WO2021136274A1 - Formulation containing anti-trop2 antibody-drug conjugate and preparation method and application thereof - Google Patents

Formulation containing anti-trop2 antibody-drug conjugate and preparation method and application thereof Download PDF

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WO2021136274A1
WO2021136274A1 PCT/CN2020/140885 CN2020140885W WO2021136274A1 WO 2021136274 A1 WO2021136274 A1 WO 2021136274A1 CN 2020140885 W CN2020140885 W CN 2020140885W WO 2021136274 A1 WO2021136274 A1 WO 2021136274A1
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parts
buffer
antibody
preparation
histidine
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PCT/CN2020/140885
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French (fr)
Chinese (zh)
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吴用
陈燕宇
杨依丽
刘翠华
汤伟佳
李胜峰
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百奥泰生物制药股份有限公司
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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
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5355—Non-condensed oxazines and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • 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
    • A61K47/50—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—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 the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • 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
    • 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
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells

Definitions

  • the invention belongs to the field of biological preparations, and relates to a preparation containing an antibody-drug conjugate.
  • Antibody-Drug Conjugates are highly effective and specific drugs for the treatment of cancer and other conditions obtained by coupling antibodies and drugs, in which the antibody part specifically binds to target cells On the antigen, the drug can exert its cytotoxicity or other therapeutic effects on target cells.
  • anti-Trop2 antibody-drug conjugate binds to the Trop2 receptor
  • the anti-Trop2-ADC binds to the Trop2 receptor
  • the anti-Trop2-ADC begins to internalize with the receptor as a mediator, and the subsequent lysosomal degradation process causes it to release cytotoxicity in the cell Metabolites. Cytotoxic metabolites in turn kill tumor cells.
  • the drug coupling itself will reduce the stability of the antibody and change its physical and chemical properties.
  • the purpose of the present invention is to provide a stable anti-Trop2 antibody-drug conjugate preparation during storage, so as to solve the problems existing in the prior art.
  • the present invention provides a liquid formulation, which includes: an anti-Trop2 antibody-drug conjugate; and also includes one or more of a stabilizer, a buffer, and a surfactant.
  • the pH of the liquid formulation is 4-7.
  • the pH of the liquid formulation is 4.5 to 6.0.
  • the pH of the liquid formulation is 4.5 to 5.6.
  • the present invention provides a liquid formulation comprising: an anti-Trop2 antibody-drug conjugate, wherein the concentration of the conjugate is 10 mg/ml-50 mg/ml; it also includes stabilizers, buffers, and One or more of surfactants, the concentration of the stabilizer is 20mg/ml ⁇ 80mg/ml, the concentration of the buffering agent is 5mM ⁇ 60mM; the concentration of the surfactant is 0.1mg/ml ⁇ 0.5 mg/ml; in some embodiments, the liquid formulation has a pH value of 4.5 to 6.0.
  • the present invention provides a solid preparation, which is lyophilized from the liquid preparation.
  • the present invention provides a solid preparation, comprising: an anti-Trop2 antibody-drug conjugate, wherein the mass parts of the conjugate is about 10 parts to about 50 parts; and it also includes a stable anti-Trop2 antibody-drug conjugate.
  • an anti-Trop2 antibody-drug conjugate wherein the mass parts of the conjugate is about 10 parts to about 50 parts; and it also includes a stable anti-Trop2 antibody-drug conjugate.
  • One or more of agents, buffers and surfactants is about 20 parts to about 80 parts; the mass parts of the buffering agent is 1 part to 10 parts;
  • the mass parts of the surfactant is about 0.1 part to about 0.5 part.
  • the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ia:
  • X is hydrogen or halogen
  • R 2 is H or C1-C6 alkyl
  • R 4 is -OH or -SH
  • R 5 is C1-C6 alkyl or benzyl
  • R 6 is C1-C6 alkyl, phenyl or benzyl
  • R 7 is hydrogen, C1-C6 alkyl or amino acid side chain
  • R 8 is hydrogen or C1-C6 alkyl
  • n 0, 1, 2, 3, 4, 5, 6, 7 or 8;
  • p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the range between any two of these values (including endpoints) or any value therein;
  • Anti-Trop2 is an anti-Trop2 antibody.
  • the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ib:
  • X is hydrogen or halogen
  • R 2 is H or C1-C6 alkyl
  • R 4 is -OH or -SH
  • R 5 is C1-C6 alkyl or benzyl
  • R 6 is C1-C6 alkyl, phenyl or benzyl
  • R 7 is hydrogen, C1-C6 alkyl or amino acid side chain
  • R 8 is hydrogen or C1-C6 alkyl
  • p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the range between any two of these values (including endpoints) or any value therein;
  • Anti-Trop2 is an anti-Trop2 antibody.
  • the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ic:
  • p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the range between any two of these values (including endpoints) or any value therein ;
  • Anti-Trop2 is an anti-Trop2 antibody.
  • p is 2-6; in some embodiments, p is 2-3; in some embodiments, p is about 2.1.
  • the light chain amino acid sequence of the anti-Trop2 antibody is shown in SEQ ID NO: 1 or SEQ ID NO: 3, and the heavy chain amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • the anti-Trop2 antibody is expressed in CHO cells.
  • the anti-Trop2 antibody is antibody A, antibody B, and/or antibody C; in some embodiments, the anti-Trop2 antibody is antibody C.
  • the amino acid sequence of the light chain of the antibody A is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2, and the antibody A is expressed in suspended CHO cells, for example, cultured by CHO-K1 cells Chinese hamster ovary cells adapted to growth in suspension;
  • the light chain amino acid sequence of antibody B is shown in SEQ ID NO: 3, and the heavy chain amino acid sequence is shown in SEQ ID NO: 4, and antibody B is expressed in suspended CHO cells, for example, CHO-K1 cells are cultured to adapt to suspension Growing Chinese Hamster Ovary Cells;
  • antibody C is expressed in CHO cells in which the Fut8 gene ( ⁇ -(1,6)-fucosyltransferase gene) has been knocked out, such as CHO-BAT-KF fut8(-/-) cells , CN109096399A has disclosed the above-mentioned cells.
  • Fut8 gene ⁇ -(1,6)-fucosyltransferase gene
  • the anti-Trop2 antibody-drug conjugate is ADC-1, which is a compound represented by Ic, wherein p is 2.1, and the anti-Anti-Trop2 antibody is antibody C.
  • the mass parts of the anti-Trop2 antibody-drug conjugate is 20 parts to 30 parts; in some embodiments, the mass parts of the anti-Trop2 antibody-drug conjugate is 23 parts to 27 parts; In some embodiments, the mass parts of the anti-Trop2 antibody-drug conjugate is 25 parts.
  • the stabilizer is trehalose and/or sucrose; in some embodiments, the stabilizer is trehalose.
  • the mass parts of the stabilizer is 40 parts to 80 parts; in some embodiments, the mass parts of the stabilizer is 50 parts to 70 parts; in some embodiments, the mass parts of the stabilizer is 50 parts to 70 parts.
  • the mass parts of the stabilizer is 54 or 60 parts.
  • the buffer is a histidine-succinate buffer or a histidine-citrate buffer; in some embodiments, the buffer is a histidine-succinate buffer.
  • the mass parts of the buffer is 1 to 10 parts; in some embodiments, the mass parts of the buffer is 3 to 7 parts; in some embodiments, the The mass parts of the buffer is 3 parts to 4 parts.
  • the buffer comprises 1.18 parts by mass of succinic acid and 2.1 parts of histidine hydrochloride, wherein part or all of the succinic acid may be in the form of a corresponding amount of salt, and part or all of histidine hydrochloride The acid may be present in the corresponding amount of histidine.
  • the buffer comprises 1.18 parts by mass of succinic acid and 2.1 parts of histidine hydrochloride monohydrate.
  • the surfactant is Tween 20 (ie, polysorbate 20) or Tween 80 (ie, polysorbate 80); in some embodiments, the surfactant is Tween 80.
  • the parts by mass of the surfactant is 0.2 to 0.4 parts; in some embodiments, the parts by mass of the surfactant is 0.25 to 0.35 parts; in some embodiments, the part by mass of the surfactant is The parts by mass is about 0.3 parts.
  • the solid preparation includes: 10-50 parts by mass of the anti-Trop2 antibody-drug conjugate, 1-10 parts by mass of buffer, and 20 parts by mass. ⁇ 80 parts of stabilizer, and 0.1 parts to 0.5 parts of surfactant by mass; in some embodiments, the solid preparation includes: 20 parts to 30 parts of anti-Trop2 antibody-drug couple Conjugate, 3 to 7 parts by mass of buffer, 40 to 80 parts by mass of stabilizer, and 0.2 to 0.4 parts by mass of surfactant; in some embodiments, The solid preparation comprises: 20-30 parts by mass of ADC-1, 3-7 parts by mass of histidine-succinic acid buffer, and 40-80 parts by mass of seaweed Sugar, and 0.2 to 0.4 parts of Tween 80 by mass; in some embodiments, the solid preparation includes: 23 to 27 parts by mass of ADC-1, and 3 to 27 parts by mass of ADC-1 4 parts of histidine-succinic acid buffer, 50 parts to 70 parts of trehalose by mass, and 0.25
  • the solid preparation is a lyophilized preparation, which is obtained by lyophilizing a liquid preparation containing the anti-Trop2 antibody-drug conjugate.
  • the lyophilized formulation is a powder.
  • some embodiments provide a liquid formulation comprising the anti-Trop2 antibody-drug conjugate described above, the liquid formulation further comprising one or more of the buffer, stabilizer, and surfactant.
  • the liquid formulation can be used to prepare the above-mentioned lyophilized formulation, or a reconstituted formulation obtained after the above-mentioned lyophilized formulation is reconstituted.
  • the pH of the liquid formulation is 4.6 to 5.6; in some embodiments, the pH of the liquid formulation is 4.6 to 5.2; in some embodiments, the pH of the liquid formulation is 4.9.
  • the liquid formulation includes: an anti-Trop2 antibody-drug conjugate, wherein the concentration of the conjugate is 10 mg/ml to 50 mg/ml; it also includes stabilizers, buffers, and surfactants.
  • concentration of the stabilizer is 20mg/ml ⁇ 80mg/ml;
  • the buffer is a histidine buffer, a succinic acid buffer, a citric acid buffer or a combination thereof;
  • concentration of the active agent is 0.1 mg/ml to 0.5 mg/ml.
  • the concentration of the buffer is 1 mg/ml to 10 mg/ml; in some embodiments, the concentration of the buffer is 3 mg/ml to 7 mg/ml; in some embodiments Wherein, the concentration of the buffer is 3 mg/ml to 4 mg/ml.
  • the buffer comprises succinic acid at a concentration of 1.18 mg/ml and histidine hydrochloride at 2.1 mg/ml. In some embodiments, the buffer comprises succinic acid at a concentration of 1.18 mg/ml (ie, 10 mM) and histidine hydrochloride monohydrate at 2.1 mg/ml (ie, 10 mM).
  • the concentration of the buffer in the liquid formulation is 10 mM to 60 mM; in some embodiments, the concentration of the buffer in the liquid formulation is 20 mM to 40 mM; in some embodiments, the concentration of the buffer is 20 mM to 40 mM; In the liquid formulation, the concentration of the buffer is about 20 mM. In some embodiments, in the liquid formulation, the buffer is about 10 mM succinate buffer and about 10 mM histidine buffer.
  • the liquid formulation comprises: 10mg/ml-50mg/ml anti-Trop2 antibody-drug conjugate, 5mM-30mM buffer, 20mg/ml-80mg/ml stabilizer, and 0.1mg /ml ⁇ 0.5mg/ml surfactant.
  • the liquid formulation comprises: 25mg/ml of anti-Trop2 antibody-drug conjugate, 1.18mg/ml of succinic acid, 2.1mg/ml of histidine hydrochloride, 54mg/ml of trehalose, 0.3mg/ml Tween 80, pH is 4.6-5.2.
  • the liquid formulation comprises: 25mg/ml of anti-Trop2 antibody-drug conjugate, 1.18mg/ml of succinic acid, 2.1mg/ml of histidine hydrochloride monohydrate, 60mg/ml of Trehalose dihydrate, 0.3mg/ml Tween 80, pH 4.6-5.2.
  • the liquid formulation comprises: 25mg/ml of anti-Trop2 antibody-drug conjugate, 1.18mg/ml of succinic acid, 2.1mg/ml of histidine hydrochloride, 158mM of trehalose, 0.3mg /ml Tween 80, adjust the pH to 4.6-5.2 with sodium hydroxide.
  • the liquid formulation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride, 54 mg/ml trehalose, 0.3 mg/ml Tween 80, pH is 4.6-5.2. In some embodiments, the liquid formulation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride monohydrate, 54 mg/ml trehalose, 0.3 mg /ml Tween 80, the pH is 4.6-5.2.
  • the liquid formulation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride, 60 mg/ml trehalose dihydrate, 0.3 mg /ml Tween 80, the pH is 4.6-5.2. In some embodiments, the liquid formulation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride monohydrate, 60 mg/ml trehalose dihydrate , 0.3mg/ml Tween 80, pH is 4.6-5.2.
  • the liquid formulation includes 25 mg/ml anti-Trop2 antibody-drug conjugate, 10 mM succinic acid, 10 mM histidine hydrochloride, 158 mM trehalose, and 0.3 mg/ml Tween 80 , Adjust the pH to 4.6-5.2 with sodium hydroxide.
  • the present invention provides a method for preparing the solid preparation, which comprises:
  • Preparation take the anti-Trop2 antibody-drug conjugate, stabilizer, buffer, and surfactant, dissolve it in water to a specified amount, and adjust the pH value to the specified value to obtain a liquid preparation;
  • the step of freeze-drying includes pre-freezing, annealing, primary drying, and secondary drying.
  • a reconstituted preparation is also provided, wherein the reconstituted preparation is obtained by reconstitution of the lyophilized preparation (ie, solid preparation) with a pharmaceutically acceptable solvent.
  • the reconstituted preparation includes: an anti-Trop2 antibody-drug conjugate, wherein the concentration of the conjugate is 10 mg/ml to 50 mg/ml; it also includes stabilizers, buffers and surfactants The concentration of the stabilizer is 20mg/ml ⁇ 80mg/ml; the buffer is a histidine buffer, a succinic acid buffer, a citric acid buffer or a combination thereof; The concentration of the surfactant is 0.1 mg/ml to 0.5 mg/ml.
  • the concentration of the buffer in the reconstituted preparation is 10 mM to 60 mM; in some embodiments, the concentration of the buffer in the reconstituted preparation is 20 mM to 40 mM; in some embodiments, In the reconstituted preparation, the concentration of the buffer is about 20 mM.
  • the reconstituted formulation comprises: 10 mg/ml-50 mg/ml anti-Trop2 antibody-drug conjugate, 5 mM-30 mM buffer, 20 mg/ml-80 mg/ml stabilizer, and 0.1 Surfactant with mg/ml ⁇ 0.5mg/ml.
  • the reconstituted preparation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride, 54 mg/ml trehalose, 0.3 mg/ml Tween 80, the pH is 4.6-5.2.
  • the reconstituted preparation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride monohydrate, 60 mg/ml trehalose dihydrate
  • the product 0.3mg/ml Tween 80, pH is 4.6-5.2.
  • the solid preparation of the present invention can effectively solve the problems of ADC instability and easy aggregation, and has excellent long-term stability and accelerated stability.
  • the solid preparation of the present invention is freeze-dried, reconstituted, and reconstituted and diluted, various indicators have little change, the binding activity is also within the normal range, the stability is good, and it is suitable for long-term storage.
  • the lyophilized preparation is reconstituted, the ADC remains stable and is not easy to form aggregates or particles.
  • the invention provides the application of the solid preparation in the preparation of medicines for preventing and/or treating Trop2-positive diseases.
  • the present invention provides a kit, which includes the liquid preparation or solid preparation, and instructions for instructing patients in need of administration.
  • Another method for preventing and/or treating Trop2-positive disease includes injecting an effective amount of the reconstituted preparation or liquid preparation into a patient in need.
  • the Trop2-positive disease includes, but is not limited to, triple-negative breast cancer, glioblastoma, medulloblastoma, non-small cell lung cancer, small cell lung cancer, epithelial cancer, breast cancer, head and neck cancer, Kidney cancer, ovarian cancer, stomach cancer, Kaposi's sarcoma, pancreatic cancer and lung cancer, cervical cancer, colorectal cancer, esophageal cancer, oral squamous cell carcinoma, prostate cancer, thyroid cancer, bladder cancer, glioma, hepatobiliary duct Cancer, colorectal cancer, T cell lymphoma.
  • the Trop2-positive disease includes, but is not limited to, non-small cell lung cancer, small cell lung cancer, and epithelial cancer.
  • Figure 1 The variation trend of SEC-HPLC monomer purity of different samples over time
  • Figure 1 The trend of SEC-HPLC aggregates of different samples over time
  • Figure 2 The trend of SEC-HPLC fragments of different samples over time
  • Figure 3 The trend of the content of the main HIC-HPLC peak (D2) of different samples over time;
  • Figure 6 The change trend of SEC-HPLC polymer content in different pH samples over time
  • Figure 7 The change trend of HIC-HPLC main peak content of different pH samples over time
  • FIG. 8 SEC-HPLC monomer purity of samples in different buffers over time
  • Figure 10 The change trend of SEC-HPLC fragments of samples in different buffers over time
  • Figure 11 The trend of HIC-HPLC main peak content of samples in different buffers over time
  • FIG. 12 SEC-HPLC monomer purity of different stabilizer samples over time
  • Figure 13 The change trend of SEC-HPLC aggregates of different stabilizer samples over time
  • Figure 14 The change trend of HIC-HPLC main peak content of different stabilizer samples over time
  • Figure 15 Number of particles ( ⁇ 25 ⁇ m) (pieces/ml) after the compatibility of different surfactant samples
  • Figure 16 Number of particles ( ⁇ 10 ⁇ m) (pieces/ml) after the compatibility of different surfactant samples.
  • the "%" related to the components of the liquid preparation refers to the weight volume (w/v) percentage, where the weight unit may be g and the volume unit may be ml.
  • the solution containing 1% stabilizer means that 100ml of the solution contains 1g stabilizer, or the stabilizer content is 0.01g/ml.
  • an "effective amount” or “therapeutically effective amount” as used herein refers to a sufficient amount of an agent being administered that will reduce one or more symptoms of the disease or condition being treated to a certain degree. The result can be a reduction and/or alleviation of the signs, symptoms, or causes of the disease, or any other desired changes in the biological system.
  • an "effective amount” for therapeutic use is the amount required to provide a clinically significant reduction in disease symptoms without undue adverse side effects including the formulation as disclosed herein.
  • the “effective amount” or “therapeutically effective amount” may vary depending on the patient, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.
  • the term "acceptable” or “pharmaceutically acceptable” with respect to formulations, solid preparations or ingredients means that it does not have a lasting deleterious effect on the general health of the patient being treated or does not eliminate the compound’s It is biologically active or characteristic, and is relatively non-toxic.
  • ADC can form a wide variety of pharmaceutically acceptable salts, including but not limited to: acid addition salts formed with organic acids, including but not limited to aliphatic monocarboxylic and dicarboxylic acids, phenyl substituted Alkanoic acid, hydroxyalkanoic acid, alkanedioic acid, aromatic acid, aliphatic and aromatic sulfonic acid, amino acid, etc., such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, propylene Diacid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.; formed by reaction with inorganic acids Acid addition salts, such inorganic acids include hydrochloric acid, hydro
  • the term "individual” or “patient” as used herein refers to an animal that is the target of treatment, observation, or experiment.
  • the patient may be (but not limited to) a mammal, including (but not limited to) a human.
  • the “parts by mass” in the present invention refers to the value in the case of equal unit mass, and the unit mass is the mass of each part, which can be any suitable mass.
  • the unit mass is 1g/ serving, at this time 20 servings is 20g; the unit mass can also be 1kg/ serving or 1mg/ serving.
  • the unit mass is not limited to an integer, for example, it can be 0.5 g/part, and 20 parts is 10 g.
  • buffer in some documents, is also called a buffer system or a buffer system, which includes but is not limited to organic acid salts such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid or benzene Dicarboxylic acid and its salts; Tris, thomethamine hydrochloride, or phosphate buffer.
  • organic acid salts such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid or benzene Dicarboxylic acid and its salts
  • Tris Tris, thomethamine hydrochloride, or phosphate buffer.
  • amino acids and their salts can also be used as buffers.
  • the buffer in the present invention can be one, such as histidine buffer, succinic acid buffer, citric acid buffer, etc.; it can also be multiple, and when it involves multiple buffers, "-" is used to indicate
  • histidine-succinic acid buffer means composed of histidine buffer and succinic acid buffer
  • histidine-citrate buffer means composed of histidine buffer and citric acid buffer, etc.
  • histidine-succinic acid buffer it does not mean that the buffer is composed of histidine monomers and succinic acid monomers, but that the buffer is composed of the acid (or Base) and/or salt, and the acid (or base) and/or salt constituting the succinic acid buffer; for example, it can be composed of histidine and sodium succinate, can be composed of histidine hydrochloride and succinic acid, and can be composed of It is composed of histidine hydrochloride and sodium succinate, and can be composed of histidine, histidine hydrochloride, succinic acid, and sodium succinate.
  • the histidine-succinic acid buffer consists of histidine hydrochloride and sodium succinate.
  • the amount of buffer in the present invention refers to the total amount of buffer pairs in the buffer system constituting the buffer.
  • the molar concentration is used as the unit of the amount of the buffer, and its value refers to the molar concentration of the buffer pair in the buffer system of the buffer.
  • a histidine-succinic acid buffer composed of histidine hydrochloride and succinic acid
  • a given concentration of histidine-succinic acid buffer (such as 20mM) is a combination of histidine hydrochloride and succinic acid Combined concentration (e.g. histidine hydrochloride is 10mM, succinic acid is 10mM.)
  • trehalose can be anhydrous trehalose or trehalose hydrate, such as trehalose dihydrate.
  • trehalose dihydrate a trehalose hydrate
  • "5.4% trehalose” is 5.4g trehalose (or a corresponding amount of trehalose hydrate (such as 6g trehalose dihydrate)) dissolved in a solvent to form a 100ml solution, unless otherwise specified.
  • the drugs suitable for coupling the linker are various known or foreseeable cytotoxic agents in the art, including anti-mitotic cytotoxic drugs and DNA lytic cytotoxic drugs.
  • the drug is an anti-mitotic cytotoxic drug.
  • the drug in the anti-Trop2 antibody-drug conjugate (hereinafter referred to as ADC) can be maytansine or its derivatives, or other tumor cytotoxic compounds, such as camptothecin or its derivatives. Substances, auristatin analogs, etc.
  • the drug is DM1.
  • Maytansinol analogs and derivatives may include maytansine and maytansine analogs, which can be isolated from natural sources according to known methods and manufactured using biotechnology (see, e.g., Yu et al., 99PNAS7968-7973 (2002)), Or synthetically prepared according to known methods (see: Cassady et al., Chem. Pharm. Bull. 52(1) 1-26 (2004)). Depending on the type of linker, many locations on maytansinol can be used as attachment locations.
  • the C-3 position has a hydroxyl group
  • the C-14 position is a hydroxymethyl modification
  • the C-15 position is a hydroxyl modification
  • the C-20 position has a hydroxyl group.
  • the ADC antibody can specifically act on the trophoblast cell surface glycoprotein antigen 2.
  • the anti-Trop2 antibody is antibody A, antibody B, and/or antibody C.
  • the light chain of antibody A includes the amino acid sequence shown in SEQ ID NO: 1
  • the heavy chain includes the amino acid sequence shown in SEQ ID NO: 2
  • the light chain of antibody B and antibody C includes SEQ ID NO: The amino acid sequence shown in 3
  • the heavy chain includes the amino acid sequence shown in SEQ ID NO: 4.
  • the amino acid sequence of antibody A and the amino acid sequence of antibody B are expressed in suspended CHO cells.
  • the cell line is the Chinese hamster ovary cell line CHO-K1 (ATCC#CCL-61) after acclimatization to adapt to suspension growth. Come.
  • the amino acid sequence of antibody C is expressed in CHO-BAT-KF fut8(-/-) cells.
  • the anti-Trop2 antibody is antibody C.
  • the anti-Trop2 antibody is coupled with the drug through a linker to obtain ADC.
  • Linkers suitable for use in the present invention include cleavable linkers and non-cleavable linkers. Among them, the cleavable linker usually has a special sensitive structure. This linker is stable in a normal environment and can be broken under a tumor environment to release drugs to play a therapeutic role. Common cleavable linkers include protease-sensitive linkers. Most of them contain dipeptide bonds that can be broken by proteases, and most commonly are val-citrulline linkers (val-cit); there are also acid-sensitive linkers.
  • linkers containing hydrazone bonds Most are linkers containing hydrazone bonds; the other is a glutathione-sensitive linker, which usually contains glutathione reducible disulfide bonds.
  • the non-cleavable linker remains intact after being metabolized in the cell. Antibody-drug conjugates containing such linkers need to undergo lysosomal degradation to release cytotoxic molecules. Compared with the cleavable linker, the non-cleavable linker has good stability in the circulatory system.
  • the linker is a non-cleavable linker.
  • the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ia or Ib:
  • X is hydrogen or halogen
  • R 2 is H or C1-C6 alkyl
  • R 4 is -OH or -SH
  • R 5 is C1-C6 alkyl or benzyl
  • R 6 is C1-C6 alkyl, phenyl or benzyl
  • R 7 is hydrogen, C1-C6 alkyl or amino acid side chain
  • R 8 is hydrogen or C1-C6 alkyl
  • n 0, 1, 2, 3, 4, 5, 6, 7 or 8;
  • p 1-10;
  • Anti-Trop2 is an anti-Trop2 antibody.
  • the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ic:
  • Anti-Trop2 is an anti-Trop2 antibody.
  • Anti-Trop2 is antibody C.
  • the drug-to-antibody coupling ratio is the molar ratio of the drug to the antibody in the conjugate.
  • DAR is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in some embodiments, DAR is selected from 2, 3, 4, 5, or 6; in some In an embodiment, DAR is selected from 2.
  • p is the average DAR of the ADC. In some embodiments, p is 1-10; in some embodiments, p is 2-6; in some embodiments, p is 2-3; in some embodiments, p is about 2.1.
  • the ADC is the structure represented by Formula Ic, wherein Anti-Trop2 is antibody C, p is about 2.1, and the ADC is ADC-1 described herein.
  • the present invention provides a solid preparation comprising: ADC, wherein the mass parts of ADC is about 10 parts to about 50 parts; further comprising one or more of stabilizers, buffers and surfactants Species; the mass parts of the stabilizer is about 20 parts to about 80 parts; the buffer is histidine buffer, succinic acid buffer, citric acid buffer or a combination thereof; the mass parts of the surfactant is about 0.1 Servings ⁇ about 0.5 servings.
  • the mass parts of ADC is about 20 parts to about 30 parts; in some embodiments, the mass parts of ADC is 23 parts to 27 parts; in some embodiments, the mass parts of ADC is About 25 servings. In some embodiments, the parts by mass of the ADC are about 10 parts, about 20 parts, about 23 parts, about 25 parts, about 27 parts, about 30, about 40 parts, about 50 parts, or any two parts by mass The range between, including endpoint values.
  • the ADC is ADC-1; in some embodiments, the mass parts of ADC-1 is about 20 parts to about 30 parts; in some embodiments, the mass parts of ADC-1 is about 23 parts. Parts to about 27 parts; in some embodiments, the mass parts of ADC-1 is about 25 parts.
  • the mass parts of the stabilizer is about 40 parts to about 80 parts; in some embodiments, the mass parts of the stabilizer is about 50 parts to about 70 parts; in some embodiments, the stabilizer The number of parts by mass is about 54 parts. In some embodiments, the mass parts of the stabilizer is about 20 parts, about 40 parts, about 50 parts, about 54 parts, about 60 parts, about 70 parts, about 80 parts, or any two parts by mass. Range, including endpoint values.
  • the stabilizer is one or more of trehalose and its hydrates and sucrose and its hydrates; in some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is 50 parts to 70 parts by mass of trehalose; in some embodiments, the stabilizer is about 54 parts by mass of trehalose or about 60 parts by mass of trehalose dihydrate.
  • the buffer is a histidine buffer, a succinic acid buffer, a citric acid buffer, or a combination thereof.
  • the buffer is a histidine-succinate buffer or a histidine-citrate buffer; in some embodiments, the buffer is a histidine-succinate buffer.
  • buffers for example, histidine buffer, citrate buffer, succinic acid buffer
  • acids such as hydrochloric acid
  • bases such as NaOH or KOH
  • the buffer system can be a system of a weak acid and its salt, or a system of a weak base and its salt, depending on the specific buffer used.
  • the buffering agent in the form of acid and/or salt when the mass parts or molar concentration of the buffering agent is mentioned, it is all based on its total acid radical ions.
  • the buffer system is a succinic acid (HOOCCH 2 CH 2 COOH) buffer
  • succinic acid (HOOCCH 2 CH 2 COOH) buffer no matter which succinic acid exists in the form of HOOCCH 2 CH 2 COOH, HOOCCH 2 CH 2 COO - and/or - OOCCH 2 CH 2 COO -, amber
  • the amount of acid buffer refers to the total amount of HOOCCH 2 CH 2 COOH, HOOCCH 2 CH 2 COO - and - OOCCH 2 CH 2 COO - based on the acid radical ion - OOCCH 2 CH 2 COO - .
  • the buffer system is a histidine-succinic acid buffer
  • the mass parts of the buffer is the sum of the mass parts of succinic acid and the mass parts of histidine hydrochlor
  • the parts by mass of the buffer is about 1 part to about 10 parts; in some embodiments, the parts by mass of the buffer is about 3 parts to about 7 parts; in some embodiments, the buffer The parts by mass of is about 3 parts to about 4 parts.
  • the buffer comprises about 1.18 parts by mass of succinic acid and about 2.1 parts by mass of histidine hydrochloride. In some embodiments, the buffer comprises about 1.18 parts by mass of succinic acid and about 2.1 parts of histidine hydrochloride monohydrate.
  • the parts by mass of the surfactant is about 0.2 parts to about 0.4 parts; in some embodiments, the parts by mass of the surfactant is about 0.25 parts to about 0.35 parts; in some embodiments, The mass parts of the surfactant is about 0.3 parts. In some embodiments, the mass parts of the surfactant is about 0.1 parts, about 0.2 parts, about 0.25 parts, 0.3 parts, about 0.35 parts, about 0.4 parts, about 0.5 parts, or between any two parts by mass. Range, including endpoint values.
  • the surfactant is Tween 20 or Tween 80; in some embodiments, the surfactant is Tween 80. In some embodiments, the surfactant is about 0.25 to about 0.35 parts by mass of Tween 80; in some embodiments, the surfactant is about 0.3 parts by mass of Tween 80.
  • the solid preparation includes: about 10 parts to about 50 parts by mass of ADC (such as ADC-1), and about 1 part to about 10 parts by mass of a buffer (such as histidine- Succinic acid buffer or histidine-citric acid buffer), about 20 parts to about 80 parts of stabilizer (such as trehalose or sucrose or its hydrate), and about 0.1 parts to about 0.1 parts by mass.
  • ADC such as ADC-1
  • a buffer such as histidine- Succinic acid buffer or histidine-citric acid buffer
  • stabilizer such as trehalose or sucrose or its hydrate
  • about 0.1 parts to about 0.1 parts by mass about 0.5 parts of surfactant (such as Tween 20 or Tween 80).
  • the solid preparation includes: about 20 parts to about 30 parts by mass of ADC (such as ADC-1), and about 3 parts to about 7 parts of buffer (such as histidine- Succinic acid buffer or histidine-citric acid buffer), a stabilizer (such as trehalose or sucrose or its hydrate) with a mass part of about 40 to about 80 parts, and a mass part of about 0.2 to about About 0.4 parts of surfactant (such as Tween 20 or Tween 80).
  • ADC such as ADC-1
  • buffer such as histidine- Succinic acid buffer or histidine-citric acid buffer
  • a stabilizer such as trehalose or sucrose or its hydrate
  • surfactant such as Tween 20 or Tween 80.
  • the solid preparation includes: about 23 parts to about 27 parts by mass of ADC (such as ADC-1), and about 3 parts to about 4 parts of buffer (such as histidine- Succinic acid buffer or histidine-citric acid buffer), about 50 parts to about 70 parts of stabilizer (such as trehalose or sucrose or its hydrate), and about 0.25 parts to about 0.25 parts by mass.
  • ADC such as ADC-1
  • buffer such as histidine- Succinic acid buffer or histidine-citric acid buffer
  • stabilizer such as trehalose or sucrose or its hydrate
  • 0.25 parts to about 0.25 parts by mass such as trehalose or sucrose or its hydrate
  • surfactant such as Tween 20 or Tween 80.
