Stabilized solution preparation of pharmaceutical glp-1r antibody fusion protein

Abstract

The invention discloses a stable solution preparation of medicinal GLP-1R antibody fusion protein, which comprises a therapeutically effective dose of GLP-1R antibody fusion protein, amino acid, a surfactant and a buffer system, the final concentration of the amino acid is 1-500mM, the final concentration of the surfactant is 0.01%-0.5%, and the pH value of the stable solution preparation is 5.0-8.0. The invention has the advantages of stable performance, long in-vivo half-life period and good treatment effect, and can be used for treating diabetes, obesity and related diseases thereof.

Description

(19) State Intellectual Property Office (12) Invention Patent Application (10) Application Publication Number (43) Application Publication Date (21) Application Number 202610244442.3 (22) Application Date 2016.02.03 (66) Domestic Priority Data 201510071304.1 2015.02.11 CN (62) Divisional Application Data 201610077253.8 2016.02.03 (71) Applicant Hongyun Huaning (Hangzhou) Biomedical Co., Ltd. Address 302, 3rd Floor, Building 2, Dongguan High-tech Industrial Park, No. 288 Qiuyi Road, Binjiang District, Hangzhou City, Zhejiang Province, 310052 (72) Inventors Zhang Chengzhang, Hua Fankesuo, Guo Yongjing, Shuqian (74) Patent Agency Beijing Junhe Law Firm 11517 Patent Attorney Wu Yu, Gu Yunfeng (51) Int.Cl. A61K 38 / 26(2006.01) A61K 47 / 68(2017.01) A61K 9 / 08(2006.01) A61K 47 / 12(2006.01) A61K 47 / 26(2006.01) A61K 47 / 18(2017.01) A61P 3 / 04(2006.01) A61P 3 / 10(2006.01) A61P 1 / 00(2006.01) (54) Invention Title: A Stable Solution Formulation of Pharmaceutical GLP-1R Antibody Fusion Protein (57) Abstract: This invention discloses a stable solution formulation of pharmaceutical GLP-1R antibody fusion protein, comprising a therapeutically effective dose of GLP-1R antibody fusion protein, amino acids, surfactants, and a buffer system. The final concentration of the amino acid is 1-500 mM, the final concentration of the surfactant is 0.01%-0.5%, and the pH value of the stable solution preparation is 5.0 to 8.0. This invention has stable performance, a long half-life in vivo, and good therapeutic effects, and can be used for the treatment of diabetes, obesity, and related diseases. Claims: 1 page; Description: 11 pages; Sequence Listing (electronic publication) CN 121927035 A 2026.04.28 CN 1 21 92 70 35 A 1. A stable solution preparation of a pharmaceutical GLP-1R antibody fusion protein, characterized in that it comprises a therapeutically effective dose of GLP-1R antibody fusion protein, amino acids, a surfactant, and a buffer system, wherein the final concentration of the amino acid is 1-500 mM, the final concentration of the surfactant is 0.01-0.5%, and the pH value of the stable solution preparation is 5.0 to 8.0. 2. The stable solution formulation according to claim 1, characterized in that: the final concentration of the amino acid is 80-200 mM, the final concentration of the surfactant is 0.01%-0.2%, and the buffer system is a citrate buffer solution.The stable solution formulation has a pH value of 5.5 to 7.0. 3. The stable solution formulation according to claim 2, characterized in that: the concentration of the citrate buffer is 5-30 mM. 4. The stable solution formulation according to claim 2, characterized in that: the amino acid is L-arginine, the final concentration of L-arginine is 100-180 mM, and the surfactant is Tween-80, the final concentration of Tween-80 is 0.05%-0.15%. 5. The stable solution formulation according to claim 1, characterized in that: the final concentration of the therapeutically effective dose of GLP-1R antibody fusion protein is 0.1 mg / mL-100 mg / mL. 6. The stable solution formulation according to claim 5, characterized in that: the final concentration of the therapeutically effective dose of GLP-1R antibody fusion protein is 5 mg / mL-40 mg / mL. 7. The stable solution formulation according to any one of claims 1-6, characterized in that: the amino acid sequence of the light chain variable domain of the GLP-1R antibody fusion protein is selected from one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the amino acid sequence of the heavy chain variable domain is selected from one of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. 8. The stable solution formulation according to claim 7, characterized in that: the amino acid sequence of the light chain constant domain of the GLP-1R antibody fusion protein is SEQ ID NO:10 or SEQ ID NO:11, and the amino acid sequence of the heavy chain constant domain is SEQ ID NO:12. 9. The stable solution formulation according to claim 1, characterized in that: it is used to treat diabetes or obesity. 10. The stable solution formulation according to claim 1, characterized in that: it is used to treat irritable bowel syndrome and other diseases that benefit from lowering plasma glucose, inhibiting gastric and / or intestinal motility, inhibiting gastric and / or intestinal emptying, or inhibiting food intake. Claims 1 / 1 Page 2 CN 121927035 A Stable Solution Formulation of a Pharmaceutical GLP-1R Antibody Fusion Protein Technical Field

[0001] This invention relates to the field of biomedical technology, and particularly to a stable solution formulation of a pharmaceutical GLP-1R antibody fusion protein.

