Anti-B7H3 antibody-imine conjugate preparation as well as preparation method and application thereof

Abstract

The invention relates to an anti-B7H3 antibody-imine conjugate preparation as well as a preparation method and application thereof. The preparation contains trehalose, polysorbate 20, histidine, histidine hydrochloride and an anti-B7H3 antibody-imine brurin conjugate or a pharmaceutically acceptable salt or solvent compound of the anti-B7H3 antibody-imine brurin conjugate. According to the anti-B7H3 antibody-imine conjugate and the preparation method thereof, a specific preparation formula is designed for the anti-B7H3 antibody-imine conjugate, the stability of the preparation is remarkably improved by virtue of synergistic cooperation of specific component auxiliary materials and a buffer system, and the preparation process is further scientifically and systematically verified, so that the development of the good anti-B7H3 antibody-imine conjugate preparation and the preparation process thereof is proved, and the preparation method has the advantages that the preparation process is simple and convenient, and the application prospect is wide. The purity, the coupling rate and free drugs of the finished product are kept stable, the conditions of molecular degradation and aggregation are reduced, the process stability is high, large-scale application can be realized, and the application of the anti-B7H3 antibody-imine conjugate is promoted.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an anti-B7H3 antibody-eribulin conjugate formulation, its preparation method, and its application. Background Technology

[0002] Antibody-drug conjugate (ADC) technology uses linkers to conjugate monoclonal antibodies to drugs. Currently, most ADCs are composed of antibodies targeting tumor antigens linked to highly cytotoxic small-molecule chemical drugs via linkers. Utilizing the specific binding characteristic of antibodies to target antigens, the small-molecule drug is delivered directly to tumor cells to exert its tumor-killing effect.

[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. It may have multiple corresponding receptor ligands, but no single major ligand or receptor has been identified. Although it has been shown to have immunosuppressive or immunoactivating effects, its primary biological function remains unknown. Furthermore, in various tumors, high expression of B7-H3 in tumor tissue is negatively correlated with prognosis and survival to some extent. B7-H3 is widely expressed in tissues such as the heart, liver, pancreas, prostate, small intestine, and colon, but at very low levels. It is also expressed in immune cells, but at very low levels, and is inducible rather than constitutive. B7-H3 is expressed in various malignant tumors, including melanoma, glioma, lung cancer, pancreatic cancer, kidney cancer, colon cancer, ovarian cancer, breast cancer, gastric cancer, endometrial cancer, and some hematologic malignancies.

[0004] The target B7-H3 is highly expressed on the surface of various solid tumor cells but lowly expressed in normal tissues and cells. Drugs targeting B7-H3 are already in clinical trials. Typical representative drugs include: early large-molecule drugs targeting B7-H3 (excluding cell therapy) included the radionuclide-conjugated antibody iodine-131 (Omburtamab), followed by several camptothecin ADCs such as MGD009, MGA271, MGC018, and DS-7300. ABBV-155 (Mirzotamab Clezutoclax) is an antibody-drug conjugate targeting B7-H3, conjugated with the BCL inhibitor; MGA271 (Enoblituzumab) is a monoclonal antibody drug targeting B7-H3; and MGC018 is an antibody-drug conjugate targeting B7-H3, conjugated with the small molecule ducardioid.

[0005] In practical applications and storage, antibody-drug conjugates (ADCs) require specific formulations. In these formulations, molecular degradation and aggregate formation reduce drug stability and therapeutic efficacy, leading to side effects such as immunogenicity or venous complications. Therefore, reducing antibody molecule degradation and aggregation during ADC formulation preparation is crucial for ensuring drug stability. Developing specific formulations and preparation methods to effectively guarantee the stability and therapeutic efficacy of ADCs is therefore of great significance for their application. Summary of the Invention

[0006] To address the shortcomings of existing technologies and practical needs, this invention provides an anti-B7H3 antibody-eribulin conjugate formulation, its preparation method, and its application. A novel formulation and preparation process are designed to improve product stability, maintaining stable purity, conjugation rate, and free drug levels. Furthermore, it reduces molecular degradation and aggregation, thus facilitating the application of the anti-B7H3 antibody-eribulin conjugate.

