WO2024002357A1 - 抗pcsk9抗体的制剂及其应用 - Google Patents

抗pcsk9抗体的制剂及其应用 Download PDF

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Publication number
WO2024002357A1
WO2024002357A1 PCT/CN2023/105006 CN2023105006W WO2024002357A1 WO 2024002357 A1 WO2024002357 A1 WO 2024002357A1 CN 2023105006 W CN2023105006 W CN 2023105006W WO 2024002357 A1 WO2024002357 A1 WO 2024002357A1
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pcsk9 antibody
pcsk9
antibody preparation
seq
buffer
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PCT/CN2023/105006
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English (en)
French (fr)
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夏瑜
王诏
夏羽
李百勇
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康融东方（广东）医药有限公司
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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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • 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]

Definitions

  • the present invention relates to the field of biopharmaceuticals. Relates to a preparation of an anti-PCSK9 antibody and its application. Specifically, the present invention relates to a formulation of anti-PCSK9 monoclonal antibodies.
  • PCSK9 proproprotein Convertase Subtilisin Kexin Type 9, proprotein convertase subtilisin/kexin9 type
  • PCSK9 binds to the LDL receptor (LDL-R) on the surface of liver cells, mediates the intracellular degradation of LDL-R, and increases plasma LDL-C levels.
  • PCSK9 inhibitors anti-PCSK9 antibodies
  • PCSK9 can interfere with the binding of PCSK9 to LDL-R, recycle LDL-R to the liver surface, promote the liver to express more LDL-R, and reduce plasma LDL-C levels.
  • Anti-PCSK9 antibodies are indicated for the treatment of heterozygous familial hypercholesterolemia or clinical atherosclerotic cardiovascular disease in patients requiring additional lowering of low-density lipoprotein cholesterol (LDL-C) and as a statin intolerant agent. Adjuvant drugs for drug-like treatment. Anti-PCSK9 antibodies may also improve cardiovascular disease through other mechanisms, including reducing inflammation and oxidative stress within atherosclerotic plaques and inhibiting prothrombotic pathways, which is particularly important in patients with acute coronary syndrome.
  • LDL-C low-density lipoprotein cholesterol
  • PCSK9 The clinical significance of inhibiting PCSK9 is that loss-of-function mutations in PCSK9 reduce LDL-C levels and significantly reduce the risk of cardiovascular events. In contrast, gain-of-function mutations in PCSK9 increase LDL-C levels and the risk of cardiovascular events. HMG-CoA reductase mutations and PCSK9 mutations reduce the effect of LDL-C and have a cumulative effect on reducing cardiovascular event rates, that is, the effects of anti-PCSK9 antibodies and statins can be additive.
  • a meta-analysis included 24 randomized trials (n 10,159), covering a variety of clinical conditions, including: familial hypercholesterolemia; other hypercholesterolemia; statin-intolerant hypercholesterolemia; intensive, Non-intensive statin therapy and no statin therapy.
  • PCSK9 inhibitors approved for marketing in the world namely Amgen’s evolocumab (Evolocumab, trade name: Repatha) and alirocumab (trade name: Praluent) jointly developed by Sanofi and Regeneron, and both products are anti-PCSK9 monoclonal antibodies.
  • Amgen s evolocumab (Evolocumab, trade name: Repatha)
  • alirocumab trade name: Praluent
  • Anti-PCSK9 antibodies can meet the clinical needs of people with dyslipidemia and poorly controlled dyslipidemia. Therefore, it is necessary to conduct in-depth research on this target antibody to obtain a preparation that is more stable, more effective, and convenient for clinical use.
  • the purpose of the present invention is to provide a stable anti-PCSK9 antibody preparation.
  • the present invention relates to the following aspects:
  • Anti-PCSK9 antibody preparation which comprises an anti-PCSK9 antibody or an antigen-binding fragment thereof, at least one buffer, at least one stabilizer and at least one surfactant, wherein the anti-PCSK9 antibody comprises SEQ ID NO:8
  • the heavy chain variable region shown includes HCDR1, HCDR2 and HCDR3, and the antibody includes the light chain variable region shown in SEQ ID NO: 10 including LCDR1, LCDR2 and LCDR3, preferably, according to the IMGT numbering system
  • the The anti-PCSK9 antibody includes HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2 and HCDR3 shown in SEQ ID NO:3, and LCDR1 shown in SEQ ID NO:4, SEQ ID NO: LCDR2 shown in 5 and LCDR3 shown in SEQ ID NO:6, where the sequence shown in SEQ ID NO:5 is GVI,
  • the antigen-binding fragment is selected from the group consisting of Fab fragment, Fab' fragment, F(ab)' fragment, Fv fragment, isolated CDR region, single chain Fv molecule (scFv), F(ab') 2 , Fd, dAb, Fab /c, diabodies and domain antibodies.
  • the buffer is selected from 1-20mM sodium citrate, 1-20mM histidine and/or histidine hydrochloride, preferably the content of histidine is 1-7.4mM, Preferably 3-5.4mM, more preferably 4-4.4mM, most preferably 4.2mM, preferably the content of histidine hydrochloride is 5-16.6mM, preferably 8-13.6mM, more preferably 10-11.6mM, most preferably 10.8mM, preferably
  • the pH value of the anti-PCSK9 antibody preparation is 5.0-6.0, preferably 5.2-5.8, and the most preferred pH value is 5.5.
  • the stabilizer is selected from sugars, Amino acids, polyols, antioxidants, preservatives, cyclodextrins, polyethylene glycols (such as PEG 3000, PEG3350, PEG4000, PEG6000), albumins (such as human serum albumin (HSA), bovine serum albumin (BSA) ), one or more salts (such as sodium chloride, calcium chloride), integrators (such as EDTA), preferably the stabilizer is selected from one or more types of sugars, and the sugars are optional From sucrose and/or trehalose:
  • the content of sucrose is 5-100 mg/mL, preferably 20-100 mg/mL, 40-80 mg/mL, more preferably 54 mg/mL-66 mg/mL, 55-65 mg/mL or 57 mg/mL.
  • the content of trehalose is 5-100mg/mL, preferably 5-55mg/mL, 15-45mg/mL, more preferably 25-35mg/mL, 27mg/mL-33mg/ mL or 28.5 mg/mL-31.5 mg/mL, most preferably 30 mg/mL.
  • the total concentration of sucrose and trehalose is 85-95 mg/mL, preferably 90 mg/mL.
  • the anti-PCSK9 antibody preparation according to any one of items 1-3, wherein the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters (Tween), such as polysorbate 20 and polysorbate 80 , the preferred content of polysorbate 80 is 0.02%-0.08% (w/w), more preferably 0.025%-0.075% or 0.04%-0.06% (w/w), and most preferably 0.05% (w/w).
  • the surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid esters (Tween), such as polysorbate 20 and polysorbate 80
  • the preferred content of polysorbate 80 is 0.02%-0.08% (w/w), more preferably 0.025%-0.075% or 0.04%-0.06% (w/w), and most preferably 0.05% (w/w).
  • anti-PCSK9 antibody preparation according to any one of items 1 to 4, wherein the anti-PCSK9 antibody preparation further contains water for injection.
  • anti-PCSK9 antibody preparation according to any one of items 1-5, wherein the anti-PCSK9 antibody preparation contains 100 mg/mL anti-PCSK9 antibody, 4.2mM histidine, 10.8mM histidine hydrochloride, and 60 mg/mL sucrose. , 30 mg/mL trehalose, 0.05% (w/w) polysorbate 80, pH 5.5.
  • the anti-PCSK9 antibody preparation further comprises a preservative, preferably, a preservative is used in an amount of about 0.001% to about 2% (w/v) , preferably, the preservative is selected from ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben, propylparaben or benzalkonium chloride.
  • a preservative is used in an amount of about 0.001% to about 2% (w/v) , preferably, the preservative is selected from ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methylparaben, propylparaben or benzalkonium chloride.
  • anti-PCSK9 antibody preparation according to any one of items 1 to 6, wherein the anti-PCSK9 antibody preparation further contains an antibacterial agent and an antifungal agent, for example, chlorobutanol, sorbic acid.
  • anti-PCSK9 antibody preparation according to any one of items 1 to 8, wherein the anti-PCSK9 antibody preparation is in a form suitable for injection (preferably subcutaneous injection).
  • Methods for preparing anti-PCSK9 antibody preparations comprising conjugating anti-PCSK9 antibodies or antigens thereof
  • the fragments are mixed with at least one buffer, at least one stabilizer and at least one surfactant, and the pH of the preparation is adjusted with acid or alkali to 5.0-6.0, preferably 5.2-5.8, and the most preferred pH value is 5.5.
  • the surfactant is a non-ionic surfactant (such as polysorbate 80), and the non-ionic surfactant is added last and then adjusted to volume.
  • the expression “pharmaceutical preparation” also means anti-PCSK9 antibody preparations.
  • the anti-PCSK9 antibody or antigen-binding fragment thereof contains any one or more CDR region sequences selected from the following or has at least 85% sequence thereof (for example, at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) homologous amino acid sequences with the same activity:
  • the anti-PCSK9 antibody includes a heavy chain variable region and a light chain variable region
  • the antibody includes HCDR1, HCDR2 and HCDR3 included in the heavy chain variable region shown in SEQ ID NO:8
  • the anti-PCSK9 antibody includes LCDR1, LCDR2 and LCDR3 included in the light chain variable region shown in SEQ ID NO:10.
  • the anti-PCSK9 antibody includes HCDR1 shown in SEQ ID NO:1, SEQ ID NO: HCDR2 shown in 2 and HCDR3 shown in SEQ ID NO:3, and LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5 and LCDR3 shown in SEQ ID NO:6.
  • amino acid sequence is shown in Table 1 below:
  • the DNA sequence of the heavy chain variable region of the anti-PCSK9 antibody is as follows (369bp):
  • the encoded heavy chain variable region sequence is as follows (123aa):
  • the DNA sequence of the light chain variable region of the anti-PCSK9 antibody is as follows (333bp):
  • the encoded light chain variable region sequence is as follows (111aa):
  • the anti-PCSK9 antibody preparation of the present invention is a pharmaceutical preparation suitable for subcutaneous injection.
  • the anti-PCSK9 antibody preparation includes anti-PCSK9 antibody, buffer, carbohydrates, surfactants or consists of them, optionally also including water for injection.
