WO2020088492A1 - 含有抗pcsk9抗体的稳定制剂 - Google Patents

含有抗pcsk9抗体的稳定制剂 Download PDF

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WO2020088492A1
WO2020088492A1 PCT/CN2019/114233 CN2019114233W WO2020088492A1 WO 2020088492 A1 WO2020088492 A1 WO 2020088492A1 CN 2019114233 W CN2019114233 W CN 2019114233W WO 2020088492 A1 WO2020088492 A1 WO 2020088492A1
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antibody
amino acid
acid sequence
antigen
histidine
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PCT/CN2019/114233
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English (en)
French (fr)
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刘洪川
刘沛想
张静
丁申皓
张梅
吴纯
冯辉
武海
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上海君实生物医药科技股份有限公司
苏州君盟生物医药科技有限公司
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Application filed by 上海君实生物医药科技股份有限公司, 苏州君盟生物医药科技有限公司 filed Critical 上海君实生物医药科技股份有限公司
Priority to KR1020217016186A priority Critical patent/KR20210096105A/ko
Priority to US17/290,078 priority patent/US20210393777A1/en
Priority to CN201980071607.XA priority patent/CN112955180A/zh
Priority to JP2021523466A priority patent/JP7502286B2/ja
Priority to EP19878080.1A priority patent/EP3878467A4/en
Publication of WO2020088492A1 publication Critical patent/WO2020088492A1/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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—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
    • 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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • 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
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • 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 invention relates to the field of therapeutic antibody preparations.
  • the present invention relates to the field of pharmaceutical preparations, which contain a humanized antibody that specifically binds to human proprotein convertase subtilisin 9 (PCSK9).
  • PCSK9 human proprotein convertase subtilisin 9
  • PCSK9 Proprotein convertase subtilisin 9
  • LDL low-density lipoprotein
  • WO2017088782 discloses that a variety of anti-PCSK9 antibodies can significantly reduce the LDL concentration in blood by antagonizing the biological activity of PCSK9, and has important prospects in the treatment of hypercholesterolemia and other related diseases.
  • therapeutic anti-PCSK9 antibodies are affected by physical and chemical instabilities such as aggregation, denaturation, cross-linking, deamidation, isomerization, oxidation, and shear during manufacturing or storage ( Wang. Et al., J. Pharm. Sci. 96: 1-26, 2007 (Wang et al., Journal of Pharmaceutical Sciences, Volume 96, pages 1-26, 2007)). Therefore, it is very challenging to develop an antibody preparation that can keep the physical and chemical properties of the antibody stable.
  • Hypercholesterolemia and other related diseases are generally chronic diseases, and it is very important to provide the convenience of the patients out of hospital or self-administration.
  • Protein therapeutics can generally only be administered by parenteral routes, where subcutaneous injection (SC) or intramuscular injection (IM) administration routes can reduce treatment costs and improve the convenience of patients and healthcare providers during administration.
  • SC or IM injections usually 0.5 to 2 mL
  • the small volume required for SC or IM injections (usually 0.5 to 2 mL) presents additional formulation challenges, because administration requires high concentration antibody preparations, typically between 100 mg and 1 g protein / dose, to achieve therapeutic levels.
  • Highly concentrated protein preparations usually increase protein aggregation, precipitation, and increase viscosity, resulting in negative consequences during processing, manufacturing, and storage.
  • the increased viscosity may also have a negative effect on the application of the preparation, such as pain and burning symptoms and drugs Restrictions on delivery device options (Shire et al., J. Pharm. Sci. 93: 1390-1402, 2004 (Shire et al., Journal of Pharmaceutical Sciences, Volume 93, pages 1390-1402, 2004).
  • the present invention provides a pharmaceutical preparation containing a human antibody that specifically binds to human proprotein convertase subtilisin 9 (PCSK9).
  • PCSK9 human proprotein convertase subtilisin 9
  • the present invention provides a stable antibody preparation comprising: (1) a buffer; (2) a stabilizer; (3) an anti-PCSK9 antibody or antigen-binding fragment.
  • the stable antibody preparation may further include a nonionic surfactant.
  • the buffer is histidine buffer.
  • the histidine buffer is made of L-histidine and L-histidine monohydrochloride.
  • the concentration of the histidine buffer is about 20 nM.
  • the stabilizer comprises one or more selected from arginine or a salt thereof, sorbitol, mannitol or sucrose.
  • the stabilizer is an arginine salt.
  • the concentration of arginine or its salt is about 50 mM to about 200 mM. In some specific embodiments, the concentration of arginine or its salt is about 60 mM, 130 mM, 160 mM, or 165 mM.
  • the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: HCDR1 has the amino acid sequence SEQ ID NO: 1; HCDR2 has the amino acid sequence SEQ ID NO: 2; HCDR3 has the amino acid sequence SEQ ID NO: 3; LCDR1 has the amino acid sequence SEQ ID NO: 4; LCDR2 has the amino acid sequence SEQ ID NO: 5; and LCDR3 has the amino acid sequence SEQ ID NO: 6.
  • said antibody or antigen binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH has the amino acid sequence SEQ ID NO: 7, VL has amino acids Sequence SEQ ID NO: 8.
  • the antibody or antigen-binding fragment comprises a heavy chain (HC) and a light chain (LC), wherein HC has an amino acid sequence of SEQ ID NO: 9, LC has an amino acid sequence of SEQ ID NO: 10.
  • the concentration of the antibody or antigen-binding fragment is about 100 mg / mL to about 200 mg / mL. In a specific embodiment, the concentration of said antibody or antigen-binding fragment is about 150 mg / mL.
  • the antibody preparation provided by the present invention comprises polysorbate 20 or polysorbate 80 at a concentration of about 0.01% to about 0.05%. In a specific embodiment, the antibody preparation provided by the present invention comprises polysorbate 20 at a concentration of about 0.02%.
  • the antibody preparation provided by the present invention comprises: (1) about 20 mM histidine formulation buffer; (2) about 50 mM to about 200 mM arginine or its salt stabilizer; (3) about 100 mg / mL to about 200 mg / mL of anti-PCSK9 antibody or antigen-binding fragment thereof; and (4) about 0.02% polysorbate 20; wherein the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where: HCDR1 has the amino acid sequence SEQ ID NO: 1; HCDR2 has the amino acid sequence SEQ ID NO: 2; HCDR3 has the amino acid sequence SEQ ID NO: 3; LCDR1 has the amino acid sequence SEQ ID NO: 4; LCDR2 has the amino acid sequence SEQ ID NO: 5; and LCDR3 has the amino acid sequence SEQ ID NO: 6.
  • the antibody preparation provided by the present invention has at least 94% of the antibodies having a natural conformation after storage at 40 ° C for 28 days.
  • the antibody preparations provided by the present invention have a predominantly charged variant with at least 45% antibody after 28 days of storage at 40 ° C.
  • the antibody preparation provided by the present invention has at least 98% of the antibody having a natural conformation after storage at 2-8 ° C for 12 months.
  • the antibody preparation provided by the present invention has a predominantly charged variant with at least 87% antibody after 12 months of storage at 2-8 ° C.
  • the invention provides a delivery device comprising any of the antibody preparations provided by the invention.
  • the invention provides a pre-filled syringe comprising any of the antibody preparations provided by the invention.
  • the present invention provides a delivery device comprising any one of the antibody preparations provided by the present invention, or a method of pre-filled syringe for treating, preventing or ameliorating any disease or disorder related to PCSK9 activity.
  • Figure 1 The preparation containing JS002 inhibits the binding and uptake of LDL by cells.
  • Figure 2 Multiple subcutaneous injections of JS002 to observe the effect of 78 days on LDL-C in rhesus monkeys with hyperlipidemia.
  • the present invention is characterized by a stable aqueous liquid pharmaceutical preparation containing an anti-PCSK9 antibody or antigen-binding portion thereof, which has improved properties compared to preparations recognized in the art.
  • the high-concentration formulation provided by the present invention has unexpected characteristics, that is, has high stability and low viscosity.
  • “Therapeutic active antibody” or “therapeutic antibody” refers to an antibody that can be used for therapeutic purposes, that is, to treat a disorder in a subject. It should be noted that although therapeutic proteins can be used for therapeutic purposes, the present invention is not limited to such uses because the proteins can also be used for in vitro studies.
  • pharmaceutical formulation or “preparation” is a product in a form that makes the biological activity of the active ingredient effective and does not contain other ingredients that have unacceptable toxicity to the subject to whom the formulation is administered.
  • the preparation is sterile.
  • liquid formulation refers to a formulation in a liquid state, and is not intended to refer to a lyophilized formulation that is resuspended in weight.
  • the liquid formulation of the present invention is stable during storage, and its stability does not depend on lyophilization (or other state changing methods, such as spray drying).
  • aqueous liquid preparation refers to a liquid preparation using water as a solvent.
  • the aqueous liquid formulation is a formulation that does not require lyophilization, spray drying, and / or freezing to maintain stability (eg, chemical and / or physical stability and / or biological activity).
  • excipient refers to an agent that can be added to a formulation to provide desired properties (e.g. consistency, increased stability) and / or to adjust osmotic pressure.
  • desired properties e.g. consistency, increased stability
  • excipients include but are not limited to sugars, polyols, amino acids, surfactants and polymers.
  • the term "buffering agent that provides a pH of about 5.5 to about 6.5” refers to an agent that, through the action of its acid / base conjugate component, makes the solution containing the agent resistant to pH changes.
  • the buffer used in the formulation of the present invention may have a pH in the range of about 5.5 to about 6.5, or a pH in the range of about 5.5 to about 6.0. In one embodiment, the pH is about 6.0.
  • examples of "buffering agents” that control the pH within this range include acetate (eg sodium acetate), succinate (eg sodium succinate), gluconic acid, histidine and / or salts thereof, Methionine, citrate, phosphate, citrate / phosphate, imidazole, combinations thereof, and other organic acid buffers.
  • the buffer is not a protein.
  • the buffer is histidine and / or a salt thereof, preferably L-histidine and / or a salt thereof.
  • the concentration of the buffer in the formulation can be in the range of 5-100 mM, or about 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM, 60 mM, 65 mM, 70 mM, 75 mM, 80 mM, 85 mM, 90 mM, 95 mM, or 100 mM or within a range composed of any two of the above values as the end point, such as 10-30 mM or 15-25 mM.
  • the buffer concentration is about 20 mM.
  • “Histidine buffer” is a buffer containing histidine and / or salts thereof. Salts of histidine include one or more of histidine hydrochloride, histidine acetate, histidine phosphate and histidine sulfate.
  • the histidine buffer is: a histidine buffer made of 1-10 mM L-histidine and 10-20 mM L-histidine monohydrochloride.
  • the histidine preparation is: a histidine buffer pH 6.0 made from 4.5 mM L-histidine and 15.5 mM L-histidine monohydrochloride.
  • the histidine preparation is a histidine buffer with a pH of 5.5 made of 9.5 mM L-histidine and 10.5 mM L-histidine monohydrochloride.
  • the histidine buffer consists of histidine and histidine hydrochloride in a molar ratio of 1: 1 to 1: 4.
  • surfactant generally includes protecting proteins, such as antibodies, from air / solution interface induced stress, solution / surface induced stress to reduce antibody aggregation or minimize the formation of particulates in the formulation Chemical reagents.
  • exemplary surfactants include, but are not limited to, nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-polypropylene copolymers, poly Ethylene-polypropylene glycol, polyoxyethylene-stearate, polyoxyethylene alkyl ethers, such as polyoxyethylene monolauryl ether, alkylphenyl polyoxyethylene ether (Triton-X), polyoxyethylene- Polyoxypropylene copolymer (poloxamer, Pluronic), sodium dodecyl sulfate (SDS).
  • nonionic surfactants such as polyoxyethylene sorbitan fatty acid esters (such as polysorbate 20 and polysorbate 80), polyethylene-pol
  • the nonionic surfactant is polysorbate 20. In one embodiment, the concentration of polysorbate 20 is about 0 to 0.1% (w / v). In one embodiment, the concentration of polysorbate 20 is about 0.02% (w / v). In one embodiment, the nonionic surfactant is polysorbate 80. In one embodiment, the concentration of polysorbate 80 is about 0 to 0.1% (w / v). In one embodiment, the concentration of polysorbate 80 is about 0.02% (w / v).
  • stabilizer can reduce aggregation of antibodies and other proteins.
  • exemplary stabilizers include, but are not limited to: human serum albumin (hsa), bovine serum albumin (bsa), ⁇ -casein, globulin, ⁇ -lactalbumin, LDH, lysozyme, myoglobin, egg white Protein and RNAaseA.
  • Stabilizers also include amino acids and their metabolites and their salts such as hydrochloride, such as: arginine, glycine, alanine ( ⁇ -alanine, ⁇ -alanine), betaine, leucine , Lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, ⁇ -aminobutyric acid (GABA), opines (opamine, octopus) Alkali, strombine) and trimethylamine N-oxide (TMAO).
