Pharmaceutical composition containing an antibody-drug conjugate and its use

Abstract

The present disclosure relates to a pharmaceutical composition containing an antibody-drug conjugate and its use. Specifically, the present disclosure relates to a pharmaceutical composition containing an antibody-drug conjugate in a buffer solution. The pharmaceutical composition related to the present disclosure exhibits good stability.

Description

Technical Field

[0001] The present disclosure belongs to the field of pharmaceutical preparations, and more particularly relates to a pharmaceutical composition containing an antibody-drug conjugate and its use as an anticancer agent.

Background Art

[0002] The description herein only provides background information related to the present disclosure and does not necessarily mean prior art.

[0003] An antibody-drug conjugate (ADC) binds a monoclonal antibody or antibody fragment to a biologically active cytotoxic agent via a stable chemical linker compound, maximizing the specificity of the antibody for tumor cell surface antigens and the high efficiency of the cytotoxic agent while avoiding the disadvantages including the low efficacy of the former and the excessive toxicity and side effects of the latter. This means that compared with conventional chemotherapeutic drugs, antibody-drug conjugates can accurately target tumor cells while minimizing the impact on normal cells (Mullard A, (2013) Nature Reviews Drug Discovery, 12: 329-332; DiJoseph JF, Armellino DC, (2004) Blood, 103: 1807-1814).

[0004] There are several types of cytotoxic small molecules used for antibody-drug conjugates. One of them is a camptothecin derivative, which exerts an antitumor effect by inhibiting topoisomerase I. Documents reporting the use of the camptothecin derivative exatecan (chemical name: (1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo [d] pyrano [3'4':6,7] imidazo [1,2-b] quinoline-10,13(9H,15H)-dione) in antibody-drug conjugates (ADCs) include WO2014057687; Clinical Cancer Research (2016) 22(20):5097-5108; Cancer Sci (2016) 107:1039-1046, etc. However, further development of more effective ADC drugs is still needed.

[0005] Furthermore, since ADCs have a more complex heterogeneous structure than antibodies, it poses a great challenge for ADC formulations for therapeutic purposes.

Summary of the Invention

[0006] The present disclosure describes a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, and the antibody-drug conjugate has the structure shown below;

[0007]

Chemical formula

[0008] The above-mentioned antibody-drug conjugate was prepared with reference to the preparation of FADC-2 in Example 9 of the antibody conjugate on pages 45-47 in the description of WO2020063673A1;

[0009] In the formula, h1702DS is an anti-B7H3 antibody (the source thereof refers to the antibody h1702DS on page 44 of WO2020063673A1), and contains a heavy chain represented by SEQ ID NO: 1 (SEQ ID NO: 14 of WO2020063673A1) and a light chain represented by SEQ ID NO: 2 (SEQ ID NO: 16 of WO2020063673A1):

[0010] Amino acid sequence of the heavy chain (SEQ ID NO: 1) of h1702DS QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVAVISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSARLYASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0011] Amino acid sequence of the light chain (SEQ ID NO: 2) of h1702DS DTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDIWVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTEC;

[0012] n ranges from 1 to 10, preferably from 1 to 8, more preferably from 3 to 5, and even more preferably is about 4;

[0013] The buffer of the pharmaceutical composition is succinic acid buffer or histidine buffer, preferably succinic acid-sodium succinate, histidine-acetate, or histidine-hydrochloride buffer.

[0014] The concentration of the buffer of the pharmaceutical composition is about 15 mM to about 50 mM, preferably about 20 mM to about 40 mM, and more preferably about 30 mM.

[0015] The pH of the pharmaceutical composition is about 4.5 to about 6.5, preferably about 5.0 to about 6.0, and more preferably about 5.5 to about 5.6.

[0016] In an optional embodiment, the pH of the buffer in the pharmaceutical composition is about 4.5 to about 6.5. Non-limiting examples are about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, and any range between the values of these points, etc., preferably about 5.0 to about 6.0, and more preferably about 5.5 to about 5.6.

[0017] In an optional embodiment, the succinic acid buffer in the pharmaceutical composition is succinic acid-sodium succinate, and the histidine buffer is preferably histidine-acetate or histidine-hydrochloride buffer.

[0018] In an optional embodiment, the pharmaceutical composition also contains a surfactant. The surfactant may be polysorbate, polysorbate 20, polysorbate 80, poloxamer, Triton, sodium dodecyl sulfonate, sodium lauryl sulfonate, sodium caprylyl glucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleic sulfobetaine, stearyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearyl sarcosine, linoleic betaine, myristyl betaine, cetyl betaine, lauramidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmitamidopropyl betaine, isostearamidopropyl betaine, myristamidopropyldimethylamine, palmitamidopropyldimethylamine, isostearamidopropyldimethylamine, sodium methyl cocoyl sarcosinate, sodium methyl oleoyl sarcosinate, polyethylene glycol, polypropylene glycol, a copolymer of ethylene and propylene glycol, etc. Preferred surfactants are polysorbate 80 or polysorbate 20, more preferably polysorbate 80.

[0019] In an optional embodiment, the concentration of the surfactant in the pharmaceutical composition is about 0.01 mg / mL to about 1.0 mg / mL, preferably about 0.05 mg / mL to about 0.5 mg / mL, more preferably about 0.1 mg / mL to about 0.4 mg / mL or about 0.2 mg / mL to about 0.3 mg / mL, most preferably about 0.2 mg / mL, and non-limiting examples include 0.02 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.15 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.8 mg / mL, and any range between the values of these points.

[0020] In an optional embodiment, the aforementioned pharmaceutical composition also contains sugar. The "sugar" included in the present disclosure is the conventional composition (CH2O) nand derivatives thereof including monosaccharides, disaccharides, trisaccharides, polysaccharides, sugar alcohols, reducing sugars, and non-reducing sugars. The sugars may be glucose, sucrose, trehalose, lactose, fructose, maltose, dextran, glycerin, erythritol, glycerol, arabitol, xylitol, sorbitol, mannitol, melibiose, melezitose, melitriose, manninotriose, stachyose, maltose, lactulose, maltulose, sorbitol, maltitol, lactitol, isomaltulose, etc. Preferred sugars are non-reducing disaccharides, more preferably trehalose, mannitol, or sucrose, and most preferably sucrose or trehalose.

[0021] In an optional embodiment, the concentration of the sugar in the aforementioned pharmaceutical composition is about 25 mg / mL to about 80 mg / mL, preferably about 30 mg / mL to about 60 mg / mL, more preferably about 40 mg / mL. Non-limiting examples include 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, and any range between the values of these points, preferably 40 mg / mL.

[0022] In an optional embodiment, the aforementioned pharmaceutical composition also contains amino acids and their salts, which are preferably glycine and arginine hydrochloride, more preferably glycine.

[0023] In an optional embodiment, the concentration of glycine in the aforementioned pharmaceutical composition is about 5 mg / mL to about 10 mg / mL, about 5.3 mg / mL to about 9.8 mg / mL, and about 6 mg / mL to about 9 mg / mL, preferably about 7 mg / mL to about 8 mg / mL. Non-limiting examples include 6 mg / mL, 6.5 mg / mL, 7 mg / mL, 7.2 mg / mL, 7.6 mg / mL, 7.8 mg / mL, 8 mg / mL, 8.5 mg / mL, 9 mg / mL, and any range between the values of these points, and most preferably about 7.6 mg / mL.

[0024] In an optional embodiment, the aforementioned pharmaceutical composition further contains arginine hydrochloride, and the concentration is about 40 mM to about 80 mM, preferably about 50 mM to about 70 mM. Non-limiting examples include 40 mM, 45 mM, 50 mM, 55 mM, 58 mM, 60 mM, 65 mM, 70 mM, 75 mM, and 80 mM, and most preferably about 58 mM.

