A liquid preparation of CD47 monoclonal antibody and its preparation method

Abstract

The present invention belongs to the technical field of protein and polypeptide drugs, and specifically relates to a liquid preparation of CD47 monoclonal antibody, including CD47 monoclonal antibody, stabilizer, solubilizer and buffer. After adding the stabilizer to the CD47 antibody solution, the stabilizer can be surrounded by the antibody protein, so that the movement of macromolecular substances is hindered, the spatial structure of protein molecules is maintained, and the precipitation of protein molecules is organized. After adding the solubilizer to the CD47 antibody solution, the uniformity of the protein in the solution is increased, and the aggregation of antibodies in the preparation and the degradation of CD47 antibodies are further reduced. The preparation has a simple formula, a stable protein system, is convenient for large-scale production, storage and transportation, and is convenient for clinical administration.

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 liquid 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 ubiquitously expressed transmembrane glycoprotein with an immunoglobulin-like domain and five transmembrane regions, which serves as a cellular ligand for SIRPα through the NH 2 The terminal V-like domain binds to SIRPα, which is mainly expressed in myeloid cells, including macrophages, granulocytes, dendritic cells (DCs), 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 liquid preparation of CD47 monoclonal antibody. After adding a stabilizer to the CD47 antibody solution, the stabilizer can be surrounded by the antibody protein, so that the movement of macromolecular substances is hindered, the spatial structure of protein molecules is maintained, and the precipitation of protein molecules is organized. After adding a solubilizing agent to the CD47 antibody solution, the uniformity of the protein in the solution is increased, and the antibody aggregation and CD47 antibody degradation in the preparation are further reduced. The preparation has a simple formula, a stable protein system, is convenient for large-scale production, storage and transportation, and is convenient for clinical administration.

[0007] The technical solution of the present invention is as follows:

[0008] A liquid preparation of CD47 monoclonal antibody, characterized in that the preparation comprises CD47 monoclonal antibody, a buffer, a stabilizer and a solubilizing agent.

[0009] Preferably, the buffer is one of acetic acid / sodium acetate, citric acid / sodium citrate, histidine / histidine hydrochloride or tris(hydroxymethylaminomethane) (Tris); more preferably citric acid / sodium citrate.

[0010] Preferably, the stabilizer is selected from one or more of sucrose, trehalose, mannitol, threitol and chitosan; more preferably trehalose or chitosan.

[0011] Preferably, the solubilizing agent is selected from one or more of egg yolk lecithin, aspartic acid, and asparagine; more preferably, egg yolk lecithin.

[0012] The contents of the components of a liquid preparation of CD47 monoclonal antibody are as follows:

[0013]

[0014] Preferably, the contents of the components of the preparation are as follows:

[0015]

[0016] In a preferred experimental scheme: the preparation includes the following content:

[0017]

[0018]

[0019] The CD47 monoclonal antibody described in the present invention refers to a monoclonal antibody that can specifically bind to CD47.

[0020] The present invention also provides a method for preparing the CD47 monoclonal antibody liquid preparation, the method comprising the following steps:

[0021] Weigh the prescribed amount of buffer, add injection water to dissolve, adjust the pH, ultrafilter and concentrate the CD47 antibody protein, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of stabilizer and solubilizer, stir evenly and obtain the product.

[0022] Preferably, the preparation method of the CD47 monoclonal antibody liquid preparation comprises the following steps: weighing a prescribed amount of buffer, adding injection water to dissolve, adjusting the pH to 5.0-7.5, ultrafiltration and concentrating the CD47 antibody protein stock solution with the buffer system obtained above, replacing the original buffer system of the CD47 antibody protein, and then adding a prescribed amount of stabilizer and solubilizer, stirring evenly, and then adjusting the CD47 protein concentration to 40-60 mg / ml with the buffer system, and sterile filtering through a 0.22 μm filter membrane before filling.

[0023] The present invention does not limit the source of CD47 antibody protein, and it can be prepared by referring to the existing technology or methods known in the art. Experiments have shown that the technical solution of the present invention can improve the stability of CD47 monoclonal antibody liquid preparations.

