GLP-1-fc-FGF21 double-target fusion protein composition, and injection and use thereof

Abstract

A GLP-1-Fc-FGF21 double-target fusion protein composition and the use thereof. The composition comprises a GLP-1-Fc-FGF21 fusion protein and a buffer solution. In the composition, by means of adding the buffer solution, the generation of visible particles can be effectively controlled, and the stability of the GLP-1-Fc-FGF21 fusion protein is improved, so that the shelf life of a product can be prolonged. The composition can be used for treating and / or preventing metabolic diseases, such as diabetes, hyperlipidemia, obesity and diseases related to fatty liver.

Description

GLP-1-Fc-FGF21 dual-target fusion protein composition and injection and use thereof

[0001] CROSS-REFERENCE TO RELATED APPLICATION

[0002] This application claims the priority and benefits of Chinese Patent Application No.202310741972.5, filed with the State Intellectual Property Office of China on June 20,2023, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention belongs to the field of biopharmaceutical technology. Specifically, the present invention relates to a GLP-1-Fc-FGF21 dual-target fusion protein composition and injection and their uses. More specifically, the present invention relates to a composition and injection and their uses and an injection device or container. Background Art

[0004] Glucagon-like peptide-1 (GLP-1) is an incretin hormone secreted by L cells in the small intestine. It stimulates pancreatic beta cells to secrete insulin, thereby maintaining insulin balance in patients. GLP-1 acts indirectly through insulin and only works for type 2 diabetes, limiting its application and effectiveness. There are also reports that GLP-1 has a potential risk of causing thyroid cancer.

[0005] FGF21 is a member of the FGF family (fibroblast growth factors, FGFs). FGF21 can promote glucose absorption by adipocytes and enhance insulin sensitivity. Compared to insulin, FGF21 does not cause side effects such as hypoglycemia and is more effective in protecting pancreatic β-islet cells, promoting their regeneration and repair. Furthermore, since it lacks mitogenic activity, it does not pose a potential risk of tumors. FGF21 has the potential to become a treatment for type 1 diabetes. Furthermore, FGF21 also has a significant lipid-lowering effect, making it a promising lipid-lowering agent.

[0006] The GLP-1-Fc-FGF21 dual-target fusion protein can be used to treat metabolic diseases such as diabetes, hyperlipidemia, obesity, and fatty liver disease. However, this dual-target fusion protein can undergo physical and chemical changes during storage, such as denaturation, aggregation, precipitation, or fragmentation, which can affect its stability during storage.

[0007] Therefore, there is an urgent need to develop a GLP-1-Fc-FGF21 dual-target fusion protein composition with high stability.

[0008] Summary of the Invention

[0009] The present invention aims to solve at least one of the technical problems existing in the prior art to a certain extent. To this end, the present invention provides a GLP-1-Fc-FGF21 dual-target fusion protein composition, which has strong stability and can extend the shelf life of the product.

[0010] In one aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises a GLP-1-Fc-FGF21 fusion protein and a buffer. The addition of the buffer to the composition of the present invention effectively controls the formation of visible foreign matter and aggregates, improves the stability of the GLP-1-Fc-FGF21 fusion protein, and thereby extends the shelf life of the product.

[0011] According to an embodiment of the present invention, the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequences shown in SEQ ID NOs: 1 to 12.

[0012] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 1 to 100 mg / mL.

[0013] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 80 mg / mL.

[0014] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 70 mg / mL.

[0015] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 5 to 70 mg / mL.

[0016] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 65 mg / mL.

[0017] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 60 mg / mL.

[0018] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 55 mg / mL.

[0019] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 10-65 mg / mL.

[0020] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 20-60 mg / mL.

[0021] According to an embodiment of the present invention, the pH value of the composition is 6.2 to 8.0, preferably 6.4 to 8.0.

[0022] According to an embodiment of the present invention, the buffer comprises at least one of a histidine buffer, a citrate buffer, a phosphate buffer and a Tris buffer.

[0023] According to an embodiment of the present invention, the buffer comprises a histidine buffer or a citrate buffer.

[0024] According to an embodiment of the present invention, the concentration of the buffer in the composition is 5-30 mM.

[0025] According to an embodiment of the present invention, the concentration of the buffer in the composition is 5-25 mM.

[0026] According to an embodiment of the present invention, the concentration of the buffer in the composition is 8-25 mM.

[0027] According to an embodiment of the present invention, the concentration of the buffer in the composition is 5-20 mM.

[0028] According to an embodiment of the present invention, the concentration of the buffer in the composition is 8-12 mM.

[0029] According to an embodiment of the present invention, the concentration of the buffer in the composition is 10-20 mM.

[0030] According to an embodiment of the present invention, the composition further comprises at least one of an osmotic pressure regulator, a surfactant, a preservative and a metal chelating agent.

[0031] According to an embodiment of the present invention, the osmotic pressure regulator includes at least one of mannitol, sucrose, sodium chloride and trehalose.

[0032] According to an embodiment of the present invention, the concentration of the osmotic pressure regulator in the composition is 30-100 mg / mL or 1-10 mg / mL.

[0033] According to an embodiment of the present invention, the surfactant includes at least one of polysorbate 80, polysorbate 20 and poloxamer 188.

[0034] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 0.1-10 mg / mL.

[0035] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 0.1-1.5 mg / mL.

[0036] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 6-10 mg / mL.

[0037] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 0.1-0.7 mg / mL.

[0038] According to an embodiment of the present invention, the preservative is at least one of phenol and m-cresol.

[0039] According to an embodiment of the present invention, the concentration of the preservative in the composition is 0.1-10 mg / mL, preferably 0.1-5.0 mg / mL, more preferably 1.0-5.0 mg / mL.

[0040] According to an embodiment of the present invention, the metal chelating agent is at least one of edetate disodium and edetate calcium sodium.

[0041] According to an embodiment of the present invention, the concentration of the metal chelator in the composition is 0.01 to 1 mg / mL, preferably 0.05 to 0.5 mg / mL.

[0042] According to an embodiment of the present invention, the buffer is a histidine buffer, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 25-100 mg / mL, preferably 25-95 mg / mL, 25-90 mg / mL, or 25-85 mg / mL.

[0043] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 25-80 mg / mL, and the buffer is a histidine buffer.

[0044] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 25-55 mg / mL, and the buffer is a histidine buffer.

[0045] According to an embodiment of the present invention, the concentration of the histidine buffer in the composition is 5-30 mM.

[0046] According to an embodiment of the present invention, the concentration of the histidine buffer in the composition is 5-25 mM.

[0047] According to an embodiment of the present invention, the concentration of the histidine buffer in the composition is 8-25 mM.

[0048] According to an embodiment of the present invention, the concentration of the histidine buffer in the composition is 8-20 mM.

[0049] According to an embodiment of the present invention, the concentration of the histidine buffer in the composition is 10-20 mM.

[0050] According to an embodiment of the present invention, the concentration of the histidine buffer in the composition is 8-12 mM.

[0051] According to an embodiment of the present invention, the pH value of the composition is 6.7-7.5, preferably 6.9-7.5.

[0052] According to an embodiment of the present invention, the osmotic pressure regulator is mannitol, and the concentration of the osmotic pressure regulator in the composition is 30-50 mg / mL; or the osmotic pressure regulator is sucrose, and the concentration of the osmotic pressure regulator in the composition is 60-100 mg / mL, preferably 80-100 mg / mL; or the osmotic pressure regulator is trehalose, and the concentration of the osmotic pressure regulator in the composition is 40-100 mg / mL, preferably 40-70 mg / mL, and more preferably 50-70 mg / mL.

[0053] According to an embodiment of the present invention, the surfactant is polysorbate 80, and the concentration of the surfactant in the composition is 0.1 to 2 mg / mL, preferably 0.1 to 1.5 mg / mL, and preferably 0.4 to 1.3 mg / mL; or the surfactant is polysorbate 20, and the concentration of the surfactant in the composition is 0.1 to 1.5 mg / mL, preferably 0.1 to 0.5 mg / mL.

[0054] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2-25 mg / mL, and the buffer is a citrate buffer.

[0055] According to an embodiment of the present invention, the concentration of the citrate buffer in the composition is 5-30 mM.

[0056] According to an embodiment of the present invention, the concentration of the citrate buffer in the composition is 5-25 mM.

[0057] According to an embodiment of the present invention, the concentration of the citrate buffer in the composition is 8-25 mM.

[0058] According to an embodiment of the present invention, the concentration of the citrate buffer in the composition is 8-20 mM.

[0059] According to an embodiment of the present invention, the concentration of the citrate buffer in the composition is 10-20 mM.

[0060] According to an embodiment of the present invention, the concentration of the citrate buffer in the composition is 8-12 mM.

[0061] According to an embodiment of the present invention, the pH value of the composition is 6.4-8.0.

[0062] According to an embodiment of the present invention, the osmotic pressure regulator is mannitol, and the concentration of the osmotic pressure regulator in the composition is 30-50 mg / mL; or the osmotic pressure regulator is sucrose, and the concentration of the osmotic pressure regulator in the composition is 60-100 mg / mL, preferably 80-100 mg / mL; or the osmotic pressure regulator is trehalose, and the concentration of the osmotic pressure regulator in the composition is 40-100 mg / mL, preferably 40-70 mg / mL, and more preferably 50-70 mg / mL.

[0063] According to an embodiment of the present invention, the osmotic pressure regulator is sodium chloride, and the concentration of the osmotic pressure regulator in the composition is 1 to 10 mg / mL, preferably 3 to 9 mg / mL.

[0064] According to an embodiment of the present invention, the surfactant includes at least one of polysorbate 80, polysorbate 20 and poloxamer 188, preferably polysorbate 80.

[0065] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 0.1-10.0 mg / mL.

[0066] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 0.1-1.5 mg / mL.

[0067] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 0.1-1.0 mg / mL.

[0068] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 6.0-10.0 mg / mL.

[0069] According to an embodiment of the present invention, the concentration of the surfactant in the composition is 0.1-0.5 mg / mL.

[0070] According to an embodiment of the present invention, the surfactant is poloxamer 188, and the concentration of the surfactant in the composition is 6 to 10 mg / mL; or the surfactant is polysorbate 80 or polysorbate 20, and the concentration of the surfactant in the composition is 0.1 to 1.5 mg / mL, preferably 0.1 to 1.0 mg / mL or 0.1 to 0.5 mg / mL.

[0071] According to an embodiment of the present invention, the dosage form of the composition is an injection.

