A pharmaceutical composition containing an antibody drug conjugate and use thereof

Abstract

The application discloses a kind of drug composition containing antibody drug conjugate and application thereof.The application provides a kind of drug composition 1, it includes compound C, histidine buffer, saccharide stabilizer, surfactant and water, the pH of the described drug composition 1 is 6.0-6.5;Wherein, the compound C is A-(B) n The drug composition of the application can be long-term stably stored in liquid form without freeze-drying, can effectively inhibit the aggregation and degradation of ADC molecules, has excellent biological activity and safety, greatly improves the convenience of clinical application while reducing the production cost.Meanwhile, the drug composition of the application can also maintain good stability after freeze-drying treatment and reconstitution, providing more options for clinical application.

Description

Technical Field

[0001] This invention relates to a pharmaceutical composition containing an antibody-drug conjugate and its application. Background Technology

[0002] This invention relates to a pharmaceutical composition of an antibody drug conjugate (ADC), its preparation method, and its uses. ADCs are novel antibody drugs formed by conjugating an antibody or antibody fragment targeting a specific antigen to a payload via a linker. ADC molecules combine the targeting properties of antibody drugs with the potent effects of small molecule drugs, showing promising clinical application prospects. Currently, more than a dozen ADC products have been approved for marketing worldwide. Due to the unique and complex structure of ADC molecules, the development of ADC products faces many challenges. For example, inhibiting ADC molecule focusing and degradation to ensure its safety and efficacy has always been a technical challenge in formulation development. ADC molecules exhibit high heterogeneity; therefore, the development of different ADC products often has personalized characteristics.

[0003] ADC conjugation methods include non-site-specific conjugation and site-specific conjugation. Non-site-specific conjugation involves directly coupling the payload to amino acid residues on the antibody using chemical methods without modification or alteration of the antibody; the number of payloads and the conjugation sites are unpredictable. Site-specific conjugation, on the other hand, involves modifying or altering the antibody to achieve payload connection at specific sites, improving the uniformity of the ADC. WO2012153193 discloses an ADC with an amino acid motif recognizable by isoprenoid transferases, and WO2015182984 discloses an ADC containing a self-detaching group and an amino acid motif recognizable by isoprenoid transferases. These prior art techniques achieve site-specific conjugation by introducing additional amino acid sequences at the antibody chain terminus, thereby enabling precise control of the drug-antibody ratio (DAR) in the ADC molecule, improving the pharmacokinetic properties of the ADC molecule, and increasing its safety and efficacy. However, no stable, clinically applicable drug formulations of ADC molecules prepared based on the above-mentioned site-specific conjugation techniques have been reported in the prior art. In particular, all currently marketed ADC products are lyophilized powder injections. There is an urgent need to develop a drug formulation that can be stably stored in liquid form for a long period of time in order to reduce production costs and improve the convenience of clinical application. Summary of the Invention

[0004] The technical problem this invention aims to solve is that existing antibody-drug conjugate (ADC) products are all lyophilized powder injections. Therefore, this invention provides a pharmaceutical composition containing an antibody-drug conjugate and its application. This pharmaceutical composition does not require lyophilization and can be stored stably in liquid form for a long period. It effectively inhibits the aggregation and degradation of ADC molecules and exhibits excellent bioactivity and safety, significantly improving the convenience of clinical application while reducing production costs. Furthermore, the pharmaceutical composition of this invention maintains good stability even after lyophilization and reconstitution, providing more options for clinical applications.

[0005] The present invention provides a pharmaceutical composition 1 comprising compound C, a histidine buffer, a carbohydrate stabilizer, a surfactant, and water, wherein the pH of the pharmaceutical composition 1 is 6.0-6.5;

[0006] Wherein, compound C is A-(B). n ;

[0007]

[0008] The A mentioned is trastuzumab-LC-G7CVIM (i.e., an antibody formed by adding 11 amino acid residues G7CVIM to the C-terminus of the light chain of trastuzumab);

[0009] The B in the above-mentioned B replaces the hydrogen on the cysteine ​​thiol group in G7CVIM of the above-mentioned A (i.e., it is linked by a thioether bond);

[0010] n is 1-2.

[0011] In this invention, the hydrogen atom on the cysteine ​​thiol group in G7CVIM of A is the underlined hydrogen atom:

[0012]

[0013] In one embodiment, some parameters of the pharmaceutical composition 1 are as described below, and the remaining parameters are as described in any other embodiment (hereinafter referred to as "in one embodiment"):

[0014] The pharmaceutical composition 1 is a pharmaceutical composition 1 for treating HER2-positive cancer.

[0015] In one embodiment, the pharmaceutical composition 1 is a pharmaceutical composition 1 for treating HER2-positive breast cancer, non-small cell lung cancer, gastric cancer, or urothelial carcinoma.

[0016] In one embodiment, the pharmaceutical composition 1 is not lyophilized or reconstituted.

[0017] In one embodiment, the pharmaceutical composition 1 is lyophilized and reconstituted.

[0018] In one embodiment, compound C is a pure substance or a mixture.

[0019] In one embodiment, compound C is a pure substance, and n is 1 or 2, representing antibody-drug conjugates with DAR of 1 or 2, respectively.

[0020] In one embodiment, compound C is a mixture, and n is 1-2, but not 1 or 2, representing a mixture of antibody-drug conjugates with a DAR of 1 or 2.

[0021] In this invention, an antibody-drug conjugate with a DAR of 1 refers to a conjugate in which only one of the B molecules replaces the hydrogen on one cysteine ​​thiol group in G7CVIM of A; an antibody-drug conjugate in which DAR of 2 refers to a conjugate in which only two of the B molecules replace the hydrogen on two cysteine ​​thiol groups in G7CVIM of A.

[0022] In one particular scheme, n is between 1.7 and 2.0.

[0023] In one particular scheme, n is 1.9.

[0024] In one embodiment, compound C was prepared according to the method disclosed in Example 79 of WO2017089890A1.

[0025] In this invention, in the pharmaceutical composition 1, the mass-volume concentration of compound C is the conventional mass-volume concentration in the art.

[0026] In one embodiment, in the pharmaceutical composition 1, the mass-volume concentration of compound C is 1-50 mg / mL, or 1-25 mg / mL, or 1-10 mg / mL, or 2.5-7.5 mg / mL, or even 5 mg / mL.

[0027] In this invention, the histidine buffer refers to a buffer containing histidine and / or its salts and having a buffering effect. Histidine used in this art is typically a natural amino acid, namely L-histidine.

[0028] In one embodiment, the histidine buffer comprises histidine and / or histidine hydrochloride monohydrate.

[0029] In one embodiment, the histidine buffer comprises histidine and / or histidine hydrochloride.

[0030] In this invention, in the pharmaceutical composition 1, the molar volume concentration of the histidine buffer, based on the total amount of histidine, is a conventional molar volume concentration in the art.

[0031] In one embodiment, in the pharmaceutical composition 1, the molar volume concentration of the histidine buffer, based on the total amount of histidine, is 1-25 mM, or 5-15 mM, or 7.5-12.5 mM, or even 10 mM.

[0032] In this invention, the "total histidine" refers to the total amount of histidine in histidine salt and free histidine.

[0033] In this invention, the sugar stabilizer is a conventional sugar stabilizer in the art, including but not limited to monosaccharides, oligosaccharides, non-reducing sugars, reducing sugars, sugar alcohols, sugar acids, etc., such as glucose, fructose, galactose, mannose, sorbitol, ribose, deoxyribose, sucrose, lactose, maltose, melitriose, trehalose, raffinose, arabinose, rhamnose, mannitol, xylitol, erythritol, threitol, sorbitol, glycerol, etc.

[0034] In one embodiment, the sugar stabilizer is one or more of sucrose, trehalose, mannitol (i.e., mannitol) and sorbitol (i.e., sorbitol), wherein trehalose and / or mannitol are preferred, and mannitol is most preferred.

[0035] In this invention, the mass-volume concentration of the carbohydrate stabilizer in the pharmaceutical composition 1 is the conventional mass-volume concentration in the art.

[0036] In one embodiment, the mass-volume concentration of the carbohydrate stabilizer in the pharmaceutical composition 1 is 1-10 g / 100 mL, for example, 1 g / 100 mL, 1.5 g / 100 mL, 2 g / 100 mL, 2.5 g / 100 mL, 3 g / 100 mL, 3.5 g / 100 mL, 4 g / 100 mL, 4.5 g / 100 mL, 5 g / 100 mL, 5.5 g / 100 mL, 6 g / 100 mL, 6.5 g / 100 mL, 7 g / 100 mL, 7.5 g / 100 mL, 8 g / 100 mL, 8.5 g / 100 mL, 9 g / 100 mL, 9.5 g / 100 mL, or 10 g / 100 mL.

[0037] In one embodiment, the mass-volume concentration of the carbohydrate stabilizer in the pharmaceutical composition 1 is 1 g / 100 mL to 8 g / 100 mL, or 2 g / 100 mL to 6 g / 100 mL, or 3 g / 100 mL to 5 g / 100 mL, or 2 g / 100 mL to 4 g / 100 mL. Preferably, it is 3 g / 100 mL.

[0038] In this invention, the surfactant is a conventional surfactant in the art, including but not limited to sodium dodecyl sulfate, polysorbate, sodium dioctyl sulfosuccinate, lecithin, stearyl alcohol, cetearyl alcohol, cholesterol, polyoxyethylene castor oil, polyoxyethylene fatty acid glycerides, poloxamer, polyoxyethylene castor oil derivatives, etc.

[0039] In one embodiment, the surfactant is polysorbate and / or poloxamer, and may be polysorbate.

[0040] In this invention, the polysorbate is a conventional polysorbate in the art.

[0041] In one embodiment, the polysorbate is one or more of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80, and may be polysorbate 20 and / or polysorbate 80, or polysorbate 80 (i.e. polysorbate 80 or Tween 80).

[0042] In this invention, the poloxamer is the conventional poloxamer in the art.

[0043] In one embodiment, the poloxamer is one or more of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407, and may be poloxamer 124 and / or poloxamer 188, or poloxamer 188.

[0044] In one embodiment, the surfactant is polysorbate 80.

[0045] In this invention, the mass-volume concentration of the surfactant in the pharmaceutical composition 1 is a conventional mass-volume concentration in the art.

[0046] In one embodiment, the surfactant in the pharmaceutical composition 1 has a mass-volume concentration of 0.01-0.10 g / 100 mL, for example, 0.01 g / 100 mL, 0.02 g / 100 mL, 0.03 g / 100 mL, 0.04 g / 100 mL, 0.05 g / 100 mL, 0.06 g / 100 mL, 0.07 g / 100 mL, 0.08 g / 100 mL, 0.09 g / 100 mL, or 0.10 g / 100 mL.

[0047] In one embodiment, the surfactant in the pharmaceutical composition 1 has a mass-volume concentration of 0.02 g / 100 mL - 0.08 g / 100 mL, or 0.03 g / 100 mL - 0.07 g / 100 mL, or 0.04 g / 100 mL - 0.06 g / 100 mL, or 0.03 g / 100 mL - 0.05 g / 100 mL. Preferably, it is 0.04 g / 100 mL.

[0048] In one embodiment, the pH of the pharmaceutical composition 1 may be adjusted using a pH adjuster.

[0049] In one embodiment, the pH of the pharmaceutical composition 1 is 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. Preferably, it is 6.3.

[0050] In one embodiment, the pH of the pharmaceutical composition 1 is 6.1-6.4, or 6.2-6.4. Preferably, it is 6.3.

