Pharmaceutical composition, preparation and preparation method and use of antibody-drug conjugate

Abstract

The present invention discloses a pharmaceutical composition, a preparation, a preparation method and a use of an antibody-drug conjugate. The present invention provides a pharmaceutical composition comprising the following components: 5 to 25 mg / mL of F0124-ADC; 7 to 90 mg / mL of a protein protective agent; 0.05 to 1.0 mg / mL of a surfactant; 5 to 20 mM of a buffer salt; water; the pH value of the pharmaceutical composition is 5.0 to 5.5; wherein F0124-ADC is an antibody conjugate. The pharmaceutical composition greatly improves the production, storage and use stability of the drug, avoids the premature release of small molecule active substances; the long-term stability experiment verifies that the formula can meet the clinical needs of F0124-ADC; and improves the safety of clinical use.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical preparations, and in particular relates to a pharmaceutical composition containing a F0124-ADC antibody-coupled drug, a preparation method thereof, and uses thereof. Background Art

[0002] Cancer is a major disease that poses a serious threat to human survival and social development. It is one of the most serious public health problems in China and even in the world. According to the 2020 U.S. Cancer Statistics Report (Cancer Statistics, 2020), the United States is expected to have 1,806,590 new cancer cases and 606,520 cancer deaths in 2020 [1]. According to the 2019 Cancer Report released by the National Cancer Center of China, there were approximately 3.929 million new cases of malignant tumors in the country in 2015, with an incidence rate of 285.83 / 100,000 (305.47 / 100,000 for males and 265.21 / 100,000 for females). In the same year, there were approximately 2.338 million deaths from malignant tumors, with a mortality rate of 170.05 / 100,000 (210.10 / 100,000 for males and 128.00 / 100,000 for females). Common malignant tumors in China include lung cancer, stomach cancer, colorectal cancer, liver cancer and female breast cancer, and the top 10 cancers account for about 76.70% of all new cases of malignant tumors. Lung cancer, liver cancer, stomach cancer, esophageal cancer and colorectal cancer are the main causes of death from cancer, and the top 10 cancers account for about 83.00% of all deaths from malignant tumors. Therefore, the World Health Organization (WHO) has made conquering cancer a top priority.

[0003] Breast cancer is the most common malignant tumor in women worldwide, of which triple-negative breast cancer (TNBC) accounts for 10% to 20% of all breast cancer pathological types. It is more common in premenopausal women, is highly invasive, easily metastatic, and has a very poor prognosis. The survival period after diagnosis is usually no more than 20 months, and the 5-year survival rate is less than 15%. TNBC is not sensitive to hormone therapy and targeted therapy, and is generally treated as a breast cancer with a poor prognosis. The standard treatment is adjuvant chemotherapy containing anthracyclines or paclitaxel after surgery. Therefore, researchers are still trying to find new mechanisms to combat metastatic triple-negative breast cancer after drug resistance.

[0004] Urothelial carcinoma (UC) is a multi-source malignant tumor originating from the urothelium, including renal pelvic cancer, ureteral cancer, bladder cancer and urethral cancer. It is the most common urinary system tumor. Urothelial carcinoma can be divided into non-muscle-invasive urothelial carcinoma and muscle-invasive urothelial carcinoma. 10%-15% of patients with muscle-invasive urothelial carcinoma have metastasis at the time of diagnosis. The 5-year survival rate for high-risk patients with T3-T4 and (or) N+Mo is only 25%-35%.

[0005] Human trophoblast cell surface glycoprotein antigen (Trop-2), also known as tumor-associated calcium signal transducer 2 (TACSTD2), is the protein product of the TACSTD2 gene and is a type I cell surface glycoprotein that is highly expressed in human cancers. It has been found that Trop-2 is of great significance in the process of embryonic development and tumor cell proliferation and metastasis. Epidemiology has shown that Trop-2 is associated with the development and prognosis of a variety of tumors and can become a target for antibody drug development. Trop-2 antibodies have a high efficiency of binding to Trop-2 endocytosis and are also suitable for the development of antibody-drug conjugates (ADCs). However, Trop-2 is also highly expressed in many healthy tissues, including the kidneys, pancreas, and liver. ADC drugs targeting Trop-2 need to take into account both efficacy and safety.

[0006] Immunomedics Inc launches first commercial product TRODELVY in the U.S. market TM (Sacituzumabgovitecan-hziy, codenamed IMMU-132). TRODELVY is composed of an antibody targeting Trop-2 and SN-38, the active metabolite of the chemotherapy drug irinotecan, connected by a pH-sensitive linker, with a drug-antibody ratio of up to 7.6:1. The U.S. FDA accelerated the approval of TRODELVY for marketing in April 2020 for the treatment of patients with metastatic triple-negative breast cancer (mTNBC) who have received at least two previous treatments. The approval was based on tumor objective response rate (ORR) and duration of response (DoR) data in a multicenter, single-arm, Phase II clinical trial. The ORR of 108 patients with triple-negative breast cancer who had previously received 3 lines of treatment (median, range: 2-10) was 33.3% (95% CI: 24.6%-43.1%), and the median DoR was 7.7 months (95% CI: 4.9-10.8 months).

[0007] DS-1062 is a Trop-2 ADC drug developed by Daiichi Sankyo Co., Ltd. of Japan using its independent ADC technology platform. Its small molecule is a derivative of the camptothecin drug Exatecan. In January 2018, Daiichi Sankyo launched a phase I clinical dose escalation study, and the first patient was dosed in February 2018. PF-06664178 is a Trop-2-ADC drug developed by Pfizer. Its small molecule is a new auristatin derivative Aur0101, which has stronger activity than MMAE. In January 2018, the results of a phase I clinical study showed that the drug had no effect in the treatment of advanced solid tumors. At present, Pfizer has clearly terminated the follow-up research of the drug. The domestic companies that have made rapid progress in the development of Trop-2 target ADC drugs are Bio-Thera, Duoxi Biotech and Kelun Pharmaceutical. Bio-Thera and Duoxi Biotech applied for clinical trials in China in 2019, and the Trop-2 target ADC drug developed by Kelun Pharmaceutical was approved by the FDA to enter clinical research in August 2019.

[0008] Antibody drug conjugates (ADCs) connect monoclonal antibodies to biologically active cytotoxins through chemical connectors, making full use of the antibody's specificity for tumor cell-specific or highly expressed antigens and the high efficiency of cytotoxins to avoid toxic side effects on normal cells. This means that compared with traditional chemotherapy drugs, antibody drug conjugates can accurately bind to tumor cells and reduce the killing effect on normal cells. The therapeutic concept of ADC is to use antibodies as a medium to deliver cytotoxic drugs to tumor cells by binding to target surface antigens and internalizing them.

[0009] However, antibody drugs, especially ADCs, have a larger molecular weight and a more complex structure compared to other chemical drugs. They are easily degraded, polymerized, or undergo undesirable chemical modifications and become unstable. ADCs have a more complex heterogeneous structure than uncoupled antibodies, and the introduction of chemical drugs and linkers greatly changes the physical and chemical properties of ADCs. In order to make ADCs suitable for production, and to maintain stability during storage and subsequent use, and to achieve better effects, the study of stable preparations of ADCs is particularly important. In the development of ADC preparation prescriptions, in addition to considering the stability of naked antibodies, it is also necessary to understand the chemical degradation mechanism of small molecule drugs-linkers, consider the quality attributes of small molecule drugs and linkers together, and find conditions and dosage forms under which naked antibodies, small molecule drugs, and linkers can be stably stored. Under physiological conditions, the ADC drug F0124-ADC described in the present invention releases the drug active SN-38 spontaneously, with a half-life of about 16-20 hours. Providing a sufficiently stable drug (preparation) composition of F0124-ADC is an urgent problem to be solved in the further development of the ADC drug. Summary of the invention

[0010] The present invention aims to solve the problem that the prior art lacks a drug (preparation) composition with good solubility and stability for F0124-ADC antibody-drug conjugates, and provides a drug combination, a freeze-dried preparation, a preparation method and uses of an antibody-drug conjugate.