  • the solid preparation includes: about 25 parts by mass of ADC (such as ADC-1), and about 3.3 parts (or about 3.28 parts) of buffer (such as histidine-succinic acid) by mass. Buffer or histidine-citric acid buffer), about 54 parts (or about 60 parts) of stabilizers (such as trehalose or sucrose or its hydrate) in parts by mass, and about 0.3 parts by mass Surfactant (such as Tween 20 or Tween 80).
  • ADC such as ADC-1
  • buffer such as histidine-succinic acid
  • Buffer or histidine-citric acid buffer such as trehalose or sucrose or its hydrate
  • Surfactant such as Tween 20 or Tween 80.
  • the solid preparation includes: about 20 parts to about 30 parts by mass of ADC-1, about 3 parts to about 7 parts by mass of citric acid-histidine hydrochloride buffer, and parts by mass
  • the amount of sucrose is about 40 parts to about 80 parts, and the amount of Tween 20 is about 0.2 parts to about 0.4 parts by mass.
  • the solid preparation comprises: about 20 parts to about 30 parts by mass of ADC-1, about 3 parts to about 7 parts by mass of histidine-succinic acid buffer, and parts by mass It is about 40 parts to about 80 parts of trehalose, and about 0.2 parts to about 0.4 parts of Tween 80 by mass.
  • the solid preparation includes: about 23 parts to about 27 parts by mass of ADC-1, about 3 parts to about 4 parts by mass of histidine-succinic acid buffer, and parts by mass of about 3 to about 4 parts of ADC-1. It is 50 parts to 70 parts of trehalose, and about 0.25 parts to about 0.35 parts of Tween 80 by mass.
  • the solid preparation includes: about 25 parts by mass of ADC-1, about 1.18 parts by mass of succinic acid, about 2.1 parts by mass of histidine hydrochloride, and about 54 parts of trehalose, and about 0.3 parts of Tween 80 by mass.
  • the solid preparation includes: about 25 parts by mass of ADC-1, about 1.18 parts by mass of succinic acid, about 2.1 parts by mass of histidine hydrochloride, and about 60 parts of trehalose dihydrate, and about 0.3 parts of Tween 80 by mass.
  • the solid preparation includes: about 25 parts by mass of ADC-1, about 1.18 parts by mass of succinic acid, about 2.1 parts by mass of histidine hydrochloride monohydrate, and about 2.1 parts by mass of ADC-1. About 60 parts of trehalose dihydrate, and about 0.3 parts of Tween 80 by mass.
  • the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.5-6.0. In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.6. In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.2.
  • the above-mentioned solid preparation also contains a certain amount of Na + or K + , which is used to form a buffer system with a buffer to adjust the pH, such as about 0.1 part to about 1 part of Na +, about 0.2 part to about 0.5 parts of Na + , or about 0.25 parts to about 0.3 parts of Na + .
  • the solid formulation is a powder. In some embodiments, the solid formulation is a lyophilized formulation.
  • the specification of the solid preparation (that is, the dose that can be packed into a container (such as a vial or bag)) is about 40 to about 200 mg of ADC, about 80 to about 120 mg of ADC, and about 92 to about 108mg ADC, or about 100mg ADC.
  • the strength of the solid formulation is about 40 mg, about 80 mg, about 92 mg, about 100 mg, about 108 mg, about 120 mg, about 200 mg ADC, or a range between any two numbers, including endpoints.
  • the solid formulation includes: about 40 to about 200 mg of ADC, about 4 to about 40 mg of buffer, 80 to 320 mg of stabilizer, and about 0.4 to about 2 mg of surfactant by mass.
  • the solid formulation includes: about 80 to about 120 mg of ADC-1, about 12 to about 28 mg of histidine-succinic acid buffer, about 160 to about 320 mg of trehalose, and about 0.8 to about 1.6 mg of Tween 80.
  • the solid preparation includes: about 92 to about 108 mg of ADC-1 by mass, about 12 to about 16 mg of histidine-succinic acid buffer, about 200 to about 280 mg of trehalose, and mass
  • the serving size is about 1 to about 1.4 mg of Tween 80.
  • the solid formulation includes about 100 mg of ADC-1, about 4.72 mg of succinic acid, about 8.4 mg of histidine hydrochloride, about 216 mg of trehalose, and about 1.2 mg of Tween 80.
  • the solid formulation includes: about 100 mg of ADC-1, about 4.72 mg of succinic acid, about 8.4 mg of histidine hydrochloride, about 240 mg of trehalose dihydrate, and about 1.2 mg of Tween 80 .
  • the solid formulation includes: about 100 mg ADC-1, about 4.72 mg succinic acid, about 8.4 mg histidine hydrochloride monohydrate, about 240 mg trehalose dihydrate, and about 1.2 mg Tween 80.
  • the solid preparation described above further contains a certain amount of Na + or K + , such as about 0.5 mg to about 4 mg of Na + , about 1 mg to about 2 mg of Na + , or about 1 mg to about 1.2 mg of Na + Na + .
  • the dose contained in each container is dissolved in a sufficient amount of a solvent for injection (such as water for injection or physiological saline) to obtain about 4 ml of a solution.
  • a solvent for injection such as water for injection or physiological saline
  • the solid formulation is a lyophilized formulation, which is obtained by lyophilizing a liquid formulation containing ADC, and the liquid formulation also contains one or more of a buffer, a stabilizer, and a surfactant, and a solvent, Such as water, such as water for injection.
  • the invention provides a liquid formulation.
  • the liquid formulation can be used to prepare the above-mentioned lyophilized formulation.
  • the liquid formulation is a reconstituted formulation obtained after reconstituted the above-mentioned lyophilized formulation.
  • the liquid formulation includes: ADC, wherein the concentration of ADC is about 10 mg/ml to about 50 mg/ml; further includes one or more of stabilizers, buffers and surfactants; stabilizers
  • the concentration of the surfactant is about 20 mg/ml to about 80 mg/ml
  • the buffer is histidine buffer, succinic acid buffer, citric acid buffer or a combination thereof
  • the concentration of the surfactant is about 0.1 mg/ml to about 0.5 mg/ml.
  • the ADC concentration of the liquid formulation is about 20 mg/ml to about 30 mg/ml; in some embodiments, the ADC concentration is about 23 mg/ml to about 27 mg/ml; in some embodiments, the ADC The concentration is about 25mg/ml. In some embodiments, the concentration of ADC is about 10 mg/ml, about 20 mg/ml, about 23 mg/ml, about 25 mg/ml, about 27 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml Or the range between any two concentrations, including endpoints.
  • the ADC of the liquid formulation is ADC-1; in some embodiments, the concentration of ADC-1 is about 20 mg/ml to about 30 mg/ml; in some embodiments, the concentration of ADC-1 is about From 23 mg/ml to about 27 mg/ml; in some embodiments, the concentration of ADC-1 is about 25 mg/ml.
  • stabilizers are used to protect ADCs in liquid formulations.
  • the stabilizer is selected from sugars, in some embodiments is a non-reducing sugar and/or sorbitol, and in some embodiments is selected from sucrose and/or trehalose. In some embodiments, it is trehalose.
  • these stabilizers make the preparation form a glassy state during the process, including crystallizable components therein. On the one hand, it eliminates the possibility of crystallization of the buffer components, thereby eliminating pH shifts and protecting the protein.
  • the formed glass state also plays a key role in the stability of the protein at low temperature and loss of water, but at the same time, in some embodiments of the present application, the stabilizer also acts as a filler for the freeze-dried formulation. Provide a certain structure for the freeze-dried preparation, and further improve the stability of ADC during long-term storage.
  • the filler can also be glycine, mannitol, and the like.
  • the concentration of stabilizer is controlled to maintain the stability of the protein and form during the freeze-drying process. Good "cake" shape.
  • the concentration of the stabilizer of the liquid formulation is about 40 mg/ml to about 80 mg/ml; in some embodiments, the concentration of the stabilizer is about 50 mg/ml to about 70 mg/ml; in some embodiments , The concentration of stabilizer is about 54mg/ml.
  • the concentration of the stabilizer is about 20 mg/ml, about 40 mg/ml, about 50 mg/ml, about 54 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, or any two The range between concentrations, including endpoints.
  • the stabilizer is trehalose at a concentration of about 50 mg/ml to about 70 mg/ml; in some embodiments, the stabilizer is trehalose at a concentration of about 54 mg/ml; in some embodiments, the stabilizer is Trehalose dihydrate with a concentration of about 60mg/ml.
  • trehalose In the solid state, trehalose usually exists as trehalose dihydrate. Therefore, in some embodiments, trehalose dihydrate is used when formulating the preparation, or other forms of trehalose can also be used.
  • the stabilizer is trehalose at a concentration of about 106 mM to about 212 mM; in some embodiments, the stabilizer is trehalose at a concentration of about 132 mM to about 185 mM; in some embodiments, the stabilizer is trehalose at a concentration of about 158 mM. Trehalose.
  • the buffer is selected from the group consisting of histidine buffer, citric acid buffer and succinic acid buffer, or a combination of any two of the above buffers, such as histidine buffer and succinic acid buffer.
  • the buffering agent mixed with the agent, or the buffering agent of the above three combinations.
  • the buffer is a combination of a succinic acid buffer and a histidine buffer.
  • the buffer is a histidine-succinic acid buffer.
  • the concentration of the buffer is the sum of the concentration of succinic acid and the concentration of histidine hydrochloride.
  • the concentration of the buffer when the concentration is too low, the buffering capacity will be weak, and no obvious protective effect will be achieved; when the concentration is too high, it will easily precipitate during the freezing process, which will affect the pH value in the solution and thus affect the ADC The stability.
  • the concentration of the buffer is about 1 mg/ml to about 10 mg/ml; in some embodiments, the concentration of the buffer is about 3 mg/ml to about 7 mg/ml; in some embodiments, the buffer The concentration is about 3mg/ml to about 4mg/ml.
  • the buffer comprises succinic acid at a concentration of about 1.18 mg/ml and histidine hydrochloride at a concentration of about 2.1 mg/ml. In some embodiments, the buffer comprises succinic acid at a concentration of about 1.18 mg/ml and histidine hydrochloride monohydrate at a concentration of about 2.1 mg/ml.
  • the concentration of the buffer of the liquid formulation is about 10 mM to about 60 mM; in some embodiments, the concentration of the buffer is about 16 mM to about 40 mM; in some embodiments, the concentration of the buffer is about 16 mM ⁇ About 24mM.
  • the buffer comprises a succinic acid buffer at a concentration of about 10 mM and a histidine buffer at a concentration of about 10 mM. In some embodiments, the buffer comprises succinic acid at a concentration of about 10 mM and histidine hydrochloride at a concentration of about 10 mM.
  • the liquid formulation contains a surfactant.
  • the surfactant is a nonionic surfactant, such as Tween 20, Tween 80, and poloxamer (such as poloxamer 188) and the like.
  • the surfactant is Tween 20 and/or Tween 80.
  • the surfactant is Tween 80.
  • the addition of these surfactants reduces surface tension and restricts protein polymerization.
  • the concentration of the surfactant of the liquid formulation is 0.2 mg/ml to about 0.4 mg/ml; in some embodiments, the concentration of the surfactant is about 0.25 mg/ml to about 0.35 mg/ml; In some embodiments, the concentration of the surfactant is about 0.3 mg/ml. In some embodiments, the concentration of the surfactant is about 0.1 mg/ml, about 0.2 mg/ml, about 0.25 mg/ml, about 0.3 mg/ml, about 0.35 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml or the range between any two concentrations, including endpoints. In some embodiments, the surfactant improves the stability of the ADC.
  • the surfactant concentration is Tween 80 at a concentration of about 0.25 mg/ml to about 0.35 mg/ml; in some embodiments, the surfactant is Tween 80 at a concentration of about 0.3 mg/ml.
  • the liquid formulation is used with a pharmaceutically acceptable solvent as a carrier, such as sterile pharmaceutical grade water for injection or saline, sterile water for injection, water for injection for bacteriostasis and other water for injection.
  • a pharmaceutically acceptable solvent such as sterile pharmaceutical grade water for injection or saline, sterile water for injection, water for injection for bacteriostasis and other water for injection.
  • the components in the liquid preparation before lyophilization such as ADC, buffer, stabilizer, and/or surfactant
  • the calculated amount is calculated according to the prescription.
  • each component is separately dissolved and dispersed with water for injection, and then the resulting solution is mixed to adjust the concentration and pH value.
  • the liquid formulation includes: about 10 mg/ml to about 50 mg/ml ADC (such as ADC-1), about 10 mM to about 60 mM buffer (such as histidine-succinic acid buffer or histidine -Citric acid buffer), stabilizers (such as trehalose or sucrose) from about 20mg/ml to about 80mg/ml, and surfactants (such as Tween 20 or vomiting agent) from about 0.1 mg/ml to about 0.5 mg/ml Wen 80).
  • ADC such as ADC-1
  • 10 mM to about 60 mM buffer such as histidine-succinic acid buffer or histidine -Citric acid buffer
  • stabilizers such as trehalose or sucrose
  • surfactants such as Tween 20 or vomiting agent
  • the liquid formulation includes: about 20 mg/ml to about 30 mg/ml ADC (such as ADC-1), about 16 mM to about 40 mM buffer (such as histidine-succinate buffer or histidine -Citrate buffer), stabilizers (such as trehalose or sucrose) from about 40mg/ml to about 80mg/ml, and surfactants (such as Tween 20 or vomiting agent) from about 0.2 mg/ml to about 0.4 mg/ml Wen 80).
  • ADC such as ADC-1
  • ADC-1 such as ADC-1
  • 40 mM buffer such as histidine-succinate buffer or histidine -Citrate buffer
  • stabilizers such as trehalose or sucrose
  • surfactants such as Tween 20 or vomiting agent
  • the liquid formulation includes: about 23 mg/ml to about 27 mg/ml ADC (such as ADC-1), about 16 mM to about 24 mM buffer (such as histidine-succinic acid buffer or histidine -Citrate buffer), stabilizers (such as trehalose or sucrose) from about 50mg/ml to about 70mg/ml, and surfactants (such as Tween 20 or vomiting agent) from about 0.25 mg/ml to about 0.35 mg/ml Wen 80).
  • ADC such as ADC-1
  • 16 mM to about 24 mM buffer such as histidine-succinic acid buffer or histidine -Citrate buffer
  • stabilizers such as trehalose or sucrose
  • surfactants such as Tween 20 or vomiting agent
  • the liquid formulation includes: about 25 mg/ml ADC (such as ADC-1), about 20 mM buffer (such as histidine-succinic acid buffer or histidine-citrate buffer), about 54 or 60 mg/ml stabilizer (such as trehalose or sucrose or its hydrate), and about 0.3 mg/ml surfactant (such as Tween 20 or Tween 80).
  • ADC such as ADC-1
  • 20 mM buffer such as histidine-succinic acid buffer or histidine-citrate buffer
  • stabilizer such as trehalose or sucrose or its hydrate
  • surfactant such as Tween 20 or Tween 80.
  • the liquid formulation includes: about 20 mg/ml to about 30 mg/ml ADC-1, about 16 mM to about 40 mM citric acid to histidine hydrochloride buffer, about 40 mg/ml to about 80 mg/ml Sucrose, and about 0.2mg/ml to about 0.4mg/ml Tween 20.
  • the liquid formulation includes: ADC-1 at about 20 mg/ml to about 30 mg/ml, histidine-succinic acid buffer at about 16 mM to about 40 mM, and seaweed at about 40 mg/ml to about 80 mg/ml Sugar, and about 0.2mg/ml to about 0.4mg/ml Tween 80.
  • the liquid formulation includes: about 23 mg/ml to about 27 mg/ml ADC-1, about 16 mM to about 24 mM histidine-succinic acid buffer, about 50 mg/ml to about 70 mg/ml seaweed Sugar, and about 0.25mg/ml to about 0.35mg/ml Tween 80.
  • the liquid formulation includes: about 20 mg/ml-40 mg/ml ADC-1, about 5-20 mM succinic acid and/or salt, about 5-20 mM histidine and/or salt, about 40 mg/ml ml ⁇ 80mg/ml trehalose, and about 0.1mg/ml ⁇ 0.5mg/ml Tween 80.
  • the liquid formulation includes: about 20mg/ml-30mg/ml ADC-1, about 5-15mM succinic acid and/or salt, about 5-15mM histidine and/or salt, about 50mg/ml ml ⁇ 70mg/ml trehalose, and about 0.2mg/ml ⁇ 0.4mg/ml Tween 80.
  • the liquid formulation includes: about 25 mg/ml ADC-1, about 10 mM succinic acid and/or salt, about 10 mM histidine and/or salt, about 54 mg/ml trehalose, and about 0.3 Tween 80 mg/ml.
  • the liquid formulation includes: about 25 mg/ml ADC-1, about 10 mM succinic acid, about 10 mM histidine hydrochloride, about 54 mg/ml trehalose, and about 0.3 mg/ml Tween 80 .
  • the pH of the liquid formulation is about 4.5 to about 6.0; in some embodiments, the pH of the liquid formulation is about 4.5 to about 5.6; in some embodiments, the pH of the liquid formulation is about 4.6 ⁇ 5.2; In some embodiments, the pH of the liquid formulation is 4.9 ⁇ 0.3. In some embodiments, the pH is adjusted by adding an appropriate amount of NaOH or KOH.
  • the liquid formulation includes: ADC, wherein the concentration of the conjugate is about 10 mg/ml to about 50 mg/ml; further includes one or more of stabilizers, buffers and surfactants; stabilizers
  • concentration of the surfactant is about 20 mg/ml to about 80 mg/ml
  • the buffer is histidine buffer, succinic acid buffer, citric acid buffer or a combination thereof
  • concentration of the surfactant is about 0.1 mg/ml to about 0.5 mg/ml.
  • the concentration of the buffer in the liquid formulation is from about 5 mM to about 30 mM; in some embodiments, the concentration of the buffer in the liquid formulation is from about 10 mM to about 20 mM; in some embodiments, the concentration of the buffer is from about 10 mM to about 20 mM. Among them, the concentration of the buffer is about 10 mM.
  • the liquid formulation comprises: about 10 mg/ml to about 50 mg/ml ADC, about 5 mM to about 30 mM buffer, about 20 mg/ml to about 80 mg/ml stabilizer, and about 0.1 mg/ml ⁇ About 0.5mg/ml of surfactant.
  • the liquid formulation comprises: ADC-1 at about 25 mg/ml, succinic acid at about 1.18 mg/ml, histidine hydrochloride at about 2.1 mg/ml, trehalose dihydrate at about 60 mg/ml, About 0.3mg/ml of Tween 80, the pH is about 4.6 to about 5.2.
  • the liquid formulation comprises: about 25 mg/ml ADC-1, about 1.18 mg/ml succinic acid, about 2.1 mg/ml histidine hydrochloride monohydrate, about 60 mg/ml trehalose dihydrate Hydrate, about 0.3 mg/ml of Tween 80, with a pH of about 4.6 to about 5.2.
  • the present invention provides a method for preparing a solid preparation, which comprises:
  • Preparation take ADC, stabilizer, buffer, surfactant, dissolve in water to a specified amount, and adjust the pH value to a specified value, such as 4.5 to 6.0, to obtain a liquid preparation;
  • Lyophilization or freeze-drying is a common method for preparing solid preparations from liquid preparations.
  • the preparations containing water and/or other solvents are usually rapidly cooled (for example, -30°C or lower) to freezing, and then the pressure is reduced in a sealed condition.
  • the frozen water and/or other solvents in the formulation are directly sublimated from the solid phase to the gas phase, thereby removing the water or other solvents to obtain a solid formulation that is usually a powder.
  • Lyophilization is usually carried out using a lyophilizer.
  • the step of freeze-drying includes pre-freezing, annealing, primary drying, and secondary drying.
  • the present invention provides the application of solid preparations in the preparation of medicines for preventing and/or treating Trop2-positive diseases.
  • Trop2-positive diseases include, but are not limited to, proliferative, inflammatory, or immune diseases or disorders.
  • Trop2-positive diseases include, but are not limited to, triple-negative breast cancer, glioblastoma, medulloblastoma, epithelial cancer, breast cancer, head and neck cancer, kidney cancer, ovarian cancer, gastric cancer, Kaposi Sarcoma, pancreatic and lung cancer, cervical cancer, colorectal cancer, esophageal cancer, oral squamous cell carcinoma, prostate cancer, thyroid cancer, bladder cancer, glioma, hepatobiliary cancer, colorectal cancer, T cell lymphoma.
  • the above-mentioned solid preparation is used in the preparation of a medicament for the prevention and/or treatment of epithelial cancer.
  • the solid preparation of the present invention before administering the medicine to the patient, the solid preparation is dissolved with a pharmaceutically acceptable solvent, reconstituted to obtain a reconstituted preparation, and then administered.
  • Solvents include sterile pharmaceutical grade water for injection or saline, sterile water for injection, and antibacterial water for injection.
  • a method for treating cancer which comprises administering an effective amount of a reconstituted preparation or liquid preparation of ADC to a patient in need of treatment.
  • a pharmaceutically acceptable solvent sterile pharmaceutical grade water for injection or saline, sterile water for injection, bacteriostatic water for injection, etc.
  • the present invention provides a reconstituted preparation, wherein the reconstituted preparation is obtained by reconstituting the above-mentioned solid preparation with a pharmaceutically acceptable solvent.
  • the reconstituted preparation can be used to prevent and/or treat Trop2-positive diseases, such as proliferative, inflammatory or immune diseases or disorders, such as epithelial cancer.
  • the reconstituted reconstituted formulation includes: ADC, wherein the concentration of the conjugate is about 10 mg/ml to about 50 mg/ml; and further includes one or more of stabilizers, buffers, and surfactants. Species; the concentration of stabilizer is about 20mg/ml to about 80mg/ml; the buffer is histidine buffer, succinic acid buffer, citric acid buffer or a combination thereof; the concentration of surfactant is about 0.1mg/ ml ⁇ 0.5mg/ml.
  • the concentration of the buffer in the reconstituted formulation is about 5 mM to about 30 mM; in some embodiments, the concentration of the buffer in the reconstituted formulation is about 10 mM to about 20 mM; in some embodiments, In the reconstituted preparation, the concentration of the buffer is about 20 mM.
  • the reconstituted formulation comprises: about 10 mg/ml to about 50 mg/ml ADC, about 5 mM to about 30 mM buffer, about 20 mg/ml to about 80 mg/ml stabilizer, and about 0.1 mg/ml ml to about 0.5mg/ml surfactant.
  • the reconstituted formulation comprises: about 25 mg/ml ADC-1, about 1.18 mg/ml succinic acid, about 2.1 mg/ml histidine hydrochloride, about 60 mg/ml trehalose dihydrate , About 0.3mg/ml Tween 80, pH is about 4.6 to about 5.2, part or all of succinic acid can be in the form of a corresponding amount of salt, and part or all of histidine hydrochloride can be in the form of a corresponding amount of histidine .
  • the reconstituted preparation comprises: about 25 mg/ml ADC-1, about 1.18 mg/ml succinic acid, about 2.1 mg/ml histidine hydrochloride monohydrate, about 60 mg/ml trehalose Dihydrate, about 0.3 mg/ml of Tween 80, with a pH of about 4.6 to about 5.2.
  • the solid preparation is dissolved and reconstituted with a solvent (such as sterile water for injection) to obtain a reconstituted preparation.
  • a solvent such as sterile water for injection
  • the commonly used ADC concentration after reconstitution is about 25 mg/ml.
  • an isotonic solution such as 0.9% NaCl solution for injection
  • the ADC concentration after dilution is about 0.1 mg/ml to about 5 mg/ml or about 0.5 mg/ml to about 5 mg/ml.
  • Both the reconstituted reconstituted preparations and the diluted preparations are injection preparations, generally intravenous infusion.
  • the amount of ADC is about 0.2 mg/kg to about 10 mg/kg.
  • the unit dose of ADC is about 0.2 mg/kg, or about 0.5 mg/kg, or about 1 mg/kg, or about 2 mg/kg, or about 4 mg/kg, or about 6 mg/kg, or About 8 mg/kg, or about 10 mg/kg, or a range between any two doses, including endpoints.
  • the administration period is 14-28 days, such as 20-22 days, or 21 days, that is, every 14-28 days, such as every 20-22 days, or every 21 days, the patient is administered once.
  • the ADC unit dose is 14-28 days, such as 20-22 days, or 21 days, that is, every 14-28 days, such as every 20-22 days, or every 21 days, the patient is administered once.
  • the patient is a Trop2-positive advanced solid tumor patient.
  • low temperature (4°C) and room temperature (25°C) stability experiments are performed on the prepared solid preparations.
  • the solid preparation prepared by the invention can be stored at low temperature and room temperature for 3 months, 6 months, 9 months, 12 months, 18 months or even 24 months, and remains stable. Compared with month 0, ADC has a very small amount of protein polymerization; the average DAR remains stable; there is no sign of protein degradation; after reconstitution with sterile water for injection, it also maintains good performance in terms of insoluble particles.
  • 0.9% normal saline is used to compatibly dilute the reconstituted preparation after reconstitution into a high-concentration preparation (about 5 mg/ml) and a low-concentration preparation (about 0.5 mg/ml).
  • a high-concentration preparation about 5 mg/ml
  • a low-concentration preparation about 0.5 mg/ml.
  • stability tests at low temperature (4°C) and room temperature (25°C) are performed on the reconstituted preparation after the solid preparation is reconstituted.
  • the reconstituted preparation prepared by the present invention can be placed at low temperature and room temperature for 6 hours, 24 hours or even 48 hours, and remains stable.
  • the present invention provides an anti-Trop2 antibody drug conjugate solid preparation.
  • the solid preparation can be prepared by lyophilizing and removing moisture from ADC at low temperature, so that ADC remains stable during storage.
  • ADC-1 is obtained by coupling with antibody C through L-3AA-MDC (see patent CN 201310081867.X for the preparation method).
  • Antibody C was diluted to 8.0 mg/ml with solution A (20 mM sodium phosphate, 100 mM NaCl and 2 mM EDTA, pH 7.4), and then completely reduced with excess tris(2-carboxyethyl)phosphine (TCEP). After incubating at 37°C for more than one hour, the solution B (10 mM succinic acid, 2 mM EDTA, pH 7.4) was used for ultrafiltration to concentrate the solution. The sulfhydryl antibody value is determined by measuring the absorbance.
  • the sulfhydryl concentration is determined by the reaction product of the sulfhydryl and DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich), and then measuring the absorbance at 412nm. . Subsequently, using excess copper sulfate (CuSO 4 ) or dehydroascorbic acid (dHAA) for oxidation, the disulfide bonds between the antibody chains are reconnected, while the cysteine at the mutation site is retained.
  • CuSO 4 copper sulfate
  • dHAA dehydroascorbic acid
  • the concentration of DMA during the coupling reaction is 10%-30%.
  • the ratio of L-3AA-MDC to the number of mercapto groups is 1-2:1.0 (molar equivalent).
  • Add L-3AA-MDC to the reduced antibody and after stirring for 3 hours at room temperature, add 5mM cysteine and continue stirring for 0.5-3 hours.
  • the reaction mixture is purified by a cation exchange column, and the product is concentrated by ultrafiltration, and then filtered with a 0.22 micron filter, and stored at -80°C.
  • the concentration of ADC-1 can be measured by ultraviolet absorption, the aggregation rate can be measured by size exclusion chromatography, and the ratio of coupled drugs can be measured by reversed-phase high performance liquid chromatography.
  • the reagents and instruments used are conventional reagents and instruments in the art, which can be obtained commercially; the method used is a conventional technical method in the art, and those skilled in the art will follow the example The content of the method can be implemented without any doubt and the corresponding results can be obtained.
  • Wavelength 280nm; column temperature 40°C; flow rate 0.3ml/min, equilibrate for about 30min until the baseline is stable.
  • the capillary electrophoresis instrument uses Beckman's PA800plus. After the sample is injected, the chromatogram is recorded, the data is integrated, and the area normalization method is used to calculate the percentage of the monomer peak.
  • Blocking Wash the plate 3 times with an automatic plate washer, 300 ⁇ L of washing solution per well, then add 250 ⁇ L of blocking solution to each reaction well, and incubate at 37°C for 2h.
  • the ADC concentration is 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.2ng/ml, 15.6ng/ml, 7.8ng/ml, 3.9ng/ml, 1.9ng/ml Gradient solution.
  • Color development Take out the ELISA plate and repeat the washing for 8 times, with 300 ⁇ L washing solution per well. Add TMB color developing solution to the reaction wells, 100 ⁇ L per well, and develop color at room temperature (25 ⁇ 2°C) in the dark for 10-15 minutes.
  • Termination Add 50 ⁇ L of 2M sulfuric acid solution to each reaction well to terminate the color reaction, and then read within 30min.
  • the anti-Trop2-drug conjugates in the following examples take ADC-1 as an example.
  • His histidine buffer
  • HPS succinic acid buffer
  • His5.5, His6.0, His6.5, HPS4.5, HPS5.0 and HPS5.5 six sets of solution samples, wherein the concentration of anti-Trop2 antibody in each set of solutions is 12.5mg/ml.
  • the samples were divided into aliquots and placed at 40°C for a high temperature test. Samples were taken on the 0th day, 1st week, 2nd week, and 3rd week for SEC-HPLC and HIC-HPLC detection.
  • HIC stability of the anti-Trop2 antibody conjugate is better when the pH is less than or equal to 5.5, and when the pH is more than 5.5, the HIC main peak content of the sample decreases rapidly and the stability is poor.
  • the stability of the six groups of samples in terms of the main peak content of HIC-HPLC has obvious differences. From the results of HIC-HPLC main peak content data mapping, it can be seen that HPS45, HPS50, HPS55, His55 are better than His60 and His65, HPS45>HPS50>HPS55>His55>His60>His65, indicating that the sample is in a buffer with pH ⁇ 5.5. The content of the main peak of HIC is stable.
  • the stability of the anti-Trop2 antibody conjugate is better when the pH range is 4.5 ⁇ pH ⁇ 5.5 under high temperature (40°C) conditions.
  • the anti-Trop2 antibody conjugate has good stability in histidine or succinic acid buffer around pH 5.0. Now the anti-Trop2 antibody conjugate is subjected to a pH optimization screening test. Prepared formulations with 6 pH gradients: histidine-succinic acid buffer (pH4.6), histidine-succinic acid buffer (pH4.8), histidine-succinic acid buffer (pH5.0) ), histidine-succinic acid buffer (pH5.2), histidine-succinic acid buffer (pH5.4), histidine-succinic acid buffer (pH5.6). The molar concentration ratio of histidine buffer to succinic acid buffer is 1:1.
  • the concentration of the anti-Trop2 antibody conjugate was adjusted to 10mg/ml, the samples were placed at 40°C for high temperature test, and samples were taken on day 0, week 1, week 2, week 3, and week 4. SEC-HPLC, HIC-HPLC detection, the results are shown in Figure 5, Figure 6 and Figure 7.
  • HIC-HPLC data results From the analysis of HIC-HPLC data results, it can be seen that under high temperature conditions, with the extension of time, the content of the main HIC-HPLC peak of the protein decreases. Under the same experimental conditions, the six groups of samples have significant differences in the stability of the main peak content of HIC-HPLC. From the HIC-HPLC main peak content change trend graph in Figure 7, it can be seen that the target protein is unstable at high temperature. As time goes by, the main peak content of the six groups of samples decreases to varying degrees. When the pH is between 4.6 and 5.2, The main peak of HIC-HPLC of the sample decreased slowly, and the stability was better.
  • the pH value is a key factor in the formulation of the anti-Trop2-ADC preparation of this product.
  • Samples show different stability in buffers with different pH values, but when the pH value fluctuates within a certain range, it will not have a significant impact on the quality of the target protein.
  • the pH value of the formulation must provide an effective range, not a fixed value. Based on the above research, it is preliminarily determined that the stability of the sample against Trop2-ADC is better in the pH range of 4.6 to 5.2 in the histidine-succinic acid buffer system.
  • the anti-Trop2 antibody conjugate of the present invention has better stability under pH 4.5-5.5 range conditions, so a buffer with a buffering capacity around pH 5.0 is selected for the screening test.
  • a buffer with a buffering capacity around pH 5.0 is selected for the screening test.
  • succinate-sodium succinate buffer (HPS) citric acid-sodium citrate buffer (NMS)
  • succinate-citrate buffer (HPSNMS) histidine-succinate buffer Solution
  • HisHPS histidine-succinate buffer Solution
  • HisNMS histidine-citrate buffer
  • the concentration of the anti-Trop2 antibody conjugate was 11mg/ml
  • the pH value of the solution was 5.0
  • the solution was divided into aliquots and placed at 40°C for high temperature test.