  • the components of the anti-PCSK9 antibody preparation are preferably as shown in Table 2 below:
  • the "antibody” described in the present invention refers to an immunoglobulin, which is a tetrapeptide chain structure composed of two identical heavy chains and two identical light chains connected by inter-chain disulfide bonds.
  • the antibodies of the present invention include murine antibodies, chimeric antibodies, and humanized antibodies, with humanized antibodies being preferred.
  • antibody fragment refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen (eg, PCSK9). It has been shown that fragments of full-length antibodies can be utilized to perform the antigen-binding function of the antibody. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.
  • examples of the antigen-binding fragment include Fab fragment, Fab' fragment, F(ab)' fragment, Fv fragment, isolated CDR region, single-chain Fv molecule (scFv), F(ab') 2 , Fd, dAb, Fab/c, diabodies and domain antibodies.
  • CDR refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contribute to antigen binding.
  • One of the most commonly used definitions of the six CDRs is provided by Kabat E.A. et al., (1991) Sequences of proteins of immunological cal interest. NIH Publication 91-3242).
  • Kabat definition of CDR applies only to LCDR1, LCDR2, and LCDR3 of the light chain variable domains, and to HCDR1, HCDR2, and HCDR3 of the heavy chain variable domains.
  • the antibody of the present invention may further comprise a light chain constant region, and the light chain constant region includes human or murine kappa, lambda chains or variants thereof.
  • the antibody of the present invention may further comprise a heavy chain constant region, and the heavy chain constant region includes human or murine IgG1, IgG2, IgG3, IgG4 or variants thereof.
  • chimeric antibody refers to an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies.
  • a chimeric antibody you must first establish a hybridoma that secretes mouse-derived specific monoclonal antibodies, then clone the variable region gene from the mouse hybridoma cells, and then clone the constant region gene of the human antibody as needed, and combine the mouse variable region gene with It is connected to the human constant region gene to form a chimeric gene and then inserted into an expression vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic system or a prokaryotic system.
  • humanized antibody also known as CDR-grafted antibody, refers to transplanting mouse CDR sequences into the human antibody variable region framework, that is, different types of human germline antibodies Antibodies produced in framework sequences. Chimeric antibodies can be overcome due to Carry large amounts of murine protein components, thereby inducing heterologous reactions.
  • framework sequences can be obtained from public DNA databases or published references that include germline antibody gene sequences.
  • pharmaceutical preparation or “formulation” means a preparation that is in a form that permits the biological activity of the active ingredient to be clearly effective and that does not contain additional components that would be toxic to the subject to whom the preparation is to be administered.
  • liquid as used herein in connection with a formulation according to the present invention means a formulation that is liquid at a temperature of at least about 20°C to about 80°C at atmospheric pressure.
  • stabilizer means a pharmaceutically acceptable excipient which protects the active pharmaceutical ingredient and/or formulation against chemical and/or physical degradation during manufacture, storage and application.
  • the formulations of the present invention are “stable” formulations, which means formulations in which the protein (e.g., antibody) contained therein substantially retains its physical and chemical stability, and thus its biological activity, after storage .
  • stable formulations means formulations in which the protein (e.g., antibody) contained therein substantially retains its physical and chemical stability, and thus its biological activity, after storage .
  • the stability standards are as follows: the visual test result is a colorless to light yellow clear liquid; the main peak of the size exclusion chromatography (SE-HPLC) test should not be less than 95%; the reduction capillary electrophoresis (rCE-SDS) test The results showed that the sum of heavy and light chains should not be less than 95%; the main peak of the non-reducing capillary electrophoresis (nrCE-SDS) test result should not be less than 85%; the biological activity test (ELISA) result should be 50%-150% of the reference product.
  • SE-HPLC size exclusion chromatography
  • rCE-SDS reduction capillary electrophoresis
  • PCSK9 protein PCSK9
  • PCSK9 protein PCSK9
  • PCSK9 PCSK9
  • variants isoforms
  • species homologues of human PCSK9 and analogs that share at least one epitope in common with PCSK9.
  • NCBI Reference Sequence Genbank ID: NP_000567.1.
  • anti-PCSK9 antibody refers to the ability to bind the PCSK9 protein with sufficient affinity such that the antibody can be used as a diagnostic and/or therapeutic in targeting the PCSK9 protein agent.
  • binding to PCSK9 protein refers to the binding of an antibody to PCSK9 protein in a BIAcore assay (Pharmacia Biosensor AB, Uppsala, Sweden) or in an ELISA.
  • surfactant refers to pharmaceutically acceptable excipients used to protect protein formulations against physical stress, such as agitation and shear.
  • pharmaceutically acceptable surfactants include: polyoxyethylene sorbitan fatty acid ester (Tween).
  • polyoxyethylene sorbitan-fatty acid esters are polysorbate 20 and polysorbate 80.
  • buffer refers to a pharmaceutically acceptable excipient that stabilizes the pH of a pharmaceutical formulation.
  • Suitable buffers are well known in the art and can be found in the literature.
  • Preferred pharmaceutically acceptable buffers include, but are not limited to: histidine buffer, citrate buffer, succinate buffer, acetate buffer, arginine buffer, phosphate buffer or their mixture.
  • Preferred buffers include histidine/histidine hydrochloride buffers, which are pH adjusted with acids or bases known in the art.
  • the above-mentioned buffer solution is usually used in an amount of 1mM-100mM, preferably 10mM-30mM, and most preferably 20mM.
  • the pH of the pharmaceutical formulation can be adjusted to pH 5.0-6.0 using acids or bases known in the art (e.g. hydrochloric, acetic, phosphoric, sulfuric and citric acids, sodium and potassium hydroxide) , especially adjusted to pH 5.2-5.8, most especially adjusted to pH 5.5.
  • acids or bases known in the art (e.g. hydrochloric, acetic, phosphoric, sulfuric and citric acids, sodium and potassium hydroxide) , especially adjusted to pH 5.2-5.8, most especially adjusted to pH 5.5.
  • pharmaceutical formulations of the present invention may also include an antioxidant as a second stabilizer.
  • Antioxidants are pharmaceutically acceptable excipients that prevent oxidation of the active pharmaceutical ingredient. Antioxidants include, but are not limited to: integrators such as EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, Cysteine, methionine, ethanol, benzyl alcohol, and n-acetylcysteine.
  • integrators such as EDTA, citric acid, ascorbic acid, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), sodium sulfite, para-aminobenzoic acid, glutathione, propyl gallate, Cysteine, methionine, ethanol, benzyl alcohol, and n
  • sugar refers to monosaccharides or oligosaccharides.
  • Monosaccharides are monomeric carbohydrates that cannot be hydrolyzed by acids, including monosaccharides and their derivatives, such as amino sugars. Examples of monosaccharides include glucose, fructose, galactose, mannose, sorbose, ribose, deoxyribose, and neuraminic acid.
  • Oligosaccharides are branched or straight-chain carbohydrates composed of more than one monomeric sugar unit linked via glycosidic bonds. The monomeric sugar units within the oligosaccharide may be the same or different.
  • oligosaccharides are disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, and so on. Unlike polysaccharides, monosaccharides and oligosaccharides are water-soluble. Examples of oligosaccharides include sucrose, trehalose, lactose, maltose and raffinose. Specifically, the sugar is selected from sucrose and trehalose.
  • amino acid refers to a pharmaceutical compound having an amino moiety located ⁇ -position of the carboxyl group. acceptable organic molecules.
  • amino acids include arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine Acid, tryptophan, methionine, serine, proline.
  • Amino acids are generally used in amounts from 5mM to 200mM, especially from 5mM to 100mM, more particularly from 5mM to 70mM.
  • cryoprotectant means a pharmaceutically acceptable excipient that protects an unstable active ingredient (eg, a protein) against destabilizing conditions during the freeze-drying process, subsequent storage and reconstitution.
  • Cryoprotectants include, but are not limited to, sugars, polyols (eg, sugar alcohols), and amino acids.
  • the cryoprotectant can be selected from: sugars such as sucrose, trehalose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, neuraminic acid, amino sugars such as glucosamine , galactosamine, N-methylglucamine ("glucosamine”), polyols such as mannitol and sorbitol, and amino acids such as arginine and glycine or mixtures thereof.
  • the cryoprotectant is preferably a disaccharide, specifically sucrose and trehalose. The present invention surprisingly found that the stability of disaccharides to preparations is better than that of monosaccharides, polyols, and amino acids.
  • compositions may also contain tonicity agents.
  • tonicity agent as used herein means a pharmaceutically acceptable tonicity agent used to adjust the tonicity of a formulation.
  • the formulation may be hypotonic, isotonic or hypertonic, preferably isotonic.
  • Isotonicity generally refers to the relative osmotic pressure of a solution, often relative to the osmotic pressure of human serum.
  • An isotonic preparation is a liquid or a liquid reconstituted from a solid form (eg from a lyophilized form) and means a solution that has the same tonicity as some other solution to which it is compared (such as physiological saline solutions and serum).
