Method for producing long-acting drug conjugates by producing novel intermediates

Abstract

To provide a novel method for preparing a long-acting drug conjugate and a long-acting drug conjugate prepared using the method.SOLUTION: The present invention provides a compound having a structure depicted in a following chemical formula, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, binding to a physiologically active polypeptide. In the chemical formula, n is 200-250.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a novel intermediate for producing a long-acting drug conjugate, a composition containing the same, and a method for producing a long-acting drug conjugate using the same. [Background technology]

[0002] Because physiologically active polypeptides have low stability, are easily denatured, degraded by proteolytic enzymes in the blood, and are easily eliminated through the kidneys and liver, protein drugs containing physiologically active polypeptides as pharmacological components must be frequently administered to patients to maintain their blood concentration and potency. However, in the case of protein drugs that are mainly administered to patients in the form of injections, frequent injections to maintain the blood concentration of the physiologically active polypeptide cause significant pain to patients and result in high treatment costs. To solve these problems, continuous efforts have been made to improve the blood stability of protein drugs and maximize their efficacy by maintaining high and prolonged blood drug concentrations. Such sustained-release formulations of protein drugs must not only improve the stability of the protein drug, but also maintain a sufficiently high potency of the drug itself and not induce an immune response in patients.

[0003] To stabilize proteins and prevent their contact with protease enzymes and their loss due to elongation, a conventional method involves chemically attaching a highly soluble polymer, such as polyethylene glycol (PEG), to the surface of a protein drug. However, while the PEG method can increase the molecular weight of PEG and extend the in vivo duration of peptide drugs, the increased molecular weight leads to a significant decrease in the potency of the peptide drug and a decrease in reactivity with peptides, resulting in a decrease in yield.

[0004] In response to this, conjugates of immunoglobulin fragments and physiologically active polypeptides have been used as a method for increasing blood half-life, and research has been conducted from various angles to improve the manufacturing method (Korean Patent Publication No. 10-2014-0109342 (Patent Document 1)).

[0005] In particular, there is a continuing need to develop efficient manufacturing processes for long-acting drug conjugates by shortening the manufacturing process. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Korean Patent Publication No. 10-2014-0109342 [Patent Document 2] International Patent Publication No. 97 / 34631 [Patent Document 3] International Patent Publication No. 96 / 32478 [Non-patent literature]

[0007] [Non-Patent Document 1] H. Neurath, RL Hill, The Proteins, Academic Press, New York, 1979 Summary of the Invention [Problem to be solved by the invention]

[0008] One object of the present invention is to provide novel intermediates for the preparation of long-acting drug conjugates.

[0009] Another object of the present invention is to provide a composition for producing a sustained-release drug conjugate, which comprises the intermediate.

[0010] It is still another object of the present invention to provide a method for preparing a long-acting drug conjugate using the intermediate.

[0011] It is still another object of the present invention to provide a long-acting drug conjugate prepared by the above method. [Means for solving the problem]

[0012] One aspect of the present invention is a novel intermediate.

[0013] In one embodiment, the present invention relates to a compound having the structure of the following chemical formula (1), a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[0014] X-L1-(OCH2CH2)nO-L2-R···(1)

[0015] In the chemical formula (1), X is an immunoglobulin Fc region; L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 10 to 2400; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof.

[0016] The present invention relates to a compound, its stereoisomer, solvate, or pharmaceutically acceptable salt thereof according to the above specific example, wherein in said chemical formula (1), L1 is linear or branched C1-C6 alkylene, L2 is -a1-NHCO- or -a1-NHCO-a2-, a1 and a2 are each independently C1-C6 linear or branched alkylene, n is 200-250, and R is maleimide, or a compound, its stereoisomer, solvate, or pharmaceutically acceptable salt thereof.

[0017] The present invention relates to a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, wherein X is an immunoglobulin Fc region comprising a hinge sequence at its N-terminus.

[0018] The present invention relates to a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any one of the preceding specific examples, wherein X is an immunoglobulin Fc region comprising a hinge sequence mutated to have only one cysteine ​​residue by deletion of a portion of the amino acid sequence shown below:

[0019] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

[0020] The present invention relates to a compound, a stereoisomer, a solvate or a pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, wherein the hinge sequence comprises the amino acid sequence of SEQ ID NO: 8 (Ser-Cys-Pro) or SEQ ID NO: 9 (Pro-Ser-Cys-Pro).

[0021] The present invention relates to a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, wherein said L1 is bonded to an amine or thiol reactive group located at the terminus of X.

[0022] The present invention relates to a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any one of the above embodiments, wherein the compound has the structure of the following chemical formula (2):

[0023] TIFF0007752664000001.tif46146...(2)

[0024] In the chemical formula (2), n is 200-250.