[0072] In another aspect of the present invention, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, mannitol, and optionally disodium edetate or sodium calcium edetate, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL; the concentration of the histidine buffer in the injection is 8 to 25 mM; the concentration of the mannitol in the injection is 35 to 45 mg / mL; the concentration of the polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the injection is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0073] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0074] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, mannitol, and optionally edetate disodium or edetate calcium sodium, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein has the following sequence: NO: The amino acid sequence shown in any one of items 1 to 12, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-95 mg / mL, 25-90 mg / mL, 25-85 mg / mL, 25-80 mg / mL, 25-75 mg / mL, 25-70 mg / mL, 25-65 mg / mL, 25-60 mg / mL, 25-55 mg / mL, or 25-50 mg / mL; the concentration of the histidine buffer in the composition is 8-25 mM; the concentration of the mannitol in the composition is 35-45 mg / mL; the concentration of the polysorbate 80 in the composition is 0.4-1.3 mg / mL, or the concentration of the polysorbate 20 in the composition is 0.1-0.5 mg / mL; the concentration of the metal chelator in the composition is 0.05-0.5 mg / mL; and the pH value of the injection is 6.9-7.5. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0075] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0076] In another aspect of the present invention, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, mannitol, and optionally edetate disodium or edetate sodium calcium, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO: 1, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 50 mg / mL; the concentration of the histidine buffer in the injection is 10 to 20 mM; the concentration of the mannitol in the injection is 35 to 45 mg / mL; the concentration of the polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the injection is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0077] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0078] In another aspect of the present invention, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, sucrose or trehalose, and optionally edetate disodium or edetate calcium sodium, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL; the concentration of the histidine buffer in the injection is 8 to 25 mM; the concentration of the sucrose in the injection is 85 to 95 mg / mL or the concentration of the trehalose in the injection is 55 to 65 mg / mL; the concentration of the polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0079] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0080] In another aspect of the present invention, an injection is provided. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, sucrose or trehalose, and optionally edetate disodium or edetate calcium sodium, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein has a GLP-1-Fc-FGF21 fusion protein having ... NO: The amino acid sequence shown in any one of items 1 to 12, wherein the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-95 mg / mL, 25-90 mg / mL, 25-85 mg / mL, 25-80 mg / mL, 25-75 mg / mL, 25-70 mg / mL, 25-65 mg / mL, 25-60 mg / mL, 25-55 mg / mL, or 25-50 mg / mL; the concentration of the histidine buffer in the composition is 8-20 mM; the concentration of the sucrose in the composition is 85-95 mg / mL; the concentration of the trehalose in the injection is 55-65 mg / mL; the concentration of the polysorbate 80 in the composition is 0.4-1.3 mg / mL, or the concentration of the polysorbate 20 in the composition is 0.1-0.5 mg / mL; and the pH value of the injection is 6.9-7.5. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0081] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0082] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, sucrose, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 50 mg / mL; the concentration of the histidine buffer in the injection is 10 to 20 mM; the concentration of the sucrose in the injection is 85 to 95 mg / mL; the concentration of the polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0083] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0084] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, trehalose, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 50 mg / mL; the concentration of the histidine buffer in the injection is 10 to 20 mM; the concentration of the trehalose in the injection is 55 to 65 mg / mL; the concentration of the polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0085] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0086] In another aspect of the present invention, an injection is provided. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, sucrose or trehalose, and optionally edetate disodium or edetate calcium sodium, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein comprises SEQ ID NO: The amino acid sequence shown in any one of items 1 to 12, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, 25 to 50 mg / mL; the concentration of the citrate buffer in the injection is 8-25 mM, preferably 10 to 20 mM; the concentration of sucrose in the injection is 85 to 95 mg / mL; the concentration of trehalose in the injection is 55 to 65 mg / mL; the concentration of polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of polysorbate 20 The concentration in the injection is 0.1 to 0.5 mg / mL; the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the production of visible foreign matter and improve the physical and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0087] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0088] In another aspect of the present invention, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: GLP-1-Fc-FGF21 fusion protein, citrate buffer, mannitol and optionally edetate disodium or edetate calcium sodium, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, or 25 to 50 mg / mL; the citrate buffer is The concentration of mannitol in the injection is 8 to 25 mM, preferably 10 to 20 mM; the concentration of mannitol in the injection is 35 to 45 mg / mL; the concentration of polysorbate 80 in the injection is 0.4 to 1.5 mg / mL, or 0.4 to 1.3 mg / mL, or the concentration of polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the injection is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the production of visible foreign matter and improve the physical and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0089] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium. In another aspect of the present invention, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, mannitol, polysorbate 80, and optionally edetate disodium or edetate calcium sodium; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the injection is 5 to 20 mM; the concentration of mannitol in the injection is 40 to 50 mg / mL; the concentration of polysorbate 80 in the injection is 0.1 to 1.5 mg / mL; the concentration of the metal chelator in the injection is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.4 to 8.0. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0090] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0091] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, mannitol, polysorbate 80, and optionally disodium edetate or calcium sodium edetate; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the injection is 8 to 20 mM, 10 to 20 mM, or 10 to 15 mM; the concentration of mannitol in the injection is 40 to 50 mg / mL; the concentration of polysorbate 80 in the injection is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the injection is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.4 to 8.0. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0092] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0093] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, mannitol, polysorbate 80, and optionally edetate disodium or edetate sodium calcium; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the injection is 8 to 20 mM, 10 to 20 mM, or 10 to 15 mM; the concentration of mannitol in the injection is 40 to 50 mg / mL; the concentration of polysorbate 80 in the injection is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the injection is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.4 to 8.0. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0094] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0095] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, polysorbate 80, and optionally edetate disodium or edetate calcium sodium, and sucrose or trehalose; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the injection is 5 to 20 mM; the concentration of the sucrose in the injection is 80 to 95 mg / mL; the concentration of the trehalose in the injection is 55 to 65 mg / mL; the concentration of the polysorbate 80 in the injection is 0.1 to 1.5 mg / mL; and the pH value of the injection is 6.4 to 8.0. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0096] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0097] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, and polysorbate 80, as well as sucrose or trehalose, and optionally edetate disodium or edetate sodium calcium; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the injection is 8 to 20 mM, 10 to 20 mM, or 10 to 15 mM; the concentration of sucrose in the injection is 80 to 95 mg / mL; the concentration of trehalose in the injection is 55 to 65 mg / mL; the concentration of polysorbate 80 in the injection is 0.1 to 0.5 mg / mL; and the pH value of the injection is 6.4 to 8.0. The injection of the present invention can effectively control the generation of visible foreign matter, improve the physical stability and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0098] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0099] In an optional embodiment of the present application, the injection solutions of the above aspects may further include a preservative.

[0100] In an optional embodiment of the present application, the preservative is at least selected from phenol, m-cresol, benzyl alcohol, and phenoxyethanol.

[0101] In an optional embodiment of the present application, the concentration of the phenol in the composition or injection is 0 to 7 mg / mL, preferably 3 to 7 mg / mL.

[0102] In an optional embodiment of the present application, the concentration of m-cresol in the composition or injection is 0-4 mg / mL, preferably 2-4 mg / mL.

[0103] In another aspect of the present invention, the present invention provides a use of the aforementioned composition or the aforementioned injection in preparing a medicament for treating and / or preventing metabolic diseases.

[0104] According to an embodiment of the present invention, the metabolic diseases include diabetes, hyperlipidemia, obesity and diseases related to fatty liver.

[0105] According to an embodiment of the present invention, the fatty liver-related diseases include non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

[0106] In another aspect, the present invention provides an injection device or container. According to an embodiment of the present invention, the injection device or container comprises: the aforementioned composition or the aforementioned injection solution.

[0107] According to an embodiment of the present invention, the injection device or container includes at least one selected from a container bottle and a syringe.

[0108] According to an embodiment of the present invention, the syringe includes at least one selected from an automatic syringe and a pre-filled syringe.

[0109] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0111] FIG1 is a graph showing the growth trend of high temperature SEC-aggregates of different preparations in step 7 of Example 1 of the present invention;

[0112] FIG2 is a graph showing the growth trend of high temperature RP-HPLC purity (total related substances) of different preparations in step 7 of Example 1 of the present invention. DETAILED DESCRIPTION

[0113] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0114] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0115] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0116] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present invention, but not excluding other contents.

[0117] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0118] As used herein, the term "treatment" refers to a method for obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a composition, injection or drug to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a composition or injection as described herein to an individual in need.

[0119] The present invention provides a composition and an injection solution, use thereof, and an injection device or container, which will be described in detail below.

[0120] Composition

[0121] In one aspect, the present invention provides a composition. According to an embodiment of the present invention, the composition comprises a GLP-1-Fc-FGF21 fusion protein and a buffer. The addition of the buffer to the composition of the present invention effectively controls the formation of visible foreign matter and aggregates, improves the stability of the GLP-1-Fc-FGF21 fusion protein, and thereby extends the shelf life of the product.

[0122] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NOs: 1 to 12.

[0123] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12.

[0124] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO: 1.

[0125] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO: 2.

[0126] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO: 3.

[0127] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO:4.

[0128] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO:5.

[0129] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO:6.

[0130] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO:7.

[0131] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO:8.

[0132] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO:9.

[0133] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO:10.

[0134] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO:11.

[0135] In some optional embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO:12.

[0136] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 1 to 100 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 100 mg / mL, or a range value between any two values ​​as endpoints.Such as 2 to 99 mg / mL, 2 to 95 mg / mL, 2 to 90 mg / mL, 2 to 85 mg / mL, 2 to 80 mg / mL, 2 to 75 mg / mL, 2 to 70 mg / mL, 2 to 65 mg / mL, 2 to 60 mg / mL, 2 to 55 mg / mL, 5 to 99 mg / mL, 5 to 95 mg / mL, 5 to 90 mg / mL, 5 to 85 mg / mL, 5 to 80 mg / mL, 5 to 75 mg / mL, 5 to 70 mg / mL, 5 to 65 mg / mL, 5 to 60 mg / mL, 5 to 55 mg / mL, 10 to 99 mg / mL, 10 to 95 mg / mL, 10 to 90 mg / mL, 10 to 85 mg / mL, 10 to 80 mg / mL, 10 to 75 mg / mL, 10 to 70 mg / mL, 10 to 65 mg / mL, 10 to 60 mg / mL, 10 to 55 mg / mL, 25 to 99 mg / mL, 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, 30 to 99 mg / mL, 30 to 95 mg / mL, 30 to 90 mg / mL, 30 to 85 mg / mL, 30 to 80 mg / mL, 30 to 75 mg / mL, 30 to 70 mg / mL, 30 to 65 mg / mL, 30 to 60 mg / mL, 30 to 55 mg / mL, 35 to 99 mg / mL, 35 to 95 mg / mL, 35 to 90 mg / mL, 35 to 85 mg / mL, 35 to 80 mg / mL, 35 to 75 mg / mL, 35 to 70 mg / mL, 35 to 65 mg / mL, 35 to 60 mg / mL, 35 to 55 mg / mL, 40 to 99 mg / mL, 40 to 95 mg / mL, 40 to 90 mg / mL, 40 to 85 mg / mL, 40 to 80 mg / mL, 40 to 75 mg / mL, 40 to 70 mg / mL, 40 to 65 mg / mL, 40 to 60 mg / mL, 40 to 55 mg / mL.,

[0137] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2-55 mg / mL, for example, 2-25 mg / mL, 3-25 mg / mL, 10-25 mg / mL, 10-20 mg / mL, 15-25 mg / mL, 15-20 mg / mL, 25-100 mg / mL, 25-95 mg / mL, 25-90 mg / mL, 25-85 mg / mL, 25-80 mg / mL, 25-75 mg / mL, 25-70 mg / mL, 25-65 mg / mL, 25-60 mg / mL, 25-55 mg / mL, 25-50 mg / mL, 25-40 mg / mL, 40-55 mg / mL, 40-50 mg / mL, 45-55 mg / mL, 45-50 mg / mL.

[0138] In some optional embodiments of the present invention, the pH value of the composition is 6.4-8.0, for example, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0, or a range between any two values ​​as endpoints. This can further improve the stability of the fusion protein, for example, 6.2-8.0, 6.9-7.5.

