Formulation of Anti-Her2 Antibody-Drug Conjugate

Abstract

To provide a pharmaceutical preparation of anti-Her2 antibody drug conjugate.SOLUTION: A pharmaceutical preparation includes a mixture of non-reducing sugar, recombinant humanized anti-Her2 monoclonal antibody-MMAE composite, and surfactant. The pharmaceutical preparation has characteristics such as reduction of particulate substance, reduction of aggregate formation, improvement of conjugate stability, and improvement of an external appearance of frozen dry powder. A water-containing liquid pharmaceutical preparation of antibody-drug conjugate contains antibody-drug conjugate, non-reducing sugar, amino acid and chaotropic agent, where the non-reducing sugar is selected from mannitol, sucrose, trehalose or a combination thereof, the amino acid is selected from histidine, alanine, arginine, glycine, glutaminic acid or a combination thereof, and the chaotropic agent is selected from glycerol, Tween80 or a combination thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a formulation of an anti-Her2 antibody-drug conjugate and belongs to the field of antitumor drugs 。

Background Art

[0002] Biological therapies targeting tumors have been attracting increasing attention in cancer treatment research Among them, monoclonal antibodies have high target specificity and few side effects, but when used alone their effectiveness is relatively limited. Currently, the most successful anti-tumor monoclonal antibody drugs target, for example, non-Hodgkin lymphoma (NHL), chronic lymphocytic leukemia (CLL), etc 。 ErbB2, also known as Her2 / neu, is the second member of the EGFR family and ErbB2 forms heterodimers with the other three members of the EGFR family to play a biological role The neu gene encoding ErbB2 was first isolated from rat neuroblastoma The homologous gene of the neu gene in human somatic cells is called Her2 and is located on the long arm of chromosome 17 (17q21.1). The encoded product ErbB2 consists of 1255 amino acids, has a molecular weight of about 185 kDa, and among them amino acids 720-987 belong to the tyrosine kinase active region. In addition to acting through the PI3K and MAPK signaling pathways, ErbB2 reduces the expression of cyclin D and c-myc, and further reduces the expression of the cyclin-dependent kinase (cdk) inhibitor p27kipl Thereby inhibiting the activity of cdk2 and causing cell proliferation. With the expansion and deepening of research n) D and c-myc, and further reducing the expression of the cyclin-dependent kinase (cdk) inhibitor p27kipl in-dependentkinase, cdk) inhibitor p27kipl, reducing the expression of thereby inhibiting the activity of cdk2 and causing cell proliferation. As research expands and deepens , since it has been discovered that HER2 is expressed and overexpressed in various tumors, effective drugs targeting HER2 are clinically needed for treating malignant tumors There are currently three commercially available monoclonal antibodies targeting Her2 (see Table 1) ).

[0003]

Table 1

[0004] Based on the characteristics of antibody targeting, a new generation of biological targeting drugs, antibody-drug conjugates (ADCs), has emerged. An ADC consists of three parts: an antibody, a cytotoxin, and a linker that connects both. After coupling a monoclonal antibody with a cytotoxin, the antibody-drug conjugate uses the targeting property of the monoclonal antibody to specifically recognize the receptor on the surface of cancer cells, bind to the receptor, enter the cell, release the cytotoxic substance using intracellular proteases, prevent the proliferation of cancer cells, and kill cancer cells. In the prior art, generally, mammalian cell culture is used to produce monoclonal antibodies, and the highly purified antibodies are conjugated to the cytotoxin MMAE via a linker to obtain an antibody-drug conjugate (ADC). Antibody-drug coupling technology integrates small molecule toxin drugs and biological proteins, combines the advantages of both, and becomes a new generation of therapeutic products, significantly enhancing the efficacy of drugs and reducing toxic side effects. Currently, there are four ADC drugs approved by the US FDA (see Table 2).

[0005]

Table 2

[0006] Similar to other biopolymeric drugs, ADC drugs are susceptible to degradation, for example, oxidation, deamidation, and fragmentation , as well as the formation of microparticles and aggregates. Furthermore, the drug coupling itself can reduce stability and may change the physicochemical properties of the antibody. For example, when DM1 is conjugated to trastuzumab, an anti- HER2 antibody, the stability of the CH2 domain of the antibody is lost (Non-Patent Document 1: Physicochemical Stability of the Antibody-Drug Conjugate Trastuzu mab-DM1: Changes due to Modification and Conjugation Processes. Aditya A. Wakank ar et al., Bioconjugate Chemistry 2010: 21 (9) , 1588-1595). ADC drugs are generally hydrophobic and may be more insoluble than the unconjugated antibody as a whole, making them prone to aggregation, microparticle formation, and surface adsorption. To provide a stable ADC drug during transportation and storage, each additive in the formulation needs to be carefully selected. Currently, the components of the additives of commercially available ADC drugs are shown in Table 3. The types of additive components used in currently commercially available ADC formulations are still very limited, and since there are many options for related formulation components, developing an ADC additive formulation combination that provides stability during transportation and storage requires a great deal of labor and a long time for screening into a large number of additive formulation compositions and identifying suitable concentrations . .

[0007] ​​​​​

Table 3

[0008] The above four ADC formulation additive components have independent characteristics in each formulation. Mono Due to the fragile stability and complex structure of monoclonal antibody drugs, manufacturing such drugs and storing them become very difficult. Due to the heterogeneous structure of antibodies, especially the complementary determining regions (CDRs) and Fc glycosylation, each case of the formulation development of different monoclonal antibodies needs to be carried out separately (Non-Patent Document 2: Monoclonal antibod ies: formulations of marketed products a nd recent advances in novel delivery sys tem, Yanan Cui et al., Drug Development and Ind ustrial Pharmacy, Vol. 43, No. 4, pp. 519-530, 2017 year). Moreover, since ADC drugs are further related to coupling and toxin molecules, their formulation de velopment has even higher uniqueness.

