WO2019001560A1 - 一种pcsk-9抗体药物组合物及其用途 - Google Patents

一种pcsk-9抗体药物组合物及其用途 Download PDF

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Publication number
WO2019001560A1
WO2019001560A1 PCT/CN2018/093573 CN2018093573W WO2019001560A1 WO 2019001560 A1 WO2019001560 A1 WO 2019001560A1 CN 2018093573 W CN2018093573 W CN 2018093573W WO 2019001560 A1 WO2019001560 A1 WO 2019001560A1
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Prior art keywords
antibody
pcsk
pharmaceutical composition
buffer
seq
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PCT/CN2018/093573
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English (en)
French (fr)
Inventor
田晨敏
李皓
刘洵
Original Assignee
江苏恒瑞医药股份有限公司
上海恒瑞医药有限公司
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Application filed by 江苏恒瑞医药股份有限公司, 上海恒瑞医药有限公司 filed Critical 江苏恒瑞医药股份有限公司
Priority to CA3067953A priority Critical patent/CA3067953A1/en
Priority to RU2020102952A priority patent/RU2782792C2/ru
Priority to JP2019572725A priority patent/JP2020532491A/ja
Priority to AU2018291702A priority patent/AU2018291702A1/en
Priority to EP18823350.6A priority patent/EP3647325A4/en
Priority to CN201880019136.3A priority patent/CN110431153B/zh
Priority to US16/627,075 priority patent/US20200223941A1/en
Publication of WO2019001560A1 publication Critical patent/WO2019001560A1/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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • 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
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • 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
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • 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 invention belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition comprising a PCSK-9 antibody and an antigen-binding fragment thereof, and the use thereof as a medicament.
  • Hypercholesterolemia is a disorder of lipid metabolism characterized by elevated serum cholesterol levels, which is mainly caused by elevated serum cholesterol levels, leading to cholesterol accumulation in blood vessels and atherosclerosis.
  • a large number of clinical and experimental studies have confirmed that lipid metabolism abnormalities are closely related to the occurrence and development of coronary heart disease. Therefore, lowering the concentration of cholesterol in the blood has become a major means of treating and preventing atherosclerosis.
  • the main lipid-lowering drugs are mainly statins.
  • Liptor the world's most widely used cholesterol-lowering drug, is also the best-selling drug in the history of medicine. By blocking the enzymes that produce cholesterol in the liver, it reduces cholesterol production and increases the liver's intake of more cholesterol from the blood. , thereby reducing the concentration of cholesterol in the blood.
  • Lipitor also has its shortcomings. First, from the data, Lipitor can reduce LDL by 30%-40%, but there are still many patients still unable to reach effective blood lipid lowering (low-density lipoprotein concentration ⁇ 50mg/dL), the second patient's response rate to Lipitor is also different. For these reasons, patients need a more effective drug to lower blood lipids.
  • FM Familial hypercholesterolemia
  • LDL-c low density lipoprotein-cholesterol
  • Low-density lipoprotein receptor (LDLR) gene mutation causes defects or deficiency, LDL-c can not be smoothly transported to the liver to clear, resulting in elevated blood LDL-c levels.
  • LDLR gene apolipoprotein B100 gene
  • PCSK9 proprotein convertase subtilisin/kexin type9 genes.
  • PCSK9 The proprotein convertase subtilisin 9 (PCSK9) is a proprotein convertase belonging to the proteinase K subfamily of the secreted subtilisin family.
  • the encoded protein is synthesized as a soluble zymogen and processed in the endoplasmic reticulum by autocatalytic intramolecular processing.
  • the results showed that PCSK9 promoted the degradation of LDL receptors and increased plasma LDL cholesterol content, while LDL receptors mediate LDL endocytosis in the liver, which is the main pathway for clearing LDL from the circulatory system.
  • ADH hypercholesterolemia
  • PCSK9 mutations are diverse in form and can be divided into two categories based on the different effects of mutations on the regulation of LDK-C levels by PCSK9: loss of function and function acquisition. Among them, loss-of-function mutations are associated with low blood cholesterol levels and prevent coronary atherosclerotic heart disease. The rate of PCSK9 mutations in low cholesterol in African populations is higher than in other races.
  • PCSK9 function-acquired mutants increase plasma cholesterol levels by increasing PCSK9 function and decreasing LDLR expression, leading to severe hypercholesterolemia and premature coronary atherosclerotic heart disease, and currently found PCSK9 function-acquired mutations Including: D374Y, S127R, F216L, N157K, R306S and so on. Among them, compared with PCSK9 wild type, the LDLR on the surface of D374Y mutant cells was reduced by 36%, and the S127R mutation was reduced by 10%.
  • antibody drugs have a large molecular weight, a complicated structure, and are easily degraded, polymerized, or undergo undesired chemical modification and the like, and become unstable.
  • stable formulation studies of antibody drugs are particularly important.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a PCSK-9 antibody or antigen-binding fragment thereof, and a buffer, preferably a histidine or succinate or phosphate or citrate buffer, more preferably The histidine buffer is most preferably a histidine-hydrochloride buffer.
  • the pharmaceutical composition wherein the concentration of the PCSK-9 antibody or antigen-binding fragment thereof is from about 1 mg/ml to 150 mg/ml, preferably from about 30 mg/ml to 100 mg/ml, most preferably 50- 60 mg/ml, more preferably 50 mg/ml.
  • Non-limiting examples include 45 mg/ml, 46 mg/ml, 47 mg/ml, 48 mg/ml, 49 mg/ml, 50 mg/ml, 51 mg/ml, 52 mg/ml, 53 mg/ml, 54 mg/ml, 55 mg/ml, 56 mg/ml, 57 mg/ml, 58 mg/ml, 59 mg/ml, 60 mg/ml.
  • the concentration of the buffer is from about 5 mM to 50 mM, preferably from about 5 mM to 30 mM, and non-limiting examples include 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM. 30 mM, more preferably 10 mM to 15 mM, most preferably 10 mM.
  • the pH of the buffer in the pharmaceutical composition is from about 5.5 to 6.5, preferably from about 6.0 to 6.5, and the non-limiting examples include about 6.0, about 6.1, about 6.2, about 6.3, About 6.4, about 6.5.
  • the pharmaceutical composition further comprises a sugar.
  • the "sugar” of the present invention comprises a conventional composition (CH 2 O) n and derivatives thereof, including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, non-reducing sugars and the like.
  • Preferred sugars are non-reducing disaccharides, more preferably trehalose or sucrose.
  • the concentration of the sugar in the pharmaceutical composition is from about 10 mg/ml to about 75 mg/ml, preferably from 20 mg/ml to about 60 mg/ml, more preferably from 20 mg/ml to about 40 mg/ml, most Preferably it is 25 mg/ml, non-limiting examples include 20 mg/ml, 21 mg/ml, 22 mg/ml, 23 mg/ml, 23 mg/ml, 24 mg/ml, 25 mg/ml, 26 mg/ml, 27 mg/ml, 28 mg/ Ml, 29 mg/ml, 30 mg/ml, 31 mg/ml, 32 mg/ml, 33 mg/ml, 34 mg/ml, 35 mg/ml, 36 mg/ml, 37 mg/ml, 38 mg/ml, 39 mg/ml, 40 mg/ml.
  • the pharmaceutical composition further comprises a surfactant.
  • a surfactant May be selected from polysorbate 20, polysorbate 80, polyhydroxyl, Triton, sodium dodecyl sulfate, sodium lauryl sulfonate, sodium octyl sulphate, lauryl-, myristyl- , linoleyl-, stearyl-sulfobetaine, lauryl-, myristyl-, linoleyl-, stearyl-sarcosine, linoleyl-, myristyl-, cetyl -betaine, lauramide-propyl-, cocamidopropyl-, linoleidopropyl-, myristylpropyl-, palmitoylpropyl-, isostearamido-propyl- Betaine, myristylpropyl-propyl, palmitoylpropyl-, isostearamido-
  • the concentration of the surfactant in the pharmaceutical composition is from about 0.05 mg/ml to 1.0 mg/ml, preferably from 0.1 mg/ml to 0.4 mg/ml, and non-limiting examples include 0.1 mg/ml. Ml, 0.15 mg/ml, 0.2 mg/ml, 0.25 mg/ml, 0.3 mg/ml, 0.35 mg/ml, 0.4 mg/ml, further preferably 0.1 mg/ml to 0.3 mg/ml, more preferably 0.1 mg /ml to 0.2 mg/ml, most preferably 0.2 mg/ml.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • pH of the pharmaceutical composition is about 6.0-6.5.
  • the pharmaceutical composition comprises:
  • the histidine buffer is preferably a histidine-hydrochloride buffer.
  • the pharmaceutical composition comprises:
  • the histidine buffer is preferably a histidine-hydrochloride buffer.
  • the pharmaceutical composition comprises:
  • the histidine buffer is preferably a histidine-hydrochloride buffer.
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the pharmaceutical composition comprises:
  • the PCSK-9 antibody or antigen-binding fragment of the pharmaceutical composition has HCDR1, HCDR2 and HCDR3 as set forth in SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively. ,with
  • LCDR1, LCDR2, and LCDR3 shown in SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17, respectively.
  • the antibody or antigen-binding fragment in a pharmaceutical composition may be selected from the group consisting of a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.
  • the light chain of the PCSK-9 antibody in the pharmaceutical composition has at least 85%, 86%, 87%, 88%, 89% of the antibody light chain amino acid sequence of h001-4-YTE, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, heavy chain amino acid sequence of the PCSK-9 antibody and h001
  • the antibody heavy chain of -4-YTE has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% sequence identity
  • the h001-4-YTE antibody light chain sequence is set forth in SEQ ID NO: 30
  • the h001-4-YTE antibody heavy chain sequence is set forth in SEQ ID NO:32.
  • the present invention also provides a method for preparing the aforementioned pharmaceutical composition, which comprises the step of replacing a PCSK-9 antibody or an antigen-binding fragment thereof with a buffer, preferably a histidine buffer, more preferably a histamine.
  • Acid-hydrochloride buffer preferably at a concentration of about 5 mM to 30 mM, and non-limiting examples include 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26 mM, 28 mM, 30 mM, more preferably 10 mM to 15 mM; the buffer pH is about 6.0 to 6.5, and non-limiting examples include 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.
  • the stock solution further, the method for preparing the aforementioned pharmaceutical composition, further comprising the step of adding sucrose and polysorbate 80 to the solution obtained in the replacement step, and then adjusting the volume to a buffer, wherein the buffer concentration is preferably about 10 mM to 20 mM, without limitation. Examples include 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM; the buffer pH is about 6.0 to 6.5, and non-limiting examples include 6.0, 6.1, 6.2, 6.3, 6.4, 6.5.
  • the present invention also provides a method of preparing a lyophilized preparation containing a PCSK-9 antibody, which comprises the step of freeze-drying the aforementioned pharmaceutical composition.
  • the method of preparing a lyophilized formulation comprising a PCSK-9 antibody comprises the steps of pre-freezing, primary drying, and secondary drying, in sequence.
  • the pre-freezing is from 5 ° C to -40 ° C to -50 ° C, preferably -45 ° C, which does not require vacuum.
  • the primary drying temperature is -14 ° C to 0 ° C, preferably -5 ° C; the degree of vacuum is 0.1 mBar - 0.5 mBar, and most preferably 0.3 mBar.
  • the secondary drying temperature is 20 ° C to 30 ° C, preferably 25 ° C, and the degree of vacuum is from 0.1 mBar to 0.5 mBar, preferably 0.3 mBar, to a vacuum of 0.005 mBar to 0.02 mBar, preferably 0.01 mBar.
  • the present invention also provides a lyophilized preparation containing the PCSK-9 antibody prepared by the aforementioned method for preparing a lyophilized preparation containing a PCSK-9 antibody.
  • the lyophilized formulation is stable at 2-8 ° C for at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40 ° C for at least 7 days, at least 14 days or at least 28 days.
  • the present invention also provides a method for preparing a reconstituted solution containing a lyophilized preparation of a PCSK-9 antibody, which comprises the step of reconstituting the aforementioned lyophilized preparation, wherein the solution for reconstitution is selected from, but not limited to, water for injection, physiological saline or Glucose solution.
  • the present invention also provides a reconstituted solution of a lyophilized preparation containing a PCSK-9 antibody prepared by the aforementioned method of reconstituting a lyophilized preparation containing a PCSK-9 antibody.
  • the reconstituted solution containing the PCSK-9 antibody of the present invention comprises a component having a concentration of 2-5 times the concentration of the component contained in the pharmaceutical composition before lyophilization, preferably 3 times.
  • the reconstituted solution comprising a PCSK-9 antibody, wherein the concentration of the PCSK-9 antibody or antigen-binding fragment thereof is from about 120 mg/ml to 200 mg/ml, most preferably 150 mg/ml .
  • the reconstituted solution comprising a PCSK-9 antibody, wherein the pharmaceutical composition has a pH of from about 6.0 to about 6.5, preferably about 6.4.
  • the pH of the reconstituted solution is related to the pH of the buffer used in the preparation of the stock solution. When the pH of the stock solution is 6.0, the pH of the final reconstituted solution is 6.3 ⁇ 1.
  • the reconstituted solution comprising a PCSK-9 antibody, wherein the buffer concentration is from about 15 mM to 45 mM, preferably 30 mM.
  • the reconstituted solution comprising a PCSK-9 antibody further comprises a disaccharide, preferably from trehalose or sucrose, most preferably sucrose.
  • the reconstituted solution comprising a PCSK-9 antibody, wherein the sugar concentration is from about 55 mg/ml to 95 mg/ml, preferably 75 mg/ml.
  • the reconstituted solution comprising a PCSK-9 antibody further comprises a surfactant, preferably a polysorbate, more preferably a polysorbate 80.
  • the reconstituted solution comprising a PCSK-9 antibody, wherein the concentration of the surfactant is from about 0.4 mg/ml to 0.8 mg/ml, preferably 0.6 mg/ml.
  • the invention further provides an article or kit comprising a container containing any of the stable pharmaceutical compositions described herein.
  • the vial is a neutral borosilicate glass controlled injection vial.
  • the present invention also provides the use of the aforementioned pharmaceutical composition or a reconstituted solution of a lyophilized preparation or a lyophilized preparation for the preparation of a medicament for treating a PCSK-9-related disease or condition, wherein the disease or condition is preferably cholesterol-related
  • the disease is more preferably hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and general dyslipidemia, most preferably hypercholesterolemia , dyslipidemia, atherosclerosis, CVD, or coronary heart disease.
  • the present invention also provides a method of treating and preventing a PCSK-9-related disease or condition comprising administering to a patient in need thereof a therapeutically effective amount of a reconstituted solution of the aforementioned pharmaceutical composition or lyophilized formulation or lyophilized formulation, wherein said
  • the disease is preferably a cholesterol-related disease, more preferably hypercholesterolemia, heart disease, metabolic syndrome, diabetes, coronary heart disease, stroke, cardiovascular disease, Alzheimer's disease, and general dyslipidemia, most Hypercholesterolemia, dyslipidemia, atherosclerosis, CVD or coronary heart disease are preferred.
  • the present invention also provides an article comprising a container containing the aforementioned pharmaceutical composition or a reconstituted solution of a lyophilized preparation or a lyophilized preparation.
  • Buffer refers to a buffer that is resistant to changes in pH by the action of its acid-base conjugate component.
  • Examples of the buffer which controls the pH in an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, Glycyl glycine and other organic acid buffers.
  • Hetidine buffer is a buffer containing histidine ions.
  • examples of the histidine buffer include histidine-hydrochloride, histidine-acetate, histidine-phosphate, histidine-sulfate, etc., preferably histidine-hydrochloride buffer. liquid.
  • the histidine-hydrochloride buffer is prepared by histidine and hydrochloric acid or histidine and histidine hydrochloride.
  • citrate buffer is a buffer that includes citrate ions.
  • the citrate buffer include citric acid-sodium citrate, citric acid-potassium citrate, citric acid-calcium citrate, citric acid-magnesium citrate, and the like.
  • a preferred citrate buffer is citric acid-sodium citrate.
  • succinate buffer is a buffer comprising succinate ions.
  • succinate buffer examples include sodium succinate-succinate, potassium succinate-succinate, calcium succinate-succinate, and the like.
  • a preferred succinate buffer is sodium succinate-succinate.
  • Phosphate buffer is a buffer that includes phosphate ions.
  • examples of the phosphate buffer solution include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and the like.
  • a preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.
  • Acetate buffer is a buffer that includes acetate ions.