[0051] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and the water.

[0052] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, the water, and the pH adjuster.

[0053] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the carbohydrate stabilizer is one or more selected from sucrose, trehalose, mannitol, and sorbitol, and the surfactant is polysorbate; wherein the mass-volume concentration of the compound C is 1-50 mg / mL, the molar volume concentration of the histidine buffer is 1-25 mM based on the total histidine content, the mass-volume concentration of the carbohydrate stabilizer is 1-10 g / 100 mL, and the mass-volume concentration of the surfactant is 0.01-0.10 g / 100 mL.

[0054] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 20 and / or polysorbate 80; wherein the mass-volume concentration of the compound C is 1-25 mg / mL, the molar volume concentration of the histidine buffer is 5-15 mM based on the total histidine content, the mass-volume concentration of the carbohydrate stabilizer is 1-8 g / 100 mL, and the mass-volume concentration of the surfactant is 0.02-0.08 g / 100 mL.

[0055] In one embodiment, the pharmaceutical composition 1 comprises compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, wherein the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of compound C is 1-10 mg / mL, the molar volume concentration of the histidine buffer is 7.5-12.5 mM (based on total histidine), the mass-volume concentration of the carbohydrate stabilizer is 2-6 g / 100 mL, and the mass-volume concentration of the surfactant is 0.03-0.07 g / 100 mL.

[0056] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 2.5-7.5 mg / mL, the molar volume concentration of the histidine buffer is 7.5-12.5 mM based on the total histidine content, the mass-volume concentration of the carbohydrate stabilizer is 2-4 g / 100 mL, and the mass-volume concentration of the surfactant is 0.03-0.05 g / 100 mL.

[0057] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 2.5-7.5 mg / mL, the molar volume concentration of the histidine buffer is 7.5-12.5 mM based on the total histidine content, the mass-volume concentration of the carbohydrate stabilizer is 2-4 g / 100 mL, and the mass-volume concentration of the surfactant is 0.03-0.05 g / 100 mL.

[0058] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.2-6.4; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 2.5-7.5 mg / mL, the molar volume concentration of the histidine buffer is 7.5-12.5 mM based on the total histidine content, the mass-volume concentration of the carbohydrate stabilizer is 2-4 g / 100 mL, and the mass-volume concentration of the surfactant is 0.03-0.05 g / 100 mL.

[0059] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.2-6.4; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 2.5-7.5 mg / mL, the molar volume concentration of the histidine buffer is 10 mM based on the total amount of histidine, the mass-volume concentration of the carbohydrate stabilizer is 2-4 g / 100 mL, and the mass-volume concentration of the surfactant is 0.03-0.05 g / 100 mL.

[0060] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.2-6.4; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 5 mg / mL, the molar volume concentration of the histidine buffer is 10 mM based on the total amount of histidine, the mass-volume concentration of the carbohydrate stabilizer is 3 g / 100 mL, and the mass-volume concentration of the surfactant is 0.04 g / 100 mL.

[0061] In one embodiment, the pharmaceutical composition 1 comprises 5 mg / mL of compound C, 10 mM of histidine buffer, 3 g / 100 mL of mannitol, and 0.04 g / 100 mL of polysorbate 80, the pH of the pharmaceutical composition 1 is 6.3, and the histidine buffer comprises histidine and histidine hydrochloride.

[0062] In one embodiment, the pharmaceutical composition 1 comprises 5 mg / mL of compound C, 0.97 mg / mL of histidine, 0.80 mg / mL of histidine hydrochloride, 30.0 mg / mL of mannitol, and 0.40 mg / mL of polysorbate 80, and the pH of the pharmaceutical composition 1 is 6.3.

[0063] In one embodiment, the pharmaceutical composition 1 comprises 5 mg / mL of compound C, 0.97 mg / mL of histidine, 0.80 mg / mL of histidine hydrochloride, 30.0 mg / mL of mannitol, and 0.40 mg / mL of polysorbate 80, wherein the pH of the pharmaceutical composition 1 is 6.3, and the n in compound C is 1.7 to 2.0.

[0064] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the carbohydrate stabilizer is one or more selected from sucrose, trehalose, mannitol, and sorbitol, and the surfactant is polysorbate; wherein the mass-volume concentration of the compound C is 2.5-7.5 mg / mL, the molar volume concentration of the histidine buffer is 10 mM based on the total histidine content, the mass-volume concentration of the carbohydrate stabilizer is 3-5 g / 100 mL, and the mass-volume concentration of the surfactant is 0.04-0.06 g / 100 mL.

[0065] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 2.5-7.5 mg / mL, the molar volume concentration of the histidine buffer is 10 mM based on the total amount of histidine, the mass-volume concentration of the carbohydrate stabilizer is 3-5 g / 100 mL, and the mass-volume concentration of the surfactant is 0.04-0.06 g / 100 mL.

[0066] In one embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, and water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 5 mg / mL, the molar volume concentration of the histidine buffer is 10 mM based on the total amount of histidine, the mass-volume concentration of the carbohydrate stabilizer is 3-5 g / 100 mL, and the mass-volume concentration of the surfactant is 0.04-0.06 g / 100 mL.

[0067] In one embodiment, the pharmaceutical composition 1 comprises 5 mg / mL of compound C, 0.97 mg / mL of histidine, 0.80 mg / mL of histidine hydrochloride, 30.0 mg / mL of mannitol, 0.40 mg / mL of polysorbate 80, and water, and the pH of the pharmaceutical composition 1 is 6.3.

[0068] In one embodiment, the pharmaceutical composition 1 comprises 5 mg / mL of compound C, 0.97 mg / mL of histidine, 0.80 mg / mL of histidine hydrochloride, 30.0 mg / mL of mannitol, 0.40 mg / mL of polysorbate 80, and water. The pH of the pharmaceutical composition 1 is 6.3, and n in compound C is 1.7 to 2.0.

[0069] In one embodiment, the pharmaceutical composition 1 is placed at 40°C for 4 weeks, and the DAR2% is ≥50%, preferably ≥55%, more preferably ≥60%.

[0070] In one embodiment, the pharmaceutical composition 1 is placed at 40°C for 4 weeks, and the DAR2% is ≥50%, preferably ≥55%, more preferably ≥60%, under the following test conditions:

[0071] The chromatographic column was TOSOH TSKgel ButyNPR, the flow rate was 0.5 mL / min, and the mobile phase was A (50 mM potassium phosphate, 1.5 M ammonium sulfate, pH 6.8). The mobile phase was 100% A for 0 min to 18 min.

[0072] In one embodiment, the pharmaceutical composition 1 is placed at 40°C for 4 weeks, and the DAR is ≥1.7, preferably ≥1.8, and more preferably ≥1.9.

[0073] In one embodiment, the pharmaceutical composition 1 is placed at 40°C for 4 weeks, and the DAR is ≥1.7, preferably ≥1.8, more preferably ≥1.9, under the following test conditions:

[0074] The chromatographic column was TOSOH TSKgel ButyNPR, the flow rate was 0.5 mL / min, and the mobile phase was A (50 mM potassium phosphate, 1.5 M ammonium sulfate, pH 6.8). The mobile phase was 100% A for 0 min to 18 min.

[0075] The present invention provides a pharmaceutical composition 2, which is obtained by freeze-drying the above-mentioned pharmaceutical composition 1.

[0076] In one embodiment, some parameters of the pharmaceutical composition 2 are as described below, and the remaining parameters are as described in any other embodiment (hereinafter referred to as "in one embodiment"):

[0077] The pharmaceutical composition 2 is a pharmaceutical composition 2 for treating HER2-positive cancer.

[0078] In one embodiment, the pharmaceutical composition 2 is a pharmaceutical composition 2 for treating HER2-positive breast cancer, non-small cell lung cancer, gastric cancer, or urothelial carcinoma.

[0079] In one embodiment, the freeze-drying process can be described as follows:

[0080] (1) Pre-freezing: Atmospheric pressure, temperature -45℃;

[0081] (2) First drying: vacuum degree 25Pa, temperature -15℃;

[0082] (3) Secondary drying: vacuum degree 25 Pa, temperature 30℃.

[0083] In one embodiment, the freeze-drying process can be described as follows:

[0084] (1) Pre-freezing: At atmospheric pressure, temperature -45℃, time 3h;

[0085] (2) One-time drying: vacuum degree 25Pa, temperature -15℃, time 39h;

[0086] (3) Secondary drying: vacuum degree 25Pa, temperature 30℃, time 5h.

[0087] The present invention provides a pharmaceutical composition 3, which comprises compound C, a histidine buffer, a carbohydrate stabilizer and a surfactant, wherein the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0-6.5;

[0088] Wherein, compound C is A-(B). n ;

[0089]

[0090] The A mentioned is trastuzumab-LC-G7CVIM (i.e., an antibody formed by adding 11 amino acid residues G7CVIM to the C-terminus of the light chain of trastuzumab);

[0091] The B in the above-mentioned B replaces the hydrogen on the cysteine ​​thiol group in G7CVIM of the above-mentioned A (i.e., it is linked by a thioether bond);

[0092] n is 1-2.

[0093] In one embodiment, some parameters of the pharmaceutical composition 3 are as described below, and the remaining parameters are as described in any other embodiment (hereinafter referred to as "in one embodiment"):

[0094] The pharmaceutical composition 3 is a pharmaceutical composition 3 for treating HER2-positive cancer.

[0095] In one embodiment, the pharmaceutical composition 3 is a pharmaceutical composition 3 for treating HER2-positive breast cancer, non-small cell lung cancer, gastric cancer, or urothelial carcinoma.

[0096] In one embodiment, compound C is as described in pharmaceutical composition 1.

[0097] In one embodiment, after the pharmaceutical composition 3 is dissolved in water, the mass-volume concentration of compound C is 1-50 mg / mL, or 1-25 mg / mL, or 1-10 mg / mL, or 2.5-7.5 mg / mL, or even 5 mg / mL.

[0098] In one embodiment, the histidine buffer is as described in pharmaceutical composition 1.

[0099] In one embodiment, the histidine buffer is 1-25 mmol, or 5-15 mmol, or 7.5-12.5 mmol, or even 10 mmol, based on a compound C content of 1-50 g (e.g., 1-25 g, or 1-10 g, or 2.5-7.5 g, or 5 g).

[0100] In one embodiment, the carbohydrate stabilizer is as described in pharmaceutical composition 1.

[0101] In one embodiment, the sugar stabilizer is 10-100g, for example, 10g, 15g, 20g, 25g, 30g, 35g, 40g, 45g, 50g, 55g, 60g, 65g, 70g, 75g, 80g, 85g, 90g, 95g, or 100g, based on the amount of compound C being 1-50g (e.g., 1-25g, 1-10g, 2.5-7.5g, or 5g).

[0102] In one embodiment, with compound C weighing 1-50g (e.g., 1-25g, or 1-10g, or 2.5-7.5g, or 5g), the sugar stabilizer weighing 10g-80g, or 20g-60g, or 30g-50g, or 20g-40g is preferred.

[0103] In one embodiment, the surfactant is as described in pharmaceutical composition 1.

[0104] In one embodiment, the surfactant is 0.1-1g, for example, 0.1g, 0.2g, 0.3g, 0.4g, 0.5g, 0.6g, 0.7g, 0.8g, 0.9g, or 1g, based on a compound C content of 1-50g (e.g., 1-25g, 1-10g, 2.5-7.5g, or 5g).

