Pharmaceutical composition containing antibody drug conjugate and application thereof

Abstract

The invention discloses a pharmaceutical composition containing an antibody drug conjugate and application of the pharmaceutical composition. The invention provides a pharmaceutical composition 1 which comprises a compound C, a histidine buffer agent, a saccharide stabilizer, a surfactant and water, and the pH value of the pharmaceutical composition 1 is 6.0-6.5; wherein the compound C is A-(B) n. The pharmaceutical composition provided by the invention can be stably stored in a liquid form for a long time without freeze-drying, can effectively inhibit ADC molecule aggregation and degradation, has excellent biological activity and safety, and greatly improves the convenience of clinical application while reducing the production cost. Meanwhile, the pharmaceutical composition disclosed by the invention can maintain good stability after being subjected to freeze-drying treatment and redissolution, so that more choices are provided for clinical application.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition containing an antibody-drug conjugate and application thereof. Background Art

[0002] The present invention relates to a pharmaceutical composition of an antibody drug conjugate (ADC), a preparation method and uses thereof. ADC is a new type of antibody drug formed by coupling an antibody or antibody fragment targeting a specific antigen with a payload through a linker. ADC molecules have both the targeting properties of antibody drugs and the potent effects of small molecule drugs, and have good clinical application prospects. Currently, more than a dozen ADC products have been approved for marketing worldwide. In view of the particularity and complexity of the ADC molecular structure, the development of ADC products faces many challenges. For example, inhibiting the focusing and degradation of ADC molecules to ensure their safety and effectiveness has always been a technical difficulty in prescription development. ADC molecules have high heterogeneity, so the development of different ADC products often has personalized characteristics.

[0003] The coupling methods of ADC include non-site coupling and site-specific coupling. Non-site-specific coupling is to directly couple the payload to the amino acid residues on the antibody by chemical methods without transforming or modifying the antibody, and the number of coupled payloads and the coupling position cannot be determined. Site-specific coupling improves the uniformity of ADC by transforming or modifying the antibody to achieve the connection of the payload at a specific site. WO2012153193 discloses an ADC having an amino acid motif that can be recognized by isoprenoid transferase, and WO2015182984 discloses an ADC containing a self-shedding group and having an amino acid motif that can be recognized by isoprenoid transferase. The above-mentioned prior art achieves the purpose of site-specific coupling by introducing an additional amino acid sequence at the end of the antibody chain, thereby achieving precise control of the drug-antibody ratio (DAR) in the ADC molecule, which can improve the pharmacokinetic properties of the ADC molecule and increase its safety and effectiveness. However, there is no report in the prior art about the stable pharmaceutical preparations for clinical use of the ADC molecules prepared based on the above-mentioned site-specific coupling technology. In particular, all ADC products currently on the market are lyophilized powder injections, and there is an urgent need to develop a drug preparation that can be stored stably in liquid form for a long time, in order to reduce production costs and improve the convenience of clinical application. Summary of the invention

[0004] The technical problem to be solved by the present invention is that the existing ADC products are all freeze-dried powder injections. To this end, the present invention provides a pharmaceutical composition containing an antibody drug conjugate and its application. The pharmaceutical composition of the present invention does not need to be freeze-dried, that is, it can be stored stably in liquid form for a long time, can effectively inhibit the aggregation and degradation of ADC molecules, and has excellent biological activity and safety. It greatly improves the convenience of clinical application while reducing production costs. At the same time, the pharmaceutical composition of the present invention can also maintain good stability after freeze-drying and re-dissolution, providing more options for clinical application.

[0005] The present invention provides a pharmaceutical composition 1, which comprises a 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, the compound C is A-(B) n ;

[0007]

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

[0009] The B replaces the hydrogen on the cysteine ​​sulfhydryl group in G7CVIM in A (i.e. connected by a thioether bond);

[0010] n is 1-2.

[0011] In the present invention, the hydrogen on the cysteine ​​sulfhydryl group in G7CVIM in A is the following underlined hydrogen:

[0012]

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

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

[0015] In a certain 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 a certain embodiment, the pharmaceutical composition 1 is not freeze-dried or reconstituted.

[0017] In a certain embodiment, the pharmaceutical composition 1 is freeze-dried and reconstituted.

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

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

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

[0021] In the present invention, an antibody-drug conjugate with a DAR of 1 means that only one of the Bs replaces the hydrogen on one cysteine ​​thiol group in G7CVIM in A; an antibody-drug conjugate with a DAR of 2 means that only two of the Bs replace the hydrogen on two cysteine ​​thiol groups in G7CVIM in A.

[0022] In one embodiment, n is 1.7-2.0.

[0023] In one embodiment, n is 1.9.

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

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

[0026] In a certain embodiment, in the pharmaceutical composition 1, the mass volume concentration of the 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 the present invention, the histidine buffer refers to a buffer containing histidine and / or its salt and having a buffering effect. The histidine used in the art is usually 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 the present invention, in the pharmaceutical composition 1, the molar volume concentration of the histidine buffer is the conventional molar volume concentration in the art, based on the total amount of histidine.

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

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

[0033] In the present invention, the carbohydrate stabilizer is a conventional carbohydrate stabilizer in the art, and the carbohydrate stabilizer includes but is not limited to monosaccharides, oligosaccharides, non-reducing sugars, reducing sugars, sugar alcohols, sugar acids, etc., for example, glucose, fructose, galactose, mannose, sorbitol, ribose, deoxyribose, sucrose, lactose, maltose, melezitose, trehalose, raffinose, arabinose, rhamnose, mannitol, xylitol, erythritol, threitol, sorbitol, glycerol, etc.

[0034] In a certain embodiment, the carbohydrate stabilizer is one or more of sucrose, trehalose, mannitol (i.e., mannitol) and sorbitol (i.e., sorbitol), among which trehalose and / or mannitol are preferred, and mannitol is most preferred.

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

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

[0037] In a certain embodiment, in the pharmaceutical composition 1, the mass volume concentration of the carbohydrate stabilizer is 1g / 100mL-8g / 100mL, 2g / 100mL-6g / 100mL, 3g / 100mL-5g / 100mL, and 2g / 100mL-4g / 100mL, preferably 3g / 100mL.

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

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

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

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

[0042] In the present invention, the poloxamer is a 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 can be poloxamer 124 and / or poloxamer 188, or poloxamer 188.

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

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

[0046] In one embodiment, in the pharmaceutical composition 1, the mass volume concentration of the surfactant is 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 a certain embodiment, in the pharmaceutical composition 1, the mass volume concentration of the surfactant is 0.02g / 100mL-0.08g / 100mL, 0.03g / 100mL-0.07g / 100mL, 0.04g / 100mL-0.06g / 100mL, and 0.03g / 100mL-0.05g / 100mL. Among them, 0.04g / 100mL is preferred.

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

[0049] In a certain embodiment, the pH of the pharmaceutical composition 1 is 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5, wherein 6.3 is preferred.

[0050] In a certain embodiment, the pH of the pharmaceutical composition 1 is 6.1-6.4, or 6.2-6.4, and preferably 6.3.

[0051] In a certain embodiment, the pharmaceutical composition 1 is composed of the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the water.

[0052] In a certain embodiment, the pharmaceutical composition 1 is composed of the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant, the water and the pH adjuster.

[0053] In a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the carbohydrate stabilizer is one or more of 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 amount of histidine, 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 a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the 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, and the molar volume concentration of the histidine buffer is 5-15 mM based on the total amount of histidine, 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 a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the 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 1-10 mg / mL, and the molar volume concentration of the histidine buffer is 7.5-12.5 mM, 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. Calculated on the total amount of histidine,

[0056] In a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the 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, and the molar volume concentration of the histidine buffer is 7.5-12.5 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.