[0025] In an optional embodiment, the antibody-drug conjugate in the pharmaceutical composition has a protein concentration of about 1 mg / mL to about 100 mg / mL. Non-limiting examples include 1 mg / mL, 10 mg / mL, 11 mg / mL, 12 mg / mL, 13 mg / mL, 14 mg / mL, 15 mg / mL, 16 mg / mL, 17 mg / mL, 18 mg / mL, 19 mg / mL, 20 mg / mL, 21 mg / mL, 22 mg / mL, 23 mg / mL, 24 mg / mL, 25 mg / mL, 26 mg / mL, 27 mg / mL, 28 mg / mL, 29 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, and any range between these values. Preferably, it is about 10 mg / mL to about 70 mg / mL, more preferably about 20 mg / mL to about 50 mg / mL, even more preferably about 20 mg / mL to about 30 mg / mL or about 30 mg / mL to about 50 mg / mL, and still more preferably about 20 mg / mL or about 30 mg / mL or about 50 mg / mL. Specifically, non-limiting examples include 50.1 mg / mL, 50.2 mg / mL, 50.3 mg / mL, 50.4 mg / mL, 50.5 mg / mL, 50.6 mg / mL, 50.7 mg / mL, 50.8 mg / mL, 50.81 mg / mL, 50.82 mg / mL, 50.83 mg / mL, 50.84 mg / mL, 50.85 mg / mL, 50.86 mg / mL, 50.87 mg / mL, 50.88 mg / mL, 50.89 mg / mL, 50.9 mg / mL, 50.9 mg / mL, 50.91 mg / mL, 50.92 mg / mL, 50.93 mg / mL, 50.94 mg / mL, 50.95 mg / mL, 50.96 mg / mL, 50.97 mg / mL, 50.98 mg / mL, 50.99 mg / mL, 51 mg / mL, and any range between these values. The protein concentration is the concentration of the antibody fraction in the antibody-drug conjugate.

[0026] In an optional embodiment, the concentration of the buffer in the pharmaceutical composition is from about 15 mM to about 50 mM, non-limiting examples include 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 30 mM, 40 mM, 50 mM, and any range between the values of these points, preferably from about 15 mM to about 50 mM, more preferably from about 20 mM to about 40 mM, and most preferably about 30 mM.

[0027] In an optional embodiment, the range of the drug-loading dose (drug-to-antibody ratio; DAR) may be the average number of cytotoxic drugs bound to each antibody h1702DS. In non-limiting examples, such an average number is from about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, or any range between the values of these points. The number of bound cytotoxic drugs is preferably from about 3 to 8, from about 4 to 8, from about 5 to 7, and from about 4 to 6, more preferably from about 3 to 5, and most preferably 4.1, where n is a decimal or an integer.

[0028] In an optional embodiment, the drug-loading dose (DAR) is about 4.

[0029] In an optional embodiment, the pharmaceutical composition is: (a) an antibody-drug conjugate from about 10 mg / mL to about 70 mg / mL, (b) polysorbate from about 0.1 mg / mL to about 0.8 mg / mL, (c) sugar from about 25 mg / mL to about 80 mg / mL, (d) glycine from about 5 mg / mL to about 10 mg / mL, and (e) succinate buffer from about 15 mM to about 50 mM, and the pH of the composition is from about 5.0 to about 6.0;

[0030] Preferably, the pharmaceutical composition comprises the following components: (a) an antibody-drug conjugate from about 20 mg / mL to about 70 mg / mL, (b) polysorbate from about 0.2 mg / mL to about 0.8 mg / mL, (c) sugar from about 25 mg / mL to about 80 mg / mL, (d) glycine from about 7 mg / mL to about 8 mg / mL, and (e) succinate buffer from about 10 mM to about 40 mM, and the pH of the composition is from about 5.0 to about 6.0;

[0031] More preferably, the pharmaceutical composition comprises the following components: (a) an antibody-drug conjugate at about 20 mg / mL to about 50 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL to about 0.8 mg / mL, (c) sucrose at about 40 mg / mL to about 80 mg / mL, (d) glycine at about 7 mg / mL to about 8 mg / mL, and (e) succinic acid-sodium succinate at about 20 mM to about 40 mM, and the pH of the composition is about 5.0 to about 6.0;

[0032] Even more preferably, the pharmaceutical composition comprises the following components: (a) an antibody-drug conjugate at about 20 mg / mL to about 30 mg / mL, (b) 0.2 mg of polysorbate 80, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) succinic acid-sodium succinate at about 30 mM, and the pH of the composition is about 5.5 to about 5.6; (a) an antibody-drug conjugate at about 30 mg / mL to about 50 mg / mL, (b) 0.2 mg of polysorbate 80, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) succinic acid-sodium succinate at about 30 mM, and the pH of the composition is about 5.5 to about 5.6;

[0033] Still even more preferably, the pharmaceutical composition comprises the following components: (a) an antibody-drug conjugate at about 20 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) succinic acid-sodium succinate at about 30 mM, and the pH of the composition is about 5.5 to about 5.6. (a) an antibody-drug conjugate at about 30 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) succinic acid-sodium succinate at about 30 mM, and the pH of the composition is about 5.5 to about 5.6. (a) An antibody-drug conjugate at about 50 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) sodium succinate-succinic acid at about 30 mM, and the pH of the composition is about 5.5 to about 5.6.

[0034] In any optional embodiment, the pharmaceutical composition described in any of the above is a liquid formulation.

[0035] The present disclosure also relates to a lyophilized formulation containing an antibody-drug conjugate, characterized in that the aforementioned pharmaceutical composition can be formed upon reconstitution.

[0036] The present disclosure also relates to a method for preparing a lyophilized formulation containing an antibody-drug conjugate, which includes the lyophilization step of the aforementioned pharmaceutical composition.

[0037] In an optional embodiment, the lyophilization step in a method for preparing a lyophilized formulation containing an antibody-drug conjugate includes a pre-freezing step, a primary drying step, and a secondary drying step in sequence. Lyophilization is carried out by freezing the formulation and then sublimating water at a temperature suitable for primary drying. Under this condition, the temperature of the product is below the eutectic point or the collapse temperature of the formulation. Typically, the temperature range for primary drying is about -30°C to 25°C (assuming the product remains frozen during primary drying). The formulation, the size and type of the container containing the sample (such as a glass vial), and the volume of the liquid determine the time required for drying, which may range from several hours to several days (such as 40 - 60 hours). The secondary drying phase can be carried out at about 0 - 40°C, mainly depending on the type and size of the container and the type of protein used. The duration of secondary drying is determined by the desired residual moisture level of the product and is typically at least about 5 hours. Generally, the moisture content of the formulation lyophilized under low pressure is less than about 5%, preferably less than about 3%. The pressure may be the same as the pressure applied during the primary drying step, but the pressure for secondary drying is preferably lower than the pressure for primary drying. The lyophilization conditions may vary depending on the formulation and the size of the vial.

[0038] In an optional example of the present disclosure, 5 mL of the drug substance of the composition is lyophilized using the following lyophilization procedure: the pre-freezing temperatures are 5°C and -45°C in sequence, the temperature for primary drying is -20°C, the vacuum degree is 10 Pa, the temperature for secondary drying is 25°C, and the vacuum degrees are 10 Pa and 1 Pa in sequence.

[0039] In some embodiments, the lyophilized formulation is stable at 2 - 8°C for at least 16 days, at least 1 month, at least 3 months, at least 6 months, at least 12 months, at least 18 months, or at least 24 months. In some embodiments, the lyophilized formulation is stable at 40°C for at least 7 days, at least 14 days, at least 28 days, or at least 30 days.

[0040] The present disclosure also relates to a lyophilized formulation containing an antibody-drug conjugate, obtained through lyophilization of the aforementioned pharmaceutical composition containing the antibody-drug conjugate.

[0041] The present disclosure further relates to a reconstitution solution containing an antibody-drug conjugate, characterized by being obtained by reconstituting the aforementioned lyophilized formulation.

