has certain limitations for diabetics.
  • the inventor of the present application has unexpectedly found that the sugar-free antibody preparation of the present invention can not only maintain the osmotic pressure of the antibody preparation to obtain excellent physiological compatibility, but also maintain the physical stability and chemistry of the preparation Stability and biological activity, tolerate severe conditions during storage and transportation, which is particularly conducive to long-term storage of drugs, and can expand the scope of clinical medication, has great clinical value.
  • the above CDR can be obtained by analyzing the amino acid sequence of the heavy chain variable region or the light chain variable region of the following heavy chain variable region according to the IMGT definition through the VBASE2 database according to the technical means well known to those skilled in the art.
  • Flowcam GWF-8JD uses Flowcam GWF-8JD to determine the number of particles in the sample, use a pipette to draw about 1ml of the sample, place the tip of the sample on the sample port, and determine the number of insoluble particles ⁇ 10 ⁇ m and ⁇ 25 ⁇ m in the sample.
  • the column temperature was kept at room temperature, and the TSKgel G3000SWXL column was used to inject the processed sample.
  • the mobile phase was eluted for about 20 minutes, and the size and size distribution of the sample was determined by molecular size exclusion chromatography.
  • the sample curve was obtained based on the UV absorption at 280 nm, and the relative percentage peak area of the total area was reported as the result.
  • the processed sample was injected and eluted with a mobile phase, and the distribution of different charged ion components in the sample was determined by ion exchange chromatography.
  • the sample curve was obtained based on the UV absorption at 280 nm, and the relative percentage peak area of the total area was reported as the result.
  • CE-SDS sodium dodecyl sulfate capillary electrophoresis
  • ELISA enzyme-linked immunoassay
  • Prescription 1 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 100 mM sodium chloride, 20 mM citric acid-sodium citrate, pH 5.2.
  • Prescription 2 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 100 mM sodium chloride, 20 mM citric acid-sodium citrate, pH 5.6.
  • Prescription 3 Anti-PD-L1 humanized monoclonal antibody 20mg/ml, 100mM sodium chloride, 20mM citric acid-sodium citrate, pH 6.0.
  • Prescription 4 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 100 mM sodium chloride, 20 mM citric acid-sodium citrate, pH 6.4.
  • Prescription 5 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 100 mM sodium chloride, 20 mM citric acid-sodium citrate, pH 6.8.
  • the preparation method of antibody preparation of prescription 1-5 is as follows:
  • Configure buffer solution add 100 mM sodium chloride and 20 mM citric acid to water for injection, and adjust to the required pH value in the above prescription with sodium hydroxide.
  • the antibody stock solution (Sichuan Kelun Botai Biomedicine Co., Ltd., batch number 1) was replaced by ultrafiltration, dialyzed into the above buffer solution, and the antibody concentration was adjusted to 20 mg/ml.
  • the configured sample is sterilized and filtered through a filter with a pore size of 0.22 ⁇ m, filled into a vial, and capped to obtain a sample.
  • the effect of the pH value of the preparation on the stability of the monoclonal antibody was investigated by the DSF method.
  • the results showed that the Tm values of the samples with different pH values were all greater than 65°C, indicating that the pH value had no significant effect on the thermal stability of the protein in the range of pH5.6-6.8 (table 3).
  • the sample was placed at 40°C for 3 days for accelerated experiments.
  • the SEC-HPLC results showed that the monomer was greater than 99.0%, there was no significant decrease, the oligomer was not detected, the high polymer was less than 0.20%, there was no significant increase, each The sample stability is good (Table 4).
  • Prescription 1 Prescription 2
  • Prescription 3 Prescription 4
  • Prescription 5 DSF Tm(°C) 65.31 66.25 66.25 66.33 66.40
  • Prescription 6 anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 140 mM sodium chloride, 20 mM histidine-histidine hydrochloride, polysorbate 80 with a mass volume ratio of 0.02%, pH 5.8.
  • Configure buffer solution add 140 mM sodium chloride and 20 mM histidine to the water for injection, and adjust to pH 5.8 with hydrochloric acid.
  • the antibody stock solution (Lot No. 1) was replaced by ultrafiltration, dialyzed to the above buffer solution, the antibody concentration was adjusted to 20 mg/ml, and an appropriate amount of polysorbate 80 storage solution was added.
  • the configured sample is sterilized and filtered through a filter with a pore size of 0.22 ⁇ m, filled into a vial, and capped to obtain a sample.
  • Prescription 7 anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 140 mM sodium chloride, 20 mM histidine-histidine hydrochloride, polysorbate 80 with a mass volume ratio of 0.02%, pH 5.0.
  • Prescription 8 anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 140 mM sodium chloride, 20 mM histidine-histidine hydrochloride, polysorbate 80 with a mass volume ratio of 0.02%, pH 7.0.
  • Prescription 9 anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 140 mM sodium chloride, 20 mM histidine-histidine acetate, polysorbate 80 with a mass volume ratio of 0.02%, pH 5.8.
  • Example 2 To the water for injection, 140 mM sodium chloride and 20 mM histidine were added, and the pH was adjusted to 5.8 with acetic acid, and the remaining preparation methods were the same as in Example 2.
  • Prescription 10 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 140 mM sodium chloride, 20 mM acetic acid-sodium acetate, polysorbate 80 with a mass volume ratio of 0.02%, pH 5.8.
  • Example 2 To the water for injection was added 140 mM sodium chloride and 20 mM sodium acetate, and the pH was adjusted to 5.8 with acetic acid. The rest of the preparation method was the same as in Example 2.
  • Prescription 11 anti-PDL-1 humanized monoclonal antibody 20 mg/ml, 140 mM sodium chloride, 20 mM citric acid-sodium citrate, polysorbate 80 with a mass volume ratio of 0.02%, pH 5.8.
  • Example 2 140 mM sodium chloride and 20 mM sodium citrate were added to the water for injection, and the pH was adjusted to 5.8 with citric acid. The rest of the preparation method was the same as in Example 2.
  • Prescription 12 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 140 mM sodium chloride, 20 mM histidine-histidine hydrochloride, polysorbate 20 with a mass volume ratio of 0.02%, pH 5.8.
  • polysorbate 80 was replaced with polysorbate 20, and the remaining preparation methods were the same as in Example 2.
  • the osmotic pressure test results of Examples 2-7 are shown in the table below. The results show that the osmotic pressure of the samples of Examples 2-7 can be stabilized in the range of 280-320 mOsmol/kg, and the stability is good.
  • the insoluble particles of the samples of Examples 2-7 were detected by Flowcam, and the results are shown in the table below.
  • the results showed that the number of particles ⁇ 10 ⁇ m in all directions was less than 1000, and the number of particles ⁇ 25 ⁇ m was less than 200, indicating that prescriptions 6-11 still had no obvious aggregation after being placed at 40°C for 28 days, meeting the medicinal requirements.
  • the monomer content in the samples of Examples 2-8 was detected by molecular size exclusion chromatography. The results are shown in the table below. The results showed that the monomer content of the samples of prescriptions 6-12 were all greater than 95%, and they remained stable at 40°C for 28 days.
  • the main peak content of the samples of Examples 2-8 was detected by ion exchange chromatography. The results are shown in the table below. The results showed that the content of the main peak of prescriptions 6-12 remained stable and satisfies the quality requirements after being left at 40°C for 28 days.
  • the main peak purity of the samples of Examples 2-8 was detected by capillary electrophoresis. The results are shown in the table below. The results show that after being left at 40°C for 28 days, the main peak of the sample 6-12 has high purity, the content remains stable, and meets the quality requirements.
  • Example 3 97.0 95.8 89.2
  • Example 4 97.0 95.3 94.0
  • Example 5 96.8 95.9 93.4
  • Example 6 97.0 95.7 93.1
  • Example 7 97.0 95.1 91.3
  • Example 8 97.2 95.0 93.4
  • Prescription 6 anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 140 mM sodium chloride, 20 mM histidine-histidine hydrochloride, polysorbate 80 with a mass volume ratio of 0.02%, pH 5.8.
  • the antibody preparation for Prescription 6 is scaled up to 10L, and the preparation method is as follows:
  • Configure ultrafiltration buffer solution add sodium chloride and histidine to the water for injection, and adjust the pH value to 5.8 with hydrochloric acid, and configure into a solution containing 140 mM sodium chloride and 20 mM histidine-histidine hydrochloride.
  • the antibody stock solution (Lot No. 2) uses Millipore ultrafiltration membrane packs, selects the appropriate quantity of membrane packs according to the load, and uses the Thermo Pellicon ultrafiltration system to perform ultrafiltration fluid exchange.
  • the protein was dialyzed into the above buffer solution, the antibody concentration was adjusted to 20 mg/ml, and an appropriate amount of polysorbate 80 was added.
  • the sterilized filtration of the configured sample is carried out under laminar flow conditions.
  • the filter adopts Millipak series 0.22 ⁇ m filter (Millipore Corporation) to filter. If necessary, a pre-filtration membrane Opticap filter can be connected in series before the sterilization filter to increase the filter capacity.
