WO2020233534A1 - Antibody-drug conjugate preparation, preparation method therefor and use thereof - Google Patents

Abstract

Provided are an antibody-drug conjugate (ADC) preparation (comprising a solid preparation, a liquid preparation and a reconstituted preparation), a preparation method therefor and a use thereof. In some embodiments, the solid preparation provided contains an antibody-drug conjugate composed of an antibody, a linker and a drug module, and also contains one or more of a buffer, a protective agent and a surfactant. In some embodiments, the solid preparation is a lyophilized preparation, wherein same is obtained by freeze-drying a liquid preparation of an ADC composed of an antibody, a linker and a drug module, and the liquid preparation also contains one or more of a buffer, a protective agent and a surfactant. The above-mentioned preparation can be used for preparing drugs for treating cancers, such as EGFR or HER2 positive cancer.

Description

Antibody-drug conjugate preparation, preparation method and application Technical field

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

Background technique

Antibody-Drug Conjugates (ADC) are highly effective and specific drugs for the treatment of cancer and other conditions obtained by coupling antibodies and drugs, in which the antibody part specifically binds to target cells On the antigen, the drug can exert its cytotoxicity or other therapeutic effects on target cells.

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 microparticles formed in protein drugs will cause risks in clinical applications. For example, the microparticles in the injected drug formulation can cause vein damage or venous blockage in the injected patient. In addition, protein aggregation is the degradation of the drug One of the main ways, but also may produce undesirable immunogenicity.

For ADC drugs, the drug coupling itself will reduce the stability of the antibody and change its physical and chemical properties. Since most of the conjugated drugs are hydrophobic small molecule drugs, the ADC conjugate as a whole may have a lower solubility than the unconjugated antibody. At the same time, in addition, ADC has a more complicated heterogeneous structure than unconjugated antibody, which makes ADC easier to aggregate, forming particles and surface adsorption. Therefore, the instability and aggregation of ADC greatly increase the risk in clinical application.

Summary of the invention

Based on this, it is necessary to provide an antibody-drug conjugate solid preparation for the instability and easy aggregation of ADC, which has excellent long-term stability and accelerated stability.

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

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; some embodiments , The antibody is BAT-A.

In some embodiments, the drug binding number (or drug-to-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.

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

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.

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

Or a pharmaceutically acceptable salt or solvate thereof,

among them

X is hydrogen or halogen;

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

R ^{1 is} selected from H, -OH, -OC(=O)R ⁵ and -OR ⁵ groups;

R ² is H or C ¹ -C ⁶ alkyl;

R ³ is methyl, -CH ₂ OH or -CH ₂ OC(=O)R ⁶ ;

R ⁴ is -OH or -SH;

R ⁵ is C ¹ -C ⁶ alkyl or benzyl;

R ⁶ is C ¹ -C ⁶ alkyl, phenyl or benzyl;

R ⁷ is hydrogen, C ¹ -C ⁶ alkyl or amino acid side chain;

R ⁸ is hydrogen or C ¹ -C ⁶ alkyl;

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

p is 2-8;

Ab is an antibody, preferably an anti-HER2 antibody, more preferably a BAT-A antibody.

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

among them,

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

p is DAR or average DAR.

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

In some embodiments, the ADC is ADC1 described herein.

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

In some embodiments, the buffer in the solid formulation includes one or more of histidine buffer, citrate buffer, and succinic acid buffer. In some embodiments, the buffer is a succinic acid buffer. It should be understood that in the present application, buffers (for example, histidine buffer, citrate buffer, succinic acid buffer) are used in combination with acids (such as hydrochloric acid) or bases (such as NaOH or KOH) to form a buffer system. The buffer system can be a weak acid and its salt system, or a weak base and its salt system, depending on the specific buffer used. Unless otherwise specified, no matter the buffer in the present application is in the form of acid and/or salt, when the mass parts or molar concentration of the buffer is mentioned, it is calculated based on its total acid ion. For example, when the buffer system is a succinic acid (HOOCCH ₂ CH ₂ COOH) buffer, no matter which succinic acid exists in the form of HOOCCH ₂ CH ₂ COOH, HOOCCH ₂ CH ₂ COO ^- and/or ^- OOCCH ₂ CH ₂ COO ^- , amber The amount of acid buffer refers to the total amount of HOOCCH ₂ CH ₂ COOH, HOOCCH ₂ CH ₂ COO ^- and ^- OOCCH ₂ CH ₂ COO ^- based on the acid radical ion ^- OOCCH ₂ CH ₂ COO ^- .

In some embodiments, the protective agent in the solid formulation is selected from one or two of sucrose and trehalose. In some embodiments, the protective agent is selected from trehalose. In some embodiments, the protective agent is selected from sucrose.

In some embodiments, the surfactant in the solid preparation is selected from one or two 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.

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

In some embodiments, the solid preparation contains about 1 part to about 100 parts of ADC1 by mass. In some embodiments, the solid preparation contains about 10 parts to about 40 parts of ADC1 by mass. In some embodiments, the solid preparation contains about 15 parts to about 25 parts of ADC1 by mass. In some embodiments, the solid preparation contains about 20 parts by mass of ADC1.

In some embodiments, the mass parts of the buffer in the solid preparation is about 0.6 parts to about 2.4 parts based on acid radical ions. In some embodiments, the mass parts of the buffer in the solid formulation is about 0.9 parts to about 1.5 parts. In some embodiments, the mass parts of the buffer in the solid preparation is about 1.16 parts. In some embodiments, the mass parts of the buffer in the solid preparation is about 1.9 parts. In some embodiments, the mass parts of the buffer in the solid preparation is about 1.56 parts.

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

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

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

In some embodiments, the solid preparation contains about 4 parts to about 95 parts of trehalose by mass. In some embodiments, the solid preparation contains about 19 parts to about 85 parts of trehalose by mass. In some embodiments, the solid preparation contains about 40 parts to about 70 parts of trehalose by mass. In some embodiments, the solid preparation contains about 55 parts to about 60 parts of trehalose by mass. In some embodiments, the solid preparation contains about 60 parts by mass of trehalose.

In some embodiments, the mass part of the surfactant in the solid preparation is about 0.05 part to about 1 part. In some embodiments, the mass part of the surfactant in the solid preparation is about 0.1 part to about 0.4 part. In some embodiments, the mass parts of the surfactant in the solid preparation is about 0.2 parts.

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

The "parts by mass" mentioned in the present invention refers to the value in the case of equal unit mass, and the unit mass is the mass of each part, which can be any suitable mass. For example, when each serving is 1g, the unit mass is 1g/ serving, at this time 20 servings is 20g; the unit mass can also be 1kg/ serving or 1 mg/ serving. In addition, the unit mass is not limited to an integer, for example, it can be 0.5 g/part, in which case 20 parts is 10 g.

In some embodiments, the solid preparation comprises:

The mass parts are about 1 part to about 100 parts of ADC (such as ADC1);

Based on acid radical ions, the mass parts are about 0.6 to about 2.4 parts of buffer (such as histidine buffer, citrate buffer, or succinic acid buffer, such as succinic acid buffer);

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

The mass part is about 0.05 part to about 1 part of surfactant (such as Tween 20 or Tween 80, such as Tween 80).

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.4-6.0, 4.6-5.6 or 4.8-5.2.