  • week 1, week 2 Samples were taken in the 3rd and 4th weeks and tested by SEC-HPLC and HIC-HPLC respectively.
  • ADC-1 solutions containing five stabilizers including sucrose, trehalose, mannitol, glycine, and sorbitol were prepared respectively. See Table 2 for specific formulations.
  • the weight percentage of trehalose in this embodiment is the percentage calculated by the weight of trehalose dihydrate.
  • test plan is as follows:
  • trehalose which is commonly used in biological monoclonal antibody preparations, is selected as the stabilizer in the formulation of this preparation, and sucrose can be used as an alternative stabilizer.
  • Example 5 Screening test of surfactant content—compatibility test with physiological saline
  • the solid preparation of the present invention needs to be administered clinically with physiological saline. Therefore, this compatibility test is performed to determine the concentration of the surfactant, which can effectively improve the stability of the protein.
  • ADC sample solutions containing different concentrations and different types of surfactants are prepared.
  • the specific formula is shown in Table 6, and the ADC concentration in each group of solutions is 25 mg/ml.
  • the protein solution is tested for insoluble particles. The test results are shown in Table 7, Figure 15 and Figure 16.
  • a specific component and content of the liquid preparation of this product are: 10mM succinic acid, 10mM histidine hydrochloride (or 10mM histidine hydrochloride monohydrate), 6% trehalose dihydrate (That is, 158mM), 0.03% Tween 80, and adjust the pH to 4.9 ⁇ 0.3 with sodium hydroxide.
  • the preparation method is as follows: take 100g of anti-Trop2 antibody-maytansine conjugate, 4.72g succinic acid, 8.4g histidine hydrochloride (herein histidine hydrochloride refers to histidine hydrochloride monohydrate), trehalose two 240g hydrate and 1.2g Tween 80, add water to prepare a 4L solution, and adjust the pH to 4.9 ⁇ 0.3 with NaOH.
  • the above liquid preparation is freeze-dried to obtain a solid preparation.
  • Example 6 The solid preparation prepared in Example 6 was used for the experiment. After the solution was frozen at -80°C for 24 hours, it was placed at 25°C for 24 hours as a freeze-thaw cycle. SEC-HPLC, HIC-HPLC, CE-SDS (NR), binding activity and other tests were performed on the frozen and thawed samples. The results are shown in Table 8 and Table 9.
  • Table 8 SEC-HPLC and HIC-HPLC results of the solid preparation sample of Example 6 frozen and thawed 5 times
  • DR in the sample name indicates freeze-thaw, and the number indicates the number of times.
  • DR1 means freeze-thaw once.
  • Table 9 The results of CE-SDS (NR) and binding activity of the solid preparation sample of Example 6 frozen and thawed 5 times
  • HHL means that the antibody contains two heavy chains and one light chain
  • fraction means that it is a fragment.
  • Example 6 The solid preparation prepared in Example 6 was reconstituted to a reconstituted concentration of 25 mg/ml. The reconstituted solution was placed at 25°C and 4°C for testing. At 25°C, samples were taken at 6h, 24h, and 48h for testing; at 4°C, samples were taken at 6h, 24h, and 48h for testing. Test items include appearance, visible foreign matter, pH, ADC concentration, insoluble particles, SEC-HPLC, IEC-HPLC, CE-SDS(R), CE-SDS(NR), HIC-HPLC, PR-HPLC, toxin residue, biological For activity, binding activity, etc., the test results are compared with those at 0h, and the results are shown in Table 12 and Table 13.
  • the appearance standard is white or slightly yellow lumpy loose body; the visible foreign body standard is no visible foreign body detected; the toxin residue standard is bacterial endotoxin ⁇ 2EU/ml; N/A means no test result.
  • the appearance standard is white or slightly yellow lumpy loose body; the visible foreign body standard is no visible foreign body detected; the toxin residue standard is bacterial endotoxin ⁇ 2EU/ml; N/A means no test result.
  • the reconstituted sample was placed at 25°C and 4°C for 48 hours.
  • the appearance of the sample visible foreign matter, pH, ADC concentration, SEC-HPLC, IEC-HPLC, CE-SDS(R ), CE-SDS (NR), HIC-HPLC, PR-HPLC, toxin residue, biological activity, binding activity and other quality attributes have not changed significantly, indicating that the reconstituted sample can be placed at 25°C and 4°C 48 hours.
  • the reconstituted sample should be used immediately. If it is not used immediately, it can be stored in a refrigerator at 2-8°C for 6 hours after reconstitution. It should not be frozen or shaken.
  • This product is intravenously injected in clinical practice, and it needs to be diluted with sodium chloride injection before it can be used on patients. Therefore, compatibility tests and stability tests of the drug delivery process are required.
  • Example 6 The solid preparation of Example 6 was tested for compatibility with 0.9% sodium chloride injection.
  • Example 6 Take the solid preparation of Example 6 (specification: 100mg/bottle), reconstitute it with 4ml of sterile water for injection, mix well and let stand, extract a certain amount of medicine, and transfer to an injection containing 0.9% sodium chloride In the container, mix the diluted solution by gently inverting, and the final concentration of the diluted solution is between 0.5mg/ml and 5mg/ml.
  • Test items include appearance, visible foreign matter, pH value, ADC concentration after dilution, insoluble particles, osmotic pressure, SEC-HPLC monomer purity, IEC-HPLC, CE-SDS(R), CE-SDS(NR), HIC-HPLC , PR-HPLC, toxin residue, relative biological activity, binding activity, the test results are compared with the 0h. The results are shown in Table 14, Table 15.
  • Example 6 The solid preparations prepared in Example 6 were placed at 25°C and 4°C respectively for testing. At 25°C, samples were taken in the first month, February, March, June, September, and December, and reconstituted with water for injection and then tested. The reconstitution concentration was 25mg/ml; at 4°C, respectively Samples were taken in March, June, September, December, 18th, and 24 months, and re-dissolved with water for injection and re-tested. The reconstitution concentration was 25 mg/ml.
  • Test items include appearance, visible foreign matter, pH, ADC concentration, insoluble particles, SEC-HPLC, IEC-HPLC, CE-SDS(R), CE-SDS(NR), HIC-HPLC, PR-HPLC, toxin residue, biological For activity, binding activity, etc., the test results are compared with those at 0h, and the results are shown in Table 16 and Table 17.
  • the appearance standard is white or slightly yellow lumpy loose body; the visible foreign body standard is no visible foreign body detected; the toxin residue standard is bacterial endotoxin ⁇ 2EU/ml; N/A means no test result.
  • the appearance standard is white or slightly yellow lumpy loose body; the visible foreign body standard is no visible foreign body detected; the toxin residue standard is bacterial endotoxin ⁇ 2EU/ml; N/A means no test result.

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Abstract

Provided are a formulation containing an anti-Trop2 antibody-drug conjugate (ADC) and a preparation method and an application thereof. The formulation comprises: an anti-Trop2 antibody-drug conjugate, and one or more of a stabilizer, a buffering agent, and a surfactant. The formulation can effectively resolve problems of instability and easy aggregation of ADCs, and has excellent long-term stability and stability of acceleration.

Description

含抗Trop2抗体-药物偶联物的制剂及其制备方法和应用Preparation containing anti-Trop2 antibody-drug conjugate and preparation method and application thereof 技术领域Technical field
本发明属于生物制剂领域，涉及一种含抗体-药物偶联物的制剂。The invention belongs to the field of biological preparations, and relates to a preparation containing an antibody-drug conjugate.
背景技术Background technique
抗体-药物偶联物(Antibody-Drug Conjugates，ADC)是由抗体和药物偶联得到的具有高度有效性和特异性的用于治疗癌症和其它病况的药物，其中抗体部分特异性结合在靶细胞上的抗原，使得药物可发挥其对靶细胞的细胞毒性或其它治疗效果。抗Trop2抗体-药物偶联物(抗Trop2-ADC)结合到Trop2受体后，抗Trop2-ADC开始以受体为媒介进行内化，之后的溶酶体降解过程使其在细胞内释放细胞毒性代谢物。细胞毒性代谢物进而杀伤肿瘤细胞。对于ADC药物，药物的偶联本身会降低抗体的稳定性，改变其物理化学性质。Antibody-Drug Conjugates (ADC) are highly effective and specific drugs for the treatment of cancer and other conditions obtained by coupling antibodies and drugs, in which the antibody part specifically binds to target cells On the antigen, the drug can exert its cytotoxicity or other therapeutic effects on target cells. After the anti-Trop2 antibody-drug conjugate (anti-Trop2-ADC) binds to the Trop2 receptor, the anti-Trop2-ADC begins to internalize with the receptor as a mediator, and the subsequent lysosomal degradation process causes it to release cytotoxicity in the cell Metabolites. Cytotoxic metabolites in turn kill tumor cells. For ADC drugs, the drug coupling itself will reduce the stability of the antibody and change its physical and chemical properties.
本领域对稳定的制剂依然有需求。There is still a need for stable formulations in the field.
发明内容Summary of the invention
本发明的目的是提供一种在储藏过程中稳定的含抗Trop2抗体-药物偶联物制剂，以解决现有技术存在的问题。The purpose of the present invention is to provide a stable anti-Trop2 antibody-drug conjugate preparation during storage, so as to solve the problems existing in the prior art.
一方面，本发明提供一种液体制剂，包括：抗Trop2抗体-药物偶联物；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种。在一些实施方案中，所述液体制剂pH值为4～7。在一些实施方案中，所述液体制剂pH值为4.5～6.0。在一些实施方案中，所述液体制剂pH值为4.5～5.6。In one aspect, the present invention provides a liquid formulation, which includes: an anti-Trop2 antibody-drug conjugate; and also includes one or more of a stabilizer, a buffer, and a surfactant. In some embodiments, the pH of the liquid formulation is 4-7. In some embodiments, the pH of the liquid formulation is 4.5 to 6.0. In some embodiments, the pH of the liquid formulation is 4.5 to 5.6.
在一些实施方案中，本发明提供一种液体制剂，包括：抗Trop2抗体-药物偶联物，其中所述偶联物的浓度为10mg/ml～50mg/ml；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种，所述稳定剂的浓度为20mg/ml～80mg/ml，所述缓冲剂的浓度为5mM～60mM；所述表面活性剂的浓度为0.1mg/ml～0.5mg/ml；在一些实施方案中，所述液体制剂pH值为4.5～6.0。In some embodiments, the present invention provides a liquid formulation comprising: an anti-Trop2 antibody-drug conjugate, wherein the concentration of the conjugate is 10 mg/ml-50 mg/ml; it also includes stabilizers, buffers, and One or more of surfactants, the concentration of the stabilizer is 20mg/ml～80mg/ml, the concentration of the buffering agent is 5mM～60mM; the concentration of the surfactant is 0.1mg/ml～ 0.5 mg/ml; in some embodiments, the liquid formulation has a pH value of 4.5 to 6.0.
一方面，在一些实施方案中，本发明提供一种固体制剂，由所述液体制剂冻干而成。In one aspect, in some embodiments, the present invention provides a solid preparation, which is lyophilized from the liquid preparation.
一方面，在一些实施方案中，本发明提供一种固体制剂，包括：抗Trop2抗体-药物偶联物，其中所述偶联物的质量份数为约10份～约50份；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种；所述稳定剂的质量份数为约20份～约80份；所述缓冲剂的质量份数为1份～10份；所述表面活性剂的质量份数为约0.1份～约0.5份。In one aspect, in some embodiments, the present invention provides a solid preparation, comprising: an anti-Trop2 antibody-drug conjugate, wherein the mass parts of the conjugate is about 10 parts to about 50 parts; and it also includes a stable anti-Trop2 antibody-drug conjugate. One or more of agents, buffers and surfactants; the mass parts of the stabilizer is about 20 parts to about 80 parts; the mass parts of the buffering agent is 1 part to 10 parts; The mass parts of the surfactant is about 0.1 part to about 0.5 part.
在一些实施方案中，所述抗Trop2抗体-药物偶联物包含如式Ia的化合物：In some embodiments, the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ia:
Figure PCTCN2020140885-appb-000001
Figure PCTCN2020140885-appb-000001
或其药学上可接受的盐或溶剂合物,Or a pharmaceutically acceptable salt or solvate thereof,
其中among them
X为氢或卤素；X is hydrogen or halogen;
Y选自氢、C1-C6烷基、C3-C6环烷基和-C(＝O)R 5； Y is selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl and -C(=O)R 5 ;
R 1选自H、-OH、-OC(＝O)R 5和-OR 5基团； R 1 is selected from H, -OH, -OC(=O)R 5 and -OR 5 groups;
R 2为H或C1-C6烷基； R 2 is H or C1-C6 alkyl;
R 3为甲基、-CH2OH或-CH2OC(＝O)R 6； R 3 is methyl, -CH2OH or -CH2OC(=O)R 6 ;
R 4为-OH或–SH； R 4 is -OH or -SH;
R 5为C1-C6烷基或苄基； R 5 is C1-C6 alkyl or benzyl;
R 6为C1-C6烷基、苯基或苄基； R 6 is C1-C6 alkyl, phenyl or benzyl;
R 7为氢、C1-C6烷基或氨基酸侧链； R 7 is hydrogen, C1-C6 alkyl or amino acid side chain;
R 8为氢或者C1-C6烷基； R 8 is hydrogen or C1-C6 alkyl;
n为0、1、2、3、4、5、6、7或8；n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
p为1-10，如1、2、3、4、5、6、7、8、9、10，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值；p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the range between any two of these values (including endpoints) or any value therein;
Anti-Trop2为抗Trop2抗体。Anti-Trop2 is an anti-Trop2 antibody.
在一些实施方案中，所述抗Trop2抗体-药物偶联物包含如式Ib的化合物：In some embodiments, the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ib:
Figure PCTCN2020140885-appb-000002
Figure PCTCN2020140885-appb-000002
或其药学上可接受的盐或溶剂合物,Or a pharmaceutically acceptable salt or solvate thereof,
其中among them
X为氢或卤素；X is hydrogen or halogen;
Y选自氢、C1-C6烷基、C3-C6环烷基和-C(＝O)R 5； Y is selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl and -C(=O)R 5 ;
R 1选自H、-OH、-OC(＝O)R 5和-OR 5基团； R 1 is selected from H, -OH, -OC(=O)R 5 and -OR 5 groups;
R 2为H或C1-C6烷基； R 2 is H or C1-C6 alkyl;
R 3为甲基、-CH 2OH或-CH 2OC(＝O)R 6； R 3 is methyl, -CH 2 OH or -CH 2 OC(=O)R 6 ;
R 4为-OH或–SH； R 4 is -OH or -SH;
R 5为C1-C6烷基或苄基； R 5 is C1-C6 alkyl or benzyl;
R 6为C1-C6烷基、苯基或苄基； R 6 is C1-C6 alkyl, phenyl or benzyl;
R 7为氢、C1-C6烷基或氨基酸侧链； R 7 is hydrogen, C1-C6 alkyl or amino acid side chain;
R 8为氢或者C1-C6烷基； R 8 is hydrogen or C1-C6 alkyl;
p为1-10，如1、2、3、4、5、6、7、8、9、10，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值；p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the range between any two of these values (including endpoints) or any value therein;
Anti-Trop2为抗Trop2抗体。Anti-Trop2 is an anti-Trop2 antibody.
在一些实施方案中，所述抗Trop2抗体-药物偶联物包含如式Ic的化合物：In some embodiments, the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ic:
Figure PCTCN2020140885-appb-000003
Figure PCTCN2020140885-appb-000003
或其药学上可接受的盐或溶剂合物，Or a pharmaceutically acceptable salt or solvate thereof,
其中，p为1-10，如1、2、3、4、5、6、7、8、9、10，或这些数值中的任何两个值之间的范围(包括端点)或其中任何值；Wherein, p is 1-10, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or the range between any two of these values (including endpoints) or any value therein ；
Anti-Trop2为抗Trop2抗体。Anti-Trop2 is an anti-Trop2 antibody.
在一些实施方案中，p为2-6；在一些实施方案中，p为2-3；在一些实施方案中，p约为2.1。In some embodiments, p is 2-6; in some embodiments, p is 2-3; in some embodiments, p is about 2.1.
在一些实施方案中，所述抗Trop2抗体的轻链氨基酸序列如SEQ ID NO:1或SEQ ID NO:3所示，重链氨基酸序列如SEQ ID NO:2或SEQ ID NO:4所示。在一些实施方案中，所述抗Trop2抗体表达于CHO细胞中。In some embodiments, the light chain amino acid sequence of the anti-Trop2 antibody is shown in SEQ ID NO: 1 or SEQ ID NO: 3, and the heavy chain amino acid sequence is shown in SEQ ID NO: 2 or SEQ ID NO: 4. In some embodiments, the anti-Trop2 antibody is expressed in CHO cells.
在一些实施方案中，所述抗Trop2抗体为抗体A、抗体B和/或抗体C；在一些实施方案中，所述抗Trop2抗体为抗体C。In some embodiments, the anti-Trop2 antibody is antibody A, antibody B, and/or antibody C; in some embodiments, the anti-Trop2 antibody is antibody C.
所述抗体A的轻链氨基酸序列如SEQ ID NO:1所示，重链氨基酸序列如SEQ ID NO:2所示，并且抗体A表达于悬浮的CHO细胞中，比如由CHO-K1细胞经培养适应悬浮生长的中国仓鼠卵巢细胞；The amino acid sequence of the light chain of the antibody A is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2, and the antibody A is expressed in suspended CHO cells, for example, cultured by CHO-K1 cells Chinese hamster ovary cells adapted to growth in suspension;
抗体B的轻链氨基酸序列如SEQ ID NO:3所示，重链氨基酸序列如SEQ ID NO:4所 示，并且抗体B表达于悬浮的CHO细胞中，比如由CHO-K1细胞经培养适应悬浮生长的中国仓鼠卵巢细胞；The light chain amino acid sequence of antibody B is shown in SEQ ID NO: 3, and the heavy chain amino acid sequence is shown in SEQ ID NO: 4, and antibody B is expressed in suspended CHO cells, for example, CHO-K1 cells are cultured to adapt to suspension Growing Chinese Hamster Ovary Cells;
抗体C的轻链氨基酸序列如SEQ ID NO:3所示，重链氨基酸序列如SEQ ID NO:4所示，并且抗体C的岩藻糖含量为0-5％。在一些实施方案中，抗体C表达于Fut8基因(α-(1,6)-岩藻糖基转移酶基因)被敲除的CHO细胞中，比如CHO-BAT-KF fut8(-/-)细胞，CN109096399A已公开上述细胞。The light chain amino acid sequence of antibody C is shown in SEQ ID NO: 3, the heavy chain amino acid sequence is shown in SEQ ID NO: 4, and the fucose content of antibody C is 0-5%. In some embodiments, antibody C is expressed in CHO cells in which the Fut8 gene (α-(1,6)-fucosyltransferase gene) has been knocked out, such as CHO-BAT-KF fut8(-/-) cells , CN109096399A has disclosed the above-mentioned cells.
在一些实施方案中，抗Trop2抗体-药物偶联物为ADC-1，为Ic所示化合物，其中，p为2.1，抗Anti-Trop2抗体为抗体C。In some embodiments, the anti-Trop2 antibody-drug conjugate is ADC-1, which is a compound represented by Ic, wherein p is 2.1, and the anti-Anti-Trop2 antibody is antibody C.
在一些实施方案中，抗Trop2抗体-药物偶联物的质量份数为20份～30份；在一些实施方案中，抗Trop2抗体-药物偶联物的质量份数为23份～27份；在一些实施方案中，抗Trop2抗体-药物偶联物的质量份数为25份。In some embodiments, the mass parts of the anti-Trop2 antibody-drug conjugate is 20 parts to 30 parts; in some embodiments, the mass parts of the anti-Trop2 antibody-drug conjugate is 23 parts to 27 parts; In some embodiments, the mass parts of the anti-Trop2 antibody-drug conjugate is 25 parts.
在一些实施方案中，所述稳定剂为海藻糖和/或蔗糖；在一些实施方案中，所述稳定剂为海藻糖。In some embodiments, the stabilizer is trehalose and/or sucrose; in some embodiments, the stabilizer is trehalose.
在一些实施方案中，所述稳定剂的质量份数为40份～80份；在一些实施方案中，所述稳定剂的质量份数为50份～70份；在一些实施方案中，所述稳定剂的质量份数为54或60份。In some embodiments, the mass parts of the stabilizer is 40 parts to 80 parts; in some embodiments, the mass parts of the stabilizer is 50 parts to 70 parts; in some embodiments, the mass parts of the stabilizer is 50 parts to 70 parts. The mass parts of the stabilizer is 54 or 60 parts.
在一些实施方案中，所述缓冲剂为组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂；在一些实施方案中，所述缓冲剂为组氨酸-琥珀酸缓冲剂。In some embodiments, the buffer is a histidine-succinate buffer or a histidine-citrate buffer; in some embodiments, the buffer is a histidine-succinate buffer.
在一些实施方案中，所述缓冲剂的质量份数为1份～10份；在一些实施方案中，所述缓冲剂的质量份数为3份～7份；在一些实施方案中，所述缓冲剂的质量份数为3份～4份。In some embodiments, the mass parts of the buffer is 1 to 10 parts; in some embodiments, the mass parts of the buffer is 3 to 7 parts; in some embodiments, the The mass parts of the buffer is 3 parts to 4 parts.
在一些实施方案中，所述缓冲剂包含质量份数为1.18份的琥珀酸和2.1份的盐酸组氨酸，其中部分或全部琥珀酸可以以相应量盐的形式存在，部分或全部盐酸组氨酸可以以相应量组氨酸形式存在。在一些实施方案中，所述缓冲剂包含质量份数为1.18份的琥珀酸和2.1份的盐酸组氨酸一水合物。In some embodiments, the buffer comprises 1.18 parts by mass of succinic acid and 2.1 parts of histidine hydrochloride, wherein part or all of the succinic acid may be in the form of a corresponding amount of salt, and part or all of histidine hydrochloride The acid may be present in the corresponding amount of histidine. In some embodiments, the buffer comprises 1.18 parts by mass of succinic acid and 2.1 parts of histidine hydrochloride monohydrate.
在一些实施方案中，所述表面活性剂为吐温20(即聚山梨酯20)或吐温80(即聚山梨酯80)；在一些实施方案中，所述表面活性剂为吐温80。In some embodiments, the surfactant is Tween 20 (ie, polysorbate 20) or Tween 80 (ie, polysorbate 80); in some embodiments, the surfactant is Tween 80.
在一些实施方案中，表面活性剂的质量份数为0.2份～0.4份；在一些实施方案中，表面活性剂的质量份数为0.25份～0.35份；在一些实施方案中，表面活性剂的质量份数为约0.3份。In some embodiments, the parts by mass of the surfactant is 0.2 to 0.4 parts; in some embodiments, the parts by mass of the surfactant is 0.25 to 0.35 parts; in some embodiments, the part by mass of the surfactant is The parts by mass is about 0.3 parts.
在一些实施方案中，所述固体制剂包括：质量份数为10份～50份的抗Trop2抗体-药物偶联物，质量份数为1份～10份的缓冲剂，质量份数为20份～80份的稳定剂，以及质量份数为0.1份～0.5份的表面活性剂；在一些实施方案中，所述固体制剂包括：质量份数为20份～30份的抗Trop2抗体-药物偶联物，质量份数为3份～7份的缓冲剂，质量份数为40份～80份的稳定剂，以及质量份数为0.2份～0.4份的表面活性剂；在一些实施方案中，所述固体制剂包括：质量份数为20份～30份的ADC-1，质量份数为3份～7份的组氨酸-琥珀酸缓冲剂，质量份数为40份～80份的海藻糖，以及质量份数为0.2份～0.4份的吐温80；在一些 实施方案中，所述固体制剂包括：质量份数为23份～27份的ADC-1，质量份数为3份～4份的组氨酸-琥珀酸缓冲剂，质量份数为50份～70份的海藻糖，以及质量份数为0.25份～0.35份的吐温80；在一些实施方案中，所述固体制剂包括：质量份数为25份的ADC-1，质量份数为1.18份的琥珀酸、2.1份盐酸组氨酸，质量份数为54份的海藻糖，以及质量份数为约0.3份的吐温80；在一些实施方案中，所述固体制剂包括：质量份数为25份的ADC-1，质量份数为1.18份的琥珀酸、2.1份盐酸组氨酸一水合物，质量份数为60份的海藻糖二水合物(或质量份数为54份的海藻糖)，以及质量份数为0.3份的吐温80。In some embodiments, the solid preparation includes: 10-50 parts by mass of the anti-Trop2 antibody-drug conjugate, 1-10 parts by mass of buffer, and 20 parts by mass. ~80 parts of stabilizer, and 0.1 parts to 0.5 parts of surfactant by mass; in some embodiments, the solid preparation includes: 20 parts to 30 parts of anti-Trop2 antibody-drug couple Conjugate, 3 to 7 parts by mass of buffer, 40 to 80 parts by mass of stabilizer, and 0.2 to 0.4 parts by mass of surfactant; in some embodiments, The solid preparation comprises: 20-30 parts by mass of ADC-1, 3-7 parts by mass of histidine-succinic acid buffer, and 40-80 parts by mass of seaweed Sugar, and 0.2 to 0.4 parts of Tween 80 by mass; in some embodiments, the solid preparation includes: 23 to 27 parts by mass of ADC-1, and 3 to 27 parts by mass of ADC-1 4 parts of histidine-succinic acid buffer, 50 parts to 70 parts of trehalose by mass, and 0.25 to 0.35 parts of Tween 80 by mass; in some embodiments, the solid formulation Including: 25 parts by mass ADC-1, 1.18 parts by mass succinic acid, 2.1 parts histidine hydrochloride, 54 parts by mass trehalose, and about 0.3 parts by mass Temperature 80; In some embodiments, the solid preparation includes: 25 parts by mass of ADC-1, 1.18 parts by mass of succinic acid, 2.1 parts of histidine hydrochloride monohydrate, and parts by mass of 60 parts of trehalose dihydrate (or 54 parts by mass of trehalose), and 0.3 parts of Tween 80 by mass.
在一些实施方案中，所述固体制剂为冻干制剂，其通过将包含所述抗Trop2抗体-药物偶联物的液体制剂冻干获得。在一些实施方案中，所述冻干制剂为粉末。In some embodiments, the solid preparation is a lyophilized preparation, which is obtained by lyophilizing a liquid preparation containing the anti-Trop2 antibody-drug conjugate. In some embodiments, the lyophilized formulation is a powder.
一方面，一些实施方案提供包含上文所述抗Trop2抗体-药物偶联物的液体制剂，所述液体制剂中还包含所述缓冲剂、稳定剂以及表面活性剂中的一种或多种。所述液体制剂可以用来制备上述冻干制剂，或为将上述冻干制剂复溶后得到的复溶制剂。In one aspect, some embodiments provide a liquid formulation comprising the anti-Trop2 antibody-drug conjugate described above, the liquid formulation further comprising one or more of the buffer, stabilizer, and surfactant. The liquid formulation can be used to prepare the above-mentioned lyophilized formulation, or a reconstituted formulation obtained after the above-mentioned lyophilized formulation is reconstituted.
在一些实施方案中，所述液体制剂的pH值为4.6～5.6；在一些实施方案中，所述液体制剂的pH值为4.6～5.2；在一些实施方案中，所述液体制剂的pH值为4.9。In some embodiments, the pH of the liquid formulation is 4.6 to 5.6; in some embodiments, the pH of the liquid formulation is 4.6 to 5.2; in some embodiments, the pH of the liquid formulation is 4.9.
在一些实施方案中，所述液体制剂包括：抗Trop2抗体-药物偶联物，其中所述偶联物的浓度为10mg/ml～50mg/ml；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种；所述稳定剂的浓度为20mg/ml～80mg/ml；所述缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的组合；所述表面活性剂的浓度为0.1mg/ml～0.5mg/ml。In some embodiments, the liquid formulation includes: an anti-Trop2 antibody-drug conjugate, wherein the concentration of the conjugate is 10 mg/ml to 50 mg/ml; it also includes stabilizers, buffers, and surfactants. The concentration of the stabilizer is 20mg/ml～80mg/ml; the buffer is a histidine buffer, a succinic acid buffer, a citric acid buffer or a combination thereof; the surface The concentration of the active agent is 0.1 mg/ml to 0.5 mg/ml.
在一些实施方案中，所述液体制剂中，缓冲剂的浓度为1mg/ml～10mg/ml；在一些实施方案中，所述缓冲剂的浓度为3mg/ml～7mg/ml；在一些实施方案中，所述缓冲剂的浓度为3mg/ml～4mg/ml。在一些实施方案中，所述缓冲剂包含浓度为1.18mg/ml的琥珀酸和2.1mg/ml的盐酸组氨酸。在一些实施方案中，所述缓冲剂包含浓度为1.18mg/ml(即10mM)的琥珀酸和2.1mg/ml(即10mM)的盐酸组氨酸一水合物。In some embodiments, in the liquid formulation, the concentration of the buffer is 1 mg/ml to 10 mg/ml; in some embodiments, the concentration of the buffer is 3 mg/ml to 7 mg/ml; in some embodiments Wherein, the concentration of the buffer is 3 mg/ml to 4 mg/ml. In some embodiments, the buffer comprises succinic acid at a concentration of 1.18 mg/ml and histidine hydrochloride at 2.1 mg/ml. In some embodiments, the buffer comprises succinic acid at a concentration of 1.18 mg/ml (ie, 10 mM) and histidine hydrochloride monohydrate at 2.1 mg/ml (ie, 10 mM).
在一些实施方案中，所述液体制剂中，缓冲剂的浓度为10mM～60mM；在一些实施方案中，所述液体制剂中，缓冲剂的浓度为20mM～40mM；在一些实施方案中，所述液体制剂中，缓冲剂的浓度为约20mM。在一些实施方案中，所述液体制剂中，缓冲剂为约10mM琥珀酸缓冲液和约10mM组氨酸缓冲液。In some embodiments, the concentration of the buffer in the liquid formulation is 10 mM to 60 mM; in some embodiments, the concentration of the buffer in the liquid formulation is 20 mM to 40 mM; in some embodiments, the concentration of the buffer is 20 mM to 40 mM; In the liquid formulation, the concentration of the buffer is about 20 mM. In some embodiments, in the liquid formulation, the buffer is about 10 mM succinate buffer and about 10 mM histidine buffer.
在一些实施方案中，所述液体制剂包含：10mg/ml～50mg/ml的抗Trop2抗体-药物偶联物，5mM～30mM的缓冲剂，20mg/ml～80mg/ml的稳定剂，以及0.1mg/ml～0.5mg/ml的表面活性剂。在一些实施方案中，所述液体制剂包含：25mg/ml的抗Trop2抗体-药物偶联物，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸，54mg/ml的海藻糖，0.3mg/ml的吐温80，pH为4.6-5.2。在一些实施方案中，所述液体制剂包含：25mg/ml的抗Trop2抗体-药物偶联物，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸一水合物，60mg/ml的海藻糖二水合物，0.3mg/ml的吐温80，pH为4.6-5.2。在一些实施方案中，所述液体制剂包含：25mg/ml的抗Trop2抗体-药物偶联物，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸，158mM的海藻糖，0.3mg/ml的吐温80，用氢氧化钠调pH至4.6-5.2。在一些实施方案中，所述液体制剂 包含：25mg/ml的ADC-1，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸，54mg/ml的海藻糖，0.3mg/ml的吐温80，pH为4.6-5.2。在一些实施方案中，所述液体制剂包含：25mg/ml的ADC-1，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸一水合物，54mg/ml的海藻糖，0.3mg/ml的吐温80，pH为4.6-5.2。在一些实施方案中，所述液体制剂包含：25mg/ml的ADC-1，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸，60mg/ml的海藻糖二水合物，0.3mg/ml的吐温80，pH为4.6-5.2。在一些实施方案中，所述液体制剂包含：25mg/ml的ADC-1，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸一水合物，60mg/ml的海藻糖二水合物，0.3mg/ml的吐温80，pH为4.6-5.2。在一些实施方案中，所述液体制剂包括，25mg/ml的抗Trop2抗体-药物偶联物，10mM的琥珀酸，10mM的盐酸组氨酸，158mM的海藻糖，0.3mg/ml的吐温80，用氢氧化钠调pH至4.6-5.2。In some embodiments, the liquid formulation comprises: 10mg/ml-50mg/ml anti-Trop2 antibody-drug conjugate, 5mM-30mM buffer, 20mg/ml-80mg/ml stabilizer, and 0.1mg /ml～0.5mg/ml surfactant. In some embodiments, the liquid formulation comprises: 25mg/ml of anti-Trop2 antibody-drug conjugate, 1.18mg/ml of succinic acid, 2.1mg/ml of histidine hydrochloride, 54mg/ml of trehalose, 0.3mg/ml Tween 80, pH is 4.6-5.2. In some embodiments, the liquid formulation comprises: 25mg/ml of anti-Trop2 antibody-drug conjugate, 1.18mg/ml of succinic acid, 2.1mg/ml of histidine hydrochloride monohydrate, 60mg/ml of Trehalose dihydrate, 0.3mg/ml Tween 80, pH 4.6-5.2. In some embodiments, the liquid formulation comprises: 25mg/ml of anti-Trop2 antibody-drug conjugate, 1.18mg/ml of succinic acid, 2.1mg/ml of histidine hydrochloride, 158mM of trehalose, 0.3mg /ml Tween 80, adjust the pH to 4.6-5.2 with sodium hydroxide. In some embodiments, the liquid formulation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride, 54 mg/ml trehalose, 0.3 mg/ml Tween 80, pH is 4.6-5.2. In some embodiments, the liquid formulation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride monohydrate, 54 mg/ml trehalose, 0.3 mg /ml Tween 80, the pH is 4.6-5.2. In some embodiments, the liquid formulation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride, 60 mg/ml trehalose dihydrate, 0.3 mg /ml Tween 80, the pH is 4.6-5.2. In some embodiments, the liquid formulation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride monohydrate, 60 mg/ml trehalose dihydrate , 0.3mg/ml Tween 80, pH is 4.6-5.2. In some embodiments, the liquid formulation includes 25 mg/ml anti-Trop2 antibody-drug conjugate, 10 mM succinic acid, 10 mM histidine hydrochloride, 158 mM trehalose, and 0.3 mg/ml Tween 80 , Adjust the pH to 4.6-5.2 with sodium hydroxide.
