WO2022017401A1 - Formulation lyophilisée d'anticorps bispécifiques anti-her2/pd1 et son procédé de préparation - Google Patents

Formulation lyophilisée d'anticorps bispécifiques anti-her2/pd1 et son procédé de préparation Download PDF

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WO2022017401A1
WO2022017401A1 PCT/CN2021/107474 CN2021107474W WO2022017401A1 WO 2022017401 A1 WO2022017401 A1 WO 2022017401A1 CN 2021107474 W CN2021107474 W CN 2021107474W WO 2022017401 A1 WO2022017401 A1 WO 2022017401A1
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concentration
preparation
histidine
freeze
protein
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PCT/CN2021/107474
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Chinese (zh)
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杨泗兴
杨芳
赵安杰
翁志兵
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
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/16—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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • 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
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific

Definitions

  • the invention belongs to the field of biopharmaceuticals, and in particular relates to a freeze-dried preparation of an anti-HER2/PD1 bispecific antibody and a preparation method thereof.
  • HER2 human epidermal growth factor receptor2
  • HER2 has receptor tyrosine protein kinase activity and is a member of the human epidermal growth factor receptor family. It is only expressed at a low level in a few normal tissues of adults. However, studies have shown that HER2 is overexpressed in a variety of tumors, such as about 30% of breast cancer patients and 16% of gastric cancer patients. Tumor growth and enhanced tumor invasive and metastatic capabilities are important indicators of poor prognosis in such patients.
  • Human programmed cell death receptor-1 (PD-1) is a type I membrane protein composed of 288 amino acids, the extracellular segment is the Ig variable (V-type) domain responsible for binding ligands, and the intracellular segment is The cytoplasmic tail responsible for binding signal transduction molecules.
  • the PD-1 cytoplasmic tail contains two tyrosine-based signal transduction motifs, ITIM (immunoreceptor tyrosine inhibition motif) and ITSM (immunoreceptor tyrosine switching motif), respectively.
  • ITIM immunoimmunoreceptor tyrosine inhibition motif
  • ITSM immunommunoreceptor tyrosine switching motif
  • Body 2 programmed cell death-Ligand 2 binding can inhibit the activity of T lymphocytes and related in vivo cellular immune responses.
  • Numerous studies have shown that the interaction of PD-1 and PD-L1 not only maintains the balance of the immune system in the body, but also is the main mechanism that causes PD-L1-positive tumor cells to evade immune surveillance. By blocking the PD-1/PD-L1 signaling pathway, it can activate the immune system and restore the immune killing function of T cells.
  • Bispecific antibodies refer to antibody molecules that can simultaneously bind to two (or more) different epitopes. Compared with traditional monoclonal antibodies, bispecific antibodies have a unique mechanism of action: 1) Bispecific antibodies can bind 2 or more different antigen molecules or different epitopes of the same molecule at the same time, and combination drugs often do not. have this effect. 2) Mediate cell-to-cell interactions, bispecific antibodies can bind to two antigens on effector cells and target cells, respectively, to build a bridge between effector cells and target cells, and promote cell-to-cell interactions, such as mediating The killing of tumor cells by immune cells. Therefore, bispecific antibodies have unique advantages that traditional monoclonal antibodies do not have.
  • PCT patent application WO2020/103629A1 discloses a self-developed anti-HER2/PD1 bispecific antibody, which is an anti-HER2/PD1 bispecific antibody expressed in CHO cells using DNA recombinant technology. It consists of a single-chain antibody with two PD1 ends connected (ScFv-IgG C-terminal fusion). ScFv-IgG C-terminal fusion bispecific antibodies are very easy to cause molecular aggregation and precipitation because the C-terminal ScFv expands and contracts in solution state and presents a respiratory chain state; at the same time, it is easy to expose internal amino acids during expansion and contraction, resulting in oxidation and deamination Occurs, forming charge isomers with poor stability. Therefore, it is necessary to improve the stability through formulation optimization.
  • the purpose of the present invention is to provide a lyophilized preparation of anti-HER2/PD1 bispecific antibody and a preparation method thereof.
  • the lyophilized preparation consists of an anti-HER2/PD1 bispecific antibody, a buffer, a protein protectant, and a surfactant.
  • the preparation method includes the processes of medicinal liquid preparation, medicinal liquid subpackaging, freeze-drying and the like.
  • a first aspect of the present invention provides a lyophilized preparation of an anti-HER2/PD1 bispecific antibody, the lyophilized preparation comprising an anti-HER2/PD1 bispecific antibody, a buffer, a protein protectant, and a surfactant.