[0011] The present invention solves the above technical problems through the following technical solutions.

[0012] The development of ADC drug formulations is similar to that of traditional antibody drugs, but the form of antibody-small molecule coupling will introduce new quality attributes. Therefore, the properties of both monoclonal antibodies and small molecules need to be taken into account in the selection of formulations. First, the main framework of ADC is a monoclonal antibody, so the stability of the monoclonal antibody needs to be considered during the formulation research process; secondly, the effect of small molecule drugs on the physicochemical properties of the entire ADC must be fully considered. For example, since the coupled payload is usually a highly hydrophobic small molecule, it is easy to reduce the overall solubility of the ADC drug, which directly affects the drug properties. During the process development of the F0124-ADC preparation, it was found that SN-38 was very easy to spontaneously release during the production, storage and use of the antibody-coupled drug of F0124-ADC, thereby affecting the production and storage stability of the antibody-coupled drug. At the same time, the premature shedding of SN-38 will bring safety risks to the clinical application of the drug. The present invention provides a stable pharmaceutical composition containing F0124-ADC antibody-coupled drug.

[0013] The present invention provides a pharmaceutical composition comprising the following components:

[0014] 5-25 mg / mL of F0124-ADC;

[0015] 7-90 mg / mL protein protectant;

[0016] 0.05-1.0 mg / mL surfactant;

[0017] 5-20 mM buffer salt;

[0018] water;

[0019] The pH value of the pharmaceutical composition is 5.0 to 5.5;

[0020] Among them, F0124-ADC is an antibody conjugate, and its general structural formula is Ab-(LD) n ; Ab is the anti-human Trop2 antibody hRS7, its active fragment or variant thereof; L is a linker; D is the cytotoxic drug SN-38; the Ab is connected to D via the L; n is 2 to 8.

[0021] In one embodiment of the present invention, the structural formula of the F0124-ADC is shown in formula (I), which is referred to as ADC-I:

[0022]

[0023] (I)

[0024] Wherein Ab is the anti-human Trop2 antibody hRS7; n can be 7-8, preferably 7.6.

[0025] In the present invention, the F0124-ADC can be prepared by conventional methods in the art. For example, the above ADC-I can be prepared by the synthesis method disclosed in the literature Clinical Cancer Research, 2011, 17 (10), 3157-3169.

[0026] In the present invention, the pharmaceutical composition is composed of the following components:

[0027] 5-25 mg / mL of F0124-ADC;

[0028] 7-90 mg / mL protein protectant;

[0029] 0.05-1.0 mg / mL surfactant;

[0030] 5-20 mM buffer salt;

[0031] water;

[0032] The pH value of the pharmaceutical composition is 5.0 to 5.5;

[0033] Among them, the definition of F0124-ADC is as described in any of the previous schemes.

[0034] The pH value of the pharmaceutical composition may be 5.0 or 5.5.

[0035] In the pharmaceutical composition, the concentration of F0124-ADC may be 10-20 mg / mL, such as 10 mg / mL or 20 mg / mL.

[0036] In the pharmaceutical composition, the concentration of the protein protectant may be 7 to 60 mg / mL, such as 7 mg / mL, 30 mg / mL, 50 mg / mL or 60 mg / mL.

[0037] In the pharmaceutical composition, the concentration of the surfactant may be 0.05 to 0.6 mg / mL, for example, 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL or 0.6 mg / mL.

[0038] In the pharmaceutical composition, the concentration of the buffer salt may be 10 mM to 20 mM, such as 10 mM or 20 mM.

[0039] The buffer salt may be citrate buffer salt, acetate buffer salt, succinate buffer salt, histidine buffer salt, phosphate buffer salt or Bis-Tris buffer salt, such as succinate buffer salt; the Bis-Tris is bis(2-hydroxyethyl)amino(trihydroxymethyl)methane.

[0040] The citrate buffer salt may be a buffer salt comprising citrate ions, such as citric acid-sodium citrate buffer salt, citric acid-potassium citrate buffer salt, citric acid-calcium citrate buffer salt or citric acid-sodium hydroxide buffer salt, for example citric acid-sodium citrate buffer salt.

[0041] The acetate buffer salt may be a buffer salt including acetate ions, such as acetic acid-sodium acetate buffer salt, acetic acid-potassium acetate buffer salt, acetic acid-sodium hydroxide buffer salt or sodium acetate-hydrochloric acid buffer salt, for example acetic acid-sodium acetate buffer salt.

[0042] The succinate buffer salt may be a buffer salt comprising succinate ions, such as succinate-sodium succinate buffer salt, succinate histidine buffer salt, succinate-potassium succinate buffer salt or succinate-calcium succinate buffer salt, and another example is succinate-sodium succinate.

[0043] The histidine buffer salt can be histidine-hydrochloric acid buffer salt or histidine-histidine hydrochloride buffer salt, such as histidine-hydrochloric acid buffer salt.

[0044] The phosphate buffer salt can be a buffer salt including dihydrogen phosphate ions or a buffer salt including monohydrogen phosphate ions, such as potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer salt, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer salt, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer salt or sodium dihydrogen phosphate-dipotassium hydrogen phosphate buffer salt, and another example is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer salt.

[0045] The Bis-Tris buffer salt can be Bis-Tris-hydrochloric acid buffer salt.

[0046] The protein protecting agent may be a carbohydrate used as a suitable additive or excipient in pharmaceutical formulations in the art, and the carbohydrate may be a sugar substance, such as one or more of sucrose, maltose and dihydrated α, α-trehalose, such as sucrose.

[0047] The surfactant may be one or more of a nonionic surfactant, an anionic surfactant and a zwitterionic surfactant.

[0048] The nonionic surfactant can be one or more of polysorbate, poloxamer, triton, myristyl amide propyl-dimethylamine, palmitamide propyl-dimethylamine, isostearyl amide propyl-dimethylamine, polyethylene glycol, copolymer of ethylene and propylene glycol, and polypropylene glycol, such as polysorbate; the polysorbate is such as polysorbate 20 or polysorbate 80; the poloxamer is such as poloxamer 188; the copolymer of ethylene and propylene glycol is such as Pluronics; the Pluronics is such as PF68 ( F-68).

[0049] The anionic surfactant can be one or more of sodium dodecyl sulfate (SDS), sodium lauryl sulfate, sodium octyl glucoside, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearoyl-sarcosine, sodium methyl cocoyl taurate and disodium methyl oleyl taurate.

[0050] The zwitterionic surfactant may be one or more of lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearyl-sulfobetaine, linoleyl-betaine, myristyl-betaine, or cetyl-betaine, lauroylamidopropyl-betaine, cocoamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitopropyl-betaine and isostearamidopropyl-betaine, such as lauroylamidopropyl-betaine.

[0051] In the present invention, the pharmaceutical composition comprises or consists of the following components:

[0052] 10-20 mg / mL of F0124-ADC;

[0053] 7-60 mg / mL protein protectant;

[0054] 0.09-0.6 mg / mL surfactant;

[0055] 10-20 mM buffer salt;

[0056] water;

[0057] The pH value of the pharmaceutical composition is 5.0 to 5.5;

[0058] Among them, the definition of F0124-ADC is as described in any of the previous schemes.