本发明提供一种制备所述的固体制剂的方法，其中，包括：The present invention provides a method for preparing the solid preparation, which comprises:
1)配制：取抗Trop2抗体-药物偶联物、稳定剂、缓冲剂、表面活性剂，溶解于水中至规定量，并调整pH值至规定值，得到液体制剂；1) Preparation: take the anti-Trop2 antibody-drug conjugate, stabilizer, buffer, and surfactant, dissolve it in water to a specified amount, and adjust the pH value to the specified value to obtain a liquid preparation;
2)冻干：将液体制剂进行冻干，得到所述的固体制剂。2) Lyophilization: freeze-dry the liquid preparation to obtain the solid preparation.
在一些实施方案中，冻干的步骤包括预冻、退火、一次干燥、二次干燥。In some embodiments, the step of freeze-drying includes pre-freezing, annealing, primary drying, and secondary drying.
并提供一种复溶制剂，其中，所述复溶制剂通过用药学上可接受的溶剂对所述的冻干制剂(即固体制剂)进行重构获得。A reconstituted preparation is also provided, wherein the reconstituted preparation is obtained by reconstitution of the lyophilized preparation (ie, solid preparation) with a pharmaceutically acceptable solvent.
在一些实施方案中，所述复溶制剂包括：抗Trop2抗体-药物偶联物，其中所述偶联物的浓度为10mg/ml～50mg/ml；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种；所述稳定剂的浓度为20mg/ml～80mg/ml；所述缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的组合；所述表面活性剂的浓度为0.1mg/ml～0.5mg/ml。In some embodiments, the reconstituted preparation includes: an anti-Trop2 antibody-drug conjugate, wherein the concentration of the conjugate is 10 mg/ml to 50 mg/ml; it also includes stabilizers, buffers and surfactants The concentration of the stabilizer is 20mg/ml～80mg/ml; the buffer is a histidine buffer, a succinic acid buffer, a citric acid buffer or a combination thereof; The concentration of the surfactant is 0.1 mg/ml to 0.5 mg/ml.
在一些实施方案中，所述复溶制剂中，缓冲剂的浓度为10mM～60mM；在一些实施方案中，所述复溶制剂中，缓冲剂的浓度为20mM～40mM；在一些实施方案中，所述复溶制剂中，缓冲剂的浓度为约20mM。In some embodiments, the concentration of the buffer in the reconstituted preparation is 10 mM to 60 mM; in some embodiments, the concentration of the buffer in the reconstituted preparation is 20 mM to 40 mM; in some embodiments, In the reconstituted preparation, the concentration of the buffer is about 20 mM.
在一些实施方案中，所述复溶制剂包含：10mg/ml～50mg/ml的抗Trop2抗体-药物偶联物，5mM～30mM的缓冲剂，20mg/ml～80mg/ml的稳定剂，以及0.1mg/ml～0.5mg/ml的表面活性剂。在一些实施方案中，所述复溶制剂包含：25mg/ml的ADC-1，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸，54mg/ml的海藻糖，0.3mg/ml的吐温80，pH为4.6-5.2。在一些实施方案中，所述复溶制剂包含：25mg/ml的ADC-1，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸一水合物，60mg/ml的海藻糖二水合物，0.3mg/ml的吐温80，pH为4.6-5.2。In some embodiments, the reconstituted formulation comprises: 10 mg/ml-50 mg/ml anti-Trop2 antibody-drug conjugate, 5 mM-30 mM buffer, 20 mg/ml-80 mg/ml stabilizer, and 0.1 Surfactant with mg/ml～0.5mg/ml. In some embodiments, the reconstituted preparation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride, 54 mg/ml trehalose, 0.3 mg/ml Tween 80, the pH is 4.6-5.2. In some embodiments, the reconstituted preparation comprises: 25 mg/ml ADC-1, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride monohydrate, 60 mg/ml trehalose dihydrate The product, 0.3mg/ml Tween 80, pH is 4.6-5.2.
本发明的固体制剂可有效解决ADC不稳定、易聚集的问题，并具有优异的长期稳定性和加速稳定性。The solid preparation of the present invention can effectively solve the problems of ADC instability and easy aggregation, and has excellent long-term stability and accelerated stability.
本发明的固体制剂在冻干后、复溶后、以及复溶配伍稀释后，各项指标变化不大，结合活性也在正常范围内，稳定性好，适合长期保存。在冻干制剂在重构后，ADC保持稳定，不易形成聚集体或颗粒。After the solid preparation of the present invention is freeze-dried, reconstituted, and reconstituted and diluted, various indicators have little change, the binding activity is also within the normal range, the stability is good, and it is suitable for long-term storage. After the lyophilized preparation is reconstituted, the ADC remains stable and is not easy to form aggregates or particles.
本发明提供所述的固体制剂在制备用于预防和/或治疗Trop2阳性疾病的药物中的应用。 在一些实施方案中，本发明提供一种试剂盒，试剂盒包括所述液体制剂或固体制剂，以及用于指导有需要患者给药的说明书。另提供一种预防和/或治疗Trop2阳性疾病的方法，包括向需要的患者注射有效量的所述复溶制剂或液体制剂。在一些实施方案中，所述Trop2阳性疾病包括但不限于三阴性乳腺癌、胶质母细胞瘤、成神经管细胞瘤、非小细胞肺癌、小细胞肺癌、上皮癌、乳腺癌、头颈癌、肾癌、卵巢癌、胃癌、卡波西肉瘤、胰腺癌和肺癌、***、结肠直肠癌、食管癌、口腔鳞状细胞癌、***癌、甲状腺癌、膀胱癌、神经胶质瘤、肝胆管癌、结肠直肠癌、T细胞淋巴瘤。在一些实施方案中，所述Trop2阳性疾病包括但不限于非小细胞肺癌、小细胞肺癌、上皮癌。The invention provides the application of the solid preparation in the preparation of medicines for preventing and/or treating Trop2-positive diseases. In some embodiments, the present invention provides a kit, which includes the liquid preparation or solid preparation, and instructions for instructing patients in need of administration. Another method for preventing and/or treating Trop2-positive disease includes injecting an effective amount of the reconstituted preparation or liquid preparation into a patient in need. In some embodiments, the Trop2-positive disease includes, but is not limited to, triple-negative breast cancer, glioblastoma, medulloblastoma, non-small cell lung cancer, small cell lung cancer, epithelial cancer, breast cancer, head and neck cancer, Kidney cancer, ovarian cancer, stomach cancer, Kaposi's sarcoma, pancreatic cancer and lung cancer, cervical cancer, colorectal cancer, esophageal cancer, oral squamous cell carcinoma, prostate cancer, thyroid cancer, bladder cancer, glioma, hepatobiliary duct Cancer, colorectal cancer, T cell lymphoma. In some embodiments, the Trop2-positive disease includes, but is not limited to, non-small cell lung cancer, small cell lung cancer, and epithelial cancer.
附图说明Description of the drawings
图1：不同样品SEC-HPLC单体纯度随时间的变化趋势；Figure 1: The variation trend of SEC-HPLC monomer purity of different samples over time;
图1：不同样品SEC-HPLC聚体随时间的变化趋势；Figure 1: The trend of SEC-HPLC aggregates of different samples over time;
图2：不同样品SEC-HPLC片段随时间的变化趋势；Figure 2: The trend of SEC-HPLC fragments of different samples over time;
图3：不同样品HIC-HPLC主峰(D2)含量随时间的变化趋势；Figure 3: The trend of the content of the main HIC-HPLC peak (D2) of different samples over time;
图5：不同pH样品SEC-HPLC单体纯度随时间的变化趋势；Figure 5: SEC-HPLC monomer purity of samples with different pH changes over time;
图6：不同pH样品SEC-HPLC聚体含量随时间的变化趋势；Figure 6: The change trend of SEC-HPLC polymer content in different pH samples over time;
图7：不同pH样品HIC-HPLC主峰含量随时间的变化趋势；Figure 7: The change trend of HIC-HPLC main peak content of different pH samples over time;
图8：样品在不同缓冲液中SEC-HPLC单体纯度随时间的变化趋势；Figure 8: SEC-HPLC monomer purity of samples in different buffers over time;
图9：样品在不同缓冲液中SEC-HPLC聚体随时间的变化趋势；Figure 9: The trend of SEC-HPLC aggregates of samples in different buffers over time;
图10：样品在不同缓冲液中SEC-HPLC片段随时间的变化趋势；Figure 10: The change trend of SEC-HPLC fragments of samples in different buffers over time;
图11：样品在不同缓冲液中HIC-HPLC主峰含量随时间的变化趋势；Figure 11: The trend of HIC-HPLC main peak content of samples in different buffers over time;
图12：不同稳定剂样品SEC-HPLC单体纯度随时间的变化趋势；Figure 12: SEC-HPLC monomer purity of different stabilizer samples over time;
图13：不同稳定剂样品SEC-HPLC聚体随时间的变化趋势；Figure 13: The change trend of SEC-HPLC aggregates of different stabilizer samples over time;
图14：不同稳定剂样品HIC-HPLC主峰含量随时间的变化趋势；Figure 14: The change trend of HIC-HPLC main peak content of different stabilizer samples over time;
图15：不同表面活性剂样品配伍后的微粒数(≥25μm)(个/ml)；Figure 15: Number of particles (≥25μm) (pieces/ml) after the compatibility of different surfactant samples;
图16：不同表面活性剂样品配伍后的微粒数(≥10μm)(个/ml)。Figure 16: Number of particles (≥10μm) (pieces/ml) after the compatibility of different surfactant samples.
具体实施方案Specific implementation plan
下面将通过具体实施方案来说明本发明，但本发明的内容不限于此。The present invention will be described below through specific embodiments, but the content of the present invention is not limited thereto.
需要说明的是，本发明中，涉及到液体制剂组分的“％”指重量体积(w/v)百分比，其中重量单位可以为g，体积单位可以为ml。比如溶液中含1％稳定剂表示100ml溶液中含有1g稳定剂，或稳定剂含量为0.01g/ml。It should be noted that in the present invention, the "%" related to the components of the liquid preparation refers to the weight volume (w/v) percentage, where the weight unit may be g and the volume unit may be ml. For example, the solution containing 1% stabilizer means that 100ml of the solution contains 1g stabilizer, or the stabilizer content is 0.01g/ml.
术语“约”当在数值之前使用时指示这个值可以在合理的范围内变化，诸如在规定值的±10％、±5％或±1％以内。“约x”包括“x”。The term "about" when used before a value indicates that the value can vary within a reasonable range, such as within ±10%, ±5%, or ±1% of the specified value. "About x" includes "x".
如本文所用的术语“有效量”或“治疗有效量”指的是正在施用的药剂的足够量，其将使正在治疗的疾病或病状的一个或多个症状减轻至一定程度。结果可以是疾病的征象、 症状或病因的减少和/或缓和，或生物***的任何其它所需的改变。举例来说，用于治疗用途的“有效量”是提供疾病症状的临床上显著的减少而无过度的不良副作用所需要的包括如本文所公开的制剂的量。“有效量”或“治疗有效量”可能因患者，正在治疗的病状，正在治疗的病状的严重性，以及处方医师的判断而异。The term "effective amount" or "therapeutically effective amount" as used herein refers to a sufficient amount of an agent being administered that will reduce one or more symptoms of the disease or condition being treated to a certain degree. The result can be a reduction and/or alleviation of the signs, symptoms, or causes of the disease, or any other desired changes in the biological system. For example, an "effective amount" for therapeutic use is the amount required to provide a clinically significant reduction in disease symptoms without undue adverse side effects including the formulation as disclosed herein. The "effective amount" or "therapeutically effective amount" may vary depending on the patient, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.
如本文所用的关于配方、固体制剂或成分的术语“可接受的”或“医药学上可接受的”意味着对所治疗的患者的一般健康状况不具有持续的有害影响或并不消除化合物的生物活性或特性，并且是相对无毒的。As used herein, the term "acceptable" or "pharmaceutically acceptable" with respect to formulations, solid preparations or ingredients means that it does not have a lasting deleterious effect on the general health of the patient being treated or does not eliminate the compound’s It is biologically active or characteristic, and is relatively non-toxic.
ADC可以形成广泛多种医药学上可接受的盐，包括但不限于：与有机酸形成的酸加成盐，这种有机酸包括但不限于脂肪族单羧酸和二羧酸、苯基取代的烷酸、羟基烷酸、烷二酸、芳香酸、脂肪族和芳香族磺酸、氨基酸等等，例如乙酸、三氟乙酸、丙酸、乙醇酸、丙酮酸、草酸、马来酸、丙二酸、琥珀酸、富马酸、酒石酸、柠檬酸、苯甲酸、肉桂酸、扁桃酸、甲磺酸、乙磺酸、对甲苯磺酸、水杨酸，等等；与无机酸反应形成的酸加成盐，这种无机酸包括盐酸、氢溴酸、硫酸、硝酸、磷酸、氢碘酸、氢氟酸、亚磷酸，等等；及与金属离子(例如，碱金属离子(例如钠或钾)、碱土金属离子(例如钙或镁)或铝离子)或者与有机碱如二乙醇胺、三乙醇胺、N-甲基葡糖胺等等形成的盐。ADC can form a wide variety of pharmaceutically acceptable salts, including but not limited to: acid addition salts formed with organic acids, including but not limited to aliphatic monocarboxylic and dicarboxylic acids, phenyl substituted Alkanoic acid, hydroxyalkanoic acid, alkanedioic acid, aromatic acid, aliphatic and aromatic sulfonic acid, amino acid, etc., such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, propylene Diacid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.; formed by reaction with inorganic acids Acid addition salts, such inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, etc.; and metal ions (e.g., alkali metal ions (e.g., sodium or Potassium), alkaline earth metal ions (e.g. calcium or magnesium) or aluminum ions) or salts formed with organic bases such as diethanolamine, triethanolamine, N-methylglucamine and the like.
如本文所用的术语“个体”或“患者”指的是作为治疗、观测或实验的目标的动物。仅举例来说，患者可以是(但不限于)哺乳动物，包括(但不限于)人类。The term "individual" or "patient" as used herein refers to an animal that is the target of treatment, observation, or experiment. For example only, the patient may be (but not limited to) a mammal, including (but not limited to) a human.
本发明所述的“质量份数”是指相等单位质量情况下的数值，单位质量即每份的质量，可以是任何适合的质量。如每份是1g时，单位质量就是1g/份，此时20份即为20g；单位质量也可以是1kg/份或1mg/份。此外，单位质量不仅限于整数，例如可以为0.5g/份，此时20份即为10g。The "parts by mass" in the present invention refers to the value in the case of equal unit mass, and the unit mass is the mass of each part, which can be any suitable mass. For example, when each serving is 1g, the unit mass is 1g/ serving, at this time 20 servings is 20g; the unit mass can also be 1kg/ serving or 1mg/ serving. In addition, the unit mass is not limited to an integer, for example, it can be 0.5 g/part, and 20 parts is 10 g.
术语“缓冲剂”，在一些文献中，也被称为缓冲***或缓冲体系，其包括但不限于有机酸盐，如琥珀酸、醋酸、柠檬酸，抗坏血酸，葡糖酸，碳酸，酒石酸或苯二甲酸及其盐；Tris，thomethamine盐酸盐，或磷酸盐缓冲剂。此外，氨基酸及其盐也可以用作缓冲剂。本发明中的缓冲剂，可以是一种，如组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂等；也可以是多种，当涉及多种缓冲剂组成时，采用“-”表示，如组氨酸-琥珀酸缓冲剂，表示由组氨酸缓冲剂和琥珀酸缓冲剂组成；组氨酸-柠檬酸缓冲剂，表示由组氨酸缓冲剂和柠檬酸缓冲剂组成等。应当注意，以组氨酸-琥珀酸缓冲剂为例，并非表示该缓冲剂由组氨酸单体和琥珀酸单体组成，而是表示该缓冲剂由组成组氨酸缓冲剂的酸(或碱)和/或盐，以及组成琥珀酸缓冲剂的酸(或碱)和/或盐组成；如可以由组氨酸和琥珀酸钠组成，可以由盐酸组氨酸和琥珀酸组成，可以由盐酸组氨酸和琥珀酸钠组成，可以由组氨酸、盐酸组氨酸、琥珀酸和琥珀酸钠组成等。在一些实施方案中，组氨酸-琥珀酸缓冲剂由盐酸组氨酸和琥珀酸钠组成。The term "buffer", in some documents, is also called a buffer system or a buffer system, which includes but is not limited to organic acid salts such as succinic acid, acetic acid, citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid or benzene Dicarboxylic acid and its salts; Tris, thomethamine hydrochloride, or phosphate buffer. In addition, amino acids and their salts can also be used as buffers. The buffer in the present invention can be one, such as histidine buffer, succinic acid buffer, citric acid buffer, etc.; it can also be multiple, and when it involves multiple buffers, "-" is used to indicate For example, histidine-succinic acid buffer means composed of histidine buffer and succinic acid buffer; histidine-citrate buffer means composed of histidine buffer and citric acid buffer, etc. It should be noted that taking histidine-succinic acid buffer as an example, it does not mean that the buffer is composed of histidine monomers and succinic acid monomers, but that the buffer is composed of the acid (or Base) and/or salt, and the acid (or base) and/or salt constituting the succinic acid buffer; for example, it can be composed of histidine and sodium succinate, can be composed of histidine hydrochloride and succinic acid, and can be composed of It is composed of histidine hydrochloride and sodium succinate, and can be composed of histidine, histidine hydrochloride, succinic acid, and sodium succinate. In some embodiments, the histidine-succinic acid buffer consists of histidine hydrochloride and sodium succinate.
本发明中缓冲剂的量，是指组成缓冲剂的缓冲体系中缓冲对的总量。在一些实施方案中，采用摩尔浓度作为缓冲剂的量的单位，其数值指缓冲剂的缓冲体系中缓冲对的摩尔浓度。如，由盐酸组氨酸和琥珀酸组成的组氨酸-琥珀酸缓冲剂作为缓冲剂时，给定浓度的组 氨酸-琥珀酸缓冲剂(如20mM)是盐酸组氨酸和琥珀酸的组合浓度(如盐酸组氨酸为10mM，琥珀酸为10mM。)The amount of buffer in the present invention refers to the total amount of buffer pairs in the buffer system constituting the buffer. In some embodiments, the molar concentration is used as the unit of the amount of the buffer, and its value refers to the molar concentration of the buffer pair in the buffer system of the buffer. For example, when a histidine-succinic acid buffer composed of histidine hydrochloride and succinic acid is used as a buffer, a given concentration of histidine-succinic acid buffer (such as 20mM) is a combination of histidine hydrochloride and succinic acid Combined concentration (e.g. histidine hydrochloride is 10mM, succinic acid is 10mM.)
本发明所述的制剂可以用所述辅料或其水合物配制。比如海藻糖，可以是无水海藻糖，也可以是海藻糖水合物，如海藻糖二水合物。如所述的“5.4％海藻糖”，为5.4g海藻糖(或相应量的海藻糖水合物(如6g海藻糖二水合物))溶解在溶剂里形成100ml溶液，有特别说明的除外。The formulation of the present invention can be formulated with the adjuvant or its hydrate. For example, trehalose can be anhydrous trehalose or trehalose hydrate, such as trehalose dihydrate. As mentioned, "5.4% trehalose" is 5.4g trehalose (or a corresponding amount of trehalose hydrate (such as 6g trehalose dihydrate)) dissolved in a solvent to form a 100ml solution, unless otherwise specified.
适于偶联连接子的药物是本领域各种已知的或可预见的细胞毒性试剂，包括抗有丝***细胞毒药物和DNA裂解细胞毒药物。在一些实施方案中，药物为抗有丝***细胞毒药物。在一些实施方案中，抗Trop2抗体-药物偶联物(以下简称ADC)中的药物可以是美登素或其衍生物，也可以是其它有肿瘤细胞毒性的化合物，如喜树碱或其衍生物，澳瑞他汀类似物等。在一些实施方案中，药物为DM1。美登醇类似物及衍生物可包括美登素及美登素类似物，它们可以按照已知方法从天然源分离、使用生物技术制造(见如：Yu等，99PNAS7968-7973(2002))、或按照已知方法合成制备(见如：Cassady等，Chem.Pharm.Bull.52(1)1-26(2004))。取决于连接子的类型，美登醇上的很多位置可用作连接位置。例如，对于形成酯键，C-3位置具有羟基、C-14位置为羟甲基修饰、C-15位置为羟基修饰以及C-20位置具有羟基都是合适的。The drugs suitable for coupling the linker are various known or foreseeable cytotoxic agents in the art, including anti-mitotic cytotoxic drugs and DNA lytic cytotoxic drugs. In some embodiments, the drug is an anti-mitotic cytotoxic drug. In some embodiments, the drug in the anti-Trop2 antibody-drug conjugate (hereinafter referred to as ADC) can be maytansine or its derivatives, or other tumor cytotoxic compounds, such as camptothecin or its derivatives. Substances, auristatin analogs, etc. In some embodiments, the drug is DM1. Maytansinol analogs and derivatives may include maytansine and maytansine analogs, which can be isolated from natural sources according to known methods and manufactured using biotechnology (see, e.g., Yu et al., 99PNAS7968-7973 (2002)), Or synthetically prepared according to known methods (see: Cassady et al., Chem. Pharm. Bull. 52(1) 1-26 (2004)). Depending on the type of linker, many locations on maytansinol can be used as attachment locations. For example, for forming an ester bond, it is suitable that the C-3 position has a hydroxyl group, the C-14 position is a hydroxymethyl modification, the C-15 position is a hydroxyl modification, and the C-20 position has a hydroxyl group.
在一些实施方案中，ADC的抗体，其能特异性的作用于滋养层细胞表面糖蛋白抗原2。在一些实施方案中，抗Trop2抗体为抗体A、抗体B和/或抗体C。在一些实施方案中，抗体A的轻链包括SEQ ID NO:1所示的氨基酸序列，重链包括SEQ ID NO:2所示的氨基酸序列；抗体B和抗体C的轻链包括SEQ ID NO:3所示的氨基酸序列，重链包括SEQ ID NO:4所示的氨基酸序列。在一些实施方案中，抗体A的氨基酸序列和抗体B的氨基酸序列表达于悬浮的CHO细胞中，该细胞系为中华仓鼠卵巢细胞系CHO-K1(ATCC#CCL-61)驯化后适应悬浮生长而来。抗体C的氨基酸序列表达于CHO-BAT-KF fut8(-/-)细胞中。在一些实施方案中，抗Trop2抗体为抗体C。In some embodiments, the ADC antibody can specifically act on the trophoblast cell surface glycoprotein antigen 2. In some embodiments, the anti-Trop2 antibody is antibody A, antibody B, and/or antibody C. In some embodiments, the light chain of antibody A includes the amino acid sequence shown in SEQ ID NO: 1, the heavy chain includes the amino acid sequence shown in SEQ ID NO: 2; the light chain of antibody B and antibody C includes SEQ ID NO: The amino acid sequence shown in 3, the heavy chain includes the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the amino acid sequence of antibody A and the amino acid sequence of antibody B are expressed in suspended CHO cells. The cell line is the Chinese hamster ovary cell line CHO-K1 (ATCC#CCL-61) after acclimatization to adapt to suspension growth. Come. The amino acid sequence of antibody C is expressed in CHO-BAT-KF fut8(-/-) cells. In some embodiments, the anti-Trop2 antibody is antibody C.
各序列编号对应的序列如表1所示。The sequence corresponding to each sequence number is shown in Table 1.
表1Table 1
Figure PCTCN2020140885-appb-000004
Figure PCTCN2020140885-appb-000004
Figure PCTCN2020140885-appb-000005
Figure PCTCN2020140885-appb-000005
抗Trop2抗体与药物通过连接子偶联，得到ADC。适用于本发明的连接子包括可断裂连接子和不可断裂连接子。其中，可断裂连接子通常具有特殊的敏感型结构，这种连接子在正常环境下稳定，在肿瘤环境下，能够断裂，进而释放药物，以起到治疗作用。常见的可断裂连接子有蛋白酶敏感型的连接子多含有蛋白酶可识别断裂的二肽键，多见为缬氨酸-瓜氨酸连接子(val-cit)；还有酸敏感型连接子，多为含有腙键的连接子；另外一种为谷胱甘肽敏感型连接子，通常含有谷胱甘肽可还原的二硫键。不可断裂连接子在细胞内代谢后仍保持完整。含有此类连接子的抗体-药物偶联物，需要经过溶酶体降解，才能释放出细胞毒分子。与可断裂连接子相比，不可断裂连接子在循环***中，具有良好的稳定性。在一些实施方案中，连接子为不可断裂连接子。The anti-Trop2 antibody is coupled with the drug through a linker to obtain ADC. Linkers suitable for use in the present invention include cleavable linkers and non-cleavable linkers. Among them, the cleavable linker usually has a special sensitive structure. This linker is stable in a normal environment and can be broken under a tumor environment to release drugs to play a therapeutic role. Common cleavable linkers include protease-sensitive linkers. Most of them contain dipeptide bonds that can be broken by proteases, and most commonly are val-citrulline linkers (val-cit); there are also acid-sensitive linkers. Most are linkers containing hydrazone bonds; the other is a glutathione-sensitive linker, which usually contains glutathione reducible disulfide bonds. The non-cleavable linker remains intact after being metabolized in the cell. Antibody-drug conjugates containing such linkers need to undergo lysosomal degradation to release cytotoxic molecules. Compared with the cleavable linker, the non-cleavable linker has good stability in the circulatory system. In some embodiments, the linker is a non-cleavable linker.
在一些实施方案中，抗Trop2抗体-药物偶联物包含如式Ia或Ib的化合物：In some embodiments, the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ia or Ib:
Figure PCTCN2020140885-appb-000006
Figure PCTCN2020140885-appb-000006
或其药学上可接受的盐或溶剂合物，Or a pharmaceutically acceptable salt or solvate thereof,
其中among them
X为氢或卤素；X is hydrogen or halogen;
Y选自氢、C1-C6烷基、C3-C6环烷基和-C(＝O)R 5； Y is selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl and -C(=O)R 5 ;
R 1选自H、-OH、-OC(＝O)R 5和-OR 5基团； R 1 is selected from H, -OH, -OC(=O)R 5 and -OR 5 groups;
R 2为H或C1-C6烷基； R 2 is H or C1-C6 alkyl;
R 3为甲基、-CH2OH或-CH2OC(＝O)R 6； R 3 is methyl, -CH2OH or -CH2OC(=O)R 6 ;
R 4为-OH或–SH； R 4 is -OH or -SH;
R 5为C1-C6烷基或苄基； R 5 is C1-C6 alkyl or benzyl;
R 6为C1-C6烷基、苯基或苄基； R 6 is C1-C6 alkyl, phenyl or benzyl;
R 7为氢、C1-C6烷基或氨基酸侧链； R 7 is hydrogen, C1-C6 alkyl or amino acid side chain;
R 8为氢或者C1-C6烷基； R 8 is hydrogen or C1-C6 alkyl;
n为0、1、2、3、4、5、6、7或8；n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
p为1-10；p is 1-10;
Anti-Trop2为抗Trop2抗体。Anti-Trop2 is an anti-Trop2 antibody.
在一些实施方案中，抗Trop2抗体-药物偶联物包含如式Ic的化合物：In some embodiments, the anti-Trop2 antibody-drug conjugate comprises a compound of formula Ic:
Figure PCTCN2020140885-appb-000007
Figure PCTCN2020140885-appb-000007
或其药学上可接受的盐或溶剂合物，其中，p为1-10；Anti-Trop2为抗Trop2抗体。在一些实施方案中，Anti-Trop2为抗体C。Or a pharmaceutically acceptable salt or solvate thereof, wherein p is 1-10; Anti-Trop2 is an anti-Trop2 antibody. In some embodiments, Anti-Trop2 is antibody C.
药物抗体偶联比(DAR)为偶联物中药物与抗体的摩尔比。在一些实施方案中，DAR选自1、2、3、4、5、6、7、8、9或10；在一些实施方案中，DAR选自2、3、4、5或6；在一些实施方案中，DAR选自2。The drug-to-antibody coupling ratio (DAR) is the molar ratio of the drug to the antibody in the conjugate. In some embodiments, DAR is selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; in some embodiments, DAR is selected from 2, 3, 4, 5, or 6; in some In an embodiment, DAR is selected from 2.
p为ADC的平均DAR。在一些实施方案中，p为1-10；在一些实施方案中，p为2-6；在一些实施方案中，p为2-3；在一些实施方案中，p约为2.1。在一些实施方案中，ADC为式Ic所示的结构，其中Anti-Trop2为抗体C，p约为2.1，该ADC为文中描述的ADC-1。p is the average DAR of the ADC. In some embodiments, p is 1-10; in some embodiments, p is 2-6; in some embodiments, p is 2-3; in some embodiments, p is about 2.1. In some embodiments, the ADC is the structure represented by Formula Ic, wherein Anti-Trop2 is antibody C, p is about 2.1, and the ADC is ADC-1 described herein.
在一些实施方案中，本发明提供一种固体制剂，包括：ADC，其中ADC的质量份数为约10份～约50份；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种；稳定剂的质量份数为约20份～约80份；缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的组合；表面活性剂的质量份数为约0.1份～约0.5份。In some embodiments, the present invention provides a solid preparation comprising: ADC, wherein the mass parts of ADC is about 10 parts to about 50 parts; further comprising one or more of stabilizers, buffers and surfactants Species; the mass parts of the stabilizer is about 20 parts to about 80 parts; the buffer is histidine buffer, succinic acid buffer, citric acid buffer or a combination thereof; the mass parts of the surfactant is about 0.1 Servings ~ about 0.5 servings.
在一些实施方案中，ADC的质量份数为约20份～约30份；在一些实施方案中，ADC的质量份数为23份～27份；在一些实施方案中，ADC的质量份数为约25份。在一些实施方案中，ADC的质量份数约为约10份、约20份、约23份、约25份、约27份、约30、约40份、约50份或任意两个质量份数之间的范围，包括端点值。In some embodiments, the mass parts of ADC is about 20 parts to about 30 parts; in some embodiments, the mass parts of ADC is 23 parts to 27 parts; in some embodiments, the mass parts of ADC is About 25 servings. In some embodiments, the parts by mass of the ADC are about 10 parts, about 20 parts, about 23 parts, about 25 parts, about 27 parts, about 30, about 40 parts, about 50 parts, or any two parts by mass The range between, including endpoint values.
在一些实施方案中，ADC为ADC-1；在一些实施方案中，ADC-1的质量份数为约20份～约30份；在一些实施方案中，ADC-1的质量份数为约23份～约27份；在一些实施方案中，ADC-1的质量份数为约25份。In some embodiments, the ADC is ADC-1; in some embodiments, the mass parts of ADC-1 is about 20 parts to about 30 parts; in some embodiments, the mass parts of ADC-1 is about 23 parts. Parts to about 27 parts; in some embodiments, the mass parts of ADC-1 is about 25 parts.