[0025] The present invention relates to a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, wherein said X is an immunoglobulin Fc region derived from IgG, IgA, IgD, IgE, or IgM.

[0026] The present invention relates to a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, wherein said X is an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0027] The present invention relates to a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, wherein X is a dimeric immunoglobulin Fc region.

[0028] The present invention relates to a compound, a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, wherein said X is an immunoglobulin Fc region comprising the amino acid sequence of SEQ ID NO: 10.

[0029] The present invention relates to a compound, its stereoisomer, solvate or pharmaceutically acceptable salt thereof according to any one of the preceding embodiments, wherein said compound has a size of 40 to 250 kDa.

[0030] Another embodiment of the present invention is a composition for producing a long-acting drug conjugate, comprising the compound, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof.

[0031] In one embodiment, the present invention relates to a composition comprising a compound having the structure of the following chemical formula (1), a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein the drug is a physiologically active polypeptide:

[0032] X-L1-(OCH2CH2)nO-L2-R···(1)

[0033] In the chemical formula (1), X is an immunoglobulin Fc region; L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 10 to 2400; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof.

[0034] The composition according to the above embodiment, wherein in said chemical formula (1): L1 is a straight or branched C1-C6 alkylene; L2 is -α1-NHCO- or -α1-NHCO-α2-; a1 and a2 are each independently a C1 to C6 straight or branched chain alkylene; n is 200-250; R is maleimide.

[0035] The composition according to any one of the preceding embodiments, wherein the bioactive polypeptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, G protein-coupled receptor (G-CRR), and the like. receptor), interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage-activating factors, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy-inhibitory factors, necrotic glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, metastatic growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, hyperglycosylated erythropoietic factors, angiopoietins, hemoglobin, thrombin, thrombin receptor-activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activators, fibrin-binding peptides, urokinase, streptokinase, hirudin, protein C, and C -reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, atriopeptin, chondroinductive factor, elcatonin, connective tissue active factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, incretin, GIP (Gastric Inhibitory Protein)The present invention relates to a composition of matter selected from the group consisting of a GLP-1 / GIP dual active substance, a GLP-1 / GIP / glucagon triple active substance, a cell surface antigen, a virus-derived vaccine antigen, a monoclonal antibody, a polyclonal antibody, and an antibody fragment.

[0036] The present invention relates to a composition according to the above embodiment, wherein R of the compound of the composition binds to a cysteine ​​of the drug.

[0037] The composition according to the previous embodiment, wherein X is an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0038] The composition according to the aforementioned embodiment, wherein X is an immunoglobulin Fc region comprising the following amino acid sequence:

[0039] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

[0040] Another embodiment of the present invention relates to a method for producing a long-acting biologically active polypeptide conjugate.

[0041] As a specific example, the present invention relates to a method for producing a long-acting physiologically active polypeptide conjugate, which comprises the step of linking a single-pegylated immunoglobulin Fc region, prepared by linking a linker of the following chemical formula (3) to the N-terminus of an immunoglobulin Fc region containing a hinge sequence, to a physiologically active polypeptide to produce a conjugate.

[0042] [Chemical formula (3)] CHO-L1-(OCH2CH2)nO-L2-R···(3)

[0043] In the formula (3), L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 10 to 2400; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof.

[0044] The present invention relates to a production method according to the above-mentioned specific example, wherein the single-pegylated immunoglobulin Fc region is produced by linking the linker of chemical formula (3) to the N-terminus of the immunoglobulin Fc region in the presence of a reducing agent at pH 4.0 to 8.0.

[0045] The present invention relates to a method according to any one of the above-mentioned embodiments, wherein the conjugate is produced by linking a linker of a single PEGylated immunoglobulin Fc region to a physiologically active polypeptide at pH 5.5 to 8.0.

[0046] The present invention relates to the method according to any one of the above specific examples, wherein the step of producing the conjugate comprises reacting a single PEGylated immunoglobulin Fc region and a physiologically active polypeptide at a molar ratio of 1:1 to 1:3.

[0047] The present invention relates to a method for producing a single-pegylated immunoglobulin Fc region according to any one of the above embodiments, comprising the steps of: linking the linker of formula (3) to the N-terminus of an immunoglobulin Fc region to produce a single-pegylated immunoglobulin Fc region; and linking the linker of the single-pegylated immunoglobulin Fc region produced in the above step to a physiologically active polypeptide to produce a conjugate.

[0048] The method according to any of the previous embodiments, wherein the linker of the single PEGylated immunoglobulin Fc region is linked to a cysteine ​​of a biologically active polypeptide.

