WO2021254221A1 - 一种稳定的抗IL-4Rα单克隆抗体液体制剂 - Google Patents

一种稳定的抗IL-4Rα单克隆抗体液体制剂 Download PDF

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WO2021254221A1
WO2021254221A1 PCT/CN2021/099056 CN2021099056W WO2021254221A1 WO 2021254221 A1 WO2021254221 A1 WO 2021254221A1 CN 2021099056 W CN2021099056 W CN 2021099056W WO 2021254221 A1 WO2021254221 A1 WO 2021254221A1
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liquid preparation
concentration
monoclonal antibody
histidine
polysorbate
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French (fr)
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杨泗兴
徐鹏翔
黄浩旻
朱祯平
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三生国健药业（上海）股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • 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
    • A61K47/26—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
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/02—Nasal agents, e.g. decongestants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • 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

Definitions

  • the invention belongs to the field of biopharmaceuticals, and specifically relates to a stable liquid preparation of anti-IL-4R ⁇ monoclonal antibody.
  • Atopic Dermatitis is a common chronic inflammatory recurrent disease of the skin. This disease is the most common inflammatory skin disease in the world, and it affects about 15%-30% of children and 2%-10% of adults worldwide. According to statistics, there are more than 2.3 million adolescents in my country suffering from atopic dermatitis (eczema), and there are 2 to 3 million children suffering from children.
  • Dupixent for the treatment of moderate to severe eczema (atopic dermatitis) in adult patients.
  • Dupixent is intended for patients whose eczema is not adequately controlled after topical medication, or patients who are not recommended to use topical medication.
  • Dupixent is administered by subcutaneous injection.
  • the active ingredient is an antibody that binds to the interleukin-4 receptor alpha subunit (IL-4R ⁇ ), a protein that causes inflammation. By combining with this protein, Dupixent can inhibit the inflammatory response that plays a role in the development of atopic dermatitis.
  • IL-4R ⁇ interleukin-4 receptor alpha subunit
  • Dupixent's preparation is 150mg/ml injection, which is provided by EMA Assessment report Dupixent (European Medicines Agency, Science Medicines Health, EMA/512262/2017, p16, https://www.ema.europa.eu/en/documents/assessment -report/dupixent-epar-public-assessment- report_en.pdf ) It can be seen that it can be stored at 2-8°C for 15 months, and at 25°C for up to 14 days, the stability is poor.
  • PCT patent application WO2020/048312 discloses a type of self-developed recombinant anti-IL-4R ⁇ humanized monoclonal antibody, which is a recombinant humanized monoclonal antibody expressed in CHO cells using DNA recombination technology, and is composed of anti-IL-4R ⁇
  • the variable region of the heavy chain and the variable region of the light chain of the murine antibody are constructed by humanization. It is necessary to conduct in-depth research on these new recombinant anti-IL-4R ⁇ humanized monoclonal antibodies to develop stable preparations suitable for clinical applications.
  • the purpose of the present invention is to provide a stable high-concentration liquid anti-IL-4R ⁇ monoclonal antibody preparation.
  • the liquid preparation is composed of anti-IL-4R ⁇ monoclonal antibody, histidine, arginine hydrochloride, trehalose, glacial acetic acid, and polysorbate 80. It can play a role in stabilizing the protein, so that the target protein can be stored at 2-8°C for at least 24 months, and at 25°C for at least 6 months, which has excellent stability.
  • the first aspect of the present invention provides a stable liquid preparation of anti-IL-4R ⁇ monoclonal antibody, said liquid preparation comprising anti-IL-4R ⁇ monoclonal antibody, histidine, arginine hydrochloride, trehalose, ice Acetic acid, polysorbate 80, wherein the concentration of the anti-IL-4R ⁇ monoclonal antibody is 100-200mg/ml, and the anti-IL-4R ⁇ monoclonal antibody comprises any one of SEQ ID NO: 1-3 The heavy chain shown in SEQ ID NO: 4 and the light chain shown in SEQ ID NO: 4.
  • the concentration of the anti-IL-4R ⁇ monoclonal antibody is 150 mg/ml
  • the anti-IL-4R ⁇ monoclonal antibody includes the heavy chain shown in SEQ ID NO: 1 and the heavy chain shown in SEQ ID NO: 4 Light chain.
  • the histidine concentration is 20-30 mM, preferably, the histidine concentration is 20 mM.
  • the concentration of arginine hydrochloride is 25-75 mM, preferably, the concentration of arginine hydrochloride is 25 mM.
