Pharmaceutically stable solution formulation with GIPR antibody fusion protein and use thereof

Abstract

Disclosed in the present invention is a pharmaceutically stable solution formulation with a GIPR antibody fusion protein, comprising a therapeutically effective amount of the GIPR antibody fusion protein; a pharmaceutically acceptable pH buffer at 1-500 mM, with a pH buffering range of 4.0-9.0; a first pharmaceutically acceptable protective agent at 1-500 mM; and a second pharmaceutically acceptable protective agent at 1-500 mM. The pharmaceutically stable solution formulation further comprises one or more pharmaceutically acceptable antioxidants at 1-500 mM, and 0.01%-10% (w / v) of one or more pharmaceutically acceptable surfactants. The pharmaceutically stable solution formulation with the GIPR antibody fusion protein does not need to be diluted, and can be used for subcutaneous injection administration. Also provided herein is use of the pharmaceutically stable solution formulation with the GIPR antibody fusion protein in treating, preventing, or ameliorating diseases or symptoms such as overweight or obesity, type 2 diabetes, and non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

Description

Pharmaceutically stable solution formulation of a GIPR antibody fusion protein and uses thereof INVENTION

[0001] A pharmaceutically stable solution formulation of a GIPR antibody fusion protein is disclosed. BACKGROUND

[0002] Obesity is caused by long-term energy intake exceeding energy consumption, leading to excessive accumulation and / or abnormal distribution of fat in the body, usually accompanied by weight gain. In the 2016 WHO report, it is estimated that there are about 1.9 billion overweight adults (18 years and older) and about 650 million obese adults in the world; more than 100 million children are obese. According to the statistics of the Centers for Disease Control and Prevention (CDC), the incidence of obesity in the United States increased from 30.5% to 42.4% from 1999-2000 to 2017-2018, and the incidence of severe obesity increased from 4.7% to 9.2%.

[0003] Overweight and obesity can lead to serious health consequences, often accompanied by a surge in the risk of other metabolic diseases, including type 2 diabetes, nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). In addition, the incidence of complications such as hypertension, dyslipidemia, cardiovascular disease, sleep apnea syndrome, etc. will also increase, seriously affecting the health and quality of life of patients.

[0004] The main treatment measures for overweight and obesity include behavior intervention, diet adjustment, drug treatment and surgical treatment. Currently, several weight loss drugs have been marketed at home and abroad (including orlistat tablets (Xenical), phentermine and topiramate extended-release combination (Qsymia), naltrexone hydrochloride and bupropion hydrochloride extended-release tablets (Contrave), liraglutide injection (Saxenda), setmelanotide injection (Imcivree) and semaglutide injection (Wegovy), tizepatide), among which orlistat tablets (Xenical) and tizepatide have been approved for marketing in China.

[0005] Due to the safety problems of marketed weight loss drugs, inconvenient administration (such as liraglutide which needs to be administered once a day), high price (such as semaglutide), etc., there is a huge unmet need in clinical practice, and there is an urgent need to develop new safe, efficient and convenient weight loss drugs.

[0006] Long-acting glucagon-like peptide-1 receptor (GLP-1R) agonists are considered as promising next-generation weight loss drugs. Glucagon-like peptide-1 (GLP-1) is an incretin secreted by the small intestinal epithelial L-cells in response to food stimuli. Endogenous GLP-1 is rapidly degraded by dipeptidyl peptidase 4 (DPP-4) with a half-life of about 2 minutes. GLP-1 receptors are mainly expressed on the surface of pancreatic beta cells, pancreatic alpha cells, and cells in the intestinal tract and central nervous system (CNS). GLP-1 can promote insulin secretion and inhibit glucagon secretion in a glucose-dependent manner, and can also inhibit gastric emptying, gastric acid secretion, and gastric motility, leading to appetite reduction and food intake reduction. Long-acting GLP-1 receptor agonists are also the best therapeutic drugs for type 2 diabetes mellitus (Tomlinson et al., 2015, Expert Opin. Investig. Drugs 25: 1744-7658; Gallwitz, 2015, Eur. Endocr. 11: 21-25). At the same time, long-acting GLP-1 drugs are also being tried in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD). The results show that they have a significant effect on improving liver tissue morphology, down-regulating the ratio of glutamic-pyruvic transaminase / glutamic-oxaloacetic transaminase, and reducing liver fat content in patients with NAFLD (Samson et al., 2013, J. Diabetes Complications 27: 401-6; Portillo-Sanchez and Cusi, 2016, Clin. Diabetes Endocrinol. 2: 9).

[0007] Gastric Inhibitory Polypeptide (GIP) is an incretin secreted from K cells in the upper small intestine in response to food intake (Tseng et al., 1996, J. Clin. Invest. 98:2440-2445; Ravn et al., 2013, J. Biol. Chem. 288:19760-72). Gastric Inhibitory Polypeptide Receptor (GIPR) is distributed in pancreatic beta cells, adipose tissue, and central nervous system, etc. (Peter et al., 2013, J. Biol. Chem. 288:19760-72). Experimental evidence shows that GIP / GIPR signaling pathway is closely related to fat metabolism in at least these tissues (Yip and Wolfe, 2000, Life Sci. 66:91-103). Experimental data also show that the circulating GIP concentration in obese or diabetic patients is elevated (Flatt et al., 1984, J. Endocrinol. 101:249-256; Rask-Flot et al., 2003, J. Clin. Endocrinol. Metab. 88:2706-2713). After blocking GIPR signaling using GIPR inhibitors, a significant decrease in body weight and a reduction in insulin resistance symptoms in high-fat diet-induced obese mice can be observed, and even the type 2 diabetes caused by high-fat diet is reversed (Ravn et al., 2013, J. Biol. Chem. 288:19760-72). etc. (Peter et al., 2013, J. Biol. Chem. 288:19760-72). Experimental evidence shows that GIP / GIPR signaling pathway is closely related to fat metabolism in at least these tissues (Yip and Wolfe, 2000, Life Sci. 66:91-103). Experimental data also show that the circulating GIP concentration in obese or diabetic patients is elevated (Flatt et al., 1984, J. Endocrinol. 101:249-256; Rask-Flot et al., 2003, J. Clin. Endocrinol. Metab. 88:2706-2713). After blocking GIPR signaling using GIPR inhibitors, a significant decrease in body weight and a reduction in insulin resistance symptoms in high-fat diet-induced obese mice can be observed, and even the type 2 diabetes caused by high-fat diet is reversed (Ravn et al., 2013, J. Biol. Chem. 288:19760-72).

[0008] GIPR antibody fusion protein fuses a long-acting GLP-1R agonist with a GIPR antagonist, which can achieve the effect of simultaneously improving insulin resistance and excessive accumulation of fat (i.e., obesity). That is, by the GLP-1 part to improve glucose metabolism, reduce appetite, and reduce weight; by the GIPR antibody part to reduce further accumulation of fat and improve liver function. The fat-reducing effect of the GIPR antibody part and the weight-reducing effect of the GLP-1 part are superimposed, and the patient suffering from one or more of obesity, type 2 diabetes, nonalcoholic fatty liver disease, or nonalcoholic steatohepatitis is treated by dual action.

[0009] In order to fully meet the needs of patients and increase compliance for its clinical indications, the pharmaceutical preparation of GIPR antibody fusion protein should be in the form of a solution suitable for subcutaneous injection as the first choice. Due to the high sensitivity of GIPR antibody fusion protein to various environmental factors (e.g., temperature, silicone oil, oxygen, etc.), its uniformity and biological activity are very susceptible to the influence during long-term storage, which in turn affects the therapeutic effect and practical application, so the known common solution dosage form cannot meet its requirements.

[0010] After intensive studies, the present inventors have found that a pharmaceutical stable solution formulation of a GIPR antibody fusion protein, which can maintain stability for a long period of time, can be prepared by using a specific buffer and a specific surfactant. SUMMARY

[0011] In one aspect, provided herein is a pharmaceutical stable solution formulation of a GIPR antibody fusion protein, which can maintain stability for a long period of time under suitable storage conditions, and has an effect of increasing the in vivo half-life of the contained GIPR antibody fusion protein.

[0012] In some embodiments, provided herein is a pharmaceutical stable solution formulation of a GIPR antibody fusion protein, which comprises a GLP-1 peptide and a GIPR antibody moiety, wherein the GLP-1 peptide and the GIPR antibody moiety are linked, optionally directly linked or linked via a linker, and the GIPR antibody fusion protein has one or more of the following characteristics:

[0013] a) the GLP-1 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 2, or a derivative, variant, fragment, and mutant of the amino acid sequence;

[0014] b) the GIPR antibody moiety comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or SEQ ID NO: 4, or a derivative, variant, fragment, and mutant of the amino acid sequence;

[0015] c) the GIPR antibody moiety comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, or a derivative, variant, fragment, and mutant of the amino acid sequence;

[0016] d) the GIPR antibody fusion protein is configured in such a manner that a) is fused to the N-terminus of b) or c) or both b) and c) simultaneously by a linker comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12; or a) is directly fused to the N-terminus of b) or c) or both b) and c) simultaneously without a linker;

[0017] e) the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO: 12;

[0018] f) the GLP-1 peptide is linked to the N-terminus of the light chain and / or to the N-terminus of the heavy chain of the GIPR antibody moiety; or

[0019] g) the GIPR antibody fusion protein comprises a combination of amino acid sequences selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0020] In some embodiments, in the GIPR antibody fusion protein provided herein, the ratio between the number of the GLP-1 and the number of the GIPR antibody is 2:1 or 4:1.

[0021] In some embodiments, the use of the pharmaceutical stable solution formulation of the GIPR antibody fusion protein in the treatment, prevention or amelioration of overweight or obesity and its related conditions, type 2 diabetes and its related conditions, and non-alcoholic fatty liver disease or non-alcoholic steatohepatitis and other diseases or symptoms is also provided herein.

[0022] The pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein aims to solve the problems of impaired uniformity and biological activity and less than ideal in vivo half-life of the GIPR antibody fusion protein during long-term storage. After in-depth research on how to improve the in vitro and in vivo stability of the pharmaceutical stable solution formulation of the GIPR antibody fusion protein, from among pharmaceutically acceptable excipients, such as inorganic salts, basic compounds, buffer systems, sugars, amino acids and their derivatives, surfactants, etc., components with suitable physicochemical properties are selected, and a suitable pH buffer range is selected to form the pharmaceutical stable solution formulation of the GIPR antibody fusion protein described herein. Among them, the creative combination of pH buffers, protective agents, antioxidants and surfactants made by the present application has a particularly remarkable effect, which improves the long-term stability of the GIPR antibody fusion protein during storage, and is the core content of the solvent formulation formula described herein. In the research process, the melting temperature and solubility were selected as the indexes, and the specific research methods are described in the examples, but these examples should not be regarded as a limitation of the present patent.

[0023] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0024] wherein said first pharmaceutically acceptable protective agent and said second pharmaceutically acceptable protective agent are independently selected from the group consisting of arginine, arginine hydrochloride, histidine, histidine hydrochloride, cystine, cysteine, methionine, aspartic acid, lysine, glycine, tryptophan, leucine, isoleucine, phenylalanine, threonine, glutamic acid, serine, and derivatives thereof, sucrose, trehalose, sorbitol, and mannitol. Preferably, said first pharmaceutically acceptable protective agent and said second pharmaceutically acceptable protective agent are independently selected from the group consisting of a) arginine or arginine hydrochloride, b) histidine or histidine hydrochloride, c) methionine or a derivative thereof, and d) aspartic acid or a derivative thereof. More preferably, said first pharmaceutically acceptable protective agent and said second pharmaceutically acceptable protective agent are selected from the group consisting of a) arginine or arginine hydrochloride; and b) aspartic acid or a derivative thereof. Even more preferably, said first pharmaceutically acceptable protective agent and said second pharmaceutically acceptable protective agent are selected from the group consisting of a) arginine or arginine hydrochloride; and b) aspartic acid or a derivative thereof, wherein said arginine or arginine hydrochloride is present in said formulation at a concentration of 1-500 mM, optionally 1-400 mM, 1-300 mM, 1-200 mM, 1-100 mM, 75-120 mM, 75-175 mM, or 100-200 mM; and said aspartic acid or a derivative thereof is present in said formulation at a concentration of 1-500 mM, optionally 1-400 mM, 1-300 mM, 1-200 mM, 1-100 mM, 100-200 mM, 1-28 mM, or 1-50 mM.

[0025] In some embodiments, in the pharmaceutically stable solution formulation of the GIPR antibody fusion proteins provided herein, wherein said pharmaceutically acceptable pH buffering agent is selected from the group consisting of histidine, histidine hydrochloride, arginine, arginine hydrochloride, aspartic acid, sodium hydroxide, potassium hydroxide, sodium phosphate monobasic, alkaline compounds of sodium phosphate dibasic, phosphate, acetate, citrate, barbiturate, tris, borate, histidate, and succinate. Preferably, said pharmaceutically acceptable pH buffering agent comprises histidine or histidine hydrochloride, and is present in said formulation at a concentration of 1-500 mM, optionally 1-400 mM, 1-300 mM, 1-200 mM, 1-140 mM, 1-100 mM, 100-200 mM, 25-100 mM, or 1-50 mM, and provides a pH buffering range of pH 4.0-9.0, optionally, pH 5.0-8.0, pH 5.5-7.5, or pH 6.0-7.0.

[0026] In some embodiments, in the pharmaceutically stable solution formulation of the GIPR antibody fusion proteins provided herein, the pharmaceutically acceptable antioxidant is selected from one or more of the group consisting of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, diethylenetriaminepentaacetic acid, methionine, cysteine and its derivatives, and amino acid or metal chelators. Preferably, the pharmaceutically acceptable antioxidant is present in the formulation at a concentration of 1-500 mM, alternatively 1-400 mM, 1-300 mM, 1-200 mM, 1-100 mM, or 1-50 mM. More preferably, the pharmaceutically acceptable antioxidant comprises methionine, which is present in the formulation at a concentration of 1-500 mM, alternatively 1-400 mM, 1-300 mM, 1-200 mM, 1-100 mM, 1-50 mM, or 1-20 mM.

[0027] In some embodiments, in the pharmaceutically stable solution formulation of the GIPR antibody fusion proteins provided herein, the first pharmaceutically acceptable protective agent and the second pharmaceutically acceptable protective agent are, respectively, a) arginine or arginine hydrochloride; and b) aspartic acid or its derivatives; the pH buffer comprises histidine or a histidine salt (alternatively, histidine hydrochloride), and the antioxidant comprises methionine.