[0009] The patent application (Patent Document 1: CN105008398A or Patent Document 2: WO201507 4528A1) discloses a humanized RC48 antibody-drug conjugate, where the humanized RC48 antibody is a monoclonal antibody targeting Her2 (an antibody secreted by Chinese Hamster Ovary cells (CHO cells) deposited in the China Center for Type Culture Collection under the accession number C20131 70, or an antibody derived therefrom), and the poison is selected from Monomethyl Auristatin E (MMAE), and the poison is an auristatin It belongs to the derivative. The linker is Maleimido-Caproyl-Valine-C itrulline-p-AminoBenzyloxy (mc-VC-PAB), and is selected. Among the recombinant humanized anti-Her2 monoclonal antibody-MMAE conjugates, the monoclonal antibody and MMAE are linked to the linker via cysteine. The drug achieves excellent therapeutic effects against Her2-positive tumors.

Prior Art Documents

Patent Documents

[0010]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0011]

Non-Patent Document 1

Non-Patent Document 2

[0012] As shown in the test, after purification, the recombinant humanized anti-Her2 monoclonal antibody-MMAE conjugate has low solubility in normal buffers and shows visible insoluble particles to the naked eye, indicating that it does not meet the standards of injection solutions. However, in order to achieve an effective therapeutic dose, the protein concentration needs to reach 5 mg / ml or higher. Therefore, further research is needed on how to solve the problem of insoluble particles on the premise of ensuring a certain guaranteed concentration. Furthermore, when lyophilizing the protein solution, in order to improve the appearance of the lyophilized powder and prevent protein damage during the lyophilization process, it is necessary to add a lyoprotectant. In order to avoid the slow decomposition of the protein lyophilized powder during long-term storage, it is necessary to add an appropriate protein stabilizer. The method of identifying an appropriate protein stabilizer also needs to be developed and determined through a large number of tests.

[0013] Therefore, the present invention conducts extensive screening and research on the concentration range of additives applicable to biological preparations, ​​​​​​​​​​The combination of additives was obtained, and the anti-Her2 monoclonal antibody-MMAE conjugate was freeze-dried. It is sufficiently dissolved before and after injection, and insoluble particles and visible foreign matter meet the standards for human injections. Both remain stable over long periods during freeze-drying and storage, and do not polymerize or react after reconstitution. To achieve the technical effect of being resistant to degradation or decomposition and maintaining good biological activity, The purpose is. [Means for solving the problem]

[0014] The present invention relates to a water-containing liquid pharmaceutical formulation of an antibody-drug conjugate, The non-reducing sugar comprises mannitol, a non-reducing sugar, an amino acid, and a chaotropic agent. glycerol, sucrose, trehalose, or a combination thereof, and the amino acid is selected from the group consisting of hydroxyl, hydroxypropyl ... Styrene, alanine, arginine, glycine, glutamic acid, or a combination thereof The chaotropic agent is selected from glycerin, Tween 80, or a combination thereof. The present invention provides a water-containing liquid pharmaceutical formulation selected from the group consisting of:

[0015] In some embodiments, the concentration of mannitol is 100 to 300 mmol / L. Preferably, the concentration is 190 to 300 mmol / L, and more preferably, the concentration is 200 to 2 60mmol / L, and most preferably 240 to 260mmol / L. The concentration of the sugar is 0 to 100 mmol / L, preferably 40 to 100 mmol / L. More preferably, it is 60 to 100 mmol / L, and most preferably, it is 40 to 6 0mmol / L. In some embodiments, the histidine is histidine hydrochloride and has a concentration of 0 to 100 mM. It is 100 mmol / L, preferably 5 - 50 mmol / L, more preferably , 5 - 20 mmol / L, most preferably 10 mmol / L, and the arginine is arginine hydrochloride, with a concentration of 0 - 160 mmol / L, preferably 20 - 100 mmol / L, more preferably 30 - 90 mmol / L, and most preferably about 35 mmol / L. In some embodiments, the content of the glycerin is 0 - 1%, preferably 0.2 - 0.5% (w / v), and the Tween 80 is 0 - 0.02% by mass ( w / v).

[0016] Furthermore, the antibody of the antibody - drug conjugate is an anti - HER2 monoclonal antibody , and the drug conjugated with the antibody is MMAE or MMAF and their derivatives, or DM1, DM4 and their derivatives. Furthermore, the anti - HER2 antibody - drug conjugate is anti - HER2 monoclonal antibody -vc - MMAE, and the anti - HER2 monoclonal antibody and MMAE are linked by linker v c, where the structure of the linker linking MMAE is as follows :

[0017] [Chemical formula]

[0018] Furthermore, the anti - HER2 monoclonal antibody includes a heavy chain and a light chain, (i) The heavy chain includes CDR1 - 3 having amino acid sequences represented by SEQ ID NO:1, 2 and 3 respectively, and / or (ii) The light chain includes amino acids represented by SEQ ID NO:4, 5 and 6 respectively (ii) The light chain has amino acid sequences represented by SEQ ID NO:4, 5 and 6 respectively It includes CDR1-3 having an array.

[0019] Furthermore, the monoclonal antibody is preferably a chimeric antibody or a humanized antibody. . In some embodiments, the concentration of the anti-HER2 monoclonal antibody-vc-MMAE is 5-30 mg / ml. In some embodiments, the non-reducing sugar is 240-260 mmol / L of mannitol and / or 40 mmol / L - 60 mmol / L of sucrose, the amino acid is 8-12 mmol / L of histidine hydrochloride, and the chaotropic agent is 0-0.0 2% (w / v) of Tween 80. In some embodiments, the non-reducing sugar is about 260 mmol / L of mannitol and about 40 mmol / L of sucrose, the amino acid is about 10 mmol / L of histidine hydrochloride, the concentration of the antibody-drug conjugate is about 10 mg / ml, and the chaotropic agent is about 0.02% (w / v) of Tween 80.