  • examples of the acetate buffer include acetic acid-sodium acetate, acetate histidine, acetic acid-potassium acetate, calcium acetate acetate, acetic acid-magnesium acetate, and the like.
  • a preferred acetate buffer is acetic acid-sodium acetate.
  • “Pharmaceutical composition” means a mixture comprising one or more compounds described herein, or a physiologically/pharmaceutically acceptable salt or prodrug thereof, with other chemical components, such as physiological/pharmaceutically acceptable Carrier and excipients.
  • the purpose of the pharmaceutical composition is to maintain the stability of the active ingredient of the antibody, promote the administration to the organism, and facilitate the absorption of the active ingredient to exert biological activity.
  • “pharmaceutical composition” and “formulation” are not mutually exclusive.
  • Freeze-dried formulation means a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step of a pharmaceutical composition or a liquid or solution formulation in liquid or solution form.
  • the freeze drying of the present invention includes pre-freezing, primary drying, and secondary drying.
  • the purpose of pre-freezing is to freeze the product to obtain a crystalline solid.
  • the pre-freezing temperature and the pre-freezing speed are two important process parameters.
  • the pre-freezing temperature is set to -45 ° C
  • the pre-freezing speed is set to 1 ° C / min.
  • One drying, also known as main drying is the main stage of sample freeze drying. The goal is to remove the ice from the product while maintaining the shape of the product, minimizing damage to the product. If the temperature and vacuum of one drying are not properly selected, it will cause the product to collapse. Higher temperatures and vacuums will accelerate lyophilization efficiency, but at the same time increase the risk of product collapse.
  • the temperature of the primary drying of the present invention may be a temperature conventional in the art, for example, -27 ° C to 0 ° C, preferably -14 ° C to -5 ° C.
  • Secondary drying also known as analytical drying, is the primary step in removing bound water from an article by pumping an ultimate vacuum (0.01 mbar) and elevated temperature (20-40 °C). Since most biologics are sensitive to temperature, the secondary drying temperature is chosen to be at the low end of the temperature range, ie 25 °C.
  • the freeze-drying time is related to the freezer, the lyophilized formulation dose, and the lyophilized drug container. Such adjustments in time are well known to those skilled in the art.
  • the pharmaceutical composition of the present invention is capable of achieving a stable effect: a pharmaceutical composition in which the antibody substantially retains its physical stability and/or chemical stability and/or biological activity after storage, preferably, a drug
  • the composition retains substantially its physical and chemical stability and its biological activity upon storage.
  • the shelf life is generally selected based on the predetermined shelf life of the pharmaceutical composition.
  • a stable pharmaceutical antibody preparation is one in which no significant change is observed in the following conditions: storage at a refrigerating temperature (2-8 ° C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years.
  • stable liquid preparations include liquid preparations which exhibit desirable characteristics after storage at temperatures including 25 ° C and 40 ° C including periods of 1 month, 3 months, and 6 months.
  • Typical acceptable criteria for stability are as follows: typically no more than about 10%, preferably no more than about 5%, of antibody monomers are degraded by SEC-HPLC.
  • the pharmaceutical antibody formulation is colorless or clear to slightly milky white.
  • the concentration, pH and osmolality of the formulation have a variation of no more than ⁇ 10%.
  • a truncation of no more than about 10%, preferably no more than about 5% is generally observed.
  • Aggregation is generally formed no more than about 10%, preferably no more than about 5%.
  • the antibody did not show significant aggregation increase, precipitation and/or denaturation if visually examined for color and/or clarity, or by UV light scattering, size exclusion chromatography (SEC) and dynamic light scattering (DLS). Then the antibody "retains its physical stability" in the pharmaceutical preparation. Changes in protein conformation can be assessed by fluorescence spectroscopy, which determines the tertiary structure of the protein, and by FTIR spectroscopy, which determines the secondary structure of the protein.
  • the antibody does not show a significant chemical change, the antibody "retains its chemical stability" in the pharmaceutical formulation.
  • Chemical stability can be assessed by detecting and quantifying chemically altered forms of the protein.
  • Degradation processes that frequently alter the chemical structure of a protein include hydrolysis or truncation (evaluated by methods such as size exclusion chromatography and SDS-PAGE), oxidation (by peptide mapping such as mass spectrometry or MALDI/TOF/MS) Method to evaluate), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing, peptide spectroscopy, isoaspartic acid measurement, etc.) and isomerization (by measuring the content of isoaspartic acid, Peptide mapping, etc. to evaluate).
  • An antibody "retains its biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within a predetermined range of biological activity exhibited in the preparation of the pharmaceutical formulation.
  • the biological activity of an antibody can be determined, for example, by antigen binding assays.
  • antibody refers to an immunoglobulin, which is a tetrapeptide chain structure in which two identical heavy chains and two identical light chains are linked by interchain disulfide bonds.
  • the antibody light chain of the present invention may further comprise a light chain constant region comprising a human or murine kappa, lambda chain or a variant thereof.
  • the antibody heavy chain of the present invention may further comprise a heavy chain constant region comprising IgG1, IgG2, IgG3, IgG4 or a variant thereof of human or murine origin.
  • variable region The sequences of about 110 amino acids near the N-terminus of the antibody heavy and light chains vary greatly, being the variable region (Fv region); the remaining amino acid sequences near the C-terminus are relatively stable and are constant regions.
  • the variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions determine the specificity of the antibody, also known as the complementarity determining region (CDR).
  • CDR complementarity determining region
  • Each of the light chain variable region (LCVR) and the heavy chain variable region (HCVR) consists of three CDR regions and four FR regions, and the order from the amino terminus to the carboxy terminus is: FR1, CDR1, FR2, CDR2. FR3, CDR3, FR4.
  • the three CDR regions of the light chain refer to LCDR1, LCDR2, and LCDR3; the three CDR regions of the heavy chain refer to HCDR1, HCDR2, and HCDR3.
  • the CDR amino acid residues of the LCVR region and the HCVR region of the antibody or antigen-binding fragment of the present invention conform to the known Kabat numbering rules (LCDR1-3, HCDE2-3) in number and position, or conform to the numbering rules of kabat and chothia. (HCDR1).
  • the antibody of the present invention includes a murine antibody, a chimeric antibody, a humanized antibody, preferably a humanized antibody.
  • murine antibody is in the present invention a monoclonal antibody to human PCSK-9 prepared according to the knowledge and skill in the art.
  • the test subject is injected with the PCSK-9 antigen at the time of preparation, and then the hybridoma expressing the antibody having the desired sequence or functional properties is isolated.
  • chimeric antibody is an antibody obtained by fusing a variable region of a murine antibody with a constant region of a human antibody, and can alleviate an immune response induced by a murine antibody.
  • a hybridoma that secretes a murine-specific monoclonal antibody is first established, and then the variable region gene is cloned from the mouse hybridoma cell, and the mouse variable region gene is cloned as needed, and the mouse variable region is cloned.
  • the gene is ligated into a human vector after being ligated into a human chimeric gene, and finally the chimeric antibody molecule is expressed in a eukaryotic industrial system or a prokaryotic industrial system.
  • the antibody light chain of the PCSK-9 chimeric antibody further comprises a light chain constant region of a human kappa, lambda chain or variant thereof.
  • the antibody heavy chain of the PCSK-9 chimeric antibody further comprises a heavy chain constant region of human IgGl, IgG2, IgG3, IgG4 or variants thereof.
  • the constant region of a human antibody may be selected from the heavy chain constant region of human IgGl, IgG2, IgG3 or IgG4 or variants thereof, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or without ADCC after amino acid mutation (antibody-dependent) Cell-mediated cytotoxicity, antibody-dependent cell-mediated cytotoxicity) toxic IgG4.
  • humanized antibody also known as CDR-grafted antibody, refers to the transplantation of mouse CDR sequences into human antibody variable region frameworks, ie different types of human germline An antibody produced in an antibody framework sequence. It is possible to overcome the strong antibody variable antibody response induced by chimeric antibodies by carrying a large amount of mouse protein components.
  • framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references.
  • the germline DNA sequences of human heavy and light chain variable region genes can be found in the "VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, EA, etc.
  • humanized antibodies of the invention also include humanized antibodies that are further affinity matured by phage display.
  • PCSK-9 antibody is an antibody that specifically binds to PCSK-9, including but not limited to the h001 series of PCSK-9 humanized antibodies of PCT/CN2016/111053. Among them, h001 series PCSK-9 humanized antibody was screened by human PCSK-9 immunized mice and obtained by humanized transformation.
  • the "antigen-binding fragment” as used in the present invention refers to a Fab fragment having antigen-binding activity, a Fab' fragment, a F(ab')2 fragment, and a ScFv fragment which binds to human PCSK-9 and others utilizing energy and human A fragment of a human PCSK-9 formed by the VH and VL regions of a PCSK-9-conjugated antibody; comprising one or more CDRs of the antibody of the invention selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: Area.
  • the Fv fragment contains the antibody heavy chain variable region and the light chain variable region, but has no constant region and has the smallest antibody fragment of the entire antigen binding site.
  • Fv antibodies also comprise a polypeptide linker between the VH and VL domains and are capable of forming the desired structure for antigen binding.
  • the two antibody variable regions can also be joined by a different linker into a single polypeptide chain, referred to as a single chain antibody or a single chain Fv (sFv).
  • binding to PCSK-9 refers to the ability to interact with human PCSK-9.
  • antigen binding site refers to a three-dimensional spatial site recognized by the present PCSK-9 antibody or antigen-binding fragment, either continuously or discontinuously on the antigen.
  • the antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in a non-human CDR region.
  • the human FR germline sequence can be obtained from the ImMunoGeneTics (IMGT) website http://imgt.cines.fr by comparing the IMGT human antibody variable region germline gene database and MOE software, or from the Immunoglobulin Journal, 2001 ISBN 014441351. obtain.
  • the engineered antibodies or antigen-binding fragments of the invention can be prepared and purified by conventional methods.
  • cDNA sequences encoding heavy and light chains can be cloned and recombined into GS expression vectors.
  • the recombinant immunoglobulin expression vector can stably transfect CHO cells.
  • mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site of the Fc region.
  • Stable clones were obtained by expressing antibodies that specifically bind to human PCSK-9. Positive clones were expanded in serum-free medium in a bioreactor to produce antibodies.
  • the culture medium from which the antibody is secreted can be purified by a conventional technique.
  • purification is carried out using an A or G Sepharose FF column containing an adjusted buffer.
  • the non-specifically bound components are washed away.
  • the bound antibody was eluted by a pH gradient method, and the antibody fragment was detected by SDS-PAGE and collected.
  • the antibody can be concentrated by filtration in a conventional manner. Soluble mixtures and multimers can also be removed by conventional methods such as molecular sieves, ion exchange.
  • the resulting product needs to be frozen immediately, such as -70 ° C, or lyophilized.
  • Constantly modified refers to amino acids in other amino acid substitution proteins having similar characteristics (eg, charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that Changes are made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitution of structurally or functionally similar amino acids is unlikely to disrupt biological activity.
  • Amino acid sequence "identity” refers to sequence similarity between two proteins or polypeptides. When the positions in both comparison sequences are occupied by the same amino acid residue, for example if one position of both polypeptides is occupied by the same amino acid residue, then the molecules are identical at that position.
  • Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al, respectively. (1977) Nucleic Acids Res. 25: 3389-3402.
  • Software for performing BLAST analyses is publicly available at the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • administering when applied to an animal, human, experimental subject, cell, tissue, organ or biological fluid, refers to an exogenous drug, therapeutic agent, diagnostic agent or composition and animal, human, subject Contact of the test subject, cell, tissue, organ or biological fluid.
  • administering can refer to, for example, therapeutic, pharmacokinetic, diagnostic, research, and experimental methods.
  • Treatment of the cells includes contact of the reagents with the cells, and contact of the reagents with the fluid, wherein the fluids are in contact with the cells.
  • administeristering and “treating” also means treating, for example, cells in vitro and ex vivo by reagents, diagnostics, binding compositions, or by another cell.
  • Treatment when applied to a human, veterinary or research subject, refers to therapeutic treatment, prophylactic or preventive measures, research and diagnostic applications.
  • Treatment means administering to a patient a therapeutic agent for internal or external use, for example a composition comprising any of the binding compounds of the present invention, the patient having one or more symptoms of the disease, and the therapeutic agent is known to have Therapeutic effect.
  • a therapeutic agent is administered in a subject or population to be treated to effectively alleviate the symptoms of one or more diseases to induce such symptoms to degenerate or to inhibit the progression of such symptoms to any clinically measurable extent.
  • the amount of therapeutic agent also referred to as "therapeutically effective amount" effective to alleviate the symptoms of any particular disease can vary depending on a variety of factors, such as the patient's disease state, age and weight, and the ability of the drug to produce a desired effect in the patient.
  • Whether the symptoms of the disease have been alleviated can be assessed by any clinical test method commonly used by a physician or other professional health care provider to assess the severity or progression of the condition. While embodiments of the invention (e.g., methods of treatment or preparations) may be ineffective in ameliorating the symptoms of each target disease, any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • any statistical test methods known in the art such as Student's t-test, chi-square test, according to Mann and Whitney U-test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test determined that the target disease symptoms should be alleviated in a statistically significant number of patients.
  • an "effective amount” includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical condition.
  • An effective amount also means an amount sufficient to allow or facilitate the diagnosis.
  • An effective amount for a particular patient or veterinary subject can vary depending on factors such as the condition to be treated, the overall health of the patient, the methodological route and dosage of the administration, and the severity of the side effects.
  • An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
  • Tm value refers to the heat denaturation temperature of the protein, that is, the temperature at which half of the protein is unfolded, and the spatial structure of the protein is destroyed at this time, so the higher the Tm value, the higher the thermal stability of the protein.
  • Replacement refers to the replacement of a solvent system in which an antibody protein is solubilized, for example, a buffer system using a stable formulation is physically manipulated to replace a high salt or hypertonic solvent system containing an antibody protein such that the antibody protein is present in a stable formulation.
  • the so-called physical modes of operation include, but are not limited to, ultrafiltration, dialysis, or reconstitution after centrifugation.
  • the UniProt Proprotein convertase subtilisin/kexin type 9 (human PCSK9, Uniprot number: Q8MBP7) was used as a template for the PCSK9 of the present invention, and the amino acid sequences of the antigen and the detection protein of the present invention were designed, and the fusion was further based on the PCSK9 protein.
  • Labels such as his-tag or immuno-promoting peptides such as PADRE peptides were cloned into pTT5 vector (Biovector, Cat#: 102762) or pTargeT vector (promega, A1410), transiently expressed in 293 cells or stably expressed in CHO-S. Purification, obtaining the antigen encoding the present invention and the protein for detection.
  • PCSK9-His6 for immunogenic mice or detection reagents
  • the horizontal line is the signal peptide and the italic part is the His6-tag (6 histidine tag).
  • PCSK9 PCSK9-PADRE-His6 with PADRE peptide and His tag, as an immunogen, the PADRE peptide contained can promote immunity;
  • the horizontal line is the signal peptide
  • the double-line part is the linker
  • the dotted line is the PADRE peptide
  • the italic part is the His6-tag.
  • PCSK9 and his-tag fusion protein with PCV cleavage site PCSK9-TEV-His6, N-PCSK9 (N-terminal pCSK9 domain) can be obtained by TEV digestion as an immunogen;
  • the horizontal line is the signal peptide
  • the double-lined part is the TEV cleavage site
  • the italic part is the His6-tag.
  • PCSK9-D374Y mutant protein with his tag PCSK9-D374Y-His6 as a detection reagent
  • the horizontal line is the signal peptide and the italic part is the His6-tag.
  • PCSK9 PCSK9 protein inserted into biotin-accepting peptide BP15 and his tag: PCSK9-BP15-His6, as a detection reagent, the position of BP15 peptide can be biotinylated during expression, exempting in vitro biotin labeling and possible conformational changes;
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the biotin acceptor peptide
  • the italicized portion is the His6-tag.
  • PCSK9-Y PCSK9D374Y mutant protein inserted into biotin acceptor peptide and his tag: PCSK9-D374Y-BP15-His6, detection protein;
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the biotin acceptor peptide
  • the italicized portion is the His6-tag.