[0028] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion proteins provided herein further comprises one or more pharmaceutically acceptable surfactants, optionally, the surfactant is selected from one or more of the group consisting of polysorbate (optionally, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and polyoxyethylene sorbitan fatty acid ester), poloxamer (optionally, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407), N,N-dimethylacetamide, hydroxypropyl-beta-cyclodextrin, and polyoxyethylene polyoxypropylene ether block copolymer. Preferably, the pharmaceutically acceptable surfactant is present in the formulation at a concentration of 0.01-10% (w / v), optionally, 0.01-9%, 0.01-8%, 0.01-7%, 0.01-6%, 0.01-5%, 0.01-4%, 0.01-3%, 0.01-2%, 0.01-1%, 0.01-0.8%, 0.01-0.6%, 0.01-0.5%, 0.01-0.4%, 0.01-0.3%, 0.01-0.25%, 0.025-0.25%, 0.05-0.25%, 0.1-0.25%, or 0.15-0.25% (w / v).More preferably, the pharmaceutically acceptable surfactant combination comprises a poloxamer, and hydroxypropyl-beta-cyclodextrin or N,N-dimethylacetamide, wherein the poloxamer is present in the formulation in an amount ranging from 0.01% to 10% (w / v), optionally, 0.01% to 9%, 0.01% to 8%, 0.01% to 7%, 0.01% to 6%, 0.01% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, 0.01% to 0.8%, 0.01% to 0.6%, 0.01% to 0.5%, 0.01% to 0.4%, 0.01% to 0.3%, 0.01% to 0.25%, 0.025% to 0.25%, 0.05% to 0.25%, 0.1% to 0.25%, or 0.15% to 0.25% (w / v); the hydroxypropyl-beta-cyclodextrin is present in the formulation in an amount ranging from 0.01% to 10% (w / v), optionally, 0.01% to 9%, 0.01% to 8%, 0.01% to 7%, 0.01% to 6%, 0.01% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, 0.01% to 0.8%, 0.01% to 0.6%, 0.01% to 0.5%, 0.01% to 0.4%, 0.01% to 0.3%, 0.01% to 0.25%, 0.025% to 0.25%, 0.05% to 0.25%, 0.1% to 0.25%, or 0.15% to 0.25% (w / v); and the N,N-dimethylacetamide is present in the formulation in an amount ranging from 0.01% to 10% (w / v), optionally, 0.01% to 9%, 0.01% to 8%, 0.01% to 7%, 0.01% to 6%, 0.01% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, 0.01% to 0.8%, 0.01% to 0.6%, 0.01% to 0.5%, 0.01% to 0.4%, 0.01% to 0.3%, 0.01% to 0.25%, 0.025% to 0.25%, 0.05% to 0.25%, 0.1% to 0.25%, or 0.15% to 0.25% (w / v).

[0029] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0030] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0031] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0032] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0033] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0034] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0035] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0036] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0037] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0038] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0039] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0040] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0041] In some embodiments, the formulation has a pH of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0; preferably, the formulation has a pH of 6.4. In some embodiments, the arginine is present in the formulation at a concentration ranging from 100-105mM, 100-110mM, 100-115mM, 100-120mM, or 110-120mM; preferably, the arginine is present in the formulation at a concentration ranging from 110-120mM. In some embodiments, the aspartic acid is present in the formulation at a concentration ranging from 25-26mM, 25-27mM, 25-28mM, 25-29mM, or 25-30mM; preferably, the aspartic acid is present in the formulation at a concentration ranging from 25-28mM. In some embodiments, the methionine is present in the formulation at a concentration ranging from 1-5mM, 1-10mM, 1-15mM, 1-20mM, or 15-20mM; preferably, the methionine is present in the formulation at a concentration ranging from 15-20mM.

[0042] In some embodiments, the pharmaceutical stable solution formulation of a GIPR antibody fusion protein provided herein comprises the following components:

[0043] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0044] In some embodiments, the pharmaceutical stable solution formulation of a GIPR antibody fusion protein provided herein comprises the following components:

[0045] In some embodiments, the pharmaceutical stable solution formulation of a GIPR antibody fusion protein provided herein comprises the following components:

[0046] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0047] In some embodiments, the pharmaceutical stable solution formulation of a GIPR antibody fusion protein provided herein comprises the following components:

[0048] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0049] In some embodiments, the formulation has a pH of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0; preferably, the formulation has a pH of 6.4. In some embodiments, the arginine has a concentration in the formulation ranging from 100-105mM, 100-110mM, 100-115mM, 100-120mM, or 110-120mM; preferably, the arginine has a concentration in the formulation ranging from 110-120mM. In some embodiments, the aspartic acid has a concentration in the formulation ranging from 25-26mM, 25-27mM, 25-28mM, 25-29mM, or 25-30mM; preferably, the aspartic acid has a concentration in the formulation ranging from 25-28mM. In some embodiments, the methionine has a concentration in the formulation ranging from 1-5mM, 1-10mM, 1-15mM, 1-20mM, or 15-20mM; preferably, the methionine has a concentration in the formulation ranging from 15-20mM. In some embodiments, the N,N-dimethylacetamide has a concentration in the formulation ranging from 0.05%-1% (w / v), 0.1%-1% (w / v), 0.15%-1% (w / v), 0.2%-1% (w / v), 0.25%-1% (w / v), 0.3%-1% (w / v), 0.35%-1% (w / v), 0.4%-1% (w / v), 0.45%-1% (w / v), or 0.5%-1% (w / v); preferably, the N,N-dimethylacetamide has a concentration in the formulation ranging from 0.5%-1% (w / v).

[0050] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0051] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of: SEQ ID NO: 13 and SEQ ID NO: 8.

[0052] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0053] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of: SEQ ID NO: 13 and SEQ ID NO: 8.

[0054] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0055] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0056] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0057] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0058] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0059] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0060] wherein preferably the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO: 8.

[0061] In some embodiments, the formulation has a pH of 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0; preferably, the formulation has a pH of 6.4. In some embodiments, the arginine has a concentration in the formulation ranging from 100-105mM, 100-110mM, 100-115mM, 100-120mM, or 110-120mM; preferably, the arginine has a concentration in the formulation ranging from 110-120mM. In some embodiments, the aspartic acid has a concentration in the formulation ranging from 25-26mM, 25-27mM, 25-28mM, 25-29mM, or 25-30mM; preferably, the aspartic acid has a concentration in the formulation ranging from 25-28mM. In some embodiments, the methionine has a concentration in the formulation ranging from 1-5mM, 1-10mM, 1-15mM, 1-20mM, or 15-20mM; preferably, the methionine has a concentration in the formulation ranging from 15-20mM. In some embodiments, the hydroxypropyl-β-cyclodextrin has a concentration in the formulation ranging from 1%-5% (w / v), 2%-5% (w / v), 3%-5% (w / v), or 4%-5% (w / v); preferably, the hydroxypropyl-β-cyclodextrin has a concentration in the formulation ranging from 4%-5% (w / v).

[0062] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components:

[0063] wherein preferably, the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of: SEQ ID NO: 13 and SEQ ID NO: 8.

[0064] In some embodiments above, the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of: SEQ ID NO: 13 and SEQ ID NO: 8; preferably, the GIPR antibody fusion protein has a concentration in the formulation of 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, or 140 mg / mL; more preferably, the GIPR antibody fusion protein has a concentration in the formulation of 20 mg / mL, 40 mg / mL, 80 mg / mL, 120 mg / mL, or 140 mg / mL.

[0065] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein has a pH ranging from 5.0 to 8.0; preferably, the pH ranges from 5.0 to 7.0 or 6.0 to 7.0; more preferably, the pH ranges from 6.0 to 7.0.

[0066] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein comprises the following components: GIPR antibody fusion protein 1-200 mg / mL; pharmaceutically acceptable pH buffer 1-500 mM, pH buffer range 4.0-9.0; first pharmaceutically acceptable protective agent 1-500 mM; second pharmaceutically acceptable protective agent 1-500 mM, the pharmaceutical stable solution formulation further comprises pharmaceutically acceptable antioxidant 1-500 mM; and one or more pharmaceutically acceptable surfactants 0.01%-10% (w / v). The pharmaceutical stable solution formulation of the GIPR antibody fusion protein disclosed in the present application does not need to be diluted and is suitable for subcutaneous injection administration. The pharmaceutical stable solution formulation of the GIPR antibody fusion protein can be kept stable for a long time under suitable storage conditions and has the effect of increasing the in vivo half-life of the GIPR antibody fusion protein, and can be effectively used for the treatment, prevention or improvement of diseases such as overweight or obesity, type 2 diabetes, and non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

[0067] In some embodiments, the present application provides a pre-filled syringe or a vial, characterized in that it comprises the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein.

[0068] In some embodiments, the pre-filled syringe or the vial described above, characterized in that the pharmaceutical stable solution formulation contained therein is a sterile preparation suitable for subcutaneous injection administration.

[0069] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein is used for the treatment, prevention or improvement of non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.

[0070] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein is used for the treatment, prevention or improvement of type 2 diabetes and type 2 diabetes-related conditions.

[0071] In some embodiments, the pharmaceutical stable solution formulation of the GIPR antibody fusion protein provided herein is used for the treatment, prevention or improvement of obesity and obesity-related conditions.

[0072] In some embodiments, the pharmaceutically stable solution formulation of the GIPR antibody fusion protein provided herein is used to simultaneously treat, prevent, or improve two or more of the following conditions: non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, obesity, and type 2 diabetes.

[0073] In some embodiments, the pharmaceutically stable solution formulation of the GIPR antibody fusion protein provided herein ensures that it remains stable during storage and transportation, is convenient for clinical use, and is acceptable to the human body.

[0074] Brief description of the attached diagram

[0075] Figure 1 shows typical subvisible particle image results at point T0 (from top to bottom: E10-F1, E10-F2, E10-F3).

[0076] Figure 2 shows typical subvisible particle image results at point T0 (from top to bottom: E10-F4, E10-F5, E10-F2 (PFS)).

[0077] Figure 3 shows typical subvisible particle image results from the shaking experiment (from top to bottom: E10-F1, E10-F2, E10-F3).

[0078] Figure 4 shows typical subvisible particle image results from the shaking experiment (from top to bottom: E10-F4, E10-F5, E10-F2 (PFS)).

[0079] Figure 5 shows typical subvisible particle image results from repeated freeze-thaw experiments (from top to bottom: E10-F1, E10-F2).

[0080] Figure 6 shows typical subvisible particle image results from repeated freeze-thaw experiments (from top to bottom: E10-F3, E10-F4).

[0081] Figure 7 shows typical subvisible particle image results after repeated freeze-thaw cycles (from top to bottom: E10-F5, E10-F2 (PFS)).

[0082] Figure 8 shows typical subvisible particle image results from the high-temperature experiment (from top to bottom: E10-F1, E10-F2, E10-F3).

[0083] Figure 9 shows typical subvisible particle image results from high-temperature experiments (from top to bottom: E10-F4, E10-F5, E10-F2 (PFS)).

[0084] Figure 10 shows typical subvisible particle image results from the accelerated experiment (from top to bottom: E10-F1, E10-F2, E10-F3).

[0085] Figure 11 shows typical sub-visible particle image results for the accelerated experiment (from top to bottom, E10-F4, E10-F5, E10-F2 (PFS)).

[0086] Figure 12 shows typical sub-visible particle image results for the long-term experiment (3 months) (from top to bottom, E10-F1, E10-F2).

[0087] Figure 13 shows typical sub-visible particle image results for the long-term experiment (3 months) (from top to bottom, E10-F5, E10-F2 (PFS)).

[0088] Figure 14 shows typical sub-visible particle image results for the long-term experiment (6 months) (from top to bottom, E10-F1, E10-F2, E10-F3).

[0089] Figure 15 shows typical sub-visible particle image results for the long-term experiment (6 months) (from top to bottom, E10-F4, E10-F5, E10-F2 (PFS)).

[0090] Figure 16 shows typical sub-visible particle image results at T0 (from left to right, E12-F1, E12-F2).

[0091] Figure 17 shows typical sub-visible particle image results for high temperature stability (8 weeks) (from left to right, E12-F1, E12-F2).

[0092] Figure 18 shows typical sub-visible particle image results for accelerated stability (6 months) (from left to right, E12-F1, E12-F2).

[0093] Figure 19 shows typical sub-visible particle image results for long-term stability (6 months) (from left to right, E12-F1, E12-F2).

[0094] Figure 20 shows typical sub-visible particle image results at T0 (from top to bottom, E13-F1 to E13-F6).

[0095] Figure 21 shows typical sub-visible particle image results for high temperature stability (6 weeks) (from top to bottom, E13-F1 to E13-F6).

[0096] Figure 22 shows typical sub-visible particle image results for accelerated stability (6 months) (from top to bottom, E13-F1 to E13-F6).

[0097] Figure 23 shows typical sub-visible particle image results for long-term stability (6 months) (from top to bottom, E13-F1 to E13-F6). DETAILED DESCRIPTION

[0098] DEFINITIONS

[0099] Unless otherwise defined, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Generally, nomenclature used in connection with, and techniques of, the pharmaceutical, biological, biochemical, cell and tissue culture, biological, molecular biological, immunological, microbiological, genetic and protein nucleic acid chemical arts, and hybridization described herein are those well-known and commonly used in the art.

[0100] Polypeptide sequences are indicated herein using standard one-letter or three-letter abbreviations. Polypeptide sequences are written with the first amino acid residue (N') bearing an amino group on the left and the last amino acid residue (C') bearing a carboxyl group on the right, e.g., the GLP-1 fragment sequence referred to herein: SEQ ID NO: 1 or SEQ ID NO: 2.

[0101] Specific portions of a polypeptide can be indicated by the number of the amino acid residue, e.g., amino acids 80 to 130, or by the actual residues at that position, e.g., Lys80 to Lys130 or K80 to K130. A specific polypeptide can also be described by explaining its difference from a reference sequence.

[0102] The terms "peptide," "polypeptide," "protein," and "protein" refer to molecules comprising two or more amino acids linked by peptide bonds, and in most cases, these terms can be used interchangeably without changing the intended meaning. These terms encompass, for example, protein analogs of natural or artificial sequences (e.g., protein fragments, protein mutants, protein derivatives, protein variants) and proteins that are covalently or non-covalently modified post- transcriptionally. Peptides, polypeptides, proteins, and proteins can be monomeric or multimeric, such as an antibody, which is a tetrameric protein.

[0103] "Polypeptide fragment," "protein fragment," or "protein fragment" refers to a polypeptide having an amino-terminal and / or carboxyl-terminal deletion compared to the corresponding full-length protein. The fragment can be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150, or 200 amino acids in length. The fragment can be, for example, at most 1000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids in length. The fragment can further comprise one or more additional amino acids at one or both of its ends, e.g., an amino acid sequence from a different natural protein (e.g., an Fc or leucine zipper domain) or an artificial amino acid sequence (e.g., an artificial linker sequence).

[0104] The analogs of a protein include "protein mutants", i.e., analogs of a protein formed by changing one or more amino acids in the native sequence (e.g., conservative amino acid substitution, i.e., substitution of an amino acid that does not destroy the helix present in the parent sequence or interfere with other secondary structures that are necessary to impart the characteristics of the parent sequence or its function, thereby not significantly changing the structural characteristics of the parent sequence).

[0105] The analogs of a protein also include "protein derivatives", i.e., proteins modified chemically, e.g., by conjugation with other chemical moieties such as polyethylene glycol, albumin (e.g., human serum albumin), phosphorylation, and glycosylation. The proteins herein can include modifications added for any reason and by any method, e.g., to (1) reduce hydrolytic sensitivity, (2) reduce oxidative sensitivity, (3) alter the affinity for forming protein complexes, (4) alter binding affinity, and (5) impart other physical, chemical, or functional properties.