[0057] In a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the 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, and the molar volume concentration of the histidine buffer is 7.5-12.5 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.

[0058] In a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate 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 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 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.

[0059] In a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate 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 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 a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate 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 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 a certain embodiment, 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, 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 the 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 a certain embodiment, the pharmaceutical composition 1 comprises 5 mg / mL of the 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, the pH of the pharmaceutical composition 1 is 6.3, and n in the compound C is 1.7 to 2.0.

[0064] In a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the water, and the pH of the pharmaceutical composition 1 is 6.0-6.5; wherein the carbohydrate stabilizer is one or more of 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, and 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.

[0065] In a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the 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, and 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 a certain embodiment, the pharmaceutical composition 1 comprises the compound C, the histidine buffer, the carbohydrate stabilizer, the surfactant and the 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 is composed of 5 mg / mL of the 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 a certain embodiment, the pharmaceutical composition 1 is composed of 5 mg / mL of the 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 the compound C is 1.7 to 2.0.

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

[0070] In a certain embodiment, the pharmaceutical composition 1 is placed at 40°C for 4 weeks, and DAR2% is ≥50%, preferably ≥55%, and more preferably ≥60%. The test conditions are as follows:

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

[0072] In a certain 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, and more preferably ≥1.9. The test conditions are as follows:

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

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

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

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

[0078] In a certain 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 a certain embodiment, the lyophilization can be as follows:

[0080] (1) Pre-freezing: normal pressure, temperature -45°C;

[0081] (2) Primary drying: vacuum degree 25 Pa, temperature -15 °C;

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

[0083] In a certain embodiment, the lyophilization can be as follows:

[0084] (1) Prefreezing: normal pressure, temperature -45°C, time 3h;

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

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

[0087] The present invention provides a pharmaceutical composition 3, which comprises a 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, the compound C is A-(B) n ;

[0089]

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

[0091] The B replaces the hydrogen on the cysteine ​​sulfhydryl group in G7CVIM in A (i.e. connected by a thioether bond);

[0092] n is 1-2.

[0093] In a certain scheme, some parameters of the pharmaceutical composition 3 are as follows, and the remaining parameters are as described in any other scheme (hereinafter referred to as "in a certain scheme"):

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

[0095] In a certain 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, the compound C is as described in Pharmaceutical Composition 1.

[0097] In a certain embodiment, after the pharmaceutical composition 3 is dissolved in water, the mass volume concentration of the 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 a certain embodiment, based on the compound C being 1-50 g (e.g., 1-25 g, 1-10 g or 2.5-7.5 g, or 5 g), the histidine buffer is 1-25 mmol, 5-15 mmol, 7.5-12.5 mmol, or even 10 mmol.

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

[0101] In a certain embodiment, based on the compound C being 1-50g (e.g., 1-25g, for example, 1-10g or 2.5-7.5g, and for example, 5g), the carbohydrate 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.

[0102] In a certain embodiment, based on the compound C being 1-50g (e.g. 1-25g, 1-10g or 2.5-7.5g, or 5g), the carbohydrate stabilizer is 10g-80g, 20g-60g, 30g-50g, or 20g-40g, preferably 30g.

[0103] In one embodiment, the surfactant is as described in Pharmaceutical Composition 1.

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

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

[0106] In a certain embodiment, the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0, 6.1, 6.2, 6.3, 6.4 or 6.5, wherein 6.3 is preferred.

[0107] In a certain embodiment, the pH of the pharmaceutical composition 3 after being dissolved in water is 6.1-6.4, or 6.2-6.4, and preferably 6.3.

[0108] In a certain embodiment, the pharmaceutical composition 3 is composed of the compound C, the histidine buffer, the carbohydrate stabilizer and the surfactant.

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

[0110] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate 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 histidine hydrochloride, the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein, based on 5 g of the compound C and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

[0111] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate 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 histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein, based on 5 g of the compound C and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

[0112] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after being dissolved 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 5 g of the compound C and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

[0113] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, histidine, histidine hydrochloride, mannitol and polysorbate 80. The pH of the pharmaceutical composition 3 after being dissolved in water is 6.3, wherein, based on 5 g of the compound C, the histidine is 0.97 g, the histidine hydrochloride is 0.80 g, the mannitol is 30 g, and the polysorbate 80 is 0.40 g.

[0114] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, histidine, histidine hydrochloride, mannitol and polysorbate 80. The pH of the pharmaceutical composition 3 after being dissolved in water is 6.3, wherein, based on 5 g of the compound C, the histidine is 0.97 g, the histidine hydrochloride is 0.80 g, the mannitol is 30 g, the polysorbate 80 is 0.40 g, and n in the compound C is 1.7 to 2.0.

[0115] In a certain embodiment, the pharmaceutical composition 3 is composed of the compound C, histidine, histidine hydrochloride, mannitol and polysorbate 80. The pH of the pharmaceutical composition 3 after being dissolved in water is 6.3, wherein, based on 5 g of the compound C, the histidine is 0.97 g, the histidine hydrochloride is 0.80 g, the mannitol is 30 g, and the polysorbate 80 is 0.40 g.

[0116] In a certain embodiment, the pharmaceutical composition 3 is composed of the compound C, histidine, histidine hydrochloride, mannitol and polysorbate 80. The pH of the pharmaceutical composition 3 after being dissolved in water is 6.3, wherein, based on 5 g of the compound C, the histidine is 0.97 g, the histidine hydrochloride is 0.80 g, the mannitol is 30 g, the polysorbate 80 is 0.40 g, and n in the compound C is 1.7 to 2.0.

[0117] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0-6.5; wherein the carbohydrate stabilizer is one or more of sucrose, trehalose, mannitol and sorbitol, and the surfactant is polysorbate; wherein, after the pharmaceutical composition 3 is dissolved 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 and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

[0118] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, Place The sugar stabilizer and the surfactant, the pH of the pharmaceutical composition 3 after being dissolved 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 the pharmaceutical composition 3 is dissolved 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 and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

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

[0120] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after being dissolved 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 the pharmaceutical composition 3 is dissolved in water, the mass volume concentration of the compound C is 5 mg / mL; based on 5 g of the compound C and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g, and the surfactant is 0.3-0.5 g.

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

[0122] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate 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 histidine hydrochloride, the carbohydrate stabilizer is trehalose and / or mannitol, and the surfactant is polysorbate 80; wherein, based on 5 g of the compound C and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 30-50 g, and the surfactant is 0.4-0.6 g.

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

[0124] In a certain embodiment, the pharmaceutical composition 3 comprises the compound C, the histidine buffer, the carbohydrate stabilizer and the surfactant, and the pH of the pharmaceutical composition 3 after being dissolved in water is 6.0-6.5; wherein the carbohydrate stabilizer is one or more of sucrose, trehalose, mannitol and sorbitol, and the surfactant is polysorbate; wherein, after the pharmaceutical composition 3 is dissolved 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 and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 30-50 g, and the surfactant is 0.4-0.6 g.

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

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

[0127] In a certain embodiment, the pharmaceutical composition 3 is composed of the compound C, histidine, histidine hydrochloride, mannitol and polysorbate 80. The pH of the pharmaceutical composition 3 after being dissolved in water is 6.3, wherein after the pharmaceutical composition 3 is dissolved in water, the mass volume concentration of the compound C is 5 mg / mL; based on 5 g of the compound C, the histidine is 0.97 g, the histidine hydrochloride is 0.80 g, the mannitol is 30 g, and the polysorbate 80 is 0.40 g.

