A CD47 monoclonal antibody preparation and preparation method thereof

Abstract

The present invention belongs to the technical field of protein and polypeptide drugs, and specifically relates to a CD47 monoclonal antibody preparation and a preparation method thereof. The preparation comprises CD47 monoclonal antibodies, amino acids, stabilizers, surfactants and buffers. The CD47 antibody injection obtained by the prescription and preparation process of the present invention has a low content of polymers and small molecule fragments, high stability, is suitable for large-scale production, and is conducive to storage and transportation.

Description

Technical Field

[0001] The present invention belongs to the technical field of protein and polypeptide drugs, and in particular relates to a CD47 monoclonal antibody preparation and a preparation method thereof. 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 NH2-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. Currently, there are few reports on CD47 monoclonal antibody preparations. Therefore, it is necessary to develop a stable CD47 monoclonal antibody preparation to meet clinical drug needs. Summary of the invention

[0006] Based on the deficiencies of the prior art, the present invention provides a CD47 monoclonal antibody preparation. After adding amino acids and stabilizers to the CD47 antibody solution, they can surround the antibody protein, hinder the movement of macromolecular substances, maintain the spatial structure of protein molecules, and prevent the precipitation of protein molecules, thereby increasing protein stability. Adding surfactants can further reduce the aggregation of antibodies in the preparation, the formation and adsorption of particles in the preparation, and other problems. The preparation has a simple formula, a stable protein system, is easy to mass produce, store and transport, and is convenient for clinical administration.

[0007] The technical solution adopted by the present invention to solve the technical problem is as follows:

[0008] The CD47 monoclonal antibody of the present invention refers to a monoclonal antibody that can specifically bind to CD47. A CD47 monoclonal antibody preparation, characterized in that the preparation comprises CD47 monoclonal antibody, a buffer, an amino acid, a stabilizer, and a surfactant.

[0009] The amino acid is one or more of glutamic acid, aspartic acid, glycine, tyrosine and tryptophan.

[0010] The amino acids are a mixture of glutamic acid and aspartic acid.

[0011] The molar ratio of glutamic acid to aspartic acid is 1:2-5.

[0012] The stabilizer is one or more of sucrose, trehalose, mannitol and threitol.

[0013] Preferably, the stabilizer is threitol.

[0014] The surfactant is a nonionic surfactant.

[0015] Preferably, the nonionic surfactant is a polysorbate or a poloxamer.

[0016] Further preferably, the nonionic surfactant is polysorbate 80.

[0017] The buffer is one of acetic acid / sodium acetate, citric acid / sodium citrate, histidine / histidine hydrochloride, and tris-hydrochloric acid (Tris-HCl).

[0018] Preferably, the buffer is tris-hydrochloric acid (Tris-HCl).

[0019] The preparation comprises the following components:

[0020]

[0021] The present invention also provides a method for preparing a CD47 monoclonal antibody preparation, which is prepared by the following steps:

[0022] Weigh the prescribed amount of buffer salt and amino acid, add injection water to dissolve, and adjust the pH; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein mother liquor to replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of stabilizer and surfactant, and stir evenly to obtain.

[0023] Preferably, the preparation method of the CD47 monoclonal antibody preparation is prepared by the following steps:

[0024] Weigh the prescribed amount of buffer salt and amino acid, add injection water to dissolve, and adjust the pH to 6.0-7.5; use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein mother liquor, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of stabilizer and surfactant, stir evenly, add the prepared buffer system to adjust the protein concentration to 25 mg / ml-50 mg / ml, and obtain a semi-finished product liquid; sterile filter the semi-finished product liquid with a 0.22 μm filter membrane and then fill it.