  • FcLR extracellular domain fragment of pCSK9 receptor protein with Flag tag and His tag LDLR-ECD-Flag-His6, detection reagent
  • the horizontal line is the signal peptide
  • the double-line part is the Flag label
  • the italic part is the His6-tag
  • LDLR-Fc a shortened form of the LDLR extracellular domain fragment and the hIgG1-Fc fusion protein (having binding activity to PCSK9): LDLR-sECD-Fc (hIgG1) as a detection reagent
  • the cross-hatched portion is a signal peptide
  • the double-scored portion is a shortened form of an LDLR extracellular domain fragment (LDLR-sECD) having a binding activity to PCSK9
  • the italicized portion is a hIgG1-Fc portion
  • the cross-hatched portion is the signal peptide
  • the double-lined portion is the more shortened form of the LDLR extracellular domain fragment (LDLR-ssECD) with PCSK9 binding activity
  • the italicized portion is the hIgG1-Fc portion.
  • Example 2 Purification of recombinant protein of recombinant protein of PCSK9 and LDLR, purification of hybridoma antibody and recombinant antibody
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, and the buffer was exchanged for PBS, and imidazole was added to a final concentration of 5 mM.
  • the nickel column was equilibrated with PBS solution containing 5 mM imidazole and rinsed 2-5 column volumes.
  • the displaced supernatant sample was placed on an IMAC column.
  • the column was washed with PBS containing 5 mM imidazole until the A280 reading dropped to baseline.
  • the column was washed with PBS + 10 mM imidazole, the non-specifically bound heteroprotein was removed, and the effluent was collected.
  • the protein of interest was eluted with PBS containing 300 mM imidazole, and the eluted peak was collected.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex 200 (GE), and the mobile phase was PBS. Depolymerized peaks were collected and the eluted peaks were collected.
  • the obtained protein was identified by electrophoresis, peptide mapping, LC-MS as correct and sub-equipment.
  • PCSK9-His6 His-tagged PCSK9-His6 (SEQ ID NO: 1), PCSK9-PADRE-His6 (SEQ ID NO: 2), PCSK9-TEV-His6 (SEQ ID NO: 3) PCSK9-D374Y-His6 (SEQ ID NO) : 4), PCSK9-BP15-His6 (SEQ ID NO: 5), PCSK9-D374Y-BP15-His6 (SEQ ID NO: 6) is used as an immunogen or detection reagent for the antibody of the present invention.
  • PCSK9-TEV-His6 was purified and digested by TEV enzyme, and the digested product was used to remove TEV enzyme, uncut intact PCSK9-TEV-His6 or excised His-tagged C-terminal domain fragment by IMAC column.
  • the IMAC effluent was concentrated to obtain a PCSK9 fragment (abbreviated as N-PCSK9) leaving only the N-terminal domain, and used as an immunogen for mouse immunization.
  • the sample was centrifuged at high speed to remove impurities and concentrated to an appropriate volume.
  • the flag affinity column was equilibrated with 0.5 x PBS and washed 2-5 column volumes.
  • the supernatant cells were subjected to supernatant analysis and the supernatant samples were applied to the column.
  • the column was rinsed with 0.5 x PBS until the A 280 reading dropped to baseline.
  • the column was washed with PBS containing 0.3 M NaCl, and the protein was washed and collected.
  • the protein of interest was eluted with 0.1 M acetic acid (pH 3.5-4.0) and collected to adjust the pH to neutral.
  • the collected eluate was concentrated and further purified by gel chromatography Superdex 200 (GE), and the mobile phase was PBS.
  • LDLR-ECD-Flag-His6 SEQ ID NO: 7 with FLAG/His6 tag was obtained for performance testing of the antibodies of the present invention.
  • the cell expression supernatant sample was centrifuged at high speed to remove impurities, and concentrated to an appropriate volume and applied to a Protein A column. Rinse the column with PBS until the A280 reading drops to baseline.
  • the protein of interest was eluted with 100 mM sodium acetate pH 3.0 and neutralized with 1 M Tris-HCl (tris-hydroxymethylaminomethane-hydrochloric acid). The eluted sample was appropriately concentrated and further purified by PBS-equilibrated gel chromatography Superdex 200 (GE). The peak of the depolymerized product was collected and used.
  • This method was used to purify LDLR-sECD-Fc (hIgG1) (SEQ ID NO: 8) and LDLR-ssECD-Fc (hIgG1) (SEQ ID NO: 9). Both can be used as PCSK9 antibody functional tests.
  • Anti-human PCSK9 monoclonal antibodies are produced by immunizing mice.
  • Experimental SJL white mice female, 6 weeks old (Beijing Weitong Lihua Experimental Animal Technology Co., Ltd., animal production license number: SCXK (Beijing) 2012-0001).
  • Feeding environment SPF level. After the mice were purchased, the laboratory environment was kept for 1 week, 12/12 hours light/dark cycle adjustment, temperature 20-25 ° C; humidity 40-60%. Mice that have adapted to the environment are immunized (A/B) in two regimens, 6-10 per group.
  • the immunizing antigen is His-tagged human PCSK9-His6 (SEQ ID NO: 1), PCSK9-PADRE-His6 (SEQ ID NO: 2), and N-PCSK9 (SEQ ID NO: 3).
  • Protocol A was emulsified with Freund's adjuvant (sigma Lot Num: F5881/F5506): the first use of Freund's complete adjuvant (CFA), and the rest of the booster with Freund's incomplete adjuvant (IFA).
  • the ratio of antigen to adjuvant was 1:1, 100 ⁇ g/only (first aid), 50 ⁇ g/only (boost boost).
  • Scheme B was cross-immunized with Titermax (sigma Lot Num: T2684) and Alum (Thremo Lot Num: 77161).
  • the ratio of antigen to adjuvant (titermax) was 1:1, and the ratio of antigen to adjuvant (Alum) was 3:1, 10-20 ⁇ g/only (first exempt), and 5 ⁇ g/only (boosting).
  • mice in the serum were selected to have high antibody titers (see Test Methods 1 and 2 below, combined with the ELISA method of PCSK9) and spleen cell fusion was performed in mice with titer-to-platform, and the selected mice were spurted 72 hours before the fusion, PCSK9- His 610 ⁇ g/only, intraperitoneal injection.
  • Spleen lymphocytes and myeloma cell Sp2/0 cells were optimized using an optimized PEG-mediated fusion step ( CRL-8287 (TM ) was fused to obtain hybridoma cells.
  • HAT complete medium RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • RPMI-1640 medium containing 20% FBS, 1 ⁇ HAT and 1 ⁇ OPI
  • 96-well cell culture plates 1 ⁇ 10 5 /150 ⁇ l). /well
  • HAT complete medium was added, 50 ⁇ l/well, and incubated at 37 ° C, 5% CO 2 .
  • the medium was HT complete medium (RPMI-1640 medium containing 20% FBS, 1 ⁇ HT and 1 ⁇ OPI), 200 ⁇ l/well. Incubate at 37 ° C, 5% CO 2 .
  • ELISA assays in combination with PCSK9 or PCSK9-Y were performed according to cell growth density (see Test Examples 1 and 2).
  • the positive well cells combined with ELISA were subjected to a blocking ELISA assay for binding of PCSK9 or PCSK9-Y to LDLR (see Test Examples 3 and 4), and the positive wells were exchanged and expanded into 24-well plates according to cell density.
  • the cell line transferred into the 24-well plate was subjected to retesting and then subjected to seed conservation and first subcloning.
  • the first subcloning screen (see Test Examples 1 and 2) was positive for conservation and a second subcloning.
  • the second subcloning was positive (see Test Examples 1 and 2) for conservation and protein expression. Multiple fusions were obtained to obtain hybridoma cells that blocked the binding of PCSK9 or PCSK9-Y to LDLR (see Test Examples 3 and 4).
  • the hybridoma clone mAb-001 was screened by blocking assay and binding assay, and the antibody was further prepared by serum-free cell culture, and the antibody was purified according to the purification example for use in the test example.
  • the murine anti-variable region sequences of the hybridoma clone mAb-001 were determined as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • the two murine antibodies were selected by aligning the IMGT human antibody heavy light chain variable region germline gene database and MOE software, respectively, to select the heavy and light chain variable region germline gene with high homology to mAb-001 as a template.
  • the CDRs were each transplanted into the corresponding human template to form a variable region sequence in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
  • the amino acid residues are determined and annotated by the Kabat numbering system.
  • the humanized light chain template of the murine antibody mAb-001 was IGKV1-39*01 and hjk2.1, and the humanized heavy chain template was IGHV1-2*02 and hjh2. After humanization, the humanized antibody h001- was obtained.
  • the sequence of the variable region of 1 is as follows:
  • the sequence is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and the italicized FR sequence in the sequence, underlined as the CDR sequence.
  • variable region of the humanized antibody h001-1 consists of the light chain h001_VL1 and the heavy chain h001_VH.1A. Other analogies.
  • the above humanized sequences were combined for antibodyization, the heavy chain constant region was derived from human IgG1, and the light chain constant region was derived from human kappa chain.
  • the corresponding humanized antibody was obtained, and the verified PCSK9 antibody has higher binding activity to PCSK9 and PCSK9-Y; and can effectively block the binding between PCSK9/PCSK9-Y and LDLR.
  • the method for constructing and expressing an anti-human PCSK9 humanized antibody of the present invention is as follows:
  • Primer design use the online software DNAWorks(v3.2.2)(works/) to design multiple primers to synthesize VH/VK gene fragment containing recombinant: 5'-30bp Signal peptide+VH/VK+30bp CH1/CL-3 '.
  • Primer design principle The target gene 2 is different from the target gene 1 by 2 aa, and the primer of the mutation site is also set, as shown in Fig. 1.
  • the expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) was designed using some special restriction enzymes, such as BsmBI, to distinguish the recognition sequence from the restriction site, as shown in Figure 2. Show. The vector was digested with BsmBI, and the gel was recovered for use.
  • BsmBI signal peptide and constant region gene
  • VH/VK contains the gene fragment required for recombination and BsmBI digestion and expression vector pHr (with signal peptide and constant region gene (CH1-FC/CL) fragment) was added to DH5 ⁇ competent cells in a ratio of 3:1, ice at 0 °C Bath for 30 min, heat shock at 42 °C for 90 seconds, add 5 volumes of LB medium, incubate for 45 min at 37 ° C, coat LB-Amp plate, incubate overnight at 37 ° C, pick monoclonal and send to sequencing to obtain each clone.
  • pHr signal peptide and constant region gene
  • the antibodies of the invention may be, but are not limited to, the above design constructs.
  • the antibody and its mutant design were carried out by taking h001-4 as an example to obtain the IgG1 form of 1h001-4-WT: h001-4, that is, the humanized sequence combination h001-4, which binds to the heavy chain constant region derived from human IgG1, and The light chain constant region of the human kappa chain; the 2h001-4-YTE: h001-4-IgG1-YTE form, ie the humanized sequence combination h001-4, binds to the heavy chain constant region of the mutated human IgG1 (YTE mutation), A light chain constant region from a human kappa chain.
  • the mutated human IgG1 may also be a mutation of another form.
  • the heavy chain constant region is derived from human IgG1 and the light chain constant region is derived from the human kappa light chain:
  • Heavy chain amino acid sequence (human IgG1):
  • h001-4-YTE light chain is h001-4-kappa: SEQ ID NO: 30
  • the h001-4-YTE heavy chain amino acid sequence is:
  • the first step take a certain amount of purified PCSK-9 antibody (such as h001-4-YTE) stock solution, solvent replacement with antibody-free buffer (such as 10mM, pH 6.0 histidine-hydrochloride buffer) (preferably ultrafiltration), the protein was concentrated to 60 mg/mL ( ⁇ 2 mg/mL) by 6-volume replacement of the ultrafiltration membrane. A volume of sucrose mother liquor was added and mixed to give a final sucrose concentration of 25 mg/mL. Add a certain volume of Tween-80 mother liquor and mix until the final Tween-80 concentration is 0.2 mg/mL.
  • solvent replacement with antibody-free buffer such as 10mM, pH 6.0 histidine-hydrochloride buffer
  • the volume was adjusted to a concentration of 50 mg/mL ( ⁇ 5 mg/mL) with 10 mM pH 6.0 histidine buffer, and the final product pH was about 6.3 ⁇ 0.1 (other formulations to be tested or stable formulations were prepared by reference to similar procedures).
  • the product is filtered and then controlled for sampling and testing.
  • the stock solution was passed through a 0.22 ⁇ m PVDF filter and the filtrate was collected.
  • the second step adjust the loading to 3.6ml, fill the filtrate in a 6ml vial, half-plug, respectively, at the beginning of filling, filling, at the end of filling, sampling and control the difference in loading.
  • the third step the liquid medicine after filling and filling is filled into a freeze-drying box and lyophilized.
  • the lyophilization procedures are pre-freezing, primary drying and secondary drying. After the lyophilization procedure is finished, the vacuum is stoppered.
  • the time used for lyophilization can be adjusted according to the actual situation.
  • the type of lyophilizer, the loading of lyophilized pharmacy, and the container of lyophilized medicinal products will affect the lyophilization time.
  • Such adjustments in time are well known to those skilled in the art.
  • the fourth step open the capping machine, add the aluminum cover, and carry out the capping.
  • Step 5 Visual inspection to confirm that the product has no defects such as collapse and inaccurate loading.
  • Print and paste the vial label print the tray label, fold the tray, box, and sticker box labels.
  • An anti-PCSK-9 antibody h001-4-YTE preparation with a protein concentration of 1 mg/ml was prepared using the following buffer:
  • the thermal stability of the anti-PCSK-9 antibody in each preparation was measured by differential scanning calorimetry (DSC).
  • DSC differential scanning calorimetry
  • Tm midpoint temperature
  • Table 5 The results are shown in Table 5. Therefore, a buffer between pH 6.0 and 6.5 was selected: histidine-hydrochloride, sodium dihydrogen phosphate-disodium hydrogen phosphate, sodium succinate-succinate, citric acid-sodium citrate for subsequent studies.
  • N/A means that this ingredient is not added.
  • An anti-PCSK-9 antibody preparation having a protein concentration of 1 mg/ml was prepared using the following buffer:
  • the thermal stability of the anti-PCSK-9 antibody in each preparation was measured by differential scanning calorimetry (DSC).
  • DSC differential scanning calorimetry
  • Tm thermal denaturation midpoint temperature
  • N/A means that this ingredient is not added.
  • An anti-PCSK-9 antibody preparation having a PCSK-9 antibody protein concentration of 150 mg/ml was prepared using the following buffers of different sugar types:
  • the osmotic pressure value indicates that the osmotic pressure meets the minimum subcutaneous injection requirement when sucrose is ⁇ 70 mg/ml.
  • the PCSK-9 antibody preparation of the invention is prepared by using a rare and concentrated solution (three times volume of the original solution for lyophilization, one volume of water for reconstitution by injection, and the invention is uniformly freeze-dissolved according to the invention), and the final prescription ( After reconstitution, the concentration of sugar in the preparation is 75 mg/ml, which satisfies the osmotic pressure requirement of the injection, and is convenient to set the sugar concentration in the original solution to 25 mg/ml, and the sugar concentration of 25 mg/ml is easy to freeze-dry. Therefore, the sugar concentration in the final formulation of the preparation was selected to be 75 mg/ml, and the sugar concentration in the original solution was set to 25 mg/ml.
  • N/A means that this ingredient is not added.
  • An anti-PCSK-9 antibody preparation having a PCSK-9 antibody protein concentration of 150 mg/ml was prepared using the following buffers of different concentrations of surfactant:
  • An anti-PCSK-9 antibody preparation containing a PCSK-9 antibody protein concentration of 50 mg/ml and containing 25 mg/ml sucrose was prepared in a buffer containing the following different concentrations of surfactant, different pH, and different ionic strength:
  • Each formulation was filtered and lyophilized in a 6 mL neutral borosilicate glass controlled injection vial filled with 3.6 mL/bottle, which was sealed with a halogenated butyl rubber stopper using a freeze-dried sterile powder.
  • the samples were stored at 40 ° C for stability analysis, and the data were analyzed by the difference of SEC monomers. The results showed that the ionic strength was pH 6.0, 10 mM at a protein concentration of 50 mg/ml and 25 mg/ml sucrose.
  • the histidine-hydrochloride buffer and the polysorbate 80 containing 0.2 mg/ml were formulated to stabilize the PCSK-9 antibody.
  • the concentration of each component in the final preparation was determined to be 3 times of the lyophilized stock solution (protein concentration 150 mg/ml, 75 mg/ml sucrose, 30 mM histidine-hydrochloride buffer, 0.6 Mg/ml polysorbate 80), pH 6.3 ⁇ 0.1.
  • An anti-PCSK-9 antibody having a PCSK-9 antibody protein concentration of 50 mg/ml, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80 was prepared using a buffer containing 10 mM histidine-hydrochloride at pH 6.0. preparation.