[0139] In some optional embodiments of the present invention, the buffer comprises at least one of a histidine buffer, a citrate buffer, a phosphate buffer and a Tris buffer.

[0140] In this document, without special explanation, "citrate buffer" or "citrate buffer" can be selected from citric acid, sodium citrate, or a mixture of citric acid and sodium citrate, all of which are within the scope of protection of this application.

[0141] In some optional embodiments of the present invention, the phosphate buffer is selected from a combination of disodium hydrogen phosphate and sodium dihydrogen phosphate or a combination of dipotassium hydrogen phosphate and potassium dihydrogen phosphate.

[0142] In some optional embodiments of the present invention, the histidine buffer comprises histidine and / or histidine hydrochloride.

[0143] In some optional embodiments of the present invention, the histidine buffer comprises histidine and histidine hydrochloride.

[0144] In some optional embodiments of the present invention, the histidine buffer comprises histidine or histidine hydrochloride.

[0145] In some optional embodiments of the present invention, the citrate buffer comprises citric acid and / or sodium citrate.

[0146] In some optional embodiments of the present invention, the citrate buffer comprises citric acid and sodium citrate.

[0147] In some optional embodiments of the present invention, the citrate buffer comprises citric acid or sodium citrate.

[0148] In some optional embodiments of the present invention, the Tris buffer comprises Tris and / or Tris-HCl.

[0149] In some optional embodiments of the present invention, the Tris buffer comprises Tris and Tris-HCl.

[0150] In some optional embodiments of the present invention, the Tris buffer comprises Tris or Tris-HCl.

[0151] In some optional embodiments of the present invention, the buffer comprises a histidine buffer or a citrate buffer.

[0152] In some optional embodiments of the present invention, the concentration of the buffer in the composition is 5-30mM, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 11.29mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM or 30mM, or a range value between any two values ​​as endpoint values ​​(for example, 5-25mM, 8-25mM, 8-12mM, 10-20mM).

[0153] In some optional embodiments of the present invention, the composition further comprises at least one of an osmotic pressure regulator, a surfactant, a preservative, and a metal chelator, thereby further improving the stability of the fusion protein.

[0154] In some optional embodiments of the present invention, the osmotic pressure regulator includes at least one of mannitol, sucrose, sodium chloride and trehalose.

[0155] In some optional embodiments of the present invention, the concentration of the osmotic pressure regulator in the composition is 30-100 mg / mL, for example, 30 mg / mL, 35 mg / mL, 40 mg / mL, 41 mg / mL, 41.4 mg / mL, 41.5 mg / mL, 42 mg / mL, 42.4 mg / mL, 42.5 mg / mL, 43 mg / mL, 43.4 mg / mL, 43.5 mg / mL, 44 mg / mL, 44.4 mg / mL, 44.5 mg / mL, 45 mg / mL, 45.4 mg / mL, 45.5 mg / mL, 46 mg / mL

[0015] In some embodiments, the present invention provides an intravenous initiation dose of at least 1 mg / mL, 46.4 mg / mL, 46.5 mg / mL, 47 mg / mL, 47.4 mg / mL, 47.5 mg / mL, 48 mg / mL, 48.4 mg / mL, 48.5 mg / mL, 49 mg / mL, 49.4 mg / mL, 49.5 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL or 100 mg / mL, or a range between any two values ​​as endpoints.

[0156] In some optional embodiments of the present invention, the concentration of the osmotic pressure regulator in the composition is 1 to 10 mg / mL, for example, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL or 10 mg / mL, or a range value between any two values ​​as endpoint values.

[0157] In some optional embodiments of the present invention, the surfactant includes at least one of polysorbate 80, polysorbate 20 and poloxamer 188.

[0158] In some optional embodiments of the present invention, the concentration of the surfactant in the composition is 0.1 to 10 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL

[0015] In some embodiments, the present invention provides a range of: a) a molecular weight of at least one molecular weight of the present invention and b) a molecular weight of at least one molecular weight of the present invention; c) a molecular weight of at least one molecular weight of the present invention; d) a molecular weight of at least one molecular weight of the present invention; d) a molecular weight of at least one molecular weight of the present invention; d) a molecular weight of at least one molecular weight of the present invention; d) a molecular weight of at least one molecular weight of the present invention; d) a molecular weight of at least one molecular weight of the present invention;

[0159] Illustratively, the concentration of the surfactant in the composition is 0.1 to 1.5 mg / mL or 0.1 to 1 mg / mL.

[0160] In some optional embodiments of the present invention, the preservative is at least one of phenol and m-cresol.

[0161] According to an embodiment of the present invention, the concentration of the preservative in the composition is 0.1 to 10 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, 10.0 mg / mL, such as 1.0 to 5.0 mg / mL.

[0162] In some optional embodiments of the present invention, the preservative is phenol.

[0163] According to an embodiment of the present invention, the concentration of phenol in the composition is 1.0 to 5.0 mg / mL, for example, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, 10.0 mg / mL, such as 2 to 7 mg / mL, 3.0 to 4.0 mg / mL.

[0164] In some optional embodiments of the present invention, the preservative is m-cresol.

[0165] According to an embodiment of the present invention, the concentration of meta-cresol in the composition is 1.0 to 5.0 mg / mL, for example, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, 10.0 mg / mL, such as 3.0 to 4.0 mg / mL.

[0166] In some optional embodiments of the present invention, the preservatives are phenol and m-cresol.

[0167] According to an embodiment of the present invention, the concentration of phenol in the composition is 1.0 to 5.0 mg / mL (such as 1.0 mg / mL, 1.5 mg / mL, The concentration of m-cresol in the composition is 0.1 to 1.0 mg / mL (e.g., 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL).

[0168] In some optional embodiments of the present invention, the metal chelator is at least one of edetate disodium (EDTA-2Na) and edetate calcium sodium (EDTA-CaNa2).

[0169] In some optional embodiments of the present invention, the metal chelator is edetate disodium (EDTA-2Na).

[0170] In some optional embodiments of the present invention, the metal chelator is edetate calcium sodium (EDTA-CaNa2).

[0171] In some optional embodiments of the present invention, the concentration of the metal chelator in the composition is 0.01-1 mg / mL, for example, 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.115 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL , 0.19 mg / mL, 0.2 mg / mL, 0.25 mg / mL, 0.3 mg / mL, 0.35 mg / mL, 0.4 mg / mL, 0.45 mg / mL, 0.5 mg / mL, 0.55 mg / mL, 0.6 mg / mL, 0.65 mg / mL, 0.7 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 0.95 mg / mL or 1.0 mg / mL, or a range value between any two values ​​as the endpoint values ​​(0.05-0.5 mg / mL, 0.08-0.12 mg / mL, 0.1-0.12 mg / mL).

[0172] Illustratively, the concentration of the metal chelator in the composition is 0.08 to 0.12 mg / mL. In embodiments of the present invention, regardless of whether the citric acid system or the histidine buffer system is used, the stability of the fusion protein formulation is good regardless of whether EDTA sodium salt is added. Therefore, whether or not EDTA sodium salt is added is within the scope of the present invention.

[0173] In some optional embodiments of the present invention, the buffer is a histidine buffer, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 25 to 100 mg / mL, for example, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, or a range value between any two values ​​as endpoints (e.g., 25 to 75 mg / mL). mg / mL, 25~70mg / mL, 25~65mg / mL, 25~60mg / mL, 25~55mg / mL, 25~50mg / mL, 25~40mg / mL, 40~80mg / mL, 40~75mg / mL, 40~70mg / mL, 40~65mg / mL, 40~60mg / mL, 40~55mg / mL, 40~50mg / mL, 45~80mg / mL, 45~75mg / mL, 45~70mg / mL, 45~65mg / mL, 45~60mg / mL, 45~55mg / mL, 45~50mg / mL).

[0174] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 25-55 mg / mL, and the buffer is a histidine buffer. The inventors unexpectedly discovered that the addition of a histidine buffer to a final concentration of the fusion protein of 25-55 mg / mL effectively controls the production of visible foreign matter in the composition, thereby improving the physical stability of the fusion protein and reducing potential safety risks. It also effectively controls the rate of chemical changes, maintaining stability under high temperature, accelerated, long-term, repeated freeze-thaw, shock, and simulated transportation conditions, resulting in a composition with a longer shelf life.

[0175] In some optional embodiments of the present invention, the concentration of the histidine buffer in the composition is 5-30mM, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM, 30mM, or a range value between any two values ​​as endpoints (e.g., 5-25 mg / mL, 8-25 mg / mL, 8-20 mg / mL, 10-20 mg / mL, 8-12 mg / mL).

[0176] In some optional embodiments of the present invention, the concentration of the histidine buffer in the composition is 10-20 mM.

[0177] In some optional embodiments of the present invention, the pH value of the composition is 6.7-7.5, preferably 6.9-7.5.

[0178] In some optional embodiments of the present invention, the osmotic pressure regulator is mannitol, and the concentration of the osmotic pressure regulator in the composition is 30-50 mg / mL; or the osmotic pressure regulator is sucrose, and the concentration of the osmotic pressure regulator in the composition is 60-100 mg / mL, preferably 80-100 mg / mL; or the osmotic pressure regulator is trehalose, and the concentration of the osmotic pressure regulator in the composition is 40-100 mg / mL, preferably 40-70 mg / mL, 50-70 mg / mL, 55-65 mg / mL; or the osmotic pressure regulator is sodium chloride, and the concentration of the osmotic pressure regulator in the composition is 1-10 mg / mL, preferably 3-9 mg / mL.

[0179] In some optional embodiments of the present invention, the surfactant is polysorbate 80, and the concentration of the surfactant in the composition is 0.1-2 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL, 1.5 mg / mL, 1.6 mg / mL, 1.7 mg / mL, 1.8 mg / mL, 1.9 mg / mL or 2.0 mg / mL, or a range value between any two values ​​as endpoints (for example, 0.1-1.5 mg / mL, 0.2-2 mg / mL, 0.4-1.3 mg / mL); or

[0180] In some optional embodiments of the present invention, the surfactant is polysorbate 20, and the concentration of the surfactant in the composition is 0.1 to 1.5 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.1 mg / mL, 1.2 mg / mL, 1.3 mg / mL, 1.4 mg / mL or 1.5 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 0.1 to 0.5 mg / mL).

[0181] In some optional embodiments of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 25 mg / mL, and the buffer is a citrate buffer. The inventors unexpectedly discovered that the addition of a citrate buffer to a final concentration of the fusion protein of 2 to 25 mg / mL effectively controls the formation of visible foreign matter in the composition, improves the physical and chemical stability of the fusion protein, reduces potential safety risks, and extends the shelf life of the composition.

[0182] In some optional embodiments of the present invention, the concentration of the citrate buffer in the composition is 5-30mM, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM, 14mM, 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM, 25mM, 26mM, 27mM, 28mM, 29mM or 30mM, or a range value between any two values ​​as endpoint values ​​(for example, 5-25mM, 8-25mM, 8-20mM, 10-20mM, 8-12mM, 5-15mM, 10-15mM).

[0183] In some optional embodiments of the present invention, the pH value of the composition is 6.4-8.0.

[0184] In some optional embodiments of the present invention, the osmotic pressure regulator is mannitol, and the concentration of the osmotic pressure regulator in the composition is 30-50 mg / mL; or the osmotic pressure regulator is sucrose, and the concentration of the osmotic pressure regulator in the composition is 60-100 mg / mL, preferably 80-100 mg / mL; or the osmotic pressure regulator is trehalose, and the concentration of the osmotic pressure regulator in the composition is 40-100 mg / mL, preferably 40-70 mg / mL, and more preferably 50-70 mg / mL.