[0106] The analogs of a protein also include "protein variants", i.e., analogs of a protein formed by insertion, deletion, and / or substitution of one or more amino acid residues in the amino acid sequence of the protein. "Protein variants" include "fusion proteins" or "fusion proteins", which are "protein variants" formed by linking two or more "proteins", "protein", "peptides", "polypeptides", "protein domains" together via peptide bonds. The proteins involved in forming such "protein variants" are collectively referred to herein as "fusion components". The two or more "fusion components" usually have independent encoding genes and spatial structures, and usually can perform their respective biological functions without interference from other fusion components, but there are cases where interference or synergistic effects occur between them. In the process of forming fusion proteins, the proportions of different fusion components are not always the same, i.e., there are cases where the proportion of one fusion component is higher or lower than that of other fusion components, for example, one Fc protein can form a GLP-1 fusion protein with two GLP-1 polypeptides, where the ratio of GLP-1 polypeptide to Fc protein is 2:1.

[0107] The term "antibody" refers to a specific "protein." Unless otherwise indicated, "antibody" herein refers to a tetrameric protein comprising two full-length heavy chains and two full-length light chains, as well as derivatives, fragments, mutants, and variants thereof, proteins comprising a moiety that directly binds to an antigen, a moiety that allows for antigen binding, or a scaffold or framework moiety that facilitates binding of the antibody to the antigen. Examples of antibodies include intact antibodies, antibody fragments (e.g., antigen-binding portions of antibodies), antibody derivatives, and antibody analogs. The antibody can comprise, for example, a naturally occurring protein scaffold that is optionally selected or an artificial protein scaffold with grafted complementarity determining regions (CDRs) or CDR derivatives. The scaffold includes, but is not limited to, a derivatized scaffold comprising an antibody that is introduced, for example, to stabilize the three-dimensional structure of the antibody, and a wholly synthetic scaffold comprising, for example, a biocompatible polymer. See, e.g., Korndorfer et al., 2003, Proteins 53: 121-129; Roque et al., 2004, Biotechnol. Prog. 20: 639-654. In addition, the antibody can be a peptibody ("PAMs") or comprise a scaffold that mimics an antibody, which utilizes fibronectin as a scaffold. The antibody can have, for example, the structure of a native immunoglobulin. "Immunoglobulin" is a tetrameric molecule. In a native immunoglobulin, the tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector action. Human antibody light chains are classified as kappa and lambda light chains. Heavy chains are classified as mu, delta, alpha, or epsilon, and define a different class of antibodies, e.g., IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are joined by a "J" region of about 12 or more amino acids, with the heavy chain also including a "D" region of about 10 more amino acids. See, Fundamental Immunology Ch. 7 (Paul ed., 2d ed. Raven Press, 1989). The variable regions of each light / heavy chain pair operate as the antibody binding site. Thus, an intact immunoglobulin has two binding sites. Native immunoglobulins show a general structure wherein three hypervariable CDRs are connected by four relatively conserved framework regions (FRs). From N- to C-terminus, both light and heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.The assignment of amino acids to the various domains is consistent with the definitions of Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991.

[0108] Unless otherwise indicated, "antibody" herein also refers to an intact immunoglobulin or an antigen-binding portion thereof that competes with the intact antibody for specific binding. Antigen-binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include and especially include, Fab, Fab', F(ab')2, Fv, domain antibodies (dAbs), fragments including complementarity determining regions (CDRs), single chain antibodies (scFv), chimeric antibodies, diabodies, triabodies, tetrabodies, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. The CDRs and FRs of a given antibody are identified using the methods described by Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed. U.S. Dept. of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991. One or more CDRs can be covalently or noncovalently incorporated into a molecule to make it an antibody. An antibody can have the CDRs incorporated into a larger polypeptide chain. The CDRs can be covalently linked to another polypeptide chain, or noncovalently incorporated into the CDRs. The CDRs allow the antibody to specifically bind to a particular, relevant antigen.

[0109] The term "antigen-binding portion" "antigen-binding region" or "antigen-binding site" is a portion of an antibody that contains amino acid residues that interact with an antigen and contribute to the specificity and affinity of the antibody for the antigen. For an antibody that specifically binds to its antigen, this will include at least a portion of, the entire, or multiple CDR sequences. An antibody can have one or more binding sites. If more than one binding site, the binding sites can bind to the same or different antigens. For example, a native human immunoglobulin generally has two identical binding sites, while a "bispecific" or "bifunctional" antibody has two different binding sites.

[0110] The term "epitope" is the region on an antigen to which an antibody binds. An epitope can comprise a non-contiguous part of an antigen such as amino acid residues in the primary sequence of a polypeptide that are brought into close proximity with one another in the tertiary and quaternary structure of the polypeptide so as to be bound by an antibody.

[0111] The term "humanized antibody" is an antibody that has been made by grafting the CDR sequences of a mouse antibody molecule between human antibody FRs, and also includes a modified human antibody that has been made by other similar principles to reproduce the affinity characteristics of a murine antibody.

[0112] The term "gastric inhibitory polypeptide receptor (GIPR)" is a type B receptor belonging to the family of G protein-coupled receptors with seven transmembrane domains, which is coupled to one or more intracellular signaling pathways through a heterotrimeric guanine nucleotide-binding protein (G protein) (Drucker et al., 2006, Cell Metab. 3: 153-65). Current research has found that GIPR is mainly expressed on the surface of pancreatic beta cells and adipocytes (Ravn et al., 2013, J. Biol. Chem. 288: 19760-72), and is involved in the process of glucose and lipid metabolism in the human body, so it is closely related to diabetes, obesity and related diseases (Skaw et al., 2016, Diabetes Obes. Metab. 18: 847-854). As used herein, "human GIPR" and "hGIPR" both refer to the human gastric inhibitory polypeptide receptor and can be used interchangeably. As used herein, "murine GIPR" and "mGIPR" both refer to the murine gastric inhibitory polypeptide receptor and can be used interchangeably. The amino acid sequence information of GIPR is referred to the Uniprot database of European Bioinformatics Institute:

[0113] Human (Homo sapiens) amino acid; Accession No. AAB35419.2;

[0114] Rhesus macaque amino acid; Accession No. XP_014979775;

[0115] Mus musculus amino acid; Accession No. Q0P543.

[0116] A "Gastric Inhibitory Peptide Receptor Antibody (GIPR antibody)" is an antibody that specifically binds to GIPR on the cell membrane, and the antibody can also have the characteristic of inhibiting or blocking the conduction of GIP signal in these cells. More specifically, the antibody provided herein is an antibody that specifically binds to human GIPR, and the antibody can also bind to GIPR of other species (e.g., monkey), and can have the characteristic of blocking the signal transduction of GIP in humans or other species. The GIPR antibody described herein is an intact antibody and its derivatives, variants, fragments, and muteins, which are proteins comprising a portion that directly binds to GIPR, a portion that allows the antibody to bind to GIPR, or a scaffold or framework portion that can facilitate the binding of the antibody to GIPR.

[0117] A "Gastric Inhibitory Peptide Receptor Antibody Fusion Protein (GIPR antibody fusion protein)" is a fusion protein comprising the GIPR antibody described herein. More specifically, this particular fusion protein is a variant of the GIPR antibody protein comprising a GLP-1 sequence and its derivatives, variants, fragments, and mutants. Further specifically, this particular fusion protein is characterized in that it comprises the following sequences, characteristics:

[0118] a) a GLP-1 sequence and its derivatives, variants, fragments, and mutants selected from SEQ ID NO: 1 or SEQ ID NO: 2;

[0119] b) a light chain sequence and its derivatives, variants, fragments, and mutants selected from SEQ ID NO: 3 or SEQ ID NO: 4;

[0120] c) a heavy chain sequence and its derivatives, variants, fragments, and mutants selected from SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8;

[0121] d) the GIPR antibody fusion protein is formed by fusing a) to the N-terminus of b) or c) or both b) and c) through a linker sequence selected from SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12; or by fusing a) directly to the N-terminus of b) or c) or both b) and c) without a linker.

[0122] e) the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the following: SEQ ID NO: 13 and SEQ ID NO: 8.

[0123] In addition to the above, the "Gastric Inhibitory Peptide Receptor Antibody" (GIPR antibody) described herein is characterized in that it comprises the following sequences, characteristics:

[0124] a) comprises a combination of antibody CDR amino acid sequences selected from the group consisting of SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20 or SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20.

[0125] b) comprises a combination of antibody light and heavy chain variable region amino acid sequences selected from the group consisting of SEQ ID NO: 21 and SEQ ID NO: 23 or SEQ ID NO: 22 and SEQ ID NO: 23.

[0126] Table 1 includes the above sequences.

[0127] Table 1 GIPR antibody fusion protein related amino acid sequences

[0128] A "pharmaceutically stable solution formulation of a GIPR antibody fusion protein" refers to a solution type pharmaceutical formulation that is capable of maintaining stability for a long period of time, which can be used for injection alone or in combination with other pharmaceutical agents into the human or animal body, after mixing a GIPR antibody fusion protein with pharmaceutically acceptable pH buffering agents, pharmaceutically acceptable protective agents, pharmaceutically acceptable antioxidants, and pharmaceutically acceptable surfactants, and other pharmaceutically acceptable auxiliary components.

[0129] "Stable" or "stability" refers to the ability of a solution formulation to maintain the biological activity, structure, sequence, modification, molecular weight of its main utility component without significant changes, and the changes of all other quality parameters remain within the preset parameter range. The main utility component of the pharmaceutical stable solution formulation described herein is a GIPR antibody fusion protein, and the pharmaceutical stable solution formulation can maintain stability for at least 6 months at 25°C. More precisely, its stability maintenance period at 25±2°C is 6 months to 8 months, 6 months to 12 months, 6 months to 18 months, 6 months to 24 months, 8 months to 12 months, 8 months to 18 months, 8 months to 24 months, 12 months to 18 months, 12 months to 24 months, 18 months to 24 months, 24 months to 30 months, or 30 months to 36 months.

[0130] Further, the pharmaceutical stable solution formulation has a stability maintenance period of 6 months to 12 months, 6 months to 18 months, 6 months to 24 months, 8 months to 12 months, 8 months to 18 months, 8 months to 24 months, 12 months to 18 months, 12 months to 24 months, 18 months to 24 months, 24 months to 30 months, or 30 months to 36 months at 5 ± 3 °C.

[0131] "Pharmaceutically acceptable" means suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use within the scope of sound medical judgment. Thus, "pharmaceutically acceptable pH buffering agent", "pharmaceutically acceptable protective agent", "pharmaceutically acceptable antioxidant", and "pharmaceutically acceptable surfactant" mean that the specific material is compatible with the other ingredients of each type of formulation within the scope of sound medical judgment for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, commensurate with a reasonable benefit / risk ratio, and effective for their intended use.

[0132] In addition to the above, the "pharmaceutically acceptable pH buffering agent" used in the pharmaceutical stable solution formulation described herein can comprise one or more of a wide range of organic or inorganic compounds selected from, but not limited to, sodium hydroxide, potassium hydroxide, sodium dihydrogen phosphate, disodium hydrogen phosphate, citric acid, salts of citric acid, ascorbic acid, salts of ascorbic acid, gluconic acid, salts of gluconic acid, carbonic acid, salts of carbonic acid, tartaric acid, salts of tartaric acid, succinic acid, salts of succinic acid, acetic acid, salts of acetic acid, phtalic acid, salt of phtalic acid, phosphorc acid, phosphate, salt of boric acid, salt of barbituric acid, hydrochloric acid, Tris, Thomethamine, and amino acids and amino acid salts, including but not limited to Histidine Hydrochloride, Histidine, Arginine Hydrochloride, Arginine, Glycine. Preferred pH buffering agents are present in the formulation in a concentration range of 1-25, 1-50, 1-75, 1-100, 1-125, 1-150, 1-175, 1-200, 1-225, 1-250, 1-275, 1-300, 1-325, 1-350, 1-375, 1-400, 1-425, 1-450, 1-475, 1-500, 25-50, 25-75, 25-100,25-125、25-150、25-175、25-200、25-225、25-250、25-275、25-300、25-325、25-350、25-375、25-400、25-425、25-450、25-475、25-500、50-75、50-100、50-125、50-150、50-175、50-200、50-225、50-250、50-275、50-300、50-325、50-350、50-375、50-400、50-425、50-450、50-475、50-500、50-75、50-100、50-125、50-150、50-175、50-200、50-225、50-250、50-275、50-300、50-325、50-350、50-375、50-400、50-425、50-450、50-475、50-500、75-100、75-125、75-150、75-175、75-200、75-225、75-250、75-275、75-300、75-325、75-350、75-375、75-400、75-425、75-450、75-475、75-500、100-125、100-140、100-150、100-175、100-200、100-225、100-250、100-275、100-300、100-325、100-350、100-375、100-400、100-425、100-450、100-475、100-500、125-150、125-175、125-200、125-225、125-250、125-275、125-300、125-325、125-350、125-375、125-400、125-425、125-450、125-475、125-500、150-175、150-200、150-225、150-250、150-275、150-300、150-325、150-350、150-375、150-400、150-425、150-450、150-475、150-500、175-200、175-225、175-250、175-275、175-300、175-325、175-350、175-375、175-400、175-425、175-450、175-475、175-500、200-225、200-250、200-275、200-300、200-325、200-350、200-375, 200-400, 200-425, 200-450, 200-475, 200-500, 225-250, 225-275, 225-300, 225-325, 225-350, 225-375, 225-400, 225-425, 225-450, 225-475, 225-500, 250-275, 250-300, 250-325, 250-350, 250-375, 250-400, 250-425, 250-450, 250-475, 250-500, 275-300, 275-325, 275-350, 275-375, 275-400, 275-425, 275-450, 275-475, 275-500, 300-325, 300-350, 300-375, 300-400, 300-425, 300-450, 300-475, 300-500, 325-350, 325-375, 325-400, 325-425, 325-450, 325-475, 325-500, 350-375, 350-400, 350-425, 350-450, 350-475, 350-500, 375-400, 375-425, 375-450, 375-475, 375-500, 400-425, 400-450, 400-475, 400-500, 425-450, 425-475, 425-500, 450-475, 450-500, 475-500 mM.

