A CD47 monoclonal antibody freeze-dried powder preparation and its preparation process

Abstract

The present invention belongs to the technical field of protein and polypeptide drugs, and specifically relates to a CD47 monoclonal antibody lyophilized powder preparation. The lyophilized powder preparation comprises CD47 monoclonal antibody, dextran, β-cyclodextrin, a protective agent, a solubilizing agent and a buffer. The CD47 monoclonal antibody obtained after lyophilization by the prescription and lyophilization process of the present invention has high stability, is suitable for large-scale production, and is conducive to storage and transportation.

Description

Technical Field

[0001] The invention belongs to the technical field of protein and polypeptide drugs, and particularly relates to a CD47 monoclonal antibody lyophilized powder preparation. Background Art

[0002] CD47, also known as integrin-associated protein (IAP), ovarian cancer antigen OA3, Rh-related antigen and MER6, is a multi-span transmembrane receptor belonging to the immunoglobulin superfamily. Its expression and activity have been associated with many diseases and disorders. It is a widely expressed transmembrane glycoprotein with an immunoglobulin-like domain and five transmembrane regions, which acts as a cellular ligand for SIRPα, binding to SIRPα through the NH 2-terminal V-like domain of signal regulatory protein α (SIRPα). SIRPα is mainly expressed in bone marrow cells, including macrophages, granulocytes, dendritic cells (DC), mast cells, and their precursor cells, including hematopoietic stem cells.

[0003] CD47 plays a crucial role in the tumor immune escape mechanism. Existing research results have made it clear that CD47 is involved in the occurrence and development of tumors, and it is regarded as an important target for tumor immunotherapy. Different studies have shown that almost all tumor cells and tissues highly express CD47. CD47 highly expressed on the surface of tumor cells binds to SIRPα on the surface of macrophages, releasing a "don't eat me" signal, causing macrophages in the tumor tissue infiltration area to not only live in harmony with tumor cells, but also promote the proliferation of blood vessels in the tumor, inhibit the role of effector T cells, and promote the proliferation and growth of tumor cells.

[0004] At present, multiple anti-CD47 antibodies have been reported, for example, U.S. Patent US2015 / 0183874A1 reports a human IgG1 chimeric monoclonal antibody derived from B6H12 and a humanized B6H12 antibody produced by CDR transplantation, which has lower immunogenicity than known antibodies. U.S. Patent US 9045541 reports an anti-CD47 antibody that does not significantly cause hemagglutination, and the antibody is significantly effective in tumor models compared to known antibodies, such as improving the ability of macrophages to phagocytize tumor cells. The new CD47 antibody reported in Chinese Patent CN201780003451 has low immunogenicity in humans and causes low levels or no blood cell clearance or red blood cell aggregation.

[0005] Since CD47 antibody protein has a tendency to aggregate easily, its stability is easily affected during the storage process of the injection preparation, so it is considered to be made into a lyophilized preparation. At present, there are few reports on the lyophilized preparation of CD47 monoclonal antibody. Therefore, it is necessary to develop a stable lyophilized preparation of CD47 monoclonal antibody to meet the clinical drug demand.

[0006] During the entire freeze-drying process of biological freeze-dried products, various stresses often directly or indirectly lead to unstable factors of protein drugs. Excellent protein protectants can not only protect protein drugs well during the freeze-drying process, but also inhibit protein denaturation during the storage period of the finished product. At present, there is no universal freeze-drying protectant. For the freeze-drying of any new drug biological preparation, choosing a suitable freeze-drying excipient is an extremely important task. Summary of the invention

[0007] The object of the present invention is to provide a lyophilized preparation of CD47 monoclonal antibody, which has a simple formulation, a stable protein system, is convenient for large-scale production, storage and transportation, and is convenient for clinical administration. The lyophilized CD47 monoclonal antibody of the present invention can not only increase the glass transition temperature of the protein, but also inhibit the crystallization of various components during the lyophilization process, thereby improving its stability.

[0008] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0009] A CD47 monoclonal antibody freeze-dried powder preparation comprises CD47 monoclonal antibody, dextran, beta-cyclodextrin, a protective agent, a solubilizing agent and a buffer.

[0010] The protective agent is selected from one of γ-aminobutyric acid, aspartic acid, sorbitol and mannitol.

[0011] Preferably, the protective agent is γ-aminobutyric acid.

[0012] The solubilizing agent is a nonionic surfactant.

[0013] Preferably, the nonionic surfactant is polysorbate.

[0014] Further preferably, the solubilizing agent is selected from Tween 20 or Tween 80.