[0007] To achieve this objective, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides an anti-B7H3 antibody-eribulin conjugate formulation, said formulation containing an anti-B7H3 antibody-eribulin conjugate or a pharmaceutically acceptable salt or solvent compound thereof, as well as trehalose, polysorbate 20, histidine, and histidine hydrochloride.

[0009] This invention targets anti-B7H3 antibody-eribulin conjugates, designing specific formulations and utilizing specific excipients and buffer systems in synergistic action to significantly improve formulation stability. The purity, conjugation rate, and free drug of the formulation remain stable, while molecular degradation and aggregation are reduced. The process exhibits strong stability, enabling large-scale application and promoting the application of anti-B7H3 antibody-eribulin conjugates.

[0010] Preferably, the mass ratio of the anti-B7H3 antibody-iribulin conjugate, trehalose, polysorbate 20, histidine, and histidine hydrochloride in the formulation 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.

[0011] In this invention, the anti-B7H3 antibody-eribulin conjugate formulation can be a liquid formulation or a powder formulation, which can be adjusted according to actual needs. In a specific embodiment of this invention, the concentration of the anti-B7H3 antibody-eribulin conjugate in the liquid formulation 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.

[0012] Preferably, the pH of the formulation is 5.2 to 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, and preferably 5.3 to 5.9.

[0013] In this invention, pH 5.2 to pH 6.0 conditions can maintain relatively stable SEC purity of sample, pH 5.2 to pH 6.4 conditions can maintain relatively stable nrCE purity of ADC sample, and there is no significant difference in other stability aspects between samples within the pH 5.2 to pH 6.8 range. Considering all stability results, controlling the pH to pH 5.6 ± 0.3 can further effectively maintain the stability results in all aspects.

[0014] It is understood that the focus of this invention is on designing the overall formulation, including the synergistic effect of excipients and buffer systems. Theoretically, all known anti-B7H3 antibody-eribulin conjugates in the art are applicable to this invention. For example, the structure of an anti-B7H3 antibody-eribulin conjugate is shown below (Formula I).

[0015]

[0016] TL represents anti-B7H3 antibody, and n is an integer or decimal from 1 to 20. The average n of the conjugates was 3.5.

[0017] Antibody heavy chain sequence:

[0018] Variable region (SEQ ID NO.1):

[0019] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAVSWVRQAPGKGLEWV ASISGGGIYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHG GAGYFDYWGQGTLVTVSS;

[0020] Constant region (SEQ ID NO.2):

[0021] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0022] Antibody light chain sequence:

[0023] Variable region (SEQ ID NO.3):

[0024] DSQMTQSPSSSLSASVGDRVTITTCRGSESVHSYLAWYQQKPGKAPKLLVY NAKTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYGSPPWTFGGG TKVEIK;

[0025] Constant region (SEQ ID NO.4):

[0026] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

[0027] In a second aspect, the present invention provides a method for preparing the anti-B7H3 antibody-eribulin conjugate formulation described in the first aspect, the preparation method comprising:

[0028] The anti-B7H3 antibody-eribulin conjugate, trehalose, polysorbate 20, histidine, and histidine hydrochloride were mixed to obtain the anti-B7H3 antibody-eribulin conjugate formulation.

[0029] Preferably, the preparation method specifically includes:

[0030] 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.

[0031] Preferably, the concentration of the histidine and histidine hydrochloride buffer solution is 1.0 to 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, and the pH is 5.2 to 6.8, preferably 5.3 to 5.9.

[0032] Preferably, the concentration of trehalose in the excipient solution containing trehalose and polysorbate 20 is 50–1000 mmol / L (e.g., 51, 52, 55, 60, 65, 70, 75, 80, 85, 90, 92, 96, 97, 98, or 99 mmol / L, etc.), the concentration of polysorbate 20 is 0.1–0.3 mg / mL, and the solvent is the histidine and histidine hydrochloride buffer solution.