  • Suitable tonicity agents include, but are not limited to, sodium chloride, potassium chloride, glycerol and any component selected from amino acids, sugars, especially glucose. Tonicity agents are typically used in amounts from about 5mM to about 500mM. Within stabilizers and tonicity agents there is a group of compounds that can act in two ways, i.e. they can be both stabilizers and tonicity agents at the same time. Examples can be found in the following collections: sugars, amino acids, polyols, cyclodextrins, polyglycols and salts. An example of a sugar that can be both a stabilizer and a tonicity agent is trehalose.
  • the pharmaceutical preparations may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization operations and by the inclusion of different antibacterial and antifungal agents (eg, methylparaben, chlorobutanol, phenol, sorbic acid, etc.). Preservatives are typically used in amounts from about 0.001% to about 2% (w/v). Preservatives include, but are not limited to: ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propylparaben, and benzalkonium chloride.
  • preservatives include, but are not limited to: ethanol, benzyl alcohol, phenol, m-cresol, p-chloro-m-cresol, methyl or propylparaben, and benzalkonium chloride.
  • the pharmaceutical preparation of the anti-PCSK9 antibody according to the present invention can be used to treat heterozygous familial hypercholesterolemia.
  • Sterolemia or clinical atherosclerotic cardiovascular disease for adults who require additional lowering of low-density lipoprotein cholesterol (LDL-C) and as an adjunct to statin intolerance therapy.
  • Anti-PCSK9 antibodies may improve cardiovascular disease through other mechanisms, including reducing inflammation and oxidative stress within atherosclerotic plaques and inhibiting prothrombotic pathways.
  • the anti-PCSK9 antibody pharmaceutical formulation according to the present invention can be administered by intravenous (i.v.), subcutaneous (s.c.) or any other parenteral administration means (such as those known in the pharmaceutical field).
  • the pharmaceutical formulations according to the invention can be administered subcutaneously without the need for an in-line filter.
  • Preparations for in vivo administration must be sterile. This can be effectively achieved through non-terminal sterilization aseptic production processes.
  • the anti-PCSK9 antibody pharmaceutical formulation according to the present invention can be prepared by methods known in the art, such as ultrafiltration-diafiltration, dialysis, addition and mixing, reconstitution and combinations thereof. Examples of the preparation of formulations according to the invention can be found below.
  • the inventors In order to prepare the monoclonal antibody MAB1, the inventors artificially designed a series of antibody sequences based on the existing PCSK9 protein sequence (NP_777596.2) and its three-dimensional protein crystal structure. After extensive screening and testing, a MAB1 that specifically binds to PCSK9 was finally obtained.
  • the amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody and their coding sequences are as follows SEQ ID NO: 1-4.
  • the DNA sequence of the designed MAB1 heavy chain VH is as follows (369bp):
  • VH protein sequence is as follows (123aa):
  • the encoded VL protein sequence is as follows (111aa):
  • the CDR sequence of the antibody is as follows:
  • HCDR1 GTFFSSYS(SEQ ID NO: 1);
  • HCDR3 AREYDFWSAYYDAFDV(SEQ ID NO:3);
  • LCDR1 SRNIGGGND (SEQ ID NO: 4);
  • LCDR3 QSFDGSLSGSV (SEQ ID NO: 6).
  • Plasmids pUC57simple-MAB1H and pUC57simple-MAB1L were digested with enzymes (HindIII&EcoRI) respectively, and the heavy chain and light chain recovered by electrophoresis were subcloned into pcDNA3.1 vector, and the recombinant plasmids were extracted and co-transfected into 293F cells. After the cells were cultured for 7 days, the culture medium was centrifuged at high speed and the supernatant was concentrated and then loaded onto the HiTrap MabSelect SuRe column. The protein was eluted in one step with Elution Buffer and the target sample was recovered and the medium was changed to PBS to obtain the anti-PCSK9 antibody.
  • the expression anti-PCSK9 antibody means MAB1 antibody.
  • the composition of the anti-PCSK9 antibody preparation is as follows: the preparation is composed of a solute and a solvent.
  • the solute is anti-PCSK9 antibody, histidine, histidine hydrochloride, sucrose, trehalose and polysorbate 80, and the solvent is water for injection;
  • the anti-PCSK9 antibody is in
  • the concentration of histidine in the preparation is 100mg/mL
  • the concentration of histidine in the preparation is 4.2mM
  • the concentration of histidine hydrochloride in the preparation is 10.8mM
  • the concentration of sucrose in the preparation is 60mg/mL
  • the concentration of trehalose in the preparation The concentration of polysorbate 80 is 30 mg/mL
  • the concentration of polysorbate 80 in the formulation is 0.05% (w/v)
  • the pH is 5.5.
  • the non-ionic surfactant polysorbate 80 in the solute is added last and then the volume is adjusted. Other solutes are added in no order.
  • the anti-PCSK9 antibody preparation is aseptically packed into vials and capped with rubber stoppers and aluminum plastic caps or the anti-PCSK9 antibody preparation is aseptically packed into liquid storage bags (Sartorius Flexboy) to obtain the finished product of the preparation.
  • Stability experiments were conducted on the anti-PCSK9 antibody preparation prepared in Example 1, using size-exclusion chromatography SEC-HPLC and capillary isoelectric focusing electrophoresis, respectively, using the change in the percentage content of the main component and the main peak content of the protein purity as the means of judgment.
  • Method icIEF, reduced CE-SDS, non-reduced CE-SDS, activity and appearance of the preparation were observed to characterize the chemical stability of the anti-PCSK9 antibody in the preparation under various environmental conditions, and examine the preparation under high temperature, light, freeze-thaw, vibration, etc. Stability of conditional factors.
  • Table 2 Chemical stability of anti-PCSK9 antibodies in formulations under various environmental conditions Note: A: represents 3 cycles of freezing and thawing; B: represents 2 cycles of freezing and thawing; C: represents 1 cycle of freezing and thawing; -: indicates not observed
  • the anti-PCSK9 antibody preparation prepared in Preparation Example 1 was analyzed using size exclusion chromatography SEC-HPLC, capillary isoelectric focusing electrophoresis icIEF, reduced CE-SDS, non-reducing CE-SDS, and activity (ELISA). The following experiments were carried out. Conditions: -20°C/25°C repeated freezing and thawing for 3 cycles; shaking at 300rpm for 3 days; storage under high temperature conditions of 40°C for 30 days; storage under 4500 ⁇ 500Lux light for 15 days. Changes in protein content and protein purity and changes in the purity of the charge suppressor. The results are shown in Tables 1 to 4.
  • the changes in protein content and protein purity of anti-PCSK9 antibody preparations decreased with the increase of storage time under high temperature conditions of 40°C.
  • the main peak was 92.3%, and the antibody monomer was degraded no more than 10%.
  • it was a light yellow clear liquid, consistent with normal conditions.
  • the protein concentration of the formulation is 98.2 mg/mL, which is no more than +/-10% of the protein concentration under normal conditions. It shows that the preparation is still a stable liquid antibody preparation after being stored at a high temperature of 40°C for 30 days.
  • Tables 1-4 indicate that the formulation has adequate stability under the storage conditions described above. It shows that under various conditions that accelerate the chemical degradation and physical changes of anti-PCSK9 antibodies, the preparation formula can alleviate the chemical degradation rate of the anti-PCSK9 antibodies contained and improve the physical and chemical stability of the anti-PCSK9 antibodies, making the single Clonal antibodies can be stably present in the formulation of the preparation.
  • AK102 represents anti-PCSK9 antibody-MAB1 antibody, and the concentration is 100 mg/mL.
  • Solution pH is one of the most important parameters affecting the physical and chemical stability of biopharmaceuticals. pH can adjust the charge distribution on the protein surface, thereby affecting the intramolecule and intermolecular forces of biopharmaceuticals. It also affects the rate of chemical modification of antibody drugs, which is one of the most critical attributes of biopharmaceutical preparations.
  • the protein melting temperature (Tm) reflects the thermal stability of biopharmaceuticals in aqueous solutions. The higher the Tm, the lower the possibility of conformational changes under actual storage conditions. Examine the Tm values of anti-PCSK9 antibodies in buffers with different pH. The formulas in Table 8 are prepared by using 5 ⁇ L of the anti-PCSK9 antibody in the formula in Table 7, adding 5 ⁇ L SYPRO Orange (20X) and 90 ⁇ L buffer. The Tm value of the sample was measured using a method similar to differential scanning fluorescence (DSF). The results are shown in Table 8.
  • the preparations with different formulas in Table 7 were placed in a sample test chamber at 40 ⁇ 2°C for accelerated destruction.
  • the acceleration time was 3 days.
  • the stability of the different preparations was examined through appearance and SEC-HPLC.
  • formula F11 did not undergo relevant detection and analysis. From the SEC-HPLC and CE-SDS results, the stability of F12 (trehalose) was significantly better than that of formula F13 (sorbitol). The results suggest that AK102 has a higher concentration of trehalose in trehalose. There is better stability in the formula.
  • a mixture of sucrose and trehalose was selected as the protective agent for AK102, and the mixing ratio was studied.
  • Formulas of mixed sugar protective agents with different proportions were designed, as shown in Table 14.
  • the concentration of AK102 samples in the formula was all 100 mg. /ml, the same as the final proposed packaging concentration.