[0002] Cross-Reference to Related Applications This application is a divisional application of Chinese Patent Application No. 201610077253.8, filed on February 3, 2016, entitled "A Stable Solution Formulation of a Pharmaceutical GLP-1R Antibody Fusion Protein".The original application claims priority and benefit from 201510071304.1, filed on February 11, 2015.

[0003] Reference to the sequence listing This application contains a sequence listing submitted electronically in XML format, named 087222_8021CN02_SeqL_ST26.xml, created on February 24, 2026, and approximately 13,231 bytes in size. The sequence listing contained in this XML formatted file is part of the specification and is incorporated herein by reference in its entirety. Background Art

[0004] GLP-1 analogs are used in clinical trials for the treatment of type II diabetes and obesity (Gallwitz B, European Endocrinol, 2015;11:21-5). GLP-1 induces a variety of biological effects, such as stimulating insulin secretion, inhibiting glucagon secretion, inhibiting gastric emptying, inhibiting gastric or intestinal motility, and inducing weight loss (Lund A et al, Eur J Intern Med 2014; 25:407-14). A notable feature of GLP-1 is its ability to stimulate insulin secretion without the associated risk of hypoglycemia, which is seen in the use of insulin therapy or certain types of oral therapy that work by promoting insulin secretion. The fact that naturally occurring GLP-1 is rapidly eliminated from the body and has a very short in vivo half-life limits the effectiveness of peptide therapies involving GLP-1.

[0005] Several approaches have been developed to prolong the in vivo half-life of GLP-1 and its analogues while maintaining biological activity (Verspohl EJ, Pharmacol Rev, 2012;64:A-AX), including fusing GLP-1 and its analogues with the Fc portion of immunoglobulin or human serum albumin (HSA). Our novel approach involves fusing GLP-1 with the intact GLP-1R antibody molecule (IgG). IgG prolongs the circulating half-life of the fusion molecule in vivo, which is 21 days in humans. While preserving the biological activity of GLP-1, the GLP-1R antibody fusion protein offers the stability advantage provided by the antibody molecule. Furthermore, the fusion protein formed from the GLP-1 and GLP-1R antibodies provides molecular targeting through the antibody molecule, thereby increasing the likelihood of GLP-1 binding to GLP-1R. Additionally, the immunogenicity of the antibody molecule is lower than other protein fusion partners, which is beneficial for a drug that requires long-term or even lifelong use.Low immunogenicity is an essential property.

[0006] These fusion proteins are generally produced in mammalian cells, such as CHO, SP2 / O, or NSO. However, the inventors observed that GLP-1R antibody fusion proteins produced in CHO cells are easily degraded by endogenous proteases or under specific cell culture physicochemical conditions, and this trend is more pronounced and rapid at low pH. Furthermore, it was found that GLP-1R antibody fusion proteins are more prone to polymerization at pH values ​​above 7. In addition, the inventors also found that GLP-1R antibody fusion proteins have low solubility under refrigerated conditions (2-8°C) in a single buffer solution (e.g., citrate buffer), which does not meet pharmaceutical dosage requirements. These problems are overcome in this invention by controlling pH, using specific combinations of formulation additives, and using specific concentrations of GLP-1R antibody fusion proteins. Instructions 1 / 11 Page 3 CN 121927035 A Summary of the Invention

[0007] The purpose of this invention is to provide a stable solution preparation of pharmaceutical GLP-1R fusion protein, which has stable performance, a long half-life in vivo, and good therapeutic effect, and can be used for the treatment of diabetes, obesity and related diseases.

[0008] The technical solution adopted by this invention to solve its technical problem is: A stable solution preparation of pharmaceutical GLP-1R antibody fusion protein, comprising a therapeutically effective dose of GLP-1R antibody fusion protein, amino acids, surfactants and a buffer system, wherein the final concentration of the amino acids is 1-500mM, the final concentration of the surfactant is 0.01%-0.5%, and the pH value of the stable solution preparation is 5.0 to 8.0.

[0009] To overcome the problems of instability of GLP-1R antibody fusion protein solution formulations at low pH values, easy polymerization at high pH values, and insufficient solubility of GLP-1R antibody fusion protein in a single buffer solution, this invention has developed a physically and chemically stable solution formulation containing GLP-1R antibody fusion protein, which further includes a buffer system, amino acids as stabilizers and osmotic pressure regulators, and surfactants. This stable solution formulation is stably stored at 25°C for at least 6 months. A further preferred stable solution formulation of this invention comprises GLP-1R antibody fusion protein at a final concentration of approximately 0.1-100 mg / mL, citrate buffer at a concentration of 5-30 mM, Tween-80 at a final concentration of 0.01%-0.2%, and L-arginine at a final concentration of 80-200 mM, with a pH of 5 to 8. This stable solution formulation improves the solubility of GLP-1R antibody fusion protein and its stability under special conditions, especially at high temperatures. The present invention also includes a method for treating diabetes, obesity, and related conditions, the method comprising administering the GLP-1R antibody fusion protein formulation described herein.