[0105] In one embodiment, based on the amount of compound C being 1-50g (e.g., 1-25g, or 1-10g, or 2.5-7.5g, or 5g), the surfactant is 0.2g-0.8g, or 0.3g-0.7g, or 0.4g-0.6g, or 0.3g-0.5g. Preferably, it is 0.4g.

[0106] In one embodiment, the pH of the pharmaceutical composition 3 after dissolving in water is 6.0, 6.1, 6.2, 6.3, 6.4, or 6.5. Preferably, it is 6.3.

[0107] In one embodiment, the pH of the pharmaceutical composition 3 after dissolving in water is 6.1-6.4, or 6.2-6.4. Preferably, it is 6.3.

[0108] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant.

[0109] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the carbohydrate stabilizer is one or more selected from sucrose, trehalose, mannitol, and sorbitol, and the surfactant is polysorbate; wherein, based on 5g of the compound C, the histidine buffer is 10mmol, the carbohydrate stabilizer is 20-40g, and the surfactant is 0.3-0.5g.

[0110] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein, based on 5g of the compound C, the histidine buffer is 10mmol, the carbohydrate stabilizer is 20-40g, and the surfactant is 0.3-0.5g.

[0111] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein, based on 5g of the compound C, the histidine buffer is 10mmol, the carbohydrate stabilizer is 20-40g, and the surfactant is 0.3-0.5g.

[0112] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.2-6.4; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein, based on 5g of the compound C, the histidine buffer is 10mmol, the carbohydrate stabilizer is 20-40g, and the surfactant is 0.3-0.5g.

[0113] In one embodiment, the pharmaceutical composition 3 comprises the compound C, histidine, histidine hydrochloride, mannitol, and polysorbate 80. The pH of the pharmaceutical composition 3 after dissolving in water is 6.3. Based on 5g of compound C, the composition comprises 0.97g of histidine, 0.80g of histidine hydrochloride, 30g of mannitol, and 0.40g of polysorbate 80.

[0114] In one embodiment, the pharmaceutical composition 3 comprises compound C, histidine, histidine hydrochloride, mannitol, and polysorbate 80. The pH of the pharmaceutical composition 3 after dissolving in water is 6.3. Based on 5g of compound C, the composition comprises 0.97g of histidine, 0.80g of histidine hydrochloride, 30g of mannitol, and 0.40g of polysorbate 80. In compound C, n is 1.7–2.0.

[0115] In one embodiment, the pharmaceutical composition 3 comprises compound C, histidine, histidine hydrochloride, mannitol, and polysorbate 80. The pH of the pharmaceutical composition 3 after dissolving in water is 6.3. The composition comprises, based on 5g of compound C, 0.97g of histidine, 0.80g of histidine hydrochloride, 30g of mannitol, and 0.40g of polysorbate 80.

[0116] In one embodiment, the pharmaceutical composition 3 comprises compound C, histidine, histidine hydrochloride, mannitol, and polysorbate 80. The pH of the pharmaceutical composition 3 after dissolving in water is 6.3. Based on 5g of compound C, the composition comprises 0.97g of histidine, 0.80g of histidine hydrochloride, 30g of mannitol, and 0.40g of polysorbate 80. In compound C, n is 1.7–2.0.

[0117] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the carbohydrate stabilizer is one or more selected from sucrose, trehalose, mannitol, and sorbitol, and the surfactant is polysorbate; wherein, after dissolving in water, the mass-volume concentration of the compound C is 2.5-7.5 mg / mL; based on 5 g of the compound C, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

[0118] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein, after dissolving in water, the mass-volume concentration of the compound C is 2.5-7.5 mg / mL; based on 5 g of the compound C, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

[0119] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein, after dissolving in water, the mass-volume concentration of the compound C is 5 mg / mL; based on 5 g of the compound C, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

[0120] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.2-6.4; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein, after dissolving in water, the mass-volume concentration of the compound C is 5 mg / mL; based on 5 g of the compound C, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

[0121] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the carbohydrate stabilizer is one or more selected from sucrose, trehalose, mannitol, and sorbitol, and the surfactant is polysorbate; wherein, based on 5g of the compound C, the histidine buffer is 10mmol, the carbohydrate stabilizer is 30-50g, and the surfactant is 0.4-0.6g.

[0122] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein, based on 5g of the compound C, the histidine buffer is 10mmol based on the total histidine content, the carbohydrate stabilizer is 30-50g, and the surfactant is 0.4-0.6g.

[0123] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein, based on 5g of the compound C, the histidine buffer is 10mmol based on the total amount of histidine, the carbohydrate stabilizer is 30-50g, and the surfactant is 0.4-0.6g.

[0124] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant, wherein the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the carbohydrate stabilizer is one or more selected from sucrose, trehalose, mannitol, and sorbitol, and the surfactant is polysorbate; wherein, after dissolving in water, the mass-volume concentration of the compound C is 2.5-7.5 mg / mL; based on 5 g of the compound C, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 30-50 g, and the surfactant is 0.4-0.6 g.

[0125] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant; the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; the mass-volume concentration of the compound C after dissolving in water is 2.5-7.5 mg / mL; wherein, based on 5 g of the compound C, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 30-50 g, and the surfactant is 0.4-0.6 g.

[0126] In one embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, and the surfactant; the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; the mass-volume concentration of the compound C after dissolving in water is 5 mg / mL; wherein, based on 5 g of the compound C, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 30-50 g, and the surfactant is 0.4-0.6 g.

[0127] In one embodiment, the pharmaceutical composition 3 comprises compound C, histidine, histidine hydrochloride, mannitol, and polysorbate 80. The pH of the pharmaceutical composition 3 after dissolving in water is 6.3. The mass-volume concentration of compound C after dissolving in water is 5 mg / mL. Based on 5 g of compound C, the amount of histidine is 0.97 g, the amount of histidine hydrochloride is 0.80 g, the amount of mannitol is 30 g, and the amount of polysorbate 80 is 0.40 g.

[0128] In one embodiment, the pharmaceutical composition 3 comprises compound C, histidine, histidine hydrochloride, mannitol, and polysorbate 80. The pH of the pharmaceutical composition 3 after dissolving in water is 6.3. The mass-volume concentration of compound C after dissolving in water is 5 mg / mL. Based on 5 g of compound C, the amount of histidine is 0.97 g, the amount of histidine hydrochloride is 0.80 g, the amount of mannitol is 30 g, the amount of polysorbate 80 is 0.40 g, and the n in compound C is 1.7–2.0.

[0129] The present invention also provides the use of the above-mentioned pharmaceutical composition 1, pharmaceutical composition 2 or pharmaceutical composition 3 in the preparation of a drug, wherein the drug is a drug for treating HER2-positive cancer.

[0130] In the aforementioned applications, the HER2-positive cancer may be breast cancer, non-small cell lung cancer, gastric cancer, or urothelial carcinoma.

[0131] In the described applications, pharmaceutical composition 1, pharmaceutical composition 2, or pharmaceutical composition 3 may be used in combination with other anticancer agents. These other anticancer agents include, for example, PD-1 inhibitors. PD-1 inhibitors include, for example, monoclonal antibodies.

[0132] The present invention also provides a method for treating HER2-positive cancer, comprising administering to a patient a therapeutically effective amount of the above-described pharmaceutical composition 1, pharmaceutical composition 2, or pharmaceutical composition 3.

[0133] In the treatment methods described, the HER2-positive cancer may be breast cancer, non-small cell lung cancer, gastric cancer, or urothelial carcinoma.

[0134] In the aforementioned treatment method, the patient may also be simultaneously administered a therapeutically effective amount of other anticancer agents. These other anticancer agents include, for example, PD-1 inhibitors. PD-1 inhibitors include, for example, monoclonal antibodies.

[0135] The term "drug-to-antibody ratio (DAR)" refers to the average number of small molecule toxic drugs conjugated to antibodies.

[0136] The term "treatment" refers to eliminating the cause of an illness or relieving symptoms.

[0137] The term "patient" refers to any animal that requires treatment for a disease, typically a mammal such as a human. Mammals include, but are not limited to: cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans.

[0138] The term "therapeutic effective dose" refers to the amount given to a patient that is sufficient to effectively treat the disease. Therapeutic effective doses will vary depending on the type of drug, the type of disease, the severity of the disease, the patient's age, etc., but may be adjusted as appropriate by those skilled in the art.

[0139] Without violating common sense in the field, the above conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0140] The significant advantages of this invention are as follows: the pharmaceutical composition of this invention can be stored stably in liquid form for a long period without lyophilization, effectively inhibiting the aggregation and degradation of ADC molecules, and exhibiting excellent bioactivity and safety. This greatly improves the convenience of clinical application while reducing production costs. Furthermore, the pharmaceutical composition of this invention maintains good stability even after lyophilization and reconstitution, providing more options for clinical applications. Attached Figure Description

[0141] Figure 1 Initial-reduced and non-reduced SDS-PAGE detection results

[0142] Figure 2 25℃, two weeks - SDS-PAGE results for reduced and non-reduced SDS-PAGE

[0143] Figure 3 40℃, two weeks - SDS-PAGE results of reduced and non-reduced samples Detailed Implementation

[0144] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0145] Unless otherwise specified, the solvent used in the following examples is water.

[0146] (I) Buffer System Selection

[0147] 1. Prescription preparation

[0148] Based on the formulations of different buffer systems shown in Table 1, we screened out the types and pH ranges of buffer systems that would help stabilize the active pharmaceutical ingredient (compound C).

[0149] Table 1: Composition of candidate buffer systems in the screening study of buffer systems

[0150]

[0151] 2. Preparation method

[0152]

[0153] Among them, antibody A is shown above, which is trastuzumab-LC-G7CVIM (i.e., the antibody formed by adding 11 amino acid residues G7CVIM to the C-terminus of the light chain of trastuzumab).

[0154] As shown above, group B replaces the hydrogen on the cysteine ​​thiol group in G7CVIM of antibody A, i.e., it is linked by a thioether bond.

[0155] n is 1.9.

[0156] The solution of the active pharmaceutical ingredient compound C was replaced with one of the nine buffer solutions shown in Table 1. After adjusting the concentration of compound C to 5 mg / mL, the sample was aseptically filtered and dispensed into 2 mL sterile vials, 0.55 mL per vial.

[0157] 3. Detection Method

[0158] The aliquoted samples were placed in constant temperature pharmaceutical storage chambers at 2–8℃, 25±2℃, and 40±2℃, respectively. Samples were taken and tested at 0, 1, 2, and 4 weeks. The test items included appearance, concentration (A280), pH value, molecular isomers (SEC-HPLC, SDS-PAGE (NR&R)), charge isomers (CEX-HPLC), number of small molecule complete couplings, i.e., the proportion of drug isomers with a total of 2 (Drug-2%) (HIC-HPLC), average protein particle size, and polydispersity index (PdI) (DLS).

[0159] 3.1 Appearance

[0160] Chinese Pharmacopoeia 2020 General Chapter 0102 Injection.

[0161] 3.2 Concentration (A280)

[0162] Chinese Pharmacopoeia 2020 Edition, Part III, General Chapter 0401, Ultraviolet-Visible Spectrophotometry.

[0163] 3.3, pH

[0164] Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0631, pH Value Determination.

[0165] 3.4 SDS-PAGE (NR&R)

[0166] NR indicates non-reduction, which uses iodoacetamide pretreatment; R indicates reduction, which uses β-mercaptoethanol pretreatment.

[0167] Electrophoresis time is 4-5 hours, voltage is 40-60V. Electrophoresis is stopped as soon as bromophenol blue appears, and staining is performed with Coomassie Brilliant Blue rapid staining solution.

