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WO2023077685A1 - 包含抗人白介素-33单克隆抗体的浓缩溶液的制备方法及液体制剂 - Google Patents

包含抗人白介素-33单克隆抗体的浓缩溶液的制备方法及液体制剂 Download PDF

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WO2023077685A1
WO2023077685A1 PCT/CN2022/075787 CN2022075787W WO2023077685A1 WO 2023077685 A1 WO2023077685 A1 WO 2023077685A1 CN 2022075787 W CN2022075787 W CN 2022075787W WO 2023077685 A1 WO2023077685 A1 WO 2023077685A1
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amino acid
concentration
cdr
monoclonal antibody
liquid preparation
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PCT/CN2022/075787
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English (en)
French (fr)
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薛刚
朱华杰
戴长松
李帅
许芹
郭彩明
陈卫
吴亦亮
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江苏荃信生物医药股份有限公司
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Priority claimed from CN202111302350.XA external-priority patent/CN113842457B/zh
Priority claimed from CN202111300668.4A external-priority patent/CN114014929B/zh
Application filed by 江苏荃信生物医药股份有限公司 filed Critical 江苏荃信生物医药股份有限公司
Publication of WO2023077685A1 publication Critical patent/WO2023077685A1/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
    • 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
    • A61K47/22—Heterocyclic compounds, e.g. ascorbic acid, tocopherol or pyrrolidones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • 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
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons

Definitions

  • This application relates to the field of biotechnology. Specifically, the present application relates to a liquid preparation containing anti-human interleukin-33 monoclonal antibody, and a method for preparing a concentrated solution of anti-human interleukin-33 monoclonal antibody.
  • monoclonal antibody injections are mainly used in the treatment of tumors and autoimmune-related diseases. Due to their high targeting and safety, this type of product brings more innovative treatments to relevant patients. Choice, for some autoimmune-related diseases that are difficult to treat with chemical drugs and small molecule drugs, such as asthma, atopic dermatitis, nasal polyps and chronic sinusitis, various monoclonal antibody injections provide more choices.
  • the dosage form of biological agents has gradually changed from freeze-dried dosage form to water injection dosage form, and the route of administration has also changed from intravenous administration to subcutaneous injection dosage form, which greatly reduces the treatment time and can be realized at home. Self-injection improves patient compliance.
  • the dosage of monoclonal antibody injection is usually in the range of 100-600mg, and the volume of subcutaneous injection is generally limited to less than 2mL, in such cases, highly concentrated protein preparations must be prepared, and the protein content can usually reach 100mg/ mL or greater concentration.
  • the high concentration of monoclonal antibody injection brings many challenges to the manufacturability of the production process, process scale-up, and final patient administration.
  • the most important challenge is the ultra-high viscosity. Due to the biopolymer properties of monoclonal antibodies, as the protein concentration increases, the interaction between protein molecules (such as hydrophobicity, charge interaction, etc.) gradually increases, showing a highly viscous solution. . Even in some extreme cases, a gel-like substance will be formed.
  • This kind of feed liquid brings great challenges to the ultrafiltration membrane and ultrafiltration equipment itself, such as the tangential flow rate caused by the rapid increase in pressure difference during final concentration.
  • the application provides a liquid preparation (concentrated solution) containing anti-human interleukin-33 monoclonal antibody and its preparation method
  • the liquid preparation contains a protein concentration of 120-300mg/mL
  • the liquid preparation has lower viscosity, higher stability, less aggregation of anti-human interleukin-33 monoclonal antibody, and can be used for Injections, especially hypodermic injections.
  • Interleukin-33 (interleukin 33, IL-33) is a protein encoded by the human gene IL-33, which is a member of the interleukin 1 family. Efficiently drives production of the cofactor Th2.
  • IL-33 is one of the ligands of ST2. In the inactive state, IL-33, in its precursor form (270 amino acid residues in full length), binds to the nucleus in a cellular homeostasis through binding to chromatin-binding motifs.
  • IL-33 When the cells are damaged, IL-33 can be released as an endogenous danger signal to the outside of the cell, mainly through the binding receptor ST2 to transmit the signal, and play the role of immune regulation as a cytokine.
  • QX007N can specifically bind to IL-33 and prevent immune cells from releasing pro-inflammatory cytokines, thereby preventing asthma exacerbation and improving asthma control.
  • the application provides a liquid preparation comprising anti-human interleukin-33 monoclonal antibody, characterized in that,
  • the liquid preparation comprises anti-human interleukin-33 monoclonal antibody and amino acid additives;
  • the protein concentration of the anti-human interleukin-33 monoclonal antibody is 120-300 mg/mL, preferably 120-200 mg/mL, more preferably 120-160 mg/mL;
  • the amino acid concentration of the amino acid additive is 10-500 mM ;
  • the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions and three light chain complementarity determining regions, and the three heavy chain complementarity determining regions are CDR-H1, CDR-H2 and CDR-H3, so The three light chain complementarity determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein:
  • CDR-H1 has the amino acid sequence shown in SEQ ID NO: 1;
  • CDR-H2 has the amino acid sequence shown in SEQ ID NO: 2;
  • CDR-H3 has the amino acid sequence shown in SEQ ID NO: 3;
  • CDR-L1 has the amino acid sequence shown in SEQ ID NO: 4;
  • CDR-L2 has the amino acid sequence shown in SEQ ID NO: 5;
  • CDR-L3 has the amino acid sequence shown in SEQ ID NO:6.
  • the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 7;
  • the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8.
  • the amino acid additive is selected from one or two or more of arginine hydrochloride, histidine, lysine, methionine and proline.
  • the amino acid additive includes one or two or three of arginine hydrochloride, histidine and methionine;
  • the amino acid additive includes arginine hydrochloride, and the concentration of arginine hydrochloride in the liquid preparation is 100-200 mM;
  • the amino acid additive includes histidine, and the concentration of histidine in the liquid preparation is 10-50 mM;
  • the amino acid additive includes methionine, and the concentration of methionine in the liquid preparation is 2-50 mM.
  • the liquid preparation further includes a surfactant, the content of the surfactant is 0.01-5 mg/mL, preferably 0.1-2 mg/mL, more preferably 0.2-1 mg/mL;
  • the surfactant is polysorbate 80.
  • the concentration of the anti-human interleukin-33 monoclonal antibody in the liquid preparation is above 180 mg/mL, the viscosity of the liquid preparation is below 15 cP;
  • the viscosity of the liquid preparation is below 10 cP.
  • the liquid preparation further contains sucrose, and the concentration of the sucrose is 20-140 mg/mL, preferably 50-90 mg/mL.
  • the liquid preparation further comprises sorbitol, and the concentration of the sorbitol is 10-200 mg/mL, preferably 20-100 mg/mL, more preferably 50-80 mg/mL.
  • the liquid preparation further comprises sodium chloride, and the concentration of the sodium chloride is 20-300 mg/mL, preferably 100-200 mg/mL.
  • the pH of the liquid preparation is 5.5-6.5.
  • the present application also provides a method for preparing a concentrated solution of anti-human interleukin-33 monoclonal antibody, which includes the following steps:
  • Concentration by ultrafiltration Concentrate the solution containing anti-human interleukin-33 monoclonal antibody to obtain concentrated samples;
  • Ultrafiltration replacement using a replacement buffer solution to replace the concentrated sample to obtain an ultrafiltration replacement concentrate
  • Secondary ultrafiltration concentration adding the amino acid additive mother liquor to the ultrafiltration displacement concentrated solution, mixing evenly to obtain a mixed solution, and performing ultrafiltration concentration on the mixed solution to obtain the concentrated solution;
  • the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions and three light chain complementarity determining regions, and the three heavy chain complementarity determining regions are CDR-H1, CDR-H2 and CDR-H3, so The three light chain complementarity determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein:
  • CDR-H1 has the amino acid sequence shown in SEQ ID NO: 1;
  • CDR-H2 has the amino acid sequence shown in SEQ ID NO: 2;
  • CDR-H3 has the amino acid sequence shown in SEQ ID NO: 3;
  • CDR-L1 has the amino acid sequence shown in SEQ ID NO: 4;
  • CDR-L2 has the amino acid sequence shown in SEQ ID NO: 5;
  • CDR-L3 has the amino acid sequence shown in SEQ ID NO:6.
  • the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 7;
  • the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 8.
  • the concentration conditions are as follows: the flow rate is 120-300 L/m 2 ⁇ h, and the transmembrane pressure difference is 0.6-1.5 bar;
  • the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated sample is 20-60 mg/mL, preferably 30-50 mg/mL.
  • the amount of the replacement buffer solution is more than 7 times the weight of the concentrated sample
  • the concentration of the replacement buffer solution is 5-50 mM, preferably 20 mM;
  • the pH of the replacement buffer solution is 5.5-6.5, preferably 5.7-6.3;
  • the replacement buffer solution is selected from one of histidine-hydrochloric acid buffer solution, citric acid buffer solution, phosphate buffer solution and sodium acetate-acetate buffer solution.
  • the concentration of the amino acid additive in the mixed solution is 50-300mmol/L, preferably 50-200mmol/L, more preferably 100-150mmol/L.
  • the protein concentration in the concentrated solution is 120-300 mg/mL, preferably 120-200 mg/mL, more preferably 120-160 mg/mL.
  • the amino acid additive is selected from one or two or more of arginine hydrochloride, histidine, lysine, methionine and proline.
  • the amino acid additive includes one or two or three of arginine hydrochloride, histidine and methionine;
  • the amino acid additive includes arginine hydrochloride, and the concentration of arginine hydrochloride in the mixed solution is 100-200 mM;
  • the amino acid additive includes histidine, and the concentration of histidine in the mixed solution is 10-50 mM;
  • the amino acid additive includes methionine, and the concentration of methionine in the mixed solution is 2-50 mM.
  • the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated solution is above 180 mg/mL, the viscosity of the concentrated solution is below 15 cP;
  • the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated solution is above 150 mg/mL, the viscosity of the concentrated solution is below 10 cP.
  • the solution containing the anti-human interleukin-33 monoclonal antibody is subjected to affinity chromatography, low pH inactivation, anion chromatography, and cationic layer to the cell fermentation broth expressing the anti-human interleukin-33 monoclonal antibody. obtained by analysis and nanofiltration.
  • the liquid preparation containing anti-human IL-33 monoclonal antibody prepared by the preparation method of the present application contains anti-human interleukin-33 monoclonal antibody, and it has a lower viscosity , can be easily injected with a syringe, so it can be used as an injection, especially a subcutaneous injection.
  • the anti-human IL-33 monoclonal antibody of the present application has comparable affinity to human interleukin-33, and its neutralizing activity at the cellular level is comparable to that of Etokimab/ANB020 comparable.
  • the monoclonal antibody drug (Itepekimab/REGN3500) targeting interleukin-33 developed by Sanofi is intended for the treatment of chronic obstructive pulmonary disease (clinical phase III), asthma (clinical phase II) and other atopic dermatitis diseases.
  • Etokimab/ANB020 developed by AnaptysBio is used for chronic sinusitis (clinical phase II).
  • the monoclonal antibody of the present application shows neutralizing activity comparable to that of Etokimab/ANB020 (expressed and prepared according to the patent disclosed sequence) at the cellular level, and it is expected to show good clinical effects in the prevention and treatment of related diseases.
  • Fig. 1 is a graph showing the results of nucleic acid electrophoresis for constructing the QX007N (HZD78-70) transient expression plasmid.
  • M Marker
  • Band 1 PCR product 78VH-Hu25
  • Band 2 pQX2.1, HindIII/NheI
  • Band 3 PCR product 78VK-Hu3-CK
  • Band 4 pQX1, HindIII/BamHI.
  • FIG. 2 is a flowchart of transient expression.
  • Figure 3 is the electrophoretic detection diagram of QX007N (HZD78-70).
  • Fig. 4 is a graph showing that QX007N (HZD78-70) and Etokimab/ANB020 neutralize recombinant human interleukin-33-induced NF- ⁇ B/AP-1 signal transduction activity in HEK Blue TM IL-33 cells.
  • Figure 5 is a graph showing that QX007N(HZD78-70) and Etokimab/ANB020 neutralize natural human interleukin-33-induced NF- ⁇ B/AP-1 signaling activity in HEK Blue TM IL-33 cells.
  • Figure 6 is a graph showing the activity of QX007N (HZD78-70) and Etokimab/ANB020 neutralizing recombinant human interleukin-33 to induce KU812 cells to release IL-5.
  • Figure 7 is a graph showing the activity of QX007N (HZD78-70) and Etokimab/ANB020 neutralizing recombinant human interleukin-33 to induce the release of IFN- ⁇ from human whole blood.
  • the application provides a liquid preparation comprising anti-human interleukin-33 monoclonal antibody, characterized in that,
  • the liquid preparation comprises anti-human interleukin-33 monoclonal antibody and amino acid additives;
  • the protein concentration of the anti-human interleukin-33 monoclonal antibody is 120-300 mg/mL, preferably 120-200 mg/mL, more preferably 120-160 mg/mL;
  • the amino acid concentration of the amino acid additive is 10-500 mM ;
  • the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions and three light chain complementarity determining regions, and the three heavy chain complementarity determining regions are CDR-H1, CDR-H2 and CDR-H3, so The three light chain complementarity determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein:
  • CDR-H1 has the amino acid sequence shown in SEQ ID NO: 1 (SYHMI);
  • CDR-H2 has the amino acid sequence shown in SEQ ID NO: 2 (VIYPNSNIYYATWAKG);
  • CDR-H3 has the amino acid sequence shown in SEQ ID NO: 3 (TIYVHVYSALSI);
  • CDR-L1 has the amino acid sequence shown in SEQ ID NO: 4 (QASESVLNEVS);
  • CDR-L2 has the amino acid sequence shown in SEQ ID NO: 5 (FASKLAS);
  • CDR-L3 has the amino acid sequence shown in SEQ ID NO: 6 (QQDWSMDNIDNA).
  • CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 represent heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, Light chain CDR3.
  • the protein concentration of the anti-human interleukin-33 monoclonal antibody can be, for example, 120 mg/mL, 130 mg/mL, 140 mg/mL, 150 mg/mL, 160 mg/mL, 170 mg/mL, 180 mg/mL, 190 mg/mL , 200mg/mL, 210mg/mL, 220mg/mL, 230mg/mL, 240mg/mL, 250mg/mL, 260mg/mL, 270mg/mL, 280mg/mL, 290mg/mL, 300mg/mL, etc.