[0015] The buffer is histidine / acetate.

[0016] The CD47 monoclonal antibody lyophilized powder preparation contains the following components in the solution before lyophilization:

[0017]

[0018] Preferably, the CD47 monoclonal antibody lyophilized powder preparation contains the following components in the solution before lyophilization:

[0019]

[0020]

[0021] The CD47 monoclonal antibody is prepared by the following process:

[0022] Weigh the prescribed amount of buffer salt and dissolve it with an appropriate amount of water for injection to adjust the pH. Use the buffer obtained above to ultrafilter and concentrate the CD47 antibody protein mother solution to replace the original buffer system of the CD47 antibody protein. Then accurately measure the prescribed amount of dextran, β-cyclodextrin, protective agent, and solubilizer and add them to the protein solution. Add the buffer to the fixed volume and mix well to obtain the semi-finished product liquid. Aseptically filter the semi-finished product liquid with a 0.22 μm filter membrane and fill it after passing the endotoxin test.

[0023] The present invention also provides a freeze-drying process for the CD47 monoclonal antibody freeze-dried powder preparation, which comprises freeze-drying in the following steps:

[0024] 1) Prefreezing: freeze-dried samples at -2°C for 0.5-1h, then cool to -40°C and keep for 1-2h.

[0025] 2) Annealing: freeze-dried samples are maintained at -15℃~-30℃ for 0.5-1h, then cooled to -40℃ and maintained for 3-5h for freezing.

[0026] 3) Sublimation drying: vacuum 0.1mBar, heat to -21℃~-32℃, maintain for 40-55h,

[0027] 4) Desorption and drying: Raise the temperature to 20-30°C and maintain for 8-10 hours for desorption and drying, and freeze drying is completed.

[0028] Preferably, the freeze-drying process of the CD47 monoclonal antibody freeze-dried powder preparation comprises freeze-drying in the following steps:

[0029] 1) Prefreezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for prefreezing, set the time to 10-30 minutes to reach the preset temperature, maintain it for 0.5-1 hour, then cool it to -40°C, set the time to 5-10 minutes to reach the preset temperature, maintain it for 1-2 hours to freeze,

[0030] 2) Annealing: Raise the temperature of the partition system to -28°C, set the time to 15-20min to reach the preset temperature, maintain for 0.5-1h, then lower the temperature to -40°C, set the time to 5-10min to reach the set temperature, maintain for 3-4h,

[0031] 3) Sublimation drying: Raise the temperature of the partition system to -29℃~-32℃, set the time to 40~60min to reach the preset temperature, maintain for 40-55h, and maintain the vacuum degree at 0.1mBar to complete sublimation drying.

[0032] 4) Analytical drying: Raise the temperature of the partition system to 20-30°C, set the time to 80-120min to reach the preset temperature, maintain for 8-10h for drying, and maintain the vacuum degree at 0.2mBar, and freeze drying is completed.

[0033] The CD47 monoclonal antibody freeze-dried powder prepared by the invention has low moisture content, high antibody activity and better stability. DETAILED DESCRIPTION

[0034] The present invention is further described below by way of examples. It should be correctly understood that the examples of the present invention are provided only for illustrating the present invention, rather than limiting the present invention, and therefore, simple modifications of the present invention under the premise of the method of the present invention are within the scope of protection claimed by the present invention.

[0035] Example 1

[0036] 1) Prescription

[0037]

[0038] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother liquor with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of dextran, β-cyclodextrin, γ-aminobutyric acid, and Tween 80, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0039] 3) Freeze-drying process:

[0040] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 10 minutes to reach the preset temperature, maintain it for 0.5 hours, then cool it to -40°C, set the time to 5 minutes to reach the preset temperature, maintain it for 1 hour to freeze,

[0041] b) Annealing: Raise the temperature of the partition system to -15°C, set the time to 15 minutes to reach the preset temperature, maintain for 0.5 hours, then lower the temperature to -40°C, set the time to 5 minutes to reach the preset temperature, maintain for 3 hours,

[0042] c) Sublimation drying: Raise the temperature of the partition system to -21°C, set the time to 40 minutes to reach the preset temperature, maintain it for 40 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0043] d) Desorption and drying: The temperature of the partition system is raised to 20°C, and the time is set to 80 minutes to reach the preset temperature, and the temperature is maintained for 8 hours for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0044] Example 2

[0045] 1) Prescription

[0046]

[0047]

[0048] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother liquor with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of dextran, β-cyclodextrin, γ-aminobutyric acid, and Tween 80, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0049] 3) Freeze-drying process:

