Antibody-drug conjugate preparation, preparation method and application

Abstract

The present invention provides antibody-drug conjugate (ADC) preparations (including solid preparations, liquid preparations and reconstituted preparations), preparation methods and applications thereof. In some embodiments, the solid preparation provided by the present invention comprises an antibody-drug conjugate consisting of an antibody, a linker and a drug module, and further comprises one or more of a buffer, a protective agent and a surfactant. In some embodiments, the solid preparation is a lyophilized preparation, which is obtained by lyophilizing a liquid preparation comprising an ADC consisting of an antibody, a linker and a drug module, wherein the liquid preparation further comprises one or more of a buffer, a protective agent and a surfactant. The preparation of the present invention can be used to prepare a drug for treating cancer such as EGFR or HER2-positive cancer.

Description

Technical Field

[0001] The present invention relates to the field of biopharmaceuticals, and in particular to antibody-drug conjugate preparations, preparation methods and applications. Background Art

[0002] Antibody-Drug Conjugates (ADCs) are highly effective and specific drugs for treating cancer and other conditions obtained by coupling antibodies and drugs, in which the antibody portion specifically binds to antigens on target cells, allowing the drug to exert its cytotoxic or other therapeutic effects on target cells.

[0003] Like other protein drugs, antibodies are prone to degradation such as oxidation, deamidation and fragmentation, as well as the formation of particles and aggregates. The particles formed in protein drugs will cause risks in clinical applications. For example, the particles in the injected drug formulation may cause damage to the veins of the injected patients or venous congestion. In addition, protein aggregation is one of the main pathways of drug degradation and may also produce undesirable immunogenicity.

[0004] For ADC drugs, drug conjugation itself will reduce the stability of antibodies and change their physical and chemical properties. Because most conjugated drugs are hydrophobic small molecule drugs, the solubility of ADC conjugates as a whole may be lower than that of unconjugated antibodies. At the same time, ADC has a more complex and heterogeneous structure than unconjugated antibodies, making ADC easier to aggregate, form particles and surface adsorption. Therefore, the instability and easy aggregation of ADC greatly increase the risks in clinical applications. Summary of the invention

[0005] Based on this, it is necessary to provide an antibody-drug conjugate solid preparation with excellent long-term stability and accelerated stability to address the instability and easy aggregation problems of ADC.

[0006] On the one hand, the present invention provides an antibody-drug conjugate (ADC) solid preparation, the solid preparation comprising an antibody-drug conjugate, wherein the antibody-drug conjugate is composed of an antibody, a linker and a drug module, and the solid preparation further comprises one or more of a buffer, a protective agent and a surfactant. The present invention also provides a liquid preparation that can be used to prepare an antibody-drug conjugate (ADC) solid preparation, and an injectable liquid preparation obtained by dissolving the solid preparation.

[0007] In some embodiments, the antibody is a humanized monoclonal antibody; in some embodiments, the antibody is an anti-EGFR antibody or an anti-HER2 antibody; in some embodiments, the antibody is an anti-HER2 antibody; in some embodiments, the antibody is BAT-A.

[0008] In some embodiments, the drug binding number (or drug antibody coupling ratio (DAR)) of the antibody is selected from 2-8. In some embodiments, the drug binding number of the antibody is selected from 3-5. In some embodiments, the drug binding number of the antibody is selected from 2, 4, 6 or 8. In some embodiments, the average drug binding number of the antibody is selected from 2-8. In some embodiments, the average drug binding number of the antibody is selected from 3-5, such as 3.3-3.7, or 3.5.

[0009] In some embodiments, the drug moiety is selected from one or more of N-methyl-L-alanyl maytansinol, DM1, DM4, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0010] In some embodiments, the linker is selected from SMCC, MC, MPA and MPEO; in some embodiments, the linker is SMCC or MC. In some embodiments, the linker is SMCC. In some embodiments, the linker is MC.

[0011] In some embodiments, the antibody-drug conjugate comprises a compound of Formula Ia:

[0012]

[0013] or a pharmaceutically acceptable salt or solvate thereof,

[0014] in

[0015] X is hydrogen or halogen;

[0016] Y is selected from hydrogen, C 1 -C 6 Alkyl, C3-C6 cycloalkyl and -C(=O)R 5 ;

[0017] R 1 Selected from H, -OH, -OC(=O)R 5 AND-OR 5 Group;

[0018] R 2 H or C 1 -C 6 alkyl;

[0019] R 3 is methyl, -CH2OH or -CH2OC(=O)R 6 ;

[0020] R 4 is -OH or –SH;

[0021] R 5 C 1 -C 6 Alkyl or benzyl;

[0022] R 6 C 1 -C 6 Alkyl, phenyl or benzyl;

[0023] R 7 For hydrogen, C 1 -C 6 Alkyl or amino acid side chains;

[0024] R 8 is hydrogen or C 1 -C 6 alkyl;

[0025] n is 0, 1, 2, 3, 4, 5, 6, 7 or 8;

[0026] p is 2-8;

[0027] Ab is an antibody, preferably an anti-HER2 antibody, and more preferably a BAT-A antibody.

[0028] In some embodiments, the antibody-drug conjugate is selected from the following structures:

[0029]

[0030] in,

[0031] Ab is an antibody, preferably an anti-HER2 antibody, more preferably a BAT-A antibody;

[0032] p is DAR or average DAR.

[0033] In some embodiments, Ab is an anti-HER2 antibody. In some embodiments, Ab is a BAT-A antibody or a BAT-B antibody.

[0034] In some embodiments, the ADC is ADC1 described herein.

[0035] In some embodiments, p is selected from 2 to 8. In some embodiments, p is selected from 2, 4, 6 or 8. In some embodiments, p is the average DAR and is selected from 2 to 8. In some embodiments, p is the average DAR and is selected from 3 to 5, such as 3.3 to 3.7, or 3.5.

[0036] In some embodiments, the buffer in the solid preparation comprises one or more of a histidine buffer, a citrate buffer, and a succinate buffer. In some embodiments, the buffer is a succinate buffer. It should be understood that the buffer (e.g., histidine buffer, citrate buffer, succinate buffer) in the present application is used in conjunction with an acid (e.g., hydrochloric acid) or a base (e.g., 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 use of the buffer. Unless otherwise specified, in the present application, whether the buffer is in the form of an acid and / or a salt, when the mass fraction or molar concentration of the buffer is mentioned, it is measured by its total acid radical ions. For example, when the buffer system is a succinate (HOOCCH2CH2COOH) buffer, whether the succinate is in the form of HOOCCH2CH2COOH, HOOCCH2CH2COO - and / or - OOCCH2CH2COO - The amount of succinic acid buffer refers to HOOCCH2CH2COOH, HOOCCH2CH2COO - and - OOCCH2CH2COO - The total amount of its acid ions - OOCCH2CH2COO - count.

[0037] In some embodiments, the protective agent in the solid preparation is selected from one or both of sucrose and trehalose. In some embodiments, the protective agent is selected from trehalose. In some embodiments, the protective agent is selected from sucrose.

[0038] In some embodiments, the surfactant in the solid preparation is selected from one or both of Tween 20 and Tween 80. In some embodiments, the protective agent is selected from Tween 80. In some embodiments, the protective agent is selected from Tween 20.

[0039] In some embodiments, the mass fraction of ADC in the solid preparation is about 1 part to about 100 parts. In some embodiments, the mass fraction of ADC in the solid preparation is about 10 parts to about 40 parts; in some embodiments, the mass fraction of ADC in the solid preparation is about 15 parts to about 25 parts; in some embodiments, the mass fraction of ADC in the solid preparation is about 20 parts.

[0040] In some embodiments, the solid formulation contains about 1 to about 100 parts by mass of ADC1. In some embodiments, the solid formulation contains about 10 to about 40 parts by mass of ADC1. In some embodiments, the solid formulation contains about 15 to about 25 parts by mass of ADC1. In some embodiments, the solid formulation contains about 20 parts by mass of ADC1.

[0041] In some embodiments, the mass fraction of the buffer in the solid preparation is about 0.6 parts to about 2.4 parts in terms of acid ions. In some embodiments, the mass fraction of the buffer in the solid preparation is about 0.9 parts to about 1.5 parts. In some embodiments, the mass fraction of the buffer in the solid preparation is about 1.16 parts. In some embodiments, the mass fraction of the buffer in the solid preparation is about 1.9 parts. In some embodiments, the mass fraction of the buffer in the solid preparation is about 1.56 parts.

[0042] In some embodiments, the solid preparation contains about 0.6 parts to about 2.4 parts of succinic acid buffer (in terms of acid ions) by mass. The content or concentration of succinic acid buffer in the preparation herein refers to the total content or total concentration of succinic acid and its anions. In some embodiments, the solid preparation contains about 0.9 parts to about 1.5 parts of succinic acid buffer (in terms of acid ions) by mass. In some embodiments, the solid preparation contains about 1.16 parts to about 1.18 parts of succinic acid buffer (in terms of acid ions) by mass. In some embodiments, the solid preparation contains about 1.16 parts of succinic acid buffer (in terms of acid ions) by mass.

[0043] In some embodiments, the solid preparation contains about 0.6 parts to about 2.4 parts of citric acid buffer (in terms of acid ions) by mass. The content or concentration of citric acid buffer in the preparation herein refers to the total content or total concentration of citric acid and its anions. In some embodiments, the solid preparation contains about 1.8 parts to about 2.0 parts of citric acid buffer (in terms of acid ions) by mass. In some embodiments, the solid preparation contains about 1.9 parts of citric acid buffer (in terms of acid ions) by mass.

[0044] In some embodiments, the mass fraction of the protective agent in the solid preparation is about 4 parts to about 95 parts. In some embodiments, the mass fraction of the protective agent in the solid preparation is about 19 parts to about 85 parts. In some embodiments, the mass fraction of the protective agent in the solid preparation is about 40 parts to about 70 parts. In some embodiments, the mass fraction of the protective agent in the solid preparation is about 55 parts to about 60 parts. In some embodiments, the mass fraction of the protective agent in the solid preparation is about 60 parts.

[0045] In some embodiments, the solid formulation contains about 4 to about 95 parts of trehalose by weight. In some embodiments, the solid formulation contains about 19 to about 85 parts of trehalose by weight. In some embodiments, the solid formulation contains about 40 to about 70 parts of trehalose by weight. In some embodiments, the solid formulation contains about 55 to about 60 parts of trehalose by weight. In some embodiments, the solid formulation contains about 60 parts of trehalose by weight.

[0046] In some embodiments, the mass fraction of the surfactant in the solid formulation is about 0.05 parts to about 1 parts. In some embodiments, the mass fraction of the surfactant in the solid formulation is about 0.1 parts to about 0.4 parts. In some embodiments, the mass fraction of the surfactant in the solid formulation is about 0.2 parts.

[0047] In some embodiments, the solid formulation contains about 0.05 to about 1 part by mass of Tween 80. In some embodiments, the solid formulation contains about 0.1 to about 0.4 parts by mass of Tween 80. In some embodiments, the solid formulation contains about 0.2 parts by mass of Tween 80.

[0048] The "number of parts by mass" in the present invention refers to the value under the condition of equal unit mass. The unit mass is the mass of each part, which can be any suitable mass. For example, when each part is 1g, the unit mass is 1g / part, and 20 parts are 20g; the unit mass can also be 1kg / part or 1mg / part. In addition, the unit mass is not limited to integers, for example, it can be 0.5g / part, and 20 parts are 10g.