[0020] In some embodiments, the non-reducing sugar is about 240 mmol / L of mannitol and about 60 mmol / L of sucrose, the amino acid is about 10 mmol / L of histidine hydrochloride, the concentration of the antibody-drug conjugate is about 10 mg / ml, and the chaotropic agent is about 0.02% (w / v) of Tween 80. Furthermore, the pH of the formulation is 4.5-7, preferably 5.6-6.8, more preferably 5.6-6.5, 5.6-6.4, 5.6-6.3, 6.1-6.4, 6.1-6.3. The pH of the formulation is adjusted with NaOH or hydrochloric acid. In some embodiments, the lyophilized pharmaceutical formulation is obtained by freezing and drying the above water-containing liquid pharmaceutical formulation. Furthermore, the water-containing liquid pharmaceutical formulation before lyophilization of the drug contains about 260 mmol / L of mannitol, about 40 mmol / L of sucrose, about 10 mmol / L of histidine hydrochloride, about 0.02% (w / v) of Tween 80, and about 10 mg / ml of anti-HER2 monoclonal antibody-vc-MMAE, and has a pH of 5.6 to 6.8. Furthermore, the water-containing liquid pharmaceutical formulation before lyophilization of the drug contains about 240 mmol / L of mannitol, about 60 mmol / L of sucrose, about 10 mmol / L of histidine hydrochloride, 0.02% of Tween 80, and about 10 mg / ml of anti-HER2 monoclonal antibody-vc-MMAE, and has a pH of 5.6 to 6.8. Furthermore, the non-reducing sugars contained in the water-containing liquid pharmaceutical formulation before lyophilization of the drug are mannitol and sucrose, and their concentrations are about 47.36 mg / ml and about 13.69 mg / ml respectively, the amino acid is histidine hydrochloride, and its concentration is about 2.10 mg / ml, and the chaotropic agent is Tween 80, and its content is about 0.02% (w / v). Furthermore, the non-reducing sugars contained in the water-containing liquid pharmaceutical formulation before lyophilization of the drug are mannitol and sucrose, and their concentrations are about 43.72 mg / ml and about 20.54 mg / ml respectively, the amino acid is histidine hydrochloride, and its concentration is about 2.10 mg / ml, and the chaotropic agent is Tween 80, and its content is about 0.02% (w / v).

[0021]

[0022] The present invention further provides the use of the above pharmaceutical preparation in the preparation of a drug for treating diseases caused by abnormal expression of Her2 The disease caused by the abnormal expression is more preferably cancer The cancer is more preferably Her2-positive cancer, and the Her2-positive cancer is more preferably breast cancer, ovarian cancer, gastric cancer, urothelial cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, lung cancer or bladder cancer (see Non-Patent Document 3 Human E pidermal Growth Factor Receptor 2 (HER2) in Cancers:Overexpression and Therapeut ic Implications, Nida Iqbal and Naveed Iq bal, Molecular Biology International, Volu me 2014, Article ID 852748; Patent Document 3: CN20181 0998055.4).

[0023] The present invention further provides a method for preparing an antibody-drug conjugate pharmaceutical preparation. The method includes (1) preparing any of the above preparations, and (2) evaluating the stability of the antibody-drug conjugate in the preparation (2) evaluating the stability of the antibody-drug conjugate in the preparation In certain embodiments, for example, the following items are provided: (Item 1) An aqueous liquid pharmaceutical preparation of an antibody-drug conjugate, comprising an antibody-drug conjugate, a non-reducing sugar, an amino acid, and a chaotropic agent, wherein the non-reducing sugar is selected from mannitol, sucrose, trehalose, or a combination thereof, the amino acid is selected from histidine, alanine, arginine, glycine, glutamic acid, and hydrochloride salts of the above amino acids, or a combination thereof, and the chaotropic agent is selected from glycerol, Tween80, or a combination thereof. (Item 2) The concentration of the mannitol is 100 to 300 mmol / L, preferably 190 to 300 mmol / L, more preferably 200 to 260 mmol / L, and most preferably 240 to 260 mmol / L. The concentration of the sucrose is 0 to 100 mmol / L, preferably 40 to 100 mmol / L, more preferably 60 to 100 mmol / L, and most preferably 40 to 60 mmol / L. The aqueous liquid pharmaceutical preparation according to Item 1. (Item 3) The histidine is histidine hydrochloride, and its concentration is 0 to 100 mmol / L, preferably 5 to 50 mmol / L, more preferably 5 to 20 mmol / L, and most preferably 10 mmol / L. The arginine is arginine hydrochloride, and its concentration is 0 to 160 mmol / L, preferably 20 to 100 mmol / L, more preferably 30 to 90 mmol / L, and most preferably 35 mmol / L. The aqueous liquid pharmaceutical preparation according to Item 1 or 2. (Item 4) The content of the glycerol is 0 to 1%, preferably 0.2 to 0.5% (w / v). The Tween80 is 0 to 0.02% (w / v) by mass. The aqueous liquid pharmaceutical preparation according to any one of Items 1 to 3. (Item 5) The antibody of the antibody-drug conjugate is an anti-HER2 monoclonal antibody, and the drug conjugated to the antibody is MMAE or MMAF and its derivatives, or DM1, DM4 and its derivatives. The aqueous liquid pharmaceutical preparation according to any one of items 1 to 4. (Item 6) The anti-HER2 antibody-drug conjugate is anti-HER2 monoclonal antibody-vc-MMAE, and the anti-HER2 monoclonal antibody and MMAE are linked by a linker vc. Here, the structure in which the linker and MMAE are linked is [Chemical formula] The aqueous liquid pharmaceutical preparation according to item 5. (Item 7) The anti-HER2 monoclonal antibody contains a heavy chain and a light chain. (i) The heavy chain contains CDR1-3 having amino acid sequences represented by SEQ ID NO: 1, 2 and 3 respectively, and / or (ii) The light chain contains CDR1-3 having amino acid sequences represented by SEQ ID NO: 4, 5 and 6 respectively. The monoclonal antibody is preferably a chimeric antibody or a humanized antibody. The aqueous liquid pharmaceutical preparation according to item 5 or 6. (Item 8) The concentration of the anti-HER2 monoclonal antibody-vc-MMAE is 5 to 30 mg / ml. The aqueous liquid pharmaceutical preparation according to any one of items 1 to 7. (Item 9) The non-reducing sugar is mannitol at 240 to 260 mmol / L and / or sucrose at 40 mmol / L to 60 mmol / L, the amino acid is histidine hydrochloride at 8 to 12 mmol / L, and the chaotropic agent is Tween80 at 0 to 0.02% (w / v). The aqueous liquid pharmaceutical preparation according to item 8. (Item 10) The non-reducing sugar is about 260 mmol / L of mannitol and about 40 mmol / L of sucrose, the amino acid is about 10 mmol / L of histidine hydrochloride, the concentration of the antibody-drug conjugate is about 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of Tween 80. The liquid pharmaceutical preparation containing water according to item 9. (Item 11) The non-reducing sugar is about 240 mmol / L of mannitol and about 60 mmol / L of sucrose, the amino acid is about 10 mmol / L of histidine hydrochloride, the concentration of the antibody-drug conjugate is about 10 mg / ml, and the chaotropic agent is about 0.02% (w / v) of Tween 80. The liquid pharmaceutical preparation containing water according to item 10. (Item 12) The pH of the preparation is 4.5 to 7, preferably 5.6 to 6.8, more preferably 5.6 to 6.5, 5.6 to 6.4, 5.6 to 6.3, 6.1 to 6.4, 6.1 to 6.3. The liquid pharmaceutical preparation containing water according to any one of items 1 to 11. (Item 13) A lyophilized pharmaceutical preparation of an antibody-drug conjugate obtained by lyophilizing the liquid pharmaceutical preparation containing water according to any one of items 1 to 12. (Item 14) The liquid pharmaceutical preparation containing water contains about 260 mmol / L of mannitol, about 40 mmol / L of sucrose, about 10 mmol / L of histidine hydrochloride, about 0.02% (w / v) of Tween 80, and about 10 mg / ml of anti-HER2 monoclonal antibody-vc-MMAE, and the pH is 5.6 to 6.8. The lyophilized pharmaceutical preparation according to item 13. (Item 15) The liquid pharmaceutical preparation containing water contains about 240 mmol / L of mannitol, about 60 mmol / L of sucrose, about 10 mmol / L of histidine hydrochloride, about 0.02% of Tween 80, and about 10 mg / ml of anti-HER2 monoclonal antibody-vc-MMAE, and the pH is 5.6 to 6.8. The lyophilized pharmaceutical preparation according to item 13. (Item 16) The non-reducing sugars contained in the aqueous liquid pharmaceutical preparation are mannitol and sucrose, their concentrations being about 47.36 mg / ml and about 13.69 mg / ml respectively, the amino acid is histidine hydrochloride, its concentration being about 2.10 mg / ml, and the chaotropic agent is Tween 80, its content being about 0.02% (w / v). The lyophilized pharmaceutical preparation according to item 13. (Item 17) The non-reducing sugars contained in the aqueous liquid pharmaceutical preparation are mannitol and sucrose, their concentrations being about 43.72 mg / ml and about 20.54 mg / ml respectively, the amino acid is histidine hydrochloride, its concentration being about 2.10 mg / ml, and the chaotropic agent is Tween 80, its content being about 0.02% (w / v). The lyophilized pharmaceutical preparation according to item 13. (Item 18) Use in the preparation of a drug for treating a disease caused by abnormal expression of Her2 in the pharmaceutical preparation according to any one of Items 1 to 17, wherein the disease caused by the abnormal expression is more preferably cancer, and the cancer is more preferably Her2-positive cancer, and the Her2-positive cancer is more preferably breast cancer, ovarian cancer, gastric cancer, urothelial cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, lung cancer or bladder cancer. (Item 19) (1) Preparing the preparation according to any one of Items 1 to 17, and (2) Evaluating the stability of the antibody-drug conjugate in the preparation. A method for preparing an antibody-drug conjugate pharmaceutical preparation comprising.