  • hydrochloride such as: arginine, glycine, alanine ( ⁇ -alanine, ⁇ -alanine), betaine, leucine , Lysine, glutamic acid, aspartic acid, proline, 4-hydroxyproline, sarcosine, ⁇ -aminobutyric acid (GABA), opines (opamine, octopus) Alkali, strombine) and trimethylamine
  • Stabilizers also include sugars, polyols and their metabolites, such as NaCl, KCl, MgCl 2 , CaCl 2 , sucrose, mannitol, sorbitol, etc.
  • the stabilizer is mannitol.
  • the stabilizer is sucrose.
  • the stabilizer is sorbitol.
  • the stabilizer is an amino acid or salt thereof.
  • the amino acid is arginine or arginine hydrochloride.
  • the concentration of arginine or arginine hydrochloride is about 20 to 200 mM.
  • the concentration of arginine or arginine hydrochloride is about 50 to 200 mM. In one embodiment, the concentration of arginine or arginine hydrochloride is about 60 mM, 130 mM, 160 mM, or 165 mM.
  • viscosity as used herein may be “kinematic viscosity” or “absolute viscosity”.
  • Kininematic viscosity is a measure of the resistance flow of a fluid under the influence of gravity.
  • absolute viscosity kinematic viscosity x density
  • the dimension of kinematic viscosity is L2 / T, where L is the length and T is the time.
  • kinematic viscosity is expressed in centistokes (cSt).
  • the SI unit of kinematic viscosity is mm2 / s, or lcSt. Absolute viscosity is expressed in units of centipoise (cP).
  • the term "low level viscosity” as used herein will mean an absolute viscosity of less than about 15 centipoise (cP). For example, if the absolute viscosity exhibited by the formulation is about 15 cP, about 14 cP, about 13 cP, about 12 cP, about 11 cP, about 10 cP, about 9 cP, about 8 cP, or lower when measured using standard viscosity measurement techniques Liquid formulations will be considered to have "low viscosity”.
  • the term “medium level viscosity” as used herein will mean an absolute viscosity between about 35 cP and about 15 cP.
  • the formulation exhibits an absolute viscosity of about 34 cP, about 33 cP, about 32 cP, about 31 cP, about 30 cP, about 29 cP, about 28 cP, about 27 cP, about 26 cP, about 25 cP, about 24cP, about 23cP, about 22cP, about 21cP, about 20cP, about 19cP, 18cP, about 17cP, about 16cP, or about 15.lcP, the liquid formulation of the present invention will be considered to have a "medium viscosity".
  • the liquid pharmaceutical formulations of the present invention may exhibit low to medium levels of viscosity in certain embodiments.
  • the present invention has made a surprising discovery that by formulating an antibody at a concentration of about 100-200 mM with 60-165 mM of arginine or a salt thereof, a liquid formulation containing a low viscosity can be obtained. In one embodiment, it was further found that arginine or its salt can significantly reduce the viscosity of formulations containing other tonicity agents such as sucrose, sorbitol, mannitol, and the like.
  • Isotonic means that the preparation has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250 to 350 mOsm. The isotonicity can be measured using a osmometer with a vapor pressure or freezing point drop.
  • a “stable" preparation is one in which the antibody substantially maintains its physical stability and / or chemical stability and / or biological activity during the manufacturing process and / or storage. Even if the contained antibody fails to maintain 100% of its chemical structure or biological function after a certain period of storage, the pharmaceutical preparation can be stable. In some cases, after a certain period of storage, it can maintain about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the antibody structure or function, and can also be considered as " stable”.
  • Various analytical techniques for measuring protein stability are available in this technical field and are reviewed in "Peptide and Protein Drug Delivery” (Peptide and Protein Delivery) 247-301, Vincent Lee Editor, Marcel Dekker, Inc ., New York, NY, Pubs. (1991)), and Jones, A. (1993) Adv. Drug Delivery. Rev. 10: 29-90 (both are incorporated by reference).
  • the stability of the protein is determined as the percentage of monomeric protein in the solution with a low percentage of degradation (eg, fragmentation) and / or aggregated protein.
  • the formulation can be stably stored at room temperature, about 25-30 ° C or 40 ° C for at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months , At least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months , Or longer, up to no more than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% antibody in aggregated form.
  • Stability can be measured by determining the percentage of antibody (“acidic form”) that migrates in a fraction that is more acidic than the main fraction of the antibody ("mainly charged form”) during ion exchange (and other methods), where stability and The percentage of antibodies in acidic form is inversely proportional.
  • the percentage of "acidified” antibodies can be measured by ion exchange chromatography (eg, cation exchange high performance liquid chromatography [CEX-HPLC]).
  • an acceptable degree of stability means that when the formulation is stored at a certain temperature for a certain period of time, the detectable acidic form of the antibody does not exceed at most about 49%, 45%, 40%, 35 %, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1%.
  • a certain period of storage before measuring stability may be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, or longer.
  • a certain temperature that allows storage of the pharmaceutical preparation may be any temperature in the range of about -80 ° C to about 45 ° C, for example, storage at about -80 ° C, about -30 ° C, about -20 ° C, about 0 ° C , About 2-8 ° C, about 5 ° C, about 25 ° C, or about 40 ° C.
  • the antibody shows substantially no signs of aggregation, precipitation, and / or denaturation when visually inspected for color and / or clarity or measured by UV light scattering or by pore size exclusion chromatography, for example, the antibody is in the pharmaceutical preparation "Maintain its physical stability".
  • Aggregation is the process by which individual molecules or complexes covalently or non-covalently associate to form aggregates. The aggregation can proceed to the extent that visible precipitates are formed.
  • the stability of the formulation can be evaluated by methods well known in the art, including measuring the apparent extinction (absorbance or optical density) of the sample. Such extinction measurement is related to the turbidity of the formulation.
  • the turbidity of the formulation is partly the inherent property of the protein dissolved in the solution, and is usually measured by turbidimetry and measured by turbidimetric turbidity units (NTU).
  • Turbidity levels that vary with, for example, the concentration of one or more components in a solution (eg, protein and / or salt concentration) are also referred to as the "emulsion” or “emulsion appearance" of the formulation.
  • the turbidity level can be calculated with reference to a standard curve generated using a suspension of known turbidity.
  • the reference standard used to determine the turbidity level of the pharmaceutical composition may be based on the European Pharmacopoeia standard (European Pharmacopoeia), fourth edition, "Directorate for the Quality of Medicine” (Directorate for the Quality of Medicine) of the Council of Europe (EDQM), France).
  • a clear solution is defined as a solution having a turbidity lower than or equal to a reference suspension having a turbidity of about 3 according to the European Pharmacopoeia standard.
  • Turbidimetric measurements of turbidimetry can detect Rayleigh scattering in the absence of associated or non-ideal effects, which usually change linearly with concentration. Other methods for evaluating physical stability are well known in the art.
  • Chemical stability can be assessed by, for example, detecting or quantifying the chemically altered form of antibodies.
  • Chemical changes may include size changes (eg, shortening), which can be evaluated using, for example, pore size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS).
  • Other types of chemical changes include changes in charge (eg, as a result of deamidation or oxidation), which can be evaluated by, for example, ion exchange chromatography.
  • the antibody in the pharmaceutical preparation is biologically active for its intended purpose, the antibody "maintains its biological activity" in the pharmaceutical preparation.
  • the affinity of the anti-PCSK9 antibody contained in the preparation to bind to PCSK9 is the antibody binding affinity before storage At least 90%, 95% or more, it can be considered that the formulation of the present invention is stable. Binding affinity can also be measured using, for example, ELISA or plasma resonance techniques.
  • a “therapeutically effective amount” or “effective amount” of an antibody refers to an amount effective in the prevention or treatment or alleviation of the symptoms of the disorder that the antibody can effectively treat.
  • subject or "patient” is intended to include mammalian organisms.
  • subjects / patients include human and non-human mammals, such as non-human primates, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human.
  • PCSK9 is a human proprotein convertase and belongs to the protease K subfamily that secretes the subtilase family.
  • the existing literature has demonstrated that PCSK9 can increase plasma LDL levels by binding to low-density lipoprotein particle receptors and promoting their degradation.
  • antibody as used herein should be understood to include intact antibody molecules and antigen-binding fragments thereof.
  • antigen-binding portion or “antigen-binding fragment” of an antibody (or simply “antibody portion” or “antibody fragment”) refers to an antibody that retains the ability to specifically bind to human PCSK9 or its epitope Or multiple clips.
  • full-length antibody refers to an immunoglobulin molecule comprising four peptide chains, two heavy (H) chains (approximately 50-70 kDa at full length) and two light (L) chains (approximately at full length) 25kDa) are connected to each other by disulfide bonds.
  • Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • the heavy chain constant region is composed of three domains CH1, CH2 and CH3.
  • Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region consists of a domain CL.
  • VH and VL regions can be further subdivided into complementarity determining regions (CDRs) with high variability and regions whose spacing is more conservatively referred to as framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH or VL region 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 of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors (including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system).
  • CDR refers to the complementarity determining region within the variable sequence of an antibody.
  • HCDR1, HCDR2 and HCDR3 or LCDR1, LCDR2 and LCDR3 for each heavy and light chain variable region.
  • the exact boundaries of these CDRs have different definitions according to different systems.
  • Kabat ibid.
  • This CDRs may be referred to as Kabat CDRs. Chothia et al.
  • CDR boundaries may not strictly follow one of the systems described in this article, but still overlap with Kabat CDRs, although they may be shortened or lengthened due to the prediction of specific residues or groups of residues or Even the entire CDR does not significantly affect antigen binding.
  • the methods used herein can utilize CDRs defined according to any of these systems, although certain embodiments use CDRs defined by Kabat or Chothia.
  • the anti-PCSK9 antibody or antigen-binding fragment thereof according to the present invention includes any anti-PCSK9 antibody described in International Publication No. WO2017088782.
  • the antibody used in the methods and compositions of the invention includes 6 CDRs from antibody JS002.
  • Antigen-binding fragments include fragments or derivatives of antibodies, usually including at least one fragment of the antigen-binding region or variable region (eg, one or more CDRs) of the parent antibody, which maintains at least some of the binding of the parent antibody Specificity.
  • antigen-binding fragments include, but are not limited to, Fab, Fab ', F (ab') 2 and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, such as sc-Fv; nanobody formed from antibody fragments And multispecific antibodies.
  • the binding fragment or derivative When the binding activity of an antigen is expressed on a molar concentration basis, the binding fragment or derivative generally retains at least 10% of its antigen binding activity.
  • the binding fragment or derivative retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the antigen binding affinity of the parent antibody. It is also contemplated that the antigen-binding fragment of an antibody may include conservative or non-conservative amino acid substitutions that do not significantly alter its biological activity (referred to as “conservative variants” or “functionally conservative variants” of the antibody).
  • the anti-PCSK9 antibody or antigen-binding fragment thereof of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, wherein: HCDR1 has the amino acid sequence SEQ ID NO: 1; HCDR2 has the amino acid sequence SEQ ID NO: 2; HCDR3 has the amino acid sequence SEQ ID NO: 3; LCDR1 has the amino acid sequence SEQ ID NO: 4; LCDR2 has the amino acid sequence SEQ ID NO: 5; and LCDR3 has the amino acid sequence SEQ ID NO: 6.
  • the anti-PCSK9 antibody or antigen-binding fragment of the present invention comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH has the amino acid sequence SEQ ID NO: 7; and VL Has the amino acid sequence SEQ ID NO: 8.
  • JS002 is a humanized complete antibody that specifically binds to human PCSK9 and includes heavy and light chains, wherein the amino acid sequence of the heavy chain is The amino acid sequence of SEQ ID NO: 9 and the light chain is SEQ ID NO: 10.
  • the preparation described in the present invention is a liquid preparation containing a high concentration of active antibody and having high stability and low viscosity.
  • the present invention has found that formulations containing arginine salts are significantly less viscous than formulations containing tonicity agents.
  • the arginine salt contained in the preparation solution can significantly reduce the preparation solution containing the tonicity agent.
  • the preparation of the present invention comprises an antibody or antigen-binding fragment thereof that specifically binds to human PCSK9, a buffer and a stabilizer.
  • the pH of the formulation of the present invention is in the range of 5.5 to 6.5.
  • the anti-PCSK9 antibody or antigen-binding fragment thereof generally ranges from 100 mg / mL to about 200 mg / mL. In some embodiments, the formulation of the present invention contains 150 ⁇ 10 mg / mL of anti-PCSK9 antibody or antigen-binding fragment.
  • the anti-PCSK9 antibody or antigen-binding fragment contained in the preparation of the present invention comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, wherein: HCDR1 has the amino acid sequence SEQ ID NO: 1; HCDR2 has The amino acid sequence SEQ ID NO: 2; HCDR3 has the amino acid sequence SEQ ID NO: 3; LCDR1 has the amino acid sequence SEQ ID NO: 4; LCDR2 has the amino acid sequence SEQ ID NO: 5; LCDR3 has the amino acid sequence SEQ ID NO: 6.