[0049] The present invention relates to a method for producing a single-pegylated immunoglobulin Fc region according to any one of the above-mentioned embodiments, comprising the steps of: linking a linker represented by Chemical Formula (3) to the N-terminus of the immunoglobulin Fc region to produce a single-pegylated immunoglobulin Fc region; purifying the single-pegylated immunoglobulin Fc region produced in the previous step by anion exchange chromatography in a buffer solution of pH 6.0 to 8.5; and linking the linker of the single-pegylated immunoglobulin Fc region purified in the previous step to a physiologically active polypeptide to produce a conjugate.

[0050] The method of production according to any of the previous embodiments, characterized in that no ultra / diafiltration is carried out after the step of producing said single PEGylated immunoglobulin Fc region.

[0051] The method according to any one of the previous embodiments, further comprising purifying the conjugate by hydrophobic interaction chromatography.

[0052] The present invention relates to a production method according to any one of the above specific examples, wherein in the chemical formula (3), L1 is a linear or branched C1-C6 alkylene; L2 is -a1-NHCO- or -a1-NHCO-a2-; a1 and a2 are each independently a C1-C6 linear or branched alkylene; n is 200-250; and R is maleimide.

[0053] The method according to any one of the above embodiments, wherein the linker has a structure of the following chemical formula (4):

[0054] JPEG0007752664000002.jpg34131...(4)

[0055] In the chemical formula (4), n is 200-250.

[0056] The method according to any one of the preceding embodiments, wherein the linker has a size of 1 to 100 kDa.

[0057] The method according to any of the preceding embodiments, wherein the physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, G protein-coupled receptor (G-CRR), and the like. receptor), interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage-activating factors, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy-inhibitory factors, necrotic glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, metastatic growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, hyperglycosylated erythropoietic factors, angiopoietins, hemoglobin, thrombin, thrombin receptor-activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activators, fibrin-binding peptides, urokinase, streptokinase, hirudin, protein C, and C -reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, atriopeptin, chondroinductive factor, elcatonin, connective tissue active factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, incretin, GIP (Gastric Inhibitory Protein)and a method for producing the antibody, wherein the antibody is selected from the group consisting of a GLP-1 / GIP dual active substance, a GLP-1 / GIP / glucagon triple active substance, a cell surface antigen, a virus-derived vaccine antigen, a monoclonal antibody, a polyclonal antibody, and an antibody fragment.

[0058] The method according to any one of the preceding embodiments, wherein the bioactive polypeptide is a GLP-1 / GIP / glucagon triple active substance, glucagon, or an analog thereof.

[0059] The present invention relates to a method for producing a biologically active polypeptide according to any one of the above-mentioned specific examples, wherein the biologically active polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. The method according to any one of the preceding embodiments, wherein the hinge sequence is mutated to have only one cysteine ​​residue by deleting a portion of the hinge sequence having the following amino acid sequence:

[0060] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

[0061] The method according to any one of the preceding embodiments, wherein the hinge sequence comprises the amino acid sequence of SEQ ID NO: 8 (Ser-Cys-Pro) or SEQ ID NO: 9 (Pro-Ser-Cys-Pro).

[0062] The method of any one of the preceding embodiments, wherein the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

[0063] Another embodiment of the present invention is a long-acting drug conjugate produced by the composition or production method. [Effects of the Invention]

[0064] The method for producing a long-acting drug conjugate using the novel intermediate of the present invention can produce a long-acting drug conjugate in high yield despite omitting some of the existing purification steps, thereby increasing the productivity of the long-acting drug conjugate. [Brief explanation of the drawings]

[0065] [Figure 1] FIG. 1 shows the results of MALDI-TOF analysis of the structure of a singly PEGylated immunoglobulin Fc region. DETAILED DESCRIPTION OF THE INVENTION

[0066] Specific details for carrying out the present invention will be described below.

[0067] However, each description and embodiment disclosed herein may be applied to different descriptions and embodiments. That is, all combinations of various elements disclosed herein fall within the scope of the present invention. It should be noted that the scope of the present invention is not limited to the specific description provided below.

[0068] Additionally, those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein and such equivalents are intended to be encompassed by the present invention.

[0069] Throughout this specification, the usual one-letter and three-letter codes for naturally occurring amino acids are used, as well as commonly accepted three-letter codes for other amino acids such as Aib (α-aminoisobutyric acid), Sar (N-methylglycine), α-methyl-glutamic acid, etc. Also, amino acids referred to in this specification by abbreviation are written according to the IUPAC-IUB nomenclature system.