[0128] In a certain embodiment, the pharmaceutical composition 3 is composed of the compound C, histidine, histidine hydrochloride, mannitol and polysorbate 80. The pH of the pharmaceutical composition 3 after being dissolved in water is 6.3, wherein after the pharmaceutical composition 3 is dissolved in water, the mass volume concentration of the compound C is 5 mg / mL; based on 5 g of the compound C, the histidine is 0.97 g, the histidine hydrochloride is 0.80 g, the mannitol is 30 g, the polysorbate 80 is 0.40 g, and n in the compound C is 1.7 to 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 application, the HER2-positive cancer may be breast cancer, non-small cell lung cancer, gastric cancer or urothelial carcinoma.

[0131] In the application, the pharmaceutical composition 1, the pharmaceutical composition 2 or the pharmaceutical composition 3 can be used in combination with other anticancer agents, such as PD-1 inhibitors, and the PD-1 inhibitors, such as monoclonal antibodies.

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

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

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

[0135] The term “drug-to-antibody ratio (DAR)” refers to the drug-to-antibody ratio, which refers to the average amount of antibody coupled to a small molecule toxic drug.

[0136] The term "treating" refers to removing the cause or alleviating the symptoms.

[0137] The term "patient" refers to any animal that needs to be treated for a disease, usually a mammal, such as a human. Mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

[0138] The term "therapeutically effective amount" refers to an amount administered to a patient that is sufficient to effectively treat a disease. The therapeutically effective amount will vary depending on the type of drug, the type of disease, the severity of the disease, the age of the patient, etc., but can be adjusted by those skilled in the art as appropriate.

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

[0140] The positive and progressive effects of the present invention are: the pharmaceutical composition of the present invention can be stored stably in liquid form for a long time without freeze-drying, can effectively inhibit the aggregation and degradation of ADC molecules, and has excellent biological activity and safety, greatly improving the convenience of clinical application while reducing production costs. At the same time, the pharmaceutical composition of the present invention can also maintain good stability after freeze-drying and reconstitution, providing more options for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0143] Figure 3 : 40℃, two weeks - reduced and non-reduced SDS-PAGE results DETAILED DESCRIPTION

[0144] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

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

[0146] (I) Buffer system screening

[0147] 1. Prescription configuration

[0148] According to the prescriptions of different buffer systems shown in Table 1, the types of buffer systems and buffer systems with pH ranges that are conducive to the stability of the active ingredient of the drug (Compound C) were screened out.

[0149] Table 1: Composition of alternative buffers for buffer system screening study

[0150]

[0151] 2. Preparation method

[0152]

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

[0154] Group B is as shown above, which replaces the hydrogen on the cysteine ​​sulfhydryl group in G7CVIM in the antibody A, i.e., connected by a thioether bond;

[0155] n is 1.9.

[0156] The solution of the active pharmaceutical ingredient Compound C was replaced with 9 buffer solutions shown in Table 1. After the concentration of Compound C was adjusted to 5 mg / mL, the sample was sterile filtered and dispensed into 2 mL sterile vials, 0.55 mL per vial.

[0157] 3. Detection method

[0158] The packaged samples were placed in constant temperature pharmaceutical storage boxes at 2-8°C, 25±2°C and 40±2°C, and samples were taken for testing at 0 week, 1 week, 2 weeks and 4 weeks. The test items included appearance, concentration (A280), pH value, molecular isomers (SEC-HPLC, SDS-PAGE (NR&R)), charge heterogeneity (CEX-HPLC), small molecule complete coupling number, i.e. the ratio of isomers with a drug number of 2 (Drug-2%) (HIC-HPLC), protein average particle size and polydispersity index (PdI) (DLS).

[0159] 3.1 Appearance

[0160] Chinese Pharmacopoeia 2020 Edition 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 Determination Method.

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

[0166] NR refers to non-reduced, pre-treated with iodoacetamide, and R refers to reduced, pre-treated with β-mercaptoethanol.

[0167] The electrophoresis time is 4-5 hours, and the voltage is 40-60 V. The electrophoresis is terminated when the bromophenol blue just runs out, and the samples are stained with Coomassie Brilliant Blue Rapid Staining Solution.

[0168] 3.5 DLS

[0169] The test sample was diluted with formulation buffer and filtered into a disposable cuvette using a 0.22 μm filter. The cuvette was placed in an 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] Method: 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] Calculation results:

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

[0175] Main peak % = B / (A+B+C)*100%

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

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

[0178] A high molecular weight polymer refers to a polymer formed by polymerizing monomers into a molecular weight of 2, 3 or more times. The monomer refers to the ADC molecule itself, and the low molecular weight substance refers to the detached subunit fragment.

[0179] 3.7 Charge Heterogeneities

[0180] Instrument: High performance liquid HPLC (Agilent)

[0181] Method: Chromatographic column Thermo ProPac SCX-10, column temperature: 35 degrees, wavelength 214nm, mobile phase A (20mM phosphate buffer pH6.2), mobile phase B (20mM phosphate buffer + 200mM sodium chloride pH6.2), flow rate 1mL / min, 0min-35min is 100% mobile phase A, 35min-37min is 100% mobile phase B.

[0182] Calculation results:

[0183] The peak before the main peak is the acidic component peak, and the peak after the main peak is the basic component peak

[0184] Acidic peak % = acidic peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0185] Main peak % = main peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0186] Basic peak % = basic peak area / (acidic peak area + main peak area + basic peak area) * 100%.

[0187] 3.8. Small molecule complete coupling number

[0188] Instrument: High performance liquid HPLC (Agilent)

[0189] Method: Chromatographic column TOSOH TSKgel ButyNPR, flow rate 0.5mL / min, mobile phase A (50mM potassium phosphate, 1.5M ammonium sulfate, pH6.8), mobile phase B (50mM potassium phosphate, 20% isopropanol, pH6.8), 0min-23min is 100% mobile phase A, 23min-26min is 100% mobile phase B.

[0190] Calculation results:

[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 DAR0, DAR1 and DAR2 components all peaked at 18 minutes.

[0195] 4. Test results

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

[0197] Table 2:

[0198]

[0199]

[0200] Note: CS: colorless, slightly opalescent; CS*: colorless, slightly opalescent, with some particles; CS**: colorless, slightly opalescent, with a lot of particles;

[0201] 4.2, The results of 5°C 4W SEC are shown in Table 3;

[0202] Table 3:

[0203]

[0204] 4.3. The results of 2W SEC at 25℃ and 40℃ are shown in Tables 4 and 5;

[0205] Table 4:

[0206]

[0207] Table 5:

[0208]

[0209]

[0210] 4.4, 5℃ 4W CEX data are shown in Table 6;

[0211] Table 6:

[0212]

[0213] 4.5, 25℃and 40℃2W CEX data are shown in Tables 7 and 8;

[0214] Table 7:

[0215]

[0216] Table 8:

[0217]

[0218]

[0219] The data of 4.6 and 5℃ 4W HIC are shown in Tables 9 and 10;

[0220] Table 9:

[0221]

[0222] Table 10:

[0223]

[0224] 4.7, 25℃and 40℃2W HIC data are shown in Tables 11 and 12;

[0225] Table 11:

[0226]

[0227]

[0228] Table 12:

[0229]

[0230] 4.8. Analysis of test results:

[0231] 1. Appearance results showed that no obvious particles appeared after the samples were placed in histidine-histidine hydrochloride buffer pH 5.0-7.0 at 5℃, 25℃, and 40℃ for 4 weeks, while visible foreign matter appeared in histidine-histidine hydrochloride buffer pH 7.5 and citric acid-sodium citrate buffer pH 5.5-6.5 after being placed at 5℃, 25℃, and 40℃ for 2 weeks.

[0232] 2. The concentration, DLS and pH test results showed that there was no significant difference among the 9 prescriptions at three temperatures (5℃, 25℃ and 40℃).