Abstract

涉及抗PCSK9抗体的制剂及其在医药上的应用，该制剂含有抗PCSK9抗体、缓冲液。所述抗PCSK9抗体的制剂可以有效地抑制抗PCSK9抗体的聚集，从而阻止抗体产品的降解，得到稳定的注射药物制剂。

Description

抗PCSK9抗体的制剂及其应用 技术领域：
本发明涉及生物制药领域。涉及一种抗PCSK9抗体的制剂及其应用。具体地，本发明涉及一种抗PCSK9单克隆抗体的制剂。
背景技术：
PCSK9(proproprotein Convertase Subtilisin Kexin Type 9，前蛋白转化酶枯草杆菌蛋白酶/kexin9型)是由PCSK9基因编码的丝氨酸蛋白酶，主要由肝脏产生。PCSK9与肝细胞表面的LDL受体(LDL-R)结合，介导LDL-R在细胞内的降解，血浆LDL-C水平升高。PCSK9抑制剂(抗PCSK9抗体)能干扰PCSK9与LDL-R的结合，回收LDL-R至肝脏表面，并促进肝脏表达更多的LDL-R，降低血浆LDL-C水平。
抗PCSK9抗体用于治疗杂合子家族性高胆固醇血症或临床动脉粥样硬化性心血管疾病，用于需要额外降低低密度脂蛋白胆固醇(LDL-C)的患者，还可作为不耐受他汀类药物治疗的辅助药物。抗PCSK9抗体还可能通过其他机制来改善心血管病症，包括减轻动脉粥样硬化斑块内的炎症和氧化应激，以及抑制促血栓形成途径，对于急性冠脉综合征患者格外重要。
抑制PCSK9的临床意义在于PCSK9的功能丧失性突变降低了LDL-C水平，显著降低了心血管事件的风险。相反，PCSK9的功能获得性突变升高了LDL-C水平和心血管事件的风险。HMG-CoA还原酶突变和PCSK9突变降低了LDL-C作用对降低心血管事件率有累积作用，即抗PCSK9抗体与他汀类药物的药效可相加。
一篇meta分析纳入24项随机试验(n＝10,159)，涵盖了多种临床情况，包括：家族性高胆固醇血症；其他高胆固醇血症；不耐受他汀类的高胆固醇血症；强化、非强化他汀类治疗和不用他汀类治疗。分析发现：抗PCSK9抗体减少了全因死亡率(OR 0.45,95％CI 0.23-0.86)、心血管死亡率(OR 0.50，CI 0.23-1.10)和心肌梗死(OR 0.49，CI 0.26-0.93)。纳入试验的结果的异质性无统计学意义，这表明抗PCSK9抗体与他汀类药物一样，对各种临床情况和基于心血管疾病风险有相似的治疗效果。
全球只有两款PCSK9抑制剂获批上市，分别是安进的依洛尤单抗 (Evolocumab，商品名：Repatha)和赛诺菲、再生元联合开发的阿利珠单抗(Alirocumab，商品名：Praluent)，且两个产品都是抗PCSK9单克隆抗体，目前还没有小分子化药的PCSK9抑制剂上市。中国成人血脂异常的患病率在大幅上升，根据《中国心血管病报告2018》显示，2012年我国成人血脂异常患病率已经达到40.4％。中国成人血脂异常知晓率、治疗率与控制率还有很大提升空间。抗PCSK9抗体能够满足血脂异常、控制不佳人群的临床需求缺口。因此，有必要对该靶点抗体进行深入研究，以得到更加稳定、药效更高、方便临床使用的制剂。
发明内容：
本发明的目的在于提供一种稳定的抗PCSK9抗体制剂。
具体地，本发明涉及以下方面：
1.抗PCSK9抗体制剂，其包含抗PCSK9抗体或其抗原结合片段，至少一种缓冲液，至少一种稳定剂和至少一种表面活性剂，其中所述抗PCSK9抗体包含SEQ ID NO:8所示的重链可变区包含的HCDR1，HCDR2和HCDR3，以及所述抗体包含SEQ ID NO:10所示的轻链可变区包含的LCDR1，LCDR2和LCDR3，优选地，根据IMGT编号系统，所述抗PCSK9抗体包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3，和SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3，其中SEQ ID NO:5所示的序列为GVI，
优选所述抗原结合片段选自Fab片段、Fab'片段、F(ab)'片段、Fv片段、分离的CDR区、单链Fv分子(scFv)，F(ab')2、Fd、dAb、Fab/c、双价抗体和结构域抗体。
2.项目1所述的抗PCSK9抗体制剂，其中所述缓冲液选自组氨酸缓冲液、柠檬酸盐缓冲液、琥珀酸盐缓冲液、醋酸盐缓冲液、精氨酸缓冲液、磷酸盐缓冲液的一种或多种，优选所述缓冲液选自1-20mM柠檬酸钠，1-20mM组氨酸和/或盐酸组氨酸，优选组氨酸的含量为1-7.4mM，优选3-5.4mM，更优选4-4.4mM，最优选4.2mM，优选盐酸组氨酸的含量为5-16.6mM，优选8-13.6mM，更优选10-11.6mM，最优选10.8mM，优选所述抗PCSK9抗体制剂的pH值为5.0-6.0，优选5.2-5.8，最优选pH值是5.5。
3.项目1-2任一项所述的抗PCSK9抗体制剂，其中所述稳定剂选自糖， 氨基酸，多元醇，抗氧化剂，防腐剂，环糊精，聚乙二醇(例如PEG 3000、PEG3350、PEG4000、PEG6000)，白蛋白(例如人血清白蛋白(HSA)，牛血清白蛋白(BSA))，盐(例如氯化钠，氯化钙)，整合剂(例如EDTA)的一种或多种，优选所述稳定剂选自糖类的一种或多种，所述的糖类可选自蔗糖和/或海藻糖：优选蔗糖的含量为5-100mg/mL，优选20-100mg/mL，40-80mg/mL，更优选54mg/mL-66mg/mL，55-65mg/mL或57mg/mL-63mg/mL，最优选60mg/mL，海藻糖的含量为5-100mg/mL，优选5-55mg/mL，15-45mg/mL，更优选25-35mg/mL，27mg/mL-33mg/mL或28.5mg/mL-31.5mg/mL，最优选30mg/mL，优选地，当蔗糖和海藻糖同时使用是，蔗糖和海藻糖的总浓度为85-95mg/mL，优选90mg/mL。
4.项目1-3任一项所述的抗PCSK9抗体制剂，其中所述表面活性剂选自聚氧乙烯脱水山梨糖醇脂肪酸酯(吐温)，例如聚山梨酯20和聚山梨酯80，优选聚山梨酯80的含量为0.02％-0.08％(w/w)，更优选0.025％-0.075％或0.04％-0.06％(w/w)，最优选0.05％(w/w)。
5.项目1-4任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂还包含注射用水。
6.项目1-5任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体或其抗原结合片段的浓度是10-150mg/mL，优选50-150mg/mL或80-120mg/mL，更优选90-110mg/mL，最优选100mg/mL。
7.项目1-5任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂包含100mg/mL的抗PCSK9抗体，4.2mM组氨酸，10.8mM盐酸组氨酸，60mg/mL蔗糖，30mg/mL海藻糖，0.05％(w/w)聚山梨酯80，pH为5.5。
8.项目1-6任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂还包含防腐剂，优选地，以约0.001％至约2％(w/v)的量使用防腐剂，优选地，防腐剂选自乙醇、苯甲醇、苯酚、间甲酚、对氯-间甲酚、对羟基苯甲酸甲酯、对羟基苯甲酸丙醋或苯扎氯铵。
9.项目1-6任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂还包含抗细菌剂和抗真菌剂，例如，三氯叔丁醇、山梨酸。
10.项目1-8任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂为适于注射(优选皮下注射)的形式。
11.制备抗PCSK9抗体制剂的方法，包括将抗PCSK9抗体或其抗原结合 片段与至少一种缓冲液，至少一种稳定剂和至少一种表面活性剂混合，用酸或碱调整制剂的pH为5.0-6.0，优选5.2-5.8，最优选pH值是5.5。
12.项目11所述的方法，其中所述抗PCSK9抗体或其抗原结合片段如项目1或6所述，所述缓冲液如项目2所述，所述稳定剂如项目3所述，所述表面活性剂如项目4所述。
13.权利要求11或12所述的方法，其中所述表面活性剂为非离子型表面活性剂(例如聚山梨酯80)，所述非离子型表面活性剂最后加入再进行定容。
在本发明中，表述“药物制剂”也表示抗PCSK9抗体制剂。
本发明所述的药物制剂中，所述的抗PCSK9抗体或其抗原结合片段包含任意1个或多个选自以下的CDR区序列或与其具有至少85％序列(例如至少86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％)同源性且具有相同活性的氨基酸序列：
在具体实施方案中，所述抗PCSK9抗体包含重链可变区和轻链可变区，所述抗体包含SEQ ID NO:8所示的重链可变区包含的HCDR1，HCDR2和HCDR3，以及所述抗PCSK9抗体包含SEQ ID NO:10所示的轻链可变区包含的LCDR1，LCDR2和LCDR3，优选地，所述抗PCSK9抗体包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3，和SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3。
所述氨基酸序列如下表1所示：
表1：抗PCSK9抗体包含的CDRs序列
抗PCSK9抗体的重链可变区的DNA序列如下(369bp)：
其编码的重链可变区序列如下(123aa)：
抗PCSK9抗体的轻链可变区的DNA序列如下(333bp)：
其编码的轻链可变区序列如下(111aa)：
本发明所述的抗PCSK9抗体制剂是一种适于皮下注射的药物制剂。
在本发明的一个实施方案中，所述抗PCSK9抗体制剂包括抗PCSK9抗体、缓冲液、糖类，表面活性剂或由其组成，任选还包括注射用水。
在本发明的一个实施方案中，所述抗PCSK9抗体制剂成分优选如下表2所示：
表2：抗PCSK9抗体制剂的成分
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。
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。
术语抗体的"抗原结合片段"(或简称"抗体片段")是指抗体的保持特异性结合抗原(例如，PCSK9)的能力的一个或多个片段。己显示可利用全长抗体的片段来进行抗体的抗原结合功能。可通过重组DNA技术或通过酶促或化学断裂完整免疫球蛋白来产生抗原结合部分。在本发明中，所述抗原结合片段的实例包括Fab片段、Fab'片段、F(ab)'片段、Fv片段、分离的CDR区、单链Fv分子(scFv)，F(ab')2、Fd、dAb、Fab/c、双价抗体和结构域抗体。