[0025] The CD47 monoclonal antibody preparation prepared by the present invention can be used for intravenous administration, can maintain good stability during storage, has lower polymers and small molecule fragments, and has a lower number of insoluble particles. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1

[0028] 1) Prescription

[0029]

[0030] 2) Preparation process: Weigh the prescribed amount of Tris, glutamic acid, and aspartic acid, dissolve them in water for injection, and adjust the pH to 7.5 with hydrochloric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein mother liquor, replace the original buffer system of the CD47 antibody protein, and then add the prescribed amount of threitol and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 25 mg / ml to obtain a semi-finished product liquid; the semi-finished product liquid is aseptically filtered with a 0.22 μm filter membrane and then filled.

[0031] Example 2

[0032] 1) Prescription

[0033]

[0034] 2) Preparation process: weigh the prescribed amount of Tris, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 7.5 with hydrochloric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein mother liquor, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of threitol and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; the semi-finished liquid is aseptically filtered with a 0.22 μm filter membrane and then filled.

[0035] Example 3

[0036] 1) Prescription

[0037]

[0038] 2) Preparation process: weigh the prescribed amount of Tris, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 7.1 with hydrochloric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein mother liquor, replace the original buffer system of the CD47 antibody protein, and finally concentrate it to an antibody concentration of 40 mg / ml, then add the prescribed amount of threitol and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 40 mg / ml to obtain a semi-finished product liquid; the semi-finished product liquid is aseptically filtered with a 0.22 μm filter membrane and then filled.

[0039] Example 4

[0040] 1) Prescription

[0041]

[0042]

[0043] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 6.0 with citric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein mother liquor, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of sucrose and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; the semi-finished liquid is aseptically filtered with a 0.22 μm filter membrane and then filled.

[0044] Example 5

[0045] 1) Prescription

[0046]

[0047] 2) Preparation process: Weigh the prescribed amount of acetic acid / sodium acetate, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 5.8 with acetic acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of trehalose and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; sterile filter the semi-finished liquid with a 0.22 μm filter membrane and then fill it.

[0048] Example 6

[0049] 1) Prescription

[0050]

[0051]

[0052] 2) Preparation process: Weigh the prescribed amount of histidine / histidine hydrochloride, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 6.0 with hydrochloric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of mannitol and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; the semi-finished liquid is aseptically filtered with a 0.22 μm filter membrane and then filled.

[0053] Example 7

[0054] 1) Prescription

[0055]

[0056] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 6.5 with citric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of trehalose and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 15 mg / ml to obtain a semi-finished liquid; sterile filter the semi-finished liquid with a 0.22 μm filter membrane and then fill it.

[0057] Example 8

[0058] 1) Prescription

[0059]

[0060]

[0061] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 6.5 with citric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of trehalose and Tween-20, stir evenly, and then add the buffer system to the antibody concentration of 60 mg / ml to obtain a semi-finished liquid; the semi-finished liquid is aseptically filtered with a 0.22 μm filter membrane and then filled.

[0062] Example 9

[0063] 1) Prescription

[0064]

[0065] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, accurately weigh the prescribed amount of glycine and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 6.5 with citric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, add the prescribed amount of mannitol and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; sterile filter the semi-finished liquid with a 0.22 μm filter membrane and then fill it.

[0066] Example 10

[0067] 1) Prescription

[0068]

[0069] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, accurately weigh the prescribed amount of tryptophan and tyrosine, add them to the buffer solution and stir to dissolve; adjust the pH to 6.0 with lemon; use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, add the prescribed amount of mannitol and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; sterile filter the semi-finished liquid with a 0.22 μm filter membrane and then fill it.

[0070] Embodiment 11

[0071] 1) Prescription

[0072]

[0073] 2) Preparation process: Weigh the prescribed amount of phosphoric acid / sodium phosphate, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid and aspartic acid, add them to the buffer solution and stir to dissolve; adjust the pH to 8.0 with phosphoric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of sorbitol and poloxamer 188, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; the semi-finished liquid is aseptically filtered with a 0.22 μm filter membrane and then filled.

[0074] Example 12

[0075] 1) Prescription

[0076]

[0077] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, then accurately weigh the prescribed amount of glutamic acid, add it to the buffer solution and stir to dissolve; adjust the pH to 6.5 with citric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of trehalose and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; the semi-finished liquid is aseptically filtered with a 0.22 μm filter membrane and then filled.