[0033] Preferably, the preparation method further includes a freeze-drying step.

[0034] Thirdly, the present invention provides a pharmaceutical composition comprising the anti-B7H3 antibody-eribulin conjugate formulation described in the first aspect.

[0035] Preferably, the pharmaceutical composition further contains a pharmaceutically acceptable diluent or carrier.

[0036] Fourthly, the present invention provides the use of the anti-B7H3 antibody-eribulin conjugate formulation described in the first aspect or the pharmaceutical composition described in the third aspect in the preparation of a medicament for treating tumors.

[0037] Preferably, the tumor includes at least one of the following solid tumors or bloodstream tumors: 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.

[0038] Compared with the prior art, the present invention has at least the following beneficial effects:

[0039] This invention targets anti-B7H3 antibody-elebulin conjugates, designing specific formulations and utilizing specific excipients and buffer systems to synergistically improve formulation stability. Furthermore, the preparation process is scientifically and systematically validated, demonstrating that the invention develops a well-formulated anti-B7H3 antibody-elebulin conjugate formulation and its preparation process. The purity, conjugation rate, and free drug of the finished product remain stable, while molecular degradation and aggregation are reduced. The process exhibits strong stability and is suitable for large-scale application, thus promoting the application of anti-B7H3 antibody-elebulin conjugates. Detailed Implementation

[0040] The technical solution of the present invention will be further illustrated below through specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0041] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0042] This invention designs specific formulations and preparation methods for pharmaceutically acceptable salts or solvates of anti-B7H3 antibody-eribulin conjugates to ensure product purity, conjugation rate, and stability, reduce molecular degradation and aggregation, and promote its application. Specifically, this includes designing specific excipients, buffer systems, and formulation pH.

[0043] In a specific embodiment of the present invention, the technical solution of the present invention will be further illustrated using the anti-B7H3 antibody with heavy chain and light chain as described below as an example.

[0044] Antibody heavy chain sequence:

[0045] Variable region (SEQ ID NO.1):

[0046] EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYAVSWVRQAPGKGLEWV ASISGGGIYIYYPDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARHG GAGYFDYWGQGTLVTVSS;

[0047] Constant region (SEQ ID NO.2):

[0048] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0049] Antibody light chain sequence:

[0050] Variable region (SEQ ID NO.3):

[0051] DSQMTQSPSSSLSASVGDRVTITTCRGSESVHSYLAWYQQKPGKAPKLLVY NAKTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYGSPPWTFGGG TKVEIK;

[0052] Constant region (SEQ ID NO.4):

[0053] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC.

[0054] The structure of the anti-B7H3 antibody-iribulin conjugate is shown below, and the preparation method is as described in CN117917248A.

[0055]

[0056] TL represents anti-B7H3 antibody, and n is an integer or decimal from 1 to 20. The average n of the conjugates was 3.5.

[0057] Example 1

[0058] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0059] Prepare a dialysis buffer system with pH 5.6 using a 10 mmol / L histidine / histidine hydrochloride buffer solution (weigh 1.21 g of histidine and 4.66 g of histidine hydrochloride, dissolve in water for injection, and then bring the volume to 3 L with water for injection, followed by sterile filtration). Prepare an excipient solution with pH 5.6 using a 10 mmol / L histidine / histidine hydrochloride 5×(200 mmol / L trehalose + 0.2 mg / mL polysorbate 20) solution (weigh 18.92 g of trehalose and 0.049 g of polysorbate 20, dissolve in the above histidine / histidine hydrochloride buffer solution, and then bring the volume to 50 mL with the above histidine / histidine hydrochloride buffer solution, followed by sterile filtration).

[0060] Take the B7H3 antibody-iribulin conjugate solution prepared according to CN117917248A, and use 0.1m... 2 The ultrafiltration membrane was dialyzed (inlet pressure 10-15 psi, reflux pressure 8-12 psi, 10 times the liquid replacement volume) into the above histidine / histidine hydrochloride buffer solution, and the conjugate concentration was adjusted to about 12.5 mg / mL. It was then mixed with the excipient solution at a volume ratio of 4:1 to obtain the anti-B7H3 antibody-iribulin conjugate stock solution (liquid formulation).