在一些实施方案中，稳定剂的质量份数为约40份～约80份；在一些实施方案中，稳定剂的质量份数为约50份～约70份；在一些实施方案中，稳定剂的质量份数为约54份。在一些实施方案中，稳定剂的质量份数为约20份、约40份、约50份、约54份、约60份、约70份、约80份或任意两个质量份数之间的范围，包括端点值。In some embodiments, the mass parts of the stabilizer is about 40 parts to about 80 parts; in some embodiments, the mass parts of the stabilizer is about 50 parts to about 70 parts; in some embodiments, the stabilizer The number of parts by mass is about 54 parts. In some embodiments, the mass parts of the stabilizer is about 20 parts, about 40 parts, about 50 parts, about 54 parts, about 60 parts, about 70 parts, about 80 parts, or any two parts by mass. Range, including endpoint values.
在一些实施方案中，稳定剂为海藻糖及其水合物和蔗糖及其水合物中的一种或多种；在一些实施方案中，稳定剂为海藻糖。在一些实施方案中，稳定剂为质量份数50份～70份的海藻糖；在一些实施方案中，稳定剂为质量份数约54份的海藻糖或约60份的海藻糖二水合物。In some embodiments, the stabilizer is one or more of trehalose and its hydrates and sucrose and its hydrates; in some embodiments, the stabilizer is trehalose. In some embodiments, the stabilizer is 50 parts to 70 parts by mass of trehalose; in some embodiments, the stabilizer is about 54 parts by mass of trehalose or about 60 parts by mass of trehalose dihydrate.
在一些实施方案中，缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的 组合。在一些实施方案中，缓冲剂为组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂；在一些实施方案中，缓冲剂为组氨酸-琥珀酸缓冲剂。应理解，本申请中缓冲剂(例如组氨酸缓冲剂、枸橼酸缓冲剂、琥珀酸缓冲剂)与酸(如盐酸)或碱(如NaOH或KOH)配合使用，形成缓冲体系。缓冲体系可以是弱酸及其盐的体系，也可以是弱碱及其盐的体系，视具体使用缓冲剂而定。本申请中无论所述缓冲剂处于酸和/或盐的形式，当提及缓冲剂的质量份数或摩尔浓度时，均以其总酸根离子计。比如，当缓冲体系为琥珀酸(HOOCCH 2CH 2COOH)缓冲剂，无论其中琥珀酸以HOOCCH 2CH 2COOH，HOOCCH 2CH 2COO -和/或 -OOCCH 2CH 2COO -的形式存在，琥珀酸缓冲剂的量指HOOCCH 2CH 2COOH，HOOCCH 2CH 2COO -和 -OOCCH 2CH 2COO -的总量以其酸根离子 -OOCCH 2CH 2COO -计。当缓冲体系为组氨酸-琥珀酸缓冲剂，缓冲剂的质量份数为琥珀酸的质量份数与盐酸组氨酸的质量份数的总和。 In some embodiments, the buffer is a histidine buffer, a succinic acid buffer, a citric acid buffer, or a combination thereof. In some embodiments, the buffer is a histidine-succinate buffer or a histidine-citrate buffer; in some embodiments, the buffer is a histidine-succinate buffer. It should be understood that in the present application, buffers (for example, histidine buffer, citrate buffer, succinic acid buffer) are used in combination with acids (such as hydrochloric acid) or bases (such as NaOH or KOH) to form a buffer system. The buffer system can be a system of a weak acid and its salt, or a system of a weak base and its salt, depending on the specific buffer used. In this application, regardless of the buffering agent in the form of acid and/or salt, when the mass parts or molar concentration of the buffering agent is mentioned, it is all based on its total acid radical ions. For example, when the buffer system is a succinic acid (HOOCCH 2 CH 2 COOH) buffer, no matter which succinic acid exists in the form of HOOCCH 2 CH 2 COOH, HOOCCH 2 CH 2 COO - and/or - OOCCH 2 CH 2 COO -, amber The amount of acid buffer refers to the total amount of HOOCCH 2 CH 2 COOH, HOOCCH 2 CH 2 COO - and - OOCCH 2 CH 2 COO - based on the acid radical ion - OOCCH 2 CH 2 COO - . When the buffer system is a histidine-succinic acid buffer, the mass parts of the buffer is the sum of the mass parts of succinic acid and the mass parts of histidine hydrochloride.
在一些实施方案中，缓冲剂的质量份数为约1份～约10份；在一些实施方案中，缓冲剂的质量份数为约3份～约7份；在一些实施方案中，缓冲剂的质量份数为约3份～约4份。在一些实施方案中，缓冲剂包含质量份数约为1.18份的琥珀酸和约2.1份的盐酸组氨酸。在一些实施方案中，缓冲剂包含质量份数约为1.18份的琥珀酸和约2.1份的盐酸组氨酸一水合物。In some embodiments, the parts by mass of the buffer is about 1 part to about 10 parts; in some embodiments, the parts by mass of the buffer is about 3 parts to about 7 parts; in some embodiments, the buffer The parts by mass of is about 3 parts to about 4 parts. In some embodiments, the buffer comprises about 1.18 parts by mass of succinic acid and about 2.1 parts by mass of histidine hydrochloride. In some embodiments, the buffer comprises about 1.18 parts by mass of succinic acid and about 2.1 parts of histidine hydrochloride monohydrate.
在一些实施方案中，表面活性剂的质量份数为约0.2份～约0.4份；在一些实施方案中，表面活性剂的质量份数为约0.25份～约0.35份；在一些实施方案中，表面活性剂的质量份数约为约0.3份。在一些实施方案中，表面活性剂的质量份数为约0.1份、约0.2份、约0.25份、0.3份、约0.35份、约0.4份、约0.5份或任意两个质量份数之间的范围，包括端点值。In some embodiments, the parts by mass of the surfactant is about 0.2 parts to about 0.4 parts; in some embodiments, the parts by mass of the surfactant is about 0.25 parts to about 0.35 parts; in some embodiments, The mass parts of the surfactant is about 0.3 parts. In some embodiments, the mass parts of the surfactant is about 0.1 parts, about 0.2 parts, about 0.25 parts, 0.3 parts, about 0.35 parts, about 0.4 parts, about 0.5 parts, or between any two parts by mass. Range, including endpoint values.
在一些实施方案中，表面活性剂为吐温20或吐温80；在一些实施方案中，表面活性剂为吐温80。在一些实施方案中，表面活性剂为质量份数约0.25份～约0.35份的吐温80；在一些实施方案中，表面活性剂为质量份数约0.3份的吐温80。In some embodiments, the surfactant is Tween 20 or Tween 80; in some embodiments, the surfactant is Tween 80. In some embodiments, the surfactant is about 0.25 to about 0.35 parts by mass of Tween 80; in some embodiments, the surfactant is about 0.3 parts by mass of Tween 80.
在一些实施方案中，固体制剂包括：质量份数为约10份～约50份的ADC(比如ADC-1)，质量份数为约1份～约10份的缓冲剂(比如组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂)，质量份数为约20份～约80份的稳定剂(比如海藻糖或蔗糖或其水合物)，以及质量份数为约0.1份～约0.5份的表面活性剂(比如吐温20或吐温80)。In some embodiments, the solid preparation includes: about 10 parts to about 50 parts by mass of ADC (such as ADC-1), and about 1 part to about 10 parts by mass of a buffer (such as histidine- Succinic acid buffer or histidine-citric acid buffer), about 20 parts to about 80 parts of stabilizer (such as trehalose or sucrose or its hydrate), and about 0.1 parts to about 0.1 parts by mass. About 0.5 parts of surfactant (such as Tween 20 or Tween 80).
在一些实施方案中，固体制剂包括：质量份数为约20份～约30份的ADC(比如ADC-1)，质量份数为约3份～约7份的缓冲剂(比如组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂)，质量份数为约40份～约80份的稳定剂(比如海藻糖或蔗糖或其水合物)，以及质量份数为约0.2份～约0.4份的表面活性剂(比如吐温20或吐温80)。In some embodiments, the solid preparation includes: about 20 parts to about 30 parts by mass of ADC (such as ADC-1), and about 3 parts to about 7 parts of buffer (such as histidine- Succinic acid buffer or histidine-citric acid buffer), a stabilizer (such as trehalose or sucrose or its hydrate) with a mass part of about 40 to about 80 parts, and a mass part of about 0.2 to about About 0.4 parts of surfactant (such as Tween 20 or Tween 80).
在一些实施方案中，固体制剂包括：质量份数为约23份～约27份的ADC(比如ADC-1)，质量份数为约3份～约4份的缓冲剂(比如组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂)，质量份数为约50份～约70份的稳定剂(比如海藻糖或蔗糖或其水合物)，以及质量份数为约0.25份～约0.35份的表面活性剂(比如吐温20或吐温80)。In some embodiments, the solid preparation includes: about 23 parts to about 27 parts by mass of ADC (such as ADC-1), and about 3 parts to about 4 parts of buffer (such as histidine- Succinic acid buffer or histidine-citric acid buffer), about 50 parts to about 70 parts of stabilizer (such as trehalose or sucrose or its hydrate), and about 0.25 parts to about 0.25 parts by mass. About 0.35 parts of surfactant (such as Tween 20 or Tween 80).
在一些实施方案中，固体制剂包括：质量份数为约25份的ADC(比如ADC-1)，质 量份数为约3.3份(或约3.28份)的缓冲剂(比如组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂)，质量份数为约54份(或约60份)的稳定剂(比如海藻糖或蔗糖或其水合物)，以及质量份数为约0.3份的表面活性剂(比如吐温20或吐温80)。In some embodiments, the solid preparation includes: about 25 parts by mass of ADC (such as ADC-1), and about 3.3 parts (or about 3.28 parts) of buffer (such as histidine-succinic acid) by mass. Buffer or histidine-citric acid buffer), about 54 parts (or about 60 parts) of stabilizers (such as trehalose or sucrose or its hydrate) in parts by mass, and about 0.3 parts by mass Surfactant (such as Tween 20 or Tween 80).
在一些实施方案中，固体制剂包括：质量份数为约20份～约30份的ADC-1，质量份数为约3份～约7份的柠檬酸-盐酸组氨酸缓冲剂，质量份数为约40份～约80份的蔗糖，以及质量份数为约0.2份～约0.4份的吐温20。In some embodiments, the solid preparation includes: about 20 parts to about 30 parts by mass of ADC-1, about 3 parts to about 7 parts by mass of citric acid-histidine hydrochloride buffer, and parts by mass The amount of sucrose is about 40 parts to about 80 parts, and the amount of Tween 20 is about 0.2 parts to about 0.4 parts by mass.
在一些实施方案中，固体制剂包括：质量份数为约20份～约30份的ADC-1，质量份数为约3份～约7份的组氨酸-琥珀酸缓冲剂，质量份数为约40份～约80份的海藻糖，以及质量份数为约0.2份～约0.4份的吐温80。In some embodiments, the solid preparation comprises: about 20 parts to about 30 parts by mass of ADC-1, about 3 parts to about 7 parts by mass of histidine-succinic acid buffer, and parts by mass It is about 40 parts to about 80 parts of trehalose, and about 0.2 parts to about 0.4 parts of Tween 80 by mass.
在一些实施方案中，固体制剂包括：质量份数为约23份～约27份的ADC-1，质量份数为约3份～约4份的组氨酸-琥珀酸缓冲剂，质量份数为50份～70份的海藻糖，以及质量份数为约0.25份～约0.35份的吐温80。In some embodiments, the solid preparation includes: about 23 parts to about 27 parts by mass of ADC-1, about 3 parts to about 4 parts by mass of histidine-succinic acid buffer, and parts by mass of about 3 to about 4 parts of ADC-1. It is 50 parts to 70 parts of trehalose, and about 0.25 parts to about 0.35 parts of Tween 80 by mass.
在一些实施方案中，固体制剂包括：质量份数为约25份的ADC-1，质量份数为约1.18份的琥珀酸，质量份数为约2.1份盐酸组氨酸，质量份数为约54份的海藻糖，以及质量份数为约0.3份的吐温80。In some embodiments, the solid preparation includes: about 25 parts by mass of ADC-1, about 1.18 parts by mass of succinic acid, about 2.1 parts by mass of histidine hydrochloride, and about 54 parts of trehalose, and about 0.3 parts of Tween 80 by mass.
在一些实施方案中，固体制剂包括：质量份数为约25份的ADC-1，质量份数为约1.18份的琥珀酸，质量份数为约2.1份盐酸组氨酸，质量份数为约60份的海藻糖二水合物，以及质量份数为约0.3份的吐温80。In some embodiments, the solid preparation includes: about 25 parts by mass of ADC-1, about 1.18 parts by mass of succinic acid, about 2.1 parts by mass of histidine hydrochloride, and about 60 parts of trehalose dihydrate, and about 0.3 parts of Tween 80 by mass.
在一些实施方案中，固体制剂包括：质量份数为约25份的ADC-1，质量份数为约1.18份的琥珀酸，质量份数为约2.1份盐酸组氨酸一水合物，质量份数为约60份的海藻糖二水合物，以及质量份数为约0.3份的吐温80。In some embodiments, the solid preparation includes: about 25 parts by mass of ADC-1, about 1.18 parts by mass of succinic acid, about 2.1 parts by mass of histidine hydrochloride monohydrate, and about 2.1 parts by mass of ADC-1. About 60 parts of trehalose dihydrate, and about 0.3 parts of Tween 80 by mass.
在一些实施方案中，所述固体制剂的pH(比如在加水或生理盐水溶解后)约为4.5-6.0。在一些实施方案中，所述固体制剂的pH(比如在加水或生理盐水溶解后)约为4.6-5.6。在一些实施方案中，所述固体制剂的pH(比如在加水或生理盐水溶解后)约为4.6-5.2。In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.5-6.0. In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.6. In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.2.
在一些实施方案中，以上所述固体制剂还包含一定量的Na +或K +，用于与缓冲剂形成缓冲体系，调节pH，比如约0.1份-约1份的Na+，约0.2份-约0.5份的Na +，或约0.25份-约0.3份的Na +。 In some embodiments, the above-mentioned solid preparation also contains a certain amount of Na + or K + , which is used to form a buffer system with a buffer to adjust the pH, such as about 0.1 part to about 1 part of Na +, about 0.2 part to about 0.5 parts of Na + , or about 0.25 parts to about 0.3 parts of Na + .
在一些实施方案中，固体制剂为粉末。在一些实施方案中，固体制剂为冻干制剂。In some embodiments, the solid formulation is a powder. In some embodiments, the solid formulation is a lyophilized formulation.
在一些实施方案中，固体制剂的规格(即可以装入一个容器(比如一个药瓶或者药袋)的剂量)为约40～约200mg的ADC，约80～约120mg的ADC，约92～约108mg的ADC，或约100mg的ADC。在一些实施方案中，固体制剂的规格为约40mg、约80mg、约92mg、约100mg、约108mg、约120mg、约200mg的ADC，或任何两个数字之间的范围，包括端点值。In some embodiments, the specification of the solid preparation (that is, the dose that can be packed into a container (such as a vial or bag)) is about 40 to about 200 mg of ADC, about 80 to about 120 mg of ADC, and about 92 to about 108mg ADC, or about 100mg ADC. In some embodiments, the strength of the solid formulation is about 40 mg, about 80 mg, about 92 mg, about 100 mg, about 108 mg, about 120 mg, about 200 mg ADC, or a range between any two numbers, including endpoints.
在一些实施方案中，固体制剂包括：约40～约200mg的ADC，约4～约40mg的缓冲剂，80～320mg的稳定剂，以及质量份数为约0.4～约2mg的表面活性剂。In some embodiments, the solid formulation includes: about 40 to about 200 mg of ADC, about 4 to about 40 mg of buffer, 80 to 320 mg of stabilizer, and about 0.4 to about 2 mg of surfactant by mass.
在一些实施方案中，固体制剂包括：约80～约120mg的ADC-1，约12～约28mg的组 氨酸-琥珀酸缓冲剂，约160～约320mg的海藻糖，以及约0.8～约1.6mg的吐温80。In some embodiments, the solid formulation includes: about 80 to about 120 mg of ADC-1, about 12 to about 28 mg of histidine-succinic acid buffer, about 160 to about 320 mg of trehalose, and about 0.8 to about 1.6 mg of Tween 80.
在一些实施方案中，固体制剂包括：质量份数为约92～约108mg的ADC-1，约12～约16mg的组氨酸-琥珀酸缓冲剂，约200～约280mg的海藻糖，以及质量份数为约1～约1.4mg的吐温80。In some embodiments, the solid preparation includes: about 92 to about 108 mg of ADC-1 by mass, about 12 to about 16 mg of histidine-succinic acid buffer, about 200 to about 280 mg of trehalose, and mass The serving size is about 1 to about 1.4 mg of Tween 80.
在一些实施方案中，固体制剂包括：约100mg的ADC-1，约4.72mg的琥珀酸，约8.4mg的盐酸组氨酸，约216mg的海藻糖，以及约1.2mg的吐温80。In some embodiments, the solid formulation includes about 100 mg of ADC-1, about 4.72 mg of succinic acid, about 8.4 mg of histidine hydrochloride, about 216 mg of trehalose, and about 1.2 mg of Tween 80.
在一些实施方案中，固体制剂包括：约100mg的ADC-1，约4.72mg的琥珀酸，约8.4mg的盐酸组氨酸，约240mg的海藻糖二水合物，以及约1.2mg的吐温80。In some embodiments, the solid formulation includes: about 100 mg of ADC-1, about 4.72 mg of succinic acid, about 8.4 mg of histidine hydrochloride, about 240 mg of trehalose dihydrate, and about 1.2 mg of Tween 80 .
在一些实施方案中，固体制剂包括：约100mg的ADC-1，约4.72mg的琥珀酸，约8.4mg的盐酸组氨酸一水合物，约240mg的海藻糖二水合物，以及约1.2mg的吐温80。在一些实施方案中，以上所述固体制剂还包含一定量的Na +或K +，比如约0.5mg～约4mg的Na +，约1mg～约2mg的Na +，或约1mg～约1.2mg的Na +。 In some embodiments, the solid formulation includes: about 100 mg ADC-1, about 4.72 mg succinic acid, about 8.4 mg histidine hydrochloride monohydrate, about 240 mg trehalose dihydrate, and about 1.2 mg Tween 80. In some embodiments, the solid preparation described above further contains a certain amount of Na + or K + , such as about 0.5 mg to about 4 mg of Na + , about 1 mg to about 2 mg of Na + , or about 1 mg to about 1.2 mg of Na + Na + .
在一些实施方案中，在向患者给药时，将每一个容器含的剂量溶于足够量的注射用溶剂(比如注射用水或生理盐水)以得到约4ml溶液。In some embodiments, when administering to a patient, the dose contained in each container is dissolved in a sufficient amount of a solvent for injection (such as water for injection or physiological saline) to obtain about 4 ml of a solution.
在一些实施方案中，固体制剂为冻干制剂，其通过将包含ADC的液体制剂冻干获得，液体制剂中还包含缓冲剂、稳定剂以及表面活性剂中的一种或多种，及溶剂，比如水，如注射用水。In some embodiments, the solid formulation is a lyophilized formulation, which is obtained by lyophilizing a liquid formulation containing ADC, and the liquid formulation also contains one or more of a buffer, a stabilizer, and a surfactant, and a solvent, Such as water, such as water for injection.
在一些实施方案中，本发明提供一种液体制剂。在一些实施方案中，所述液体制剂可以用来制备上述冻干制剂。在一些实施方案中，所述液体制剂为将上述冻干制剂复溶后得到的复溶制剂。In some embodiments, the invention provides a liquid formulation. In some embodiments, the liquid formulation can be used to prepare the above-mentioned lyophilized formulation. In some embodiments, the liquid formulation is a reconstituted formulation obtained after reconstituted the above-mentioned lyophilized formulation.
在一些实施方案中，所述液体制剂包括：ADC，其中ADC的浓度为约10mg/ml～约50mg/ml；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种；稳定剂的浓度为约20mg/ml～约80mg/ml；缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的组合；表面活性剂的浓度为约0.1mg/ml～约0.5mg/ml。In some embodiments, the liquid formulation includes: ADC, wherein the concentration of ADC is about 10 mg/ml to about 50 mg/ml; further includes one or more of stabilizers, buffers and surfactants; stabilizers The concentration of the surfactant is about 20 mg/ml to about 80 mg/ml; the buffer is histidine buffer, succinic acid buffer, citric acid buffer or a combination thereof; the concentration of the surfactant is about 0.1 mg/ml to about 0.5 mg/ml.
在一些实施方案中，液体制剂的ADC的浓度为约20mg/ml～约30mg/ml；在一些实施方案中，ADC的浓度为约23mg/ml～约27mg/ml；在一些实施方案中，ADC的浓度为约25mg/ml。在一些实施方案中，ADC的浓度约为约10mg/ml、约20mg/ml、约23mg/ml、约25mg/ml、约27mg/ml、约30mg/ml、约40mg/ml、约50mg/ml或任意两个浓度之间的范围，包括端点值。In some embodiments, the ADC concentration of the liquid formulation is about 20 mg/ml to about 30 mg/ml; in some embodiments, the ADC concentration is about 23 mg/ml to about 27 mg/ml; in some embodiments, the ADC The concentration is about 25mg/ml. In some embodiments, the concentration of ADC is about 10 mg/ml, about 20 mg/ml, about 23 mg/ml, about 25 mg/ml, about 27 mg/ml, about 30 mg/ml, about 40 mg/ml, about 50 mg/ml Or the range between any two concentrations, including endpoints.
在一些实施方案中，液体制剂的ADC为ADC-1；在一些实施方案中，ADC-1的浓度为约20mg/ml～约30mg/ml；在一些实施方案中，ADC-1的浓度为约23mg/ml～约27mg/ml；在一些实施方案中，ADC-1的浓度为约25mg/ml。In some embodiments, the ADC of the liquid formulation is ADC-1; in some embodiments, the concentration of ADC-1 is about 20 mg/ml to about 30 mg/ml; in some embodiments, the concentration of ADC-1 is about From 23 mg/ml to about 27 mg/ml; in some embodiments, the concentration of ADC-1 is about 25 mg/ml.
在一些实施方案中，采用稳定剂对液体制剂中的ADC进行保护。在一些实施方案中稳定剂选择糖类，在一些实施方案中为非还原性糖和/或山梨醇，在一些实施方案中为选蔗糖和/或海藻糖。在一些实施方案中为海藻糖。在制剂预冻过程中，这些稳定剂使制剂在此过程中形成玻璃态，包括其中可结晶成分，一方面消除了缓冲剂成分结晶的可能性，从而消 除pH的偏移而起到保护蛋白的作用，另一方面形成的玻璃态对蛋白在低温和失水时的稳定性也起到关键的作用，但同时，在本申请的一些实施方案中，稳定剂同时充当冻干制剂的填充剂，为冻干制剂提供一定的结构，进一步提高在长期贮存时ADC的稳定性。填充剂除了是上述的稳定剂外，还可以是甘氨酸、甘露醇等。In some embodiments, stabilizers are used to protect ADCs in liquid formulations. In some embodiments, the stabilizer is selected from sugars, in some embodiments is a non-reducing sugar and/or sorbitol, and in some embodiments is selected from sucrose and/or trehalose. In some embodiments, it is trehalose. During the pre-freezing process of the preparation, these stabilizers make the preparation form a glassy state during the process, including crystallizable components therein. On the one hand, it eliminates the possibility of crystallization of the buffer components, thereby eliminating pH shifts and protecting the protein. On the other hand, the formed glass state also plays a key role in the stability of the protein at low temperature and loss of water, but at the same time, in some embodiments of the present application, the stabilizer also acts as a filler for the freeze-dried formulation. Provide a certain structure for the freeze-dried preparation, and further improve the stability of ADC during long-term storage. In addition to the aforementioned stabilizers, the filler can also be glycine, mannitol, and the like.
对于稳定剂的浓度，过低起不到足够的保护效果。无限增高的稳定剂浓度则导致冷冻干燥过程中一次干燥时间的延长或失败和二次干燥后水分的无法清除，故稳定剂的浓度控制在能维持蛋白的稳定性并能在冷冻干燥过程中形成良好的“蛋糕”状形态。在一些实施方案中，液体制剂的稳定剂的浓度为约40mg/ml～约80mg/ml；在一些实施方案中，稳定剂的浓度为约50mg/ml～约70mg/ml；在一些实施方案中，稳定剂的浓度为约54mg/ml。在一些实施方案中，稳定剂的浓度约为约20mg/ml、约40mg/ml、约50mg/ml、约54mg/ml、约60mg/ml、约70mg/ml、约80mg/ml或任意两个浓度之间的范围，包括端点值。在一些实施方案中，稳定剂为浓度约50mg/ml～约70mg/ml的海藻糖；在一些实施方案中，稳定剂为浓度约54mg/ml的海藻糖；在一些实施方案中，稳定剂为浓度约60mg/ml的海藻糖二水合物。Regarding the concentration of stabilizer, too low a protective effect cannot be achieved. The infinitely increased concentration of stabilizer leads to the extension or failure of the primary drying time during the freeze-drying process and the inability to remove water after the secondary drying. Therefore, the concentration of the stabilizer is controlled to maintain the stability of the protein and form during the freeze-drying process. Good "cake" shape. In some embodiments, the concentration of the stabilizer of the liquid formulation is about 40 mg/ml to about 80 mg/ml; in some embodiments, the concentration of the stabilizer is about 50 mg/ml to about 70 mg/ml; in some embodiments , The concentration of stabilizer is about 54mg/ml. In some embodiments, the concentration of the stabilizer is about 20 mg/ml, about 40 mg/ml, about 50 mg/ml, about 54 mg/ml, about 60 mg/ml, about 70 mg/ml, about 80 mg/ml, or any two The range between concentrations, including endpoints. In some embodiments, the stabilizer is trehalose at a concentration of about 50 mg/ml to about 70 mg/ml; in some embodiments, the stabilizer is trehalose at a concentration of about 54 mg/ml; in some embodiments, the stabilizer is Trehalose dihydrate with a concentration of about 60mg/ml.
固体状态下，海藻糖通常以海藻糖二水合物存在的，所以在一些实施方式中，配制制剂时采用海藻糖二水合物，也可以采用其他形式的海藻糖配制。In the solid state, trehalose usually exists as trehalose dihydrate. Therefore, in some embodiments, trehalose dihydrate is used when formulating the preparation, or other forms of trehalose can also be used.
在一些实施方案中，稳定剂为浓度约106mM～约212mM的海藻糖；在一些实施方案中，稳定剂为浓度约132mM～约185mM的海藻糖；在一些实施方案中，稳定剂为浓度约158mM的海藻糖。In some embodiments, the stabilizer is trehalose at a concentration of about 106 mM to about 212 mM; in some embodiments, the stabilizer is trehalose at a concentration of about 132 mM to about 185 mM; in some embodiments, the stabilizer is trehalose at a concentration of about 158 mM. Trehalose.
在一些实施方案中，缓冲剂选自组氨酸缓冲剂、柠檬酸缓冲剂和琥珀酸缓冲剂，也可以是上述缓冲剂中的任意两种的组合，如组氨酸缓冲剂和琥珀酸缓冲剂混合的缓冲剂，亦或是上述三种组合的缓冲剂。液体制剂在冻干形成冻干制剂的过程中，需要经过低温冷冻，再进行抽真空处理，升华冷冻中的水分，再恢复温度和气压，从而得到冻干制剂。期间，随着水从液体变成固体的过程中，可能会引发缓冲剂的析出和pH的改变，进而影响了ADC的稳定性。这些缓冲剂在目标pH值下有较好的缓冲能力，且冷冻过程中，pH变化不大，降低ADC的降解。在一些实施方案中，缓冲剂为琥珀酸缓冲剂和组氨酸缓冲剂的组合。在一些实施方案中，缓冲剂为组氨酸-琥珀酸缓冲剂。在一些实施方案中，缓冲剂为组氨酸-琥珀酸缓冲剂时，缓冲剂的浓度为琥珀酸的浓度与盐酸组氨酸的浓度的总和。In some embodiments, the buffer is selected from the group consisting of histidine buffer, citric acid buffer and succinic acid buffer, or a combination of any two of the above buffers, such as histidine buffer and succinic acid buffer. The buffering agent mixed with the agent, or the buffering agent of the above three combinations. In the process of freeze-drying the liquid preparation to form a freeze-dried preparation, it needs to be frozen at a low temperature, and then subjected to a vacuum treatment to sublime the moisture in the freezing, and then restore the temperature and air pressure to obtain the freeze-dried preparation. During the process, as the water changes from liquid to solid, it may cause the precipitation of the buffer and the change of pH, which in turn affects the stability of the ADC. These buffers have good buffering capacity at the target pH value, and the pH does not change much during the freezing process, which reduces the degradation of ADC. In some embodiments, the buffer is a combination of a succinic acid buffer and a histidine buffer. In some embodiments, the buffer is a histidine-succinic acid buffer. In some embodiments, when the buffer is a histidine-succinic acid buffer, the concentration of the buffer is the sum of the concentration of succinic acid and the concentration of histidine hydrochloride.
对于缓冲剂的浓度，浓度过低时，缓冲能力较弱，起不到明显的保护效果；而浓度过高时，在冷冻的过程中，容易析出，从而影响溶液中的pH值，进而影响ADC的稳定性。Regarding the concentration of the buffer, when the concentration is too low, the buffering capacity will be weak, and no obvious protective effect will be achieved; when the concentration is too high, it will easily precipitate during the freezing process, which will affect the pH value in the solution and thus affect the ADC The stability.
在一些实施方案中，缓冲剂的浓度为约1mg/ml～约10mg/ml；在一些实施方案中，缓冲剂的浓度为约3mg/ml～约7mg/ml；在一些实施方案中，缓冲剂的浓度为约3mg/ml～约4mg/ml。在一些实施方案中，缓冲剂包含浓度为约1.18mg/ml的琥珀酸和约2.1mg/ml的盐酸组氨酸。在一些实施方案中，缓冲剂包含浓度为约1.18mg/ml的琥珀酸和约2.1mg/ml的盐酸组氨酸一水合物。In some embodiments, the concentration of the buffer is about 1 mg/ml to about 10 mg/ml; in some embodiments, the concentration of the buffer is about 3 mg/ml to about 7 mg/ml; in some embodiments, the buffer The concentration is about 3mg/ml to about 4mg/ml. In some embodiments, the buffer comprises succinic acid at a concentration of about 1.18 mg/ml and histidine hydrochloride at a concentration of about 2.1 mg/ml. In some embodiments, the buffer comprises succinic acid at a concentration of about 1.18 mg/ml and histidine hydrochloride monohydrate at a concentration of about 2.1 mg/ml.
在一些实施方案中，液体制剂的缓冲剂的浓度为约10mM～约60mM；在一些实施方案中，缓冲剂的浓度为约16mM～约40mM；在一些实施方案中，缓冲剂的浓度为约16mM～ 约24mM。在一些实施方案中，缓冲剂包含浓度为约10mM的琥珀酸缓冲剂和约10mM的组氨酸缓冲剂。在一些实施方案中，缓冲剂包含浓度为约10mM的琥珀酸和约10mM的盐酸组氨酸。In some embodiments, the concentration of the buffer of the liquid formulation is about 10 mM to about 60 mM; in some embodiments, the concentration of the buffer is about 16 mM to about 40 mM; in some embodiments, the concentration of the buffer is about 16 mM ~ About 24mM. In some embodiments, the buffer comprises a succinic acid buffer at a concentration of about 10 mM and a histidine buffer at a concentration of about 10 mM. In some embodiments, the buffer comprises succinic acid at a concentration of about 10 mM and histidine hydrochloride at a concentration of about 10 mM.
在一些实施方案中，液体制剂含表面活性剂。在一些实施方案中，表面活性剂为非离子表面活性剂，如吐温20、吐温80和泊洛沙姆(如泊洛沙姆188)等。在一些实施方案中，表面活性剂为吐温20和/或吐温80。在一些实施方案中，表面活性剂为吐温80。在一些实施方案中，这些表面活性剂的加入，降低表面张力，限制蛋白的聚合。In some embodiments, the liquid formulation contains a surfactant. In some embodiments, the surfactant is a nonionic surfactant, such as Tween 20, Tween 80, and poloxamer (such as poloxamer 188) and the like. In some embodiments, the surfactant is Tween 20 and/or Tween 80. In some embodiments, the surfactant is Tween 80. In some embodiments, the addition of these surfactants reduces surface tension and restricts protein polymerization.
在一些实施方案中，液体制剂的表面活性剂的浓度为0.2mg/ml～约0.4mg/ml；在一些实施方案中，表面活性剂的浓度为约0.25mg/ml～约0.35mg/ml；在一些实施方案中，表面活性剂的浓度约为0.3mg/ml。在一些实施方案中，表面活性剂的浓度为约0.1mg/ml、约0.2mg/ml、约0.25mg/ml、约0.3mg/ml、约0.35mg/ml、约0.4mg/ml、约0.5mg/ml或任意两个浓度之间的范围，包括端点值。在一些实施方案中，所述表面活性剂提高ADC的稳定性。In some embodiments, the concentration of the surfactant of the liquid formulation is 0.2 mg/ml to about 0.4 mg/ml; in some embodiments, the concentration of the surfactant is about 0.25 mg/ml to about 0.35 mg/ml; In some embodiments, the concentration of the surfactant is about 0.3 mg/ml. In some embodiments, the concentration of the surfactant is about 0.1 mg/ml, about 0.2 mg/ml, about 0.25 mg/ml, about 0.3 mg/ml, about 0.35 mg/ml, about 0.4 mg/ml, about 0.5 mg/ml or the range between any two concentrations, including endpoints. In some embodiments, the surfactant improves the stability of the ADC.