  • the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH has the amino acid sequence SEQ ID NO: 7, VL has the amino acid sequence SEQ ID NO: 8 . More preferably, the antibody or antigen-binding fragment comprises a heavy chain (HC) and a light chain (LC), wherein HC has the amino acid sequence SEQ ID NO: 9, and LC has the amino acid sequence SEQ ID NO: 10.
  • the buffer may be acetate buffer or histidine buffer, preferably histidine buffer.
  • the pH of the buffer is in the range of 5.5-6.0.
  • the histidine buffer of the present invention contains histidine and salts of histidine described herein.
  • the histidine buffer used in the present invention contains 1-10 mM histidine and 10-20 mM histidine salt (such as monohydrochloride).
  • the molar ratio of histidine to histidine salt is in the range of 1: 1 to 1: 4.
  • the concentration of the buffer solution may be 10-50 mM, preferably 15-25 mM, such as 20 mM.
  • the stabilizer may be selected from one or more of arginine or a salt thereof, sorbitol, mannitol, and sucrose, preferably an arginine salt.
  • arginine salts include arginine hydrochloride.
  • Other arginine salts suitable as stabilizers in pharmacy can also be used in the present invention.
  • the concentration of the stabilizer in the formulation of the present invention may be in the range of 50 mM to 300 mM, such as 50-200 mM, 130-200 mM, or 160-250 mM. When arginine salt is used alone, its concentration in the formulation may be in the range of 50-200 mM, such as 130-200 mM.
  • the stabilizer is a mixture of arginine salt and mannitol or sorbitol.
  • concentration of arginine salt in the preparation may be in the range of 50-150 mM
  • concentration of mannitol or sorbitol may be in the range of 50-200 mM
  • the total concentration of the stabilizers can be in the range of 50 mM to 300 mM, such as 50 mM to 250 mM.
  • the formulation of the present invention comprises: (1) an anti-PCSK9 antibody or antigen-binding fragment thereof; (2) a histidine buffer with a pH of about 5.5-6.5; and (3) arginine salt.
  • the formulations described herein also include the nonionic surfactants described herein.
  • the concentration of the nonionic surfactant in the formulation does not exceed 0.1% (w / v), such as 0.01-0.05% (w / v).
  • Preferred nonionic surfactants include polysorbate 20 and / or polysorbate 80, with a particularly preferred concentration of about 0.02%.
  • the formulation of the present invention comprises: (1) about 100 mg / mL to about 200 mg / mL of anti-PCSK9 antibody or antigen-binding fragment thereof; (2) about 10-50 mM histidine buffer, The pH is about 5.5-6.5; (3) about 50 mM to about 200 mM arginine salt; and (4) about 0% to about 0.1% nonionic surfactant.
  • the formulation of the present invention comprises: (1) about 150 ⁇ 10 mg / mL of anti-PCSK9 antibody or antigen-binding fragment thereof; (2) about 20 mM histidine buffer, pH about 5.5- 6.5; (3) about 60 ⁇ 5mM, 130 ⁇ 5mM or 160 ⁇ 5mM arginine salt; and (4) about 0% to about 0.1% nonionic surfactant.
  • the formulation of the present invention comprises: (1) about 150 ⁇ 10 mg / mL of anti-PCSK9 antibody or antigen-binding fragment thereof; (2) about 20 mM histidine buffer, pH about 5.5- 6.5; (3) about 160 ⁇ 5mM arginine salt; and (4) about 0.02% polysorbate 20.
  • the formulation of the present invention comprises: (1) about 150 ⁇ 10 mg / mL of anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, where: HCDR1 has the amino acid sequence SEQ ID NO: 1; HCDR2 has the amino acid sequence SEQ ID NO: 2; HCDR3 has the amino acid sequence SEQ ID NO: 3; LCDR1 has the amino acid sequence SEQ ID NO: 4; LCDR2 has the amino acid sequence SEQ ID NO: 5; and LCDR3 has the amino acid sequence SEQ ID NO: 6; (2) about 20 mM histidine buffer, pH about 5.5-6.5; (3) about 160 ⁇ 5 mM arginine; and (4) about 0.02% polysorbate 20.
  • the formulation of the present invention has a pH of 5.5-6.5 and comprises: (1) about 150 ⁇ 10 mg / mL of anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the antibody comprises a heavy chain Variable region (VH) and light chain variable region (VL), where VH has the amino acid sequence SEQ ID NO: 7, VL has the amino acid sequence SEQ ID NO: 8; (2) about 20mM histidine buffer, pH about 5.5-6.5; (3) about 160 ⁇ 5mM arginine; and (4) about 0.02% polysorbate 20.
  • VH heavy chain Variable region
  • VL light chain variable region
  • the formulation of the present invention comprises: (1) about 150 ⁇ 10 mg / mL of anti-PCSK9 antibody or antigen-binding fragment thereof, wherein the antibody is a full-length antibody, wherein the amino acid sequence of the heavy chain Is SEQ ID NO: 9 and the amino acid sequence of the light chain is SEQ ID NO: 10; (2) about 20 mM histidine buffer, pH about 5.5-6.5; (3) about 160 ⁇ 5 mM arginine; and (4) About 0.02% of polysorbate 20.
  • the pharmaceutical preparation of the present invention may be contained in any container suitable for storing drugs and other pharmaceutical compositions.
  • the pharmaceutical preparation of the present invention may be contained in a sealed and sterilized plastic or glass container having a volume, such as a vial, ampoule, syringe, cartridge, or bottle.
  • a vial such as a vial, ampoule, syringe, cartridge, or bottle.
  • Different types of vials can be used to contain the formulations of the present invention, including, for example, transparent and opaque (eg, amber) glass or plastic vials.
  • syringes can be used to contain and / or administer the pharmaceutical formulation of the present invention.
  • the pharmaceutical preparations can be administered to patients via parenteral routes such as injection (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or percutaneous, mucosal, nasal, respiratory, and / or oral administration.
  • parenteral routes such as injection (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or percutaneous, mucosal, nasal, respiratory, and / or oral administration.
  • parenteral routes such as injection (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or percutaneous, mucosal, nasal, respiratory, and / or oral administration.
  • parenteral routes such as injection (eg, subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or percutaneous, mucosal, nasal, respiratory, and / or oral administration.
  • a variety of reusable pens and / or auto-injector delivery devices can be used to deliver the pharmaceutical formulation of the present invention subcutaneously.
  • Examples of disposable pen and / or auto-injector delivery devices that can be used for subcutaneous delivery of the pharmaceutical composition of the present invention include, but are not limited to, SOLOSTAR TM pen (sanofi-aventis), FLEXPEN TM (Novo Nordisk), and KWIKPEN TM (Eli Lilly), SURECLICK TM autoinjectors (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP) and HUMIRA TM pens (Abbott Labs, Abbott Park, IL) are just a few examples here.
  • micro-infusion set means designed to slowly administer a large volume (eg, about 2.5 mL or more) over a longer period of time (eg, about 10, 15, 20, 25, 30 minutes, or longer).
  • Subcutaneous delivery device for therapeutic preparations For example, see U.S. 6,629,949, US 6,659,982 and Meehan, etc., J. Controlled Release 46: 107-116 (1996).
  • Micro-infusion sets are particularly useful for delivering large doses of therapeutic proteins contained in high concentration (eg, about 100, 125, 150, 175, 200 mg / ML or higher) and / or viscous solutions.
  • the pharmaceutical preparations of the present invention can be used to treat, prevent or ameliorate any diseases or disorders related to PCSK9 activity, including PCSK9-mediated diseases or disorders.
  • exemplary pharmaceutical preparations of the present invention can be treated or prevented, non-limiting variety of diseases and disorders include dyslipidemia, such as hypercholesterolemia, familial hypercholesterolemia, 1 3/4 hyperlipidemia, familial 1 3/4 hyperlipidemia, abnormal 3 hyperlipoproteinemia, familial hyperlipoproteinemia abnormal 3, 1 3/4 hypertriglyceridemia, and familial hypertriglyceridemia.
  • the present invention also includes the use of the antibody preparation of the present invention in the preparation of a medicament for treating, preventing or ameliorating any disease or disorder related to PCSK9 activity described herein. Also included are antibody preparations described herein that treat, prevent or ameliorate any disease or disorder associated with PCSK9 activity described herein.
  • the buffer system and pH closely affect the stability of the antibody.
  • Each antibody with unique physical and chemical properties has the most suitable buffer type and pH. Screening an optimal buffer system and pH to make the anti-PCSK9 antibody disclosed in the present invention has the best stability for clinical application.
  • SEC-HPLC Size exclusion chromatography
  • CEX-HPLC cation exchange chromatography
  • pH Buffer system Accessories 1 5.0 20mM sodium acetate buffer 130mM arginine hydrochloride 2 5.5 20mM sodium acetate buffer 130mM arginine hydrochloride 3 6.0 20mM histidine buffer 130mM arginine hydrochloride
  • the antibody in the SEC-HPLC test, the antibody can be kept relatively stable in the pH range of 5.5 to 6.0.
  • the monomer content of the sample After being placed at a high temperature of 40 ° C for 4 weeks, the monomer content of the sample is above 94%, and the monomer purity decline rate It is also below 1.5% / week; the main charge of the sample accounts for more than 45%, and the rate of decrease of the main charge is below 10% / week.
  • the buffer system is histidine buffer and the pH is 6.0 (prescription number 3)
  • the sample has the highest monomer purity (approximately 97%) after a high temperature of 40 ° C for 4 weeks, and the monomer purity decline rate is only 0.57% /week. Based on these results, a 20 mM histidine buffer with a pH of 5.5-6.0 was selected for the development of a liquid JS002 formulation.
  • sucrose, arginine hydrochloride, sorbitol or mannitol for a comparative test. That is, the above-mentioned different excipients or combinations thereof are added to a 20 mM histidine buffer (pH 5.5 or 6.0) containing about 150 mg / mL JS002, and the specific prescription information is shown in Table 3.
  • Each of the prescription preparations was placed at 40 ° C after being packed, and taken out at the second and fourth weeks for analysis and detection of antibody stability and viscosity of the prepared solution.
  • JS002 monomer content by molecular exclusion high performance liquid chromatography (SEC-HPLC), weak cation high performance liquid chromatography (CEX-HPLC) to detect the main peak content of JS002 charge, and test the viscosity of the preparation by the standard method in the fourth week .
  • SEC-HPLC molecular exclusion high performance liquid chromatography
  • CEX-HPLC weak cation high performance liquid chromatography
  • Table 4 when the prescription contains one or a combination of sucrose, arginine hydrochloride, sorbitol or mannitol, the antibody has strong thermal stability, that is, after the preparation is placed at a high temperature of 40 ° C for 4 weeks, the sample The monomer content is more than 98%, and the main charge accounts for more than 51%.
  • the stability of the antibody is the strongest and the viscosity is the lowest.
  • the formulation group containing only arginine salt (1)
  • the rate of decrease in the purity of the antibody monomer is obviously the lowest, as low as 0.1% / week, about 25-30% of the highest sucrose group, the monomer purity of the antibody is as high as 98.8%, and can improve the influence of sucrose, sorbitol or mannitol on the stability of the antibody to a certain extent;
  • the rate of decline of the main charge is the lowest, as low as 6.25% / week, and the main charge is as high as 61.2%;
  • the viscosity of the formulation the viscosity of the formulation containing only arginine is lower than 5cP (Table 5), which is significantly lower than other groups
  • the prescription especially only about 20-25% of the suc
  • High-concentration antibody solutions are generally more likely to cause antibody aggregation, precipitation, etc., which reduces the stability of the antibody, and the increase in solution viscosity leads to the difficulty of administration by injection (especially subcutaneous or intramuscular injection).
  • arginine salt can not only ensure the stability of antibody JS002, but also significantly reduce the viscosity of the liquid.
  • the buffer is a histidine buffer (pH 5.5 or 6.0) and contains only one stabilizer of arginine salt, the stability of the antibody and the viscosity of the solution are optimal.
  • surfactants to liquid formulations is commonly used to protect proteins such as antibodies from stress induced by the air / solution interface and solution / surface induced stress during storage to reduce antibody aggregation or minimize the formation of particulates in the formulation Reagents, which are conducive to the stability of antibody physicochemical properties.
  • Liquid pharmaceutical products containing therapeutic antibodies usually need to be stored at 2-8 ° C, so it is important that the formulation maintains high stability during long-term storage. Based on the above screening results, we designed 4 prescriptions to study the long-term stability of the formulation.
  • the four prescription formulations prepared as shown in Table 7 were stored in transparent vials and placed at 2-8 ° C for several months, and then analyzed and tested on each sample.