[0061] The liquid formulation was freeze-dried and dispensed into 10mL vials, totaling 7 vials. The freeze-drying process is shown in Table 1. The freeze-dried formulation of anti-B7H3 antibody-eribulin conjugate was prepared.

[0062] Table 1

[0063]

[0064] Example 2

[0065] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0066] Prepare a dialysis buffer system with pH 5.3 using a 10 mmol / L histidine / histidine hydrochloride buffer solution (weigh 0.47 g histidine and 3.56 g histidine hydrochloride, dissolve in water for injection, and bring the volume to 2 L with water for injection, then filter sterilely). Prepare an excipient solution with pH 5.3 using a 10 mmol / L histidine / histidine hydrochloride 5×(200 mmol / L trehalose + 0.2 mg / mL polysorbate 20) solution (weigh 18.92 g trehalose and 0.050 g polysorbate 20, dissolve in the above histidine / histidine hydrochloride buffer solution, and bring the volume to 50 mL with the above histidine / histidine hydrochloride buffer solution, then filter sterilely).

[0067] Liquid and lyophilized formulations of anti-B7H3 antibody-iribulin conjugate were prepared according to Example 1, with only the buffer and excipient solutions being replaced.

[0068] Example 3

[0069] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0070] Prepare a dialysis buffer system with pH 5.9 using a 10 mmol / L histidine / histidine hydrochloride buffer solution (weigh 1.27 g histidine and 2.48 g histidine hydrochloride, dissolve in water for injection, and bring the volume to 2 L with water for injection, then filter sterilely). Prepare an excipient solution with pH 5.9 using a 10 mmol / L histidine / histidine hydrochloride 5×(200 mmol / L trehalose + 0.2 mg / mL polysorbate 20) solution (weigh 18.92 g trehalose and 0.050 g polysorbate 20, dissolve in the above histidine / histidine hydrochloride buffer solution, and bring the volume to 50 mL with the above histidine / histidine hydrochloride buffer solution, then filter sterilely).

[0071] Liquid and lyophilized formulations of anti-B7H3 antibody-iribulin conjugate were prepared according to Example 1, with only the buffer and excipient solutions being replaced.

[0072] Example 4

[0073] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0074] Prepare a dialysis buffer system of pH 5.6 with 10 mmol / L histidine / histidine hydrochloride buffer (weigh 1.21 g histidine and 4.66 g histidine hydrochloride, dissolve in water for injection, and bring the volume to 3 L with water for injection, then filter sterilely). Prepare an excipient solution of pH 5.6 with 10 mmol / L histidine / histidine hydrochloride 5×(200 mmol / L trehalose + 0.1 mg / mL polysorbate 20) (weigh 18.92 g trehalose and 0.026 g polysorbate 20, dissolve in the above histidine / histidine hydrochloride buffer, and bring the volume to 50 mL with the above histidine / histidine hydrochloride buffer, then filter sterilely).

[0075] Liquid and lyophilized formulations of anti-B7H3 antibody-iribulin conjugate were prepared according to Example 1, with only the buffer and excipient solutions being replaced.

[0076] Example 5

[0077] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0078] Prepare a dialysis buffer system with pH 5.6 using a 10 mmol / L histidine / histidine hydrochloride buffer solution (weigh 1.21 g of histidine and 4.66 g of histidine hydrochloride, dissolve in water for injection, and then bring the volume to 3 L with water for injection, followed by sterile filtration). Prepare an excipient solution with pH 5.6 using a 10 mmol / L histidine / histidine hydrochloride 5×(200 mmol / L trehalose + 0.3 mg / mL polysorbate 20) solution (weigh 18.92 g of trehalose and 0.077 g of polysorbate 20, dissolve in the above histidine / histidine hydrochloride buffer solution, and then bring the volume to 50 mL with the above histidine / histidine hydrochloride buffer solution, followed by sterile filtration).

