WO2021249551A1 - Préparation liquide d'anticorps monoclonaux anti-pd-1 - Google Patents

Préparation liquide d'anticorps monoclonaux anti-pd-1 Download PDF

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WO2021249551A1
WO2021249551A1 PCT/CN2021/099811 CN2021099811W WO2021249551A1 WO 2021249551 A1 WO2021249551 A1 WO 2021249551A1 CN 2021099811 W CN2021099811 W CN 2021099811W WO 2021249551 A1 WO2021249551 A1 WO 2021249551A1
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concentration
liquid preparation
buffer
formulation
liquid
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PCT/CN2021/099811
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Chinese (zh)
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杨泗兴
廖敏
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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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • 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

Definitions

  • the present invention relates to the field of antibody pharmaceutical preparations, in particular to a liquid preparation of anti-PD-1 monoclonal antibody.
  • Programmed death receptor 1 is an important immunosuppressive molecule, and its ligand is programmed death receptor-ligand 1 (PD-L1).
  • the immune system will react to foreign antigens gathered in the lymph nodes or spleen to promote the proliferation of antigen-specific T cells.
  • One way for tumor cells to escape the destruction of T cells as an antigen is to produce PD-L1 on its surface.
  • T cells recognizes PD-L1, it can transmit inhibitory signals, and T cells cannot detect tumor cells. And send an attack signal to tumor cells.
  • the mechanism of PD-1 immunotherapy is to design specific protein antibodies against PD-1, prevent the recognition process of PD-1 and PD-L1, and partially restore the function of T cells, so that T cells can kill tumor cells.
  • anti-PD-1 antibodies only need to bind to PD-1 and do not require ADCC
  • most of the anti-PD-1 monoclonal antibody molecules are designed as IgG4 type antibodies, such as Keytruda and Opdivo.
  • the IgG4 type of antibody has poor stability and is more likely to produce aggregates and insoluble particles. Therefore, a good formulation is required to protect it.
  • the purpose of the present invention is to provide a stable IgG4 type anti-PD-1 monoclonal antibody liquid preparation.
  • the purpose of the present invention is also to provide the use of the liquid preparation for preparing drugs for treating diseases related to PD-1 signaling pathway; and to provide a preparation method of the liquid preparation.
  • the present invention provides the following technical solutions:
  • the first aspect of the present invention provides an anti-PD-1 monoclonal antibody liquid preparation, the preparation comprising: an anti-PD-1 monoclonal antibody with a concentration of 20-40 mg/ml and a buffer with a concentration of 10-30 mM ⁇ Protein protectant with a concentration of 40-100mg/ml, and a surfactant with a concentration of 0.05-1mg/ml;
  • the anti-human PD-1 monoclonal antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2;
  • the buffer is selected from acetate buffer or group Amino acid-histidine hydrochloride buffer;
  • the protein protective agent is selected from trehalose, sucrose or mannitol;
  • the surfactant is selected from polysorbate 20 or polysorbate 80;
  • the pH of the liquid preparation is 5.0 -7.0.
  • the antibody is an IgG4 antibody.
  • the concentration of the anti-PD-1 monoclonal antibody is 25 mg/ml.
  • the buffer is histidine-histidine hydrochloride buffer or acetate buffer, and the concentration of the buffer is 10 mM.
  • the buffer is a histidine-histidine hydrochloride buffer, and the concentration of the buffer is 10 mM.
  • the protein protective agent is trehalose, sucrose or mannitol, and the protein protective agent has a concentration of 40-95 mg/ml.
  • the protein protective agent has a concentration of 80 mg/ml-95 mg/ml.
  • the surfactant is polysorbate 80, and the concentration of the surfactant is 0.1 mg/ml.
  • the pH of the liquid preparation is 5.0-6.0.
  • the liquid preparation does not contain an antioxidant (for example, methionine).
  • an antioxidant for example, methionine
  • the formulation includes:
  • Anti-PD-1 monoclonal antibody with a concentration of 20-40 mg/ml, a buffer with a concentration of 10-30 mM, a protein protectant with a concentration of 40-100 mg/ml, and a surfactant with a concentration of 0.1 mg/ml;
  • the buffer is histidine-histidine hydrochloride buffer
  • the protein protective agent is selected from trehalose, sucrose or mannitol;
  • the surfactant is polysorbate 80;
  • the pH of the liquid formulation is 5.0-6.0.