  • the stability was evaluated by the following parameters: (a) visual appearance; (b) photoresist method to detect insoluble particles (OD405nm); (c) pH; (d) CE-SDS method to detect antibody molecular weight; (d) SEC-HPLC Measure the content of antibody monomer (quality standard: ⁇ 97.0%), polymer (quality standard: ⁇ 3.0%) or fragments (quality standard: ⁇ 1.0%); (e) CEX-HPLC measure the main charge of antibody (quality standard: ⁇ 70.0%), acidic charge (quality standard: ⁇ 30.0%) or alkaline charge content (quality standard: ⁇ 15.0%); (f) ELISA method to detect antibody binding activity (quality standard: 70% -130 of the reference product) %); And (g) the biological activity of the antibody (HepG2 cell uptake LDL test, quality standard: 70%
  • Example 5 Room temperature storage stability of high concentration anti-PCSK9 antibody
  • Liquid pharmaceutical products containing therapeutic antibodies usually need to be stored at 2-8 ° C until the end of the storage period. Therefore, during the time from the purchase of the drug to the use, the patient needs to be refrigerated. Depending on the proposed medication regimen, in the case of self-administered medications this may lead to storage times that are the responsibility of the patient for up to several weeks. Therefore, drugs that do not need to be stored under refrigerated conditions show a significant increase in patient convenience for home care products and a reduction in the impact of drug quality under incorrect storage, thereby reducing the monitoring of complaints and temperature deviations.
  • the formulation disclosed in the present invention (prescription number: 20) has higher stability against protein degradation, and the resulting degradation kinetic parameters measured at 25 ° C meet the requirements of environmental storage for up to 6 months.
  • ForteBio affinity determination was performed according to existing methods. In short, take 4mg JS002 (prescription number: 29) and Evolocumab (140mg / ml / branch, purchased from AMGEN, batch number: 1063135), and exchange the solution 100 times with phosphate buffer in a 10KD ultrafiltration tube. After the solution, the protein content was measured at an absorption value of 280 nm, and the concentration was adjusted to 2 mg / mL. Take out biotin, equilibrate to room temperature, weigh 2mg Sulfo-NHS-Biotin into 300 ⁇ L ultrapure water, which is 10mM biotin mother liquor.
  • biotin 1: 6. Mix well and react at room temperature for 1-2h. After the biotin reaction was completed, the phosphate buffer solution was used for 100-fold exchange in a 10KD ultrafiltration tube. After the exchange, the protein content was measured at an absorption value of 280 nm, and the concentration was adjusted within the range of 1-2 mg / mL.
  • the biotinylated protein is divided into 0.1mL per tube and stored at -80 ° C, and freeze-thaw no more than once.
  • biotinylated JS002 and Evolocumab antibodies (5 ⁇ g / mL) were coupled to the streptavidin (SA) bioprobe respectively, and the test buffer (0.1% BSA, 0.02% Tween-20 and 1xPBS) was equilibrated for 300s. Then, PCSK9 with different concentrations was injected into the JS002 plate and the Evolocumab plate respectively, combined for 300s, and the dissociation time was 1800s.
  • test results are shown in Table 10. Fortebio results show that the binding affinity with PCSK9, JS002 is significantly better than the same target drug Evolocumab.
  • Example 7 Study on biological activity of JS002 cells (HepG2 cell LDL uptake method)
  • HepG2 cells exposed to human PCSK9-D347Y from JS002 (prescription number: 29) at the cellular level, and the same target drug Evolocumab (140 mg / ml / branch, purchased from AMGEN, Lot number: 1063135) for comparison.
  • human hepatoma cell line (HepG2) cells (ATCC, batch number: 62591368) were plated at a density of 2.0 * 10 4 cells / well (80 ⁇ L per well), and incubated at 37 ° C., 7% CO 2 overnight.
  • the JS002 antibody and Evolocumab were respectively subjected to concentration gradient dilution (the initial concentration was 20 ⁇ g / mL, 2-fold concentration gradient dilution), and 10 ⁇ L of antibody dilution was added to HepG2 cells, incubated for half an hour, and the antigen was diluted to 1 ⁇ g / mL, and 10 ⁇ L
  • the antigen dilution is added to HepG2 cells, and the antigen, antibody and cells are incubated for 4-6h.
  • Add fluorescently labeled LDL (3 ⁇ g / mL) incubate with cells for 16-18h, and then detect the amount of fluorescence taken up in the cells by microplate reader.
  • Example 8 Hyperlipidemia rhesus monkey JS002 lipid-lowering effect study
  • atorvastatin calcium group (1.2 mg / kg given at weeks 1-4, the 5th -8 weeks, 2.4 mg / kg, 4 animals)
  • JS002 high-dose group (12 mg / kg, 5 animals
  • JS002 low-dose group (4 mg / kg, 5 animals)
  • placebo group (5 animals).
  • statin calcium group was orally administered for 56 consecutive days with a washout period of 21 days; the JS002 high-dose group and the JS002 low-dose group were given 2 injections every 4 weeks for 78 consecutive days; the administration period and washing Periodically observe the main efficacy analysis indicators (LOL-C, TC, HDL-C, ApoAL, ApoB and TG), secondary efficacy indicators (weight), and safety indicators (blood biochemistry, blood routine indicators and clinical observation) Changes, comprehensive analysis of the above indicators to evaluate the lipid-lowering effect of JS002 (prescription number: 20) on hyperlipidemia rhesus monkeys.
  • test product JS002 high-dose group 12mg / kg (equivalent to the clinically planned dose of 420mg / 70kg people, every 4 weeks needle, subcutaneous injection twice), for hyperlipidemia rhesus monkey LOL-C It has a very significant reduction effect, and the level of D2-D71 reduction can be maintained 40% -70% after LDL-C administration compared with baseline; JS002 low dose group 4mg / kg (equivalent to the clinically planned dose of 140mg / 70kg, every 4 Mondays) Needle, subcutaneous injection twice), compared with baseline, the level of D2-D57 after LDL-C administration can be maintained at 20% -70%, and the effect of D57-D78 is significantly weakened and rebounded.