  • the amino acid concentration of the amino acid additive can be 10mM, 30mM, 50mM, 70mM, 90mM, 110mM, 130mM, 150mM, 170mM, 190mM, 210mM, 230mM, 250mM, 270mM, 290mM, 310mM, 330mM, 350mM, 370mM, 390mM, mM, 410mM, 430mM, 450mM, 470mM, 490mM, 500mM, etc.
  • monoclonal antibody means an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies (eg, containing naturally occurring mutations or arising during the production of monoclonal antibody preparations), such variants typically exist in minor amounts.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • monoclonal antibody preparations have each monoclonal antibody directed against a single determinant on the antigen.
  • monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques including, but not limited to, the hybridoma method, recombinant DNA methods, phage display methods, and the use of all human immunoglobulin loci comprising human immunoglobulin loci. or portions of transgenic animals, such methods and other exemplary methods of making monoclonal antibodies are described herein.
  • “monoclonal antibodies” are generally human antibodies, which can be prepared using techniques well known to those skilled in the art, for example, human antibodies are generally described in van Dijk, M.A. and van de Winkel, J.G., Curr. Opin. Pharmacol 5:368-374 (2001) and Lonberg, N., Curr. Opin. Immunol. 20:450-459 (2008).
  • Antibodies can be prepared by administering an immunogen to transgenic animals that have been modified to stimulate the production of fully human antibodies or intact antibodies with human variable regions in response to antigenic challenge, these animals typically contain some or all of the human immunoglobulin loci, which replace endogenous immunoglobulin loci, either present extrachromosomally or integrated randomly in the animal. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated.
  • transgenic animals see Lonberg, N., Nat. Biotech. 23:1117 -1125 (2005). See also, for example, XENOMOUSE TM technology described in US Patent Nos. 6,075,181 and 6,150,584; US Patent No.
  • Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma cells have been described for the production of human monoclonal antibodies (see, e.g., Kozbor, D., J. Immunol. 133:3001-3005 (1984); Brodeur, B.R. et al. , Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York (1987), pp.51-63; and Boerner, P. et al., J. Immunol. 147:86-95 (1991)). Human antibodies produced via human B-cell hybridoma technology are also described in Li, J. et al., Proc. Natl. Acad. Sci.
  • Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries, and such variable domain sequences can then be combined with desired human constant domains.
  • Human antibodies can also be selected on the basis from antibody libraries, ie, human antibodies can be isolated by screening combinatorial libraries for antibodies possessing the desired activity or activities. For example, various methods are known in the art for producing phage display libraries and screening such libraries for antibodies possessing desired binding characteristics. This approach is reviewed, e.g., in Hoogenboom, H.R. et al., Methods in Molecular Biology 178:1-37 (2001), and further described, e.g., in McCafferty, J.
  • repertoires of VH and VL genes are cloned separately by polymerase chain reaction (PCR) and recombined randomly in a phage library, which is then screened for antigen-binding phage, as described in Winter , G. et al., Ann. Rev. Immunol. 12:433-455 (1994).
  • Phage typically display antibody fragments as single-chain Fv (scFv) fragments or as Fab fragments.
  • scFv single-chain Fv
  • Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas.
  • the naive repertoire can be cloned (e.g.
  • naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and rearranging in vitro using PCR primers containing random sequences encoding the highly variable CDR3 region, as described by Hoogenboom, H.R. and Winter, G., J. Mol. Biol. 227:381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373 and U.S. Patent Publication Nos. 0292936 and 2009/0002360.
  • the antibody may also be a multispecific antibody, such as a bispecific antibody.
  • Bispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. Techniques for generating multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305:537-540 (1983). ); WO 93/08829; and Traunecker, A. et al., EMBO J. 10:3655-3659 (1991 )), and "knot-in-hole” engineering (seeing, e.g., U.S. Patent No. 5,731,168).
  • Monoclonal antibodies described herein also include engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies” (see, eg, US 2006/0025576).
  • Antibodies herein also include WO 2009/080251, WO 2009/080252, WO 2009/080253, WO 2009/080254, WO 2010/112193, WO 2010/115589, WO 2010/136172, WO 2010/145792, and WO 201 0/145793, A multispecific antibody as described in WO 2011/117330, WO 2012/025525, WO 2012/025530, WO 2013/026835, WO 2013/026831, WO 2013/164325, or WO 2013/174873.
  • the monoclonal antibodies described herein may also be antibody variants, eg, it may be desirable to improve the binding affinity and/or other biological properties of the antibody.
  • Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions, and/or insertions and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, so long as the final construct possesses the desired characteristics, such as antigen binding.
  • antibody variants having one or more amino acid substitutions, sites of interest for substitution mutations include HVR and FR, e.g., amino acid substitutions can be introduced into an antibody of interest Products are screened for desired activity, eg, retained/improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.
  • the mammalian cells used for the in vitro fermentation production of the monoclonal antibody include but are not limited to various hybridoma cells and Chinese hamster ovary cells (CHO), preferably CHO cells.
  • affinity means the strength of the sum of non-covalent interactions between a single binding site of a molecule (eg, antibody) and its binding partner (eg, antigen).
  • binding affinity means an intrinsic binding affinity that reflects a 1:1 interaction between members of a binding pair (eg, antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be expressed by the equilibrium dissociation constant ( KD ). Affinity can be measured by common methods known in the art.
  • TFF tangential flow ultrafiltration
  • NFF tangential Flow Filtration
  • the particle accumulation on the surface of the tangential flow filter membrane is less, and the filtration speed is stable, which is suitable for the separation of large volume samples.
  • the molecules larger than the membrane pore size are intercepted and gradually concentrated, and the substances smaller than the membrane pore size pass through the membrane and are separated from the macromolecular solution to realize the separation of macromolecules and small molecules. Therefore, tangential flow ultrafiltration is often used in the concentration of biological products, dialysis, replacement of buffer solutions, separation of molecules of different sizes, etc.
  • human interleukin-33 means that human interleukin-33 located in the nucleus is hydrolyzed by protease to form mature human interleukin-33, which is secreted to the outside of the cell and exerts the biological activity of human interleukin-33, which has the following sequence: SEQ ID NO: 9 Amino acid sequence shown.
  • anti-human interleukin-33 monoclonal antibody means a monoclonal antibody capable of binding human interleukin-33 with sufficient affinity such that the monoclonal antibody can be used as a diagnostic targeting human interleukin-33 and/or therapeutic agents.
  • the anti-human interleukin-33 monoclonal antibody does not bind to a protein that is not related to the target.
  • “irrelevant protein” refers to other proteins except the human interleukin-33 as the target;
  • the binding ability of interleukin-33 is taken as 100%, the binding ability of the anti-human IL-33 monoclonal antibody of the present application to the irrelevant protein is less than 10%, such as 9%, 8%, 7%, 6%, 5%, %, 4%, 3%, 2%, 1% or 0.
  • the anti-human IL-33 monoclonal antibody of the present application can bind to interleukin-33 of humans and cynomolgus monkeys, but may not bind to interleukin-33 of other animal species.
  • other animal species refers to animal species other than humans and cynomolgus monkeys, such as pigs, dogs, rabbits, rats, mice, guinea pigs, etc.;
  • does not bind means that the anti-human IL-33 of the present application is regarded as 100% of the binding ability of the anti-human IL-33 as its target human interleukin-33, and the anti-human IL-33 of the present application
  • the binding ability of human IL-33 monoclonal antibody to interleukin-33 of other animal species is less than 5%, such as 4%, 3%, 2%, 1% or 0%.
  • the anti-human IL-33 monoclonal antibody of the present application has an equilibrium dissociation constant (K D ) of ⁇ 1 ⁇ M, ⁇ 100 nM, ⁇ 50 nM, ⁇ 40 nM.
  • the anti-human IL-33 monoclonal antibody of the present application is equivalent to or better than similar monoclonal antibody products on the market in terms of many biological activities.
  • the biological activities such as neutralizing recombinant/natural human interleukin-33 induce the activity of NF- ⁇ B/AP-1 signal transduction in cells, neutralizing interleukin-33 inducing the activity of KU812 cells to release IL-5, neutralizing interleukin-33 Induce human whole blood to release IFN- ⁇ and so on.
  • amino acid sequence of the heavy chain of the anti-human IL-33 monoclonal antibody of the present application is shown in SEQ ID NO: 10; the amino acid sequence of the light chain is shown in SEQ ID NO: 11.
  • SEQ ID NO: 10 and 11 are humanized sequences.
  • the anti-human interleukin-33 monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein,
  • the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 7 (EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEWVGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYVHVYSALSIWGQGTLVTVSS);
  • the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8 (AFQMTQSPSSVSASVGDRVTITCQASESVLNEVSWYQQKPGKAPKLLIYFASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDWSMDNIDNAFGGGTKVEIK).
  • the amino acid additive is selected from one or two or more of arginine hydrochloride, histidine, lysine, methionine, and proline .
  • the amino acid additive is selected from one or two or more of arginine hydrochloride, histidine, lysine, methionine and proline.
  • the amino acid additive includes one or two or three of arginine hydrochloride, histidine and methionine.
  • the amino acid additive includes arginine hydrochloride, and the concentration of arginine hydrochloride in the liquid preparation is 100-200 mM, such as 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170mM, 180mM, 190mM, 200mM, etc.
  • the amino acid additive includes histidine
  • the concentration of histidine in the liquid preparation is 10-50 mM, such as 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, 40mM, 42mM, 44mM, 46mM, 48mM, 50mM, etc.
  • the amino acid additive includes methionine
  • the concentration of methionine in the liquid preparation is 2-50 mM, such as 2 mM, 4 mM, 6 mM, 8 mM, 10 mM, 12 mM, 14 mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, 40mM, 42mM, 44mM, 46mM, 48mM, 50mM, etc.
  • the amino acid additive includes arginine hydrochloride and histidine, and in the liquid preparation, the concentration of the arginine hydrochloride is 100-200 mM, and the concentration of the histidine is 10-200 mM. 50mM.
  • the amino acid additive includes arginine hydrochloride and methionine, and in the liquid preparation, the concentration of the arginine hydrochloride is 100-200 mM, and the concentration of the methionine is 2 ⁇ 50mM.
  • the amino acid additive includes histidine and methionine, and in the liquid preparation, the concentration of the histidine is 10-50 mM, and the concentration of the methionine is 2-50 mM. 50mM.
  • the amino acid additive includes arginine hydrochloride, histidine and methionine, and in the liquid preparation, the concentration of the arginine hydrochloride is 100-200 mM, preferably 150 mM, so The concentration of histidine is 10-50mM, preferably 20mM, and the concentration of methionine is 2-50mM, preferably 10mM.
  • the amino acid additive is composed of arginine hydrochloride, histidine and methionine, and in the liquid preparation, the concentration of the arginine hydrochloride is 100-200 mM, preferably 150 mM, The concentration of the histidine is 10-50 mM, preferably 20 mM, and the concentration of the methionine is 2-50 mM, preferably 10 mM.
  • the liquid preparation further comprises a surfactant
  • the content of the surfactant is 0.01-5 mg/mL, preferably 0.1-2 mg/mL, more preferably 0.2-1 mg/mL, for example, 0.01mg/mL, 0.1mg/mL, 0.2mg/mL, 0.3mg/mL, 0.4mg/mL, 0.5mg/mL, 0.6mg/mL, 0.7mg/mL, 0.8mg/mL, 0.9mg/mL , 1mg/mL, 2mg/mL, 3mg/mL, 4mg/mL, 5mg/mL, etc.
  • the type of surfactant in the present application is not limited, for example, it may be polysorbate 80.
  • the viscosity of the liquid preparation is below 15 cP.
  • the viscosity of the liquid preparation is below 10 cP.
  • the liquid preparation further comprises sucrose
  • the concentration of the sucrose is 20-140 mg/mL, preferably 50-90 mg/mL, such as 20 mg/mL, 30 mg/mL, 40 mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120mg/mL, 140mg/mL, etc.
  • the liquid preparation further comprises sorbitol, and the concentration of the sorbitol is 10-200 mg/mL, preferably 20-100 mg/mL, more preferably 50-80 mg/mL, for example, 10 mg /mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120mg/mL, 140mg/mL, 160mg/mL , 180mg/mL, 200mg/mL, etc.
  • concentration of the sorbitol is 10-200 mg/mL, preferably 20-100 mg/mL, more preferably 50-80 mg/mL, for example, 10 mg /mL, 20mg/mL, 30mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80m
  • the liquid preparation further comprises sodium chloride
  • the concentration of the sodium chloride is 20-300 mg/mL, preferably 100-200 mg/mL, for example, 20 mg/mL, 30 mg/mL, 40mg/mL, 50mg/mL, 60mg/mL, 70mg/mL, 80mg/mL, 90mg/mL, 100mg/mL, 120mg/mL, 140mg/mL, 160mg/mL, 180mg/mL, 200mg/mL, 220mg/mL mL, 240mg/mL, 260mg/mL, 280mg/mL, 300mg/mL, etc.
  • the pH of the liquid preparation is 5.5-6.5, such as 5.5, 5.6, 5.7, 5.8, 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, etc.
  • the present application also provides a method for preparing a concentrated solution of anti-human interleukin-33 monoclonal antibody, which includes the following steps:
  • Concentration by ultrafiltration Concentrate the solution containing anti-human interleukin-33 monoclonal antibody to obtain concentrated samples;
  • Ultrafiltration replacement using a replacement buffer solution to replace the concentrated sample to obtain an ultrafiltration replacement concentrate
  • Secondary ultrafiltration concentration adding the amino acid additive mother liquor to the ultrafiltration displacement concentrated solution, mixing evenly to obtain a mixed solution, and performing ultrafiltration concentration on the mixed solution to obtain the concentrated solution;
  • the anti-human interleukin-33 monoclonal antibody comprises three heavy chain complementarity determining regions and three light chain complementarity determining regions, and the three heavy chain complementarity determining regions are CDR-H1, CDR-H2 and CDR-H3, so The three light chain complementarity determining regions are CDR-L1, CDR-L2 and CDR-L3, wherein:
  • CDR-H1 has the amino acid sequence shown in SEQ ID NO: 1 (SYHMI);
  • CDR-H2 has the amino acid sequence shown in SEQ ID NO: 2 (VIYPNSNIYYATWAKG);
  • CDR-H3 has the amino acid sequence shown in SEQ ID NO: 3 (TIYVHVYSALSI);
  • CDR-L1 has the amino acid sequence shown in SEQ ID NO: 4 (QASESVLNEVS);
  • CDR-L2 has the amino acid sequence shown in SEQ ID NO: 5 (FASKLAS);
  • CDR-L3 has the amino acid sequence shown in SEQ ID NO: 6 (QQDWSMDNIDNA).
  • CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 represent heavy chain CDR1, heavy chain CDR2, heavy chain CDR3, light chain CDR1, light chain CDR2, Light chain CDR3.
  • the concentration condition is: the flow rate is 120-300L/m 2 ⁇ h, for example, it can be 120L/m 2 ⁇ h, 135L/m 2 ⁇ h, 150L/m 2 h, 165L/m 2 h, 180L/m 2 h, 190L/m 2 h, 200L/m 2 h, 225L/m 2 h, 250L/m 2 h, 275L/m 2 h, 300L/m 2 ⁇ h, etc.; the transmembrane pressure difference (TMP) is 0.6 ⁇ 1.5bar, such as 0.6bar, 0.7bar, 0.8bar, 0.9bar, 1.0bar, 1.1bar, 1.2bar, 1.3bar, 1.4bar, 1.5bar, etc.;
  • the concentration of the anti-human interleukin-33 monoclonal antibody in the concentrated sample is 20-60 mg/mL, such as 20 mg/mL, 30 mg/mL, 40 mg/mL. mL, 50 mg/mL, 60 mg/mL, etc., preferably 30 to 50 mg/mL.
  • the amount of the replacement buffer solution is more than 7 times the weight of the concentrated sample, for example, it can be 7 times, 8 times, 9 times, 10 times, etc.
  • the application in the ultrafiltration replacement step, does not make any limitation on the concentration of the replacement buffer solution, and those skilled in the art can choose according to needs.
  • the concentration of the replacement buffer solution is 5-50 mM, such as 5 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, etc., preferably 20 mM.
  • the application in the ultrafiltration replacement step, does not impose any limitation on the pH of the replacement buffer solution, as long as it is slightly acidic.
  • the pH of the replacement buffer solution in the ultrafiltration replacement step, is 5.5-6.5, such as 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5 etc., preferably 5.7 to 6.3.
  • the replacement buffer solution is selected from one of histidine-hydrochloric acid buffer solution, citric acid buffer solution, phosphate buffer solution and sodium acetate-acetic acid buffer solution, Preferably it is a histidine-hydrochloric acid buffer solution.
  • the citric acid buffer solution may be a citric acid-sodium citrate buffer solution.
  • the phosphate buffer solution may be disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution.
  • a peristaltic pump is used to pump the replacement buffer solution (buffer solution to be replaced) into the concentrated sample, and continue ultrafiltration, while adjusting the pumping speed of the replacement buffer solution to be consistent with the permeation flow rate, when the replacement buffer solution The replacement is completed when the pumped volume is more than 7 times the weight of the concentrated sample.
  • the concentration of the amino acid additive in the mixed solution is 50-300mmol/L, such as 50mmol/L, 100mmol/L, 150mmol/L, 200mmol/L , 250mmol/L, 300mmol/L, etc., preferably 50-200mmol/L, more preferably 100-150mmol/L;
  • the protein concentration in the concentrated solution is 120-300mg/mL, such as 120mg/mL, 130mg /mL, 140mg/mL, 150mg/mL, 160mg/mL, 170mg/mL, 180mg/mL, 190mg/mL, 200mg/mL, 210mg/mL, 220mg/mL, 230mg/mL, 240mg/mL, 250mg/mL , 260 mg/mL, 270 mg/mL, 280 mg/mL, 290 mg/mL, 300 mg/mL
  • the amino acid additive is selected from one or two or more of arginine hydrochloride, histidine, lysine, methionine and proline.
  • the amino acid additive includes one or two or three of arginine hydrochloride, histidine and methionine.
  • the amino acid additive includes arginine hydrochloride, and the concentration of arginine hydrochloride in the mixed solution is 100-200 mM, such as 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170mM, 180mM, 190mM, 200mM, etc.
  • the amino acid additive includes histidine
  • the concentration of histidine in the mixed solution is 10-50 mM, such as 10 mM, 12 mM, 14 mM, 16 mM, 18 mM, 20 mM, 22 mM, 24 mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, 40mM, 42mM, 44mM, 46mM, 48mM, 50mM, etc.
  • the amino acid additive includes methionine
  • the concentration of methionine in the mixed solution is 2-50mM, such as 2mM, 4mM, 6mM, 8mM, 10mM, 12mM, 14mM, 16mM, 18mM, 20mM, 22mM, 24mM, 26mM, 28mM, 30mM, 32mM, 34mM, 36mM, 38mM, 40mM, 42mM, 44mM, 46mM, 48mM, 50mM, etc.
  • the amino acid additive includes arginine hydrochloride, histidine and methionine, and in the mixed solution, the concentration of the arginine hydrochloride is 100-200 mM, and the histidine The concentration of the methionine is 10-50mM, and the concentration of the methionine is 2-50mM.
  • the amino acid additive is composed of arginine hydrochloride, histidine and methionine, and in the mixed solution, the concentration of the arginine hydrochloride is 100-200 mM, and the histidine The acid concentration is 10-50mM, and the methionine concentration is 2-50mM.
  • the secondary ultrafiltration concentration step calculate the protein concentration in the ultrafiltration replacement concentrated solution obtained, and calculate the theoretical volume of the ultrafiltration replacement concentrated solution (ultrafiltration initial protein total amount/ultrafiltration Replace the protein concentration in the concentrated solution), and add the amino acid additive mother liquor and mix evenly, preferably, the volume of the amino acid additive is 1/9 of the volume of the concentrated solution replaced by ultrafiltration, then continue to concentrate by ultrafiltration, and carry out overconcentration according to the concentration required by the stock solution (usually about 30% higher than the concentration of the stock solution), to obtain a concentrated solution, wherein the concentration of the anti-human interleukin-33 monoclonal antibody is 100-200 mg/mL, and the viscosity of the concentrated solution is ⁇ 15 cP.
  • the viscosity of the concentrated solution is below 15 cP.
  • the viscosity of the concentrated solution is below 10 cP.
  • the solution containing the anti-human interleukin-33 monoclonal antibody is subjected to affinity chromatography, low pH inactivation, anion chromatography, It was obtained by cation chromatography and nanofiltration.
  • the affinity chromatography uses Protein A for affinity chromatography.
  • the materials of the ultrafiltration membrane package used include but are not limited to improved polyethersulfone (PES), polyvinylidene fluoride (PVDF), cellulose acetate (CA), etc.
  • the pore size is usually 30kDa or 50kDa, preferably, the ultrafiltration membrane package is Merck Millipore's Pellicon2/Pellicon3 (A-type sieve, 30kDa), and the ultrafiltration membrane package of Sartorius and Pall.
  • the preparation method of the present application can reduce the viscosity of high-concentration antibody drug solution and improve its stability in the ultrafiltration liquid replacement process.
  • the method is simple and easy, and can be scaled up for production. Rate.
  • the verification of high-concentration antibodies of different subtypes in this application proves that the method of adding amino acid additive mother liquor to reduce viscosity in the present invention has strong applicability and good stability.
  • CDR-H1 (SEQ ID No: 1), CDR-H2 (SEQ ID No: 2) and CDR-H3 (SEQ ID No: 3)) were grafted into the framework region of IGHV3-66*01; IGKV1-12*01 was selected as the light chain CDR grafting template, and the The CDR regions of the 78# cloned light chain (i.e.
  • CDR-L1 (SEQ ID No: 4), CDR-L2 (SEQ ID No: 5) and CDR-L3 (SEQ ID No: 6)) were transplanted into IGKV1-12*01
  • the humanized heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7; the humanized light chain variable region has the amino acid sequence shown in SEQ ID NO: 8.
  • the above-mentioned gene of the heavy chain variable region (SEQ ID NO: 7) and the gene of the full length of the light chain (SEQ ID NO: 11) are obtained by PCR amplification.
  • the heavy chain expression plasmid pQX2.1 was double-digested with HindIII and NheI; the transient expression plasmid pQX1 was double-digested with HindIII and BamHI; the PCR amplified gene was inserted into the corresponding expression plasmid with Infusion recombinase to construct the heavy chain expression plasmid pQX2.1-78VH-Hu25 and light chain expression plasmid pQX2.2-78VK-Hu3.
  • pQX2.2 refers to the pQX1 plasmid expressing the light chain.
  • Figure 1 shows the result of the double enzyme digestion of the plasmid detected by nucleic acid electrophoresis. According to the results in Figure 1, it can be seen that the PCR amplification results of the variable region of the heavy chain and the full length of the light chain of the antibody and the results of the expression plasmids of the heavy chain and the light chain by double digestion, wherein the plasmid size of the heavy chain and the light chain is about 5000bp , the full length of the light chain is about 781bp, and the variable region of the heavy chain is about 480bp.
  • the correct heavy chain expression plasmid pQX2.1-78VH-Hu25 and the light chain expression plasmid pQX2.2-78VK-Hu3 were co-transfected into ExpiCHO-S cells.
  • ExpiCHO-S cells were diluted to 3 ⁇ 10 6 cells/ml for passage before transfection.
  • dilute the cell density to 6 ⁇ 10 6 cells/ml put 25ml cells in a 125ml shaker flask, and wait for transfection.
  • the transfection and expression process is shown in Figure 2.
  • HEK Blue TM IL-33 cells are produced by stably transfecting human embryonic kidney cells HEK 293 with human IL1RL1 gene, and the responses of TNF- ⁇ and IL-1 ⁇ are blocked, so HEK-Blue TM IL-33 cells are resistant to IL -33 has a specific reaction.
  • the binding of interleukin-33 to IL-1RL1/IL-1RAcP on the cell surface triggers a signal cascade reaction, leading to NF- ⁇ B/AP-1 signal transduction and production of secreted alkaline phosphatase (secreted alkaline phosphatase, SEAP), which is produced by This is to detect the biological activity of interleukin-33 or to perform antibody screening.
  • the neutralizing activity of QX007N(HZD78-70) on human interleukin-33 was determined by using HEK Blue TM IL-33 cells.
  • HEK Blue TM IL-33 cells were seeded into 96 wells at 4 ⁇ 10 4 cells per well, and cultured overnight at 37°C and 5% CO 2 .
  • HEK Blue TM IL-33 cells were plated into 96 wells at 4 ⁇ 104 cells per well, cultured overnight at 37°C and 5% CO 2 , and the antibody was diluted to a concentration range of 0 to 1000ng/ml, Add the diluent and natural human interleukin-33, mix well and add to the cells for 24 hours at 37°C and 5% CO 2 , collect the cell culture supernatant, and add it to the QUANTI-Blue TM detection reagent at a ratio of 1:10.
  • Fig. 5 show that QX007N (HZD78-70) can inhibit natural human interleukin-33 from inducing NF- ⁇ B/AP-1 signal transduction in HEK Blue TM IL-33 cells, with an IC 50 of 3.91 ng/ml.
  • the human IL-5 DuoSet ELISA (R&D, DY205) was used to detect the expression level of IL-5 in the culture supernatant, and the OD 450nm value was detected by a Varioskan LUX multi-functional microplate reader, and the SoftMax Pro software was used for 4-parameter curve fitting analysis data (Figure 6), and then analyze the neutralizing activity of the antibody.
  • the neutralizing activity of QX007N was further characterized by using monocytes in human whole blood as the basis for determination and IFN- ⁇ as the measurement index.
  • the OD 450nm value was detected by a microplate reader, and the SoftMax Pro software was used to analyze the data using 4-parameter curve fitting ( Figure 7), and then the antagonistic activity of the antibody was analyzed.
  • Fig. 7 show that QX007N (HZD78-70) can neutralize the activity of human interleukin-33 to induce the release of IFN- ⁇ from human whole blood, and its IC50 is 16 ng/ml.
  • Example 1 Using CHO cells as host cells, fermentation was carried out on a 2L-scale bioreactor to produce the antibody QX007N obtained in Example 1, and the clarified fermentation broth was obtained by centrifuge or deep membrane bag filtration, and protein A chromatography was used to identify the target protein. Capture, and then go through low pH virus inactivation, anion and cation chromatography to remove impurities, and then obtain the intermediate sample to be ultrafiltered.
  • Ultrafiltration and concentration the above-mentioned intermediates are used for ultrafiltration, and the ultrafiltration equipment is Labscale small ultrafiltration instrument of Merck Millipore (holding two 50cm 2 Pellicon XL membrane bags, with a cut-off of 30kDa). With a flow rate of 120-300 L/m 2 ⁇ h and TMP maintained at 0.6-1.5 bar, the above-mentioned intermediate sample was concentrated to about 30-50 mg/mL to obtain a concentrated sample.
  • Ultrafiltration replacement use 20mM histidine-hydrochloric acid buffer solution as the replacement buffer solution, the pH value is 6.0, use a peristaltic pump to pump the histidine-hydrochloric acid buffer solution into the concentrated sample and mix to obtain a mixed solution. Continue the ultrafiltration of the mixed solution, and at the same time adjust the pumping speed of the histidine-hydrochloric acid buffer solution to be consistent with the permeation flow rate, that is, keep the weight of the sample constant until the volume of the histidine-hydrochloric acid buffer solution is 7 times the weight of the concentrated sample That is, the replacement is completed to obtain an ultrafiltration replacement concentrate.
  • Secondary ultrafiltration concentration take a sample to measure the concentration of protein in the ultrafiltration displacement concentrate, and calculate the theoretical volume of the ultrafiltration displacement concentrate at this time, and add 1/9 of the amino acid additive mother liquor of the theoretical volume of the ultrafiltration displacement concentrate, Mix well so that the buffer system is as shown in Table 2, and then use Merck Millipore's 30kDa ultrafiltration centrifuge tube to concentrate until the protein concentration is as shown in Table 2 to obtain a concentrated solution.