  • the antibody was filled into a 6 mL vial at 3.6 mL/vial, lyophilized at a drying temperature of -14 ° C and -5 ° C, respectively, and sealed with a lyophilized rubber stopper.
  • the reconstituted sample was compared before and after lyophilization. The results show that -5 ° C is the preferred primary drying temperature of the freeze-drying process.
  • H001-4-YTE was formulated at 50 mg/ml in pH 6.0, 10 mM histidine-hydrochloride, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80.
  • the preparations were filled in glass bottles, liquid storage bags and 316L stainless steel tanks, respectively, and left at 2-8 ° C for 24 hours. Analysis of protein content and purity indicated that H001-4-YTE was stable within 24 hours.
  • the formulation is compatible with 316L stainless steel cans, glass bottles and liquid storage bags.
  • H001-4-YTE was formulated at 50 mg/ml in pH 6.0, 10 mM histidine-hydrochloride buffer, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80. The formulations were passed through a 0.22 ⁇ m PES filter and a PVDF filter and sampled at 30 min and 1 h. Analysis of protein content, appearance and purity indicated that H001-4-YTE was stable within 1 hour of contact with the filter. The formulation is compatible with both PES and PVDF filters.
  • the pharmaceutical composition of the present invention can be used as a stock solution of a pharmaceutical preparation or directly as an injection.
  • a lyophilized preparation is prepared by a lyophilization process, and the lyophilized preparation is reconstituted into an injection solution for clinical use.
  • the invention prepares a lyophilized preparation according to a process of thinning and concentrated, that is, lyophilizes a low-concentration concentration of the pharmaceutical preparation to obtain a lyophilized preparation, and re-dissolves the lyophilized preparation into a higher concentration of the pharmaceutical composition for use.
  • the lyophilized formulation has a longer shelf stability than the liquid formulation.
  • the stock solution of the pharmaceutical preparation used in the lyophilized preparation the higher the concentration of each component, the longer the lyophilization time.
  • the concentration of each component of the drug preparation stock solution is usually 2-5 times the concentration of each component of the injection solution after reconstitution, and the preferred embodiment of the present invention is 3 times.
  • the stable pharmaceutical formulation provided by the present invention comprises: a PCSK-9 antibody protein (non-limiting embodiment such as h001-4-YTE) and a combination of any of the following stable buffers:
  • antibody h001-4-YTE 150mg / ml, 75mg / ml sucrose, 0.6mg / ml of polysorbate 80, and 30mM histidine-hydrochloride buffer, the final pH of 6.4;
  • antibody h001-4-YTE 50mg / ml, 25mg / ml sucrose, 0.2mg / ml of polysorbate 80, and 10mM histidine-hydrochloride buffer, the final pH of 6.4;
  • antibody h001-4-YTE 50mg / ml, 25mg / ml sucrose, 0.2mg / ml of polysorbate 80, and 10mM histidine-hydrochloride buffer, the final pH of 6.3;
  • antibody h001-4-YTE 50mg / ml, 25mg / ml sucrose, 0.2mg / ml of polysorbate 80, and 10mM histidine-hydrochloride buffer, the final pH of 6.2;
  • antibody h001-4-YTE 50mg / ml, 25mg / ml sucrose, 0.2mg / ml of polysorbate 80, and 10mM histidine-hydrochloride buffer, the final pH of 6.1;
  • antibody h001-4-YTE 50mg / ml, 25mg / ml sucrose, 0.2mg / ml of polysorbate 80, and 10mM histidine-hydrochloride buffer, the final pH of 6.0;
  • antibody h001-4-YTE 50mg / ml, 25mg / ml sucrose, 0.2mg / ml of polysorbate 80, and 10mM histidine-hydrochloride buffer, the final pH of 6.5;
  • antibody h001-4 50mg / ml, 25mg / ml sucrose, 0.2mg / ml of polysorbate 80, and 10mM histidine-hydrochloride buffer, the final pH of 6.4;
  • antibody h001-4-YTE 50mg / ml, 25mg / ml sucrose, 0.1mg / ml of polysorbate 80, and 20mM histidine-hydrochloride buffer, the final pH of 6.3;
  • antibody h001-4-YTE 50mg / ml, 25mg / ml sucrose, 0.2mg / ml of polysorbate 80, and 15mM histidine-hydrochloride buffer, the final pH of 6.2;
  • Antibody h001-4-YTE 50 mg/ml, 25 mg/ml sucrose, 0.2 mg/ml polysorbate 80, and 20 mM histidine-hydrochloride buffer, final pH 6.4.
  • antibody h001-4-YTE 30 mg/ml, 10 mg/ml sucrose, 0.05 mg/ml polysorbate 80, and 5 mM histidine-hydrochloride buffer having a pH of 5.5;
  • antibody h001-4-YTE 45 mg/ml, 20 mg/ml sucrose, 0.1 mg/ml polysorbate 80, and 8 mM histidine-hydrochloride buffer having a pH of 6.0;
  • Antibody h001-4-YTE 55 mg/ml, 40 mg/ml sucrose, 0.3 mg/ml polysorbate 80, and 15 mM histidine-hydrochloride buffer having a pH of 6.2.
  • the pharmaceutical composition of the present invention can be prepared into a corresponding lyophilized preparation by a lyophilization process, and the lyophilized preparation can be reconstituted with an injection water to obtain the following drug groups and substances for clinical use:
  • antibody PCSK-9 antibody protein 150 mg / ml, 75 mg / ml sucrose, 0.6 mg / ml polysorbate 80, and 20 mM histidine-hydrochloride buffer, the final pH of 6.3 ⁇ 0.1;
  • Antibody PCSK-9 antibody protein 200 mg/ml, 95 mg/ml sucrose, 0.8 mg/ml polysorbate 80, and 30 mM histidine-hydrochloride buffer, final pH 6.5.

Abstract

本发明提供了一种药物组合物，其包含在组氨酸缓冲液中的PCSK9抗体或其抗原结合片段。除此之外，该药物组合物还可含有糖和非离子型表面活性剂。

Description

一种PCSK-9抗体药物组合物及其用途
本申请要求申请日为2017年6月30日的中国专利申请CN201710519829.6的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于药物制剂领域，具体涉及一种包含PCSK-9抗体及其抗原结合片段的药物组合物，以及其作为药物的用途。
背景技术
高胆固醇血症是一种以血清胆固醇水平升高为主要特征的脂类代谢异常疾病，其主要表现为血清胆固醇水平升高，导致胆固醇在血管聚集，形成动脉粥样硬化。大量的临床及实验研究结果都证实，脂质代谢异常和冠心病的发生、发展有着密切的关系。因此，降低血液中的胆固醇浓度成为了目前治疗和预防动脉粥样硬化的一个主要手段。
随着我国国民生活水平的快速提高，血脂异常也成为了危害我国城乡居民的主要因素。据2012年统计数据，我国每年死亡人数中约有40％死于心血管疾病。目前，我国成人血脂异常患病率为18.6％，估计全国血脂异常现患人数1.6亿。不同类型的血脂异常现患率分别为：高胆固醇血症2.9％，高甘油三酯血症11.9％，低高密度脂蛋白血症7.4％；另有3.9％的人血胆固醇边缘升高。2012年***疾病预防控制专家委员会慢性病防治分委会达成的“慢性病防治中国专家共识”中提到，我国有3300万高胆固醇血症患者，而从局部地区看，我国血脂异常发病率情况远比上述数据要严重。
目前，临床上调脂药物主要以他汀类为主。立普妥(Liptor)作为全世界应用最广泛的降胆固醇药物，也是医药史上最畅销药物，通过阻断肝脏生产胆固醇的酶作用，减少胆固醇的生产，从而增加肝脏从血液中摄取更多的胆固醇，进而减低血液中胆固醇浓度。但是立普妥也有其不足之处，首先从数据看，立普妥可以降低30％-40％的低密度脂蛋白，但仍然有很多病人依然无法到达有效的降低血脂(低密度脂蛋白浓度<50mg/dL)，其次病人对立普妥的响应率也有人种差异。这些原因，致使病人需要一个更为有效的降低血脂的药物。
家族性高胆固醇血症(Familial hypercholesterolemia,FM)是一种常染色体单基因显性遗传性疾病，其临床特征为血总胆固醇和低密度脂蛋白胆固醇(low density lipoprotein-cholesterol,LDL-c)显著升高、睑黄瘤、角膜弓以及早发的心血管疾病。低密 度脂蛋白受体(LDL receptor,LDLR)基因突变致其缺陷或缺乏，LDL-c不能顺利转运到肝脏清除，以致血中LDL-c水平升高。目前已明确3种基因与FM的发生有关，它们分别是LDLR基因、载脂蛋白B100基因和PCSK9(proprotein convertase subtilisin/kexin type9)基因。
前蛋白转化酶枯草溶菌素9(PCSK9)是一种前蛋白转化酶，属于分泌的枯草杆菌酶家族的蛋白酶K亚族。该编码蛋白是作为可溶性酶原合成的，在内质网中经过自身催化分子内加工。实验结果显示，PCSK9促进LDL受体的降解从而增加血浆中LDL胆固醇含量，而LDL受体介导肝内的LDL胞吞过程，后者是从循环***清除LDL的主要途径。研究发现，12.5％的高胆固醇血症(ADH)患者检测有PCSK9基因突变。PCSK9突变形式多样，根据突变对PCSK9调节LDL-C水平的不同影响可分为两类：功能缺失型和功能获得型。其中功能缺失型突变与低血胆固醇水平有关，有预防冠状动脉粥样硬化性心脏病发生的作用，非洲人群中低胆固醇的PCSK9突变率高于其他种族。PCSK9功能获得型突变体通过增加PCSK9的功能、降低LDLR的表达而升高血浆胆固醇水平，可以导致严重高胆固醇血症和早发冠状动脉粥样硬化性心脏病，目前发现的PCSK9功能获得型突变包括：D374Y、S127R、F216L、N157K、R306S等。其中，与PCSK9野生型相比，D374Y突变体细胞表面的LDLR减少了36％，S127R突变有相应减少了10％。
但是，抗体药物其分子量大，结构复杂，容易降解、聚合或发生不希望发生的化学修饰等而变得不稳定。为了使抗体适合于给药，并且在储存及随后使用过程中能保持稳定性，发挥更好的效果，抗体药物的稳定制剂研究显得尤为重要。
尽管，目前有多家公司在研发包含PCSK-9抗体及药物配制剂，如：CN103717237A、CN104364266A等。但对于新的PCSK-9抗体，仍需要研制更适于给药的包含PCSK-9的药物(制剂)组合物。
发明内容
本发明提供一种药物组合物，其包含PCSK-9抗体或其抗原结合片段，以及缓冲液，所述缓冲液优选为组氨酸或琥珀酸盐或磷酸盐或柠檬酸盐缓冲液，更优选为组氨酸缓冲液，最优选为组氨酸-盐酸盐缓冲液。