[0233] The SEC results at 3.5℃ for 4W and 25℃ for 2W showed that the SEC main peak content tended to decrease slightly with the increase of pH, and the purity difference between the prescriptions was not large. The SEC results at 40℃ for 2 weeks showed that the SEC main peak content of the samples in histidine-histidine hydrochloride buffer pH 5.5, histidine-histidine hydrochloride buffer pH 6.0, and histidine-histidine hydrochloride buffer pH 6.5 was higher, and the aggregation and degradation were the lowest. Histidine-histidine hydrochloride buffer is better than citric acid-sodium citrate buffer. The relationship between the SEC main peak content between different prescriptions is as follows:

[0234] SEC main peak %: histidine buffer > citric acid buffer, H55 / H60 / H65 > H50 / H70 > H75.

[0235] The 4.25℃ and 40℃ 2W CEX results show that when the pH is greater than 6.0, the acid peak content increases with the increase of pH, and the main peak decreases. When the pH value is less than 6.0, the base peak content increases with the decrease of pH, and the CEX main peak decreases. Comparing the CEX main peak content between different prescriptions, the relationship is as follows:

[0236] CEX main peak %: histidine buffer > citric acid buffer, H50 / H55 / H60 > H65 > H70 > H75.

[0237] 5. From the HIC test results at 5℃, 4W and 25℃ accelerated 2W, it can be concluded that the DAR2% content increases with the increase of pH, and the DAR2% content between different prescriptions. Compared with the citric acid-sodium citrate buffer, the drug small molecules are more difficult to separate from the protein macromolecules in the histidine-histidine hydrochloride buffer. The relationship between the DAR2% content of different prescriptions is as follows:

[0238] DAR2%: histidine buffer > citric acid buffer, H60 / H65 / H70 / H75 > H50 / H55.

[0239] 6. NR&R_SDS-PAGE results show that compared with other formulations, H70 / H75 / C60 / C65 has slightly obvious degradation bands after being placed at 40℃ for 2 weeks, such as Figure 1 , Figure 2 and Figure 3 shown.

[0240] Taking into account the test results of SEC, CEX, HIC, appearance, etc., it can be seen that 10mmol / L histidine-histidine hydrochloride at pH 6.0 / 6.5 is the optimal buffer system.

[0241] 5. Screening conclusion

[0242] According to the results of the accelerated stability test of the buffer system screening study, the histidine buffer system is superior to the citrate buffer system, and the 10mmol / L histidine-histidine hydrochloride buffer system can play a buffering role in stabilizing the pH value of the solution. The pH range that helps to reduce the degree of dissociation of small molecule drugs is 6.0-7.5, but the pH range that helps to increase the stability of protein molecule isomers and charge isomers is 5.0-6.5. Taking into account the characteristics of antibody-drug conjugates, combined with the stability of recombinant HER2 humanized monoclonal antibodies and small molecule drugs, a 10mmol / L histidine-histidine hydrochloride buffer system with a pH value of 6.0-6.5 was selected for subsequent research.

[0243] (II) Research on screening of auxiliary materials - I

[0244] The purpose of excipient screening research is to select an excipient system that is beneficial to the stability of the active ingredients of the drug through a single-factor experimental design based on the determined buffer system.

[0245] The “%” of each excipient in Table 13 refers to the corresponding number of grams of solute contained in every 100 mL of solvent. For example, “3.0%” means “3 g of solute per 100 mL of solvent”.

[0246] 1. Table 13 Excipient Screening Study-I Alternative Prescription Composition

[0247] Table 13:

[0248]

[0249]

[0250] 2. Preparation method

[0251] Take a solution of the active ingredient compound C (whose structure is shown above), ultrafiltrate and replace the liquid, prepare it according to the prescription shown in Table 13, adjust the concentration of compound C to 5 mg / mL, and then aseptically filter the sample and dispense it into 2 mL sterile vials, 1.0 mL per bottle.

[0252] 3. Detection method

[0253] The samples were placed at 25°C 60% RH and 40°C 75% RH for accelerated stability studies. The test items included appearance, concentration (A280), pH value, molecular isomers (SEC-HPLC), charge isomers (CEX-HPLC), Drug-2% (HIC-HPLC), Size, subvisible particles (flowcam) and SDS-PAGE (NR&R).

[0254] 3.1 Appearance

[0255] Chinese Pharmacopoeia 2020 Edition General Chapter 0102 Injection.

[0256] 3.2 Concentration (A280)

[0257] Chinese Pharmacopoeia 2020 Edition Part III General Chapter 0401 Ultraviolet-Visible Spectrophotometry.

[0258] 3.3 pH

[0259] Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0631 pH Determination Method.

[0260] 3.4. Size, sub-visible particles (flowcam)

[0261] Chinese Pharmacopoeia 2020 Edition General Chapter 0903 Insoluble Particles Test Method.

[0262] 3.5. SDS-PAGE (NR&R)

[0263] NR refers to non-reduced, pre-treated with iodoacetamide, and R refers to reduced, pre-treated with β-mercaptoethanol.

[0264] The electrophoresis time is 4-5 hours, and the voltage is 40-60V. The electrophoresis is terminated when the bromophenol blue just runs out, and the samples are stained with Coomassie Brilliant Blue Rapid Staining Solution. 3.6. Molecular Isomers

[0265] Instrument: High performance liquid chromatography HPLC (Agilent)

[0266] Method: 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.

[0267] Calculation results:

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

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

[0270] LMW%=C / (A+B+C)*100%

[0271] A is the sum of the peak areas of high molecular weight polymers, B is the peak area of ​​monomers, and C is the sum of the peak areas of low molecular weight substances.

[0272] A high molecular weight polymer refers to a polymer formed by polymerizing monomers into a molecular weight of 2, 3 or more times. The monomer refers to the ADC molecule itself, and the low molecular weight substance refers to the detached subunit fragment.

[0273] 3.7 Charge Heterogeneities

[0274] Instrument: High performance liquid HPLC (Agilent)

[0275] Method: Chromatographic column Thermo ProPac SCX-10, column temperature: 35 degrees, wavelength 214nm, mobile phase A (20mM phosphate buffer pH6.2), mobile phase B (20mM phosphate buffer + 200mM sodium chloride pH6.2), flow rate 1mL / min, 0min-35min is 100% mobile phase A, 35min-37min is 100% mobile phase B.

[0276] Calculation results:

[0277] The peak before the main peak is the acidic component peak, and the peak after the main peak is the basic component peak

[0278] Acidic peak % = acidic peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0279] Main peak % = main peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0280] Basic peak % = basic peak area / (acidic peak area + main peak area + basic peak area) * 100%.

[0281] 3.8. Small molecule complete coupling number

[0282] Instrument: High performance liquid HPLC (Agilent)

[0283] Method: Chromatographic column TOSOH TSKgel ButyNPR, flow rate 0.5mL / min, mobile phase A (50mM potassium phosphate, 1.5M ammonium sulfate, pH6.8), mobile phase B (50mM potassium phosphate, 20% isopropanol, pH6.8), 0min-23min is 100% mobile phase A, 23min-26min is 100% mobile phase B.

[0284] Calculation results:

[0285] DAR0% = DAR0 component peak area / total peak area * 100%

[0286] DAR1% = DAR1 component peak area / total peak area * 100%

[0287] DAR2%=DAR2 component peak area / total peak area*100%.

[0288] The results showed that DAR0, DAR1 and DAR2 components all peaked at 18 minutes.

[0289] 4. Test results

[0290] 4.1. The data of SEC at 40°C are shown in Tables 14 and 15:

[0291] Table 14:

[0292]

[0293]

[0294] Table 15:

[0295]

[0296]

[0297] 4.2, SEC data at 25°C are shown in Tables 16 and 17;

[0298] Table 16:

[0299]

[0300]

[0301] Table 17:

[0302]

[0303] 4.3, 40℃ CEX data are shown in Tables 18 and 19;

[0304] Table 18:

[0305]

[0306] Table 19:

[0307]

[0308]

[0309] Note: Since the main peak of CEX is relatively small after 2W acceleration at 40℃, the data has no reference significance, so no further testing is performed. No test results are given and are indicated by “ / ”.