  • the trehalose concentration is 50-80 mg/ml, preferably, the trehalose concentration is 70 mg/ml.
  • the concentration of glacial acetic acid is 0.4-1.7 mg/ml, and preferably, the concentration of glacial acetic acid is 0.89 mg/ml.
  • the concentration of the polysorbate 80 is 0.3-1.5 mg/ml, and preferably, the concentration of the polysorbate 80 is 0.3 mg/ml.
  • the pH range of the liquid preparation is 5.0-6.3, and preferably, the pH of the liquid preparation is 5.8.
  • the second aspect of the present invention provides the use of the liquid formulation in the preparation of drugs for treating diseases related to IL-4R ⁇ overexpression.
  • the diseases related to IL-4R ⁇ overexpression include atopic dermatitis, asthma, allergic reactions, eosinophilic esophagitis, skin infections, nasal polyps and the like.
  • the present invention also provides a method for treating diseases related to IL-4R ⁇ overexpression, the method comprising administering a therapeutically effective amount of the liquid preparation of the present invention to a patient suffering from atopic dermatitis, asthma, and allergies. Reactions, eosinophilic esophagitis, skin infections, nasal polyps and other diseases.
  • the present invention provides a stable liquid preparation of anti-IL-4R ⁇ monoclonal antibody.
  • the present invention greatly improves the defect of poor stability of the existing anti-IL-4R ⁇ monoclonal antibody preparation by optimizing the formula.
  • the finished liquid formula pharmaceutical product of the present invention can be stored at 2-8°C for at least 24 months, and at 25°C for at least 6 months, and has excellent stability. Therefore, the liquid formulation of the present invention can provide anti-IL-4R ⁇ monoclonal antibody formulation stability, and has a wide range of industrial application prospects.
  • Figure 1 shows the results of DOE's investigation of pH and polysorbate 80 concentration.
  • Figure 2 shows the results analysis of insoluble particles of polysorbate 80 at different concentrations.
  • Figure 3 shows the analysis of the long-term stability of SEC purity.
  • Figure 4 shows the result analysis of the long-term stability of IEC purity.
  • the anti-human IL-4R ⁇ monoclonal antibody used in the present invention is derived from the humanized anti-human IL-4R ⁇ monoclonal antibody 4-2-Humanized-IgG4, 4-2-Humanized-IgG1, 4- 2-Humanized-IgG1-SELF, which contains the same variable region of the heavy chain, different constant regions of the heavy chain, and the same light chain.
  • the amino acid sequences of the heavy and light chains are shown below.
  • the protein sample used in the following examples is 4-2-Humanized-IgG4, which includes a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 4.
  • Mobile phase 200mM phosphate buffer, pH 6.8 ⁇ 0.1. Use after filtering with 0.22 ⁇ m filter membrane and ultrasonic degassing.
  • Chromatographic column TSK G3000SWxl, 7.8 ⁇ 300mm 5 ⁇ m, TOSOH 08541.
  • High performance liquid chromatograph Waters Alliance e2695 2489 UV/Visible light detector, Dionex Ultimate 3000VWD-3400 (RS) Detector or other suitable HPLC systems equipped with UV detectors.
  • Chromatography software is used for integration, and the peak area normalization method is used to calculate the peak area percentage of each peak.
  • Acceptance criteria for system suitability 6-pin system suitability samples, the resolution of polymer and monomer are both ⁇ 1.5, the retention time of the main peak is RSD ⁇ 1.0%, the peak area of the main peak is RSD ⁇ 2.0%, and the asymmetry of the main peak is all ⁇ 2.0, the number of theoretical plates is ⁇ 4000.
  • Test product report result The SEC purity report of the sample is the peak area percentage of the monomer main peak, and the aggregate content is the peak area percentage of the aggregate peak.
  • Mobile phase A 20mM phosphate buffer, pH 6.5 ⁇ 0.05. Use after filtering with 0.22 ⁇ m filter membrane and ultrasonic degassing.
  • Mobile phase B 20mM phosphate buffer + 200mM sodium chloride, pH 6.5 ⁇ 0.05. Use after filtering with 0.22 ⁇ m filter membrane and ultrasonic degassing.
  • Chromatographic column Propac WCX-10, 4 ⁇ 250mm, Thermo Dionex 054993.
  • High performance liquid chromatograph Waters Alliance e2695, Dionex Ultimate 3000 series or other suitable HPLC systems equipped with UV detectors.