[0049] In some embodiments, the solid dosage form comprises:

[0050] About 1 to about 100 parts by weight of ADC (such as ADC1);

[0051] A buffer (such as a histidine buffer, a citrate buffer, or a succinate buffer, such as a succinate buffer) in an amount of about 0.6 parts to about 2.4 parts by weight based on acid ions;

[0052] About 4 to about 95 parts by weight of a protective agent (such as sucrose or trehalose, such as trehalose); and

[0053] The mass fraction is about 0.05 parts to about 1 parts of a surfactant (such as Tween 20 or Tween 80, for example Tween 80).

[0054] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.4-6.0, 4.6-5.6 or 4.8-5.2.

[0055] In some embodiments, the solid dosage form comprises:

[0056] About 10 to about 40 parts by weight of ADC (such as ADC1);

[0057] A buffer (such as a histidine buffer, a citrate buffer, or a succinate buffer, such as a succinate buffer) in an amount of about 0.9 parts to about 1.5 parts by weight based on acid ions;

[0058] About 19 to about 85 parts by weight of a protective agent (such as sucrose or trehalose, such as trehalose); and

[0059] The mass fraction is about 0.05 parts to about 1 parts of a surfactant (such as Tween 20 or Tween 80, for example Tween 80).

[0060] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.4-5.6, 4.6-5.6 or 4.8-5.2.

[0061] In some embodiments, the solid dosage form comprises:

[0062] About 15 to about 25 parts by weight of ADC (e.g., ADC1);

[0063] A buffer (such as a histidine buffer, a citrate buffer, or a succinate buffer, such as a succinate buffer) in an amount of about 0.9 parts to about 1.5 parts by weight based on acid ions;

[0064] About 40 to about 70 parts by weight of a protective agent (such as sucrose or trehalose, such as trehalose); and

[0065] The mass fraction is about 0.1 part to about 0.4 part of a surfactant (such as Tween 20 or Tween 80, for example Tween 80).

[0066] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.6-5.6 or 4.8-5.2.

[0067] In some embodiments, the solid dosage form comprises:

[0068] About 20 parts by weight of ADC (e.g., ADC1);

[0069] About 1.16 parts by weight of a buffer (such as a histidine buffer, a citrate buffer, or a succinate buffer, such as a succinate buffer) based on acid ions;

[0070] About 60 parts by weight of a protective agent (such as sucrose or trehalose, such as trehalose); and

[0071] The mass fraction is about 0.2 parts of a surfactant (such as Tween 20 or Tween 80, for example Tween 80).

[0072] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.8-5.2.

[0073] In some embodiments, the solid preparation described above further comprises a certain amount of Na + or K + , used to form a buffer system with a buffer to adjust the pH, such as about 0.1 to about 1 part of Na + , about 0.2 to about 0.5 parts of Na + , or about 0.25 parts to about 0.3 parts of Na + .

[0074] In some embodiments, the solid formulation is a powder.

[0075] In some embodiments, the specification of the solid preparation (i.e., the dosage that can be loaded into a container (such as a medicine bottle or a medicine bag)) is about 5 mg-about 500 mg ADC, about 50 mg-about 200 mg ADC, or about 75 mg-about 125 mg ADC. In some embodiments, the specification of the solid preparation is about 10 mg ADC, about 20 mg ADC, about 50 mg ADC, about 80 mg ADC, about 100 mg ADC, about 150 mg ADC, about 200 mg ADC, about 300 mg ADC, about 400 mg ADC or about 500 mg ADC, or a range between any two numbers, including endpoints.

[0076] In some embodiments, the solid dosage form comprises:

[0077] about 5 mg to about 500 mg of ADC;

[0078] About 2.5 mg to about 25 mg of a buffer, calculated as acid ions;

[0079] about 50 mg to about 500 mg of a protective agent; and

[0080] About 0.25 mg to about 2 mg of a surfactant.

[0081] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.4-6.0, 4.6-5.6 or 4.8-5.2, such as 5.0.

[0082] In some embodiments, the solid dosage form comprises:

[0083] about 50 mg to about 200 mg of ADC;

[0084] About 2.5 mg to about 20 mg of a buffer, calculated as acid ions;

[0085] about 150 mg to about 450 mg of a protective agent; and

[0086] About 0.5 mg to about 2 mg of a surfactant.

[0087] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.4-5.6, 4.6-5.6 or 4.8-5.2, such as 5.0.

[0088] In some embodiments, the solid dosage form comprises:

[0089] about 75 mg to about 125 mg of ADC;

[0090] About 5 mg to about 10 mg of a buffer, calculated as acid ions;

[0091] about 250 mg to about 350 mg of a protective agent; and

[0092] About 0.5 mg to about 1.5 mg of a surfactant.

[0093] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.6-5.6 or 4.8-5.2, such as 5.0.

[0094] In some embodiments, the solid dosage form comprises:

[0095] About 100 mg of ADC;

[0096] About 5.8 mg to about 5.9 mg of a buffer, calculated as acid ions;

[0097] about 300 mg of a protective agent; and

[0098] About 1 mg of surfactant.

[0099] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.8-5.2.

[0100] In some embodiments, the solid dosage form comprises:

[0101] About 50 mg to about 200 mg of ADC1;

[0102] About 2.5 mg to about 25 mg of succinic acid buffer, calculated as acid radical ion;

[0103] about 150 mg to about 450 mg of trehalose; and

[0104] About 0.25 mg to about 2 mg of Tween 80.

[0105] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.6-5.6 or 4.8-5.2, such as 5.0.

[0106] In some embodiments, the solid dosage form comprises:

[0107] About 75 mg to about 125 mg of ADC1;

[0108] About 5 mg to about 10 mg of succinic acid buffer, calculated as acid radical ion;

[0109] about 250 mg to about 350 mg of trehalose; and

[0110] About 0.5 mg to about 2 mg of Tween 80.

[0111] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.6-5.6 or 4.8-5.2, such as 5.0.

[0112] In some embodiments, the solid dosage form comprises:

[0113] About 100 mg of ADC1;

[0114] About 5.8 mg to 5.9 mg of succinic acid buffer, calculated as acid radical ion;

[0115] about 300 mg of trehalose; and

[0116] About 1 mg of Tween 80.

[0117] In some embodiments, the pH of the solid preparation (eg, after dissolution in water or saline) is about 4.6-5.6 or 4.8-5.2, such as 5.0.

[0118] In some embodiments, the solid preparation described above further comprises a certain amount of Na + or K + , such as about 0.5 mg to about 4 mg of Na +, about 1mg-2mg of Na + , or about 1.2 mg to about 1.5 mg of Na + .

[0119] Some embodiments provide a lyophilized powder comprising 100 mg ADC1, 5.9 mg succinic acid, 300 mg trehalose, 1 mg polysorbate 80, and adjusted to pH 5.0 with sodium hydroxide.

[0120] In some embodiments, when administered to a patient, the dose contained in each container is dissolved in a sufficient amount of an injection solvent (such as water for injection or physiological saline) to obtain 2-10 ml of solution.

[0121] In some embodiments, when administered to a patient, the dose contained in each container is dissolved in a sufficient amount of an injection solvent (such as water for injection or saline) to obtain 5 ml of solution.

[0122] In some embodiments, when administered to a patient, the dose contained in each container is dissolved in a sufficient amount of an injection solvent (such as water for injection or saline) to obtain 7 ml of solution.

[0123] In some embodiments, the solid preparation is a lyophilized preparation, which is obtained by lyophilizing a liquid preparation comprising an antibody-drug conjugate composed of an antibody, a linker, and a drug module, wherein the liquid preparation further comprises one or more of a buffer, a protective agent, and a surfactant, and a solvent, such as water, such as water for injection.

[0124] In some embodiments, provided herein is a liquid formulation comprising an antibody-drug conjugate (ADC) composed of an antibody, a linker, and a drug module as described herein, wherein the liquid formulation further comprises one or more of a buffer, a protective agent, and a surfactant, and a solvent, such as water, such as water for injection. The liquid formulation can be used to prepare a lyophilized formulation as described herein.

[0125] In some embodiments, the concentration of ADC in the above-mentioned liquid preparation is about 1 mg / ml-about 100 mg / ml. In some embodiments, the concentration of ADC in the liquid preparation is about 10 mg / ml-about 40 mg / ml. In some embodiments, the concentration of ADC in the liquid preparation is about 15 mg / ml-about 25 mg / ml. In some embodiments, the concentration of ADC in the liquid preparation is about 20 mg / ml.

[0126] In some embodiments, in the liquid preparation, the buffer comprises one or more of a histidine buffer, a citrate buffer, and a succinate buffer. In some embodiments, the buffer is a succinate buffer.

[0127] In some embodiments, in the liquid formulation, the concentration of the buffer is 5mM-20mM. In some embodiments, the concentration of the buffer is 8mM-13mM. In some embodiments, the concentration of the buffer is 10mM.

[0128] In some embodiments, in the liquid formulation, the concentration of the buffer is about 0.5 mg / ml to about 5 mg / ml. In some embodiments, the concentration of the buffer is about 1 mg / ml to about 2 mg / ml. In some embodiments, the concentration of the buffer is about 1.16 mg / ml to about 1.18 mg / ml. In some embodiments, the concentration of the buffer is about 1.16 mg / ml.

[0129] In some embodiments, the liquid formulation contains a succinate buffer at a concentration of about 0.5 mg / ml to about 5 mg / ml. In some embodiments, the liquid formulation contains a succinate buffer at a concentration of about 1 mg / ml to about 2 mg / ml. In some embodiments, the liquid formulation contains a succinate buffer at a concentration of about 1.16 mg / ml to about 1.18 mg / ml. In some embodiments, the liquid formulation contains a succinate buffer at a concentration of about 1.16 mg / ml.

[0130] In some embodiments, the liquid preparation contains a citric acid buffer at a concentration of about 0.6 mg / ml to about 2.4 mg / ml. The content or concentration of citric acid buffer in the preparation herein refers to the total content or total concentration of citric acid and its anions. In some embodiments, the liquid preparation contains a citric acid buffer at a concentration of about 1.8 mg / ml to about 2.0 mg / ml. In some embodiments, the liquid preparation contains a citric acid buffer at a concentration of about 1.9 mg / ml.

[0131] In some embodiments, the pH of the liquid preparation is within the range of 4.4-6.0. In some embodiments, the pH of the liquid preparation is within the range of 4.4-5.6. In some embodiments, the pH of the liquid preparation is within the range of 4.6-5.6 or 4.8-5.2. In some embodiments, the pH of the liquid preparation is 5.0. In some embodiments, the pH is adjusted by adding an appropriate amount of NaOH or KOH.

[0132] In some embodiments, the protective agent is selected from one or both of sucrose and trehalose. In some embodiments, the protective agent is trehalose. In some embodiments, the protective agent is sucrose.

[0133] In some embodiments, the concentration of the protective agent in the liquid preparation is 10mM-250mM. In some embodiments, the concentration of the protective agent in the liquid preparation is 50mM-225mM. In some embodiments, the concentration of the protective agent in the liquid preparation is 105mM-185mM, and in some embodiments, the concentration of the protective agent in the liquid preparation is 140mM-180mM.

[0134] In some embodiments, the concentration of the protective agent in the liquid formulation is about 10 mg / ml to about 100 mg / ml. In some embodiments, the concentration of the protective agent in the liquid formulation is about 30 mg / ml to about 90 mg / ml. In some embodiments, the concentration of the protective agent in the liquid formulation is about 50 mg / ml to about 70 mg / ml. In some embodiments, the concentration of the protective agent in the liquid formulation is about 60 mg / ml.