[0133] The pharmaceutical stable solution formulation of the GIPR antibody fusion proteins described herein has a "pH range" of about 4.0 to 9.0, which provides acceptable stability for the pharmaceutical stable solution formulation to maintain solubility and activity of the GIPR antibody fusion protein to promote insulin secretion, and is a range tolerable for human administration. The pH can be adjusted to the intended pH by the addition of an acid, such as HC1, or the addition of a base, such as NaOH, or a combination of pH buffering agents can be added to achieve the intended buffering concentration and the intended pH. In addition to the above, a preferred pH buffering range is 5 to 7.75, 5 to 7.5, 5 to 7.25, 5 to 7.0, 5 to 6.75, 5 to 6.5, 5 to 6.25, 5 to 6.0, 5 to 5.75, 5 to 5.5, 5 to 5.25, 5.25 to 8.0, 5.25 to 7.75, 5.25 to 7.5, 5.25 to 7.25, 5.25 to 7.0, 5.25 to 6.75, 5.25 to 6.5, 5.25 to 6.25, 5.25 to 6.0, 5.25 to 5.75, 5.25 to 5.5, 5.5 to 8.0, 5.5 to 7.75, 5.5 to 7.5, 5.5 to 7.25, 5.5 to 7.0, 5.5 to 6.75, 5.5 to 6.5, 5.5 to 6.25, 5.5 to 6.0, 5.5 to 5.75, 5.75 to 8.0, 5.75 to 7.75, 5.75 to 7.5, 5.75 to 7.25, 5.75 to 7.0, 5.75 to 6.75, 5.75 to 6.5, 5.75 to 6.25, 5.75 to 6.0, 6.0 to 8.0, 6.0 to 7.75, 6.0 to 7.5, 6.0 to 7.25, 6.0 to 7.0, 6.0 to 6.75, 6.0 to 6.5, 6.0 to 6.25, 6.25 to 8.0, 6.25 to 7.75, 6.25 to 7.5, 6.25 to 7.25, 6.25 to 7, 6.25 to 6.75, 6.25 to 6.5, 6.5 to 8.0, 6.5 to 7.75, 6.5 to 7.5, 6.5 to 7.25, 6.5 to 7, 6.5 to 7, 6.5 to 6.75, 6.75 to 8, 6.75 to 7.75, 6.75 to 7.5, 6.75 to 7.25, 6.75 to 7.0, 7 to 8.0, 7 to 7.75, 7 to 7.5, 7 to 7.25, 7.25 to 8.0, 7.25 to 7.75, 7.25 to 7.5, 7.5 to 8.0, 7.5 to 7.75, 7.75 to 8.0. Further preferred pH buffering ranges for the pharmaceutical stable solution formulation described herein are 6.0-7.0.

[0134] As a supplement to the above, the "pharmaceuticalally acceptable protectant" used in the pharmaceutically stable solution formulations described in this text may include, but is not limited to, one or more of the following groups: arginine, arginine hydrochloride, histidine, histidine hydrochloride, cystine, cysteine, methionine, aspartic acid, lysine, glycine, tryptophan, leucine, isoleucine, phenylalanine, threonine, glutamic acid, serine and its derivatives, sucrose, trehalose, sorbitol, and mannitol; and the selected amino acids may be added in the form of amino acid salts, such as arginine hydrochloride, and the added amino acids may also be D-conformation amino acids, such as D-arginine, or L-conformation amino acids, such as L-arginine. The preferred pharmaceutically acceptable concentration range of the protective agent is 1-20, 1-25, 1-50, 1-75, 1-100, 1-125, 1-150, 1-175, 1-200, 1-225, 1-250, 1-275, 1-300, 1-325, 1-350, 1-375, 1-400, 1-425, 1-450, 1-475, 1-500, 25-28, 25-50, 25-75, 25-100, 25-125, 25-150, 25-175, 25-2 00, 25-225, 25-250, 25-275, 25-300, 25-325, 25-350, 25-375, 25-400, 25-425, 25-450, 25-475, 25-500, 50-75, 50-100, 50-125, 50-150, 50-175, 50-200, 50-225, 50-250, 50-275, 50-300, 50-325, 50-350, 50-375, 50-400, 50-425, 5 0-450, 50-475, 50-500, 50-75, 50-100, 50-125, 50-150, 50-175, 50-200, 50-225, 50-250, 50-275, 50-300, 50-325, 50-350, 50-375, 50-400, 50-425, 50-450, 50-475, 50-500, 75-100, 75-125, 75-150, 75-175, 75-200, 75-225, 75-25 0, 75-275, 75-300, 75-325, 75-350, 75-375, 75-400, 75-425, 75-450, 75-475, 75-500, 100-120, 100-125, 100-140, 100-150, 100-175, 100-200, 100-225, 100-250, 100-275, 100-300, 100-325, 100-350, 100-375, 100-400, 100-425100-450、100-475、100-500、125-150、125-175、125-200、125-225、125-250、125-275、125-300、125-325、125-350、125-375、125-400、125-425、125-450、125-475、125-500、150-175、150-200、150-225、150-250、150-275、150-300、150-325、150-350、150-375、150-400、150-425、150-450、150-475、150-500、175-200、175-225、175-250、175-275、175-300、175-325、175-350、175-375、175-400、175-425、175-450、175-475、175-500、200-225、200-250、200-275、200-300、200-325、200-350、200-375、200-400、200-425、200-450、200-475、200-500、225-250、225-275、225-300、225-325、225-350、225-375、225-400、225-425、225-450、225-475、225-500、250-275、250-300、250-325、250-350、250-375、250-400、250-425、250-450、250-475、250-500、275-300、275-325、275-350、275-375、275-400、275-425、275-450、275-475、275-500、300-325、300-350、300-375、300-400、300-425、300-450、300-475、300-500、325-350、325-375、325-400、325-425、325-450、325-475、325-500、350-375、350-400、350-425、350-450、350-475、350-500、375-400、375-425、375-450、375-475、375-500、400-425、400-450、400-475、400-500、425-450、425-475、425-500、450-475、450-500、475-500mM。、

[0135] As a supplement to the above, the "pharmaceutically acceptable antioxidant" used in the pharmaceutically stable solution formulations described in this text may include one or more amino acids or metal chelating agents selected from, but not limited to, the following group: ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetic acid, diethylenetriaminepentaacetic acid, methionine, cysteine ​​and their derivatives, and the selected amino acids may be added in the form of amino acid salts, such as arginine hydrochloride, and the added amino acids may also be D-conformation amino acids. The preferred concentration range of the antioxidant is 1-20, 1-25, 1-50, 1-75, 1-100, 1-125, 1-150, 1-175, 1-200, 1-225, 1-250, 1-275, 1-300, 1-325, 1-350, 1-375, 1-400, 1-425, 1-450, 1-475, 1-500, 25-50, 25-75, 25-100, 25-125, 25-150, 25-175, 25-200, 25-225, 25-250, 25-275, 25-3 00, 25-325, 25-350, 25-375, 25-400, 25-425, 25-450, 25-475, 25-500, 50-75, 50-100, 50-125, 50-150, 50-175, 50-200, 50-225, 50-250, 50-275, 50-300, 50-325, 50-350, 50-375, 50-400, 50-425, 50-450, 50-475, 50-500, 50-75, 50-100, 50-125, 50-15 0, 50-175, 50-200, 50-225, 50-250, 50-275, 50-300, 50-325, 50-350, 50-375, 50-400, 50-425, 50-450, 50-475, 50-500, 75-100, 75-125, 75-150, 75-175, 75-200, 75-225, 75-250, 75-275, 75-300, 75-325, 75-350, 75-375, 75-400, 75-425, 75-450, 75-4 75, 75-500, 100-120, 100-125, 100-140, 100-150, 100-175, 100-200, 100-225, 100-250, 100-275, 100-300, 100-325, 100-350, 100-375, 100-400, 100-425, 100-450, 100-475, 100-500, 125-150, 125-175, 125-200, 125-225, 125-250, 125-275, 125-300125-325、125-350、125-375、125-400、125-425、125-450、125-475、125-500、150-175、150-200、150-225、150-250、150-275、150-300、150-325、150-350、150-375、150-400、150-425、150-450、150-475、150-500、175-200、175-225、175-250、175-275、175-300、175-325、175-350、175-375、175-400、175-425、175-450、175-475、175-500、200-225、200-250、200-275、200-300、200-325、200-350、200-375、200-400、200-425、200-450、200-475、200-500、225-250、225-275、225-300、225-325、225-350、225-375、225-400、225-425、225-450、225-475、225-500、250-275、250-300、250-325、250-350、250-375、250-400、250-425、250-450、250-475、250-500、275-300、275-325、275-350、275-375、275-400、275-425、275-450、275-475、275-500、300-325、300-350、300-375、300-400、300-425、300-450、300-475、300-500、325-350、325-375、325-400、325-425、325-450、325-475、325-500、350-375、350-400、350-425、350-450、350-475、350-500、375-400、375-425、375-450、375-475、375-500、400-425、400-450、400-475、400-500、425-450、425-475、425-500、450-475、450-500、475-500mM。、

[0136]

[0137] In the pharmaceutically stable solution formulation of the GIPR antibody fusion protein described herein, the "final concentration of the GIPR antibody fusion protein" is approximately 1 to approximately 200 mg / mL. Preferably, the concentration (mg / mL) of the GIPR antibody fusion protein ranges from approximately 0.1-1, 1-5, 5-10, 5-20, 10-20, 20-30, 20-40, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, 90-100, 100-110, 100-120, 120-130, 130-140, 140-150, 150-160, 160-170, 170-180, 180-190, or 190-200 mg / mL. Particularly preferred concentrations (mg / mL) of the GIPR antibody fusion protein are approximately 0.1, 0.25, 0.5, 1, 2, 3, 4, 5, 6.5, 8, 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, and 11. 5. Approximately 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, 1-100, 1-120, 1-130, 1-140, 1-150, 1-160, 1-170, 1-180 or 1-190 mg / mL.

[0138] The pharmaceutically stable solution formulation of the GIPR antibody fusion protein described herein can be administered via any effective route known to a physician with conventional skills. Topical parenteral administration is one such method. Parenteral administration is generally understood in medical literature as the injection of the dosage form into the body via a sterile syringe or some other mechanical device such as an infusion pump. Topical parenteral routes can include intravenous, intramuscular, subcutaneous, and intraperitoneal administration. Subcutaneous administration is the preferred route of this patent.

[0139] The terms "treatment," "prevention," or "improvement" all refer to positive changes in one or more symptoms. These changes, depending on their degree, can include reducing or mitigating the severity of symptoms or a specific aspect of the symptoms (e.g., lowering the value of a disease indicator), or eradicating the clinical manifestations of the symptoms. The pharmaceutically stable solution formulation of the GIPR antibody fusion protein described in this article does not need to produce a complete cure or eradicate all symptoms or manifestations of the disease to be considered effective therapeutically. As is generally accepted in the relevant field, a drug as a therapeutic agent can reduce the severity of a given disease state without eliminating all manifestations of the disease to be considered an effective therapeutic agent. Similarly, prophylactic medication does not need to be completely effective in preventing the onset of symptoms to constitute an effective preventative agent. It is sufficient to reduce the impact of the disease (e.g., by reducing the number or severity of its symptoms, or by improving the effect of another treatment, or by producing another effective effect), or to reduce the likelihood of the disease occurring or worsening in the subject.

[0140] The pharmaceutically stable solution formulation of the GIPR antibody fusion protein described in this invention can be used to treat individuals with non-alcoholic fatty liver disease or non-alcoholic steatohepatitis, non-insulin-dependent diabetes mellitus or at risk of developing non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, or obesity. In the stable solution formulation described in the context, the effective amount of the GIPR antibody fusion protein is the amount that, when administered to individuals requiring GIPR and GLP-1 stimulation, produces the desired therapeutic and / or preventative effects without causing undesirable side effects.

[0141] Preferred pharmaceutically stable solutions of GIPR antibody fusion proteins are administered weekly to monthly. Depending on the disease being treated, more frequent administration of the pharmaceutically stable solution of GIPR antibody fusion proteins may be required, such as two to three times per week.

[0142] The pharmaceutically stable solution formulation of the GIPR antibody fusion protein will now be described by way of non-limiting examples.

[0143] Example

[0144] The following specific embodiments are provided in this document. These examples are merely for further illustrating the present invention and are not intended to limit the present invention.

[0145] Example 1: Screening suitable pH ranges for formulations of GIPR antibody fusion proteins (SEQ ID NO:13 and SEQ ID NO:8) by thermal melting temperature (Tm) assay.

[0146] This invention first uses a thermal fusion temperature assay to preliminarily determine the pH range suitable for the formulation of GIPR antibody fusion proteins. The tested pH range was 3-9. Different pH values ​​were provided by different buffer salt systems, namely citrate-citrate, phosphate, and Tris hydrochloric acid, which are the most common buffer salt systems for specific pH ranges.

[0147] Formulation Design: Under consistent conditions of 1 mg / mL protein concentration and 35 mM buffer salt concentration, different formulations were created by adjusting the pH value to a specific value. 1x SYPRO Orange dye was added to each formulation, and a gradual temperature ramp was set. Fluorescence intensity-time curves were recorded to calculate the thermal melting temperature of the GIPR antibody fusion protein under specific formulation conditions, and the differences in Tm1 and Tm2 under different pH conditions were compared. Tm1 and Tm2 represent the peak temperatures at which different domains of the GIPR antibody fusion protein completely disintegrate. Each formulation condition was tested in duplicate. The experimental design is as follows:

[0148] Experimental plan:

[0149] Analysis of experimental results on hot melt temperature:

[0150] The highest Tm1 and Tm2 values ​​of the GIPR antibody fusion protein were observed at pH 7, followed by pH 6. This indicates that the optimal thermal stability pH for the GIPR antibody fusion protein is between 6 and 7, and the formulation of this invention preferably uses a pH within this range. Because this example only examined the effect of pH on the thermal stability of the GIPR antibody fusion protein and did not consider the effect of the buffer salt system itself on molecular thermal stability, Example 3 further investigated the performance of different buffer salt systems within this pH range. The results are shown in Table 2.

[0151] Table 2. Tm values ​​of GIPR antibody fusion proteins at different pH values

[0152] Example 2: Effects of several common formulation additives on the solubility of GIPR antibody fusion proteins (SEQ ID NO:13 and SEQ ID NO:8)

[0153] Because the hydrophobicity of GIPR antibody fusion proteins negatively impacts their solubility and thermal stability, this invention aims to find a formulation that can significantly increase both the solubility and thermal stability of GIPR antibody fusion proteins. This invention uses the PEG precipitation method to evaluate the effects of several commonly used formulation components on the solubility of GIPR antibody fusion proteins. The tested components include arginine hydrochloride, sodium chloride, succinic acid, and polysorbate 80 (Tween-80), representing several common types of protective agents or surfactants.

[0154] Formulation Design: Under consistent conditions of 1 mg / mL protein concentration, 20 mM citrate-sodium citrate buffer system, and pH 6, different concentrations of arginine hydrochloride, sodium chloride, succinic acid, and polysorbate 80 were added to complete the formulation. The formulation was mixed with an equal volume of PEG3350 stock solution to form PEG3350 of different final concentrations (w / v). The absorbance of the mixture at 500 nm was measured, and an absorbance value of 0.1 was used as the criterion for precipitation. The experimental design is as follows:

[0155] Experimental plan:

[0156] Analysis of PEG precipitation method detection results:

[0157] Table 3 shows the minimum PEG concentration (%, w / v) required for precipitation under different additive conditions. The addition of arginine hydrochloride, sodium chloride, and succinic acid to the formulation significantly promoted the solubility of GIPR antibody fusion protein, while polysorbate 80 did not show a significant promoting effect on the solubility of GIPR antibody fusion protein.