  • System suitability sample Dilute the reference product with mobile phase to a concentration of 1.0mg/ml, centrifuge at 13000rpm for 10min, take the supernatant and transfer it to the sample bottle, and put it into the HPLC sample tray.
  • Test product Dilute the test product concentration to 1.0 mg/ml with mobile phase, centrifuge at 13000 rpm for 10 min, take the supernatant and transfer it to the sample bottle, and put it into the HPLC sample tray.
  • Chromatographic conditions column temperature 30 ⁇ 2°C; sample temperature 10 ⁇ 2°C; detection wavelength UV 214nm; injection volume 20 ⁇ L; flow rate 1.0ml/min.
  • the mobile phase gradient is as follows:
  • Purity analysis Use the peak area normalization method to calculate the peak area percentages of the main peak, acid peak area and alkali peak area on the sample spectrum. The IEC purity result is reported as the area percentage of the main peak.
  • the 10 mM citric acid system, the 20 mM histidine-histidine hydrochloride system, and the 20 mM histidine-acetic acid system were prepared respectively, and the pH was 5.5.
  • the three groups of solutions were placed at 40°C for 15 days, and samples were taken at 0, 2, 5, 9, and 15 days to detect SEC purity and IEC purity.
  • Example 2 The effect of arginine/arginine hydrochloride on preparation viscosity
  • Arginine/arginine hydrochloride has the effect of reducing the viscosity of the high-concentration protein solution and protecting the protein.
  • the protein concentration is 150mg/ml and the viscosity is relatively high. Therefore, arginine hydrochloride was added for the experiment to explore its Reduction and protection of protein viscosity.
  • this example uses DOE design to investigate the effects of pH and polysorbate 80 on the formulation.
  • Use glacial acetic acid to adjust the pH range to 5.0-6.6, and the dosage of polysorbate 80 to range from 0-3mg/ml.
  • the protein concentration of other ingredients is 150mg/ml, histidine 20mM, arginine hydrochloride 25mM, and trehalose 50mg/ml.
  • the prepared samples were placed at 40°C for 30 days for inspection, and samples were taken at 0, 7, 14, 21, and 30 days to detect SEC purity, IEC purity and insoluble particles ( ⁇ 10 ⁇ m).
  • This example examines the effect of polysorbate 80 on insoluble particles. As shown in Table 5, 6 batches of samples were prepared in this example, with a protein concentration of 150 mg/ml, histidine 20 mM, arginine hydrochloride 25 mM, trehalose 50 mg/ml, polysorbate 800-0.5 mg/ml, and glacial acetic acid Adjust the pH to 5.8. Detect insoluble particles after preparation.
  • Numbering Polysorbate 80 content (mg/ml) >2 ⁇ m(pcs/ml) >10 ⁇ m(pcs/ml) >25 ⁇ m(pcs/ml) 4-1 0 60,651.85 6,499.36 369.67 4-2 0.1 716.2 447.63 73.46 4-3 0.2 178.95 64.24 4.59 4-4 0.3 233.97 37.06 4.63 4-5 0.4 96.36 36.71 6.88 4-6 0.5 151.42 22.94 2.29
  • the polysorbate 80 preferably has a concentration of 0.3 mg/ml or more.
  • This example examines the influence of the dosage of trehalose and histidine on the preparation.
  • the protein concentration is 150mg/ml, 25mM arginine hydrochloride, and 0.3mg/ml polysorbate 80 is added, and the pH is adjusted to 5.8 with glacial acetic acid.
  • the samples were placed at 40°C for 14 days, and samples were taken at 0, 7, and 14 days to detect SEC purity and IEC purity.
  • the preferred concentration range of histidine is 20-30 mM.
  • the preferred concentration range of trehalose is It is 50-80mg/ml.
  • glacial acetic acid was added to 50 ml of histidine (20 mM) and arginine hydrochloride (25 mM) solution, and the pH was measured according to the added amount in Table 8. The concentration of glacial acetic acid corresponding to each pH is calculated.
  • 3 batches of drugs were prepared.
  • the specific prescriptions were anti-IL-4R ⁇ monoclonal antibody 150mg/ml, histidine 20mM, arginine hydrochloride 25mM, trehalose 70mg/ml, glacial acetic acid 0.89mg/ml, polysorbate 80 0.3mg/ml, pH5.8.
  • Three batches of drugs were placed at 25°C for accelerated stability inspection for 6 months, and samples were taken at 0, 1, 2, 3, and June to detect SEC purity, IEC purity and insoluble particles.