[0135] In some embodiments, the protective agent is 140 mM-180 mM trehalose, or 150 mM-190 mM sucrose.

[0136] In some embodiments, the protective agent is 160 mM trehalose, or 175 mM sucrose.

[0137] In some embodiments, the liquid preparation contains trehalose at a concentration of about 10 mg / ml to about 100 mg / ml. In some embodiments, the liquid preparation contains trehalose at a concentration of about 30 mg / ml to about 90 mg / ml. In some embodiments, the liquid preparation contains trehalose at a concentration of about 50 mg / ml to about 70 mg / ml. In some embodiments, the liquid preparation contains trehalose at a concentration of about 60 mg / ml.

[0138] In some embodiments, the surfactant is selected from one or more of Tween 20 and Tween 80. In some embodiments, the surfactant is Tween 80. In some embodiments, the surfactant is Tween 20.

[0139] In some embodiments, the concentration of the surfactant in the liquid preparation is 0.05 mg / ml-0.4 mg / ml. In some embodiments, the concentration of the surfactant in the liquid preparation is 0.1 mg / ml-0.4 mg / ml. In some embodiments, the concentration of the surfactant in the liquid preparation is 0.2 mg / ml.

[0140] In some embodiments, the liquid formulation contains Tween 80 at a concentration of about 0.05 mg / ml to about 0.4 mg / ml. In some embodiments, the liquid formulation contains Tween 80 at a concentration of about 0.1 mg / ml to about 0.4 mg / ml. In some embodiments, the liquid formulation contains Tween 80 at a concentration of about 0.2 mg / ml.

[0141] In some embodiments, the liquid formulation comprises:

[0142] about 1 mg / ml to about 100 mg / ml of ADC;

[0143] About 5mM-20mM buffer;

[0144] About 50mM-225mM of a protective agent;

[0145] about 0.05 mg / ml to about 0.4 mg / ml of a surfactant; and

[0146] water;

[0147] The pH of the liquid preparation is about 4.4-6.0 or 4.6-5.6.

[0148] In some embodiments, the liquid formulation comprises:

[0149] About 10 mg / ml to about 40 mg / ml of ADC;

[0150] About 5mM-20mM buffer;

[0151] about 105 mM to 185 mM of a protective agent;

[0152] about 0.1 mg / ml to about 0.4 mg / ml of a surfactant; and

[0153] water;

[0154] The pH of the liquid formulation is about 4.4-5.6 or 4.6-5.6.

[0155] In some embodiments, the liquid formulation comprises:

[0156] about 15 mg / ml to about 25 mg / ml of ADC;

[0157] About 8mM-13mM buffer;

[0158] About 105mM-185mM of a protective agent;

[0159] about 0.1 mg / ml to about 0.4 mg / ml of a surfactant; and

[0160] water;

[0161] The pH of the liquid formulation is about 4.6-5.4.

[0162] In some embodiments, the liquid formulation comprises:

[0163] About 20 mg / ml ADC;

[0164] about 10 mM buffer;

[0165] About 140mM-180mM of protective agent;

[0166] about 0.2 mg / ml of a surfactant; and

[0167] water;

[0168] The pH of the liquid formulation is about 4.8-5.2.

[0169] In some embodiments, the liquid formulation comprises:

[0170] About 15 mg / ml to about 25 mg / ml of ADC1;

[0171] about 1 mg / ml to about 2 mg / ml or about 5 mM to about 20 mM succinate buffer;

[0172] about 30 mg / ml to about 90 mg / ml or about 105 mM to about 185 mM trehalose;

[0173] About 0.05 mg / ml to about 0.4 mg / ml of Tween 80; and

[0174] water;

[0175] The pH of the liquid formulation is about 4.6-5.6.

[0176] In some embodiments, the liquid formulation comprises:

[0177] About 15 mg / ml to about 25 mg / ml of ADC1;

[0178] about 1 mg / ml to about 2 mg / ml or about 5 mM to about 20 mM succinate buffer;

[0179] about 50 mg / ml to about 70 mg / ml or about 140 mM to about 180 mM trehalose;

[0180] About 0.1 mg / ml to about 0.4 mg / ml of Tween 80; and

[0181] water;

[0182] The pH of the liquid formulation is about 4.8-5.2.

[0183] In some embodiments, the liquid formulation comprises:

[0184] About 20 mg / ml of ADC1;

[0185] about 1.16 mg / ml-1.18 mg / ml or about 10 mM succinate buffer;

[0186] about 60 mg / ml or about 160 mM trehalose;

[0187] About 0.2 mg / ml Tween 80; and

[0188] water;

[0189] The pH of the liquid formulation is about 5.

[0190] In some embodiments, the liquid preparation described above further comprises a certain amount of Na + or K + To achieve the pH, for example, about 0.1 mg / ml to about 0.8 mg / ml, about 0.2 mg / ml to about 0.4 mg / ml, or about 0.24 mg / ml to about 0.3 mg / ml of Na + For example, about 4mM-35mM, about 8mM-17mM, about 10mM-13mM Na + or K + .

[0191] On the other hand, the present invention also provides a method for preparing the above-mentioned solid preparation which is a lyophilized preparation, the method comprising cooling the liquid preparation to a solid state, and applying a vacuum to remove substances that can sublime in a vacuum (such as solvent water at room temperature) to obtain a solvent-free lyophilized preparation at room temperature.

[0192] In some embodiments, the method comprises the following steps:

[0193] a) cooling the liquid preparation to -35°C or lower;

[0194] b) raising the temperature of the preparation treated in step a) to between -20°C and -7°C and maintaining the temperature isothermally;

[0195] c) applying a vacuum at a temperature between -35°C and -8°C to the preparation treated in step b).

[0196] In some embodiments, the method further comprises the following steps:

[0197] d) increasing the temperature of the preparation treated in step c) to between 25°C and 40°C; and maintaining it isothermally.

[0198] In some embodiments, the method comprises the following steps:

[0199] a) cooling the liquid formulation to -35°C or lower at a rate of 0.5°C / min to 3°C / min and maintaining the temperature isothermally for at least 120 min;

[0200] b) heating the isothermally maintained preparation to between -20°C and -7°C at a rate of 0.5°C / min to 3°C / min, and isothermally maintaining the preparation for at least 120 min;

[0201] c) applying a vacuum to the preparation treated in step b) at a temperature between -30°C and -8°C using a pressure between 50 mTorr and 200 mTorr;

[0202] d) increasing the temperature of the preparation treated in step c) to between 25°C and 40°C at a rate of 0.5°C / min to 3°C / min; and maintaining the temperature isothermally for at least 7 hours.

[0203] More specifically, the method comprises the following steps: cooling the liquid preparation to -35°C or lower at a rate of 0.5°C / min to 3°C / min, and after solidification, isothermally maintaining for at least 120 minutes (cooling step). Subsequently, the preparation is heated to between -20°C and -7°C at a rate of 0.5°C / min to 3°C / min and isothermally maintained for at least 120 minutes (annealing step). Thereafter, solid water is sublimated at a temperature between -30°C and -8°C using a pressure between 50mTorr and 200mTorr (primary drying). Finally, the temperature is increased to between 25°C and 40°C at a rate of 0.5°C / min to 3°C / min and isothermally maintained for at least 7 hours (secondary drying), until the residual moisture does not exceed 2% by weight, thereby obtaining a freeze-dried preparation.

[0204] In another aspect, the present invention provides a use of the above-mentioned solid preparation or liquid preparation in the preparation of a medicament for cancer. In some embodiments, the cancer is EGFR or HER2 positive cancer.

[0205] In some embodiments, the cancer is metastatic colorectal cancer, head and neck cancer, breast cancer, gastric cancer, ovarian cancer, colon cancer, or non-small cell lung cancer.

[0206] In another aspect, the present invention provides a reconstituted preparation obtained by reconstituting the solid preparation described above with a pharmaceutically acceptable solvent. The reconstituted preparation can be used to treat cancer, such as metastatic colorectal cancer, head and neck cancer, breast cancer, gastric cancer, ovarian cancer, colon cancer or non-small cell lung cancer.

[0207] In some embodiments, the present invention provides a lyophilized powder injection containing the ADC described herein (such as ADC1, ADC2 or ADC3) for injection in a single-use bottle. In some embodiments, the lyophilized powder does not contain a preservative. In some embodiments, each bottle contains 100 mg of the ADC (such as ADC1, ADC2 or ADC3). In some embodiments, each bottle contains 140 mg of the ADC (such as ADC1, ADC2 or ADC3). In some embodiments, the lyophilized powder is formulated into a solution containing 20 mg / mL ADC (such as ADC1, ADC2 or ADC3) after formulation. In some embodiments, the solvent used for formulation is water for injection. In some embodiments, after formulation, each single-use bottle contains 20 mg / mL of the ADC (such as ADC1, ADC2 or ADC3), 0.02% (w / v) polysorbate 80, 10 mM succinic acid and 6% (w / v) trehalose, with a pH of 4.8-5.4. In some embodiments, the solution containing 20 mg / mL ADC (such as ADC1, ADC2 or ADC3) is diluted and then administered by intravenous infusion. In some embodiments, the solution containing 20 mg / mL ADC (such as ADC1, ADC2 or ADC3) is diluted with an isotonic solution (such as 0.9% NaCl solution for injection), and then administered by intravenous infusion after dilution. The solid preparation of the present invention can effectively solve the problem of ADC instability and easy aggregation, and has excellent long-term stability and accelerated stability. Further, when the solid preparation is a lyophilized preparation, the lyophilized preparation is freeze-dried by removing the water in the ADC at low temperature by freeze-drying the liquid preparation containing the antibody-drug conjugate, so that the ADC remains stable during storage. The addition of a protective agent can effectively protect the ADC during the freeze-drying process. The buffer is used to maintain a stable pH of the preparation and reduce the degradation of the ADC. The addition of a surfactant can reduce the surface tension of the lyophilized preparation after reconstitution, so that the ADC remains stable and is not easy to form aggregates or particles.

[0208] Other features and advantages of the present invention will be described in detail in the following detailed description. BRIEF DESCRIPTION OF THE DRAWINGS

[0209] Figure 1 The freeze-dried sample of Example 2 was placed at 50° C. for 10 days and analyzed by size exclusion chromatography (SEC) to obtain the percentage of the main peak substance.

[0210] Figure 2 The lyophilized sample of Example 2 was placed at 50° C. for 10 days, and the coupling ratio of the drug was determined using a reverse phase chromatography (RP) method.

[0211] Figure 3The change in the main peak percentage of the liquid preparation sample of Example 3 analyzed using the SEC method under illumination conditions is shown.

[0212] Figure 4 The change in the main peak percentage of the liquid preparation sample of Example 3 analyzed using the SEC method under high temperature conditions is shown.

[0213] Figure 5 The change in the percentage of the main peak of the freeze-dried preparation sample of Example 3 analyzed using the SEC method under light conditions is shown.

[0214] Figure 6 The change in peak percentage under high temperature conditions is shown by analyzing the liquid preparation sample of Example 4 using the SEC method.

[0215] Figure 7 The change in peak percentage under high temperature conditions is shown by analyzing the freeze-dried preparation sample of Example 4 using the SEC method.

[0216] Figure 8 The change in the main peak percentage of the freeze-dried preparation sample of Example 5 analyzed using the SEC method under high temperature conditions is shown.

[0217] Fig. 9 The change in the main peak percentage of the liquid preparation sample of Example 6 analyzed using the SEC method under high temperature conditions is shown.