[0070] Alanine Ala,A Arginine Arg,R Asparagine Asn, N Aspartic acid Asp, D Cysteine ​​Cys, C Glutamic acid Glu, E Glutamine Gln, Q Glycine Gly, G Histidine His, H Isoleucine Ile, I Leucine (Leu), L Lysine (Lys), K Methionine Met,M Phenylalanine Phe,F Proline Pro, P Serine Ser, S Threonine Thr, T Tryptophan Trp, W Tyrosine Tyr, Y Valine Val, V

[0071] One aspect of the present invention provides a compound having the structure of the following chemical formula (1), a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof:

[0072] X-L1-(OCH2CH2)nO-L2-R···(1)

[0073] In the chemical formula (1), X is an immunoglobulin Fc region; L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 10 to 2400; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof.

[0074] In the present invention, the compound having the structure of the chemical formula (1), its stereoisomer, solvate, or pharmaceutically acceptable salt thereof is a novel substance produced for the preparation of a long-acting conjugate, and may be referred to as an "intermediate" or "intermediate material" in this application.

[0075] The method for producing a long-acting drug conjugate using the intermediate of the present invention can omit the purification steps of ultra / diafiltration and hydrophobic interaction chromatography, and has the effect of producing a long-acting drug conjugate in high yield despite the omission of the purification steps.

[0076] Specifically, the intermediate is in a form in which an immunoglobulin Fc region and a linker are linked, and in the intermediate of chemical formula (1), X may be the immunoglobulin Fc region and L1-(OCH2CH2)nO-L2-R may be the linker. Specifically, in chemical formula (1), L1 is linear or branched C1-C6 alkylene; L2 is -a1-NHCO- or -a1-NHCO-a2-; a1 and a2 are each independently C1-C6 linear or branched alkylene; n is 200 to 250; and R may be, but is not limited to, maleimide.

[0077] L1 is a site that binds to the immunoglobulin Fc region and may be, but is not limited to, a straight-chain or branched C1-C6 alkylene. L1 may bind to an amine or thiol reactive group located at the end of X or a lysine residue, but is not limited to these. R is a site that links an intermediate to a physiologically active polypeptide and may, specifically, have a reactive group (e.g., thiol, maleimide, aldehyde, and succinimidyl) that can be linked to an amine group such as cysteine ​​or the N-terminus of a physiologically active polypeptide or a lysine residue.

[0078] In the present invention, the intermediate may have a structure represented by the following chemical formula (2), but is not limited thereto.

[0079] TIFF0007752664000003.tif43136...(2)

[0080] In the chemical formula (2), n is 1 to 3,000, 10 to 2,000, 50 to 1,000, 100 to 700, 150 to 300, or 200 to 250.

[0081] Specifically, the intermediate may have a size of 40 to 250 kDa, 80 to 200 kDa, or 100 to 150 kDa, but is not limited thereto.

[0082] In the present invention, X may be an immunoglobulin Fc region comprising a hinge sequence at the N-terminus, and specifically, the hinge sequence may be mutated to have only one cysteine ​​residue by partially deleting the amino acid sequence shown below, but is not limited thereto.

[0083] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

[0084] As used herein, the term "immunoglobulin Fc region" may refer to a region including heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3), excluding the heavy and light chain variable regions of an immunoglobulin, and the immunoglobulin Fc region may be a component constituting a part of a long-acting drug conjugate.

[0085] Specifically, X may be an immunoglobulin Fc region derived from IgG, IgA, IgD, IgE, or IgM, and more specifically, may be an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4, but is not limited thereto.

[0086] In the present invention, the immunoglobulin Fc region may comprise a specific hinge sequence at the N-terminus.

[0087] As used herein, the term "hinge sequence" refers to a site located in a heavy chain that forms a dimer of immunoglobulin Fc regions via inter-disulfide bonds.

[0088] As used herein, the term "N-terminus" refers to the amino terminus of a protein or polypeptide and may include the extreme amino terminus, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids from the extreme amino terminus. The immunoglobulin Fc region of the present invention may include, but is not limited to, a hinge sequence at the N-terminus.

[0089] For the purposes of the present invention, the hinge sequence may be one in which the 8th or 11th cysteine ​​residue in the hinge sequence of SEQ ID NO: 7 is deleted and the hinge sequence contains only one cysteine ​​residue. The hinge sequence of the present invention may be, but is not limited to, one consisting of 3 to 12 amino acids containing only one cysteine ​​residue. More specifically, the hinge sequence of the present invention may have the following sequence: Glu-Ser-Lys-Tyr-Gly-Pro-Pro -Pro-Ser-Cys-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser -Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser, Glu-Ser- Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro, Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys, Glu-Lys-Ty r-Gly-Pro-Pro-Cys, Glu-Ser-Pro-Ser-Cys-Pro, Glu-Pro-Ser-Cys-Pro, Pro-Ser-Cys-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro- Ser-Cys-Pro, Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro, Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys, Lys-Tyr-Gly-Pro-Pro-Cys-Pro, Glu-Ser-Lys-Pro-Ser-Cys-Pro, Glu-Ser-Lys-Pro-Ser-Cys-Pro, Glu-Ser-Pro-Ser-Cys-Pro, Glu-Pro-Ser-Cys. More specifically, the hinge sequence may comprise the amino acid sequence of SEQ ID NO: 8 (Ser-Cys-Pro) or SEQ ID NO: 9 (Pro-Ser-Cys-Pro), but is not limited thereto.