[0158] Of the three components—arginine hydrochloride, sodium chloride, and succinic acid—arginine hydrochloride and sodium chloride provided the most significant increase in solubility, with similar effects. Due to limitations in osmotic pressure, higher concentrations of these components were not tested. Results are shown in Table 3.

[0159] Table 3. Effects of several common formulation additives on the solubility of GIPR antibody fusion proteins.

[0160] Example 3: Effects of pH and buffer system on the stability of GIPR antibody fusion protein (SEQ ID NO:13 and SEQ ID NO:8) formulations

[0161] This invention first investigated the effects of pH and buffer systems on the stability of GIPR antibody fusion proteins. To examine the stability of the formulation under different pH values ​​and buffer systems, experiments were conducted under the following conditions to assess the impact of different pH values ​​and buffer systems on formulation stability:

[0162] Formulation Design: Maintaining a consistent formulation with a protein concentration of 10 mg / mL, 140 mM arginine hydrochloride as an isotonic regulator, and 0.1% polysorbate 80 as a surfactant, we selected two buffer salt systems (citric acid-sodium citrate and histidine-histidine hydrochloride) and their corresponding pH values. The experimental design is as follows:

[0163] Experimental plan:

[0164] x = Appearance, visible foreign matter, protein content, SEC-HPLC; y = Reduced CE-SDS

[0165] Analysis of high temperature test results:

[0166] After 14 days of high-temperature treatment, a certain number of small white or translucent particles were found in all six groups of prescription samples, which were inferred to be visible foreign matter formed by protein aggregation. This indicates that high temperature has a significant impact on the protein stability of the GIPR antibody fusion protein.

[0167] After high-temperature storage, the protein content and SEC-HPLC purity showed no significant changes, but the reduced CE peaks decreased to some extent, and the light chain peaks showed some breakage. Among them, the E3-F6 group maintained the best performance. The results are shown in Table 4.

[0168] Table 4. Effects of pH and buffer system on the stability of GIPR antibody fusion protein (high temperature)

[0169] Note: "*" indicates the presence of a certain number of white or translucent fine particles. "HTT14D", "L7D", "FT3", and "HTT8W" respectively represent 14 days of high temperature, 7 days of sunlight, 3 freeze-thaw cycles, and 8 weeks of high temperature, and the same applies below.

[0170] Analysis of light exposure test results:

[0171] After 7 days of light exposure, the appearance of all 6 groups of samples remained stable, with a certain increase in opalescence, but no visible particles were observed.

[0172] The protein content and SEC-HPLC purity remained stable after 7 days of light exposure, but the reduction of CE decreased to some extent. Among them, the citric acid system of groups E3-F1 to E3-F3 showed a less obvious decreasing trend with increasing pH, while groups E3-F4 to E3-F6 did not show a corresponding trend and had relatively no significant differences. The results are shown in Table 5.

[0173] Table 5. Effects of pH and buffer system on the stability of GIPR antibody fusion protein (light).

[0174] Analysis of freeze-thaw test results:

[0175] After three freeze-thaw cycles, the appearance of the six samples remained stable, but the visible foreign matter varied. E3-F1 and E3-F6 remained stable, E3-F4 showed a few small white particles, while the other three groups showed a large number of white particles.

[0176] Three freeze-thaw cycles had no significant effect on the protein content and SEC-HPLC purity of the samples. The results are shown in Table 6.

[0177] Table 6. Effects of pH and buffer system on the stability of GIPR antibody fusion protein (freeze-thaw).

[0178] Summary of Example 3:

[0179] Within the pH range of 6.0-6.4, the citric acid system more readily produces visible white particles under different conditions, and its purity decreases significantly under light exposure. The histidine system is more stable than the citric acid system under various influencing conditions, and at pH 6.4, it exhibits a significant advantage in CE-SDS purity under high-temperature conditions. This demonstrates that histidine is superior to other pH buffering systems in this test. Therefore, a 20mM histidine system at pH 6.4, with 140mM arginine hydrochloride, was chosen as the basis for the next round of formulation screening.

[0180] Example 4: Effects of Protectants and Surfactants on the Stability of GIPR Antibody Fusion Protein (SEQ ID NO:13 and SEQ ID NO:8) Formulations

[0181] This invention screened protective agents (arginine / methionine) and surfactants (polysorbate 80 / poloxam 188) to improve the stability of GIPR antibody fusion protein drug formulations, and investigated their effects on the stability of GIPR antibody fusion proteins. The experimental design is as follows:

[0182] The test plan is as follows:

[0183] x = appearance, visible foreign matter, insoluble particles, SEC-HPLC; y = reduced CE-SDS; z = charge isomer.

[0184] Analysis of vibration test results:

[0185] Comparing E4-F1 (containing polysorbate 80) and E4-F2 (containing poloxamer 188), E4-F2 (containing poloxamer 188) is better in terms of appearance and insoluble particulate matter.

[0186] Comparing E4-F1, E4-F2 (without methionine) and E4-F3, E4-F4, and E4-F5 (containing methionine), E4-F1 sample produced fewer than 5 protein-like particles after 3 days of shaking, while the other groups remained stable. There was no significant difference in insoluble particles ≥10μm and ≥25μm among the groups. SEC-HPLC purity remained stable across all groups, with no significant difference in purity regardless of the presence or absence of methionine.

[0187] Compared with the formulations using surfactant polysorbate 80 (E4-F1 and E4-F3), the formulations using surfactant poloxamer 188 (E4-F2, E4-F4, E4-F5) showed no significant differences in appearance among the groups except for the E4-F1 group, where fewer than 5 protein particles were produced after 3 days of shaking.

[0188] In the poloxamer 188 system, E4-F4 and E4-F5 contained 90 mM and 20 mM of methionine, respectively. After shaking for 3 days, there were no significant differences in various indicators between the two groups.

[0189] In summary, after 3 days of oscillation, there were no significant differences among prescriptions E4-F1 to E4-F6, except for E4-F1. See Table 7 for the results.

[0190] Table 7. Effects of Protectants and Surfactants on the Stability of GIPR Antibody Fusion Protein Formulations (with vibration)

[0191] Note: "0 or 5 white particles" indicates that there is a difference between the two parallel samples observed. One sample has no visible particles, while the other has 5 visible white particles. The same applies if such descriptions appear in the table below.

[0192] Analysis of high temperature test results:

[0193] E4-F1 (containing polysorbate 80) has a better appearance than E4-F2 (containing poloxamer 188), but E4-F2 is better in terms of purity and insoluble particulate matter.

[0194] Comparing E4-F1, E4-F2 (without methionine) and E4-F3, E4-F4, E4-F5 (with methionine), no difference was observed in appearance after 8 weeks at high temperature; however, in terms of charge isomerism, the product without methionine was more effective than the product with methionine.

[0195] Compared to the surfactants poloxamer 188 (E4-F2, E4-F4, E4-F5), polysorbate 80 formulations (E4-F1, E4-F3) showed better appearance after 8 weeks at high temperature. However, considering the previous two points, the E4-F1 formulation (using polysorbate 80 without methionine) showed the most significant decrease in SEC purity, and the overall number of insoluble particles was significantly higher than other groups. Therefore, the E4-F1 formulation is not recommended.

[0196] After 8 weeks of high-temperature treatment, the two groups of E4-F3 and E4-F4 formulations containing the same concentration of methionine showed no significant differences in any of the indicators.

[0197] Under the formulation containing poloxamer 188, E4-F4 and E4-F5 contained 90 mM and 20 mM of methionine, respectively. After 8 weeks at high temperature, the appearance of E4-F4 was relatively better than that of E4-F5, while there were no significant differences between the two groups in other indicators.

[0198] E4-F6 contains no histidine. After 8 weeks at high temperature, its appearance, reduced CE-SDS, and charge isomer results were all unsatisfactory, and its purity and main peak content decreased more rapidly compared to other groups. The results are shown in Tables 8 and 9.

[0199] Table 8. Effects of protective agents and surfactants on the stability of GIPR antibody fusion protein formulations (high temperature, E4-F1~E4-F3)

[0200] Table 9. Effects of protective agents and surfactants on the stability of GIPR antibody fusion protein formulations (high temperature, E4-F4~E4-F6)

[0201] Example 4 Experiment Summary:

[0202] Formulations with added methionine (E4-F3, E4-F4, E4-F5) showed relatively better overall stability. Furthermore, with the addition of 0.2% poloxamer, the differences in various indicators between E4-F5 (20mM methionine) and E4-F4 (90mM methionine) were acceptable; therefore, 20mM methionine was chosen. Additionally, the experimental results of the E4-F6 group indicated that the histidine-histidine hydrochloride buffer system plays a crucial role in protein stability. Compared to Tween-80, poloxamer 188 performed slightly better in terms of insoluble microparticles and GIPR antibody solubility, but formulations containing Tween-80 had an advantage in appearance. Considering the importance of each indicator, we believe poloxamer 188 is more suitable for subsequent formulation development, but Tween-80 is also a viable option. Therefore, a formulation consisting of 20 mM histidine-histidine hydrochloride, 140 mM arginine hydrochloride, 20 mM methionine, 0.2% poloxamer 188, and pH 6.4 was selected for further research.

[0203] Example 5: Further optimization of the protectant (which also serves as a pH regulator and protein protectant)

[0204] Considering that the formulation obtained in the above study still shows a small amount of fine protein-like particles in the product's appearance after 8 weeks at high temperature, it is necessary to further select protective agents to increase the solubility of proteins and improve the problem of visible foreign matter.

[0205] Prescription design:

[0206] Experimental protocol

[0207] x = appearance, visible foreign matter, insoluble particles, SEC-HPLC, reduced CE-SDS; y = charge isomers.

[0208] Results analysis:

[0209] After being placed at 37℃ for 8 weeks, formulations E5-F1, E5-F2, and E5-F3 (at different pH values) all produced a certain number of visible white particles, with E5-F1 showing a more pronounced effect. The purity of its reduced CE-SDS decreased to below 90%, and the charge isomer spectrum was inconsistent with the typical spectrum. Compared to E5-F3, E5-F2 had better SEC-HPLC purity but poorer reduced CE-SDS purity. The basic peak of the charge isomer in E5-F2 increased more, while the acidic peak in E5-F3 increased more.

[0210] After being stored at 37℃ for 8 weeks, the various indicators of formulations E5-F2, E5-F4, and E5-F5 (different protective agents) showed certain differences. E5-F5 had the fewest visible particles, and its SEC-HPLC purity was worse than that of E5-F2 and E5-F4, but its reduction of CE-SDS was relatively better, and its charge isomers were also better preserved compared to the other two groups. Based on the analysis, the E5-F5 formulation will be used as a basis for the development of subsequent formulations at different concentrations. The results are shown in Tables 10 and 11.

[0211] Table 10 Results of further optimization experiments of the protective agent (high temperature, E5-F1~E5-F3)

[0212] Table 11 Results of further optimization experiments of the protective agent (high temperature, E5-F4~E5-F5)

[0213] Summary of Example 5:

[0214] E5-F5 (with added aspartic acid, without hydrochloric acid) showed significantly better results than other formulations in terms of appearance and visible foreign matter, indicating that the presence of hydrochloric acid in the formulation and the reduction of arginine hydrochloride concentration both easily lead to protein particle formation. E5-F1, with its lower pH, showed severe fragmentation in the reduced CE-SDS results, indicating that low pH is detrimental to protein stability. Based on these results, we used histidine, aspartic acid, and arginine as pH adjusters and protectants. After slightly adjusting the aspartic acid concentration based on the above experiments, we obtained a more robust formulation: 20 mM histidine, 147 mM aspartic acid, 140 mM arginine, 20 mM methionine, 0.2% poloxamer 188, pH 6.4.

[0215] Example 6: Prescription Confirmation

[0216] Prescription design

[0217] Note: *, After adjusting to a final pH of 6.4, the concentration of aspartic acid in this experiment was 148.3 mM.

[0218] Stability test protocol

[0219] x = Appearance, visible foreign matter, insoluble particles, SEC-HPLC; y = Protein content, reduced CE-SDS, charge isomers; z = Biological activity.

[0220] Results analysis:

[0221] After 3 days of shaking, the appearance of the sample remained stable, but the number of insoluble particles increased and the purity of SEC-HPLC decreased slightly, indicating that shaking can still increase the rate of protein aggregation to a certain extent and affect its stability.

[0222] After 8 weeks of high-temperature treatment, several small white particles appeared in the sample, and both the SEC-HPLC purity and the reduced CE-SDS purity showed a significant decrease, although they still met the quality standards. While the spectrum of the charge isomers was similar to the typical spectrum, the main peak decreased significantly, while the acidic and basic peaks increased markedly. High temperature has a significant destructive effect on proteins.

[0223] As the acceleration time increased, all indicators gradually decreased. After 6 months of acceleration, visible foreign matter no longer met the requirements, and the main peak of charge isomers also decreased significantly. The remaining indicators still met the quality standards.

[0224] After a prolonged period of 6 months, the amount of visible foreign matter increased, with a greater number of fine, translucent to white protein-like particles no larger than 50 μm appearing. The purity of SEC-HPLC and the purity of reduced CE-SDS remained stable, while the main peak of the charge isomer showed a slight decrease. The results are shown in Table 12.

[0225] Summary of Example 6:

[0226] According to the stability test results, the E6-F1 formulation (GIPR antibody fusion protein 20 mg / mL, histidine 20 mM, aspartic acid 147 mM, arginine 140 mM, methionine 20 mM, poloxamer 188 0.2%) can remain stable under vibration, long-term stability, accelerated stability and high temperature stability test conditions, but there is still a risk of failure due to visible foreign matter.

[0227] Table 12 E6-F1 Prescription Confirmation Results

[0228] Example 7: Prescription Re-optimization

[0229] During the formulation development of the GIPR antibody fusion protein (SEQ ID NO:13 and SEQ ID NO:8), at least three rounds of screening and optimization were conducted on the pH / Buffer system, excipients, and surfactants, resulting in a relatively stable formulation: pH 6.4, 20 mM histidine, 140 mM arginine, 148.3 mM aspartic acid, 20 mM methionine, and 0.2% poloxamer 188. Under this formulation system, samples under long-term (6 months), accelerated (6 months), and high-temperature (2 months) conditions showed a few fine particles, but all still met the quality standards. Therefore, to obtain a better formulation, this study optimized the surfactant based on this formulation, selecting poloxamer 338 and further exploring its concentration optimization.

[0230] Prescription design

[0231] Stability scheme

[0232] x = Appearance, visible foreign matter; y = Protein content, reduced CE-SDS, charge isomers, insoluble particles, SEC-HPLC.

[0233] Results analysis:

[0234] After two months of storage at high temperatures, no visible foreign matter was observed in E7-F2 (0.1% P338), E7-F3 (0.05% P338), and E7-F4 (0.02%), representing a further improvement compared to the E7-F1 formulation. Compared to the data from day 0, there were no significant differences in pH or protein content. The SEC and CE peaks decreased, but the decrease was comparable to the original formulation. The percentage of charge isomers also decreased, but remained within acceptable ranges and met quality standards.

[0235] After being placed under accelerated and long-term conditions for 3 months, no obvious foreign matter was found in any of the prescriptions, which was a further improvement compared to the E7-F1 prescription.