In some embodiments, the solid preparation comprises:

The mass parts are about 10 parts to about 40 parts of ADC (such as ADC1);

A buffer (such as a histidine buffer, a citrate buffer, or a succinic acid buffer, such as a succinic acid buffer) of about 0.9 parts to about 1.5 parts by mass based on acid radical ions;

A protective agent (such as sucrose or trehalose, such as trehalose) of about 19 parts to about 85 parts by mass; and

The mass part is about 0.05 part to about 1 part of surfactant (such as Tween 20 or Tween 80, such as Tween 80).

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.4-5.6, 4.6-5.6 or 4.8-5.2.

In some embodiments, the solid preparation comprises:

The mass parts are about 15 parts to about 25 parts of ADC (such as ADC1);

A buffer (such as a histidine buffer, a citrate buffer, or a succinic acid buffer, such as a succinic acid buffer) of about 0.9 parts to about 1.5 parts by mass based on acid radical ions;

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

The mass part is about 0.1 part to about 0.4 part of surfactant (such as Tween 20 or Tween 80, such as Tween 80).

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.6 or 4.8-5.2.

In some embodiments, the solid preparation comprises:

ADC with about 20 parts by mass (such as ADC1);

Based on acid radical ions, the mass part is about 1.16 parts of buffer (such as histidine buffer, citrate buffer, or succinic acid buffer, such as succinic acid buffer);

About 60 parts by mass of protective agent (such as sucrose or trehalose, such as trehalose); and

The surface active agent (such as Tween 20 or Tween 80, such as Tween 80) is about 0.2 parts by mass.

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.8-5.2.

In some embodiments, the above-mentioned solid preparation also contains a certain amount of Na ⁺ or K ⁺ , which is used to form a buffer system with a buffer to adjust the pH, such as about 0.1 part to about 1 part of Na ⁺ , about 0.2 part to about 0.5 parts of Na ⁺ , or about 0.25 parts to about 0.3 parts of Na ⁺ .

In some embodiments, the solid preparation is a powder.

In some embodiments, the specification of the solid preparation (that is, the dose that can be packed into a container (such as a medicine bottle or a medicine bag)) is about 5 mg to about 500 mg ADC, about 50 mg to about 200 mg ADC, or about 75 mg. About 125mg ADC. In some embodiments, the specifications of the solid preparation are 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 the range between any two numbers, including the end point.

In some embodiments, the solid preparation comprises:

About 5mg to about 500mg ADC;

As acid radical ion, about 2.5mg to about 25mg of buffer;

About 50 mg to about 500 mg of protective agent; and

About 0.25 mg to about 2 mg of surfactant.

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.4-6.0, 4.6-5.6 or 4.8-5.2, such as 5.0.

In some embodiments, the solid preparation comprises:

About 50 mg to about 200 mg of ADC;

As acid radical ion, about 2.5mg to about 20mg of buffer;

About 150 mg to about 450 mg of protective agent; and

About 0.5 mg to about 2 mg of surfactant.

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.4-5.6, 4.6-5.6 or 4.8-5.2, such as 5.0.

In some embodiments, the solid preparation comprises:

About 75 mg to about 125 mg of ADC;

As acid radical ion, about 5mg to about 10mg of buffer;

About 250 mg to about 350 mg of protective agent; and

About 0.5 mg to about 1.5 mg of surfactant.

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.6 or 4.8-5.2, such as 5.0.

In some embodiments, the solid preparation comprises:

About 100mg ADC;

As acid radical ion, about 5.8mg to about 5.9mg of buffer;

About 300 mg of protective agent; and

About 1 mg of surfactant.

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.8-5.2.

In some embodiments, the solid preparation comprises:

About 50mg-about 200mg ADC1;

As acid radical ion, about 2.5mg to about 25mg of succinic acid buffer;

About 150 mg to about 450 mg of trehalose; and

About 0.25 mg to about 2 mg of Tween 80.

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.6 or 4.8-5.2, such as 5.0.

In some embodiments, the solid preparation comprises:

About 75mg to about 125mg ADC1;

As acid radical ion, about 5mg to about 10mg of succinic acid buffer;

About 250 mg to about 350 mg of trehalose; and

About 0.5 mg to about 2 mg of Tween 80.

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.6 or 4.8-5.2, such as 5.0.

In some embodiments, the solid preparation comprises:

About 100mg ADC1;

As acid radical ion, about 5.8mg-5.9mg of succinic acid buffer;

About 300 mg of trehalose; and

About 1mg of Tween 80.

In some embodiments, the pH of the solid preparation (for example, after being dissolved in water or physiological saline) is about 4.6-5.6 or 4.8-5.2, such as 5.0.

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

Some embodiments provide a lyophilized powder, which contains 100 mg ADC1, 5.9 mg succinic acid, 300 mg trehalose, 1 mg polysorbate 80, and sodium hydroxide adjusted to pH 5.0.

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

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

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

In some embodiments, the solid preparation is a lyophilized preparation, which is obtained by lyophilizing a liquid preparation containing an antibody-drug conjugate composed of an antibody, a linker, and a drug module, and the liquid preparation further includes a buffer. One or more of agents, protective agents and surfactants, and solvents, such as water, such as water for injection.

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, and the liquid formulation further includes a buffer, a protective agent, and a surface One or more of the active agents, and a solvent, such as water, such as water for injection. The liquid formulation can be used to prepare the lyophilized formulation described herein.

In some embodiments, the concentration of ADC in the above liquid formulation is about 1 mg/ml to about 100 mg/ml. In some embodiments, the concentration of ADC in the liquid formulation is about 10 mg/ml to about 40 mg/ml. In some embodiments, the concentration of ADC in the liquid formulation is about 15 mg/ml to about 25 mg/ml. In some embodiments, the concentration of ADC in the liquid formulation is about 20 mg/ml.

In some embodiments, in the liquid formulation, the buffer includes one or more of a histidine buffer, a citrate buffer, and a succinic acid buffer. In some embodiments, the buffer is a succinic acid buffer.

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 10 mM.

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 from 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.

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

In some embodiments, the liquid formulation contains citrate 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 concentration of citric acid and its anions. In some embodiments, the liquid formulation contains citrate buffer at a concentration of about 1.8 mg/ml to about 2.0 mg/ml. In some embodiments, the liquid formulation contains citrate buffer at a concentration of about 1.9 mg/ml.

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

In some embodiments, the protective agent is selected from one or two of sucrose and trehalose. In some embodiments, the protective agent is trehalose. In some embodiments, the protective agent is sucrose.

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

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.

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

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

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

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 Tween 20.

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

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.

In some embodiments, the liquid formulation comprises:

ADC of about 1mg/ml to about 100mg/ml;

About 5mM-20mM buffer;

About 50mM-225mM protective agent;

About 0.05 mg/ml to about 0.4 mg/ml surfactant; and

water;

The pH of the liquid formulation is about 4.4-6.0 or 4.6-5.6.