Effect of the Invention

[0024] The anti-Her2 monoclonal antibody-MMAE conjugate of the present invention can be fully dissolved before and after lyophilization, and the insoluble particles and visible foreign matters meet the standards for human injection solutions. During the lyophilization and storage processes, it is stably maintained for a long time and is not likely to polymerize or decompose even after reconstitution. dissolve, and the insoluble particles and visible foreign matters meet the standards for human injection solutions, and during the freeze-drying drying and storage processes, it is stably maintained for a long time, and is not likely to polymerize or decompose even after reconstitution. <, to achieve the technical effect of maintaining good biological activity.

Brief Description of the Drawings

[0025]

Figure 1

Figure 2

Embodiments for Carrying Out the Invention

[0026] Example 1: Purification of Antibody The mouse monoclonal antibody mRC48 and the related humanized antibody RC48 are described in the patent application ( CN105008398A or WO2015074528A1) and are obtained based on the related methods, and contain the human IgG1κ heavy chain constant region and the heavy chain variable region RC48-VH , as well as the human IgG1κ light chain constant region and the light chain variable region RC48-VL. Each of the above-expanded fragments was subcloned into the expression vector pcDNA3.0 respectively. The constructed different plasmids were transfected into suspension CHO cells (Invitrogen). Cultured under standard conditions. When the nutrients in the medium were depleted and the cells stopped growing further, they were placed in a tank and the cells were separated by centrifugation or filtration, and the supernatant was obtained. Collect it. The antibody protein is present in the supernatant and is loaded onto a Protein A affinity chromatography column for the first-step purification. The eluted target protein is then loaded onto a cation-exchange chromatography column for the second-step purification, and the peak of the target protein is collected. Subsequently, it is loaded onto the third column for the third-step purification in the target protein permeation mode, and the purified protein is concentrated by ultrafiltration after passing the inspection of each index. When the protein concentration reaches about 20 - 30 mg / ml, this is the antibody protein stock solution, which can be stored at -80°C for a long period. Load it onto a chromatography column for the first-step purification, and load the eluted target protein onto a cation-exchange chromatography column for the second-step purification. Load the eluted target protein onto a cation-exchange chromatography column for the second-step purification. Perform the purification, and collect the peak of the target protein. Then, load it onto the third column for the third-step purification in the target protein permeation mode, and the purified protein is concentrated by ultrafiltration after passing the inspection of each index. When the protein concentration reaches about 20 - 30 mg / ml, this is the antibody protein stock solution, which can be stored at -80°C for a long period. Load it onto the third column for the third-step purification in the target protein permeation mode, and the purified protein is concentrated by ultrafiltration after passing the inspection of each index. When the protein concentration reaches about 20 - 30 mg / ml, this is the antibody protein stock solution, which can be stored at -80°C for a long period. The purified protein is concentrated by ultrafiltration after passing the inspection of each index. When the protein concentration reaches about 20 - 30 mg / ml, this is the antibody protein stock solution, which can be stored at -80°C for a long period. 0 - 30 mg / ml, and this is the antibody protein stock solution, which can be stored at -80°C for a long period. Here, the CDR sequences of the RC48 antibody are as follows. Here, the CDR sequences of the RC48 antibody are as follows.