[0010] Preferably,The final concentration of the amino acid is 80-200 mM, the final concentration of the surfactant is 0.01%-0.2%, the buffer system is citrate buffer, and the pH of the stable solution preparation is 5.5 to 7.0.

[0011] Preferably, the concentration of the citrate buffer is 5-30 mM.

[0012] Preferably, the amino acid is L-arginine, the final concentration of L-arginine is 100-180 mM, the surfactant is Tween-80, and the final concentration of Tween-80 is 0.05%-0.15%.

[0013] Preferably, the final concentration of the therapeutically effective dose of GLP-1R antibody fusion protein is 0.1 mg / mL-100 mg / mL.

[0014] Preferably, the final concentration of the therapeutically effective dose of GLP-1R antibody fusion protein is 5 mg / mL-40 mg / mL.

[0015] Preferably, the amino acid sequence of the light chain variable domain of the GLP-1R antibody fusion protein is selected from one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the amino acid sequence of the heavy chain variable domain is selected from one of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9.

[0016] Preferably, the amino acid sequence of the light chain constant domain of the GLP-1R antibody fusion protein is SEQ ID NO:10 or SEQ ID NO:11, and the amino acid sequence of the heavy chain constant domain is SEQ ID NO:12.

[0017] The GLP-1R antibody fusion protein of the present invention comprises GLP-1 or an analogue thereof, which is fused to the N-terminus of the light chain of the GLP-1R antibody via a peptide linker at its C-terminus. This antibody fusion protein has similar or better biological activity and a prolonged half-life compared to GLP-1 and its analogues. The preferred GLP-1R antibody fusion protein has a light chain variable domain sequence comprising all or part of SEQ ID NO: 1, 2, 3, 4, 5, or 6; a light chain constant domain sequence comprising all or part of SEQ ID NO: 10 or 11; a heavy chain variable domain sequence comprising all or part of SEQ ID NO: 7, 8, or 9; and a heavy chain constant domain sequence comprising all or part of SEQ ID NO: 12. More preferably, the light chain variable domain sequence of the GLP-1R antibody fusion protein is substantially SEQ ID NO: 1, 2, 3, 4, 5, or 6; the light chain constant domain sequence is substantially SEQ ID NO: 10 or 11; and the heavy chain variable domain sequence is substantially SEQ ID NO: 7, 8, or 9.Its heavy chain constant domain sequence is essentially SEQ ID NO:12. The most preferred GLP-1R antibody fusion protein has a light chain variable domain sequence that is exactly as specified on page 2 / 11 of the specification, CN 121927035 A SEQ ID NO:1, 2, 3, 4, 5 or 6, its light chain constant domain sequence that is exactly as specified in SEQ ID NO:10 or 11, its heavy chain variable domain sequence that is exactly as specified in SEQ ID NO:7, 8 or 9, and its heavy chain constant domain sequence that is exactly as specified in SEQ ID NO:12.

[0018] The stable solution formulation of the present invention is used to treat diabetes or obesity.

[0019] The stable solution formulation of the present invention is used to treat irritable bowel syndrome and other diseases that benefit from lowering plasma glucose, inhibiting gastric and / or intestinal motility, inhibiting gastric and / or intestinal emptying, or inhibiting food intake.

[0020] The beneficial effects of the present invention are: stable performance, long half-life in vivo, good therapeutic effect, and can be used for the treatment of diabetes, obesity, irritable bowel syndrome and related diseases. Detailed Embodiments

[0021] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0022] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the field. Unless otherwise specified, the methods in the following embodiments are conventional methods in the field.

[0023] The preparation of the GLP-1R antibody fusion protein of the present invention is described in the applicant's prior patent application: CN104371019A, WO 2015 / 021871.

[0024] Biological activity refers to the ability of the GLP-1R antibody fusion protein to bind to GLP-1R and activate GLP-1R in vivo to induce a response. The response includes, but is not limited to, promoting insulin secretion, inhibiting glucagon secretion, suppressing appetite, reducing weight, inducing satiety, inhibiting pancreatic β-cell apoptosis, and inducing pancreatic β-cell proliferation.

[0025] The stable solution formulation of pharmaceutical-grade GLP-1R antibody fusion protein comprises GLP-1R antibody fusion protein in a buffer solution, which further contains amino acids and surfactants as stabilizers and osmotic regulators. The stable solution formulation maintains stability at 25°C for at least 6 months, with more precise stability maintenance periods at 25°C of 6 to 8 months, 6 to 12 months, 6 to 18 months, 6 to 24 months, 8 to 12 months, 8 to 18 months, 8 to 24 months, 12 to 18 months, 12 to 24 months, and 18 to 24 months.