[0059] In the present invention, the pharmaceutical composition may include or consist of the components shown in any of the following schemes:

[0060] Scheme 1: 20 mg / mL F0124-ADC, 50 mg / mL sucrose, 0.2 mg / mL polysorbate 20, 10 mM succinate buffer, water, the pH value of the pharmaceutical composition is 5.0;

[0061] Scheme 2: 20 mg / mL F0124-ADC, 50 mg / mL sucrose, 0.2 mg / mL polysorbate 20, 10 mM succinate buffer, water, the pH value of the pharmaceutical composition is 5.5;

[0062] Scheme 3: 20 mg / mL F0124-ADC, 60 mg / mL sucrose, 0.1 mg / mL polysorbate 20, 10 mM succinate buffer, water, the pH value of the pharmaceutical composition is 5.0;

[0063] Scheme 4: 20 mg / mL F0124-ADC, 60 mg / mL sucrose, 0.1 mg / mL polysorbate 20, 10 mM succinate buffer, water, the pH value of the pharmaceutical composition is 5.5;

[0064] Scheme 5: 20 mg / mL F0124-ADC, 30 mg / mL sucrose, 0.1 mg / mL polysorbate 20, 10 mM acetate buffer, water, the pH value of the pharmaceutical composition is 5.0;

[0065] Scheme 6: 10 mg / mL F0124-ADC, 7 mg / mL trehalose, 0.09 mg / mL polysorbate 20, 20 mM succinate buffer salt, water, the pH value of the pharmaceutical composition is 5.0;

[0066] Scheme 7: 20 mg / mL F0124-ADC, 60 mg / mL sucrose, 0.1 mg / mL polysorbate 80, 10 mM succinate buffer salt, water, the pH value of the pharmaceutical composition is 5.0;

[0067] Scheme 8: 20 mg / mL F0124-ADC, 60 mg / mL sucrose, 0.6 mg / mL polysorbate 20, 10 mM succinate buffer salt, water, the pH value of the pharmaceutical composition is 5.0;

[0068] In the schemes 1 to 8, the structure of F0124-ADC is preferably a compound (ADC-I) as shown in formula (I):

[0069]

[0070] (I)

[0071] Wherein Ab is the anti-human Trop2 antibody hRS7; n is 7-8, preferably 7.6.

[0072] The present invention also provides a freeze-dried preparation, which is prepared by freeze-drying the pharmaceutical composition as described in any of the above schemes.

[0073] The present invention also provides a freeze-dried preparation, which contains the above-mentioned pharmaceutical composition after being dissolved. The dissolution process preferably uses one or more of purified water, water for injection and physiological saline, and more preferably uses water for injection.

[0074] The present invention also provides an application of a pharmaceutical composition or a freeze-dried preparation in preparing a drug for treating Trop-2 positive tumors. The pharmaceutical composition and the freeze-dried preparation are as described above.

[0075] In the application, the Trop-2 positive tumor may be breast cancer or urothelial carcinoma; the breast cancer may be triple negative breast cancer (TNBC), and the urothelial carcinoma may be renal pelvic cancer, ureteral cancer, bladder cancer or urethral cancer.

[0076] The present invention also provides a method for treating and / or preventing Trop-2 positive tumors, comprising administering a therapeutically effective amount of the pharmaceutical composition or freeze-dried preparation as described above to a patient in need thereof.

[0077] In the method for treating and / or preventing Trop-2 positive tumors, the Trop-2 positive tumor may be breast cancer or urothelial carcinoma; the breast cancer may be triple negative breast cancer (TNBC), and the urothelial carcinoma may be renal pelvic cancer, ureteral cancer, bladder cancer or urethral cancer.

[0078] The present invention also provides a pharmaceutical product, which comprises a container, wherein the pharmaceutical composition or freeze-dried preparation as described above is contained in the container. The container is preferably a glass bottle or a liquid storage bag.

[0079] In order to make the present invention more easily understood, some technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0080] "Pharmaceutical composition" means a mixture containing one or more compounds described herein or their physiologically / pharmaceutically acceptable salts or prodrugs and other chemical components, such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration to an organism, facilitate the absorption of the active ingredient, and thus exert biological activity. Herein, "pharmaceutical composition" and "preparation" are not mutually exclusive.

[0081] The pharmaceutical composition of the present invention can achieve a stable effect: the antibody therein substantially retains its physical stability and / or chemical stability and / or biological activity after storage. Preferably, the pharmaceutical composition substantially retains its physical and chemical stability and its biological activity after storage. The storage period is generally selected based on the predetermined shelf life of the pharmaceutical composition. There are currently a variety of analytical techniques for measuring protein stability, which can measure stability after storage at a selected temperature for a selected period of time.

[0082] "Freeze-dried preparation" means a pharmaceutical composition in liquid or solution form or a preparation obtained after a liquid or solution preparation has been subjected to a freeze-drying step in vacuo.

[0083] "Antibody drug conjugate (ADC)" is a monoclonal antibody or antibody fragment connected to a biologically active cytotoxin or a small molecule drug with cell-killing activity through a stable chemical linker compound, which fully utilizes the antibody's specificity for binding to tumor cell-specific or highly expressed antigens and the high efficiency of cytotoxins to avoid toxic side effects on normal cells. This means that compared with traditional chemotherapy drugs, antibody drug conjugates can accurately bind to tumor cells and reduce the impact on normal cells.

[0084] “Antibody-drug conjugate F0124-ADC” refers to an antibody-drug conjugate of anti-Trop2 antibody conjugated to SN38.

[0085] "pH buffer" refers to a buffer that tolerates changes in pH through the action of its acid-base conjugate components.

[0086] WFI: water for injection, water for injection.

[0087] The term "treatment" or its equivalent, when used, for example, for cancer, refers to a procedure or process used to reduce or eliminate the number of cancer cells in a patient or to alleviate the symptoms of cancer. "Treatment" of cancer or another proliferative disorder does not necessarily mean that cancer cells or other disorders will actually be eliminated, that the number of cells or disorders will actually be reduced, or that the symptoms of cancer or other disorders will actually be alleviated. In general, a method of treating cancer will be performed even if it has only a low probability of success, but it is still believed to induce an overall beneficial course of action, taking into account the patient's medical history and estimated survival expectancy.

[0088] An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of a medical disease. An effective amount also means an amount sufficient to allow or facilitate diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition to be treated, the patient's general health, the method, route and dosage of administration, and the severity of side effects. An effective amount may be the maximum dose or dosage regimen that avoids significant side effects or toxic effects.

[0089] The term "prevention" refers to reducing the risk of developing a disease.

[0090] The term "patient" refers to any animal that has been or is about to be treated, preferably a mammal, most preferably a human. Mammals include, but are not limited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, humans, etc.

[0091] "Tm value" refers to the thermal denaturation temperature of protein, that is, the temperature when half of the protein unfolds. At this time, the spatial structure of the protein is destroyed. Therefore, the higher the Tm value, the higher the thermal stability of the protein.

[0092] Without violating the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0093] The reagents and raw materials used in the present invention are commercially available.

[0094] The positive and progressive effects of the present invention are as follows: the pharmaceutical composition (preparation) containing the F0124-ADC antibody-drug conjugate of the present invention greatly improves the production, storage and use stability of the drug, and avoids the premature release of small molecule active substances; the long-term stability experiment verifies that the formula can meet the clinical needs of F0124-ADC; and the safety of clinical use is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 The DAR value variation trends of the eight prescription solutions in Example 3 when placed at 2-8°C respectively.

[0096] Figure 2 The DAR value variation trends of the eight prescription solutions in Example 3 when placed at 25°C are shown.

[0097] Figure 3 The residual small molecules variation trends of the eight prescription solutions in Example 3 when placed at 2-8°C.

[0098] Figure 4 The residual small molecules variation trends of the eight prescription solutions in Example 3 when placed at 25°C are shown.

