Anti-b7h3 antibody-eribulin conjugate formulation, preparation method therefor, and use thereof

Abstract

An anti-B7H3 antibody-eribulin conjugate formulation, a preparation method therefor, and use thereof. The formulation contains trehalose, polysorbate 20, histidine, histidine hydrochloride, and an anti-B7H3 antibody-eribulin conjugate or a pharmaceutically acceptable salt or solvate thereof. A specific formulation formula is designed for the anti-B7H3 antibody-eribulin conjugate. By means of using the synergistic cooperation of specific component excipients and a buffer system, the stability of the formulation is significantly improved. Moreover, the preparation process is further verified scientifically and systematically, which proves that the well-developed anti-B7H3 antibody-eribulin conjugate formulation and the preparation process thereof maintain the purity, coupling rate, and free drug of the finished product stable, reduce molecular degradation and aggregates, have strong process stability, and can be applied on a large scale, thereby promoting the application of the anti-B7H3 antibody-eribulin conjugate.

Description

An anti-B7H3 antibody-eribulin conjugate preparation and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and relates to an anti-B7H3 antibody-eribulin conjugate preparation and a preparation method and application thereof. BACKGROUND

[0002] Antibody drug conjugate (ADC) technology is to couple a monoclonal antibody and a drug together through a linker. At present, most ADCs are formed by coupling an antibody targeting a tumor antigen and a small-molecule chemical drug with high cytotoxicity through a linker, and the small-molecule drug is targeted to tumor cells to kill tumors by taking advantage of the specific binding of the antibody to the target antigen.

[0003] B7-H3 protein, also known as CD276, is a type I transmembrane protein of the immunoglobulin superfamily. B7-H3 is expressed on the surface of immune cells or tumor cells, and its corresponding receptor ligand can be multiple, and no major ligand or receptor has been found, and the main biological function is unknown. At the same time, in a variety of tumors, the high expression of B7-H3 in tumor tissues is negatively correlated with the prognosis of survival period to a certain extent. B7-H3 is widely expressed in tissues such as heart, liver, pancreas, prostate, small intestine and colon, but the expression amount is very low. It is also expressed in immune cells, but the expression amount is also very low, and it is not constitutive expression but inducible expression. B7-H3 is expressed in a variety of malignant tumors, including melanoma, glioma, lung cancer, pancreatic cancer, renal cancer, colon cancer, ovarian cancer, breast cancer, gastric cancer, endometrial cancer and some hematological tumors.

[0004] The target B7-H3 is highly expressed on the surface of a variety of solid tumor cells and is lowly expressed in normal tissues and cells, and drugs targeting B7-H3 are already in the clinical stage. Typical representative drugs include: early-stage large-molecule drugs (not including cell therapy) targeting B7-H3, such as nuclide-conjugated antibody iodine 131-Omburtamab, and then MGD009, MGA271, MGC018 and DS-7300 and other camptothecin ADCs. ABBV-155 (Mirzotamab Clezutoclax) is an antibody conjugate targeting B7-H3, and the conjugated small molecule is BCL inhibitor MGA271 (Enoblituzumab) is a monoclonal antibody drug targeting B7-H3. MGC018 is an antibody conjugate targeting B7-H3, and the conjugated small molecule is duocarmycin.

[0005] In practical application, i.e. in the preservation process, the antibody conjugate drug needs to be prepared into a specific preparation. In the antibody drug preparation, molecular degradation and aggregate formation both reduce the stability of the drug and the therapeutic effect, etc., and produce side effects, such as causing immunogenicity or intravenous disorders in patients. Therefore, when preparing the antibody drug, it is necessary to reduce the degradation and aggregation of the antibody molecules and to provide the stability of the drug preparation. Therefore, it is of great significance to develop a specific preparation formula and preparation method for effectively ensuring the stability of the antibody conjugate drug, i.e. the therapeutic effect, for the application of the antibody conjugate drug. SUMMARY

[0006] The present application provides an anti-B7H3 antibody-eribulin conjugate preparation and its preparation method and application, a new preparation formula and preparation process are designed to improve the stability of the product, the purity, coupling rate and free drug are kept stable, and the molecular degradation and aggregate conditions are reduced, which is beneficial to the application of the anti-B7H3 antibody-eribulin conjugate.

[0007] In the first aspect, the present application provides an anti-B7H3 antibody-eribulin conjugate preparation, which contains an anti-B7H3 antibody-eribulin conjugate or a pharmaceutically acceptable salt or solvate thereof, and trehalose, polysorbate 20, histidine and hydrochloric acid histidine.