[0310] 4.4, 25℃ CEX data are shown in Tables 20 and 21;

[0311] Table 20:

[0312]

[0313] Table 21:

[0314]

[0315]

[0316] 4.5, 40℃ HIC data are shown in Tables 22 and 23;

[0317] Table 22:

[0318]

[0319]

[0320] Table 23:

[0321]

[0322]

[0323] 4.6, 25℃ HIC data are shown in Tables 24 and 25;

[0324] Table 24:

[0325]

[0326]

[0327] Table 25:

[0328]

[0329]

[0330] 4.7. Analysis of test results:

[0331] 1. In the accelerated stability studies at 25℃4W and 40℃4W, the appearance, concentration, pH, size, and sub-visible particles (flowcam) test results showed no significant differences between the prescriptions.

[0332] 2.SEC results show that the aggregation trend of all samples is more obvious than the degradation trend. The relationship between the main peak contents of SEC at 25℃ and 40℃ for 13 prescriptions is as follows:

[0333] SEC main peak%: 0.04% polysorbate 80>0.06% polysorbate 80, concentration 3.0%<5.0%

[0334] Mannitol / sorbitol>sucrose / trehalose>arginine hydrochloride / sucrose+NaCl / sucrose+arginine hydrochloride, pH 6.5 <pH6.0。

[0335] 3. The CEX results show that during the storage process of the stability study, the alkaline peaks of all samples increased faster than the acidic peaks. The CEX main peak content of the 13 prescriptions at 25°C and 40°C accelerated conditions is as follows:

[0336] CEX main peak %: 0.04% polysorbate 80 ≈ 0.06% polysorbate 80, concentration 3.0% ≈ 5.0%

[0337] Sucrose / trehalose / mannitol / sorbitol>arginine hydrochloride / sucrose+NaCl / sucrose+arginine hydrochloride, pH 6.5 <pH6.0。

[0338] 4. HIC results show that during the stable placement process, DAR2% and unkonwn% both decrease with the increase of acceleration time. The DAR2% content of 13 prescriptions at 25℃ and 40℃ acceleration conditions is as follows:

[0339] DAR2%: 0.04% polysorbate 80 > 0.06% polysorbate 80, 3.0% ≈ 5.0%

[0340] Mannitol / sorbitol>sucrose / trehalose>arginine hydrochloride / sucrose+NaCl / sucrose+arginine hydrochloride, pH6.5>pH6.0.

[0341] 5. In the accelerated stability studies at 25°C for 4 weeks and 40°C for 4 weeks, the SDS-PAGE (NR&R) test results showed that there was no significant difference between the prescriptions. However, it was observed that after the accelerated study at 40°C for 4 weeks, the aggregation and degradation bands of the samples increased and were more obvious.

[0342] 5. Screening conclusion

[0343] According to the results of the accelerated stability test of excipient screening study-I, the surfactant was determined to be 0.04% polysorbate 80, and the alternative stabilizers were 3% mannitol and 3% trehalose. Based on the characteristics of antibody-drug conjugates and the comprehensive consideration of the stability of recombinant HER2 humanized monoclonal antibodies and small molecule drugs, the next round of excipient screening studies will further confirm the optimal pH value.

[0344] (III) Research on screening of auxiliary materials - II

[0345] This round of screening aims to determine the optimal pH and stabilizers that are conducive to the stability of the active ingredients of the drug.

[0346] The “%” of each excipient in Table 26 refers to the corresponding number of grams of solute contained in every 100 mL of solvent. For example, “3.0%” means “3 g of solute per 100 mL of solvent”.

[0347] The dosage of histidine / histidine hydrochloride 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.

[0348] 1. Table 26-Excipient Screening Study-II Alternative Formulation Composition

[0349]

[0350]

[0351] 2. Preparation method

[0352] Take a solution of the active ingredient compound C (whose structure is shown above), ultrafiltrate and replace the liquid, prepare it according to the prescription shown in Table 26, adjust the concentration of compound C to 5 mg / mL, and then aseptically filter the sample and dispense it into 2 mL sterile vials, 1.0 mL per bottle.

[0353] 3. Detection method

[0354] The samples were placed at high temperatures of 25°C 60% RH and 40°C 75% RH, as well as low temperatures of 5°C, ultra-low temperatures of -40°C, and -80°C for stability testing. Sampling was taken for testing at different time points. 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.

[0355] 3.1 Appearance

[0356] Chinese Pharmacopoeia 2020 Edition General Chapter 0102 Injection.

[0357] 3.2 Concentration (A280)

[0358] Chinese Pharmacopoeia 2020 Edition Part III General Chapter 0401 Ultraviolet-Visible Spectrophotometry.

[0359] 3.3 pH

[0360] Chinese Pharmacopoeia 2020 Edition Part IV General Chapter 0631 pH Determination Method.

[0361] 3.4. Size, sub-visible particles (flowcam)

[0362] Chinese Pharmacopoeia 2020 Edition General Chapter 0903 Insoluble Particles Test Method.

[0363] 3.5. SDS-PAGE (NR&R)

[0364] NR refers to non-reduced, pre-treated with iodoacetamide, and R refers to reduced, pre-treated with β-mercaptoethanol.

[0365] The electrophoresis time is 4-5 hours, and the voltage is 40-60 V. The electrophoresis is terminated when the bromophenol blue just runs out, and the samples are stained with Coomassie Brilliant Blue Rapid Staining Solution.

[0366] 3.6、NR_CE-purity%

[0367] Add 75ul SDS sample buffer, 25ul sample, 5ul iodoacetamide 250mmol solution to a 1.5ml EP tube, and finally add 2ul 10kDa internal standard solution and mix well. Heat in a water bath at 70℃ for 10min, and cool to room temperature. Centrifuge at 10000rpm for 3min. Analyze using capillary electrophoresis (PDA detector, non-coated capillary inner diameter 50um, tube length 30.2cm, effective length 20cm)

[0368] 3.7, R_CE-(LC+HC)%

[0369] Add 75ul SDS sample buffer, 25ul sample, 5ul β-mercaptoethanol, and finally 2ul 10kDa internal standard solution to a 1.5ml EP tube and mix well. Heat in a water bath at 70℃ for 10min and cool to room temperature. Centrifuge at 10000rpm for 3min. Analyze using capillary electrophoresis (PDA detector, non-coated capillary inner diameter 50um, tube length 30.2cm, effective length 20cm)

[0370] 3.8 AP activity: cell method

[0371] The BT-474 cells after trypsinization were adjusted to a cell density of 1×10 5 ~2×10 5cells / mL, 100 μL / well was added to the cell culture plate, and incubated in a 37°C, 5% CO2 incubator for 14h~20h; the reference sample and the test sample (concentration range is 9000ng / mL~17.6ng / mL) diluted 2 times with the basal culture medium were transferred to the cell culture plate with cells at 50 μL / well; incubated in a 37°C, 5% CO2 incubator for 72±2h; 20 μL of CCK-8 solution was added to each well, oscillated to mix, and incubated in a 37°C, 5% CO2 incubator for 3.5±30min; centrifuged at 1000rpm for 1min to remove bubbles, and the measurement wavelength was selected as 450nm on the microplate reader for reading.

[0372] 3.9 Molecular Isomers

[0373] Instrument: High performance liquid chromatography HPLC (Agilent)

[0374] Method: 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.

[0375] Calculation results:

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

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

[0378] LMW%=C / (A+B+C)*100%

[0379] A is the sum of the peak areas of high molecular weight polymers, B is the peak area of ​​monomers, and C is the sum of the peak areas of low molecular weight substances.