  • the viscosity of the concentrated solution is measured by the ⁇ VISC viscometer of Rui Osen, and the operation steps are as follows:
  • the viscosity value of the sample liquid can be significantly reduced, because the amino acid protective agent has additional resistance Aggregation, all amino acid protective agents are preferred as viscosity regulators.
  • the subcutaneous injection concentration of QX007N monoclonal antibody injection can reach at least 150mg/mL.
  • the ultrafiltration displacement concentrate obtained in the ultrafiltration displacement step is divided into two parts and the concentration process is carried out separately:
  • the concentrated solution 1 and the concentrated solution 2 were mixed with the surfactant polysorbate 80 mother liquor, and filtered through a 0.2 ⁇ m filter membrane to obtain the original solution 1 (U1F2) and the original solution 2 (U2F2).
  • the SEC-HPLC method is used for analysis, and it is determined that the monomer of the active component QX007N in the sample is greater than 95%, the aggregate is less than 2%, and basically does not contain host foreign proteins.
  • sample analysis method setting the flow rate is 1.0mL/min, the analysis time is 15min, the injection volume is 50mg; the detection wavelength is 280nm; the system suitability must be checked before sample injection (use the reference sample for continuous sample injection, the number of sample injections Not less than 5 times, take the last 5 times for calculation), then analyze 1 blank sample, and then analyze 2 samples.
  • the viscosity value of the second step of concentration in the ultrafiltration process decreased from 31.5cP to 10.5cP after adding arginine, and arginine hydrochloride also acts as a protective agent to inhibit the formation of aggregates to a certain extent.
  • the viscosity value of the high-concentration anti-human IL-33 monoclonal antibody is significantly reduced after adding arginine hydrochloride, which will not cause excessive back pressure, which is beneficial to the control of the flow rate and membrane pressure of the ultrafiltration process, and improves the operability of the process sex.

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Abstract

本申请公开一种抗人白介素-33单克隆抗体的液体制剂，其包含蛋白质浓度为120～300mg/mL的抗人白介素-33单克隆抗体和氨基酸浓度为10～500mM的氨基酸添加剂；抗人白介素-33单克隆抗体包含三个重链互补决定区和三个轻链互补决定区，三个重链互补决定区为CDR-H1、CDR-H2和CDR-H3，三个轻链互补决定区为CDR-L1、CDR-L2和CDR-L3，其中：CDR-H1具有如SEQ ID NO：1所示的氨基酸序列；CDR-H2具有如SEQ ID NO：2所示的氨基酸序列；CDR-H3具有如SEQ ID NO：3所示的氨基酸序列；CDR-L1具有如SEQ ID NO：4所示的氨基酸序列；CDR-L2具有如SEQ ID NO：5所示的氨基酸序列；CDR-L3具有如SEQ ID NO：6所示的氨基酸序列。还公开一种抗人白介素-33单克隆抗体浓缩溶液的制备方法。本申请的液体制剂的粘度较低，稳定性高，可以用作注射剂。

Description

包含抗人白介素-33单克隆抗体的浓缩溶液的制备方法及液体制剂 技术领域
本申请涉及生物技术领域。具体地，本申请涉及一种包含抗人白介素-33单克隆抗体的液体制剂，以及一种抗人白介素-33单克隆抗体浓缩溶液的制备方法。
背景技术
单克隆抗体注射液作为一个快速增长的市场，主要应用在肿瘤治疗和自身免疫相关疾病的治疗，该类产品由于其较高的靶向性和安全性，为相关患者带来更多创新疗法的选择，针对一些化药小分子药物较难治疗的自身免疫相关的疾病，如哮喘、特异性皮炎、鼻息肉慢性鼻窦炎等，各类单抗注射液提供了更多的选择。另一方面为了降低生物制剂的临床使用成本，生物制剂剂型逐渐由冻干剂型向水针剂型转变，给药途径也由静脉给药方式向皮下注射剂型转变，大大降低了治疗时间并可实现在家自我注射，提高患者的依从性。由于单克隆抗体注射液的给药剂量通常在100～600mg范围，而皮下注射药液体积一般限制在2mL以下，在所述情况下，必须制备高度浓缩的蛋白制剂，通常蛋白含量可达到100mg/mL或者更大的浓度。
高浓度的单克隆抗体注射液给生产工艺的可制造、工艺放大、及最终的患者施用带来了许多挑战。最主要的一个挑战是超高的粘度，由于单克隆抗体的生物高分子特性，随着蛋白浓度提高，蛋白分子间相互作用力(如疏水、电荷作用等)逐渐增强，表现为高粘性的溶液。甚至在某些极端情况下会形成凝胶状物质，这种性状的料液对于超滤膜和超滤设备本身带来不小的挑战，比如最终浓缩时压差的急速上升导致的切向流速减小，浓差极化逐渐失控，直至出现蛋白沉淀将膜堵塞的现象，如此必然会造成回收率降低或工艺失败。另一方面，即使通过改进设备或膜包类型获得最终的高浓度蛋白溶液，也难以将其投入到实际的临床应用中，因为皮下给药时需要使用一次性无菌注射器吸取或者采用预充针的最终包装形式，而过高的粘度将导致注射时需要高于成年人的推注力的上限，无法实现自我皮下注射的应用。超滤浓缩高浓度 单克隆抗体药液的另一个难题是在高度浓缩时蛋白样品容易聚集形成可溶性聚体，进一步会聚集形成蛋白沉淀。
因此，针对，需要开发一种能够有效减少单克隆抗体聚集并提高其稳定性的低粘度、高浓度的包含抗人白介素-33单克隆抗体的液体制剂。
发明内容
为了解决现有技术中存在的问题，本申请提供了一种包含抗人白介素-33单克隆抗体的液体制剂(浓缩溶液)及其制备方法，所述液体制剂包含蛋白质浓度为120～300mg/mL的抗人白介素-33单克隆抗体和氨基酸浓度为10～500mM的氨基酸添加剂，所述液体制剂具有较低的粘度、较高的稳定性、抗人白介素-33单克隆抗体聚集少，可以用于注射剂，尤其是皮下注射剂。
QX007N为自主研发的靶向白介素-33的重组人源化单克隆抗体，白介素-33(interleukin 33，IL-33)是由人源基因IL-33编码的蛋白，其是白介素1家族成员，能有效驱动辅助因子Th2的产生。IL-33是ST2的配体之一。在非活性状态下，IL-33以前体形式(全长270个氨基酸残基)，通过与染色质结合基序以一种细胞稳态结合在细胞核当中。而当细胞受到损伤时，IL-33可作为一种内源的危险信号而被释放至胞外，主要通过结合受体ST2传递信号，作为细胞因子发挥免疫调节等作用。QX007N可特异性的与IL-33结合，阻止免疫细胞释放促炎细胞因子，从而预防哮喘恶化、改善哮喘控制等方面。
目前国内外尚未有上市的靶向人IL-33单克隆抗体药物，其中再生元和赛诺菲合作开发的REGN3500和AnaptysBio公司的Etokimab进度最快，目前正在进行2/3期临床研究，拟给药方式为静脉或皮下注射。从降低生物制剂的临床使用成本、提高患者依从性的角度来看，抗人IL-33单克隆抗体药物的优选剂型高蛋白水针，用于皮下注射剂，如将QX007N制备成皮下注射液，则该皮下注射液中的蛋白含量要高达100～150mg/mL。
本申请的具体技术方案如下：
本申请提供一种包含抗人白介素-33单克隆抗体的液体制剂，其特征在于，
所述液体制剂包含抗人白介素-33单克隆抗体和氨基酸添加剂；
其中，所述抗人白介素-33单克隆抗体的蛋白质浓度为120～300mg/mL，优选为120～200mg/mL，更优选为120～160mg/mL；所述氨基酸添加剂的氨基酸浓度为10～500mM；
所述抗人白介素-33单克隆抗体包含三个重链互补决定区和三个轻链互补决定区，所述三个重链互补决定区为CDR-H1、CDR-H2和CDR-H3，所述三个轻链互补决定区为CDR-L1、CDR-L2和CDR-L3，其中：
CDR-H1具有如SEQ ID NO：1所示的氨基酸序列；
CDR-H2具有如SEQ ID NO：2所示的氨基酸序列；
CDR-H3具有如SEQ ID NO：3所示的氨基酸序列；
CDR-L1具有如SEQ ID NO：4所示的氨基酸序列；
CDR-L2具有如SEQ ID NO：5所示的氨基酸序列；
CDR-L3具有如SEQ ID NO：6所示的氨基酸序列。
在本申请中，所述抗人白介素-33单克隆抗体包含重链可变区和轻链可变区，其中，
所述重链可变区具有如SEQ ID NO：7所示的氨基酸序列；
所述轻链可变区具有如SEQ ID NO：8所示的氨基酸序列。
在本申请中，所述氨基酸添加剂选自盐酸精氨酸、组氨酸、赖氨酸、甲硫氨酸、脯氨酸中的一种或两种或三种以上。
在本申请中，所述氨基酸添加剂包括盐酸精氨酸、组氨酸和甲硫氨酸中的一种或两种或三种；
优选地，所述氨基酸添加剂包括盐酸精氨酸，所述液体制剂中盐酸精氨酸的浓度为100～200mM；
优选地，所述氨基酸添加剂包括组氨酸，所述液体制剂中组氨酸的浓度为10～50mM；
优选地，所述氨基酸添加剂包括甲硫氨酸，所述液体制剂中甲硫氨酸的浓度为2～50mM。
在本申请中，所述液体制剂还包含表面活性剂，所述表面活性剂的含量为0.01～5mg/mL，优选为0.1～2mg/mL，更优选为0.2～1mg/mL；
优选地，所述表面活性剂为聚山梨酯80。
在本申请中，在所述液体制剂中所述抗人白介素-33单克隆抗体的浓度在180mg/mL以上时，所述液体制剂的粘度在15cP以下；
可选地，在所述液体制剂中所述抗人白介素-33单克隆抗体的浓度在150mg/mL以上时，所述液体制剂的粘度在10cP以下。
在本申请中，所述液体制剂还包含蔗糖，所述蔗糖的浓度为20～140mg/mL，优选为50～90mg/mL。
在本申请中，所述液体制剂还包含山梨醇，所述山梨醇的浓度为10～200mg/mL，优选为20～100mg/mL，更优选为50～80mg/mL。
在本申请中，所述液体制剂还包含氯化钠，所述氯化钠的浓度为20～300mg/mL，优选为100～200mg/mL。
在本申请中，所述液体制剂的pH为5.5～6.5。
本申请还提供一种抗人白介素-33单克隆抗体浓缩溶液的制备方法，其包括下述步骤：
超滤浓缩：浓缩含有抗人白介素-33单克隆抗体的溶液，得到浓缩样品；
超滤置换：利用置换缓冲溶液置换所述浓缩样品，得到超滤置换浓缩液；
二次超滤浓缩：将氨基酸添加剂母液加入至所述超滤置换浓缩液中，混合均匀得到混合溶液，将所述混合溶液进行超滤浓缩后得到所述浓缩溶液；
所述抗人白介素-33单克隆抗体包含三个重链互补决定区和三个轻链互补决定区，所述三个重链互补决定区为CDR-H1、CDR-H2和CDR-H3，所述三个轻链互补决定区为CDR-L1、CDR-L2和CDR-L3，其中：
CDR-H1具有如SEQ ID NO：1所示的氨基酸序列；
CDR-H2具有如SEQ ID NO：2所示的氨基酸序列；
CDR-H3具有如SEQ ID NO：3所示的氨基酸序列；
CDR-L1具有如SEQ ID NO：4所示的氨基酸序列；
CDR-L2具有如SEQ ID NO：5所示的氨基酸序列；
CDR-L3具有如SEQ ID NO：6所示的氨基酸序列。
在本申请中，所述抗人白介素-33单克隆抗体包含重链可变区和轻链可变区，其中，
所述重链可变区具有如SEQ ID NO：7所示的氨基酸序列；
所述轻链可变区具有如SEQ ID NO：8所示的氨基酸序列。
在本申请中，在超滤浓缩步骤中，浓缩条件为：流速为120～300L/m 2·h，跨膜压差为0.6～1.5bar；
优选地，在超滤浓缩步骤中，所述浓缩样品中的抗人白介素-33单克隆 抗体的浓度为20～60mg/mL，优选为30～50mg/mL。