[0008] The present application designs a specific preparation formula for the anti-B7H3 antibody-eribulin conjugate, uses specific component excipients and buffer systems to cooperate synergistically, significantly improves the stability of the preparation, the purity, coupling rate and free drug of the preparation are kept stable, and the molecular degradation and aggregate conditions are reduced, the process stability is strong, it can be applied on a large scale, and promotes the application of the anti-B7H3 antibody-eribulin conjugate.

[0009] Preferably, the mass ratio of the anti-B7H3 antibody-eribulin conjugate, trehalose, polysorbate 20, histidine and hydrochloric acid histidine in the anti-B7H3 antibody-eribulin conjugate preparation is 1:(5.0-10.0):(0.01-0.05):(0.01-0.20):(0.01-0.20), including but not limited to 1:6:0.02:0.05:0.05, 1:8:0.04:0.1:0.08, 1:9:0.03:0.15:0.18 or 1:7:0.03:0.08:0.15, etc.

[0010] In the present application, the anti-B7H3 antibody-eribulin conjugate preparation can be a liquid preparation, or a powder preparation, which can be adjusted according to actual needs. In the specific embodiments of the present application, the concentration of the anti-B7H3 antibody-eribulin conjugate in the liquid preparation is 8.5-12.5 mg / mL, the concentration of trehalose is 50-1000 mmol / L, the concentration of polysorbate 20 is 0.1-0.3 mg / mL, and the solvent is 8-12 mmol / L histidine and histidine hydrochloride buffer.

[0011] Preferably, the pH of the anti-B7H3 antibody-eribulin conjugate preparation is 5.2-6.8, including but not limited to 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 6, 6.2, 6.4, 6.6, or 6.7, etc., preferably 5.3-5.9.

[0012] In the present application, the condition of pH 5.2-pH 6.0 can keep the SEC purity of the sample relatively stable, the condition of pH 5.2-pH 6.4 can keep the nrCE purity of the ADC sample relatively stable, and there is no obvious difference in other stability aspects of the sample within the range of pH 5.2-pH 6.8. In combination with the stability results, the control of pH at 5.6±0.3 can further effectively maintain the stability results in various aspects.

[0013] It can be understood that the present application focuses on designing the overall formulation of the preparation, including the synergistic cooperation of the excipients and the buffer system. The anti-B7H3 antibody-eribulin conjugate known in the art is theoretically suitable for the present application. For example, the structure of the anti-B7H3 antibody-eribulin conjugate is shown as follows (Formula I).

[0014] In the formula, TL is an anti-B7H3 antibody, and n is an integer or a decimal number of 1-20. The average n of the conjugate is 3.5.

[0015] Antibody heavy chain sequence:

[0016] Variable region (SEQ ID NO. 1):

[0017] Constant region (SEQ ID NO. 2):

[0018] Antibody light chain sequence:

[0019] Variable region (SEQ ID NO. 3):

[0020] Constant region (SEQ ID NO. 4):

[0021] In a second aspect, the present application provides a preparation method of the anti-B7H3 antibody-eribulin conjugate preparation of the first aspect, and the preparation method comprises:

[0022] The anti-B7H3 antibody-eribulin conjugate, trehalose, polysorbate 20, histidine and histidine hydrochloride are mixed to obtain the anti-B7H3 antibody-eribulin conjugate preparation.

[0023] Preferably, the preparation method specifically comprises:

[0024] The solution containing the anti-B7H3 antibody-eribulin conjugate is dialyzed in a histidine and histidine hydrochloride buffer solution by using an ultrafiltration membrane to obtain a dialysate, and the dialysate is mixed with a solution containing trehalose and polysorbate 20 excipients to obtain the anti-B7H3 antibody-eribulin conjugate preparation.

[0025] Preferably, the concentration of the histidine and histidine hydrochloride buffer solution is 1.0-100 mmol / L, including but not limited to 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 92, 95, 97, or 99 mmol / L, etc., and the pH is 5.2-6.8, preferably the pH is 5.3-5.9.

[0026] Preferably, in the solution containing trehalose and polysorbate 20 excipients, the concentration of the trehalose is 50-1000 mmol / L (for example, it can be 51, 52, 55, 60, 65, 70, 75, 80, 85, 90, 92, 96, 97, 98 or 99 mmol / L, etc.), the concentration of the polysorbate 20 is 0.1-0.3 mg / mL, and the solvent is the histidine and histidine hydrochloride buffer solution.