[0027]

Table 4

[0028] Example 2: Coupling of Antibody and MMAE Coupling of the humanized antibody RC48 and the drug molecule Dissolve the stock solutions of TCEP (Tris-2-carboxyethyl-phosphine) and DTPA (Diethylene triamine pentacetate acid) in the coupling buffer respectively. First, dilute them with the coupling buffer and mix with the monoclonal antibody at a volume ratio of 1:1 (v:v = 1:1). The final molar ratio of TCEP to the antibody is 1.9:1, and the final concentration of DTPA is 1 mmol / L. React by stirring at 25°C for 2.5 hours. The reproducibility of TCEP reduction is good, and the number of free thiol groups after reduction may reach 3.5 - 4.5. A (Diethylene triamine pentacetate acid) in the coupling buffer respectively. First, dilute them with the coupling buffer and mix with the monoclonal antibody at a volume ratio of 1:1 (v:v = 1:1). The final molar ratio of TCEP to the antibody is 1.9:1, and the final concentration of DTPA is 1 mmol / L. React by stirring at 25°C for 2.5 hours. The reproducibility of TCEP reduction is good, and the number of free thiol groups after reduction may reach 3.5 - 4.5. Dissolve the stock solutions of TCEP (Tris-2-carboxyethyl-phosphine) and DTPA (Diethylene triamine pentacetate acid) in the coupling buffer respectively. First, dilute them with the coupling buffer and mix with the monoclonal antibody at a volume ratio of 1:1 (v:v = 1:1). The final molar ratio of TCEP to the antibody is 1.9:1, and the final concentration of DTPA is 1 mmol / L. React by stirring at 25°C for 2.5 hours. The reproducibility of TCEP reduction is good, and the number of free thiol groups after reduction may reach 3.5 - 4.5. Dissolve the stock solutions of TCEP (Tris-2-carboxyethyl-phosphine) and DTPA (Diethylene triamine pentacetate acid) in the coupling buffer respectively. First, dilute them with the coupling buffer and mix with the monoclonal antibody at a volume ratio of 1:1 (v:v = 1:1). The final molar ratio of TCEP to the antibody is 1.9:1, and the final concentration of DTPA is 1 mmol / L. React by stirring at 25°C for 2.5 hours. The reproducibility of TCEP reduction is good, and the number of free thiol groups after reduction may reach 3.5 - 4.5. The final molar ratio of TCEP to the antibody is 1.9:1, and the final concentration of DTPA is 1 mmol / L. React by stirring at 25°C for 2.5 hours. The reproducibility of TCEP reduction is good, and the number of free thiol groups after reduction may reach 3.5 - 4.5. React by stirring at 25°C for 2.5 hours. The reproducibility of TCEP reduction is good, and the number of free thiol groups after reduction may reach 3.5 - 4.5. The reproducibility of TCEP reduction is good, and the number of free thiol groups after reduction may reach 3.5 - 4.5. After reduction with TCEP, the antibody can directly perform the following coupling. 10 mmol / L of the drug (vc-MMAE, vc-MMAF, mc-MMAF) was prepared and dissolved in DMSO (d imethyl sulfoxide, dimethyl sulfoxide), and slowly added at a molar ratio of 1.1:1 with the thiol group of the drug, and stirred at 25 °C for 2 hours for reaction. D etect the concentration of free thiol groups (close to 0) at 412 nm by the TNB method, purify to remove residual unreacted drugs and free small molecules such as DMSO, and detect the coupling situation by SDS-PAGE electrophoresis , SEC, and HPLC methods. The reproducibility of the coupling reaction is , good, and the released free thiol groups can be completely coupled, and the coupling degree is 3.5 - 4.5.

[0029] Example 3: Study on the effect of pH on the dissolution and purity of recombinant humanized anti-Her-2 monoclonal antibody-MMAE conjugate (RC48 -vc-MMAE) Experimental conditions: 2 g of recombinant humanized anti-Her2 monoclonal antibody-MMAE conjugate protein for injection Ultrafiltered with a buffer solution of 0.1 mol / L citric acid, 0.02 mol / L tris(hydroxymethyl)aminomethane, 0.02 mol / L sodium dihydrogen phosphate, and 0.15 mol / L sodium chloride , and the dilution factor is 10 times or more, and evenly divided into 13 parts 4 , and each part was concentrated to a protein concentration of 10 mg / ml with a centrifugal ultrafiltration tube with a pore size of 30 kD, and the pH value was adjusted to 4.2, 4.6, 5.0, 5. 4, 5.6, 5.8, 6.0, 6.2, 6.4, 6.6, 6.8, 7.0, 7.4 with hydrochloric acid or sodium hydroxide solution Adjusted until, observed the clarity, detected the concentration change (the results are shown in Table 5), and clarified the sample Sterilized with a syringe filter with a membrane pore size of 0.22 μm sterilized in a clean bench respectively Filtered into 20 ml vials, three for each pH value, and sealed the bottle mouth with a sterilized rubber stopper and tightened the aluminum cap. Stored at 25°C, collected with a sterilized syringe at 0 hour, 1st day, 3rd day, 7th day, and performed SEC-HPLC purity inspection (the results are shown in Table 6). According to the research, changing the pH of the protein under experimental conditions has a great impact on the solubility of the protein. When the pH is below 5.4, the protein precipitates. When the pH is above 5.6, the solubility of the protein is good. When the pH is below 6.8, the protein aggregates do not increase when stored for 3 days, but slightly increase when stored for 7 days. When the pH is above 7.0, the aggregates increase with time, and the higher the pH, the more the aggregates increase (the specific results are shown in Table 5 and Table 6). Example 4: Screening of Additives Through a large amount of information analysis and test screening in the initial stage, first, sucrose, mannitol, glycerin, histidine, arginine, polysorbate 80 (i.e., Tween 80), etc. were identified as additives, and while further screening candidate additives, the following tests were carried out Furthermore As the pH increases, the aggregates increase, and the higher the pH, the more the aggregates increase (the specific results are shown in Table 5 and Table 6).