[0042] In an optional embodiment, the reconstitution solution: (a) contains an antibody-drug conjugate at about 10 mg / mL to about 70 mg / mL, (b) contains polysorbate at about 0.1 mg / mL to about 0.8 mg / mL, (c) contains sugar at about 25 mg / mL to about 80 mg / mL, (d) contains glycine at about 5 mg / mL to about 10 mg / mL, and (e) contains a succinic acid buffer at about 15 mM to about 50 mM, and the pH of the reconstitution solution is about 5.0 to 6.0;

[0043] Preferably, the reconstitution solution contains the following components: (a) contains an antibody-drug conjugate at about 20 mg / mL to about 70 mg / mL, (b) contains polysorbate at about 0.2 mg / mL to about 0.8 mg / mL, (c) contains sugar at about 25 mg / mL to about 80 mg / mL, (d) contains glycine at about 7 mg / mL to about 8 mg / mL, and (e) contains a succinic acid buffer at about 10 mM to about 40 mM, and the pH of the composition is about 5.0 to 6.0;

[0044] More preferably, the reconstitution solution contains the following components: (a) contains an antibody-drug conjugate at about 20 mg / mL to about 50 mg / mL, (b) contains polysorbate 80 at about 0.2 mg / mL to about 0.8 mg / mL, (c) contains sucrose at about 40 mg / mL to about 60 mg / mL, (d) contains glycine at about 7 mg / mL to about 8 mg / mL, and (e) contains succinic acid-sodium succinate at about 20 mM to about 40 mM, and the pH of the reconstitution solution is about 5.0 to 6.0;

[0045] Even more preferably, the reconstitution solution contains the following components: (a) An antibody-drug conjugate at about 20 mg / mL to about 30 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) sodium succinate-succinic acid at about 30 mM, and the pH of the composition is about 5.5 to 5.6, or (a) An antibody-drug conjugate at about 30 mg / mL to about 50 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) sodium succinate-succinic acid at about 30 mM, and the pH of the composition is about 5.5 to 5.6.

[0046] Even more preferably, the reconstitution solution comprises the following components: (a) An antibody-drug conjugate at about 20 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) sodium succinate-succinic acid at about 30 mM, and the pH of the composition is about 5.5 to 5.6; (a) An antibody-drug conjugate at about 30 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) sodium succinate-succinic acid at about 30 mM, and the pH of the composition is about 5.5 to 5.6; (a) An antibody-drug conjugate at about 50 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) sodium succinate-succinic acid at about 30 mM, and the pH of the composition is about 5.5 to 5.6.

[0047] The present disclosure also relates to a method for preparing the above reconstitution solution, which method includes steps for reconstituting the aforementioned lyophilized formulation, and the solution used for reconstitution includes, but is not limited to, water for injection, physiological saline, or glucose solution.

[0048] The present disclosure is further related to a product comprising a container, which contains the aforementioned pharmaceutical composition, lyophilized formulation, or reconstitution solution. In some embodiments, the container is an injection-neutral borosilicate glass vial.

[0049] The present disclosure also relates to the use of the aforementioned pharmaceutical composition, lyophilized formulation, reconstitution solution, or product in the preparation of a medicament for the treatment or prevention of tumors.

[0050] The present disclosure also relates to a method for treating a disease, including providing the aforementioned pharmaceutical composition, lyophilized formulation, reconstitution solution, or product.

[0051] The present disclosure also relates to the aforementioned pharmaceutical composition, lyophilized formulation, reconstitution solution, or product as a medicament for the treatment or prevention of preferably tumor diseases.

[0052] In optional embodiments, the disease or tumor is a cancer associated with the expression of HER2, HER3, B7H3, or EGFR.

[0053] In optional embodiments, the cancer is breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma.

[0054] As can be understood by those skilled in the art, one, several, or all of the various characteristics described in the present disclosure can be further combined to form other embodiments of the present disclosure. The above embodiments of the present disclosure and other embodiments obtained by the combinations are further illustrated by the following detailed description.

[0055] For the development of ADC formulations, even under low concentration conditions, the intermolecular disulfide bonds cleaved during conjugation may oxidize, and the highly hydrophobic conjugated toxin also affects the stability of the antibody to some extent. As the concentration increases, this effect inevitably intensifies, making the ADC more prone to aggregation / degradation and unable to maintain stability even in low-concentration formulations. Through reasonable formulation design, appropriate high-concentration formulations were screened to maintain the stability of the ADC. Furthermore, the high-concentration ADC formulation of the present invention can significantly shorten the manufacturing time, reduce the cost of consumables (vials, stoppers, aluminum caps, etc.), improve the convenience of clinical administration for medical staff, and expand a wider safe therapeutic concentration range in clinical diagnosis and treatment.

Mode for Carrying Out the Invention

[0056] The present disclosure relates to a pharmaceutical composition having stable performance that promotes manufacturing and administration more. Here, the undesirable instability can include any one or more of the following: aggregation, deamidation (such as Asn deamidation), oxidation (such as Met oxidation), isomerization (such as Asp isomerization), clipping / hydrolysis / fragmentation (such as fragmentation of the hinge region), succinimide formation, unpaired cysteine, and dissociation of the toxin. Specifically, the pharmaceutical composition described in the present disclosure contains an antibody-drug conjugate and a buffer.

[0057] Term For easier understanding of the present disclosure, certain technical terms and scientific terms are specifically defined below. Unless specifically defined in this specification, all other technical terms and scientific terms used in this specification have meanings that can be generally understood by those skilled in the art of the present disclosure.

[0058] The present disclosure incorporates the entire content of WO2020063673 by reference into this application.

[0059] An antibody-drug conjugate (ADC) is a compound in which an antibody is conjugated to a biologically active cell toxin or a small molecule drug having cell-killing activity via a linker unit.

[0060] The "drug loading amount" is also known as the drug-to-antibody ratio (DAR), i.e., the average number of drugs bound to each antibody in the ADC. The number of drugs bound to each antibody may range from about 1 to about 10, and in some examples may range from about 1 to about 8, preferably 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, 3 to 4, 3 to 5, 5 to 6, 5 to 7, 5 to 8, and 6 to 8. For example, the drug loading amount may be an average of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. The general formula of the ADC in the present disclosure includes a set of antibodies bound to drugs within a certain range as described above. In an embodiment of the present disclosure, the drug loading amount may be represented as n. The drug loading amount may be determined using conventional methods such as ultraviolet (UV) / visible spectroscopy, mass spectrometry, enzyme-linked immunosorbent assay (ELISA), and high performance liquid chromatography (HPLC).

[0061] The term "linker unit" or "linking fragment" or "linking unit" means a chemical structure fragment or bond that binds to an antibody or its antigen-binding fragment at one end and to a drug at the other end, and may be linked to other linkers before being linked to the drug.

[0062] Linkers containing extension portions, spacers, and amino acid units may be synthesized using methods known in the art, such as those described in US Patent Application Publication No. 2005-0238649. The linker may be a "cleavable linker" that facilitates the release of the drug intracellularly. For example, acid-labile linkers (such as hydrazones), protease-sensitive (such as peptidase-sensitive) linkers, photo-labile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992); US Patent No. 5,208,020) may be used.

[0063] To control the loading amount of the cytotoxic drug, the following non-limiting methods may be used: (1) Control of the molar ratio of the binder to the monoclonal antibody, (2) Control of the reaction time and temperature, (3) Selection of various reaction reagents.

[0064] The three-letter and one-letter codes of the amino acids used in the present disclosure are as described in J. Biol. Chem., 243, 3558 (1968).

[0065] The "antibody" described in the present disclosure is used in the broadest sense and includes various antibody structures including, but not limited to, full-length antibodies and antibody fragments (or antigen-binding fragments or antigen-binding fractions) as long as they exhibit the desired antigen-binding activity. Typically, a natural full antibody is composed of two identical heavy chains and two identical light chains linked by interchain disulfide bonds, forming a four-peptide chain structure.