  • the concentration of the anti-HER2/PD1 bispecific antibody is 5-20 mg/ml
  • the anti-HER2/PD1 bispecific antibody comprises the heavy chain shown in SEQ ID NO: 1 and the heavy chain shown in SEQ ID NO: : the light chain shown in 2.
  • the concentration of the anti-HER2/PD1 bispecific antibody is 10-15 mg/ml, and more preferably, the concentration of the anti-HER2/PD1 bispecific antibody is 10 mg/ml.
  • the buffer is histidine-histidine hydrochloride buffer
  • concentration of the histidine-histidine hydrochloride buffer is 10-20 mM.
  • concentration of the histidine-histidine hydrochloride buffer is 10 mM.
  • the protein protective agent is trehalose or sucrose, arginine hydrochloride, the concentration of the trehalose or sucrose is 10-50 mg/ml, and the concentration of the arginine hydrochloride is 25-50 mM.
  • the protein protecting agent is trehalose and arginine hydrochloride, the concentration of trehalose is 50 mg/ml, and the concentration of arginine hydrochloride is 50 mM.
  • the surfactant is polysorbate 80 or polysorbate 20, the concentration of polysorbate 80 or polysorbate 20 is 0.1-0.5mg/ml, preferably, the polysorbate 80 or polysorbate 20 at a concentration of 0.2-0.5 mg/ml.
  • the surfactant is polysorbate 80, and the concentration of the polysorbate 80 is 0.4 ⁇ 0.05mg/ml.
  • the pH of the freeze-dried preparation is 6.8 ⁇ 0.5.
  • freeze-dried preparation includes:
  • Anti-HER2/PD1 bispecific antibody at a concentration of 10-15mg/ml
  • Histidine-Histidine HCl buffer at a concentration of 10-20 mM
  • Polysorbate 80 at a concentration of 0.2-0.5 mg/ml
  • the pH of the lyophilized formulation was 6.8 ⁇ 0.5.
  • freeze-dried preparation does not contain antioxidants.
  • the second aspect of the present invention provides the preparation method of the freeze-dried preparation, the preparation method includes the processes of liquid medicine preparation, liquid medicine sub-packaging, freeze-drying and the like.
  • the medicinal solution is prepared as a stock solution or a medicinal solution of a preparation.
  • the preparation of the medicinal liquid includes the ultrafiltration, concentration, and liquid exchange process and the dilution process of the protein solution.
  • the ultrafiltration concentration liquid exchange process comprises the following steps: first, adding a protein protective agent to the protein solution to be ultrafiltration concentrated and ultrafiltration concentrated, the protein protective agent is trehalose or sucrose, arginine hydrochloride, so The concentration of described trehalose or sucrose is 10-50mg/ml, preferably 10mg/ml, and the concentration of described arginine hydrochloride is 25-50mM, preferably 50mM; Secondly, the protein solution after the ultrafiltration concentration is carried out.
  • the liquid change solution comprises a histidine-histidine hydrochloride buffer and a protein protective agent, and the concentration of the histidine-histidine hydrochloride buffer is 10-20 mM, preferably 10 mM, and the protein
  • the protective agent is trehalose or sucrose, arginine hydrochloride, the concentration of the trehalose or sucrose is 10-50mg/ml, preferably 20mg/ml, the concentration of the arginine hydrochloride is 25-50mM, preferably 50mM.
  • the dilution process is to add an appropriate amount of buffer, protein protective agent and surfactant to the protein solution after the liquid exchange, so that the protein solution reaches the components and dosage of the lyophilized preparation provided in the first aspect of the present invention.
  • the dosage of the medicinal liquid is 5-160mg/bottle, preferably, the dosage of the medicinal liquid is 10-50mg/bottle, more preferably, the dosage of the medicinal liquid is 50mg/ bottle.
  • the freeze-drying includes the steps of pre-freezing, main drying, secondary drying and final drying, the pre-freezing temperature is 5°C, the pre-freezing temperature is -40°C, and the pre-freezing time is not less than 3h ;
  • the main drying temperature is -20--12°C, the time is not less than 21h, and the vacuum degree is 200-250 ⁇ bar;
  • the secondary drying temperature is -5-15°C, and the time is not less than 12.5h,
  • the vacuum degree is 100-250 ⁇ bar;
  • the final drying temperature is 30°C, the time is not less than 3.3h, and the vacuum degree is 20-60 ⁇ bar.