[0218] Fig.10 The change in the main peak percentage of the freeze-dried preparation sample of Example 6 analyzed using the SEC method under high temperature conditions is shown.

[0219] Fig.11 The change in the main peak percentage of the freeze-dried preparation sample of Example 8 analyzed using the SEC method under high temperature conditions is shown.

[0220] Fig.12 The HIC method is used to analyze the change in the percentage of uncoupled antibody in the lyophilized preparation samples of Example 8 under high temperature conditions.

[0221] Fig.13 The change in the main peak percentage of the reconstituted preparation sample of Example 10 under high temperature conditions is shown using the SEC method. DETAILED DESCRIPTION

[0222] As used herein, the terms "comprise", "include" or "contain" or grammatical variations thereof are intended to mean that compositions and methods, etc. include the listed elements, but do not exclude others. "Substantially consisting of..." or grammatical variations thereof, when used to define compositions and methods, should mean excluding other elements that have any fundamental effect on the combination for the intended use, but do not exclude elements that do not substantially affect the characteristics of the composition or method. "Consisting of..." or grammatical variations thereof should mean excluding elements that are not specifically listed. Embodiments defined by each of these transitional terms are within the scope of the present invention. For example, when a formulation is described as comprising ingredients A, B and C, a formulation consisting essentially of A, B and C and a formulation consisting of A, B and C are independently within the scope of the present invention.

[0223] The term "about" when used before a numerical value indicates that this value can vary within a reasonable range, such as within ±10%, ±5%, or ±1% of the stated value. "About x" includes "x."

[0224] As used herein, the term "effective amount" or "therapeutically effective amount" refers to a sufficient amount of an agent being administered that will alleviate to some degree one or more symptoms of the disease or condition being treated. The result can be a reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desired alteration of a biological system. For example, an "effective amount" for therapeutic uses is the amount of a formulation as disclosed herein required to provide a clinically significant reduction in disease symptoms without excessive adverse side effects. The "effective amount" or "therapeutically effective amount" may vary depending on the subject, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0225] The term "acceptable" or "pharmaceutically acceptable" with respect to a formulation, composition or ingredient as used herein means having no persistent detrimental effect on the general health of the subject being treated or does not abrogate the biological activity or properties of the compound, and is relatively nontoxic.

[0226] ADCs can form a wide variety of pharmaceutically acceptable salts, including, but not limited to, acid addition salts with organic acids, including, but not limited to, aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, amino acids, and the like, such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like; acid addition salts with inorganic acids, including hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like; and salts with metal ions, such as alkali metal ions (e.g., sodium or potassium), alkaline earth metal ions (e.g., calcium or magnesium), or aluminum ions, or with organic bases such as diethanolamine, triethanolamine, N-methylglucamine, and the like.

[0227] As used herein, the term "individual," "subject," or "patient" refers to an animal that is the target of treatment, observation, or experiment. By way of example only, the subject may be, but is not limited to, a mammal, including, but not limited to, a human.

[0228] The antibody of the present invention can be any currently known antibody suitable for preparing antibody-drug conjugates, which can have a complete antibody structure, or can include antibody fragments (polyclonal antibodies and monoclonal antibodies), such as Fab, Fab', F(ab)'2 and Fv, etc. ADC herein includes pharmaceutically acceptable salts thereof.

[0229] Through monoclonal antibody technology, antibodies can specifically bind to antigens, such as tumor-specific antigens. The tumor antigens described herein refer to targets expressed in tumor cells. Tumor antigens can be used to identify tumor cells, and can also be potential indicators for tumor treatment, or targets for tumor treatment. Therefore, the selection of specific antibodies is mainly based on the type of disease, as well as the cells and tissues used as targets.

[0230] Examples of tumor antigens well known in the art include, but are not limited to, EGFR, HER2, CD20, CD30, CD33, CD47, CD52, CD133, CEA, VEGF, VEGFR, etc.

[0231] In some embodiments, the antibody may be a humanized monoclonal antibody.

[0232] In some embodiments, the antibody is an anti-EGFR antibody. In some embodiments, the antibody is BAT-C, which has enhanced antibody-dependent cell-mediated cytotoxicity. The antibody BAT-C comprises an anti-EGFR light chain amino acid sequence of SEQ ID NO: 1 and an anti-EGFR heavy chain amino acid sequence of SEQ ID NO: 2.

[0233] In other embodiments, the antibody is other types of anti-EGFR antibodies, including but not limited to: cetuximab, which is a human-mouse chimeric monoclonal antibody against EGFR; panitumumab, which is a fully humanized monoclonal antibody; nimotuzumab. EGFR is overexpressed in many tumor tissues, such as metastatic colorectal cancer and head and neck cancer.

[0234] In some embodiments, the antibody is an anti-HER2 antibody that can specifically act on human epidermal growth factor receptor 2. The anti-HER2 antibody can be modified, such as one or more amino acid sequences are changed, increased or decreased, to achieve the corresponding purpose, such as enhancing antibody-dependent cell-mediated cytotoxicity. In some embodiments of the present invention, the antibody is BAT-B. The light chain of BAT-B is SEQ ID NO:3; the heavy chain is SEQ ID NO:4. In other embodiments of the present invention, examples of anti-HER2 antibodies also include but are not limited to Pertuzumab. In other embodiments of the present invention, an example of an anti-HER2 antibody is BAT-A, whose light chain is SEQ ID NO:5 and heavy chain is SEQ ID NO:6.

[0235] The sequences corresponding to the sequence numbers are shown in Table 1.

[0236] Table 1

[0237]

[0238]

[0239] In some embodiments of the present invention, the number of small molecule drugs bound to one antibody in the antibody-drug conjugate, i.e., the drug binding number of the antibody, or the drug antibody coupling ratio (DAR), is selected from 2 to 8, or 2, 4, 6, or 8; when the average number of small molecule drugs bound is considered, i.e., the average number of drugs bound to the antibody, or the average drug antibody coupling ratio (average DAR), the value is selected from about 2 to about 8, or about 3 to about 5, or about 3.3-about 3.7, or about 3.5. In some embodiments, the small molecule drug is coupled to the sulfhydryl group formed by the disulfide bond formed between the cysteines contained in the antibody (such as antibody BAT-C, antibody BAT-B or antibody BAT-A) itself after reduction, so the number of drug binding is selected from 2, 4, 6, and 8.

[0240] The drug module suitable for coupling the linker is a variety of 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. Such drugs may include, for example, maytansinol, maytansinol analogs and derivatives, auristatin analogs, etc.

[0241] Maytansine analogs and derivatives may include maytansine and maytansine analogs, which can be isolated from natural sources according to known methods, manufactured using biotechnology (see, e.g., Yu et al., 99 PNAS 7968-7973 (2002)), or synthesized according to known methods (see, e.g., Cassady et al., Chem. Pharm. Bull. 52 (1) 1-26 (2004)). Depending on the type of linker, many positions on maytansine can be used as linking positions. For example, for forming an ester bond, it is suitable to have a hydroxyl group at the C-3 position, a hydroxymethyl modification at the C-14 position, a hydroxyl modification at the C-15 position, and a hydroxyl group at the C-20 position.

[0242] In some embodiments, the connection is formed at the C-3 position of maytansinol or maytansinol analogs and derivatives. In other embodiments, the maytansinol or maytansinol analogs and derivatives drug module has the following structure:

[0243]

[0244]

[0245] In some further embodiments, the maytansinol or maytansinol analogs and derivatives drug moiety has a structure disclosed in Patent 201310081867.X.

[0246] In some embodiments, auristatin analogs conventionally used in the art may also be used, examples of which include, but are not limited to, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). These compounds may be linked to antibodies via their N-terminus or C-terminus (WO02 / 088172). In some embodiments, the monomethyl auristatin E and monomethyl auristatin F drug moieties have the following structures:

[0247]

[0248] The linkers applicable to 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 under normal conditions and can be broken under tumor conditions to release small molecule drugs to play a therapeutic role. Common cleavable linkers include protease-sensitive linkers that contain dipeptide bonds that can be recognized and broken by proteases, most of which are valine-citrulline linkers (val-cit); there are also acid-sensitive linkers, which are mostly linkers containing hydrazone bonds; another type is glutathione-sensitive linkers, which usually contain glutathione-reducible disulfide bonds. Non-cleavable linkers remain intact after intracellular metabolism. Antibody-drug conjugates containing such linkers need to be degraded by lysosomes to release cytotoxic molecules. Compared with cleavable linkers, non-cleavable linkers have good stability in the circulatory system. Non-cleavable linkers include MC, SMCC, MPA, MPEO, etc. In some embodiments, the linker is a non-cleavable linker.

[0249] In some embodiments of the present invention, the linker is selected from a molecular fragment having the following structure:

[0250]

[0251] In some embodiments, the solid preparation of the present invention is a lyophilized preparation obtained by lyophilizing a liquid preparation containing one or more of ADC, a buffer, a protective agent, and a surfactant.

[0252] In some embodiments, the concentration of ADC in the liquid preparation before lyophilization is about 1 mg / ml to about 100 mg / ml, preferably about 10 mg / ml to about 40 mg / ml, more preferably about 15 mg / ml to about 25 mg / ml, and particularly preferably about 20 mg / ml.

[0253] In some embodiments, a buffer is used to maintain a stable pH of the preparation and reduce the degradation of ADC. In the process of freeze-drying the liquid preparation to form a freeze-dried preparation, it is necessary to undergo low-temperature freezing, then vacuum treatment, sublimate the water in the freeze, and then restore the temperature and air pressure to obtain a freeze-dried preparation. During this period, as the water changes from liquid to solid, the precipitation of the buffer and the change of pH may be caused, thereby affecting the stability of the ADC. Therefore, providing a suitable buffer, a suitable concentration thereof, and a suitable pH range is very important for preparing a stable, non-degradable and aggregated ADC freeze-dried preparation.

[0254] For the selection of the type of buffer, it is preferred to select a buffer with good buffering capacity at the target pH value and a small pH change during the freezing process. In some embodiments, the buffer is selected from histidine buffer, citrate buffer and succinate buffer, or a combination of any two of the above buffers, such as a buffer mixed with histidine buffer and succinate buffer, or a buffer combined with the above three. In some embodiments, the buffer is succinate buffer.

[0255] Regarding the concentration of the buffer, if the concentration is too low, the buffering capacity is weak and it is likely that no significant protective effect will be achieved; while if the concentration is too high, it is easy to precipitate during the freezing process, thereby affecting the pH value in the solution and further affecting the stability of the ADC. Therefore, the concentration of the buffer in the liquid preparation before lyophilization of the present invention is preferably between about 5mM and about 20mM, more preferably about 8mM to about 13mM, and particularly preferably about 10mM.

[0256] The pH value in the aqueous solvent can directly affect the stability between small molecule drugs and antibodies and is a key parameter. In order to achieve stability between antibodies, small molecule drugs and antibodies, the pH value of the liquid preparation used in the present invention is between 4.4 and 6.0, preferably between about 4.4 and about 5.6, more preferably between about 4.8 and about 5.2, and particularly preferably about 5.0.