在本申请中，在超滤置换步骤中，所述置换缓冲溶液的用量为所述浓缩样品重量的7倍以上；
优选地，在超滤置换步骤中，所述置换缓冲溶液的浓度为5～50mM，优选为20mM；
优选地，在超滤置换步骤中，所述置换缓冲溶液的pH为5.5～6.5，优选为5.7～6.3；
优选地，在超滤置换步骤中，所述置换缓冲溶液选自组氨酸-盐酸缓冲溶液、柠檬酸缓冲溶液、磷酸盐缓冲溶液和醋酸钠-醋酸缓冲溶液中的一种。
在本申请中，在二次超滤浓缩步骤中，所述混合溶液中氨基酸添加剂的浓度为50～300mmol/L，优选为50～200mmol/L，更优选为100～150mmol/L。
在本申请中，在二次超滤浓缩步骤中，所述浓缩溶液中的蛋白质浓度为120～300mg/mL，优选为120～200mg/mL，更优选为120～160mg/mL。
在本申请中，所述氨基酸添加剂选自盐酸精氨酸、组氨酸、赖氨酸、甲硫氨酸、脯氨酸中的一种或两种或三种以上。
在本申请中，所述氨基酸添加剂包括盐酸精氨酸、组氨酸和甲硫氨酸中的一种或两种或三种；
优选地，所述氨基酸添加剂包括盐酸精氨酸，所述混合溶液中盐酸精氨酸的浓度为100～200mM；
优选地，所述氨基酸添加剂包括组氨酸，所述混合溶液中组氨酸的浓度为10～50mM；
优选地，所述氨基酸添加剂包括甲硫氨酸，所述混合溶液中甲硫氨酸的浓度为2～50mM。
在本申请中，在所述浓缩溶液中所述抗人白介素-33单克隆抗体的浓度在180mg/mL以上时，所述浓缩溶液的粘度在15cP以下；
可选地，在所述浓缩溶液中所述抗人白介素-33单克隆抗体的浓度在150mg/mL以上时，所述浓缩溶液的粘度在10cP以下。
在本申请中，所述含有抗人白介素-33单克隆抗体的溶液通过对表达抗人白介素-33单克隆抗体的细胞发酵液进行亲和层析、低pH灭活、阴离子层析、阳离子层析和纳滤得到。
发明的效果
本申请的制备方法制备得到的包含抗人IL-33单克隆抗体的液体制剂(抗人白介素-33单克隆抗体浓缩溶液)，包含抗人白介素-33单克隆抗体，并且其具有较低的粘度，可用注射器轻松推注，因此可以用作注射剂，尤其是皮下注射剂。
本申请的抗人IL-33单克隆抗体，其与现有的抗人IL-33单克隆抗体(Etokimab/ANB020)相比，结合人白介素-33的亲和力相当，在细胞水平的中和活性与Etokimab/ANB020相当。
赛诺菲公司研发的靶向白介素-33的单克隆抗体药物(Itepekimab/REGN3500)拟用于慢性阻塞性肺病(临床III期)、哮喘(临床II期)等特应性皮炎疾病的治疗。AnaptysBio公司研发的Etokimab/ANB020用于慢性鼻窦炎(临床II期)。
本申请的单克隆抗体在细胞水平显示出与Etokimab/ANB020(根据专利公开序列表达制备)相当的中和活性，其有望在预防和治疗相关疾病方面展现出良好的临床效果。
附图说明
图1是显示构建QX007N(HZD78-70)瞬转表达质粒的核酸电泳结果的图。其中，M：Marker；条带1：PCR产物78VH-Hu25；条带2：pQX2.1，HindIII/NheI；条带3：PCR产物78VK-Hu3-CK；条带4：pQX1，HindIII/BamHI。
图2是瞬转表达流程图。
图3是QX007N(HZD78-70)的电泳检测图。
图4是显示QX007N(HZD78-70)和Etokimab/ANB020中和重组人白介素-33诱导HEK Blue TM IL-33细胞中NF-κB/AP-1信号转导的活性图。
图5是显示QX007N(HZD78-70)和Etokimab/ANB020中和天然人白介素-33诱导HEK Blue TM IL-33细胞中NF-κB/AP-1信号转导的活性图。
图6是显示QX007N(HZD78-70)和Etokimab/ANB020中和重组人白介素-33诱导KU812细胞释放IL-5的活性图。
图7是显示QX007N(HZD78-70)和Etokimab/ANB020中和重组人白介素-33诱导人全血释放IFN-γ的活性图。
具体实施方式
以下对本申请的示范性实施例做出说明，其中包括本申请实施例的各种细节以助于理解，应当将它们认为仅仅是示范性的。因此，本领域普通技术人员应当认识到，可以对这里描述的实施例做出各种改变和修改，而不会背离本申请的范围和精神。同样，为了清楚和简明，以下的描述中省略了对公知功能和结构的描述。
本申请提供一种包含抗人白介素-33单克隆抗体的液体制剂，其特征在于，
所述液体制剂包含抗人白介素-33单克隆抗体和氨基酸添加剂；
其中，所述抗人白介素-33单克隆抗体的蛋白质浓度为120～300mg/mL，优选为120～200mg/mL，更优选为120～160mg/mL；所述氨基酸添加剂的氨基酸浓度为10～500mM；
所述抗人白介素-33单克隆抗体包含三个重链互补决定区和三个轻链互补决定区，所述三个重链互补决定区为CDR-H1、CDR-H2和CDR-H3，所述三个轻链互补决定区为CDR-L1、CDR-L2和CDR-L3，其中：
CDR-H1具有如SEQ ID NO：1(SYHMI)所示的氨基酸序列；
CDR-H2具有如SEQ ID NO：2(VIYPNSNIYYATWAKG)所示的氨基酸序列；
CDR-H3具有如SEQ ID NO：3(TIYVHVYSALSI)所示的氨基酸序列；
CDR-L1具有如SEQ ID NO：4(QASESVLNEVS)所示的氨基酸序列；
CDR-L2具有如SEQ ID NO：5(FASKLAS)所示的氨基酸序列；
CDR-L3具有如SEQ ID NO：6(QQDWSMDNIDNA)所示的氨基酸序列。
在本申请中，CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2、CDR-L3分别表示重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2、轻链CDR3。
具体的，所述抗人白介素-33单克隆抗体的蛋白质浓度例如可为120mg/mL、130mg/mL、140mg/mL、150mg/mL、160mg/mL、170mg/mL、180mg/mL、190mg/mL、200mg/mL、210mg/mL、220mg/mL、230mg/mL、240mg/mL、250mg/mL、260mg/mL、270mg/mL、280mg/mL、290mg/mL、300mg/mL等。
所述氨基酸添加剂的氨基酸浓度例如可为10mM、30mM、50mM、70mM、 90mM、110mM、130mM、150mM、170mM、190mM、210mM、230mM、250mM、270mM、290mM、310mM、330mM、350mM、370mM、390mM、410mM、430mM、450mM、470mM、490mM、500mM等。
在本申请中，“单克隆抗体”表示得自基本上同源的抗体的群体的抗体，即，构成所述群体的各个抗体是相同的和/或结合相同表位，除了可能的变体抗体(例如，含有天然存在的突变或在单克隆抗体制品的生产过程中产生)以外，这样的变体通常以微量存在。与通常包括针对不同决定簇(表位)的不同抗体的多克隆抗体制品不同，单克隆抗体制品的每种单克隆抗体针对抗原上的单个决定簇。因而，修饰语“单克隆”指示所述抗体得自基本上同源的抗体群体的特征，并且不应解释为需要通过任何特定方法生产所述抗体。例如，要根据本发明使用的单克隆抗体可以通过多种技术来制备，所述技术包括、但不限于杂交瘤方法、重组DNA方法、噬菌体展示方法、和使用包含人免疫球蛋白基因座的全部或部分的转基因动物的方法，本文描述了这样的方法和其它示例性的制备单克隆抗体的方法。
在本申请中，“单克隆抗体”一般为人抗体，其可以使用本领域技术人员公知的技术来制备，例如，人抗体一般描述于van Dijk,M.A.and van de Winkel,J.G.,Curr.Opin.Pharmacol.5:368-374(2001)及Lonberg,N.,Curr.Opin.Immunol.20:450-459(2008)。
可以通过向已经经过修饰而对抗原攻击刺激生产完整人抗体或具有人类可变区的完整抗体的转基因动物施用免疫原来制备抗体，这些动物通常含有一部分或全部的人类免疫球蛋白基因座，其替换了内源免疫球蛋白基因座，或者存在于染色体外或随机整合于动物体内。在此类转基因小鼠中，内源免疫球蛋白基因座一般已经失活，关于自转基因动物获得人抗体的方法的综述，参见Lonberg,N.,Nat.Biotech.(自然生物技术)23:1117-1125(2005)。还可参见例如美国专利No.6,075,181和No.6,150,584描述的XENOMOUSE TM技术；美国专利No.5,770,429描述的
Figure PCTCN2022075787-appb-000001
技术；美国专利No.7,041,870描述的
Figure PCTCN2022075787-appb-000002
技术，和美国专利申请公开文本No.US 2007/0061900描述的
Figure PCTCN2022075787-appb-000003
技术。可以例如通过与不同人恒定区组合进一步修饰来自由此类动物生成的完整抗体的人可变区。
还可以通过基于杂交瘤的方法来制备人抗体。已描述了用于生产人单克隆抗体的人骨髓瘤和小鼠-人杂交骨髓瘤细胞(参见例如Kozbor,D.,J. Immunol.133:3001-3005(1984)；Brodeur,B.R.et al.,Monoclonal Antibody Production Techniques and Applications,Marcel Dekker,Inc.,New York(1987),pp.51-63；及Boerner,P.et al.,J.Immunol.147:86-95(1991))。经由人B细胞杂交瘤技术生产的人抗体也记载于Li,J.etal.,Proc.Natl.Acad.Sci.USA103:3557-3562(2006)。其他方法包括那些记载于例如美国专利No.7,189,826(其描述了自杂交瘤细胞系生成单克隆人IgM抗体)以及Ni,Xiandai Mianyixue,26(4)；265-268(其描述了人-人杂交瘤)的。人杂交瘤技术(Trioma技术)也记载于Vollmers,H.P.and Brandlein,S.,Histology and Histopathology 20:927-937(2005)及Vollmers,H.P.and Brandlein,S.,Methods and Findings in Experimentaland Clinical Pharmacology 27:185-191(2005)。
还可通过分离选自来源于人的噬菌体展示文库的Fv克隆可变结构域序列来生成人抗体，然后，可以将此类可变域序列与期望的人恒定域组合。
还可以基于自抗体文库选择人抗体，即可以通过对组合文库筛选具有期望的一种或多种活性的抗体来分离人抗体。例如，用于生产噬菌体展示文库及对此类文库筛选拥有期望结合特征的抗体的多种方法是本领域已知的。这种方法综述于例如Hoogenboom,H.R.et al.,Methods in Molecular Biology 178:1-37(2001)，并且进一步记载于例如McCafferty,J.et al.,Nature 348:552-554(1990)；Clackson,T.et al.,Nature 352:624-628(1991)；Marks,J.D.et al.,J.Mol.Biol.222:581-597(1992)；Marks,J.D.and Bradbury,A.,Methods in Molecular Biology 248:161-175(2003)；Sidhu,S.S.et al.,J.Mol.Biol.338:299-310(2004)；Lee,C.V.et al.,J.Mol.Biol.340:1073-1093(2004)；Fellouse,F.A.,Proc.Natl.Acad.Sci.USA 101:12467-12472(2004)；及Lee,C.V.et al.,J.Immunol.Methods 284:119-132(2004)。
在某些噬菌体展示方法中，通过聚合酶链反应(PCR)分别克隆VH和VL基因的全集，并在噬菌体文库中随机重组，然后在所述噬菌体文库中筛选抗原结合性噬菌体，如记载于Winter,G.et al.,Ann.Rev.Immunol.12:433-455(1994)。噬菌体通常以单链Fv(scFv)片段或以Fab片段展示抗体片段。来自经免疫来源的文库提供针对免疫原的高亲和力抗体，而不需要构建杂交瘤。或者，可以(例如自人)克隆未免疫全集以在没有任何免疫的情况中提供针对一大批非自身和还有自身抗原的抗体的单一 来源，如由Griffiths,A.D.et al.,EMBO J,12:725-734(1993)描述的。最后，也可以通过从干细胞克隆未重排的V基因区段，并使用含有随机序列的PCR引物编码高度可变的CDR3区并在体外实现重排来合成生成未免疫文库，如由Hoogenboom,H.R.and Winter,G.,J.Mol.Biol.227:381-388(1992)所描述的。描述人抗体噬菌体文库的专利公开文本包括例如：美国专利No.5,750,373及美国专利公开文本No.2005/0079574、2005/0119455、2005/0266000、2007/0117126、2007/0160598、2007/0237764、2007/0292936和2009/0002360。
所述抗体也可以是多特异性抗体，例如双特异性抗体。双特异性抗体是对至少两种不同位点具有结合特异性的单克隆抗体。用于生成多特异性抗体的技术包括但不限于具有不同特异性的两对免疫球蛋白重链-轻链的重组共表达(参见Milstein,C.and Cuello,A.C.,Nature305:537-540(1983)；WO 93/08829；及Traunecker,A.et al.,EMBO J.10:3655-3659(1991))、和“节-入-穴”工程化(参见例如美国专利No.5,731,168)。也可以通过用于生成抗体Fc-异二聚体分子的工程化静电操纵效应(WO 2009/089004)；交联两种或更多种抗体或片段(参见例如美国专利No.4,676,980及Brennan,M.et al.,Science 229:81-83(1985))；使用亮氨酸拉链来生成双特异性抗体(参见例如Kostelny,S.A.et al.,J.Immunol.148:1547-1553(1992))；使用用于生成双特异性抗体片段的“双抗体”技术(参见例如Holliger,P.et al.,Proc.Natl.Acad.Sci.USA 90:6444-6448(1993))；及使用单链Fv(scFv)二聚体(参见例如Gruber,M.et al.,J.Immunol.152:5368-5374(1994))；及制备三特异性抗体(如例如Tutt,A.et al.,J.Immunol.147:60-69(1991)中所描述的)来生成多特异性抗体。