  • the sterilization filter is connected to a peristaltic pump with a silicone tube, and then sterilized and filtered through a 0.22 ⁇ m pore size filter.
  • the filtered sample solution was collected and filled into vials, respectively, and capped and capped to obtain samples.
  • the prescription 6 was subjected to a freeze-thaw stability study at -20°C for 48 hours.
  • the investigation items were OD405, SEC-HPLC and IEC-HPLC.
  • the results are shown in Table 11. The results show that the prescription anti-PD-L1 humanized monoclonal antibody liquid preparation has good stability in freezing and thawing for 4 cycles, and can withstand the severe conditions of the preparation during storage and transportation.
  • Table 12 Test data of prescription 6's long-term stability study at 5 ⁇ 3°C
  • Prescription 6 has been placed at 5°C for 36 months for a long period of time.
  • the appearance is clear and transparent.
  • the protein content, pH, osmolality, CE-SDS reduction and non-reduction, SEC-HPLC, IEC-HPLC and relative binding activity have not changed significantly. Good stability.
  • Prescription 6 is placed under accelerated conditions of 25 ⁇ 2°C for 6 months. The appearance is clear and transparent. There is no significant change in protein content, pH, osmotic pressure and relative binding activity. CE-SDS reduction and non-reduction, SEC-HPLC and IEC- HPLC purity decreased slightly, protein stability was good, and it met the quality requirements.
  • Prescription 16 Anti-PD-L1 humanized monoclonal antibody 60 mg/ml, 10 mM histidine-histidine hydrochloride, 140 mM sodium chloride, polysorbate 80 with a mass volume ratio of 0.02%, pH 5.7.
  • the antibody concentration in the buffer was 60 mg/ml, and the rest of the preparation method was the same as in Example 2.
  • the results of the stability test at 40°C are shown in Table 14.
  • the appearance of the sample of prescription 16 is a microstrip opalescent clear transparent liquid; and, at 40°C, the OD405 is stable over time, indicating that the prescription has stability.
  • Prescription 17 anti-PD-L1 humanized monoclonal antibody 60 mg/ml, 20 mM histidine-histidine hydrochloride, 140 mM sodium chloride, 0.02% w/v polysorbate 80, pH 5.8, the preparation method is the same Example 2.
  • Prescription 18 anti-PD-L1 humanized monoclonal antibody 90 mg/ml, 20 mM histidine-histidine hydrochloride, 140 mM sodium chloride, 0.02% w/v polysorbate 80, pH 5.8, the preparation method is the same Example 2.
  • Prescription 19 Anti-PD-L1 humanized monoclonal antibody 120 mg/ml, 20 mM histidine-histidine hydrochloride, 140 mM sodium chloride, 0.02% w/v polysorbate 80, pH 5.8, the preparation method is the same Example 2.
  • NT means not detected.
  • the preparation of the present application overcomes the problems of stability, viscosity, osmotic pressure and turbidity, and is beneficial to long-term storage of drugs and clinical medication.
  • Table 16 Stability test results of prescription 18 and prescription 19 under shaking, 40°C, 25°C and 5°C.
  • NT means not detected.
  • Prescription 13 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 140 mM sucrose, 20 mM histidine-histidine hydrochloride, polysorbate 80 with a mass volume ratio of 0.02%, pH 5.8.
  • the 140 mM sodium chloride in the buffer was replaced with 140 mM sucrose, and the remaining preparation methods were the same as in Example 2.
  • Prescription 14 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 105 mM sucrose, 35 mM sodium chloride, 20 mM histidine-histidine hydrochloride, polysorbate 80 with a mass volume ratio of 0.02%, pH5.8.
  • the 140 mM sodium chloride in the buffer was replaced with 105 mM sucrose and 35 mM sodium chloride, and the remaining preparation methods were the same as in Example 2.
  • Prescription 15 Anti-PD-L1 humanized monoclonal antibody 20 mg/ml, 70 mM sucrose, 70 mM sodium chloride, 20 mM histidine-histidine hydrochloride, polysorbate 80 with a mass volume ratio of 0.02%, pH5.8.
  • the 140 mM sodium chloride in the buffer was replaced with 70 mM sodium chloride and 70 mM sucrose, and the remaining preparation methods were the same as in Example 2.
  • the osmotic pressures of prescriptions 13-15 are 199mOsmol/kg, 232mOsmol/kg and 258mOsmol/kg, which are lower than the normal human plasma osmotic pressure.
  • the antibody preparation is a hypotonic solution, and intravenous administration may cause hemolysis , Does not meet the needs of clinical medicine.
  • the results show that the antibody preparations of prescriptions 1-5 of the present invention all have good stability. This also shows that when the pH value is in the range of 5.6-6.8, the PD-L1 antibody of the present invention has a better stability.
  • the antibody preparations of prescriptions 6-12 were subjected to osmotic pressure monitoring, insoluble particle monitoring, SEC-HPLC testing, IEC-HPLC testing, and CE-SDS testing for 28 days at 40°C, respectively.
  • the antibody preparations of prescriptions 6-12 of the invention have good stability, no obvious aggregation, and all meet the medicinal requirements. And, the results show that the stability of the antibody of the present invention is the best when the pH is 5.8, and when the buffer pair in the formulation is histidine-histidine hydrochloride, its stability effect is relatively superior to other buffer pairs.
  • the present inventors conducted further detection and analysis of the antibody preparation of the prescription 6 having a relatively better effect. Specifically, its long-term stability at 5°C ⁇ 3°C for 12 months and its stability at 5°C for 36 months were analyzed. The protein concentration, SEC-HPLC, CE-SDS, IEC-HPLC, Binding activity, etc.; and, the stability at -20°C, 48 hours of freeze-thaw was analyzed, and OD405, SEC-HPLC, and IEC-HPLC were tested respectively.
  • the results show that the antibody preparation of prescription 6 can be stably stored at 5 °C ⁇ 3 °C for at least 12 months, and the same product quality as the small-scale preparation can still be achieved after scale-up production; and, after repeated freeze-thaw cycles of 4 cycles , Good stability, can withstand the severe conditions of the formulation during storage and transportation; in addition, long-term storage at 5 °C for 36 months, the appearance is clear and transparent, protein content, pH, osmolarity, CE-SDS reduction and non-reduction, SEC-HPLC, IEC-HPLC and relative binding activity did not change significantly, and the stability was good.
  • the present inventors tried the antibody preparation under high antibody concentration-prescription 16-19, and tried the stability of the antibody preparation when the PD-L1 antibody concentration was 60 mg/ml, 90 mg/ml, and 120 mg/ml, respectively. Sex.
  • the results show that under the condition of 40°C, the appearance of the antibody preparation of prescription 16 is a microstrip opalescent clear transparent liquid, and OD405 is stable; under different temperature conditions, the viscosity of the antibody preparation of prescription 17-19 over time No obvious changes occurred; the antibody preparations of prescriptions 18 and 19 were shaken continuously at 1000 rpm for 2 h.
  • the stability was good. They could be placed at 5°C and 25°C for 2 months and at 40°C for 13 days. Therefore, in the present invention, the antibody preparation at a high concentration of antibody concentration can overcome the problems of stability, viscosity, osmotic pressure, and turbidity, and is conducive to long-term storage of drugs and clinical use of drugs.
  • the inventors screened the concentration of NaCl in the antibody preparation-prescription 13-15 (comparative example). The results showed that when the NaCl concentration was 0 mM, 35 mM, and 70 mM, the osmotic pressure was 199 mOsmol/kg, 232 mOsmol/kg, and 258 mOsmol/kg, respectively, which were lower than the normal human plasma osmotic pressure. There is a risk of hemolysis caused by intravenous administration. Therefore, it does not meet the needs of clinical medicine.