[0257] In some embodiments, a protective agent is used to protect the ADC in the liquid preparation. The addition of a protective agent can change the collapse temperature, provide freeze-drying protection for the ADC during the freeze-drying process, and reduce the impact of crystal water on the ADC. In some embodiments, the protective agent selects a sugar, in some embodiments, a non-reducing sugar and / or sorbitol, in some embodiments, sucrose and / or trehalose. In some embodiments, it is trehalose. During the pre-freezing process of the product, an appropriate non-reducing sugar can cause the product to form a glassy state in this process, including the crystallizable components therein, on the one hand, eliminating the possibility of crystallization of the buffer component, thereby eliminating the pH offset and playing a role in protecting the protein, and on the other hand, the formed glassy state also plays a key role in the stability of the protein at low temperature and dehydration, but the infinitely increased reducing sugar concentration will lead to an extension or failure of the primary drying time during the freeze-drying process and the inability to remove the water after the secondary drying, so the concentration of the reducing sugar should be controlled to maintain the stability of the protein and form a good "cake" shape during the freeze-drying process.

[0258] For the concentration of the protective agent, if it is too low, it may not have a better protective effect; if it is too high, it will increase the difficulty of freeze-drying. Therefore, in some embodiments, the concentration of the protective agent is about 10mM to about 250mM, preferably about 50mM to about 225mM, more preferably about 105mM to about 185mM, more preferably about 140mM to about 180mM, and particularly preferably about 160mM or about 175mM. Further, when trehalose is used as a protective agent, the preferred concentration is about 140mM to about 180mM, and the more preferred concentration is about 160mM; when sucrose is used as a protective agent, the preferred concentration is about 150mM to about 190mM, and the more preferred concentration is about 175mM.

[0259] At the same time, in some embodiments of the present application, the protective agent can also serve as a filler for the lyophilized preparation, providing a certain structure for the lyophilized preparation, and further improving the stability of the ADC during long-term storage. In addition to the above-mentioned protective agent, the filler can also be glycine, mannitol, etc.

[0260] In some embodiments, the addition of surfactant is used to reduce surface tension and limit the polymerization of protein. In some embodiments, the surfactant is a nonionic surfactant, such as Tween 20, Tween 80 and poloxamer. In some embodiments, the surfactant is Tween 20 and / or Tween 80. In some embodiments, the surfactant is Tween 80.

[0261] For the concentration of the surfactant, too low or too high may not improve the stability of the ADC. In some embodiments, the concentration of the surfactant is about 0.05 mg / ml to about 0.4 mg / ml, preferably about 0.1 mg / ml to about 0.4 mg / ml, and more preferably about 0.2 mg / ml.

[0262] In some embodiments, the liquid preparation is used with a pharmaceutically acceptable solvent as a carrier, such as sterile pharmaceutical grade water for injection or saline, sterile water for injection, antibacterial water for injection and other water for injection. In some embodiments, when the components in the liquid preparation before freeze-drying, such as ADC, buffer, stabilizer and / or surfactant, are formulated into a solution of a specific concentration using water for injection, the required amount of solid is calculated according to the prescription amount, and a certain amount of water for injection is added to dissolve after mixing; if there is a viscous liquid in the prescription, the required amount of viscous liquid is calculated according to the prescription amount, and a certain amount of water for injection is added to disperse after mixing; the above solutions are then mixed and the pH is adjusted. In some embodiments, each component is dissolved and dispersed with water for injection, and the obtained solution is mixed to adjust the concentration and pH value.

[0263] The present invention also provides the antibody-drug conjugate solid preparation of the present invention for the treatment of cancer. In some embodiments, the preparation is used for the treatment of EGFR or HER2 positive cancer, in particular for the treatment of EGFR positive metastatic colorectal cancer or head and neck cancer, or HER2 positive breast cancer, gastric cancer, ovarian cancer, colon cancer or non-small cell carcinoma. When the antibody-drug conjugate solid preparation of the present invention is used, before administering the drug to the subject, the antibody-drug conjugate solid preparation is first dissolved with a pharmaceutically acceptable solvent, reconstituted to obtain a reconstituted preparation, and then administered. The solvent includes sterile pharmaceutical grade water for injection or saline, sterile water for injection, antibacterial water for injection, etc.

[0264] On the one hand, a method for treating cancer is provided herein, comprising administering an effective amount of a reconstituted preparation of an antibody-drug conjugate to a patient in need of treatment. In some embodiments, the reconstituted preparation obtained by reconstructing the solid preparation of any one of the above embodiments in a pharmaceutically acceptable solvent (sterile pharmaceutical grade water for injection or saline, sterile water for injection, antibacterial water for injection, etc.). In some embodiments, the concentration of the ADC of the reconstituted preparation is 1 mg / ml-100 mg / ml, the buffer is one or more of a histidine buffer, a citrate buffer, and a succinate buffer at a concentration of 5 mM-20 mM, a protective agent is sucrose and / or trehalose at a concentration of 10 mM-250 mM, a surfactant is 0.05 mg / ml-0.4 mg / ml of Tween 80 and / or Tween 20, and the pH of the reconstituted preparation is 4.4-6.0. In some embodiments, the concentration of the ADC in the reconstituted preparation is 15 mg / ml-25 mg / ml, the buffer is a succinic acid buffer with a concentration of 8 mM-13 mM, the protective agent is sucrose or trehalose with a concentration of 105 mM-185 mM, the surfactant is 0.1 mg / ml-0.4 mg / ml Tween 80, and the pH of the reconstituted preparation is 4.8-5.4. In some embodiments, the concentration of the ADC in the reconstituted preparation is about 20 mg / ml, the buffer is a succinic acid buffer with a concentration of about 10 mM, the protective agent is trehalose with a concentration of about 160 mM, the surfactant is about 0.2 mg / ml Tween 80, and the pH of the reconstituted preparation is about 4.8-5.4, such as about 5.0. In some embodiments, the reconstituted formulation comprises ADC1 at a concentration of about 20 mg / ml, succinate buffer at about 1.16 mg / ml to about 1.18 mg / ml, trehalose at about 60 mg / ml, Tween 80 at about 0.2 mg / ml, and Na + , the pH of the reconstituted formulation is about 4.8-5.4, such as about 5.0.

[0265] In some embodiments, when used, the solid preparation is dissolved and reconstituted with a solvent (such as sterile water for injection) to obtain a reconstituted preparation, and the commonly used ADC concentration after reconstitution is about 20 mg / ml. If necessary, the reconstituted preparation can be diluted with an isotonic solution (such as 0.9% NaCl solution for injection). In some embodiments, the ADC concentration after dilution is about 0.1 mg / ml-about 5 mg / ml or about 0.5 mg / ml-about 1 mg / ml. The reconstituted preparation after reconstitution and the diluted preparation are both injection preparations, generally intravenous infusion.

[0266] In some embodiments, for HER2-positive cancers, such as breast cancer, gastric cancer, ovarian cancer, etc., the dosage of ADC is 1 mg / kg-10 mg / kg. In some embodiments, for HER2-positive cancers, such as breast cancer, gastric cancer, ovarian cancer, etc., the unit dose of ADC is about 3.6 mg / kg. In some embodiments, the unit dose of ADC is about 1.2 mg / kg-6.0 mg / kg; in some embodiments, the unit dose of ADC is about 1.2 mg / kg, or 2.4 mg / kg, or 3.6 mg / kg, or 4.8 mg / kg, or 6.0 mg / kg; in some embodiments, the unit dose of ADC is about 4.8 mg / kg. In some embodiments, the unit dose of ADC is about 3.6 mg / kg.

[0267] In some embodiments, the administration cycle is 14-28 days, such as 20-22 days, or 21 days, that is, the patient is administered the ADC unit dose once every 14-28 days, such as every 20-22 days, or every 21 days.

[0268] In some embodiments, the patient is a patient with a HER2-positive advanced solid tumor.

[0269] In some embodiments, the prepared solid preparation is subjected to stability tests at low temperature, room temperature and high temperature. The solid preparation prepared by the present invention can be stored at low temperature and room temperature for 1 month, 2 months, 3 months, 6 months, 9 months, 12 months, 18 months or even 24 months and remain stable. Compared with 0 months, the ADC has very little protein aggregation; 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.

[0270] In some embodiments, the reconstituted preparation is diluted with 0.9% saline. The ADC solution after dilution is placed at room temperature for 1 hour, 2 hours, 4 hours, 8 hours or even 24 hours, and shows good stability. Size exclusion chromatography (SEC-HPLC, or SEC) determination found that the average percentage of the main peak remained greater than 97%, and good performance was also maintained in terms of insoluble particles.

[0271] In order to develop a stable, non-aggregating anti-HER2 ADC preparation, the present invention provides an antibody-drug conjugate solid preparation. In some embodiments, the solid preparation can be prepared by lyophilizing and removing the water in the ADC at low temperature, so that the ADC remains stable during storage. The addition of a protective agent can effectively protect the ADC during the lyophilization process. The buffer is used to maintain a stable pH of the preparation and reduce the degradation of the ADC. The addition of a surfactant can reduce the surface tension of the lyophilized preparation after reconstitution, so that the ADC remains stable and is not easy to form aggregates or particles.

[0272] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0273] The antibody preparation composition of the present invention and its use are further described in detail below in conjunction with specific examples. In order to more clearly understand the technical content of the present invention, the following examples are specifically described in detail. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0274] In order to evaluate the stability of ADC in liquid and lyophilized formulations, the identity, purity and potency of ADC are usually tested. Purity tests include, but are not limited to, SDS-PAGE, CESDS, isoelectric focusing, immunoelectrophoresis, protein blotting, reverse phase chromatography (RP-HPLC, or RP), SEC, ion exchange chromatography and affinity chromatography. In addition, there are other tests, including visual performance, such as color, transparency, particles, pH, moisture and reconstitution time.

[0275] In some embodiments, the main steps of preparing a synthetic antibody drug conjugate include:

[0276] After diluting the antibody, it is incompletely reduced with dithiothreitol (DTT), incubated, eluted and exchanged with Sephadex G 25 resin, the number of thiol groups of the antibody is determined, and the drug-linker is added to the reduced antibody, stirred at room temperature, and cysteine ​​is added and continued to stir to obtain; the molar equivalent ratio of the drug-linker to the number of thiol groups of the antibody is (1-2):1, and in some embodiments, the molar equivalent ratio of the drug-linker to the number of thiol groups of the antibody is 1.5:1.

[0277] In some embodiments, the diluted antibody is diluted to 7-9 mg / mL.

[0278] In some embodiments, the reaction mixture is ultrafiltered and then purified using a G25 gel filtration column equilibrated in a phosphate buffer at pH 7.4±0.1.

[0279] In some embodiments, the prepared anti-HER2-antibody drug conjugate is filtered through a 0.22 μm filter and stored at 80±5° C.

[0280] In some embodiments, the drug / antibody ratio can be obtained by measuring the number of unreacted sulfhydryl groups on the drug-linker using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB).

[0281] In some embodiments, the anti-HER2 antibody drug conjugate prepared by the method described above has a DAR of 2-8; in some embodiments, the DAR is 3-5; in some embodiments, the DAR is 3.3-3.7; in some embodiments, the DAR is 3.5.

[0282] In some embodiments, the drug-linker concentration can be measured by UV absorption, the aggregation rate can be measured by size exclusion chromatography, and the residual free drug can be measured by reversed-phase high performance liquid chromatography.

[0283] In some embodiments, the anti-HER2 antibody is diluted to 8.0 mg / mL with solution B (50 mM potassium phosphate, 50 mM NaCl and 2 mM EDTA, pH 8.0), and then incompletely reduced with DTT (6 molar equivalents). After incubation at 37 ° C for 60 minutes, the exchange is eluted with solution A (50 mM potassium phosphate, 50 mM NaCl and 2 mM EDTA, pH 6.5) through Sephadex G25 resin. The thiol antibody value is determined by measuring the absorbance, and the concentration of the thiol group is determined by measuring the absorbance value at 412 nm by measuring the reactant of the thiol group and DTNB. The concentration of diethylformamide (DMA) during the coupling reaction is 10%. The ratio of the number of thiol groups of the drug-linker to the anti-HER2 antibody is 1.5:1.0 (molar equivalent). The drug-linker is added to the reduced anti-HER2 antibody, stirred at room temperature for 3 hours, and then 5 mM cysteine ​​is added and stirred for 1 hour.