在可选的实施方案中，药物组合物其中所述PCSK-9抗体或其抗原结合片段浓度为大约1mg/ml至150mg/ml，优选为大约30mg/ml至100mg/ml，最优选为50-60mg/ml，更优选为50mg/ml。非限制性实施例包括45mg/ml、46mg/ml、47mg/ml、48mg/ml、49mg/ml、50mg/ml、51mg/ml、52mg/ml、53mg/ml、54mg/ml、55mg/ml、56mg/ml、57mg/ml、58mg/ml、 59mg/ml、60mg/ml。
在可选的实施方案中，缓冲液的浓度为约5mM至50mM，优选为大约5mM至30mM，非限制性实施例包括10mM、12mM、14mM、16mM、18mM、20mM、22mM、24mM、26mM、28mM、30mM，更优选为10mM至15mM，最优选为10mM。
在可选的实施方案中，药物组合物中所述缓冲液的pH值约为5.5到6.5，优选为大约6.0至6.5，非限制性实施例包括约6.0、约6.1、约6.2、约6.3、约6.4、约6.5。
进一步的，在可选的实施方案中，药物组合物还包含糖。本发明的“糖”包含常规组合物(CH 2O) n及其衍生物，包括单糖、二糖、三糖、多糖、糖醇、还原性糖、非还原性糖等等。可选自葡萄糖、蔗糖、海藻糖、乳糖、果糖、麦芽糖、右旋糖苷、甘油、赤藻糖醇、丙三醇、***糖醇、sylitol、山梨糖醇、甘露醇、密里二糖、松三糖、蜜三糖、甘露三糖、水苏糖、麦芽糖、乳果糖、麦芽酮糖、山梨醇、麦芽糖醇、乳糖醇、异-麦芽酮糖等等。优选的糖是非还原性二糖，更优选为海藻糖或蔗糖。
在可选的实施方案中，药物组合物中糖的浓度为约10mg/ml至约75mg/ml，优选为20mg/ml至约60mg/ml，更优选为20mg/ml至约40mg/ml，最优选为25mg/ml，非限制性实施例包括20mg/ml、21mg/ml、22mg/ml、23mg/ml、23mg/ml、24mg/ml、25mg/ml、26mg/ml、27mg/ml、28mg/ml、29mg/ml、30mg/ml、31mg/ml、32mg/ml、33mg/ml、34mg/ml、35mg/ml、36mg/ml、37mg/ml、38mg/ml、39mg/ml、40mg/ml。
在可选的实施方案中，药物组合物还包含表面活性剂。可选自聚山梨醇酯20、聚山梨醇酯80、聚羟亚烃、Triton、十二烷基磺酸钠、月桂基磺酸钠、辛基糖甙钠、月桂基-、肉豆蔻基-、亚油基-、硬脂基-磺基甜菜碱、月桂基-、肉豆蔻基-、亚油基-、硬脂基-肌氨酸、亚油基-、肉豆蔻基-、鲸蜡基-甜菜碱、月桂酰胺基丙基-、柯卡酰胺基丙基-、亚油酰胺基丙基-、肉豆蔻酰胺基丙基-、棕榈酰胺基丙基-、异硬脂酰胺基丙基-甜菜碱、肉豆蔻酰胺基丙基-、棕榈酰胺基丙基-、异硬脂酰胺基丙基-二甲基胺、甲基可可酰基钠、甲基油基牛磺酸钠、聚乙二醇、聚丙二醇、乙烯与丙烯二醇的共聚物等等。优选的表面活性剂是聚山梨醇酯80或聚山梨醇酯20，更优选为聚山梨醇酯80。
在可选的实施方案中，药物组合物中表面活性剂的浓度为约0.05mg/ml至1.0mg/ml，优选为0.1mg/ml至0.4mg/ml，非限制性实施例包括0.1mg/ml、0.15mg/ml、0.2mg/ml、0.25mg/ml、0.3mg/ml、0.35mg/ml、0.4mg/ml，进一步优选为0.1mg/ml至0.3mg/ml，更优选为0.1mg/ml至0.2mg/ml，最优选为0.2mg/ml。
在可选的实施方案中，所述药物组合物包含：
(a)1-150mg/ml的PCSK-9抗体或其抗原结合片段，
(b)5-30mM的组氨酸缓冲液，pH约为5.5-6.5，更优选为大约6.0-6.5
(c)10-75mg/ml的蔗糖，和
(d)0.05-0.6mg/ml的聚山梨醇酯80，。
在可选的实施方案中，所述药物组合物包含：
(a)50mg/ml的PCSK-9抗体或其抗原结合片段；
(b)5-20mM的组氨酸缓冲液；
(c)25mg/ml的蔗糖；和
(d)0.1-0.3mg/ml的聚山梨醇酯80。
在可选的实施方案中，所述药物组合物包含：
(a)1-150mg/ml的PCSK-9抗体或其抗原结合片段，和
(b)5-30mM的组氨酸缓冲液；
且所述药物组合物的pH约为6.0-6.5。
在可选的实施方案中，药物组合物包含：
(a)1-150mg/ml的PCSK-9抗体或其抗原结合片段；
(b)5-30mM的组氨酸缓冲液；
(c)10-75mg/ml的蔗糖；和
(d)0.05-0.6mg/ml的聚山梨醇酯80，且所述药物组合物的pH约为6.0-6.5。组氨酸缓冲液优选组氨酸-盐酸盐缓冲液。
在可选的实施方案中，药物组合物包含：
(a)50mg/ml的PCSK-9抗体或其抗原结合片段；
(b)5-20mM的组氨酸缓冲液；
(c)25mg/ml的蔗糖；和
(d)0.1-0.3mg/ml的聚山梨醇酯80，且所述药物组合物的pH约为6.0-6.5。组氨酸缓冲液优选组氨酸-盐酸盐缓冲液。
在可选的实施方案中，药物组合物包含：
(a)50mg/ml的PCSK-9抗体或其抗原结合片段；
(b)10mM的组氨酸缓冲液；
(c)25mg/ml的蔗糖；和
(d)0.2mg/ml的聚山梨醇酯80，且所述药物组合物的pH为6.3±0.1。组氨酸缓冲液优选组氨酸-盐酸盐缓冲液。
在可选的实施方案中，所述药物组合物包含：
(a)150mg/ml的PCSK-9抗体或其抗原结合片段；(b)30mM的组氨酸-盐酸盐缓冲液；(c)75mg/ml的蔗糖；和(d)0.6mg/ml的聚山梨醇酯80；药物组合物的最终PH6.3。
在可选的实施方案中，所述药物组合物包含：
(a)50mg/ml的PCSK-9抗体或其抗原结合片段；(b)10mM的组氨酸-盐酸盐缓冲液，pH 6.0；(c)25mg/ml的蔗糖；和(d)0.2mg/ml的聚山梨醇酯80。
在可选的实施方案中，所述药物组合物包含：
(a)150mg/ml的PCSK-9抗体或其抗原结合片段；(b)20mM的组氨酸-盐酸盐缓冲液，pH 6.5；和(c)70mg/ml的蔗糖。
在可选的实施方案中，所述药物组合物包含：
(a)150mg/ml的PCSK-9抗体或其抗原结合片段；(b)20mM的组氨酸-盐酸盐缓冲液，pH 6.5；和(c)70mg/ml的α,α-二水合海藻糖。
在可选的实施方案中，所述药物组合物包含：
(a)50mg/ml的PCSK-9抗体或其抗原结合片段；(b)20mM的组氨酸-盐酸盐缓冲液，pH 6.0；(c)25mg/ml的蔗糖；和(d)0.2mg/ml的聚山梨醇酯80。
在可选的实施方案中，所述药物组合物包含：
(a)50mg/ml的PCSK-9抗体或其抗原结合片段；(b)20mM的组氨酸-盐酸盐缓冲液，pH 6.5；(c)25mg/ml的蔗糖；和(d)0.2mg/ml的聚山梨醇酯80。
在可选的实施方案中，药物组合物中所述PCSK-9抗体或抗原结合片段具有分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的HCDR1、HCDR2和HCDR3，和
分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3。
在可选的实施方案中，药物组合物中所述抗体或抗原结合片段可选自鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
在可选的实施方案中，药物组合物中所述PCSK-9抗体的轻链与h001-4-YTE的抗体轻链氨基酸序列具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性，所述PCSK-9抗体的重链氨基酸序列与h001-4-YTE的抗体重链具有至少85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％的序列同一性，所述h001-4-YTE抗体轻链序列如SEQ ID NO：30所示，h001-4-YTE抗体重链序列如SEQ ID NO：32所示。
本发明还提供制备前述药物组合物的方法，其中包括：PCSK-9抗体或其抗原结合片 段原液经缓冲液置换的步骤，所述缓冲液类型优选为组氨酸缓冲液，更优选为组氨酸-盐酸盐缓冲液，所述缓冲液浓度优选为大约5mM至30mM，非限制性实施例包括5mM、6mM、7mM、8mM、9mM、10mM、12mM、14mM、16mM、18mM、20mM、22mM、24mM、26mM、28mM、30mM，更优选为10mM至15mM；所述缓冲液pH约为6.0至6.5，非限制性实施例包括6.0、6.1、6.2、6.3、6.4、6.5。
原液进一步地，制备前述药物组合物的方法，还包括向置换步骤所得溶液中加入蔗糖和聚山梨醇80，再经缓冲液定容的步骤，其中缓冲液浓度优选为大约10mM至20mM，非限制性实施例包括10mM、12mM、14mM、16mM、18mM、20mM；所述缓冲液pH约为6.0至6.5，非限制性实施例包括6.0、6.1、6.2、6.3、6.4、6.5。
本发明还提供一种制备含PCSK-9抗体的冻干制剂的方法，其中包括将前述药物组合物经冷冻干燥的步骤。
在可选的实施方案中，制备含PCSK-9抗体的冻干制剂的方法中所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。所述的预冻为从5℃冻至-40℃至-50℃，最优为-45℃，对真空度不作要求。所述的一次干燥温度为-14℃至0℃，最优为-5℃；真空度0.1mBar-0.5mBar，最优为0.3mBar。二次干燥温度20℃-30℃，最优为25℃,真空度从0.1mBar-0.5mBar，最优为0.3mBar，降至真空度0.005mBar-0.02mBar，最优为0.01mBar。
本发明还提供一种经前述制备含PCSK-9抗体的冻干制剂的方法制备的含PCSK-9抗体的冻干制剂。
在一些实施方案中，该冻干制剂于2-8℃稳定至少3个月，至少6个月，至少12个月，至少18个月或至少24个月。在一些实施方案中，该冻干制剂于40℃稳定至少7天，至少14天或至少28天。
本发明还提供制备含有PCSK-9抗体的冻干制剂的复溶溶液的方法，其中包括将前述冻干制剂经复溶的步骤，其复溶所用溶液选自但不限于注射用水、生理盐水或葡萄糖溶液。
本发明还提供一种经前述含有PCSK-9抗体的冻干制剂的复溶溶液的方法制备的含有PCSK-9抗体的冻干制剂的复溶溶液。
在可选的实施方案中，本发明所述的含有PCSK-9抗体的复溶溶液，其所含组分的浓度为冻干前药物组合物所含组分浓度的2-5倍，优选为3倍。
在可选的实施方案中，所述的含有PCSK-9抗体的复溶溶液，其中所述PCSK-9抗体或其抗原结合片段浓度为大约120mg/ml至200mg/ml，最优选为150mg/ml。
在可选的实施方案中，所述的含有PCSK-9抗体的复溶溶液，其中所述药物组合物 的pH约为6.0-6.5，优选为6.4。该复溶溶液的PH与配制所用原液的缓冲液pH相关，当原液PH为6.0时，最终复溶溶液的PH为6.3±1。
在可选的实施方案中，所述的含有PCSK-9抗体的复溶溶液，其中所述缓冲液浓度为大约15mM至45mM，优选为30mM。
在可选的实施方案中，所述的含有PCSK-9抗体的复溶溶液，其中还包括二糖，所述二糖优选自海藻糖或蔗糖，最优选为蔗糖。
在可选的实施方案中，所述的含有PCSK-9抗体的复溶溶液，其中所述糖浓度为大约55mg/ml至95mg/ml，优选为75mg/ml。
在可选的实施方案中，所述的含有PCSK-9抗体的复溶溶液，其中还包括表面活性剂，所述表面活性剂优选为聚山梨醇酯，更优选为聚山梨醇酯80。
在可选的实施方案中，所述的含有PCSK-9抗体的复溶溶液，其中表面活性剂的浓度从大约0.4mg/ml至0.8mg/ml，优选为0.6mg/ml。
本发明进一步提供一种制品或试剂盒，其包含装有本文所述任何稳定的药物组合物的容器。在一些实施方案中，该玻璃瓶为中性硼硅玻璃管制注射剂瓶。
本发明还提供前述的药物组合物或冻干制剂或冻干制剂的复溶溶液在制备用于治疗PCSK-9相关的疾病或病症的药物中的用途，其中所述的疾病或病症优选胆固醇相关疾病，更优选为高胆固醇血症、心脏病、代谢综合征、糖尿病、冠状动脉心脏病、卒中、心血管疾病、阿尔茨海默病和一般性的异常脂血症，最优选高胆固醇血症、异常脂血症、动脉粥样硬化、CVD或冠状动脉心脏病。
本发明还提供前一种治疗和预防PCSK-9相关的疾病或病症的方法，包括给予所需患者治疗有效量的前述药物组合物或冻干制剂或冻干制剂的复溶溶液，其中所述的疾病优选胆固醇相关疾病，更优选为高胆固醇血症、心脏病、代谢综合征、糖尿病、冠状动脉心脏病、卒中、心血管疾病、阿尔茨海默病和一般性的异常脂血症，最优选高胆固醇血症、异常脂血症、动脉粥样硬化、CVD或冠状动脉心脏病。
本发明还提供一种制品，其包括容器，该容器中装有前述的药物组合物或冻干制剂或冻干制剂的复溶溶液。
要理解，可以组合本文所述各个实施方案的一项、一些或所有特性以形成本发明的其它实施方案。本发明的这些和其它方面对于本领域技术人员会变得显然。本发明的这些和其它实施方案通过下面的详述进一步描述。
具体实施方式
一、术语
为了更容易理解本发明，以下具体定义了某些技术和科学术语。除非在本文中另有明确定义，本文使用的所有其它技术和科学术语都具有本发明所属领域的一般技术人员通常理解的含义。
“缓冲液”指通过其酸-碱共轭组分的作用而耐受pH变化的缓冲液。将pH控制在适当范围中的缓冲液的例子包括醋酸盐、琥珀酸盐、葡萄糖酸盐、组氨酸、草酸盐、乳酸盐、磷酸盐、柠檬酸盐、酒石酸盐、延胡索酸盐、甘氨酰甘氨酸和其它有机酸缓冲液。
“组氨酸缓冲液”是包含组氨酸离子的缓冲液。组氨酸缓冲液的实例包括组氨酸-盐酸盐，组氨酸-醋酸盐，组氨酸-磷酸盐，组氨酸-硫酸盐等缓冲液，优选组氨酸-盐酸盐缓冲液。组氨酸-盐酸盐缓冲液是组氨酸与盐酸或组氨酸与组氨酸盐酸盐配制而成。
“柠檬酸盐缓冲液”是包括柠檬酸根离子的缓冲液。柠檬酸盐缓冲液的实例包括柠檬酸-柠檬酸钠、柠檬酸-柠檬酸钾、柠檬酸-柠檬酸钙、柠檬酸-柠檬酸镁等。优选的柠檬酸盐缓冲液是柠檬酸-柠檬酸钠。
“琥珀酸盐缓冲液”是包括琥珀酸离子的缓冲液。琥珀酸盐缓冲液的实例包括琥珀酸-琥珀酸钠、琥珀酸-琥珀酸钾、琥珀酸-琥珀酸钙盐等。优选的琥珀酸盐缓冲液是琥珀酸-琥珀酸钠。
“磷酸盐缓冲液”是包括磷酸离子的缓冲液。磷酸盐缓冲液的实例包括磷酸氢二钠酸-磷酸二氢钠、磷酸氢二钠酸-磷酸二氢钾等。优选的磷酸盐缓冲液是磷酸氢二钠酸-磷酸二氢钠。
“醋酸盐缓冲液”是包括醋酸根离子的缓冲液。醋酸盐缓冲液的实例包括醋酸-醋酸钠、醋酸组氨酸盐、醋酸-醋酸钾、醋酸醋酸钙、醋酸-醋酸镁等。优选的醋酸盐缓冲液是醋酸-醋酸钠。
“药物组合物”表示含有一种或多种本文所述化合物或其生理学上/可药用的盐或前体药物与其他化学组分的混合物，所述其他组分例如生理学/可药用的载体和赋形剂。药物组合物的目的是保持抗体活性成分的稳定性，促进对生物体的给药，利于活性成分的吸收进而发挥生物活性。本文中，“药物组合物”和“制剂”并不互相排斥。
“冻干制剂”表示液体或溶液形式的药物组合物或液体或溶液制剂经真空冷冻干燥步骤之后获得的制剂或药物组合物。
本发明的冷冻干燥包括预冻，一次干燥，二次干燥。预冻目的是冷冻产品，获得结晶固体。预冻温度和预冻速度是两个重要工艺参数，本发明中将预冻温度设定为-45℃，预冻速度设定为1℃/min。一次干燥也称为主干燥，是样品冷冻干燥的主要阶段。目的是 移除产品里冰的同时，保持产品形状，最小程度的减少对产品的破坏。若一次干燥的温度和真空度选择不当，会导致制品的塌陷。较高的温度和真空度均会加快冻干效率，但同时也会增加制品塌陷的风险。本发明所述一次干燥的温度可为本领域常规的温度，例如-27℃-0℃，优选为-14℃至-5℃。二次干燥也称为解析干燥，是通过抽极限真空(0.01mbar)和升高温度(20-40℃)除去制品中结合水的主要步骤。由于大多数生物制品对温度比较敏感，所以二次干燥温度选择在温度范围的低点，即25℃。冷冻干燥的时间与冷冻机，冻干制剂剂量，冻干药剂的容器有关。这种时间的调整是在本领域技术人员所熟知的。
本发明所述的药物组合物能够达到一种稳定的效果：其中的抗体在贮藏后基本上保留其物理稳定性和/或化学稳定性和/或生物学活性的药物组合物，优选地，药物组合物在贮藏后基本上保留其物理和化学稳定性以及其生物学活性。贮藏期一般基于药物组合物的预定保存期来选择。目前有多种测量蛋白质稳定性的分析技术，可测量在选定温度贮藏选定时间段后的稳定性。
稳定的药物抗体制剂是在下述情况下没有观察到显著变化的制剂：在冷藏温度(2-8℃)保存至少3个月、优选6个月、更优选1年，且甚至更优选地最多达2年。另外，稳定的液体制剂包括这样的液体制剂：其在包括25℃和40℃在内的温度保存包括1个月、3个月、6个月在内的时段后表现出期望的特征。稳定性的典型的可接受的标准如下：通过SEC-HPLC测得，通常不超过约10％、优选不超过约5％的抗体单体发生降解。通过视觉分析，药物抗体制剂是无色的，或澄清至稍微乳白色。所述制剂的浓度、pH和重量克分子渗透压浓度具有不超过±10％变化。通常观察到不超过约10％、优选不超过约5％的截短。通常形成不超过约10％、优选不超过约5％的聚集。
如果在目检颜色和/或澄清度后，或者通过UV光散射、尺寸排阻色谱法(SEC)和动态光散射(DLS)测得，抗体没有显示出显著的聚集增加、沉淀和/或变性，那么所述抗体在药物制剂中“保留它的物理稳定性”。蛋白构象的变化可以通过荧光光谱法(其确定蛋白三级结构)和通过FTIR光谱法(其确定蛋白二级结构)来评价。