Abstract

提供一种抗PD-L1抗体制剂，包含抗PD-L1抗体、缓冲液、氯化钠，并且不存在添加的糖和/或糖醇。还提供抗PD-L1抗体制剂在制备用于预防和/或治疗和/或辅助治疗和/或诊断肿瘤或者贫血病的药物中的用途。

Description

一种抗PD-L1抗体制剂 技术领域
本发明涉及治疗性抗体领域，更具体地，本发明涉及一种抗PD-L1抗体制剂及其医药用途。
背景技术
PD-1(Programmed Death 1)全称程序性死亡受体1，是一种Ⅰ型跨膜糖蛋白，属于CD28家族成员。PD-L1(Programmed Cell Death-Ligand 1)全称程序性死亡受体-配体1，是大小为40KDa的Ⅰ型跨膜糖蛋白。
正常生理条件下，抗原进入机体后，经抗原提呈细胞(APC)加工并与MHC连接。T细胞表面TCR分子经特异性识别APC所提呈的抗原肽，并同时识别与抗原肽形成复合物的MHC分子，此外T细胞表面众多免疫调节分子参与反应，产生共刺激信号，促使T细胞活化并发挥免疫效应。PD-1在活化的T细胞、B细胞、NK细胞、单核细胞和DC表面均有表达，与处于APC细胞表面的PD-L1、PD-L2等相互作用，可传导抑制性信号，抑制T细胞的活化，维持机体免疫稳态。肿瘤细胞表面往往存在高表达PD-L1，与肿瘤浸润T淋巴细胞高表达PD-1结合，导致PD-1通路持续激活，从而抑制T细胞活化，实现肿瘤的免疫逃逸。所以阻断PD-1/PD-L1结合成为肿瘤免疫治疗领域重要策略之一。PD-L1抗体能阻断肿瘤PD-L1与T细胞PD-1的结合，消除这一免疫抑制效应，使得T细胞被重新激活，识别并杀伤肿瘤细胞。
WO2017/148424公开了新的抗PD-L1单克隆抗体，所述抗体能够十分有效地阻断PD-1与PD-L1结合，对肿瘤或者贫血病显示出更好的疗效。但PD-L1抗体用于临床治疗需要解决适合给药的理化性质及储存阶段的稳定性的问题。抗体制剂是抗体在储存运输和临床治疗中的最终形式，需要兼顾其理化性质、生理性质及有效期内的稳定性，如蛋白含量、pH、渗透压、不溶性微粒、纯度、聚集体、结合活性等。由于不同的抗体在理化性质方面各有特点，稳定性也相差很大，制备制剂难以得到稳定通用的液体制剂处方。因此对于每个创新研发的抗体，需进行深入研究，寻找适合的制剂处方，以得到生理相容性好的抗体制剂，方便药品储存以及临床用药。
因此，针对抗PD-L1抗体，开发合适的制剂处方以得到生理相容性和稳定性满足需求的抗体制剂，是十分必要的。
发明内容
本申请的发明人经过大量实验和反复摸索，出人意料地发现，抗PD-L1抗体与特定处方组成的液体制剂，具有优良的生理相容性和稳定性，进一步开发 了高浓度的抗体制剂，高浓度蛋白制剂克服了稳定性差、粘性大和易混浊的难题，因此本申请公开了特别有利于药品长期储存和临床用药的液体制剂，由此完成了本发明。
液体制剂
因此，在一方面，本发明提供了一种液体制剂，其包含：
(1)浓度为约10mg/ml至约150mg/ml的抗PD-L1抗体；其中，所述抗PD-L1抗体包含分别如SEQ ID NOs:1-3所示的重链CDRs，以及分别如SEQ ID NOs:4-6所示的轻链CDRs；
(2)浓度为约5mM至约40mM的缓冲液；
(3)浓度为约80mM至约160mM的氯化钠；
其中，所述液体制剂具有约5.0至约7.0的pH；并且，所述液体制剂不存在添加的糖和/或糖醇；
并且，液体制剂具有与人血液相当的渗透压。
本文公开的抗PD-L1抗体液体制剂的特征在于不存在添加的糖和/或糖醇，因此在本文中也可被称为无糖的抗体制剂。
在某些优选的实施方案中，所述液体制剂不含有选自下列的糖和/或糖醇：蔗糖、葡萄糖、海藻糖、甘露醇和山梨醇。
在某些优选的实施方案中，所述抗PD-L1抗体具有如SEQ ID NO:7所示的重链可变区以及如SEQ ID NO:8所示的轻链可变区。在某些优选的实施方案中，所述抗PD-L1抗体具有如SEQ ID NO:9所示的重链以及如SEQ ID NO:10所示的轻链。
在另一优选例中，所述抗PD-L1抗体的浓度为10mg/ml至30mg/ml。
在另一优选例中，所述抗PD-L1抗体的浓度为10mg/ml至120mg/ml，较佳地为30mg/ml至70mg/ml。
在某些优选的实施方案中，所述抗PD-L1抗体的浓度为约15mg/ml至约25mg/ml。在某些优选的实施方案中，所述抗PD-L1抗体的浓度为约20mg/ml。
在某些优选的实施方案中，所述抗PD-L1抗体的浓度为约50mg/ml至约70mg/ml。在某些优选的实施方案中，所述抗PD-L1抗体的浓度为约60mg/ml。
在某些优选的实施方案中，所述抗PD-L1抗体的浓度为约80mg/ml至约150mg/ml。在某些优选的实施方案中，所述抗PD-L1抗体的浓度为约90mg/ml，约100mg/ml，约110mg/ml或约120mg/ml。
在某些优选的实施方案中，所述氯化钠的浓度为约100mM至约160mM。在某些优选的实施方案中，所述氯化钠的浓度为约105mM、约110mM、约115mM、约120mM、约130mM、约135mM、约140mM、约145mM、约150mM或约155mM。在某些更优选的实施方案中，所述氯化钠的浓度为约140mM。
在某些优选的实施方案中，所述缓冲液选自：柠檬酸-柠檬酸盐缓冲液、组氨酸缓冲液或乙酸-乙酸盐缓冲液。在某些优选的实施方案中，所述组氨酸缓冲液中的组氨酸单独存在或以组氨酸盐酸盐或组氨酸乙酸盐形式存在。
在某些优选的实施方案中，所述缓冲液是组氨酸缓冲液。在某些优选的实施方案中，所述组氨酸缓冲液中的组氨酸单独存在或以组氨酸-盐酸盐或组氨酸-乙酸盐形式存在。
在某些优选的实施方案中，所述缓冲液的浓度是约10mM至约20mM。在某些优选的实施方案中，所述缓冲液的浓度是约20mM。在某些优选的实施方案中，所述液体制剂包含浓度为约20mM的组氨酸缓冲液，优选地所述组氨酸缓冲液中的组氨酸单独存在或以组氨酸盐酸盐或组氨酸乙酸盐形式存在。
在另一优选例中，所述缓冲液是20mM的组氨酸-盐酸组氨酸。
在本发明中，通过缓冲液调节所述液体制剂的pH，所述缓冲液还可以进一步使用pH调节剂(例如盐酸、醋酸、磷酸等)调节pH。在某些优选的实施方案中，所述液体制剂具有约5.2至约6.8的pH。在某些优选的实施方案中，所述液体制剂具有约5.6至约6.8的pH。在某些优选的实施方案中，所述液体制剂具有约5.6至约6.0的pH。在某些示例性实施方案中，所述液体制剂的pH为约5.0、约5.2、约5.6、约5.8、约6.0、约6.4、约6.8或约7.0。在某些示例性实施方案中，所述液体制剂的pH为5.8±0.1。在某些示例性实施方案中，所述液体制剂的pH为约5.8。
在某些优选的实施方案中，其中，所述液体制剂具有约260mOsmol/kg至约380mOsmol/kg的重量摩尔渗透压浓度。在某些优选的实施方案中，所述液体制剂具有约280mOsm/kg至约380mOsmol/kg(例如280mOsmol/kg至约360mOsmol/kg)的重量摩尔渗透压浓度。在某些优选的实施方案中，所述液体制剂具有约280mOsm/kg至约330mOsmol/kg的重量摩尔渗透压浓度。在某些优选的实施方案中，所述液体制剂具有与人血液基本上相同的渗透压。
在另一优选例中，所述与人血液相当的渗透压为约280mOsmol/kg至约400mOsmol/kg的重量摩尔渗透压浓度，较佳地290mOsmol/kg至约320mOsmol/kg的重量摩尔渗透压浓度，更佳地为295mOsmol/kg至约315mOsmol/kg的重量摩尔渗透压浓度。
在某些优选的实施方案中，所述液体制剂还包含有表面活性剂。在某些优选的实施方案中，所述表面活性剂是聚山梨酯80/聚山梨酯20或泊洛沙姆188。在某些优选的实施方案中，所述表面活性剂是聚山梨酯80。
在某些优选的实施方案中，所述表面活性剂的浓度是约0.01％w/v至约0.02％w/v。在某些优选的实施方案中，所述表面活性剂的浓度是约0.02％w/v。在某些优选的实施方案中，所述液体制剂包含浓度为约0.02％w/v的聚山梨酯80。
在某些优选的实施方案中，所述液体制剂包含：
(1)浓度为约20mg/ml、约60mg/ml、约90mg/ml或约120mg/ml的所述抗PD-L1抗体；
(2)浓度为约20mM的缓冲液；所述缓冲液选自组氨酸缓冲液、柠檬酸-柠檬酸盐缓冲液和乙酸-乙酸盐缓冲液；
(3)浓度为约140mM的氯化钠；
其中，所述液体制剂具有约5.2至约6.8的pH(例如，约5.6至约6.8的pH，例如约5.6至约6.0的pH，例如约5.8的pH)；并且，所述液体制剂不含有糖和/或糖醇。