  • groups A, B, C, and D are single administrations, and the dosages are 2 mg / kg, 10 mg / kg, 50 mg / kg, and 10 mg / kg, respectively.
  • the administration of groups A, B, and C is subcutaneous injection
  • the group D was administered intravenously.
  • Group E was administered continuously with a dose of 10 mg / kg, administered once a week and continuously for four times.
  • NA means not detected.
  • test drug JS002 in cynomolgus monkeys, the serum drug exposure level increased with increasing dose, showing a non-linear pharmacokinetic characteristic.
  • the half-life t 1/2 of the elimination phase of JS002 is 56-74h, and the effective half-life within 336h is 50-139h.

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Abstract

一种含高浓度的抗PCSK9(人类前蛋白转化酶枯草溶菌素9)抗体的液体制剂，该制剂还包含一种缓冲液、一种稳定剂和一种表面活性剂。所述液体制剂具有低粘度，并且制剂在储存数月之后，抗体具有高稳定性。

Description

含有抗PCSK9抗体的稳定制剂 技术领域
本发明涉及治疗性抗体制剂领域。尤其是，本发明涉及医药制剂领域，该制剂含有一种与人类前蛋白转化酶枯草溶菌素9(PCSK9)特异性结合的人源化抗体。
背景技术
前蛋白转化酶枯草溶菌素9(PCSK9)是一种前蛋白转化酶，具有促进肝脏细胞表面低密度脂蛋白(LDL)受体的降解从而增加血浆中LDL胆固醇含量，其表达升高与人类血脂异常及心血管相关疾病密切相关。公开号为WO2017088782的专利中公开了多种抗PCSK9抗体能通过拮抗PCSK9生物活性而显著降低血液中LDL浓度，在治疗高胆固醇血症等相关疾病领域中具有重要的前景。与任何蛋白质治疗剂一样，治疗性的抗PCSK9抗体在制造或储存过程中会受到物理和化学不稳定性诸如聚集、变性、交联、脱酰胺化、异构化、氧化和剪切的影响(Wang et al.，J.Pharm.Sci.96:1-26，2007(Wang等人，《制药科学杂志》，第96卷第1-26页，2007年))。因此，开发一种能保持抗体理化性质稳定的抗体制剂具有非常大的挑战。
高胆固醇血症等相关疾病一般是一种慢性疾病，提供患者院外或可自我施用给药的便利非常重要。蛋白治疗剂一般只能通过胃肠外途径给药，其中皮下注射(SC)或肌内注射(IM)给药途径可降低治疗成本并改善给药期间患者和医疗保健提供者的便利性。而SC或IM注射剂所需的小体积(通常为0.5至2mL)提出了另外的制剂挑战，因为给药需要通常在100mg至1g蛋白质/剂之间的高浓度抗体制剂，以实现治疗水平。而高度浓缩的蛋白质制剂通常会增加蛋白质聚集、沉淀并增加粘度，从而造成加工、制造和储存期间产生负面后果，其中粘度增加还可能对制剂的施用具有负面影响，例如感觉疼痛和灼热症状和药物递送装置选项方面的限制(Shire et al.，J.Pharm.Sci.93:1390-1402，2004(Shire等人，《制药科学杂志》，第93卷第1390-1402页，2004年)。
因此，本领域存在着对于在特别是提供低粘度并且减轻患者疼痛的高浓度蛋白质制剂的需求。
发明内容
本发明提供含有与人类前蛋白转化酶枯草溶菌素9(PCSK9)特异性结合的人源抗体的医药制剂。
一方面，本发明提供了一种稳定的抗体制剂，包含：(1)一种缓冲液；(2)一种稳定剂；(3)一种抗PCSK9抗体或抗原结合片段。
作为一种优选方式，所述的稳定的抗体制剂还可以包含一种非离子型表面活性剂。
在一个实施方式中，其中所述的缓冲液为组氨酸缓冲液。在一个实施方式中，该组氨酸缓冲液是由L-组氨酸和L-组氨酸单盐酸盐制成。在一个实施方式中，该组氨酸缓冲液的浓度约20nM。
在一个实施方式中，其中所述的稳定剂包含选自精氨酸或其盐、山梨醇、甘露醇或蔗糖中的一种或以上。在一个实施方式中，其中所述的稳定剂为精氨酸盐。在一个实施方式中，精氨酸或其盐的浓度约50mM至约200mM。在一些具体的实施方式中，精氨酸或其盐的浓度约60mM、130mM、160mM或165mM。
在一个实施方式中，其中所述的抗体或抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中：HCDR1具有氨基酸序列SEQ ID NO:1；HCDR2具有氨基酸序列SEQ ID NO:2；HCDR3具有氨基酸序列SEQ ID NO:3；LCDR1具有氨基酸序列SEQ ID NO:4；LCDR2具有氨基酸序列SEQ ID NO:5；并且LCDR3具有氨基酸序列SEQ ID NO:6。在一个具体的实施方式中，其中所述的抗体或抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中VH具有氨基酸序列SEQ ID NO:7，VL具有氨基酸序列SEQ ID NO:8。在一个具体的实施方式中，其中所述的抗体或抗原结合片段包含重链(HC)和轻链(LC)，其中HC具有氨基酸序列为SEQ ID NO:9，LC具有氨基酸序列SEQ ID NO:10。在一个实施方式中，其中所述的抗体或抗原结合片段浓度约100mg/mL至约200mg/mL。在一个具体的实施方式中，其中所述的抗体或抗原结合片段的浓度约150mg/mL。
在一个实施方式中，本发明提供的抗体制剂包含浓度约0.01％至约0.05％聚山梨醇酯20或聚山梨醇酯80。在一个具体的实施方式中，本发明提供的抗体制剂包含浓度约0.02％聚山梨醇酯20。
在一个实施方式中，本发明提供的抗体制剂包含：(1)约20mM的组氨酸配制缓冲液；(2)约50mM至约200mM的精氨酸或其盐稳定剂；(3)约100mg/mL至约200mg/mL的抗PCSK9抗体或其抗原结合片段；以及(4)约0.02％聚山梨醇酯20；其中所述的抗体或抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中：HCDR1具有氨基酸序列SEQ ID NO:1；HCDR2具有氨基酸序列SEQ ID NO:2；HCDR3具有氨基酸序列SEQ ID NO:3；LCDR1具有氨基酸序列SEQ ID NO:4；LCDR2具有氨基酸序列SEQ ID NO:5；并且LCDR3具有氨基酸序列SEQ ID NO:6。
在一个实施方式中，本发明提供的抗体制剂于40℃储存28天后具有至少94％抗体的具有天然构象。
在一个实施方式中，本发明提供的抗体制剂于40℃储存28天后具有至少45％抗体的具有主要带电变体。
在一个实施方式中，本发明提供的抗体制剂于2-8℃储存12个月后具有至少98％抗体的具有天然构象。
在一个实施方式中，本发明提供的抗体制剂于2-8℃储存12个月后具有至少87％抗体的具有主要带电变体。
在一方面，本发明提供一种递送装置，其包含本发明提供的任何一种抗体制剂。
在一方面，本发明提供一种预填装注射器，其包含本发明提供的任何一种抗体制剂。
在一方面，本发明提供一种包含本发明提供的任何一种抗体制剂的递送装置，或预填装注射器用于治疗、预防或改善任何与PCSK9活性相关的疾病或失调的方法。
附图说明
图1：含有JS002的制剂抑制细胞对LDL的结合与摄取。
图2：JS002多次皮下注射给药观察78天对高脂血症恒河猴LDL-C的影响。
具体实施方式
本发明的特点在于包含抗PCSK9抗体或其抗原结合部分的稳定的水性液体药物制剂，其与本技术领域公认的制剂相比具有改进的性质。本发明提供的高浓度制剂具有出人意料的特征，即具有高稳定性和低粘度。
应理解本发明不限于具体的方法、试剂、化合物、组合物或生物系统，当然可以对以上进行变化。还应理解本申请所用术语仅为了描述具体的实施方式，并不旨在进行限制。除非该内容被另外明确说明，否则本说明书以及所附权利要求中所用的单数形式"一个"、"一种"和"该"包括复数指代。因此，例如，提及"一种多肽"包括了两种或更多种多肽等的组合。
本申请所用的"约"在指代可测量数值(如量、持续时间等)时意在涵盖相对于具体数值±20％或±10％的变化，包括±5％、±1％和±0.1％，因为这些变化适于进行所公开的方法。
除非另外定义，本申请所用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的含义。尽管与本申请公开相似或相同的任何方法和材料都可用于测试本发明的实践中，但本申请描述了优选的材料和方法。在描述并要求本发明的权利时，将使用以下术语。
“治疗活性抗体”或“治疗性抗体”是指可用于治疗目的，即用于治疗受试者中的障碍的抗体。应该指出，尽管治疗性蛋白质可用于治疗目的，但本发明不限于这样的用途，因为所述蛋白质也可以用于体外研究。
术语"药物制剂"或“制剂”是一种制品，其采用的形式使得活性成分的生物活性有效，并且不含对该制剂所施用的受试者具有不可接受的毒性的其它成分。该制剂为无菌的。
术语“液体制剂”是指处于液体状态下的制剂，且不意图指称重悬浮的冻干制剂。本发明的液体制剂在储存时稳定，并且其稳定性不依赖于冻干(或其他状态改变方法，例如喷雾干燥)。
术语“水性液体制剂”是指使用水作为溶剂的液体制剂。在一种实施方式中，水性液体制剂是不需冻干、喷雾干燥和/或冷冻来维持稳定性(例如化学和/或物理稳定性和/或生物活性)的制剂。
术语“赋形剂”是指可以向制剂添加以提供所需特性(例如稠度、提高的稳定 性)和/或调节渗透压的试剂。常用赋形剂的实例包括但不限于糖类、多元醇、氨基酸、表面活性剂和聚合物。
本文中，术语"提供约5.5至约6.5的pH的缓冲剂"是指这样的试剂，通过其酸/碱共轭组分的作用使得包含该试剂的溶液能抵抗pH变化。本发明的制剂中使用的缓冲液可具有约5.5至约6.5范围内的pH、或约5.5至约6.0范围内的pH。在一个实施方式中，pH为约6.0。
在本文中，将pH控制在该范围内的“缓冲剂”实例包括乙酸盐(例如乙酸钠)、琥珀酸盐(例如琥珀酸钠)、葡萄糖酸、组氨酸和/或其盐、甲硫氨酸、柠檬酸盐、磷酸盐、柠檬酸盐/磷酸盐、咪唑、其组合和其他有机酸缓冲剂。在一种实施方式中，缓冲剂不是蛋白质。在一个实施方式中，该缓冲剂为组氨酸和/或其盐，优选为L-组氨酸和/或其盐。通常，制剂中缓冲剂的浓度可在5-100mM的范围内，或者为约5mM、10mM、15mM、20mM、25mM、30mM、35mM、40mM、45mM、50mM、55mM、60mM、65mM、70mM、75mM、80mM、85mM、90mM、95mM或100mM或以上述任意两个数值为端点构成的范围内，如10-30mM或15-25mM。在一个实施方式中，缓冲剂浓度为约20mM。
"组氨酸缓冲剂"为包含组氨酸和/或其盐的缓冲剂。组氨酸的盐包括组氨酸盐酸盐、组氨酸乙酸盐、组氨酸磷酸盐和组氨酸硫酸盐中的一种或多种。在本发明的一个实施方式中，组氨酸缓冲剂为：由1-10mM的L-组氨酸和10-20mM的L-组氨酸单盐酸盐制成的组氨酸缓冲剂。在一个实施方式中，组氨酸制剂为：由4.5mM的L-组氨酸和15.5mM的L-组氨酸单盐酸盐制成的pH为6.0的组氨酸缓冲剂。在一个实施方式中，组氨酸制剂为：由9.5mM的L-组氨酸和10.5mM的L-组氨酸单盐酸盐制成的pH为5.5的组氨酸缓冲剂。在一些实施方案中，组氨酸缓冲液由摩尔比为1:1到1:4的组氨酸和组氨酸盐酸盐组成。
当在本文中使用时，术语“表面活性剂”一般包括保护蛋白质例如抗体免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少抗体的聚集或使制剂中颗粒物的形成最小化的试剂。示例性的表面活性剂包括但不限于非离子型表面活性剂例如聚氧乙烯脱水山梨醇脂肪酸酯(如聚山梨醇酯20和聚山梨醇酯80)、聚乙烯-聚丙烯共聚物、聚乙烯-聚丙烯二醇、聚氧乙烯-硬脂酸酯、聚氧乙烯烷基醚、例如聚氧乙烯单月桂基醚、烷基苯基聚氧乙烯醚(Triton-X)、聚氧乙烯-聚氧丙烯共聚物(泊洛沙姆，Pluronic)、十二烷基硫酸钠(SDS)。在一个实施方式中， 该非离子型表面活性剂为聚山梨醇酯20。在一个实施方式中，聚山梨醇酯20的浓度为约0至0.1％(w/v)。在一个实施方式中，聚山梨醇酯20的浓度为约0.02％(w/v)。在一个实施方式中，该非离子型表面活性剂为聚山梨醇酯80。在一个实施方式中，聚山梨醇酯80的浓度为约0至0.1％(w/v)。在一个实施方式中，聚山梨醇酯80的浓度为约0.02％(w/v)。
当在本文中使用时，术语“稳定剂”可降低抗体及其他蛋白质聚集。示例性的稳定剂包括但不限于：人血清白蛋白(hsa)、牛血清白蛋白(bsa)、α-酪蛋白、球蛋白、α-乳白蛋白、LDH、溶菌酶、肌红蛋白、卵清蛋白和RNAaseA。稳定剂还包括氨基酸和它们的代谢产物以及它们的盐如盐酸盐，如：精氨酸、甘氨酸、丙氨酸(α-丙氨酸、β-丙氨酸)、甜菜碱、亮氨酸、赖氨酸、谷氨酸、天冬氨酸、脯氨酸、4-羟基脯氨酸、肌氨酸、γ-氨基丁酸(GABA)、奥品类(opines)(丙氨奥品、章鱼碱、甘氨奥品(strombine))和三甲胺的N-氧化物(TMAO)。稳定剂还包括糖、多元醇和它们的代谢产物等，比如NaCl、KCl、MgCl 2、CaCl 2、蔗糖、甘露醇、山梨醇等。在一个实施方式中，该稳定剂为甘露醇。在一个实施方式中，该稳定剂为蔗糖。在一个实施方式中，该稳定剂为山梨醇。在一个实施方式中，该稳定剂为氨基酸或其盐。在一个实施方式中，该氨基酸为精氨酸或盐酸精氨酸。在一个实施方式中，精氨酸或盐酸精氨酸的浓度为约20至200mM。在一个实施方式中，精氨酸或盐酸精氨酸的浓度为约50至200mM。在一个实施方式中，精氨酸或盐酸精氨酸的的浓度约60mM、130mM、160mM或165mM。