[0185] According to an embodiment of the present invention, the osmotic pressure regulator is sodium chloride, and the concentration of the osmotic pressure regulator in the composition is 1 to 10 mg / mL, preferably 3 to 9 mg / mL.

[0186] In some optional embodiments of the present invention, the surfactant includes at least one of polysorbate 80, polysorbate 20 and poloxamer 188, preferably polysorbate 80.

[0187] In some optional embodiments of the present invention, the concentration of the surfactant in the composition is 0.1 to 10.0 mg / mL, for example, 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, 5.0 mg / mL, 6.0 mg / mL, 7.0 mg / mL, 8.0 mg / mL, 9.0 mg / mL or 10.0 mg / mL, or a range value between any two values ​​as endpoints (for example, 0.1 to 1.5 mg / mL, 0.1 to 1.0 mg / mL, 6.0 to 10.0 mg / mL, 0.1 to 0.5 mg / mL, 0.1 to 0.3 mg / mL).

[0188] In some optional embodiments of the present invention, the surfactant is poloxamer 188, and the concentration of the surfactant in the composition is 6 to 10 mg / mL; or the surfactant is polysorbate 80 or polysorbate 20, and the concentration of the surfactant in the composition is 0.1 to 1.5 mg / mL, preferably 0.1 to 1 mg / mL or 0.1 to 0.5 mg / mL.

[0189] In some optional embodiments of the present invention, the composition is in the form of an injection.

[0190] injection

[0191] In another aspect of the present invention, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, mannitol, and optionally disodium edetate or sodium calcium edetate, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL; the concentration of the histidine buffer in the composition is 8 to 25 mM; the concentration of the mannitol in the composition is 35 to 45 mg / mL; the concentration of the polysorbate 80 in the composition is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the composition is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the composition is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the generation of visible foreign matter and aggregates, and can improve the physical and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0192] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 95 mg / mL.

[0193] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 90 mg / mL.

[0194] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 85 mg / mL.

[0195] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 80 mg / mL.

[0196] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 75 mg / mL.

[0197] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-70 mg / mL.

[0198] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 65 mg / mL.

[0199] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-60 mg / mL.

[0200] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-55 mg / mL.

[0201] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-50 mg / mL.

[0202] In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 1. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 2. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 3. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 4. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 5. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 6. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 7. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 8. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 9. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 10. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 11. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 12.

[0203] According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, mannitol, and optionally disodium edetate or sodium calcium edetate, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in SEQ ID NO: 1, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 50 mg / mL; the concentration of the histidine buffer in the composition is 10 to 20 mM; the concentration of the mannitol in the composition is 35 to 45 mg / mL; the concentration of the polysorbate 80 in the composition is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the composition is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the composition is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5.

[0204] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0205] In an optional embodiment of the present application, the injection solutions of the above aspects may further include a preservative.

[0206] In an optional embodiment of the present application, the preservative is at least selected from phenol, m-cresol, benzyl alcohol, and phenoxyethanol.

[0207] In an optional embodiment of the present application, the concentration of phenol in the composition or injection is 0-7 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 3-7 mg / mL, 2-4 mg / mL).

[0208] In an optional embodiment of the present application, the concentration of meta-cresol in the composition or injection is 0-4 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 2-4 mg / mL).

[0209] In another aspect of the present invention, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a histidine buffer, sucrose or trehalose, and optionally disodium edetate or calcium sodium edetate, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL; the concentration of the histidine buffer in the composition is 8 to 20 mM; the concentration of the sucrose in the composition is 85 to 95 mg / mL; the concentration of the trehalose in the injection is 55 to 65 mg / mL; the concentration of the polysorbate 80 in the composition is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the composition is 0.1 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the generation of visible foreign matter and aggregates, and can improve the physical and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0210] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 95 mg / mL.

[0211] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 90 mg / mL.

[0212] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 85 mg / mL.

[0213] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 80 mg / mL.

[0214] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 75 mg / mL.

[0215] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-70 mg / mL.

[0216] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 65 mg / mL.

[0217] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-60 mg / mL.

[0218] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-55 mg / mL.

[0219] According to an embodiment of the present invention, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25-50 mg / mL.

[0220] In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 1. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 2. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 3. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 4. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 5. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 6. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 7. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 8. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 9. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 10. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 11. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 12.

[0221] According to an embodiment of the present invention, the injection comprises: GLP-1-Fc-FGF21 fusion protein, histidine buffer and sucrose, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 50 mg / mL; the concentration of the histidine buffer in the injection is 10 to 20 mM; the concentration of the sucrose in the injection is 85 to 95 mg / mL; the concentration of the polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5.

[0222] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0223] In an optional embodiment of the present application, the injection solutions of the above aspects may further include a preservative.

[0224] In an optional embodiment of the present application, the preservative is at least selected from phenol, m-cresol, benzyl alcohol, and phenoxyethanol.

[0225] In an optional embodiment of the present application, the concentration of phenol in the composition or injection is 0-7 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 3-7 mg / mL, 2-4 mg / mL).

[0226] In an optional embodiment of the present application, the concentration of meta-cresol in the composition or injection is 0-4 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 2-4 mg / mL).

[0227] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, sucrose or trehalose, and optionally edetate disodium or edetate calcium sodium, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, or 25 to 50 mg / mL; The concentration of the citrate buffer in the injection is 8-25 mM, preferably 10-20 mM; the concentration of sucrose in the injection is 85-95 mg / mL; the concentration of trehalose in the injection is 55-65 mg / mL; the concentration of polysorbate 80 in the injection is 0.4-1.3 mg / mL, or the concentration of polysorbate 20 in the injection is 0.1-0.5 mg / mL; and the pH value of the injection is 6.9-7.5. The injection of the present invention can effectively control the production of visible foreign matter and improve the physical and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0228] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0229] In an optional embodiment of the present application, the injection solutions of the above aspects may further include a preservative.

[0230] In an optional embodiment of the present application, the preservative is at least selected from phenol, m-cresol, benzyl alcohol, and phenoxyethanol.

[0231] In an optional embodiment of the present application, the concentration of phenol in the composition or injection is 0-7 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 3-7 mg / mL, 2-4 mg / mL).

[0232] In an optional embodiment of the present application, the concentration of meta-cresol in the composition or injection is 0-4 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 2-4 mg / mL).

[0233] In another aspect of the present invention, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: GLP-1-Fc-FGF21 fusion protein, citrate buffer, mannitol and optionally edetate disodium or edetate calcium sodium, and polysorbate 80 or polysorbate 20; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, or 25 to 50 mg / mL; the citrate buffer is The concentration of mannitol in the injection is 8 to 25 mM, preferably 10 to 20 mM; the concentration of mannitol in the injection is 35 to 45 mg / mL; the concentration of polysorbate 80 in the injection is 0.4 to 1.5 mg / mL, or 0.4 to 1.3 mg / mL, or the concentration of polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the injection is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.9 to 7.5. The injection of the present invention can effectively control the production of visible foreign matter and improve the physical and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0234] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0235] In an optional embodiment of the present application, the injection solutions of the above aspects may further include a preservative.

[0236] In an optional embodiment of the present application, the preservative is at least selected from phenol, m-cresol, benzyl alcohol, and phenoxyethanol.

[0237] In an optional embodiment of the present application, the concentration of phenol in the composition or injection is 0-7 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 3-7 mg / mL, 2-4 mg / mL).

[0238] In an optional embodiment of the present application, the concentration of meta-cresol in the composition or injection is 0-4 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 2-4 mg / mL).

[0239] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, mannitol, polysorbate 80, and optionally edetate disodium or edetate sodium calcium; the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the composition is 5 to 20 mM; the concentration of the mannitol in the composition is 40 to 50 mg / mL; the concentration of the polysorbate 80 in the composition is 0.1 to 1.5 mg / mL; the concentration of the metal chelator in the composition is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.4 to 8.0. The injection of the present invention can effectively control the generation of visible foreign matter and aggregates, and can improve the physical and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0240] In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 1. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 2. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 3. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 4. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 5. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 6. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 7. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 8. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 9. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 10. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 11. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 12.

[0241] In some optional embodiments of the present invention, the concentration of the citrate buffer in the injection is 8 to 20 mM. In some optional embodiments of the present invention, the concentration of the citrate buffer in the injection is 10 to 20 mM. In some optional embodiments of the present invention, the concentration of the citrate buffer in the injection is 10 to 15 mM.

[0242] According to an embodiment of the present invention, the injection comprises: GLP-1-Fc-FGF21 fusion protein, citrate buffer, mannitol, polysorbate 80 and optionally disodium edetate or calcium sodium edetate; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the injection is 5 to 25 mM, 8 to 20 mM, 10 to 20 mM, or 10 to 15 mM; the concentration of mannitol in the injection is 40 to 50 mg / mL; the concentration of polysorbate 80 in the injection is 0.1 to 0.5 mg / mL; the concentration of the metal chelator in the injection is 0.05 to 0.5 mg / mL; and the pH value of the injection is 6.4 to 8.0.

[0243] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0244] In an optional embodiment of the present application, the injection solutions of the above aspects may further include a preservative.

[0245] In an optional embodiment of the present application, the preservative is at least selected from phenol, m-cresol, benzyl alcohol, and phenoxyethanol.

[0246] In an optional embodiment of the present application, the concentration of phenol in the composition or injection is 0-7 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 3-7 mg / mL, 2-4 mg / mL).

[0247] In an optional embodiment of the present application, the concentration of meta-cresol in the composition or injection is 0-4 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 2-4 mg / mL).

[0248] In another aspect, the present invention provides an injection. According to an embodiment of the present invention, the injection comprises: a GLP-1-Fc-FGF21 fusion protein, a citrate buffer, polysorbate 80, and optionally edetate disodium or edetate calcium sodium, and sucrose or trehalose; the GLP-1-Fc-FGF21 fusion protein has an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the composition is 5 to 20 mM; the concentration of the sucrose in the composition is 85 to 95 mg / mL; the concentration of the trehalose in the composition is 55 to 65 mg / mL; the concentration of the polysorbate 80 in the composition is 0.1 to 1.5 mg / mL; and the pH value of the injection is 6.4 to 8.0. The injection of the present invention can effectively control the generation of visible foreign matter and aggregates, and can improve the physical and chemical stability of the fusion protein, thereby reducing potential safety risks and extending the shelf life.

[0249] In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 1. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 2. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 3. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 4. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 5. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 6. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence set forth in SEQ ID NO: 7. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 8. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 9. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 10. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 11. In some alternative embodiments of the present invention, the GLP-1-Fc-FGF21 fusion protein has the amino acid sequence shown in SEQ ID NO: 12.

[0250] In some optional embodiments of the present invention, the concentration of the citrate buffer in the injection is 8 to 20 mM. In some optional embodiments of the present invention, the concentration of the citrate buffer in the injection is 10 to 20 mM. In some optional embodiments of the present invention, the concentration of the citrate buffer in the injection is 10 to 15 mM.