In some embodiments, the liquid formulation comprises:

ADC of about 10mg/ml to about 40mg/ml;

About 5mM-20mM buffer;

About 105mM to 185mM protective agent;

About 0.1 mg/ml to about 0.4 mg/ml surfactant; and

water;

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

In some embodiments, the liquid formulation comprises:

ADC of about 15mg/ml to about 25mg/ml;

About 8mM-13mM buffer;

About 105mM-185mM protective agent;

About 0.1 mg/ml to about 0.4 mg/ml surfactant; and

water;

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

In some embodiments, the liquid formulation comprises:

ADC of about 20mg/ml;

About 10mM buffer;

About 140mM-180mM protective agent;

About 0.2mg/ml of surfactant; and

water;

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

In some embodiments, the liquid formulation comprises:

ADC1 from about 15mg/ml to about 25mg/ml;

About 1 mg/ml to about 2 mg/ml or about 5 mM to about 20 mM succinic acid buffer;

About 30 mg/ml to about 90 mg/ml or about 105 mM to about 185 mM trehalose;

About 0.05 mg/ml to about 0.4 mg/ml Tween 80; and

water;

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

In some embodiments, the liquid formulation comprises:

ADC1 from about 15mg/ml to about 25mg/ml;

About 1 mg/ml to about 2 mg/ml or about 5 mM to about 20 mM succinic acid buffer;

About 50mg/ml-about 70mg/ml or about 140mM-about 180mM trehalose;

About 0.1 mg/ml to about 0.4 mg/ml Tween 80; and

water;

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

In some embodiments, the liquid formulation comprises:

About 20mg/ml ADC1;

About 1.16mg/ml-1.18mg/ml or about 10mM succinic acid buffer;

About 60mg/ml or about 160mM trehalose;

About 0.2mg/ml Tween 80; and

water;

The pH of the liquid formulation is about 5.

In some embodiments, the above-mentioned liquid formulation further contains a certain amount of Na ⁺ or K ⁺ to achieve the pH, such as about 0.1 mg/ml to about 0.8 mg/ml, about 0.2 mg/ml to about 0.4 mg/ml , Or about 0.24mg/ml to about 0.3mg/ml of Na ⁺ . Another example is about 4mM-35mM, about 8mM-17mM, about 10mM-13mM Na ⁺ or K ⁺ .

On the other hand, the present invention also provides a method for preparing the above-mentioned solid preparation which is a freeze-dried preparation, the method includes cooling the liquid preparation to a solid state, and applying a vacuum to remove sublimable substances in vacuum (such as solvent water at room temperature) To obtain a solvent-free lyophilized preparation at room temperature.

In some embodiments, the method includes the following steps:

a) Cool the liquid formulation to -35°C or lower;

b) Heat the preparation treated in step a) to between -20°C and -7°C, and keep it isothermally;

c) Apply vacuum to the formulation treated in step b) at a temperature between -35°C and -8°C.

In some embodiments, the method further includes the following steps:

d) Increase the temperature of the preparation treated in step c) to between 25°C and 40°C; and keep it isothermally.

In some embodiments, the method includes the following steps:

a) Cool the liquid formulation to -35°C or lower at a rate of 0.5°C/min to 3°C/min, and keep it isothermal for at least 120min;

b) Raise the isothermally maintained formulation to between -20°C and -7°C at a rate of 0.5°C/min to 3°C/min, and keep isothermally for at least 120 minutes;

c) Apply a vacuum to the formulation treated in step b) at a temperature between -30°C and -8°C using a pressure between 50mTorr and 200mTorr;

d) Increase 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 keep isothermal for at least 7 hours.

More specifically, the above method includes the following steps: cooling the liquid formulation 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 formulation is heated to a temperature between -20°C and -7°C at a rate of 0.5°C/min to 3°C/min and kept isothermally for at least 120 minutes (annealing step). After that, at a temperature between -30°C and -8°C, the solid water is sublimated (primary drying) using a pressure between 50 mTorr and 200 mTorr. Finally, increase the temperature to between 25°C and 40°C at a rate of 0.5°C/min to 3°C/min and keep isothermally for at least 7 hours (secondary drying) until the residual moisture does not exceed 2% by weight. Get a lyophilized preparation.

In another aspect, the present invention provides the use of the above solid preparation or liquid preparation in the preparation of drugs for cancer. In some embodiments, the cancer is EGFR or HER2 positive cancer

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.

In another aspect, the present invention provides a reconstituted preparation, which is obtained by reconstituting the above-mentioned solid preparation 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.

In some embodiments, the present invention provides a freeze-dried 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 preservatives. 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). In some embodiments, the solvent used in the formulation is water for injection. In some embodiments, after preparation, 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, pH 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, an isotonic solution (such as 0.9% NaCl solution for injection) is used to dilute the solution containing 20 mg/mL ADC (such as ADC1, ADC2, or ADC3), and the diluted solution is then used for intravenous infusion. Administration. The solid preparation of the present invention can effectively solve the problems of ADC instability and easy aggregation, and has excellent long-term stability and accelerated stability. Further, when the solid formulation is a lyophilized formulation, the lyophilized formulation is lyophilized by lyophilizing the liquid formulation containing the antibody-drug conjugate to remove moisture in the ADC at a low temperature, so that the ADC remains stable during storage. The addition of protective agent can effectively protect ADC during the freeze-drying process. The buffer is used to maintain the stable pH of the formulation and reduce the degradation of ADC. The addition of surfactants can reduce the surface tension after the lyophilized preparation is reconstituted, so that the ADC remains stable, and it is not easy to form aggregates or particles.

Other features and advantages of the present invention will be described in detail in the following specific embodiments.

Description of the drawings

Figure 1 shows that the freeze-dried sample of Example 2 is placed at a temperature of 50° C. for 10 days and analyzed by size exclusion chromatography (SEC) to obtain the percentage of the main peak substance.

Figure 2 shows that the freeze-dried sample of Example 2 was placed at 50° C. for 10 days, and reversed-phase chromatography (RP) was used to determine the drug coupling ratio.

Figure 3 shows the use of SEC to analyze the change of the main peak percentage of the liquid preparation sample of Example 3 under light conditions.

Figure 4 shows the use of SEC to analyze the change of the main peak percentage of the liquid preparation sample of Example 3 under high temperature conditions.

Figure 5 shows the use of SEC to analyze the change in the percentage of the main peak of the freeze-dried preparation sample of Example 3 under light conditions.

Figure 6 shows the change in peak percentage of the liquid preparation sample of Example 4 under high temperature conditions using the SEC method.

Figure 7 shows the use of SEC to analyze the change in peak percentage of the freeze-dried preparation sample of Example 4 under high temperature conditions.

Figure 8 shows the use of SEC to analyze the change in the percentage of the main peak of the freeze-dried preparation sample of Example 5 under high temperature conditions.

Figure 9 shows the use of SEC to analyze the change of the main peak percentage of the liquid formulation sample of Example 6 under high temperature conditions.

Figure 10 shows the use of SEC to analyze the change in the percentage of the main peak of the freeze-dried preparation sample of Example 6 under high temperature conditions.

Figure 11 shows the use of SEC to analyze the change in the percentage of the main peak of the freeze-dried preparation sample of Example 8 under high temperature conditions.

Figure 12 shows the use of HIC to analyze the change in the percentage of unconjugated antibody of the freeze-dried preparation sample of Example 8 under high temperature conditions.

Figure 13 shows the use of SEC to analyze the change of the main peak percentage of the reconstituted preparation sample of Example 10 under high temperature conditions.