[0050] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0051] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0052] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0053] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0054] Example 3

[0055] 1) Prescription

[0056]

[0057] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother liquor with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of dextran, β-cyclodextrin, γ-aminobutyric acid, and Tween 80, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0058] 3) Freeze-drying process:

[0059] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 20 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 1.5 hours to freeze,

[0060] b) Annealing: Raise the temperature of the partition system to -28°C, set the time to 15 minutes to reach the preset temperature, maintain for 1 hour, then lower the temperature to -40°C, set the time to 10 minutes to reach the preset temperature, maintain for 4 hours,

[0061] c) Sublimation drying: Raise the temperature of the partition system to -29°C, set the time to 50 minutes to reach the preset temperature, maintain it for 50 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0062] d) Desorption and drying: The temperature of the partition system is raised to 25°C, and the time is set to 100 min to reach the preset temperature, and the temperature is maintained for 9 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0063] Example 4

[0064] 1) Prescription

[0065]

[0066] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the buffer obtained above, replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of dextran, β-cyclodextrin, aspartic acid, and Tween 80, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0067] 3) Freeze-drying process:

[0068] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0069] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0070] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0071] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0072] Example 5

[0073] 1) Prescription

[0074]

[0075] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of dextran, β-cyclodextrin, sorbitol, and Tween 20, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0076] 3) Freeze-drying process:

[0077] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0078] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0079] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0080] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0081] Example 6

[0082] 1) Prescription

[0083]

[0084]

[0085] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of dextran, β-cyclodextrin, mannitol, and Tween 20, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0086] 3) Freeze-drying process:

[0087] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0088] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0089] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0090] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0091] Example 7

[0092] 1) Prescription

[0093]

[0094] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, then accurately measure the prescribed amount of dextran, β-cyclodextrin, mannitol, and Tween 80 and add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0095] 3) Freeze-drying process:

[0096] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -5°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -45°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0097] b) Annealing: Raise the temperature of the partition system to -35°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -45°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0098] c) Sublimation drying: Raise the temperature of the partition system to -35°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0099] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0100] Example 8

[0101] 1) Prescription

[0102]

[0103] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, then accurately measure the prescribed amount of dextran, β-cyclodextrin, mannitol, and Tween 80 and add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0104] 3) Freeze-drying process:

[0105] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -35°C, set the time to 10 minutes to reach the preset temperature, maintain it for 3 hours to freeze,

[0106] b) Annealing: Raise the temperature of the partition system to -10°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -35°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0107] c) Sublimation drying: Raise the temperature of the partition system to -15°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0108] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0109] Example 9

[0110] 1) Prescription

[0111]

[0112] 2) Preparation process: Weigh the prescribed amount of phosphate and dissolve it with an appropriate amount of water for injection, adjust the pH with hydrochloric acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of dextran, β-cyclodextrin, trehalose, and Tween 20, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22 μm filter membrane, and fill it after passing the endotoxin test.

[0113] 3) Freeze-drying process:

[0114] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0115] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0116] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0117] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0118] Example 10

[0119] 1) Prescription

[0120]

[0121] 2) Preparation process: Weigh the prescribed amount of sodium citrate and dissolve it with an appropriate amount of water for injection, adjust the pH with citric acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of dextran, β-cyclodextrin, lactose, and poloxamer, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22 μm filter membrane, and fill it after passing the endotoxin test.

[0122] 3) Freeze-drying process:

[0123] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0124] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0125] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0126] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0127] Embodiment 11

[0128] 1) Prescription

[0129]

[0130]

[0131] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, then accurately measure the prescribed amount of dextran, β-cyclodextrin, mannitol, and Tween 80 and add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0132] 3) Freeze-drying process:

[0133] 1) Prefreezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for prefreezing, set the time to 20 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 5 hours to freeze,

[0134] 2) Sublimation drying: Raise the temperature of the partition system to -20°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.2 mBar to complete sublimation drying;

[0135] 3) Re-drying: Raise the temperature of the partition system to 30°C, set the time to 120 min to reach the preset temperature, maintain it for 10 h for drying, and maintain the vacuum degree at 0.3 mBar, and freeze-drying is completed.