在某些优选的实施方案中，本发明的液体制剂可以在5℃±3℃下稳定并可以储存至少约2周，至少约1个月，至少约2个月，至少约3个月，至少约6个月，至少约9个月，至少约1年或至少约2年。在某些优选的实施方案中，本发明的液体制剂在约40℃下稳定并可以储存至少约2周，至少约1个月，至少约2个月，至少约3个月，至少约6个月，至少约9个月，至少约1年或至少约2年。在某些优选的实施方案中，本发明的液体制剂在室温(例如约25℃)稳定并可以储存至少约2周，至少约1个月，至少约2个月，至少约3个月，至少约6个月，至少约9个月，至少约1年或至少约2年。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的组氨酸-盐酸盐缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少28天，并且在在5℃±3℃下能稳定至少12个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的组氨酸-盐酸盐缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.0。在此类实施方案中，所述液体制剂在40℃下能稳定至少28天。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的组氨酸-盐酸盐缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为7.0。在此类实施方案中，所述液体制剂在40℃下能稳定至少28天。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的组氨酸-乙酸盐缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少28天。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的乙酸-乙酸钠缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方 案中，所述液体制剂在40℃下能稳定至少28天。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的柠檬酸-柠檬酸钠缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少28天。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的以单独组氨酸形式存在的组氨酸缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少28天，在25℃下能稳定至少6个月，并且在5℃±3℃下能稳定至少36个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的组氨酸-盐酸组氨酸缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少28天，在25℃下能稳定至少6个月，并且在5℃±3℃下能稳定至少36个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为60mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为10mM的以单独组氨酸形式存在的组氨酸缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.7。在此类实施方案中，所述液体制剂在40℃下能稳定至少14天。
在某些示例性实施方案中，所述液体制剂包含：浓度为60mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为10mM的组氨酸-盐酸组氨酸缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.7。在此类实施方案中，所述液体制剂在40℃下能稳定至少14天。
在某些示例性实施方案中，所述液体制剂包含：浓度为60mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的以单独组氨酸形式存在的组氨酸缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.8。所述液体制剂在5℃下能稳定至少6个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为60mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的组氨酸-盐酸组氨酸缓冲液，浓度为0.02％w/v的聚山梨酯80；并且，所述液体制剂的pH为5.8。所述液体制剂在5℃下能稳定至少6个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为90mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的以单独组氨酸形式存在的组氨酸缓冲液，浓度为0.02％w/v聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少13天，在25℃± 2℃下能稳定至少2个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为90mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的以单独组氨酸形式存在的组氨酸缓冲液，浓度为0.02％w/v聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少13天，在25℃±2℃下能稳定至少2个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为120mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的组氨酸-盐酸盐缓冲液，浓度为0.02％w/v聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少13天，在25℃±2℃下能稳定至少2个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为120mg/ml的所述抗PD-L1抗体，浓度为140mM的氯化钠，浓度为20mM的组氨酸-盐酸盐缓冲液，浓度为0.02％w/v聚山梨酯80；并且，所述液体制剂的pH为5.8。在此类实施方案中，所述液体制剂在40℃下能稳定至少13天，在25℃±2℃下能稳定至少2个月。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为100mM的氯化钠，浓度为20mM的柠檬酸-柠檬酸钠缓冲液，并且，所述液体制剂的pH为5.6。在此类实施方案中，所述液体制剂样品的Tm值大于65℃，在40℃下稳定至少3天。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为100mM的氯化钠，浓度为20mM的柠檬酸-柠檬酸钠缓冲液，并且，所述液体制剂的pH为6.0。在此类实施方案中，所述液体制剂样品的Tm值大于65℃，在40℃下稳定至少3天。
在某些示例性实施方案中，所述液体制剂包含：浓度为20mg/ml的所述抗PD-L1抗体，浓度为100mM的氯化钠，浓度为20mM的柠檬酸-柠檬酸钠缓冲液，并且，所述液体制剂的pH为6.8。在此类实施方案中，所述液体制剂样品的Tm值大于65℃，在40℃下稳定至少3天。
在另一优选例中，所述液体制剂的规格为150mg(10ml)/瓶至2000mg(10ml)/瓶。
在另一优选例中，所述液体制剂的规格选自下组：200mg(10ml)/瓶、600mg(10ml)/瓶、900mg(10ml)/瓶、1200mg(10ml)/瓶或1500mg(10ml)/瓶。本发明的抗PD-L1抗体液体制剂可采用本领域公知的方法将各种组分按预定浓度进行组合制备。例如，可以将抗PD-L1抗体透析至包含该液体制剂中其他组分 的溶液中，并调整至所需浓度。采用0.22μm孔径的过滤器将制剂过滤除菌。对所制备的制剂进行包装以方便使用。包材可以是玻璃瓶(例如西林瓶)、金属合金容器、预填充注射器或笔式注射器。
本发明的抗PD-L1抗体液体制剂可包含在任何适于储存药物以及其他药物组合物的容器中。例如，本发明的液体制剂可包含在具有一定体积的密封及灭菌的塑料容器、金属合金容器或玻璃容器中，诸如安瓿、西林瓶或注射器。因此，在某些优选的实施方案中，本发明的液体制剂包含在玻璃小瓶(例如安瓿或西林瓶)、注射器或微量输液器中。在某些示例性实施方案中，本发明的液体制剂包含在玻璃小瓶(例如安瓿或西林瓶)中。
因此，在另一方面，本发明还提供一种制品，其包含装有本发明液体制剂的容器。在某些优选的实施方案中，合适的容器包括例如塑料容器、金属合金容器或玻璃容器，诸如安瓿、西林瓶或注射器。在某些优选的实施方案中，本发明的制品包含装有本发明液体制剂的容器，合适的容器为玻璃瓶，金属合金容器或预冲注射器。在某些优选的实施方案中，所述制品可进一步包括印有使用说明的包装插页。
本发明的抗PD-L1抗体液体制剂可通过肠胃外途径施用于受试者，诸如注射(例如，皮下、静脉内、肌内、腹膜内等)、或经皮、粘膜、鼻、呼吸道和/或口腔施用。在某些优选的实施方案中，所述液体制剂通过静脉内注射或皮下注射施用于受试者(例如人)。
液体制剂的治疗用途
本发明的抗PD-L1抗体液体制剂抗体可通过抑制和/或阻断由PD-1结合PD-L1介导的细胞内信号传导从而用于预防和/或***或贫血病。
因此，在另一方面，本发明还涉及本发明的液体制剂在制备药物中的用途，所述药物用于：
(a)预防和/或***或者贫血病；
(b)辅助***或者贫血病；
(c)诊断肿瘤或者贫血病；
(d)(a)-(c)的任意组合。