  • the third aspect of the present invention provides the use of the freeze-dried preparation described in the first aspect of the present invention for preparing a medicament for treating cancer or tumor.
  • a fourth aspect of the present invention provides a kit comprising:
  • a container for holding the lyophilized formulation is provided.
  • kit further includes instructions for use.
  • the present invention greatly improves the unstable defect of the bispecific antibody of the ScFv-IgG C-terminal fusion class by optimizing the formulation formula and the freeze-drying process.
  • Methods for evaluating stability such as storage at 5 ⁇ 3°C for 3 months, 6 months, 9 months, 12 months, 18 months, 24 months, 36 months, the tested SEC purity and IEC purity should be Comply with quality standards.
  • FIG 1 shows the results of SEC purity and IEC purity of protein solutions under different pH conditions.
  • Figure 2 is the result of the effect of protein protectant on the ultrafiltration concentration exchange medium.
  • Figure 3 shows the analysis of the DOE results.
  • Figure 4 shows the optimization results of the lyophilized formula.
  • Figure 5 is the lyophilization curve of 20191210.
  • Figure 6 is the lyophilization curve of 20191213.
  • Figure 7 is the lyophilization curve of 20200114.
  • Figure 8 is the lyophilization curve of 20200214.
  • Figure 9 is the lyophilization curve of 20200314.
  • the inventors have found through extensive and in-depth research that a freeze-dried preparation of an anti-HER2/PD1 bispecific antibody fused to the C-terminal of ScFv-IgG and a preparation method thereof are provided.
  • the lyophilized preparation comprises a specific concentration of the bispecific antibody, a buffer system (such as histidine-histidine hydrochloride buffer), a protein protectant (such as trehalose or sucrose, arginine hydrochloride) and surfactants (such as polysorbate 80 or polysorbate 20).
  • the freeze-dried preparation of the present invention significantly improves the stability of the anti-HER2/PD1 bispecific antibody fused to the C-terminal of ScFv-IgG, and the storage time of the preparation is effectively prolonged.
  • the freeze-dried preparation product of the invention has small batch difference and stable quality, and is suitable for industrial batch production. The present invention has been completed on this basis.
  • the lyophilized formulation of the antibody of the present invention can be used interchangeably, and both refer to the lyophilized preparation of the anti-HER2/PD1 bispecific antibody described in the first aspect of the present invention.
  • the terms "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “consisting essentially of,” or “consisting of.”
  • the term “about” means that the value may vary by no more than 1% from the recited value.
  • the expression “about 100” includes all values between 99 and 101 and (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the antibody freeze-dried preparation of the present invention mainly includes:
  • the concentration of the anti-HER2/PD1 bispecific antibody is 5-20 mg/ml
  • the buffer is histidine-histidine hydrochloride buffer, and the concentration of the histidine-histidine hydrochloride buffer is 10-20 mM;
  • Described protein protective agent is trehalose or sucrose, arginine hydrochloride, the concentration of described trehalose or sucrose is 10-50mg/ml, and the concentration of described arginine hydrochloride is 25-50mM;
  • Described surfactant is polysorbate 80 or polysorbate 20, the concentration of described polysorbate 80 or polysorbate 20 is 0.1-0.5mg/ml, the pH of described freeze-dried preparation is 6.8 ⁇ 0.5.
  • the concentration of each component in the antibody lyophilized preparation of the present invention refers to the same volume as before lyophilization
  • concentration of each component in the antibody lyophilized preparation of the present invention refers to the same volume as before lyophilization
  • the reconstituted concentration in a solution eg, water, or buffer.
  • the pH of the antibody lyophilized formulation of the present invention refers to the pH after reconstitution in the same volume of solution (eg, water, or buffer) as before lyophilization.
  • Lyophilized formulations are generally more chemically stable than solution formulations, thus increasing half-life.
  • Antibody lyophilized formulations can be reconstituted at different concentrations depending on clinical factors such as route of administration or dosage.
  • the amount of antibody present in the formulations of the invention is determined by taking into account the desired dosage volume and mode of administration.
  • the concentration of the antibody is 5-20 mg/ml, preferably 10-15 mg/ml, most preferably 10 mg/ml.
  • the present invention includes ranges of values that use a combination of any of the above-mentioned values as the upper and/or lower limit.
  • the buffer system used in the formulation of the present invention is a buffer system comprising histidine.
  • histidine can exist alone or in the following forms, such as histidine hydrochloride, histidine acetate, histidine phosphate, histidine sulfate and the like.