如果抗体没有显示出显著的化学改变，那么所述抗体在药物制剂中“保留它的化学稳定性”。通过检测和定量化学上改变的形式的蛋白，可以评估化学稳定性。经常改变蛋白化学结构的降解过程包括水解或截短(通过诸如尺寸排阻色谱法和SDS-PAGE等方法来评价)、氧化(通过诸如与质谱法或MALDI/TOF/MS结合的肽谱法等方法来评价)、脱酰胺作用(通过诸如离子交换色谱法、毛细管等电聚焦、肽谱法、异天冬氨酸测量等方法来评价)和异构化(通过测量异天冬氨酸含量、肽谱法等来评价)。
如果抗体在给定时间的生物活性是在制备药物制剂时表现出的生物活性的预定范围内，那么所述抗体在药物制剂中“保留它的生物活性”。抗体的生物活性可以例如通过抗原结合测定来确定。
本发明所用氨基酸三字母代码和单字母代码如J.biol.chem,243,p3558(1968)中所述。
本发明所述的“抗体”指免疫球蛋白，是由两条相同的重链和两条相同的轻链通过链间二硫键连接而成的四肽链结构。
在本发明中，本发明所述的抗体轻链可进一步包含轻链恒定区，所述的轻链恒定区包含人源或鼠源的κ、λ链或其变体。
在本发明中，本发明所述的抗体重链可进一步包含重链恒定区，所述的重链恒定区包含人源或鼠源的IgG1、IgG2、IgG3、IgG4或其变体。
抗体重链和轻链靠近N端的约110个氨基酸的序列变化很大，为可变区(Fv区)；靠近C端的其余氨基酸序列相对稳定，为恒定区。可变区包括3个高变区(HVR)和4个序列相对保守的骨架区(FR)。3个高变区决定抗体的特异性，又称为互补性决定区(CDR)。每条轻链可变区(LCVR)和重链可变区(HCVR)由3个CDR区4个FR区组成，从氨基端到羧基端依次排列的顺序为：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。轻链的3个CDR区指LCDR1、LCDR2、和LCDR3；重链的3个CDR区指HCDR1、HCDR2和HCDR3。本发明所述的抗体或抗原结合片段的LCVR区和HCVR区的CDR氨基酸残基在数量和位置符合已知的Kabat编号规则(LCDR1-3，HCDE2-3)，或者符合kabat和chothia的编号规则(HCDR1)。
本发明的抗体包括鼠源抗体、嵌合抗体、人源化抗体，优选人源化抗体。
术语“鼠源抗体”在本发明中为根据本领域知识和技能制备的对人PCSK-9的单克隆抗体。制备时用PCSK-9抗原注射试验对象，然后分离表达具有所需序列或功能特性的抗体的杂交瘤。
术语“嵌合抗体(chimeric antibody)”，是将鼠源性抗体的可变区与人抗体的恒定区融合而成的抗体，可以减轻鼠源性抗体诱发的免疫应答反应。建立嵌合抗体，要先建立分泌鼠源性特异性单抗的杂交瘤，然后从小鼠杂交瘤细胞中克隆可变区基因，再根据需要克隆人抗体的恒定区基因，将小鼠可变区基因与人恒定区基因连接成嵌合基因后***人载体中，最后在真核工业***或原核工业***中表达嵌合抗体分子。在本发明一个优选的实施方案中，所述的PCSK-9嵌合抗体的抗体轻链进一步包含人源κ、λ链或其变体的轻链恒定区。所述的PCSK-9嵌合抗体的抗体重链进一步包含人源IgG1、IgG2、IgG3、 IgG4或其变体的重链恒定区。人抗体的恒定区可选自人源IgG1、IgG2、IgG3或IgG4或其变体的重链恒定区，优选包含人源IgG2或IgG4重链恒定区，或者使用氨基酸突变后无ADCC(antibody-dependent cell-mediated cytotoxicity，抗体依赖的细胞介导的细胞毒作用)毒性的IgG4。
术语“人源化抗体(humanized antibody)”，也称为CDR移植抗体(CDR-grafted antibody)，是指将小鼠的CDR序列移植到人的抗体可变区框架，即不同类型的人种系抗体构架序列中产生的抗体。可以克服嵌合抗体由于携带大量小鼠蛋白成分，从而诱导的强烈的抗体可变抗体反应。此类构架序列可以从包括种系抗体基因序列的公共DNA数据库或公开的参考文献获得。如人重链和轻链可变区基因的种系DNA序列可以在“VBase”人种系序列数据库(在因特网www.mrccpe.com.ac.uk/vbase可获得)，以及在Kabat，E.A.等人，1991 Sequences of Proteins of Immunological Interest，第5版中找到。为避免免疫原性下降的同时，引起的活性下降，可对所述的人抗体可变区框架序列进行最少反向突变或回复突变，以保持活性。本发明的人源化抗体也包括进一步由噬菌体展示对CDR进行亲和力成熟后的人源化抗体。
“PCSK-9抗体”为与PCSK-9特异性结合的抗体，包括但不限于PCT/CN2016/111053中的h001系列PCSK-9人源化抗体。其中，h001系列PCSK-9人源化抗体，是利用人PCSK-9免疫小鼠筛选，并经人源化改造获得。
本发明中所述的“抗原结合片段”，指具有抗原结合活性的Fab片段，Fab‘片段，F(ab’)2片段，以及与人PCSK-9结合的ScFv片段及其他利用了能与人PCSK-9结合的抗体的VH和VL区形成的可结合人PCSK-9的片段；其包含本发明所述抗体的选自SEQ ID NO:1至SEQ ID NO:2中的一个或多个CDR区。Fv片段含有抗体重链可变区和轻链可变区，但没有恒定区，并具有全部抗原结合位点的最小抗体片段。一般地，Fv抗体还包含在VH和VL结构域之间的多肽接头，且能够形成抗原结合所需的结构。也可以用不同的连接物将两个抗体可变区连接成一条多肽链，称为单链抗体(single chain antibody)或单链Fv(sFv)。本发明的术语“与PCSK-9结合”，指能与人PCSK-9相互作用。本发明的术语“抗原结合位点”指抗原上连续或不连续的，由本PCSK-9抗体或抗原结合片段识别的三维空间位点。
现有技术中熟知生产和纯化抗体和抗原结合片段的方法，如冷泉港的抗体实验技术指南，5-8章和15章。发明所述的抗体或抗原结合片段用基因工程方法在非人源的CDR区加上一个或多个人源FR区。人FR种系序列可以通过比对IMGT人类抗体可变区种系基因数据库和MOE软件，从ImMunoGeneTics(IMGT)的网站http://imgt.cines.fr得到，或 者从免疫球蛋白杂志，2001ISBN012441351上获得。
本发明工程化的抗体或抗原结合片段可用常规方法制备和纯化。比如，编码重链和轻链的cDNA序列，可以克隆并重组至GS表达载体。重组的免疫球蛋白表达载体可以稳定地转染CHO细胞。作为一种更推荐的现有技术，哺乳动物类表达***会导致抗体的糖基化，特别是在Fc区的高度保守N端位点。通过表达与人PCSK-9特异性结合的抗体得到稳定的克隆。阳性的克隆在生物反应器的无血清培养基中扩大培养以生产抗体。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
“保守修饰”或“保守置换或取代”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
氨基酸序列“同一性”是指两个蛋白或多肽之间的序列相似性。当两个比较序列中的位置均被相同氨基酸残基占据时，例如如果两个多肽的一个位置都被同一个氨基酸残基占据时，那么所述分子在该位置是一致的。适于确定序列同一性百分比和序列相似性百分比的算法的实例是BLAST和BLAST2.0算法，它们分别描述于Altschul et al.(1990)J.Mol.Biol.215:403-410和Altschul et al.(1977)Nucleic Acids Res.25:3389-3402。用于执行BLAST分析的软件可在美国国家生物技术信息中心公开获取(http://www.ncbi.nlm.nih.gov/)。
“给予”和“处理”当应用于动物、人、实验受试者、细胞、组织、器官或生物流体时，是指外源性药物、治疗剂、诊断剂或组合物与动物、人、受试者、细胞、组织、器官或生物流体的接触。“给予”和“处理”可以指例如治疗、药物代谢动力学、诊断、研究和实验方法。细胞的处理包括试剂与细胞的接触，以及试剂与流体的接触，其中所述流体与细胞接触。“给予”和“处理”还意指通过试剂、诊断、结合组合物或通过另一种细胞体外和离体处理例如细胞。“处理”当应用于人、兽医学或研究受试者时，是指治疗处理、预防或预防性措施，研究和诊断应用。
“治疗”意指给予患者内用或外用治疗剂，例如包含本发明的任一种结合化合物的组合物，所述患者具有一种或多种疾病症状，而已知所述治疗剂对这些症状具有治疗作用。通常，在受治疗患者或群体中以有效缓解一种或多种疾病症状的量给予治疗剂，以诱导这类症状退化或抑制这类症状发展到任何临床可测量的程度。有效缓解任何具体疾病症状的治疗剂的量(也称作“治疗有效量”)可根据多种因素变化，例如患者的疾病状态、年龄和体重，以及药物在患者产生需要疗效的能力。通过医生或其它专业卫生保健人士通常用于评价该症状的严重性或进展状况的任何临床检测方法，可评价疾病症状是否已被减轻。尽管本发明的实施方案(例如治疗方法或制品)在缓解每个目标疾病症状方面可能无效，但是根据本领域已知的任何统计学检验方法如Student t检验、卡方检验、依据Mann和Whitney的U检验、Kruskal-Wallis检验(H检验)、Jonckheere-Terpstra检验和Wilcoxon检验确定，其在统计学显著数目的患者中应当减轻目标疾病症状。
“有效量”包含足以改善或预防医学疾病的症状或病症的量。有效量还意指足以允许或促进诊断的量。用于特定患者或兽医学受试者的有效量可依据以下因素而变化：例如，待治疗的病症、患者的总体健康情况、给药的方法途径和剂量以及副作用严重性。有效量可以是避免显著副作用或毒性作用的最大剂量或给药方案。
“Tm值”是指蛋白质热变性温度，即一半蛋白去折叠时的温度，此时蛋白的空间结构被破坏，所以Tm值越高，蛋白热稳定性越高。
“置换”是指溶解抗体蛋白的溶剂体系的置换，例如，使用稳定制剂的缓冲体系经物理操作方式将含抗体蛋白的高盐或高渗溶剂体系置换，从而使抗体蛋白存在于稳定制剂中。所称物理操作方式包括但不限于超滤、透析或离心后复溶。
二、实施例与测试例
通过以下实施例进一步详细说明本发明。这些实施例仅用于说明性目的，而并不用于限制本发明的范围。
本发明实施例中未注明具体条件的实验方法，通常按照常规条件；或按照原料或商品制造厂商所建议的条件。未注明具体来源的试剂，为市场购买的常规试剂。
实施例
PCSK-9抗原和抗体的制备
实施例1、PCSK9抗原及检测用蛋白的制备
蛋白设计及表达
以UniProt Proprotein convertase subtilisin/kexin type 9(人PCSK9，Uniprot号：Q8MBP7)作为本发明PCSK9的模板，设计本发明涉及的抗原及检测用蛋白的氨基酸序列，可选的 在PCSK9蛋白基础上融合不同的标签如his标签或促进免疫的肽段如PADRE肽，分别克隆到pTT5载体上(Biovector，Cat#:102762)或pTargeT载体上(promega,A1410)，在293细胞瞬转表达或CHO-S稳定表达，纯化，获得编码本发明抗原及检测用蛋白。
带His标签的PCSK9：PCSK9-His6，用于免疫原免疫小鼠或检测试剂
Figure PCTCN2018093573-appb-000001
SEQ ID NO：1
注释：划横线部分为信号肽，斜体部分为His6-tag(6组氨酸标签)。
带PADRE肽和His标签的PCSK9：PCSK9-PADRE-His6，作为免疫原，所含PADRE肽可以促进免疫；
Figure PCTCN2018093573-appb-000002
Figure PCTCN2018093573-appb-000003
SEQ ID NO：2
注释：划横线部分为信号肽，双划线部分为linker，点划线部分为PADRE肽，斜体部分为His6-tag。
带TEV酶切位点的PCSK9与his标签融合蛋白：PCSK9-TEV-His6，可通过TEV酶切获得N-PCSK9(N端pCSK9结构域)，作为免疫原；
Figure PCTCN2018093573-appb-000004
SEQ ID NO：3
注释：划横线部分为信号肽，双划线部分为TEV酶切位点，斜体部分为His6-tag。
PCSK9-D374Y突变蛋白，带his标签：PCSK9-D374Y-His6，作为检测试剂；
Figure PCTCN2018093573-appb-000005
Figure PCTCN2018093573-appb-000006
SEQ ID NO：4
注释：划横线部分为信号肽，斜体部分为His6-tag。
PCSK9：***生物素接受肽BP15及his标签的PCSK9蛋白：PCSK9-BP15-His6，作为检测试剂，BP15肽位置在表达过程中能够进行生物素标记，免除体外生物素标记及可能导致的构象变化；
Figure PCTCN2018093573-appb-000007
SEQ ID NO：5
注释：划横线部分为信号肽，双划线部分为生物素接受肽，斜体部分为His6-tag。
PCSK9-Y：***生物素接受肽及his标签的PCSK9D374Y突变体蛋白：PCSK9-D374Y-BP15-His6，检测蛋白；
Figure PCTCN2018093573-appb-000008
SEQ ID NO：6
注释：划横线部分为信号肽，双划线部分为生物素接受肽，斜体部分为His6-tag。
带Flag标签和His标签的pCSK9受体蛋白LDLR胞外域片段：LDLR-ECD-Flag-His6，检测试剂
Figure PCTCN2018093573-appb-000009
Figure PCTCN2018093573-appb-000010
SEQ ID NO：7
注释：划横线部分为信号肽，双划线部分为Flag标签，斜体部分为His6-tag；
LDLR-Fc：缩短形式的LDLR胞外域片段与hIgG1-Fc融合蛋白(具有与PCSK9结合活性)：LDLR-sECD–Fc(hIgG1)作为检测试剂
Figure PCTCN2018093573-appb-000011
SEQ ID NO：8
注释：划横线部分为信号肽，双划线部分为缩短形式的具有与PCSK9结合活性的LDLR胞外域片段(LDLR-sECD)，斜体部分为hIgG1-Fc部分；
更加缩短形式的LDLR胞外域片段与hIgG1-Fc融合蛋白(具有与pCSK9结合活性)：LDLR-ssECD–Fc(hIgG1)作为检测试剂
Figure PCTCN2018093573-appb-000012
SEQ ID NO：9
注释：划横线部分为信号肽，双划线部分为更加缩短形式的具有与PCSK9结合活性的LDLR胞外域片段(LDLR-ssECD)，斜体部分为hIgG1-Fc部分。
实施例2、PCSK9、LDLR相关重组蛋白的纯化重组蛋白以及杂交瘤抗体、重组抗 体的纯化
1、带His标签的重组蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，并将缓冲液换置换为PBS，加入咪唑至终浓度为5mM。用含有5mM咪唑的PBS溶液平衡镍柱，冲洗2-5倍柱体积。将置换后的上清样品上IMAC柱。用含有5mM咪唑的PBS溶液冲洗柱子，至A 280读数降至基线。后用PBS+10mM咪唑冲洗层析柱，除去非特异结合的杂蛋白，并收集流出液。再用含有300mM咪唑的PBS溶液洗脱目的蛋白，并收集洗脱峰。收集的洗脱液浓缩后用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS。去聚体峰，收集洗脱峰。所得到的蛋白经电泳，肽图，LC-MS鉴定为正确后分装备用。得到带His标签的PCSK9-His6(SEQ ID NO：1)、PCSK9-PADRE-His6(SEQ ID NO：2)、PCSK9-TEV-His6(SEQ ID NO：3)PCSK9-D374Y-His6(SEQ ID NO：4)、PCSK9-BP15-His6(SEQ ID NO：5)、PCSK9-D374Y-BP15-His6(SEQ ID NO：6)用于本发明抗体的免疫原或检测试剂。其中PCSK9-TEV-His6纯化后通过TEV酶进行酶切，酶切产物再利用IMAC柱结合去除TEV酶、未酶切完全的PCSK9-TEV-His6或切除的带His标签的C端结构域片段，IMAC流出液中浓缩获得仅留N端结构域的PCSK9片段(缩写为N-PCSK9)，作为免疫原用于小鼠免疫。
2、带His标签和Flag标签的LDLR-ECD-Flag-His6(SEQ ID NO:7)重组蛋白的纯化步骤：
将样品高速离心去除杂质，并浓缩至适当体积。利用0.5×PBS平衡flag亲和柱，冲洗2-5倍柱体积。将除杂后的细胞表达上清样品上柱。用0.5×PBS冲洗柱子，至A 280读数降至基线。用含有0.3M NaCl的PBS冲洗柱子，冲洗杂蛋白，并收集。用0.1M乙酸(pH3.5-4.0)洗脱目的蛋白，并收集，调节pH至中性。收集的洗脱液浓缩后用凝胶层析Superdex200(GE)进一步纯化，流动相为PBS。去聚体峰，收集洗脱峰收集样品经电泳，肽图，LC-MS鉴定正确后分装备用。得到带FLAG/His6标签的LDLR-ECD-Flag-His6(SEQ ID NO:7)，用于本发明抗体的性能测试。
3、LDLR的Fc融合蛋白的纯化步骤：