[0027] Preferably, the preparation method further comprises a freeze-drying step.

[0028] In a third aspect, the present application provides a pharmaceutical composition containing the anti-B7H3 antibody-eribulin conjugate preparation of the first aspect.

[0029] Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable diluent or carrier.

[0030] In a fourth aspect, the present application provides use of the anti-B7H3 antibody-eribulin conjugate preparation of the first aspect or the pharmaceutical composition of the third aspect in the preparation of a drug for treating tumors.

[0031] Preferably, the tumor comprises at least one of a solid tumor or a blood flow such as breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethral cancer, bladder cancer, liver cancer, gastric cancer, endometrial cancer, salivary gland cancer, esophageal cancer, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioblastoma, sarcoma, lymphoma or leukemia.

[0032] Compared with the prior art, the present application has at least the following beneficial effects:

[0033] The present application is directed to an anti-B7H3 antibody-eribulin conjugate, a specific formulation is designed, and specific component excipients and buffer systems are used to cooperate synergistically to significantly improve the stability of the formulation. Further, the preparation process is scientifically and systematically verified, proving that the anti-B7H3 antibody-eribulin conjugate formulation and preparation process developed in the present application are good, the purity, conjugation rate and free drug of the finished product are stable, and the molecular degradation and aggregate conditions are reduced. The process has strong stability and can be applied on a large scale, promoting the application of the anti-B7H3 antibody-eribulin conjugate. DETAILED DESCRIPTION

[0034] The technical solutions of the present application will be further described below through specific embodiments. However, the following examples are only simple examples of the present application and do not represent or limit the protection scope of the present application. The protection scope of the present application is subject to the claims.

[0035] If a specific technology or condition is not specified in the examples, the technology or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be purchased through a regular channel.

[0036] In the present application, a specific formulation and preparation method are designed for the anti-B7H3 antibody-eribulin conjugate and its pharmaceutically acceptable salt or solvate, in order to ensure the purity, conjugation rate and stability of the product, reduce the molecular degradation and aggregate conditions, promote its application, and specifically include the design of specific excipients, buffer systems, formulation pH, etc.

[0037] In the specific embodiments of the present application, the anti-B7H3 antibody with heavy chain and light chain as described below is taken as an example to further illustrate the technical solutions of the present application.

[0038] Antibody heavy chain sequence:

[0039] Variable region (SEQ ID NO. 1):

[0040] Constant region (SEQ ID NO. 2):

[0041] Antibody light chain sequence:

[0042] Variable region (SEQ ID NO. 3):

[0043] Constant region (SEQ ID NO. 4):

[0044] The structure of the anti-B7H3 antibody-eribulin conjugate is as follows, and the preparation method refers to CN117917248A.

[0045] TL is an anti-B7H3 antibody, n is an integer or a decimal number between 1 and 20, and the average n of the conjugate is 3.5 after detection.

[0046] Example 1

[0047] In this embodiment, an anti-B7H3 antibody-eribulin conjugate preparation is prepared.

[0048] A 10 mmol / L histidine / histidine hydrochloride buffer solution with a pH of 5.6 (weigh 1.21 g of histidine and 4.66 g of histidine hydrochloride, dissolve in water for injection, and then use water for injection to make up to 3 L after dissolution, and filter sterilize) is prepared, and a 10 mmol / L histidine / histidine hydrochloride 5x (200 mmol / L trehalose + 0.2 mg / mL polysorbate 20) auxiliary solution with a pH of 5.6 (weigh 18.92 g of trehalose and 0.049 g of polysorbate 20, dissolve in the above histidine / histidine hydrochloride buffer solution, and then use the above histidine / histidine hydrochloride buffer solution to make up to 50 mL after dissolution, and filter sterilize) is prepared.

[0049] The B7H3 antibody-eribulin conjugate solution prepared according to CN117917248A is taken, and dialysis is performed using a 0.1 mm 2 Ultrafiltration membrane (inlet pressure 10-15 psi, backflow pressure 8-12 psi, 10 times the volume of liquid) into the above histidine / histidine hydrochloride buffer solution, and the conjugate concentration is adjusted to about 12.5 mg / mL. Mix with the auxiliary solution at a volume ratio of 4:1 to obtain an anti-B7H3 antibody-eribulin conjugate preparation stock solution (liquid preparation).