[0090] The X may be in the form of a dimer formed by two immunoglobulin Fc chain molecules due to the presence of a hinge sequence, and the intermediate of the present invention may be in the form in which one end of the linker is linked to one chain of the dimeric immunoglobulin Fc region, but is not limited thereto. In the present invention, the X may be, but is not limited to, a dimeric immunoglobulin Fc region.

[0091] Furthermore, X of the present invention may be, but is not limited to, an immunoglobulin Fc region comprising the amino acid sequence of SEQ ID NO: 10.

[0092] On the other hand, the immunoglobulin Fc region of the present invention may be an extended Fc region that excludes only the heavy and light chain variable regions of an immunoglobulin and includes part or all of heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), as long as it has effects substantially equivalent to or improved from those of the native type. Alternatively, it may be a region in which a fairly long portion of the amino acid sequence corresponding to CH2 and / or CH3 has been deleted.

[0093] For example, the immunoglobulin Fc region of the present invention may be, but is not limited to, 1) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains selected from the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a portion of a hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0094] Furthermore, the immunoglobulin Fc region of the present invention includes not only naturally occurring amino acid sequences but also sequence derivatives thereof, where the amino acid sequence derivative means a sequence that differs from the naturally occurring amino acid sequence by deletion, insertion, non-conservative or conservative substitution of one or more amino acid residues, or a combination thereof.

[0095] For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331, which are known to be important for binding, are used as suitable sites for modification.

[0096] Various types of derivatives are possible, such as those in which disulfide bond formation sites are removed, several amino acids at the N-terminus of native Fc are removed, or a methionine residue is added to the N-terminus of native Fc. Furthermore, to eliminate effector functions, complement binding sites, such as the C1q binding site, and ADCC (antibody dependent cell-mediated cytotoxicity) sites are removed. Techniques for producing such sequence derivatives of immunoglobulin Fc regions are disclosed in International Patent Publication No. 97 / 34631 (Patent Document 2), International Patent Publication No. 96 / 32478 (Patent Document 3), and the like. Amino acid exchanges in proteins and peptides that do not overall alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, "The Proteins," Academic Press, New York, 1979). The most commonly occurring exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. In some cases, modifications such as phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation may be performed.

[0097] The sequence derivatives of the immunoglobulin Fc region may exhibit biological activity equivalent to that of the Fc region of the present invention, and may have increased structural stability of the Fc region against heat, pH, and the like.

[0098] Such Fc regions can also be obtained from native immunoglobulins isolated in vivo from animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or from recombinant immunoglobulins or derivatives thereof obtained from transformed animal cells or microorganisms. Here, the method for obtaining the native immunoglobulins may involve isolating whole immunoglobulins from the human or animal body and then treating them with protease. Treatment with papain results in cleavage into Fab and Fc, while treatment with pepsin results in cleavage into pF'c and F(ab)2. The resulting Fc or pF'c can then be separated using size-exclusion chromatography or other methods. In a more specific embodiment, the Fc region is a recombinant immunoglobulin Fc region derived from a human being obtained from a microorganism.

[0099] Furthermore, the immunoglobulin Fc region of the present invention may be in a form having native sugar chains, increased sugar chains compared to the native form, decreased sugar chains compared to the native form, or a form in which the sugar chains have been removed. Such increase, decrease, or removal of immunoglobulin Fc sugar chains can be achieved using conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Here, an immunoglobulin Fc region from which the sugar chains have been removed from the Fc has significantly reduced complement (C1q) binding ability and reduced or eliminated antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity, and therefore does not induce unnecessary immune responses in vivo. In this respect, a form that is more suited to the original purpose of the immunoglobulin Fc region as a drug carrier can be said to be a form in which the sugar chains have been removed or which has been deglycosylated.

[0100] In the present invention, "deglycosylation" refers to an Fc region from which sugars have been removed using an enzyme, and "non-glycosylated" refers to an Fc region that is produced in a prokaryote, or more specifically, in Escherichia coli, and is not glycosylated.

[0101] On the other hand, the immunoglobulin Fc region may be of human or animal origin, such as bovine, caprine, porcine, murine, rabbit, hamster, rat, guinea pig, and in a more specific embodiment, of human origin.