将细胞表达上清样品高速离心去除杂质，浓缩至适当体积后上Protein A柱。用PBS冲洗柱子，至A280读数降至基线。用100mM醋酸钠pH3.0洗脱目的蛋白，用1M Tris-HCl(三羟甲基氨基甲烷-盐酸)中和。洗脱样品适当浓缩后利用PBS平衡好的凝胶层析 Superdex200(GE)进一步纯化，去聚体的峰收集好后分装备用。此方法用来纯化LDLR-sECD–Fc(hIgG1)(SEQ ID NO:8)和LDLR-ssECD–Fc(hIgG1)(SEQ ID NO:9)。两者可用作PCSK9抗体功能性测试。
实施例3、抗人PCSK9杂交瘤单克隆抗体的制备
1、免疫
抗人PCSK9单克隆抗体通过免疫小鼠产生。实验用SJL白小鼠，雌性，6周龄(北京维通利华实验动物技术有限公司，动物生产许可证号：SCXK(京)2012-0001)。饲养环境：SPF级。小鼠购进后，实验室环境饲养1周，12/12小时光/暗周期调节，温度20-25℃；湿度40-60％。将已适应环境的小鼠按两种方案免疫(A/B)，每组6-10只。免疫抗原为带His标签的人PCSK9-His6(SEQ ID NO:1)、PCSK9-PADRE-His6(SEQ ID NO:2)及N-PCSK9(SEQ ID NO:3)。
方案A用弗氏佐剂(sigma Lot Num：F5881/F5506)乳化：首免用弗氏完全佐剂(CFA)，其余加强免疫用弗氏不完全佐剂(IFA)。抗原与佐剂比例为1:1，100μg/只(首免)，50μg/只(加强免疫)。第0天腹膜内(IP)注射100μg/只的乳化后抗原，首免后每两周一次，共6-8周。
方案B用Titermax(sigma Lot Num：T2684)与Alum(Thremo Lot Num：77161)交叉免疫。抗原与佐剂(titermax)比例为1:1，抗原与佐剂(Alum)比例为3:1，10-20μg/只(首免)，5μg/只(加强免疫)。第0天腹膜内(IP)注射20/10μg/只的乳化后抗原，首免后每周一次，Titermax和Alum交替使用，共6-11周。免疫四周后，根据背部结块和腹部肿胀情况，选择背部或腹膜内注射抗原。
2、细胞融合
选择血清中抗体滴度高(见后面的测试例1和2，结合PCSK9的ELISA方法)并且滴度趋于平台的小鼠进行脾细胞融合，融合前72小时冲刺免疫所选小鼠，PCSK9-His610μg/只，腹腔注射。采用优化的PEG介导的融合步骤将脾淋巴细胞与骨髓瘤细胞Sp2/0细胞(
Figure PCTCN2018093573-appb-000013
CRL-8287 TM)进行融合得到杂交瘤细胞。融合好的杂交瘤细胞用HAT完全培养基(含20％FBS、1×HAT和1×OPI的RPMI-1640培养基)重悬，分装于96孔细胞培养板中(1×10 5/150μl/孔)，37℃，5％CO 2孵育。融合后的第5天加入HAT完全培养基，50μl/孔，37℃，5％CO 2孵育。融合后第7天～8天，根据细胞生长密度，全换液，培养基为HT完全培养基(含20％FBS、1×HT和1×OPI的RPMI-1640培养基)，200μl/ 孔，37℃，5％CO 2孵育。
3、杂交瘤细胞筛选
融合后第10-11天，根据细胞生长密度，进行结合PCSK9或PCSK9-Y的ELISA方法检测(见测试例1和2)。并将结合ELISA检测的阳性孔细胞进行PCSK9或PCSK9-Y与LDLR结合的阻断ELISA检测(见测试例3和4)，阳性孔换液，并根据细胞密度及时扩大至24孔板中。移入24孔板的细胞株经过复测后进行保种和第一次亚克隆。第一次亚克隆筛选(见测试例1和2)为阳性的进行保种，并进行第二次亚克隆。第二次亚克隆为阳性(见测试例1和2)的进行保种和蛋白表达。多次融合获得有阻断PCSK9或PCSK9-Y与LDLR结合效果(见测试例3和4)的杂交瘤细胞。
通过阻断实验和结合实验筛选得到杂交瘤克隆mAb-001，用无血清细胞培养法进一步制备抗体，按纯化实例纯化抗体，供在检测例中使用。
其中测得杂交瘤克隆mAb-001的鼠抗可变区序列如下：
>mAb-001VH
Figure PCTCN2018093573-appb-000014
SEQ ID NO：10
>mAb-001VL
Figure PCTCN2018093573-appb-000015
SEQ ID NO：11
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
表1 PCSK-9抗体mAb-001的重链及轻链CDR区序列
Figure PCTCN2018093573-appb-000016
Figure PCTCN2018093573-appb-000017
实施例4、抗人PCSK9杂交瘤单克隆抗体的人源化
1、杂交瘤克隆mAb-001人源化框架选择
通过比对IMGT人类抗体重轻链可变区种系基因数据库和MOE软件，分别挑选与mAb-001同源性高的重轻链可变区种系基因作为模板，将这两个鼠源抗体的CDR分别移植到相应的人源模板中，形成次序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4的可变区序列。其中氨基酸残基由Kabat编号***确定并注释。
鼠源抗体mAb-001的人源化轻链模板为IGKV1-39*01和hjk2.1，人源化重链模板为IGHV1-2*02和hjh2，人源化后得到人源化抗体h001-1的可变区序列如下：
>h001-1VH
Figure PCTCN2018093573-appb-000018
TTVTVSS SEQ ID NO：18
>h001-1VL
Figure PCTCN2018093573-appb-000019
SEQ ID NO：24
注：顺序为FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4，序列中斜体为FR序列，下划线为CDR序列。
2、杂交瘤克隆mAb-001的模板选择和回复突变设计，见下表2；杂交瘤克隆回复突变后的人源化序列组合见表4。
表2 杂交瘤克隆回复突变设计
Figure PCTCN2018093573-appb-000020
Figure PCTCN2018093573-appb-000021
注：如S66D表示依照Kabat编号***，将66位S突变回D。
Grafted代表鼠抗体CDR植入人种系FR区序列，各突变可变区具体序列如下表3：
表3 各突变体可变区序列
Figure PCTCN2018093573-appb-000022
Figure PCTCN2018093573-appb-000023
注：序列中横线部分为CDR区。
表4 鼠抗mAb-001人源化序列组合
h001_VL.1 h001_VL.1A h001_VL.1B h001_VL.1C
h001_VH.1 h001-1 h001-2 h001-3 h001-4
h001_VH.1A h001-5 h001-6 h001-7 h001-8
h001_VH.1B h001-9 h001-10 h001-11 h001-12
h001_VH.1C h001-13 h001-14 h001-15 h001-16
h001_VH.1D h001-17 h001-18 h001-19 h001-20
h001_VH.1E h001-21 h001-22 h001-23 h001-24
注：该表表示各种序列及其突变序列组合所得的人源化抗体可变区部分的组合。如h001-1表示，人源化抗体h001-1的可变区由轻链h001_VL1、重链h001_VH.1A组成。其它类推。
3、将以上的人源化序列组合进行抗体化，重链恒定区来自人IgG1，轻链恒定区来自人kappa链。得到相应的人源化抗体，经验证得到的PCSK9抗体与PCSK9和PCSK9-Y有较高的结合活性；并且能有效阻断PCSK9/PCSK9-Y与LDLR之间的结合。
实施例5、构建和表达抗人PCSK9人源化抗体IgG1及IgG1-YTE形式
本发明构建和表达抗人PCSK9人源化抗体的方法如下：
1、引物设计：利用在线软件DNAWorks(v3.2.2)(works/)设计多条引物合成VH/VK含重组所需基因片段：5’-30bp Signal peptide+VH/VK+30bp CH1/CL-3’。引物设计原则：目的基因2与目的基因1有2个aa不一样，则另设突变位点所在引物，如图1所示。
2、片段拼接：按照TaKaRa公司Primer STAR GXL DNA聚合酶操作说明书，用上面设计的多条引物，分两步PCR扩增得到VH/VK含重组所需基因片段。
3、表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)构建及酶切
利用一些特殊的限制性内切酶,如BsmBI，识别序列与酶切位点不同的特性设计构建表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)，如图2所示。BsmBI酶切载体，切胶回收备用。
4、重组构建表达载体VH-CH1-FC-pHr/VK-CL-pHr
VH/VK含重组所需基因片段与BsmBI酶切回收表达载体pHr(带信号肽及恒定区基因(CH1-FC/CL)片段)按3:1比例分别加入DH5α感受态细胞中，0℃冰浴30min，42℃热 击90秒，加入5倍体积LB medium，37℃孵育45min，涂布LB-Amp平板，37℃培养过夜，挑取单克隆送测序得到各目的克隆。
本发明的抗体可以，但不限于以上设计构建方式。以h001-4为例进行抗体及其突变体设计，得到①h001-4-WT：h001-4的IgG1形式，即人源化序列组合h001-4，结合来自人IgG1的重链恒定区，与来自人kappa链的轻链恒定区；②h001-4-YTE：h001-4-IgG1-YTE形式，即人源化序列组合h001-4，结合突变的人IgG1(YTE突变)的重链恒定区，与来自人kappa链的轻链恒定区。突变的人IgG1也可以是别种形式的突变。
构建和表达抗人PCSK9人源化抗体(IgG1及h001-4的IgG1-YTE形式)序列如下：
h001-4IgG1形式，重链恒定区来自人IgG1，轻链恒定区来自人kappa轻链：
重链氨基酸序列(人IgG1)：
Figure PCTCN2018093573-appb-000024
SEQ ID NO：28
重链DNA序列：
Figure PCTCN2018093573-appb-000025
Figure PCTCN2018093573-appb-000026
SEQ ID NO：29
h001-4-kappa
轻链氨基酸序列：
Figure PCTCN2018093573-appb-000027
SEQ ID NO：30
轻链DNA序列：
Figure PCTCN2018093573-appb-000028
Figure PCTCN2018093573-appb-000029
SEQ ID NO：31
h001-4-YTE轻链为h001-4-kappa:SEQ ID NO：30
h001-4-YTE重链氨基酸序列为：
Figure PCTCN2018093573-appb-000030
SEQ ID NO：32
h001-4-YTE重链DNA序列：
Figure PCTCN2018093573-appb-000031
Figure PCTCN2018093573-appb-000032
SEQ ID NO：33
备注：下划线部分为信号肽DNA序列
示例性抗体药物组合物(制剂)制备工艺
第一步：取一定量的纯化的PCSK-9抗体(如h001-4-YTE)原液，用不含抗体的缓冲液(如10mM，pH 6.0组氨酸-盐酸盐缓冲液)进行溶剂置换(优选超滤)，经超滤膜6倍体积置换，蛋白浓缩到60mg/mL(±2mg/mL)。加入一定体积的蔗糖母液，混匀，使最终蔗糖浓度为25mg/mL。加入一定体积的吐温-80母液，混匀，使最终吐温-80浓度为0.2mg/mL。加10mM pH 6.0组氨酸缓冲液定容，使蛋白浓度为50mg/mL(±5mg/mL)，最终产品pH约为6.3±0.1(其他待测试制剂或稳定性制剂参照相似步骤进行配制)。
产品经过滤后中控取样检测无菌。将原液过0.22μm PVDF滤芯，收集滤液。
第二步：调节装量至3.6ml，将滤液灌装于6ml西林瓶中，半加塞，分别于灌装开始、灌装中间、灌装结束时取样中控检测装量差异。
第三步：将灌装加塞后的药液装入冻干箱中，冻干。冻干程序为预冻、一次干燥和二次干燥。冻干程序结束后，真空加塞。
Figure PCTCN2018093573-appb-000033
冻干所用的时间可随实际情况调整，冻干机的型号，冻干药剂的装载量，冻干药剂的容器，都会影响冻干时间。这种时间的调整是在本领域技术人员所熟知的。
第四步：开启轧盖机，加铝盖，进行轧盖。
第五步：目检，确认产品无塌陷、装量不准等缺陷。打印、粘贴西林瓶标签；打印纸盒标签，折叠纸盒，装盒，贴纸盒标签。
实施例6、缓冲体系的筛选
用下列缓冲液，配制蛋白浓度为1mg/ml的抗PCSK-9抗体h001-4-YTE制剂：
1)20mM柠檬酸-柠檬酸钠，pH 4.0
2)20mM柠檬酸-柠檬酸钠，pH 4.5
3)20mM柠檬酸-柠檬酸钠，pH 5.0
4)20mM柠檬酸-柠檬酸钠，pH 5.5
5)20mM柠檬酸-柠檬酸钠，pH 6.0
6)20mM磷酸二氢钠-磷酸氢二钠，pH 6.0
7)20mM磷酸二氢钠-磷酸氢二钠，pH 6.5
8)20mM磷酸二氢钠-磷酸氢二钠，pH 7.0
9)20mM磷酸二氢钠-磷酸氢二钠，pH 7.5
10)20mM三羟甲基氨基甲烷-盐酸，pH 7.5
11)20mM三羟甲基氨基甲烷-盐酸，pH 8.0
12)20mM三羟甲基氨基甲烷-盐酸，pH 8.5
用差示扫描量热法(differential scanning calorimetry,DSC)测量抗PCSK-9抗体在每种制剂中的热稳定性。药品的热变性中点温度(Tm)分析显示，抗PCSK-9抗体在pH≥6.0时热稳定性较好。结果如表5所示。因此选择pH6.0-6.5之间的缓冲液：组氨酸-盐酸 盐、磷酸二氢钠-磷酸氢二钠、琥珀酸-琥珀酸钠、柠檬酸-柠檬酸钠，进行后续研究。
表5 H001-4-YTE缓冲体系-pH值DSC筛选结果
Figure PCTCN2018093573-appb-000034
注：N/A表示未添加该成分。
用下列缓冲液，配制蛋白浓度为1mg/ml的抗PCSK-9抗体制剂：
1)20mM组氨酸-盐酸盐，pH 6.0
2)20mM组氨酸-盐酸盐，pH 6.5
3)20mM磷酸二氢钠-磷酸氢二钠，pH 6.0
4)20mM磷酸二氢钠-磷酸氢二钠，pH 6.5
5)20mM琥珀酸-琥珀酸钠，pH 6.0
6)20mM柠檬酸-柠檬酸钠，pH 6.0
7)20mM柠檬酸-柠檬酸钠，pH 6.5
用差示扫描量热法(differential scanning calorimetry,DSC)测量抗PCSK-9抗体在每种制剂中的热稳定性。药品的热变性中点温度(Tm)分析显示(见表6)，抗PCSK-9抗体在组氨酸、磷酸盐和琥珀酸盐缓冲体系中的稳定性略优于柠檬酸盐缓冲体系。
表6 H001-4-YTE缓冲体系-pH值DSC筛选结果
Figure PCTCN2018093573-appb-000035
注：N/A表示未添加该成分。
实施例7、制剂中糖的筛选
用下列不同糖种类的缓冲液，制备PCSK-9抗体蛋白浓度为150mg/ml的抗PCSK-9抗体制剂：
1)20mM组氨酸-盐酸盐，70mg/ml蔗糖，pH 6.5
2)20mM组氨酸-盐酸盐，70mg/mlα,α-二水合海藻糖，pH 6.5
过滤每种制剂并以0.5mL/瓶填充入2mL中性硼硅玻璃管制注射剂瓶中，水针压塞轧盖封口。取样品分别进行40℃高温、低温光照以及冻融循环(样品置于-20℃条件下24h后取出置于室温使样品完全融化混匀为一个循环)实验，结果表明蔗糖与海藻糖对抗PCSK-9抗体的稳定性作用相似，我们选择蔗糖作为抗PCSK-9抗体的稳定剂，结果见表7。
表7 H001-4-YTE不同糖种类加速实验结果
Figure PCTCN2018093573-appb-000036
Figure PCTCN2018093573-appb-000037
用下列不同蔗糖浓度的缓冲液，制备PCSK-9抗体蛋白浓度为150mg/ml的抗体制剂：
1)20mM组氨酸-盐酸盐，0mg/ml蔗糖，pH 6.5
2)20mM组氨酸-盐酸盐，10mg/ml蔗糖，pH 6.5
3)20mM组氨酸-盐酸盐，40mg/ml蔗糖，pH 6.5
4)20mM组氨酸-盐酸盐，70mg/ml蔗糖，pH 6.5
渗透压值表明，当蔗糖≥70mg/ml时渗透压符合最低皮下注射要求。本发明的PCSK-9抗体制剂采用稀冻浓溶(三倍体积原液进行冻干，一倍体积注射用水复溶，未经特别说明本发明均采用该比例冻溶)工艺制备，当终处方(复溶后制剂)中糖浓度定为75mg/ml时即满足注射的渗透压要求，又方便原液中糖浓度的设定，为25mg/ml，同时该25mg/ml的糖浓度易于冻干。因此制剂终处方中的糖浓度选择为75mg/ml，原液中糖浓度定为25mg/ml。
表8 H001-4-YTE糖浓度渗透压测定结果
Figure PCTCN2018093573-appb-000038
注：N/A表示未添加该成分。
实施例8、制剂中表面活性剂的筛选