在一些实施方案中，表面活性剂浓度为约0.25mg/ml～约0.35mg/ml的吐温80；在一些实施方案中，表面活性剂为浓度约0.3mg/ml的吐温80。In some embodiments, the surfactant concentration is Tween 80 at a concentration of about 0.25 mg/ml to about 0.35 mg/ml; in some embodiments, the surfactant is Tween 80 at a concentration of about 0.3 mg/ml.
在一些实施方案中，采用所述液体制剂以药学上可接受的溶剂作为载体，例如无菌药用级注射用水或盐水、无菌注射用水、抑菌注射用水等注射用水。在一些实施方案中，采用注射用水对冻干前的液体制剂中的组分，如ADC、缓冲剂、稳定剂和/或表面活性剂进行配制成特定浓度的溶液时，按处方量计算得到所需量的固体，混合后加入一定量的注射用水溶解；若处方中有粘稠液体，再按处方量计算得到所需量的粘稠液体，混合后加入一定量的注射用水进行分散；再将上述的溶液混合，调节pH即得。在一些实施方案中，各组分分别用注射用水进行溶解分散，再将得到的溶液进行混合，调节浓度和pH值即得。In some embodiments, the liquid formulation is used with a pharmaceutically acceptable solvent as a carrier, such as sterile pharmaceutical grade water for injection or saline, sterile water for injection, water for injection for bacteriostasis and other water for injection. In some embodiments, when the components in the liquid preparation before lyophilization, such as ADC, buffer, stabilizer, and/or surfactant, are formulated into a solution with a specific concentration using water for injection, the calculated amount is calculated according to the prescription. For the required amount of solids, add a certain amount of water for injection to dissolve after mixing; if there is a viscous liquid in the prescription, calculate the required amount of viscous liquid according to the prescription amount, and add a certain amount of water for injection to disperse after mixing; Mix the above solutions and adjust the pH. In some embodiments, each component is separately dissolved and dispersed with water for injection, and then the resulting solution is mixed to adjust the concentration and pH value.
在一些实施方案中，液体制剂包括：约10mg/ml～约50mg/ml的ADC(比如ADC-1)，约10mM～约60mM的缓冲剂(比如组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂)，约20mg/ml～约80mg/ml的稳定剂(比如海藻糖或蔗糖)，以及约0.1mg/ml～约0.5mg/ml的表面活性剂(比如吐温20或吐温80)。In some embodiments, the liquid formulation includes: about 10 mg/ml to about 50 mg/ml ADC (such as ADC-1), about 10 mM to about 60 mM buffer (such as histidine-succinic acid buffer or histidine -Citric acid buffer), stabilizers (such as trehalose or sucrose) from about 20mg/ml to about 80mg/ml, and surfactants (such as Tween 20 or vomiting agent) from about 0.1 mg/ml to about 0.5 mg/ml Wen 80).
在一些实施方案中，液体制剂包括：约20mg/ml～约30mg/ml的ADC(比如ADC-1)，约16mM～约40mM的缓冲剂(比如组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂)，约40mg/ml～约80mg/ml的稳定剂(比如海藻糖或蔗糖)，以及约0.2mg/ml～约0.4mg/ml的表面活性剂(比如吐温20或吐温80)。In some embodiments, the liquid formulation includes: about 20 mg/ml to about 30 mg/ml ADC (such as ADC-1), about 16 mM to about 40 mM buffer (such as histidine-succinate buffer or histidine -Citrate buffer), stabilizers (such as trehalose or sucrose) from about 40mg/ml to about 80mg/ml, and surfactants (such as Tween 20 or vomiting agent) from about 0.2 mg/ml to about 0.4 mg/ml Wen 80).
在一些实施方案中，液体制剂包括：约23mg/ml～约27mg/ml的ADC(比如ADC-1)，约16mM～约24mM的缓冲剂(比如组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂)，约50mg/ml～约70mg/ml的稳定剂(比如海藻糖或蔗糖)，以及约0.25mg/ml～约0.35mg/ml的表面活性剂(比如吐温20或吐温80)。In some embodiments, the liquid formulation includes: about 23 mg/ml to about 27 mg/ml ADC (such as ADC-1), about 16 mM to about 24 mM buffer (such as histidine-succinic acid buffer or histidine -Citrate buffer), stabilizers (such as trehalose or sucrose) from about 50mg/ml to about 70mg/ml, and surfactants (such as Tween 20 or vomiting agent) from about 0.25 mg/ml to about 0.35 mg/ml Wen 80).
在一些实施方案中，液体制剂包括：约25mg/ml的ADC(比如ADC-1)，约20mM的缓冲剂(比如组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂)，约54或60mg/ml的稳定剂(比如海藻糖或蔗糖或其水合物)，以及约0.3mg/ml的表面活性剂(比如吐温20或吐 温80)。In some embodiments, the liquid formulation includes: about 25 mg/ml ADC (such as ADC-1), about 20 mM buffer (such as histidine-succinic acid buffer or histidine-citrate buffer), about 54 or 60 mg/ml stabilizer (such as trehalose or sucrose or its hydrate), and about 0.3 mg/ml surfactant (such as Tween 20 or Tween 80).
在一些实施方案中，液体制剂包括：约20mg/ml～约30mg/ml的ADC-1，约16mM～约40mM的柠檬酸～盐酸组氨酸缓冲剂，约40mg/ml～约80mg/ml的蔗糖，以及约0.2mg/ml～约0.4mg/ml的吐温20。In some embodiments, the liquid formulation includes: about 20 mg/ml to about 30 mg/ml ADC-1, about 16 mM to about 40 mM citric acid to histidine hydrochloride buffer, about 40 mg/ml to about 80 mg/ml Sucrose, and about 0.2mg/ml to about 0.4mg/ml Tween 20.
在一些实施方案中，液体制剂包括：约20mg/ml～约30mg/ml的ADC-1，约16mM～约40mM的组氨酸-琥珀酸缓冲剂，约40mg/ml～约80mg/ml的海藻糖，以及约0.2mg/ml～约0.4mg/ml的吐温80。In some embodiments, the liquid formulation includes: ADC-1 at about 20 mg/ml to about 30 mg/ml, histidine-succinic acid buffer at about 16 mM to about 40 mM, and seaweed at about 40 mg/ml to about 80 mg/ml Sugar, and about 0.2mg/ml to about 0.4mg/ml Tween 80.
在一些实施方案中，液体制剂包括：约23mg/ml～约27mg/ml的ADC-1，约16mM～约24mM的组氨酸-琥珀酸缓冲剂，约50mg/ml～约70mg/ml的海藻糖，以及约0.25mg/ml～约0.35mg/ml的吐温80。In some embodiments, the liquid formulation includes: about 23 mg/ml to about 27 mg/ml ADC-1, about 16 mM to about 24 mM histidine-succinic acid buffer, about 50 mg/ml to about 70 mg/ml seaweed Sugar, and about 0.25mg/ml to about 0.35mg/ml Tween 80.
在一些实施方案中，液体制剂包括：约20mg/ml～40mg/ml的ADC-1，约5～20mM的琥珀酸和/或盐，约5～20mM组氨酸和/或盐，约40mg/ml～80mg/ml的海藻糖，以及约0.1mg/ml～0.5mg/ml的吐温80。In some embodiments, the liquid formulation includes: about 20 mg/ml-40 mg/ml ADC-1, about 5-20 mM succinic acid and/or salt, about 5-20 mM histidine and/or salt, about 40 mg/ml ml～80mg/ml trehalose, and about 0.1mg/ml～0.5mg/ml Tween 80.
在一些实施方案中，液体制剂包括：约20mg/ml～30mg/ml的ADC-1，约5～15mM的琥珀酸和/或盐，约5～15mM组氨酸和/或盐，约50mg/ml～70mg/ml的海藻糖，以及约0.2mg/ml～0.4mg/ml的吐温80。In some embodiments, the liquid formulation includes: about 20mg/ml-30mg/ml ADC-1, about 5-15mM succinic acid and/or salt, about 5-15mM histidine and/or salt, about 50mg/ml ml～70mg/ml trehalose, and about 0.2mg/ml～0.4mg/ml Tween 80.
在一些实施方案中，液体制剂包括：约25mg/ml的ADC-1，约10mM的琥珀酸和/或盐，约10mM组氨酸和/或盐，约54mg/ml的海藻糖，以及约0.3mg/ml的吐温80。In some embodiments, the liquid formulation includes: about 25 mg/ml ADC-1, about 10 mM succinic acid and/or salt, about 10 mM histidine and/or salt, about 54 mg/ml trehalose, and about 0.3 Tween 80 mg/ml.
在一些实施方案中，液体制剂包括：约25mg/ml的ADC-1，约10mM的琥珀酸，约10mM盐酸组氨酸，约54mg/ml的海藻糖，以及约0.3mg/ml的吐温80。In some embodiments, the liquid formulation includes: about 25 mg/ml ADC-1, about 10 mM succinic acid, about 10 mM histidine hydrochloride, about 54 mg/ml trehalose, and about 0.3 mg/ml Tween 80 .
在一些实施方案中，液体制剂的pH值为约4.5～约6.0；在一些实施方案中，液体制剂的pH值为约4.5～约5.6；在一些实施方案中，液体制剂的pH值为约4.6～5.2；在一些实施方案中，液体制剂的pH值为4.9±0.3。在一些实施方案中，pH是通过加入适量NaOH或KOH调节的。In some embodiments, the pH of the liquid formulation is about 4.5 to about 6.0; in some embodiments, the pH of the liquid formulation is about 4.5 to about 5.6; in some embodiments, the pH of the liquid formulation is about 4.6 ~5.2; In some embodiments, the pH of the liquid formulation is 4.9±0.3. In some embodiments, the pH is adjusted by adding an appropriate amount of NaOH or KOH.
在一些实施方案中，液体制剂包括：ADC，其中偶联物的浓度为约10mg/ml～约50mg/ml；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种；稳定剂的浓度为约20mg/ml～约80mg/ml；缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的组合；表面活性剂的浓度为约0.1mg/ml～约0.5mg/ml。In some embodiments, the liquid formulation includes: ADC, wherein the concentration of the conjugate is about 10 mg/ml to about 50 mg/ml; further includes one or more of stabilizers, buffers and surfactants; stabilizers The concentration of the surfactant is about 20 mg/ml to about 80 mg/ml; the buffer is histidine buffer, succinic acid buffer, citric acid buffer or a combination thereof; the concentration of the surfactant is about 0.1 mg/ml to about 0.5 mg/ml.
在一些实施方案中，液体制剂中，缓冲剂的浓度为约5mM～约30mM；在一些实施方案中，液体制剂中，缓冲剂的浓度为约10mM～约20mM；在一些实施方案中，液体制剂中，缓冲剂的浓度为约10mM。In some embodiments, the concentration of the buffer in the liquid formulation is from about 5 mM to about 30 mM; in some embodiments, the concentration of the buffer in the liquid formulation is from about 10 mM to about 20 mM; in some embodiments, the concentration of the buffer is from about 10 mM to about 20 mM. Among them, the concentration of the buffer is about 10 mM.
在一些实施方案中，液体制剂包含：约10mg/ml～约50mg/ml的ADC，约5mM～约30mM的缓冲剂，约20mg/ml～约80mg/ml的稳定剂，以及约0.1mg/ml～约0.5mg/ml的表面活性剂。在一些实施方案中，液体制剂包含：约25mg/ml的ADC-1，约1.18mg/ml的琥珀酸，约2.1mg/ml的盐酸组氨酸，约60mg/ml的海藻糖二水合物，约0.3mg/ml的吐温80，pH为约4.6-约5.2。在一些实施方案中，液体制剂包含：约25mg/ml的ADC-1，约1.18mg/ml的琥 珀酸，约2.1mg/ml的盐酸组氨酸一水合物，约60mg/ml的海藻糖二水合物，约0.3mg/ml的吐温80，pH为约4.6-约5.2。In some embodiments, the liquid formulation comprises: about 10 mg/ml to about 50 mg/ml ADC, about 5 mM to about 30 mM buffer, about 20 mg/ml to about 80 mg/ml stabilizer, and about 0.1 mg/ml ~ About 0.5mg/ml of surfactant. In some embodiments, the liquid formulation comprises: ADC-1 at about 25 mg/ml, succinic acid at about 1.18 mg/ml, histidine hydrochloride at about 2.1 mg/ml, trehalose dihydrate at about 60 mg/ml, About 0.3mg/ml of Tween 80, the pH is about 4.6 to about 5.2. In some embodiments, the liquid formulation comprises: about 25 mg/ml ADC-1, about 1.18 mg/ml succinic acid, about 2.1 mg/ml histidine hydrochloride monohydrate, about 60 mg/ml trehalose dihydrate Hydrate, about 0.3 mg/ml of Tween 80, with a pH of about 4.6 to about 5.2.
另一方面，本发明提供一种制备的固体制剂的方法，其中，包括：In another aspect, the present invention provides a method for preparing a solid preparation, which comprises:
1)配制：取ADC、稳定剂、缓冲剂、表面活性剂，溶解于水中至规定量，并调整pH值至规定值，如4.5～6.0，得到液体制剂；1) Preparation: take ADC, stabilizer, buffer, surfactant, dissolve in water to a specified amount, and adjust the pH value to a specified value, such as 4.5 to 6.0, to obtain a liquid preparation;
2)冻干：将液体制剂进行冻干，得到的固体制剂。2) Lyophilization: freeze-dry the liquid preparation to obtain a solid preparation.
冻干或冷冻干燥是从液体制剂制备固体制剂的常用方法，通常将含有水和/或其他溶剂的制剂快速降温(比如-30℃或更低)至冷冻，然后在密封的情况下降低压力，使制剂中的冷冻水和/或其他溶剂直接从固相升华到气相，从而去除水或其他溶剂而得到通常为粉末的固体制剂。冻干通常使用冻干机进行。在一些实施方案中，冻干的步骤包括预冻、退火、一次干燥、二次干燥。Lyophilization or freeze-drying is a common method for preparing solid preparations from liquid preparations. The preparations containing water and/or other solvents are usually rapidly cooled (for example, -30°C or lower) to freezing, and then the pressure is reduced in a sealed condition. The frozen water and/or other solvents in the formulation are directly sublimated from the solid phase to the gas phase, thereby removing the water or other solvents to obtain a solid formulation that is usually a powder. Lyophilization is usually carried out using a lyophilizer. In some embodiments, the step of freeze-drying includes pre-freezing, annealing, primary drying, and secondary drying.
另一方面，本发明提供固体制剂在制备用于预防和/或治疗Trop2阳性疾病的药物中的应用。在一些实施方案中，Trop2阳性疾病包括但不限于增殖性、炎症性或免疫性疾病或病症。在一些实施方案中，Trop2阳性疾病包括但不限于三阴性乳腺癌、胶质母细胞瘤、成神经管细胞瘤、上皮癌、乳腺癌、头颈癌、肾癌、卵巢癌、胃癌、卡波西肉瘤、胰腺癌和肺癌、***、结肠直肠癌、食管癌、口腔鳞状细胞癌、***癌、甲状腺癌、膀胱癌、神经胶质瘤、肝胆管癌、结肠直肠癌、T细胞淋巴瘤。在一些实施方案中，上述固体制剂在制备用于预防和/或治疗上皮癌的药物中的应用。在应用本发明的固体制剂时，给予患者药物前，先用药学上可接受的溶剂对固体制剂进行溶解，重构得到复溶制剂，再进行给药。溶剂包括无菌药用级注射用水或盐水、无菌注射用水、抑菌注射用水等。On the other hand, the present invention provides the application of solid preparations in the preparation of medicines for preventing and/or treating Trop2-positive diseases. In some embodiments, Trop2-positive diseases include, but are not limited to, proliferative, inflammatory, or immune diseases or disorders. In some embodiments, Trop2-positive diseases include, but are not limited to, triple-negative breast cancer, glioblastoma, medulloblastoma, epithelial cancer, breast cancer, head and neck cancer, kidney cancer, ovarian cancer, gastric cancer, Kaposi Sarcoma, pancreatic and lung cancer, cervical cancer, colorectal cancer, esophageal cancer, oral squamous cell carcinoma, prostate cancer, thyroid cancer, bladder cancer, glioma, hepatobiliary cancer, colorectal cancer, T cell lymphoma. In some embodiments, the above-mentioned solid preparation is used in the preparation of a medicament for the prevention and/or treatment of epithelial cancer. When applying the solid preparation of the present invention, before administering the medicine to the patient, the solid preparation is dissolved with a pharmaceutically acceptable solvent, reconstituted to obtain a reconstituted preparation, and then administered. Solvents include sterile pharmaceutical grade water for injection or saline, sterile water for injection, and antibacterial water for injection.
另一方面，本文提供一种治疗癌症的方法，包括向需要治疗的患者给予有效量的ADC的复溶制剂或液体制剂。在一些实施方案中，通过在药学上可接受的溶剂(无菌药用级注射用水或盐水、无菌注射用水、抑菌注射用水等)中重构前述实施方案中任一个的固体制剂获得的复溶制剂。On the other hand, provided herein is a method for treating cancer, which comprises administering an effective amount of a reconstituted preparation or liquid preparation of ADC to a patient in need of treatment. In some embodiments, obtained by reconstituting the solid formulation of any one of the preceding embodiments in a pharmaceutically acceptable solvent (sterile pharmaceutical grade water for injection or saline, sterile water for injection, bacteriostatic water for injection, etc.) Reconstituted preparations.
本发明提供的一种复溶制剂，其中，复溶制剂通过用药学上可接受的溶剂对上述的固体制剂进行重构获得。该复溶制剂可以用来预防和/或治疗Trop2阳性疾病，比如增殖性、炎症性或免疫性疾病或病症，比如上皮癌。The present invention provides a reconstituted preparation, wherein the reconstituted preparation is obtained by reconstituting the above-mentioned solid preparation with a pharmaceutically acceptable solvent. The reconstituted preparation can be used to prevent and/or treat Trop2-positive diseases, such as proliferative, inflammatory or immune diseases or disorders, such as epithelial cancer.
在一些实施方案中，重构的复溶制剂包括：ADC，其中偶联物的浓度为约10mg/ml～约50mg/ml；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种；稳定剂的浓度为约20mg/ml～约80mg/ml；缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的组合；表面活性剂的浓度为约0.1mg/ml～约0.5mg/ml。In some embodiments, the reconstituted reconstituted formulation includes: ADC, wherein the concentration of the conjugate is about 10 mg/ml to about 50 mg/ml; and further includes one or more of stabilizers, buffers, and surfactants. Species; the concentration of stabilizer is about 20mg/ml to about 80mg/ml; the buffer is histidine buffer, succinic acid buffer, citric acid buffer or a combination thereof; the concentration of surfactant is about 0.1mg/ ml ~ 0.5mg/ml.
在一些实施方案中，复溶制剂中，缓冲剂的浓度为约5mM～约30mM；在一些实施方案中，复溶制剂中，缓冲剂的浓度为约10mM～约20mM；在一些实施方案中，复溶制剂中，缓冲剂的浓度为约20mM。In some embodiments, the concentration of the buffer in the reconstituted formulation is about 5 mM to about 30 mM; in some embodiments, the concentration of the buffer in the reconstituted formulation is about 10 mM to about 20 mM; in some embodiments, In the reconstituted preparation, the concentration of the buffer is about 20 mM.
在一些实施方案中，复溶制剂包含：约10mg/ml～约50mg/ml的ADC，约5mM～约30mM的缓冲剂，约20mg/ml～约80mg/ml的稳定剂，以及约0.1mg/ml～约0.5mg/ml的表面活性剂。 在一些实施方案中，复溶制剂包含：约25mg/ml的ADC-1，约1.18mg/ml的琥珀酸，约2.1mg/ml的盐酸组氨酸，约60mg/ml的海藻糖二水合物，约0.3mg/ml的吐温80，pH为约4.6-约5.2，其中部分或全部琥珀酸可以以相应量盐的形式存在，部分或全部盐酸组氨酸可以以相应量组氨酸形式存在。在一些实施方案中，复溶制剂包含：约25mg/ml的ADC-1，约1.18mg/ml的琥珀酸，约2.1mg/ml的盐酸组氨酸一水合物，约60mg/ml的海藻糖二水合物，约0.3mg/ml的吐温80，pH为约4.6-约5.2。In some embodiments, the reconstituted formulation comprises: about 10 mg/ml to about 50 mg/ml ADC, about 5 mM to about 30 mM buffer, about 20 mg/ml to about 80 mg/ml stabilizer, and about 0.1 mg/ml ml to about 0.5mg/ml surfactant. In some embodiments, the reconstituted formulation comprises: about 25 mg/ml ADC-1, about 1.18 mg/ml succinic acid, about 2.1 mg/ml histidine hydrochloride, about 60 mg/ml trehalose dihydrate , About 0.3mg/ml Tween 80, pH is about 4.6 to about 5.2, part or all of succinic acid can be in the form of a corresponding amount of salt, and part or all of histidine hydrochloride can be in the form of a corresponding amount of histidine . In some embodiments, the reconstituted preparation comprises: about 25 mg/ml ADC-1, about 1.18 mg/ml succinic acid, about 2.1 mg/ml histidine hydrochloride monohydrate, about 60 mg/ml trehalose Dihydrate, about 0.3 mg/ml of Tween 80, with a pH of about 4.6 to about 5.2.
在一些实施方案中，使用时，用溶剂(如无菌注射用水)对固体制剂进行溶解重构，得到复溶制剂，常用的重构后的ADC浓度为约25mg/ml。必要时，可用等渗溶液(如0.9％注射用NaCl溶液)对复溶制剂进行稀释使用。在一些实施方案中，稀释后ADC浓度为约0.1mg/ml-约5mg/ml或约0.5mg/ml-约5mg/ml。重构后的复溶制剂以及稀释后的制剂均为注射制剂，一般为静脉输注。In some embodiments, during use, the solid preparation is dissolved and reconstituted with a solvent (such as sterile water for injection) to obtain a reconstituted preparation. The commonly used ADC concentration after reconstitution is about 25 mg/ml. If necessary, an isotonic solution (such as 0.9% NaCl solution for injection) can be used to dilute the reconstituted preparation. In some embodiments, the ADC concentration after dilution is about 0.1 mg/ml to about 5 mg/ml or about 0.5 mg/ml to about 5 mg/ml. Both the reconstituted reconstituted preparations and the diluted preparations are injection preparations, generally intravenous infusion.
在一些实施方案中，针对Trop2阳性疾病，如三阴性乳腺癌、上皮癌等，ADC的用量为约0.2mg/kg～约10mg/kg。在一些实施方案中，ADC的单位剂量约为约0.2mg/kg，或约0.5mg/kg，或约1mg/kg，或约2mg/kg，或约4mg/kg，或约6mg/kg，或约8mg/kg，或约10mg/kg，或任意两个剂量之间的范围值，包括端点值。In some embodiments, for Trop2-positive diseases, such as triple-negative breast cancer, epithelial cancer, etc., the amount of ADC is about 0.2 mg/kg to about 10 mg/kg. In some embodiments, the unit dose of ADC is about 0.2 mg/kg, or about 0.5 mg/kg, or about 1 mg/kg, or about 2 mg/kg, or about 4 mg/kg, or about 6 mg/kg, or About 8 mg/kg, or about 10 mg/kg, or a range between any two doses, including endpoints.
在一些实施方案中，给药周期为14-28天，比如20-22天，或21天，即分别每14-28天，比如每20-22天，或每21天，给予患者一次所述的ADC单位剂量。In some embodiments, the administration period is 14-28 days, such as 20-22 days, or 21 days, that is, every 14-28 days, such as every 20-22 days, or every 21 days, the patient is administered once. The ADC unit dose.
在一些实施方案中，患者为Trop2阳性晚期实体瘤患者。In some embodiments, the patient is a Trop2-positive advanced solid tumor patient.
在一些实施方案中，对制备得到的固体制剂进行低温(4℃)和室温(25℃)的稳定性实验。通过本发明制备得到的固体制剂，能够在低温和室温下贮存3个月、6个月、9个月、12个月、18个月甚至24个月，保持稳定。与0个月相比，ADC发生极少数的蛋白聚合；平均DAR保持稳定；蛋白没有降解迹象；采用无菌注射用水重构后，在不溶性微粒方面也保持良好的表现。In some embodiments, low temperature (4°C) and room temperature (25°C) stability experiments are performed on the prepared solid preparations. The solid preparation prepared by the invention can be stored at low temperature and room temperature for 3 months, 6 months, 9 months, 12 months, 18 months or even 24 months, and remains stable. Compared with month 0, ADC has a very small amount of protein polymerization; the average DAR remains stable; there is no sign of protein degradation; after reconstitution with sterile water for injection, it also maintains good performance in terms of insoluble particles.
在一些实施方案中，采用0.9％的生理盐水对重构后的复溶制剂进行配伍稀释成高浓度制剂(约5mg/ml)和低浓度制剂(约0.5mg/ml)。进行配伍稀释后的ADC溶液室温放置2小时、6小时甚至24小时，均表现出良好的稳定性。分子排阻色谱(SEC-HPLC，或SEC)测定发现平均百分比主峰保持大于99％，在不溶性微粒方面也保持良好的表现。In some embodiments, 0.9% normal saline is used to compatibly dilute the reconstituted preparation after reconstitution into a high-concentration preparation (about 5 mg/ml) and a low-concentration preparation (about 0.5 mg/ml). After the compatibility and dilution of the ADC solution is placed at room temperature for 2 hours, 6 hours or even 24 hours, it shows good stability. Size exclusion chromatography (SEC-HPLC, or SEC) measurement found that the average percentage of the main peak remained greater than 99%, and it also maintained good performance in terms of insoluble particles.
在一些实施方案中，对固体制剂复溶后的复溶制剂进行低温(4℃)和室温(25℃)的稳定性实验。通过本发明制备得到的复溶制剂，能够在低温和室温下放置6小时、24小时甚至48小时，保持稳定。In some embodiments, stability tests at low temperature (4°C) and room temperature (25°C) are performed on the reconstituted preparation after the solid preparation is reconstituted. The reconstituted preparation prepared by the present invention can be placed at low temperature and room temperature for 6 hours, 24 hours or even 48 hours, and remains stable.
为了开发出稳定的、不易聚集的抗Trop2抗体药物偶联物制剂，本发明提供一种抗Trop2抗体药物偶联物固体制剂。在一些实施方案中，该固体制剂可通过冻干经低温除去ADC中的水分制得，使ADC在保存时保持稳定。In order to develop a stable anti-Trop2 antibody drug conjugate preparation that is not easy to aggregate, the present invention provides an anti-Trop2 antibody drug conjugate solid preparation. In some embodiments, the solid preparation can be prepared by lyophilizing and removing moisture from ADC at low temperature, so that ADC remains stable during storage.
在一些实施方案中，ADC-1通过L-3AA-MDC(制备方法见专利CN 201310081867.X)与抗体C偶联得到。将抗体C用溶液A(20mM磷酸钠，100mM NaCl和2mM EDTA，pH为7.4)稀释至8.0mg/ml，然后用过量的三(2-羧乙基)膦(TCEP)完全还原。在37℃孵 育一小时以上，用溶液B(10mM琥珀酸，2mM EDTA，pH为7.4)超滤浓缩换液。巯基抗体值通过测定吸光度确定，通过巯基与DTNB(5,5'-二硫代双(2-硝基苯甲酸)，Aldrich公司)的反应物，然后测定412nm处的吸收值来确定巯基的浓度。随后，使用过量的硫酸铜(CuSO 4)或脱氢抗坏血酸(dHAA)进行氧化，抗体链间二硫键重新连接，而突变位点的半胱氨酸则被保留下来。 In some embodiments, ADC-1 is obtained by coupling with antibody C through L-3AA-MDC (see patent CN 201310081867.X for the preparation method). Antibody C was diluted to 8.0 mg/ml with solution A (20 mM sodium phosphate, 100 mM NaCl and 2 mM EDTA, pH 7.4), and then completely reduced with excess tris(2-carboxyethyl)phosphine (TCEP). After incubating at 37°C for more than one hour, the solution B (10 mM succinic acid, 2 mM EDTA, pH 7.4) was used for ultrafiltration to concentrate the solution. The sulfhydryl antibody value is determined by measuring the absorbance. The sulfhydryl concentration is determined by the reaction product of the sulfhydryl and DTNB (5,5'-dithiobis(2-nitrobenzoic acid), Aldrich), and then measuring the absorbance at 412nm. . Subsequently, using excess copper sulfate (CuSO 4 ) or dehydroascorbic acid (dHAA) for oxidation, the disulfide bonds between the antibody chains are reconnected, while the cysteine at the mutation site is retained.
偶联反应时DMA的浓度为10％-30％。L-3AA-MDC与巯基数的比率为1-2:1.0(摩尔当量)。将L-3AA-MDC加入已还原的抗体中，室温搅拌3小时后，加入5mM半胱氨酸继续搅拌0.5-3小时。反应混合物经阳离子交换柱纯化，产品经超滤浓缩换液后用0.22微米的滤器过滤，-80℃保存。ADC-1可以通过紫外吸收测得浓度，通过尺寸排阻色谱测定聚集率，通过反向高效液相色谱法测定偶联药物比率。The concentration of DMA during the coupling reaction is 10%-30%. The ratio of L-3AA-MDC to the number of mercapto groups is 1-2:1.0 (molar equivalent). Add L-3AA-MDC to the reduced antibody, and after stirring for 3 hours at room temperature, add 5mM cysteine and continue stirring for 0.5-3 hours. The reaction mixture is purified by a cation exchange column, and the product is concentrated by ultrafiltration, and then filtered with a 0.22 micron filter, and stored at -80°C. The concentration of ADC-1 can be measured by ultraviolet absorption, the aggregation rate can be measured by size exclusion chromatography, and the ratio of coupled drugs can be measured by reversed-phase high performance liquid chromatography.
以下实施例中，如无特别说明，所使用的试剂和仪器为本领域常规试剂和仪器，可通过商购的方式获得；所使用的方法为本领域常规技术方法，本领域技术人员根据实施例的内容可以毫无疑义地实施所述方法并获得相应的结果。In the following examples, unless otherwise specified, the reagents and instruments used are conventional reagents and instruments in the art, which can be obtained commercially; the method used is a conventional technical method in the art, and those skilled in the art will follow the example The content of the method can be implemented without any doubt and the corresponding results can be obtained.
以下实验中所使用的方法可以如下所示：The method used in the following experiment can be as follows:
SEC-HPLC的分析方法：SEC-HPLC analysis method:
1)用液相色谱***和TOSOH生物科技TSK-GEL G3000SWXL色谱柱(柱规格为7.8*300mm，5μm)。柱温设定为室温，流速0.5ml/min，平衡30-60min至基线平稳。1) Use liquid chromatography system and TOSOH Biotechnology TSK-GEL G3000SWXL chromatographic column (column specification is 7.8*300mm, 5μm). The column temperature is set to room temperature, the flow rate is 0.5ml/min, and the balance is 30-60min until the baseline is stable.
2)进样，记录色谱图，积分后按照峰面积归一法计算供试液的单体或多聚体百分含量。2) Inject the sample, record the chromatogram, and calculate the percentage of monomer or polymer in the test solution according to the peak area normalization method after integration.
HIC-HPLC的分析方法：HIC-HPLC analysis method:
1)液相色谱***和色谱柱1) Liquid chromatography system and chromatographic column
波长：280nm；柱温40℃；流速0.3ml/min，平衡约30min至基线平稳。Wavelength: 280nm; column temperature 40°C; flow rate 0.3ml/min, equilibrate for about 30min until the baseline is stable.
2)样品检测和数据处理2) Sample testing and data processing
采用液相数据分析软件对检测的色谱图进行积分，以空白对照溶液基线为参照，积分起始时间至终止时间为18分钟至48分钟。采用面积归一化法，用D表示DAR，数值表示抗体偶联的药物个数(即DAR的值)计算未偶联抗体(D0)、偶联一个药物的抗体(D1)、偶联两个药物的抗体(D2)的峰面积的百分含量。Use liquid phase data analysis software to integrate the detected chromatogram, with the baseline of the blank control solution as the reference, the integration start time to end time is 18 minutes to 48 minutes. Using area normalization method, use D to represent DAR, and the value to represent the number of drugs conjugated to the antibody (that is, the value of DAR) to calculate unconjugated antibody (D0), antibody conjugated with one drug (D1), and two conjugated The percentage of the peak area of the drug's antibody (D2).