  • the formulation has one or more characteristics selected from the following group:
  • the liquid preparation is stored at 25 ⁇ 2°C for 6 months, and the SEC purity is ⁇ 97%, preferably ⁇ 98%, more preferably ⁇ 98.5%; and/or IEC purity ⁇ 40% , Preferably ⁇ 42%, more preferably ⁇ 44%.
  • the liquid preparation is stored at 5 ⁇ 3°C for 24 months, and the SEC purity is ⁇ 97%, preferably ⁇ 98%, more preferably ⁇ 98.5%; and/or IEC purity ⁇ 40% , Preferably ⁇ 42%, more preferably ⁇ 44% or ⁇ 46%.
  • the liquid preparation is stored at 25 ⁇ 2°C for 6 months.
  • the SEC purity has a change range of ⁇ 2%, preferably ⁇ 1%, more preferably Ground ⁇ 0.5%; IEC purity variation range ⁇ 6%, preferably ⁇ 5%, more preferably ⁇ 4%.
  • the liquid formulation is for injection administration.
  • the liquid formulation is used for intravenous, intramuscular, intraperitoneal or subcutaneous injection administration.
  • the second aspect of the present invention provides the use of the anti-PD-1 monoclonal antibody liquid preparation for preparing drugs for treating diseases related to PD-1 signaling pathway.
  • the PD-1 signaling pathway related diseases include cancer or autoimmune diseases.
  • the third aspect of the present invention provides a preparation method of the anti-PD-1 monoclonal antibody liquid preparation, which includes the following steps: (a) The concentration of the anti-PD-1 monoclonal antibody is 20-40 mg/ml. A buffer solution of 10-30 mM, a protein protectant with a concentration of 40-100 mg/ml and a surfactant with a concentration of 0.1 mg/ml are mixed; (b) the pH of the mixed mixture is adjusted to 5.0-6.0;
  • the anti-human PD-1 monoclonal antibody comprises a heavy chain as shown in SEQ ID NO: 1 and a light chain as shown in SEQ ID NO: 2;
  • the buffer is selected from acetate buffer or group Amino acid-histidine hydrochloride buffer;
  • the protein protective agent is selected from trehalose, sucrose or mannitol;
  • the surfactant is selected from polysorbate 20 or polysorbate 80;
  • the pH of the liquid preparation is 5.0 -6.0.
  • the concentration of the anti-PD-1 monoclonal antibody is 25 mg/ml
  • the concentration of the buffer is 10 mM
  • the concentration of the protein protectant is 80 mg/ml-95 mg/ml
  • the surfactant The concentration is 0.1 mg/ml
  • the pH of the liquid preparation is 5.0-6.0.
  • the fourth aspect of the present invention provides a kit including:
  • a container for containing the liquid preparation is provided.
  • kit further includes instructions for use.
  • the beneficial effect of the present invention is that the liquid anti-PD-1 monoclonal antibody preparation of the present invention can protect the anti-PD-1 monoclonal antibody and improve the stability of the existing IgG4-type anti-PD-1 monoclonal antibody.
  • the preparation can be stored at 2-8°C for at least 60 months, and at 25°C for at least 6 months. It has excellent long-term stability and is suitable for clinical application as an injection.
  • Figure 1 Long-term stability SEC purity (1A) and IEC purity (1B) detection chart.
  • anti-PD-1 antibodies are combined with a specific concentration of buffer system (such as acetate buffer system or histidine buffer system), protein protective agents (such as trehalose or sucrose) and surfactants (Such as polysorbate 80 or polysorbate 20) composition of liquid formulations under the condition of pH 5.0-6.0, not only maintains excellent stability in appearance, protein concentration, turbidity, purity, etc., but also chemically stable formulations
  • buffer system such as acetate buffer system or histidine buffer system
  • protein protective agents such as trehalose or sucrose
  • surfactants Sud as polysorbate 80 or polysorbate 20
  • liquid formulation refers to a preparation in the form that allows the biological activity of the active ingredient to be effective and does not contain other ingredients that are unacceptably toxic to the subject who will administer the formulation .