  • the liquid preparation formula of the present invention has a good protective effect on the target protein at 25°C.
  • 3 batches of drugs were prepared.
  • the specific prescriptions were anti-IL-4R ⁇ monoclonal antibody 150mg/ml, histidine 20mM, arginine hydrochloride 25mM, trehalose 70mg/ml, glacial acetic acid 0.89mg/ml, polysorbate 80 0.3mg/ml, pH5.8.
  • the insoluble particles, SEC purity, and IEC purity all meet the standard requirements. From the analysis of the results of Figure 3 and Figure 4, it can be seen that the validity period of the drug indicated by the SEC purity is 78 months, and the validity period of the drug indicated by the IEC purity is 87 months. Therefore, under the protection of the formulation of the present invention, the anti-IL-4R ⁇ monoclonal antibody The injection can be stored for at least 24 months at 2-8°C.

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Abstract

本发明提供了一种稳定的抗IL-4Rα单克隆抗体液体制剂。本发明的液体制剂药品成品在2-8℃至少可保存24个月，25℃至少可保存6个月，具有稳定性。

Description

一种稳定的抗IL-4Rα单克隆抗体液体制剂 技术领域
本发明属于生物制药领域，具体地说，涉及一种稳定的抗IL-4Rα单克隆抗体液体制剂。
背景技术
特应性皮炎(Atopic Dermatitis，简称AD)是一种常见的皮肤慢性炎症性复发性疾病。该病症是世界范围内最常见的炎性皮肤病，全球约有15％-30％的儿童和2％-10％的成人发病。据统计，我国约有超过230万青少年受到特应性皮炎(湿疹)的困扰，儿童患者也达到200万-300万。
2017年美国FDA批准Dupixent(dupilumab)用于治疗中重度湿疹(特应性皮炎)成年患者。Dupixent旨在用于局部用药后湿疹得不到充分控制的患者，或不建议使用局部用药的患者。Dupixent以皮下注射使用，活性成分是一种可与引起炎症的一种蛋白--白介素-4受体α亚基(IL-4Rα)相结合的抗体。通过与这种蛋白相结合，Dupixent能够抑制在特应性皮炎发展中起作用的炎症反应。
Dupixent的制剂为150mg/ml的注射液，由EMA Assessment report Dupixent(European Medicines Agency,Science Medicines Health,EMA/512262/2017,p16, https://www.ema.europa.eu/en/documents/assessment-report/dupixent-epar-public-assessment-r eport_en.pdf)可知，其在2-8℃可保存15个月，25℃最多保存14天，稳定性较差。
PCT专利申请WO2020/048312中公开了一类自主研发的重组抗IL-4Rα人源化单克隆抗体，系采用DNA重组技术在CHO细胞中表达的重组人源化单克隆抗体，由抗IL-4Rα鼠源抗体的重链可变区和轻链可变区经人源化构建而成。有必要针对这类新的重组抗IL-4Rα人源化单克隆抗体进行深入研究，以开发适合临床应用的稳定制剂。
发明内容
为了解决现有的抗IL-4Rα抗体制剂稳定性差的问题，本发明的目的在于提供了一种稳定的高浓度的抗IL-4Rα单克隆抗体液体制剂。该液体制剂由抗IL-4Rα单克隆抗体、组氨酸、盐酸精氨酸、海藻糖、冰醋酸、聚山梨酯80组成。可以起到稳定蛋白的作用，使得目的蛋白在2-8℃至少保存24个月，25℃至少保存6个月，具有优异的稳定性。