[0078] Embodiment 13

[0079] 1) Prescription

[0080]

[0081] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, then accurately weigh the prescribed amount of aspartic acid, add it to the buffer solution and stir to dissolve; adjust the pH to 6.5 with citric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of trehalose and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; sterile filter the semi-finished liquid with a 0.22 μm filter membrane and then fill it.

[0082] Comparative Example 1

[0083] 1) Prescription

[0084]

[0085] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, and adjust the pH to 6.5 with citric acid; use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, and then add the prescribed amount of trehalose and polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; the semi-finished liquid is sterile filtered with a 0.22 μm filter membrane and then filled.

[0086] Comparative Example 2

[0087] 1) Prescription

[0088]

[0089]

[0090] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, then accurately weigh the prescribed amount of aspartic acid, add it to the buffer solution and stir to dissolve; adjust the pH to 6.5 with citric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of polysorbate 80, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; sterile filter the semi-finished liquid with a 0.22 μm filter membrane and then fill it.

[0091] Comparative Example 3

[0092] 1) Prescription

[0093]

[0094] 2) Preparation process: Weigh the prescribed amount of citric acid / sodium citrate, dissolve it with water for injection, then accurately weigh the prescribed amount of aspartic acid, add it to the buffer solution and stir to dissolve; adjust the pH to 6.5 with citric acid; use the above-obtained buffer system to ultrafilter and concentrate the CD47 antibody protein solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of trehalose, stir evenly, and then add the buffer system to the antibody concentration of 50 mg / ml to obtain a semi-finished liquid; sterile filter the semi-finished liquid with a 0.22 μm filter membrane and then fill it.

[0095] Verification Example

[0096] The CD47 monoclonal antibody injections obtained in Example 1-13 and Comparative Example 1-3 were tested for antibody stability under shaking (300 rpm, 25°C) and repeated freezing and thawing (-80°C to room temperature thawing), respectively. After shaking for 7 days and repeated freezing and thawing for 5 rounds, the insoluble particles and purity (SEC-HPLC) of the antibody solutions were tested.

[0097] Table 1. CD47 monoclonal antibody injection stability test

[0098]

[0099]

[0100]

[0101] Note: A-3D = shaking for 3 days, A-7D = shaking for 7 days, FT-5C = repeated freezing and thawing for 5 cycles.

[0102] After oscillation and repeated freezing and thawing, the CD47 monoclonal antibody injection prepared by the present invention has low contents of aggregates and small molecule fragments, less insoluble particles, far below the limit level, and has high stability.

Claims

1. A CD47 monoclonal antibody preparation, characterized in that: The preparation is composed of water for injection and the following components: composition: CD47 monoclonal antibody 25-50mg / ml Amino Acids 10-35mM Stabilizer 200-250 mM Surfactant 0.02%-0.06% Buffer 10-35 mM pH 6.0-7.5 The amino acid is a mixture of glutamic acid and aspartic acid, and the molar ratio of glutamic acid to aspartic acid is 1:2-5; the stabilizer is threitol, the surfactant is polysorbate-80, and the buffer is tris(hydroxymethyl)aminomethane / hydrochloric acid.

2. A method for preparing the CD47 monoclonal antibody preparation according to claim 1, characterized in that: Prepared by the following steps: Weigh the prescribed amount of buffer and amino acid, add water for injection, and adjust the pH; use the obtained buffer system to ultrafilter and concentrate the CD47 monoclonal antibody protein mother solution, replace the original buffer system of the CD47 monoclonal antibody protein, then add the prescribed amount of stabilizer and surfactant, stir evenly, add the prepared buffer system to adjust the concentration of the CD47 monoclonal antibody protein, and obtain a semi-finished product liquid; and sterile filter the semi-finished product liquid with a 0.22 μm filter membrane and then fill it.

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