[0136] Comparative Example 1

[0137] 1) Prescription

[0138]

[0139] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother liquor with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of β-cyclodextrin, mannitol and Tween 80, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0140] 3) Freeze-drying process:

[0141] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0142] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0143] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0144] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0145] Comparative Example 2

[0146] 1) Prescription

[0147]

[0148] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the buffer obtained above, replace the original buffer system of the CD47 antibody protein, and then accurately measure the prescribed amount of dextran, mannitol, and Tween 80 and add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0149] 3) Freeze-drying process:

[0150] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0151] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0152] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0153] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0154] Comparative Example 3

[0155] 1) Prescription

[0156]

[0157]

[0158] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the buffer obtained above, replace the original buffer system of the CD47 antibody protein, and then accurately measure the prescribed amount of mannitol and Tween 80 and add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0159] 3) Freeze-drying process:

[0160] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0161] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0162] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0163] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0164] Comparative Example 4

[0165] 1) Prescription

[0166]

[0167] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the buffer obtained above, replace the original buffer system of the CD47 antibody protein, and then accurately measure the prescribed amount of dextran, β-cyclodextrin, and mannitol and add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0168] 3) Freeze-drying process:

[0169] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0170] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0171] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0172] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0173] Comparative Example 5

[0174] 1) Prescription

[0175]

[0176] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother solution with the buffer obtained above, replace the original buffer system of the CD47 antibody protein, and then accurately measure the prescribed amount of dextran, β-cyclodextrin, and Tween 80 and add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0177] 3) Freeze-drying process:

[0178] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0179] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0180] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0181] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0182] Comparative Example 6

[0183] 1) Prescription

[0184]

[0185] 2) Preparation process: Weigh the prescribed amount of histidine and dissolve it with an appropriate amount of water for injection, adjust the pH with acetic acid, ultrafilter and concentrate the CD47 antibody protein mother liquor with the above-obtained buffer to replace the original buffer system of the CD47 antibody protein, accurately measure the prescribed amount of trehalose, lactose, mannitol and Tween 80, add them to the protein solution, mix well to obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22μm filter membrane, and fill it after passing the endotoxin test.

[0186] 3) Freeze-drying process:

[0187] a) Pre-freezing: First, place the vial containing CD47 monoclonal antibody solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 30 minutes to reach the preset temperature, maintain it for 1 hour, then cool it to -40°C, set the time to 10 minutes to reach the preset temperature, maintain it for 2 hours to freeze,

[0188] b) Annealing: Raise the temperature of the partition system to -30°C, set the time to reach the preset temperature for 20 minutes, maintain for 1 hour, then lower the temperature to -40°C, set the time to reach the preset temperature for 10 minutes, maintain for 5 hours,

[0189] c) Sublimation drying: Raise the temperature of the partition system to -32°C, set the time to 60 minutes to reach the preset temperature, maintain it for 55 hours, and maintain the vacuum degree at 0.1 mBar to complete the sublimation drying.

[0190] d) Desorption and drying: The temperature of the partition system is raised to 30°C, and the time is set to 120 min to reach the preset temperature, and the temperature is maintained for 10 h for drying, while the vacuum degree is maintained at 0.2 mBar, and the freeze-drying is completed.

[0191] Verification Example

[0192] 1. Differential Scanning Calorimetry (DSC)

[0193] The melting temperatures T of the semi-finished solutions of the CD47 monoclonal antibodies of Examples 1-11 and Comparative Examples 1-6 were measured using a MicroCalTM VP-Capillary DSC instrument under the temperature controlled by the instrument program. m Value. After the sample was diluted with the corresponding buffer solution to a CD47 monoclonal antibody protein concentration of 1 mg / mL, 350 μL of the sample was added to the sample well of the 96-well plate, and 350 μL of the corresponding buffer was added to the buffer well. The scanning temperature was set to 20-90°C and the scanning rate was 60°C / hr. MicroCal VP-Capillary DSC automatic analysis software was used for data analysis. The results are shown in Table 1.

[0194] Table 1. T of CD47 monoclonal antibody m Value Result

[0195] sample <![CDATA[T m onset / ℃]]> <![CDATA[T m1 / ℃]]> <![CDATA[T m2 / ℃]]> Example 1 43.64 66.57 73.39 Example 2 43.54 66.64 73.42 Example 3 43.88 67.12 73.48 Example 4 43.01 66.04 73.17 Example 5 43.05 66.02 73.23 Example 6 43.03 66.07 73.31 Example 7 41.38 62.14 73.28 Example 8 41.25 62.19 73.37 Example 9 41.85 62.62 73.28 Example 10 41.83 62.67 73.33 Embodiment 11 40.84 61.55 71.21 Comparative Example 1 36.35 57.91 71.37 Comparative Example 2 36.19 57.68 71.26 Comparative Example 3 33.37 55.21 70.98 Comparative Example 4 35.06 56.57 71.38 Comparative Example 5 35.35 56.96 71.56 Comparative Example 6 35.18 55.91 71.48

[0196] T m The value represents the midpoint temperature of the protein thermal transition. For multi-domain proteins, there can be multiple T m T value is an important indicator of protein thermal stability. An upward shift represents an increase in stability and can be used to assess the formation tendency of oligomers and aggregates. m onset is the temperature at which unfolding starts, T m1 is the first transition temperature, T m2 The results in Table 1 show that after the prescriptions of Comparative Examples 1-6 were changed, the T m onset Low.