[0066] The engineered antibodies or antigen-binding fragments in the present disclosure may be prepared and purified using conventional methods. For example, cDNA sequences encoding the heavy and light chains may be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector may be stably introduced into CHO cells. As a more preferred prior art, mammalian expression systems cause glycosylation of antibodies, especially at the highly conserved N-terminal site within the Fc region. Positive clones are grown in a serum-free medium in a bioreactor to produce the antibody. The culture medium containing the secreted antibody may be purified using conventional techniques. For example, purification may be performed using an A or G Sepharose FF column containing a conditioned buffer. Non-specifically bound components are washed away. Next, the bound antibody is eluted using a pH gradient method, and the antibody fragments may be detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and collected. The antibody may be filtered and concentrated using conventional methods. Soluble mixtures and polymers may also be removed using conventional methods such as molecular sieving and ion exchange. The resulting product needs to be immediately frozen at, for example, -70 °C or lyophilized.

[0067] "Buffer solution" means a buffer solution that can withstand pH changes due to the influence of acid-base complex components. Examples of buffer solutions that control the pH within an appropriate range include acetate, succinate, gluconate, histidine, oxalate, lactate, phosphate, citrate, tartrate, fumarate, glycylglycine, and other organic acid buffer solutions.

[0068] "Histidine buffer solution" is a buffer solution containing histidine ions. Examples of histidine buffer solutions include histidine-hydrochloride, histidine-acetate, histidine-phosphate, and histidine-sulfate, preferably histidine-acetate buffer solution, which is prepared from histidine and acetic acid, while histidine hydrochloride buffer solution is prepared from histidine and hydrochloric acid.

[0069] "Citrate buffer" is a buffer solution containing citrate ions. Examples of citrate buffers include citrate-sodium citrate, citrate-potassium citrate, citrate-calcium citrate, and citrate-magnesium citrate. A preferred citrate buffer is citrate-sodium citrate.

[0070] "Succinate buffer" is a buffer solution containing succinate ions. Examples of succinate buffers include succinate-sodium succinate, succinate-potassium succinate, and succinate-calcium succinate. A preferred succinate buffer is succinate-sodium succinate. For example, succinate-sodium succinate may be prepared from succinic acid and sodium hydroxide, or from succinic acid and sodium succinate.

[0071] "Phosphate buffer" is a buffer solution containing phosphate ions. Examples of phosphate buffers include disodium hydrogen phosphate-sodium dihydrogen phosphate, disodium hydrogen phosphate-potassium dihydrogen phosphate, and disodium hydrogen phosphate-citric acid. A preferred phosphate buffer is disodium hydrogen phosphate-sodium dihydrogen phosphate.

[0072] "Acetate buffer" is a buffer solution containing acetate ions. Examples of acetate buffers include acetic acid-sodium acetate, histidine acetate, acetic acid-potassium acetate, acetic acid-calcium acetate, and acetic acid-magnesium acetate. A preferred acetate buffer is acetic acid-sodium acetate.

[0073] "Pharmaceutical composition" means a mixture containing one or more of the antibody-drug conjugates described herein or their physiologically / pharmaceutically acceptable salts or prodrugs, together with other chemical components such as physiologically / pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to maintain the stability of the active antibody component, facilitate administration to an organism, and promote the absorption of the active component to exert biological activity.

[0074] In the present disclosure, "pharmaceutical composition" and "formulation" are not mutually exclusive.

[0075] Unless otherwise specified, the solution form of the pharmaceutical composition described in the present disclosure contains water as a solvent.

[0076] "Lyophilized formulation" means a formulation or pharmaceutical composition obtained after a vacuum freeze-drying step from a pharmaceutical composition in the form of a liquid or solution or a formulation in the form of a liquid or solution.

[0077] As used herein, the terms "about" or "approximately" mean that a value is within the tolerance of a specific value determined by those skilled in the art, and the numerical portion may depend on how it is measured or determined (i.e., the limitations of the measurement system). For example, in each embodiment of this field, "about" may mean that the standard deviation is within 1 or greater than 1. Alternatively, "about" or "substantially contains" may mean a range of up to 20%. Further, especially in the case of biological systems or methods, this term may mean up to 1 digit, or up to 5 times the value. Unless otherwise specified, when a specific value appears in the present application and claims, "about" or "substantially contains" is meant to assume that the specific value is within the tolerance.

[0078] The pharmaceutical compositions described in the present disclosure can achieve a stabilizing effect: in the case of a pharmaceutical composition in which the antibody-drug conjugate 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 biological activity after storage. The storage period is generally selected based on the predetermined storage period of the pharmaceutical composition. Currently, there are various analytical techniques available for determining protein stability that can determine the stability after storage at a selected temperature over a specified period.

[0079] A stable formulation is a formulation that shows no significant change even when maintained under the following conditions: at a refrigerated temperature (2 - 8°C) for at least 3 months, preferably 6 months, more preferably 1 year, and even more preferably up to 2 years. Furthermore, a stable liquid formulation is a liquid formulation that exhibits the desired properties even after being stored for periods such as 1 month, 3 months, and 6 months at a temperature such as 25°C. Typical examples of stability: When measured by size exclusion chromatography - high performance liquid chromatography (SEC - HPLC), usually, no more than about 10%, preferably no more than about 5% of the antibody monomer aggregates or degrades. By visual analysis, the formulation is a pale yellow to almost colorless transparent liquid, or colorless or transparent to slightly milky white. The concentration, pH, and osmotic pressure of the formulation show fluctuations of no more than ±10%. A decrease of no more than about 10%, preferably no more than about 5% is usually observed. Aggregation generally forms at no more than about 10%, preferably no more than about 5%.

[0080] An antibody - drug conjugate "maintains physical stability" in a pharmaceutical formulation if it shows no significant increase in aggregation, precipitation, and / or denaturation after visual inspection of color and / or transparency, or upon measurement by ultraviolet light scattering, size exclusion chromatography (SEC), and dynamic light scattering (DLS). Changes in the protein structure may be evaluated by fluorescence spectroscopy (to determine the tertiary structure of the protein) and Fourier transform infrared (FTIR) spectroscopy (to determine the secondary structure of the protein).

[0081] When an antibody-drug conjugate shows no significant chemical changes, the antibody "retains chemical stability" in a pharmaceutical formulation. Chemical stability may be evaluated by detecting and quantifying proteins having a chemically changed form. Degradation methods that frequently change the chemical structure of a protein include hydrolysis or cleavage (evaluated by methods such as size exclusion chromatography and capillary sodium dodecyl sulfate electrophoresis [CE-SDS]), oxidation (evaluated by methods such as peptide mapping combined with mass spectrometry or matrix-assisted laser desorption ionization time-of-flight mass spectrometry [MALDI / TOF / MS]), deamidation (evaluated by methods such as ion exchange chromatography, capillary isoelectric focusing electrophoresis, peptide mapping, and measurement of isoaspartic acid), and isomerization (evaluated by measurement of the isoaspartic acid content and peptide mapping).

[0082] When the biological activity of an antibody-drug conjugate at a given time point is within a predetermined range of the biological activity shown during the preparation of a pharmaceutical formulation, the antibody-drug conjugate "retains biological activity" in the pharmaceutical formulation.

[0083] "Optional" or "optionally" means that the event or situation described later may or may not occur, and the description includes both the case where the event or situation occurs and the case where it does not occur. For example, "optionally comprising one to three variable regions of an antibody heavy chain" means that there may or may not be a variable region of an antibody heavy chain having a specific sequence.

[0084] "Substitute" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3, in a group are independently replaced by the corresponding number of substituents. It is self-evident that the substituents exist only at possible chemical positions, and those skilled in the art can determine (experimentally or theoretically) which substitutions are possible or impossible without undue effort. For example, when an amino group or a hydroxyl group having a free hydrogen binds to a carbon atom having an unsaturated (such as olefin) bond, it may become unstable.

[0085] The preparation of conventional pharmaceutical compositions is described in the Chinese Pharmacopoeia.