[0026] The buffer system used in this invention is composed of one or more of the following organic or inorganic compounds, including but not limited to: citric acid and 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, salts of phtalic acid, phosphoric acid, phosphate, hydrochloric acid, Tris, thomethamine, and amino acids, including but not limited to histidine, arginine, and glycine.

[0027] In this invention, an osmotic pressure regulator is defined as a substance that, upon addition, can increase the osmotic pressure of a formulation. The osmotic pressure regulator involved in this invention is a free amino acid, including but not limited to the following: arginine, histidine, methionine, lysine, ornithine, leucine, isoleucine, alanine, glycine, glutamic acid, and aspartic acid. The preferred basic amino acid is arginine, histidine, lysine, or a combination thereof. The amino acid can be added in the form of an amino acid salt, and the added amino acid can be a D-conformation amino acid, such as D-arginine, or an L-conformation amino acid, such as L-arginine.

[0028] Within the scope of this invention, the addition of amino acids is not limited to acting as osmotic pressure regulators, but also as stabilizers.It includes, but is not limited to, arginine, histidine, methionine, lysine, ornithine, leucine, isoleucine, alanine, glycine, glutamine, glutamic acid, asparagine, aspartic acid, phenylalanine, tyrosine, serine, proline, and tryptophan. The amino acids, used as stabilizers and osmotic pressure regulators, are preferably present at concentrations of 1 to 500 mM in this invention.

[0029] Surfactants are defined in this invention as organic compounds with an amphoteric structure, i.e., they contain groups with different soluble properties; typically, such compounds contain both oil-soluble hydrocarbon groups and water-soluble ionic groups. The surfactants involved in this invention include, but are not limited to, sorbitan fatty acid esters; for example,Sorbitan monocaprylate, sorbitan monolaurate, sorbitan monopalmitate; sorbitan trioleate; glycerine fatty acid esters (e.g., glycerine monocaprylate, glycerine monomyristate, glycerine monostearate); polyglycerine fatty acid esters (e.g., decaglycerine monostearate, decaglyceryl distearate, decaglyceryl monolinoleate); polyoxyethylene sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate). (monolarate), of which polyoxyethylene (20) sorbitan monolaurate is Tween-20, polyoxyethylene sorbitan monopalmitate is Tween-40, polyoxyethylene sorbitan monooleate is Tween-80, and polyoxyethylene sorbitan monostearate is polyoxyethylene (80) sorbitan monooleate.Among them, polyoxyethylene (60) sorbitan monostearate is Tween-60; polyoxyethylene sorbitan trioleate is Tween-85; polyoxyethylene sorbitan tristearate is Tween-65; polyoxyethylene sorbitol fatty acid esters (e.g., polyoxyethylene sorbitol tetrastearate, polyoxyethylene sorbitol tetraoleate); polyoxyethylene glycerine fatty acid esters (e.g., polyoxyethylene glyceryl monostearate). Monostearate), polyoxyethylene glycol fatty acid esters (e.g., polyoxyethylene polyethylene glycol distearate); polyoxyethylene alkyl ethers (e.g., polyoxyethylene lauryl ether); polyoxyethylene polyoxypropylene alkyl ethers (e.g., polyoxyethylene polyoxypropylene glycol), polyoxyethylene polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether; polyoxyethylene alkylphenyl ethers (e.g., polyoxyethylene polyoxypropylene glycol, polyoxypropylene propyl ether, polyoxyethylene polyoxypropylene cetyl ether, polyoxyethylene polyoxypropylene cetyl ether, polyoxyethylene polyoxypropylene cetyl ether, polyoxyethylene polyoxypropylene cetyl ether, polyoxyethylene polyoxypropylene alkyl phenyl ether, polyoxyethylene polyoxypropylene alkyl ...Examples include: nonylphenyl ether; hydrogenated castor oils; beeswax derivatives (e.g., sorbitol beeswax); lanolin derivatives and fatty acid amides (e.g., stearic acid amide); and C10-C18 alkyl sulfates (e.g., sodium cetyl sulfate, sodium lauryl sulfate, sodium oleyl sulfate). Ammonium polyoxyethylene C10-C16 alkyl ether sulfate with an average of 2 to 4 moles of ethylene oxide units added, such as sodium polyoxyethylene lauryl sulfate; C1-C18 alkyl sulfosuccinate ester salts, such as sodium lauryl sulfosuccinate ester; and natural surfactants such as lecithin, glycerophospholipids, and sphingophospholipids.For example, sphingomyelin and sucrose esters of C12-C18 fatty acids. The surfactant formulation used in this invention may contain one or more of the surfactant components described above. Suitable surfactants are polyoxyethylene sorbitan fatty acid ester compounds, such as Tween-20, Tween-40, Tween-60, and Tween-80.