[0050] The liquid preparation is taken for freeze-drying, and the liquid preparation is divided into 10 mL vials, 7 vials are divided, and the freeze-drying process is shown in Table 1. The anti-B7H3 antibody-eribulin conjugate freeze-dried preparation is prepared.

[0051] Table 1

[0052] Example 2

[0053] The anti-B7H3 antibody-eribulin conjugate formulation was prepared in this example.

[0054] A 10 mmol / L histidine / histidine hydrochloride buffer solution (histidine 0.47 g, histidine hydrochloride 3.56 g, dissolved with injection water, and then diluted with injection water to 2 L, and sterilized filtered) was prepared at pH 5.3. A 10 mmol / L histidine / histidine hydrochloride 5x (200 mmol / L trehalose + 0.2 mg / mL polysorbate 20) auxiliary solution (trehalose 18.92 g, polysorbate 20 0.050 g, dissolved with the above histidine / histidine hydrochloride buffer solution, and then diluted with the above histidine / histidine hydrochloride buffer solution to 50 mL, and sterilized filtered) was prepared at pH 5.3.

[0055] The anti-B7H3 antibody-eribulin conjugate liquid formulation and lyophilized formulation were prepared according to the preparation method of Example 1, except that the buffer and auxiliary solution were replaced.

[0056] Example 3

[0057] The anti-B7H3 antibody-eribulin conjugate formulation was prepared in this example.

[0058] A 10 mmol / L histidine / histidine hydrochloride buffer solution (histidine 1.27 g, histidine hydrochloride 2.48 g, dissolved with injection water, and then diluted with injection water to 2 L, and sterilized filtered) was prepared at pH 5.9. A 10 mmol / L histidine / histidine hydrochloride 5x (200 mmol / L trehalose + 0.2 mg / mL polysorbate 20) auxiliary solution (trehalose 18.92 g, polysorbate 20 0.050 g, dissolved with the above histidine / histidine hydrochloride buffer solution, and then diluted with the above histidine / histidine hydrochloride buffer solution to 50 mL, and sterilized filtered) was prepared at pH 5.9.

[0059] The anti-B7H3 antibody-eribulin conjugate liquid formulation and lyophilized formulation were prepared according to the preparation method of Example 1, except that the buffer and auxiliary solution were replaced.

[0060] Example 4

[0061] The anti-B7H3 antibody-eribulin conjugate formulation was prepared in this example.

[0062] Prepare 10 mmol / L histidine / histidine hydrochloride buffer of pH = 5.6 (weigh histidine 1.21 g, histidine hydrochloride 4.66 g, dissolve in water for injection, after dissolving, use water for injection to constant volume to 3 L, filter sterilization), prepare 10 mmol / L histidine / histidine hydrochloride 5x (200 mmol / L trehalose + 0.1 mg / mL polysorbate 20) auxiliary solution of pH = 5.6 (weigh 18.92 g of trehalose, 0.026 g of polysorbate 20, dissolve in the above histidine / histidine hydrochloride buffer, after dissolving, use the above histidine / histidine hydrochloride buffer to constant volume to 50 mL, filter sterilization).

[0063] Prepare the liquid formulation and lyophilized formulation of the anti-B7H3 antibody-eribulin conjugate according to Example 1, only replace the buffer and auxiliary solution.

[0064] Example 5

[0065] Prepare the anti-B7H3 antibody-eribulin conjugate formulation in this example.

[0066] Prepare 10 mmol / L histidine / histidine hydrochloride buffer of pH = 5.6 (weigh histidine 1.21 g, histidine hydrochloride 4.66 g, dissolve in water for injection, after dissolving, use water for injection to constant volume to 3 L, filter sterilization), prepare 10 mmol / L histidine / histidine hydrochloride 5x (200 mmol / L trehalose + 0.1 mg / mL polysorbate 20) auxiliary solution of pH = 5.6 (weigh 18.92 g of trehalose, 0.026 g of polysorbate 20, dissolve in the above histidine / histidine hydrochloride buffer, after dissolving, use the above histidine / histidine hydrochloride buffer to constant volume to 50 mL, filter sterilization).

[0067] Prepare the liquid formulation and lyophilized formulation of the anti-B7H3 antibody-eribulin conjugate according to Example 1, only replace the buffer and auxiliary solution.

[0068] Example 6

[0069] Prepare the anti-B7H3 antibody-eribulin conjugate formulation in this example.