[0086] The term "carrier" as used for the drugs in the present disclosure means a system that can change the way the drug enters the human body and its distribution in the body, control the release rate of the drug, and deliver the drug to the target organ. The drug carrier release and targeting system can reduce the degradation and loss of the drug, reduce side effects, and improve bioavailability. For example, polymeric surfactants functioning as carriers can self-organize due to their unique amphiphilic structures and form various types of aggregates, preferably micelles, microemulsions, gels, liquid crystals, and vesicles. These aggregates can encapsulate drug molecules while showing good permeability to membranes, making them excellent drug carriers.

[0087] "Administration" and "treatment," when used in relation to an animal, human, subject, cell, tissue, organ, or body fluid, mean contact between an exogenous drug, therapeutic agent, diagnostic agent, or composition and the animal, human, subject, cell, tissue, organ, or body fluid. "Administration" and "treatment" may mean, for example, therapy, pharmacokinetics, diagnosis, research, and experimental methods. Treatment of a cell includes contact between a reagent and the cell and between the reagent and a fluid, where the fluid contacts the cell. "Administration" and "treatment" also mean treatment by other forms of in vitro and ex vivo treatment, such as a reagent, diagnostic agent, composite composition, or cell. "Treatment," when used in relation to a human, animal, or research subject, means therapeutic treatment, prophylactic or preventive means, research, and diagnostic use.

[0088] "Treatment" means administration of a therapeutic agent for oral or topical use in a patient, such as a composition containing any of the composite compounds of the present disclosure, where the patient has one or more disease symptoms and the therapeutic agent is known to have a therapeutic effect on those symptoms. Generally, the therapeutic agent is administered in an amount effective to reduce one or more disease symptoms in the patient or population being treated, to induce regression of such symptoms, or to inhibit progression of the symptoms to a clinically measurable extent. The amount of a therapeutic agent effective to reduce any particular disease symptom (also referred to as a "therapeutically effective amount") may vary depending on several factors, such as the patient's disease state, age, and weight, as well as the ability of the drug to produce the desired effect in the patient. Whether a disease symptom has been reduced may be evaluated by any clinical test method commonly used by physicians and other medical personnel to assess the severity or progression of the symptom. Embodiments of the present disclosure (such as treatment methods or products) may not be effective to reduce each targeted disease symptom, but any statistical test method known in the art, such as a Student's t-test, chi-square test, Mann-Whitney U test, Kruskal-Wallis test (H test), Jonckheere-Terpstra test, and Wilcoxon test, can determine that those embodiments reduce the targeted disease symptoms in a statistically significant number of patients.

[0089] "Effective amount" means an amount sufficient to ameliorate or prevent the symptoms or conditions of a medical disorder. An effective amount also means an amount sufficient to enable or facilitate a diagnosis. The effective amount for a particular patient or veterinary subject may vary depending on factors such as the condition being treated, the overall health of the patient, the route and dosage of administration, and the severity of side effects. The effective amount may be the maximum dosage or dosing schedule that avoids significant side effects or toxic effects.

[0090] "Replacement" means replacement of the solvent system in which an antibody protein is dissolved, such as replacing a high-salt or high-osmotic pressure solvent system containing the antibody protein by physical manipulation using a buffer system of a stable formulation to enable the presence of the antibody protein in the stable formulation. The so-called physical manipulation includes, but is not limited to, ultrafiltration, dialysis, and reconstitution after centrifugation.

[0091] Specific embodiments The following further describes the present disclosure in combination with examples, but these examples do not limit the scope of the present disclosure. Experimental methods for which specific conditions are not described in the examples of the present disclosure are generally carried out under conventional conditions such as those described in the "Antibody Experimental Manual" and "Molecular Cloning Manual" of Cold Spring Harbor Laboratory, or conditions recommended by raw material or product manufacturers. Reagents for which specific sources are not indicated are commercially available conventional reagents.

[0092] Biological evaluation I. Preparation of anti-B7H3-ADC formulation ADC-1 The structure of the anti-B7H3-ADC in the present disclosure is as follows:

[0093]

Chemical formula

[0094] First, the naked antibody solution h1702DS (6.43 L, 1.39 mmol) is diluted with an acetic acid-sodium acetate buffer solution (0.98 L, 30 mM) at pH 5.0, and then an ethylenediaminetetraacetic acid (EDTA) solution (0.57 L, 29 mmol), a histidine-acetic acid-tris solution (1.47 L, 20 mM) at pH 8.2, and a tris solution (0.2 L, 1 M) are added in sequence, mixed well, and then a tris(2-carboxyethyl)phosphine (TCEP) solution (1.78 L, 3.06 mmol) is added and mixed well. The resulting solution is reacted at 12°C for 2 hours, and after the reaction is completed, acetic acid is added to adjust the pH to 6.0.

[0095] First, 0.57 L of a dimethyl sulfoxide (DMSO) solvent is added to the above reaction solution, and then a compound 5-A solution (prepared with reference to 5-A in Example 5 of the specification of WO2020063673A1; in this case, the compound 5-A is dissolved in DMSO, 6.58 g, 6.12 mmol) is added dropwise to the reaction solution and mixed well. The resulting solution is reacted at 12°C for 1 hour. After the reaction is completed, acetic acid is added to adjust the pH of the reaction solution to 5.0. The reaction solution is purified by cation chromatography (column resin Capto S impAct, elution phase 50 mM acetic acid-sodium acetate + 240 mM NaCl, pH 5.5), and then ultrafiltration exchange is performed to yield an exemplary product ADC-1 of the antibody-drug conjugate B7H3-ADC in the present disclosure.

[0096] The DAR value was detected by RP-HPLC and n was 4.1.

[0097] II. Formulation The equipment used for the preparation and detection of the formulation and the method for the calculation of the results are as follows:

[0098] 1) Size exclusion chromatography (SEC): A method for the analysis of the separation of solutes based on the relative relationship between the pore size of the gel pores and the molecular coil size of the polymer sample.

[0099] SEC% (Percentage of SEC monomer content) = monomer / total × 100% (The monomer is the peak area of the main peak monomer in the sample, and the total is the sum of the areas of all peaks).

[0100] SEC measuring instrument: Agilent 1260, column: Waters, XBridge BEH200Å SEC (300×7.8mm, 3.5μm)

[0101] 2) Capillary electrophoresis (CE): A type of electrophoresis that transfers the gel to a capillary as the supporting medium, and it is also a separation method based on the molecular weight of the sample under a certain voltage.

[0102] Percentage of reduced CE purity = main peak / total × 100% (The main peak is the peak area of the light chain main peak + heavy chain main peak in the sample, and the total is the sum of the areas of all peaks).

[0103] CE measuring instrument: Beckman model Plus800

[0104] 3) Turbidity determination: The degree to which light is blocked when passing through the aqueous layer indicates the ability of the aqueous layer to scatter and absorb light, which is related not only to the content of suspended solids but also to the composition, size, shape, and surface reflectivity of the particles. By comparing the absorbance values of the same protein sample at the same concentration and wavelength (near-ultraviolet and visible wavelength ranges), the higher the absorbance value, the higher the turbidity, indicating a more significant tendency of protein aggregation in the sample. The measuring instrument is a multifunctional microplate reader (Molecular Devices M5). The same amount of sample was added to a 96-well plate, and the absorbance value was read.

[0105] 4) Osmotic pressure determination: The osmotic pressure was determined using the freezing point method. Based on the direct proportional relationship between the freezing point depression of the solution and the molar concentration, the freezing point of the solution was determined using a highly sensitive temperature-sensitive component, and the electrical quantity was converted into osmotic pressure. Equipment manufacturer: Loser, model OM815.

[0106] 5) Protein concentration determination: The concentration of the antibody-drug conjugate in the present disclosure was measured by the protein concentration, that is, the concentration of the antibody fraction in the antibody-drug conjugate.