用下列不同浓度表面活性剂的缓冲液，制备PCSK-9抗体蛋白浓度为150mg/ml的抗PCSK-9抗体制剂：
1)20mM组氨酸-盐酸盐，pH 6.5
2)20mM组氨酸-盐酸盐，0.2mg/ml聚山梨醇酯20，pH 6.5
3)20mM组氨酸-盐酸盐，0.4mg/ml聚山梨醇酯80，pH 6.5
过滤每种制剂并以0.5mL/瓶填充入2mL中性硼硅玻璃管制注射剂瓶中，水针塞压塞轧盖封口。样品置于25℃恒温摇床上，以300rpm的速度振摇。外观结果表明，表面活性剂有效防止了抗PCSK-9抗体的聚集。而聚山梨醇酯20与聚山梨醇酯80对 H001-4-YTE作用无明显差异，我们选择聚山梨醇酯80作为抗PCSK-9抗体的稳定剂。
表9 表面活性剂对于25℃，300rpm振摇的H001-4-YTE聚集的影响
Figure PCTCN2018093573-appb-000039
实施例9、制剂中缓冲体系的筛选与确认
用pH 6.0或6.5的含20mM组氨酸-盐酸盐或20mM琥珀酸-琥珀酸钠的缓冲液制备含25mg/ml蔗糖，0.2mg/ml聚山梨醇酯80，抗体蛋白浓度为50mg/ml的制剂。过滤每种制剂并以3.6mL/瓶填充入6mL中性硼硅玻璃管制注射剂瓶中进行冻干，所述玻璃瓶用冷冻干燥无菌粉末用卤化丁基橡胶塞封口。将冻干品保存于2-8℃、25℃和40℃后，使用注射用水复溶，进行稳定性分析。结果显示抗PCSK-9抗体在2-8℃和25℃均非常稳定。但第0天复溶外观显示与His体系相比pH6.0琥珀酸缓冲液乳光明显(表中未显示)，因此选择组氨酸体系作为抗PCSK-9抗体的缓冲体系。
表10 H001-4-YTE冻干粉在不同温度下的稳定性
Figure PCTCN2018093573-appb-000040
Figure PCTCN2018093573-appb-000041
实施例10、制剂成分的综合筛选
用含下列不同浓度表面活性剂、不同pH、不同离子强度的缓冲液中，制备PCSK-9抗体蛋白浓度为50mg/ml，含25mg/ml蔗糖的抗PCSK-9抗体制剂：
1)10mM组氨酸-盐酸盐，0.1mg/ml聚山梨醇酯80，pH 6.5
2)10mM组氨酸-盐酸盐，0.3mg/ml聚山梨醇酯80，pH 6.0
3)10mM组氨酸-盐酸盐，0.2mg/ml聚山梨醇酯80，pH 5.5
4)15mM组氨酸-盐酸盐，0.3mg/ml聚山梨醇酯80，pH 5.5
5)10mM组氨酸-盐酸盐，0.2mg/ml聚山梨醇酯80，pH 6.0
6)15mM组氨酸-盐酸盐，0.3mg/ml聚山梨醇酯80，pH 6.5
7)15mM组氨酸-盐酸盐，0.2mg/ml聚山梨醇酯80，pH 6.0
8)5mM组氨酸-盐酸盐，0.3mg/ml聚山梨醇酯80，pH 5.5
9)15mM组氨酸-盐酸盐，0.1mg/ml聚山梨醇酯80，pH 5.5
10)5mM组氨酸-盐酸盐，0.1mg/ml聚山梨醇酯80，pH 6.0
11)5mM组氨酸-盐酸盐，0.2mg/ml聚山梨醇酯80，pH 6.5
过滤每种制剂并以3.6mL/瓶填充入6mL中性硼硅玻璃管制注射剂瓶中进行冻干，所述玻璃瓶用冷冻干燥无菌粉末用卤化丁基橡胶塞封口。将样品保存于40℃条件下进行稳定性分析，以SEC单体的差值进行数据分析，结果显示在蛋白浓度为50mg/ml，含25mg/ml蔗糖条件下，使用离子强度为pH 6.0，10mM组氨酸-盐酸盐缓冲液，且含0.2mg/ml的聚山梨醇酯80配制PCSK-9抗体更利于PCSK-9抗体的稳定。冻干复溶后，经测定，其最终制剂中各组分浓度为冻干前原液的3倍(蛋白浓度为150mg/ml，75mg/ml蔗糖，30mM组氨酸-盐酸盐缓冲液，0.6mg/ml的聚山梨醇酯80)，pH为6.3±0.1。
表11 H001-4-YTE高温实验结果
Figure PCTCN2018093573-appb-000042
Figure PCTCN2018093573-appb-000043
实施例11、一次干燥温度的优化
用pH 6.0的含10mM组氨酸-盐酸盐的缓冲液，制备PCSK-9抗体蛋白浓度为50mg/ml，含25mg/ml蔗糖，0.2mg/ml聚山梨醇酯80的抗PCSK-9抗体制剂。将抗体以3.6mL/瓶填充入6mL西林瓶中，分别以-14℃和-5℃的一次干燥温度进行冻干，并用冻干胶塞封口。复溶样品进行冻干前后对比。结果表明，-5℃为冻干工艺较优的一次干燥温度。
表12 H001-4-YTE用不同一次干燥工艺制备样品冻干前后对比
检测项目 冻干前 -14℃复溶后 -5℃复溶后
浓度 52.3mg/ml 152.5mg/ml 148.0mg/ml
pH 6.32 6.36 6.32
外观 澄清透明 澄清透明 澄清透明
SEC单体(％) 99.8 99.8 99.9
CE非还原(％) 96.3 96.2 96.3
IEC主峰(％) 61.3 61.4 60.5
水分 N/A 0.5％ 0.6％
实施例12、制剂与不同材质容器的相容性测定
H001-4-YTE以50mg/ml配制在pH 6.0，10mM组氨酸-盐酸盐，25mg/ml蔗糖，0.2mg/ml聚山梨醇酯80中。将制剂分别填充在玻璃瓶、储液袋和316L不锈钢罐中，2-8℃放置24小时。蛋白含量和纯度分析表明，H001-4-YTE在24小时内是稳定的。该制剂与316L不锈钢罐，玻璃瓶和储液袋可以相容。
表13 在不同接触材料中H001-4-YTE的稳定性
Figure PCTCN2018093573-appb-000044
实施例13、制剂与不同滤膜的相容性测定
H001-4-YTE以50mg/ml配制在pH 6.0，10mM组氨酸-盐酸盐缓冲液，25mg/ml蔗糖，0.2mg/ml聚山梨醇酯80中。将制剂分别过0.22μm PES滤膜和PVDF滤膜并于30min和1h取样检测。蛋白含量、外观和纯度分析表明，H001-4-YTE在与滤膜接触的1小时内是稳定的。该制剂与PES和PVDF滤膜均可以相容。
表14 在不同滤膜材质中H001-4-YTE的稳定性
Figure PCTCN2018093573-appb-000045
实施例14、其它可选择制剂配方
本发明的药物组合物可以做为药物制剂原液，或直接做为注射液。当本发明的药物组合物做为药物制剂原液时，经冻干工艺，制备成冻干制剂，冻干制剂经复溶后成注射液，可供临床使用。本发明按照稀冻浓溶的工艺制备冻干制剂，即对低组分浓度的药物制剂原液进行冻干，得到冻干制剂，使用时将冻干制剂重新溶解成较高浓度的药物组合物供临床使用。冻干制剂的保存稳定性比液体制剂更长。冻干制剂所用的药物制剂原液，各组分浓度越高，冻干时间越长。综合冻干工艺的各项指标设置，通常药物制剂原液各组分浓度为复溶后注射液各组分浓度的2-5倍，本发明的优选实施例为3倍。
本发明提供的稳定的药物制剂包含：PCSK-9抗体蛋白(非限制性实施例如h001-4-YTE)和任选自以下的稳定缓冲液的组合：
(1)抗体h001-4-YTE 150mg/ml，75mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和30mM组氨酸-盐酸盐缓冲液，最终pH为6.4；
(2)抗体h001-4-YTE 150mg/ml，75mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和30mM组氨酸-盐酸盐缓冲液，最终pH为6.2；
(3)抗体h001-4-YTE 150mg/ml，75mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和30mM组氨酸-盐酸盐缓冲液，最终pH为6.3；
(4)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.4；
(5)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.3；
(6)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.2；
(7)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.1；
(8)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.0；
(9)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.5；
(10)抗体h001-4 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.3；
(11)抗体h001-4 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.2；
(12)抗体h001-4 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.4；
(13)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.1mg/ml的聚山梨醇酯80，和20mM组氨酸-盐酸盐缓冲液，最终pH为6.3；
(14)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和15mM组氨酸-盐酸盐缓冲液，最终pH为6.2；
(15)抗体h001-4-YTE 50mg/ml，25mg/ml蔗糖，0.2mg/ml的聚山梨醇酯80，和20mM组氨酸-盐酸盐缓冲液，最终pH为6.4。
(16)抗体h001-4-YTE 30mg/ml，10mg/ml蔗糖，0.05mg/ml的聚山梨醇酯80，和pH为5.5的5mM组氨酸-盐酸盐缓冲液；
(17)抗体h001-4-YTE 70mg/ml，75mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和pH为6.5的30mM组氨酸-盐酸盐缓冲液；
(18)抗体h001-4-YTE 45mg/ml，20mg/ml蔗糖，0.1mg/ml的聚山梨醇酯80，和pH为6.0的8mM组氨酸-盐酸盐缓冲液；
(19)抗体h001-4-YTE 55mg/ml，40mg/ml蔗糖，0.3mg/ml的聚山梨醇酯80，和pH为6.2的15mM组氨酸-盐酸盐缓冲液。
本发明的药物组合物可经冻干工艺制备成相应的冻干制剂，该冻干制剂可用注射药水复溶，得到以下供临床使用的药物组和物：
(1)抗体PCSK-9抗体蛋白150mg/ml，75mg/ml蔗糖，0.6mg/ml的聚山梨醇酯80，和20mM组氨酸-盐酸盐缓冲液，最终pH为6.3±0.1；
(2)抗体PCSK-9抗体蛋白120mg/ml，55mg/ml蔗糖，0.4mg/ml的聚山梨醇酯80，和10mM组氨酸-盐酸盐缓冲液，最终pH为6.0；
(3)抗体PCSK-9抗体蛋白200mg/ml，95mg/ml蔗糖，0.8mg/ml的聚山梨醇酯80，和30mM组氨酸-盐酸盐缓冲液，最终pH为6.5。
虽然以上描述了本发明的具体实施方式，但是本领域的技术人员应当理解，这些仅是举例说明，在不背离本发明的原理和实质的前提下，可以对这些实施方式做出多种变更或修改。因此，本发明的保护范围由所附权利要求书限定。

Claims (28)

  1. 一种药物组合物，其包含PCSK-9抗体或其抗原结合片段，以及缓冲液，所述缓冲液优选为组氨酸或琥珀酸盐或磷酸盐或柠檬酸盐缓冲液，更优选为组氨酸缓冲液，最优选为组氨酸-盐酸盐缓冲液。
  2. 权利要求1的药物组合物，其中所述PCSK-9抗体或其抗原结合片段浓度为大约1mg/ml至150mg/ml，优选为大约30mg/ml至100mg/ml，最优选为45至55mg/ml，更优选为50mg/ml。
  3. 权利要求1或2所述的药物组合物，其中所述药物组合物的pH约为5.5至6.5，优选为大约6.0至6.5，最优选为6.0。
  4. 权利要求1至3任一项所述的药物组合物，其中所述缓冲液浓度为大约5mM至30mM，优选为大约5mM至20mM，进一步优选为5mM至15mM，最优选为10mM。
  5. 权利要求1至4任一项所述的药物组合物，其中还包括二糖，所述二糖优选自海藻糖或蔗糖，最优选为蔗糖。
  6. 权利要求5所述的药物组合物，其中所述糖浓度为大约10mg/ml至75mg/ml，优选为大约20mg/ml至40mg/ml，最优选为25mg/ml。
  7. 权利要求1至6任一项所述的药物组合物，其中还包括表面活性剂，所述表面活性剂优选为聚山梨醇酯，更优选为聚山梨醇酯80。
  8. 权利要求7所述的药物组合物，其中表面活性剂的浓度从大约0.05mg/ml至0.6mg/ml，优选为0.1mg/ml至0.4mg/ml，进一步优选为0.1mg/ml至0.3mg/ml，最优选为0.2mg/ml。
  9. 权利要求1至8任一项所述的药物组合物，其中所述PCSK-9抗体或其抗原结合片段具有分别如SEQ ID NO:12、SEQ ID NO:13和SEQ ID NO:14所示的HCDR1、HCDR2和HCDR3，和
    分别如SEQ ID NO:15、SEQ ID NO:16和SEQ ID NO:17所示的LCDR1、LCDR2和LCDR3。
  10. 权利要求9所述的药物组合物，其中所述PCSK-9抗体或其抗原结合片段具有选自如SEQ ID NO:18-23所示的重链可变区，和选自如SEQ ID NO:24-27所示的轻链可变区。
  11. 权利要求1至10任一项所述的药物组合物，其中所述PCSK-9抗体的轻链与SEQ ID NO：30所示的抗体轻链氨基酸序列具有至少95％的序列同一性，所述PCSK-9抗体 的重链氨基酸序列与SEQ ID NO：28或32所示的抗体重链具有至少95％的序列同一性。
  12. 制备权利要求1至11任一项所述的药物组合物的方法，包括：PCSK-9抗体或其抗原结合片段原液经缓冲液置换的步骤，所述缓冲液优选组氨酸缓冲液，所述缓冲液浓度优选为大约5mM至30mM，所述缓冲液pH约为6.0至6.5。
  13. 权利要求12所述的方法，其还包括向置换步骤所得溶液中加入蔗糖和聚山梨醇80，再经缓冲液定容的步骤，所述缓冲液浓度优选为大约10mM至20mM，所述缓冲液pH约为6.0至6.5。
  14. 制备含PCSK-9抗体的冻干制剂的方法，其中包括将权利要求1至11任一项所述的药物组合物经冷冻干燥的步骤。
  15. 如权利要求14所述制备含PCSK-9抗体的冻干制剂的方法，其中所述冷冻干燥依次包括预冻、一次干燥和二次干燥的步骤。
  16. 一种由权利要求14至15任一项所述的方法制备的含PCSK-9抗体的冻干制剂。
  17. 制备含有PCSK-9抗体的复溶溶液的方法，其中包括将权利要求16所述的冻干制剂经复溶的步骤，其复溶所用溶剂优选为注射用水。
  18. 一种由权利要求17所述的方法制备的含有PCSK-9抗体的复溶溶液。
  19. 如权利要求18所述的含有PCSK-9抗体的复溶溶液，其中所述PCSK-9抗体或其抗原结合片段浓度为大约120mg/ml至200mg/ml，最优选为150mg/ml。
  20. 如权利要求18所述的含有PCSK-9抗体的复溶溶液，其中所述药物组合物的pH约为6.0-6.5，优选为6.3。
  21. 如权利要求18所述的含有PCSK-9抗体的复溶溶液，其中所述缓冲液浓度为大约15mM至45mM，优选为30mM。
  22. 如权利要求18所述的含有PCSK-9抗体的复溶溶液，其中还包括二糖，所述二糖优选自海藻糖或蔗糖，最优选为蔗糖。
  23. 如权利要求22所述的含有PCSK-9抗体的复溶溶液，其中所述糖浓度为大约55mg/ml至95mg/ml，优选为75mg/ml。
  24. 如权利要求18所述的含有PCSK-9抗体的复溶溶液，其中还包括表面活性剂，所述表面活性剂优选为聚山梨醇酯，更优选为聚山梨醇酯80。
  25. 如权利要求24所述的含有PCSK-9抗体的复溶溶液，其中表面活性剂的浓度从大约0.4mg/ml至0.8mg/ml，优选为0.6mg/ml。
  26. 权利要求1至11任一项所述的药物组合物或权利要求16所述的冻干制剂或权利要求18-25任一项所述的冻干制剂的复溶溶液在制备用于治疗PCSK-9相关的疾病或病 症的药物中的用途，其中所述的疾病或病症优选胆固醇相关疾病，更优选为高胆固醇血症、心脏病、代谢综合征、糖尿病、冠状动脉心脏病、卒中、心血管疾病、阿尔茨海默病和一般性的异常脂血症，最优选高胆固醇血症、异常脂血症、动脉粥样硬化、CVD或冠状动脉心脏病。
  27. 一种治疗和预防PCSK-9相关的疾病或病症的方法，包括给予所需患者治疗有效量的如权利要求1至11任一项所述的药物组合物或权利要求16所述的冻干制剂或权利要求18-25任一项所述的冻干制剂的复溶溶液，其中所述的疾病优选胆固醇相关疾病，更优选为高胆固醇血症、心脏病、代谢综合征、糖尿病、冠状动脉心脏病、卒中、心血管疾病、阿尔茨海默病和一般性的异常脂血症，最优选高胆固醇血症、异常脂血症、动脉粥样硬化、CVD或冠状动脉心脏病。
  28. 一种制品，其包括容器，该容器中装有权利要求1至11任一项所述的药物组合物或权利要求16所述的冻干制剂或权利要求18-25任一项所述的冻干制剂的复溶溶液。
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