Detailed ways

As used herein, the terms "comprising", "comprising" or "containing" or their grammatical variants are intended to mean that the compositions, methods, etc. include the listed elements, but do not exclude others. "Consisting essentially of" or its grammatical variants when used to define compositions and methods shall mean excluding other elements that have any fundamental impact on the combination used for the intended purpose, but does not exclude that it will not substantially affect the combination The characteristic element of a thing or method. "Consisting of" or its grammatical variants shall mean excluding elements not specifically listed. The embodiments defined by each of these transition terms are within the scope of the present invention. For example, when a formulation formulation is described as containing ingredients A, B, and C, a formulation formulation consisting essentially of A, B and C and a formulation formulation consisting of A, B and C are independently within the scope of the present invention .

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

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

The term "acceptable" or "pharmaceutically acceptable" as used herein in relation to a formulation, composition or ingredient means that it does not have a lasting harmful effect or does not eliminate the general health of the subject being treated The biological activity or properties of the compound, and it is relatively non-toxic.

ADC can form a wide variety of pharmaceutically acceptable salts, including but not limited to: acid addition salts formed with organic acids, such organic acids including but not limited to aliphatic monocarboxylic and dicarboxylic acids, phenyl substituted Alkanoic acid, hydroxyalkanoic acid, alkanedioic acid, aromatic acid, aliphatic and aromatic sulfonic acid, amino acid, etc., such as acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, propylene Diacid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, etc.; formed by reaction with inorganic acids Acid addition salts, such inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, etc.; and metal ions (for example, alkali metal ions (such as sodium or Potassium), alkaline earth metal ions (e.g. calcium or magnesium) or aluminum ions) or salts formed with organic bases such as diethanolamine, triethanolamine, N-methylglucamine and the like.

The term "individual", "subject" or "patient" as used herein refers to an animal that is the target of treatment, observation, or experiment. For example only, the subject may be (but not limited to) a mammal, including (but not limited to) a human.

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

Through monoclonal antibody technology, antibodies can specifically bind to antigens, such as tumor-specific antigens. The tumor antigen mentioned herein refers to a target expressed in tumor cells. Tumor antigens can be used to identify tumor cells, can also become a potential indicator of tumor treatment, or become a target of tumor treatment. Therefore, the selection of specific antibodies mainly depends on the type of disease and the cells and tissues that are the target.

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 and the like.

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

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. Antibody BAT-C contains 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.

In other embodiments, the antibody is another kind of anti-EGFR antibody, including but not limited to: Cetuximab, which is a human-mouse chimeric monoclonal antibody against EGFR; Panitumumab, which is a A fully humanized monoclonal antibody; Nimotuzumab. EGFR is overexpressed in many tumor tissues, such as metastatic colorectal cancer and head and neck cancer.

In some embodiments, the antibody is an anti-HER2 antibody, which can specifically act on human epidermal growth factor receptor 2. The anti-HER2 antibody may be modified, such as one or more amino acid sequence changes, increases or decreases, to achieve corresponding purposes, such as enhancing antibody-dependent cell-mediated cytotoxicity. In some embodiments of the 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, the light chain of which is SEQ ID NO: 5, and the heavy chain of which is SEQ ID NO: 6.

The sequence corresponding to each sequence number is shown in Table 1.

Table 1

In some embodiments of the present invention, the binding number of the small molecule drug corresponding to an antibody in the antibody-drug conjugate, that is, the drug binding number of the antibody, or called the drug-antibody coupling ratio (DAR), is selected from 2 To 8, or 2, 4, 6, or 8; when considering the average number of small molecule drugs bound, that is, the average number of antibody drug binding, or called the average drug-antibody coupling ratio (average DAR), its value It is selected from about 2 to about 8, or about 3 to about 5, or about 3.3 to about 3.7, or about 3.5. In some embodiments, the small molecule drug is coupled to the sulfhydryl group formed after reduction of the disulfide bond formed between the cysteines contained in the antibody (such as antibody BAT-C, antibody BAT-B or antibody BAT-A). Therefore, the combined number of drugs is selected from 2, 4, 6, and 8.

The drug modules suitable for coupling linkers are various known or foreseeable cytotoxic agents in the art, including anti-mitotic cytotoxic drugs and DNA lytic cytotoxic drugs. In some embodiments, the drug is an anti-mitotic cytotoxic drug. Such drugs may include, for example, maytansinol, maytansinol analogs and derivatives, auristatin analogs, and the like.

Maytansinol analogs and derivatives may include maytansine and maytansine analogs, which can be isolated from natural sources according to known methods and manufactured using biotechnology (see for example: Yu et al., 99PNAS 7968-7973 (2002)) Or synthetically prepared according to known methods (see: Cassady et al., Chem.Pharm.Bull.52(1)1-26(2004)). Depending on the type of linker, many locations on maytansinol can be used as attachment locations. For example, for forming an ester bond, it is suitable 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.

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

N-methyl-L-alanyl maytansinol

In still other embodiments, maytansinol or maytansinol analogs and derivative drug modules have the structure disclosed in patent 201310081867.X.

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 can 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 modules have the following structure:

The linkers suitable for the present invention include cleavable linkers and non-cleavable linkers. Among them, the cleavable linker usually has a special sensitive structure. This linker is stable in a normal environment, and can be broken in a tumor environment to release small molecule drugs for therapeutic effects. Common cleavable linkers include protease-sensitive linkers, which contain dipeptide bonds that can be recognized and broken by proteases, most commonly val-cit; there are also acid-sensitive linkers. Most are linkers containing hydrazone bonds; the other is glutathione-sensitive linkers, which usually contain glutathione reducible disulfide bonds. The non-cleavable linker remains intact after being metabolized in the cell. Antibody-drug conjugates containing such linkers need to undergo lysosomal degradation to release cytotoxic molecules. Compared with cleavable linkers, non-cleavable linkers have good stability in the circulatory system. Non-breakable linkers include MC, SMCC, MPA, MPEO, etc. In some embodiments, the linker is a non-cleavable linker.

In some embodiments of the present invention, the linker is selected from molecular fragments having the following structure:

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

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

In some embodiments, a buffer is used to maintain a stable pH of the formulation and reduce degradation of ADC. In the process of freeze-drying the liquid preparation to form a freeze-dried preparation, it needs to be frozen at a low temperature, and then subjected to a vacuum treatment to sublime the moisture in the freezing, and then restore the temperature and air pressure to obtain the freeze-dried preparation. During the process, as the water changes from liquid to solid, it may cause the precipitation of the buffer and the change of pH, thereby affecting the stability of ADC. Therefore, it is very important to provide a suitable buffer, its suitable concentration, and a suitable pH range for preparing stable, non-degradable and aggregated ADC freeze-dried formulations.

For the selection of buffer types, it is preferred to choose buffers that have good buffering capacity at the target pH and have little change in pH during the freezing process. In some embodiments, the buffer is selected from the group consisting of histidine buffer, citrate buffer and succinic acid buffer, or a combination of any two of the above buffers, such as histidine buffer and succinic acid The buffer is a mixed buffer or a buffer of the above three combinations. In some embodiments, the buffer is a succinic acid buffer.