[0030] The stable solution formulation of pharmaceutical GLP-1R fusion protein contains a final concentration of about 0.1 to about 100 mg / mL of GLP-1R antibody fusion protein. The preferred concentration (mg / mL) of the GLP-1R antibody fusion protein is in the range of about 0.1 to 1, 1 to 5, 5 to 10, 5 to 20, 10 to 20, 20 to 30, 20 to 40, 30 to 40, 40 to 50, 50 to 60, 60 to 70, 70 to 80, 80 to 90, and 90 to 100 mg / mL. Particularly preferred concentrations (mg / mL) of the GLP-1R antibody fusion protein are about 0.1, about 0.25, about 0.5, about 1, about 2, about 3, about 4, about 5, about 6.5, about 8, about 10, about 12.5, about 15, about 17.5, about 20, about 22.5, about 25, about 27.5, about 30, about 32.5, about 35, about 37.5, about 40, about 45, about 50, about 55, about 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, and about 100 mg / mL.

[0031] A preferred buffer system for the stable solution formulation is citrate.Its concentration range is about 5 to 30 mM. More preferred citrate concentration (mM) ranges are about 5 to 25, 5 to 20, 5 to 15, 5 to 12.5, 5 to 10, 7.5 to 30, 7.5 to 25, 7.5 to 20, 7.5 to 15, 7.5 to 12.5, 7.5 to 10, 8 to 30, 8 to 25, 8 to 20, 8 to 15, 8 to 12.5, 8 to 11, 8 to 10, 9 to 30, 9 to 25, 9 to 20, 9 to 15, 9 to 12.5, 10 to 30, 10 to 25, 10 to 20, 10 to 17.5, 10 to 15, 10 The pH range is approximately 12.5, 12.5 to 30, 12.5 to 25, 12.5 to 20, 12.5 to 15, 15 to 30, 15 to 25, 15 to 20, 17.5 to 30, 17.5 to 25, 17.5 to 22.5, 17.5 to 20, 20 to 30, 20 to 27.5, 20 to 25, 20 to 22.5, 22.5 to 30, 22.5 to 27.5, 22.5 to 25, 25 to 30, 25 to 27.5, 27.5 to 30. More preferably, the citrate concentration is about 5 to 20 mM. Particularly preferred, the citrate concentration is about 10, 10.0, 20, or 20.0 mM.

[0032] The pH adjustment range of the stable solution formulation of the pharmaceutical GLP-1R fusion protein of the present invention is about 5 to 8. This pH range provides acceptable stability to the formulation, maintaining the solubility of the GLP-1R antibody fusion protein and its insulin-secreting activity, and is acceptable for parenteral administration. This can be achieved by adding an acid, such as HCl, or a base, such as NaOH.Instructions for use, page 5 / 11, CN 121927035 A: Adjust the pH to the desired pH, or a combination of citrate buffer and citric acid can be added to obtain the desired buffer concentration and desired pH. Preferred pH ranges are 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. 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. A further preferred pH range is 6 to 7, and a particularly preferred pH value is about 6.5 or 6.50.

[0033] In this invention, L-arginine is preferably used as an osmotic pressure regulator and stabilizer in the stable solution formulation of pharmaceutical GLP-1R fusion protein, with a concentration range of 1 to 500 mM, to stabilize the GLP-1R antibody fusion protein and increase its solubility.The osmotic pressure of the formulation is adjusted to make it suitable for parenteral administration. The preferred concentration range of L-arginine is 80 to 200 mM. More preferably, the concentration ranges are 80 to 190, 80 to 180, 80 to 170, 80 to 160, 80 to 150, 80 to 140, 80 to 130, 80 to 120, 80 to 110, 80 to 100, 80 to 90, 90 to 200, 90 to 190, 90 to 180, 90 to 170, 90 to 160, 90 to 150, 90 to 140, 90 to 130, 90 to 120, 90 to 110, 90 to 100, 100 to 200, 100 to 190, and 100 to 180. 100-170, 100-160, 100-150, 100-140, 100-130, 100-120, 100-110, 110-200, 110-190, 110-180, 110-170, 110-160, 110-150, 110-140, 110-130, 110-120, 120-200 120-190, 120-180, 120-170, 120-160, 120-150, 120-140, 120-130, 130-200, 130-190, 130-180, 130-170, 130-160, 130-150, 130-140, 140-200, 140-190, 140-180 140 to 170, 140 to 160, 140 to 150, 150 to 200, 150 to 190, 150 to 180, 150 to 170, 150 to 160, 160 to 200, 160 to 190, 160 to 180, 160 to 170, 170 to 200, 170 to 190, 170 to 180, 180 to 190, 190 to 200. More preferably, the L-arginine concentration is 100 to 180 mM. Particularly preferred is about 138 mM or about 138.0 mM.