  • the subject includes mammals, preferably humans.
  • antibody stability refers to an antibody that substantially retains its physical stability and/or chemical stability and/or biological activity after storage.
  • the shelf life is generally selected based on the intended shelf life of the preparation.
  • Various analytical techniques for measuring antibody stability are known in the art.
  • the stability can be determined for a selected time at a selected temperature.
  • the formulation is stable at room temperature or 30°C to 40°C for at least 1 month and/or stable at about 2-8°C for at least 2 years.
  • the antibody in the formulation retains it The physical stability.
  • the antibody retains its chemical stability.
  • the chemical stability can be assessed by detecting and quantifying the chemically modified form of the antibody.
  • Chemical changes may involve size changes (e.g., shearing), for example, it can be assessed by using size exclusion chromatography, SDS-PAGE, and/or matrix-assisted laser resolution ionization/time-of-flight mass spectrometry (MALDI/TOF MS) .
  • Other types of chemical changes include charge changes (for example, changes due to deamidation), which can be assessed by ion exchange chromatography, for example.
  • the antibody in a formulation has the biological activity for its intended use, the antibody retains its biological activity in the formulation. For example, if the biological activity of the antibody in the preparation is within about 70% to 130% (within the error range of the measurement) of the biological activity exhibited during the preparation of the preparation, it is considered to have maintained its biological activity. Biological activity (e.g., determined by antigen binding assay).
  • the term "monoclonal antibody (monoclonal antibody)” refers to an antibody obtained from a substantially homogeneous population, that is, the single antibodies contained in the population are the same, except for a few naturally occurring mutations that may exist. Monoclonal antibodies are highly specific to a single antigenic site. Moreover, unlike conventional polyclonal antibody preparations (usually a mixture of different antibodies directed against different antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they can be synthesized by culturing hybridomas without being contaminated by other immunoglobulins.
  • humanized means that its CDRs are derived from non-human species (preferably mouse) antibodies, and the remaining parts of the antibody molecule (including framework regions and constant regions) are derived from human antibodies.
  • framework residues can be changed to maintain binding affinity.
  • the antibody liquid preparation of the present invention mainly includes:
  • the content of the antibody present in the preparation of the present invention is determined by considering the required dosage volume and mode of administration.
  • the concentration of the antibody is 20-40 mg/ml, preferably 25-30 mg/ml, most preferably 25 mg/ml.
  • the present invention includes a range of values using a combination of any of the above-mentioned values as the upper limit and/or the lower limit.
  • the buffer system used in the preparation of the present invention may be an acetate buffer system or a buffer system containing histidine.
  • a buffer system containing histidine is preferred, in which histidine can exist alone or in the following forms, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine Sulfate etc.
  • the histidine in the buffer system exists alone or in the form of histidine hydrochloride.
  • the protein protective agent of the present invention is mainly composed of polyols, where "polyols” are substances with multiple hydroxyl groups, and include sugars (reducing sugars and non-reducing sugars), sugar alcohols and sugar acids.
  • “Reducing sugars” are sugars that include hemiacetal groups, which can reduce metal ions or covalently react with lysine and other amino groups in proteins, while “non-reducing sugars” are sugars that do not have the above-mentioned characteristics of reducing sugars. Examples of reducing sugars include fructose, mannose, maltose, lactose, arabinose, and xylose.
  • Non-reducing sugars include sucrose, trehalose, sorbose, melezitose and raffinose.
  • sugar alcohols include mannitol, xylitol, erythritol, threitol, sorbitol and glycerin.
  • sugar acids L-gluconic acid and its metal salts are included. If the formulation is required to be freeze-thaw stable, the polyol is preferably not crystallized at freezing temperatures (e.g., -20°C) so that it destabilizes the antibody in the formulation.
  • the amount of polyol can be changed according to the required isotonicity of the formulation.
  • the formulations of the invention are preferably isotonic.
  • the amount of polyol added can also be changed according to the molecular weight of the polyol.
  • the preferred polyols of the present invention are sugar alcohols.