为了实现本发明的目的，本发明采用以下技术方案：
本发明的第一个方面提供了一种稳定的抗IL-4Rα单克隆抗体液体制剂，所述的液体制剂包括抗IL-4Rα单克隆抗体、组氨酸、盐酸精氨酸、海藻糖、冰醋酸、聚山梨酯80，其中，所述的抗IL-4Rα单克隆抗体浓度为100-200mg/ml，所述的抗IL-4Rα单克隆抗体包含如SEQ ID NO：1-3中任一所示的重链和如SEQ ID NO：4所示的轻链。
其中，所述的抗IL-4Rα单克隆抗体浓度为150mg/ml，所述的抗IL-4Rα单克隆抗体包含如SEQ ID NO：1所示的重链和如SEQ ID NO：4所示的轻链。
其中，所述的组氨酸浓度为20-30mM，优选的，所述的组氨酸浓度为20mM。
其中，所述的盐酸精氨酸浓度为25-75mM，优选的，所述的盐酸精氨酸浓度为25mM。
其中，所述的海藻糖浓度为50-80mg/ml，优选的，所述的海藻糖浓度为70mg/ml。
其中，所述的冰醋酸浓度为0.4-1.7mg/ml，优选的，所述的冰醋酸浓度为0.89mg/ml。
其中，所述的聚山梨酯80浓度为0.3-1.5mg/ml，优选的，所述的聚山梨酯80浓度为0.3mg/ml。
其中，所述的液体制剂pH范围为5.0-6.3，优选的，所述的液体制剂pH为5.8。
本发明的第二个方面提供了所述的液体制剂用于制备治疗IL-4Rα过表达相关的疾病的药物中的用途。
其中，所述的IL-4Rα过表达相关的疾病包括特应性皮炎、哮喘、过敏反应、嗜酸性食道炎、皮肤感染、鼻息肉症等。
本发明也提供一种治疗IL-4Rα过表达相关的疾病的方法，所述方法包括将治疗有效量的本发明所述的液体制剂给予患者，所述患者患有特应性皮炎、哮喘、过敏反应、嗜酸性食道炎、皮肤感染、鼻息肉症等疾病。
有益效果：本发明提供了一种稳定的抗IL-4Rα单克隆抗体液体制剂。本发明通过优化配方极大的改进了现有抗IL-4Rα单克隆抗体制剂稳定性差的缺陷。本发明的液体配方制剂药品成品在2-8℃至少可保存24个月，25℃至少可保存6个月，具有优异的稳定性。因此，本发明的液体配方制剂能够提供抗IL-4Rα单克隆抗体的制剂稳定性，具有广泛的工业应用前景。
附图说明
图1为DOE考察pH和聚山梨酯80浓度结果分析。
图2为不同浓度聚山梨酯80的不溶性微粒结果分析。
图3为SEC纯度长期稳定性结果分析。
图4为IEC纯度长期稳定性结果分析。
具体实施方式
本发明中使用的抗人IL-4Rα单克隆抗体来源自WO2020/048312中公开的人源化抗人IL-4Rα单克隆抗体4-2-Humanized-IgG4、4-2-Humanized-IgG1、4-2-Humanized-IgG1-SELF，其包含相同的重链可变区、不同的重链恒定区以及相同的轻链，重链和轻链氨基酸序列如下所示。
4-2-Humanized-IgG4重链的氨基酸序列(SEQ ID NO：1)
Figure PCTCN2021099056-appb-000001
4-2-Humanized-IgG1重链的氨基酸序列(SEQ ID NO：2)
Figure PCTCN2021099056-appb-000002
4-2-Humanized-IgG1-SELF重链的氨基酸序列(SEQ ID NO：3)
Figure PCTCN2021099056-appb-000003
Figure PCTCN2021099056-appb-000004
4-2-Humanized-IgG4、4-2-Humanized-IgG1、4-2-Humanized-IgG1-SELF轻链的氨基酸序列(SEQ ID NO：4)
Figure PCTCN2021099056-appb-000005
为了示例性的目的，以下实施例中使用的蛋白样品为4-2-Humanized-IgG4，包含如SEQ ID NO：1所示的重链和如SEQ ID NO：4所示的轻链。
以下实施例中使用的检测方法说明如下：
SEC纯度、聚体检测方法：
流动相：200mM磷酸盐缓冲液，pH 6.8±0.1。经0.22μm滤膜过滤、超声脱气后使用。色谱柱：TSK G3000SWxl，7.8×300mm 5μm，TOSOH 08541。高效液相色谱仪：Waters Alliance e2695 2489紫外/可见光检测器，Dionex Ultimate 3000VWD-3400(RS)Detector或其他适合配有紫外检测器的HPLC***。