术语"CDR"是指抗体的可变结构域内主要促成抗原结合的6个高变区之一。所述6个CDR的最常用的定义之一由Kabat E.A.等人，(1991)Sequences of proteins of immunol ogi cal interest.NIH Publication 91-3242)提供。如本文中使用的，CDR的Kabat定义只应用于轻链可变结构域的LCDR1、LCDR2和LCDR3，以及重链可变结构域的HCDR1、HCDR2和HCDR3。
在本发明中，本发明所述的抗体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K、λ链或其变体。
在本发明中，本发明所述的抗体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。
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，将鼠可变区基因与人恒定区基因连接成嵌合基因后插入表达载体中，最后在真核系统或原核系统中表达嵌合抗体分子。
术语"人源化抗体(humanized antibody)"，也称为CDR移植抗体(CDR-grafted antibody)，是指将鼠的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 携带大量鼠蛋白成分，从而诱导的异源性反应。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。
术语"药物制剂"或“制剂”表示这样的制品：其处于允许活性成分的生物活性明确有效的形式，且其不含有对所述制剂要施用的受试者有毒的额外组分。
本文中结合根据本发明的制剂使用的术语"液体"表示，在大气压下在至少约20℃至约80℃的温度为液体的制剂。
术语"稳定剂"表示药学可接受的赋形剂，其在制造，储存和应用过程中保护活性药物成分和/或制剂免受化学和/或物理降解。由Cleland，J.L.,M.F.Powell，等人(1993)."The development of stable protein fomulations：a c10se look at protein aggregation,deamidation,and oxidation."Crit Rev Ther Drug Carrier Syst 10(4)：307-77,Wang,W.(1999)."Instability,stabilization,and formulation of liquid protein pharmaceuticals."Int J Pharm 185(2)：129-88.,Wang,W.(2000)."Lyophilization and development of solid protein pharmaceuticals咀1t J Ph arm203(1-2)：1-60和Chi，E.Y.,S.Kri shnan，等人(2003)."Physical stability of proteins in aqueous solution：mechanism and driving forces in nonnative protein aggregation."Pharm Res 20(9)：1325-36综述了蛋白质药物的化学和物理降解途径。
优选地，本发明的制剂是"稳定的"制剂，其代表这样的制剂：其中所含的蛋白(例如抗体)在贮存后基本上保留它的物理和化学稳定性，且因而保留它的生物活性。
例如，本发明的抗体制剂在冷藏温度2-8℃下至少12个月，特别是2年，并且更特别是3年观察不到显著改变。例如，稳定性的标准如下：目视法检测结果为无色至淡黄色澄明液体；分子排阻色谱(SE-HPLC)检测结果主峰不得低于95％；还原型毛细管电泳(rCE-SDS)检测结果重链轻链之和不得低于95％；非还原型毛细管电泳(nrCE-SDS)检测结果主峰不得低于85％；生物学活性检测(ELISA)结果为参考品50％-150％。
术语"蛋白PCSK9"、"PCSK9"互换使用，包括人PCSK9的变体、同种型(isoform)、物种同系物，及与PCSK9具有至少一个共同表位的类似物。完整的PCSK9序列可以根据NCBI Reference Sequence：Genbank ID：NP_000567.1查到。
术语"抗PCSK9抗体"、"针对PCSK9的抗体"和"针对PCSK9蛋白的抗体"指能够以足够的亲和力结合PCSK9蛋白，使得所述抗体可在靶向PCSK9蛋白中用作诊断剂和/或治疗剂。本文中使用的术语"结合PCSK9蛋白"是指，在BIAcore测定(Pharmacia Biosensor AB,Uppsala，瑞典)中或在ELISA中抗体对PCSK9蛋白的结合。
本文中使用的术语"表面活性剂"表示，用于保护蛋白制剂抵抗物理应力(如搅拌和剪切)的药学上可接受的赋形剂。药学上可接受的表面活性剂的例子包括：聚氧乙烯脱水山梨糖醇脂肪酸酯(吐温)。聚氧乙烯脱水山梨糖醇-脂肪酸酯的例子是聚山梨酯20和聚山梨酯80。
本文中使用的术语"缓冲液"表示稳定药物制剂的pH的药学上可接受的赋形剂。合适的缓冲液是本领域众所周知的，且可以在文献中找到。优选的药学上可接受的缓冲液包括但不限于：组氨酸缓冲液、柠檬酸盐缓冲液、琥珀酸盐缓冲液、醋酸盐缓冲液、精氨酸缓冲液、磷酸盐缓冲液或其混合物。优选的缓冲液包含组氨酸/盐酸组氨酸缓冲液，其用本领域己知的酸或碱进行pH调节。上述的缓冲液通常以1mM-100mM、优选10mM-30mM、最优选20mM的量使用。独立于使用的缓冲液，用本领域己知的酸或碱(例如盐酸、乙酸、磷酸、硫酸和柠檬酸、氢氧化钠和氢氧化钾)可以将药物制剂的pH调至pH5.0-6.0，特别是调至pH 5.2-5.8，最特别地是调至pH 5.5。
在某些实施方案中，本发明药物制剂还可以包含抗氧化剂作为第二种稳定剂。"抗氧化剂"是阻止活性药物成分的氧化的药学上可接受的赋形剂。抗氧化剂包括、但不限于：整合剂诸如EDTA、柠檬酸、抗坏血酸、丁羟甲苯(BHT)、丁羟茴香醚(BHA)、亚硫酸钠、对氨基苯甲酸、谷胱甘肽、没食子酸丙酯、半胱氨酸、甲硫氨酸、乙醇、苯甲醇和正乙酰基半胱氨酸。
本文中使用的术语"糖"表示单糖或寡糖。单糖是不可被酸水解的单体碳水化合物，包括单糖和它们的衍生物，例如氨基糖。单糖的例子包括葡萄糖、果糖、半乳糖、甘露糖、山梨糖、核糖、脱氧核糖、神经氨酸。寡糖是由超过一个经由糖苷键连接的单体糖单元组成的支链或直链碳水化合物。在寡糖内的单体糖单元可以是相同的或不同的。取决于单体糖单元的数目，寡糖是二糖、三糖、四糖、五糖，诸如此类。与多糖不同，单糖和寡糖是水溶性的。寡糖的例子包括蔗糖、海藻糖、乳糖、麦芽糖和棉子糖。具体地，糖选自蔗糖和海藻糖。
本文中使用的术语"氨基酸"表示，具有位于羧基的α-位的氨基部分的药学 上可接受的有机分子。氨基酸的例子包括精氨酸、甘氨酸、鸟氨酸、赖氨酸、组氨酸、谷氨酸、天冬氨酸、异亮氨酸、亮氨酸、丙氨酸、苯丙氨酸、酪氨酸、色氨酸、甲硫氨酸、丝氨酸、脯氨酸。通常以下述量使用氨基酸：5mM-200mM的量，特别是5mM-100mM的量，更特别是5mM-70mM的量。
术语"稳定剂"也包括冷冻保护剂。术语"冷冻保护剂"表示，在冷冻干燥过程、随后的贮存和重构中保护不稳定的活性成分(例如蛋白)抵抗失稳条件的药学上可接受的赋形剂。冷冻保护剂包括、但不限于糖、多元醇(例如糖醇)和氨基酸。具体地，冷冻保护剂可以选自：糖类诸如蔗糖、海藻糖、乳糖、葡萄糖、甘露糖、麦芽糖、半乳糖、果糖、山梨糖、棉子糖、神经氨酸，氨基糖类诸如葡糖胺、半乳糖胺、N-甲基葡糖胺("葡甲胺")，多元醇类诸如甘露醇和山梨糖醇，和氨基酸类诸如精氨酸和甘氨酸或其混合物。所述的冷冻保护剂优选二糖，具体地，优选蔗糖、海藻糖。本发明惊奇地发现，二糖对制剂的稳定性优于单糖，多元醇，氨基酸。
药物制剂还可以含有张度剂。本文中使用的术语"张度剂"表示用于调节制剂的张度的药学上可接受的张度剂。所述制剂可以是低渗的、等渗的或高渗的，优选地是等渗的。等渗性通常涉及溶液的相对渗透压，经常是相对于人血清的渗透压。等渗制剂是液体或从固体形式(例如从低压冻干形式)重构的液体，且表示具有与它所对比的某种其它溶液(诸如生理学的盐溶液和血清)相同的张度的溶液。合适的张度剂包括、但不限于氯化钠、氯化钾、甘油和选自以下的任意组分：氨基酸，糖，尤其是葡萄糖。通常以约5mM至约500mM的量使用张度剂。在稳定剂和张度剂内，存在一组可以以两种方式起作用的化合物，即它们可以同时为稳定剂和张度剂。其例子可以在以下集合中找到：糖、氨基酸、多元醇、环糊精、聚二醇和盐。可以同时为稳定剂和张度剂的糖的一个例子是海藻糖。
所述药物制剂还可以含有佐剂诸如防腐剂、润湿剂、乳化剂和分散剂。通过灭菌操作和通过包含不同的抗细菌剂和抗真菌剂(例如，对羟基苯甲酸甲酯、三氯叔丁醇、苯酚、山梨酸等)，可以确保阻止微生物的存在。通常以约0.001％至约2％(w/v)的量使用防腐剂。防腐剂包括、但不限于：乙醇、苯甲醇、苯酚、间甲酚、对氯-间甲酚、对羟基苯甲酸甲酯或对羟基苯甲酸丙醋、苯扎氯铵。
根据本发明的抗PCSK9抗体的药物制剂可以用于治疗杂合子家族性高胆 固醇血症或临床动脉粥样硬化性心血管疾病，用于需要额外降低低密度脂蛋白胆固醇(LDL-C)的成人，还可作为不耐受他汀类药物治疗的辅助药物。抗PCSK9抗体可通过其他机制来改善心血管病症，包括减轻动脉粥样硬化斑块内的炎症和氧化应激，以及抑制促血栓形成途径。
通过静脉内(i.v.)、皮下(s.c.)或任意其它胃肠外施用方式(如药学领域中己知的那些方式)，可以施用根据本发明的抗PCSK9抗体药物制剂。
考虑到它们的高稳定性，根据本发明的药物制剂可以在不需要过滤器(in-line filter)的情况下皮下施用。
用于体内施用的制剂必须是无菌的。这通过非终端灭菌的无菌生产工艺可以有效实现。
通过本领域己知的方法，例如超滤-透析过滤、透析、添加和混合、重构及其组合，可以制备根据本发明的抗PCSK9抗体药物制剂。可以在下文中找到根据本发明的制剂的制备例子。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著，黄培堂等译的《分子克隆实验指南》，第三版，科学出版社)或按照产品说明书进行。所用试剂或仪器未注明生产厂商者，为可以通过市场购买获得的常规产品。
制备例1.人源化抗PCSK9抗体的制备
1.抗体的设计
为制备单克隆抗体MAB1，本发明人根据已有的PCSK9蛋白序列(NP_777596.2)及其蛋白三维晶体结构等，人工设计了一系列的抗体序列。通过大量的筛选和检测，最终得到了一种与PCSK9特异性结合的MAB1。该单克隆抗体重链和轻链可变区的氨基酸序列及其编码序列如下面的SEQ ID NO:1-4。
设计的MAB1重链VH的DNA序列如下(369bp)：