[0168] 3.5, DLS

[0169] After diluting the test sample with the reagent buffer, filter it through a 0.22 μm filter into a disposable cuvette, and then place the cuvette into the instrument (Protein Dynamic and Static Light Scattering Analysis System / Malvern) for detection.

[0170] 3.6 Molecular isomers

[0171] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0172] Methods: TOSOH TSK G300 column, flow rate 0.5 mL / min, wavelength 280 nm, mobile phase 100 mM sodium dihydrogen phosphate + 0.5% sodium chloride, pH 6.8.

[0173] Result calculation:

[0174] HMW% = A / (A + B + C) * 100%

[0175] Peak percentage = B / (A+B+C)*100%

[0176] LMW% = C / (A + B + C) * 100%

[0177] A represents the sum of peak areas of high molecular weight polymers, B represents the peak area of ​​monomers, and C represents the sum of peak areas of low molecular weight substances.

[0178] High molecular weight polymers refer to polymers formed by the polymerization of monomers into molecules with a molecular weight of 2, 3 or more. Monomers refer to the ADC molecule itself, while low molecular weight substances refer to the detached subunit fragments.

[0179] 3.7. Charged heterostructures

[0180] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0181] Methods: Thermo ProPac SCX-10 column, column temperature: 35 degrees Celsius, wavelength: 214 nm, mobile phase A (20 mM phosphate buffer, pH 6.2), mobile phase B (20 mM phosphate buffer + 200 mM sodium chloride, pH 6.2), flow rate: 1 mL / min, 0 min-35 min was 100% mobile phase A, 35 min-37 min was 100% mobile phase B.

[0182] Result calculation:

[0183] The peaks preceding the main peak are acidic component peaks, and the peaks following the main peak are alkaline component peaks.

[0184] Acidity peak % = Acidity peak area / (Acidity peak area + Main peak area + Basic peak area) * 100%

[0185] Main peak % = Main peak area / (Acid peak area + Main peak area + Basic peak area) * 100%

[0186] Basic peak % = Basic peak area / (Acid peak area + Main peak area + Basic peak area) * 100%.

[0187] 3.8. Number of Complete Couplings of Small Molecules

[0188] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0189] Methods: Chromatographic column: TOSOH TSKgel ButyNPR, flow rate: 0.5 mL / min, mobile phase A (50 mM potassium phosphate, 1.5 M ammonium sulfate, pH 6.8), mobile phase B (50 mM potassium phosphate, 20% isopropanol, pH 6.8), 0 min-23 min was 100% mobile phase A, 23 min-26 min was 100% mobile phase B.

[0190] Result calculation:

[0191] DAR0% = (DAR0 component peak area / total peak area) * 100%

[0192] DAR1% = (DAR1 component peak area / total peak area) * 100%

[0193] DAR2% = (DAR2 component peak area / total peak area) * 100%.

[0194] The results showed that the DAR0, DAR1, and DAR2 components all showed peaks at 18 min.

[0195] 4. Test Results

[0196] 4.1 The appearance inspection results at 5℃ / 25℃ / 40℃ are shown in Table 2;

[0197] Table 2:

[0198]

[0199] Note: CS: colorless, slightly milky white; CS*: colorless, slightly milky, with some particles; CS**: colorless, slightly milky, with a large number of particles;

[0200] 4.2 The results of 4W SEC at 5℃ are shown in Table 3;

[0201] Table 3:

[0202]

[0203] 4.3 The results of 2W SEC at 25℃ and 40℃ are shown in Tables 4 and 5;

[0204] Table 4:

[0205]

[0206] Table 5:

[0207]

[0208]

[0209] 4.4 and 5℃ 4W CEX data are shown in Table 6;

[0210] Table 6:

[0211]

[0212] The data for 4.5, 25℃ and 40℃ 2W CEX are shown in Tables 7 and 8;

[0213] Table 7:

[0214]

[0215] Table 8:

[0216]

[0217]

[0218] The data for 4.6 and 5℃ 4W HIC are shown in Tables 9 and 10;

[0219] Table 9:

[0220]

[0221] Table 10:

[0222]

[0223] 4.7 The data for 2W HIC at 25℃ and 40℃ are shown in Tables 11 and 12;

[0224] Table 11:

[0225]

[0226]

[0227] Table 12:

[0228]

[0229] 4.8 Analysis of Test Results:

[0230] 1. Appearance results showed that after the samples were placed in histidine-histidine hydrochloride buffer at pH 5.0–7.0 for 4 weeks at 5℃, 25℃, and 40℃, no obvious visible particles appeared. However, visible foreign matter appeared in histidine-histidine hydrochloride buffer at pH 7.5 and citrate-sodium citrate buffer at pH 5.5–6.5 after being placed at 5℃, 25℃, and 40℃ for 2 weeks.

[0231] 2. Concentration, DLS, and pH test results showed no significant differences among the nine formulations at three temperatures (5℃, 25℃, and 40℃).

[0232] SEC results at 3.5℃ (4W) and 25℃ (2W) showed that the SEC peak content decreased slightly with increasing pH, with little difference in purity between formulations. However, SEC results at 40℃ for 2 weeks showed higher SEC peak content and the lowest aggregation and degradation in histidine-histidine hydrochloride buffers at pH 5.5, pH 6.0, and pH 6.5. Histidine-histidine hydrochloride buffer was superior to citrate-sodium citrate buffer. The relationship between SEC peak content among different formulations is as follows:

[0233] SEC main peak percentage: histidine buffer > citrate buffer, H55 / H60 / H65 > H50 / H70 > H75.

[0234] The 2W CEX results at 4.25℃ and 40℃ showed that when pH was greater than 6.0, the acid peak content increased with increasing pH, thus decreasing the main peak. Conversely, when pH was less than 6.0, the alkali peak content increased with decreasing pH, thus decreasing the CEX main peak. The relationship between the CEX main peak content of different formulations is as follows:

[0235] CEX main peak percentage: histidine buffer > citrate buffer, H50 / H55 / H60 > H65 > H70 > H75.

[0236] 5. The HIC detection results at 5℃, 4W, and 25℃ accelerated 2W show that the DAR2% content increases with increasing pH, and the DAR2% content varies among different formulations. Compared to citrate-sodium citrate buffer, in histidine-histidine hydrochloride buffer, small drug molecules are less likely to separate from large protein molecules. The relationship of DAR2% content among different formulations is as follows:

[0237] DAR2%: Histidine buffer > Citrate buffer, H60 / H65 / H70 / H75 > H50 / H55.

[0238] 6. NR&R SDS-PAGE results showed that, compared to other formulations, H70 / H75 / C60 / C65 exhibited slightly more noticeable degradation bands after being stored at 40°C for 2 weeks. Figure 1 , Figure 2 and Figure 3 As shown.

[0239] Taking into account the test results of SEC, CEX, HIC, and appearance, it can be concluded that 10 mmol / L histidine-histidine hydrochloride at pH 6.0 / 6.5 is the optimal buffer system.

[0240] 5. Screening Conclusion

[0241] Based on the results of accelerated stability testing in buffer system screening studies, the histidine buffer system was superior to the citrate buffer system, and the 10 mmol / L histidine-histidine hydrochloride buffer system could effectively stabilize the solution pH. The pH range of 6.0–7.5 is beneficial for reducing the dissociation of small molecule drugs, while the pH range of 5.0–6.5 is beneficial for increasing the stability of protein isoforms and charge isoforms. Considering the characteristics of antibody-drug conjugates, combined with the stability of recombinant HER2 humanized monoclonal antibody and small molecule drugs, a 10 mmol / L histidine-histidine hydrochloride buffer system with a pH of 6.0–6.5 was selected for further research.

[0242] (II) Research on Excipient Screening - I

[0243] The purpose of excipient screening studies is to select an excipient system that is conducive to the stability of the active pharmaceutical ingredient by using single-factor experimental design, based on an established buffer system.

[0244] In Table 13, the "%" for each excipient refers to the corresponding number of grams of solute contained in 100 mL of solvent. For example, "3.0%" means "3g of solute in 100 mL of solvent".

[0245] 1. Table 13 Excipient Screening Study - I. Composition of Alternative Formulations

[0246] Table 13:

[0247]

[0248]

[0249] 2. Preparation method

[0250] Take a solution of the active pharmaceutical ingredient compound C (its structure is shown above), ultrafilter and change the medium, prepare according to the prescription shown in Table 13, adjust the concentration of compound C to 5 mg / mL, filter the sample aseptically and dispense it into 2 mL sterile vials, 1.0 mL per vial.

[0251] 3. Detection Method

[0252] Accelerated stability studies were conducted on samples at 25°C and 60% RH and at 40°C and 75% RH. The assays included appearance, concentration (A280), pH, molecular isomers (SEC-HPLC), charge isomers (CEX-HPLC), drug-2% (HIC-HPLC), size, subvisible particles (flowcam), and SDS-PAGE (NR&R).

[0253] 3.1 Appearance

[0254] Chinese Pharmacopoeia 2020 General Chapter 0102 Injection.

[0255] 3.2 Concentration (A280)

[0256] Chinese Pharmacopoeia 2020 Edition, Part III, General Chapter 0401, Ultraviolet-Visible Spectrophotometry.

[0257] 3.3, pH

[0258] Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0631, pH Value Determination.

[0259] 3.4 Size, Subvisible Particles (flowcam)

[0260] Chinese Pharmacopoeia 2020 Edition, General Chapter 0903, Insoluble Particulate Matter Test Method.

[0261] 3.5 SDS-PAGE (NR&R)

[0262] NR indicates non-reduction, which uses iodoacetamide pretreatment; R indicates reduction, which uses β-mercaptoethanol pretreatment.

[0263] Electrophoresis time is 4–5 hours, voltage is 40–60V. Electrophoresis is stopped as soon as bromophenol blue appears, and staining is performed with Coomassie Brilliant Blue rapid staining solution. 3.6 Molecular Isomers

[0264] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0265] Methods: TOSOH TSK G300 column, flow rate 0.5 mL / min, wavelength 280 nm, mobile phase 100 mM sodium dihydrogen phosphate + 0.5% sodium chloride, pH 6.8.

[0266] Result calculation:

[0267] HMW% = A / (A + B + C) * 100%

[0268] Peak percentage = B / (A+B+C)*100%

[0269] LMW% = C / (A + B + C) * 100%

[0270] A represents the sum of peak areas of high molecular weight polymers, B represents the peak area of ​​monomers, and C represents the sum of peak areas of low molecular weight substances.

[0271] High molecular weight polymers refer to polymers formed by the polymerization of monomers into molecules with a molecular weight of 2, 3 or more. Monomers refer to the ADC molecule itself, while low molecular weight substances refer to the detached subunit fragments.

[0272] 3.7. Charged heterostructures

[0273] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0274] Methods: Thermo ProPac SCX-10 column, column temperature: 35 degrees Celsius, wavelength: 214 nm, mobile phase A (20 mM phosphate buffer, pH 6.2), mobile phase B (20 mM phosphate buffer + 200 mM sodium chloride, pH 6.2), flow rate: 1 mL / min, 0 min-35 min was 100% mobile phase A, 35 min-37 min was 100% mobile phase B.

[0275] Result calculation:

[0276] The peaks preceding the main peak are acidic component peaks, and the peaks following the main peak are alkaline component peaks.

[0277] Acidity peak % = Acidity peak area / (Acidity peak area + Main peak area + Basic peak area) * 100%

[0278] Main peak % = Main peak area / (Acid peak area + Main peak area + Basic peak area) * 100%

[0279] Basic peak % = Basic peak area / (Acid peak area + Main peak area + Basic peak area) * 100%.