CE-SDS(NR)的分析方法：CE-SDS (NR) analysis method:
毛细管电泳仪采用Beckman的PA800plus，样品进样后，记录色谱图，积分处理数据，采用面积归一化方法计算单体峰的百分含量。The capillary electrophoresis instrument uses Beckman's PA800plus. After the sample is injected, the chromatogram is recorded, the data is integrated, and the area normalization method is used to calculate the percentage of the monomer peak.
相对结合活性检测方法：Relative binding activity detection method:
1)包被：取适量96孔酶标板，向中间60个反应孔中每孔加入包被液100μL(Trop2蛋白用PBS稀释至0.5μg/ml，混匀，现配现用)，4℃包被过夜(16h～20h)。1) Coating: Take an appropriate amount of 96-well microtiter plate, add 100μL of coating solution to each of the 60 reaction wells in the middle (dilute the Trop2 protein to 0.5μg/ml with PBS, mix well, and use it now), 4℃ Coated overnight (16h-20h).
2)封闭：用自动洗板机重复洗板3遍，每孔300μL洗板液，然后向每个反应孔中加入封闭液250μL，37℃孵育2h。2) Blocking: Wash the plate 3 times with an automatic plate washer, 300μL of washing solution per well, then add 250μL of blocking solution to each reaction well, and incubate at 37°C for 2h.
3)样品配制：3) Sample preparation:
3.1)对照品稀释3.1) Reference dilution
取对照品适量，加入PBS稀释至1mg/ml，总体积400μL混匀；从中移取20μL加入预先加有480μL PBS的EP管中；混匀后从中移取25μL加入预先加有975μL PBS的EP管中，混匀即得1μg/ml的对照品溶液。混匀后将溶液加至稀释板第2列孔中，每孔260μL，3个复孔，每个稀释度用移液枪吹打混匀。得ADC浓度为1000ng/ml、500ng/ml、250ng/ml、125ng/ml、62.5ng/ml、31.2ng/ml、15.6ng/ml、7.8ng/ml、3.9ng/ml、1.9ng/ml的梯度溶液。Take an appropriate amount of the control substance, add PBS to dilute to 1mg/ml, and mix with a total volume of 400μL; pipette 20μL from it and add it to the EP tube pre-added with 480μL PBS; after mixing, take 25μL from it and add it to the EP tube pre-added with 975μL PBS In, mix well to get 1μg/ml reference solution. After mixing, add the solution to the second column of the dilution plate, 260μL per well, 3 replicate holes, pipette and mix for each dilution. The ADC concentration is 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 31.2ng/ml, 15.6ng/ml, 7.8ng/ml, 3.9ng/ml, 1.9ng/ml Gradient solution.
3.2)待测样品：3.2) Samples to be tested:
取供试品原液或三支注射剂适量，分别用PBS稀释至1mg/ml，总体积400μL。原液取3次平行稀释3份；注射剂每支取适量进行稀释，共3份稀释液。然后同对照品方法进行稀释。Take an appropriate amount of the original solution of the test substance or three injections, and dilute them with PBS to 1mg/ml, with a total volume of 400μL. Dilute 3 parts of the original solution 3 times in parallel; for each injection, take an appropriate amount to dilute, and a total of 3 diluents. Then dilute with the control method.
4)加样：将封闭好的酶标板取出，重复洗板3遍，每孔300μL洗板液。取各浓度的对照品溶液和供试品溶液各100μL加入相应的反应孔，每个浓度设三个复孔，37℃孵育1h。4) Adding samples: Take out the sealed ELISA plate, repeat the plate washing 3 times, 300μL plate washing solution per well. Take 100μL of each concentration of the reference substance solution and the test substance solution into the corresponding reaction wells, set up three multiple wells for each concentration, and incubate at 37°C for 1h.
5)加酶标二抗：取出酶标板，反复洗板5遍，每孔300μL洗板液。向反应孔中加入HRP标记的羊抗人IgG-Fc抗体稀释液，每孔100μL，37℃孵育1h。5) Add enzyme-labeled secondary antibody: take out the enzyme-labeled plate, wash the plate 5 times, 300μL of washing solution per well. Add HRP-labeled goat anti-human IgG-Fc antibody diluent to the reaction well, 100 μL per well, and incubate at 37°C for 1 hour.
6)显色：取出酶标板，重复洗板8遍，每孔300μL洗板液。向反应孔中加入TMB显色液，每孔100μL，室温(25±2℃)避光显色10～15min。6) Color development: Take out the ELISA plate and repeat the washing for 8 times, with 300 μL washing solution per well. Add TMB color developing solution to the reaction wells, 100 μL per well, and develop color at room temperature (25±2°C) in the dark for 10-15 minutes.
7)终止：向每个反应孔中加入2M硫酸溶液50μL终止显色反应，然后于30min内进行读数。7) Termination: Add 50μL of 2M sulfuric acid solution to each reaction well to terminate the color reaction, and then read within 30min.
8)读板：设定波长为450nm，用酶标仪自带分析软件进行读数，对所得读数以四参数方程曲线模型进行拟合，方程为y＝(A-D)/(1+(x/C)^B)+D，其中A为吸光值下限，B代表曲线斜率，C为最大反应值一半对应的抗体浓度(EC 50)，D为吸光值上限。 8) Reading plate: set the wavelength to 450nm, read with the analysis software of the microplate reader, and fit the obtained readings with a four-parameter equation curve model, the equation is y=(AD)/(1+(x/C) )^B)+D, where A is the lower limit of absorbance, B represents the slope of the curve, C is the antibody concentration (EC 50 ) corresponding to half of the maximum response value, and D is the upper limit of absorbance.
9)结果计算：按如下公式结算结果9) Result calculation: Settlement result according to the following formula
Figure PCTCN2020140885-appb-000008
Figure PCTCN2020140885-appb-000008
以下实施例的抗Trop2-药物偶联物以ADC-1为例。The anti-Trop2-drug conjugates in the following examples take ADC-1 as an example.
实施例1：pH值范围筛选Example 1: Screening of pH range
选取pH覆盖4.5～6.5范围的两种缓冲液：组氨酸缓冲液(His)和琥珀酸缓冲液(HPS)，制备出pH为His5.5、His6.0、His6.5、HPS4.5、HPS5.0、HPS5.5的六组溶液样品，其中，每组溶液中抗Trop2抗体的浓度为12.5mg/ml。将样品分装后置于40℃条件下进行高温试验，于第0天、第1周、第2周、第3周取样，进行SEC-HPLC、HIC-HPLC检测。Choose two buffers whose pH covers the range of 4.5-6.5: histidine buffer (His) and succinic acid buffer (HPS), and prepare the pH as His5.5, His6.0, His6.5, HPS4.5, HPS5.0 and HPS5.5 six sets of solution samples, wherein the concentration of anti-Trop2 antibody in each set of solutions is 12.5mg/ml. The samples were divided into aliquots and placed at 40°C for a high temperature test. Samples were taken on the 0th day, 1st week, 2nd week, and 3rd week for SEC-HPLC and HIC-HPLC detection.
从图1～图3可以看出，六组样品在高温条件下考察3周后，单体纯度均有所下降，聚体和片段含量增加，说明聚合和片段化是抗Trop2抗体偶联物的主要降解途径之一。在相同试验条件下，六组样品在SEC-HPLC单体纯度方面的稳定性有显著差异；图中，His55 表示His5.5，His60表示His6.0，His65表示His6.5，HPS45表示HPS4.5，HPS50表示His5.0，HPS55表示His5.5。It can be seen from Figure 1 to Figure 3 that the purity of the monomers of the six groups of samples decreased after 3 weeks of investigation under high temperature conditions, and the content of aggregates and fragments increased, indicating that the polymerization and fragmentation are anti-Trop2 antibody conjugates. One of the main degradation pathways. Under the same test conditions, the six groups of samples have significant differences in the stability of SEC-HPLC monomer purity; in the figure, His55 represents His5.5, His60 represents His6.0, His65 represents His6.5, and HPS45 represents HPS4.5. , HPS50 means His5.0, HPS55 means His5.5.
具体而言，从SEC-HPLC单体纯度数据作图结果可以看出，6组样品的SEC单体纯度均呈现下降趋势，但是各组的下降幅度有明显区别，His55>HPS45>His60>HPS50>His65>HPS55，表明样品在pH≤6.0的His缓冲液中单体纯度稳定性较好。Specifically, from the results of SEC-HPLC monomer purity data mapping, it can be seen that the SEC monomer purity of the 6 groups of samples all show a downward trend, but the decline of each group is obviously different, His55>HPS45>His60>HPS50> His65>HPS55, indicating that the monomer purity stability of the sample in His buffer with pH≤6.0 is better.
从SEC聚体数据作图结果可以看出，His缓冲液的三个pH从低到高，聚体的增加明显，HPS缓冲液也是同样情况，说明pH低较好；6组样品的SEC聚体均呈现上升趋势，HPS45组和His55组的上升趋势较缓，优于His60优于其他组，His55＝HPS45>His60>HPS50>His65>HPS55，表明样品在pH≤6.0的His缓冲液中产生的聚体较少。It can be seen from the results of SEC aggregate data mapping that the three pHs of His buffer range from low to high, and the increase in aggregates is obvious. The same is true for HPS buffer, indicating that low pH is better; the SEC aggregates of the 6 groups of samples Both showed an upward trend, the HPS45 group and His55 group showed a slower upward trend, better than His60 than other groups, His55=HPS45>His60>HPS50>His65>HPS55, indicating that the sample produced in the His buffer pH≤6.0 aggregates Less body.
从SEC片段数据作图结果可以看出，HPS45明显较其他组差，表明样品在pH 4.5的HPS缓冲液中产生的片段较多，故样品在琥珀酸缓冲液中pH应选择大于4.5的范围。From the results of SEC fragment data mapping, it can be seen that HPS45 is significantly worse than other groups, indicating that the sample produces more fragments in the HPS buffer at pH 4.5, so the pH of the sample in the succinic acid buffer should be selected to be greater than 4.5.
从图4可知，抗Trop2抗体偶联物在pH≤5.5时，HIC稳定性较好，当pH＞5.5时，样品的HIC主峰含量下降较快，稳定性较差。相同试验条件下，六组样品在HIC-HPLC主峰含量方面的稳定性有明显差异。从HIC-HPLC主峰含量数据作图结果可以看出，HPS45、HPS50、HPS55、His55优于His60和His65，HPS45>HPS50>HPS55>His55>His60>His65，表明样品在pH≤5.5的缓冲液中，HIC主峰含量稳定性较好。It can be seen from Figure 4 that the HIC stability of the anti-Trop2 antibody conjugate is better when the pH is less than or equal to 5.5, and when the pH is more than 5.5, the HIC main peak content of the sample decreases rapidly and the stability is poor. Under the same experimental conditions, the stability of the six groups of samples in terms of the main peak content of HIC-HPLC has obvious differences. From the results of HIC-HPLC main peak content data mapping, it can be seen that HPS45, HPS50, HPS55, His55 are better than His60 and His65, HPS45>HPS50>HPS55>His55>His60>His65, indicating that the sample is in a buffer with pH≤5.5. The content of the main peak of HIC is stable.
综合SEC-HPLC和HIC-HPLC的结果，高温(40℃)条件下，pH范围在4.5<pH<5.5时，抗Trop2抗体偶联物的稳定性较好。Based on the results of SEC-HPLC and HIC-HPLC, the stability of the anti-Trop2 antibody conjugate is better when the pH range is 4.5<pH<5.5 under high temperature (40℃) conditions.
实施例2：pH值优化筛选试验Example 2: pH value optimization screening test
已确定抗Trop2抗体偶联物在pH5.0附近的组氨酸或琥珀酸缓冲液中稳定性较好，现对抗Trop2抗体偶联物进行pH值优化筛选试验。制备出6个pH梯度的制剂处方：组氨酸-琥珀酸缓冲液(pH4.6)、组氨酸-琥珀酸缓冲液(pH4.8)、组氨酸-琥珀酸缓冲液(pH5.0)、组氨酸-琥珀酸缓冲液(pH5.2)、组氨酸-琥珀酸缓冲液(pH5.4)、组氨酸-琥珀酸缓冲液(pH5.6)。组氨酸缓冲液与琥珀酸缓冲液的摩尔浓度比为1:1。抗Trop2抗体偶联物浓度均调整至10mg/ml，将样品置于40℃条件下进行高温试验，于第0天、第1周、第2周、第3周、第4周取样，分别进行SEC-HPLC、HIC-HPLC检测，结果见图5、图6和图7。It has been determined that the anti-Trop2 antibody conjugate has good stability in histidine or succinic acid buffer around pH 5.0. Now the anti-Trop2 antibody conjugate is subjected to a pH optimization screening test. Prepared formulations with 6 pH gradients: histidine-succinic acid buffer (pH4.6), histidine-succinic acid buffer (pH4.8), histidine-succinic acid buffer (pH5.0) ), histidine-succinic acid buffer (pH5.2), histidine-succinic acid buffer (pH5.4), histidine-succinic acid buffer (pH5.6). The molar concentration ratio of histidine buffer to succinic acid buffer is 1:1. The concentration of the anti-Trop2 antibody conjugate was adjusted to 10mg/ml, the samples were placed at 40°C for high temperature test, and samples were taken on day 0, week 1, week 2, week 3, and week 4. SEC-HPLC, HIC-HPLC detection, the results are shown in Figure 5, Figure 6 and Figure 7.
经过4周的高温试验，六组样品的单体纯度都有下降，聚体含量也有增加。相同试验条件下，六组样品在SEC-HPLC单体纯度方面的稳定性差异明显。After 4 weeks of high temperature test, the monomer purity of the six groups of samples has decreased, and the polymer content has also increased. Under the same experimental conditions, the stability of the six groups of samples in terms of SEC-HPLC monomer purity is significantly different.
从图5的SEC-HPLC单体纯度数据图可知，六组样品的变化趋势基本一致。在高温条件下放置4周后，样品的单体纯度下降明显。同一时间点，在单体纯度方面，六组样品有差异。pH5.6组样品SEC-HPLC单体纯度较差。From the SEC-HPLC monomer purity data chart in Figure 5, it can be seen that the changing trends of the six groups of samples are basically the same. After being placed under high temperature conditions for 4 weeks, the monomer purity of the sample decreased significantly. At the same time, the six groups of samples were different in terms of monomer purity. The SEC-HPLC monomer purity of pH5.6 group samples was poor.
从图6的SEC-HPLC聚体数据图可知，六组样品的变化趋势一致，都有不同程度的上升。在高温条件下放置4周后，样品的聚体增长明显。同一时间点，在SEC-HPLC聚体方 面，六组样品有显著差异。pH在4.6-5.2之间，SEC-HPLC聚体增长幅度较小。From the SEC-HPLC aggregate data graph in Figure 6, it can be seen that the six groups of samples have the same trend of change, and all have different degrees of increase. After being placed under high temperature conditions for 4 weeks, the aggregates of the sample increased significantly. At the same time point, there are significant differences among the six groups of samples in terms of SEC-HPLC aggregates. When the pH is between 4.6-5.2, the growth of SEC-HPLC aggregates is small.
从HIC-HPLC数据结果分析可知，在高温条件下，随着时间的延长，蛋白的HIC-HPLC主峰含量有所下降。相同试验条件下，六组样品在HIC-HPLC主峰含量方面的稳定性有显著差异。从图7的HIC-HPLC主峰含量变化趋势图可以看出，目的蛋白在高温下不稳定，随着时间的延长，六组样品的主峰含量均有不同程度的下降，pH在4.6-5.2时，样品的HIC-HPLC主峰下降较缓，稳定性较好。From the analysis of HIC-HPLC data results, it can be seen that under high temperature conditions, with the extension of time, the content of the main HIC-HPLC peak of the protein decreases. Under the same experimental conditions, the six groups of samples have significant differences in the stability of the main peak content of HIC-HPLC. From the HIC-HPLC main peak content change trend graph in Figure 7, it can be seen that the target protein is unstable at high temperature. As time goes by, the main peak content of the six groups of samples decreases to varying degrees. When the pH is between 4.6 and 5.2, The main peak of HIC-HPLC of the sample decreased slowly, and the stability was better.
综合SEC-HPLC及HIC-HPLC的结果可知，缓冲液的pH在4.6～5.2时，样品抗Trop2-ADC的稳定性较好。Based on the results of SEC-HPLC and HIC-HPLC, it can be seen that when the pH of the buffer is between 4.6 and 5.2, the stability of the sample against Trop2-ADC is better.
从实验结果可知，pH值是本品抗Trop2-ADC制剂处方中的一个关键因子。样品在不同pH值的缓冲液中表现出不同的稳定性，但当pH值在某一个范围内波动时，并不会对目的蛋白的质量产生显著性的影响。且考虑到长期稳定性考察要求以及产业化要求，制剂pH值必须提供一个有效的范围，而非一个固定值。综合以上研究初步判定，在组氨酸-琥珀酸缓冲体系中pH 4.6～5.2范围内，样品抗Trop2-ADC的稳定性较好。It can be seen from the experimental results that the pH value is a key factor in the formulation of the anti-Trop2-ADC preparation of this product. Samples show different stability in buffers with different pH values, but when the pH value fluctuates within a certain range, it will not have a significant impact on the quality of the target protein. And considering the long-term stability inspection requirements and industrialization requirements, the pH value of the formulation must provide an effective range, not a fixed value. Based on the above research, it is preliminarily determined that the stability of the sample against Trop2-ADC is better in the pH range of 4.6 to 5.2 in the histidine-succinic acid buffer system.
实施例3：缓冲剂筛选试验Example 3: Buffer screening test
已确定本发明的抗Trop2抗体偶联物在pH 4.5-5.5范围条件下稳定性较好，故选取缓冲能力范围在pH5.0附近的缓冲液进行筛选试验。制备5种不同的缓冲液：琥珀酸-琥珀酸钠缓冲液(HPS)、柠檬酸-柠檬酸钠缓冲液(NMS)、琥珀酸-柠檬酸缓冲液(HPSNMS)、组氨酸-琥珀酸缓冲液(HisHPS)、组氨酸-柠檬酸缓冲液(HisNMS)。各组溶液中，抗Trop2抗体偶联物的浓度为11mg/ml，溶液pH值为5.0，分装，置于40℃条件下进行高温试验，于第0天、第1周、第2周、第3周、第4周取样，分别进行SEC-HPLC、HIC-HPLC检测。It has been determined that the anti-Trop2 antibody conjugate of the present invention has better stability under pH 4.5-5.5 range conditions, so a buffer with a buffering capacity around pH 5.0 is selected for the screening test. Prepare 5 different buffers: succinate-sodium succinate buffer (HPS), citric acid-sodium citrate buffer (NMS), succinate-citrate buffer (HPSNMS), histidine-succinate buffer Solution (HisHPS), histidine-citrate buffer (HisNMS). In each group of solutions, the concentration of the anti-Trop2 antibody conjugate was 11mg/ml, the pH value of the solution was 5.0, and the solution was divided into aliquots and placed at 40℃ for high temperature test. On day 0, week 1, week 2, Samples were taken in the 3rd and 4th weeks and tested by SEC-HPLC and HIC-HPLC respectively.
经过4周的高温试验，五组样品的聚体、片段各有不同程度的增加，单体纯度则都有下降。相同试验条件下，五组样品在SEC-HPLC单体纯度方面的稳定性有显著差异。After 4 weeks of high temperature test, the aggregates and fragments of the five groups of samples increased to varying degrees, while the purity of the monomers decreased. Under the same experimental conditions, the five groups of samples have significant differences in the stability of SEC-HPLC monomer purity.
从图8的SEC-HPLC单体纯度数据图分析可知，五组样品的变化趋势基本一致，但是下降幅度有显著差异。在高温条件下放置4周后，在单体纯度方面，HisHPS缓冲液优于HisNMS缓冲液优于HPS缓冲液优于其他组。From the analysis of the SEC-HPLC monomer purity data graph in Fig. 8, it can be seen that the change trend of the five groups of samples is basically the same, but the decline range is significantly different. After being placed at high temperature for 4 weeks, in terms of monomer purity, HisHPS buffer is better than HisNMS buffer and HPS buffer is better than other groups.
从图9的SEC-HPLC聚体数据图分析可知，五组样品的聚体含量变化趋势基本一致。在高温条件下放置4周后，各组中多聚体略有增加，从增长幅度来看，HisHPS缓冲液优于HisNMS缓冲液优于HPS缓冲液优于其他组。From the analysis of the SEC-HPLC aggregate data graph in Figure 9, it can be seen that the change trend of the aggregate content of the five groups of samples is basically the same. After being placed under high temperature conditions for 4 weeks, the multimers in each group increased slightly. From the perspective of the growth rate, HisHPS buffer is better than HisNMS buffer and HPS buffer is better than other groups.
从图10的SEC-HPLC片段数据图分析可知，五组样品的片段变化趋势基本一致。在高温条件下放置4周后，样品的片段均有所增加，其中，HisHPS缓冲液优于HisNMS缓冲液优于其他组。From the analysis of the SEC-HPLC fragment data graph in Figure 10, it can be seen that the fragment change trends of the five groups of samples are basically the same. After being placed at high temperature for 4 weeks, the fragments of the samples increased. Among them, HisHPS buffer was better than HisNMS buffer better than other groups.
从HIC-HPLC数据分析结果可知，在高温条件下，随着时间的延长，5组样品的主峰含量均有不同程度的下降。相同试验条件下，五组样品在HIC-HPLC主峰含量方面的稳定性有显著差异(图11)。其中，NMS缓冲液、HPSNMS缓冲液、HisNMS缓冲液较好，优 于其他组。From the results of HIC-HPLC data analysis, it can be seen that under high temperature conditions, with the extension of time, the main peak content of the five groups of samples has decreased to varying degrees. Under the same experimental conditions, the five groups of samples had significant differences in the stability of the main peak content of HIC-HPLC (Figure 11). Among them, NMS buffer, HPSNMS buffer, and HisNMS buffer are better and better than other groups.
综合SEC-HPLC及HIC-HPLC的结果可知，高温(40℃)环境下，样品在HisHPS缓冲液中，SEC-HPLC稳定性很好，HIC-HPLC稳定性也不错；而NMS缓冲液，在SEC-HPLC稳定性方面表现较差，故选择HisHPS作为抗Trop2-ADC的制剂缓冲体系。Based on the results of SEC-HPLC and HIC-HPLC, it can be seen that under high temperature (40℃) environment, the stability of SEC-HPLC is very good when the sample is in HisHPS buffer, and the stability of HIC-HPLC is also good; while the stability of NMS buffer is good in SEC -The stability of HPLC is poor, so HisHPS is chosen as the buffer system for anti-Trop2-ADC preparations.
实施例4：稳定剂种类筛选试验Example 4: Screening test of stabilizer types
分别制备含蔗糖、海藻糖、甘露醇、甘氨酸、山梨醇等五种稳定剂的ADC-1溶液，具体配方见表2。本实施例中海藻糖重量百分比是以海藻糖二水合物的重量计算的百分比。ADC-1 solutions containing five stabilizers including sucrose, trehalose, mannitol, glycine, and sorbitol were prepared respectively. See Table 2 for specific formulations. The weight percentage of trehalose in this embodiment is the percentage calculated by the weight of trehalose dihydrate.
表2：不同稳定剂制剂配方Table 2: Formulations of different stabilizer preparations
Figure PCTCN2020140885-appb-000009
Figure PCTCN2020140885-appb-000009
试验方案如下：The test plan is as follows:
1、高温实验1. High temperature experiment
在高温(40℃)条件下，加有稳定剂的样品与未加稳定剂的样品进行对比试验，考察样品质量的变化情况，于第0天(0d)、第1周(1w)、第2周(2w)、第3周(3w)、第4周(4w)取样进行SEC-HPLC、HIC-HPLC检测。结果见表3。Under the condition of high temperature (40℃), the sample with stabilizer was added and the sample without stabilizer was tested to compare the quality of the sample. On the 0th day (0d), the first week (1w), and the second Week (2w), 3rd week (3w), 4th week (4w) samples were taken for SEC-HPLC and HIC-HPLC detection. The results are shown in Table 3.
表3：不同稳定剂样品SEC-HPLC、HIC-HPLC随着时间的变化情况Table 3: SEC-HPLC and HIC-HPLC changes of different stabilizer samples over time
Figure PCTCN2020140885-appb-000010
Figure PCTCN2020140885-appb-000010
Figure PCTCN2020140885-appb-000011
Figure PCTCN2020140885-appb-000011
*样品名称：“-”前的数字表示表2中的配方序号，“-”后的数字和字母表示实验时间，如4w表示4周.*Sample name: The number before "-" indicates the recipe number in Table 2, and the numbers and letters after "-" indicate the experimental time, such as 4w for 4 weeks.
从表3中数据分析可知，经过4周的高温试验，六组样品的SEC-HPLC聚体段随着时间的增长各有不同程度的增加(图13)，单体纯度都有下降(图12)。相同试验条件下，六组样品在SEC-HPLC单体纯度方面的稳定性有略微差异。From the data analysis in Table 3, it can be seen that after 4 weeks of high temperature test, the SEC-HPLC polymer segment of the six groups of samples increased to varying degrees with time (Figure 13), and the monomer purity decreased (Figure 12). ). Under the same experimental conditions, the stability of the six groups of samples in terms of SEC-HPLC monomer purity was slightly different.
从图12的SEC-HPLC单体纯度数据图分析可知，六组样品的变化趋势基本一致，但是下降幅度略有不同，除4号和6号配方略低外，其他组单体纯度差异不大。From the analysis of the SEC-HPLC monomer purity data graph in Figure 12, it can be seen that the change trend of the six groups of samples is basically the same, but the decline is slightly different. Except for the slightly lower formula of No. 4 and No. 6, the purity of the other groups is not much different. .
从图13的SEC-HPLC聚体数据作图分析可知，六组样品的聚体含量变化趋势基本一致。从图13分析可知，在高温条件下放置4周后，样品的多聚体略有增加，从增长幅度来看，4号和6号略差。From the SEC-HPLC aggregate data mapping analysis in Figure 13, it can be seen that the change trend of aggregate content of the six groups of samples is basically the same. From the analysis in Figure 13, it can be seen that after being placed under high temperature conditions for 4 weeks, the polymer of the sample increased slightly. From the perspective of the growth rate, No. 4 and No. 6 were slightly worse.
从图14的HIC-HPLC主峰含量变化趋势图可以看出，在40℃高温条件下，随着时间的延长，样品的HIC-HPLC主峰含量均有所下降，相同试验条件下，六组样品在HIC-HPLC主峰含量方面的稳定性有较大差异。随着时间的增加，6组样品的主峰含量均有不同程度的下降，从下降幅度来看，1、2号较好，4号最差。From the HIC-HPLC main peak content change trend chart in Figure 14, it can be seen that under the high temperature condition of 40℃, with the extension of time, the HIC-HPLC main peak content of the samples decreased. Under the same test conditions, the six groups of samples were There is a big difference in the stability of HIC-HPLC main peak content. With the increase of time, the main peak content of the 6 groups of samples all decreased to varying degrees. From the perspective of the decline, No. 1 and No. 2 are better, and No. 4 is the worst.
2、冻干后样品的高温试验2. High temperature test of samples after freeze-drying
对加有稳定剂的样品进行冻干，在高温(40℃)条件下进行对比试验，考察样品质量的变化情况，于第30天(30d)取样进行SEC-HPLC、HIC-HPLC检测，并与未高温的样品进行对比，结果见表4。The samples with stabilizers were freeze-dried, and a comparative test was conducted under high temperature (40°C) conditions to investigate the changes in the quality of the samples. On the 30th day (30d), samples were taken for SEC-HPLC and HIC-HPLC detection, and compared with The samples without high temperature were compared, and the results are shown in Table 4.
表4：不同稳定剂样品SEC-HPLC、HIC-HPLC随着时间的变化情况Table 4: SEC-HPLC and HIC-HPLC changes of different stabilizer samples over time
Figure PCTCN2020140885-appb-000012
Figure PCTCN2020140885-appb-000012
从表4中数据分析可知，经过30天的高温试验，五组样品的SEC聚体随着时间的增长有不同程度的增加，单体纯度则都有下降。相同试验条件下，五组样品在SEC-HPLC单体纯度方面的稳定性差异明显，3号和4号处方的SEC-HPLC稳定性较差，表5的微粒数据也显示3号和4号较差。从HIC-HPLC数据来看，4号和5号处方稳定性较差。From the data analysis in Table 4, it can be seen that after 30 days of high temperature test, the SEC polymer of the five groups of samples increased to varying degrees with the increase of time, while the purity of the monomers all decreased. Under the same experimental conditions, the five groups of samples showed significant differences in the stability of SEC-HPLC monomer purity. The SEC-HPLC stability of prescription No. 3 and No. 4 was poor. The particle data in Table 5 also showed that No. 3 and No. 4 were relatively stable. difference. Judging from the HIC-HPLC data, formulations No. 4 and No. 5 have poor stability.
表5：不同稳定剂样品复溶后的微粒情况Table 5: The particles of different stabilizer samples after reconstitution
Figure PCTCN2020140885-appb-000013
Figure PCTCN2020140885-appb-000013
综合以上实验结果，选取生物单抗制剂中比较常用的海藻糖作为本制剂处方中的稳定剂，蔗糖可作为备选稳定剂。Based on the above experimental results, trehalose, which is commonly used in biological monoclonal antibody preparations, is selected as the stabilizer in the formulation of this preparation, and sucrose can be used as an alternative stabilizer.
实施例5：表面活性剂含量筛选试验—与生理盐水相容性试验Example 5: Screening test of surfactant content—compatibility test with physiological saline
本发明的固体制剂临床上需要与生理盐水配伍给药，故进行此相容性试验，以确定表面活性剂浓度为多少时，能有效提高蛋白的稳定性。The solid preparation of the present invention needs to be administered clinically with physiological saline. Therefore, this compatibility test is performed to determine the concentration of the surfactant, which can effectively improve the stability of the protein.
本实施例制备含不同浓度、不同种类表面活性剂的ADC样品溶液，具体配方见表6，各组溶液中的ADC浓度为25mg/ml。分别取相同体积含不同浓度表面活性剂的样品溶液，按配伍比例(4ml样品：100ml生理盐水)加入到0.9％氯化钠溶液中，轻微、充分混匀后观察其性状，并对放置2h后的蛋白溶液进行不溶性微粒检测。检测结果见表7，图15和 图16。In this embodiment, ADC sample solutions containing different concentrations and different types of surfactants are prepared. The specific formula is shown in Table 6, and the ADC concentration in each group of solutions is 25 mg/ml. Take the same volume of sample solutions containing different concentrations of surfactants, add them to the 0.9% sodium chloride solution according to the compatibility ratio (4ml sample: 100ml physiological saline), and observe the properties after mixing slightly and thoroughly, and leave it for 2h. The protein solution is tested for insoluble particles. The test results are shown in Table 7, Figure 15 and Figure 16.
表6：不同配方蛋白溶液Table 6: Different formula protein solutions
Figure PCTCN2020140885-appb-000014
Figure PCTCN2020140885-appb-000014
％表示质量体积比，g/100ml% Means mass volume ratio, g/100ml
表7：ADC样品溶液在生理盐水中的微粒变化情况Table 7: Changes of particles in ADC sample solution in saline
Figure PCTCN2020140885-appb-000015
Figure PCTCN2020140885-appb-000015
对表7中微粒均值数据进行作图，得图15和图16：Plotting the average particle data in Table 7, we get Figure 15 and Figure 16:
从图15和图16中结果可以看出，未加表面活性剂的Z0组微粒数最高，而加有表面活性剂的样品的微粒数普遍较低，且表面活性剂含量高一些时，微粒数略低。但当表面活性剂浓度到达一定程度后，样品的微粒数变化不大。辅料中表面活性剂的量也不宜过高，结合市售单抗制剂中表面活性剂的用量，本品选择常用的吐温80，含量可定为0.03％。It can be seen from the results in Figure 15 and Figure 16 that the number of particles in the Z0 group without surfactant is the highest, while the number of particles in the sample with surfactant is generally lower, and when the surfactant content is higher, the number of particles Slightly lower. But when the surfactant concentration reaches a certain level, the number of particles in the sample does not change much. The amount of surfactant in the excipients should not be too high. Combined with the amount of surfactant in the commercially available monoclonal antibody preparations, the commonly used Tween 80 is selected for this product, and the content can be set as 0.03%.