本文所用的术语“粘度”可以是“运动粘度”或“绝对粘度”。“运动粘度”是对流体在重力影响下所产生的抵抗性流动的一种测量指标。“绝对粘度”，有时称为动态粘度或简单粘度，是运动粘度与流体密度的乘积(绝对粘度＝运动粘度X密度)。运动粘度的量纲是L2/T，其中L是长度，T是时间。通常，运动粘度以厘沲(cSt)表示。运动粘度的国际单位制单位是mm2/s，即lcSt。绝对粘度以厘泊(cP)单位表示。绝对粘度的国际单位制单位是毫帕斯卡-秒(mPa-s)，其中1cP＝lmPa-s。
对于本发明的液体型制剂，本文所用的术语“低水平粘度”将表示低于约15厘泊(cP)的绝对粘度。例如，如果当使用标准粘度测量技术测量时，该制剂展示的绝对粘度为约15cP、约14cP、约13cP、约12cP、约11cP、约10cP、约9cP、约8cP，或更低，则本发明液体型制剂将被认为是具有“低粘度”。对于本发明液体型制剂，本文所用的术语“中等水平粘度”将表示介于约35cP和约15cP之间的绝 对粘度。例如，如果当使用标准粘度测量技术测量时，该制剂展示的绝对粘度为约34cP、约33cP、约32cP、约31cP、约30cP、约29cP、约28cP、约27cP、约26cP、约25cP、约24cP、约23cP、约22cP、约21cP、约20cP、约19cP、18cP、约17cP、约16cP，或约15.lcP，则本发明液体型制剂将被认为是具有“中等粘度”。本发明液体型医药制剂在某些实施方式中可展示低水平至中等水平的粘度。在一个实施方式中，本发明作出了惊人的发现，通过将浓度为约100-200mM的抗体与60-165mM的精氨酸或其盐一起配制，可以获得含低粘度的液体型制剂。在一个实施方式中，还进一步发现，精氨酸或其盐可明显地降低含有其它张度剂如蔗糖、山梨醇、甘露醇等的制剂粘度。
"等渗"是指该制剂具有与人血液基本相同的渗透压。等渗制剂一般具有约250至350mOsm的渗透压。可使用蒸汽压或冰点下降式的渗透压计测量等渗性。
“稳定的”制剂是其中的抗体在制造过程期间和/或储存时基本上保持其物理稳定性和/或化学稳定性和/或生物活性的制剂。即使所含的抗体在经过一定时间储存之后未能保持其100％的化学结构或生物功能，医药制剂也可以是稳定的。在某些情况下，在经过一定时间储存之后，能维持约90％、约95％、约96％、约97％、约98％或约99％的抗体结构或功能，也可被认为是“稳定的”。用于测量蛋白质稳定性的各种分析技术在本技术领域中是可得的，并综述在《肽和蛋白质药物递送》(Peptide and Protein Drug Delivery)247-301，Vincent Lee主编，Marcel Dekker，Inc.，New York，N.Y.，Pubs.(1991))，和Jones，A.(1993)Adv.Drug Delivery Rev.10：29-90中(二者引入作为参考)。
制剂在一定温度下经过一定时间的储存之后，通过测定其中剩余的天然抗体的百分比(及其它方法)，可以测量其稳定性。除其它方法外，天然抗体的百分比可以通过尺寸排阻色谱法(例如尺寸排阻高效液相色谱法[SE-HPLC])来测量，“天然的”指未聚集的和未降解的。在一些实施方式中，蛋白质的稳定性按照具有低百分比的降解(例如片段化)和/或聚集蛋白质的溶液中单体蛋白质的百分数来确定。在一种实施方式中，制剂可以在室温、约25-30℃或40℃下稳定储存至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长，最多不超过约6％、5％、4％、3％、2％、1％、0.5％，或0.1％聚集形式的抗体。
通过测定在离子交换期间在比抗体主馏分(“主要荷电形式”)较为酸性的馏分中迁移的抗体(“酸性形式”)的百分比(及其它方法)，可以测量稳定性，其中稳定性与酸性形式抗体的百分比成反比。除其它方法外，“酸化”抗体的百分比可以通过离子交换色谱法(例如阳离子交换高效液相色谱法[CEX-HPLC])来测量。在一个实施方式中，可接受程度的稳定性意为当制剂在一定温度下经过一定时间的储存之后，其中可检测出的酸性形式的抗体最多不超过约49％、45％、40％、35％、30％、25％、20％、15％、10％、5％、4％、3％、2％、1％、0.5％或0.1％。在测量稳定性之前储存的一定时间可以是至少2周、至少28天、至少1个月、至少2个月、至少3个月、至少4个月、至少5个月、至少6个月、至少7个月、至少8个月、至少9个月、至少10个月、至少11个月、至少12个月、至少18个月、至少24个月，或更长。当评估稳定性时，容许储存医药制剂的一定温度可以是约-80℃至约45℃范围内的任何温度，例如储存于约-80℃、约-30℃、约-20℃、约0℃、约2-8℃、约5℃、约25℃，或约40℃。
如果抗体在颜色和/或澄清度目测检查时或通过UV光散射或通过孔径排阻层析测量时基本上不显示出例如聚集、沉淀和/或变性的迹象，则所述抗体在该药物制剂中“保持其物理稳定性”。聚集是单个分子或复合物共价或非共价缔合以形成聚集体的过程。聚集可以进行到形成可见沉淀物的程度。
制剂的稳定性例如物理稳定性可以通过本技术领域中公知的方法来评估，包括测量样品的表观消光度(吸光度或光密度)。这样的消光测量与制剂的浊度相关。制剂的浊度部分地是溶解在溶液中的蛋白质的固有性质，并且通常通过比浊法来测量，并用比浊法浊度单位(NTU)来量度。
随着例如溶液中一种或多种组分的浓度(例如蛋白质和/或盐浓度)而变化的浊度水平也被称为制剂的“乳浊”或“乳浊外观”。浊度水平可以参照使用已知浊度的悬液产生的标准曲线来计算。用于测定药物组合物的浊度水平的参比标准品可以基于《欧洲药典》标准(《欧洲药典》(European Pharmacopoeia)，第四版，“欧洲药品质量委员会指令”(Directorate for the Quality of Medicine of the Council of Europe)(EDQM)，Strasbourg，France)。根据《欧洲药典》标准，澄清溶液被定义为浊度低于或等于按照《欧洲药典》标准具有约3的参比悬液的浊度的溶液。比浊法的浊度测量可以检测在不存在缔合或非理想效应的情况下的瑞利散射，其通常随浓度线性变化。用于评估物理稳定性的其他方法在本技术领域中是公知的。
如果抗体在给定时间点的化学稳定性使得抗体被认为仍保持如下文中所定义的其生物活性，则所述抗体在药物制剂中“保持其化学稳定性”。可以通过例如检测或定量抗体的化学改变的形式来评估化学稳定性。化学改变可以包括尺寸改变(例如剪短)，其可以使用例如孔径排阻层析、SDS-PAGE和/或基质辅助的激光解吸电离/飞行时间质谱(MALDI/TOF MS)来评估。其他类型的化学改变包括电荷改变(例如作为脱酰胺或氧化的结果而发生)，其可以通过例如离子交换层析来评估。
如果药物制剂中的抗体对于其预期目的来说是生物活性的，则所述抗体在药物制剂中“保持其生物活性”。例如，如果制剂于例如5℃、25℃、45℃等温度下储存一定时间(例如1至12个月)之后，该制剂所含抗PCSK9抗体与PCSK9结合的亲和力为所述储存之前抗体结合亲和力的至少90％、95％或以上，则可认为本发明之制剂是稳定的。结合亲和力也可用例如ELISA或等离子共振技术测定。
在本发明的情形中，在药理学意义上，抗体的“治疗有效量”或“有效量”是指在抗体可以有效治疗的障碍的症状的预防或治疗或减轻方面有效的量。
术语“受试者”或“患者”意图包括哺乳动物生物体。受试者/患者的实例包括人类和非人类哺乳动物，例如非人类灵长动物、狗、奶牛、马、猪、绵羊、山羊、猫、小鼠、兔、大鼠和转基因非人类动物。在本发明的特定实施方式中，受试者是人类。
抗PCSK9抗体
本发明所述的制剂包含一种与人PCSK9特异性结合的抗体或其抗原结合片段。本文所用的术语“PCSK9”为一种人类前蛋白转化酶，属于分泌枯草杆菌酶家族的蛋白酶K亚族。现有文献已证明通过与低密度脂蛋白颗粒受体结合和促进其降解，PCSK9可提高血浆LDL水平。
本文所用的术语“抗体”应被理解为包括完整抗体分子及其抗原结合片段。本文所用的术语抗体的“抗原结合部分”或“抗原结合片段”(或简称为“抗体部分”或“抗体片段”)是指抗体中保持了与人PCSK9或其表位特异性结合能力的一个或多个片段。
本文所用的术语“全长抗体”，指包含四条肽链的免疫球蛋白分子，两条重(H)链(全长时约50-70kDa)和两条轻(L)链(全长时约25kDa)通过二硫键互相连 接。每一条重链由重链可变区(在本文中缩写为VH)和重链恒定区(在本文中缩写为CH)组成。重链恒定区由3个结构域CH1、CH2和CH3组成。每一条轻链由轻链可变区(在本文中缩写为VL)和轻链恒定区组成。轻链恒定区由一个结构域CL组成。VH和VL区可被进一步细分为具有高可变性的互补决定区(CDR)和其间隔以更保守的称为框架区(FR)的区域。每一个VH或VL区由按下列顺序：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。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导免疫球蛋白对宿主组织或因子(包括免疫系统的各种细胞(例如，效应细胞)和经典补体系统的第一组分(Clq))的结合。
当在本文中使用时，术语“CDR”是指抗体可变序列内的互补决定区。在重链和轻链的各个可变区中存在3个CDR，其对于各个重链和轻链可变区被命名为HCDR1、HCDR2和HCDR3或LCDR1、LCDR2和LCDR3。这些CDR的准确边界按照不同的系统有不同的定义。由Kabat(同上)描述的系统不仅提供了可适用于抗体的任何可变区的明确的残基编号系统，而且提供了定义3个CDR的准确残基边界。这些CDR可以被称为Kabat CDR。Chothia等发现，KabatCDR内的某些子部分采取几乎一致的肽骨架构型，尽管在氨基酸序列水平上具有大的多样性(Chothia等，(1987)Mol.Biol.196:901-917；Chothia等，(1989)Nature 342:877-883)。定义与Kabat CDR交叠的CDR的其他边界已由Padlan(1995)FASEB J.9:133-139和MacCalIum(1996)J.Mol.Biol.262(5):732-45描述。再其他的CDR边界定义可能不严格遵从本文中描述的系统之一，但仍然与Kabat CDR交叠，尽管它们可能被缩短或加长，这是由于根据预测或实验发现特定残基或残基组或甚至整个CDR不显著影响抗原结合。本文中使用的方法可以利用按照任何这些系统所定义的CDR，尽管某些实施方式使用了Kabat或Chothia定义的CDR。
本发明所述的抗PCSK9抗体或其抗原结合片段包括国际公开号为WO2017088782中描述的任意一个抗PCSK9抗体。在一种实施方式中，在本发明的方法和组合物中使用的抗体包括来自于抗体JS002的6个CDR。
本文所述的“抗原结合片段”包括抗体的片段或衍生物，通常包括亲代抗体的抗原结合区或可变区(例如一个或多个CDR)的至少一个片段，其保持亲代抗体的至少一些结合特异性。抗原结合片段的实例包括但不限于Fab，Fab'，F(ab')2 和Fv片段；双抗体；线性抗体；单链抗体分子，例如sc-Fv；由抗体片段形成的纳米抗体(nanobody)和多特异性抗体。当抗原的结合活性在摩尔浓度基础上表示时，结合片段或衍生物通常保持其抗原结合活性的至少10％。优选结合片段或衍生物保持亲代抗体的抗原结合亲和力的至少20％、50％、70％、80％、90％、95％或100％或更高。还预期抗体的抗原结合片段可包括不明显改变其生物活性的保守或非保守氨基酸取代(称为抗体的“保守变体”或“功能保守变体”)。
在一个实施方式中，本发明所述的抗PCSK9抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中：HCDR1具有氨基酸序列SEQ ID NO:1；HCDR2具有氨基酸序列SEQ ID NO:2；HCDR3具有氨基酸序列SEQ ID NO:3；LCDR1具有氨基酸序列SEQ ID NO:4；LCDR2具有氨基酸序列SEQ ID NO:5；并且LCDR3具有氨基酸序列SEQ ID NO:6。
在一个实施方式中，本发明所述的抗PCSK9抗体或抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中VH具有氨基酸序列SEQ ID NO:7；和VL具有氨基酸序列SEQ ID NO:8。
在本文实施例中所用的非限制性、示范性抗体被称为“JS002”，其是与人PCSK9特异性结合的人源化完全抗体，包含重链和轻链，其中重链的氨基酸序列为SEQ ID NO:9和轻链的氨基酸序列为SEQ ID NO:10。
医药制剂
本发明所述的制剂是一种包含高浓度活性抗体且具有高稳定性、低粘度的液体性制剂。特别地，本发明发现含有精氨酸盐的制剂粘度明显低于含有张度剂的配制液。另外，在配制液中含有的精氨酸盐能明显降低含有张度剂的配制液。
本发明的制剂包含特异性结合人PCSK9的抗体或其抗原结合片段、缓冲液和稳定剂。优选地，本发明制剂的pH在5.5～6.5的范围内。
本发明的制剂中，抗PCSK9的抗体或其抗原结合片段通常在100mg/mL至约200mg/mL的范围。在一些实施方案中，本发明的制剂中含有150±10mg/mL的抗PCSK9的抗体或抗原结合片段。在优选的实施方案中，本发明制剂中所含有的抗PCSK9的抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中：HCDR1具有氨基酸序列SEQ ID NO:1；HCDR2具有氨基酸序列SEQ ID NO:2；HCDR3具有氨基酸序列SEQ ID NO:3；LCDR1具有氨基酸序列SEQ  ID NO:4；LCDR2具有氨基酸序列SEQ ID NO:5；LCDR3具有氨基酸序列SEQ ID NO:6。更优选地，所述抗体或抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中VH具有氨基酸序列SEQ ID NO:7，VL具有氨基酸序列SEQ ID NO:8。更优选地，所述抗体或抗原结合片段包含重链(HC)和轻链(LC)，其中HC具有氨基酸序列SEQ ID NO:9，LC具有氨基酸序列SEQ ID NO:10。