For the concentration of the buffering agent, if the concentration is too low, the buffering capacity will be weak, and it may not have obvious protective effect; while the concentration is too high, it is easy to precipitate during the freezing process, which affects the pH value in the solution, and then affects The stability of ADC. Therefore, in the present invention, the concentration of the buffer of the liquid preparation before lyophilization is preferably between about 5 mM and about 20 mM, more preferably at a concentration of about 8 mM to about 13 mM, and particularly preferably at a concentration of about 10 mM.

For the pH value in the aqueous solvent, it can directly affect the stability between small molecule drugs and antibodies, which is a key parameter. In order to stabilize the antibody, small molecule drug and antibody, 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, and more preferably between about 4.8 and about 5.2. , Particularly preferably about 5.0.

In some embodiments, a protective agent is used to protect the ADC in the liquid formulation. The addition of protective agent can change the collapse temperature, provide freeze-drying protection for ADC during the freeze-drying process, and reduce the influence of crystal water on ADC. In some embodiments, the protective agent is selected from sugars, in some embodiments it is non-reducing sugar and/or sorbitol, and in some embodiments it is selected from sucrose and/or trehalose. In some embodiments, it is trehalose. In the pre-freezing process of the product, the appropriate non-reducing sugar will make the product form a glassy state during the process, including the crystallizable components. On the one hand, it eliminates the possibility of crystallization of the buffer components, thereby eliminating pH shifts. The role of protein protection, on the other hand, the formed glass state also plays a key role in the stability of the protein at low temperature and loss of water, but the infinitely increased concentration of reducing sugars will cause the first drying time to prolong or fail during the freeze drying process And the water cannot be removed after secondary drying, so the concentration of reducing sugar should be controlled to maintain the stability of the protein and form a good "cake" shape during the freeze-drying process.

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

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

In some embodiments, the addition of a surfactant is used to reduce the surface tension and limit the polymerization of the 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.

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

In some embodiments, the liquid formulation uses a pharmaceutically acceptable solvent as a carrier, such as sterile pharmaceutical grade water for injection or saline, sterile water for injection, bacteriostatic water for injection and other water for injection. In some embodiments, when the components in the liquid preparation before lyophilization, such as ADC, buffer, stabilizer, and/or surfactant, are formulated into a solution with a specific concentration using water for injection, the calculated amount is calculated according to the prescription. For the required amount of solids, add a certain amount of water for injection to dissolve after mixing; if there is a viscous liquid in the prescription, calculate the required amount of viscous liquid according to the prescription amount, and add a certain amount of water for injection to disperse after mixing; Mix the above solutions and adjust the pH. In some embodiments, each component is separately dissolved and dispersed with water for injection, and then the resulting solution is mixed to adjust the concentration and pH value.

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, especially 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 applying the antibody-drug conjugate solid preparation of the present invention, before administering the drug to the subject, the antibody-drug conjugate solid preparation is dissolved in a pharmaceutically acceptable solvent, and the reconstituted preparation is obtained by reconstitution. Do the drug. Solvents include sterile pharmaceutical grade water for injection or saline, sterile water for injection, and antibacterial water for injection.

In one aspect, provided herein is a method of treating cancer, comprising administering an effective amount of a reconstituted preparation of an antibody-drug conjugate to a patient in need of treatment. In some embodiments, it is obtained by reconstituting the solid preparation of any one of the foregoing embodiments in a pharmaceutically acceptable solvent (sterile pharmaceutical grade water for injection or saline, sterile water for injection, bacteriostatic water for injection, etc.) Reconstituted preparations. In some embodiments, the ADC concentration of the reconstituted preparation is 1 mg/ml-100 mg/ml, and the buffer is one of histidine buffer, citric acid buffer and succinic acid buffer at a concentration of 5mM-20mM Or more, the protective agent is sucrose and/or trehalose at a concentration of 10mM-250mM, the surfactant is Tween 80 and/or Tween 20 at a concentration of 0.05mg/ml-0.4mg/ml, and the pH of the reconstituted preparation is 4.4 -6.0. In some embodiments, the ADC concentration of the reconstituted preparation is 15mg/ml-25mg/ml, the buffer is a succinic acid buffer with a concentration of 8mM-13mM, the protective agent is sucrose or trehalose with a concentration of 105mM-185mM, and the surface activity The dosage is 0.1mg/ml-0.4mg/ml Tween 80, and the pH of the reconstituted preparation is 4.8-5.4. In some embodiments, the ADC concentration of the reconstituted formulation 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, and the surfactant is about 0.2 mg/ml The pH of the reconstituted Tween 80 is about 4.8-5.4, such as about 5.0. In some embodiments, the reconstituted formulation contains ADC1 at a concentration of about 20 mg/ml, succinic acid buffer at a concentration of about 1.16 mg/ml to about 1.18 mg/ml, trehalose at about 60 mg/ml, and trehalose at a concentration of about 0.2 mg/ml. Tween 80, and Na ⁺ , the pH of the reconstituted preparation is about 4.8-5.4, such as about 5.0.

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

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 ADC unit dose 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.

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

In some embodiments, the patient is a HER2-positive advanced solid tumor patient.

In some embodiments, stability experiments at low temperature, room temperature, and high temperature are performed on the prepared solid preparation. 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 keep stable. Compared with month 0, ADC has a very small amount of protein polymerization; the average DAR remains stable; there is no sign of protein degradation; after reconstitution with sterile water for injection, it also maintains good performance in terms of insoluble particles.

In some embodiments, 0.9% normal saline is used to compatibly dilute the reconstituted preparation after reconstitution. The ADC solution after compatibility and dilution shows good stability when placed at room temperature for 1 hour, 2 hours, 4 hours, 8 hours or even 24 hours. Size exclusion chromatography (SEC-HPLC, or SEC) determined that the average percentage of the main peak remained greater than 97%, and it also maintained good performance in terms of insoluble particles.

In order to develop a stable anti-HER2 ADC formulation that is not prone to aggregation, the present invention provides an antibody-drug conjugate solid formulation. In some embodiments, the solid preparation can be prepared by lyophilizing and removing moisture from ADC at low temperature, so that ADC remains stable during storage. The addition of protective agent can effectively protect ADC during the freeze-drying process. The buffer is used to maintain the stable pH of the formulation and reduce the degradation of ADC. The addition of surfactants can reduce the surface tension after the lyophilized preparation is reconstituted, so that the ADC remains stable, and it is not easy to form aggregates or particles.

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

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

In order to evaluate the stability of ADCs in liquid preparations and freeze-dried preparations, the identification, purity, and efficacy of ADCs are usually tested. Purity testing includes, but is not limited to, SDS-PAGE, CESDS, isoelectric focusing, immunoelectrophoresis, western blot, 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.

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

After the antibody is diluted, it is incompletely reduced with dithiothreitol (DTT). After incubation, it is eluted and exchanged with Sephadex G 25 resin to determine the number of sulfhydryl groups of the antibody. Add the drug-linker to the reduced antibody, stir at room temperature, and add Cysteine is continuously stirred to obtain; the molar equivalent ratio of the drug-linker to the number of sulfhydryl groups of the antibody is (1-2):1. In some embodiments, the molar equivalent of the drug-linker to the number of sulfhydryl groups of the antibody The ratio is 1.5:1.

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

In some embodiments, after the above-mentioned reaction mixture is ultrafiltered, it is purified with a gel filtration column equilibrated with G25 in a phosphate buffer with a pH value of 7.4±0.1.