[0079] Liquid and lyophilized formulations of anti-B7H3 antibody-iribulin conjugate were prepared according to Example 1, with only the buffer and excipient solutions being replaced.

[0080] Example 6

[0081] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0082] Compared with Example 1, the only difference is that the 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.6 is replaced with an equal volume of 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.2 (weigh 0.37 g of histidine and 3.69 g of histidine hydrochloride, dissolve in water for injection, and after dissolution, bring the volume to 2 L with water for injection and filter sterilely).

[0083] Example 7

[0084] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0085] Compared with Example 1, the only difference is that the 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.6 is replaced with an equal volume of 10 mmol / L histidine / histidine hydrochloride buffer at pH 6.0 (weigh 1.46 g of histidine and 2.22 g of histidine hydrochloride, dissolve in water for injection, and after dissolution, bring the volume to 2 L with water for injection and filter sterilely).

[0086] Example 8

[0087] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0088] Compared with Example 1, the only difference is that the 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.6 is replaced with an equal volume of 10 mmol / L histidine / histidine hydrochloride buffer at pH 6.4 (weigh 2.14 g of histidine and 1.30 g of histidine hydrochloride, dissolve in water for injection, and after dissolution, bring the volume to 2 L with water for injection and filter sterilely).

[0089] Example 9

[0090] This embodiment describes the preparation of an anti-B7H3 antibody-iribulin conjugate formulation.

[0091] Compared with Example 1, the only difference is that the 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.6 is replaced with an equal volume of 10 mmol / L histidine / histidine hydrochloride buffer at pH 6.8 (weigh 2.64 g of histidine and 0.63 g of histidine hydrochloride, dissolve in water for injection, and then bring the volume up to 2 L with water for injection after dissolution).

[0092] Comparative Example 1

[0093] This comparative study prepared an anti-B7H3 antibody-eribulin conjugate formulation.

[0094] The only difference from Example 1 is that the 200 mmol / L trehalose in the excipient solution is replaced with 200 mmol / L sucrose.

[0095] Comparative Example 2

[0096] This comparative study prepared an anti-B7H3 antibody-eribulin conjugate formulation.

[0097] The only difference from Example 1 is that 200 mmol / L trehalose is replaced with 200 mmol / L mannitol and 53 mmol / L trehalose in the excipient solution.

[0098] Comparative Example 3

[0099] This comparative study prepared an anti-B7H3 antibody-eribulin conjugate formulation.

[0100] The only difference from Example 1 is that the 0.2 mg / mL polysorbate 20 in the excipient solution is replaced with 0.2 mg / mL polysorbate 80.

[0101] Comparative Example 4

[0102] This comparative study prepared an anti-B7H3 antibody-eribulin conjugate formulation.

[0103] The only difference from Example 1 is that the 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.6 is replaced with an equal volume of 10 mmol / L citric acid / sodium citrate buffer at pH 5.6.

[0104] Comparative Example 5

[0105] This comparative study prepared an anti-B7H3 antibody-eribulin conjugate formulation.

[0106] The only difference from Example 1 is that the 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.6 is replaced with an equal volume of 10 mmol / L citric acid / sodium citrate buffer at pH 5.2.

[0107] Comparative Example 6

[0108] This comparative study prepared an anti-B7H3 antibody-eribulin conjugate formulation.

[0109] The only difference from Example 1 is that the 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.6 is replaced with an equal volume of 10 mmol / L citric acid / sodium citrate buffer at pH 4.4.

[0110] Comparative Example 7

[0111] This comparative study prepared an anti-B7H3 antibody-eribulin conjugate formulation.

[0112] The only difference from Example 1 is that the 10 mmol / L histidine / histidine hydrochloride buffer at pH 5.6 is replaced with a 10 mmol / L citric acid / sodium citrate buffer at pH 4.8.

[0113] Test case

[0114] This test case examines the anti-B7H3 antibody-eribulin conjugate formulations prepared in each of the embodiments and comparative examples.