[0107] Since the toxin in the antibody-drug conjugate is also absorbed at the characteristic absorption wavelengths of proteins, 280 nm and 370 nm, the following formula is used to calculate the aforementioned protein concentration:

[0108]

Number

[0109] That is:

[0110]

Number

[0111] In the formula, A 280nm : The average absorbance value at wavelength 280 nm of a single sample of the test substance solution when the path length is 1 cm; A 370nm : The average absorbance value at wavelength 370 nm of a single sample of the test substance solution when the path length is 1 cm; E mAb-280 : The mass absorption coefficient of the protein at wavelength 280 nm, which is 1.532 g -1 cm -1 L; E 薬物-280 : The mass absorption coefficient of the toxin at wavelength 280 nm, which is 5.17 g -1 cm -1 L; E 薬物-370 : The mass absorption coefficient of the toxin at wavelength 370 nm, which is 17.89 g -1 cm-1 It is L; R: The ratio of the absorbance coefficient of the toxin at 370 nm to that at 280 nm is 3.46; C mAb : Protein concentration, mg / mL; l: Path length, cm (in this case, the path length is 1 cm).

[0112] When the test solution is diluted, the protein concentration is: C (mg / mL) = C mAb × N, where N is the dilution factor.

[0113] Equipment for determining protein concentration: UV-visible spectrophotometer, model: Nano Drop One.

[0114] 6) Calculation of drug loading dose (reverse-phase high-performance liquid chromatography, RP-HPLC)

[0115] 1. Reagents and equipment: HPLC system: Waters H-Class ultra-high-performance liquid chromatography (UPLC) system Detector: TUV detector (measurement wavelength: 280 nm) Chromatography column: BioResolve RPmAb polyphenyl (2.7 μm 4.6 * 150 mm)

[0116] 2. Detection conditions: Column temperature: 80 °C Flow rate: 1.0 mL / min Mobile phase A: Aqueous solution of 0.1% formic acid + 0.025% trifluoroacetic acid (TFA) Mobile phase B: Acetonitrile solution of 0.1% FA + 0.025% trifluoroacetic acid (TFA) Gradient procedure: 27.0% B - 45.0% B (0.00 min - 12.00 min), 45.0% B - 80.0% B (12.00 min - 13.00 min), 80.0% B - 80.0% B (13.00 min - 15.00 min), 27.0% B - 27.0% B (15.04 min - 20.00 min), Injection volume: 5.0 μL

[0117] 3. Data analysis When compared with the antibody light chain (L0) and heavy chain (H0) not bound to the drug, the light chain bound to the drug (light chain bound to one drug: L1) and the heavy chain bound to the drug (heavy chain bound to one drug: H1, heavy chain bound to two drugs: H2, heavy chain bound to three drugs: H3, heavy chain bound to four drugs: H4) showed an increase in hydrophobicity proportional to the number of drugs bound, and the retention time became longer. Therefore, elution can be carried out in the order of L0, L1, H0, H1, H2, H3, and H4. As a comparison, the results of the retention times of L0 to H0 were used as a reference to assign the detected peaks to any of L0, L1, H0, H1, H2, H3, and H4.

[0118] Due to the absorption of UV by the drug linker, the peak areas obtained according to the number of drugs bound were corrected based on the following formula using the molar absorption coefficients of the light chain, heavy chain, and drug linker. The calculation formula is as follows: Light chain (εLC-280) / (εLC-280 + number of drugs bound × εdrug-280) Heavy chain (εHC-280) / (εHC-280 + number of drugs bound × εdrug-280) Note: εLC-280: Molar extinction coefficient of the light chain at 280 nm; εHC-28: Molar extinction coefficient of the heavy chain at 280 nm; εdrug-280: Molar extinction coefficient of the toxin at 280 nm.

[0119]

Table 1

[0120] Note: Total of the corrected peak areas of LC = corrected peak area of L0 + corrected peak area of L1 Total of the corrected peak areas of HC = corrected peak area of H0 + corrected peak area of H1 + corrected peak area of H2 + corrected peak area of H3 + corrected peak area of H4, and the drug loading dose of this ADC is calculated as follows:

[0121] [Number] [Examples]

[0122] Example 1: Buffer System and pH Screening of Anti-B7H3-ADC Antibody Preparation Using the following buffers, an anti-B7H3-ADC preparation containing 60 mg / ml sucrose, 0.2 mg / ml polysorbate 80 (abbreviation: PS80), and ADC-1 with a protein concentration of 20 mg / mL was prepared. The buffers are as follows: 1) 10 mM citric acid - sodium citrate (abbreviation: CA), pH 5.0; 2) 10 mM succinic acid - sodium succinate (abbreviation: SA), pH 5.0; 3) 10 mM SA, pH 5.5; 4) 10 mM histidine - hydrochloride (abbreviation: His), pH 5.5; 5) 10 mM His, pH 6.0; 6) 10 mM His, pH 6.5; 7) 10 mM histidine - acetate (abbreviation: His-AA), pH 5.0; 8) 10 mM His-AA, pH 5.5; 9) 10 mM disodium hydrogen phosphate - sodium dihydrogen phosphate (abbreviation: PB), pH 6.5.

[0123] The prepared preparations were filtered, filled, sealed, and capped. The stability of the samples maintained under forced degradation conditions (40 °C M1, that is, standing at a high temperature of 40 °C for 1 month, shaking at 300 rpm for D10, that is, shaking for 10 days) was investigated. The stability of the preparations was investigated using appearance, SEC, and reduced CE-SDS as evaluation indicators. The experimental results are shown in Table 2.

[0124] Under the forced degradation conditions of oscillation D10, the appearance of the protein formulation in the SA and His-AA buffer systems was superior to that in the CA, His, and PB systems. The SA, His-AA, CA, and His buffer systems showed no significant difference in purity and were all superior to the PB system. Under the forced degradation conditions of 40°C M1, the protein formulations in the SA and His-AA buffer systems had an appearance superior to that in the CA and PB systems. Considering comprehensively, SA and His-AA were preferred as the buffer systems for the B7H3-ADC formulation for subsequent formulation screening, and the preferred pH value was 5.0 - 5.5, more preferably 5.5.

[0125]

Table 2

[0126] Note: D0 indicates the start of the experiment; "M" indicates months, for example, M1 indicates 1 month.

[0127] Example 2: Screening of Surfactants in Anti-B7H3-ADC Antibody Formulations Using a 10 mM SA buffer system at pH 5.0, anti-B7H3-ADC formulations containing 60 mg / ml of sucrose, ADC-1 with a protein concentration of 20 mg / mL, and various types and concentrations of surfactants were prepared. Specifically: 1) 0.2 mg / mL of polysorbate 80 (abbreviation: PS80); 2) 0.4 mg / mL of PS80; 3) 0.2 mg / mL of polysorbate 20 (abbreviation: PS20).

[0128] The prepared formulations were filtered, filled, sealed, and capped. The samples were maintained under conditions of oscillation (25°C, 300 rpm, 6 days), freeze-thaw (-35°C to 2 - 8°C for 5 cycles), and high temperature (40°C M1). The stability of the formulations was investigated using appearance, SEC, and reducing capillary electrophoresis (R-CE) as evaluation indicators. The experimental results are shown in Table 3.

[0129] The experimental results showed no significant differences in appearance and purity among the 0.2 mg / mL PS80 group, 0.4 mg / mL PS80 group, and 0.2 mg / mL PS20 group under the storage conditions.

[0130]

Table 3

[0131] Note: In the table, "D" indicates days, for example, D6 indicates 6 days, and D0 indicates the start of the experiment; "M" indicates months, for example, M1 indicates 1 month, and "FT 5 cycles" indicates 5 freeze-thaw cycles.

[0132] Example 3: Screening of sugars in anti-B7H3-ADC antibody formulations Using a 10 mM SA buffer system at pH 5.0, an anti-B7H3-ADC formulation containing 0.2 mg / ml of polysorbate 80 and an ADC-1 with a protein concentration of 20 mg / mL was prepared. The various sugars are as follows: 1) 60 mg / mL of sucrose; 2) 60 mg / mL of trehalose; 3) 50 mg / mL of mannitol.