在某些实施方案中，本发明的液体制剂与另外的药学活性剂分开、联合、同时、或相继施用，用于预防和/或治疗和/或辅助***或者贫血病。在某些实施方案中，另外的药学活性剂例如为化疗药物。在某些优选的实施方案中，所述肿瘤选自乳腺癌、肺癌例如非小细胞性肺癌和肺鳞癌、肝癌、胃癌、肠癌例如结肠癌或者直肠癌、食管癌、卵巢癌、***、肾癌、***癌、膀胱癌、胰腺癌、Merkel细胞癌、胆管癌、鼻咽癌、和头颈鳞癌、神经胶质瘤、黑素瘤白血病和淋巴瘤。
在某些优选的实施方案中，所述淋巴瘤为霍奇金淋巴瘤或非霍奇金淋巴瘤。在某些优选的实施方案中，所述非霍奇金淋巴瘤为外周T细胞淋巴瘤、血管免疫母细胞T细胞淋巴瘤、NK/T细胞淋巴瘤和B细胞非霍奇金淋巴瘤中的一种或多种。
术语定义
在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的药剂学生物化学、核酸化学、免疫学实验室等操作步骤均为相应领域内广泛使用的常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
如本文中所使用的，术语“抗体”(Ab)应包括但不限于特异性结合抗原并且包含通过二硫键相互连接的至少两个重(H)链和两个轻(L)链的免疫球蛋白。每个重链包含重链可变区(本文缩写为VH)和重链恒定区。重链恒定区包含三个恒定域CH1、CH2和CH3。每个轻链包含轻链可变区(本文缩写为VL)和轻链恒定区。轻链恒定区包含一个恒定域CL。VH和VL区还可被细分为具有高变性的区域(称为互补决定区(CDR))，其间散布有较保守的区域(称为构架区(FR))。各VH和VL由按下列顺序：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4从氨基末端至羧基末端排列的3个CDR和4个FR组成。各重链/轻链对的可变区(VH和VL)分别形成抗原结合部位。基于恒定区的氨基酸序列和可变区的骨架区的氨基酸序列，重链可分类为μ、δ、γ、α或ε，并且分别将抗体的同种型定义为IgM、IgD、IgG、IgA和IgE，“轻链”分为两种严格的类型：kappa(κ)和lambda(λ)。
术语“抗体”包括例如天然存在和非天然存在的抗体；单克隆和多克隆抗体；嵌合和人源化抗体；人或非人抗体；全合成抗体。非人抗体可以通过重组方法进行人源化以降低其在人中的免疫原性。除非特别另外指出，本文使用的“抗体”应当理解为包含完整的抗体分子，既免疫球蛋白IgG。
术语“抗PD-L1抗体”是指能够特异性结合PD-L1的抗体。术语“特异性结合”是指抗体与抗原形成在生理条件下相对稳定的复合物。特异性结合可通过约1×10 -9M或更小的平衡解离常数(K D)来表征。用于确定两个分子是否特异性结合的方法是本领域公知的，例如生物发光干涉测量法或表面等离子共振技术(例如Biacore)。适用于本文公开的液体制剂中的抗PD-L1抗体(完整的抗体分子)呈现于PCT专利公开号WO2017/148424中，其通过引用以其整体并入本文。在某些优选的实施方案中，本文所述的抗PD-L1抗体包含分别如SEQ ID NO:1-3所示的重链CDR，以及分别如SEQ ID NO:4-6所示的轻链CDR。在某些优选的实施方案中，本文所述的抗PD-L1抗体具有如SEQ ID NO:7所示的重链可变区以及如SEQ ID NO:8所示的轻链可变区。在某些优选的实施方案中， 本文所述的抗PD-L1抗体具有如SEQ ID NO:9所示的重链以及如SEQ ID NO:10所示的轻链。重链末位Lys是容易缺失的，但这种缺失并不影响生物活性，参见Dick,L.W.等人，Biotechnol.Bioeng.，100:1132-1143。
如本文中所使用的，术语“程序性死亡-1(PD-1)”是指属于CD28家族的免疫抑制受体。PD-1在体内主要在以前活化的T细胞上表达，并结合两种配体PD-L1和PD-L2。完整的hPD-1序列可以在GenBank登录号U64863下找到。相应地，术语“程序性死亡配体-1(PD-L1)”是PD-1的两种细胞表面糖蛋白配体之一(另一种是PD-L2)，其在结合PD-1时下调T细胞活化和细胞因子分泌。本文所用的术语“PD-L1”包括人PD-L1(h PD-L1)，h PD-L1的变体、同工型和物种同源物，以及具有至少一个与h PD-L1共同的表位的类似物。完整的h PD-L1序列可以在GenBank登录号Q9NZQ7下找到。
如本文中所使用的，术语“液体制剂”表示至少一种活性成分(例如抗体，其能够在受试者中发挥生物活性)以及至少一种非活性成分的组合，所述非活性成分当与活性成分组合时，适合于施用给受试者。通常，液体制剂不含有对会施用该制剂的受试者有不可接受的毒性的成分。在本文中，受试者可以包括哺乳动物，优选为人。
如本文中所使用的，表述抗体的“稳定性”是指一种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。贮藏期一般基于制剂的预定货架期来选择。用于测量抗体稳定性的分析技术是本领域所公知的，可以在选定温度下用选定时间测定稳定性(例如，在40℃下储存至少28天并检测期间的抗体稳定性，或者在5℃±3℃下储存至少12个月并检测期间的抗体稳定性)。
对于物理稳定性而言，如果在通过对颜色和/或透明度进行肉眼检查，或通过UV光散射或通过大小排阻层析测定时基本没有表现出明显的聚集，沉淀和/或变性的迹象，则认为制剂中的抗体保持它的物理稳定性。
对于化学稳定性而言，可以通过检测和定量所述抗体的化学改变形式来进行评估。化学改变可能涉及大小改变(例如，剪切)，例如，可以通过采用大小排阻层析，SDS-PAGE和/或矩阵辅助的激光解析离子化/飞行时间质谱分析(MALDI/TOF MS)进行评估。其他类型的化学改变包括电荷改变(例如，因为脱酰胺导致的改变)，例如，可以通过离子交换层析对它进行评估。
对于生物学活性而言，如果一种制剂中的抗体具有它预期用途的生物学活性，则所述抗体在制剂中保持它的生物学活性。例如，如果所述制剂中的抗体的生物学活性为在制备所述制剂时所表现出的生物学活性的大约70％～130％(例如80％～130％)内，就认为保持了它的生物学活性(例如，通过抗原结合测定确定)。
如本文中所使用的，术语“缓冲液”是指通过其酸-碱共轭成分的作用来抵 抗pH的改变的缓冲溶液。此类缓冲液的例子包括乙酸盐、琥珀酸盐、葡糖酸盐、组氨酸、柠檬酸盐、甘氨酰甘氨酸及其他有机酸缓冲液。在某些优选的实施方案中，适用于本文公开的液体制剂中的缓冲液选自柠檬酸-柠檬酸盐缓冲液、组氨酸缓冲液和乙酸-乙酸盐缓冲液。
术语“组氨酸缓冲液”是指包含组氨酸的缓冲液，该缓冲液中的组氨酸可以以单独存在，或以以下形式存在：例如，组氨酸盐酸盐、组氨酸乙酸盐、组氨酸磷酸盐或组氨酸硫酸盐。当组氨酸不是以单独存在时，氨酸缓冲液(例如组氨酸盐酸盐缓冲液)可以通过用相应的酸(例如盐酸)滴定L-组氨酸(游离碱，固体)进行制备。当组氨酸以单独形式存在时，后续制备步骤可根据实际情况加入pH调节剂以获得适宜pH值范围的液体制剂。在某些优选的实施方案中，适用于本文公开的液体制剂中的组氨酸缓冲液中的组氨酸单独存在或以组氨酸盐酸盐或组氨酸乙酸盐形式存在。
术语“柠檬酸-柠檬酸盐缓冲液”是包括柠檬酸和柠檬酸盐的混合物。在某些优选的实施方案中，所述柠檬酸-柠檬酸盐缓冲液是柠檬酸-柠檬酸钠缓冲液，也即，包含柠檬酸和柠檬酸钠的混合物。柠檬酸-柠檬酸盐缓冲液(例如柠檬酸-柠檬酸钠缓冲液)可以通过向柠檬酸中加入成盐所需的碱(例如氢氧化钠)进行制备。
术语“乙酸-乙酸盐缓冲液”是包括乙酸和乙酸盐的混合物。在某些优选的实施方案中，所述乙酸-乙酸盐缓冲液是乙酸-乙酸钠缓冲液，也即，包含乙酸和乙酸钠的混合物。乙酸-乙酸盐缓冲液(例如乙酸-乙酸钠缓冲液)可以通过向乙酸中加入成盐所需的碱(例如氢氧化钠)进行制备。
如本文中所使用的，术语“糖”或“糖醇”具有本领域技术人员通常所理解的含义。糖包括一般组成(CH 2O)n及其衍生物，包括单糖、二糖、三糖、多糖、还原糖、非还原糖等，其在本文中的非限制性实例包括葡萄糖、蔗糖、海藻糖、乳糖、果糖、麦芽糖、葡聚糖(dextran)等。糖醇可以从相应的糖制取获得，即将糖分子上的醛基或酮基还原成羟基从而获得糖醇；糖醇具有通式H(CHOH)nH，其在本文中的非限制性实例包括，赤藓醇、苏糖醇、***糖醇、木糖醇、山梨糖醇、甘露糖醇、麦芽糖醇、乳糖醇等。
本发明的抗PD-L1抗体液体制剂不含有糖和/或糖醇，因此在本文中该制剂亦可被称为无糖的抗体制剂。在某些优选的实施方案中，本发明的抗PD-L1抗体液体制剂不含有选自下列的糖和/或糖醇：蔗糖、海藻糖、葡萄糖、甘露醇和山梨醇。本领域已知，通常抗体制剂中需要加入糖和/或糖醇(例如，蔗糖、海藻糖、葡萄糖、甘露醇和山梨醇等)作为抗体的稳定剂，此外，糖和/或糖醇同时也可作为有效的渗透压调节剂，能够维持该制剂的稳定(例如等渗，即具有与人血液基本上相同的渗透压)。
如本文中所使用的，术语“重量摩尔渗透压浓度”是指溶解的溶质颗粒在 水溶液中的渗透压的量度。溶质颗粒包括两种离子以及非离子化分子。渗透压表示为溶解在1kg溶剂(即水)中的渗透活性颗粒的浓度(即渗透压摩尔)。本文以毫渗透压摩尔/千克水(mOsmol/kg)为单位表示重量摩尔渗透压浓度。本领域技术人员已知，期望水平的重量摩尔渗透压浓度可以通过添加一种或多种渗透压调节剂(诸如糖和/或糖醇)来实现。
如本文中所使用的，术语“等渗”意指目的制剂具有与人血液基本上相同的渗透压。在某些优选的实施方案中，所述等渗的制剂一般将具有约260-330mOsmol/kg的渗透压。等渗性可以使用例如蒸气压或冰冻型渗透压计进行测量。