本文中所述的单克隆抗体还包括具有三个或更多个功能性抗原结合位点的工程化改造抗体，包括“章鱼抗体”(参见例如US 2006/0025576)。
本文中的抗体还包括WO 2009/080251、WO 2009/080252、WO2009/080253、WO 2009/080254、WO 2010/112193、WO 2010/115589、WO2010/136172、WO 2010/145792、及WO 2010/145793、WO 2011/117330、WO 2012/025525、WO 2012/025530、WO 2013/026835、WO2013/026831、WO 2013/164325、或WO 2013/174873中记载的多特异性抗体。
本文中所述的单克隆抗体也可以是抗体变体，例如，可能期望改善抗体的结合亲和力和/或其它生物学特性。可以通过将适宜的修饰引入编码抗体的核苷酸序列中，或者通过肽合成来制备抗体的氨基酸序列变体。此类修饰包 括例如对抗体的氨基酸序列内的残基的删除、和/或插入和/或替代。可以进行删除、插入、和替代的任何组合以得到最终的构建体，只要最终的构建体拥有期望的特征，例如抗原结合。因此，在某些实施方案中，提供了具有一个或多个氨基酸置换的抗体变体，用于置换突变的感兴趣的位点包括HVR和FR，例如，可将氨基酸置换引入感兴趣的抗体中并筛选具有所需活性的产物，例如，保留/改善的抗原结合性，降低的免疫原性，或改善的ADCC或CDC。
本申请中，所述单克隆抗体体外发酵生产使用的哺乳动物细胞包括但不限于目前通用的各种杂交瘤细胞、中国仓鼠卵巢细胞(CHO)，优选的是CHO细胞。
本申请中，“亲和力”表示分子(例如，抗体)的单个结合位点和它的结合配偶体(例如，抗原)之间的非共价相互作用的总和的强度。除非另外指出，否则本说明书中使用的“结合亲和力”表示反映结合对(例如，抗体和抗原)的成员之间的1∶1相互作用的固有结合亲和力。分子X对它的配偶体Y的亲和力通常可以由平衡解离常数(K D)表示。通过本领域已知的常见方法，可以测量亲和力。
本申请中，“超滤”是指切相流超滤(Tangential Flow Filtration，TFF)是指液体流动方向切向(平行)于滤膜表面的过滤形式，相比流动方向与滤膜垂直方向的传统过滤方式(NFF)，切向流滤膜表面的颗粒堆积较少，过滤速度稳定，适用于大体积样品的分离。其中，大于膜孔径的分子被截留并逐步浓缩，小于膜孔径的物质透过膜，从大分子溶液中分离出来，实现大分子和小分子的分离。因而切向流超滤常用于生物制品的浓缩、透析、置换缓冲溶液、不同尺寸分子的分离等。
本申请中，人白介素-33表示位于细胞核内的人白介素-33经蛋白酶水解形成成熟的人白介素-33，分泌至细胞外，发挥人白介素-33的生物活性，其具有如SEQ ID NO：9所示的氨基酸序列。
SEQ ID NO：9：
Figure PCTCN2022075787-appb-000004
本申请中，“抗人白介素-33单克隆抗体”表示这样的单克隆抗体：其能够以足够的亲和力结合人白介素-33，使得所述单克隆抗体可用作靶向人白介素-33的诊断剂和/或治疗剂。
本申请中，抗人白介素-33单克隆抗体与靶标无关的蛋白不结合。这里，“无关的蛋白”是指除作为靶标的人白介素-33以外的其它蛋白；这里，“不结合”是指：在将本申请的抗人IL-33单克隆抗体与作为其靶标的人白介素-33的结合能力作为100％的情况下，本申请的抗人IL-33单克隆抗体与所述无关蛋白的结合能力小于10％，例如9％、8％、7％、6％、5％、4％、3％、2％、1％或者0。
本申请的抗人IL-33单克隆抗体与人、食蟹猴的白介素-33可以结合，与其他动物种属的白介素-33可以不结合。这里，“其它动物种属”是指除人、食蟹猴以外的动物种属，例如猪、犬、兔、大鼠、小鼠、豚鼠等；这里，在判定本申请的抗人IL-33单克隆抗体的种属特异性时，“不结合”是指：在将本申请的抗人IL-33与作为其靶标的人白介素-33的结合能力作为100％的情况下，本申请的抗人IL-33单克隆抗体与其它动物种属的白介素-33的结合能力小于5％，例如4％、3％、2％、1％或者0。
本申请的抗人IL-33单克隆抗体具有≤1μM、≤100nM、≤50nM、≤40nM的平衡解离常数(K D)。
实验结果显示，本申请的抗人IL-33单克隆抗体可以特异性结合人白介素-33。
本申请的抗人IL-33单克隆抗体在诸多生物活性方面与上市同类单抗产品相当、或优于上市同类单抗产品。所述生物活性例如中和重组/天然人白介素-33诱导细胞中NF-κB/AP-1信号转导的活性、中和白介素-33诱导KU812细胞释放IL-5的活性、中和白介素-33诱导人全血释放IFN-γ等。
在一个实施方式中，本申请的抗人IL-33单克隆抗体的重链的氨基酸序列如SEQ ID NO：10所示；轻链的氨基酸序列如SEQ ID NO：11所示。
SEQ ID NO：10：
Figure PCTCN2022075787-appb-000005
Figure PCTCN2022075787-appb-000006
SEQ ID NO：11：
Figure PCTCN2022075787-appb-000007
其中，SEQ ID NO：10和11均为经人源化的序列。
在一个具体实施方式中，所述抗人白介素-33单克隆抗体包含重链可变区和轻链可变区，其中，
所述重链可变区具有如SEQ ID NO：7(EVQLVESGGGLVQPGGSLRLSCAASGFSLSSYHMIWVRQAPGKGLEWVGVIYPNSNIYYATWAKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARTIYVHVYSALSIWGQGTLVTVSS)所示的氨基酸序列；
所述轻链可变区具有如SEQ ID NO：8(AFQMTQSPSSVSASVGDRVTITCQASESVLNEVSWYQQKPGKAPKLLIYFASKLASGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQDWSMDNIDNAFGGGTKVEIK)所示的氨基酸序列。
在一个具体实施方式中，所述氨基酸添加剂(氨基酸保护剂)选自盐酸精氨酸、组氨酸、赖氨酸、甲硫氨酸、脯氨酸中的一种或两种或三种以上。
在一个具体实施方式中，所述氨基酸添加剂选自盐酸精氨酸、组氨酸、赖氨酸、甲硫氨酸、脯氨酸中的一种或两种或三种以上。
在一个具体实施方式中，所述氨基酸添加剂包括盐酸精氨酸、组氨酸和甲硫氨酸中的一种或两种或三种。
在一个具体实施方式中，所述氨基酸添加剂包括盐酸精氨酸，所述液体制剂中盐酸精氨酸的浓度为100～200mM，例如可为100mM、110mM、120mM、 130mM、140mM、150mM、160mM、170mM、180mM、190mM、200mM等。
在一个具体实施方式中，所述氨基酸添加剂包括组氨酸，所述液体制剂中组氨酸的浓度为10～50mM，例如可为10mM、12mM、14mM、16mM、18mM、20mM、22mM、24mM、26mM、28mM、30mM、32mM、34mM、36mM、38mM、40mM、42mM、44mM、46mM、48mM、50mM等。
在一个具体实施方式中，所述氨基酸添加剂包括甲硫氨酸，所述液体制剂中甲硫氨酸的浓度为2～50mM，例如可为2mM、4mM、6mM、8mM、10mM、12mM、14mM、16mM、18mM、20mM、22mM、24mM、26mM、28mM、30mM、32mM、34mM、36mM、38mM、40mM、42mM、44mM、46mM、48mM、50mM等。
在一个具体实施方式中，所述氨基酸添加剂包括盐酸精氨酸和组氨酸，所述液体制剂中，所述盐酸精氨酸的浓度为100～200mM，所述组氨酸的浓度为10～50mM。
在一个具体实施方式中，所述氨基酸添加剂包括盐酸精氨酸和甲硫氨酸，所述液体制剂中，所述盐酸精氨酸的浓度为100～200mM，所述甲硫氨酸的浓度为2～50mM。
在一个具体实施方式中，所述氨基酸添加剂包括组氨酸和甲硫氨酸，所述液体制剂中，所述组氨酸的浓度为10～50mM，所述甲硫氨酸的浓度为2～50mM。
在一个具体实施方式中，所述氨基酸添加剂包括盐酸精氨酸、组氨酸以及甲硫氨酸，所述液体制剂中，所述盐酸精氨酸的浓度为100～200mM，优选为150mM，所述组氨酸的浓度为10～50mM，优选为20mM，所述甲硫氨酸的浓度为2～50mM，优选为10mM。
在一个具体实施方式中，所述氨基酸添加剂由盐酸精氨酸、组氨酸以及甲硫氨酸组成，所述液体制剂中，所述盐酸精氨酸的浓度为100～200mM，优选为150mM，所述组氨酸的浓度为10～50mM，优选为20mM，所述甲硫氨酸的浓度为2～50mM，优选为10mM。
在一个具体实施方式中，所述液体制剂还包含表面活性剂，所述表面活性剂的含量为0.01～5mg/mL，优选为0.1～2mg/mL，更优选为0.2～1mg/mL，例如可为0.01mg/mL、0.1mg/mL、0.2mg/mL、0.3mg/mL、0.4mg/mL、0.5mg/mL、 0.6mg/mL、0.7mg/mL、0.8mg/mL、0.9mg/mL、1mg/mL、2mg/mL、3mg/mL、4mg/mL、5mg/mL等。本申请中的表面活性剂的种类不做限定，例如可为聚山梨酯80。
在一个具体实施方式中，在所述液体制剂中所述抗人白介素-33单克隆抗体的浓度在180mg/mL以上时，所述液体制剂的粘度在15cP以下。
在一个具体实施方式中，在所述液体制剂中所述抗人白介素-33单克隆抗体的浓度在150mg/mL以上时，所述液体制剂的粘度在10cP以下。
在一个具体实施方式中，所述液体制剂还包含蔗糖，所述蔗糖的浓度为20～140mg/mL，优选为50～90mg/mL，例如可为20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL、120mg/mL、140mg/mL等。
在一个具体实施方式中，所述液体制剂还包含山梨醇，所述山梨醇的浓度为10～200mg/mL，优选为20～100mg/mL，更优选为50～80mg/mL，例如可为10mg/mL、20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL、120mg/mL、140mg/mL、160mg/mL、180mg/mL、200mg/mL等。
在一个具体实施方式中，所述液体制剂还包含氯化钠，所述氯化钠的浓度为20～300mg/mL，优选为100～200mg/mL，例如可为20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL、70mg/mL、80mg/mL、90mg/mL、100mg/mL、120mg/mL、140mg/mL、160mg/mL、180mg/mL、200mg/mL、220mg/mL、240mg/mL、260mg/mL、280mg/mL、300mg/mL等。
在一个具体实施方式中，所述液体制剂的pH为5.5～6.5，例如可为5.5、5.6、5.7、5.8、5.9、6、6.1、6.2、6.3、6.4、6.5等。
本申请还提供一种抗人白介素-33单克隆抗体浓缩溶液的制备方法，其包括下述步骤：
超滤浓缩：浓缩含有抗人白介素-33单克隆抗体的溶液，得到浓缩样品；
超滤置换：利用置换缓冲溶液置换所述浓缩样品，得到超滤置换浓缩液；
二次超滤浓缩：将氨基酸添加剂母液加入至所述超滤置换浓缩液中，混合均匀得到混合溶液，将所述混合溶液进行超滤浓缩后得到所述浓缩溶液；
所述抗人白介素-33单克隆抗体包含三个重链互补决定区和三个轻链互补决定区，所述三个重链互补决定区为CDR-H1、CDR-H2和CDR-H3，所 述三个轻链互补决定区为CDR-L1、CDR-L2和CDR-L3，其中：
CDR-H1具有如SEQ ID NO：1(SYHMI)所示的氨基酸序列；
CDR-H2具有如SEQ ID NO：2(VIYPNSNIYYATWAKG)所示的氨基酸序列；
CDR-H3具有如SEQ ID NO：3(TIYVHVYSALSI)所示的氨基酸序列；
CDR-L1具有如SEQ ID NO：4(QASESVLNEVS)所示的氨基酸序列；
CDR-L2具有如SEQ ID NO：5(FASKLAS)所示的氨基酸序列；
CDR-L3具有如SEQ ID NO：6(QQDWSMDNIDNA)所示的氨基酸序列。
在本申请中，CDR-H1、CDR-H2、CDR-H3、CDR-L1、CDR-L2、CDR-L3分别表示重链CDR1、重链CDR2、重链CDR3、轻链CDR1、轻链CDR2、轻链CDR3。
在一个具体实施方式中，在超滤浓缩步骤中，浓缩条件为：流速为120～300L/m 2·h，例如可为120L/m 2·h、135L/m 2·h、150L/m 2·h、165L/m 2·h、180L/m 2·h、190L/m 2·h、200L/m 2·h、225L/m 2·h、250L/m 2·h、275L/m 2·h、300L/m 2·h等；跨膜压差(TMP)为0.6～1.5bar，例如可为0.6bar、0.7bar、0.8bar、0.9bar、1.0bar、1.1bar、1.2bar、1.3bar、1.4bar、1.5bar等；
在一个具体实施方式中，在超滤浓缩步骤中，所述浓缩样品中的抗人白介素-33单克隆抗体的浓度为20～60mg/mL，例如可为20mg/mL、30mg/mL、40mg/mL、50mg/mL、60mg/mL等，优选为30～50mg/mL。
在一个具体实施方式中，在超滤置换步骤中，所述置换缓冲溶液的用量为所述浓缩样品重量的7倍以上，例如可为7倍、8倍、9倍、10倍等。
在本申请中，在超滤置换步骤中，对于置换缓冲溶液的浓度，本申请不作任何限制，本领域技术人员可以根据需要进行选择。在一个具体实施方式中，在超滤置换步骤中，所述置换缓冲溶液的浓度为5～50mM，例如可为5mM、10mM、20mM、30mM、40mM、50mM等，优选为20mM。