[0115] 1. Stability study under repeated freeze-thaw cycles

[0116] The anti-B7H3 antibody-eribulin conjugate liquid formulations prepared in Example 1 and Comparative Examples 1-3 were used for stability testing through five freeze-thaw cycles. The freeze-thaw cycle was performed as follows: the sample was frozen at -80±10℃ for at least 4 hours, completely thawed at room temperature (25±5℃), and then allowed to stand for 1 hour. Anti-B7H3 antibody-eribulin conjugates without formulation preparation were used as a control. The testing protocol is shown in Table 2. Test items: W = CE-SDS; X = osmotic pressure; Y = pH; Z = appearance, protein concentration, SEC-HPLC, MFI, and HIC-HPLC.

[0117] Table 2

[0118]

[0119] The results are shown in Table 3. As can be seen from Table 3, in the high-temperature stability test at 40℃, the specific combination of mannose and polysorbate 20 designed in this invention as excipients works synergistically to effectively resist B7H3 antibody-erebrine conjugate, ensuring more stable SEC purity and insoluble particle concentration, and protecting the product appearance. The repeated freeze-thaw stability test at -80℃ further shows that the combination of mannose and polysorbate 20 as excipients in this invention can effectively resist B7H3 antibody-erebrine conjugate products.

[0120] Table 3

[0121]

[0122] Further analysis of the product stability of Example 1 and Comparative Examples 4-7 revealed that, for both buffer systems, under the same pH conditions, the histidine / histidine hydrochloride buffer system was superior to the citric acid / sodium citrate buffer system in maintaining the stability of product insoluble particle concentration, SEC purity, nrCE purity, rCE purity, and coupling rate.

[0123] In summary, this invention designs a specific combination of mannose and polysorbate 20 as excipients, along with a specific histidine / histidine hydrochloride buffer system, to synergistically prepare an anti-B7H3 antibody-erebrine conjugate formulation. This formulation effectively protects the anti-B7H3 antibody-erebrine conjugate and improves its stability in various aspects.

[0124] 2. Further analysis was conducted on the stability of the prepared anti-B7H3 antibody-iribulin conjugate liquid and lyophilized formulations.

[0125] Comparing Examples 1 and 6-9, the histidine buffer systems at different pH values ​​showed that pH 5.2–6.0 maintained relatively stable SEC purity in the sample, while pH 5.2–6.4 maintained relatively stable nrCE purity in the ADC sample. Other stability parameters showed no significant differences between samples within the pH 5.2–6.8 range. Considering all stability results, controlling the pH to 5.6 ± 0.3 further effectively maintained stability in all aspects.

[0126] The test scheme for lyophilized samples is shown in Table 4. Test items: X = osmotic pressure; Y = appearance, pH, protein concentration, SEC-HPLC, HIC-HPLC, MFI, CE-SDS, moisture, reconstitution time, free drug, and binding activity.

[0127] Table 4

[0128] initial 40℃ for 2 weeks 40℃ for 4 weeks X, Y Y Y

[0129] The results of Examples 1-5 were analyzed, including the experimental results of the influence of high temperature at 30°C on liquid formulations and repeated freeze-thaw cycles (-80°C freezing) and the influence of high temperature at 40°C on lyophilized formulations.

[0130] (1) For liquid formulations, the initial osmotic pressure of the five groups of samples with pH ranges of 5.3 to 5.9 and polysorbate 20 concentrations of 0.1 to 0.3 mg / mL was within acceptable ranges. After being placed at 30°C for two weeks, the pH, protein concentration, and appearance of the samples showed no significant changes. The purity of SEC, nrCE-SDS, rCE-SDS, coupling rate, and free drug remained stable. The level of insoluble particles was very low, and the binding activity was within the acceptable range of 100 ± 30%. There were no significant differences among the five groups of samples. Therefore, for liquid formulations with pH ranges of 5.3 to 5.9, polysorbate 20 concentrations of 0.1 to 0.3 mg / mL, and high temperature (30°C) as an influence factor, the stability of the samples was not significantly different, and the stability was good.