[0284] In some embodiments, the ADC is ADC1. ADC1 has the following structure

[0285]

[0286] Wherein Ab is BAT-A, p is about 3.3-3.7 (average value), preferably 3.5. The drug-linker intermediate is synthesized according to the method disclosed in patent 201310081867.X.

[0287] In some embodiments, the ADC is ADC2, wherein ADC2 has the following structure:

[0288]

[0289] Wherein Ab is BAT-A and p is about 3.3-3.7 (average), such as about 3.5.

[0290] In some embodiments, the ADC is ADC3. Wherein ADC3 has the following structure

[0291]

[0292] Wherein Ab is Pertuzumab and p is about 3.3-3.7 (average), such as about 3.5.

[0293] In some embodiments, ADC2 is prepared using the antibody BAT-A according to the method disclosed in patent KR1020070037575A.

[0294] In some embodiments, ADC3 uses pertuzumab and is prepared according to the method used for ADC1.

[0295] The freeze-dried liquid preparation of the present invention is prepared by conventional methods and technical means in the art.

[0296] Example 1

[0297] Preparation of ADC1

[0298] Antibody BAT-A was diluted to 8.0 mg / ml with solution B (50 mM potassium phosphate, 50 mM NaCl and 2 mM EDTA, pH 8.0) and then incompletely reduced with DTT (6 molar equivalents). After incubation at 37°C for 60 minutes, the exchange was eluted with solution A (50 mM potassium phosphate, 50 mM NaCl and 2 mM EDTA, pH 6.5) through Sephadex G 25 resin. The thiol antibody value was determined by measuring the absorbance, and the concentration of the thiol was determined by measuring the absorbance at 412 nm from the reactant of the thiol with DTNB. The concentration of diethylformamide (DMA) was 10% during the coupling reaction. The drug-linker intermediate (synthesis method see patent 201310081867.X) was added to the reduced anti-HER2 antibody at a ratio of 1.5:1.0 (molar equivalent) between the number of thiol groups of the drug-linker intermediate and the anti-HER2 antibody. After stirring at room temperature for 3 hours, 5mM cysteine ​​was added and stirred for 1 hour. The reaction mixture was ultrafiltered, purified with a gel filtration column, and then filtered with a 0.22μm filter to obtain ADC1. The number of unreacted thiol groups was determined using DTNB to obtain a drug / antibody ratio of approximately 3.5 / 1, that is, the DAR of ADC1 was approximately 3.5.

[0299] Among them, the structure of the drug-linker intermediate is as follows:

[0300]

[0301] Example 2

[0302] Research on types of protective agents and buffers.

[0303] This example contains 10 formulations, with different protective agents, buffers and pH values. The composition of the liquid formulation of the antibody-drug conjugate lyophilized formulation is shown in Table 2:

[0304] Table 2

[0305]

[0306] The lyophilized preparations obtained by freeze-drying the liquid preparations prepared by the above 10 prescriptions were subjected to a high temperature test at 50°C. At 0, 2, 4, 6 and 10 days, the lyophilized preparations of each prescription were reconstituted with 5 ml of sterile water for injection to obtain reconstituted preparations. The reconstituted preparations after reconstitution were subjected to ADC stability analysis, including analysis using molecular exclusion chromatography (SEC) to determine the degree of ADC aggregation and degradation in each prescription. The results are as follows: Figure 1 As shown; and, using reverse phase chromatography (RP) to determine the drug coupling ratio, the results are as follows Figure 2 As shown. Since the drug is hydrophobic, it can cause the polarity of the light and heavy chains to change after being coupled to the antibody. The reverse phase method can be used to separate the ratio of the light and heavy chains that are connected to the drug and those that are not connected to the drug, thereby calculating the coupling ratio of the ADC drug. This method can determine the drug shedding position and ratio by comparison.

[0307] Figure 1 It can be seen that, relative to non-reducing sugar preparations, the main peak of polyhydroxy alcohols decreased significantly more than other prescription preparations. Therefore, the selection of non-reducing sugars as protective agents plays an important role in the preparation of stable ADC lyophilized preparations, especially sucrose and trehalose. In addition, there is no obvious trend between different buffers in the lyophilized samples, and the purity does not change significantly after high temperature treatment. In the lyophilized preparations, the sample has a low stress response to high temperature, indicating that the ADC lyophilized preparation can remain stable in buffers such as histidine buffer, citrate buffer, and succinate buffer.

[0308] Figure 2 It can be seen that in each prescription preparation, the drug coupling number is not significantly affected after high temperature treatment.

[0309] Example 3

[0310] Further research on the types of protective agents and buffers.

[0311] This example contains 4 formulations, the pH of the liquid preparations is 5.0, and the protective agent and buffer are different. The composition of the liquid preparation of the antibody-drug conjugate lyophilized preparation is shown in Table 3:

[0312] Table 3

[0313]

[0314] The liquid preparations prepared by the above four prescriptions were subjected to 4000Lx light intensity and 50℃ high temperature experiments. The stability of the liquid preparations was analyzed at 0, 1, 3, 5 and 10 days respectively, and the purity of ADC was determined by SEC. The results are as follows Figure 3 and Figure 4 shown.

[0315] The liquid preparations prepared by the above four prescriptions were freeze-dried to prepare freeze-dried preparations, and then subjected to high temperature experiments at 60°C. The stability of the freeze-dried preparations was analyzed at 0, 5, 10, 15 and 40 days, and the purity of ADC was determined by SEC. The results are as follows: Figure 5 shown.

[0316] from Figure 3 and Figure 4 It can be further seen that the limiting effect of citrate buffer and histidine buffer on protein aggregation is weaker than that of succinate buffer; the protective effect of succinate buffer on ADC stability in liquid preparations is better than that of citrate buffer and histidine buffer; non-reducing sugars have a certain protective effect on ADC, among which trehalose is better than sucrose.

[0317] from Figure 5 It can be seen that the protective effect of succinate buffer on ADC stability in lyophilized preparations is also better than that of citrate buffer and histidine buffer; similarly, trehalose is better than sucrose. In comparison, formulation 4 has the best effect on protein aggregation restriction.

[0318] Example 4

[0319] Buffer concentration studies

[0320] The buffer can stabilize the pH value of the liquid preparation and control the range of the buffer concentration, which plays an important role in the stability of the ADC. In this embodiment, there are 4 prescriptions, each with a different buffer concentration, and the pH of the liquid preparation is controlled at about 5.0 with sodium hydroxide, and its composition is shown in Table 4.

[0321] Table 4

[0322]

[0323] The above four prescriptions were prepared into liquid preparations before lyophilization, and then lyophilized to obtain lyophilized preparations. The above liquid preparations were incubated at 50°C, and stability analysis was performed on the liquid preparations at 0, 1, 3, 5 and 10 days. The purity of ADC was determined by SEC, and the results were as follows: Figure 6 shown.

[0324] The above lyophilized preparations were incubated at 60°C and stability analysis was performed on the lyophilized preparations at 0, 5, 10, 15 and 30 days. The purity of ADC was determined by SEC. The results are as follows: Figure 7 shown.

[0325] The results show that the concentration range of the buffer is between 5mM and 20mM, and there is no significant difference in the stability of the protective protein. Considering that the succinic acid buffer is a crystalline buffer, a significant pH shift will occur at a high concentration; while at a low concentration, the ability to maintain the pH stabilization system is weak. Therefore, the present invention particularly prefers a buffer with a concentration of 10mM.

[0326] Example 5

[0327] Study on the concentration of protective agent

[0328] There are 4 prescriptions in this example, and the difference between the prescriptions lies in the concentration of the protective agent, and their compositions are shown in Table 5. The pH of the solution preparations is controlled at about 5.0 with sodium hydroxide.

[0329] Table 5

[0330]

[0331] The above four prescriptions were prepared into liquid preparations before lyophilization, and then lyophilized to obtain lyophilized preparations. The lyophilized preparations were incubated at 50°C, and stability analysis was performed on the lyophilized preparations at 0, 5, 15, 30 and 60 days. The purity of ADC was determined by SEC, and the results were as follows: Figure 8 shown.

[0332] The results show that the protective agent has a relatively good protective effect on the protein in the range of 105mM to 185mM concentration. As the concentration of the protective agent increases, the protective agent has a better effect on the restriction of protein polymerization, that is, the protective effect on the stability of the protein is better. When the concentration is between 160mM and 185mM, there is no obvious difference in the effect of restricting protein polymerization. In view of the effect of the protective agent on the freeze-drying time, the present invention particularly prefers a protective agent with a concentration of 160mM.

[0333] Example 6

[0334] Study on pH Value of Aqueous Solutions

[0335] There are 7 prescriptions in this embodiment, and the composition of each prescription is shown in Table 6.

[0336] Table 6

[0337]

[0338] The above 7 prescriptions were prepared into liquid preparations before lyophilization, and the liquid preparations were placed in a high temperature test at 50°C. The stability of the liquid preparations was analyzed at 0 and 3 days. The purity of ADC was determined by SEC. The results are as follows: Fig. 9 As shown; the content of uncoupled protein in ADC was determined by hydrophobic chromatography (HIC) method, and the results were as follows Fig.10As shown. Since parameters such as pH will affect the stability between drug molecules and antibodies, showing the phenomenon of drug shedding, measuring the content of uncoupled antibodies in the sample can reflect the stability of the drug.

[0339] The results show that the protein exhibits good stability when the solution pH is between 4.6 and 5.6. Further analysis of the results shows that as the pH in the solution increases, the ability of protein polymerization restriction becomes stronger, while the content of uncoupled antibodies increases, and the rate of change tends to be flat when the pH is 4.8 to 5.2. Comparing the effects of pH changes on protein polymerization and uncoupled antibodies, the requirements of protein polymerization restriction can also be suitable for limiting drug shedding. The pH of the liquid preparation of the present invention is preferably between 4.8 and 5.2, and more preferably 5.0.

[0340] Example 7

[0341] Research on surfactants

[0342] Surfactants often play an important role in limiting protein aggregation in freeze-dried formulations, such as limiting the possible polymerization of proteins at the ice-water interface during the freezing process, and also limiting the possible polymerization of freeze-dried proteins during the reconstitution process. In general, surfactants can minimize the unpredictable aggregation of proteins during the process.

[0343] Four prescriptions were designed in this embodiment, and the composition of each prescription is shown in Table 7. The pH value of the liquid preparation was controlled at about 5.0 with sodium hydroxide.

[0344] Table 7

[0345]

[0346] The above four prescriptions were prepared into liquid preparations before freeze-drying, and the liquid preparations were repeatedly frozen and thawed 5 times. The samples of the liquid preparations that were repeatedly frozen and thawed were judged by the insoluble particles in the experiment. In addition, the samples were re-dissolved after freeze-drying, and the re-dissolution time of the samples and the insoluble particles of the re-dissolved samples were tested to judge the experiment.

[0347] The insoluble particles of the repeatedly frozen and thawed samples are shown in Table 8 below, and the insoluble particles of the lyophilized samples after reconstitution are shown in Table 9 below.