[0099] Figure 5 The CE-SDS high molecular impurity change trends of the eight prescription solutions in Example 3 were placed at 2-8°C.

[0100] Figure 6 The CE-SDS high molecular impurity change trends of the eight prescription solutions in Example 3 when placed at 25°C are shown.

[0101] Figure 7 The SEC polymer variation trends of the eight prescription solutions in Example 3 were placed at 2-8°C.

[0102] Figure 8 The SEC polymer change trends of the eight prescription solutions in Example 3 placed at 25°C are shown.

[0103] Fig. 9 The SEC polymer change trends after freeze-thaw of the eight prescription solutions in Example 3.

[0104] Fig.10 The DAR value variation trend of the eight freeze-dried products in Example 4 at 25°C is shown.

[0105] Fig.11 The DAR value variation trend of the eight freeze-dried products in Example 4 at 40°C is shown.

[0106] Fig.12 The variation trend of small molecule residues of eight freeze-dried products of the prescriptions in Example 4 at 25°C is shown.

[0107] Fig.13 The small molecule residue variation trend of the eight freeze-dried products of Example 4 at 40°C.

[0108] Fig.14 The SEC polymer variation trends of the eight freeze-dried products of Example 4 at 25°C are shown.

[0109] Fig.15 The SEC polymer variation trends of the eight freeze-dried products of Example 4 at 40°C are shown. DETAILED DESCRIPTION

[0110] 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.

[0111] abbreviation:

[0112] MES: 2-(N-morpholino)ethanesulfonic acid.

[0113] Materials and Methods

[0114] F0124-ADC: can be prepared by conventional methods in the art. For example, ADC-I can be prepared by the synthesis method disclosed in the literature Clinical Cancer Research, 2011, 17(10), 3157-3169.

[0115] Main instruments and reagents: freeze dryer TELSTAR LyoBeta 6PL; high performance liquid analysis system Waters E2695; chromatographic column TOSOH TSKgel G3000SWXL (5μm, 7.8mm×300mm); capillary electrophoresis analysis system Beckman Coulter PA800 plus; NanoTemper protein stability analyzer Prometheus NT.48; coulometric Karl Fischer titrator Metrohm 831 KF Coulometer; multifunctional microplate reader Molecular Devices SpectraMax m2e. Succinic acid was purchased from Nanjing Chemical Reagent Co., Ltd.; sucrose was purchased from Merck; histidine was purchased from Shanghai Xiehe Amino Acid Co., Ltd.; polysorbate 20 was purchased from JTBaker.

[0116] Differential scanning fluorescence (DSF): The hydrophobic components of proteins under physiological conditions are wrapped inside the molecule to form a hydrophilic surface to maintain stability. As the external temperature increases, the protein denatures and exposes the hydrophobic area. The hydrophobic fluorescent dye binds to the exposed hydrophobic area and then emits fluorescence. Running on a real-time quantitative PCR system can obtain specific fluorescence images (melting curves) of the protein to be tested or in different buffer environments. The relative change of Tm (melting temperature) calculated from this can reflect the speed of exposure (denaturation speed) of the hydrophobic area of ​​the protein under different proteins or different buffer conditions, that is, the higher the Tm, the higher the temperature required for protein denaturation, and the better the protein stability. NanoDSF technology is used to detect the thermal stability of antibodies. The heating rate is set to 1℃ / min, the starting temperature is 25℃, and the ending temperature is 99℃. Tm and Tagg are determined based on the changes in the ultraviolet absorption ratio of each antibody and the changes in the scattering intensity of the antibody to light.

[0117] Size exclusion chromatography (SEC purity analysis): chromatographic column, TSKgel G3000SWXL (5 μm, 7.8 mm × 300 mm); mobile phase, 0.2 mol / L K 2 HPO 4 , 0.2mol / L KCl, 15% isopropanol, pH 5.5; flow rate, 0.6ml / min; sample load: 200μg; column temperature, 25℃; sample pool temperature, 5℃; detection wavelength, 280nm. The experimental results were processed using a Waters E2695 high performance liquid analysis system, and the purity was calculated using the area normalization method.

[0118] Capillary electrophoresis (CE-SDS) purity analysis: dilute the sample to 1 mg / ml with SDS sample buffer (0.1 mol / L Tris-HCl buffer, containing 1% SDS, pH 9.0), take 95 μl of the diluted protein solution, 2 μl of 10KDa internal standard, and 5 μl of β-mercaptoethanol, add all of them into a 0.5 ml centrifuge tube, vortex and mix, place in a constant temperature mixer at 70°C for 10 minutes, cool to room temperature, take 100 μl, and use Beckman Coulter PA800 plus capillary electrophoresis analysis system to process the sample, bandwidth: 10 nm, sample cell temperature: 10°C, capillary temperature: 25°C, detection wavelength: 220 nm. The original spectrum was imported into Empower software and calculated by area normalization method.

[0119] Water content analysis: Refer to the coulometric titration method in the first method of General Rules 0832 of the 2015 edition of the Pharmacopoeia of the People's Republic of China, based on the Karl-Fischer reaction, and use the permanent stop titration method (General Rules 0701 of the 2015 edition of the Pharmacopoeia of the People's Republic of China) to determine the sample, and determine the water content based on the principle that iodine and sulfur dioxide react quantitatively with water in pyridine and methanol solutions. Quickly crush the sample, accurately weigh about 20 mg of the sample and add it to the moisture analyzer for reaction. Measure 3 bottles of sample per batch, and measure each bottle twice.

[0120] Relative binding activity analysis: hTrop2 antigen was coated in a 96-well ELISA plate at 108 ng / ml and left to stand overnight at 2-8°C. 3% BSA was used for blocking at room temperature for 2 hours. The gradient diluted F0124-ADC sample (20000 ng / ml~1.4 ng / ml) was mixed with the biotinylated FDA 018 antibody in equal volumes and added to the ELISA plate to compete for binding to hTrop2 antigen. The plate was incubated at 200 rpm for 1 hour. HRP-labeled streptavidin was used as the enzyme linker and incubated for 1 hour under the same conditions as above. TMB was used for color development, and 1 mol / L H 2 SO 4Terminate. For the microplate reader m2e, read at 450nm (detection) / 650nm (reference) wavelength. Select the four-parameter fitting method to analyze the data.

[0121] Biological activity analysis: Karpas299 cells were diluted to 5×10 4 / ml, 100 μl / well was seeded in a 96-well cell culture plate. Then, a gradient dilution of F0124-ADC sample (50 ng / ml to 0.3 ng / ml) was added. Incubate at 37°C with 5% CO 2 After incubation in an incubator for 77 hours, 30 μl / well of Alamar Blue dye was added and incubated for another 19 hours. After mixing at room temperature for half an hour, the microplate reader m2e was used to read the wavelengths of 530 nm (excitation) / 590 nm (emission). The four-parameter fitting curve was selected for data analysis of the results.

[0122] In the following examples, "succinate buffer salt" refers to succinic acid-sodium succinate buffer salt; "histidine buffer salt" refers to histidine-hydrochloric acid buffer salt; "acetate buffer salt" refers to acetic acid-sodium acetate buffer salt.