其编码的VH蛋白序列如下(123aa)：
EVQLVESGGGLVQPGRSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSGISSSSSYISYADSVQGRFTISRDNGKNSLYLQMNSLRAEDTALYFCAREYDFWSAYYDAFDVWGQGTMVTVSS(SEQ ID NO:8)MAB1轻链VL的DNA序列如下(333bp)：
其编码的VL蛋白序列如下(111aa)：
所述抗体的CDR序列如下：
HCDR1：GFTFSSYS(SEQ ID NO：1)；
HCDR2：ISSSSSYI(SEQ ID NO：2)；
HCDR3：AREYDFWSAYYDAFDV(SEQ ID NO：3)；
LCDR1：SRNIGGGND(SEQ ID NO：4)；
LCDR2：GVI(SEQ ID NO：5)；以及
LCDR3：QSFDGSLSGSV(SEQ ID NO：6)。
2.抗体的表达和纯化
将MAB1的重链cDNA序列(VH的编码序列如SEQ ID NO：7所示；恒定区是Ig gamma-1 chain C region,ACCESSION:P01857)和轻链的cDNA序列(VL的编码序列如SEQ ID NO：9所示；恒定区为Ig lambda-2 chain C regions；ACCESSION:P0CG05.1)分别克隆到pUC57simple(金斯瑞公司提供)载体中，分别获得pUC57simple-MAB1H和pUC57simple-MAB1L质粒。分别将质粒pUC57simple-MAB1H和pUC57simple-MAB1L进行酶切(HindIII&EcoRI)，电泳回收得到的重链和轻链分别亚克隆到pcDNA3.1载体中，提取重组质粒共转染293F细胞。细胞培养7天后，将培养液通过高速离心、上清浓缩后上样至HiTrap MabSelect SuRe柱，用Elution Buffer一步洗脱蛋白并回收目标样品并换液至PBS，获得抗PCSK9抗体。在实施例中，表述抗PCSK9抗体表示MAB1抗体。
实施例1：制剂制备工艺
抗PCSK9抗体制剂的组成如下：该制剂由溶质和溶剂组成，溶质为抗PCSK9抗体、组氨酸、盐酸组氨酸、蔗糖、海藻糖和聚山梨酯80，溶剂为注射用水；抗PCSK9抗体在制剂中的浓度为100mg/mL，组氨酸在制剂中的浓度为4.2mM，盐酸组氨酸在制剂中的浓度为10.8mM，蔗糖在制剂中的浓度为60mg/mL，海藻糖在制剂中的浓度为30mg/mL，聚山梨酯80在制剂中的浓度为0.05％(w/v)，pH为5.5。
在配制抗PCSK9抗体制剂时，溶质中非离子型表面活性剂聚山梨酯80最后加入再进行定容，其他溶质的添加则无先后顺序。将抗PCSK9抗体制剂无菌分装至西林瓶中，加盖橡胶塞和铝塑盖或将抗PCSK9抗体制剂无菌分装至储液袋(赛多利斯Flexboy)中，获得该制剂的成品。
实施例2：抗PCSK9抗体制剂的稳定性实验
对实施例1制备的抗PCSK9抗体制剂进行稳定性实验，以主成分的百分含量的变化和蛋白纯度的主峰含量作为判定手段，分别通过分子排阻色谱法SEC-HPLC、毛细管等电聚焦电泳法icIEF、还原型CE-SDS、非还原型CE-SDS、活性以及观察制剂的外观表征制剂中抗PCSK9抗体在各环境条件下的化学稳定性，考察制剂在高温、光照、冻融、震动等条件因素的稳定性。
表1：各环境条件下制剂中抗PCSK9抗体的化学稳定性