[0348] Table 8

[0349]

[0350] Table 9

[0351]

[0352] The results show that the addition of surfactant has a significant effect on the stability of ADC, and samples without surfactant show more polymerization tendency. Both Tween 20 and Tween 80 show the ability to limit protein polymerization, and the difference between the two is not large. In addition, for different concentrations of surfactant, there is no significant difference in the effect on the stability of ADC. Considering that too much surfactant is not conducive to the application of the preparation, the concentration of the surfactant of the present invention is preferably between 0.2 mg / ml and 0.4 mg / ml, more preferably 0.2 mg / ml.

[0353] Example 8

[0354] Study of ADC concentration

[0355] Three prescriptions were designed in this embodiment, and the composition of each prescription is shown in Table 10. Sodium hydroxide was used to control the pH value in the liquid preparation to about 5.0.

[0356] Table 10

[0357]

[0358] The above three prescriptions were prepared into liquid preparations before lyophilization, and then lyophilized to obtain lyophilized preparations. The prepared lyophilized preparations were stored at 40°C. The lyophilized preparations were reconstituted after 0, 1 and 2 months, respectively, and stability analysis was performed. The purity of ADC was determined by SEC, and the results were as follows: Fig.11 As shown; the HIC method was used to determine the content of uncoupled protein in ADC, and the results were as follows Fig.12 shown.

[0359] The results showed that when the ADC concentration was between 15 mg / ml and 25 mg / ml, there was no significant difference in the effect of high temperature on its stability.

[0360] Example 9

[0361] Long-term stability and accelerated stability studies

[0362] In this example, the following formulation was used for stability study.

[0363]

[0364] A liquid preparation was prepared according to the above prescription, and the pH was adjusted to 5.0 with sodium hydroxide. The liquid preparation was lyophilized to obtain a lyophilized preparation.

[0365] The lyophilized formulation samples were evaluated by long-term and accelerated stability procedures. The lyophilized formulation samples were placed at 5±3°C at 0, 3 and 6 months, or at 9, 12, 18 and 24 months, and placed at 25±2°C at 0, 1, 2, 3 and 6 months. The lyophilized formulations were reconstituted and the composition and concentration after reconstitution were 20 mg / mL ADC, 10 mM succinate buffer, 160 mM trehalose and 0.2 mg / ml Tween 80. Stability analysis was performed after reconstitution. SEC, HIC, insoluble particulates, UV / Vis, appearance, color and turbidity after reconstitution were tested.

[0366] All samples remained stable by all test methods after at least 6 months storage at 5±3°C and 25±2°C. All samples remained stable by all test methods after at least 24 months storage at 5±3°C. Samples stored at 5±3°C showed no significant changes at least at the 24-month time point, and samples stored at 25±2°C showed no significant changes at least at the 6-month time point compared to the start of the study.

[0367] Specifically, regarding protein aggregation, SEC analysis showed that the percentage of aggregates remained at approximately 0.2% for samples stored at 5±3°C for at least 24 months and at 25±2°C for at least 6 months. Compared to storage at 0 months, the change in the main material isoform for samples stored at 5±3°C was within 0.2% after at least 24 months, and the insoluble particulates also remained good.

[0368] In addition, the average DAR, DAR and free drug remained almost constant after at least 24 months at 5°C and at least 6 months at 25°C. There was no sign of degradation after at least 24 months at 5°C and at least 6 months at 25°C. Most importantly, the biological function of ADC can be maintained for at least 24 months at 5°C and at least 6 months at 25°C as demonstrated by bioassay of cytotoxicity. Therefore, in the formulation of the present invention, the lyophilized formulation has acceptable stability for ADC application.

[0369] The example data of the stability program of the lyophilized preparation stored at 5±3°C for 6 months are shown in Table 11a; the example data of the stability program of the lyophilized preparation stored at 5±3°C for 24 months are shown in Table 11b; the example data of the stability program of the lyophilized preparation stored at 25±2°C for 6 months are shown in Table 12.

[0370] Table 11a

[0371]

[0372] Table 11b

[0373]

[0374]

[0375] Table 12

[0376]

[0377] Example 10

[0378] Study on the stability of compatibility

[0379] The lyophilized preparation prepared in Example 9 was subjected to a compatibility stability study. First, the lyophilized preparation was reconstituted with sterile water for injection to obtain a reconstituted preparation with an ADC concentration of 20 mg / ml. The reconstituted preparation was then diluted with 0.9% saline to a solution with a concentration of 0.5 mg / ml and 1 mg / ml, respectively. The solution was placed at room temperature and stability was evaluated by SEC and determination of insoluble particles at 0, 1, 2, 4, 8 and 24 hours.

[0380] SEC results as Fig.13 The results showed that the average percentage of the main peak of the sample placed at room temperature for 24 hours remained greater than 97%, and there was no significant difference between the 1 mg / mL sample and the 0.5 mg / ml sample, and the 0.9% saline diluent did not cause the effect of protein aggregation.

[0381] The results of the insoluble particle test showed that no matter whether it was 0.5 mg / ml or 1 mg / ml sample, dilution with 0.9% physiological saline and storage at room temperature for 24 hours did not cause adverse effects on the insoluble particles of the sample, as shown in Table 13. This shows that saline can be used as a diluent for the lyophilized preparation of the present invention.

[0382] Table 13

[0383]

[0384] Embodiment 11

[0385] In this example, the ADC2 lyophilized preparation was prepared using the following formulation.

[0386]

[0387] A liquid preparation was prepared according to the above prescription, and the pH was adjusted to 5.0 with sodium hydroxide. The liquid preparation was lyophilized to obtain a lyophilized preparation.

[0388] Example 12

[0389] In this example, the following prescription was used to prepare the ADC3 lyophilized preparation.

[0390]

[0391] A liquid preparation was prepared according to the above prescription, and the pH was adjusted to 5.0 with sodium hydroxide. The liquid preparation was lyophilized to obtain a lyophilized preparation.

[0392] Example 13

[0393] In this embodiment, a solid preparation comprising the following formulation composition (by mass fraction) is prepared:

[0394]

[0395]

[0396] Embodiment 14

[0397] In this embodiment, a solid preparation comprising the following formulation composition (by mass fraction) is prepared:

[0398]

[0399] Embodiment 15

[0400] In this embodiment, a solid preparation comprising the following formulation composition (by mass fraction) is prepared:

[0401]

[0402] Example 16

[0403] Preparation of reconstituted preparations: The three lyophilized preparations obtained by freeze-drying in Example 8 were placed in vials respectively. Sterile water for injection was slowly injected into the vials using a sterile syringe. The vials were gently rotated until completely dissolved. Vigorous shaking was prohibited. The reconstituted preparations were prepared into ADC1 concentrations of 15 mg / ml, 25 mg / ml, and 20 mg / ml, respectively.

[0404] Embodiment 17

[0405] Preparation of injection preparation: Take 250 ml of 0.9% sodium chloride for injection infusion bag / bottle, calculate the required amount of the reconstituted preparation prepared in Example 16 based on the dosage of 3.6 mg / kg, use a sterile syringe to extract the corresponding volume of the reconstituted preparation from the vial, add it to the infusion bag / bottle, gently invert the infusion bag / bottle to mix the solution, repeat several times to avoid bubbles, and the diluted injection preparation is obtained.

[0406] Embodiment 18

[0407] The lyophilization cycle of the lyophilized formulation of the present invention can be performed as follows.

[0408] In one embodiment, the formulated liquid formulation is cooled to -35°C at a rate of 0.5°C / min; isothermally maintained for 120 min. Next, the temperature is increased to -20°C at a rate of 0.5°C / min; isothermally maintained for 120 min; then, a vacuum is applied at a pressure of 50 mTorr at a temperature of -30°C. Finally, the temperature is increased to 25°C at a rate of 0.5°C / min; isothermally maintained for 7 hours. When the residual moisture in the formulation does not exceed 2% by weight, a lyophilized formulation is prepared.

[0409] In one embodiment, the formulated liquid formulation is cooled to -35°C at a rate of 1°C / min; isothermally maintained for at least 120 min. Next, the temperature is raised to -7°C at a rate of 3°C / min; isothermally maintained for at least 120 min; then, a vacuum is applied at a temperature of -8°C using a pressure of 200 mTorr. Finally, the temperature is increased to 30°C at a rate of 1°C / min; isothermally maintained for at least 7 hours. When the residual moisture in the formulation does not exceed 2% by weight, a lyophilized formulation is prepared.

[0410] Embodiment 19

[0411] The preparation of the solution, the prescription components and the dosage per bottle are as follows:

[0412]

[0413] The above components are dissolved in water for injection, wherein the pH is adjusted to 5.0 using sodium hydroxide.

[0414] The above solution was added to vials, 5 ml was added to each vial, and freeze-dried using the method of Example 18 to obtain a freeze-dried solid preparation. The composition and content of each bottle of solid preparation were as follows:

[0415]

[0416] When a solid preparation is used, the solid preparation is formulated into a reconstituted preparation with an ADC1 concentration of 20 mg / ml using water for injection using the method of Example 16.

[0417] In the above embodiments, in the formulation of sodium ions, the sodium ions and the succinic acid buffer form a buffer system.

[0418] Embodiment 20

[0419] Take 250 ml of 0.9% sodium chloride for injection infusion bag / bottle, calculate the required reconstituted preparation prepared in Example 19 according to the dosage of ADC1 of 1.2 mg / kg, 2.4 mg / kg, 3.6 mg / kg, 4.8 mg / kg and 6.0 mg / kg, respectively, use a sterile syringe to extract the corresponding volume of the reconstituted preparation from the vial, add it to the infusion bag / bottle, gently invert the infusion bag / bottle to mix the solution, repeat several times to avoid bubbles, and obtain the diluted injection preparation.

[0420] The formulated injectable formulation is administered by intravenous infusion to patients with HER2-positive advanced solid tumors once every three weeks.

[0421] Example 21 Clinical Study

[0422] In this study, ADC1 was administered once every 3 weeks at a dose of 1.2 mg / kg, 2.4 mg / kg, 3.6 mg / kg, 4.8 mg / kg or 6.0 mg / kg to treat patients with HER2-positive advanced solid tumors. The pharmacokinetic parameters (PK) after the first administration are shown in Table 14:

[0423] Table 14

[0424] Dosage (mg / kg) 1.2 2.4 3.6 4.8 6.0 <![CDATA[C max (μg / mL)]]> 27.2 38.5 76.6 92.7 140 <![CDATA[AUC (0-t) (μg / mL·h)]]> 1190 2417 3930 5190 6450

[0425] The PK after the fourth administration is shown in Table 15:

[0426] Table 15

[0427] Dosage (mg / kg) 1.2 2.4 3.6 4.8 6.0 <![CDATA[C max (μg / mL)]]> 22.6 36.6 70.0 95.0 102.7 <![CDATA[AUC (0-t) (μg / mL·h)]]> 1226 2177 4066 6030 6121

[0428] A total of 29 subjects were enrolled in this study, of whom 28 had imaging results. The preliminary efficacy assessment 10 months after treatment found that 1 patient (3.7%) had a complete response (CR), 5 patients (18.5%) had a partial response (PR), and 5 patients (18.5%) had stable disease.