本发明的制剂中，缓冲液可以是乙酸盐缓冲液或组氨酸缓冲液，优选为组氨酸缓冲液。优选地，缓冲液的pH在5.5-6.0的范围。优选地，本发明的组氨酸缓冲液含有组氨酸和本文所述的组氨酸的盐。通常，用于本发明的组氨酸缓冲液含有1-10mM的组氨酸和10-20mM的组氨酸盐(如单盐酸盐)。优选地，组氨酸缓冲液中，组氨酸与组氨酸的盐的摩尔比在1:1到1:4的范围内。本发明的制剂中，缓冲液的浓度可以为10-50mM，优选为15-25mM，如20mM。
本发明的制剂中，稳定剂可选自精氨酸或其盐、山梨醇、甘露醇和蔗糖中的一种或多种，优选精氨酸盐。示例性的精氨酸盐包括盐酸精氨酸。药剂学中适用作稳定剂的其它精氨酸盐也可用于本发明。通常，本发明制剂中稳定剂的浓度可在50mM至300mM的范围内，如50-200mM、130-200mM或160-250mM。当单独使用精氨酸盐时，制剂中其浓度可在50-200mM、如130-200mM的范围内。在一些实施方案中，稳定剂为精氨酸盐与甘露醇或山梨醇的混合物。当使用精氨酸盐与甘露醇或山梨醇的组合时，制剂中精氨酸盐的浓度可在50-150mM的范围内，甘露醇或山梨醇的浓度可在50-200mM的范围内，两种稳定剂的总浓度可在50mM至300mM、如50mM至250mM的范围内。
在一些实施方式中，本发明所述的制剂包含：(1)抗PCSK9的抗体或其抗原结合片段；(2)组氨酸缓冲液，pH约为5.5-6.5；和(3)精氨酸盐。
在本发明的一些实施方式中，本发明所述的制剂还包含本文所述的非离子型表面活性剂。通常，含有时，制剂中非离子型表面活性剂的浓度不超过0.1％(w/v)，如0.01-0.05％(w/v)。优选的非离子型表面活性剂包括聚山梨醇酯20和/或聚山梨醇酯80，特别优选的浓度约为0.02％。
在一个实施方式中，本发明所述的制剂包含：(1)约100mg/mL至约200mg/mL的抗PCSK9的抗体或其抗原结合片段；(2)约10-50mM组氨酸缓冲液，pH约为5.5-6.5；(3)约50mM至约200mM的精氨酸盐；以及(4)约0％至约0.1％的非离子表面活性剂。
在一个实施方式中，本发明所述的制剂包含：(1)约150±10mg/mL的抗PCSK9的抗体或其抗原结合片段；(2)约20mM组氨酸缓冲液，pH约为5.5-6.5；(3)约60±5mM、130±5mM或160±5mM的精氨酸盐；以及(4)约0％至约0.1％的非离子表面活性剂。
在一个实施方式中，本发明所述的制剂包含：(1)约150±10mg/mL的抗PCSK9的抗体或其抗原结合片段；(2)约20mM组氨酸缓冲液，pH约为5.5-6.5；(3)约160±5mM的精氨酸盐；以及(4)约0.02％的聚山梨酯20。
在一个实施方式中，本发明所述的制剂包含：(1)约150±10mg/mL的抗PCSK9的抗体或其抗原结合片段，其中所述的抗体包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中：HCDR1具有氨基酸序列SEQ ID NO:1；HCDR2具有氨基酸序列SEQ ID NO:2；HCDR3具有氨基酸序列SEQ ID NO:3；LCDR1具有氨基酸序列SEQ ID NO:4；LCDR2具有氨基酸序列SEQ ID NO:5；并且LCDR3具有氨基酸序列SEQ ID NO:6；(2)约20mM组氨酸缓冲液，pH约为5.5-6.5；(3)约160±5mM的精氨酸；以及(4)约0.02％的聚山梨酯20。
在一个实施方式中，本发明所述的制剂具有5.5-6.5的pH并且包含：(1)约150±10mg/mL的抗PCSK9的抗体或其抗原结合片段，其中所述的抗体包含包含重链可变区(VH)和轻链可变区(VL)，其中VH具有氨基酸序列SEQ ID NO:7，VL具有氨基酸序列SEQ ID NO:8；(2)约20mM组氨酸缓冲液，pH约为5.5-6.5；(3)约160±5mM的精氨酸；以及(4)约0.02％的聚山梨酯20。
在一个实施方式中，本发明所述的制剂包含：(1)约150±10mg/mL的抗PCSK9的抗体或其抗原结合片段，其中所述的抗体为全长抗体，其中重链的氨基酸序列为SEQ ID NO:9和轻链的氨基酸序列为SEQ ID NO:10；(2)约20mM组氨酸缓冲液，pH约为5.5-6.5；(3)约160±5mM的精氨酸；以及(4)约0.02％的聚山梨酯20。
药物制剂的容器及施用方法
本发明的药物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，本发明的药物制剂可包含在具有一定体积的密封及灭菌的塑料或玻璃容器中，诸如小瓶、安瓿、注射器、药筒或瓶子。不同类型的小瓶可用于包含本发明的制剂，例如包括透明和不透明(例如，琥珀色)玻璃或塑料小瓶。同样地， 可应用不同类型的注射器来容纳和/或施用本发明的药物制剂。
药物制剂可通过肠胃外途径施用于患者，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。可应用多种可重复使用的笔和/或自动注射器递送装置来皮下递送本发明的药物制剂。可用于皮下递送本发明药物组合物的一次性笔和/或自动注射器递送装置的实例包括但不限于S0L0STAR TM笔(sanofi-aventis)、FLEXPEN TM(Novo Nordisk)、以及KWIKPEN TM(Eli Lilly)、SURECLICK TM自动注射器(Amgen，Thousand Oaks，CA)、PENLETTM(Haselmeier，Stuttgart，德国)、EPIPEN(Dey，L.P.)和HUMIRA TM笔(Abbott Labs，Abbott Park，IL)，此处仅是几个例子。
微量输液器用于递送本发明制剂的用途也包含在本文之内。本文使用的术语“微量输液器”表示设计用于在较长时间(例如，约10、15、20、25、30分钟或更长)内缓慢施用大体积(例如，约2.5mL或更多)治疗制剂的皮下递送装置。例如参见U.S.6，629，949、US 6，659，982和Meehan等，J.Controlled Release 46:107-116(1996)。微量输液器尤其可用于递送包含在高浓度(例如，约100、125、150、175、200mg/ML或更高)和/或粘稠溶液中的大剂量治疗蛋白。
医药制剂的治疗用途
除其它用途外，本发明的医药制剂可用于治疗、预防或改善任何与PCSK9活性相关的疾病或失调，包括PCSK9介导的疾病或失调。可以本发明的医药制剂治疗或预防的示范性、非限制性疾病和失调包括各种血脂异常症，例如高胆固醇血症、家族性高胆固醇血症、1 3/ 4脂血症、家族性1 3/ 4脂血症、异常3脂蛋白血症、家族性异常3脂蛋白血症、1 3/ 4甘油三酯血症和家族性高甘油三酯血症。
本发明也包括本发明的抗体制剂在制备治疗、预防或改善本文所述的任何与PCSK9活性相关的疾病或失调的药剂中的应用。还包括的是治疗、预防或改善本文所述的任何与PCSK9活性相关的疾病或失调的本文所述的抗体制剂。
下面通过实施例对本发明进行详细描述，但并不意味着对本发明任何不利限制。本文已经详细地描述了本发明，其中也公开了其具体实施例方式，对本领域的技术人员而言，在不脱离本发明精神和范围的情况下针对本发明具体实施方式进行各种变化和改进将是显而易见的。
实施例1：缓冲液体系和pH筛选实验
液体型抗体制剂中，缓冲液体系和pH密切影响抗体的稳定性，每种具有独特理化性质的抗体都具有最适宜的缓冲液的种类和pH。筛选一种最佳缓冲液体系和pH，使本发明公开的抗PCSK9抗体具有最佳的稳定性以适宜临床应用。
本研究是以约150mg/mL的浓度JS002，辅料为130mM盐酸精氨酸进行的。使用透析袋进行透析换液使JS002蛋白处于相应的处方中，样品放置在密闭的离心管中进行缓冲液筛选。我们筛选了乙酸钠缓冲液和组氨酸缓冲液(组氨酸与盐酸组氨酸的摩尔比为1：1)两种类型，pH从5.0到6.0水平(如表1所示)。将样品在40℃/环境RH下放置，分别在第2周和第4周取出进行分析检测。蛋白降解的主要途径是聚集物、裂解产品和带电变体的形成。采用尺寸排阻色谱法(SEC-HPLC)测定天然形式(蛋白单体)与聚集形式JS002所占的百分比，采用阳离子交换色谱法(CEX-HPLC)测定酸性与碱性形式mAb所占的百分比。以试验起始(0W)、放置两周(2W)和放置四周(4W)的SEC-HPLC单体含量和CEX-HPLC主峰含量，拟合直线并计算下降斜率(％/周)考察不同缓冲液体系和pH对JS002抗体稳定性的影响，结果汇总见表2所示。
表1：缓冲液体系和pH筛选实验中的处方信息
处方编号 pH 缓冲体系 辅料
1 5.0 20mM乙酸钠缓冲液 130mM盐酸精氨酸
2 5.5 20mM乙酸钠缓冲液 130mM盐酸精氨酸
3 6.0 20mM组氨酸缓冲液 130mM盐酸精氨酸
表2：缓冲液体系和pH筛选实验中稳定性结果汇总
Figure PCTCN2019114233-appb-000001
Figure PCTCN2019114233-appb-000002
由表2所示，SEC-HPLC实验检测中，抗体在制剂pH 5.5～6.0范围内均可保持相对稳定，高温40℃放置4周后，样品单体含量在94％以上、单体纯度下降速率也在1.5％/周以下；样品主要电荷占45％以上、主要电荷的下降速率在10％/周以下。当缓冲体系为组氨酸缓冲液并且pH为6.0(处方编号3)时，高温40℃放置4周后样品的单体纯度最高(约达97％)，单体纯度的下降速率仅为0.57％/周。基于这些结果，选择pH值为5.5-6.0的20mM组氨酸缓冲剂用于液体型JS002制剂的开发。
实施例2：稳定剂筛选实验
为了进一步探究不同赋形剂对抗体稳定性和粘度的影响，我们选取蔗糖、盐酸精氨酸、山梨醇或甘露醇之一或其组合的制剂进行了比较测试。即将上述不同的赋形剂或其组合分别加入含约150mg/mL JS002的20mM组氨酸缓冲液(pH为5.5或6.0)，具体处方信息如表3所示。各处方制剂分装后放置于40℃条件下，分别在第2周、第4周取出进行分析检测抗体稳定性和配制液粘度。通过分子排阻高效液相色谱法(SEC-HPLC)检测JS002单体含量变化、弱阳离子高效液相色谱法(CEX-HPLC)检测JS002电荷主峰含量，以及在第四周按标准方法检测制剂粘度。如表4所示，当处方中含有蔗糖、盐酸精氨酸、山梨醇或甘露醇一种或其组合时，抗体均有较强的热稳定性，即制剂高温40℃放置4周后，样品单体含量在98％以上，带主要电荷占51％以上。
特别地，综合各项数据分析，当只含精氨酸盐的制剂，抗体的稳定性最强且粘度最低。具体地，高温40℃放置4周后，仅含精氨酸盐的制剂组：(1)对于抗 体结构的稳定性，抗体单体纯度的下降速率明显最低，低至0.1％/周，约为最高的蔗糖组的25-30％，抗体的单体纯度高达98.8％，并且能一定程度改善蔗糖、山梨醇或甘露醇对抗体稳定性的影响；(2)对如抗体电荷的稳定性，抗体主要电荷的下降速率最低，低至6.25％/周，主要电荷高达61.2％；(3)对于制剂的粘度，仅含精氨酸的制剂粘度低于5cP(表5)，明显低于其它各组处方，特别是仅为含蔗糖制剂组的约20-25％。另外，精氨酸盐明显改善含蔗糖、山梨醇或甘露醇制剂的粘度，约降低50％。
高浓度的抗体溶液一般更容易引起抗体聚集、沉淀等，使抗体稳定性降低，并且溶液粘度增加导致注射(特别是皮下或肌内注射)施用难度。综合以上实验，我们发现，当制剂液体制剂中，精氨酸盐不仅能保证抗体JS002的稳定性，还能显著降低液体粘度。特别是，当缓冲液为组氨酸缓冲液(pH为5.5或6.0)，仅含精氨酸盐的一种稳定剂时，抗体稳定性和溶液的粘度效果最优。
表3：稳定剂筛选实验中的处方信息
Figure PCTCN2019114233-appb-000003
Figure PCTCN2019114233-appb-000004
表4：稳定剂筛选实验结果汇总
Figure PCTCN2019114233-appb-000005
Figure PCTCN2019114233-appb-000006
表5：各处方制剂的粘度
处方编号 4 5 6 7 8 9 10 11 12 13 14 15 16 17
粘度(cP) 25.3 22.5 4.8 4.3 16.0 18.4 14.1 17.9 12.2 11.9 7.8 8.1 8.0 8.0
实施例3：表面活性剂筛选实验
液体制剂中添加表面活性剂常用于保护蛋白质例如抗体在储存过程中免受空气/溶液界面诱导的应力、溶液/表面诱导的应力的影响以减少抗体的聚集或使制剂中颗粒物的形成最小化的试剂，其利于抗体理化性质的稳定。在含有20mM组氨酸缓冲液(pH为6.0)、130mM盐酸精氨酸和150mg/ml的JS002的制剂中分别加入不同浓度的(0-0.5％)的聚山梨酯20或聚山梨酯80，40℃放置2周后分析检测。如表6所示，SEC-HPLC单体含量和CEX-HPLC主峰含量测定结果显示，含有不同浓度的(0-0.5％)的聚山梨酯20或聚山梨酯80制剂中抗体JS002的热稳定性无明显区别，但都保持高稳定性。
表6：表面活性剂筛选
Figure PCTCN2019114233-appb-000007
Figure PCTCN2019114233-appb-000008
实施例4：制剂长期稳定性研究
含有治疗性抗体的液体药物制品通常需要储存在2-8℃条件下，所以制剂能在长期储存中保持高稳定性非常重要。根据上述的筛选结果，我们设计4个处方进行制剂的长期稳定性研究。
配制如表7所示的4种处方制剂分别储存于透明小瓶中，放置2～8℃条件下若干个月后，对各样品进行分析测试。通过以下参数评估稳定性：(a)目视外观；(b)光阻法检测不溶性微粒(OD405nm)；(c)pH；(d)CE-SDS法检测抗体的分子量；(d)SEC-HPLC测量抗体单体(质量标准：≥97.0％)、聚体(质量标准：≤3.0％)或片段的含量(质量标准：≤1.0％)；(e)CEX-HPLC测量抗体主要电荷(质量标准：≥70.0％)、酸性电荷(质量标准：≤30.0％)或碱性电荷含量(质量标准：≤15.0％)；(f)ELISA法检测抗体结合活性(质量标准：参比品的70％-130％)；以及(g)抗体的生物学活性(HepG2细胞摄取LDL实验，质量标准：参比品的70％-130％)。如表8所示，4种制剂处方在2-8℃条件下，1-12月储存过程具有非常好的稳定性。