[0070] Compared with Example 1, the only difference is that 10 mmol / L histidine / histidine hydrochloride buffer of pH = 5.6 is replaced by an equal amount of 10 mmol / L histidine / histidine hydrochloride buffer of pH = 5.2 (weigh histidine 0.37 g, histidine hydrochloride 3.69 g, dissolve in water for injection, after dissolving, use water for injection to constant volume to 2 L, filter sterilization).

[0071] Example 7

[0072] The anti-B7H3 antibody-eribulin conjugate preparation is prepared in this example.

[0073] The difference compared with Example 1 is only that the 10 mmol / L histidine / histidine hydrochloride buffer solution of pH = 5.6 is replaced by an equivalent amount of 10 mmol / L histidine / histidine hydrochloride buffer solution of pH = 6.0 (histidine 1.46 g, histidine hydrochloride 2.22 g are weighed, dissolved with water for injection, and after dissolution, water for injection is used to make up to 2 L, and sterilized by filtration).

[0074] Example 8

[0075] The anti-B7H3 antibody-eribulin conjugate preparation is prepared in this example.

[0076] The difference compared with Example 1 is only that the 10 mmol / L histidine / histidine hydrochloride buffer solution of pH = 5.6 is replaced by an equivalent amount of 10 mmol / L histidine / histidine hydrochloride buffer solution of pH = 6.4 (histidine 2.14 g, histidine hydrochloride 1.30 g are weighed, dissolved with water for injection, and after dissolution, water for injection is used to make up to 2 L, and sterilized by filtration).

[0077] Example 9

[0078] The anti-B7H3 antibody-eribulin conjugate preparation is prepared in this example.

[0079] The difference compared with Example 1 is only that the 10 mmol / L histidine / histidine hydrochloride buffer solution of pH = 5.6 is replaced by an equivalent amount of 10 mmol / L histidine / histidine hydrochloride buffer solution of pH = 6.8 (histidine 2.64 g, histidine hydrochloride 0.63 g are weighed, dissolved with water for injection, and after dissolution, water for injection is used to make up to 2 L).

[0080] Comparative Example 1

[0081] The anti-B7H3 antibody-eribulin conjugate preparation is prepared in this example.

[0082] The difference compared with Example 1 is only that the 200 mmol / L trehalose in the excipient solution is replaced by 200 mmol / L sucrose.

[0083] Comparative Example 2

[0084] The anti-B7H3 antibody-eribulin conjugate preparation is prepared in this example.

[0085] The difference compared with Example 1 is only that the 200 mmol / L trehalose in the excipient solution is replaced by 200 mmol / L mannitol and 53 mmol / L trehalose.

[0086] Comparative Example 3

[0087] The comparative example 1 was prepared to prepare an anti-B7H3 antibody-eribulin conjugate formulation.

[0088] The difference compared with example 1 is only that 0.2 mg / mL polysorbate 20 in the auxiliary material solution is replaced by 0.2 mg / mL polysorbate 80.

[0089] Comparative example 4

[0090] The comparative example 1 was prepared to prepare an anti-B7H3 antibody-eribulin conjugate formulation.

[0091] The difference compared with example 1 is only that 10 mmol / L histidine / histidine hydrochloride buffer with pH = 5.6 is replaced by an equal amount of 10 mmol / L citric acid / citric acid sodium buffer with pH = 5.6.

[0092] Comparative example 5

[0093] The comparative example 1 was prepared to prepare an anti-B7H3 antibody-eribulin conjugate formulation.

[0094] The difference compared with example 1 is only that 10 mmol / L histidine / histidine hydrochloride buffer with pH = 5.6 is replaced by an equal amount of 10 mmol / L citric acid / citric acid sodium buffer with pH = 5.2.

[0095] Comparative example 6

[0096] The comparative example 1 was prepared to prepare an anti-B7H3 antibody-eribulin conjugate formulation.

[0097] The difference compared with example 1 is only that 10 mmol / L histidine / histidine hydrochloride buffer with pH = 5.6 is replaced by an equal amount of 10 mmol / L citric acid / citric acid sodium buffer with pH = 4.4.

[0098] Comparative example 7

[0099] The comparative example 1 was prepared to prepare an anti-B7H3 antibody-eribulin conjugate formulation.

[0100] The difference compared with example 1 is only that 10 mmol / L histidine / histidine hydrochloride buffer with pH = 5.6 is replaced by 10 mmol / L citric acid / citric acid sodium buffer with pH = 4.8.

[0101] Test example

[0102] The anti-B7H3 antibody-eribulin conjugate formulations prepared in each example and comparative example were tested in this test example.