[0030]

Table 5

[0031]

Table 6

[0032] First, through a large amount of information analysis and test screening in the initial stage, sucrose, mannitol, glycerin, histidine, arginine, polysorbate 80 (i.e., Tween 80), etc. were identified as additives, and while further screening candidate additives, the following tests were carried out Through a large amount of information analysis and test screening in the initial stage, first, sucrose, mannitol, glycerin, histidine, arginine, polysorbate 80 (i.e., Tween 80), etc. were identified as additives, and while further screening candidate additives, the following tests were carried out Furthermore Furthermore, the following tests were carried out

[0033]

Table 7

[0034]

Table 8

[0035] Explanation: “-” means that the number of white dots is less than 3, “+” means that the number of white dots is 3 to 5, and “++” means that the number of white dots exceeds 5.

[0036]

Table 9

[0037] From the above results, it was found that the addition of arginine has no obvious effect on the improvement of visible foreign substances in the protein. Adding sucrose, glycerin, and polysorbate 80 can all improve the state of the protein. Therefore, an appropriate amount of sucrose was added to the formulation prescription to protect the protein, and glycerin and polysorbate 80 were added to promote the dissolution of the protein.

[0038] Example 5: Vacuum freeze-drying Take out the protein stock solution from the refrigerator at -80°C and dissolve it, and accurately dilute it with “1× formulation buffer solution” until the protein concentration reaches 10 mg / ml. Aliquot it into standard 20-ml vials without bacteria and pyrogens at 6 ml per vial, and perform vacuum freeze-drying. Freeze-drying conditions: Preliminary freezing: temperature -45°C, 5 hours; Primary drying: temperature -26°C, 40 hours, vacuum degree 10 - 15 Pa; Drying twice: temperature 25℃, 10 hours, vacuum 10~15Pa. After freeze-drying, remove the rubber stopper from the freeze-dryer under vacuum and tighten the aluminum cap. Ta.

[0039] Example 6: Formulation Screening According to the formulations in Table 10, the appearance and molding stability of the freeze-dried powders of the formulations were investigated. The freeze-dried samples were stored at 4°C and 37°C and rated as 0, 1, 3, and 7 in appearance, respectively. The appearance was observed on the 2nd day and the formulations with the best appearance were screened. The results are shown in Table 11.

[0040] [Table 10]

[0041] [Table 11]

[0042] Explanation: 1) "- -" means that the agglomerates shrink to less than half of their volume before freeze-drying. and "-" means that the agglomerates have shrunk but their volume exceeds half that before freeze-drying. "+" indicates that only the peripheral area has shrunk slightly, and the volume of the aggregate is almost the same as before freeze-drying. "++" means that there was no shrinkage, but the volume of the aggregates was the same as before freeze-drying. It means something.

[0043] The acceptance criteria for the appearance of freeze-dried powder are: uniform color, dense pores, and retention before freeze-drying. The volume and shape of the material remain essentially unchanged, and it exhibits a mass or sponge-like agglomerate structure. From Table 11, it can be seen that the formulation containing glycerin slightly shrunk after being left at 37°C for 1 day, After 7 days, the appearance of the ADC formulation was found to have shrunk and become significantly concave. It was found that glycerin was excluded as such.

[0044] Example 7: Determination of Freeze-Dried Powder Formulation and Inspection of Stability By visual inspection of the freeze-dried powder, Formulations B7 - B12 were excluded, and further screening of additional additives was carried out by moisture content inspection, inspection for visible foreign matters and insoluble particles visible to the naked eye, and stability investigation of stored samples (Formulations B1 - B6). The freeze-dried powders of each formulation were stored at 37, 25, and 4 °C respectively, sampled at different time points, and inspected for SDS-PAGE, reverse-phase HPLC, biological activity by ligand binding method, moisture content, appearance, pH value, visible foreign matters, and insoluble particles.

[0045]

Table 12

[0046] Explanation: “ - - ” means that the aggregate shrinks to less than half of its volume before freeze-drying, “ - ” means that it shrinks, but the volume of the aggregate exceeds half of its volume before freeze-drying, “ + ” means that only the edge shrinks slightly and the volume of the aggregate is almost the same as before freeze-drying, and “ ++ ” means that there is no shrinkage and the volume of the aggregate is the same as before freeze-drying.

[0047] It was found from Table 12 that the appearances of the freeze-dried powders of Formulations B1 - B6 all met the requirements.

[0048] Three samples were taken for each formulation, the moisture content was measured according to the specified method, the average value was taken, and the results are shown in Table 13.

[0049]

Table 13

[0050] The water contents of Formulations B1, B2, B3, B4, B5, and B6 were all less than 3%, and they passed the test. Five samples were randomly collected from each formulation, redissolved according to the specified method, and then examined for visible foreign matters. Among them, three samples were collected, and the insoluble particles were measured according to the specified method, and the results are shown in Table 14 .

[0051]

Table 14

[0052] From the above results, it was found that in the inspection of visible foreign matters and insoluble particles, Formulations B1, B3, and B5 did not meet the requirements and were excluded.

[0053]

Table 15

[0054]

Table 16

[0055]

Table 17

[0056] Note: As the appearance standard, "--" means that the agglomerate shrinks to less than half of its volume before lyophilization; "-" means that it shrinks, but the volume of the agglomerate exceeds half of its volume before lyophilization; "+" means that only the edge shrinks slightly and the volume of the agglomerate is almost the same as before lyophilization. ; "-" means that it shrinks, but the volume of the agglomerate exceeds half of its volume before lyophilization. ; "+" means that only the edge shrinks slightly and the volume of the agglomerate is almost the same as before lyophilization. means; "++" is not contracted at all, but the volume of the agglomerate is the same as before lyophilization means.

[0057] According to the above results, the stability of Formulation B2 and Formulation B4 at different temperatures (4°C, 25°C, 37°C) is good, but it was found that Formulation B6 was excluded because there were too many insoluble particles. Through the above tests, Formulation B2 and B4 reached excellent levels in terms of the appearance inspection of the lyophilized powder, the measurement of the moisture content of the lyophilized powder , the inspection of visible foreign matters and insoluble particles after redissolution of the lyophilized powder , and stability.