[0123] Example 1 Preparation of solution preparations with different prescriptions

[0124] The prescriptions of each pharmaceutical composition are shown in Table 1. The preparation methods of solution preparations of different prescriptions include the following steps:

[0125] (1) Preparation of diafiltration buffer for replacement: according to the composition and concentration of the buffer salt in the prescription of each pharmaceutical composition, succinic acid or histidine is dissolved in water for injection and mixed evenly. For the buffer salt composition of succinic acid-sodium succinate buffer salt, sodium hydroxide is added to adjust the pH value to meet the prescription requirements; and for the buffer salt composition of histidine-hydrochloric acid buffer salt, concentrated hydrochloric acid is added to adjust the pH value to meet the prescription requirements;

[0126] (2) Preparation of dilution buffer: According to the prescription of each pharmaceutical composition, succinic acid or histidine, protective agent and surfactant other than the antibody conjugate are dissolved in water for injection, mixed evenly, and the pH value is adjusted to meet the prescription requirements; for each pharmaceutical composition prescription, except for the addition of the antibody drug conjugate, the types and concentrations of buffer salts, protective agents and surfactants in the dilution buffer are consistent with the prescription of the pharmaceutical composition, for example, the dilution buffer of the composition prescription numbered 1 in Table 1 contains 10 mM succinate buffer salt, 50 mg / mL sucrose as a protein protective group and 0.2 mg / mL polysorbate 20 as a surfactant, and the pH value is 5.0;

[0127] (3) In order to prepare different formulations, the protein solution containing the antibody conjugate (prepared according to the synthesis method disclosed in the document Clinical Cancer Research, 2011, 17 (10), 3157-3169) is subjected to buffer exchange of the diafiltration buffer by concentrated diafiltration (UF / DF), and when necessary, concentrated by diafiltration to an antibody conjugate solution having a protein concentration of about 20-35 mg / ml. After the diafiltration operation is completed, a protein protective agent (e.g., sucrose) is dissolved in the diafiltration buffer to form a concentrated stock solution, which is added to the antibody conjugate solution. Then, a surfactant (e.g., polysorbate 20) is dissolved in the diafiltration buffer to form a 50 to 200-fold concentrated stock solution, which is added to the antibody conjugate solution. Finally, the protein concentration is adjusted to a final protein concentration of about 10 mg / ml, 15 mg / ml, 20 mg / ml, or 25 mg / ml using the corresponding dilution buffer.

[0128] (4) All solution preparations were sterile filtered through a 0.22 μm sterilizing filter and filled into 2 ml sterile cryovials under aseptic conditions and sealed for storage.

[0129] Example 2 Preparation of freeze-dried preparation

[0130] This example studies the effects of buffer salts such as succinate buffer salt, histidine buffer salt, and pH value on freeze-dried preparations.

[0131] The preparation method of freeze-dried preparation is as follows:

[0132] The solution preparation of the pharmaceutical composition in Table 1 prepared according to the method of Example 1 is aseptically divided into vials, and the vials containing the samples are placed in a LyoBeta 6PL freeze dryer to start freeze drying. Freeze drying is carried out according to the preset freeze drying parameters, precooling at 5°C for 60 minutes; prefreezing at -5°C for 90 minutes: prefreezing at -45°C for 300 minutes; the primary drying temperature is set to -22°C, the duration is not less than 3000 minutes, and the vacuum degree is controlled at 0.08-0.18 mbar; the analytical drying temperature is set to 30°C, and the duration is not less than 300 minutes. After freeze drying, vacuum plugging is performed. Capping is performed after unpacking.

[0133] Table 1. Tm values ​​and water contents of various pharmaceutical composition prescriptions and freeze-dried preparations

[0134]

[0135] Differential scanning fluorescence (DSF) is a thermal analysis method and an important tool for studying protein stability. The unfolding of protein molecules and the exposure of hydrophobic parts will cause fluorescence changes of fluorescent dyes. In this method, protein samples are incubated with fluorescent dyes, and the fluorescence changes are monitored by linear temperature increase. The stability of protein molecules in a certain formulation system is predicted by measuring the protein melting temperature (Tm value).

[0136] As shown in Table 1, under the same pH conditions, as the concentration of buffer salts increases, the Tm value of the formulation decreases, indicating that the increase in the concentration of buffer salts may be detrimental to the stability of the formulation. At the same time, within the selected range, the increase in pH can increase the Tm value of the formulation, indicating that the stability of the formulation may be enhanced. Therefore, the stability of formulations at different pH values ​​needs to be further evaluated.

[0137] Example 3 Study on the stability of ADC-I solution formulations at different pH values

[0138] This example compares the changes in DAR values ​​and small molecule residues of solution preparations with different pH prescriptions (prescriptions are shown in Table 2, prepared according to the methods described in Examples 1 and 2) at different storage temperatures of 2-8°C or 25°C (as shown in Tables 3-6, Figure 1-4 (as shown), reflecting the difference in stability of ADC-I solution preparations at different pH. Among them, the prescription of prescription E is obtained by replacing IMMU-132 in the prescription of Trodelvy, a similar foreign drug, with ADC-I. The results show that the shedding of SN-38 under prescription E (pH 6.5) and prescription C (pH 6.0) is significantly stronger than that of prescriptions A, D, F, G, H (pH 5.0) and prescription B (pH 5.5), which is manifested by a decrease in DAR value and an increase in small molecule residues. The difference between prescriptions A, D, F, G, H (pH 5.0) and prescription B (pH 5.5) is not very obvious. At the same time, with the increase in temperature, the trend of SN-38 shedding increases more obviously. Therefore, in order to ensure the long-term stability of the preparation in the solution state, it should be considered to be stored and transported at low temperatures (such as -20°C or -80°C).

[0139] Table 2. Prescriptions of pharmaceutical compositions at different pH values

[0140] prescription pH ADC-I Buffer salt Protein Protectant Surfactants A 5.0 20mg / ml 10 mM succinate 60mg / ml sucrose 0.1mg / ml Polysorbate 20 B 5.5 20mg / ml 10 mM succinate 60mg / ml sucrose 0.1mg / ml Polysorbate 20 C 6.0 20mg / ml 10 mM succinate 60mg / ml sucrose 0.1mg / ml Polysorbate 20 D 5.0 20mg / ml 10 mM acetate 30mg / ml sucrose 0.1mg / ml Polysorbate 20 E 6.5 10mg / ml 20mM MES 7mg / ml trehalose 0.09mg / ml Polysorbate 20 F 5.0 10mg / ml 20 mM succinate 7mg / ml trehalose 0.09mg / ml Polysorbate 20 G 5.0 20mg / ml 10 mM succinate 60mg / ml sucrose 0.1mg / ml Polysorbate 80 H 5.0 20mg / ml 10 mM succinate 60mg / ml sucrose 0.6mg / ml Polysorbate 20

[0141] The trehalose in Table 2 is α,α-trehalose dihydrate.

[0142] Table 3. Changes in DAR values ​​of solutions at 2-8°C

[0143] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 7.48 7.46 7.37 7.35 7.34 7.47 7.46 7.46 1 7.46 7.46 7.32 7.34 7.29 7.46 7.46 7.45 3 7.45 7.45 7.26 7.34 7.17 7.46 7.44 7.45 7 7.47 7.43 7.14 7.34 7 7.46 7.46 7.45 14 7.35 7.33 7.01 7.25 6.58 7.34 7.37 7.33 30 7.27 7.21 6.59 7.19 6.03 7.2 7.28 7.25 60 7.11 7.05 5.88 7.08 5.24 7.08 7.11 7.11

[0144] Table 4. Changes in DAR values ​​of solutions at 25°C

[0145] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 7.48 7.46 7.37 7.35 7.34 7.47 7.46 7.46 1 7.31 7.3 7.08 7.25 6.85 7.32 7.32 7.33 2 7.17 7.14 6.74 7.07 6.44 7.14 7.11 7.11 3 7 7.03 6.49 6.88 6.11 6.95 6.95 6.97 7 6.36 6.29 5.29 6.21 4.82 6.31 6.3 6.35 14 5.33 5.25 3.77 5.14 2.22 5.3 5.1 5.33

[0146] Table 5. Changes in small molecule residues in solution at 2-8°C (%)