在本申请中，在超滤置换步骤中，对于置换缓冲溶液的pH，本申请不作任何限制，其只要为偏酸性即可。在一个具体实施方式中，在超滤置换步骤中，所述置换缓冲溶液的pH为5.5～6.5，例如可为5.5、5.6、5.7、5.8、5.9、6.0、6.1、6.2、6.3、6.4、6.5等，优选为5.7～6.3。
在一个具体实施方式中，在超滤置换步骤中，所述置换缓冲溶液选自组 氨酸-盐酸缓冲溶液、柠檬酸缓冲溶液、磷酸盐缓冲溶液和醋酸钠-醋酸缓冲溶液中的一种，优选为组氨酸-盐酸缓冲溶液。具体地，所述柠檬酸缓冲溶液可以为柠檬酸-柠檬酸钠缓冲溶液。具体地，所述磷酸盐缓冲溶液可以为磷酸氢二钠-磷酸二氢钠缓冲溶液。
在一个具体实施方式中，使用蠕动泵将置换缓冲溶液(待置换的缓冲溶液)泵入浓缩样品中，继续超滤，同时调节置换缓冲溶液的泵入速度与透过流速一致，当置换缓冲溶液的泵入量为浓缩样品重量的7倍以上时即完成置换。
在一个具体实施方式中，在二次超滤浓缩步骤中，所述混合溶液中氨基酸添加剂的浓度为50～300mmol/L，例如可为50mmol/L、100mmol/L、150mmol/L、200mmol/L、250mmol/L、300mmol/L等，优选为50～200mmol/L，更优选为100～150mmol/L；所述浓缩溶液中的蛋白质浓度为120～300mg/mL，例如可为120mg/mL、130mg/mL、140mg/mL、150mg/mL、160mg/mL、170mg/mL、180mg/mL、190mg/mL、200mg/mL、210mg/mL、220mg/mL、230mg/mL、240mg/mL、250mg/mL、260mg/mL、270mg/mL、280mg/mL、290mg/mL、300mg/mL等，优选为120～200mg/mL，更优选为120～160mg/mL。
在一个具体实施方式中，所述氨基酸添加剂选自盐酸精氨酸、组氨酸、赖氨酸、甲硫氨酸、脯氨酸中的一种或两种或三种以上。
在一个具体实施方式中，所述氨基酸添加剂包括盐酸精氨酸、组氨酸和甲硫氨酸中的一种或两种或三种。
在一个具体实施方式中，所述氨基酸添加剂包括盐酸精氨酸，所述混合溶液中盐酸精氨酸的浓度为100～200mM，例如可为100mM、110mM、120mM、130mM、140mM、150mM、160mM、170mM、180mM、190mM、200mM等。
在一个具体实施方式中，所述氨基酸添加剂包括组氨酸，所述混合溶液中组氨酸的浓度为10～50mM，例如可为10mM、12mM、14mM、16mM、18mM、20mM、22mM、24mM、26mM、28mM、30mM、32mM、34mM、36mM、38mM、40mM、42mM、44mM、46mM、48mM、50mM等。
在一个具体实施方式中，所述氨基酸添加剂包括甲硫氨酸，所述混合溶液中甲硫氨酸的浓度为2～50mM，例如可为2mM、4mM、6mM、8mM、10mM、12mM、14mM、16mM、18mM、20mM、22mM、24mM、26mM、28mM、 30mM、32mM、34mM、36mM、38mM、40mM、42mM、44mM、46mM、48mM、50mM等。
在一个具体实施方式中，所述氨基酸添加剂包括盐酸精氨酸、组氨酸以及甲硫氨酸，所述混合溶液中，所述盐酸精氨酸的浓度为100～200mM，所述组氨酸的浓度为10～50mM，所述甲硫氨酸的浓度为2～50mM。
在一个具体实施方式中，所述氨基酸添加剂由盐酸精氨酸、组氨酸以及甲硫氨酸组成，所述混合溶液中，所述盐酸精氨酸的浓度为100～200mM，所述组氨酸的浓度为10～50mM，所述甲硫氨酸的浓度为2～50mM。
在一个具体实施方式中，在二次超滤浓缩步骤中，计算得到的超滤置换浓缩液中的蛋白质浓度，并计算该超滤置换浓缩液的理论体积(超滤初始蛋白总量/超滤置换浓缩液中的蛋白质浓度)，并加入氨基酸添加剂母液混合均匀，优选的，氨基酸添加剂的体积是超滤置换浓缩液体积的1/9，然后继续超滤浓缩，根据原液要求的浓度进行过浓缩(通常高于原液浓度约30％)，得到浓缩溶液，其中的抗人白介素-33单克隆抗体的浓度为100～200mg/mL，所述浓缩溶液的粘度≤15cP。若无本申请采用的上述氨基酸添加剂直接进行过浓缩，浓缩溶液粘度值会达到25～50cP，反压过高导致无法使用该型膜包和设备进行过浓缩及回收。
在一个具体实施方式中，在所述浓缩溶液中所述抗人白介素-33单克隆抗体的浓度在180mg/mL以上时，所述浓缩溶液的粘度在15cP以下。
在一个具体实施方式中，在所述浓缩溶液中所述抗人白介素-33单克隆抗体的浓度在150mg/mL以上时，所述浓缩溶液的粘度在10cP以下。
在一个具体实施方式中，所述含有抗人白介素-33单克隆抗体的溶液通过对表达抗人白介素-33单克隆抗体的细胞发酵液进行亲和层析、低pH灭活、阴离子层析、阳离子层析和纳滤得到。
在一个具体实施方式中，所述亲和层析采用Protein A进行亲和层析。具体地，在超滤浓缩步骤和超滤置换步骤中，使用的超滤膜包的材质包括但不限于改良聚醚砜(PES)、聚偏氟乙烯(PVDF)、醋酸纤维素(CA)等，其孔径通常采用30kDa或50kDa，优选的，超滤膜包为默克密理博的Pellicon2/Pellicon3(A型筛网，30kDa)，以及赛多利斯和颇尔公司的超滤膜包。
本申请的制备方法，能够在超滤换液工艺中降低高浓度抗体药液的粘度 并提高其稳定性，方法简单易行，能够进行放大生产，可以保证样品的高纯度并获得较高的回收率。同时，本申请中经过对不同亚型的高浓度抗体进行验证，证明本发明中的添加氨基酸添加剂母液降低粘度的方法具有较强的适用性及良好的稳定性。
实施例
以下，通过实施例对本申请进行更具体的说明。应当理解的是，本申请不限于这些实施例。
实施例1 抗人白介素-33单克隆抗体QX007N的制备
从上海近岸科技有限公司采购人白介素-33，用于免疫新西兰兔，运用B细胞克隆技术获得抗原结合特异性抗体克隆，进而筛选出结合人白介素-33并具有人白介素-33抑制活性的单克隆抗体。首先，用Binding ELISA检测细胞上清，挑选出与人白介素-33结合的克隆；再用HEK Blue TM IL-33报告基因细胞法进行检测，挑选出具有人白介素-33抑制活性的克隆。以上免疫和筛选过程委托给商业化公司完成。
先后挑选出12个克隆进行重组表达，并测序。经测定，78#的细胞中和活性最优，对78#进行人源化改造。利用NCBI IgBlast进行人IgG胚系序列(Germline)同源性比对，选择IGHV3-66*01作为重链CDR移植模板，将78#克隆重链的CDR区(即CDR-H1(SEQ ID No:1)、CDR-H2(SEQ ID No:2)和CDR-H3(SEQ ID No:3))移植入IGHV3-66*01的骨架区；选择IGKV1-12*01作为轻链CDR移植模板，将78#克隆轻链的CDR区(即CDR-L1(SEQ ID No:4)、CDR-L2(SEQ ID No:5)和CDR-L3(SEQ ID No:6))移植入IGKV1-12*01的骨架区；对骨架区特定位点进行回复突变，获得本申请的单克隆抗体QX007N可变区。最终，人源化后的重链可变区具有如SEQ ID NO：7所示的氨基酸序列；人源化后的轻链可变区具有如SEQ ID NO：8所示的氨基酸序列。
上述重链可变区(SEQ ID NO：7)的基因和轻链全长(SEQ ID NO：11)的基因，利用PCR扩增获得。用HindIII和NheI双酶切重链表达质粒pQX2.1；用HindIII和BamHI双酶切瞬转表达质粒pQX1；用Infusion重组酶将PCR扩增基因分别插入对应的表达质粒中，构建重链表达质粒pQX2.1-78VH-Hu25和轻链表达质粒pQX2.2-78VK-Hu3。其中，pQX2.2是 指表达轻链的pQX1质粒。
通过核酸电泳检测质粒的双酶切结果如图1所示。根据图1的结果可以看出，抗体重链可变区和轻链全长PCR扩增结果以及双酶切重链和轻链表达质粒的结果，其中，重链和轻链的质粒大小约5000bp，轻链全长约781bp，重链可变区约480bp。
将序列正确的重链表达质粒pQX2.1-78VH-Hu25和轻链表达质粒pQX2.2-78VK-Hu3共转染ExpiCHO-S细胞。转染前一天，将ExpiCHO-S细胞稀释成3×10 6个细胞/ml进行转染前传代。转染当天，将细胞密度稀释成6×10 6个细胞/ml，125ml摇瓶装25ml细胞，等待转染。转染和表达过程如图2所示。
转染后第5天，收获培养上清，用ProteinA进行一步纯化。用SDS-PAGE电泳检测纯化的抗体，将其命名为QX007N(HZD78-70)，利用蛋白电泳检测该抗体的结果如图3所示。蛋白电泳用变性还原胶检测，图3的结果显示出有两条带，两个条带的大小分别约50kDa和25kDa，与重链(49.3kDa)和轻链(23.4kDa)理论分子量一致。
实施例2 平衡解离常数(K D)的测定
用BiacoreT200检测QX007N(HZD78-70)与人白介素-33的亲和力，所有过程都在25℃进行。采用商品化Protein A芯片，通过捕获法固定适量的抗体，使得Rmax在50RU左右，捕获流速是10μl/min。将抗原进行梯度稀释，仪器流速切换成30μl/min，按照浓度从低到高的顺序依次流过参比通道和固定抗体的通道，流过缓冲溶液作为阴性对照。每一个结合、解离完成后用pH1.5甘氨酸再生芯片。用仪器自带分析软件选择Kinetics选项中1:1结合模型进行拟合，计算抗体的结合速率常数k a，解离速率常数k d以及平衡解离常数K D值。
除此之外，将QX007N(HZD78-70)与AnaptysBio公司研发的人白介素-33的单克隆抗体Etokimab/ANB020的亲和力进行比较，针对已知抗体的检测方法与对QX007N进行检测的方法相同，结果如表1所示。其中Etokimab/ANB020根据专利WO2015106080A2提供的APE4909序列，构建表达质粒，瞬转ExpiCHO-S细胞自制获得。
表1
Figure PCTCN2022075787-appb-000008
实施例3 中和人白介素-33诱导的HEK Blue TM IL-33细胞NF-κB/AP-1信号转导的活性检测
HEK Blue TM IL-33细胞是通过用人IL1RL1基因稳定转染人胚胎肾细胞HEK 293而产生的，且TNF-α和IL-1β的应答被阻断，因此HEK-Blue  TMIL-33细胞对IL-33有特异性反应。白介素-33与细胞表面IL-1RL1/IL-1RAcP结合触发信号级联反应，导致NF-κB/AP-1信号转导并产生分泌型碱性磷酸酶(secreted alkaline phos-phatase，SEAP)，由此检测白介素-33的生物活性或进行抗体筛选。
利用HEK Blue TM IL-33细胞测定QX007N(HZD78-70)对人白细胞介素-33的中和活性。将HEK Blue TM IL-33细胞以每孔4×10 4个细胞铺种到96孔内，在37℃和5％CO 2条件下培养过夜。将抗体稀释至浓度范围为0到500ng/ml，稀释液与2ng/ml的重组人白介素-33混匀孵育1h，孵育完成后加入细胞中在37℃和5％CO 2条件下培养24小时，收集细胞培养上清，以1:10比例加入QUANTI-Blue TM检测试剂(InvivoGen，rep-qbs2)中，并在37℃条件下反应1小时，使用Varioskan LUX多功能酶标仪检测OD 630nm值，采用softMaxPro软件使用四参数曲线拟合分析数据(图4)，进而分析抗体的拮抗活性。
结果显示QX007N(HZD78-70)能够抑制重组人白介素-33诱导HEK Blue TM IL-33细胞中NF-κB/AP-1信号转导，其IC 50为6.67ng/ml。
实施例4 中和天然人白介素-33诱导的HEK Blue TM IL-33细胞NF-κB/AP-1信号转导的活性检测
制备天然人白介素-33，并验证QX007N(HZD78-70)对天然人白介素-33的中和活性。培养HFL-1细胞，并用200ng/ml TNF-α诱导培养24小时，收 集细胞并利用反复冻融法裂解细胞，收集细胞裂解液上清，上清中含有人白介素-33，通过HEK Blue TM IL-33细胞验证活性。
将HEK Blue TM IL-33细胞以每孔4×10 4个细胞铺种到96孔内，在37℃和5％CO 2条件下培养过夜，抗体稀释至浓度范围为0到1000ng/ml后，添加稀释液与天然人白介素-33，混匀后加入细胞中在37℃和5％CO 2条件下培养24小时，收集细胞培养上清，以1:10比例加入QUANTI-Blue TM检测试剂中，并在37℃条件下反应1小时，使用Varioskan LUX多功能酶标仪检测OD 630nm值，采用SoftMax Pro软件使用4参数曲线拟合分析数据(图5)，进而分析抗体的中和活性。
图5的结果显示，QX007N(HZD78-70)能够抑制天然人白介素-33诱导HEK Blue TM IL-33细胞中NF-κB/AP-1信号转导，其IC 50为3.91ng/ml。
实施例5 中和人白介素-33诱导的KU812(人外周血嗜碱性白血病细胞)释放IL-5的活性检测
以人白介素-33诱导KU812(人外周血嗜碱性白血病细胞)释放IL-5作为指标，评价QX007N(HZD78-70)中和人白介素-33的活性。于96孔板中接种KU812细胞(2*10 5细胞/孔)，继而添加抗体和重组人白介素-33(终浓度4ng/ml)在37℃和5％CO 2条件下培养24小时，收集细胞培养上清采用Human IL-5 DuoSet ELISA(R&D，DY205)检测上清中IL-5的表达量，使用Varioskan LUX多功能酶标仪检测OD 450nm值，采用SoftMax Pro软件使用4参数曲线拟合分析数据(图6)，进而分析抗体的中和活性。
图6的结果显示，QX007N(HZD78-70)能够中和人白介素-33诱导的KU812(人外周血嗜碱性白血病细胞)释放IL-5的活性，其IC 50为5.87ng/ml。
实施例6 中和人白介素-33诱导人全血释放IFN-γ的活性检测
以人全血中的单核细胞作为测定基础，IFN-γ作为测定指标，进一步表征QX007N(HZD78-70)的中和活性。使用来自健康志愿者的全血铺板(100μL/孔)，继而添加抗体和重组人白介素-33(终浓度4ng/ml)在37℃和5％CO 2条件下培养24小时，使用Varioskan LUX多功能酶标仪检测OD 450nm值，采用SoftMax Pro软件使用4参数曲线拟合分析数据(图7)，进而分析抗体的拮抗活性。