[0058] As can be seen from the above experimental evidence, among substances such as non-reducing sugars, amino acids, and chaotropic agents , the selection of specific additives shows that it can have an unpredictable impact on the final formulation, and the Her2 monoclonal antibody-drug conjugate of the present invention needs to be tested for each performance through a large number of tests in order to obtain a good formulation combination . For example, adding arginine has no obvious effect on improving the visible foreign matters of the protein. Adding sucrose, glycerin and polysorbate 80 can all improve the aggregation of the protein , but adding glycerin makes the lyophilized product have a shrunk appearance and be extremely prone to depression after reconstitution. Furthermore, if the concentration of sucrose is too high or the concentration of mannitol is too low, the insoluble substances after reconstitution will increase. These results are all difficult to predict before conducting relevant tests, and moreover, the ADC formulation is related to the combined use of multiple additives, and furthermore, it is related to factors such as long-term stability tests, so the development of ADC formulations is very difficult. ​​​Through a large number of tests, a combination of Her2 ADC formulations with excellent performance in each aspect was identified, and the anti- Her2 monoclonal antibody-MMAE conjugate was sufficiently dissolved before and after lyophilization, and the insoluble particles and visible foreign matters met the standards for human injectable solutions, and were stably maintained over a long period during the lyophilization and storage processes. In particular, when placed under high temperature conditions of 25 °C or 37 °C for a long time, it still maintained good stability. The ADC formulation was also difficult to polymerize or decompose after reconstitution and maintained good biological activity. ​

Claims

1. A method for producing a lyophilized pharmaceutical formulation of an antibody-drug conjugate, comprising lyophilizing a liquid pharmaceutical formulation containing water, said antibody-drug conjugate, a non-reducing sugar, an amino acid, and a chaotropic agent, wherein the non-reducing sugar is selected from mannitol, sucrose, or a combination thereof, the amino acid is selected from histidine, histidine hydrochloride, or a combination thereof, the chaotropic agent is polysorbate 80, the pH of the formulation is 5.6 to 6.8, the antibody-drug conjugate is an anti-HER2 monoclonal antibody-vc-MMAE, the anti-HER2 monoclonal antibody is linked to MMAE by a linker vc, and the structure of the linker and MMAE linked is 【Chemical 1】 wherein the antibody of the antibody-drug conjugate comprises a heavy chain and a light chain, the heavy chain comprises CDR1-3 having amino acid sequences represented by SEQ ID NOs: 1, 2, and 3 respectively, and the light chain comprises CDR1-3 having amino acid sequences represented by SEQ ID NOs: 4, 5, and 6 respectively, the lyophilized pharmaceutical formulation has a stability of at least 99% or more as determined by SEC-HPLC after storage at 4°C for 6 months.

2. The method according to claim 1, wherein the liquid pharmaceutical formulation containing water contains mannitol and the concentration of mannitol is 100 to 300 mmol / L.

3. The method according to claim 2, wherein the concentration of mannitol is 190 to 300 mmol / L.

4. The method according to claim 3, wherein the concentration of mannitol is 200 to 260 mmol / L.

5. The method according to claim 4, wherein the concentration of mannitol is 240 to 260 mmol / L.

6. The method according to claim 1, wherein the liquid pharmaceutical formulation containing water contains sucrose and the concentration of sucrose is 0 to 100 mmol / L.

7. The method according to claim 6, wherein the concentration of sucrose is 40 to 100 mmol / L.

8. The method according to claim 7, wherein the concentration of sucrose is 60 to 100 mmol / L.

9. The method according to claim 6, wherein the concentration of sucrose is 40 to 60 mmol / L.

10. The method according to any one of claims 1 to 9, wherein the amino acid is histidine hydrochloride and its concentration is 5 to 50 mmol / L.

11. The method according to claim 10, wherein the concentration of the histidine hydrochloride is 5 to 20 mmol / L.

12. The method according to claim 11, wherein the concentration of the histidine hydrochloride is 10 mmol / L.

13. The method according to any one of claims 1 to 12, wherein the content of polysorbate 80 is 0.02% (w / v).

14. The method according to any one of claims 1 to 13, wherein the monoclonal antibody is a chimeric antibody or a humanized antibody.

15. The method according to any one of claims 1 to 14, wherein the concentration of the anti-HER2 monoclonal antibody-vc-MMAE is 5 to 30 mg / ml.

16. The method according to claim 15, wherein the non-reducing sugar is mannitol at 240 to 260 mmol / L and / or sucrose at 40 mmol / L to 60 mmol / L, the amino acid is histidine hydrochloride at 8 to 12 mmol / L, and the chaotropic agent is polysorbate 80 at 0.02% (w / v).

17. The method according to claim 15, wherein the non-reducing sugar is mannitol at 240 to 260 mmol / L and / or sucrose at 40 mmol / L to 60 mmol / L, the amino acid is histidine at 8 to 12 mmol / L, and the chaotropic agent is polysorbate 80 at 0.02% (w / v).

18. The method according to claim 15, wherein the non-reducing sugar is mannitol at 240 to 260 mmol / L and / or sucrose at 40 mmol / L to 60 mmol / L, the amino acid is histidine and histidine hydrochloride at 8 to 12 mmol / L, and the chaotropic agent is polysorbate 80 at 0.02% (w / v).

19. The method according to claim 16, wherein the non-reducing sugar is mannitol at 260 mmol / L and sucrose at 40 mmol / L, the amino acid is histidine hydrochloride at 10 mmol / L, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is polysorbate 80 at 0.02% (w / v).

20. The method according to claim 17, wherein the non-reducing sugar is 260 mmol / L of mannitol and 40 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80.

21. The method according to claim 18, wherein the non-reducing sugar is 260 mmol / L of mannitol and 40 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine and histidine hydrochloride, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80.

22. The method according to claim 16, wherein the non-reducing sugar is 240 mmol / L of mannitol and 60 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine hydrochloride, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80.

23. The method according to claim 17, wherein the non-reducing sugar is 240 mmol / L of mannitol and 60 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80.

24. The method according to claim 18, wherein the non-reducing sugar is 240 mmol / L of mannitol and 60 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine and histidine hydrochloride, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80.

25. The method according to any one of claims 1 to 24, wherein the pH of the formulation is 5.6 to 6.5, 5.6 to 6.4, 5.6 to 6.3, 6.1 to 6.4, or 6.1 to 6.

3.

26. A method for producing a water-containing pharmaceutical formulation of an antibody-drug conjugate, the method comprising reconstituting a lyophilized pharmaceutical formulation produced according to the method of any one of claims 1 to 25.