[0103] 1. Investigation of repeated freeze-thaw stability

[0104] The anti-B7H3 antibody-eribulin conjugate liquid preparation prepared in Example 1 and Comparative Examples 1-3 was used for stability investigation of repeated freeze-thawing for 5 times. The freeze-thawing operation was as follows: the sample was frozen at -80±10℃ for not less than 4h, and after complete thawing at room temperature (25±5℃) for 1h, the sample was used as a control, and the investigation scheme was as shown in Table 2, and the test items were: W = CE-SDS; X = osmotic pressure; Y = pH; Z = appearance, protein concentration, SEC-HPLC, MFI and HIC-HPLC.

[0105] Table 2

[0106] The results are shown in Table 3. According to Table 3, in the high-temperature stability investigation at 40℃, the specific mannitol and polysorbate 20 used as the excipient combination in the present application can effectively resist the anti-B7H3 antibody-eribulin conjugate, ensure the stability of SEC purity and insoluble particle concentration, and protect the appearance of the product. The repeated freeze-thawing stability investigation at -80℃ further shows that the specific mannitol and polysorbate 20 used as the excipient combination in the present application can effectively resist the anti-B7H3 antibody-eribulin conjugate product.

[0107] Table 3

[0108] Further analysis of the product stability of Example 1 and Comparative Examples 4-7 shows that for the two buffer systems, under the same pH conditions, the histidine / histidine hydrochloride buffer system is superior to the citric acid / sodium citrate buffer system in terms of maintaining the stability of product insoluble particle concentration, SEC purity, nrCE purity, rCE purity and conjugation rate.

[0109] In summary, the specific mannitol and polysorbate 20 used as the excipient combination, and the specific histidine / histidine hydrochloride buffer system in the present application can effectively protect the anti-B7H3 antibody-eribulin conjugate and improve the stability in all aspects.

[0110] 2, Further analysis of the stability of the prepared anti-B7H3 antibody-eribulin conjugate liquid preparation and freeze-dried preparation

[0111] Comparative Example 1 and Examples 6-9 show that the histidine salt buffer system with different pH values, the pH 5.2-pH 6.0 condition can maintain the SEC purity of the sample more stable, the pH 5.2-pH 6.4 condition can maintain the nrCE purity of the ADC sample more stable, and there is no obvious difference in other stability aspects within the pH 5.2-pH 6.8 range. According to the comprehensive stability results, controlling the pH to be 5.6±0.3 can further effectively maintain the stability results in all aspects.

[0112] The freeze-dried sample investigation scheme is shown in Table 4, and the test items are: X = osmotic pressure; Y = appearance, pH, protein concentration, SEC-HPLC, HIC-HPLC, MFI, CE-SDS, moisture, reconstitution time, free drug, binding activity.

[0113] Table 4

[0114] The results of Examples 1-5 were analyzed, including the effects of high temperature (30°C) on liquid formulations and repeated freezing and thawing (-80°C storage) and the results of experiments on the effects of high temperature (40°C) on freeze-dried formulations.

[0115] (1) For the liquid formulations, the initial osmotic pressures of the five groups of samples with pH in the range of 5.3-5.9 and polysorbate 20 concentration in the range of 0.1-0.3 mg / mL were all within the acceptable range. After being placed at 30°C for 2 weeks, the pH, protein concentration, and appearance of the samples showed no significant changes, the SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate, and free drug remained stable, the level of insoluble particles was very low, the binding activity was within the acceptable range of 100±30%, and there was no significant difference between the five groups of samples. Therefore, the stability of the samples subjected to the high temperature (30°C) effect factor experiment was not significantly different, and the stability was good.

[0116] (2) After being repeatedly frozen and thawed at -80°C for 5 times, the pH, protein concentration, and appearance of the five groups of samples with pH in the range of 5.3-5.9 and polysorbate 20 concentration in the range of 0.1-0.3 mg / mL showed no significant changes, the SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate, and free drug remained stable, the level of insoluble particles was very low, the binding activity was within the acceptable range of 100±30%, and there was no significant difference between the five groups of samples. Therefore, the stability of the samples subjected to the repeated freezing and thawing (-80°C) experiment was not significantly different, and the stability was good.