[0251] According to an embodiment of the present invention, the injection comprises: GLP-1-Fc-FGF21 fusion protein, citrate buffer and polysorbate 80, as well as sucrose or trehalose, and optionally disodium edetate or calcium sodium edetate; the GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in SEQ ID NO: 1, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 3 to 25 mg / mL; the concentration of the citrate buffer in the injection is 5 to 25 mM, 8 to 20 mM, 10 to 20 mM, or 10 to 15 mM; the concentration of sucrose in the injection is 80 to 95 mg / mL; the concentration of trehalose in the injection is 55 to 65 mg / mL; the concentration of polysorbate 80 in the injection is 0.1 to 0.5 mg / mL; and the pH value of the injection is 6.4 to 8.0.

[0252] According to an embodiment of the present invention, the injection does not contain edetate disodium or edetate calcium sodium.

[0253] In an optional embodiment of the present application, the injection solutions of the above aspects may further include a preservative.

[0254] In an optional embodiment of the present application, the preservative is at least selected from phenol, m-cresol, benzyl alcohol, and phenoxyethanol.

[0255] In an optional embodiment of the present application, the concentration of phenol in the composition or injection is 0-7 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 4.5 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 3-7 mg / mL, 2-4 mg / mL).

[0256] In an optional embodiment of the present application, the concentration of meta-cresol in the composition or injection is 0-4 mg / mL, for example, 0.1 mg / mL, 0.5 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, or a range value between any two values ​​as endpoint values ​​(for example, 2-4 mg / mL).

[0257] use

[0258] In another aspect of the present invention, the present invention provides a use of the aforementioned composition or the aforementioned injection in preparing a medicament for treating and / or preventing metabolic diseases.

[0259] In some optional embodiments of the present invention, the metabolic diseases include diabetes, obesity, hyperlipidemia and diseases related to fatty liver.

[0260] In some optional embodiments of the present invention, the fatty liver-related diseases include non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis and cirrhosis.

[0261] Methods for treating and / or preventing metabolic diseases

[0262] In another aspect, the present invention provides a method for treating and / or preventing metabolic diseases. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the aforementioned composition or the aforementioned injection to a subject.

[0263] The effective amount of the composition or injection of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by a person of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the urgency of the treatment, the dosage may be administered, for example, once a day, once every other day, once a week, or once every two weeks.

[0264] In some optional embodiments of the present invention, the administration route of the method includes subcutaneous injection or intravenous injection, preferably subcutaneous injection.

[0265] Injection device or container

[0266] In another aspect, the present invention provides an injection device or container. According to an embodiment of the present invention, the injection device or container comprises: the aforementioned composition or the aforementioned injection solution.

[0267] In some optional embodiments of the present invention, the injection device or container includes at least one selected from a container bottle, a vial, a cartridge, and a syringe.

[0268] In some optional embodiments of the present invention, the syringe comprises at least one selected from an automatic syringe and a pre-filled syringe.

[0269] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not indicated, the technology or conditions described in the literature in this area or the product instructions are used.The reagents or instruments used are not indicated by the manufacturer, and are all conventional products that can be obtained by commercial purchase.

[0270] It should be noted that the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein in Example 1 of the present invention is shown in SEQ ID NO: 1, and it was prepared using conventional methods in the art.

[0271] Example 1:

[0272] 1. Investigation of injections prepared with different buffer systems

[0273] 1.1 Low-concentration injection formulations A1-A3 were prepared using different buffer systems. The final concentrations of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as the fusion protein, or API) and the types and final concentrations of the excipients in the injections are detailed in Table 1. The injections were prepared by weighing the excipients and API according to the dosage in Table 1 and mixing them. If necessary, the pH of the formulation was adjusted to the appropriate value using 2% hydrochloric acid solution or 2% sodium hydroxide solution. Finally, water for injection was added to the volume. After preparation, the formulations were filtered in series using 0.45μm and 0.22μm PVDF filters, transferred to 1ml prefilled syringes, and sealed with rubber stoppers. Formulations A1-A3 were then subjected to high-temperature stability studies. The SEC-HPLC stability test results at 40°C for 0, 10, and 14 days are shown in Table 2.

[0274] Table 1: Ratios of ingredients in formulations A1 to A3

[0275] Table 2: Stability results of formulations A1 to A3 at 40°C

[0276] Note: (1) The “*” in the table above and in the following text indicates that the visible foreign matter complies with the provisions of General Principles 0904 of Part IV of the ChP 2015 Edition. The “visible foreign matter does not comply with the provisions of General Principles 0904 of Part IV of the ChP 2015 Edition”.

[0277] (2) The reason why the main peak purity of the RP-HPLC-purity (main peak) at day 0 in the above table is low is mainly because the main peak purity of the reverse phase of the API stock solution directly used in this example is not high. Therefore, the data in this example is only used for screening of formulation prescriptions and does not represent the purity of the actual fusion protein samples of this application, the same as preparations A4 to A37 in this example.

[0278] As shown in Table 2, the citrate buffer system exhibited the slowest rate of decline in the purity of the RP-HPLC main peak, and visible foreign matter met regulatory requirements after 14 days at a high temperature of 40°C. Therefore, the citrate buffer system exhibited relatively good chemical and physical stability for low-concentration fusion protein preparations.

[0279] Because different buffer systems may also have different effects on the thermal stability of proteins, the Tm and Tonset values ​​of Formulations A1-A3 in different buffer systems were further measured using DSC (differential scanning calorimetry). The temperature changes during the protein thermal denaturation process were used to study and compare the differences in the effects of various factors on protein thermal stability. The DSC test results are shown in Table 3. The Tonset and Tm values ​​are indicators of thermal stability. The Tonset value is the onset temperature of protein unfolding, and the Tm value is the midpoint temperature of the protein thermal transition. Generally speaking, the higher the Tonset and Tm values, the more stable the protein is and the less likely it is to unfold and denature in a low-temperature environment.

[0280] Table 3: DSC test results of preparations A1 to A3

[0281] As shown in Table 3, by comparing the Tonset, Tm1 and Tm2 values, for low concentration fusion protein preparations, the thermal stability in citrate (preparation A1) and phosphate buffer (preparation A2) is better than that in the histidine buffer system (preparation A3).

[0282] 1.2 High-concentration injection formulations F1-F4 were prepared using different buffer systems. The final concentrations of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) and the types and final concentrations of the excipients in the injections are detailed in Table 4. The injections were prepared by ultrafiltration and concentration of the fusion protein stock solution. After the solution was exchanged, the protein concentration was determined. The fusion protein solution was then diluted to 25 mg / ml using the appropriate buffer system. If necessary, the pH was adjusted to 7.0 using 2% hydrochloric acid or 2% sodium hydroxide solution. After preparation, the solution was filtered in series using 0.45 μm and 0.22 μm PVDF filters and transferred into 2R medium-borosilicate vials, each containing 1 ml. The vials were then fitted with rubber stoppers and sealed with aluminum-plastic composite caps. Formulations F1-F4 were then subjected to high-temperature stability studies. The SEC-HPLC results of the aggregation change rate (days / %) after 14 days at 40°C are shown in Tables 5-1, 5-2, and 5-3.

[0283] Table 4: Ratios of ingredients in formulations F1 to F4

[0284] Table 5-1: SEC-HPLC aggregation change rate of preparations F1 to F4 after being placed at high temperature of 40°C for 14 days (days / %)

[0285] Table 5-2: IEX-HPLC main peak change rate of preparations F1 to F4 after being placed at high temperature of 40°C for 14 days (days / %)

[0286] Table 5-3: RP-HPLC total impurity change rate of preparations F1 to F4 after being placed at 40°C for 14 days (days / %)

[0287] As shown in Table 5-1, after 14 days at 40°C, SEC aggregates grew faster in sodium citrate buffer (preparation F1), phosphate buffer (preparation F3), and Tris buffer (preparation F4). Histidine buffer (preparation F2) significantly reduced the growth rate of SEC aggregates (the SEC aggregate standard is 5%). RP-HPLC total impurities (total impurity standard is 40%) showed comparable changes across the four preparations. This further demonstrates that when the fusion protein concentration is greater than or equal to 25 mg / ml, the stability of the preparation prepared with histidine buffer is higher, but the stability data for the preparations prepared with different buffer systems are all within the acceptable range.

[0288] 2. Investigation of injection solutions prepared at different pH values

[0289] 2.1 Low-concentration injection formulations A4-A8 were prepared at different pH values. The final concentrations of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as the fusion protein, or API) and the types and final concentrations of the excipients in the injections are detailed in Table 6. The injections were prepared by weighing the excipients and API according to the prescribed amounts in the table below and mixing them. If necessary, the pH of the formulation was adjusted to the appropriate value using 2% hydrochloric acid solution or 2% sodium hydroxide solution. Finally, water for injection was added to the volume. After preparation, the formulation was filtered in series using 0.45μm and 0.22μm PVDF filters, transferred into 1ml pre-filled slender syringes, and sealed with rubber stoppers. Formulations A4-A8 were then subjected to high-temperature and long-term stability studies. The SEC-HPLC stability test results at 40°C for 0 days, 10 days, and 14 days are shown in Table 7. The SEC-HPLC stability test results at 4°C for 0 months, 1 month, 6 months, and 12 months are shown in Table 8.

[0290] Table 6: Ratios of ingredients in formulations A4 to A8

[0291] Table 7: Stability results of formulations A4-A8 at 40°C

[0292] Table 8: Stability results of formulations A4-A8 at 4°C

[0293] As shown in Tables 7 and 8, when the pH value is 6.0, the RP-HPLC-main peak purity of the injection under high temperature conditions of 40°C has the fastest decrease rate, the highest dimer content, and the worst stability. In addition, foreign matter that can be seen for a long time for 12 months does not meet the regulations, and the SEC-purity (monomer including shoulder peak) has the fastest decrease rate, the highest dimer content, and the worst stability. The chemical stability of the other pH value groups is good.

[0294] Because different buffer systems can also have different effects on protein thermal stability, we further measured the Tm and Tonset values ​​of formulations A4-A8 in different buffer systems using DSC (differential scanning calorimetry). This study compared the effects of various factors on protein thermal stability by analyzing the temperature changes during the protein thermal denaturation process. The DSC test results are shown in Table 9.

[0295] Table 9: DSC test results of preparations A4 to A8

[0296] From the DSC test results in Table 9, it can be seen that the thermal stability is the worst when the pH is 6.0, and the other groups are better.

[0297] The effect of pH between 6.0 and 7.0 on product stability was further refined. Injection preparations A9 to A15 were prepared using different pH values. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injection and the types and final concentrations of each excipient are specifically shown in Table 10. The preparation method of the injection is specifically referred to the preparation method of preparations A4 to A8 in step 2.1 of Example 1. Preparations A9 to A15 were then subjected to high temperature stability studies and DSC tests. The SEC-HPLC stability test results at high temperature of 40°C for 0 days, 10 days, 14 days, and 21 days are shown in Table 11, and the DSC test results are shown in Table 12.

[0298] Table 10: Ratios of ingredients in formulations A9 to A15

[0299] Table 11: Stability results of formulations A9-A15 at high temperature (40°C)

[0300] Table 12: DSC test results of preparations A9 to A15

[0301] It can be seen from Tables 11 and 12 that when the pH is 6.0 and 6.2, the Tonset and Tm1 values ​​are low, and the thermal stability is worse than that of the other groups. When the pH is 6.4 and above, the chemical stability, physical stability and thermal stability of the injection are good.