表7：用于制剂长期稳定性研究的制剂处方
Figure PCTCN2019114233-appb-000009
Figure PCTCN2019114233-appb-000010
表8：制剂处方的长期稳定性数据
Figure PCTCN2019114233-appb-000011
Figure PCTCN2019114233-appb-000012
Figure PCTCN2019114233-appb-000013
实施例5：高浓度抗PCSK9抗体的室温储存稳定性
含有治疗性抗体的液体药物制品通常需要储存在2-8℃下直至储存期结束。因此在购买药品至使用的时间内，还需要由患者进行冷藏。取决于所提出的用药方案，在自我施用药物的情况下这可能导致由患者负责的储存时间长达几周。因此，不需在冷藏条件下储存的药物显示出对于家庭护理产品来说患者方便性的显著提高和不正确储存情况下药物品质影响的降低，从而减少了投诉率和温度偏离的监测。
如表9所示，本发明公开的制剂(处方编号：20)对抗蛋白质降解具有更高稳定性，在25℃下测量的所得降解动力学参数满足环境储存长达6个月的要求。
表9：制剂加速稳定性数据汇总(25±2℃)
Figure PCTCN2019114233-appb-000014
Figure PCTCN2019114233-appb-000015
Figure PCTCN2019114233-appb-000016
实施例6：ForteBio亲和力测定(生物光干涉法)
ForteBio亲和力测定按照现有的方法进行。简言之，取4mg JS002(处方编号：29)和Evolocumab(140mg/ml/支，购自AMGEN，批号：1063135)，分别在10KD超滤管中用磷酸盐缓冲液进行100倍换液，换液后以280nm的吸收值测定蛋白含量，调整浓度为2mg/mL。取出生物素，平衡至室温，称取2mg Sulfo-NHS-Biotin加入300μL超纯水中，即为10mM的生物素母液。取2mg/mL JS002和Evolocumab各1mL置于新的EP管中，按照蛋白：生物素＝1:6，加入8μL的生物素母液。混匀，室温反应1-2h。生物素反应结束后，于10KD超滤管中用磷酸盐缓冲液进行100倍换液，换液后以280nm的吸收值测定蛋白含量，调整浓度在1-2mg/mL范围内。生物素化的蛋白分装为每管0.1mL，-80℃保存，冻融不超过1次。分别将生物素化后的JS002和Evolocumab抗体(5μg/mL)偶联到链霉亲和素(SA)生物探针上，实验缓冲液(0.1％BSA，0.02％Tween-20及1xPBS)平衡300s，然后，分别向JS002板和Evolocumab板中依次注入不同浓度稀释的PCSK9，结合300s，解离时间为1800s。根据公式KD＝koff/kon计算亲和力常数。
试验结果如表10所示。Fortebio结果显示，与PCSK9的结合亲和力，JS002明显优于同靶点药物Evolocumab。
表10：Fortebio比较JS002和Repatha对人PCSK9亲和力
  KD(M) kon(1/Ms) kdis(1/s)
JS002 2.15 -11 6.02E+04 1.30E-06
Evolocumab 6.51 -10 4.92E+04 3.21E-05
实施例7：JS002细胞生物学活性研究(HepG2细胞LDL摄取法)
本实验从细胞水平评价JS002(处方编号：29)对暴露在人PCSK9-D347Y的HepG2细胞LDL的摄取的情况，并且与已上市的同靶点药物Evolocumab(140mg/ml/支，购自AMGEN，批号：1063135)比较。简言之，将人肝癌细胞系(HepG2)细胞(ATCC，批号：62591368)以2.0*10 4个/孔的密度进行铺板(80μL每孔)，37℃，7％CO 2培养过夜。将JS002抗体及Evolocumab分别进行浓度梯度稀释(起始浓度为20μg/mL，2倍浓度梯度稀释)，取10μL抗体稀释液加入HepG2细胞中，孵育半小时，同时稀释抗原至1μg/mL，取10μL抗原稀释液加入HepG2细胞中，抗原抗体及细胞共孵育4-6h。加入荧光标记的LDL(3μg/mL)，与细胞共孵育16-18h，然后通过酶标仪检测细胞内摄取的荧光量。
实验结果如图1所示，结果表明JS002能够与细胞表面人PCSK9-D347Y结合，从而抑制其与LDLR的结合，增加LDLR对LDL的结合与摄取。并且，JS002(EC50＝92.68ng/mL)促LDL内吞的效果明显优于Evolocumab(EC50＝151.1ng/mL)。
实施例8：高血脂症恒河猴JS002降脂效果研究
本次试验入选19只高脂血症恒河猴(LDL≥l.3mmol/L)，共分为4组：阿托伐他汀钙组(第1-4周给予l.2mg/kg，第5-8周给予2.4mg/kg，4只)、JS002高剂量组(12mg/kg，5只)、JS002低剂量组(4mg/kg，5只)和安慰剂组(5只〉。阿托伐他汀钙组经口给药，连续给药56天，洗脱期21天；JS002高剂量组和JS002低剂量组，每4周一针，给药2次，连续观察78天；给药期和洗脱期定期观察主要疗效分析指标(LOL-C，TC，HDL-C，ApoAL，ApoB和TG)，次要疗效指标(体重)，及安全性指标(血液生化、血常规指标和临床观察)的变化，综合上述指标分析评价JS002(处方编号：20)对高血脂症恒河猴的降脂效果。
实验结果见图2。本次试验中安慰剂组血脂水平稳定，阳性对照阿托伐他汀钙组药效结果与临床文献报道类似，证明实验体系稳定可靠。
在本实验体系下，供试品JS002高剂量组12mg/kg(相当于临床拟用剂量420mg/70kg人，每4周一针，皮下注射2次)，对高脂血症恒河猴LOL-C具有极显著降低作用，与基线比LDL-C给药后D2-D71降低水平可维持40％-70％；JS002低剂量组4mg/kg(相当于临床拟用剂量140mg/70kg人，每4周一针，皮下注射2次)，与基线比LDL-C给药后D2-D57降低水平可维持20％-70％，D57-D78药效作用显著减弱而反弹。JS002高剂量组作用强度和持续时间均优于JS002低剂量组(图2)。整个给药期TG、FPG、ApoAl和HDL-C未见明显变化；各项安全性指标未见与给药相关的改变。
实施例9：食蟹猴药代动力学研究
试验观察健康食蟹猴单次或多次给予不同剂量JS002(处方编号：20)后的药代动力学性质。食蟹猴实验分组与给药情况见表11。其中A、B、C、D组为单次给药，给药剂量分别为2mg/kg、10mg/kg、50mg/kg和10mg/kg，其中A、B和C组给药方式为皮下注射，而D组给药方式为静脉注射。E组为连续给药，给药剂量为10mg/kg，每周给药一次，连续给药四次。
表11：动物分组与给药情况
Figure PCTCN2019114233-appb-000017
注：
1.sc:皮下注射。
2.iv:静脉注射。
3.QW:每周一次。
采样后制备血清样品，采用经方法学验证的酶联免疫吸附分析法进行定量检测，结果见表12。
表12：食蟹猴药代动力学参数(平均值±标准偏差)
Figure PCTCN2019114233-appb-000018
注：NA表示未检测。
结果显示，食蟹猴单次皮下注射不同剂量(2，10，50mg/kg)供试药物JS002后，血清药物暴露水平随剂量的增高而增加，呈非线性药代动力学特征。JS002消除相半衰期t 1/2为56-74h，336h内的有效半衰期为50-139h。
食蟹猴单次皮下注射10mg/kg受试药物JS002后的生物利用度为89.15％。
食蟹猴连续多次皮下注射10mg/kg(每周1次，连续4次)JS002后，药物在体内的暴露量显著高于同剂量单次给药组，药物在体内存在明显的蓄积。
实施例10：食蟹猴免疫毒性和免疫原性研究
本试验伴随食蟹猴皮下注射给予受试药物JS002(处方编号：20)四周重复给药毒性试验进行考察。按照CFDA的GLP规范要求，评价重组人源化抗PCSK9单克隆抗体注射液可能产生的免疫毒性和免疫原性。本实验设四个剂量组，分别为赋形剂对照组(0mg/kg)，低剂量组(30mg/kg)，中剂量组(100mg/kg)和高 剂量组000mg/kg)，每组10只动物，雌雄各半。每周给药l次，连续给药4周，恢复期4周。研究剂量范围内，食蟹猴多次皮下注射给予赋形剂和不同剂量(30、100、300mg/kg)受试药物JS002后，所有个体不同时间点血样均未检测到抗药物抗体，各组的样品阳性率和个体阳性率均为0.0％。结果提示多次皮下注射给予不同剂量(30、100、300mg/kg)受试药物JS002后，其在食蟹猴体内的免疫原性较低。对各组动物肾脏组织进行免疫复合物检测，均未见免疫复合物的沉积现象。其他免疫相关指标显示，各组动物白细胞绝对计数及分类计数、免疫球蛋白水平及AIG比值，淋巴器官/组织的大体解剖观察，胸腺、脾脏脏器重量/系数等均未见给药相荣的异常改变，淋巴细胞亚群分布，均未见给药相关的规律性改变。同时，病理组织学检查显示正常。

Claims (15)

  1. 一种稳定的抗体制剂，包含：
    (a)缓冲液；
    (b)稳定剂；和
    (c)特异性结合人PCSK9的抗体或其抗原结合片段，其中所述抗体或其抗原结合片段包含HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3，其中：
    HCDR1具有氨基酸序列SEQ ID NO:1；
    HCDR2具有氨基酸序列SEQ ID NO:2；
    HCDR3具有氨基酸序列SEQ ID NO:3；
    LCDR1具有氨基酸序列SEQ ID NO:4；
    LCDR2具有氨基酸序列SEQ ID NO:5；和
    LCDR3具有氨基酸序列SEQ ID NO:6；并且
    该制剂的pH为5.5～6.5。
  2. 如权利要求1所述的抗体制剂，其特征为所述缓冲液为乙酸盐缓冲液或组氨酸缓冲液。
  3. 如权利要求2所述的抗体制剂，其特征为所述缓冲液是由L-组氨酸和L-组氨酸单盐酸盐制成的组氨酸缓冲液。
  4. 如权利要求2所述的抗体制剂，其特征为所述组氨酸缓冲液含有1-10mM的组氨酸和10-20mM的组氨酸盐；优选地，所述组氨酸与组氨酸盐的摩尔比在1:1到1:4的范围内。
  5. 如权利要求1所述的抗体制剂，其特征为所述缓冲液的浓度为15-25mM。
  6. 如权利要求1所述的抗体制剂，其特征为所述的稳定剂为精氨酸或其盐、山梨醇、甘露醇或蔗糖，或为精氨酸或其盐与山梨醇、甘露醇或蔗糖的组合。
  7. 如权利要求6所述的抗体制剂，其特征为所述稳定剂为精氨酸盐，其 在所述制剂中的浓度为50mM到200mM；或所述稳定剂为精氨酸盐与甘露醇或山梨醇的混合物，其中，所述制剂中精氨酸盐的浓度为50-150mM，甘露醇或山梨醇的浓度为50-200mM，且两种稳定剂的总浓度为在50mM至250mM的范围内。
  8. 如权利要求1所述的抗体制剂，其特征为所述的抗体或其抗原结合片段包含重链可变区(VH)和轻链可变区(VL)，其中VH具有氨基酸序列SEQ ID NO:7，VL具有氨基酸序列SEQ ID NO:8。
  9. 如权利要求1所述的抗体制剂，其特征为其中所述抗体或其抗原结合片段包含重链(HC)和轻链(LC)，其中HC具有氨基酸序列SEQ ID NO:9；和LC具有氨基酸序列SEQ ID NO:10。
  10. 如权利要求1所述的抗体制剂，其特征为所述抗体制剂还包含浓度约0.01％至约0.05％聚山梨醇酯20或聚山梨醇酯80。
  11. 如权利要求1所述的抗体制剂，其特征为包含：
    (a)约20mM的组氨酸缓冲液；
    (b)约100mM至200mM的精氨酸盐；
    (c)约0.02％聚山梨醇酯20；和
    (d)约100mg/mL至约200mg/mL的特异性结合人PCSK9的抗体或抗原结合片段。
  12. 如权利要求1所述的抗体制剂，其特征为，所述抗体制剂选自包含以下组份的抗体制剂：
    (1)约100mg/mL至约200mg/mL的抗PCSK9的抗体或其抗原结合片段；(2)约10-50mM组氨酸缓冲液，pH约为5.5-6.5；(3)约50mM至约200mM的精氨酸盐；以及(4)约0％至约0.1％的非离子表面活性剂；或
    (1)约150±10mg/mL的抗PCSK9的抗体或其抗原结合片段；(2)约20mM组氨酸缓冲液，pH约为5.5-6.5；(3)约60±5mM、130±5mM或160±5mM的精氨酸盐；以及(4)约0％至约0.1％的非离子表面活性剂；或
    (1)约150±10mg/mL的抗PCSK9的抗体或其抗原结合片段，其中所述的抗体包含包含重链可变区(VH)和轻链可变区(VL)，其中VH具有氨基酸 序列SEQ ID NO:7，VL具有氨基酸序列SEQ ID NO:8；(2)约20mM组氨酸缓冲液，pH约为5.5-6.5；(3)约160±5mM的精氨酸；以及(4)约0.02％的聚山梨酯20；或
    (1)约150±10mg/mL的抗PCSK9的抗体或其抗原结合片段，其中所述的抗体为全长抗体，其中重链的氨基酸序列为SEQ ID NO:9，轻链的氨基酸序列为SEQ ID NO:10；(2)约20mM组氨酸缓冲液，pH约为5.5-6.5；(3)约160±5mM的精氨酸；以及(4)约0.02％的聚山梨酯20；或
    (1)150mg/ml的抗PCSK9的抗体或其抗原结合片段；(2)20mM组氨酸缓冲液，(3)160mM盐酸精氨酸；以及(4)0.02％聚山梨酯20；或
    (1)150mg/ml的抗PCSK9的抗体或其抗原结合片段；(2)20mM组氨酸缓冲液，(3)120mM盐酸精氨酸，60mM甘露醇；以及(4)0.02％聚山梨酯20；或
    (1)150mg/ml的抗PCSK9的抗体或其抗原结合片段；(2)20mM组氨酸缓冲液，(3)60mM盐酸精氨酸，160mM甘露醇；以及(4)0.02％聚山梨酯20。
  13. 一种递送装置，其包含权利要求1-12中任一项的抗体制剂。
  14. 一种预填装注射器，其包含权利要求1-12中任一项的抗体制剂。
  15. 权利要求1-12中任一项所述的抗体制剂或权利要求13所述的递送装置，或权利要求14所述的预填装注射器用于治疗、预防或改善任何与PCSK9活性相关的疾病的方法。
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