[0117] (3) The initial freeze-dried appearance of the 5 groups of samples with pH in the range of 5.3-5.9 and polysorbate 20 concentration in the range of 0.1-0.3 mg / mL had no obvious difference, and different polysorbate 20 concentrations had no effect on the appearance of the freeze-dried samples. The 0-time reconstituted samples of the 3 groups of samples with pH in the range of 5.3-5.9 had osmotic pressure in the acceptable range and had no obvious difference from the samples before freeze-drying. After being placed at 40°C for 4 weeks, the samples had no obvious change in pH and protein concentration and had no obvious difference from the samples before freeze-drying. The appearance, SEC purity, nrCE-SDS purity, rCE-SDS purity, conjugation rate and free drug were stable. The reconstitution was fast, the reconstituted solution had very low insoluble particle content, the water content was slightly increased compared with that at 0 time, but was at a low level (<1.0%), and the binding activity was in the acceptable range of 100±30%. There was no obvious difference among the 3 groups of samples. Therefore, the ADC freeze-drying formulation with pH in the range of 5.3-5.9 had no obvious difference in stability under the influence of high temperature at 40°C, and the stability was good.

[0118] In summary, the formulation was determined as follows: 10 mmol / L histidine / histidine hydrochloride (target pH=5.6, pH range: 5.3-5.9), 200 mmol / L trehalose, 0.2 mg / mL polysorbate 20 (acceptable range: 0.1-0.3 mg / mL), and target protein concentration 10 mg / mL.

[0119] 3. Stability of the stock solution and finished product in the determined preparation process was investigated

[0120] The preparation was prepared in multiple batches according to the preparation process of Reference Example 1. One batch of the preparation stock solution was selected, filtered and divided into 5 mL PC bottles, 5 mL / bottle, a total of 4 bottles, one bottle was placed at 2-8°C as a control, and the other 3 bottles were subjected to repeated freeze-thawing at -80±10°C for 1 time, 3 times and 5 times, respectively. The freeze-thawing operation was as follows: the sample was frozen at -80±10°C for no less than 4 h, completely thawed at room temperature and then placed for 1 h. The detection items were appearance, protein concentration, particle concentration (MFI), SEC purity, nrCE-SDS purity, rCE-SDS purity, conjugation rate, free drug and binding activity.

[0121] The results of the repeated freeze-thawing experiment of the batch preparation stock solution are shown in Table 5. According to the detection results of the repeated freeze-thawing experiment of the stock solution, the sample appearance, protein concentration, SEC purity, nrCE-SDS purity, rCE-SDS purity, conjugation rate and free drug had no obvious change after repeated freeze-thawing at -80°C±10°C and room temperature for 1, 3 and 5 times. The insoluble particle concentration was low, and the binding activity was within the acceptable standard (70%-130%). It was indicated that repeated freeze-thawing at -80°C±10°C and room temperature for 5 times had no effect on the quality of the stock solution.

[0122] Table 5

[0123] The short-term stability of the stock solution at 2-8°C and 30±2°C was confirmed to provide parameter support for the preparation production. The stock solution was filtered and divided into 5mL PC bottles, 5mL per bottle, a total of 10 bottles, 2 bottles were used for 0h detection, 4 bottles were placed at 2-8°C, and samples were taken for detection at 3 days and 7 days, respectively. Another 4 bottles were placed at 30°C±2°C, and samples were taken for detection at 3 days and 7 days, respectively. Detection items: appearance, protein concentration, particle concentration (MFI), SEC purity, nrCE-SDS purity, rCE-SDS purity, free drug, coupling rate and binding activity.

[0124] Table 6

[0125] From the short-term stability results of the confirmation batch stock solution in Table 6, according to the short-term stability test results of the stock solution, after the stock solution was placed at 2-8°C and 30°C±2°C for 1 week, the appearance, protein concentration, SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate and free drug had no obvious change, the insoluble particle concentration was low, and the binding activity was within the acceptable standard (70%-130%). It showed that the quality of the stock solution could be kept stable at 2-8°C and 30°C±2°C for 1 week.

[0126] The stability of the freeze-dried preparation product at 40±2°C was investigated. 56 freeze-dried preparation products were divided into 3 groups, one group of 20 was used as the initial control, and the other 2 groups of 18 were investigated at 40±2°C for 2 weeks and 4 weeks, respectively. Detection items: reconstitution time, appearance / color (after reconstitution), clarity, moisture, visible foreign matter, insoluble particles, protein concentration, pH, osmotic pressure, SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate, free drug, binding activity, cell activity.

[0127] The high temperature influencing factor investigation results of the freeze-dried product of the confirmation batch are shown in Table 7.