[0302] 2.2 High-concentration injection formulations F5-F11 were prepared using different pH values. The final concentrations of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as the fusion protein, or API) and the types and final concentrations of the excipients in the injections are detailed in Table 13. The injections were prepared by weighing the excipients and API according to the prescribed amounts in the table below and mixing them. If necessary, the pH of the formulation was adjusted to the appropriate value using 2% hydrochloric acid solution or 2% sodium hydroxide solution. Finally, the volume was adjusted to 10 ml with water for injection. After preparation, the solution was filtered in series using 0.45 μm and 0.22 μm PVDF filters, transferred into 1 ml slender prefilled syringes, each filled with 0.5 ml, and sealed with a rubber stopper. Formulations F1-F4 were then subjected to high-temperature stability studies. The SEC-HPLC results of the aggregation change rate (days / %) after 20 days at 40°C are shown in Table 14.

[0303] Table 13: Ratios of ingredients in formulations F5 to F11

[0304] Table 14: SEC-HPLC aggregation change rate of formulations F5 to F11 placed at high temperature 40°C for 20 days (days / %)

[0305] Table 14 shows that after 20 days at 40°C, within the pH range of 6.6-7.5, the growth rate of SEC aggregates slowed as the pH increased. At pH 6.6, the high-molecular-weight protein increased to 5.53% on day 20, exceeding the established upper quality standard of 5%. At pH 6.7-7.5, the growth rate of SEC aggregates remained below 5%.

[0306] Furthermore, formulations F5-F11 were subjected to room temperature shaking studies with a shaking time of 72 hours and a shaking speed of 600 rpm. The results of the shaking experiments are shown in Table 15 below.

[0307] Table 15: SEC-HPLC aggregation change rate (days / %) of formulations F5 to F11 placed under room temperature shaking conditions for 72 hours

[0308] As shown in Table 15, after 72 hours of shaking, the SEC-aggregates of preparations F5-F11 only increased slightly, and the growth rate also slowed down with the increase of pH value. When the pH value of the preparation is 6.6-7.5, the shaking can prevent the protein from forming more aggregates.

[0309] To further evaluate the effects of different pH values ​​on protein conformational and colloidal stability, formulations F5-F11 were tested using the NanoTemper Prometheus NT.48 Protein Stability Analyzer using the ThermControl module to obtain protein denaturation temperatures (Tm and Tonset) and the onset temperature for protein aggregation (Tagg). The results are shown in Table 16 below.

[0310] Table 16: Tagg and Tm value test results of preparations F5 to F11

[0311] As shown in Table 16, at a pH of 6.6, Tonset, Tm, and Tagg values ​​were lower than those of the other groups, indicating poor stability. At pH values ​​of 6.7-7.5, Tonset and Tm1 values ​​increased with increasing pH, indicating improved thermal stability. At pH values ​​of 7.0-7.5, no aggregation temperature Tagg values ​​were detected, indicating good colloidal stability.

[0312] In summary, when the pH of the preparation is within the range of 6.7-7.5, the chemical stability and physical stability of the preparation are good.

[0313] 3. Investigation of injection solutions prepared with different surfactant types

[0314] 3.1 Low-concentration injection formulations A16-A19 were prepared using different surfactants. The final concentrations of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein, API) and the types and final concentrations of the excipients in the injections are detailed in Table 17. The injections were prepared by weighing the excipients and API according to the prescribed amounts in the table below and mixing them. If necessary, the pH of the formulation was adjusted to the appropriate value using 2% hydrochloric acid solution or 2% sodium hydroxide solution. Finally, water for injection was added to the volume. After preparation, the formulation was filtered in series using 0.45μm and 0.22μm PVDF filters, transferred into 1ml slender prefilled syringes, each filled with 0.8ml, and sealed with a rubber stopper. Formulations A16-A19 were then subjected to high-temperature stability studies. The SEC-HPLC stability test results at 40°C for 0 and 10 days are shown in Table 18, and the SEC-HPLC stability test results at 40°C for 10 days are shown in Table 19.

[0315] Table 17: Ratios of ingredients in formulations A16 to A19

[0316] Table 18: Stability results of formulations A16-A19 at high temperature (40°C)

[0317] Table 19: Change rate results of various indicators of formulations A16 to A19 at high temperature 40°C

[0318] As shown in Tables 18 and 19, different types of surfactants have little effect on SEC purity and aggregates, but have a greater impact on RP-HPLC purity. Comprehensive analysis of the average daily decrease rate of RP-HPLC purity (main peak + peak 4) and the average daily increase rate of RP-HPLC purity (total related substances) shows that polysorbate 80 or polysorbate 20 is more effective as a stabilizer, but the stability of the preparation is also within the acceptable standard range when poloxamer 188 is used as a stabilizer.

[0319] 3.2 High-concentration injection formulations F12 and F13 were prepared using different surfactants. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as the fusion protein, or API) in the injections and the types and final concentrations of the excipients are detailed in Table 20. The injections were prepared as follows: the excipients and API were weighed according to the table below and mixed. If necessary, the pH of the solution was adjusted to the appropriate value using 2% hydrochloric acid solution or 2% sodium hydroxide solution. Finally, the volume was adjusted to 15 ml with water for injection. After preparation, the solution was filtered in series using 0.45 μm and 0.22 μm PVDF filters, and the solution was transferred to 1 ml slender prefilled syringes, each filled with 0.5 ml and sealed with a rubber stopper. Formulations F12 and F13 were then subjected to high-temperature stability studies. The SEC-HPLC aggregation change rate (days / %) after 28 days at 40°C is shown in Table 21, and the appearance test results are shown in Table 22.

[0320] Table 20: Ratios of ingredients in formulations F12 and F13

[0321] Table 21: SEC-HPLC aggregation change rate results of formulations F12-F13 placed at 40°C for 28 days

[0322] Table 22: Appearance test results of formulations F12 and F13 after being placed at 40°C for 28 days

[0323] It can be seen from Tables 21 and 22 that the injections prepared with polysorbate 80 and polysorbate 20 can slow down the growth of aggregates and prevent the formation of visible particles, thereby having a good stabilizing effect on the fusion protein.

[0324] 4. Investigation of injections prepared with different types of osmotic pressure regulators

[0325] 4.1 Low-concentration injection formulations A23-A25 were prepared using different types of osmotic pressure regulators. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injections and the types and final concentrations of the excipients are detailed in Table 23. The injection preparation method is detailed in Step 2.1 of the Examples herein. Formulations A23-A25 were then subjected to a high-temperature stability study. The SEC-HPLC stability test results after 0, 7, and 14 days at 40°C are shown in Table 24.

[0326] Table 23: Ratios of ingredients in formulations A23 to A25

[0327] Table 24: Stability results of formulations A23-A25 at high temperature (40°C)

[0328] As shown in Table 24, the RP-HPLC purity (total related substances) of the preparation using mannitol increased the slowest compared to the combination of sodium chloride and sodium chloride and sucrose, and there was less visible foreign matter. Therefore, the preparation prepared using mannitol as an osmotic pressure regulator has better stability.

[0329] In addition, by testing the osmotic pressure values ​​of the low-concentration injection under the dosage of mannitol, they were all equal to or close to the osmotic pressure of human plasma. Among them, the osmotic pressure of preparation A12 batch preparation was 307mOsm / kg, the osmotic pressure of preparation A16 was 310mOsm / kg, the osmotic pressure of preparation A26 was 309mOsm / kg, and the osmotic pressure of preparation A29 batch preparation was 325mOsm / kg.

[0330] 4.2 Low-concentration injection formulations A34-A35 were prepared using different types of osmotic pressure regulators. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injections and the types and final concentrations of the excipients are detailed in Table 25. The injection preparation method is detailed in Step 2.1 of the Examples herein. Formulations A34-A35 were then subjected to a high-temperature stability study. The SEC-HPLC stability test results after 0, 7, and 14 days at 40°C are shown in Table 26.

[0331] Table 24: Ratios of ingredients in formulations A34-A35

[0332] Table 25: Stability results of formulations A34-A35 at high temperature (40°C)

[0333] The stability of preparations prepared using sucrose and trehalose as osmotic pressure regulators is also better.

[0334] 4.3 High-concentration injection formulations F14 and F15 were prepared using different osmotic pressure regulators. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as the fusion protein, or API) in the injections and the types and final concentrations of the excipients are detailed in Table 26. The injections were prepared as follows: the excipients and API were weighed according to the table below and mixed. If necessary, the pH of the solution was adjusted to the appropriate value using 2% hydrochloric acid solution or 2% sodium hydroxide solution. Finally, the volume was adjusted to 9 ml with water for injection. After preparation, the solution was filtered in series using 0.45 μm and 0.22 μm PVDF filters, and the solution was transferred to 1 ml slender prefilled syringes, each filled with 0.5 ml and sealed with a rubber stopper. Formulations F14 and F15 were then subjected to high-temperature stability studies. The SEC-HPLC aggregation change rate (days / %) after 30 days at 40°C is shown in Table 27, and the visual inspection results after 14 days at 40°C are shown in Table 28.

[0335] Table 26: Ratios of ingredients in formulations F14 and F15

[0336] Table 27: SEC-HPLC aggregation change rate results of formulations F14-F15 placed at 40°C for 30 days

[0337] Table 28: Appearance test results of formulations F14 and F15 after being placed at 40°C for 14 days

[0338] To further evaluate the effects of different osmotic pressure regulators on protein conformational and colloidal stability, formulations F14 and F15 were tested using the ThermControl module of a NanoTemper Prometheus NT.48 Protein Stability Analyzer to obtain protein denaturation temperatures (Tm and Tonset) and the onset temperature of protein aggregation (Tagg). The results are shown in Table 29 below.

[0339] Table 29: Tagg and Tm value test results of preparations F14-F15

[0340] As shown in Tables 27 to 29, mannitol or sucrose can maintain good colloidal stability and conformational stability of the dual-target fusion protein preparation, and can slow down the aggregation rate of the protein, preventing the protein from forming visible particles, and have a good stabilizing effect on the fusion protein.

[0341] 5. Investigation of injections prepared with different histidine dosages

[0342] High-concentration injection formulations F16-F18 were prepared using varying amounts of histidine. The final concentrations of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as the fusion protein, API) and the types and final concentrations of the excipients in the injections are detailed in Table 30. The injections were prepared by weighing the excipients and API according to the table below and mixing them. If necessary, the pH of the solution was adjusted to the appropriate value using 2% hydrochloric acid solution or 2% sodium hydroxide solution. Finally, the volume was adjusted to 10 ml with water for injection. After preparation, the solution was filtered in series using 0.45 μm and 0.22 μm PVDF filters, transferred into 1 ml slender prefilled syringes, each filled with 0.5 ml, and sealed with a rubber stopper. Formulations F16-F18 were then subjected to a high-temperature stability study. The SEC-HPLC results of the aggregation change rate (days / %) after 30 days at 40°C are shown in Table 31.

[0343] Table 30: Ratios of ingredients in formulations F16 to F18

[0344] Table 31: SEC-HPLC aggregation change rate results of formulations F16 to F18 placed at high temperature 40°C for 30 days

[0345] From the above, it can be seen that when the dosage of histidine is 10-20 mM, the growth rate of SEC-aggregates of the fusion protein is slow, and it has a good stabilizing effect on the fusion protein.