[0147] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 0.83 0.7 0.94 0.95 0.92 0.81 0.85 0.8 1 0.64 0.68 1.11 0.81 1.21 0.69 0.71 0.66 3 0.89 0.9 1.76 0.99 1.89 0.85 0.91 0.85 7 1.31 1.24 2.82 1.55 3.01 1.33 1.4 1.38 14 2.15 1.87 4.99 2.31 5.45 2.2 2.22 2.2 30 4.08 4.57 9.59 4.3 12.54 4.15 4.18 4.18 60 8.24 9.1 22.45 8.85 32.52 8.45 8.6 8.54

[0148] Table 6. Changes in small molecule residues in solution at 25°C (%)

[0149] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 0.83 0.7 0.94 0.95 0.92 0.81 0.85 0.8 1 3.11 2.83 5.4 3.22 6.62 3.01 3.11 3.08 2 5.59 5.24 9.68 5.87 10.59 5.68 5.77 5.74 3 8.26 8.38 15.08 8.46 17.23 8.33 8.4 8.38 7 18.46 19.67 35.07 18.99 43.33 18.75 18.9 18.84 14 33.33 33.98 58.48 35.08 83.38 34.1 35.01 34.22

[0150] This example also uses CE-SDS and SEC detection (as shown in Tables 7-10, Figure 5-8 ) respectively reflect the changes of covalent aggregates and soluble aggregates of ADC-I under different formulation conditions. The results show that the trend of covalent aggregates increasing under formulation C and formulation E is faster than that of other formulations; the trend of soluble aggregates increasing under formulation B (pH 5.5) is slightly faster than that of other formulations. At the same time, the trend of aggregates increasing increases with the increase of temperature.

[0151] Table 7. Solution 2-8°C SEC aggregates (%)

[0152] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 1.3 1.4 1.3 1.4 1.3 1.3 1.3 1.3 1 1.3 1.5 1.4 1.4 1.4 1.3 1.4 1.4 3 1.3 1.6 1.3 1.4 1.5 1.4 1.4 1.5 7 1.4 1.7 1.4 1.4 1.5 1.4 1.4 1.5 14 1.5 1.8 1.4 1.5 1.5 1.4 1.5 1.6 30 1.5 2.1 1.5 1.5 1.7 1.6 1.6 1.6 60 1.6 2.4 1.6 1.6 1.6 1.6 1.7 1.7

[0153] Table 8. SEC polymers at 25°C (%)

[0154] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 1.3 1.4 1.3 1.4 1.4 1.3 1.3 1.3 1 1.4 2 1.4 1.4 1.4 1.5 1.6 1.6 2 1.6 2.3 1.6 1.6 1.7 1.5 1.6 1.7 3 1.8 2.6 1.6 1.7 1.8 2 1.9 1.9 7 2 3.4 1.8 2 1.9 2 2.2 2.1 14 2.3 4.1 1.8 2.1 2 2.3 2.5 2.5

[0155] Table 9. Solution 2-8°C CE-SDS high molecular weight impurities total (%)

[0156] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 3.5 3.7 4 4 4.1 3.7 3.6 3.6 1 4 3.7 4.1 4 4.2 3.7 3.7 3.8 3 4 3.8 4.3 4.1 4.4 3.8 3.7 3.8 7 3.9 3.9 4.4 4.1 4.5 3.8 3.8 4 14 4 4.1 4.6 4.2 4.6 3.8 3.8 3.8 30 3.9 4.2 4.5 4.1 4.7 3.9 3.8 4

[0157] Table 10. Solution 25°C CE-SDS high molecular weight impurities total (%)

[0158] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 3.5 3.7 4 4 4.1 3.7 3.6 3.6 1 4.4 4.3 4.8 4.5 4.8 4.4 4.4 4.4 2 4.6 4.7 5.2 5.2 5.2 4.8 5 4.8 3 4.8 4.9 5.6 5.1 5.5 4.8 5 4.8 7 5.9 6 6.2 6.2 6.9 6 6.1 6 14 7.2 7.2 8.4 8.1 9.2 7.4 7.8 7.7

[0159] This example also examines the trend of sample aggregate changes after 10 freeze-thaw cycles of different formulations (see Table 11, Fig. 9 The results showed that 10 freeze-thaw cycles did not result in an increase in aggregates; there was no significant difference in the trends of aggregate changes among different formulations.

[0160] Table 11. Effect of freeze-thaw times on SEC aggregation (%)

[0161] frequency Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 1.3 1.4 1.3 1.3 1.4 1.3 1.3 1.3 5 1.2 1.4 1.3 1.3 1.3 1.3 1.4 1.4 10 1.2 1.5 1.4 1.3 1.4 1.3 1.3 1.3

[0162] Example 4 Stability study of ADC-I lyophilized formulations at different pH values

[0163] This example compares the changes in DAR values ​​and small molecule residues of freeze-dried preparations with different formulations and pH values ​​listed in Table 2 when placed at different temperatures, reflecting the differences in the stability of ADC-I freeze-dried preparations at different pH values. The residual moisture of the samples after freeze-drying of different formulations was all below 3%, meeting the relevant requirements of the Chinese Pharmacopoeia for freeze-dried preparations. However, compared to the complete and uniform powder appearance of other formulations after freeze-drying, prescription E lost its complete cake-like appearance after freeze-drying and showed partial collapse, indicating that the excipient effects of the two formulations were not ideal. The results show that compared to solution preparations, freeze-dried preparations greatly improved the stability of the samples. From the DAR values ​​(as shown in Tables 12-13, Figure 10-11 ) and small molecule residues (as shown in Tables 14-15, Figure 12-13 As shown in the results, it can be seen that from the stability of the small molecule drug end, prescriptions A, D, F, G, H (pH5.0) and prescription B (pH5.5) are better than other prescriptions.

[0164] Table 12. Changes in DAR values ​​of freeze-dried products at 25°C

[0165] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 7.43 7.39 7.4 7.12 7.14 7.4 7.41 7.44 1 7.44 7.44 7.41 7.14 7.14 7.41 7.4 7.42 3 7.48 7.39 7.41 7.14 7.15 7.38 7.4 7.41 7 7.44 7.41 7.42 7.16 7.16 7.38 7.38 7.41 14 7.48 7.39 7.4 7.15 7.16 7.4 7.38 7.41 30 7.42 7.39 7.4 7.18 7.15 7.38 7.38 7.4 60 7.49 7.46 7.41 7.21 7.22 7.39 7.39 7.4

[0166] Table 13. Changes in DAR values ​​of freeze-dried products at 40°C

[0167] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 7.43 7.39 7.4 7.12 7.14 7.4 7.41 7.44 1 7.46 7.43 7.41 7.14 7.14 7.42 7.41 7.42 3 7.47 7.42 7.41 7.14 7.15 7.42 7.4 7.42 7 7.45 7.44 7.44 7.13 7.15 7.43 7.41 7.42 14 7.44 7.41 7.42 7.18 7.16 7.42 7.41 7.42 30 7.39 7.37 7.38 7.18 7.15 7.4 7.4 7.4 60 7.39 7.43 7.39 7.2 7.18 7.38 7.36 7.38

[0168] Table 14. Changes in small molecule residues of freeze-dried products at 25°C (%)

[0169] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 1.25 1.09 1.11 1.67 1.7 1.08 1.11 1.08 1 1.04 1.06 1.07 1.68 1.71 1.09 1.11 1.09 3 1.11 1.06 1.04 1.68 1.75 1.11 1.13 1.09 7 1.13 1.09 1.06 1.7 1.72 1.18 1.15 1.11 14 1.18 1.18 1.07 1.71 1.8 1.18 1.15 1.11 30 1.2 1.27 1.04 1.76 1.88 1.2 1.17 1.15 60 1.29 1.59 1.25 1.99 2.59 1.3 1.29 1.29

[0170] Table 15. Changes in small molecule residues of freeze-dried products at 40°C (%)

[0171] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 1.25 1.09 1.11 1.67 1.7 1.08 1.11 1.08 1 1.12 1.1 1.07 1.68 1.7 1.18 1.2 1.19 3 1.12 1.19 1.08 1.69 1.78 1.18 1.2 1.21 7 1.28 1.26 1.11 1.77 1.9 1.33 1.31 1.35 14 1.41 1.35 1.21 1.89 2.01 1.44 1.4 1.48 30 1.71 1.65 1.42 2.21 2.32 1.8 1.78 1.89 60 2.84 1.92 2.52 3.22 3.4 2.91 2.9 3.02

[0172] SEC results of freeze-dried samples (such as Table 16-Table 17, Figure 14-15 The results show that under accelerated conditions, there was no significant difference in the trend of increasing aggregates in all formulation conditions, and freeze-drying of the formulations effectively prevented the formation of aggregates.