In some embodiments, the prepared anti-HER2-antibody drug conjugate is filtered with a 0.22 micron filter and stored at 80±5°C.

In some embodiments, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) is used to determine the number of unreacted sulfhydryl groups of the drug-linker to obtain the drug/antibody ratio.

In some embodiments, the DAR in the anti-HER2 antibody drug conjugate prepared by the method described above is 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.

In some embodiments, the concentration of the drug-linker can be measured by ultraviolet 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.

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 incubating at 37°C for 60 minutes, the solution A (50mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 6.5) was eluted and exchanged with Sephadex G25 resin. The sulfhydryl antibody value is determined by measuring the absorbance, and the sulfhydryl concentration is determined by the reaction product of the sulfhydryl and DTNB, and then measuring the absorbance at 412nm. The concentration of diethylformamide (DMA) during the coupling reaction was 10%. The ratio of the number of sulfhydryl groups of the drug-linker to the anti-HER2 antibody is 1.5:1.0 (molar equivalent). The drug-linker was added to the reduced anti-HER2 antibody, and after stirring for 3 hours at room temperature, 5mM cysteine was added and stirring continued for 1 hour to obtain.

In some embodiments, the ADC is ADC1. Where ADC1 has the following structure

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

In some embodiments, the ADC is ADC2. Among them, ADC2 has the following structure:

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

In some embodiments, the ADC is ADC3. Among them, ADC3 has the following structure

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

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

In some embodiments, ADC3 uses Pertuzumab and is prepared according to the method used by ADC1.

The lyophilized liquid preparation of the present invention is prepared by conventional methods and technical means in the field.

Example 1

Preparation of ADC1

The 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 incubating for 60 minutes at 37°C, it was exchanged with solution A (50mM potassium phosphate, 50mM NaCl and 2mM EDTA, pH 6.5) with Sephadex G 25 resin. The sulfhydryl antibody value is determined by measuring the absorbance, and the sulfhydryl concentration is determined by the reaction product of the sulfhydryl and DTNB, and then measuring the absorbance at 412nm. The concentration of diethylformamide (DMA) during the coupling reaction was 10%. Add the drug-linker intermediate (see patent 201310081867.X for synthesis method) to the reduced anti-HER2 antibody at a ratio of 1.5:1.0 (molar equivalent) of the number of sulfhydryl groups of the drug-linker intermediate to the anti-HER2 antibody After stirring for 3 hours at room temperature, add 5mM cysteine and continue stirring for 1 hour. After ultrafiltration, the reaction mixture was purified with a gel filtration column, and then filtered with a 0.22 μm filter to obtain ADC1. Using DTNB to measure the number of unreacted sulfhydryl groups, the drug/antibody ratio is about 3.5/1, that is, the DAR of ADC1 is about 3.5.

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

Example 2

Research on the types of protective agents and buffers.

There are 10 prescriptions in this example, and the protective agent, buffer, and pH are different. The composition of the liquid preparation of the antibody-drug conjugate lyophilized preparation is shown in Table 2:

Table 2

The freeze-dried preparations obtained by freeze-drying the liquid preparations made by the above 10 prescriptions were tested at a high temperature of 50°C. On days 0, 2, 4, 6, and 10, 5 ml of sterile water for injection was used to reconstitute the lyophilized preparations of various prescriptions to obtain reconstituted preparations. Perform ADC stability analysis on the reconstituted reconstituted preparations, including analysis using size exclusion chromatography (SEC) to determine the extent of ADC aggregation and degradation in each prescription. The results are shown in Figure 1; and, reversed-phase chromatography is used (RP) The drug coupling ratio was measured, and the result is shown in Figure 2. Because the drug is hydrophobic, it can cause changes in the polarity of the light and heavy chains after being coupled to the antibody. The reversed phase method can be used to separate the ratio of the light and heavy chains with and without the drug, so as to calculate the ADC drug coupling Combination ratio, this method can determine the dropping position and ratio of the drug through comparison.

It can be seen from Figure 1 that, compared with non-reducing sugar preparations, the main peak of polyhydric alcohols is significantly lower than other prescription preparations. Therefore, choosing non-reducing sugars as protective agents plays an important role in preparing stable ADC freeze-dried preparations, especially sucrose and trehalose. In addition, there is no obvious trend among different buffers in the freeze-dried samples, and the purity changes are not obvious after high temperature treatment. In the freeze-dried preparation, the sample has a low stress response to high temperature, indicating that the ADC freeze-dried preparation can remain stable in buffers such as histidine buffer, citrate buffer, and succinic acid buffer.

As can be seen in Figure 2, the number of drug couplings does not have a significant effect on the number of drug couplings after high temperature treatment in each formulation.

Example 3

Further research on the types of protective agents and buffers.

There are 4 prescriptions in this example, the pH of the liquid preparation is all 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:

table 3

The liquid preparation made by the above 4 prescriptions was tested with 4000Lx intensity light and 50℃ high temperature. The stability of the liquid formulation was analyzed on days 0, 1, 3, 5, and 10, and the purity of ADC was determined by SEC. The results are shown in Figures 3 and 4.

The liquid preparations prepared by the above 4 prescriptions were freeze-dried to make freeze-dried preparations, and the test was conducted at a high temperature of 60°C. The stability of the freeze-dried preparation was analyzed at 0, 5, 10, 15 and 40 days, and the purity of ADC was determined by SEC. The results are shown in Figure 5.

It can be further seen from Figures 3 and 4 that citrate buffer and histidine buffer have weaker restriction on protein polymerization than succinate buffer; succinate buffer has a protective effect on ADC stability in liquid formulations It is better than citrate buffer and histidine buffer; non-reducing sugar has a certain protective effect on ADC, among which trehalose is better than sucrose.

It can be seen from Figure 5 that the succinic acid buffer in the freeze-dried preparation is also better than citrate buffer and histidine buffer in protecting ADC stability; similarly, trehalose is better than sucrose. In comparison, prescription 4 has the most excellent restriction on protein aggregation.

Example 4

Buffer concentration study

The buffer can stabilize the pH 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 example, there are 4 prescriptions, each of which has different buffer concentrations, and sodium hydroxide is used to control the pH of the liquid preparation at about 5.0. The composition is shown in Table 4.

Table 4

The above four prescriptions are formulated into a liquid preparation before lyophilization, and the lyophilized preparation is obtained after lyophilization. Incubate the above liquid formulation at a high temperature of 50°C, and conduct stability analysis of the liquid formulation at 0, 1, 3, 5 and 10 days. SEC was used to determine the purity of ADC, and the results are shown in Figure 6.

Incubate the freeze-dried preparation at a high temperature of 60°C, and analyze the stability of the freeze-dried preparation at 0, 5, 10, 15 and 30 days. SEC was used to determine the purity of ADC, and the results are shown in Figure 7.

The results show that the concentration 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, when the concentration is high, there will be a significant pH shift; while the low concentration has a weaker ability to maintain a pH stable system. Therefore, a buffer with a concentration of 10 mM is particularly preferred in the present invention.