[0380] A high molecular weight polymer refers to a polymer formed by polymerizing monomers into a molecular weight of 2, 3 or more times. The monomer refers to the ADC molecule itself, and the low molecular weight substance refers to the detached subunit fragment.

[0381] 3.10 Charge Heterogeneities

[0382] Instrument: High performance liquid HPLC (Agilent)

[0383] Method: Chromatographic column Thermo ProPac SCX-10, column temperature: 35 degrees, wavelength 214nm, mobile phase A (20mM phosphate buffer pH6.2), mobile phase B (20mM phosphate buffer + 200mM sodium chloride pH6.2), flow rate 1mL / min, 0min-35min is 100% mobile phase A, 35min-37min is 100% mobile phase B.

[0384] Calculation results:

[0385] The peak before the main peak is the acidic component peak, and the peak after the main peak is the basic component peak

[0386] Acidic peak % = acidic peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0387] Main peak % = main peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0388] Basic peak % = basic peak area / (acidic peak area + main peak area + basic peak area) * 100%.

[0389] 3.11. Small molecule complete coupling number

[0390] Instrument: High performance liquid HPLC (Agilent)

[0391] Method: Chromatographic column TOSOH TSKgel ButyNPR, flow rate 0.5mL / min, mobile phase A (50mM potassium phosphate, 1.5M ammonium sulfate, pH6.8), mobile phase B (50mM potassium phosphate, 20% isopropanol, pH6.8), 0min-23min is 100% mobile phase A, 23min-26min is 100% mobile phase B.

[0392] Calculation results:

[0393] DAR0% = DAR0 component peak area / total peak area * 100%

[0394] DAR1% = DAR1 component peak area / total peak area * 100%

[0395] DAR2%=DAR2 component peak area / total peak area*100%.

[0396] The results showed that DAR0, DAR1 and DAR2 components all peaked at 18 minutes.

[0397] 3.12. Activity

[0398] Instruments: ELISA reader (MD), centrifuge (Thermo), plate washer (MD)

[0399] Method: ELISA

[0400] Add 30 μL of coating solution to a 96-well half-well plate and incubate overnight at 2-8 degrees. Shake off the liquid in the plate, wash the plate with a plate washer, pat dry the remaining liquid in the well, add 60 μL of blocking solution to each well, and incubate for 1 hour. After dilution, wash the blocked plate, add 30 μL of sample to each well, and incubate for 1 hour. Then 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 the plate, add 30 μL of HRP colorimetric substrate TMB, incubate for 15 minutes, then add stop solution, and detect with an enzyme reader at 450 nm.

[0401] calculate:

[0402] EC 50 =10^((logIC 50 -1)-b*log(concentration)); where EC 50 is the median effective concentration, b is the slope parameter, and concentration is the drug concentration

[0403] Relative activity % = EC of reference product 50 (ng / mL) / Sample EC 50 (ng / mL)*100%.

[0404] 4. Test results

[0405] H60: 10mmol / L histidine-HCl, pH 6.0, 0.04% polysorbate 80

[0406] H63: 10mmol / L histidine-HCl, pH 6.3, 0.04% polysorbate 80

[0407] H65: 10mmol / L histidine-HCl, pH 6.5, 0.04% polysorbate 80

[0408] 4.1, -80℃ 8W SEC data are shown in Table 27:

[0409] Table 27:

[0410]

[0411]

[0412] Note: “NA” means no test, no test results.

[0413] 4.2, -40℃ 8W SEC data are shown in Tables 28 and 29:

[0414] Table 28:

[0415] Table 29:

[0416] Note: “NA” means no test, no test results.

[0417] 4.3, 5℃ 8W SEC data are shown in Tables 30 and 31:

[0418] Table 30:

[0419] Table 31:

[0420] Note: “NA” means no test, no test results.

[0421] 4.4. 25℃ 6W SEC data are shown in Tables 32 and 33:

[0422] Table 32:

[0423]

[0424]

[0425] Table 33:

[0426]

[0427] 4.5, 40℃ 4W SEC data are shown in Tables 34 and 35;

[0428] Table 34:

[0429]

[0430] Table 35:

[0431]

[0432]

[0433] 4.6, -80℃ 8W CEX data are shown in Table 36;

[0434] Table 36:

[0435] Note: “NA” means no test, no test results.

[0436] 4.7, -40℃ 8W CEX data are shown in Table 37;

[0437] Table 37:

[0438] Note: “NA” means no test, no test results.

[0439] 4.8, 5℃ 8W CEX data are shown in Table 38;

[0440] Table 38:

[0441] Note: “NA” means no test, no test results.

[0442] 4.9, 25℃ 6W SEC data are shown in Tables 39 and 40:

[0443] Table 39:

[0444] Table 40:

[0445]

[0446]

[0447] 4.10, 40℃ 4W SEC data are shown in Tables 41 and 42;

[0448] Table 41:

[0449]

[0450] Table 42:

[0451]

[0452]

[0453] 4.11, -80℃ 8W HIC data are shown in Table 43;

[0454] Table 43:

[0455] Note: “NA” means no test, no test results.

[0456] 4.12, -40℃ 8W HIC data are shown in Table 44;

[0457] Table 44:

[0458] Note: “NA” means no test, no test results.

[0459] 4.13, 5℃ 8W HIC data are shown in Tables 45 and 46;

[0460] Table 45:

[0461]

[0462] Table 46:

[0463]

[0464]

[0465] Note: “NA” means no test, no test results.

[0466] 4.14, 25℃ 6W HIC data are shown in Tables 47 and 48;

[0467] Table 47:

[0468] Table 48:

[0469]

[0470]

[0471] 4.15, 40℃ 4W HIC data are shown in Tables 49 and 50;

[0472] Table 49:

[0473]

[0474] Table 50:

[0475]

[0476]

[0477] 4.16. Activity data are shown in Tables 51 and 52;

[0478] Table 51:

[0479] Note: “NA” means no test, no test results.

[0480] Table 52:

[0481]

[0482]

[0483] Note: “NA” means no test, no test results.

[0484] 4.17, placed at -40℃ for 12 weeks, the test results of each key quality index are summarized in Table 53;

[0485] H60M: 10mmol / L histidine-HCl, pH 6.0, 0.04% polysorbate 80, 3% mannitol

[0486] H63M: 10mmol / L histidine-HCl group, HCl, pH 6.3, 0.04% polysorbate 80, 3% mannitol H65M: 10mmol / L histidine-HCl group, HCl, pH 6.5, 0.04% polysorbate 80, 3% mannitol

[0487] Table 53:

[0488]

[0489] Note: “NA” means no test, no test results.

[0490] 4.18. Analysis of test results:

[0491] 1. In the stability studies at -80℃8W, -40℃12W, 5℃8W, 25℃6W, and 40℃4W, the appearance, concentration, pH, Size, SDS-PAGE (NR&R), and sub-visible particles (flowcam) test results showed no significant differences among the six prescriptions.

[0492] 2. In the stability study at -80℃8W, -40℃12W, 5℃8W, and 25℃6W, the SEC test results showed no significant difference among the six prescriptions, and the SEC main peak content did not decrease significantly. In the stability study at 40℃4W, it was found that at pH6.3, the SEC main peak content was slightly higher than that at pH6.0 and pH6.5, and there was no significant difference between the two excipients, trehalose and mannitol.

[0493] 3. The SEC test results at -80℃8W and -40℃12W showed no significant difference among the six prescriptions, and the CEX main peak content did not decrease significantly. In the stability study at 40℃4W, 5℃8W, and 25℃6W, it was found that there was no significant difference between different pH (pH6.0, pH6.3, pH6.5) and two excipients (trehalose and mannitol).