[0131] (2) For liquid formulations, after five freeze-thaw cycles at -80℃, the pH, protein concentration, and appearance of the five groups of samples with pH ranges of 5.3 to 5.9 and polysorbate 20 concentrations of 0.1 to 0.3 mg / mL showed no significant changes. The purity of SEC, nrCE-SDS, rCE-SDS, coupling rate, and free drug remained stable, with very low levels of insoluble particles and binding activity within the acceptable range of 100±30%. There were no significant differences among the five groups of samples. Therefore, for liquid formulations with pH ranges of 5.3 to 5.9 and polysorbate 20 concentrations of 0.1 to 0.3 mg / mL, the stability of samples subjected to repeated freeze-thaw cycles at -80℃ showed no significant difference, and all samples exhibited good stability.

[0132] (3) For lyophilized formulations, the five groups of samples with pH range of 5.3 to 5.9 and polysorbate 20 concentration of 0.1 to 0.3 mg / mL showed no significant difference in appearance after initial lyophilization. Different polysorbate 20 concentrations did not affect the appearance of the lyophilized samples. The three groups of samples with pH range of 5.3 to 5.9 had osmotic pressure within acceptable range after reconstitution at pH 0, showing no significant difference from the samples before lyophilization. After being placed at 40℃ for 4 weeks, the pH and protein concentration of the samples showed no significant change, showing no significant difference from the samples before lyophilization. The appearance, SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate, and free drug remained stable. Reconstitution was relatively fast. The level of insoluble particles in the solution after reconstitution was very low. The water content increased slightly compared to pH 0, but remained at a low level (<1.0%). The binding activity was within the acceptable range of 100±30%, and there was no significant difference among the three groups of samples. Therefore, the stability of the ADC freeze-dried formulations within the pH range of 5.3 to 5.9 and the experimental samples under the influence of high temperature at 40℃ showed no significant difference, and all samples exhibited good stability.

[0133] In summary, the formulation is determined as follows: 10 mmol / L histidine / histidine hydrochloride (target pH 5.6, pH range: pH 5.3 to pH 5.9), 200 mmol / L trehalose, 0.2 mg / mL polysorbate 20 (acceptable range: 0.1 to 0.3 mg / mL), and target protein concentration of 10 mg / mL.

[0134] 3. Investigate the stability of the stock solution and the finished product used to determine the formulation preparation process.

[0135] Multiple batches of formulations were prepared according to the preparation process in Example 1. One batch of the formulation stock solution was sterilized, filtered, and dispensed into 5mL PC bottles (5mL / bottle), for a total of four bottles. One bottle was kept at 2–8℃ as a control, and the other three bottles were subjected to repeated freeze-thaw cycles at -80±10℃ once, three times, and five times, respectively. The freeze-thaw cycle was as follows: the sample was frozen at -80±10℃ for at least 4 hours, completely thawed at room temperature, and then allowed to stand for 1 hour. The following parameters were tested: appearance, protein concentration, particle concentration (MFI), SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate, free drug, and binding activity.

[0136] The results of the repeated freeze-thaw experiments on the batch formulation stock solution are shown in Table 5. According to the test results, after repeated freeze-thaw cycles of 1, 3, and 5 times at -80℃±10℃ and room temperature, there were no significant changes in sample appearance, protein concentration, SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate, and free drug. The concentration of insoluble particles was low, and the binding activity was within acceptable limits (70%-130%). This indicates that repeated freeze-thaw cycles of 5 times at -80℃±10℃ and room temperature had no effect on the quality of the stock solution.

[0137] Table 5

[0138]

[0139] The short-term stability of the stock solution was confirmed at 2–8℃ and 30±2℃ to provide parameter support for formulation production. The stock solution was sterilely filtered and dispensed into 5mL PC bottles (5mL / bottle), for a total of 10 bottles. Two bottles were used for 0-hour testing, four bottles were placed at 2–8℃ and samples were taken for testing at 3 and 7 days, respectively, and the remaining four bottles were placed at 30℃±2℃ and samples were taken for testing at 3 and 7 days, respectively. The following parameters were measured: appearance, protein concentration, particle concentration (MFI), SEC purity, nrCE-SDS purity, rCE-SDS purity, free drug, conjugation rate, and binding activity.