Example 5

Study on the concentration of protective agents

There are 4 prescriptions in this example, and the difference between the prescriptions is the concentration of the protective agent. The composition is shown in Table 5. Sodium hydroxide was used to control the pH of the solution preparations at about 5.0.

table 5

The above four prescriptions are formulated into a liquid preparation before lyophilization, and the lyophilized preparation is obtained after lyophilization. The lyophilized preparation was incubated at a high temperature of 50°C, and the stability of the lyophilized preparation was analyzed at 0, 5, 15, 30 and 60 days. SEC was used to determine the purity of ADC, and the results are shown in Figure 8.

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

Example 6

Research on pH Value of Aqueous Solution

There are 7 prescriptions in this example, and the composition of each prescription is shown in Table 6.

Table 6

The above 7 prescriptions were formulated into liquid preparations before lyophilization, the liquid preparations were placed in a high temperature test at 50°C, and stability analysis of the liquid preparations was carried out at 0 and 3 days. SEC was used to determine the purity of ADC, and the results are shown in Figure 9; the hydrophobic chromatography (HIC) method was used to determine the content of uncoupled protein in ADC, and the results are shown in Figure 10. Because the pH and other parameters will affect the stability between the drug molecule and the antibody, showing the phenomenon of drug shedding, so the stability of the drug can be seen by measuring the content of unconjugated antibody in the sample.

The results showed that the protein showed good stability when the pH of the solution was between 4.6 and 5.6. Further analysis results show that as the pH in the solution increases, the ability of protein polymerization restriction is stronger, while the content of unconjugated antibody increases, and the rate of change tends to be flat when the pH is 4.8 to 5.2. Comparing the effects of changes in pH on protein polymerization and unconjugated antibodies, taking into account the requirements of protein polymerization restrictions 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, more preferably 5.0.

Example 7

Surfactant research

Surfactants often play an important role in limiting the polymerization of proteins in freeze-dried formulations, such as limiting the possible polymerization of proteins during the freezing process on the ice-water interface, and also limiting the production of freeze-dried proteins during the reconstitution process. Possible polymerization. Taken together, surfactants can minimize the unpredictable polymerization of proteins during the process.

In this embodiment, there are 4 prescriptions, and the composition of each prescription is shown in Table 7. Sodium hydroxide is used to control the pH of the liquid preparation at about 5.0.

Table 7

The above 4 prescriptions were formulated into liquid preparations before freeze-drying, and the liquid preparations were subjected to repeated freezing and thawing 5 times. The sample of the liquid preparation repeatedly freeze-thaw is judged by the insoluble particles, and the sample is reconstituted after lyophilization, and the reconstitution time of the sample and the insoluble particles of the reconstituted sample are tested to judge the experiment.

The insoluble particles of the repeatedly freeze-thaw samples are shown in Table 8 below, and the insoluble particles of the freeze-dried samples are shown in Table 9 below after reconstitution.

Table 8

Table 9

The results show that the addition of surfactants has a significant effect on the stability of ADC, and the samples without surfactants show more polymerization tendency. Both Tween 20 and Tween 80 showed the ability to restrict protein polymerization, and there was little difference between the two. Moreover, for different concentrations of surfactants, there is no significant difference in their effects on the stability of ADC. Considering that too much surfactant is not conducive to the application of the formulation, 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.

Example 8

ADC concentration study

In this embodiment, there are 3 prescriptions, and the composition of each prescription is shown in Table 10. Sodium hydroxide is used to control the pH value in the liquid preparation at about 5.0.

Table 10

The above three prescriptions are formulated into a liquid preparation before lyophilization, and the lyophilized preparation is obtained after lyophilization. The prepared lyophilized preparation was stored at 40°C. The freeze-dried preparations were reconstituted after 0, 1, and 2 months, and their stability was analyzed. SEC was used to determine the purity of ADC, and the result is shown in Figure 11; the HIC method was used to determine the content of uncoupled protein in ADC, and the result is shown in Figure 12.

The results show that the ADC concentration is between 15mg/ml and 25mg/ml, and there is no obvious difference in the effect of high temperature on its stability.

Example 9

Long-term stability and accelerated stability studies

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

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

The freeze-dried formulation samples were evaluated with long-term and accelerated stability procedures. Lyophilized preparation 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, and At 2, 3 and 6 months, the lyophilized preparation was reconstituted. After reconstitution, each composition and concentration were 20 mg/mL ADC, 10 mM succinate buffer, 160 mM trehalose, and 0.2 mg/ml Tween 80. After reconstruction, stability analysis was performed. Tests include SEC, HIC, insoluble particles, UV/Vis, appearance, color and turbidity after reconstitution.

After storage at 5±3°C and 25±2°C for at least 6 months, all samples remain stable through all test methods. After storage at 5±3°C for at least 24 months, all samples remain stable through all test methods. Compared with the beginning of the study, samples stored at 5±3°C did not show significant changes at least 24 months, and samples stored at 25±2°C did not show significant changes at least 6 months.

Specifically, regarding protein aggregation, SEC analysis showed that the percentage of aggregates was maintained at approximately 0.2% for samples that had passed at least 24 months at 5±3°C and samples that had passed at least 6 months at 25±2°C. Compared with 0 months of storage, after at least 24 months, for the samples stored at 5±3℃, the change of the main material isotype is within 0.2%, and the same insoluble particles also maintain good performance.

In addition, after at least 24 months at 5°C and at least 6 months at 25°C, the average DAR, DAR, and free drug remained almost constant. There is no sign of degradation after at least 24 months at 5°C and at least 6 months at 25°C. The most important thing is that, through the cytotoxicity bioassay, the biological function of ADC can be maintained at 5°C for at least 24 months and at 25°C for at least 6 months. Therefore, in the formulation of the present invention, the lyophilized formulation has acceptable stability for ADC applications.

The example data of the stability program of the freeze-dried preparation stored at 5±3°C for 6 months is shown in Table 11a; the example data of the stability program of the freeze-dried preparation stored at 5±3°C for 24 months is shown in Table 11b Example data of the stability program of the freeze-dried formulation stored at 25±2°C for 6 months are shown in Table 12.

Table 11a

Table 11b

Table 12

Example 10

Study on compatibility and stability

The compatibility stability of the freeze-dried formulation prepared in Example 9 was studied. First, first reconstitute the lyophilized preparation with sterile water for injection to obtain a reconstituted preparation with an ADC concentration of 20 mg/ml. Then the reconstituted preparation was diluted with 0.9% normal saline, and diluted into solutions with concentrations of 0.5 mg/ml and 1 mg/ml respectively. Place it at room temperature, and at 0, 1, 2, 4, 8 and 24 hours, use SEC and measure insoluble particles for stability evaluation.

The SEC results are shown in Figure 13. The results showed that the average percentage of the main peak of the samples kept at room temperature for 24 hours remained greater than 97%, and there was no big difference between the 1 mg/mL sample and the 0.5 mg/ml sample. The 0.9% saline diluent did not cause protein aggregation.

The results of insoluble particles inspection showed that whether it was a 0.5mg/ml or 1mg/ml sample, diluted with 0.9% saline and stored at room temperature for 24 hours, it did not adversely affect the insoluble particles of the sample. The results are shown in Table 13. . This indicates that saline can be used as a diluent for the freeze-dried preparation of the present invention.

Table 13

Example 11

In this example, the ADC2 freeze-dried formulation was prepared using the following recipe.

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

Example 12

In this example, the ADC3 freeze-dried formulation was prepared using the following recipe.