  • the histidine in the buffer system is present alone or in the form of histidine hydrochloride.
  • the protein protectant of the present invention is mainly composed of polyols, wherein "polyols” are substances having 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 are capable of reducing metal ions or covalently react with lysine and other amino groups in proteins, while “non-reducing sugars” are sugars that do not possess the above-mentioned characteristics of reducing sugars. Examples of reducing sugars include fructose, mannose, maltose, lactose, arabinose, xylose.
  • Non-reducing sugars include sucrose, trehalose, sorbose, melezitose, and raffinose.
  • sugar alcohols include mannitol, xylitol, erythritol, threitol, sorbitol and glycerol.
  • sugar acids L-gluconic acid and its metal salts are included. If the formulation is desired to be freeze-thaw stable, the polyol preferably does not crystallize at freezing temperatures (eg, -20°C) so that it destabilizes the antibody in the formulation.
  • the amount of polyol used can vary depending on the desired isotonicity of the formulation.
  • the formulations of the present invention are preferably isotonic.
  • the amount of polyol added can also vary depending on the molecular weight of the polyol.
  • Preferred polyols of the present invention are sugar alcohols.
  • the polyol is 10-50 mg/ml trehalose or sucrose; preferably 10-50 mg/ml trehalose; more preferably 50 mg/ml trehalose.
  • the protein protectant in the present invention also includes arginine hydrochloride.
  • the concentration of arginine hydrochloride is 25-50 mM, more preferably 50 mM.
  • the present invention includes ranges of values that use a combination of any of the above-mentioned values as the upper and/or lower limit.
  • the surfactants in the formulations of the present invention are preferably nonionic surfactants such as polysorbates or poloxamers.
  • the amount of surfactant added is such that it reduces aggregation of antibodies in the formulation and/or reduces particle formation in the formulation and/or reduces adsorption.
  • Preferred surfactants of the present invention are polysorbates, such as polysorbate 80 or polysorbate 20, more preferably polysorbate 80.
  • the concentration of polysorbate 80 is 0.1-0.5 mg/ml, preferably, 0.2-0.5 mg/ml; more preferably, 0.35-0.45 mg/ml.
  • the present invention includes ranges of values that use a combination of any of the above-mentioned values as the upper and/or lower limit.
  • the present invention adjusts the pH of the formulation through a buffer system to control the pH in the range of 6.0-7.5, Between 6.6-7.0, 6.7 to 6.9, the present invention includes ranges of values using a combination of any of the above as the upper and/or lower limit.
  • the formulation pH is 6.0-7.0; more preferably, 6.3-7.3.
  • the buffer system of the present invention may further include one or more other buffer components, and the pH value of the preparation can be controlled within the above-mentioned range by combining with other buffer components.
  • Suitable other buffer components include citrate, phosphate, acetate (eg, sodium acetate), succinate (eg, sodium succinate), and the like.
  • the buffer system is histidine-hydrochloric acid, wherein the concentration of histidine is 10-20 mM, more preferably 20 mM.
  • the present invention includes ranges of values that use a combination of any of the above-mentioned values as the upper and/or lower limit.
  • the pH of the formulation is adjusted with a mineral acid such as citric, acetic or phosphoric acid.
  • the inventor found that when the concentration of histidine in the preparation of the present invention is lower than 10mM, the buffering capacity of the buffer system will be significantly limited. When the concentration of histidine is higher than 20mM, the preparation Stability does not improve.
  • One or more other pharmaceutically acceptable carriers, excipients or stabilizers may be included in the formulations of the present invention, provided they are essential to the formulation The desired characteristics of , are not adversely affected.
  • Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include other co-solvents; antioxidants, including ascorbic acid and methionine; integrators, such as EDTA; metals complexes (eg, Zn-protein complexes); biodegradable polymers, such as polyesters; and/or salt-forming counterions.
  • the formulation of the present invention can be prepared by combining various components in a certain concentration by methods known in the art.
  • the protein samples used in the following examples were derived from the anti-HER2/PD1 bispecific antibody a disclosed in PCT patent application WO2020/103629A1, the heavy and light chain amino acid sequences of which are shown below.
  • System suitability sample take the reference substance and dilute the concentration to 5.0mg/ml with mobile phase, centrifuge at 13000rpm for 10min, transfer the supernatant to the injection bottle, and put it into the HPLC sample tray.
  • Test sample Dilute the test sample with mobile phase to a concentration of 5.0 mg/ml, centrifuge at 13000 rpm for 10 min, take the supernatant and transfer it to a sample bottle, and put it into the HPLC sample tray.