[0429] Treatment-emergent adverse events (TEAEs)

[0430]

[0431] The technical features of the above-described embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above-described embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification. The above-described embodiments only express several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present invention, several adjustments and improvements can be made, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached claims. Sequence Listing <110> Bio-Thera Pharmaceuticals Co., Ltd. <120> Antibody-drug conjugate preparation, preparation method and application <130> DSP1F201062JW <160> 6 <170> SIPOSequenceListing 1.0 <210> 1 <211> 214 <212> PRT <213> Artificial Sequence <400> 1 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Phe Cys Gln His Phe Asp His Leu Pro Leu 85 90 95 Ala Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 2 <211> 447 <212> PRT <213> Artificial Sequence <400> 2 Gln Val Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu 1 5 10 15 Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Val Ser Ser Gly Asp Tyr 20 25 30 Tyr Trp Thr Trp Ile Arg Gln Ser Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly His Ile Tyr Tyr Ser Gly Asn Thr Asn Tyr Asn Pro Ser Leu Lys 50 55 60 Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys Val 85 90 95 Arg Asp Arg Val Thr Gly Ala Phe Asp Ile Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu 115 120 125 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 130 135 140 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 145 150 155 160 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 165 170 175 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 180 185 190 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 195 200 205 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His 210 215 220 Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 260 265 270 Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 3 <211> 214 <212> PRT <213> Artificial Sequence <400> 3 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 4 <211> 451 <212> PRT <213> Artificial Sequence <400> 4 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Pro Lys Ser Cys 210 215 220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly 225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340 345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly Lys 450 <210> 5 <211> 214 <212> PRT <213> Artificial Sequence <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 <210> 6 <211> 450 <212> PRT <213> Artificial Sequence <400> 6 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310 315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445 Gly Lys 450

Claims

1. An antibody-drug conjugate solid preparation, It is characterized in that The solid preparation comprises an antibody-drug conjugate, wherein the antibody-drug conjugate is composed of an antibody, a linker and a drug module, and the solid preparation further comprises one or more of a buffer, a protective agent and a surfactant; The antibody-drug conjugate has the following structure: in, Ab is antibody; p is 2-8; The antibody is an anti-HER2 antibody; the anti-HER2 antibody light chain amino acid sequence is shown in SEQ ID NO: 3 or SEQ ID NO: 5, and the anti-HER2 antibody heavy chain amino acid sequence is shown in SEQ ID NO: 4 or SEQ ID NO: 6; The mass fraction of the antibody-drug conjugate in the solid preparation is 1-100 parts; The mass fraction of the buffer in the solid preparation is 0.6 parts to 2.4 parts based on the acid ion of the buffer, and the buffer is a histidine buffer, a citric acid buffer or a succinic acid buffer; The mass fraction of the protective agent in the solid preparation is 4-95 parts, and the protective agent is sucrose or trehalose; The mass fraction of the surfactant in the solid preparation is 0.05-0.4 parts, and the surfactant is Tween 20 or Tween 80.

2. The solid preparation according to claim 1, It is characterized in that The drug binding number of the antibody is selected from 2-8.

3. The solid preparation according to claim 2, It is characterized in that The drug binding number of the antibody is 2, 4, 6 or 8.

4. The solid preparation according to claim 1, It is characterized in that The average drug binding number of the antibody is selected from 2-8.

5. The solid preparation according to any one of claims 1 to 4, It is characterized in that The solid preparation is a lyophilized preparation, which is obtained by lyophilizing a liquid preparation containing the antibody-drug conjugate, and the liquid preparation further contains one or more of the buffer, protective agent and surfactant.

6. The solid preparation according to claim 5, It is characterized in that In the liquid preparation, the concentration of the antibody-drug conjugate is 15 mg / ml-25 mg / ml; In the liquid preparation, the concentration of the buffer is 1 mg / ml-2 mg / ml or 5 mM-20 mM; the pH of the liquid preparation is in the range of 4.6-5.6; The concentration of the protective agent in the liquid preparation is 30 mg / ml-90 mg / ml or 105 mM to 185 mM; The concentration of the surfactant in the liquid preparation is 0.05 mg / ml-0.4 mg / ml.

7. A solid preparation, Features: The solid preparation comprises: 1 to 100 parts by weight of an antibody-drug conjugate; A buffer having a mass fraction of 0.6 to 2.4 parts based on acid radical ions, wherein the buffer is a histidine buffer, a citric acid buffer, or a succinic acid buffer; 4 to 95 parts by weight of a protective agent, wherein the protective agent is sucrose or trehalose; and 0.05 to 1 part by weight of a surfactant, wherein the surfactant is Tween 20 or Tween 80; or The solid preparation comprises: 10 to 40 parts by weight of an antibody-drug conjugate; 0.9 to 1.5 parts by weight of a buffer calculated as acid radical ions, wherein the buffer is a histidine buffer, a citric acid buffer, or a succinic acid buffer; 19 to 85 parts by weight of a protective agent, wherein the protective agent is sucrose or trehalose; and 0.05 to 1 part by weight of a surfactant, wherein the surfactant is Tween 20 or Tween 80; or The solid preparation comprises: 15-25 parts by weight of an antibody-drug conjugate; 0.9 to 1.5 parts by weight of a buffer calculated as acid radical ions, wherein the buffer is a histidine buffer, a citric acid buffer, or a succinic acid buffer; 40 to 70 parts by weight of a protective agent, wherein the protective agent is sucrose or trehalose; and 0.1 to 0.4 parts by weight of a surfactant, wherein the surfactant is Tween 20 or Tween 80; or The solid preparation comprises: 20 parts by weight of antibody-drug conjugate; 1.16 parts by weight of a buffer calculated as acid radical ions, wherein the buffer is a histidine buffer, a citric acid buffer, or a succinic acid buffer; 60 parts by weight of a protective agent, wherein the protective agent is sucrose or trehalose; and 0.2 parts by weight of a surfactant, wherein the surfactant is Tween 20 or Tween 80; The antibody-drug conjugate has the following structure: in, Ab is antibody; p is 2-8; Wherein, the antibody is an anti-HER2 antibody; the anti-HER2 antibody light chain amino acid sequence is shown in SEQ ID NO:3 or SEQ ID NO:5, and the anti-HER2 antibody heavy chain amino acid sequence is shown in SEQ ID NO:4 or SEQ ID NO:

6.

8. The solid preparation according to claim 7, It is characterized in that The average p value is 3.3-3.

7.

9. The solid preparation according to claim 8, It is characterized in that The average value of p is 3.

5.

10. A solid preparation, Features: The solid preparation comprises: 100 parts by mass of an antibody-drug conjugate; 5.8 to 5.9 parts by weight of succinic acid buffer calculated as acid radical ion; 300 parts by weight of trehalose; and 1 part by weight of Tween 80; The antibody-drug conjugate has the following structure: Among them, Ab was BAT-A, and the average p value was 3.3-3.7; The BAT-A is an anti-HER2 antibody, whose light chain amino acid sequence is shown in SEQ ID NO:5, and whose heavy chain amino acid sequence is shown in SEQ ID NO:

6.

11. The solid preparation according to claim 10, It is characterized in that The average value of p is 3.

5.

12. A freeze-dried powder, It is characterized in that The lyophilized powder comprises 100 parts by weight of an antibody-drug conjugate, 5.9 parts by weight of succinic acid, 300 parts by weight of trehalose, 1 part by weight of polysorbate 80, and sodium hydroxide is adjusted to a pH value of 5.0; the antibody-drug conjugate has the following structure: Among them, Ab was BAT-A, and the average p value was 3.3-3.7; The BAT-A is an anti-HER2 antibody, whose light chain amino acid sequence is shown in SEQ ID NO:5, and whose heavy chain amino acid sequence is shown in SEQ ID NO:

6.

13. The freeze-dried powder according to claim 12, It is characterized in that The average value of p is 3.

5.

14. A method for preparing the solid preparation according to claim 5 or 6, the method The following steps are involved: a) cooling the liquid preparation to -35°C or lower and maintaining it isothermally; b) raising the temperature of the preparation treated in step a) to between -20°C and -7°C and maintaining the temperature isothermally; c) applying a vacuum to the preparation treated in step b) at a temperature between -35°C and -8°C; d) increasing the temperature of the preparation treated in step c) to between 25°C and 40°C; and maintaining it isothermally.

15. A reconstituted preparation, It is characterized in that The reconstituted preparation is obtained by reconstructing the solid preparation according to any one of claims 1 to 6 with a pharmaceutically acceptable solvent.

16. A liquid preparation, Features: The liquid preparation comprises: 15mg / ml-25mg / ml of antibody-drug conjugate; 1 mg / ml-2 mg / ml or 5 mM-20 mM buffer, wherein the buffer is histidine buffer, citrate buffer or succinate buffer; 30 mg / ml-90 mg / ml or 105 mM to 185 mM of a protective agent, wherein the protective agent is sucrose or trehalose; 0.05 mg / ml-0.4 mg / ml of a surfactant, wherein the surfactant is Tween 20 or Tween 80; and water; The pH of the liquid preparation is 4.6-5.6; or The liquid preparation comprises: 15mg / ml-25mg / ml of antibody-drug conjugate; 1 mg / ml-2 mg / ml or 5 mM-20 mM buffer, wherein the buffer is histidine buffer, citrate buffer or succinate buffer; 50mg / ml-70mg / ml or 140mM-180mM of a protective agent, wherein the protective agent is sucrose or trehalose; 0.1 mg / ml-0.4 mg / ml of a surfactant, wherein the surfactant is Tween 20 or Tween 80; and water; The pH of the liquid preparation is 4.8-5.2; or The liquid preparation comprises: 20 mg / ml of antibody-drug conjugate; 1.16 mg / ml-1.18 mg / ml or 10 mM buffer, wherein the buffer is histidine buffer, citrate buffer or succinate buffer; 60 mg / ml or 160 mM protective agent, wherein the protective agent is sucrose or trehalose; 0.2 mg / ml of a surfactant, wherein the surfactant is Tween 20 or Tween 80; and water; The pH of the liquid preparation is 5; or The liquid preparation comprises: 15mg / ml-25mg / ml of antibody-drug conjugate; 1mg / ml-2mg / ml or 5mM-20mM succinate buffer; 30mg / ml-90mg / ml or 105mM to 185mM trehalose; 0.05mg / ml-0.4mg / ml Tween 80; and water; The pH of the liquid preparation is 4.6-5.6; or The liquid preparation comprises: 15mg / ml-25mg / ml of antibody-drug conjugate; 1mg / ml-2mg / ml or 5mM-20mM succinate buffer; 50mg / ml-70mg / ml or 140mM-180mM trehalose; 0.1mg / ml-0.4mg / ml Tween 80; and water; The pH of the liquid preparation is 4.8-5.2; or The liquid preparation comprises: 20 mg / ml of antibody-drug conjugate; 1.16mg / ml-1.18mg / ml or 10mM succinate buffer; 60 mg / ml or 160 mM trehalose; 0.2 mg / ml Tween 80; and water; The pH of the liquid preparation is 5; The antibody-drug conjugate has the following structure: Among them, Ab was BAT-A, and the average p value was 3.3-3.7; The BAT-A is an anti-HER2 antibody, whose light chain amino acid sequence is shown in SEQ ID NO:5, and whose heavy chain amino acid sequence is shown in SEQ ID NO:

6.

17. The liquid preparation according to claim 16, It is characterized in that The average value of p is 3.

5.

18. Use of the solid preparation according to any one of claims 1 to 11, the lyophilized powder according to claim 12 or 13, the reconstituted preparation according to claim 15, or the liquid preparation according to claim 16 or 17 in the preparation of a medicament for treating cancer.

19. The use according to claim 18, It is characterized in that The cancer is an EGFR or HER2 positive cancer.

20. The use according to claim 19, It is characterized in that The cancer is metastatic colorectal cancer, head and neck cancer, breast cancer, gastric cancer, ovarian cancer, colon cancer, or non-small cell lung cancer.

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