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

Example 13

In this example, a solid preparation containing the following prescription composition (in parts by mass) was prepared:

Example 14

In this example, a solid preparation containing the following prescription composition (in parts by mass) was prepared:

Example 15

In this example, a solid preparation containing the following prescription composition (in parts by mass) was prepared:

Example 16

Preparation of the reconstituted preparation: Place the three prescription freeze-dried preparations obtained in Example 8 in a vial, use a sterile syringe to slowly inject the sterile water for injection into the vial, and gently rotate the vial until it is complete Dissolve, do not shake vigorously, and configure into reconstituted preparations with ADC1 concentrations of 15mg/ml, 25mg/ml, and 20mg/ml respectively.

Example 17

Preparation of injection preparation: Take 250ml 0.9% sodium chloride infusion bag/bottle for injection, calculate the required reconstituted preparation in Example 16 according to the dosage of 3.6mg/kg, and use a sterile syringe to remove the reconstituted preparation from Xilin Draw the corresponding volume from the bottle, add it to the infusion bag/bottle, gently invert the infusion bag/bottle, mix the solution, repeat it many times, avoid air bubbles, and obtain the diluted injection preparation.

Example 18

The freeze-drying cycle of the freeze-dried preparation of the present invention can be performed as described below.

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

In one embodiment, the formulated liquid formulation is cooled to -35°C at a rate of 1°C/min; the temperature is kept isothermal for at least 120 minutes. Then, the temperature is increased to -7°C at a rate of 3°C/min; the isothermal temperature is maintained for at least 120 minutes; then, a vacuum is applied at a temperature of -8°C with a pressure of 200 mTorr. Finally, increase the temperature to 30°C at a rate of 1°C/min; keep isothermal for at least 7 hours. When the residual moisture in the formulation does not exceed 2% by weight, the freeze-dried formulation is prepared.

Example 19

The preparation of the solution, the components of the prescription and the dosage of each bottle are as follows:

The above components are dissolved in water for injection, and the pH is adjusted to 5.0 with sodium hydroxide.

Add the above solution to a vial, add 5ml to each vial, and lyophilize using the method in Example 18 to obtain a lyophilized solid preparation. The components and contents of each solid preparation are as follows:

When using a solid preparation, the method of Example 16 was used to formulate the solid preparation with water for injection into a reconstituted preparation with an ADC1 concentration of 20 mg/ml.

In many of the above embodiments, in the formulation involving sodium ions, sodium ions and succinic acid buffers form a buffer system.

Example 20

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

The prepared injection preparation is applied to patients with HER2-positive advanced solid tumors by intravenous infusion, and the administration cycle is once every three weeks.

Example 21 Clinical study

In this study, ADC1 was used to treat patients with HER2-positive advanced solid tumors 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 every 3 weeks. The pharmacokinetic parameters (PK) after the first administration are shown in Table 14:

Table 14

Dose (mg/kg) 1.2 2.4 3.6 4.8 6.0 C _max (μg/mL) 27.2 38.5 76.6 92.7 140 AUC _(0-t) (μg/mL·h) 1190 2417 3930 5190 6450

The PK after the 4th administration is shown in Table 15:

Table 15

Dose (mg/kg) 1.2 2.4 3.6 4.8 6.0 C _max (μg/mL) 22.6 36.6 70.0 95.0 102.7 AUC _(0-t) (μg/mL·h) 1226 2177 4066 6030 6121

A total of 29 subjects joined the study, 28 of whom had imaging results. Preliminary evaluation of efficacy 10 months after treatment found that 1 patient (3.7%) had complete remission (CR), 5 (18.5%) patients had partial remission (PR), and 5 (18.5%) patients had stable disease.

Adverse events during treatment (TEAEs)

The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above-mentioned embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, All should be considered as the scope of this specification. The above-mentioned embodiments only express several implementation modes of the present invention, and their description is relatively specific and detailed, but they should not be understood as a limitation on the scope of the invention patent. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of the present invention, several adjustments and improvements can be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be subject to the appended claims.

Claims (12)

An antibody-drug conjugate solid preparation, wherein 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 It also contains one or more of buffers, protective agents and surfactants. The solid preparation of claim 1, wherein the antibody is a humanized monoclonal antibody; Preferably, the antibody is an anti-EGFR antibody or an anti-HER2 antibody; more preferably, the antibody is an anti-HER2 antibody; Preferably, the drug binding number of the antibody is selected from 2-8, preferably 2, 4, 6 or 8; Preferably, the average drug binding number of the antibody is selected from 2-8. The solid preparation of claim 1 or 2, wherein the drug module is selected from one or more of N-methyl-L-alanyl maytansinol, DM1, DM4, MMAE and MMAF . The solid preparation according to any one of claims 1 to 3, wherein the linker is selected from SMCC, MC, MPA or MPEO. The solid preparation of claim 1, wherein the antibody-drug conjugate has the following structure:

among them, Ab is an antibody; p is 2-8. The solid preparation according to any one of claims 1 to 5, wherein the buffer in the solid preparation comprises one of histidine buffer, citrate buffer and succinic acid buffer or Multiple The protective agent in the solid preparation is selected from one or two of sucrose and trehalose; The surfactant in the solid preparation is selected from one or two of Tween 20 and Tween 80. The solid preparation according to any one of claims 1-6, wherein the mass parts of the antibody-drug conjugate in the solid preparation are 1 part to 100 parts; The mass parts of the buffer in the solid preparation are 0.6 to 2.4 parts based on the acid radical ion of the buffer; The mass parts of the protective agent in the solid preparation are 4 parts to 95 parts; The mass parts of the surfactant in the solid preparation is 0.05 parts to 0.4 parts. The solid preparation according to any one of claims 1 to 6, wherein the solid preparation is a lyophilized preparation, which is obtained by lyophilizing a liquid preparation containing the antibody-drug conjugate, and The liquid formulation also contains one or more of the buffer, protective agent and surfactant. The solid preparation according to claim 8, wherein in the liquid preparation, the concentration of the antibody-drug conjugate is 1 mg/ml-100 mg/ml; In the liquid preparation, the concentration of the buffer is 5mM-20mM; the pH of the liquid preparation is in the range of 4.4-6.0; The concentration of the protective agent in the liquid preparation is 10mM-250mM; The concentration of the surfactant in the liquid preparation is 0.05 mg/ml-0.4 mg/ml. A method for preparing the solid preparation according to claim 8 or 9, the method comprising the following steps: a) Cool the liquid formulation to -35°C or lower and keep it isothermally; b) Heat the preparation treated in step a) to between -20°C and -7°C, and keep it isothermally; c) Apply vacuum to the preparation processed in step b) at a temperature between -35°C and -8°C; d) Increase the temperature of the preparation treated in step c) to between 25°C and 40°C; and keep it isothermally. A reconstituted preparation, characterized in that the reconstituted preparation is obtained by reconstituting the solid preparation of any one of claims 1-9 with a pharmaceutically acceptable solvent. Use of the solid preparation according to any one of claims 1-9 or the reconstituted preparation according to claim 11 in the preparation of a medicine for the treatment of cancer; Preferably, the cancer is EGFR or HER2 positive cancer; or the cancer is metastatic colorectal cancer, head and neck cancer, breast cancer, stomach cancer, ovarian cancer, colon cancer or non-small cell lung cancer.

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