[0197] 2. Stability test

[0198] Three batches of samples were prepared according to Examples 1-11 and Comparative Examples 1-6, 60 bottles were taken from each batch, and the storage stability was investigated by long-term tests.

[0199] The long-term test was carried out at 2-8°C, and the key stability inspection items were tested at the end of 0 month, 3 months, 6 months, 12 months and 24 months; (the moisture content was determined by the Mettler-Zalido DL37 Karl Fischer titrator according to the coulometric method specified in the general rules of Part III of the 2015 Edition of the Chinese Pharmacopoeia, the purity was checked according to the general rule 0514 molecular grouping chromatography and the general rule 0542 capillary electrophoresis, and the activity was determined according to the bioluminescence-based reporter gene method). The results showed that there was no significant change compared with 0 month, and the comparison ratio had more stable characteristics. The results are shown in Table 2.

[0200] Table 2. Results of long-term stability test at 2-8℃

[0201]

[0202]

[0203]

[0204] The lyophilized preparations of Examples 1-11 are white loose bodies, which are clear and transparent after reconstitution, with a slight opalescence. The stability data results in Table 2 show that the moisture content of the lyophilized preparations of Examples 1-11 is less than 1.50%; the monomer content detected by SEC-HPLC remains basically unchanged after being stored at 2-8°C for 6 months, and the monomer content is ≧95.5% after being stored for 24 months, and the stability is relatively high.

Claims

1. A CD47 monoclonal antibody lyophilized powder preparation, characterized in that: The solution of the freeze-dried powder preparation before freeze-drying is composed of water for injection and the following components: Composition: CD47 monoclonal antibody 125-150 mg / ml, β-cyclodextrin 50-100 mM, dextran 20-50 mM, protective agent 10-30 mM, solubilizing agent 0.1-0.4 mg / ml, buffer 10-50 mM, pH 5.5-6.5; the buffer is histidine / acetic acid; the solubilizing agent is Tween 80; the protective agent is γ-aminobutyric acid.

2. A CD47 monoclonal antibody lyophilized powder preparation according to claim 1, characterized in that: The freeze drying process specifically adopts the following steps: 1) Prefreezing: Prefreeze the solution before lyophilization at -2℃ for 0.5-1h, cool it to -40℃ and keep it for 1-2h for freezing. 2) Annealing: Keep the pre-frozen sample at -15℃~-30℃ for 0.5-1 h, then cool it to -40℃ and keep it for 3-5 h before freezing. 3) Sublimation drying: vacuum 0.1mBar, heat to -21℃~ -32℃, maintain for 40-55h, 4) Desorption and drying: Raise the temperature to 20-30°C and maintain for 8-10 hours for desorption and drying. The freeze-drying is completed.

3. The CD47 monoclonal antibody lyophilized powder preparation according to claim 2, characterized in that: Freeze drying is performed using a freeze drying process with the following steps: 1) Pre-freezing: First, place the vial containing the pre-lyophilized solution on the freeze dryer partition, set the temperature to -2°C for pre-freezing, set the time to 10-30 minutes to reach the preset temperature, maintain it for 0.5-1 hour, then cool it down to -40°C, set the time to 5-10 minutes to reach the preset temperature, maintain it for 1-2 hours to freeze, 2) Annealing: Raise the temperature of the partition to -28°C, set the time to 15-20min to reach the preset temperature, maintain for 0.5-1h, then cool it down to -40°C, set the time to 5-10min to reach the set temperature, maintain for 3-4h, 3) Sublimation drying: Heat the partition to -29℃~ -32℃, set the time to 40~60min to reach the preset temperature, maintain it for 40-55h, and maintain the vacuum degree at 0.1mBar to complete sublimation drying. 4) Desorption and drying: Heat the partition to 20-30°C, set the time to 80-120 minutes to reach the preset temperature, maintain for 8-10 hours for drying, and maintain the vacuum degree at 0.2 mBar, and freeze-drying is completed.

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