[0034] The present invention preferably uses Tween-80 as a surfactant in a stable solution formulation of pharmaceutical GLP-1R fusion protein, with a concentration range of 0.01% to 0.5%. The preferred concentration range of Tween-80 is about 0.01% to 0.2%, and the preferred concentration range is determined by the combination with GLP-1R antibody fusion protein and arginine.In order to minimize the formation of soluble aggregates and insoluble particles. Further preferred concentration ranges for Tween-80 are approximately 0.01% to 0.2%, 0.01% to 0.15%, 0.01% to 0.1%, 0.01% to 0.05%, 0.01% to 0.025%, 0.025% to 0.2%, 0.025% to 0.15%, 0.025% to 0.1%, 0.025% to 0.075%, 0.025% to 0.05%, 0.05% to 0.2%, 0.05% to 0.15%, 0.05% to 0.1%, 0.05% to 0.075%, 0.075% to 0.2%, 0.075% to 0.15%, 0.075% to 0.1%, 0.1% to 0.2%, 0.1% to 0.15%, and 0.15% to 0.2%. More preferably, the Tween-80 concentration ranges from about 0.05% to about 0.15%. Particularly preferred, the Tween-80 concentration is about 0.1%.

[0035] A particularly preferred stable solution formulation of pharmaceutical-grade GLP-1R fusion protein comprises GLP-1R antibody fusion protein at a concentration ranging from about 5 to about 20 mg / mL, citrate buffer at a concentration of about 20 mM, Tween-80 at a concentration of about 0.1%, L-arginine at a concentration of about 138 mM, and a pH of about 6.5. Another particularly preferred stable solution formulation of pharmaceutical-grade GLP-1R fusion protein comprises GLP-1R antibody fusion protein at a concentration ranging from about 20 to about 40 mg / mL, citrate buffer at a concentration of about 20 mM, Tween-80 at a concentration of about 0.1%, L-arginine at a concentration of about 138 mM, and a pH of about 6.5. Another particularly preferred stable solution formulation comprises GLP-1R antibody fusion protein at a concentration ranging from about 5 to about 20 mg / mL, citrate buffer at a concentration ranging from about 5 to 20 mM, Tween-80 at a concentration ranging from about 0.05% to 0.15%, L-arginine at a concentration ranging from about 100 to 180 mM, and a pH range of about 6.0 to 7. Another particularly preferred stable solution formulation comprises GLP-1R antibody fusion protein at a concentration ranging from about 20 to about 40 mg / mL, citrate buffer at a concentration ranging from about 5 to 20 mM, Tween-80 at a concentration ranging from about 0.05% to 0.15%, L-arginine at a concentration ranging from about 100 to 180 mM.And the pH range is approximately 6.0 to 7.

[0036] The stable solution formulation 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 the 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.

[0037] The stable solution formulation of the present invention can be used to treat individuals with 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 GLP-1R antibody fusion protein is the amount that, when administered to an individual requiring GLP-1R stimulation, causes the expected therapeutic and / or preventive effects without causing undesirable side effects.

[0038] Preferably, the GLP-1R antibody fusion protein is administered once every two weeks or once weekly. Depending on the disease being treated, more frequent administration of the GLP-1R antibody fusion protein may be required, for example, two to three times per week.

[0039] The present invention will be described by way of non-limiting example with reference to the following examples.

[0040] Description of detection method: Reporter gene assay to detect the function of GLP-1R antibody fusion protein in activating GLP-1R in vitro.

[0041] 20,000 CHO-DHFR- cells co-expressing hGLP-1R-CRE-Luciferase were seeded into 96-well cell culture plates and cultured overnight at 37°C. The next day, the supernatant was removed, the cell surface was washed twice with serum-free medium, residual liquid was aspirated, and 100 μl of serum-free medium was added to dilute the GLP-1R antibody fusion protein sample and control, and incubated at 37°C for 4 hours. After incubation, 100 μl of Promega's Bright Glo chemiluminescent substrate was added. Finally, the cell lysate was transferred to a white 96-well plate, and the relative fluorescence intensity was read on a Molecular Devices SpectraMax L microplate reader. Data was processed using GraphPad to obtain the dose-response curve and EC50 value of logarithmic concentration versus response intensity.

[0042] The purity of the GLP-1R antibody fusion protein was determined by size exclusion high-performance liquid chromatography (SEC-HPLC).

[0043] SEC-HPLC was used to detect the formation of polymers (soluble aggregates) and the corresponding monomer loss of the GLP-1R antibody fusion protein. An Agilent 1100 series HPLC column was used, and the TSK-G3000SWxl high-performance liquid chromatography column was washed with a mobile phase containing 200 mM phosphate and pH 6.8 at a column temperature of 25°C.Until the UV absorption curve remains at the baseline. Inject 50 μL of a diluent of the stable solution formulation of pharmaceutical GLP-1R fusion protein with a concentration of 1 to 3 mg / mL (diluted with the original sample using a mobile phase). After loading, continue running at a mobile phase flow rate of 0.5 mL / min and record the UV 280 nm absorption curve. After completing the method, integrate the absorption peaks of the main peak (monomer), dimer, and polymer at UV 280 nm and calculate the proportion of the total area occupied by the main peak, which is the purity of the sample.