[0102] The immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, or IgM, or a combination thereof, or a hybrid thereof. In a more specific embodiment, the Fc region is derived from IgG or IgM, which are most abundant in human blood, and even more specifically, the Fc region is derived from IgG, which is known to improve the half-life of ligand-binding proteins. In a further specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in a most specific embodiment, the immunoglobulin Fc region is an aglycosylated Fc region derived from human IgG4, but is not limited thereto.

[0103] Meanwhile, in the present invention, the term "combination" means that, when a dimer or multimer is formed, a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin is bound to a single-chain polypeptide of a different origin. That is, a dimer or multimer can be produced from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0104] In the present invention, the term "hybrid" means that a single-chain immunoglobulin constant region contains sequences corresponding to immunoglobulin Fc fragments of two or more different origins. In the present invention, various forms of hybrids are possible. That is, hybrids of domains consisting of 1 to 4 domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc are possible, and may include a hinge.

[0105] Meanwhile, IgG can also be divided into subclasses, IgG1, IgG2, IgG3, and IgG4, and the present invention also allows for combinations or hybridizations of these subclasses. Specifically, these subclasses are IgG2 and IgG4, and most specifically, the Fc region of IgG4, which has almost no effector functions such as complement-dependent cytotoxicity (CDC).

[0106] The term "linker" as used herein refers to a moiety that links a drug (e.g., a physiologically active polypeptide) and an immunoglobulin Fc region in a long-acting drug conjugate, and the linker may be a peptidic linker or a non-peptidic linker. Specifically, the linker may be represented by the following chemical formula (3), but is not limited thereto.

[0107] CHO-L1-(OCH2CH2)nO-L2-R···(3)

[0108] In the formula (3), L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 1 to 3000, 10 to 2000, 50 to 1000, 100 to 700, 150 to 300, or 200 to 250; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof.

[0109] The linker may include polyethylene glycol and may have a specific chemical structure at both ends of the polyethylene glycol, but is not limited thereto.

[0110] Specifically, in the chemical formula (3), L1 is a linear or branched C1-C6 alkylene; L2 is -a1-NHCO- or -a1-NHCO-a2-; a1 and a2 are each independently a linear or branched C1-C6 alkylene; n is 200 to 250; R may be maleimide, and the linker may have a size of, but is not limited to, 1 to 200 kDa, 1 to 150 kDa, 1 to 100 kDa, 1 to 50 kDa, or 1 to 10 kDa.

[0111] The linker may have a structure represented by the following chemical formula (4), but is not limited thereto.

[0112] JPEG0007752664000004.jpg34131...(4)

[0113] In the chemical formula (4), n may be 1 to 3,000, 10 to 2,000, 50 to 1,000, 100 to 700, 150 to 300, or 200 to 250.

[0114] One end of the linker may be linked to an immunoglobulin Fc region, specifically the N-terminus of the immunoglobulin Fc region, more specifically a hinge sequence located at the N-terminus of the immunoglobulin Fc region, specifically a proline residue in the hinge sequence, to form an intermediate, but is not limited to this.

[0115] In the present invention, the term "pharmaceutically acceptable" means a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reaction, within the scope of medical judgment.

[0116] As used herein, the term "pharmaceutically acceptable salts" includes salts derived from pharmaceutically acceptable inorganic acids, organic acids, or bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium.

[0117] The present invention includes not only the chemical compounds or their pharmaceutically acceptable salts, but also any possible solvates that may be prepared therefrom.

[0118] Furthermore, if the compound has an asymmetric carbon center (chiral carbon) in its substituent, it may exist in the form of an enantiomer (R or S isomer), a racemate, or a diastereomer, or any mixture thereof. Furthermore, if a chemical compound contains a bridged ring, it can exist as an exo or endo isomer, but is not limited thereto.

[0119] Another aspect of the present invention provides a composition comprising a compound having the structure of the following chemical formula (1), a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein the drug is a physiologically active polypeptide:

[0120] X-L1-(OCH2CH2)nO-L2-R···(1)

[0121] In the chemical formula (1), X is an immunoglobulin Fc region; L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 1 to 3000, 10 to 2000, 50 to 1000, 100 to 700, 150 to 300, or 200 to 250; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof.

[0122] Specifically, in the chemical formula (1), L1 is a linear or branched C1 to C6 alkylene; L2 is -a1-NHCO- or -a1-NHCO-a2-; a1 and a2 are each independently a linear or branched C1 to C6 alkylene; n is 200 to 250; and R may be, but is not limited to, maleimide.

[0123] The composition of the present invention is a composition containing an intermediate, and is useful for producing a long-acting drug conjugate.