注：-：表示未观察
表2：各环境条件下制剂中抗PCSK9抗体的化学稳定性

注：A：表示冻融3个循环；B：表示冻融2个循环；C：表示冻融1个循环；
-：表示未观察
表3：各环境条件下制剂中抗PCSK9抗体的化学稳定性


注：-：表示未观察
表4：各环境条件下制剂中抗PCSK9抗体的化学稳定性

注：-：表示未观察
采用分子排阻色谱法SEC-HPLC、毛细管等电聚焦电泳法icIEF、还原型CE-SDS、非还原型CE-SDS、活性(ELISA)分析制备例1制备的抗PCSK9抗体制剂，分别在以下实验条件下：-20℃/25℃反复冻融3个循环；300rpm震荡3天；在40℃高温条件下贮存30天；在4500±500Lux光照下贮存15天的蛋白含量的变化、蛋白纯度的变化和电荷抑制体纯度的变化，结果如表1-表4所示。
根据表1的结果，抗PCSK9抗体制剂的蛋白含量的变化以及蛋白纯度在40℃高温条件下随储存时间的增加而下降。在第30天的SE-HPLC测量中主峰为92.3％，抗体单体被降解不多于10％。此外，通过视觉分析，为淡黄色澄明液体，与正常条件下一致。制剂的蛋白质浓度为98.2mg/mL，不多于+/-10％正常条件下的蛋白质浓度。说明在40℃高温条件下贮存30天，该制剂依然为稳定的液体抗体制剂。
根据表2的结果，抗PCSK9抗体制剂的蛋白含量的变化以及蛋白纯度在冻融后无明显变化。说明在冻融三个循环后，该制剂依然为稳定的液体抗体制剂。
根据表3的结果，抗PCSK9抗体制剂的蛋白含量的变化以及蛋白纯度在光照条件下随储存时间的增加而下降。在第15天的SE-HPLC测量中主峰为94.4％，抗体单体被降解不多于10％。此外，通过视觉分析，为淡黄色澄明液体，与正常条件下一致。制剂的蛋白质浓度为97.3mg/mL，不多于+/-10％正常条件下的蛋白质浓度。说明在光照条件下贮存15天，该制剂依然为稳定的液体抗体制剂。
根据表4的结果，抗PCSK9抗体制剂的蛋白含量的变化以及蛋白纯度在震荡处理后无明显变化。说明在300rpm震荡3天后，该制剂依然为稳定的液体抗体制剂。
总结：表1-4的结果表明该制剂在上述的储存条件中有充足的稳定性。表明在处于加速抗PCSK9抗体的化学降解和物理变化的各种条件下，该制剂配方可缓解所含有的抗PCSK9抗体的化学降解速率降低和提高了抗PCSK9抗体的物理化学稳定性，使得该单克隆抗体能稳定存在于制剂的配方中。
实施例3.不同制剂组成和pH的比较
(1)分别采用表5所示的不同配方组成，通过SEC-HPLC测试在不同时间制剂的纯度，结果见表6。其中AK102表示抗PCSK9抗体-MAB1抗体，浓度为100mg/mL。
表5抗PCSK9抗体混合配方筛选组成
表6抗PCSK9抗体配方筛选SEC-HPLC纯度结果

由表6可见，在本发明组分含量范围内的制剂，都保证了良好的稳定性。
(2)溶液pH是影响生物药物物理化学稳定性的最重要参数之一。pH可以调节蛋白质表面的电荷分布，从而影响生物药物分子内及分子间的作用力，同时影响抗体药物发生化学修饰的速率，是生物药物制剂的最关键属性之一。
按表7的制剂配方，进行Tm测试和高温破坏试验，比较不同配方的稳定性差异。
表7.抗PCSK9抗体的不同配方组成
(2.1)Tm值测试
蛋白融化温度(Tm)反映了生物药物在水溶液中的热稳定性，Tm越高则在实际保存条件下发生构象改变的可能性越低。考察不同pH的缓冲液中抗PCSK9抗体的Tm值。表8中的配方是分别对应采用5μL表7配方中的抗PCSK9抗体，加入5μL SYPRO Orange(20X)和90μL缓冲液配制而成。通过类差示荧光扫描技术(DSF)的方法测定样品的Tm值。结果如表8。
表8.不同pH条件下制剂的Tm值
(2.2)不同配方样品的高温加速实验
将表7不同配方的制剂置于40±2℃的样品试验箱中进行加速破坏，加速时间3天，通过外观和SEC-HPLC考察不同制剂的稳定性情况。
检测结果整理如表9和表10。
表9.不同制剂的高温试验外观变化情况