[0236] Based on the above experimental results (accelerated stability and long-term stability experiments are still ongoing), we obtained the optimized formulation: GIPR antibody fusion protein 20 mg / mL, 20 mM histidine, 147 mM aspartic acid, 140 mM arginine, 20 mM methionine, 0.1% poloxamer 338, pH 6.4. In this formulation system, sample stability was further significantly improved, the risk of visible foreign matter was greatly reduced, and medication safety was enhanced. Specific results are shown in Tables 13, 14, and 15.

[0237] Summary of Example 7:

[0238] Based on the original formulation, the type and concentration of surfactant were optimized, resulting in a superior formulation: 20 mg / mL GIPR antibody fusion protein, 20 mM histidine, 147 mM aspartic acid, 140 mM arginine, 20 mM methionine, 0.1% poloxamer 338, pH 6.4. In this formulation system, while ensuring the stability of the GIPR antibody fusion protein during storage, no visible foreign matter was observed, and other relevant quality indicators remained relatively stable, meeting quality standards and ensuring medication safety.

[0239] Table 13 Results of the prescription further optimization experiment (high temperature experiment)

[0240] Table 14 Results of the prescription optimization experiment (accelerated stability experiment)

[0241] Table 15 Results of the prescription optimization experiment (long-term stability experiment)

[0242] Example 8: Formulation Development of 40 mg / mL GIPR Antibody Fusion Protein (SEQ ID NO:13 and SEQ ID NO:8)

[0243] To accommodate different patient dosages, the protein concentration of the formulation was increased from 20 mg / ml to 40 mg / ml. However, increasing the concentration also further amplifies the protein aggregation effect. Previous formulation development revealed that increasing the histidine and aspartic acid content can reduce the aggregation effect caused by increased protein concentration, and in terms of stability, it can further improve SEC purity. Based on this, the 20 mg / ml formulation will be optimized and adjusted.

[0244] Prescription design

[0245] Stability scheme

[0246] Notes: X = Appearance, visible foreign matter; Y = Concentration, SEC-HPLC, cIEF, CE-SDS (Reduced & Non-Reduced), DLS

[0247] Results Analysis

[0248] After two months of high-temperature storage, no visible foreign matter was found in either group of samples, and there were no significant changes in appearance or concentration. The apparent particle size of DLS decreased to some extent, possibly due to some protein chain breakage under high-temperature conditions. After 2 months of high-temperature storage, the purity of SEC decreased significantly (E8-F1: Δ% = 5.3%, E8-F2: Δ% = 5.9%), with a more pronounced change in E8-F2. The purity of CE (reduced & non-reduced) decreased to some extent, with increased levels of acidic and basic peaks of charge isomers and a decreased main peak, but the difference between the two was not significant. These results suggest that reducing the arginine hydrochloride concentration may slightly decrease the purity of SEC, and that some proteins may undergo some degree of chain breakage degradation and aggregation under high-temperature conditions.

[0249] Under accelerated conditions for six months, no visible foreign matter was observed in the samples, and the appearance and concentration remained stable, with a slight decrease in particle size. The purity of SEC and reduced CE decreased by 2.2% and 3.9%, respectively, both within acceptable ranges, while the purity of non-reduced CE showed no significant change. The charge isomer spectrum was consistent with the working reference, but the content of the main peak decreased, while the content of acidic and basic peaks increased to some extent. There was no difference between E8-F1 and E8-F2.

[0250] After a six-month long-term observation period, the appearance, visible foreign matter, concentration, and particle size of the samples in both groups remained stable; the purity of SEC, purity of CE, and content of charge isomers remained relatively stable, with no significant changes. Specific results are shown in Tables 16, 17, and 18.

[0251] Summary of Example 8:

[0252] Further development based on the original formulation resulted in a 40 mg / mL formulation: 40 mg / mL GIPR antibody fusion protein, 60 mM histidine, 20 mM aspartic acid, 140 mM arginine hydrochloride, 20 mM methionine, 0.1% poloxamer 338, pH 6.4. In this formulation system, while ensuring the stability of the GIPR antibody fusion protein during storage, no visible foreign matter was observed, and other relevant quality indicators remained relatively stable, meeting quality standards and ensuring medication safety.

[0253] Table 16 Summary of High Temperature Experiment Results

[0254] Note: 1. SO, HTT1M, and HTT2M represent slight opalescence, with one month and two months of high-temperature observation, respectively. 2. The CE-SDS (reduced and non-reduced) of T0, HTT1M, and HTT2M were measured using different instruments compared to other time points.

[0255] Table 17 Summary of Accelerated Stability Results

[0256] Note: SO, ACT3M and ACT6M represent slight opalescence, observed under accelerated conditions for three months and six months, respectively.

[0257] Table 18 Summary of Long-Term Stability Results

[0258] Note: SO, LTT3M and LTT6M represent slight opalescence, respectively, and are observed under long-term conditions for three months and six months.

[0259] Example 9: Formulation development of 80 mg / mL GIPR antibody fusion protein (SEQ ID NO:13 and SEQ ID NO:8)

[0260] To accommodate different patient dosages, the protein concentration of the formulation was increased from 40 mg / ml to 80 mg / ml. However, increasing the concentration also further amplifies the protein aggregation effect. Previous formulation development revealed that optimizing the histidine and aspartic acid content can reduce the aggregation effect caused by increased protein concentration, and while maintaining other stability indicators, it can further improve SEC purity. Based on this, a formulation with an 80 mg / ml concentration will be developed.

[0261] Prescription design

[0262] Note: * indicates that the aspartic acid content can be adjusted with pH, ​​and the final concentration is 46mM.

[0263] Stability scheme

[0264] Note: 1. X = Appearance, visible foreign matter, dynamic light scattering particle size, SEC-HPLC; Z = Charge isomers, CE-SDS; 2. Y = Concentration, osmotic pressure; where the osmotic pressures of E9-F1 and E9-F2 are 427 mOsmol / kg and 443 mOsmol / kg, respectively; 3. (X, Z) represent that the relevant test items can be selectively detected according to the actual situation.

[0265] Results Analysis

[0266] After being placed under high temperature conditions for 2 months, the E9-F1 sample showed no change in appearance and no visible foreign matter was observed. The DLS particle size decreased from 88.15 nm to 69.85 nm; the SEC purity decreased from 99.1% to 93.5%, with an aggregation rate consistent with the control group (ΔHTT2M: control: 5.7%, E9-F1: 5.6%); the purity of reduced CE and non-reduced CE decreased by 5.3% and 6.7%, respectively, slightly better than the control group; the charge isomer content changed significantly, with increased acid and basic peak content and decreased main peak content, with a rate of change basically consistent with the control group. These results suggest that after adjusting the content of the formulation components, the stability of the GIPR antibody fusion protein under high temperature conditions was not significantly affected, and the results were all within acceptable ranges. After adjusting the excipients, the ionic strength of the E9-F2 (120mM Cl-) system decreased (E9-F1: 140mM Cl-), resulting in reduced intermolecular interactions and closer proximity of protein molecules at the microscopic level, leading to a larger apparent particle size. The osmotic pressure also increased further to 443 mOsmol / kg. After two months of high-temperature storage, the DLS decreased by 22.86 nm, while E9-F1 decreased by 18.1 nm, suggesting that the E9-F2 formulation has slightly poorer colloidal stability. SEC purity, reduced CE, and non-reduced CE purity decreased by 5.8%, 5.1%, and 6.6%, respectively, all without significant differences from E9-F1. The charge isomer results were also consistent with those of E9-F1.

[0267] After 6 months of accelerated storage, the appearance and concentration of the E9-F1 sample remained stable, and no visible foreign matter was observed. The DLS particle size decreased from 88.15 nm to 74.19 nm, which may be related to GLP end-chain scission. The SEC purity decreased from 99.1% to 97.0%, showing good performance. The purity of reduced CE and non-reduced CE decreased from 95.4% and 97.7% to 92.5% and 94.1%, respectively, showing good performance, even slightly better than the control group. The content of charge isomers changed significantly, with the acid peak and basic peak content increasing by 6.8% and 4.0%, respectively, and the main peak content decreasing by 10.8%. All of these results were not significantly different from the control group samples. These results suggest that the accelerated stability of the formulation after osmotic pressure optimization was not significantly affected and was within an acceptable range.

[0268] After being stored under long-term conditions for 6 months, the appearance, visible foreign matter, DLS particle size, and concentration of the E9-F1 sample did not show significant changes. The purity of SEC, reduced and non-reduced CE were 98.0%, 96.8%, and 97.7%, respectively, all remaining relatively stable. The content of acid-base isomers also did not show significant changes. The results indicate that the GIPR antibody fusion protein exhibits good long-term stability in the osmotically optimized formulation system. Specific results are shown in Tables 19, 20, and 21.

[0269] Summary of Example 9:

[0270] Replacing histidine hydrochloride and arginine in the formulation with histidine and arginine hydrochloride, although the formulation composition remained consistent with the GIPR antibody fusion protein (40 mg / mL) and protein stability was not significantly affected, the sample osmotic pressure was slightly higher, which is not conducive to later clinical applications. Therefore, the GIPR antibody fusion protein (80 mg / mL) will be further studied using the E9-F1 formulation of this experiment, namely 140 mM histidine hydrochloride, 28 mM aspartic acid, 120 mM arginine, 20 mM methionine, 0.1% poloxamer 338, pH 6.4. Its mass changes under high temperature and accelerated conditions were within acceptable ranges, and its long-term stability was good.

[0271] Table 19 Summary of High Temperature Stability Results

[0272] Note: "SO" represents slight opalescence;

[0273] Table 20 Summary of Accelerated Stability Results

[0274] Note: 1. "SO" represents slight opalescence; 2. The E9-F1 sample was prepared by direct dilution of the UFDF sample, and the E9-F2 sample was prepared by a second solution change of the UFDF sample; due to the quality problem of the dialysis bag used in the process, the protein stability was abnormal under accelerated conditions, so the E9-F2 data were not analyzed in detail.

[0275] Table 21 Summary of Long-Term Stability Results

[0276] Note: 1. "SO" represents slight opalescence; 2. The E9-F1 sample was prepared by direct dilution of the UFDF sample, and the E9-F2 sample was prepared by a second solution change of the UFDF sample; due to the quality problem of the dialysis bag used in the process, the protein stability was abnormal under long-term conditions, so the E9-F2 data were not analyzed in detail.

[0277] Example 10: Formulation optimization of 80 mg / mL GIPR antibody fusion protein (SEQ ID NO:13 and SEQ ID NO:8)

[0278] After a series of studies, we have basically obtained a relatively stable formulation for GIPR antibody fusion protein (80 mg / mL): 140 mM histidine hydrochloride, 28 mM aspartic acid, 120 mM arginine, 20 mM methionine, 0.1% poloxamer 338, pH 6.4. Based on our previous experience developing a 40 mg formulation, the GIPR antibody fusion protein is relatively sensitive to shear force and interfacial stress. To address the challenges in its later production, transportation, and use, this study optimized the type and concentration of surfactants in the formulation and preliminarily investigated the influence of different packaging materials.

[0279] Prescription design

[0280] Investigation Plan

[0281] Note: 1. X = Appearance, visible foreign matter; Y = DLS particle size, SEC-HPLC, FlowCam; Z = Concentration, pH.

[0282] icIEF, CE-SDS, biological activity;

[0283] 2. Based on historical research data on GIPR antibody fusion proteins, the purity and biological activity of non-reduced CE are less affected by surfactants, and the risk is low. Therefore, this was not detected during the actual experiment.

[0284] Results Analysis

[0285] The initial DLS particle sizes of samples E10-F1 (0.1% poloxamer 338), E10-F2 (0.2% poloxamer 338), E10-F3 (0.3% poloxamer 338), and E10-F4 (0.4% poloxamer 338) were 80.59 nm, 97.24 nm, 111.00 nm, and 137.55 nm, respectively; E10-F5 (0.2% poloxamer 188) had a particle size of 87.78 nm; and E10-F2 (PFS packaging) had a particle size of 97.51 nm. The results show that the particle size of the samples is significantly affected by the surfactant concentration, and the apparent particle size increases with increasing concentration. This phenomenon suggests that there may be some kind of association between the protein surface and the surfactant. E10-F5 and E10-F2 are surfactants of the same concentration but different types. Comparing their results, the apparent particle size is inconsistent, suggesting that poloxamer 188 and poloxamer 338 may have different morphologies when binding to proteins. E10-F2 and E10-F2 (PFS) have the same formulation but are packaged in vials and pre-filled syringes (PFS), respectively; there was no difference in particle size between the two samples. Flow cytometry particle imaging at T0 showed that the PFS-packaged E10-F2 sample contained more silicone oil sub-visible particles, while no corresponding images were observed in the vial-packaged E10-F1 to E10-F5 samples, and there was no significant difference in the results for the E10-F1 to E10-F5 samples. There were no significant differences in the initial SEC purity, reduced CE purity, and charge isomer results among the six groups of samples.

[0286] After five freeze-thaw cycles, the appearance, visible foreign matter, concentration, pH, and DLS particle size of the six samples remained stable, and the purity of SEC and reduced CE did not change significantly. However, FlowCam analysis showed that the protein morphology of samples F1–F5 changed significantly, with E10-F1 (0.1% poloxamer 338) and E10-F5 (0.2% poloxamer 188) showing significantly worse results than the other three groups (E10-F2–E10-F4); while no significant changes in protein morphology were observed in sample E10-F2 (PFS). These results suggest that repeated freeze-thaw cycles have no significant effect on the physicochemical quality of GIPR antibody fusion proteins, but may have some impact on the sub-visible morphology of the proteins, potentially posing a risk of accelerated protein aggregation. This effect can be reduced by increasing the surfactant content. Furthermore, repeated freeze-thaw cycles had no effect on samples packaged in pre-filled syringes.

[0287] After being placed under high temperature conditions for two months, the appearance, concentration, and pH of the six sample groups remained unchanged, and no visible foreign matter was observed. The particle size of the formulation containing poloxamer 338 significantly decreased, a trend consistent with historical batch variations. Based on research experience with similar products, the likely cause is partial truncation of the light chains fused with GLP under high temperature conditions. The particle size of the formulation group containing poloxamer 188 (E10-F5) showed an increasing trend. The possible reason for this is that under high temperature conditions, the partially truncated light chains undergo intermolecular interactions. The samples exhibited a tendency to aggregate; the SEC purity of E10-F1 decreased by 5.7%, while the other five groups all decreased by approximately 6.3%; the reduced CE also decreased significantly, but there was no difference between the groups; the content of charge isomers changed significantly, i.e., the content of acidic and basic peaks increased, while the content of the main peak decreased, and there was no difference among the six groups; flow cytometry particle imaging results showed that suspected protein particles were present in each sample, with E10-F2, E10-F3, and E10-F4 performing slightly better than E10-F1; the E10-F2 (PFS) pre-filled packaging material was better than the E10-F2 vial packaging material. E10-F2 (poloxam 338) was slightly better than E10-F5 (poloxam 188). These results indicate that high temperature has a significant impact on the amount of GIPR antibody fusion protein, and the concentration and type of surfactant also have a certain impact on product quality.