[0346] 6. Investigation of injections prepared with different polysorbate 80 dosages

[0347] 6.1 Low-concentration injection formulations A20-A22 and A32-A33 were prepared using varying amounts of polysorbate 80. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injections, as well as the types and final concentrations of the excipients, are detailed in Table 32. The injection preparation method is detailed in Step 2.1 of the Examples herein. Formulations A20-A22 and A32-A33 were then subjected to a high-temperature stability study. The SEC-HPLC stability test results after 40°C for 0, 7, 14, and 28 days are shown in Table 33.

[0348] Table 32: Ratios of ingredients in formulations A20-A22 and A32-A33

[0349] Table 33: Stability results of formulations A20-A22 and A32-A33 at high temperature (40°C)

[0350] Note: “ / ” means not tested.

[0351] As shown in Table 33, when the dosage of polysorbate 80 is in the range of 0.1 mg / ml to 0.5 mg / ml, the stability of the injection is better.

[0352] 6.2 High-concentration injection formulations F19-F25 were prepared using varying amounts of polysorbate 80. The final concentrations of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as the fusion protein, or API) and the types and final concentrations of the excipients in the injections are detailed in Table 34. Injection preparation method: Weigh the excipients and API according to the table below and mix. If necessary, adjust the pH of the solution to the appropriate value using 2% hydrochloric acid solution or 2% sodium hydroxide solution. Finally, add water for injection to a final volume of 15 ml. After preparation, filter the solution using 0.45 μm and 0.22 μm PVDF filters in series, transfer the solution into 1 ml slender prefilled syringes, fill each with 0.5 ml, and seal with a rubber stopper. Formulations F19-F25 were then subjected to a high-temperature stability study. The SEC-HPLC results of the aggregation change rate (days / %) after 28 days at 40°C are shown in Table 35. The results of the appearance test are shown in Table 36. The results of the insoluble particulate matter test are shown in Table 37.

[0353] Table 34: Ratios of ingredients in formulations F19 to F25

[0354] Table 35: SEC-HPLC aggregation change rate results of formulations F19 to F25 placed at high temperature 40°C for 28 days

[0355] Table 36: Appearance test results of formulations F19-F25 after being placed at 40°C for 28 days

[0356] Table 37: Results of insoluble particles test of formulations F19-F25 placed at 40°C for 28 days

[0357] From the above, it can be seen that when the dosage of polysorbate 80 is 0.4-1.3 mg / ml, it can maintain the stability of the dual-target fusion protein, the growth rate of protein aggregates is slow, the number of insoluble particles is small, and the protein is prevented from forming visible particles and the formation of insoluble particles is reduced.

[0358] 7. Investigation of injection solutions prepared with different fusion protein concentrations

[0359] 7.1 Low-concentration injection formulations A26 to A31 were prepared using different fusion protein concentrations. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injection and the types and final concentrations of each excipient are specifically shown in Table 38. The preparation method of the injection is specifically shown in Step 2.1 of the Examples of this application. Then, formulations A26 to A31 were subjected to a high-temperature stability study. The SEC-HPLC stability test results at high temperature of 40°C for 0 days, 7 days, 14 days, and 28 days are shown in Table 39. Among them, the high-temperature SEC-aggregate growth trend graph of the formulations of different concentrations is shown in Figure 1, and the high-temperature RP-HPLC-purity (total related substances) growth trend graph of the formulations of different concentrations is shown in Figure 2.

[0360] Table 38: Ratios of ingredients in formulations A26 to A31

[0361] Table 39: Stability results of formulations A26-A31 at high temperature 40°C

[0362] As can be seen from Table 39 and Figures 1 and 2, there is no significant difference in the growth rate of RP-HPLC-purity (total related substances) of preparations with different concentrations, and the growth rate of SEC-purity (aggregate) (%) increases with the increase of preparation concentration.

[0363] The long-term stability of formulation A29 (17 mg / ml) and formulation A30 (20 mg / ml) at 2-8°C was further investigated. The test results are shown in Table 40.

[0364] Table 40: Stability results of formulations A29-A30 at 2-8°C

[0365] Note: “ / ” means not tested.

[0366] As shown in Table 40, the RP-HPLC purity (main peak + peak 4) of the preparations of different concentrations did not change significantly with the change of storage time, and the SEC purity (aggregate) showed a slight increasing trend, but all were within the acceptable standard range (≤5%).

[0367] 7.2 Low-concentration injection formulations F26-F29 were prepared using different fusion protein concentrations. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injections and the types and final concentrations of the excipients are detailed in Table 41. The injection preparation method is detailed in Step 2.1 of the Examples of this application. Formulations F26-F29 were then subjected to a high-temperature stability study. SEC-HPLC stability testing was performed at 40°C for 0, 7, 14, and 28 days. The results showed a slight increase in SEC-purity (aggregate) for the formulations at different concentrations, but all were within the acceptable range (≤5%). This Example exemplifies the results for Formulation F26, as shown in Tables 42 and 43.

[0368] Table 41: Ratios of ingredients in formulations F26 to F29

[0369] Table 42: Change rate results of SEC-HPLC aggregates and RP-HPLC main peak purity at high temperature of 40°C for 28 days

[0370] Table 43: Appearance test results after being placed at 40°C for 28 days

[0371] 8. Effect of EDTA sodium salt on the stability of preparations under different buffer systems

[0372] 8.1 Low-concentration injection formulations A36-A37 containing EDTA in a citric acid system were prepared. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injections and the types and final concentrations of the excipients are detailed in Table 44. The injection preparation method is detailed in Step 2.1 of the Examples herein. Formulations A36-A37 were then subjected to a high-temperature stability study. The SEC-HPLC stability test results after 40°C for 0, 10, 14, and 28 days are shown in Table 45.

[0373] Table 44: Ratios of ingredients in formulations A36-A37

[0374] Table 45: Stability results of formulations A36-A37 at high temperature (40°C)

[0375] 8.2 High-concentration EDTA-containing injection formulations F30-F31 were prepared in a histidine system. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injections, as well as the types and final concentrations of the excipients, are detailed in Table 46. The injection preparation method is detailed in Step 2.1 of the Examples herein. Formulations F30-F31 were then subjected to a high-temperature stability study. The SEC-HPLC stability test results after 0, 7, 14, and 30 days at 40°C are shown in Table 47.

[0376] Table 46: Ratios of ingredients in formulations F30-F31

[0377] Table 47: Stability results of formulations F30-F31 at high temperature (40°C)

[0378] In summary, whether it is a citric acid system or a histidine buffer system, the stability of the preparation is better with or without the addition of EDTA sodium salt.

[0379] Example 2: Investigation of different fusion proteins

[0380] The amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 2 in this example is shown in SEQ ID NO: 2; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 3 in this example is shown in SEQ ID NO: 3; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 4 in this example is shown in SEQ ID NO: 4; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 5 in this example is shown in SEQ ID NO: 5; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 6 in this example is shown in SEQ ID NO: 6; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 7 in this example is shown in SEQ ID NO: 7; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 8 in this example is shown in SEQ ID NO: 8; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 9 in this example is shown in SEQ ID NO: NO:9; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 10 in this example is shown in SEQ ID NO:10; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 11 in this example is shown in SEQ ID NO:11; the amino acid sequence of the GLP-1-Fc-FGF21 dual-target fusion protein 12 in this example is shown in SEQ ID NO:12. GLP-1-Fc-FGF21 dual-target fusion proteins 2 to 11 (referred to as fusion proteins 2 to 11) were all prepared using conventional methods in the art.

[0381] 1. The type and final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injection, as well as the type and final concentration of each excipient, are detailed in Table 48. The preparation method for the injection is detailed in Step 2.1 of Example 1. Stability studies were then performed on formulations A38 to A31.

[0382] Table 48: Ratios of ingredients in formulations A38 to A31

[0383] 2. The type and final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (hereinafter referred to as fusion protein) in the injection, as well as the type and final concentration of each excipient, are detailed in Table 49. The preparation method for the injection is detailed in Step 2.1 of Example 1. Stability studies were then performed on formulations F32 to F53.

[0384] Table 49: Ratios of ingredients in formulations F32 to F53

[0385] Example 3: Investigation of different preservative systems

[0386] Low-concentration injection preparations were prepared using different preservative systems. The final concentration of the GLP-1-Fc-FGF21 dual-target fusion protein (abbreviated as fusion protein, or API) in the injection and the types and final concentrations of each excipient are detailed in Table 50. The injection preparation method is as follows: the excipients and API are weighed and mixed according to the prescription amount in Table 1. If necessary, the pH of the preparation is adjusted to the corresponding value using 2% hydrochloric acid solution or 2% sodium hydroxide solution, and finally, water for injection is added to the volume. After the preparation is completed, the drug solution is filtered in series using 0.45μm and 0.22μm PVDF filter membranes into 1ml slender prefilled syringes, each filled with 1ml, and sealed with a rubber stopper. The preparations were then subjected to stability studies. The results showed that all preparations exhibited good stability. The SEC-HPLC aggregation change rate (day / %) was within the acceptable standard range, and no visible particles were formed.

[0387] Table 50: Ratio of ingredients in each preparation

[0388] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0389] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A composition, characterized in that include: GLP-1-Fc-FGF21 fusion protein and buffer.

2. The composition according to claim 1, characterized in that The GLP-1-Fc-FGF21 fusion protein comprises the amino acid sequence shown in SEQ ID NOs: 1 to 12; Optionally, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 1 to 100 mg / mL; Optionally, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 80 mg / mL; Optionally, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 5 to 70 mg / mL; Optionally, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 55 mg / mL; Optionally, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 10 to 65 mg / mL; Optionally, the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 20 to 60 mg / mL; Optionally, the pH value of the composition is 6.2 to 8.0, preferably 6.4 to 8.

0.

3. The composition according to claim 2, characterized in that The buffer comprises at least one of a histidine buffer, a citrate buffer, a phosphate buffer and a Tris buffer; Preferably, the buffer comprises histidine buffer or citrate buffer; Optionally, the concentration of the buffer in the composition is 5 to 30 mM; Optionally, the concentration of the buffer in the composition is 5 to 25 mM; Optionally, the concentration of the buffer in the composition is 8 to 25 mM; Optionally, the concentration of the buffer in the composition is 5 to 20 mM; Optionally, the concentration of the buffer in the composition is 8 to 12 mM; Preferably, the concentration of the buffer in the composition is 10-20 mM.

4. The composition according to claim 1, characterized in that The composition further comprises at least one of an osmotic pressure regulator, a surfactant, a preservative, and a metal chelator; Optionally, the osmotic pressure regulator includes at least one of mannitol, sucrose, trehalose and sodium chloride; Optionally, the concentration of the osmotic pressure regulator in the composition is 30 to 100 mg / mL or 1 to 10 mg / mL; Optionally, the surfactant includes at least one of polysorbate 80, polysorbate 20 and poloxamer 188; Optionally, the concentration of the surfactant in the composition is 0.1 to 10 mg / mL, preferably 0.1 to 1.5 mg / mL or 6 to 10 mg / mL, more preferably 0.1 to 0.7 mg / mL; Optionally, the preservative is at least one of phenol and m-cresol; Optionally, the concentration of the preservative in the composition is 0.1 to 10 mg / mL, preferably 1.0 to 5.0 mg / mL; Optionally, the metal chelator is at least one of edetate disodium and edetate calcium sodium; Optionally, the concentration of the metal chelator in the composition is 0.01 to 1 mg / mL, preferably 0.05 to 0.5 mg / mL.