[0140] Table 6

[0141]

[0142] Table 6 shows the short-term stability results of the confirmed batch of the drug solution. According to the short-term stability test results, after being stored at 2–8℃ and 30℃±2℃ for one week, the appearance, protein concentration, SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate, and free drug showed no significant changes. The concentration of insoluble particles was low, and the binding activity was within acceptable limits (70%-130%). This indicates that the quality of the drug solution remained stable after being stored at 2–8℃ and 30℃±2℃ for one week.

[0143] The stability of the lyophilized formulation was investigated at 40±2℃ under accelerated conditions. Fifty-six vials of the lyophilized formulation were divided into three groups: one group of 20 vials served as the initial control, and the other two groups each contained 18 vials. These were tested at 40±2℃ for 2 weeks and 4 weeks, respectively. The following parameters were measured: reconstitution time, appearance / color (after reconstitution), clarity, moisture content, visible foreign matter, insoluble particles, protein concentration, pH, osmotic pressure, SEC purity, nrCE-SDS purity, rCE-SDS purity, coupling rate, free drug, binding activity, and cell viability.

[0144] The results of the investigation into the high-temperature effects on the confirmed batch of freeze-dried products are shown in Table 7.

[0145] Based on the results of the investigation into the effects of high temperature on the confirmed batch of lyophilized finished products, compared with the initial results, after 4 weeks of storage at 40℃, the color of the powder and the reconstituted formulation remained unchanged, the reconstitution time was short, and the osmotic pressure, protein concentration, and pH of the formulation showed no significant changes. Clarity remained relatively stable, and the number of insoluble microparticles met the requirements (no more than 6000 particles of 10μm or larger per container, and no more than 600 particles of 25μm or larger per container). Moisture content met the requirements (no higher than 3.0%), and the purity of SEC, nrCE-SDS, rCE-SDS, conjugation rate, and free drug remained stable. Binding activity and cell viability were within acceptable limits (70%–130%) and within acceptable limits (60%–140%). This indicates that the lyophilized finished product remained relatively stable after 4 weeks at 40±2℃.

[0146] Table 7

[0147]

[0148] In summary, this invention designs specific formulations and develops preparation processes for anti-B7H3 antibody-elebulin conjugates. By utilizing specific excipient components and buffer systems in synergistic effects, the stability of the formulation is significantly improved. Furthermore, the preparation process is scientifically and systematically validated, demonstrating that the anti-B7H3 antibody-elebulin conjugate formulation and its preparation process developed by this invention maintain stable purity, conjugation rate, and free drug levels in the finished product. It also reduces molecular degradation and aggregation, exhibits strong process stability, and is suitable for large-scale application, thus promoting the application of anti-B7H3 antibody-elebulin conjugates.

[0149] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A formulation of an anti-B7H3 antibody-eribulin conjugate, characterized in that, The formulation contains an anti-B7H3 antibody-eribulin conjugate or a pharmaceutically acceptable salt or solvent compound thereof, as well as trehalose, polysorbate 20, histidine, and histidine hydrochloride.

2. The anti-B7H3 antibody-eribulin conjugate formulation according to claim 1, characterized in that, 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, characterized in that, The pH of the preparation is 5.2 to 6.8, preferably 5.3 to 5.

9.

4. The method for preparing the anti-B7H3 antibody-eribulin conjugate formulation according to any one of claims 1-3, characterized in that, The preparation method includes: 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 method for preparing the anti-B7H3 antibody-eribulin conjugate formulation according to claim 4, characterized in that, 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 method for preparing the anti-B7H3 antibody-eribulin conjugate formulation according to claim 5, characterized in that, 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 method for preparing the anti-B7H3 antibody-eribulin conjugate formulation according to claim 5 or 6, characterized in that, The concentration of trehalose in the excipient solution containing trehalose and polysorbate 20 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 method for preparing the anti-B7H3 antibody-eribulin conjugate formulation according to any one of claims 4-7, characterized in that, The preparation method also includes a freeze-drying step.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains 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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