[0494] 4. In the stability study at -80℃8W, -40℃12W, 5℃8W, HIC test results showed no significant difference among the six prescriptions, and the DAR2% content did not decrease significantly. In the stability study at 25℃6W / 40℃4W, it was found that at pH6.3, the DAR2% decrease rate was lower than that at pH6.0 and pH6.5, and the DAR2% decrease rate of the prescription with mannitol was lower than that of trehalose.

[0495] 5. From the stability data at -40℃, there is no obvious change in the key quality indicators after the sample is placed at -40℃ for 12 weeks, which can be confirmed as the optimal storage temperature.

[0496] 5. Screening conclusion

[0497] According to the results of excipient screening study-II, taking into account the characteristics of ADC drugs, combined with the stability of recombinant HER2 humanized monoclonal antibodies and small molecule drugs, the optimal pH is 6.3 and the stabilizer is 3% mannitol. The 10mmol / L histidine-histidine hydrochloride buffer with a pH of 6.3 contains 0.97mg / mL histidine and 0.80mg / mL histidine hydrochloride. From the stability data at -40℃, after the sample was placed at -40℃ for 12W, there was no significant change in the key quality indicators, indicating that the liquid preparation can be stored for a long time at -40℃.

[0498] (IV) Freeze-thaw stability study

[0499] 1. Research Methods

[0500] The pharmaceutical composition was prepared according to the best prescription F4b confirmed in (III) Excipient Screening Study-II, and after sterile filtration, it was divided into 5 sterilized 20mL vials, each with 12.5mL, stoppered and capped, and packed together with 2550 samples containing placebo or the same concentration of protein substitute in a 64-well medium-sized freezing box (40 boxes), and placed at room temperature for about 16 hours. The 40 boxes of frozen samples were placed and frozen in a -40℃ refrigerator in the same way as the drug product freezing. Five samples were placed in freezing boxes at different positions in the refrigerator. After 8 days of storage, all freezing boxes were transferred to a 2-8℃ refrigerator (without opening the freezing box cover) and placed for half an hour, and then continued to be placed at 2-8℃ for about 3 hours, and then transferred to a greenhouse for further thawing. After thawing, they were stored at 2-8℃, and the key quality indicators SEC / CEX / HIC / activity were tested in time.

[0501] 1.1 Molecular isomers

[0502] Instrument: High performance liquid chromatography HPLC (Agilent)

[0503] Method: 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.

[0504] Calculation results:

[0505] HMW%=A / (A+B+C)*100%

[0506] Main peak % = B / (A+B+C)*100%

[0507] LMW%=C / (A+B+C)*100%

[0508] A is the sum of the peak areas of high molecular weight polymers, B is the peak area of ​​monomers, and C is the sum of the peak areas of low molecular weight substances.

[0509] A high molecular weight polymer refers to a polymer formed by polymerizing monomers into a molecular weight of 2, 3 or more times. The monomer refers to the ADC molecule itself, and the low molecular weight substance refers to the detached subunit fragment.

[0510] 1.2 Charge heterogeneity

[0511] Instrument: High performance liquid HPLC (Agilent)

[0512] Method: Chromatographic column Thermo ProPac SCX-10, column temperature: 35 degrees, wavelength 214nm, mobile phase A (20mM phosphate buffer pH6.2), mobile phase B (20mM phosphate buffer + 200mM sodium chloride pH6.2), flow rate 1mL / min, 0min-35min is 100% mobile phase A, 35min-37min is 100% mobile phase B.

[0513] Calculation results:

[0514] The peak before the main peak is the acidic component peak, and the peak after the main peak is the basic component peak

[0515] Acidic peak % = acidic peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0516] Main peak % = main peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0517] Basic peak % = basic peak area / (acidic peak area + main peak area + basic peak area) * 100%.

[0518] 1.3. Small molecule complete coupling number

[0519] Instrument: High performance liquid HPLC (Agilent)

[0520] Method: Chromatographic column TOSOH TSKgel ButyNPR, flow rate 0.5mL / min, mobile phase A (50mM potassium phosphate, 1.5M ammonium sulfate, pH6.8), mobile phase B (50mM potassium phosphate, 20% isopropanol, pH6.8), 0min-23min is 100% mobile phase A, 23min-26min is 100% mobile phase B.

[0521] Calculation results:

[0522] DAR0% = DAR0 component peak area / total peak area * 100%

[0523] DAR1% = DAR1 component peak area / total peak area * 100%

[0524] DAR2%=DAR2 component peak area / total peak area*100%.

[0525] The results showed that DAR0, DAR1 and DAR2 components all peaked at 18 minutes.

[0526] 1.4. Activity

[0527] Instruments: ELISA reader (MD), centrifuge (Thermo), plate washer (MD)

[0528] Method: ELISA

[0529] Add 30 μL of coating solution to a 96-well half-well plate and incubate overnight at 2-8 degrees. Shake off the liquid in the plate, wash the plate with a plate washer, pat dry the remaining liquid in the well, add 60 μL of blocking solution to each well, and incubate for 1 hour. After dilution, wash the blocked plate, add 30 μL of sample to each well, and incubate for 1 hour. Then 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 the plate, add 30 μL of HRP colorimetric substrate TMB, incubate for 15 minutes, then add stop solution, and detect with an enzyme reader at 450 nm.

[0530] calculate:

[0531] EC 50 =10^((logIC 50 -1)-b*log(concentration)); where EC 50 is the median effective concentration, b is the slope parameter, and concentration is the drug concentration

[0532] Relative activity % = EC of reference product 50 (ng / mL) / Sample EC 50 (ng / mL)*100%.

[0533] 2. Research Results

[0534] 2.1. Freeze-thaw SEC data are shown in Table 54:

[0535] Table 54:

[0536]

[0537] 2.2, freeze-thaw CEX data are shown in Table 55;

[0538] Table 55:

[0539]

[0540] 2.3, freeze-thaw HIC data are shown in Table 56;

[0541] Table 56:

[0542]

[0543] 2.4. Freeze-thaw potency data are shown in Tables 57 and 58;

[0544] Table 57:

[0545]

[0546] Table 58:

[0547]

[0548] 3 Research Conclusions

[0549] There were no significant changes in the key quality indicators, indicating that the -40°C freeze-thaw process had no significant impact on the quality of the drug product.

[0550] (V) Study on stability of freeze-dried products

[0551] 1. Freeze-drying method

[0552] The pharmaceutical composition was prepared according to the optimal prescription F4b confirmed in (III) Excipient Screening Study-II, filled at 6.3 mL / vial, and freeze-dried, including the following steps:

[0553] (1) Prefreezing: normal pressure, temperature -45°C, time 3h;

[0554] (2) Primary drying: vacuum degree 25 Pa, temperature -15 °C, time 39 h;

[0555] (3) Secondary drying: vacuum degree 25 Pa, temperature 30 °C, time 5 h.

[0556] 2. Research Methods

[0557] The freeze-dried finished product was placed at 40°C for acceleration, and samples were taken and tested at 0 hours, 2 weeks, and 4 weeks; and at 25°C for acceleration, and samples were taken and tested at 0 hours, 3 months, and 6 months. The test items include: appearance / color after reconstitution, reconstitution time, protein concentration, pH, osmotic pressure, clarity, insoluble particles, moisture, SEC-HPLC purity, CE-SDS purity, charge heterogeneity, HIC-HPLC, free drug, binding activity, and cell activity.

[0558] 2.1. Appearance / color after reconstitution

[0559] Chinese Pharmacopoeia 2020 General Chapter 0102 Injection

[0560] 2.2 Redissolution time

[0561] Add 20-25℃ sterile water for injection according to the labeled amount and shake gently. It needs to be completely dissolved within 10 minutes.