[0044] Example 1: The effect of pH on the stability of GLP-1R antibody fusion protein: pH can affect the solubility and stability of GLP-1R antibody fusion protein and is one of the most critical parameters in the formulation. We evaluated the effect of pH on the stability of GLP-1R antibody fusion protein through Example 1. Using the amount of soluble polymers (dimers and polymers) formed as a standard, we selected the proportion of soluble polymers formed (polymer %) in each sample after 1 week and 3 weeks at 45°C to compare the stability of GLP-1R antibody fusion protein at different pH values.

[0045] Stable solution formulations of pharmaceutical GLP-1R fusion protein were prepared according to Table 1: Table 1 GLP-1R antibody fusion protein formulations under different pH conditions, instructions for use, 7 / 11 pages, 9 CN 121927035 A. The stable solution formulations of pharmaceutical GLP-1R fusion protein were sterilized and filtered through a 0.22 μm polyvinylidene 1,1-difluoroethylene (PVDF) membrane. The solution was stored at 45°C in 2 mL borosilicate glass vials until analysis or for a maximum of 3 weeks. The proportion of soluble polymers in each sample was determined by SEC-HPLC. Table 2 shows that under the particularly preferred pH value of 6.5, the proportion of soluble polymers formed in the formulation was the lowest and the stability of the formulation was the best. It also shows that the stability tends to increase as the pH decreases.

[0046] Table 2 Proportion of soluble polymers formed in GLP-1R antibody fusion protein formulation under different pH conditions Example 2: Robustness study of preferred formulation We conducted a robustness study on the preferred formulation at a concentration of 10 mg / mL of GLP-1R antibody fusion protein to examine whether the deviation (±15%) of the three most important formulation components or factors (L-arginine, pH value and Tween-80) in the actual production process would have a significant impact on drug quality and stability. Based on the general rules of formulation research and the characteristics of GLP-1R antibody fusion protein,The change in monomer purity (i.e., the percentage of the main peak) was selected as the evaluation criterion.

[0047] GLP-1R antibody fusion protein formulations were prepared according to Table 3 for DOE experiments: Table 3 DOE Experiment GLP-1R Antibody Fusion Protein Formulation Sample Preparation Formulation Formulas were sterilized and filtered through a 0.22 μm polyvinylidene 1,1-difluoroethylene (PVDF) membrane. The formulations were stored in 2 mL glass vials at 40 °C until analysis or for up to 1 month. The purity values ​​of each formulation were determined by SEC-HPLC. Table 4 shows that within the range of pH 6 to 7, L-arginine 110 to 166 mM, and Tween-80 concentration 0.05% to 0.15%, the formulations showed comparable purity, with no significant differences, indicating that the preferred formulations are robust and that deviations in production do not significantly affect the stability of the actual formulations.

[0048] Table 4 Purity test results of various GLP-1R antibody fusion protein formulation samples in the DOE experiment Example 3: Effect of GLP-1R antibody fusion protein concentration on formulation stability We compared the performance of the preferred formulation at two different GLP-1R antibody fusion protein concentrations to examine whether the preferred formulation can provide acceptable stability for higher concentrations of GLP-1R antibody fusion protein, thus facilitating the practical application of higher doses of GLP-1R antibody fusion protein. We selected 10 and 20 mg / mL GLP-1R antibody fusion protein for comparison.

[0049] GLP-1R antibody fusion protein formulations were prepared according to Table 5 for the experiment: Table 5 GLP-1R antibody fusion protein formulations with different protein concentrations were prepared and sterilized by filtration through a 0.22 μm polyvinylidene 1,1-difluoroethylene (PVDF) membrane. The formulations were stored in 2 mL glass vials at 37 °C until analysis or up to 1 month. The purity values ​​of each formulation were determined by SEC-HPLC, and the biological activity was detected by reporter gene assay. Table 6 shows that the preferred formulation can provide high stability for GLP-1R antibody fusion protein at a concentration of 20 mg / mL. The change in purity at 37°C is similar to that at 10 mg / mL, with only a non-significant change. At the same time, there is no significant difference in biological activity between the two.

[0050] Table 6 Detection results of purity and biological activity of GLP-1R antibody fusion protein formulation at different protein concentrations. Instructions for use, page 9 / 11, CN 121927035 A Example 4: Vibration stability study of preferred formulation. We conducted a stability study of the preferred formulation under vibration conditions to examine whether the preferred formulation can provide stability for GLP-1R antibody fusion protein under vibration.The impact of sample transportation and daily carrying on it was assessed.