[0280] 3.8. Number of Complete Couplings of Small Molecules

[0281] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0282] Methods: Chromatographic column: TOSOH TSKgel ButyNPR, flow rate: 0.5 mL / min, mobile phase A (50 mM potassium phosphate, 1.5 M ammonium sulfate, pH 6.8), mobile phase B (50 mM potassium phosphate, 20% isopropanol, pH 6.8), 0 min-23 min was 100% mobile phase A, 23 min-26 min was 100% mobile phase B.

[0283] Result calculation:

[0284] DAR0% = (DAR0 component peak area / total peak area) * 100%

[0285] DAR1% = (DAR1 component peak area / total peak area) * 100%

[0286] DAR2% = (DAR2 component peak area / total peak area) * 100%.

[0287] The results showed that the DAR0, DAR1, and DAR2 components all showed peaks at 18 min.

[0288] 4. Test Results

[0289] 4.1 The data for 40℃ SEC are shown in Tables 14 and 15:

[0290] Table 14:

[0291]

[0292]

[0293] Table 15:

[0294]

[0295]

[0296] 4.2 SEC data at 25℃ are shown in Tables 16 and 17;

[0297] Table 16:

[0298]

[0299]

[0300] Table 17:

[0301]

[0302] 4.3 CEX data at 40℃ are shown in Tables 18 and 19;

[0303] Table 18:

[0304]

[0305] Table 19:

[0306]

[0307]

[0308] Note: Because the CEX main peak is small after 2W acceleration at 40℃, the data is not meaningful and will not be tested further. No test results are given, indicated by " / ".

[0309] 4.4 CEX data at 25℃ are shown in Tables 20 and 21;

[0310] Table 20:

[0311]

[0312] Table 21:

[0313]

[0314]

[0315] 4.5 HIC data at 40℃ are shown in Tables 22 and 23;

[0316] Table 22:

[0317]

[0318]

[0319] Table 23:

[0320]

[0321]

[0322] 4.6 HIC data at 25℃ are shown in Tables 24 and 25;

[0323] Table 24:

[0324]

[0325]

[0326] Table 25:

[0327]

[0328]

[0329] 4.7 Analysis of Test Results:

[0330] 1. In the accelerated stability studies at 25℃ 4W and 40℃ 4W, the results of appearance, concentration, pH, size, and subvisible particle (flowcam) detection showed no significant differences between the formulations.

[0331] 2. SEC results showed that the aggregation trend was more pronounced than the degradation trend in all samples. The relationship between the main peak content of the SEC for the 13 formulations under accelerated conditions at 25℃ and 40℃ is as follows:

[0332] SEC main peak percentage: 0.04% polysorbate 80 > 0.06% polysorbate 80, concentration 3.0% < 5.0%

[0333] Mannitol / sorbitol > Sucrose / trehalose > Arginine hydrochloride / Sucrose + NaCl / Sucrose + Arginine hydrochloride, pH 6.5 <pH6.0。

[0334] 3. CEX results showed that during the stability test, the alkaline peaks of all samples increased faster than the acidic peaks. The relationship between the main peak content of the 13 formulations under accelerated conditions at 25℃ and 40℃ is as follows:

[0335] CEX main peak percentage: 0.04% polysorbate 80 ≈ 0.06% polysorbate 80, concentration 3.0% ≈ 5.0%

[0336] Sucrose / trehalose / mannitol / sorbitol > arginine hydrochloride / sucrose + NaCl / sucrose + arginine hydrochloride, pH 6.5 <pH6.0。

[0337] 4. HIC results showed that during the stability study, both DAR2% and unknown percentage decreased with increasing acceleration time. The relationship between DAR2% content of the 13 formulations under acceleration at 25℃ and 40℃ is as follows:

[0338] DAR 2%: 0.04% Polysorbate 80 > 0.06% Polysorbate 80, 3.0% ≈ 5.0%

[0339] Mannitol / sorbitol > sucrose / trehalose > arginine hydrochloride / sucrose + NaCl / sucrose + arginine hydrochloride, pH 6.5 > pH 6.0.

[0340] 5. In the accelerated stability tests at 25℃ for 4W and 40℃ for 4W, the SDS-PAGE (NR&R) results showed no significant difference between the formulations. However, after the accelerated test at 40℃ for 4W, it was observed that the aggregation and degradation bands of the samples increased significantly.

[0341] 5. Screening Conclusion

[0342] Based on the results of the accelerated stability test in Excipient Screening Study-I, the surfactant was determined to be 0.04% polysorbate 80, and the alternative stabilizers were 3% mannitol and 3% trehalose. Considering the characteristics of antibody-drug conjugates and the stability of both recombinant HER2 humanized monoclonal antibodies and small molecule drugs, the next round of excipient screening studies will further confirm the optimal pH value.

[0343] (III) Research on Excipient Screening - II

[0344] This round of screening aims to determine the optimal pH and stabilizers that are conducive to the stability of the active pharmaceutical ingredient.

[0345] In Table 26, the "%" for each excipient refers to the corresponding number of grams of solute contained in 100 mL of solvent. For example, "3.0%" means "3g of solute in 100 mL of solvent".

[0346] The histidine / histidine hydrochloride dosage at pH 6.3 is as follows: the 10 mmol / L histidine-histidine hydrochloride aqueous solution contains 0.97 mg / mL histidine and 0.80 mg / mL histidine hydrochloride.

[0347] 1. Table 26 - Excipient Screening Study - II. Composition of Alternative Formulations

[0348]

[0349]

[0350] 2. Preparation method

[0351] Take a solution of the active pharmaceutical ingredient compound C (its structure is shown above), ultrafilter and change the medium, prepare according to the prescription shown in Table 26, adjust the concentration of compound C to 5 mg / mL, filter the sample aseptically and dispense it into 2 mL sterile vials, 1.0 mL per vial.

[0352] 3. Detection Method

[0353] The stability of the samples was investigated at high temperatures of 25℃ and 60%RH and 40℃ and 75%RH, as well as at low temperatures of 5℃, ultra-low temperatures of -40℃ and -80℃. Samples were taken at different time points for testing. The test items included appearance, concentration, pH value, molecular isomers (SEC-HPLC), charge isomers (CEX-HPLC), drug-2% (HIC-HPLC), activity, size, SDS-PAGE (NR&R), subvisible particles (flowcam), NR_CE-purity%, R_CE-(LC+HC)% and AP activity.

[0354] 3.1 Appearance

[0355] Chinese Pharmacopoeia 2020 General Chapter 0102 Injection.

[0356] 3.2 Concentration (A280)

[0357] Chinese Pharmacopoeia 2020 Edition, Part III, General Chapter 0401, Ultraviolet-Visible Spectrophotometry.

[0358] 3.3, pH

[0359] Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0631, pH Value Determination.

[0360] 3.4 Size, Subvisible Particles (flowcam)

[0361] Chinese Pharmacopoeia 2020 Edition, General Chapter 0903, Insoluble Particulate Matter Test Method.

[0362] 3.5 SDS-PAGE (NR&R)

[0363] NR indicates non-reduction, which uses iodoacetamide pretreatment; R indicates reduction, which uses β-mercaptoethanol pretreatment.

[0364] Electrophoresis time is 4-5 hours, voltage is 40-60V. Electrophoresis is stopped as soon as bromophenol blue appears, and staining is performed with Coomassie Brilliant Blue rapid staining solution.

[0365] 3.6, NR_CE-purity%

[0366] Add 75 μL of SDS sample buffer, 25 μL of sample, 5 μL of iodoacetamide 250 mmol solution, and finally 2 μL of 10 kDa internal standard solution to a 1.5 mL EP tube. Mix well. Heat in a water bath at 70 °C for 10 min, then cool to room temperature. Centrifuge at 10,000 rpm for 3 min. Analyze using a capillary electrophoresis apparatus (PDA detector, uncoated capillary inner diameter 50 μm, tube length 30.2 cm, effective length 20 cm).

[0367] 3.7, R_CE-(LC+HC)%

[0368] Add 75 μL of SDS sample buffer, 25 μL of sample, and 5 μL of β-mercaptoethanol to a 1.5 mL EP tube, and finally add 2 μL of 10 kDa internal standard solution. Mix well. Heat in a water bath at 70 °C for 10 min, then cool to room temperature. Centrifuge at 10,000 rpm for 3 min. Analyze using a capillary electrophoresis apparatus (PDA detector, uncoated capillary inner diameter 50 μm, tube length 30.2 cm, effective length 20 cm).

[0369] 3.8 AP activity: Cellular method

[0370] After trypsin digestion, the BT-474 cells were adjusted to a cell density of 1×10⁶ cells using complete culture medium. 5 ~2×10 5Add 100 μL / well of the cell culture medium to each cell culture plate and incubate at 37°C in a 5% CO2 incubator for 14-20 h. Transfer 50 μL / well of the reference and test samples (concentration range 9000 ng / mL to 17.6 ng / mL) serially diluted 2-fold with basal medium into cell culture plates containing cells. Incubate at 37°C in a 5% CO2 incubator for 72 ± 2 h. Add 20 μL of CCK-8 solution to each well, vortex to mix, and incubate at 37°C in a 5% CO2 incubator for 3.5 ± 30 min. Centrifuge at 1000 rpm for 1 min to remove air bubbles, and read the values ​​at 450 nm using a microplate reader.

[0371] 3.9 Molecular isomers

[0372] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0373] Methods: TOSOH TSK G300 column, flow rate 0.5 mL / min, wavelength 280 nm, mobile phase 100 mM sodium dihydrogen phosphate + 0.5% sodium chloride, pH 6.8.

[0374] Result calculation:

[0375] HMW% = A / (A + B + C) * 100%

[0376] Peak percentage = B / (A+B+C)*100%

[0377] LMW% = C / (A + B + C) * 100%

[0378] A represents the sum of peak areas of high molecular weight polymers, B represents the peak area of ​​monomers, and C represents the sum of peak areas of low molecular weight substances.

[0379] High molecular weight polymers refer to polymers formed by the polymerization of monomers into molecules with a molecular weight of 2, 3 or more. Monomers refer to the ADC molecule itself, while low molecular weight substances refer to the detached subunit fragments.

[0380] 3.10. Charged heterostructures

[0381] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0382] Methods: Thermo ProPac SCX-10 column, column temperature: 35 degrees Celsius, wavelength: 214 nm, mobile phase A (20 mM phosphate buffer, pH 6.2), mobile phase B (20 mM phosphate buffer + 200 mM sodium chloride, pH 6.2), flow rate: 1 mL / min, 0 min-35 min was 100% mobile phase A, 35 min-37 min was 100% mobile phase B.

[0383] Result calculation:

[0384] The peaks preceding the main peak are acidic component peaks, and the peaks following the main peak are alkaline component peaks.

[0385] Acidity peak % = Acidity peak area / (Acidity peak area + Main peak area + Basic peak area) * 100%

[0386] Main peak % = Main peak area / (Acid peak area + Main peak area + Basic peak area) * 100%

[0387] Basic peak % = Basic peak area / (Acid peak area + Main peak area + Basic peak area) * 100%.

[0388] 3.11. Number of Completely Coupled Small Molecules

[0389] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0390] Methods: Chromatographic column: TOSOH TSKgel ButyNPR, flow rate: 0.5 mL / min, mobile phase A (50 mM potassium phosphate, 1.5 M ammonium sulfate, pH 6.8), mobile phase B (50 mM potassium phosphate, 20% isopropanol, pH 6.8), 0 min-23 min was 100% mobile phase A, 23 min-26 min was 100% mobile phase B.