实施例6：固体制剂的制备Example 6: Preparation of solid preparation
综合以上所有试验的结果，本产品的液体制剂的一种具体成分和含量为：10mM琥珀酸、10mM盐酸组氨酸(或10mM盐酸组氨酸一水合物)、6％海藻糖二水合物(即158mM)、0.03％吐温80，用氢氧化钠调pH至4.9±0.3。其制备方式为：取抗Trop2抗体-美登素偶联 物100g，琥珀酸4.72g，盐酸组氨酸8.4g(这里盐酸组氨酸指的是盐酸组氨酸一水合物)，海藻糖二水合物240g和吐温80 1.2g，加水制备成4L溶液，以NaOH调节pH值至4.9±0.3。Based on the results of all the above tests, a specific component and content of the liquid preparation of this product are: 10mM succinic acid, 10mM histidine hydrochloride (or 10mM histidine hydrochloride monohydrate), 6% trehalose dihydrate ( That is, 158mM), 0.03% Tween 80, and adjust the pH to 4.9±0.3 with sodium hydroxide. The preparation method is as follows: take 100g of anti-Trop2 antibody-maytansine conjugate, 4.72g succinic acid, 8.4g histidine hydrochloride (herein histidine hydrochloride refers to histidine hydrochloride monohydrate), trehalose two 240g hydrate and 1.2g Tween 80, add water to prepare a 4L solution, and adjust the pH to 4.9±0.3 with NaOH.
将上述液体制剂冻干，得到固体制剂。The above liquid preparation is freeze-dried to obtain a solid preparation.
实施例7：固体制剂的性能检测Example 7: Performance testing of solid preparations
以实施例6制备的固体制剂进行实验。将溶液在-80℃冷冻24h后，在25℃放置24h为一次冻融。对冻融后的样品进行SEC-HPLC、HIC-HPLC、CE-SDS(NR)、结合活性等检测，结果见表8和表9。The solid preparation prepared in Example 6 was used for the experiment. After the solution was frozen at -80°C for 24 hours, it was placed at 25°C for 24 hours as a freeze-thaw cycle. SEC-HPLC, HIC-HPLC, CE-SDS (NR), binding activity and other tests were performed on the frozen and thawed samples. The results are shown in Table 8 and Table 9.
表8：实施例6固体制剂样品冻融5次的SEC-HPLC、HIC-HPLC结果Table 8: SEC-HPLC and HIC-HPLC results of the solid preparation sample of Example 6 frozen and thawed 5 times
Figure PCTCN2020140885-appb-000016
Figure PCTCN2020140885-appb-000016
*样品名称中的DR表示冻融，数字表示次数。如DR1表示冻融1次。*DR in the sample name indicates freeze-thaw, and the number indicates the number of times. For example, DR1 means freeze-thaw once.
从表8中数据可以看出，5次冻融后，样品的SEC和HIC数据变化不大，稳定性较好。It can be seen from the data in Table 8 that after five freeze-thaw cycles, the SEC and HIC data of the sample did not change much, and the stability was good.
表9：实施例6固体制剂样品冻融5次的CE-SDS(NR)、结合活性结果Table 9: The results of CE-SDS (NR) and binding activity of the solid preparation sample of Example 6 frozen and thawed 5 times
Figure PCTCN2020140885-appb-000017
Figure PCTCN2020140885-appb-000017
*HHLL表示是抗体含有两条重链和两条轻链，HHL表示抗体含有两条重链和一条轻链，fraction表示是片段。*HHLL means that the antibody contains two heavy chains and two light chains, HHL means that the antibody contains two heavy chains and one light chain, and fraction means that it is a fragment.
从表9中数据可以看出，5次冻融后，样品的CE-SDS(NR)数据变化不大，结合活性都在正常范围，稳定性较好。It can be seen from the data in Table 9 that after five freeze-thaw cycles, the CE-SDS (NR) data of the sample did not change much, the binding activity was in the normal range and the stability was good.
实施例8：固体制剂冻干前后稳定剂的影响Example 8: Effect of stabilizer before and after freeze-drying of solid preparation
根据表10的内容配制不同的制剂溶液，其中ADC含量为25mg/ml，pH值5.0，进行冻干，对冻干后的样品进行复溶，检测不溶性微粒、SEC-HPLC、HIC-HPLC等质量属性，检测结果见表11。Prepare different preparation solutions according to the content in Table 10. Among them, ADC content is 25mg/ml, pH value is 5.0, lyophilized, the lyophilized sample is reconstituted, and the quality of insoluble particles, SEC-HPLC, HIC-HPLC, etc. are detected Properties, the test results are shown in Table 11.
表10：冻干实验涉及的固体制剂配方Table 10: The solid preparation formula involved in the freeze-drying experiment
Figure PCTCN2020140885-appb-000018
Figure PCTCN2020140885-appb-000018
Figure PCTCN2020140885-appb-000019
Figure PCTCN2020140885-appb-000019
表11：冻干样品复溶后检测数据Table 11: Test data after reconstitution of freeze-dried samples
Figure PCTCN2020140885-appb-000020
Figure PCTCN2020140885-appb-000020
表11中，3、4号样品的SEC-HPLC单体纯度在冻干后略有下降，1号的稳定剂是6％蔗糖，2号是6％海藻糖，3号4％甘露醇，4号2％甘氨酸，5号4％山梨醇。本品制剂处方最终选择用海藻糖(2号)作为稳定剂，它在冻干前后的质量属性变化很小，复溶时间短，不溶性微粒检测结果也符合规定。In Table 11, the SEC-HPLC monomer purity of No. 3 and No. 4 samples decreased slightly after freeze-drying. No. 1 stabilizer was 6% sucrose, No. 2 was 6% trehalose, No. 3 was 4% mannitol, 4 No. 2% glycine, No. 5 4% sorbitol. The formulation of this product finally chooses trehalose (No. 2) as a stabilizer. Its quality attributes change little before and after freeze-drying, the reconstitution time is short, and the test results of insoluble particles also meet the requirements.
实施例9：样品复溶后稳定性试验Example 9: Stability test after sample reconstitution
将实施例6制备的固体制剂复溶，复溶浓度25mg/ml。将复溶后的溶液分别置于25℃ 和4℃条件下进行试验。在25℃条件下，分别于第6h、24h和48h取样检测；在4℃条件下，分别于第6h、24h和48h取样检测。检测项目包括外观、可见异物、pH、ADC浓度、不溶性微粒、SEC-HPLC、IEC-HPLC、CE-SDS(R)、CE-SDS(NR)、HIC-HPLC、PR-HPLC、毒素残留、生物活性、结合活性等，检测结果与第0h进行对比，结果见表12、表13。The solid preparation prepared in Example 6 was reconstituted to a reconstituted concentration of 25 mg/ml. The reconstituted solution was placed at 25°C and 4°C for testing. At 25°C, samples were taken at 6h, 24h, and 48h for testing; at 4°C, samples were taken at 6h, 24h, and 48h for testing. Test items include appearance, visible foreign matter, pH, ADC concentration, insoluble particles, SEC-HPLC, IEC-HPLC, CE-SDS(R), CE-SDS(NR), HIC-HPLC, PR-HPLC, toxin residue, biological For activity, binding activity, etc., the test results are compared with those at 0h, and the results are shown in Table 12 and Table 13.
表12：冻干粉复溶后稳定性试验(4℃)Table 12: Stability test of freeze-dried powder after reconstitution (4℃)
Figure PCTCN2020140885-appb-000021
Figure PCTCN2020140885-appb-000021
注：外观标准为本品为白色或微黄色块状疏松体；可见异物标准为无可见异物检出；毒素残留标准为细菌内毒素<2EU/ml；N/A表示无检测结果。Note: The appearance standard is white or slightly yellow lumpy loose body; the visible foreign body standard is no visible foreign body detected; the toxin residue standard is bacterial endotoxin <2EU/ml; N/A means no test result.
表13：冻干粉复溶后稳定性试验(25℃)Table 13: Stability test of freeze-dried powder after reconstitution (25℃)
Figure PCTCN2020140885-appb-000022
Figure PCTCN2020140885-appb-000022
Figure PCTCN2020140885-appb-000023
Figure PCTCN2020140885-appb-000023
注：外观标准为本品为白色或微黄色块状疏松体；可见异物标准为无可见异物检出；毒素残留标准为细菌内毒素<2EU/ml；N/A表示无检测结果。Note: The appearance standard is white or slightly yellow lumpy loose body; the visible foreign body standard is no visible foreign body detected; the toxin residue standard is bacterial endotoxin <2EU/ml; N/A means no test result.
结果显示，复溶后的样品在25℃和4℃放置48小时，与第0小时相比，样品的外观、可见异物、pH、ADC浓度、SEC-HPLC、IEC-HPLC、CE-SDS(R)、CE-SDS(NR)、HIC-HPLC、PR-HPLC、毒素残留、生物活性、结合活性等质量属性没有发生明显变化，说明复溶后的样品在25℃和4℃在条件下可放置48小时。从无菌安全方面考虑，复溶的样品应立即使用，如不立即使用，复溶后可在2-8℃冰箱内储存6小时，不宜冻结或摇晃。The results showed that the reconstituted sample was placed at 25°C and 4°C for 48 hours. Compared with the 0 hour, the appearance of the sample, visible foreign matter, pH, ADC concentration, SEC-HPLC, IEC-HPLC, CE-SDS(R ), CE-SDS (NR), HIC-HPLC, PR-HPLC, toxin residue, biological activity, binding activity and other quality attributes have not changed significantly, indicating that the reconstituted sample can be placed at 25°C and 4°C 48 hours. From the perspective of sterility and safety, the reconstituted sample should be used immediately. If it is not used immediately, it can be stored in a refrigerator at 2-8°C for 6 hours after reconstitution. It should not be frozen or shaken.
实施例10：给药稀释稳定性试验Example 10: Dilution stability test for administration
本品在临床上采用静脉滴注，需要用氯化钠注射液稀释后才能用于患者身上。因此，需要进行配伍试验及给药过程稳定性试验。This product is intravenously injected in clinical practice, and it needs to be diluted with sodium chloride injection before it can be used on patients. Therefore, compatibility tests and stability tests of the drug delivery process are required.
将实施例6的固体制剂与0.9％氯化钠注射液进行配伍试验。The solid preparation of Example 6 was tested for compatibility with 0.9% sodium chloride injection.
实验方法：取实施例6的固体制剂(规格：100mg/瓶)，用无菌注射用水4ml复溶，混匀后静置，抽取一定量的药品，转移至一个含0.9％氯化钠注射液的容器，通过轻轻倒置混匀稀释溶液，最终稀释溶液浓度在0.5mg/ml至5mg/ml间。Experimental method: Take the solid preparation of Example 6 (specification: 100mg/bottle), reconstitute it with 4ml of sterile water for injection, mix well and let stand, extract a certain amount of medicine, and transfer to an injection containing 0.9% sodium chloride In the container, mix the diluted solution by gently inverting, and the final concentration of the diluted solution is between 0.5mg/ml and 5mg/ml.
取本品制剂，用氯化钠注射液分别稀释至ADC浓度为0.5mg/ml和5.0mg/ml，轻轻倒置瓶子使其混合均匀，将混合溶液分别置于4℃和25℃条件下进行试验。在4℃条件下，分别于第2h，6h，24h取样检测；在25℃条件下分别于第2h，6h，24h取样检测。检测项目包括外观、可见异物、pH值、稀释后ADC浓度、不溶性微粒、渗透压、SEC-HPLC单体纯度、IEC-HPLC、CE-SDS(R)、CE-SDS(NR)、HIC-HPLC、PR-HPLC、毒素残留、相对生物学活性、结合活性，检测结果与第0h进行比较。结果见表14，表15。Take the preparation of this product and dilute it with sodium chloride injection to the ADC concentration of 0.5mg/ml and 5.0mg/ml, gently invert the bottle to make it evenly mixed, and place the mixed solution at 4℃ and 25℃ respectively. test. At 4℃, samples were taken at 2h, 6h, and 24h for testing; at 25℃, samples were taken at 2h, 6h, and 24h for testing. Test items include appearance, visible foreign matter, pH value, ADC concentration after dilution, insoluble particles, osmotic pressure, SEC-HPLC monomer purity, IEC-HPLC, CE-SDS(R), CE-SDS(NR), HIC-HPLC , PR-HPLC, toxin residue, relative biological activity, binding activity, the test results are compared with the 0h. The results are shown in Table 14, Table 15.
表14：制剂稀释稳定性试验(0.5mg/ml)Table 14: Preparation dilution stability test (0.5mg/ml)
Figure PCTCN2020140885-appb-000024
Figure PCTCN2020140885-appb-000024
Figure PCTCN2020140885-appb-000025
Figure PCTCN2020140885-appb-000025
从表14可以看出，样品经生理盐水稀释至0.5mg/ml后，其外观、可见异物、pH值、稀释后ADC浓度、不溶性微粒、渗透压、SEC-HPLC单体纯度、CE-SDS(R)、CE-SDS(NR)、HIC-HPLC、PR-HPLC、毒素残留、相对生物学活性、结合活性都没有发生明显变化。It can be seen from Table 14 that after the sample is diluted to 0.5mg/ml with saline, its appearance, visible foreign matter, pH value, ADC concentration after dilution, insoluble particles, osmotic pressure, SEC-HPLC monomer purity, CE-SDS ( R), CE-SDS (NR), HIC-HPLC, PR-HPLC, toxin residue, relative biological activity, and binding activity did not change significantly.
表15：制剂稀释稳定性试验(5mg/ml)Table 15: Preparation dilution stability test (5mg/ml)
Figure PCTCN2020140885-appb-000026
Figure PCTCN2020140885-appb-000026
Figure PCTCN2020140885-appb-000027
Figure PCTCN2020140885-appb-000027
从表14和表15可知，本品与氯化钠注射液混合稀释后，分别在4℃条件下放置24h，在25℃条件下放置24h，混合溶液澄清透明，没有可见异物，稀释液的pH值和ADC浓度均没有发生明显变化，不溶性微粒的检测数据略有波动，但没有出现逐渐增多的趋势，且检测数据均没有超过标准。说明本品能与0.9％氯化钠溶液配伍，在4℃下放置24h及25℃放置24h，本品的质量能保持稳定。It can be seen from Table 14 and Table 15 that after this product is mixed and diluted with sodium chloride injection, it is placed at 4°C for 24 hours and at 25°C for 24 hours. The mixed solution is clear and transparent, and there is no visible foreign matter. The pH of the diluted solution There was no significant change in the value and ADC concentration. The detection data of insoluble particles fluctuated slightly, but there was no gradual increase trend, and the detection data did not exceed the standard. This shows that this product can be compatible with 0.9% sodium chloride solution, placed at 4 ℃ for 24 hours and 25 ℃ for 24 hours, the quality of this product can remain stable.
实施例11：固体制剂的稳定性试验Example 11: Stability test of solid preparation
将实施例6制备的固体制剂分别置于25℃和4℃条件下进行试验。在25℃条件下，分别于第1月、2月、3月、6月、9月和12月取样，使用注射用水复溶再检测，复溶浓度25mg/ml；在4℃条件下，分别于3月、6月、9月、12月、18月和24月取样，使用注射用水复溶再检测，复溶浓度25mg/ml。检测项目包括外观、可见异物、pH、ADC浓度、不溶性微粒、SEC-HPLC、IEC-HPLC、CE-SDS(R)、CE-SDS(NR)、HIC-HPLC、PR-HPLC、毒素残留、生物活性、结合活性等，检测结果与第0h进行对比，结果见表16、表17。The solid preparations prepared in Example 6 were placed at 25°C and 4°C respectively for testing. At 25℃, samples were taken in the first month, February, March, June, September, and December, and reconstituted with water for injection and then tested. The reconstitution concentration was 25mg/ml; at 4℃, respectively Samples were taken in March, June, September, December, 18th, and 24 months, and re-dissolved with water for injection and re-tested. The reconstitution concentration was 25 mg/ml. Test items include appearance, visible foreign matter, pH, ADC concentration, insoluble particles, SEC-HPLC, IEC-HPLC, CE-SDS(R), CE-SDS(NR), HIC-HPLC, PR-HPLC, toxin residue, biological For activity, binding activity, etc., the test results are compared with those at 0h, and the results are shown in Table 16 and Table 17.
表16：冻干粉复溶后稳定性试验(4℃)Table 16: Stability test of freeze-dried powder after reconstitution (4℃)
Figure PCTCN2020140885-appb-000028
Figure PCTCN2020140885-appb-000028
Figure PCTCN2020140885-appb-000029
Figure PCTCN2020140885-appb-000029
注：外观标准为本品为白色或微黄色块状疏松体；可见异物标准为无可见异物检出；毒素残留标准为细菌内毒素<2EU/ml；N/A表示无检测结果。Note: The appearance standard is white or slightly yellow lumpy loose body; the visible foreign body standard is no visible foreign body detected; the toxin residue standard is bacterial endotoxin <2EU/ml; N/A means no test result.
表17：冻干粉复溶后稳定性试验(25℃)Table 17: Stability test of freeze-dried powder after reconstitution (25℃)
Figure PCTCN2020140885-appb-000030
Figure PCTCN2020140885-appb-000030
Figure PCTCN2020140885-appb-000031
Figure PCTCN2020140885-appb-000031
注：外观标准为本品为白色或微黄色块状疏松体；可见异物标准为无可见异物检出；毒素残留标准为细菌内毒素<2EU/ml；N/A表示无检测结果。Note: The appearance standard is white or slightly yellow lumpy loose body; the visible foreign body standard is no visible foreign body detected; the toxin residue standard is bacterial endotoxin <2EU/ml; N/A means no test result.
结果显示，固体制剂在25℃放置12个月的样品，以及在4℃条件下放置24个月的样品，与第0小时相比，样品的外观、可见异物、pH、ADC浓度、SEC-HPLC、IEC-HPLC、CE-SDS(R)、CE-SDS(NR)、HIC-HPLC、PR-HPLC、毒素残留、生物活性、结合活性等质量属性没有发生明显变化，说明本发明制备的固体制剂能够在25℃条件下放置12个月，或者在4℃条件下放置24个月，仍保持稳定。The results showed that the appearance, visible foreign matter, pH, ADC concentration, SEC-HPLC of samples of solid preparations stored at 25°C for 12 months and samples stored at 4°C for 24 months compared with the 0 hour , IEC-HPLC, CE-SDS(R), CE-SDS(NR), HIC-HPLC, PR-HPLC, toxin residue, biological activity, binding activity and other quality attributes have not changed significantly, indicating that the solid preparation prepared by the present invention It can be placed at 25°C for 12 months, or at 4°C for 24 months, and it remains stable.

Claims (20)

  1. 一种液体制剂，包括：抗Trop2抗体-药物偶联物，其中所述偶联物的浓度为10mg/ml～50mg/ml；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种，所述稳定剂的浓度为20mg/ml～80mg/ml，所述缓冲剂的浓度为5mM～60mM；所述表面活性剂的浓度为0.1mg/ml～0.5mg/ml；优选地，所述液体制剂pH值为4～7。A liquid preparation, comprising: an anti-Trop2 antibody-drug conjugate, wherein the concentration of the conjugate is 10 mg/ml-50 mg/ml; and further comprising one or more of stabilizers, buffers and surfactants The concentration of the stabilizer is 20mg/ml～80mg/ml, the concentration of the buffer is 5mM～60mM; the concentration of the surfactant is 0.1mg/ml～0.5mg/ml; preferably, the The pH value of the liquid preparation is 4-7.
  2. 根据权利要求1所述的液体制剂，其中，所述稳定剂为海藻糖和/或蔗糖；所述缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的组合，优选地，所述缓冲剂为组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂；所述表面活性剂为吐温20或吐温80。The liquid preparation according to claim 1, wherein the stabilizer is trehalose and/or sucrose; the buffer is a histidine buffer, a succinic acid buffer, a citric acid buffer or a combination thereof, preferably Preferably, the buffer is a histidine-succinic acid buffer or a histidine-citric acid buffer; the surfactant is Tween 20 or Tween 80.
  3. 根据权利要求2所述的液体制剂，其中，所述液体制剂包括，25mg/ml的抗Trop2抗体-药物偶联物，1.18mg/ml的琥珀酸，2.1mg/ml的盐酸组氨酸，158mM的海藻糖，0.3mg/ml的吐温80，用氢氧化钠调pH至4.6-5.2。The liquid preparation according to claim 2, wherein the liquid preparation comprises 25 mg/ml anti-Trop2 antibody-drug conjugate, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride, 158 mM Trehalose, 0.3mg/ml Tween 80, adjust the pH to 4.6-5.2 with sodium hydroxide.
  4. 根据权利要求2所述的液体制剂，其中，所述液体制剂包括，25mg/ml的抗Trop2抗体-药物偶联物，1.18mg/ml的琥珀酸，2.1mg/ml盐酸组氨酸一水合物，158mM的海藻糖，0.3mg/ml的吐温80，用氢氧化钠调pH至4.6-5.2。The liquid preparation according to claim 2, wherein the liquid preparation comprises 25 mg/ml anti-Trop2 antibody-drug conjugate, 1.18 mg/ml succinic acid, 2.1 mg/ml histidine hydrochloride monohydrate , 158mM trehalose, 0.3mg/ml Tween 80, adjust the pH to 4.6-5.2 with sodium hydroxide.
  5. 根据权利要求2所述的液体制剂，其中，所述液体制剂包括，25mg/ml的抗Trop2抗体-药物偶联物，10mM的琥珀酸，10mM的盐酸组氨酸，158mM的海藻糖，0.3mg/ml的吐温80，用氢氧化钠调pH至4.6-5.2。The liquid preparation according to claim 2, wherein the liquid preparation comprises 25 mg/ml of anti-Trop2 antibody-drug conjugate, 10 mM succinic acid, 10 mM histidine hydrochloride, 158 mM trehalose, 0.3 mg /ml Tween 80, adjust the pH to 4.6-5.2 with sodium hydroxide.
  6. 一种固体制剂，由权利要求1～5任一项所述的液体制剂冻干获得。A solid preparation obtained by freeze-drying the liquid preparation according to any one of claims 1 to 5.
  7. 一种固体制剂，包括：抗Trop2抗体-药物偶联物，其中所述偶联物的质量份数为10份～50份，优选20份～30份，更优选23～27份；还包括稳定剂、缓冲剂和表面活性剂中的一种或多种；所述稳定剂的质量份数为20份～80份，优选40份～80份，更优选50份～70份；所述缓冲剂的质量份数为1份～10份，优选3份～7份，更优选3份～4份；所述表面活性剂的质量份数为0.1份～0.5份，优选0.2～0.4份，更优选0.25份～0.35份。A solid preparation, comprising: an anti-Trop2 antibody-drug conjugate, wherein the mass parts of the conjugate are 10-50 parts, preferably 20-30 parts, more preferably 23-27 parts; also including stable One or more of agents, buffers and surfactants; the mass parts of the stabilizer is 20 parts to 80 parts, preferably 40 parts to 80 parts, more preferably 50 parts to 70 parts; the buffering agent The mass parts of the surfactant is 1 part to 10 parts, preferably 3 parts to 7 parts, more preferably 3 parts to 4 parts; the mass parts of the surfactant is 0.1 part to 0.5 parts, preferably 0.2 to 0.4 parts, more preferably 0.25 parts to 0.35 parts.
  8. 根据权利要求7所述的固体制剂，其中，所述稳定剂为海藻糖和/或蔗糖或其水合物；所述缓冲剂为组氨酸缓冲剂、琥珀酸缓冲剂、柠檬酸缓冲剂或它们的组合，优选地，所述缓冲剂为组氨酸-琥珀酸缓冲剂或组氨酸-柠檬酸缓冲剂；所述表面活性剂为吐温20或吐温80。The solid preparation according to claim 7, wherein the stabilizer is trehalose and/or sucrose or a hydrate thereof; the buffer is a histidine buffer, a succinic acid buffer, a citric acid buffer or these Preferably, the buffer is histidine-succinic acid buffer or histidine-citric acid buffer; the surfactant is Tween 20 or Tween 80.
  9. 根据权利要求8所述的固体制剂，其中，所述固体制剂包括：以质量份数计，25份抗Trop2抗体-药物偶联物，54份海藻糖，1.18份的琥珀酸和2.1份的盐酸组氨酸和0.3份吐温80。The solid preparation according to claim 8, wherein the solid preparation comprises: in parts by mass, 25 parts of anti-Trop2 antibody-drug conjugate, 54 parts of trehalose, 1.18 parts of succinic acid and 2.1 parts of hydrochloric acid Histidine and 0.3 parts Tween 80.
  10. 根据权利要求8所述的固体制剂，其中，所述固体制剂包括：以质量份数计，25份抗Trop2抗体-药物偶联物，60份海藻糖二水合物，1.18份的琥珀酸和2.1份的盐酸组氨酸一水合物和0.3份吐温80。The solid preparation according to claim 8, wherein the solid preparation comprises: in parts by mass, 25 parts of anti-Trop2 antibody-drug conjugate, 60 parts of trehalose dihydrate, 1.18 parts of succinic acid and 2.1 parts. Parts of histidine hydrochloride monohydrate and 0.3 parts of Tween 80.
  11. 一种复溶制剂，通过用药学上可接受的溶剂对权利要求6～10中任一项所述的固体制剂进行重构获得。A reconstituted preparation, which is obtained by reconstituting the solid preparation according to any one of claims 6 to 10 with a pharmaceutically acceptable solvent.
  12. 权利要求1～5任一项所述的液体制剂、权利要求6～10任一项所述的固体制剂或权利要求11所述的复溶制剂在制备用于预防和/或治疗Trop2阳性疾病的药物中的应用。The liquid preparation according to any one of claims 1 to 5, the solid preparation according to any one of claims 6 to 10, or the reconstituted preparation according to claim 11 are used for the prevention and/or treatment of Trop2-positive diseases. Application in medicine.
  13. 根据权利要求12所述的应用，其中，，所述Trop2阳性疾病选自三阴性乳腺癌、胶质母细胞瘤、成神经管细胞瘤、非小细胞肺癌、小细胞肺癌、上皮癌、乳腺癌、头颈癌、肾癌、卵巢癌、胃癌、卡波西肉瘤、胰腺癌和肺癌、***、结肠直肠癌、食管癌、口腔鳞状细胞癌、***癌、甲状腺癌、膀胱癌、神经胶质瘤、肝胆管癌、结肠直肠癌、T细胞淋巴瘤。The application according to claim 12, wherein the Trop2 positive disease is selected from triple negative breast cancer, glioblastoma, medulloblastoma, non-small cell lung cancer, small cell lung cancer, epithelial cancer, breast cancer , Head and neck cancer, kidney cancer, ovarian cancer, stomach cancer, Kaposi's sarcoma, pancreatic cancer and lung cancer, cervical cancer, colorectal cancer, esophageal cancer, oral squamous cell carcinoma, prostate cancer, thyroid cancer, bladder cancer, glial Tumor, liver and cholangiocarcinoma, colorectal cancer, T cell lymphoma.
  14. 一种制备权利要求6～10任一项所述的固体制剂的方法，包括：A method for preparing the solid preparation according to any one of claims 6 to 10, comprising:
    1)配制：取抗Trop2抗体-药物偶联物、稳定剂、缓冲剂、表面活性剂，溶解于水中至规定量，并调整pH值至4.5～6.0，得到液体制剂；1) Preparation: Take the anti-Trop2 antibody-drug conjugate, stabilizer, buffer, and surfactant, dissolve it in water to a specified amount, and adjust the pH to 4.5-6.0 to obtain a liquid preparation;
    2)冻干：将液体制剂进行冻干，得到所述的固体制剂。2) Lyophilization: freeze-dry the liquid preparation to obtain the solid preparation.
  15. 根据权利要求1～5任一项所述的液体制剂、权利要求6～10任一项所述的固体制剂、权利要求11所述的复溶制剂、权利要求12或13所述的应用、或权利要求14所述的方法，其中，所述抗Trop2抗体-药物偶联物为如式Ia的化合物：The liquid preparation according to any one of claims 1 to 5, the solid preparation according to any one of claims 6 to 10, the reconstituted preparation according to claim 11, the application according to claim 12 or 13, or The method of claim 14, wherein the anti-Trop2 antibody-drug conjugate is a compound of formula Ia:
    Figure PCTCN2020140885-appb-100001
    Figure PCTCN2020140885-appb-100001
    或其药学上可接受的盐或溶剂合物，其中Or a pharmaceutically acceptable salt or solvate thereof, wherein
    X为氢或卤素；X is hydrogen or halogen;
    Y选自氢、C1-C6烷基、C3-C6环烷基和-C(＝O)R 5； Y is selected from hydrogen, C1-C6 alkyl, C3-C6 cycloalkyl and -C(=O)R 5 ;
    R 1选自H、-OH、-OC(＝O)R 5和-OR 5基团； R 1 is selected from H, -OH, -OC(=O)R 5 and -OR 5 groups;
    R 2为H或C1-C6烷基； R 2 is H or C1-C6 alkyl;
    R 3为甲基、-CH 2OH或-CH 2OC(＝O)R 6； R 3 is methyl, -CH 2 OH or -CH 2 OC(=O)R 6 ;
    R 4为-OH或–SH； R 4 is -OH or -SH;
    R 5为C1-C6烷基或苄基； R 5 is C1-C6 alkyl or benzyl;
    R 6为C1-C6烷基、苯基或苄基； R 6 is C1-C6 alkyl, phenyl or benzyl;
    R 7为氢、C1-C6烷基或氨基酸侧链； R 7 is hydrogen, C1-C6 alkyl or amino acid side chain;
    R 8为氢或者C1-C6烷基； R 8 is hydrogen or C1-C6 alkyl;
    n为0、1、2、3、4、5、6、7或8；n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;
    p为1-10；p is 1-10;
    Anti-Trop2为抗Trop2抗体。Anti-Trop2 is an anti-Trop2 antibody.
  16. 根据权利要求15所述的液体制剂、所述的固体制剂、所述的复溶制剂、所述的应用、或所述的方法，其中，所述抗Trop2抗体为抗体A、抗体B和/或抗体C。The liquid preparation, the solid preparation, the reconstituted preparation, the application, or the method according to claim 15, wherein the anti-Trop2 antibody is antibody A, antibody B and/or Antibody C.
  17. 根据权利要求1～5任一项所述的液体制剂、权利要求6～10任一项所述的固体制剂、权利要求11所述的复溶制剂、权利要求12或13所述的应用、或权利要求14所述的方法，其中，所述抗Trop2抗体-药物偶联物为如式Ic的化合物：The liquid preparation according to any one of claims 1 to 5, the solid preparation according to any one of claims 6 to 10, the reconstituted preparation according to claim 11, the application according to claim 12 or 13, or The method of claim 14, wherein the anti-Trop2 antibody-drug conjugate is a compound of formula Ic:
    Figure PCTCN2020140885-appb-100002
    Figure PCTCN2020140885-appb-100002
    或其药学上可接受的盐或溶剂合物，其中，p约为2.1，Anti-Trop2为抗体C。Or a pharmaceutically acceptable salt or solvate thereof, wherein p is about 2.1 and Anti-Trop2 is antibody C.
  18. 根据权利要求15-17任一项所述的液体制剂、所述的固体制剂、所述的复溶制剂、所述的应用或所述的方法，其中The liquid preparation, the solid preparation, the reconstituted preparation, the application or the method according to any one of claims 15-17, wherein
    所述抗体A的轻链氨基酸序列如SEQ ID NO:1所示，重链氨基酸序列如SEQ ID NO:2所示；或The amino acid sequence of the light chain of the antibody A is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain is shown in SEQ ID NO: 2; or
    所述抗体B的轻链氨基酸序列如SEQ ID NO:3所示，重链氨基酸序列如SEQ ID NO:4所示；或The light chain amino acid sequence of the antibody B is shown in SEQ ID NO: 3, and the heavy chain amino acid sequence is shown in SEQ ID NO: 4; or
    所述抗体C的轻链氨基酸序列如SEQ ID NO:3所示，重链氨基酸序列如SEQ ID NO:4所示。The light chain amino acid sequence of the antibody C is shown in SEQ ID NO: 3, and the heavy chain amino acid sequence is shown in SEQ ID NO: 4.
  19. 根据权利要求18所述的液体制剂、所述的固体制剂、所述的复溶制剂、所述的应用或所述的方法，其中，所述抗体A由CHO细胞表达；或The liquid preparation, the solid preparation, the reconstituted preparation, the application or the method according to claim 18, wherein the antibody A is expressed by CHO cells; or
    所述抗体B由CHO细胞表达；或The antibody B is expressed by CHO cells; or
    所述抗体C的岩藻糖含量为0-5％。The fucose content of the antibody C is 0-5%.
  20. 根据权利要求18所述的液体制剂、所述的固体制剂、所述的复溶制剂、所述的应用或所述的方法，其中，所述抗体C由敲除α-(1,6)-岩藻糖基转移酶基因的细胞表达。The liquid preparation, the solid preparation, the reconstituted preparation, the application or the method according to claim 18, wherein the antibody C is knocked out of α-(1,6)- Cellular expression of the fucosyltransferase gene.
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