[0133] The prepared formulations were filtered, filled, sealed, and capped. The stability of the samples under forced degradation conditions (shaking D6, i.e., shaking for 6 days; FT5 cycles, i.e., 5 freeze-thaw cycles from -35°C to 2 - 8°C) was investigated, and the stability of the formulations was investigated by SEC as an evaluation index. The experimental results are shown in Table 4.

[0134] The experimental data showed that under the forced degradation conditions of shaking D6 and FT5C, the SEC purity of the sucrose group and the trehalose group was superior to that of the mannitol group. Subsequently, an anti-B7H3-ADC antibody formulation containing 20 mg / mL of antibody, 60 mg / mg / ml of sucrose, and 0.2 mg / mL of PS80 was prepared in 10 mM SA buffer at pH 5.0, and the formulation sample was lyophilized. After lyophilization, the appearance showed bottom collapse, so further optimization was continued and the concentration of sucrose was reduced from 60 mg / mL to 40 mg / mL.

[0135]

Table 4

[0136] Note: In the table, "D" indicates the day, and D0 indicates the start of the experiment.

[0137] Example 4: Screening of Osmotic Pressure Regulators in Anti-B7H3-ADC Antibody Formulations Using a 10 mM SA buffer system at pH 5.5, an anti-B7H3-ADC formulation containing 40 mg / mL of sucrose, 0.2 mg / ml of PS80, and ADC-1 with a protein concentration of 20 mg / mL was prepared. By increasing and screening the ionic strength of the osmotic pressure regulator, the osmotic pressure of the formulation was controlled within the isotonic range (270 - 330 mosm).

[0138] Using a 30 mM SA buffer system at pH 5.5, an anti-B7H3-ADC formulation containing 40 mg / mL of sucrose, 0.2 mg / ml of PS80, ADC-1 with a protein concentration of 20 mg / mL, and various types of isotonic osmotic pressure regulators was prepared. Specifically: 1) 58 mM arginine hydrochloride; 2) 7.6 mg / mL glycine;

[0139] The prepared formulations were filtered, filled, partially sealed, and lyophilized. The lyophilization procedure is shown in Table 5:

[0140]

Table 5

[0141] The stability of the lyophilized samples under the forced degradation conditions (40 °C D23, i.e., standing at a high temperature of 40 °C for 23 days) was investigated, and the stability of the formulation was investigated using appearance, SEC, and reduced R-CE as evaluation indicators. The experimental results are shown in Table 6.

[0142] Under the forced degradation conditions of 40 °C D23, there was no significant difference in purity among the groups, and the appearance of the glycine group was significantly superior to that of the arginine hydrochloride group. Since the osmotic pressure of the formulation containing 40 mg / ml of sucrose and 7.6 mg / ml of glycine in the sample was about 300 mOsm, the preferred concentration of glycine was 7.6 mg / mL. The osmotic pressure regulator glycine was used, and when its concentration was 5.3 mg / mL to 9.8 mg / mL, the osmotic pressure of the formulation was controlled within the isotonic range.

[0143]

Table 6

[0144] Note: In the table, "D" indicates days. For example, D23 indicates 23 days.

[0145] Example 5: Screening of Protein Concentration in Anti-B7H3-ADC Antibody Formulation Using a 30 mM SA buffer system at pH 5.5, anti-B7H3-ADC formulations with various protein concentrations containing 40 mg / mL of sucrose, 0.2 mg / ml of PS80, and 7.6 mg / mL of glycine were prepared. Specifically: 1) 20 mg / mL of ADC-1 2) 50 mg / mL of ADC-1 3) 60 mg / mL of ADC-1 4) 70 mg / mL of ADC-1

[0146] The prepared formulation was filtered, filled, sealed, and capped. Samples were placed under shaking (25°C, 300 rpm, for 6 days), freeze-thaw (-35°C to 2 - 8°C for 5 cycles), and high temperature (40°C M1) conditions, and the stability of the formulation was investigated using appearance, SEC, and DAR value as evaluation indicators. The experimental results are shown in Table 7.

[0147] The experimental results showed that under storage conditions, there were no significant differences in appearance, purity, and toxin load among the 20 mg / mL ADC-1, 50 mg / mL ADC-1, 60 mg / mL ADC-1, and 70 mg / mL ADC-1 groups.

[0148]

Table 7

[0149] Note: In this table, "D" indicates day, for example, D0 indicates the start of the experiment; "M" indicates month, for example, M1 indicates 1 month; "FT 5 cycles" indicates 5 freeze-thaw cycles.

[0150] Example 6: Stability Experiment of Anti-B7H3-ADC Antibody Formulation An anti-B7H3-ADC formulation containing 20 mg / mL ADC-1 of protein concentration, 30 mM succinic acid - sodium succinate (SA) pH 5.5, 40 mg / mL sucrose, 7.6 mg / mL glycine, and 0.2 mg / mL PS80 was prepared.

[0151] The prepared formulation was filtered, filled, sealed, and capped. The stability of the samples under forced degradation conditions (-35°C to 2 - 8°C for 5 cycles of freeze-thaw or shaking for 8 days) and long-term storage conditions (2 - 8°C for 16 days) was investigated, and the stability of the formulation was investigated using appearance, SEC, and reduced R-CE as evaluation indicators. The experimental results are shown in Table 8.

[0152] The experimental results showed that for appearance, the samples remained transparent under various forced degradation conditions, there was no significant difference in purity, and the stability was good.

[0153]

Table 8

[0154] Note: In the table, "D" indicates the day. For example, D0 is the start date of the experiment, and FT5C indicates 5 freeze-thaw cycles.

[0155] Example 7: Formulation Optimization Experiment of B7H3-ADC Preparation Optimization Experiment 1: For the anti-B7H3-AD preparation containing 7.6 mg / mL of glycine and 40 mg / mL of sucrose, DOE (Design of Experiments) experiments were carried out using 30 mM succinic acid-sodium succinate (SA) pH value, protein concentration of ADC-1, and concentration of PS80 as variables. To design a series of formulations, the DOE, experimental coefficients, and levels were set at pH 5-6, ADC-1 protein concentration 10-30 mg / mL, and PS80 concentration 0.1-0.3 mg / mL (see Table 9). The lyophilized samples were subjected to a forced degradation experiment at a high temperature of 40 °C for 1 month, and the evaluation indicators included appearance and SEC. See Table 9 for the results.

[0156] The results showed that the preparations of various formulations maintained at a high temperature of 40 °C for 1 month were transparent, and there was no significant difference in purity among the SEC groups. Therefore, the B7H3-ADC samples showed good stability when the pH was 5-6, the protein concentration of ADC-1 was 10-30 mg / mL, and the concentration of PS80 was 0.1-0.3 mg / mL.

[0157]

Table 9

[0158]

Table 10

[0159] Note: In the table, "D" indicates day, for example, D0 indicates the start of the experiment; "M" indicates month, for example, M1 indicates 1 month.

[0160] Optimization Experiment 2: Based on the anti-B7H3-AD formulation containing ADC-1 with a protein concentration of 50 mg / mL, 40 mg / ml of sucrose, 7.6 mg / ml of glycine, and 0.2 mg / mL of PS80 in 30 mM SA succinic acid-sodium succinate buffer at pH 5.5, a series of formulations were designed using, as variables, buffer concentration, pH, sucrose concentration, glycine concentration, PS80 concentration, and ADC-1 protein concentration (see Table 11). The lyophilized samples were subjected to a forced degradation experiment at a high temperature of 40°C for 1 month, and evaluation was carried out using appearance, SEC purity, and DAR value as evaluation indicators. Refer to Table 12 for the results.

[0161] The results showed that the formulations of various formulations maintained at a high temperature of 40°C for 1 month were transparent, and there was no significant difference in SEC purity and DAR toxin load among groups. Therefore, the B7H3-ADC samples showed good stability when the SA concentration was 20 - 30 mM, the pH was 5.0 - 5.5, the protein concentration of ADC-1 was 20 - 70 mg / mL, the sucrose concentration was 40 - 80 mg / mL, and the PS80 concentration was 0.2 - 0.8 mg / mL.