[0173] Table 16. Lyophilized product 25℃ SEC aggregates (%)

[0174] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 1.3 1.8 1.4 1.5 1.7 1.3 1.3 1.4 1 1.4 1.8 1.5 1.6 1.6 1.4 1.4 1.4 3 1.5 1.8 1.5 1.6 1.6 1.5 1.5 1.6 7 1.5 1.9 1.5 1.6 1.7 1.5 1.5 1.6 14 1.5 1.9 1.5 1.6 1.7 1.6 1.6 1.7 30 1.6 2 1.6 1.7 1.7 1.6 1.7 1.7 60 1.5 1.9 1.6 1.6 1.7 1.6 1.7 1.7

[0175] Table 17. SEC aggregates of freeze-dried products at 40°C (%)

[0176] Number of days Prescription A Prescription B Prescription C Prescription D Prescription E Prescription F Prescription G Prescription H 0 1.3 1.8 1.4 1.5 1.7 1.3 1.3 1.4 1 1.5 1.9 1.6 1.6 1.8 1.7 1.5 1.6 3 1.6 2 1.6 1.7 1.8 1.7 1.6 1.7 7 1.8 2.1 1.8 1.9 2.1 1.8 1.8 1.8 14 1.9 2.3 1.9 2 2.1 1.8 1.8 1.9 30 2.3 2.7 2.1 2.4 2.4 2.5 2.4 2.5 60 3.4 3.6 2.7 3.2 3 3.3 3.2 3.5

[0177] Example 5 Freeze / thaw stability of pharmaceutical compositions

[0178] The formula of the pharmaceutical composition is as follows: 20 mg / mL ADC-I, 10 mM succinate buffer, 60 mg / ml sucrose, 0.1 mg / ml polysorbate 20, pH 5.0. The specific preparation method is the same as that of Example 1.

[0179] During the freeze-thaw process of biomacromolecule samples, the properties of liquid water (such as ionization constant, dielectric constant, hydrogen bonding, hydrophobic interaction) change with temperature. This leads to reversible changes in protein structure. At the same time, in the process of water freezing into ice, the effects of protein cryoconcentration and dehydration will occur. Stress caused by other freezing processes include the formation of ice interface, changes in pH value and phase separation. These stresses may cause irreversible changes in protein structure, which may affect the natural structure, aggregation state and activity of antibody conjugates. The above-mentioned pharmaceutical composition was subjected to 0, 5, and 10 freeze-thaw cycles, respectively, and characterized by DAR, SEC, non-reduced CE-SDS, biological activity, relative binding activity and free small molecule residues. The characterization results are shown in Table 18 below, indicating that repeated freeze-thaw has no effect on the stability of the pharmaceutical composition.

[0180] Table 18. Analysis of repeated freeze-thaw stability of pharmaceutical compositions

[0181]

[0182] Example 6 Preparation of freeze-dried preparation

[0183] The formula of the pharmaceutical composition is as follows: 20 mg / mL ADC-I, 10 mM succinate buffer, 60 mg / ml sucrose, 0.1 mg / ml polysorbate 20, pH 5.0. The specific preparation method is the same as that of Example 2. The specific raw materials are as follows:

[0184] Table 19. Raw material composition of the pharmaceutical composition of freeze-dried preparation

[0185] Ingredient name Function Volume per vial ADC-I Active substances 100mg Succinate buffered saline Buffer 5.9 mg succinic acid + 2.25 mg sodium hydroxide sucrose Stabilizer 300mg Polysorbate 20 Surfactants 0.5mg Water for injection medium Make the final volume of the drug composition 5mL

[0186] The pharmaceutical composition was prepared into a freeze-dried preparation according to the preparation method of Example 2. The freeze-dried preparation was reconstituted with 5 mL of sterile water for injection (WFI) to obtain a 20 mg / mL ADC-I solution for injection. After the freeze-dried preparation was reconstituted with 5 mL of sterile water for injection, the concentrations of the components contained in each 1 mL of the solution were: 20 mg / mL ADC-I, 10 mM succinate buffer salt, 60 mg / ml sucrose, 0.1 mg / ml polysorbate 20, and the pH value of the reconstituted solution was 5.0.

[0187] The lyophilized formulation (formulation contains buffer, sugar, surfactant, and antibody drug conjugate) is for single use and does not contain preservatives. The reconstituted drug product is diluted with 0.9% normal saline (Sodium Chloride Injection, USP) for dosing by infusion.

[0188] Example 7 Long-term stability of freeze-dried preparations at 2-8°C

[0189] The stable freeze-dried preparation prepared in Example 6 was subjected to a long-term stability test at 2-8°C, and was reconstituted with WFI at 0, 3, 6, 9, and 12 months. It was characterized by size exclusion chromatography SEC, non-reducing CE-SDS, free small molecule content, moisture, antibody-drug conjugate content, drug-antibody conjugate ratio, biological activity, and relative binding activity. The results (as shown in Table 20) show that the quality attributes of the ADC-I finished product did not change significantly, and the stability was good.

[0190] Table 20. Long-term stability of lyophilized formulations (2-8°C)

[0191]

[0192] Example 8. Accelerated stability of freeze-dried formulations at 25°C

[0193] The stable freeze-dried formulation prepared in Example 6 was subjected to an accelerated stability test at 25°C and tested after reconstitution with WFI at 0, 1, 2, 3 and 6 months. The formulation was characterized by size exclusion chromatography SEC, non-reducing CE-SDS, free small molecule content, antibody-drug conjugate content, drug-antibody conjugate ratio, biological activity and relative binding activity.

[0194] The results (as shown in Table 21) show that when the ADC-I finished product is placed at 25°C for 6 months, there is no significant change in the physical and chemical properties such as purity (size exclusion chromatography SEC, non-reducing CE-SDS), free small molecule content, antibody-drug conjugate content and drug-antibody conjugation ratio; there is no obvious change trend in biological activity and relative binding activity.

[0195] Table 21. Accelerated stability of lyophilized formulations (25°C)

[0196]

[0197] Although the specific embodiments of the present invention are described above, it should be understood by those skilled in the art that these are only examples, and various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the protection scope of the present invention is limited by the appended claims.

Claims

1. A pharmaceutical composition, characterized in that It includes the following components: 5-25 mg / mL of F0124-ADC; 7~90mg / mL protein protective agent; 0.05~1.0mg / mL surfactant; 5~20mM buffer salt; water; The pH value of the pharmaceutical composition is 5.0-5.5; The protein protective agent is sucrose or trehalose; The surfactant is polysorbate 20 or polysorbate 80; Among them, F0124-ADC is an antibody conjugate. The structural formula of the F0124-ADC is shown in formula (I), which is referred to as ADC-I: （I）； Wherein Ab is the anti-human Trop2 antibody hRS7; n is 7-8.

2. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition meets one or more of the following conditions: (1) n is 7.6; (2) The pH value of the pharmaceutical composition is 5.0 or 5.5; (3) The concentration of F0124-ADC is 10-20 mg / mL; (4) The concentration of the protein protective agent is 7-60 mg / mL; (5) The concentration of the surfactant is 0.05-0.6 mg / mL; (6) The concentration of the buffer salt is 10 mM to 20 mM; (7) The buffer salt is selected from citrate buffer, acetate buffer, succinate buffer, histidine buffer, phosphate buffer or Bis-Tris buffer; the Bis-Tris is bis(2-hydroxyethyl)amino(trihydroxymethyl)methane.

3. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition meets one or more of the following conditions: (1) The concentration of F0124-ADC is 10 mg / mL or 20 mg / mL; (2) The concentration of the protein protective agent is 7 mg / mL, 30 mg / mL, 50 mg / mL or 60 mg / mL; (3) The concentration of the surfactant is 0.09 mg / mL, 0.1 mg / mL, 0.2 mg / mL or 0.6 mg / mL; (4) The concentration of the buffer salt is 10 mM or 20 mM; (5) The type of buffer salt is succinate buffer salt.

4. The pharmaceutical composition according to claim 2, characterized in that The pharmaceutical composition meets one or more of the following conditions: (1) The histidine salt buffer is histidine-hydrochloric acid buffer or histidine-histidine hydrochloride buffer; (2) The phosphate buffer salt is a buffer salt including dihydrogen phosphate ions or a buffer salt including monohydrogen phosphate ions; (3) The Bis-Tris buffer salt is Bis-Tris-hydrochloric acid buffer salt.

5. The pharmaceutical composition according to claim 4, characterized in that The pharmaceutical composition meets one or more of the following conditions: (1) The citrate buffer salt is citric acid-sodium citrate buffer salt, citric acid-potassium citrate buffer salt, citric acid-calcium citrate buffer salt or citric acid-sodium hydroxide buffer salt; (2) The acetate buffer salt is acetic acid-sodium acetate buffer salt, acetic acid-potassium acetate buffer salt, acetic acid-sodium hydroxide buffer salt or sodium acetate-hydrochloric acid buffer salt; (3) The succinate buffer salt is succinic acid-sodium succinate buffer salt, succinic acid histidine buffer salt, succinic acid-potassium succinate buffer salt or succinic acid-calcium succinate buffer salt; (4) The histidine salt buffer is histidine-hydrochloric acid buffer; (5) The phosphate buffer salt is potassium dihydrogen phosphate-dipotassium hydrogen phosphate buffer salt, potassium dihydrogen phosphate-disodium hydrogen phosphate buffer salt, sodium dihydrogen phosphate-disodium hydrogen phosphate buffer salt or sodium dihydrogen phosphate-dipotassium hydrogen phosphate buffer salt.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition meets one or more of the following conditions: (1) The citrate buffer salt is citric acid-sodium citrate buffer salt; (2) The acetate buffer salt is acetic acid-sodium acetate buffer salt; (3) The succinate buffer salt is succinic acid-sodium succinate buffer salt; (4) The phosphate buffer salt is sodium dihydrogen phosphate-disodium hydrogen phosphate buffer salt.

7. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition is composed of the following components: 5-25 mg / mL of F0124-ADC; 7~90mg / mL protein protectant; 0.05~1.0mg / mL surfactant; 5~20mM buffer salt; water; The pH value of the pharmaceutical composition is 5.0-5.5; The protein protective agent is sucrose or trehalose; The surfactant is polysorbate 20 or polysorbate 80; wherein the definition of F0124-ADC is as described in claim 1.

8. The pharmaceutical composition according to claim 1, characterized in that The pharmaceutical composition comprises or consists of the following components: 10-20 mg / mL of F0124-ADC; 7~60mg / mL protein protectant; 0.09~0.6mg / mL surfactant; 10~20mM buffer salt; water; The pH value of the pharmaceutical composition is 5.0-5.5; The protein protective agent is sucrose or trehalose; The surfactant is polysorbate 20 or polysorbate 80; Wherein, the definition of F0124-ADC is as described in claim 1.

9. The pharmaceutical composition according to any one of claims 1 to 8, characterized in that The pharmaceutical composition comprises or consists of the components shown in any of the following schemes: Scheme 1: 20 mg / mL F0124-ADC, 50 mg / mL sucrose, 0.2 mg / mL polysorbate 20, 10 mM succinate buffer and water, the pH value of the pharmaceutical composition is 5.0; Scheme 2: 20 mg / mL F0124-ADC, 50 mg / mL sucrose, 0.2 mg / mL polysorbate 20, 10 mM succinate buffer and water, the pH value of the pharmaceutical composition is 5.5; Scheme 3: 20 mg / mL F0124-ADC, 60 mg / mL sucrose, 0.1 mg / mL polysorbate 20, 10 mM succinate buffer and water, the pH value of the pharmaceutical composition is 5.0; Scheme 4: 20 mg / mL F0124-ADC, 60 mg / mL sucrose, 0.1 mg / mL polysorbate 20, 10 mM succinate buffer and water, the pH value of the pharmaceutical composition is 5.5; Scheme 5: 20 mg / mL F0124-ADC, 30 mg / mL sucrose, 0.1 mg / mL polysorbate 20, 10 mM acetate buffer and water, the pH value of the pharmaceutical composition is 5.0; Scheme 6: 10 mg / mL F0124-ADC, 7 mg / mL trehalose, 0.09 mg / mL polysorbate 20, 20 mM succinate buffer salt and water, the pH value of the pharmaceutical composition is 5.0; Scheme 7: 20 mg / mL F0124-ADC, 60 mg / mL sucrose, 0.1 mg / mL polysorbate 80, 10 mM succinate buffer salt and water, the pH value of the pharmaceutical composition is 5.0; Scheme 8: 20 mg / mL of F0124-ADC, 60 mg / mL of sucrose, 0.6 mg / mL of polysorbate 20, 10 mM succinate buffer salt and water. The pH value of the pharmaceutical composition is 5.

0.

10. A freeze-dried preparation, characterized in that: The freeze-dried preparation is prepared by freeze-drying the pharmaceutical composition according to any one of claims 1 to 9.

11. A freeze-dried preparation, characterized in that: The freeze-dried preparation contains A pharmaceutical composition according to any one of claims 1 to 9.

12. The freeze-dried preparation according to claim 11, characterized in that The dissolution process uses one or more of purified water, water for injection and physiological saline.

13. The freeze-dried preparation according to claim 12, characterized in that The dissolution process adopts water for injection.

14. Use of a pharmaceutical composition or a freeze-dried preparation in the preparation of a drug for treating Trop-2 positive tumors, wherein the pharmaceutical composition is as described in any one of claims 1 to 9; the freeze-dried preparation is as described in any one of claims 10 or 11 to 13.

15. The use according to claim 14, wherein the Trop-2 positive tumor is breast cancer or urothelial carcinoma.

16. The use according to claim 15, wherein the breast cancer is triple-negative breast cancer.

17. The use according to claim 15, wherein the urothelial carcinoma is renal pelvic carcinoma, ureteral carcinoma, bladder carcinoma or urethral carcinoma.

18. A pharmaceutical product, characterized in that The invention comprises a container, wherein the container contains a pharmaceutical composition or a freeze-dried preparation, wherein the pharmaceutical composition is as described in any one of claims 1 to 9; and the freeze-dried preparation is as described in any one of claims 10 or 11 to 13.

19. The pharmaceutical product according to claim 18, characterized in that The container is a glass bottle or a liquid storage bag.

Images