  • Chromatographic conditions column temperature 25 ⁇ 2°C; sample temperature 10 ⁇ 2°C; detection wavelength UV 280nm; injection volume 20 ⁇ L; flow rate 0.5ml/min.
  • the chromatographic software was used for integration, and the peak area percentage of each peak was calculated by the peak area normalization method.
  • Acceptable criteria for system suitability 6-pin system suitability samples, the resolution of both polymer and monomer are ⁇ 1.5, the retention time RSD of the main peak is less than or equal to 1.0%, the peak area RSD of the main peak is less than or equal to 2.0%, and the asymmetry of the main peak is all ⁇ 2.0, the number of theoretical plates is ⁇ 4000.
  • Report results for the test product The SEC purity of the sample is reported as the peak area percentage of the main monomer peak, and the aggregate content is the peak area percentage of the polymer peak.
  • Mobile phase A 20 mM phosphate buffer, pH 6.5 ⁇ 0.05. It was filtered through a 0.22 ⁇ m filter membrane and degassed by ultrasonic before use.
  • Mobile phase B 20 mM phosphate buffer + 200 mM sodium chloride, pH 6.5 ⁇ 0.05. It was filtered through a 0.22 ⁇ m filter membrane and degassed by ultrasonic before use.
  • 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 system with UV detector.
  • System suitability sample take the reference substance and dilute the concentration with mobile phase to 1.0mg/ml, centrifuge at 13000rpm for 10min, transfer the supernatant to the injection bottle, and put it into the HPLC sample tray.
  • Test sample dilute the test sample with mobile phase to a concentration of 1.0 mg/ml, centrifuge at 13000 rpm for 10 min, transfer the supernatant to a 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 214 nm; injection volume 20 ⁇ L; flow rate 1.0 ml/min.
  • the mobile phase gradient is as follows:
  • Purity analysis Calculate the peak area percentage of the main peak, acid peak area and base peak area on the sample spectrum by the peak area normalization method. The IEC purity results report the percent area of the main peak.
  • the IEC purity results show that there is no obvious difference between the samples in each group; the SEC purity results show that the slope of the pH5.0 and pH5.5 The descending trend is significantly greater than the slope under the conditions of pH6.0-7.0 , so the preferred pH range is 6.0-7.0, which indicates that the pH of neutral alkaline is more conducive to protein stability.
  • Embodiment 2 The investigation of the influence of arginine hydrochloride and trehalose on ultrafiltration concentration and exchange of liquid
  • the trehalose concentration could be 10-50mg /ml.
  • the preferred protein concentration of the preparation is 5-20mg/ml, which can be divided into 2ml vials to 20ml vials for freeze-dried preparations, with a volume of 1-8ml, which can cover the dose 5-160mg.
  • DOE model analysis was performed on the SEC purity slope at 5 ⁇ 3°C, the IEC purity slope at 40 ⁇ 2°C, 2 ⁇ m particles, and 10 ⁇ m particles, and the results are shown in FIG. 3 .
  • DOE adopts the central composite design-effect surface method, and the model is fitted to a continuous surface, so the set experimental parameter range can be properly extrapolated. It can be seen from the results in Fig. 3 that the preferred pH range is 6.3-7.3, and the preferred concentration range of polysorbate 80 is 0.1-0.5 mg/ml.
  • Embodiment 6 medicinal liquid preparation
  • the protein solution (initial concentration of 2.06 mg/ml) to be concentrated and exchanged by ultrafiltration was added with an appropriate amount of trehalose stock solution (containing 10 mM histidine-histidine hydrochloride buffer, 50mM arginine hydrochloride, 363mg/ml trehalose, pH 6.8 ⁇ 0.1) to make the trehalose concentration in the solution 10mg/ml, use an ultrafiltration instrument to carry out ultrafiltration and concentrate to a protein concentration of about 15mg/ml, and carry out ultrafiltration to change the liquid , the exchange solution is 10mM histidine-histidine hydrochloride buffer, 20mg/ml trehalose, 50mM arginine hydrochloride, pH 6.8 ⁇ 0.1.
  • trehalose stock solution containing 10mM histidine-histidine hydrochloride buffer, 50mM arginine hydrochloride, 363mg/ml trehalose, pH 6.8 ⁇ 0.1
  • polysorbate 80 stock solution containing 10mM histidine-histidine hydrochloride buffer, 50mM arginine hydrochloride, 50mg/ml trehalose, 20mg/ml polysorbate 80, pH 6.8 ⁇ 0.1
  • preparation excipient solution containing 10mM histidine-histidine hydrochloride buffer, 50mM arginine hydrochloride, 50mg/ml trehalose, 0.4mg/ml polysorbate 80, pH6.8 ⁇ 0.1) to dilute to protein concentration 10.0-10.5mg/ml.