[0124] Specifically, since the composition of the present invention includes an intermediate in which a linker and an immunoglobulin Fc region are linked, the composition of the present invention can be reacted with a physiologically active polypeptide to link the linker of the intermediate to the physiologically active polypeptide, thereby preparing a long-acting drug conjugate. More specifically, the R in formula (1), which corresponds to one end of the linker, can be bound to an amine group such as a cysteine ​​or N-terminal lysine residue of the physiologically active polypeptide to prepare a long-acting drug conjugate, but is not limited thereto.

[0125] In the present invention, the physiologically active polypeptide of the long-acting drug conjugate that can be prepared using the composition may be included within the scope of the present invention regardless of type, size, origin, etc., as long as it exhibits a pharmacological effect against a disease.Examples of the physiologically active polypeptide include glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, G protein-coupled receptor (G protein-coupled receptor), and the like. receptor), interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage-activating factors, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy-inhibitory factors, necrotic glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, metastatic growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, hyperglycosylated erythropoietic factors, angiopoietins, hemoglobin, thrombin, thrombin receptor-activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activators, fibrin-binding peptides, urokinase, streptokinase, hirudin, protein C, and C -reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, atriopeptin, chondroinductive factor, elcatonin, connective tissue active factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, incretin, GIP (Gastric The antibody may be, but is not limited to, a GLP-1 / GIP dual inhibitor, a GLP-1 / GIP / glucagon triple inhibitor, a cell surface antigen, a virus-derived vaccine antigen, a monoclonal antibody, a polyclonal antibody, or an antibody fragment.

[0126] More specifically, the bioactive polypeptide may be, but is not limited to, glucagon-like peptide-1 (GLP-1), glucagon, insulin, an enzyme, an incretin, a gastric inhibitory polypeptide (GIP), a GLP-1 / GIP dual activator, or a GLP-1 / GIP / glucagon triple activator.

[0127] The method for producing a long-acting drug conjugate using the intermediate of the present invention or a composition containing the intermediate involves first linking an intermediate to which a linker and an immunoglobulin Fc region are attached to a physiologically active polypeptide. Therefore, regardless of the specific type of physiologically active polypeptide, as long as the physiologically active polypeptide has an amino acid residue or reactive group that can be linked to the intermediate, a long-acting drug conjugate can be produced without restriction using the intermediate of the present invention or a composition containing the intermediate.

[0128] In the present invention, X may be, but is not limited to, an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[0129] Furthermore, X may be, but is not limited to, an immunoglobulin Fc region containing a hinge sequence mutated to have only one cysteine ​​residue by partially deleting the amino acid sequence of the following amino acids:

[0130] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

[0131] When a long-acting drug conjugate is produced using the composition of the present invention, the long-acting drug conjugate can be produced without ultra / diafiltration, and optionally, one hydrophobic interaction chromatography step can be omitted.

[0132] Specifically, when a long-acting drug conjugate is prepared by reacting a drug with an intermediate of the present invention or a composition containing the intermediate, the purity of the prepared long-acting drug conjugate may be, but is not limited to, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. The purity may be measured by methods known in the art, specifically, SE-HPLC, RP-HPLC, and IE-HPLC, or any method used in the art. More specifically, the purity of the long-acting drug conjugate prepared using the composition of the present invention may be, but is not limited to, 90% or more by SE-HPLC, 80% or more by RP-HPLC, and 85% or more by IE-HPLC.

[0133] Furthermore, the composition of the present invention may further contain, but is not limited to, buffers, stabilizers, preservatives, salts, etc. necessary for stabilizing intermediates and producing long-acting drug conjugates.

[0134] Another aspect of the present invention provides a kit for preparing a long-acting drug conjugate, comprising the composition. The kit may include, but is not limited to, reagents and instructions for preparing the long-acting drug conjugate.

[0135] Yet another aspect of the present invention provides a method for producing a long-acting drug conjugate.

[0136] The production method of the present invention is a method for producing a long-acting drug conjugate in which a drug and an immunoglobulin Fc region are linked via a linker, and specifically, may be a method for producing a long-acting drug conjugate by linking the intermediate to a drug, but is not limited thereto.

[0137] Specifically, the method is characterized by the following sequence: i) linking a linker containing polyethylene glycol (PEG) to an immunoglobulin Fc region, and then ii) linking the linker linked to the immunoglobulin Fc region to a drug (e.g., a physiologically active polypeptide or protein). That is, the method is characterized by producing a long-acting drug conjugate in a specific order, with one step of linking a linker containing PEG to an immunoglobulin Fc region to produce an intermediate, followed by two steps of linking a drug to the intermediate. Alternatively, the production method of the present invention may omit one step and only perform two steps of reacting the intermediate or a composition for producing a long-acting drug conjugate containing the intermediate with a drug to produce a long-acting drug conjugate, but is not limited to this. In this application, the above production method may be referred to as a "reverse production method."