27. An aqueous liquid pharmaceutical formulation of an antibody-drug conjugate containing an antibody-drug conjugate, a non-reducing sugar, an amino acid, and a chaotropic agent, wherein the non-reducing sugar is selected from mannitol, sucrose, or a combination thereof, the amino acid is selected from histidine, histidine hydrochloride, or a combination thereof, the chaotropic agent is polysorbate 80, the pH of the formulation is 5.6 to 6.8, the antibody-drug conjugate is an anti-HER2 monoclonal antibody-vc-MMAE, the anti-HER2 monoclonal antibody is linked to MMAE by a linker vc, and the structure of the linker and MMAE linked is 【Chemical 1】 wherein the antibody of the antibody-drug conjugate includes a heavy chain and a light chain, the heavy chain includes CDR1-3 having amino acid sequences represented by SEQ ID NOs: 1, 2, and 3 respectively, and the light chain includes CDR1-3 having amino acid sequences represented by SEQ ID NOs: 4, 5, and 6 respectively, the aqueous liquid pharmaceutical formulation has at least 99% or more stability by SEC-HPLC after storage at 4 °C for 6 months when lyophilized.

28. The aqueous liquid pharmaceutical formulation according to claim 27, wherein the aqueous liquid pharmaceutical formulation contains mannitol and the concentration of the mannitol is 100 to 300 mmol / L.

29. The aqueous liquid pharmaceutical formulation according to claim 28, wherein the concentration of the mannitol is 190 to 300 mmol / L.

30. The aqueous liquid pharmaceutical formulation according to claim 29, wherein the concentration of the mannitol is 200 to 260 mmol / L.

31. The aqueous liquid pharmaceutical formulation according to claim 30, wherein the concentration of the mannitol is 240 to 260 mmol / L.

32. The aqueous liquid pharmaceutical formulation according to claim 27, wherein the aqueous liquid pharmaceutical formulation contains sucrose and the concentration of the sucrose is 0 to 100 mmol / L.

33. The aqueous liquid pharmaceutical formulation according to claim 32, wherein the concentration of the sucrose is 40 to 100 mmol / L.

34. The aqueous liquid pharmaceutical formulation according to claim 33, wherein the concentration of the sucrose is 60 to 100 mmol / L.

35. The aqueous liquid pharmaceutical formulation according to claim 33, wherein the concentration of the sucrose is 40 to 60 mmol / L.

36. The water-containing liquid pharmaceutical preparation according to any one of claims 27 to 35, wherein the amino acid is histidine hydrochloride and its concentration is 5 to 50 mmol / L.

37. The water-containing liquid pharmaceutical preparation according to claim 36, wherein the concentration of the histidine hydrochloride is 5 to 20 mmol / L.

38. The water-containing liquid pharmaceutical preparation according to claim 37, wherein the concentration of the histidine hydrochloride is 10 mmol / L.

39. The water-containing liquid pharmaceutical preparation according to any one of claims 27 to 38, wherein the content of polysorbate 80 is 0.02% (w / v).

40. The water-containing liquid pharmaceutical preparation according to any one of claims 27 to 39, wherein the monoclonal antibody is a chimeric antibody or a humanized antibody.

41. The water-containing liquid pharmaceutical preparation according to any one of claims 27 to 40, wherein the concentration of the anti-HER2 monoclonal antibody-vc-MMAE is 5 to 30 mg / ml.

42. The water-containing liquid pharmaceutical preparation according to claim 41, wherein the non-reducing sugar is mannitol at 240 to 260 mmol / L and / or sucrose at 40 mmol / L to 60 mmol / L, the amino acid is histidine hydrochloride at 8 to 12 mmol / L, and the chaotropic agent is polysorbate 80 at 0.02% (w / v).

43. The water-containing liquid pharmaceutical preparation according to claim 41, wherein the non-reducing sugar is mannitol at 240 to 260 mmol / L and / or sucrose at 40 mmol / L to 60 mmol / L, the amino acid is histidine at 8 to 12 mmol / L, and the chaotropic agent is polysorbate 80 at 0.02% (w / v).

44. The water-containing liquid pharmaceutical preparation according to claim 41, wherein the non-reducing sugar is mannitol at 240 to 260 mmol / L and / or sucrose at 40 mmol / L to 60 mmol / L, the amino acid is histidine and histidine hydrochloride at 8 to 12 mmol / L, and the chaotropic agent is polysorbate 80 at 0.02% (w / v).

45. The non-reducing sugar is 260 mmol / L of mannitol and 40 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine hydrochloride, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80. The aqueous liquid pharmaceutical preparation according to claim 42.

46. The non-reducing sugar is 260 mmol / L of mannitol and 40 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80. The aqueous liquid pharmaceutical preparation according to claim 43.

47. The non-reducing sugar is 260 mmol / L of mannitol and 40 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine and histidine hydrochloride, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80. The aqueous liquid pharmaceutical preparation according to claim 44.

48. The non-reducing sugar is 240 mmol / L of mannitol and 60 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine hydrochloride, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80. The aqueous liquid pharmaceutical preparation according to claim 42.

49. The non-reducing sugar is 240 mmol / L of mannitol and 60 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80. The aqueous liquid pharmaceutical preparation according to claim 43.

50. The non-reducing sugar is 240 mmol / L of mannitol and 60 mmol / L of sucrose, the amino acid is 10 mmol / L of histidine and histidine hydrochloride, the concentration of the antibody-drug conjugate is 10 mg / ml, and the chaotropic agent is 0.02% (w / v) of polysorbate 80. The aqueous liquid pharmaceutical preparation according to claim 44.

51. Use of the pharmaceutical preparation according to any one of claims 27 to 50 in the preparation of a drug for treating a disease caused by abnormal expression of Her2.

52. The use according to claim 51, wherein the disease caused by the abnormal expression of Her2 is cancer.

53. The use according to claim 52, wherein the cancer is Her2-positive cancer.

54. The use according to claim 53, wherein the Her2-positive cancer is breast cancer, ovarian cancer, gastric cancer, urothelial cancer, gastroesophageal junction cancer, esophageal cancer, endometrial cancer, lung cancer or bladder cancer.

55. A method for preparing an antibody-drug conjugate pharmaceutical preparation, comprising: (1) preparing a preparation produced according to the method according to any one of claims 1 to 25, and (2) evaluating the stability of the antibody-drug conjugate of the preparation A method.

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