[0562] 2.3. Protein concentration (A280):

[0563] Chinese Pharmacopoeia 2020 Edition Part III General Rules 0401 Ultraviolet-Visible Spectrophotometry

[0564] 2.4, pH:

[0565] Chinese Pharmacopoeia 2020 Edition Part 4 General Rules 0631 pH Value Determination Method

[0566] 2.5. Osmotic pressure:

[0567] Chinese Pharmacopoeia 2020 General Chapter 0632 Osmolality Determination Method

[0568] 2.6. Clarity:

[0569] Chinese Pharmacopoeia 2020 General Chapter 0902 Clarity Test Method - Turbidimeter Method

[0570] 2.7. Insoluble particles:

[0571] Chinese Pharmacopoeia 2020 General Chapter 0903 Insoluble Particles Test Method

[0572] 2.8. Moisture:

[0573] Chinese Pharmacopoeia 2020 General Chapter 0832 Moisture Determination Method (Coulometric Method)

[0574] 2.9. CE-SDS purity:

[0575] Same as NR_CE-purity% test method

[0576] 2.10 Free drugs:

[0577] The UPLC system parameter settings are shown in Table 59;

[0578] Table 59:

[0579]

[0580]

[0581] Calculation of small molecule drug residues in samples:

[0582]

[0583] 2.11. Binding activity: ELISA method

[0584] 2 μg / mL HER2-Fc protein was coated in a 96-well half-well plate. After overnight, the plate was blocked with blocking solution, and then 2.5-fold gradient dilutions of reference and test products (concentration range: 20000 ng / mL to 5.24 ng / mL) were added. The plates were incubated at room temperature. After washing with PBS-T, 1:4000 diluted goat anti-human Fc antibody HRP enzyme-labeled secondary antibody working solution was added, and color was developed with HRP color developing substrate TMB. Finally, stop solution was added, and the detection wavelength was selected as 450 nm for reading on the microplate reader.

[0585] 2.12 Molecular Isomers

[0586] Instrument: High performance liquid chromatography HPLC (Agilent)

[0587] Method: 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.

[0588] Calculation results:

[0589] HMW%=A / (A+B+C)*100%

[0590] Main peak % = B / (A+B+C)*100%

[0591] LMW%=C / (A+B+C)*100%

[0592] A is the sum of the peak areas of high molecular weight polymers, B is the peak area of ​​monomers, and C is the sum of the peak areas of low molecular weight substances.

[0593] A high molecular weight polymer refers to a polymer formed by polymerizing monomers into a molecular weight of 2, 3 or more times. The monomer refers to the ADC molecule itself, and the low molecular weight substance refers to the detached subunit fragment.

[0594] 2.13 Charge Heterogeneity

[0595] Instrument: High performance liquid HPLC (Agilent)

[0596] Method: Chromatographic column Thermo ProPac SCX-10, column temperature: 35 degrees, wavelength 214nm, mobile phase A (20mM phosphate buffer pH6.2), mobile phase B (20mM phosphate buffer + 200mM sodium chloride pH6.2), flow rate 1mL / min, 0min-35min is 100% mobile phase A, 35min-37min is 100% mobile phase B.

[0597] Calculation results:

[0598] The peak before the main peak is the acidic component peak, and the peak after the main peak is the basic component peak

[0599] Acidic peak % = acidic peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0600] Main peak % = main peak area / (acidic peak area + main peak area + basic peak area) * 100%

[0601] Basic peak % = basic peak area / (acidic peak area + main peak area + basic peak area) * 100%.

[0602] 2.14. Small molecule complete coupling number

[0603] Instrument: High performance liquid HPLC (Agilent)

[0604] Method: Chromatographic column TOSOH TSKgel ButyNPR, flow rate 0.5mL / min, mobile phase A (50mM potassium phosphate, 1.5M ammonium sulfate, pH6.8), mobile phase B (50mM potassium phosphate, 20% isopropanol, pH6.8), 0min-23min is 100% mobile phase A, 23min-26min is 100% mobile phase B.

[0605] Calculation results:

[0606] DAR0% = DAR0 component peak area / total peak area * 100%

[0607] DAR1% = DAR1 component peak area / total peak area * 100%

[0608] DAR2%=DAR2 component peak area / total peak area*100%.

[0609] The results showed that DAR0, DAR1 and DAR2 components all peaked at 18 minutes.

[0610] 2.4 Cell Viability

[0611] Instruments: ELISA reader (MD), centrifuge (Thermo), plate washer (MD)

[0612] Method: ELISA

[0613] Add 30 μL of coating solution to a 96-well half-well plate and incubate overnight at 2-8 degrees. Shake off the liquid in the plate, wash the plate with a plate washer, pat dry the remaining liquid in the well, add 60 μL of blocking solution to each well, and incubate for 1 hour. After dilution, wash the blocked plate, add 30 μL of sample to each well, and incubate for 1 hour. Then 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 the plate, add 30 μL of HRP colorimetric substrate TMB, incubate for 15 minutes, then add stop solution, and detect with an enzyme reader at 450 nm.

[0614] calculate:

[0615] EC 50 =10^((logIC 50 -1)-b*log(concentration)); where EC 50 is the median effective concentration, b is the slope parameter, and concentration is the drug concentration

[0616] Relative activity % = EC of reference product 50 (ng / mL) / Sample EC 50 (ng / mL)*100%.

[0617] 3. Research Results

[0618] 3.1. The results of the 40°C high temperature accelerated stability test are shown in Table 60;

[0619] Table 60:

[0620]

[0621] 3.2, The results of the 25°C accelerated stability test are shown in Table 61;

[0622] Table 61:

[0623]

[0624]

[0625] 4. Research Conclusion

[0626] After being placed at 40°C for 4 weeks and at 25°C for 6 months, the freeze-dried product showed good stability.

Claims

1. A pharmaceutical composition 3 is used in the preparation of a drug, wherein the drug is a drug for treating HER2-positive cancer, the pharmaceutical composition 3 comprises compound C, a histidine buffer, a carbohydrate stabilizer and a surfactant, and the pH of the pharmaceutical composition 3 after being dissolved in water is 6.2-6.4; in, The compound C is A-(B) n ; The A is trastuzumab-LC-G7CVIM; The B replaces the hydrogen on the cysteine ​​sulfhydryl group in G7CVIM in A; n is 1-2; Wherein, the histidine buffer comprises histidine and histidine hydrochloride, the carbohydrate stabilizer is mannitol, and the surfactant is polysorbate 80; wherein, based on 5 g of the compound C and the total amount of histidine, the histidine buffer is 10 mmol, the carbohydrate stabilizer is 20-40 g or 30-50 g, and the surfactant is 0.3-0.5 g or 0.4-0.6 g.

2. The use according to claim 1, characterized in that: The pH of the pharmaceutical composition 3 after being dissolved in water is 6.3, wherein, based on 5 g of the compound C, the histidine is 0.97 g, the histidine hydrochloride is 0.80 g, the mannitol is 30 g, and the polysorbate 80 is 0.40 g.

3. The use according to claim 2, characterized in that: In the compound C, n is 1.7 to 2.

0.

4. The use according to any one of claims 1 to 3, characterized in that: After the pharmaceutical composition 3 is dissolved in water, the mass volume concentration of the compound C is 5 mg / mL.

5. The use according to any one of claims 1 to 4, characterized in that: The HER2-positive cancer is breast cancer, non-small cell lung cancer, gastric cancer or urothelial carcinoma.

6. The use according to claim 5, characterized in that: The pharmaceutical composition 3 is used in combination with other anticancer agents.

7. The use according to claim 6, characterized in that: The other anticancer agent is a PD-1 inhibitor.

8. The use according to claim 7, characterized in that: The PD-1 inhibitor is a monoclonal antibody.

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