[0391] Result calculation:

[0392] DAR0% = (DAR0 component peak area / total peak area) * 100%

[0393] DAR1% = (DAR1 component peak area / total peak area) * 100%

[0394] DAR2% = (DAR2 component peak area / total peak area) * 100%.

[0395] The results showed that the DAR0, DAR1, and DAR2 components all showed peaks at 18 min.

[0396] 3.12. Activity

[0397] Instruments: Microplate reader (MD), centrifuge (Thermo), plate washer (MD)

[0398] Method: ELISA

[0399] Add 30 μL of coating buffer to each well of a 96-well half-well plate and incubate overnight at 2-8°C. Discard the liquid from the plate, wash the plate with a plate washer, pat dry any remaining liquid in the wells, add 60 μL of blocking buffer to each well, and incubate for 1 hour. After dilution, wash the blocked plate again, add 30 μL of sample to each well, and incubate for 1 hour. Wash the plate again, add 30 μL of secondary antibody (goat anti-human IgG antibody kappa chain HRP-labeled enzyme-labeled secondary antibody working solution), and incubate for 1 hour. After washing, add 30 μL of HRP chromogenic substrate TMB, incubate for 15 min, then add stop solution and detect using a microplate reader at 450 nm.

[0400] calculate:

[0401] EC 50 =10^((logIC) 50 -1)-b*log(concentration)); where EC 50 The half-maximal effective concentration (WMC) is given by 'b', where 'b' is the slope parameter and 'concentration' is the drug concentration.

[0402] Relative activity % = Reference EC 50 (ng / mL) / sample EC 50 (ng / mL)*100%.

[0403] 4. Test Results

[0404] H60: 10 mmol / L histidine-hydrochloric acid, pH 6.0, 0.04% polysorbate 80

[0405] H63: 10 mmol / L histidine-hydrochloride, pH 6.3, 0.04% polysorbate 80

[0406] H65: 10 mmol / L histidine-hydrochloride, pH 6.5, 0.04% polysorbate 80

[0407] 4.1, SEC data for -80℃ 8W is shown in Table 27:

[0408] Table 27:

[0409]

[0410]

[0411] Note: "NA" indicates no detection or no result.

[0412] 4.2. SEC data for -40℃ 8W are shown in Tables 28 and 29:

[0413] Table 28:

[0414] Table 29:

[0415] Note: "NA" indicates no detection or no result.

[0416] 4.3, 5℃ 8W SEC data are shown in Tables 30 and 31:

[0417] Table 30:

[0418]

[0419] Table 31:

[0420]

[0421] Note: "NA" indicates no detection or no result.

[0422] 4.4, 25℃ 6W SEC data are shown in Tables 32 and 33:

[0423] Table 32:

[0424]

[0425]

[0426] Table 33:

[0427]

[0428] 4.5, 40℃ 4W SEC data are shown in Tables 34 and 35;

[0429] Table 34:

[0430]

[0431] Table 35:

[0432]

[0433]

[0434] 4.6. The CEX data for -80℃ 8W is shown in Table 36.

[0435] Table 36:

[0436]

[0437] Note: "NA" indicates no detection or no result.

[0438] 4.7. The CEX data for -40℃ 8W is shown in Table 37.

[0439] Table 37:

[0440]

[0441] Note: "NA" indicates no detection or no result.

[0442] 4.8, 5℃ 8W CEX data are shown in Table 38;

[0443] Table 38:

[0444] Note: "NA" indicates no detection or no result.

[0445] 4.9. SEC data at 25℃ and 6W are shown in Tables 39 and 40:

[0446] Table 39:

[0447]

[0448] Table 40:

[0449]

[0450]

[0451] 4.10, 40℃ 4W SEC data are shown in Tables 41 and 42;

[0452] Table 41:

[0453]

[0454] Table 42:

[0455]

[0456]

[0457] 4.11. HIC data at -80℃ and 8W are shown in Table 43;

[0458] Table 43:

[0459]

[0460] Note: "NA" indicates no detection or no result.

[0461] 4.12. HIC data at -40℃ and 8W are shown in Table 44.

[0462] Table 44:

[0463]

[0464] Note: "NA" indicates no detection or no result.

[0465] 4.13, 5℃ 8W HIC data are shown in Tables 45 and 46;

[0466] Table 45:

[0467]

[0468] Table 46:

[0469]

[0470]

[0471] Note: "NA" indicates no detection or no result.

[0472] 4.14. HIC data at 25℃ and 6W are shown in Tables 47 and 48.

[0473] Table 47:

[0474]

[0475] Table 48:

[0476]

[0477]

[0478] 4.15. HIC data at 40℃ and 4W are shown in Tables 49 and 50.

[0479] Table 49:

[0480]

[0481] Table 50:

[0482]

[0483]

[0484] 4.16. Activity data are shown in Tables 51 and 52;

[0485] Table 51:

[0486]

[0487] Note: "NA" indicates no detection or no result.

[0488] Table 52:

[0489]

[0490]

[0491] Note: "NA" indicates no detection or no result.

[0492] 4.17. The test results of each key quality indicator after being placed at -40℃ for 12 weeks are summarized in Table 53.

[0493] H60M: 10 mmol / L histidine-hydrochloride, pH 6.0, 0.04% polysorbate 80, 3% mannitol

[0494] H63M: 10 mmol / L histidine-hydrochloride, pH 6.3, 0.04% polysorbate 80, 3% mannitol. H65M: 10 mmol / L histidine-hydrochloride, pH 6.5, 0.04% polysorbate 80, 3% mannitol.

[0495] Table 53:

[0496]

[0497] Note: "NA" indicates no detection or no result.

[0498] 4.18 Analysis of Test Results:

[0499] 1. In the stability studies at -80℃ 8W, -40℃ 12W, 5℃ 8W, 25℃ 6W, and 40℃ 4W, the results of appearance, concentration, pH, size, SDS-PAGE (NR&R), and sub-visible particle (flowcam) tests showed no significant differences among the six formulations.

[0500] 2. In the stability studies at -80℃ (8W), -40℃ (12W), 5℃ (8W), and 25℃ (6W), SEC testing showed no significant differences among the six formulations, and the SEC peak content did not decrease significantly. However, in the stability study at 40℃ (4W), it was found that at pH 6.3, the SEC peak content was slightly higher than at pH 6.0 and pH 6.5, and there was no significant difference between the two excipients, trehalose and mannitol.

[0501] 3. SEC testing at -80℃ (8W) and -40℃ (12W) showed no significant differences among the six formulations, and the CEX main peak content did not decrease significantly. Stability studies at 40℃ (4W), 5℃ (8W), and 25℃ (6W) showed no significant differences among different pH values ​​(pH 6.0, pH 6.3, pH 6.5) and between the two excipients (trehalose and mannitol).

[0502] 4. In the stability studies at -80℃ (8W), -40℃ (12W), and 5℃ (8W), HIC testing showed no significant differences among the six formulations, and the DAR2% content did not decrease significantly. However, in the stability studies at 25℃ (6W) and 40℃ (4W), it was found that at pH 6.3, the rate of DAR2% decrease was less than at pH 6.0 and pH 6.5, and the formulations containing mannitol showed a slower rate of DAR2% decrease than those containing trehalose.

[0503] 5. Based on the stability data at -40℃, after the sample was placed at -40℃ for 12W, there were no significant changes in the key quality indicators, confirming that this temperature is the optimal storage temperature.

[0504] 5. Screening Conclusion

[0505] Based on the results of Excipient Screening Study-II, and considering the characteristics of the ADC drug, combined with the stability of the recombinant HER2 humanized monoclonal antibody and the small molecule drug, the optimal pH value was determined to be 6.3, and the stabilizer was 3% mannitol. A 10 mmol / L histidine-histidine hydrochloride buffer at pH 6.3 contains 0.97 mg / mL histidine and 0.80 mg / mL histidine hydrochloride. Stability data at -40℃ showed no significant changes in key quality indicators after 12 weeks of storage at -40℃, indicating that this liquid formulation can be stored long-term at -40℃.

[0506] (IV) Freeze-thaw stability study

[0507] 1. Research Methods

[0508] The drug composition was prepared according to the optimal formulation F4b confirmed in (III) Excipient Screening Study-II. After aseptic filtration, it was dispensed into 5 sterilized 20mL vials, 12.5mL per vial, capped, and packaged together with 2550 vials containing placebo or protein substitutes of the same concentration in 64-well medium-sized cryopreservation boxes (40 boxes). The samples were placed at room temperature for about 16 hours. The 40 boxes of frozen samples were placed and frozen at -40℃ in accordance with the cryopreservation method for drug products. Five samples were placed in cryopreservation boxes in different locations in the freezer. After 8 days of storage, all cryopreservation boxes were transferred to a 2-8℃ freezer (without opening the cryopreservation box lids) and placed for half an hour. Then, they were placed at 2-8℃ for about 3 hours. They were then transferred to a warm room for further thawing. After thawing, they were stored at 2-8℃ and the key quality indicators SEC / CEX / HIC / activity were tested in a timely manner.

[0509] 1.1 Molecular isomers

[0510] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0511] Methods: TOSOH TSK G300 column, flow rate 0.5 mL / min, wavelength 280 nm, mobile phase 100 mM sodium dihydrogen phosphate + 0.5% sodium chloride, pH 6.8.

[0512] Result calculation:

[0513] HMW% = A / (A + B + C) * 100%

[0514] Peak percentage = B / (A+B+C)*100%

[0515] LMW% = C / (A + B + C) * 100%

[0516] A represents the sum of peak areas of high molecular weight polymers, B represents the peak area of ​​monomers, and C represents the sum of peak areas of low molecular weight substances.

[0517] High molecular weight polymers refer to polymers formed by the polymerization of monomers into molecules with a molecular weight of 2, 3 or more. Monomers refer to the ADC molecule itself, while low molecular weight substances refer to the detached subunit fragments.

[0518] 1.2. Charge heterostructure

[0519] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0520] Methods: Thermo ProPac SCX-10 column, column temperature: 35 degrees Celsius, wavelength: 214 nm, mobile phase A (20 mM phosphate buffer, pH 6.2), mobile phase B (20 mM phosphate buffer + 200 mM sodium chloride, pH 6.2), flow rate: 1 mL / min, 0 min-35 min was 100% mobile phase A, 35 min-37 min was 100% mobile phase B.

[0521] Result calculation:

[0522] The peaks preceding the main peak are acidic component peaks, and the peaks following the main peak are alkaline component peaks.

[0523] Acidity peak % = Acidity peak area / (Acidity peak area + Main peak area + Basic peak area) * 100%

[0524] Main peak % = Main peak area / (Acid peak area + Main peak area + Basic peak area) * 100%

[0525] Basic peak % = Basic peak area / (Acid peak area + Main peak area + Basic peak area) * 100%.

[0526] 1.3. Number of complete couplings of small molecules

[0527] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0528] Methods: Chromatographic column: TOSOH TSKgel ButyNPR, flow rate: 0.5 mL / min, mobile phase A (50 mM potassium phosphate, 1.5 M ammonium sulfate, pH 6.8), mobile phase B (50 mM potassium phosphate, 20% isopropanol, pH 6.8), 0 min-23 min was 100% mobile phase A, 23 min-26 min was 100% mobile phase B.

[0529] Result calculation:

[0530] DAR0% = (DAR0 component peak area / total peak area) * 100%

[0531] DAR1% = (DAR1 component peak area / total peak area) * 100%

[0532] DAR2% = (DAR2 component peak area / total peak area) * 100%.