如本文中所使用的，术语“质量体积比”或“w/v”表示单一组分相对于含有该组分的混合物的总体积(以毫升计)的百分比重量(以克计)。例如，总体积100ml中的20mg组分为0.02％w/v。
如本文中所使用的，术语“约”是指在由本领域普通技术人员确定的特定值或组成可接受的误差范围内的值或组成，这将部分地取决于值或组成如何测量或确定，即测量***的限制。在本文中，当“约”用于描述可测量的值(例如，物质的浓度、时间、温度等)时，意味着包含给定值的±5％的范围，例如表述“约10mM”是指9.5-10.5mM。
发明的有益效果
在制备抗体制剂过程中，本领域通常需要加入糖和/或糖醇以维持抗体的稳定性，然而，糖和/或糖醇的使用对于糖尿病人而言有一定的局限性。本申请的发明人经过大量实验，出乎意料的发现，本发明的无糖抗体制剂不仅能够维持该抗体制剂的渗透压以获得优良的生理相容性，并且能够维持制剂的物理稳定性、化学稳定性及生物学活性，在储存运输期间耐受剧烈条件，从而特别有利于药品长期储存，并且能够扩展临床用药范围，具有重大的临床价值。
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将理解，下列实施例仅用于说明本发明，而不是对本发明的范围的限定。根据优选实施方案的下列详细描述，本发明的各种目的和有利方面对于本领域技术人员来说将变得显然。
序列信息
本发明涉及的部分序列的信息提供于下面的表1中。
表1：序列的描述
Figure PCTCN2019121069-appb-000001
Figure PCTCN2019121069-appb-000002
上述CDR可以通过本领域技术人员所熟知的技术手段，例如通过VBASE2数据库根据IMGT定义分析下面的重链可变区的氨基酸序列或者轻链可变区的氨基酸序列得到。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实验材料及方法
表2：实施例涉及的试剂
物料名称 厂家
聚山梨酯80 南京威尔化工有限公司
盐酸组氨酸 上海协和氨基酸有限公司
组氨酸 上海协和氨基酸有限公司
氯化钠 江苏勤奋药业有限公司
蔗糖 默克
乙酸 湖南尔康制药股份有限公司
乙酸钠 湖南尔康制药股份有限公司
枸橼酸 湖南尔康制药股份有限公司
枸橼酸钠 湖南尔康制药股份有限公司
以下实施例中的抗PD-L1人源化单克隆抗体的来源参见PCT专利公开号WO2017/148424，并且具备如SEQ ID NO:9所示的重链和如SEQ ID NO:10所示的轻链。
检测方法
(1)蛋白质浓度
采用Solo-VPE可变光程紫外分光光度计，吸取约30μl的样品置于比色皿中，通过测量UV吸光度来测定样品蛋白质浓度。
(2)Flowcam
采用Flowcam GWF-8JD测定样品中的颗粒数量，用移液枪吸取约1ml样品，将装有样品的枪头置于样品口上，测定样品中≥10μm以及≥25μm的不溶性颗粒数量。
(3)渗透压
取25μl样品置于样品管中，将样品管置于819M冰点渗透压仪
Figure PCTCN2019121069-appb-000003
探针下，测定样品渗透压。
(4)分子大小排阻层析(SEC-HPLC)
在Waters e2695HPLC上，柱温保持室温，使用TSKgel G3000SWXL柱，将处理后样品进样，流动相洗脱约20min，经分子大小排阻层析法确定样品中大小分子分布。根据280nm处UV吸收得出样品曲线，并将总面积的相对百分比峰面积作为结果报告。
(5)离子交换色谱(IEC-HPLC)
在Waters e2695HPLC上，柱温30℃，使用ProPac WCX-10柱，将处理后样品进样，以流动相洗脱，经离子交换色谱法确定样品中不同带电离子组分分布。根据280nm处UV吸收得出样品曲线，并将总面积的相对百分比峰面积作为结果报告。
(6)毛细管电泳(CE-SDS)
采用十二烷基硫酸钠毛细管电泳(CE-SDS)紫外检测方法，将样品进行处理后，在非还原(Non-reduced，NR)条件下，依据分子量大小，定量测定重组单克隆抗体产品的纯度。
(7)相对结合活性(％)
建立酶联免疫法(ELISA)检测样品的EC50(半数有效浓度)。通过比较样品与参比品的EC50，计算样品的相对结合活性，由于该方法的检测偏差为±20％，所以标准初步定为70-130％。
实施例1
处方1：抗PD-L1人源化单克隆抗体20mg/ml，100mM的氯化钠，20mM柠檬酸-柠檬酸钠，pH5.2。
处方2：抗PD-L1人源化单克隆抗体20mg/ml，100mM的氯化钠，20mM柠檬酸-柠檬酸钠，pH 5.6。
处方3：抗PD-L1人源化单克隆抗体20mg/ml，100mM的氯化钠，20mM柠檬酸-柠檬酸钠，pH 6.0。
处方4：抗PD-L1人源化单克隆抗体20mg/ml，100mM的氯化钠，20mM柠檬酸-柠檬酸钠，pH 6.4。
处方5：抗PD-L1人源化单克隆抗体20mg/ml，100mM的氯化钠，20mM柠檬 酸-柠檬酸钠，pH 6.8。
处方1-5的抗体制剂制备方法如下：
1.配置缓冲液：向注射用水中加入100mM氯化钠、20mM柠檬酸，用氢氧化钠调节至上述处方中所需的pH值。
2.抗体原液(四川科伦博泰生物医药股份有限公司，批号1)经超滤换液，透析至上述缓冲液中，调整抗体浓度至20mg/ml。
3.将配置好的样品在层流条件下经0.22μm孔径的过滤器进行除菌过滤，灌装至西林瓶，加塞轧盖即获得样品。
通过DSF法考察制剂pH值对单克隆抗体稳定性的影响，结果显示不同的pH值样品的Tm值均大于65℃，表明在pH 5.6-6.8范围内pH值对蛋白质热稳定性并无显著影响(表3)。将样品置于40℃条件下放置3天进行加速实验，SEC-HPLC结果显示单体大于99.0％，没有明显降低，低聚物未检出，高聚物均小于0.20％，无明显增加，各样品稳定性良好(表4)。
表3：不同pH值样品Tm值
样品 处方1 处方2 处方3 处方4 处方5
DSF Tm(℃) 65.31 66.25 66.25 66.33 66.40
表4：不同pH值样品加速实验结果
Figure PCTCN2019121069-appb-000004
实施例2
处方6：抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.8。
制备处方6的抗体制剂100ml，制备方法如下：
1.配置缓冲液：向注射用水中加入140mM氯化钠、20mM组氨酸，用盐酸调节至pH 5.8。
2.抗体原液(批号1)经超滤换液，透析至上述缓冲液，调整抗体浓度至20mg/ml，加入适量聚山梨酯80储存液。
3.将配置好的样品在层流条件下经0.22μm孔径的过滤器进行除菌过滤，灌装至西林瓶，加塞轧盖即获得样品。
考察处方6在40℃条件下28天样品稳定性，其检测结果分别示于表5-9中。
实施例3
处方7：抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.0。
调节缓冲液pH至5.0，其余制备方法同实施例2。
考察处方7在40℃条件下28天样品稳定性，其检测结果分别示于表5-9中。
实施例4
处方8：抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 7.0。
调节缓冲液pH至7.0，其余制备方法同实施例2。
考察处方8在40℃条件下28天样品稳定性，其检测结果分别示于表5-9中。
实施例5
处方9：抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-乙酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.8。
向注射用水中加入140mM氯化钠、20mM组氨酸，用乙酸调节至pH至5.8，其余制备方法同实施例2。
考察处方9在40℃条件下28天样品稳定性，其检测结果分别示于表5-9中。
实施例6
处方10：抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM乙酸-乙酸钠，质量体积比为0.02％的聚山梨酯80，pH 5.8。
向注射用水中加入140mM氯化钠、20mM乙酸钠，用乙酸调节pH至5.8，其 余制备方法同实施例2。
考察处方10在40℃条件下28天样品稳定性，其检测结果分别示于表5-9中。
实施例7
处方11：抗PDL-1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM枸橼酸-枸橼酸钠，质量体积比为0.02％的聚山梨酯80，pH 5.8。
向注射用水中加入140mM氯化钠、20mM枸橼酸钠，用枸橼酸调节pH至5.8，其余制备方法同实施例2。
考察处方11在40℃条件下28天样品稳定性，其检测结果分别示于表5-9中。
实施例8
处方12：抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯20，pH 5.8。
处方8中聚山梨酯80替换为聚山梨酯20，其余制备方法同实施例2。
考察处方12在40℃条件下28天样品稳定性，其部分检测结果分别示于表7-9中。
实施例9
检测上述实施例2-8样品在40℃条件下放置28天的稳定性。
9.1渗透压检测