  • Embodiment 7 freeze-drying process investigation
  • freeze-drying parameters are: pre-freezing -40°C, not less than 3h; main drying temperature -20--12°C, vacuum 200-250 ⁇ bar, time not less than 21h; secondary drying temperature -5°C-15 °C, vacuum 100-250 ⁇ bar, time not less than 12.5h; final drying temperature 30°C, vacuum 20-60 ⁇ bar, time not less than 3.3h.
  • the formula is protein 10mg/ml, L-histidine 1.34mg/ml, histidine hydrochloride 0.28mg/ml, arginine hydrochloride 10.53mg/ml (50mM), trehalose 50mg/ml, poly Sorbitan 80 0.4 ⁇ 0.05mg/ml, pH 6.8 ⁇ 0.5, using 20ml vial, filling volume 5ml, dose 50mg/bottle.
  • the lyophilization process is as shown in Table 12. After lyophilization, the samples were placed at 5 ⁇ 3°C, and samples were taken every week to detect the purity of SEC and IEC. The results are shown in Table 13.

Abstract

L'invention concerne une formulation lyophilisée d'anticorps bispécifiques anti-HER2/PD1 et son procédé de préparation. En optimisant une formule d'une formulation et d'un procédé de lyophilisation, le défaut d'instabilité d'un anticorps bispécifique à fusion avec le carbone scFv-IgG est considérablement amélioré. Selon la formule de la formulation et du procédé de préparation, la pureté SEC et la pureté IEC mesurées au moyen d'un procédé d'évaluation de stabilité connu dans l'état de la technique, par exemple, le stockage pendant 3 mois, 6 mois, 9 mois, 12 mois, 18 mois, 24 mois, ou 36 mois à 5 ± 3 °C, devraient répondre aux normes de qualité.
PCT/CN2021/107474 2020-07-22 2021-07-20 Formulation lyophilisée d'anticorps bispécifiques anti-her2/pd1 et son procédé de préparation WO2022017401A1 (fr)

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EP2196476A1 (fr) * 2008-12-10 2010-06-16 Novartis Ag Formulation d'anticorps
WO2011080209A2 (fr) * 2009-12-29 2011-07-07 F. Hoffmann-La Roche Ag Nouvelle formation d'anticorps
CN109021110A (zh) * 2018-08-07 2018-12-18 苏州塞恩塔生物技术有限公司 抗Her2/PD-1双特异性抗体及其制备方法
CN109966487A (zh) * 2017-12-28 2019-07-05 上海复宏汉霖生物制药有限公司 一种包含抗pd-l1单克隆抗体的药物配制剂
WO2019171253A1 (fr) * 2018-03-07 2019-09-12 Pfizer Inc. Compositions d'anticorps anti-pd -1
CN111196856A (zh) * 2018-11-19 2020-05-26 三生国健药业（上海）股份有限公司 抗her2/pd1双特异性抗体
CN111375057A (zh) * 2018-12-28 2020-07-07 上海复宏汉霖生物技术股份有限公司 一种包含抗Her2单克隆抗体的药物配制剂

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2196476A1 (fr) * 2008-12-10 2010-06-16 Novartis Ag Formulation d'anticorps
WO2011080209A2 (fr) * 2009-12-29 2011-07-07 F. Hoffmann-La Roche Ag Nouvelle formation d'anticorps
CN109966487A (zh) * 2017-12-28 2019-07-05 上海复宏汉霖生物制药有限公司 一种包含抗pd-l1单克隆抗体的药物配制剂
WO2019171253A1 (fr) * 2018-03-07 2019-09-12 Pfizer Inc. Compositions d'anticorps anti-pd -1
CN109021110A (zh) * 2018-08-07 2018-12-18 苏州塞恩塔生物技术有限公司 抗Her2/PD-1双特异性抗体及其制备方法
CN111196856A (zh) * 2018-11-19 2020-05-26 三生国健药业（上海）股份有限公司 抗her2/pd1双特异性抗体
CN111375057A (zh) * 2018-12-28 2020-07-07 上海复宏汉霖生物技术股份有限公司 一种包含抗Her2单克隆抗体的药物配制剂

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