  • the polyol is trehalose, sucrose or mannitol, the concentration of trehalose or sucrose is preferably 80-95 mg/ml, and the concentration of mannitol is preferably 40 mg/ml.
  • the present invention includes a range of values using a combination of any of the above-mentioned values as the upper limit and/or the lower limit.
  • the surfactant in the formulation of the present invention is preferably a nonionic surfactant, such as polysorbate or poloxamer.
  • the amount of surfactant added makes it possible to reduce the aggregation of antibodies in the formulation and/or reduce the formation of particles in the formulation and/or reduce adsorption.
  • the preferred surfactant of the present invention is polysorbate, such as polysorbate 80 or polysorbate 20, more preferably polysorbate 80. In a preferred embodiment, the concentration of polysorbate 80 is 0.1 mg/ml.
  • the present invention includes a range of values using a combination of any of the above-mentioned values as the upper limit and/or the lower limit.
  • the present invention uses a buffer system to adjust the pH of the formulation to control the pH in the range of 5.0-6.0.
  • the pH of the formulation is between 5.0 to 6.0, 5.1 to 5.9, 5.2 to 5.8, 5.3 to 5.7, Between 5.4 and 5.6, the present invention includes a range of values using any combination of the above-mentioned values as the upper and/or lower limit.
  • the formulation pH is 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0.
  • the buffer system of the present invention may further include one or more other buffer components, and the pH value of the formulation can be controlled within the above range by combining with other buffer components.
  • Other suitable buffer components include citrate, phosphate, acetate (e.g. sodium acetate), succinate (e.g. sodium succinate) and the like.
  • the buffer system is histidine-hydrochloric acid, wherein the histidine concentration is 10-30 mM, preferably 10-25 mM, more preferably 10 mM.
  • the present invention includes a range of values using a combination of any of the above-mentioned values as the upper limit and/or the lower limit.
  • the pH of the formulation is adjusted with mineral acids such as citric acid, acetic acid, or phosphoric acid.
  • the inventor found that when the concentration of histidine in the preparation of the present invention is lower than 10 mM, the buffering capacity of the buffer system will be significantly limited. When the concentration of histidine is higher than 30 mM, the preparation Stability does not improve.
  • the formulation of the present invention may include one or more other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutical1 Sciences 16th edition, Osol, A. Ed. (1980), as long as they are suitable for the formulation. There is no adverse effect on the desired characteristics.
  • Acceptable carriers, excipients or stabilizers are non-toxic to recipients at the doses and concentrations used, and include other co-solvents; antioxidants, including ascorbic acid and methionine; integrating agents, such as EDTA; metals Complexes (e.g. Zn-protein complexes); biodegradable polymers, such as polyester; and/or salt-forming counterions.
  • the preparation of the present invention can be prepared by combining various components at a certain concentration using methods known in the art.
  • 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 ultraviolet/visible light detector, Dionex Ultimate 3000 VWD-3400 (RS) Detector or other suitable HPLC systems equipped with ultraviolet detectors.
  • Chromatography software is used for integration, and the peak area normalization method is used to calculate the peak area percentage of each peak.
  • Acceptability criteria for system suitability 6-pin system suitability samples, the resolution of aggregates and monomers 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.
  • proteins in the following examples refer to anti-PD-1 antibodies.
  • the anti-PD-1 antibody used in the following examples is a recombinant anti-PD-1 humanized monoclonal antibody derived from the monoclonal antibody mAb1-25-humanized disclosed in WO2018137576A1 (application number PCT/CN2018/073575).
  • the antibody includes two identical light chains and two identical heavy chains, and is a recombinant humanized IgG4-type monoclonal antibody expressed in CHO cells using DNA recombination technology. After being humanized, the heavy chain and light chain variable regions of the anti-PD-1 murine antibody are connected to the constant regions of the human IgG4 heavy chain and kappa light chain respectively, which can specifically bind to human PD-1.
  • the amino acid sequence of the heavy chain is shown in SEQ ID: 1
  • the amino acid sequence of the light chain is shown in SEQ ID: 2.
  • the histidine buffer system refers to histidine-histidine hydrochloride buffer
  • the acetate buffer system refers to acetate buffer
  • the citric acid system refers to citrate buffer
  • the succinic acid buffer system refers to succinate. Buffer.