[0533] The results showed that the DAR0, DAR1, and DAR2 components all showed peaks at 18 min.

[0534] 1.4. Activity

[0535] Instruments: Microplate reader (MD), centrifuge (Thermo), plate washer (MD)

[0536] Method: ELISA

[0537] Add 30 μL of coating buffer to each well of a 96-well half-well plate and incubate overnight at 2-8°C. Discard the liquid from the plate, wash the plate with a plate washer, pat dry any remaining liquid in the wells, add 60 μL of blocking buffer to each well, and incubate for 1 hour. After dilution, wash the blocked plate again, add 30 μL of sample to each well, and incubate for 1 hour. Wash the plate again, add 30 μL of secondary antibody (goat anti-human IgG antibody kappa chain HRP-labeled enzyme-labeled secondary antibody working solution), and incubate for 1 hour. After washing, add 30 μL of HRP chromogenic substrate TMB, incubate for 15 min, then add stop solution and detect using a microplate reader at 450 nm.

[0538] calculate:

[0539] EC 50 =10^((logIC) 50 -1)-b*log(concentration)); where EC 50 The half-maximal effective concentration (WMC) is given by 'b', where 'b' is the slope parameter and 'concentration' is the drug concentration.

[0540] Relative activity % = Reference EC 50 (ng / mL) / sample EC 50 (ng / mL)*100%.

[0541] 2. Research Results

[0542] 2.1. The freeze-thaw SEC data are shown in Table 54:

[0543] Table 54:

[0544]

[0545] 2.2. Freeze-thaw CEX data are shown in Table 55;

[0546] Table 55:

[0547]

[0548] 2.3. The freeze-thaw HIC data are shown in Table 56.

[0549] Table 56:

[0550]

[0551] 2.4. Freeze-thaw potency data are shown in Tables 57 and 58;

[0552] Table 57:

[0553]

[0554] Table 58:

[0555]

[0556] 3. Research Conclusions

[0557] No significant changes were observed in any of the key quality indicators, indicating that the -40℃ freeze-thaw process did not have a significant impact on the quality of the drug product.

[0558] (V) Stability Study of Freeze-Dried Products

[0559] 1. Freeze-drying method

[0560] The pharmaceutical composition was prepared according to the optimal formulation F4b identified in (III) Excipient Screening Study-II, and filled into 6.3 mL / vial vials. The composition was then freeze-dried, including the following steps:

[0561] (1) Pre-freezing: At atmospheric pressure, temperature -45℃, time 3h;

[0562] (2) One-time drying: vacuum degree 25Pa, temperature -15℃, time 39h;

[0563] (3) Secondary drying: vacuum degree 25Pa, temperature 30℃, time 5h.

[0564] 2. Research Methods

[0565] The freeze-dried product was accelerated at 40℃, and samples were taken and tested at 0 hours, 2 weeks, and 4 weeks; and accelerated at 25℃, and samples were taken and tested at 0 hours, 3 months, and 6 months. Test items included: appearance / color after reconstitution, reconstitution time, protein concentration, pH, osmotic pressure, clarity, insoluble particles, moisture, SEC-HPLC purity, CE-SDS purity, charged heteroplasm, HIC-HPLC, free drug, binding activity, and cell viability.

[0566] 2.1 Appearance / Color after Reconstitution

[0567] Chinese Pharmacopoeia 2020 General Chapter 0102 Injection

[0568] 2.2 Reconstitution time

[0569] Add sterile water for injection at 20-25℃ according to the indicated amount, shake gently, and it needs to be completely dissolved within 10 minutes.

[0570] 2.3 Protein concentration (A280):

[0571] Chinese Pharmacopoeia 2020 Edition, Part III, General Chapter 0401, Ultraviolet-Visible Spectrophotometry

[0572] 2.4, pH:

[0573] Chinese Pharmacopoeia 2020 Edition, Part IV, General Chapter 0631, pH Determination

[0574] 2.5 Osmotic pressure:

[0575] Chinese Pharmacopoeia 2020 General Chapter 0632 Determination of Osmolar Concentration

[0576] 2.6 Clarity:

[0577] Chinese Pharmacopoeia 2020 General Chapter 0902 Clarity Test Method - Turbidity Meter Method

[0578] 2.7. Insoluble particles:

[0579] Chinese Pharmacopoeia 2020 Edition, General Chapter 0903, Insoluble Particulate Matter Test

[0580] 2.8. Moisture:

[0581] Chinese Pharmacopoeia 2020 Edition, General Chapter 0832, Moisture Determination (Coulometric Method)

[0582] 2.9 CE-SDS Purity:

[0583] Same as the NR_CE-purity% test method

[0584] 2.10. Free drug:

[0585] The UPLC system parameter settings are shown in Table 59;

[0586] Table 59:

[0587]

[0588]

[0589] Calculation of small molecule drug residues in samples:

[0590]

[0591] 2.11. Binding activity: ELISA method

[0592] 2 μg / mL of HER2-Fc protein was coated into 96-well half-well plates. After overnight incubation, the plates were blocked with blocking buffer, and then 2.5-fold serially diluted reference and test samples (concentration range: 20000 ng / mL to 5.24 ng / mL) were added. The plates were incubated at room temperature, washed with PBS-T, and then 1:4000 diluted goat anti-human Fc antibody HRP-labeled secondary antibody working solution was added. The plates were then developed using the HRP chromogenic substrate TMB. Finally, stop solution was added, and the plates were read at a detection wavelength of 450 nm on a microplate reader.

[0593] 2.12. Molecular isomers

[0594] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0595] Methods: TOSOH TSK G300 column, flow rate 0.5 mL / min, wavelength 280 nm, mobile phase 100 mM sodium dihydrogen phosphate + 0.5% sodium chloride, pH 6.8.

[0596] Result calculation:

[0597] HMW% = A / (A + B + C) * 100%

[0598] Peak percentage = B / (A+B+C)*100%

[0599] LMW% = C / (A + B + C) * 100%

[0600] A represents the sum of peak areas of high molecular weight polymers, B represents the peak area of ​​monomers, and C represents the sum of peak areas of low molecular weight substances.

[0601] High molecular weight polymers refer to polymers formed by the polymerization of monomers into molecules with a molecular weight of 2, 3 or more. Monomers refer to the ADC molecule itself, while low molecular weight substances refer to the detached subunit fragments.

[0602] 2.13. Charged heterostructures

[0603] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0604] Methods: Thermo ProPac SCX-10 column, column temperature: 35 degrees Celsius, wavelength: 214 nm, mobile phase A (20 mM phosphate buffer, pH 6.2), mobile phase B (20 mM phosphate buffer + 200 mM sodium chloride, pH 6.2), flow rate: 1 mL / min, 0 min-35 min was 100% mobile phase A, 35 min-37 min was 100% mobile phase B.

[0605] Result calculation:

[0606] The peaks preceding the main peak are acidic component peaks, and the peaks following the main peak are alkaline component peaks.

[0607] Acidity peak % = Acidity peak area / (Acidity peak area + Main peak area + Basic peak area) * 100%

[0608] Main peak % = Main peak area / (Acid peak area + Main peak area + Basic peak area) * 100%

[0609] Basic peak % = Basic peak area / (Acid peak area + Main peak area + Basic peak area) * 100%.

[0610] 2.14. Number of Completely Coupled Small Molecules

[0611] Instrument: High Performance Liquid Chromatography (HPLC) (Agilent)

[0612] Methods: Chromatographic column: TOSOH TSKgel ButyNPR, flow rate: 0.5 mL / min, mobile phase A (50 mM potassium phosphate, 1.5 M ammonium sulfate, pH 6.8), mobile phase B (50 mM potassium phosphate, 20% isopropanol, pH 6.8), 0 min-23 min was 100% mobile phase A, 23 min-26 min was 100% mobile phase B.

[0613] Result calculation:

[0614] DAR0% = (DAR0 component peak area / total peak area) * 100%

[0615] DAR1% = (DAR1 component peak area / total peak area) * 100%

[0616] DAR2% = (DAR2 component peak area / total peak area) * 100%.

[0617] The results showed that the DAR0, DAR1, and DAR2 components all showed peaks at 18 min.

[0618] 2.4 Cell viability

[0619] Instruments: Microplate reader (MD), centrifuge (Thermo), plate washer (MD)

[0620] Method: ELISA

[0621] Add 30 μL of coating buffer to each well of a 96-well half-well plate and incubate overnight at 2-8°C. Discard the liquid from the plate, wash the plate with a plate washer, pat dry any remaining liquid in the wells, add 60 μL of blocking buffer to each well, and incubate for 1 hour. After dilution, wash the blocked plate again, add 30 μL of sample to each well, and incubate for 1 hour. Wash the plate again, add 30 μL of secondary antibody (goat anti-human IgG antibody kappa chain HRP-labeled enzyme-labeled secondary antibody working solution), and incubate for 1 hour. After washing, add 30 μL of HRP chromogenic substrate TMB, incubate for 15 min, then add stop solution and detect using a microplate reader at 450 nm.

[0622] calculate:

[0623] EC 50 =10^((logIC) 50 -1)-b*log(concentration)); where EC 50 The half-maximal effective concentration (WMC) is given by 'b', where 'b' is the slope parameter and 'concentration' is the drug concentration.

[0624] Relative activity % = Reference EC 50 (ng / mL) / sample EC 50 (ng / mL)*100%.

[0625] 3. Research Results

[0626] 3.1 The results of the accelerated stability test at 40℃ are shown in Table 60.

[0627] Table 60:

[0628]

[0629] 3.2 The results of accelerated stability testing at 25℃ are shown in Table 61;

[0630] Table 61:

[0631]

[0632]

[0633] 4. Research Conclusions

[0634] After being placed at 40℃ for 4 weeks and at 25℃ for 6 months, the freeze-dried product showed good stability.

Claims

1. A pharmaceutical composition 1 comprising a compound C, a histidine buffer, a saccharide stabilizer, a surfactant, and water, wherein the pharmaceutical composition 1 has a pH of 6.0-6.

5. wherein The compound C is A-(B) n ; ； ； A is trastuzumab-LC-G7CVIM; the trastuzumab-LC-G7CVIM is an antibody in which 11 amino acid residues G7CVIM are added to the C-terminus of the light chain of trastuzumab; B replaces the hydrogen on the cysteine thiol in G7CVIM in A; n is 1-2; In the pharmaceutical composition 1, the mass concentration of the compound C is 2.5-7.5 mg / mL; In the pharmaceutical composition 1, the molar concentration of the histidine buffer is 7.5-12.5 mM, based on the total amount of histidine; In the pharmaceutical composition 1, the saccharide stabilizer is one or both of trehalose and mannitol; The mass concentration of the saccharide stabilizer is 2 g / 100 mL-4 g / 100 mL; In the pharmaceutical composition 1, the surfactant is polysorbate; The mass concentration of the surfactant is 0.03 g / 100 mL-0.05 g / 100 mL.

2. The pharmaceutical composition 1 according to claim 1, wherein One or more of the following conditions are met: a) the pharmaceutical composition 1 is not lyophilized, reconstituted, or the pharmaceutical composition 1 is lyophilized, reconstituted; b) the compound C is a pure substance or a mixture; c) n is 1.7-2.

0.

3. The pharmaceutical composition 1 according to claim 1, wherein One or both of the following conditions are met: (1) the histidine buffer comprises histidine and / or histidine hydrochloride monohydrate; (2) n is 1.

9.