5. The composition according to any one of claims 1 to 4, characterized in that The buffer is a histidine buffer, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, or 25 to 85 mg / mL; Optionally, the buffer is a histidine buffer, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 25 to 80 mg / mL, preferably 25 to 55 mg / mL; Optionally, the concentration of the histidine buffer in the composition is 5 to 30 mM, preferably 5 to 25 mM, 8 to 25 mM, 8 to 20 mM, more preferably 10 to 20 mM, 8 to 12 mM; Optionally, the pH value of the composition is 6.7 to 7.5, preferably 6.9 to 7.5; Optionally, the osmotic pressure regulator is mannitol, and the concentration of the osmotic pressure regulator in the composition is 30 to 50 mg / mL; or The osmotic pressure regulator is sucrose, and the concentration of the osmotic pressure regulator in the composition is 60 to 100 mg / mL, preferably 80 to 100 mg / mL; or The osmotic pressure regulator is trehalose, and the concentration of the osmotic pressure regulator in the composition is 40 to 100 mg / mL, preferably 40 to 70 mg / mL, and more preferably 50 to 70 mg / mL; Optionally, the surfactant is polysorbate 80, and the concentration of the surfactant in the composition is 0.1 to 2 mg / mL, preferably 0.1 to 1.5 mg / mL, preferably 0.4 to 1.3 mg / mL; or The surfactant is polysorbate 20, and the concentration of the surfactant in the composition is 0.1 to 1.5 mg / mL, preferably 0.1 to 0.5 mg / mL.

6. The composition according to any one of claims 1 to 4, characterized in that The concentration of the GLP-1-Fc-FGF21 fusion protein in the composition is 2 to 25 mg / mL, and the buffer is a citrate buffer; Optionally, the concentration of the citrate buffer in the composition is 5 to 30 mM, preferably 5 to 25 mM, 8 to 25 mM, 8 to 20 mM, more preferably 10 to 20 mM, 8 to 12 mM; Optionally, the pH value of the composition is 6.4 to 8.0; Optionally, the osmotic pressure regulator is mannitol, and the concentration of the osmotic pressure regulator in the composition is 30 to 50 mg / mL; or The osmotic pressure regulator is sucrose, and the concentration of the osmotic pressure regulator in the composition is 60 to 100 mg / mL, preferably 80 to 100 mg / mL; or The osmotic pressure regulator is trehalose, and the concentration of the osmotic pressure regulator in the composition is 40 to 100 mg / mL, preferably 40 to 70 mg / mL, and more preferably 50 to 70 mg / mL; The osmotic pressure regulator is sodium chloride, and the concentration of the osmotic pressure regulator in the composition is 1 to 10 mg / mL, preferably 3 to 9 mg / mL; Optionally, the surfactant comprises at least one of polysorbate 80, polysorbate 20 and poloxamer 188, preferably polysorbate 80; Optionally, the concentration of the surfactant in the composition is 0.1 to 10 mg / mL, preferably 0.1 to 1.5 mg / mL, 0.1 to 1.0 mg / mL or 6 to 10 mg / mL; Optionally, the surfactant is poloxamer 188, and the concentration of the surfactant in the composition is 6 to 10 mg / mL; or The surfactant is polysorbate 80 or polysorbate 20, and the concentration of the surfactant in the composition is 0.1 to 1.5 mg / mL, preferably 0.1 to 0.5 mg / mL.

7. The composition according to any one of claims 1 to 4, characterized in that The dosage form of the composition is a liquid preparation, preferably an injection.

8. An injection, characterized in that: include: GLP-1-Fc-FGF21 fusion protein, histidine buffer, mannitol and optionally edetate disodium or edetate sodium calcium, and polysorbate 80 or polysorbate 20; The GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, 25-50 mg / mL; The concentration of the histidine buffer in the injection solution is 8-25 mM, preferably 10-20 mM; The concentration of mannitol in the injection is 35-45 mg / mL; The concentration of the polysorbate 80 in the injection is 0.4 to 1.5 mg / mL, or 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; The concentration of the metal chelator in the injection is 0.05-0.5 mg / mL; The pH value of the injection solution is 6.9 to 7.5; Preferably, the injection does not contain edetate disodium or edetate calcium sodium.

9. An injection, characterized in that: include: GLP-1-Fc-FGF21 fusion protein, Histidine buffer, Sucrose or trehalose, Polysorbate 80 or polysorbate 20, and Optionally edetate disodium or edetate calcium sodium; The GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, 25 to 50 mg / mL; The concentration of the histidine buffer in the injection solution is 8-25 mM, preferably 10-20 mM; The concentration of sucrose in the injection solution is 85-95 mg / mL; The concentration of trehalose in the injection solution is 55-65 mg / mL; The concentration of the polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; The pH value of the injection solution is 6.9 to 7.5; Preferably, the injection does not contain edetate disodium or edetate calcium sodium.

10. An injection, characterized in that: include: GLP-1-Fc-FGF21 fusion protein, citrate buffer, sucrose or trehalose, and optionally edetate disodium or edetate sodium calcium, and polysorbate 80 or polysorbate 20; The GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, 25 to 50 mg / mL; The concentration of the citrate buffer in the injection solution is 8-25 mM, preferably 10-20 mM; The concentration of sucrose in the injection solution is 85-95 mg / mL; The concentration of trehalose in the injection solution is 55-65 mg / mL; The concentration of the polysorbate 80 in the injection is 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; The pH value of the injection solution is 6.9 to 7.5; Preferably, the injection does not contain edetate disodium or edetate calcium sodium.

11. An injection, characterized in that: include: GLP-1-Fc-FGF21 fusion protein, citrate buffer, mannitol, and optionally edetate disodium or edetate sodium calcium, and polysorbate 80 or polysorbate 20; The GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection is 25 to 100 mg / mL, preferably 25 to 95 mg / mL, 25 to 90 mg / mL, 25 to 85 mg / mL, 25 to 80 mg / mL, 25 to 75 mg / mL, 25 to 70 mg / mL, 25 to 65 mg / mL, 25 to 60 mg / mL, 25 to 55 mg / mL, 25 to 50 mg / mL; The concentration of the citrate buffer in the injection solution is 8 to 25 mM, preferably 10 to 20 mM; The concentration of mannitol in the injection is 35-45 mg / mL; The concentration of the polysorbate 80 in the injection is 0.4 to 1.5 mg / mL, or 0.4 to 1.3 mg / mL, or the concentration of the polysorbate 20 in the injection is 0.1 to 0.5 mg / mL; The concentration of the metal chelator in the injection is 0.05-0.5 mg / mL; The pH value of the injection solution is 6.9 to 7.5; Preferably, the injection does not contain edetate disodium or edetate calcium sodium.

12. An injection, characterized in that: include: GLP-1-Fc-FGF21 fusion protein, citrate buffer, mannitol, polysorbate 80, and optionally edetate disodium or edetate calcium sodium; The GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection solution is 3 to 25 mg / mL; The concentration of the citrate buffer in the injection solution is 5 to 20 mM, preferably 8 to 20 mM, preferably 10 to 15 mM; The concentration of mannitol in the injection is 40-50 mg / mL; The concentration of polysorbate 80 in the injection is 0.1 to 1.5 mg / mL; The concentration of the metal chelator in the injection is 0.05-0.5 mg / mL; The pH value of the injection is 6.4 to 8.0; Preferably, the injection does not contain edetate disodium or edetate calcium sodium.

13. An injection, characterized in that: include: GLP-1-Fc-FGF21 fusion protein, citrate buffer and polysorbate 80, and sucrose or trehalose, and optionally edetate disodium or edetate calcium sodium; The GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection solution is 3 to 25 mg / mL; The concentration of the citrate buffer in the injection is 5 to 20 mM, preferably 8 to 20 mM, more preferably 10 to 15 mM; The concentration of sucrose in the injection solution is 85-95 mg / mL; The concentration of trehalose in the injection solution is 55-65 mg / mL; The concentration of polysorbate 80 in the injection is 0.1 to 1.5 mg / mL; The pH value of the injection is 6.4 to 8.0; Preferably, the injection does not contain edetate disodium or edetate calcium sodium.

14. An injection, characterized in that: include: GLP-1-Fc-FGF21 fusion protein, histidine buffer, mannitol, polysorbate 80, and optionally edetate disodium or edetate calcium sodium; The GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection solution is 3 to 25 mg / mL; The concentration of the histidine buffer in the injection solution is 5-30 mM, preferably 10-20 mM; The concentration of mannitol in the injection is 40-50 mg / mL; The concentration of polysorbate 80 in the injection is 0.1 to 1.5 mg / mL; The concentration of the metal chelator in the injection is 0.05-0.5 mg / mL; The pH value of the injection is 6.4 to 8.0; Preferably, the injection does not contain edetate disodium or edetate calcium sodium.

15. An injection, characterized in that: include: GLP-1-Fc-FGF21 fusion protein, histidine buffer and polysorbate 80, and sucrose or trehalose, and optionally edetate disodium or edetate calcium sodium; The GLP-1-Fc-FGF21 fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NOs: 1 to 12, and the concentration of the GLP-1-Fc-FGF21 fusion protein in the injection solution is 3 to 25 mg / mL; The concentration of the histidine buffer in the injection solution is 5-30 mM, preferably 10-20 mM; The concentration of sucrose in the injection solution is 85-95 mg / mL; The concentration of trehalose in the injection solution is 55-65 mg / mL; The concentration of polysorbate 80 in the injection is 0.1 to 1.5 mg / mL; The pH value of the injection is 6.4 to 8.0; Preferably, the injection does not contain edetate disodium or edetate calcium sodium.

16. The injection according to any one of claims 8 to 15, characterized in that: Also contains preservatives.

17. The injection according to claim 16, characterized in that: The preservative is at least one selected from phenol, m-cresol, benzyl alcohol, and phenoxyethanol; Optionally, the concentration of the phenol in the composition or injection is 0 to 7 mg / mL, preferably 3 to 7 mg / mL; Optionally, the concentration of m-cresol in the composition or injection is 0-4 mg / mL, preferably 2-4 mg / mL.

18. Use of the composition according to any one of claims 1 to 7 or the injection according to any one of claims 8 to 17 in the preparation of a medicament for treating and / or preventing metabolic diseases; Optionally, the metabolic diseases include diabetes, hyperlipidemia, obesity, and diseases associated with fatty liver; Optionally, the fatty liver-related diseases include non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

19. A method for treating and / or preventing metabolic diseases, the method comprising administering to a subject the composition of any one of claims 1 to 7 or the injection of any one of claims 8 to 17 for use in preparing a medicament for treating and / or preventing metabolic diseases; Optionally, the metabolic diseases include diabetes, hyperlipidemia, obesity, and diseases associated with fatty liver; Optionally, the fatty liver-related diseases include non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

20. The composition according to any one of claims 1 to 7 or the injection according to any one of claims 8 to 17 is used for treating and / or preventing metabolic diseases; Optionally, the metabolic diseases include diabetes, hyperlipidemia, obesity, and diseases associated with fatty liver; Optionally, the fatty liver-related diseases include non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, liver fibrosis and liver cirrhosis.

21. An injection device or container, characterized in that include: The composition according to any one of claims 1 to 7 or the injection according to any one of claims 8 to 17; Optionally, the injection device or container comprises at least one selected from a container bottle, a vial, a cartridge and a syringe; Preferably, the syringe comprises at least one selected from an autoinjector and a pre-filled syringe.

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