  • reagents and raw materials used in the following examples can be purchased from commercial sources.
  • results are shown in Table 2.
  • the results show that with the increase of the pH value, the SEC purity of the protein in the solution of various prescriptions gradually decreases, and the range of pH 4.5-6.0 is better.
  • the change trend of IEC purity shows that prescription 1-2, prescription 1-3 and prescription 1-4 are better than prescription 1-1 and prescription 1-5, indicating that pH 5.0-6.0 is beneficial to IEC purity.
  • the comprehensive results show that pH 5.0-6.0 is better for protein SEC purity and IEC purity.
  • the histidine-histidine hydrochloride buffer system in Table 5 was prepared, and the protein and auxiliary materials were prepared to obtain the target prescription solution. After sterilization and filtration with 0.22 ⁇ m filter membrane, 1ml/bottle was aliquoted into 2ml vials, stoppered and capped to obtain candidate prescription samples, and placed in a 40 ⁇ 2°C stability test box. In the first week, Samples were taken for 2 weeks, 4 weeks, and 8 weeks, and the inspection indicators were SEC purity, IEC purity, and insoluble particles.
  • Example 4 The influence of the dosage of formula auxiliary materials on the formula
  • each formula solution in Table 7 respectively add protein to the corresponding excipients, and then use the separately prepared excipient solution to adjust the protein concentration to 25mg/ml to obtain the target prescription solution. After sterilization and filtration with a 0.22 ⁇ m filter membrane, 4.3ml/bottle were dispensed into 10ml vials, capped, and samples of each prescription were obtained. The stability of each candidate formulation was investigated at 40 ⁇ 2°C, and the investigation time was 2 Months, the inspection indicators are SEC purity and IEC purity.
  • the anti-PD-1 monoclonal antibody liquid preparation is prepared according to the following preparation formula: 25mg/ml anti-PD-1 monoclonal antibody, 10mM histidine-histidine hydrochloride buffer, 95mg/ml trehalose, 0.1mg/ml polysorbate Ester 80, pH 5.5 ⁇ 0.5.
  • the anti-PD-1 monoclonal antibody liquid preparation is prepared according to the following preparation formula: 25mg/ml anti-PD-1 monoclonal antibody, 10mM histidine-histidine hydrochloride buffer, 95mg/ml trehalose, 0.1mg/ml polysorbate Ester 80, pH 5.5 ⁇ 0.5. Prepare 3 batches of medicines and store them for long-term stability at 5 ⁇ 3°C. Samples will be taken to detect SEC purity, IEC purity and insoluble particles. The results are shown in Table 10.
  • Figure 1 A further analysis of the results is shown in Figure 1. It can be seen from Figure 1 that the long-term stability of the anti-PD-1 monoclonal antibody preparation of the present invention is estimated to have a validity period of 154 months. The IEC purity does not decrease during the long-term stability. The data of insoluble particles has not changed significantly, so it can support a 60-month validity period (the validity period is the period under long-term stable conditions).
  • the stability of the antibody formulation is critical. Some existing studies have shown that for different antibody products, different formulations will lead to differences in the stability of the formulations.
  • the optimized formulation has excellent stability:
  • the antibody preparation of the present invention does not contain an antioxidant (such as methionine), but still has excellent long-term stability.
  • an antioxidant such as methionine

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  • Biochemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Endocrinology (AREA)
  • Dermatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicinal Preparation (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne une préparation liquide d'anticorps monoclonaux anti-PD-1, la préparation comprenant des anticorps monoclonaux anti-PD-1, un tampon, un agent de protection protéique et un tensioactif. La préparation liquide peut protéger les anticorps monoclonaux anti-PD-1 et améliorer la stabilité des anticorps monoclonaux anti-PD-1 de type IgG4. La préparation liquide peut être stockée à 2-8 °C pendant au moins 60 mois, peut être stockée à 25 °C pendant au moins 6 mois, et est appropriée pour une application clinique sous forme d'injection.
PCT/CN2021/099811 2020-06-11 2021-06-11 Préparation liquide d'anticorps monoclonaux anti-pd-1 WO2021249551A1 (fr)

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