***适用性样品：取参考品用流动相稀释浓度至5.0mg/ml，13000rpm离心10min，取上清转移至进样瓶，放入HPLC样品盘。供试品：用流动相稀释供试品浓度至5.0mg/ml，13000rpm离心10min，取上清转移至进样瓶，放入HPLC样品盘。色谱条件：柱温25±2℃；样品温度10±2℃；检测波长UV 280nm；进样体积20μL；流速0.5ml/min。
用色谱软件进行积分，峰面积归一化法计算各个峰的峰面积百分比。***适用性可接受标准：6针***适用性样品，聚体与单体的分离度均≥1.5，主峰的保留时间RSD≤1.0％，主峰峰面积RSD≤2.0％，且主峰的不对称性均≤2.0，理论塔板数均≥4000。供试品报告结果：样品的SEC纯度报告为单体主峰的峰面积百分比，聚体含量为聚体峰的峰面积百分比。
IEC纯度检测方法：
流动相A：20mM磷酸盐缓冲液，pH 6.5±0.05。经0.22μm滤膜过滤、超声脱气后使用。流动相B：20mM磷酸盐缓冲液+200mM氯化钠，pH 6.5±0.05。经0.22μm滤膜过滤、超声脱气后使用。色谱柱：Propac WCX-10，4×250mm，Thermo Dionex 054993。高效液相色谱仪：Waters Alliance e2695，Dionex Ultimate 3000系列或其他适合配有紫外检测器的HPLC***。
***适用性样品：取参考品用流动相稀释浓度至1.0mg/ml，13000rpm离心10min，取上清转移至进样瓶，放入HPLC样品盘。供试品：用流动相稀释供试品浓度至1.0mg/ml，13000rpm离心10min，取上清转移至进样瓶，放入HPLC样品盘。色谱条件：柱温30±2℃；样品温度10±2℃；检测波长UV 214nm；进样体积20μL；流速1.0ml/min。流动相梯度如下：
Figure PCTCN2021099056-appb-000006
纯度分析：用峰面积归一化法计算样品图谱上主峰、酸峰区和碱峰区的峰面积百分比。IEC纯度结果报告为主峰的峰面积百分比。
除特别注明外，以下实施例中使用的原料组分皆市售可得。
以下实施例、实验例是对本发明进行进一步的说明，但不以任何形式限制本发明。
实施例1 缓冲体系对制剂稳定性影响
如表1所示，分别配制10mM柠檬酸体系、20mM组氨酸-盐酸组氨酸体系、20mM组氨酸-醋酸体系，pH为5.5。考察150mg/ml蛋白在不同体系中的稳定性。将三组溶液放置40℃考察15天，并于0、2、5、9、15天取样检测SEC纯度和IEC纯度。
表1、缓冲体系考察结果
Figure PCTCN2021099056-appb-000007
Figure PCTCN2021099056-appb-000008
由表1的结果可知，组氨酸-盐酸组氨酸缓冲体系和组氨酸-醋酸缓冲体系两组的SEC纯度和IEC纯度结果优于柠檬酸体系。
实施例2 精氨酸/盐酸精氨酸对制剂粘度影响
精氨酸/盐酸精氨酸对高浓度蛋白溶液具有降低粘度的影响，并保护蛋白，本实施例中蛋白浓度为150mg/ml，粘度较高，因此加入盐酸精氨酸进行试验，探索其对蛋白粘度的降低和保护作用。
如表2所示，在20mM组氨酸-盐酸组氨酸和20mM组氨酸-醋酸体系中，分别加入0-100mM的盐酸精氨酸，前者用盐酸调节pH至5.5，后者用醋酸调节pH至5.5。
表2、盐酸精氨酸影响
Figure PCTCN2021099056-appb-000009
由表2的结果可知，在组氨酸-盐酸组氨酸体系中，加入50mM以上浓度的盐酸精氨酸可明显降低粘度；在组氨酸-醋酸体系中，加入25mM以上浓度的盐酸精氨酸可显著降低粘度。由于组氨酸-醋酸体系比组氨酸-盐酸组氨酸体系缓冲能力更好，因此后续选择组氨酸-醋酸作为缓冲体系。
实施例3 pH和聚山梨酯80考察
如表3所示，本实施例采用DOE设计考察pH和聚山梨酯80对制剂的影响。使用冰醋 酸调节pH范围为5.0-6.6，聚山梨酯80用量范围为0-3mg/ml。其他成分蛋白浓度为150mg/ml，组氨酸20mM，盐酸精氨酸25mM，海藻糖50mg/ml。将配制的样品放置40℃考察30天，分别于0、7、14、21、30天取样，检测SEC纯度、IEC纯度和不溶性微粒(≥10μm)。
表3、pH和聚山梨酯80考察设计表
编号 模式 pH 聚山梨酯80
3-1 A0 6.6 1.5
3-2 0A 5.8 3.0
3-3 -- 5.0 0.0
3-4 -+ 5.0 3.0
3-5 0a 5.8 0.0
3-6 0 5.8 1.5
3-7 0 5.8 1.5
3-8 a0 5.0 1.5
3-9 +- 6.6 0.0
3-10 ++ 6.6 3.0
表4、DOE考察结果
Figure PCTCN2021099056-appb-000010