[0288] After 6 months of accelerated storage, the appearance, pH, and concentration of each sample remained stable, and no visible foreign matter was observed. The DLS particle size of sample E10-F1 (vial, 0.1% poloxamer 338) decreased by 20.21 nm. For samples E10-F2 to E10-F4, the particle size change rate increased with increasing poloxamer 338 concentration (ΔACT6M: E10-F2: 27.63 nm, E10-F3: 34.52 nm, E10-F4: 43.68 nm). For E10-F5 (0.2% poloxamer 338)... The morphology of E10-F5 (0.2% poloxamer 338) decreased by 22.68 nm, and that of E10-F2 (PFS) (pre-filled syringe, 0.2% poloxamer 338) decreased by 28.52 nm. The SEC purity of F1 decreased from 99.0% to 97.4%, while the other five groups decreased from 99.0% to 96.8%. The CE purity of E10-F1 decreased by 3.4%, which was not different from the other five groups. The content of acidic and basic isomers of E10-F1 increased by 6.0% and 8.0%, respectively, while the main peak content decreased by 14.0%, with no significant difference from the results of the other five groups. Flow cytometry particle imaging results showed that the sub-visible particle morphology of the E10-F5 (0.2% poloxamer 188) sample changed significantly, while no significant changes were observed in the other five groups. The above results indicate that, under accelerated conditions, poloxamer 188 has a weaker protective effect on the GIPR antibody fusion protein than poloxamer 338, and the concentration of poloxamer 338 and the type of packaging container have no significant effect on its accelerated stability.

[0289] After 6 months of long-term storage, the appearance, visible foreign matter, DLS particle size, concentration, and pH of all six samples remained stable; the purity and charge isomers of reduced CE also showed no significant changes; the purity of SEC decreased from 99.0% to 98.5%. Combining flow cytometry particle imaging results from 3 and 6 months, the sub-visible particle morphology of sample E10-F5 (0.2% poloxamer 188) changed significantly, while the other five groups showed no significant changes. Similarly, in vial packaging, the protein morphology of E10-F1 (0.1% poloxamer 338) was slightly inferior to the other three groups. At the same P338 concentration, the E10-F2 (PFS) group was superior to the E10-F2 (via vial) group. The results above suggest that, under long-term storage conditions, poloxamer 188 has a significantly weaker protective effect against the GIPR antibody fusion protein than poloxamer 338. Furthermore, the protective effect of poloxamer 338 exhibits a concentration-dependent effect, and the pre-filled syringe (PFS) packaging type is superior to vials. Specific results are shown in Tables 22-25 and Figures 1-15. Analysis of the overall data leads to the following conclusions: poloxamer 388 has a stronger protective effect against the GIPR antibody fusion protein than poloxamer 188; the E10-F2 formulation performs slightly better than other groups; and the E10-F2 formulation performs slightly better in pre-filled syringes than in vials.

[0290] Summary of Example 10:

[0291] Vigorous shaking conditions may accelerate protein aggregation, so this condition should be avoided as much as possible during actual sample storage.

[0292] Under repeated freeze-thaw cycles, accelerated and long-term conditions, poloxamer 188 showed significantly weaker protective effect on GIPR antibody fusion protein than poloxamer 338 at the same concentration. Sub-visible particle changes were significant, posing a risk of accelerated protein aggregation or the formation of visible foreign matter. Therefore, poloxamer 338 was still selected as the surfactant in the formulation of GIPR antibody fusion protein (80 mg / mL).

[0293] The same sample packaged in vials and pre-filled syringes (PFS) showed no difference in physicochemical quality, but there was a certain difference in the subvisible particle morphology (PFS was superior to vials), and this difference was particularly obvious during repeated freeze-thaw cycles.

[0294] Sub-visible particles in the sample may pose risks such as accelerated protein aggregation and the formation of visible foreign matter. Poloxamer 338 exhibits a concentration effect in maintaining the morphology of sub-visible particles, with concentrations above 0.1% showing better performance (freeze-thaw, high temperature, and acceleration generally show the same trend). However, as the concentration increases to 0.2% and above, the purity of SEC decreases more rapidly under high temperature conditions. This may be because excessively high concentrations of surfactant can increase the protein particle size, increasing the chance of contact between particles and accelerating aggregation to some extent, similar to high temperature conditions. Therefore, considering all aspects of balance, controlling the concentration of poloxamer 338 within the range of 0.1% to 0.2% is optimal. Considering the operational space and quality control range, a concentration of 0.15% for poloxamer 338 is more appropriate.

[0295] In summary, the formulation of the GIPR antibody fusion protein (80 mg / mL) was determined to be 140 mM histidine hydrochloride, 28 mM aspartic acid, 120 mM arginine, 20 mM methionine, 0.15% poloxamer 338, pH 6.4.

[0296] Table 22 Summary of Shaking, Freeze-Thaw and High-Temperature Test Results (Partial: E10-F1~E10-F3)

[0297] Table 23 Summary of shaking, freeze-thaw and high-temperature test results (E10-F4~E10-F5, E10-F2(PFS))

[0298] Notes: 1. “AGI3D”, “FT5C”, “HTT2M” and “SO” represent 3 days of shaking, 5 cycles of repeated freeze-thaw cycles, 2 months of high-temperature observation and slight opalescence, respectively; 2. After 3 cycles of repeated freeze-thaw cycles and 1 month of high-temperature observation, the appearance of E10-F4 to E10-F2 (PFS) did not change and no visible foreign matter was observed.

[0299] Table 24 Summary of Accelerated Stability Results

[0300] Table 25 Summary of Long-Term Stability Results

[0301] Example 11: Formulation development of 120–140 mg / mL GIPR antibody fusion protein (SEQ ID NO:13 and SEQ ID NO:8)

[0302] To accommodate different patient dosages, the formulation concentration was increased from 80 mg / mL to 120–140 mg / mL. However, increasing the concentration further amplifies protein aggregation and poses challenges to protein solubility. Therefore, based on previous formulation development, we attempted to develop a new high-concentration formulation by screening and optimizing the types and proportions of surfactants.

[0303] Prescription design

[0304] Stability scheme

[0305] Note 1. X = appearance, visible foreign matter; Y = dynamic light scattering particle size (DLS), FlowCam, SEC-HPLC, reduced CE-SDS;

[0306] 2. (Y) represents optional testing. Generally, the appearance and visible foreign matter are observed first. If the appearance and visible foreign matter do not meet the requirements, other tests and subsequent investigations of a sample can be terminated in a timely manner, depending on the situation.

[0307] Results analysis:

[0308] Examining the formulations, under shear force conditions of 100 pipette blows, no visible foreign matter was observed in formulations E11-F1 to E11-F6. Compared with the T0 data, the SEC main peak and the reduced CE main peak showed no change in purity, indicating that the high-concentration formulations could withstand certain shear force requirements in the examined formulations.

[0309] After being placed under high temperature for 2 months, the E11-F1 to E11-F6 formulations all maintained good appearance with no visible foreign matter. DLS particle size varied in each formulation; the SEC main peak and the reduced CE main peak both decreased, while SEC aggregates showed an increasing trend. The E11-F4 to E11-F6 groups (containing hydroxypropyl-β-cyclodextrin) were significantly better than the E11-F1 to E11-F3 groups (poloxamer 388). After 2 months of observation at high temperature, the SEC aggregate content in the E11-F4 to E11-F6 groups increased from 1.1% to approximately 6.0%, while in the E11-F1 to E11-F3 groups, the SEC aggregate content increased from 1.0% to approximately 8.8% to 10.0%. Moreover, the aggregate content increased synchronously with the increase in poloxamer 388 concentration. Reduced CE-SDS decreased in all six candidate formulations, from 97% to approximately 85%, with no significant differences between formulations.

[0310] After being stored under long-term conditions for 3 months, the appearance, visible foreign matter, and DLS particle size of the six candidate formulations did not change significantly; the purity of SEC and the purity of reduced CE remained relatively stable. Specific results are shown in Tables 26-29.

[0311] Summary of Example 11:

[0312] Based on the original formulation, the type and concentration of surfactant were optimized. Finally, a solubilizer, N,N-dimethylacetamide, was added to the original 80 mg / mL formulation, which increased the protein concentration to 120 mg / mL. At this protein concentration, the product remained stable under shear stress, high temperature and long-term low temperature conditions, which provided a guarantee for the continued development of high-concentration formulations.

[0313] Table 26 Summary of the test results for blowing stability

[0314] Note: "SO" represents slight opalescence; "SM100" represents 100 pipette strokes.

[0315] Table 27 Summary of High Temperature Stability Test Results (Partial: E11-F1~E11-F3)

[0316] Note 1. "SO" represents slight opalescence;

[0317] Table 28 Summary of High Temperature Stability Test Results (Partial: E11-F4~E11-F6)

[0318] Note 1. "SO" represents slight opalescence;

[0319] Table 29 Summary of Long-Term Stability Test Results

[0320] Note 1. "SO" represents slight opalescence;

[0321] Example 12: Formulation concentration exploration and stability confirmation experiments of 120 mg / mL GIPR antibody fusion protein (SEQ ID NO:13 and SEQ ID NO:8)

[0322] Previous experimental results showed that under the original formulation conditions of 80 mg / mL GIPR antibody fusion protein (i.e., 140 mM histidine hydrochloride, 28 mM aspartic acid, 120 mM arginine, 20 mM methionine, 0.15% poloxamer 338, pH 6.4), the addition of 1% N,N-dimethylacetamide (DMA) significantly improved the solubility of the GIPR antibody fusion protein and reduced the opalescence of the sample. This suggests that DMA may help reduce protein-protein interactions at a microscopic level. Meanwhile, other stability parameters of the GIPR antibody fusion protein formulation were not significantly affected. Therefore, this study will explore the relevant stability at different protein concentrations under the given DMA-containing formulation conditions, providing a reference for the development of higher concentration GIPR antibody fusion protein formulations. Accelerated and long-term stability studies (6 months) were also conducted on the high-concentration formulation to determine its robustness.

[0323] Prescription design

[0324] Stability scheme

[0325] Note: X = Appearance, visible foreign matter, DLS particle size, (FlowCam), SEC purity, charge isomers, CE-SDS; Y = Concentration, (Biological Activity 1), (Biological Activity 2); where "()" indicates optional detection based on experimental results.

[0326] Results Analysis

[0327] After being placed under high temperature conditions for 2 months, the E12-F1 (120 mg / mL) sample showed no change in appearance and no visible foreign matter was observed; the DLS particle size decreased from 74.69 nm to 72.63 nm; the SEC purity decreased from 99.3% to 89.6% (ΔHTT2M: 9.7%); the purity of reduced CE and non-reduced CE decreased by 13.4% and 6.3%, respectively; and the content of charge isomers changed significantly, namely, the content of acidic peaks and basic peaks increased, while the content of the main peak decreased. After being placed at high temperature for 2 months, the E12-F2 (140 mg / mL) sample showed no change in appearance and no visible foreign matter was observed. The DLS particle size decreased from 73.02 nm to 69.13 nm; the SEC purity decreased from 99.3% to 88.4% (ΔHTT2M: 10.9%); the purity of reduced CE and non-reduced CE decreased by 13.5% and 7.3%, respectively; and the charge isomer content changed significantly, with increased acidic and basic peak content and decreased main peak content. The aggregation rate of the E12-F2 sample was higher than that of the E12-F1 sample, but the DLS particle size, reduced and non-reduced CE purity, and charge isomer results were not significantly different from those of E12-F1. These results suggest that increasing protein concentration accelerates the aggregation of GIPR antibody fusion proteins under high temperature conditions, while other stability qualities remain within acceptable ranges.

[0328] After 6 months of accelerated storage, the appearance and visible foreign matter of the E12-F1 sample remained stable; the DLS particle size decreased from 74.69 nm to 61.79 nm; the SEC purity decreased from 99.3% to 96.6%, and the purity of reduced CE and non-reduced CE decreased from 97.2% and 97.2% to 90.6% and 94.2%, respectively; the content of charge isomers changed significantly, with the acidic peak and basic peak contents increasing by 6.6% and 9.6%, respectively, and the main peak content decreasing by 16.1%. These results suggest that, in the existing formulation system, the accelerated stability quality changes when the GIPR antibody fusion protein concentration is maintained at 120 mg / mL are generally within an acceptable range. The appearance of the E12-F2 sample remained unchanged, and no visible foreign matter was observed. The DLS particle size decreased from 73.02 nm to 62.69 nm; the SEC purity decreased from 99.3% to 96.3%; the purity of reduced CE and non-reduced CE decreased from 97.1% and 97.2% to 90.1% and 93.7%, respectively; the content of charge isomers changed significantly, with acidic and basic peak contents increasing by 5.9% and 12.3%, respectively, while the main peak content decreased by 18.2%. These results suggest that, in the existing formulation system, the accelerated stability mass of the GIPR antibody fusion protein at a concentration of 140 mg / mL is slightly lower than that at 120 mg / mL, but the difference is not significant and is within acceptable limits. Furthermore, a trend comparison of the data from 120 mg / mL and 140 mg / mL with the data from the 80 mg / mL formulation shows that the mass change trends of samples at each concentration are basically consistent, with no significant differences.

[0329] After 6 months of long-term storage under extended conditions, the appearance, visible foreign matter, and DLS particle size of samples E12-F1 and E12-F2 did not show significant changes. The SEC purity of both groups of samples decreased slightly, from 99.3% and 99.3% to 98.4% and 98.3%, respectively. The content of acid-base isomers, reduced CE, and non-reduced CE purity remained relatively stable. These results indicate that the GIPR antibody fusion protein maintains good long-term stability at concentrations of 120 mg / mL to 140 mg / mL under the existing formulation system. The viscosities of samples stored at 120 mg / mL and 140 mg / mL for 6 months were 21.4 cP and 58.8 cP, respectively, while the viscosity of 80 mg / mL was 9.8 cP. The viscosities of 80 mg / mL and 120 mg / mL are sufficient to support further development and production, while the viscosity of the 140 mg / mL formulation may require further optimization to suit commercial production. The data from this experiment at 120 mg / mL and 140 mg / mL were summarized and compared with the original prescription at 80 mg / mL. The results showed that the mass change trends of the samples at each concentration were basically consistent, with no significant differences. Detailed results are shown in Tables 30-32 and Figures 16-19.

[0330] Summary of Example 12:

[0331] In summary, within the established formulation system of this experiment, 1) the aggregation rate of the GIPR antibody fusion protein under high-temperature conditions exhibited a concentration-dependent effect, i.e., it increased with increasing concentration; 2) the quality changes of the GIPR antibody fusion protein under high-temperature and accelerated conditions (appearance, visible foreign matter, particle size, SEC purity, CE-SDS purity, and charge isomers) were all within acceptable ranges, demonstrating good long-term stability. Therefore, this formulation (i.e., 140 mM histidine hydrochloride, 28 mM aspartic acid, 120 mM arginine, 20 mM methionine, 0.15% poloxamer 338, 1.0% DMA, pH 6.4) can support the development and production of a GIPR antibody fusion protein formulation with a concentration of 120 mg / mL, while a concentration of 140 mg / mL requires further development to address the viscosity issue.