图7的结果显示，QX007N(HZD78-70)能够中和人白介素-33诱导人全血释放IFN-γ的活性，其IC50为16ng/ml。
实施例7 抗人IL-33单克隆抗体高浓度超滤浓缩
使用CHO细胞作为宿主细胞，在2L规模的生物反应器上进行发酵，生产实施例1所得到的抗体QX007N，通过离心机或深层膜包过滤获得澄清发酵液，采用Protein A层析进行目的蛋白的捕获，再经过低pH值病毒灭活、阴离子和阳离子层析去除杂质，即获得待超滤的中间体样品。
超滤浓缩：采用上述中间体进行超滤，超滤设备选用默克密理博的Labscale小型超滤仪(夹持两块50cm 2Pellicon XL膜包，截留量30kDa)。以120～300L/m 2·h的流速，TMP维持在0.6～1.5bar基础上将上述中间体样品浓缩至约30～50mg/mL，得到浓缩样品。
超滤置换：置换缓冲溶液选用20mM的组氨酸-盐酸缓冲溶液，pH值为6.0，使用蠕动泵将组氨酸-盐酸缓冲溶液泵入至所述浓缩样品中混合得到混合溶液，将所述混合溶液继续超滤，同时调节组氨酸-盐酸缓冲溶液的泵入速度与透过流速一致，即保持样品的重量恒定，待组氨酸-盐酸缓冲溶液的体积为浓缩样品重量的7倍时即完成置换得到超滤置换浓缩液。
二次超滤浓缩：取样测定超滤置换浓缩液中蛋白质的浓度，并计算此时超滤置换浓缩液的理论体积，并加入1/9所述超滤置换浓缩液理论体积的氨基酸添加剂母液，混匀，使得缓冲体系如表2所示，然后使用默克密理博的30kDa的超滤离心管浓缩至蛋白质浓度为表2的浓度得到浓缩溶液。
将所得的浓缩溶液，采用紫外分光度法测定该浓缩溶液中QX007N的浓度，测定方法如下：
1.分光光度计波长调至280nm，用空白缓冲液或水作为对照，进行校零。
2.待测用空白缓冲液或水对样品进行稀释，测定样品在280nm的吸光值(吸光值保证在0.5～1.5之间)，并按照之下公式计算样品浓度(QX007N的消光系数为1.513)。
Figure PCTCN2022075787-appb-000009
采用锐欧森的μVISC粘度计测定该浓缩溶液的粘度，其操作步骤如下：
1.取一次性专用注射器，抽取200～400微升待检样品，排尽注射器内气泡，并用无尘纸轻擦残留液体；
2.用将注射器安放至管槽中固定，点击仪器主机界面，设置剪切速率，设置完成后，点击“Run”进行检测，记录粘度值，一般多次测量取平均值；
3.对于多个样品的检测，前一个样品完成检测后，取出注射器，排空管内液体，无尘纸轻柔擦拭管口后及可进行下一个样品的吸取测量。
使用0.2μm滤膜过滤样品后，与表2中不同的缓冲液或添加剂母液混合使得蛋白浓度如表2所示，然后按照上述方法测定粘度值，其结果如表2所示。
表2
Figure PCTCN2022075787-appb-000010
由表2结果可知，随着蛋白浓度的提高，该抗人IL-33抗体的粘度值呈指数级增加，若无添加剂加入，该单抗在浓度为200mg/mL时粘度值会达到31.1cP，如此高的粘度值无法使用常规的超滤膜实现浓缩工艺。通过添加100～200mM的氨基酸或其组合可以将相同蛋白浓度条件下的抗体粘度值降至原来的50％以下，小于15cP。一般认为，粘度小于10cP的液体制剂适合用作皮下注射剂。通过加入氯化钠、氨基酸保护剂(盐酸精氨酸、赖氨酸、脯氨酸或几种氨基酸组合)上述液体制剂，可以明显降低样品药液的粘度值，由于氨基酸保护剂具有额外的抗聚集作用，所有优先选用氨基酸保护剂作为粘度调节剂，综上QX007N单抗注射液的皮下注射给药浓度至少可以达到 150mg/mL。
实施例8包含高浓度QX007N的低粘度液体制剂的制备
含有抗人白介素-33单克隆抗体QX007N的溶液(UF0)的制备、超滤浓缩步骤和超滤置换步骤的方法均与实施例7相同。
二次超滤浓缩：将超滤置换步骤得到的超滤置换浓缩液分成两份分别进行浓缩工艺：
A.从第一份中取样测定超滤置换浓缩液中蛋白质的浓度，并计算此时超滤置换浓缩液的理论体积，并加入1/9理论体积的盐酸精氨酸母液(1.5mol/L)混匀，然后使用默克密理博的30kDa的超滤离心管浓缩至蛋白质浓度为150mg/mL，记为浓缩溶液1；
B.将第二份直接采用超滤浓缩步骤的方法进行浓缩，至蛋白质浓度为150mg/mL，记为浓缩溶液2；
然后将浓缩溶液1、浓缩溶液2分别与表面活性剂聚山梨酯80母液混合均匀，使用0.2μm滤膜过滤即分别获得原液1(U1F2)和原液2(U2F2)。采用SEC-HPLC法进行分析，确定该样品中活性组分QX007N单体大于95％、聚体小于2％、基本不含宿主杂蛋白。
分别检测原液1(U1F2)和原液2(U2F2)的粘度和纯度，其中粘度的测定方法与实施例7的方法相同，纯度的测定方法如下：
1.采用高效液相色谱仪(Agilent 1260或等效仪器)、色谱柱(Waters，
Figure PCTCN2022075787-appb-000011
规格：3.5μm，7.8×300mm；或等效色谱柱)，流动相为PBS缓冲液；
2.拧开四元泵管路，在5mL/min流速条件下100％流动相冲洗管路3分钟，拧紧四元泵开关，接上色谱柱，在1.0mL/min流速条件下100％流动相冲洗色谱柱30min；
3.样品分析方法设置：流速为1.0mL/min，分析时间为15min，进样量为50mg；检测波长为280nm；进样前需先检查系统适应性(使用参考品连续进样，进样次数不少于5次，取后5次结果计算)，然后分析1针空白样品，接着分析2个样品。
4.按照聚合体、主峰及降解产物等顺序依次积分每个样品的色谱峰，所有与空白样品保留时间不一致的色谱峰均应积分，以面积归一法计算各峰比 例。
测定结果如表3所示。
表3 精氨酸超滤浓缩降低抗人IL-33单抗粘度值及保护作用
Figure PCTCN2022075787-appb-000012
由表3结果可知，超滤工艺的第二步浓缩在添加精氨酸后粘度值由原来的31.5cP下降至10.5cP，同时盐酸精氨酸也作为保护剂一定程度上抑制聚体形成。高浓度的抗人IL-33单抗加入盐酸精氨酸后其粘度值明显降低，不会引起过高的反压，有利于超滤工艺的流速和膜压力的控制，提高了工艺的可操作性。
以上所述，仅是本申请的较佳实施例而已，并非是对本申请作其它形式的限制，任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本申请技术方案内容，依据本申请的技术实质对以上实施例所作的任何简单修改、等同变化与改型，仍属于本申请技术方案的保护范围。

Claims (20)

  1. 一种包含抗人白介素-33单克隆抗体的液体制剂，其特征在于，
    所述液体制剂包含抗人白介素-33单克隆抗体和氨基酸添加剂；
    其中，所述抗人白介素-33单克隆抗体的蛋白质浓度为120～300mg/mL，优选为120～200mg/mL，更优选为120～160mg/mL；所述氨基酸添加剂的氨基酸浓度为10～500mM；
    所述抗人白介素-33单克隆抗体包含三个重链互补决定区和三个轻链互补决定区，所述三个重链互补决定区为CDR-H1、CDR-H2和CDR-H3，所述三个轻链互补决定区为CDR-L1、CDR-L2和CDR-L3，其中：
    CDR-H1具有如SEQ ID NO：1所示的氨基酸序列；
    CDR-H2具有如SEQ ID NO：2所示的氨基酸序列；
    CDR-H3具有如SEQ ID NO：3所示的氨基酸序列；
    CDR-L1具有如SEQ ID NO：4所示的氨基酸序列；
    CDR-L2具有如SEQ ID NO：5所示的氨基酸序列；
    CDR-L3具有如SEQ ID NO：6所示的氨基酸序列。
  2. 根据权利要求1所述的液体制剂，其特征在于，所述抗人白介素-33单克隆抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区具有如SEQ ID NO：7所示的氨基酸序列；
    所述轻链可变区具有如SEQ ID NO：8所示的氨基酸序列。
  3. 根据权利要求1或2所述的液体制剂，其特征在于，所述氨基酸添加剂选自盐酸精氨酸、组氨酸、赖氨酸、甲硫氨酸、脯氨酸中的一种或两种或三种以上。
  4. 根据权利要求1～3中任一项所述的液体制剂，其特征在于，所述氨基酸添加剂包括盐酸精氨酸、组氨酸和甲硫氨酸中的一种或两种或三种；
    优选地，所述氨基酸添加剂包括盐酸精氨酸，所述液体制剂中盐酸精氨酸的浓度为100～200mM；
    优选地，所述氨基酸添加剂包括组氨酸，所述液体制剂中组氨酸的浓度为10～50mM；
    优选地，所述氨基酸添加剂包括甲硫氨酸，所述液体制剂中甲硫氨酸的浓度为2～50mM。
  5. 根据权利要求1～4中任一项所述的液体制剂，其特征在于，所述液体制剂还包含表面活性剂，所述表面活性剂的含量为0.01～5mg/mL，优选为0.1～2mg/mL，更优选为0.2～1mg/mL；
    优选地，所述表面活性剂为聚山梨酯80。
  6. 根据权利要求1～5中任一项所述的液体制剂，其特征在于，在所述液体制剂中所述抗人白介素-33单克隆抗体的浓度在180mg/mL以上时，所述液体制剂的粘度在15cP以下；
    可选地，在所述液体制剂中所述抗人白介素-33单克隆抗体的浓度在150mg/mL以上时，所述液体制剂的粘度在10cP以下。
  7. 根据权利要求1～6中任一项所述的液体制剂，其特征在于，所述液体制剂还包含蔗糖，所述蔗糖的浓度为20～140mg/mL，优选为50～90mg/mL。
  8. 根据权利要求1～7中任一项所述的液体制剂，其特征在于，所述液体制剂还包含山梨醇，所述山梨醇的浓度为10～200mg/mL，优选为20～100mg/mL，更优选为50～80mg/mL。
  9. 根据权利要求1～8中任一项所述的液体制剂，其特征在于，所述液体制剂还包含氯化钠，所述氯化钠的浓度为20～300mg/mL，优选为100～200mg/mL。
  10. 根据权利要求1～9中任一项所述的液体制剂，其特征在于，所述液体制剂的pH为5.5～6.5。
  11. 一种抗人白介素-33单克隆抗体浓缩溶液的制备方法，其包括下述步骤：
    超滤浓缩：浓缩含有抗人白介素-33单克隆抗体的溶液，得到浓缩样品；
    超滤置换：利用置换缓冲溶液置换所述浓缩样品，得到超滤置换浓缩液；
    二次超滤浓缩：将氨基酸添加剂母液加入至所述超滤置换浓缩液中，混合均匀得到混合溶液，将所述混合溶液进行超滤浓缩后得到所述浓缩溶液；
    所述抗人白介素-33单克隆抗体包含三个重链互补决定区和三个轻链互补决定区，所述三个重链互补决定区为CDR-H1、CDR-H2和CDR-H3，所述三个轻链互补决定区为CDR-L1、CDR-L2和CDR-L3，其中：
    CDR-H1具有如SEQ ID NO：1所示的氨基酸序列；
    CDR-H2具有如SEQ ID NO：2所示的氨基酸序列；
    CDR-H3具有如SEQ ID NO：3所示的氨基酸序列；
    CDR-L1具有如SEQ ID NO：4所示的氨基酸序列；
    CDR-L2具有如SEQ ID NO：5所示的氨基酸序列；
    CDR-L3具有如SEQ ID NO：6所示的氨基酸序列。
  12. 根据权利要求11所述的制备方法，其特征在于，所述抗人白介素-33单克隆抗体包含重链可变区和轻链可变区，其中，
    所述重链可变区具有如SEQ ID NO：7所示的氨基酸序列；
    所述轻链可变区具有如SEQ ID NO：8所示的氨基酸序列。
  13. 根据权利要求11或12所述的制备方法，其特征在于，在超滤浓缩步骤中，浓缩条件为：流速为120～300L/m 2·h，跨膜压差为0.6～1.5bar；
    优选地，在超滤浓缩步骤中，所述浓缩样品中的抗人白介素-33单克隆抗体的浓度为20～60mg/mL，优选为30～50mg/mL。
  14. 根据权利要求11～13中任一项所述的制备方法，其特征在于，在超滤置换步骤中，所述置换缓冲溶液的用量为所述浓缩样品重量的7倍以上；
    优选地，在超滤置换步骤中，所述置换缓冲溶液的浓度为5～50mM，优选为20mM；
    优选地，在超滤置换步骤中，所述置换缓冲溶液的pH为5.5～6.5，优选为5.7～6.3；
    优选地，在超滤置换步骤中，所述置换缓冲溶液选自组氨酸-盐酸缓冲溶液、柠檬酸缓冲溶液、磷酸盐缓冲溶液和醋酸钠-醋酸缓冲溶液中的一种。
  15. 根据权利要求11～14中任一项所述的制备方法，其特征在于，在二次超滤浓缩步骤中，所述混合溶液中氨基酸添加剂的浓度为50～300mmol/L，优选为50～200mmol/L，更优选为100～150mmol/L。
  16. 根据权利要求11～15中任一项所述的制备方法，其特征在于，在二次超滤浓缩步骤中，所述浓缩溶液中的蛋白质浓度为120～300mg/mL，优选为120～200mg/mL，更优选为120～160mg/mL。
  17. 根据权利要求11～16中任一项所述的制备方法，其特征在于，所述氨基酸添加剂选自盐酸精氨酸、组氨酸、赖氨酸、甲硫氨酸、脯氨酸中的一种或两种或三种以上。
  18. 根据权利要求11～17中任一项所述的制备方法，其特征在于，所述氨基酸添加剂包括盐酸精氨酸、组氨酸和甲硫氨酸中的一种或两种或三种；
    优选地，所述氨基酸添加剂包括盐酸精氨酸，所述混合溶液中盐酸精氨 酸的浓度为100～200mM；
    优选地，所述氨基酸添加剂包括组氨酸，所述混合溶液中组氨酸的浓度为10～50mM；
    优选地，所述氨基酸添加剂包括甲硫氨酸，所述混合溶液中甲硫氨酸的浓度为2～50mM。
  19. 根据权利要求11～18中任一项所述的制备方法，其特征在于，在所述浓缩溶液中所述抗人白介素-33单克隆抗体的浓度在180mg/mL以上时，所述浓缩溶液的粘度在15cP以下；
    可选地，在所述浓缩溶液中所述抗人白介素-33单克隆抗体的浓度在150mg/mL以上时，所述浓缩溶液的粘度在10cP以下。
  20. 根据权利要求11～19中任一项所述的制备方法，其特征在于，所述含有抗人白介素-33单克隆抗体的溶液通过对表达抗人白介素-33单克隆抗体的细胞发酵液进行亲和层析、低pH灭活、阴离子层析、阳离子层析和纳滤得到。
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