注：*T0表示0时的结果；**40C3d表示40℃放置3天的结果。
表10.不同制剂的高温试验SEC-HPLC的纯度变化情况


注：*T0表示0时的结果；**40C3d表示40℃放置3天的结果。
根据Tm测定结果和高温加速实验结果，只有配方F8在加速后外观仍然澄清，其他配方均出现浑浊，不适用于AK102的保存。同时SEC-HPLC纯度显示配方F8在加速破坏3天后纯化未发生明显变化，初步评估AK102在多羟基的保护剂和pH5.5条件下稳定性较好，根据此结果，进行多羟基结构类保护剂的筛选和优化。
保护剂的筛选优化
根据抗体药物常保护剂，设计表11中的三种配方，将不同配方样品放置于40±2℃条件下进行稳定性考察，比较不同保护剂对AK102的稳定性影响，从而选出最佳的配方。
表11.AK102配方筛选组成
在样品40±2℃条件下破坏后，分别于1周、2周、4周、8周取样进行SEC-HPLC、CE-SDS分析，比较三种配方发生变化情况。检测结果见表12和13。
表12.AK102配方筛选SEC-HPLC纯度结果
表13.AK102配方筛选还原型CE-SDS纯度结果
小结：在加速8周时，配方F11未进行相关检测分析，从SEC-HPLC和CE-SDS结果看，F12(海藻糖)稳定性明显优于配方F13(山梨醇)，结果提示AK102在海藻糖配方中有更好的稳定性。
从工艺成本角度考虑，选择蔗糖和海藻糖的混合后作为AK102的保护剂，并对混合的比例进行研究，设计不同比例混合糖类保护剂配方如表14，配方中AK102样品的浓度均为100mg/ml，与最终拟定包装浓度相同。
表14.AK102混合配方筛选组成
表15.AK102配方筛选SEC-HPLC纯度结果
实施例4.不同制剂配方在不同时间点的稳定性分析
按照表16配制不同的制剂
表16.制剂样品F1-F19的配方


注：F17-F19为同样配方用于重复实验的制剂编号。
按照表16的配方配制样品F1-19超滤缓冲液1L(不含聚山梨酯80和抗体)，加入1.9mL 6M盐酸)，并使用超滤浓缩系统制备表16的蛋白溶液，加入表16对应样品中的10％聚山梨酯80(II)溶液，每个制剂制备约90ml。
使用0.22μm的滤膜进行过滤。每个配方样品制备30支(1.5mL/支)。每个配方取10支样品送检为T0点检测(T0表示配置后直接检测的时间点)，20支放入25℃稳定性试验箱进行加速试验(25℃3M表示在25℃放置3个月)。检测项目参见表17。检测结果见表18-1、18-2(T0检测结果)和表18-3、18-4(25℃3M检测结果)。
样品制剂F1-F19检测时间点及项目：





Claims (13)

  1. 抗PCSK9抗体制剂，其包含抗PCSK9抗体或其抗原结合片段，至少一种缓冲液，至少一种稳定剂和至少一种表面活性剂，其中所述抗PCSK9抗体包含SEQ ID NO:8所示的重链可变区包含的HCDR1，HCDR2和HCDR3，以及所述抗体包含SEQ ID NO:10所示的轻链可变区包含的LCDR1，LCDR2和LCDR3，优选地，按照IMGT编号系统，所述抗PCSK9抗体包含SEQ ID NO:1所示的HCDR1，SEQ ID NO:2所示的HCDR2和SEQ ID NO:3所示的HCDR3，和SEQ ID NO:4所示的LCDR1，SEQ ID NO:5所示的LCDR2和SEQ ID NO:6所示的LCDR3，其中SEQ ID NO:5所示的序列为GVI，
    优选所述抗原结合片段选自Fab片段、Fab'片段、F(ab)'片段、Fv片段、分离的CDR区、单链Fv分子(scFv)，F(ab')2、Fd、dAb、Fab/c、双价抗体和结构域抗体。
  2. 权利要求1所述的抗PCSK9抗体制剂，其中所述缓冲液选自组氨酸缓冲液、柠檬酸盐缓冲液、琥珀酸盐缓冲液、醋酸盐缓冲液、精氨酸缓冲液、磷酸盐缓冲液的一种或多种，优选所述缓冲液选自1-20mM柠檬酸钠，1-20mM组氨酸和/或盐酸组氨酸，优选组氨酸的含量为1-7.4mM，优选3-5.4mM，更优选4-4.4mM，最优选4.2mM，优选盐酸组氨酸的含量为5-16.6mM，优选8-13.6mM，更优选10-11.6mM，最优选10.8mM，优选所述抗PCSK9抗体制剂的pH值为5.0-6.0，优选5.2-5.8，最优选pH值是5.5。
  3. 权利要求1-2任一项所述的抗PCSK9抗体制剂，其中所述稳定剂选自糖，氨基酸，多元醇，抗氧化剂，防腐剂，环糊精，聚乙二醇(例如PEG 3000、PEG3350、PEG4000、PEG6000)，白蛋白(例如人血清白蛋白(HSA)，牛血清白蛋白(BSA))，盐(例如氯化钠，氯化钙)，整合剂(例如EDTA)的一种或多种，优选所述稳定剂选自糖类的一种或多种，所述的糖类可选自蔗糖和/或海藻糖：优选蔗糖的含量为5-100mg/mL，优选20-100mg/mL，40-80mg/mL，更优选54mg/mL-66mg/mL，55-65mg/mL或57mg/mL-63mg/mL，最优选60mg/mL，海藻糖的含量为5-100mg/mL，优选5-55mg/mL，15-45mg/mL，更优选25-35mg/mL，27mg/mL-33mg/mL或28.5mg/mL-31.5mg/mL，最优选30mg/mL，优选地，当蔗糖和海藻糖同时使用是，蔗糖和海藻糖的总浓度为85-95mg/mL，优选90mg/mL。
  4. 权利要求1-3任一项所述的抗PCSK9抗体制剂，其中所述表面活性剂选自聚氧乙烯脱水山梨糖醇脂肪酸酯(吐温)，例如聚山梨酯20和聚山梨酯80，优选聚山梨酯80的含量为0.02％-0.08％(w/w)，更优选0.025％-0.075％或0.04％-0.06％(w/w)，最优选0.05％(w/w)。
  5. 权利要求1-4任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂还包含注射用水。
  6. 权利要求1-5任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体或其抗原结合片段的浓度是10-150mg/mL，优选50-150mg/mL或80-120mg/mL，更优选90-110mg/mL，最优选100mg/mL。
  7. 权利要求1-5任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂包含100mg/mL的抗PCSK9抗体，4.2mM组氨酸，10.8mM盐酸组氨酸，60mg/mL蔗糖，30mg/mL海藻糖，0.05％(w/w)聚山梨酯80，pH为5.5。
  8. 权利要求1-6任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂还包含防腐剂，优选地，以约0.001％至约2％(w/v)的量使用防腐剂，优选地，防腐剂选自乙醇、苯甲醇、苯酚、间甲酚、对氯-间甲酚、对羟基苯甲酸甲酯、对羟基苯甲酸丙醋或苯扎氯铵。
  9. 权利要求1-6任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂还包含抗细菌剂和抗真菌剂，例如，三氯叔丁醇、山梨酸。
  10. 权利要求1-8任一项所述的抗PCSK9抗体制剂，其中所述抗PCSK9抗体制剂为适于注射(优选皮下注射)的形式。
  11. 制备抗PCSK9抗体制剂的方法，包括将抗PCSK9抗体或其抗原结合片段与至少一种缓冲液，至少一种稳定剂和至少一种表面活性剂混合，用酸或碱调整制剂的pH为5.0-6.0，优选5.2-5.8，最优选pH值是5.5。
  12. 权利要求11所述的方法，其中所述抗PCSK9抗体或其抗原结合片段如权利要求1或6所述，所述缓冲液如权利要求2所述，所述稳定剂如权利要求3所述，所述表面活性剂如权利要求4所述。
  13. 权利要求11或12所述的方法，其中所述表面活性剂为非离子型表面活性剂(例如聚山梨酯80)，所述非离子型表面活性剂最后加入再进行定容。
PCT/CN2023/105006 2022-06-30 2023-06-30 抗pcsk9抗体的制剂及其应用 WO2024002357A1 (zh)

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