[0024] 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

[0025] 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.

[0026] Example 1

[0027] (1) Formulation:

[0028]

[0029] (2) Preparation method

[0030] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 5.5, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of chitosan and egg yolk lecithin, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0031] Example 2

[0032] (1) Formulation:

[0033]

[0034] (2) Preparation method

[0035] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 6.0, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of trehalose and egg yolk lecithin, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0036] Example 3

[0037] (1) Formulation:

[0038]

[0039] (2) Preparation method

[0040] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 5.0, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of sucrose and asparagine, stir evenly, and then adjust the CD47 protein concentration to 60 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0041] Example 4

[0042] (1) Formulation:

[0043]

[0044]

[0045] (2) Preparation method

[0046] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 7.0, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of threitol and aspartic acid, stir evenly, and then adjust the CD47 protein concentration to 40 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0047] Example 5

[0048] (1) Formulation:

[0049]

[0050] (2) Preparation method

[0051] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 7.5, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of mannitol and aspartic acid, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0052] Example 6

[0053] (1) Formulation:

[0054]

[0055] (2) Preparation method

[0056] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 6.0, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of chitosan and egg yolk lecithin, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0057] Example 7

[0058] (1) Formulation:

[0059]

[0060] (2) Preparation method

[0061] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 8.0, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of trehalose and egg yolk lecithin, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0062] Example 8

[0063] (1) Formulation:

[0064]

[0065] (2) Preparation method

[0066] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 7.4, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of chitosan, aspartic acid and egg yolk lecithin, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane and fill it.

[0067] Example 9

[0068] (1) Formulation:

[0069]

[0070] (2) Preparation method

[0071] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 6.0, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of sorbitol and egg yolk lecithin, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0072] Example 10

[0073] (1) Formulation:

[0074]

[0075] (2) Preparation method

[0076] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 5.5, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of chitosan and Tween 80, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0077] Embodiment 11

[0078] (1) Formulation:

[0079]

[0080] (2) Preparation method

[0081] Weigh the prescribed amount of buffer, add water for injection to dissolve, adjust the pH to 5.5, use the buffer system obtained above to ultrafilter and concentrate the CD47 antibody protein stock solution, replace the original buffer system of the CD47 antibody protein, then add the prescribed amount of chitosan and Tween 20, stir evenly, and then adjust the CD47 protein concentration to 50 mg / ml with the buffer system, sterile filter through a 0.22 μm filter membrane, and then fill it.

[0082] Verification Example

[0083] The CD47 monoclonal antibody injection obtained in Example 1-11 was tested for antibody stability under shaking (300 rpm, 25°C) and repeated freezing and thawing (-80°C to room temperature thawing). After shaking for 7 days and repeated freezing and thawing for 5 rounds, the insoluble particles and purity (SEC-HPLC) of the antibody solution were tested.

[0084] Table 1. CD47 monoclonal antibody liquid preparation stability test

[0085]

[0086]

[0087] Note: A-3D = shaking for 3 days, A-7D = shaking for 7 days, FT-5C = repeated freezing and thawing for 5 cycles.

[0088] 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 liquid preparation of CD47 monoclonal antibody, characterized in that: The preparation comprises the following components: 40 mg / ml-60 mg / ml of CD47 monoclonal antibody, 200-250 mM of stabilizer, 0.04-0.06%, W / V, 10-20 mM of buffer, pH 5.0-7.5; the stabilizer is trehalose or chitosan, the solubilizer is egg yolk lecithin, and the buffer is citric acid / sodium citrate.

2. A method for preparing the preparation according to claim 1, characterized in that: The method comprises the following steps: weighing a prescribed amount of a buffer, adding injection water to dissolve it, adjusting the pH, ultrafiltrating and concentrating the purified CD47 antibody protein, replacing the original buffer system of the CD47 antibody protein, adding a prescribed amount of a stabilizer and a solubilizer, stirring evenly, and filtering aseptically through a 0.22 μm filter membrane before canning to obtain the product.

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