[0128] According to the results of the investigation on the high temperature influencing factors of the confirmed batch of freeze-dried finished product, compared with the initial results, the powder cake and the reconstituted preparation had no color change after the finished product was placed at 40°C for 4 weeks, the reconstitution time was shorter, the preparation osmotic pressure, protein concentration and pH had no obvious change, the clarity was stable, the insoluble particles met the requirements (the number of particles of 10 μm and above in each container should not exceed 6000, and the number of particles of 25 μm and above in each container should not exceed 600), the water content met the requirements (should not be higher than 3.0%), the SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate and free drug remained stable, the binding activity was within the acceptable standard (70%-130%), and the cell activity was within the acceptable standard (60%-140%). It was shown that the freeze-dried finished product could remain stable under the condition of 40±2°C acceleration for 4 weeks.

[0129] Table 7

[0130] In summary, the present application is directed to an anti-B7H3 antibody-eribulin conjugate, a specific formulation is designed, a preparation process is developed, specific component excipients and buffer systems are used to cooperate, which significantly improves the stability of the preparation, and further scientific and systematic verification of the preparation process is carried out, which proves that the present application develops a good anti-B7H3 antibody-eribulin conjugate formulation and its preparation process, the purity, coupling rate and free drug of the finished product remain stable, and the molecular degradation and aggregate conditions are reduced, the process stability is strong, it can be used in large scale, and promotes the application of anti-B7H3 antibody-eribulin conjugate.

[0131] The applicant declares that the above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and those skilled in the art should understand that any changes or replacements within the technical scope disclosed by the present application can be easily thought out by any person skilled in the art, which falls within the protection scope and disclosure scope of the present application.

Claims

1. An anti-B7H3 antibody-eribulin conjugate formulation comprising an anti-B7H3 antibody-eribulin conjugate or a pharmaceutically acceptable salt or solvent compound thereof, and trehalose, polysorbate 20, histidine, and histidine hydrochloride.

2. The anti-B7H3 antibody-eribulin conjugate formulation according to claim 1, wherein, In the formulation, the mass ratio of the anti-B7H3 antibody-iribulin conjugate or its pharmaceutically acceptable salt or solvent compound to trehalose, polysorbate 20, histidine, and histidine hydrochloride is 1:(5.0–10.0):(0.01–0.05):(0.01–0.20):(0.01–0.20).

3. The anti-B7H3 antibody-eribulin conjugate formulation according to claim 1 or 2, wherein, The pH of the anti-B7H3 antibody-iribulin conjugate formulation is 5.2-6.8, preferably 5.3-5.

9.

4. A method for preparing an anti-B7H3 antibody-eribulin conjugate formulation according to any one of claims 1-3, comprising: The anti-B7H3 antibody-eribulin conjugate, trehalose, polysorbate 20, histidine, and histidine hydrochloride were mixed to obtain the anti-B7H3 antibody-eribulin conjugate formulation.

5. The preparation method according to claim 4, wherein, The preparation method specifically includes: A solution containing the anti-B7H3 antibody-eribulin conjugate was dialyzed using an ultrafiltration membrane in a histidine and histidine hydrochloride buffer solution to obtain a dialysate. The dialysate was then mixed with a solution containing trehalose and polysorbate 20 excipients to obtain the anti-B7H3 antibody-eribulin conjugate formulation.

6. The preparation method according to claim 5, wherein, The concentration of the histidine and histidine hydrochloride buffer solution is 1.0–100 mmol / L, and the pH is 5.2–6.8, preferably 5.3–5.

9.

7. The preparation method according to claim 5 or 6, wherein, In the excipient solution containing trehalose and polysorbate 20, the concentration of trehalose is 50-1000 mmol / L, the concentration of polysorbate 20 is 0.1-0.3 mg / mL, and the solvent is the histidine and histidine hydrochloride buffer solution.

8. The preparation method according to any one of claims 4-7, wherein, The preparation method also includes a freeze-drying step.

9. A pharmaceutical composition comprising the anti-B7H3 antibody-eribulin conjugate formulation according to any one of claims 1-3.

10. The use of the anti-B7H3 antibody-eribulin conjugate formulation according to any one of claims 1-3 or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating tumors; The tumors include at least one of the following: 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, lung cancer, colon cancer, rectal cancer, colorectal cancer, bone cancer, skin cancer, thyroid cancer, pancreatic cancer, melanoma, glioma, neuroblastoma, glioma multiforme, sarcoma, lymphoma, or leukemia.

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