Figure PCTCN2021099056-appb-000011
将表4中结果进行斜率分析，各组斜率用JMP DOE模型分析如图1所示。由图1的结果可知，优选的pH范围为pH5.0-6.3，优选的聚山梨酯80用量为0-1.5mg/ml。
实施例4 不溶性微粒考察
本实施例考察聚山梨酯80对不溶性微粒的影响。如表5所示，本实施例配制6批小样，蛋白浓度150mg/ml，组氨酸20mM，盐酸精氨酸25mM，海藻糖50mg/ml，聚山梨酯800-0.5mg/ml，用冰醋酸调节pH至5.8。配制后检测不溶性微粒。
表5、不同聚山梨酯80用量的不溶性微粒结果
编号 聚山梨酯80含量(mg/ml) >2μm(个/ml) >10μm(个/ml) >25μm(个/ml)
4-1 0 60651.85 6499.36 369.67
4-2 0.1 716.2 447.63 73.46
4-3 0.2 178.95 64.24 4.59
4-4 0.3 233.97 37.06 4.63
4-5 0.4 96.36 36.71 6.88
4-6 0.5 151.42 22.94 2.29
由图2可知，不溶性微粒随聚山梨酯80的增加而降低，高于0.3mg/ml的浓度时不溶性微粒数据趋于稳定。因此聚山梨酯80优选浓度为0.3mg/ml以上。
实施例5 海藻糖及组氨酸考察
本实施例考察海藻糖和组氨酸用量对制剂的影响。按表6配制4组处方，蛋白浓度为150mg/ml，25mM盐酸精氨酸，并添加0.3mg/ml的聚山梨酯80，用冰醋酸调节pH至5.8。样品放置40℃14天，于0、7、14天取样检测SEC纯度和IEC纯度。
表6、海藻糖及组氨酸考察
序号 海藻糖(mg/ml) 组氨酸(mM)
5-1 50 20
5-2 60 20
5-3 50 30
5-4 80 20
表7、海藻糖及组氨酸考察结果
Figure PCTCN2021099056-appb-000012
Figure PCTCN2021099056-appb-000013
由表7结果可知，各组处方的SEC和IEC纯度无显著性差异，因此，组氨酸优选的浓度范围为20-30mM，考虑到注射液与人体等渗的原则，海藻糖优选的浓度范围为50-80mg/ml。
实施例6 冰醋酸考察
如表8所示，本实施例将50ml组氨酸(20mM)、盐酸精氨酸(25mM)溶液中加入冰醋酸，并按表8各加入量检测pH。通过计算得出每个pH对应的冰醋酸的浓度。
表8 冰醋酸考察
冰醋酸用量(μL/50mL) pH 计算冰醋酸浓度(mg/ml)
0 7.36 0.00
10 6.7 0.21
20 6.35 0.42
30 6.07 0.63
40 5.82 0.84
50 5.57 1.05
60 5.33 1.26
70 5.13 1.47
80 4.96 1.68
90 4.84 1.89
100 4.73 2.10
由表8的结果可知，在20mM组氨酸和25mM的盐酸精氨酸溶液中，pH5.0-6.3对应的冰醋酸浓度约为0.4-1.7mg/ml。
实施例7 加速稳定性考察
本实施例配制3批药品，具体处方为抗IL-4Rα单克隆抗体150mg/ml，组氨酸20mM，盐酸精氨酸25mM，海藻糖70mg/ml，冰醋酸0.89mg/ml，聚山梨酯80 0.3mg/ml，pH5.8。将三批药品放置25℃进行加速稳定性考察6个月，分别在0、1、2、3、6月取样，检测SEC纯度、IEC纯度和不溶性微粒。
表9、加速稳定性结果
Figure PCTCN2021099056-appb-000014
Figure PCTCN2021099056-appb-000015
由表9的结果可知，6个月加速稳定性后，不溶性微粒、SEC纯度、IEC纯度均符合质量标准要求。因此，本发明的液体制剂配方对目的蛋白在25℃具有很好的保护作用。
实施例8 长期稳定性考察
本实施例配制3批药品，具体处方为抗IL-4Rα单克隆抗体150mg/ml，组氨酸20mM，盐酸精氨酸25mM，海藻糖70mg/ml，冰醋酸0.89mg/ml，聚山梨酯80 0.3mg/ml，pH5.8。将三批药品放置2-8℃进行长期稳定性考察，每个点取样检测SEC纯度、IEC纯度和不溶性微粒。
表10、长期稳定性结果
Figure PCTCN2021099056-appb-000016
由表10的结果可知，不溶性微粒、SEC纯度、IEC纯度均符合标准规定。由图3和图4 结果分析可知，SEC纯度指示的药品有效期为78个月，IEC纯度指示的药品有效期为87个月，因此，在本发明制剂配方的保护下，抗IL-4Rα单克隆抗体注射液于2-8℃下可储存至少24个月。