[0332] Table 30 Summary of High Temperature Stability Results

[0333] Note: 1. SO represents slight opalescence; 2. *The detection spectrum is abnormal, and no retest was performed due to insufficient sample volume;

[0334] Table 31 Summary of Accelerated Stability Results

[0335] Note: 1. "SO" represents slight opalescence.

[0336] Table 32 Summary of Long-Term Stability Results

[0337] Note: 1. "SO" represents slight opalescence.

[0338] Example 13: Further optimization of the formulation of the 120 mg / mL GIPR antibody fusion protein (SEQ ID NO:13 and SEQ ID NO:8): replacing N,N-dimethylacetamide (DMA) with hydroxypropyl-β-cyclodextrin (HP-β-CD).

[0339] Through Example 12, we mainly studied the synergistic effect of 0.15% poloxamer 338 and 1.0% DMA, which can significantly increase the protein concentration to 120 mg / mL and maintain the protein's stability at short-term high temperatures, accelerating its stability over 6 months and long-term, making it an excellent high-concentration formulation. However, considering that N,N-dimethylacetamide (DMA) is not widely used in macromolecular biopharmaceuticals, and previous experimental results (as shown in Example 11) indicate, we found that the formulations containing hydroxypropyl-β-cyclodextrin (HP-β-CD) (E11-F4 to E11-F6) showed significantly better stability than those without. This demonstrates the excellent solubilization and formulation stability of GIPR antibody fusion proteins by hydroxypropyl-β-cyclodextrin (HP-β-CD). Therefore, this embodiment mainly examines the formulation of high-concentration GIPR antibody fusion protein by the synergistic effect of poloxamer 338 and hydroxypropyl-β-cyclodextrin (HP-β-CD) without N,N-dimethylacetamide (DMA), providing further assurance for the commercialization of high-concentration formulations.

[0340] Prescription design

[0341] Stability scheme

[0342] Note: X = Appearance, visible foreign matter, DLS particle size, (FlowCam), SEC purity, reduced CE-SDS;

[0343] Y = charge isomer, (biological activity 1), (biological activity 2); where “()” represents optional detection based on experimental results;

[0344] Z = concentration, viscosity

[0345] Results analysis:

[0346] The formulations were examined. After 6 weeks of high-temperature storage, the appearance remained unchanged, and no visible foreign matter was observed. The particle size of DLS decreased to some extent in all formulations; the higher the content of hydroxypropyl-β-cyclodextrin (HP-β-CD), the smaller the decrease. Lower poloxamer 338 content resulted in smaller initial T0 DLS particle size. SEC purity decreased from 99.4% to 92.4%–92.8% (ΔHTT6W: ~6.6%), with E13-F4 (5% HP-β-CD) showing better SEC purity. Reduced CE purity decreased from 96.4% to 88.2%–89.4% (ΔHTT6W: 7–8.2%), with E13-F4–E13-F6 showing better purity. The content of charge isomers changed significantly, with increased acid and basic peak content and decreased main peak content; there were no significant differences between formulations. After 6 weeks of high-temperature storage, all quality indicators were generally acceptable.

[0347] After 6 months of accelerated storage, formulations E13-F1 to E13-F6 all maintained good appearance with no visible foreign matter. DLS particle size decreased to some extent in all formulations, with a smaller decrease observed at higher levels of hydroxypropyl-β-cyclodextrin (HP-β-CD) content. SEC purity decreased from 99.4% to 97.1%–97.4% (ΔACT 6M: ~2.3%), with E13-F4 (5% HP-β-CD) showing the best SEC purity. Reduced CE purity decreased from 96.4% to 89.2%–91.3% (ΔACT 6M: ~6.4%), with E13-F1–E13-F4 showing superior purity, all >90%. The content of charge isomers changed significantly, especially the basic peak content, which increased significantly, while the main peak content decreased by approximately 20%. Among the formulations, E13-F4 was superior. After 6 months of accelerated storage, all quality indicators were generally acceptable. These results suggest that, in the existing formulation systems, the accelerated stability quality changes when the GIPR antibody fusion protein concentration is maintained at 120 mg / mL are generally within an acceptable range. Among the formulations, the E13-F4 formulation is the best and its stability data are basically equivalent to those of the E12-F1 formulation with a GIPR antibody fusion protein concentration of 120 mg / mL in Example 12.

[0348] After being stored under long-term conditions for 6 months, the appearance, visible foreign matter, and DLS particle size of the six candidate formulations did not change significantly. The SEC purity of the samples decreased slightly, from 99.3% to 98.5%–98.6%. The purity of reduced CE and the content of acid-base isomers remained relatively stable, with no significant differences among the formulations. The results indicate that the GIPR antibody fusion protein maintained good long-term stability at a concentration of 120 mg / mL under the tested formulation systems. The viscosities of the formulation samples ranged from 21.8 cP to 47.9 cP, with the E13-F4 formulation having the lowest viscosity at 21.8 cP, which was comparable to or slightly better than E12-F1 in Example 12 (120 mg / mL: 21.4 cP in the first determination and 25.0 cP in the simultaneous comparison determination). Specific results are shown in Tables 33–38 and Figures 20–23.

[0349] Summary of Example 13:

[0350] Based on the studies in Examples 11 and 12, the type and concentration of surfactant in the formulation were further optimized. The final formulation for a GIPR antibody fusion protein concentration of 120 mg / mL can also be selected by removing N,N-dimethylacetamide and replacing it with hydroxypropyl-β-cyclodextrin (HP-β-CD), thereby increasing the GIPR antibody fusion protein concentration to 120 mg / mL. Therefore, an optional formulation (i.e., 140 mM histidine hydrochloride, 28 mM aspartic acid, 120 mM arginine, 20 mM methionine, 0.15% poloxamer 338, 5% HP-β-CD, pH 6.4) can support the development and production of a GIPR antibody fusion protein formulation with a concentration of 120 mg / mL.

[0351] Table 33 Summary of high-temperature stability results (partial data E13-F1~E13-F3)

[0352] Table 34 Summary of high-temperature stability results (partial data E13-F4~E13-F6)

[0353] Table 35 Summary of Accelerated Stability Results (Partial Data E13-F1~E13-F3)

[0354] Table 36 Summary of Accelerated Stability Results (Partial Data E13-F4~E13-F6)

[0355] Table 37 Summary of long-term stability results (partial data E13-F1~E13-F3)

[0356] Table 38 Summary of long-term stability results (partial data E13-F4~E13-F6)

[0357] The above embodiments are provided to fully disclose and illustrate to those skilled in the art how to manufacture and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications that are obvious to those skilled in the art are within the scope of the claims herein.

Claims

1. A pharmaceutical stable solution formulation of a GIPR antibody fusion protein, characterized in that: The GIPR antibody fusion protein comprises a GLP-1 peptide and a GIPR antibody moiety, wherein the GLP-1 peptide and the GIPR antibody moiety are linked, optionally directly linked or linked via a linker, and the GIPR antibody fusion protein has one or more of the following characteristics: a) the GLP-1 peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 or SEQ ID NO: 2 or a derivative, variant, fragment and mutant of the amino acid sequence; b) the GIPR antibody moiety comprises a light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3 or SEQ ID NO: 4 or a derivative, variant, fragment and mutant of the amino acid sequence; c) the GIPR antibody moiety comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8 or a derivative, variant, fragment and mutant of the amino acid sequence; d) the linker comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12; e) the GLP-1 peptide is linked to the N-terminus of the light chain and / or to the N-terminus of the heavy chain of the GIPR antibody moiety; or f) the GIPR antibody fusion protein comprises an amino acid sequence combination selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO:

8.

2. The pharmaceutical stable solution formulation of claim 1, characterized in that comprise the following components: wherein the first and second pharmaceutically acceptable protective agents are independently selected from the group consisting of arginine, arginine hydrochloride, histidine, histidine hydrochloride, cystine, cysteine, methionine, aspartic acid, lysine, glycine, tryptophan, leucine, isoleucine, phenylalanine, threonine, glutamic acid, serine, salts or derivatives thereof, sucrose, trehalose, sorbitol and mannitol.

3. The pharmaceutical stable solution formulation of claim 2, wherein, The first and second pharmaceutically acceptable protective agents are independently selected from the group consisting of a) arginine or arginine hydrochloride, b) histidine or histidine hydrochloride, c) methionine or a derivative thereof, and d) aspartic acid or a derivative thereof.

4. The pharmaceutical stable solution formulation of any one of claims 1-3, wherein the first pharmaceutically acceptable protective agent and the second pharmaceutically acceptable protective agent are in combination: a) arginine or arginine hydrochloride and b) aspartic acid or a derivative thereof; wherein, The concentration of the arginine or arginine hydrochloride in the formulation is 1-500 mM, optionally 1-400 mM, 1-300 mM, 1-200 mM, 1-100 mM, 75-120 mM, 75-175 mM or 100-200 mM, and the concentration of the aspartic acid or a derivative thereof in the formulation is 1-500 mM, optionally 1-400 mM, 1-300 mM, 1-200 mM, 1-100 mM, 100-200 mM, 1-28 mM or 1-50 mM.

5. The pharmaceutical stable solution formulation of any one of claims 1-4, wherein the pH buffering agent is selected from the group consisting of histidine, histidine hydrochloride, arginine, arginine hydrochloride, aspartic acid, sodium hydroxide, potassium hydroxide, sodium phosphate monobasic, alkali compounds of sodium phosphate dibasic, phosphates, acetates, citrates, barbiturates, tris, borates, histidates, and succinates.

6. The pharmaceutical stable solution formulation of any one of claims 1-5, wherein the pH buffering agent is histidine or histidine hydrochloride at a concentration of 1-500 mM, optionally, 1-400 mM, 1-300 mM, 1-200 mM, 1-140 mM, 1-100 mM, 100-200 mM, 25-100 mM, or 1-50 mM, which provides a pH buffering range of pH 4.0-9.0, optionally, pH 5.0-8.0, pH 5.5-7.5, or pH 6.0-7.

0.

7. The pharmaceutical stable solution formulation of any one of claims 1-6, wherein the pharmaceutical stable solution formulation further comprises a pharmaceutically acceptable antioxidant, optionally, the pharmaceutically acceptable antioxidant is selected from one or more of the group consisting of ethylenediaminetetraacetic acid, disodium ethylenediaminetetraacetate, diethylenetriaminepentaacetic acid, methionine, cysteine and its derivatives, and amino acids or metal chelators.

8. The pharmaceutical stable solution formulation of any one of claims 1-7, wherein the pharmaceutically acceptable antioxidant comprises methionine at a concentration of 1-500 mM, optionally, 1-400 mM, 1-300 mM, 1-200 mM, 1-100 mM, 1-50 mM, or 1-20 mM.

9. The pharmaceutical stable solution formulation of any one of claims 1-8, wherein the pharmaceutical stable solution formulation further comprises a pharmaceutically acceptable surfactant, optionally, the surfactant is selected from one or more of the group consisting of polysorbates (optionally, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and polyoxyethylene sorbitan fatty acid esters), poloxamers (optionally, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, poloxamer 407), N,N-dimethylacetamide, hydroxypropyl-beta-cyclodextrin, and polyoxyethylene polyoxypropylene ether block copolymers.

10. The pharmaceutical stable solution formulation of any one of claims 1-9, wherein the pharmaceutically acceptable surfactant comprises a poloxamer at a concentration of 0.01% to 10% (w / v), optionally, 0.01% to 9%, 0.01% to 8%, 0.01% to 7%, 0.01% to 6%, 0.01% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, 0.01% to 0.8%, 0.01% to 0.6%, 0.01% to 0.5%, 0.01% to 0.4%, 0.01% to 0.3%, 0.01% to 0.25%, 0.025% to 0.25%, 0.05% to 0.25%, 0.1% to 0.25%, or 0.15% to 0.25% (w / v) in the formulation.

11. The pharmaceutical stable solution formulation of any one of claims 1-10, wherein the pharmaceutically acceptable surfactant further comprises N,N-dimethylacetamide at a concentration of 0.01% to 10% (w / v), optionally, 0.01% to 9%, 0.01% to 8%, 0.01% to 7%, 0.01% to 6%, 0.01% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, 0.01% to 0.8%, 0.01% to 0.6%, 0.01% to 0.5%, 0.01% to 0.4%, 0.01% to 0.3%, 0.01% to 0.25%, 0.025% to 0.25%, 0.05% to 0.25%, 0.1% to 0.25%, or 0.15% to 0.25% (w / v) in the formulation.

12. The pharmaceutical stable solution formulation of any one of claims 1-10, wherein the pharmaceutically acceptable surfactant further comprises hydroxypropyl-β-cyclodextrin at a concentration of 0.01% to 10% (w / v), optionally, 0.01% to 9%, 0.01% to 8%, 0.01% to 7%, 0.01% to 6%, 0.01% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, 0.01% to 0.8%, 0.01% to 0.6%, 0.01% to 0.5%, 0.01% to 0.4%, 0.01% to 0.3%, 0.01% to 0.25%, 0.025% to 0.25%, 0.05% to 0.25%, 0.1% to 0.25%, or 0.15% to 0.25% (w / v) in the formulation.

13. The pharmaceutical stable solution formulation according to any one of claims 1 to 12, characterized in that comprise the following components:

14. The pharmaceutical stable solution formulation according to any one of claims 1 to 12, characterized in that comprise the following components:

15. The pharmaceutical stable solution formulation according to any one of claims 1 to 12, characterized in that comprise the following components:

16. The pharmaceutical stable solution formulation according to any one of claims 1 to 12, characterized in that comprise the following components:

17. The pharmaceutical stable solution formulation of claim 16, characterized in that comprise the following components:

18. The pharmaceutical stable solution formulation of claim 16, characterized in that comprise the following components:

19. The pharmaceutical stable solution formulation according to any one of claims 1 to 12, characterized in that comprising the following components:

20. The pharmaceutical stable solution formulation of claim 19, characterized in that comprise the following components:

21. The pharmaceutical stable solution formulation of claim 19, characterized in that comprise the following components:

22. The pharmaceutical stable solution formulation according to any one of claims 1 to 12, characterized in that comprise the following components:

23. The pharmaceutical stable solution formulation of claim 22, characterized in that comprise the following components:

24. The pharmaceutical stable solution formulation of claim 22, wherein comprise the following components:

25. The pharmaceutical stable solution formulation of claims 1-24, characterized by: The GIPR antibody fusion protein comprises a combination of amino acid sequences selected from the group consisting of SEQ ID NO: 13 and SEQ ID NO:

8.

26. Use of the pharmaceutical stable solution formulation of any one of claims 1-25 for the manufacture of a medicament for treating, preventing, or ameliorating nonalcoholic fatty liver disease or nonalcoholic steatohepatitis.

27. Use of the pharmaceutical stable solution formulation according to claims 1-25 for the preparation of a medicament for the treatment, prevention or amelioration of type 2 diabetes mellitus and conditions associated with type 2 diabetes mellitus.

28. Use of the pharmaceutical stable solution formulation according to claims 1-25 for the preparation of a medicament for the treatment, prevention or amelioration of obesity and conditions associated with obesity.

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