实施例2-7的渗透压检测结果如下表所示。结果显示，在40℃条件下放置28天，实施例2-7样品的渗透压能够稳定在280-320mOsmol/kg范围内，稳定性良好。
表5：渗透压检测结果
Figure PCTCN2019121069-appb-000005
9.2不溶性颗粒检测
通过Flowcam检测实施例2-7样品的不溶性颗粒，结果见下表。结果显示，各处方样品中≥10μm的颗粒数量均小于1000，≥25μm的颗粒数量均小于200，表明处方6-11在40℃条件下放置28天后依然无明显聚集，满足药用要求。
表6：Flowcam检测结果
Figure PCTCN2019121069-appb-000006
9.3分子大小排阻层析(SEC-HPLC)检测
通过分子大小排阻层析检测实施例2-8样品中单体含量，结果见下表。结果显示，处方6-12样品单体含量均大于95％，在40℃条件下放置28天保持稳定。
表7：SEC-HPLC检测结果
Figure PCTCN2019121069-appb-000007
9.4离子交换色谱(IEC-HPLC)检测
通过离子交换色谱检测实施例2-8样品的主峰含量，结果见下表。结果显示，在40℃条件下放置28天，处方6-12的主峰含量均维持稳定，符合质量要求。
表8：IEC-HPLC检测结果
Figure PCTCN2019121069-appb-000008
Figure PCTCN2019121069-appb-000009
9.5毛细管电泳(CE-SDS)检测
通过毛细管电泳检测实施例2-8样品的主峰纯度，结果见下表。结果显示，在40℃条件下放置28天后，处方6-12样品的主峰纯度高，含量保持稳定，且符合质量要求。
表9：非还原CE-SDS检测结果
主峰纯度(％) 40℃0天 40℃14天 40℃28天
实施例2 96.3 95.9 95.0
实施例3 97.0 95.8 89.2
实施例4 97.0 95.3 94.0
实施例5 96.8 95.9 93.4
实施例6 97.0 95.7 93.1
实施例7 97.0 95.1 91.3
实施例8 97.2 95.0 93.4
实施例10
处方6：抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.8。
制备处方6的抗体制剂放大到10L，制备方法如下：
1.配置超滤缓冲液：向注射用水中加入氯化钠与组氨酸，并用盐酸调节pH值至5.8，配置成含140mM氯化钠和20mM组氨酸-盐酸组氨酸溶液。
2.抗体原液(批号2)使用Millipore超滤膜包，根据载量选取合适膜包数量并用Thermo Pellicon超滤***进行超滤换液。将蛋白透析至上述缓冲液，调整抗体浓度至20mg/ml，加入适量聚山梨酯80。
3.将配置好的样品在层流条件下进行除菌过滤，滤器采用Millipak系列0.22μm滤器(Millipore公司)，进行过滤。如有需要，可在除菌滤器前串联预过滤膜Opticap滤器以增大滤器载量。在无菌环境(超净台)中将除菌过滤器用硅胶管与蠕动泵连接，下经0.22μm孔径的过滤器进行除菌过滤。收集过滤后样品溶液，分别灌装至西林瓶，加塞轧盖即获得样品。
进行5℃±3℃长期12月稳定性研究，考察项目为蛋白浓度，SEC-HPLC、CE-SDS、IEC-HPLC，结果如表10所示。结果显示，该处方的抗PD-L1人源化单 克隆抗体液体制剂可放大生产，经放大生产仍可达到与小规模配置制剂相同的质量，抗体浓度稳定，纯度保持较高，稳定性良好，在5℃±3℃温度条件下至少可以稳定保存12个月。
表10：处方6的长期稳定性检测数据
Figure PCTCN2019121069-appb-000010
此外，对处方6进行-20℃48小时冻融稳定性研究，考察项目为OD405，SEC-HPLC以及IEC-HPLC，结果如表11所示。结果显示，该处方的抗PD-L1人源化单克隆抗体液体制剂冻融4个循环周期稳定性良好，可以耐受制剂在储存运输期间的剧烈条件。
表11：处方6的冻融稳定性检测数据
Figure PCTCN2019121069-appb-000011
按本例上述相同的方法制备另一批抗体原液(批号3)的相同处方制剂。进行处方6的5±3℃长期稳定性研究和25±2℃加速稳定性研究，考察项目为外观，蛋白浓度，pH，渗透压，不溶性微粒，SEC-HPLC、CE-SDS、IEC-HPLC，结合活性。结果如下：
表12：处方6的5±3℃长期稳定性研究检测数据
Figure PCTCN2019121069-appb-000012
Figure PCTCN2019121069-appb-000013
处方6在5℃条件下长期放置36月，外观澄清透明，蛋白含量、pH、摩尔渗透压、CE-SDS还原和非还原、SEC-HPLC、IEC-HPLC和相对结合活性均未发生明显改变，稳定性较好。
表13：处方6的25℃±2℃加速稳定性研究检测数据
Figure PCTCN2019121069-appb-000014
处方6在25±2℃加速条件下放置6个月，外观澄清透明，蛋白含量、pH、渗透压和相对结合活性均未发生明显变化，CE-SDS还原和非还原、SEC-HPLC和IEC-HPLC纯度略微下降，蛋白稳定性较好，符合质量要求。
实施例11
处方16：抗PD-L1人源化单克隆抗体60mg/ml，10mM组氨酸-盐酸组氨酸，140mM氯化钠，质量体积比为0.02％的聚山梨酯80，pH 5.7。缓冲液中的抗体浓 度为60mg/ml，其余制备方法同实施例2。
40℃条件稳定性检测结果如表14所示，处方16样品外观为微带乳光澄清透明液体；并且，在40℃条件下随时间延长，OD405稳定，表明该处方具有稳定性。
表14：处方16的稳定性检测结果
Figure PCTCN2019121069-appb-000015
实施例12
处方17：抗PD-L1人源化单克隆抗体60mg/ml，20mM组氨酸-盐酸组氨酸，140mM氯化钠，0.02％w/v聚山梨酯80，pH5.8，其制备方法同实施例2。
处方18：抗PD-L1人源化单克隆抗体90mg/ml，20mM组氨酸-盐酸组氨酸，140mM氯化钠，0.02％w/v聚山梨酯80，pH5.8，其制备方法同实施例2。
处方19：抗PD-L1人源化单克隆抗体120mg/ml，20mM组氨酸-盐酸组氨酸，140mM氯化钠，0.02％w/v聚山梨酯80，pH5.8，其制备方法同实施例2。
分别考察处方6，处方17、处方18和处方19的粘度以及不同温度条件下的粘度变化。结果如表15所示，低浓度条件下处方6的粘度较低，随着浓度增加，处方17、18和19的粘度逐渐增加。处方6和处方17在5℃放置6个月，粘度与起始时相比没有明显变化；处方18和处方19，在5℃和25℃放置3个月，粘度与起始时相比没有明显改变。高浓度制剂通常粘度较高，本申请制剂在高浓度时粘度可控且稳定，符合质量要求。
表15：处方6和17-19的粘度以及不同温度条件下的粘度值
Figure PCTCN2019121069-appb-000016
NT代表未检测。
实施例13
分别考察处方18和处方19在振荡、40℃、25℃和5℃条件下的稳定性。结果如表16所示，在1000rpm条件下连续振荡2h，处方18和处方19各检项均未发生明显改变，稳定性良好。在5℃条件下，放置2个月，处方19的各理化检项基本保持不变，稳定性良好，表明处方19制剂成品在5±3℃条件下可至少放置2个月。加速25℃条件下，放置2个月，处方18和处方19的IEC主峰略微降低，其余各检项基本不变，表明处方18和处方19制剂成品在25±2℃条件下可至少放置2个月。在40℃条件下，放置13天，处方18和处方19的IEC主峰和CE-SDS非还原主峰有小幅下降以外，其余各检项没有明显改变，符合质量要求，表明处方18和处方19制剂成品在40℃条件下可至少放置13天。本申请制剂克服稳定性、粘性、渗透压和混浊性的难题，有利于药品长期储存和临床用药。
表16：处方18和处方19在振荡、40℃、25℃和5℃条件下的稳定性检测结果。
Figure PCTCN2019121069-appb-000017
NT代表未检测。
对比例1
处方13：抗PD-L1人源化单克隆抗体20mg/ml，140mM蔗糖，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.8。缓冲液中140mM氯化钠替换为140mM蔗糖，其余制备方法同实施例2。
处方14：抗PD-L1人源化单克隆抗体20mg/ml，105mM蔗糖，35mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.8。缓冲液中140mM氯化钠替换为105mM蔗糖和35mM氯化钠，其余制备方法同实施例2。
处方15：抗PD-L1人源化单克隆抗体20mg/ml，70mM蔗糖，70mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.8。缓冲液中140mM氯化钠替换为70mM氯化钠和70mM蔗糖，其余制备方法同实施例2。
经检测，处方13-15的渗透压分别为199mOsmol/kg、232mOsmol/kg和258mOsmol/kg，均低于正常人体血浆渗透压，该抗体制剂为低渗溶液，静脉给药有发生溶血现象的风险，不满足临床用药需求。
总结在本发明的实施例中所涉及的所有处方及其各自的成分总结如表17所示。
表17各处方编号及其各自的成分
Figure PCTCN2019121069-appb-000018