Claims (18)

  1. 一种稳定的抗IL-4Rα单克隆抗体液体制剂，所述的液体制剂包括抗IL-4Rα单克隆抗体、组氨酸、盐酸精氨酸、海藻糖、冰醋酸、聚山梨酯80，其中，所述的抗IL-4Rα单克隆抗体浓度为100-200mg/ml，所述的抗IL-4Rα单克隆抗体包含如SEQ ID NO：1-3中任一所示的重链和如SEQ ID NO：4所示的轻链。
  2. 如权利要求1所述的液体制剂，其特征在于，所述的抗IL-4Rα单克隆抗体浓度为150mg/ml，所述的抗IL-4Rα单克隆抗体包含如SEQ ID NO：1所示的重链和如SEQ ID NO：4所示的轻链。
  3. 如权利要求1所述的液体制剂，其特征在于，所述的组氨酸浓度为20-30mM。
  4. 如权利要求3所述的液体制剂，其特征在于，所述的组氨酸浓度为20mM。
  5. 如权利要求1所述的液体制剂，其特征在于，所述的盐酸精氨酸浓度为25-75mM。
  6. 如权利要求5所述的液体制剂，其特征在于，所述的盐酸精氨酸浓度为25mM。
  7. 如权利要求1所述的液体制剂，其特征在于，所述的海藻糖浓度为50-80mg/ml。
  8. 如权利要求7所述的液体制剂，其特征在于，所述的海藻糖浓度为70mg/ml。
  9. 如权利要求1所述的液体制剂，其特征在于，所述的冰醋酸浓度为0.4-1.7mg/ml。
  10. 如权利要求9所述的液体制剂，其特征在于，所述的冰醋酸浓度为0.89mg/ml。
  11. 如权利要求1所述的液体制剂，其特征在于，所述的聚山梨酯80浓度为0.3-1.5mg/ml。
  12. 如权利要求11所述的液体制剂，其特征在于，所述的聚山梨酯80浓度为0.3mg/ml。
  13. 如权利要求1所述的液体制剂，其特征在于，所述的液体制剂pH范围为5.0-6.3。
  14. 如权利要求13所述的液体制剂，其特征在于，所述的液体制剂pH为5.8。
  15. 如权利要求1所述的液体制剂，其特征在于，所述液体制剂组成为：
    抗IL-4Rα单克隆抗体150mg/ml，组氨酸20mM，盐酸精氨酸25mM，海藻糖70mg/ml，冰醋酸0.89mg/ml，聚山梨酯80 0.3mg/ml，pH5.8。
  16. 如权利要求1-15所述的液体制剂用于制备治疗IL-4Rα过表达相关的疾病的药物中的用途。
  17. 如权利要求16所述的用途，其特征在于，所述的IL-4Rα过表达相关的疾病包括特应性皮炎、哮喘、过敏反应、嗜酸性食道炎、皮肤感染、鼻息肉症。
  18. 一种治疗疾病的方法，包括将治疗有效量的如权利要求1所述的液体制剂施于患者，所述患者患有与IL-4Rα过表达相关的疾病。
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Citations (3)

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Publication number Priority date Publication date Assignee Title
WO2016034648A1 (en) * 2014-09-03 2016-03-10 Medimmune Limited Stable anti-il-4r-alpha antibody formulation
CN106267189A (zh) * 2010-10-06 2017-01-04 瑞泽恩制药公司 含有抗白介素‑4 受体(il‑4r)的抗体的稳定制剂
CN110872349A (zh) * 2018-09-04 2020-03-10 三生国健药业（上海）股份有限公司 结合人il-4r的抗体、其制备方法和用途

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106267189A (zh) * 2010-10-06 2017-01-04 瑞泽恩制药公司 含有抗白介素‑4 受体(il‑4r)的抗体的稳定制剂
WO2016034648A1 (en) * 2014-09-03 2016-03-10 Medimmune Limited Stable anti-il-4r-alpha antibody formulation
CN110872349A (zh) * 2018-09-04 2020-03-10 三生国健药业（上海）股份有限公司 结合人il-4r的抗体、其制备方法和用途

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