[0162]

Table 11

[0163]

Table 12

[0164] Note: In the table, "D" indicates day and "W" indicates week. For example, D0 indicates the start of the experiment and W4 indicates 4 weeks.

[0165] Example 8: Lyophilization of Anti-B7H3-ADC Antibody Formulation The anti-B7H3-ADC antibody formulation containing ADC-1 at a protein concentration of 20 mg / mL, sucrose at 40 mg / mL, glycine at 7.6 mg / mL, and PS80 at 0.2 mg / mL was prepared in 30 mM SA succinic acid-sodium succinate buffer at pH 5.5. The formulation sample was lyophilized using a lyophilization procedure consisting of pre-freezing, primary drying, and secondary drying; refer to the lyophilization procedure in Table 13. After the completion of the lyophilization procedure, vacuum sealing was performed. Before and after lyophilization, the sample was evaluated using appearance, SEC, and R-CE as evaluation indicators; refer to Table 14 for the results. The results showed that the reconstituted solution could maintain the good performance of the liquid formulation.

[0166]

Table 13

[0167]

Table 14

Claims

1. In a pharmaceutical composition comprising an antibody-drug conjugate and a buffer, the antibody-drug conjugate has the following structure: 【Chemical 1】 (wherein, h1702DS is an anti-B7H3 antibody comprising the heavy chain shown in SEQ ID NO: 1 and the light chain shown in SEQ ID NO: 2, n ranges from 1 to 10, preferably from 1 to 8, more preferably from 3 to 5, and even more preferably is about 4, the buffer for the pharmaceutical composition is a succinate buffer or a histidine buffer, preferably succinic acid-sodium succinate or histidine-acetate), a pharmaceutical composition characterized by having the same.

2. The pharmaceutical composition according to claim 1, wherein the concentration of the buffer is about 10 mM to about 50 mM, preferably about 20 mM to about 40 mM, and more preferably about 30 mM.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pH of the pharmaceutical composition is about 4.5 to 6.5, preferably about 5.0 to 6.0, and more preferably about 5.5 to 5.

6.

4. The pharmaceutical composition according to any one of claims 1 to 3, further comprising a surfactant which is preferably polysorbate, more preferably polysorbate 80 or polysorbate 20, and most preferably polysorbate 80.

5. The pharmaceutical composition according to claim 4, wherein the concentration of the surfactant is about 0.01 mg / mL to about 1.0 mg / mL, preferably about 0.2 mg / mL to about 0.8 mg / mL, and more preferably about 0.2 mg / mL.

6. The pharmaceutical composition according to any one of claims 1 to 5, further comprising a sugar which is preferably sucrose, mannitol, or trehalose, more preferably sucrose or trehalose.

7. The pharmaceutical composition according to claim 6, wherein the concentration of the sugar is about 25 mg / mL to about 80 mg / mL, preferably about 40 mg / mL to about 60 mg / mL, and more preferably about 40 mg / mL.

8. The pharmaceutical composition according to any one of claims 1 to 7, further comprising an amino acid or amino acid salt which is glycine or arginine hydrochloride, preferably glycine.

9. The pharmaceutical composition according to claim 8, wherein the concentration of the amino acid or amino acid salt is about 5 mg / mL to about 10 mg / mL, preferably about 7 mg / mL to about 8 mg / mL, more preferably about 7.6 mg / mL.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the concentration of the antibody-drug conjugate is measured by protein concentration and is in the range of about 10 mg / mL to about 70 mg / mL, preferably about 20 mg / mL to about 50 mg / mL, more preferably about 20 mg / mL to about 30 mg / mL or about 30 mg / mL to about 50 mg / mL, and even more preferably about 20 mg / mL or about 30 mg / mL or about 50 mg / mL.

11. The pharmaceutical composition comprises the following components: (a) the antibody-drug conjugate with a protein concentration of about 20 mg / mL to about 70 mg / mL, (b) polysorbate at about 0.2 mg / mL to about 0.8 mg / mL, (c) sugar at about 25 mg / mL to about 80 mg / mL, (d) glycine at about 7 mg / mL to about 8 mg / mL, and (e) succinic acid buffer at about 10 mM to about 40 mM, and the pH of the composition is about 5.0 to 6.

0. Preferably, the pharmaceutical composition comprises the following components: (a) the antibody-drug conjugate with a protein concentration of about 20 mg / mL to about 50 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL to about 0.8 mg / mL, (c) sucrose at about 40 mg / mL to about 80 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) succinic acid-sodium succinate at about 30 mM, and the pH of the composition is about 5.5 to about 5.

6. More preferably, the pharmaceutical composition comprises the following components: (a) the antibody-drug conjugate with a protein concentration of about 20 mg / mL to about 30 mg / mL, (b) polysorbate 80 at about 0.2 mg / mL, (c) sucrose at about 40 mg / mL, (d) glycine at about 7.6 mg / mL, and (e) succinic acid-sodium succinate at about 30 mM, and the pH of the composition is about 5.5 to 5.

6. The pharmaceutical composition according to any one of claims 1 to 10, comprising (a) the antibody-drug conjugate at a protein concentration of about 30 mg / mL to about 50 mg / mL, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM of succinic acid-sodium succinate, wherein the pH of the composition is about 5.5 to 5.

6.

12. A lyophilized formulation containing the antibody-drug conjugate, characterized in that the pharmaceutical composition according to any one of claims 1 to 11 is formed upon reconstitution.

13. A method for preparing a lyophilized formulation containing the antibody-drug conjugate, characterized in that the method comprises the step of lyophilizing the pharmaceutical composition according to any one of claims 1 to 11.

14. A lyophilized formulation containing the antibody-drug conjugate, characterized in that it is obtained by lyophilizing the pharmaceutical composition according to any one of claims 1 to 11.

15. A reconstitution solution containing the antibody-drug conjugate, obtained by reconstituting the lyophilized formulation according to claim 12 or 14, wherein the reconstitution solution comprises the following components: (a) the antibody-drug conjugate at a protein concentration of about 20 mg / mL to about 70 mg / mL, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate, (c) about 25 mg / mL to about 80 mg / mL of sugar, (d) about 7 mg / mL to about 8 mg / mL of glycine, and (e) about 10 mM to about 40 mM of succinic acid buffer, and the pH of the composition is about 5.0 to 6.

0. Preferably, the reconstitution solution comprises the following components: (a) the antibody-drug conjugate at a protein concentration of about 20 mg / mL to about 50 mg / mL, (b) about 0.2 mg / mL to about 0.8 mg / mL of polysorbate 80, (c) about 40 mg / mL to about 80 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM of succinic acid-sodium succinate, and the pH of the composition is about 5.5 to 5.

6. More preferably, the reconstitution solution comprises the following components: (a) said antibody-drug conjugate having a protein concentration of about 20 mg / mL to about 30 mg / mL, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM of succinic acid-sodium succinate, wherein the pH of said composition is about 5.5 to 5.6, (a) said antibody-drug conjugate having a protein concentration of about 30 mg / mL to about 50 mg / mL, (b) about 0.2 mg / mL of polysorbate 80, (c) about 40 mg / mL of sucrose, (d) about 7.6 mg / mL of glycine, and (e) about 30 mM of succinic acid-sodium succinate, and wherein the pH of said composition is about 5.5 to 5.6, characterized in that it is a reconstitution solution.

16. A product, characterized in that it comprises a container containing the pharmaceutical composition according to any one of claims 1 to 11, the lyophilized preparation according to claim 12 or 14, or the reconstitution solution according to claim 15.

17. Preferably, the disease is cancer associated with B7H3 expression, more preferably, said cancer is breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, or lymphoma, for the preparation of a medicament for the treatment of said disease, the use of the pharmaceutical composition according to any one of claims 1 to 11, the lyophilized preparation according to claim 12 or 14, the reconstitution solution according to claim 15, or the product according to claim 16.

Images