[0051] GLP-1R antibody fusion protein formulations were prepared for experiments according to Table 7: Table 7 GLP-1R antibody fusion protein formulations for vibration stability studies were prepared and sterilized by filtration through a 0.22 μm polyvinylidene 1,1-difluoroethylene (PVDF) membrane. The formulations were stored in 2 mL glass vials at 37 °C and shaken at 70 rpm on a shaker until analysis or up to 15 days. The purity values ​​of each formulation were determined by SEC-HPLC, and the biological activity was detected by reporter gene method. The results in Table 8 show that the stability and biological activity of GLP-1R antibody fusion protein remained stable under such vibration conditions.

[0052] Table 8 Detection results of purity and biological activity of GLP-1R antibody fusion protein formulation samples for vibration stability studies Example 5: Accelerated stability study of preferred formulation We conducted an accelerated stability study on the preferred formulation to more quickly examine the trend of GLP-1R antibody fusion protein stability over time under the preferred formulation.

[0053] GLP-1R antibody fusion protein formulations were prepared for experiments according to Table 9: Table 9 GLP-1R antibody fusion protein formulations for accelerated stability studies, 10 / 11 pages, 12 CN 121927035 A. The formulations were prepared and sterilized by filtration through a 0.22 μm polyvinylidene 1,1-difluoroethylene (PVDF) membrane. The formulations were stored in 2 mL glass vials at 25 °C until analysis or for a maximum of 6 months. The purity values ​​of each formulation were determined by SEC-HPLC, and the biological activity was detected by a reporter gene method. The results in Table 10 show that the GLP-1R antibody fusion protein formulations can withstand at least 6 months of storage at 25 °C without significant changes in purity and biological activity, demonstrating excellent stability.

[0054] Table 10 Results of purity and biological activity detection of GLP-1R antibody fusion protein formulation samples in accelerated stability studies N / A = Not tested.

[0055] The above-described embodiments are only a preferred embodiment of the present invention and are not intended to limit the present invention in any way.Other variations and modifications are possible without departing from the scope of the technical solutions described in the claims. Specification 11 / 11, page 13, CN 121927035. A ABSTRACT Disclosed is a stabilized solution preparation of a pharmaceutical GLP-1R antibody fusion protein, comprising therapeutically effective amounts of the GLP-1R antibody fusion protein, an amino acid, a surfactant, and a buffer system. The final concentration of the amino acid is 1-500 mM, the final concentration of the surfactant is 0.01%-0.5%, and the pH value of the stabilized solution preparation is from 5.0 to 8.0. The stabilized solution preparation of the present invention has a long half-life in vivo and good therapeutic effect and can be used in the treatment of diabetes and obesity, and conditions associated therewith.

Claims

1. A stable solution formulation of a pharmaceutical-grade GLP-1R antibody fusion protein, characterized in that, The formulation includes a therapeutically effective dose of GLP-1R antibody fusion protein, amino acids, surfactants, and a buffer system, wherein the final concentration of the amino acids is 1-500 mM, the final concentration of the surfactant is 0.01-0.5%, and the pH of the stable solution formulation is 5.0 to 8.

0.

2. The stable solution formulation according to claim 1, characterized in that: The final concentration of the amino acid is 80-200 mM, the final concentration of the surfactant is 0.01%-0.2%, the buffer system is a citrate buffer, and the pH of the stable solution preparation is 5.5 to 7.

0.

3. The stable solution formulation according to claim 2, characterized in that: The concentration of the citrate buffer is 5-30 mM.

4. The stable solution formulation according to claim 2, characterized in that: The amino acid is L-arginine, with a final concentration of 100-180 mM, and the surfactant is Tween-80, with a final concentration of 0.05%-0.15%.

5. The stable solution formulation according to claim 1, characterized in that: The final concentration of the therapeutically effective dose of GLP-1R antibody fusion protein is 0.1 mg / mL to 100 mg / mL.

6. The stable solution formulation according to claim 5, characterized in that: The final concentration of the therapeutically effective dose of GLP-1R antibody fusion protein is 5 mg / mL to 40 mg / mL.

7. The stable solution formulation according to any one of claims 1-6, characterized in that: The amino acid sequence of the light chain variable domain of the GLP-1R antibody fusion protein is selected from one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, and the amino acid sequence of the heavy chain variable domain is selected from one of SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:

9.

8. The stable solution formulation according to claim 7, characterized in that: The amino acid sequence of the light chain constant domain of the GLP-1R antibody fusion protein is SEQ ID NO:10 or SEQ ID NO:11, and the amino acid sequence of the heavy chain constant domain is SEQ ID NO:

12.

9. The stable solution formulation according to claim 1, characterized in that: It is used to treat diabetes or obesity.

10. The stable solution formulation according to claim 1, characterized in that: It is used to treat irritable bowel syndrome and other diseases that benefit from lowering plasma glucose, inhibiting gastric and / or intestinal motility, inhibiting gastric and / or intestinal emptying, or inhibiting food intake.