4. The pharmaceutical composition 1 according to claim 1, wherein The histidine buffer comprises histidine and / or histidine hydrochloride.

5. The pharmaceutical composition 1 of claim 1, wherein: The polysorbate is one or more of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

6. The pharmaceutical composition 1 according to claim 1, wherein The pH of the pharmaceutical composition 1 is 6.0, 6.1, 6.2, 6.3, 6.4, or 6.

5.

7. The pharmaceutical composition 1 according to claim 1, wherein The pH of the pharmaceutical composition 1 is 6.2-6.

4.

8. The pharmaceutical composition 1 of claim 1, wherein: The pharmaceutical composition 1 comprises the compound C, the histidine buffer, the saccharide stabilizer, the surfactant, and the water, and the pharmaceutical composition 1 has a pH of 6.0-6.5; wherein the saccharide stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein the mass concentration of the compound C is 2.5-7.5 mg / mL, the molar concentration of the histidine buffer is 7.5-12.5 mM, based on the total amount of histidine, the mass concentration of the saccharide stabilizer is 2-4 g / 100 mL, and the mass concentration of the surfactant is 0.03-0.05 g / 100 mL.

9. The pharmaceutical composition 1 of claim 1, wherein: The pharmaceutical composition 1 comprises the compound C, the histidine buffer, the saccharide stabilizer, the surfactant and the water, and the pH of the pharmaceutical composition 1 is 6.2-6.4; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the saccharide stabilizer is mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 2.5-7.5 mg / mL, the molar concentration of the histidine buffer is 7.5-12.5 mM based on the total amount of histidine, the mass-volume concentration of the saccharide stabilizer is 2-4 g / 100 mL, and the mass-volume concentration of the surfactant is 0.03-0.05 g / 100 mL.

10. The pharmaceutical composition 1 of claim 1, wherein: The pharmaceutical composition 1 comprises the compound C, the histidine buffer, the saccharide stabilizer, the surfactant and the water, and the pH of the pharmaceutical composition 1 is 6.2-6.4; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the saccharide stabilizer is mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 2.5-7.5 mg / mL, the molar concentration of the histidine buffer is 10 mM based on the total amount of histidine, the mass-volume concentration of the saccharide stabilizer is 2-4 g / 100 mL, and the mass-volume concentration of the surfactant is 0.03-0.05 g / 100 mL.

11. The pharmaceutical composition 1 of claim 1, wherein: The pharmaceutical composition 1 comprises the compound C, the histidine buffer, the saccharide stabilizer, the surfactant and the water, and the pH of the pharmaceutical composition 1 is 6.2-6.4; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the saccharide stabilizer is mannitol, and the surfactant is polysorbate 80; wherein the mass-volume concentration of the compound C is 5 mg / mL, the molar concentration of the histidine buffer is 10 mM based on the total amount of histidine, the mass-volume concentration of the saccharide stabilizer is 3 g / 100 mL, and the mass-volume concentration of the surfactant is 0.04 g / 100 mL.

12. The pharmaceutical composition 1 of claim 1, wherein: The pharmaceutical composition 1 comprises 5 mg / mL of the compound C, 10 mM of the histidine buffer, 3 g / 100 mL of mannitol and 0.04 g / 100 mL of polysorbate 80, and the pH of the pharmaceutical composition 1 is 6.3, and the histidine buffer comprises histidine and hydrochloric acid histidine.

13. The pharmaceutical composition 1 of claim 1, wherein: The pharmaceutical composition 1 comprises 5 mg / mL of the compound C, 0.97 mg / mL of histidine, 0.80 mg / mL of hydrochloric acid histidine, 30.0 mg / mL of mannitol and 0.40 mg / mL of polysorbate 80, and the pH of the pharmaceutical composition 1 is 6.

3.

14. The pharmaceutical composition 1 of claim 1, wherein: The pharmaceutical composition 1 comprises 5 mg / mL of the compound C, 0.97 mg / mL of histidine, 0.80 mg / mL of hydrochloric acid histidine, 30.0 mg / mL of mannitol and 0.40 mg / mL of polysorbate 80, and the pH of the pharmaceutical composition 1 is 6.3, and n in the compound C is 1.7-2.

0.

15. A pharmaceutical composition 2, which is prepared by lyophilizing the pharmaceutical composition 1 of any one of claims 1-14.

16. The pharmaceutical composition 2 according to claim 15, wherein The lyophilization is as follows: (1) pre-freezing: normal pressure, temperature -45℃; (2) primary drying: vacuum degree 25 Pa, temperature -15℃; (3) secondary drying: vacuum degree 25 Pa, temperature 30℃.

17. The pharmaceutical composition 2 according to claim 16, wherein The lyophilization is as follows: (1) pre-freezing: normal pressure, temperature -45℃, time 3h; (2) primary drying: vacuum degree 25 Pa, temperature -15℃, time 39h; (3) secondary drying: vacuum degree 25 Pa, temperature 30℃, time 5h.

18. A pharmaceutical composition 3, which comprises the compound C, a histidine buffer, a sugar stabilizer and a surfactant, and the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0-6.

5. wherein The compound C is A-(B) n ; ； ； The A is trastuzumab-LC-G7CVIM; the trastuzumab-LC-G7CVIM is an antibody in which 11 amino acid residues G7CVIM are added to the C-terminus of the light chain of trastuzumab; The B replaces the hydrogen on the cysteine thiol in the G7CVIM of the A; n is 1-2; After the pharmaceutical composition 3 is dissolved in water, the mass-volume concentration of the compound C is 2.5-7.5 mg / mL; The amounts of the histidine buffer, the sugar stabilizer and the surfactant are as follows, based on 2.5-7.5 g of the compound C: The histidine buffer is 7.5-12.5 mmol, based on the total amount of histidine; The sugar stabilizer is 20 g-40 g; The surfactant is 0.3 g-0.5 g; The sugar stabilizer is one or both of trehalose and mannitol; The surfactant is polysorbate.

19. The pharmaceutical composition 3 according to claim 18, wherein It meets one or both of the following conditions: (1) the compound C is a pure substance or a mixture; and (2) n is 1.7-2.

0.

20. The pharmaceutical composition 3 according to claim 18, wherein It meets one or both of the following conditions: (1) the histidine buffer comprises histidine and / or hydrochloric acid histidine monohydrate; and (2) n is 1.

9.

21. The pharmaceutical composition 3 according to claim 18, wherein The histidine buffer comprises histidine and / or hydrochloric acid histidine.

22. The pharmaceutical composition 3 according to claim 18, wherein The polysorbate is one or more of polysorbate 20, polysorbate 40, polysorbate 60 and polysorbate 80.

23. The pharmaceutical composition 3 according to claim 18, wherein The pH of the pharmaceutical composition 3 after dissolving in water is 6.0, 6.1, 6.2, 6.3, 6.4 or 6.

5.

24. The pharmaceutical composition 3 according to claim 18, wherein The pH of the pharmaceutical composition 3 after dissolving in water is 6.2-6.

4.

25. The pharmaceutical composition 3 of claim 18, wherein: The pharmaceutical composition 3 comprises the compound C, the histidine buffer, the sugar stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after dissolving in water is 6.0-6.5; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the sugar stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein, based on 5 g of the compound C: The histidine buffer is 10 mmol based on the total amount of histidine, the sugar stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

26. The pharmaceutical composition 3 of claim 18, wherein: The pharmaceutical composition 3 comprises the compound C, the histidine buffer, the sugar stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after dissolving in water is 6.2-6.4; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the sugar stabilizer is mannitol, and the surfactant is polysorbate 80; wherein, based on 5 g of the compound C: The histidine buffer is 10 mmol based on the total amount of histidine, the sugar stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

27. The pharmaceutical composition 3 of claim 18, wherein: The pharmaceutical composition 3 comprises the compound C, histidine, hydrochloric acid histidine, mannitol and polysorbate 80, and the pH of the pharmaceutical composition 3 after dissolving in water is 6.3; wherein, based on 5 g of the compound C: The histidine is 0.97 g, the hydrochloric acid histidine is 0.80 g, the mannitol is 30 g, and the polysorbate 80 is 0.40 g.

28. The pharmaceutical composition 3 of claim 18, wherein: The pharmaceutical composition 3 comprises the compound C, histidine, hydrochloric acid histidine, mannitol and polysorbate 80, and the pH of the pharmaceutical composition 3 after dissolving in water is 6.3; wherein, based on 5 g of the compound C: The histidine is 0.97 g, the hydrochloric acid histidine is 0.80 g, the mannitol is 30 g, and the polysorbate 80 is 0.40 g, and n in the compound C is 1.7-2.

0.

29. The pharmaceutical composition 3 of claim 18, wherein: The pharmaceutical composition 3 comprises the compound C, the histidine buffer, the sugar stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0-6.5; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the sugar stabilizer is mannitol, and the surfactant is polysorbate 80; the mass-volume concentration of the compound C after the pharmaceutical composition 3 is dissolved in water is 5 mg / mL; wherein the compound C is 5 g: The histidine buffer is 10 mmol in total histidine amount, the sugar stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

30. The pharmaceutical composition 3 of claim 18, wherein: The pharmaceutical composition 3 comprises the compound C, the histidine buffer, the sugar stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0-6.5; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the sugar stabilizer is mannitol, and the surfactant is polysorbate 80; the mass-volume concentration of the compound C after the pharmaceutical composition 3 is dissolved in water is 5 mg / mL; wherein the compound C is 5 g: The histidine buffer is 10 mmol in total histidine amount, the sugar stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

31. The pharmaceutical composition 3 of claim 18, wherein: The pharmaceutical composition 3 comprises the compound C, the histidine buffer, the sugar stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0-6.5; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the sugar stabilizer is mannitol, and the surfactant is polysorbate 80; the mass-volume concentration of the compound C after the pharmaceutical composition 3 is dissolved in water is 5 mg / mL; wherein the compound C is 5 g: The histidine is 0.97 g, the hydrochloric acid histidine is 0.80 g, the mannitol is 30 g, and the polysorbate 80 is 0.40 g.

32. The pharmaceutical composition 3 of claim 18, wherein: The pharmaceutical composition 3 comprises the compound C, the histidine buffer, the sugar stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0-6.5; wherein the histidine buffer comprises histidine and hydrochloric acid histidine, the sugar stabilizer is mannitol, and the surfactant is polysorbate 80; the mass-volume concentration of the compound C after the pharmaceutical composition 3 is dissolved in water is 5 mg / mL; wherein the compound C is 5 g: The histidine is 0.97 g, the hydrochloric acid histidine is 0.80 g, the mannitol is 30 g, and the polysorbate 80 is 0.40 g; and n in the compound C is 1.7-2.

0.

33. Use of the pharmaceutical composition 1 of any one of claims 1-14, the pharmaceutical composition 2 of any one of claims 15-17, or the pharmaceutical composition 3 of any one of claims 18-32 in the manufacture of a medicament for the treatment of a HER2-positive cancer.

34. The use of claim 33, wherein the compound is ###00021### which satisfies one or both of the following conditions; (1) the HER2-positive cancer is breast cancer, non-small cell lung cancer, gastric cancer, or urothelial cancer; (2) the pharmaceutical composition 1, the pharmaceutical composition 2, or the pharmaceutical composition 3 is used in combination with another anti-cancer agent.

35. The use of claim 34, wherein the compound is ###00019### 34 the other anti-cancer agent is a PD-1 inhibitor.

36. The use of claim 35, wherein the compound is ###00019### 35 the PD-1 inhibitor is a monoclonal antibody.

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