首先，通过对处方1-5的抗体制剂进行Tm值测定以及40℃条件稳定性测定实验，其结果表明本发明处方1-5的抗体制剂均有良好的稳定性。这也表明当pH值在5.6-6.8范围内，本发明PD-L1抗体均具有一个较好的稳定性。
随后，通过对处方6-12的抗体制剂分别进行了40℃条件下防止28天的渗 透压监测、不溶性颗粒监测、SEC-HPLC检测、IEC-HPLC检测，以及CE-SDS检测，其结果表明本发明的处方6-12的抗体制剂的稳定性良好、无明显聚集，均符合药用要求。并且，结果表明，当pH为5.8时本发明抗体的稳定性最佳，并且，当制剂中的缓冲对为组氨酸-盐酸组氨酸时，其稳定性效果相对优于其他的缓冲对。
因此，本发明人对相对效果更优的处方6的抗体制剂进行了进一步的检测和分析。具体地，分析了其5℃±3℃的长期12个月的稳定性和在5℃长期存放36个月的稳定性，分别测试了蛋白浓度、SEC-HPLC、CE-SDS、IEC-HPLC、结合活性等；并且，分析了其-20℃、48小时冻融的稳定性，分别测试了OD405、SEC-HPLC以及IEC-HPLC。结果表明，处方6的抗体制剂，在5℃±3℃下至少可以稳定保存12个月，经放大生产仍可达到与小规模配制制剂相同的产品质量；并且，反复冻融4个循环周期后，稳定性良好，可以耐受制剂在储存运输期间的剧烈条件；此外，在5℃下长期放置36个月，外观澄清透明，蛋白含量、pH、摩尔渗透压、CE-SDS还原和非还原、SEC-HPLC、IEC-HPLC和相对结合活性均未发生明显改变，稳定性较好。
进一步地，本发明人尝试了高抗体浓度条件下的抗体制剂-处方16-19，分别尝试了PD-L1抗体浓度为60mg/ml、90mg/ml和120mg/ml的情况下，抗体制剂的稳定性。结果表明，在40℃的条件下，处方16的抗体制剂外观为微带乳光澄清透明液体、OD405稳定；在不同的温度条件下，随着时间的推移，处方17-19的抗体制剂的粘度没有发生明显的变化；处方18和19的抗体制剂，在1000rpm条件下连续震荡2h稳定性好，在5℃、25℃下可放置2个月，在40℃下可放置13天。因此，在本发明中，高浓度抗体浓度下的抗体制剂，可克服稳定性、粘性、渗透压和混浊性的难题，有利于药品长期储存和临床用药。
此外，本发明人对抗体制剂中NaCl的浓度进行了筛选-处方13-15(对比例)。结果表明，当NaCl浓度分别为0mM、35mM和70mM时，渗透压分别为199mOsmol/kg、232mOsmol/kg和258mOsmol/kg，均低于正常人体血浆渗透压，静脉给药有发生溶血现象的风险，因此不满足临床用药需求。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (17)

  1. 一种液体制剂，其包含：
    (1)浓度为约10mg/ml至约150mg/ml的抗PD-L1抗体；其中，所述抗PD-L1抗体包含分别如SEQ ID NO:1-3所示的重链CDRs，以及分别如SEQ ID NO:4-6所示的轻链CDRs；
    (2)浓度为约5mM至约40mM的缓冲液；
    (3)浓度为约80mM至约160mM的氯化钠；
    其中，所述液体制剂具有约5.0至约7.0的pH，并且，所述液体制剂不存在添加的糖和/或糖醇；
    并且，液体制剂具有与人血液相当的渗透压。
  2. 权利要求1的液体制剂，其中，所述抗PD-L1抗体具有如SEQ ID NO:7所示的重链可变区以及如SEQ ID NO:8所示的轻链可变区；
    优选地，所述抗PD-L1抗体具有如SEQ ID NO:9所示的重链以及如SEQ ID NO:10所示的轻链。
  3. 权利要求1或2的液体制剂，其中，所述抗PD-L1抗体的浓度为约15mg/ml至约25mg/ml、约50mg/ml至约70mg/ml，或约80mg/ml至约150mg/ml；
    优选地，所述抗PD-L1抗体的浓度为约20mg/ml、约60mg/ml、约90mg/ml或约120mg/ml。
  4. 权利要求1-3任一项的液体制剂，其中，所述液体制剂不含有选自下列的糖和/或糖醇：蔗糖、葡萄糖、海藻糖、甘露醇和山梨醇。
  5. 权利要求1-4任一项的液体制剂，其中，所述氯化钠的浓度为约100mM至约160mM,如约110mM,约120mM,约130mM,约140mM,约150mM,约155mM；
    优选地，所述氯化钠的浓度为约140mM。
  6. 权利要求1-5任一项的液体制剂，其中，所述缓冲液选自：柠檬酸-柠檬酸盐缓冲液、组氨酸缓冲液或乙酸-乙酸盐缓冲液；所述组氨酸缓冲液中的组氨酸单独存在或以组氨酸-盐酸盐或组氨酸-醋酸盐形式存在。
  7. 权利要求1-6任一项的液体制剂，其中，所述缓冲液的浓度是约10mM至约20mM。
  8. 权利要求1-7任一项的液体制剂，其中，所述液体制剂具有约5.2至约7.0的pH；
    优选地，所述液体制剂具有约5.6至约6.8的pH；
    优选地，所述液体制剂具有约5.6至约6.0的pH；优选地，所述液体制剂具有5.8±0.1的pH。
  9. 权利要求1-8任一项的液体制剂，其中，所述与人血液相当的渗透压为具有约280mOsmol/kg至约380mOsmol/kg的重量摩尔渗透压浓度；
    优选地，所述液体制剂具有约280mOsmol/kg至约360mOsmol/kg的重量摩尔渗透压浓度。
  10. 权利要求1-9任一项的液体制剂，其中，所述液体制剂还包含有表面活性剂；所述表面活性剂是聚山梨酯80、聚山梨酯20或泊洛沙姆188；
    其中，所述表面活性剂的浓度是约0.01％w/v至约0.02％w/v。
    优选地，所述表面活性剂是浓度为约0.02％w/v的聚山梨酯80。
  11. 权利要求1-10的液体制剂，所述液体制剂的成分选自下组：
    (a)抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.8；
    (b)抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.0；
    (c)抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-乙酸组氨酸，质量体积比为0.02％的聚山梨酯80，pH 5.8；
    (d)抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM乙酸-乙酸钠，质量体积比为0.02％的聚山梨酯80，pH 5.8；
    (e)抗PDL-1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM枸橼酸-枸橼酸钠，质量体积比为0.02％的聚山梨酯80，pH 5.8；
    (f)抗PD-L1人源化单克隆抗体20mg/ml，140mM氯化钠，20mM组氨酸-盐酸组氨酸，质量体积比为0.02％的聚山梨酯20，pH 5.8；
    (g)抗PD-L1人源化单克隆抗体60mg/ml，10mM组氨酸-盐酸组氨酸，140mM氯化钠，质量体积比为0.02％的聚山梨酯80，pH 5.7；
    (h)抗PD-L1人源化单克隆抗体60mg/ml，20mM组氨酸-盐酸组氨酸，140mM氯化钠，0.02％w/v聚山梨酯80，pH5.8；
    (i)抗PD-L1人源化单克隆抗体90mg/ml，20mM组氨酸-盐酸组氨酸，140mM氯化钠，0.02％w/v聚山梨酯80，pH5.8；
    (j)抗PD-L1人源化单克隆抗体120mg/ml，20mM组氨酸-盐酸组氨酸，140mM氯化钠，0.02％w/v聚山梨酯80，pH5.8；
    (k)抗PD-L1人源化单克隆抗体20mg/ml，100mM的氯化钠，20mM柠檬酸-柠檬酸钠，pH 5.6；
    (l)抗PD-L1人源化单克隆抗体20mg/ml，20mM组氨酸-盐酸组氨酸，140mM氯化钠，0.02％的聚山梨酯80，pH 7.0。
  12. 一种制品，其包含装有权利要求1-11任一项的液体制剂的容器。
  13. 如权利要求12所述的制品，其特征在于，所述容器为玻璃瓶，金属合金容器或预充注射器。
  14. 权利要求1-11任一项的液体制剂或权利要求12的制品在制备药物中的用途，所述药物用于：
    (a)预防和/或***或者贫血病；
    (b)辅助***或者贫血病；
    (c)诊断肿瘤或者贫血病；
    (d)(a)-(c)的任意组合；
  15. 如权利要求14所述的用途，其特征在于，所述药物与另外的药学活性剂分开、联合、同时、或相继施用；其中，另外的药学活性剂是化疗药物。
  16. 如权利要求14所述的用途，其特征在于，所述肿瘤选自乳腺癌、肺癌例如非小细胞性肺癌或肺鳞癌、肝癌、胃癌、肠癌例如结肠癌或者直肠癌、食管癌、卵巢癌、***、肾癌、***癌、膀胱癌、胰腺癌、Merkel细胞癌、胆管癌、鼻咽癌、头颈鳞癌、神经胶质瘤、黑素瘤白血病和淋巴瘤。
  17. 如权利要求16所述的用途，其特征在于，所述淋巴瘤为霍奇金淋巴瘤或非霍奇金淋巴瘤；更优选地，所述非霍奇金淋巴瘤为外周T细胞淋巴瘤、血管免疫母细胞T细胞淋巴瘤、NK/T细胞淋巴瘤和B细胞非霍奇金淋巴瘤中的一种或多种。
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