[0138] In the present invention, in the case of a method for producing a long-acting drug conjugate by first producing an intermediate and then conjugating it with a drug, it has been confirmed that the steps of purification by ultra / diafiltration and hydrophobic interaction chromatography can be omitted, and that a long-acting drug conjugate can be produced in high yield despite the omission of the purification steps.

[0139] In existing production methods in which a linker is first linked to a physiologically active polypeptide without forming an intermediate and then an immunoglobulin Fc region is linked to it, when linking a physiologically active polypeptide-linked linker (e.g., polyethylene glycol) to an immunoglobulin Fc region, the low pH conditions (around pH 3.0) of the equilibration and elution buffers used to purify the linker linked to the physiologically active polypeptide must be used to reduce the risk of aggregation, and the pH conditions must be adjusted using an appropriate buffer for the reaction. Therefore, after linking the linker to the physiologically active polypeptide, a separate ultra / diafiltration step is required before linking to the immunoglobulin Fc region. In contrast, the production method of the present invention, in which an intermediate is first linked to a linker and an immunoglobulin Fc region to produce an intermediate, is characterized by the fact that the buffer used to purify the immunoglobulin Fc region linked to the linker has a relatively high pH, ​​omitting the ultra / diafiltration step and instead performing the linking to the physiologically active polypeptide step.

[0140] Therefore, the production method of the present invention may not require ultra / diafiltration after the production step of the single PEGylated immunoglobulin Fc region, but is not limited to this. In the production method of a long-acting drug conjugate according to the present invention, the pH of the purified solution of the single PEGylated immunoglobulin Fc region is not significantly different from the pH of the subsequent reaction solution, so drug conjugation can be performed without ultra / diafiltration. Omission of the ultra / diafiltration step reduces the risk of aggregate impurities that may occur during the concentration step and simplifies the production process, which is expected to reduce costs when the technology is commercialized.

[0141] Furthermore, the production method of the present invention may further comprise purifying the conjugate by hydrophobic interaction chromatography, but is not limited thereto.

[0142] Specifically, the hydrophobic interaction chromatography may be performed only once, or may be performed more than once depending on the properties of the drug in the long-acting drug conjugate and the type and size of the linker.

[0143] The production method according to the present invention not only reduces the amount of expensive drug used, but also reduces the amount of unreacted immunoglobulin Fc region, thereby making it possible to omit all or part of the hydrophobic interaction chromatography purification step, thereby reducing the raw materials and costs required for the production of long-acting drug conjugates compared to existing methods.

[0144] Meanwhile, the preparation method according to the present invention has the advantage that the ultra / diafiltration and hydrophobic interaction chromatography steps performed in the conventional manufacturing process of long-acting drug conjugates are omitted, and only a final purification step (e.g., a single hydrophobic interaction chromatography) is performed, while maintaining the purity of the final conjugate compared to the conventional process. That is, the preparation method according to the present invention can maintain the final purity despite omitting some of the purification steps, thereby improving the productivity of the production of long-acting drug conjugates.

[0145] The purity of the long-acting drug conjugate of the present invention can be measured by a method known in the art, and examples of such methods may include, but are not limited to, SE-HPLC, RP-HPLC, or IE-HPLC measurement.

[0146] According to the manufacturing method of the present invention, the final purity of the long-acting drug conjugate may be 90% or more, specifically, but is not limited to, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more.

[0147] On the other hand, the production method of the present invention first produces a single PEGylated immunoglobulin Fc region, which is then linked to a physiologically active polypeptide, thereby enabling the production of a long-acting drug conjugate in a higher yield than existing methods, not only in terms of the physiologically active polypeptide but also in terms of the immunoglobulin Fc region.

[0148] In one example of the present invention, it was confirmed that the yield of long-acting drug conjugates produced by the manufacturing method of the present invention was more than twice as high as that produced by existing methods.

[0149] Specifically, the production method of the present invention relates to a production method for a long-acting drug conjugate, which includes a step of producing a conjugate by linking a single PEGylated immunoglobulin Fc region, produced by linking a linker of the following chemical formula (3) to the N-terminus of an immunoglobulin Fc region containing a hinge sequence, to a drug (biologically active polypeptide).

[0150] CHO-L1-(OCH2CH2)nO-L2-R···(3)

[0151] In the chemical formula (3), L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 10 to 2400; R may be any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof, but is not limited thereto.

[0152] More specifically, the production method may include the steps of: linking the linker of formula (3) to the N-terminus of an immunoglobulin Fc region to prepare a single PEGylated immunoglobulin Fc region; and linking the linker of the single PEGylated immunoglobulin Fc region prepared in the step to a physiologically active polypeptide to prepare a conjugate; or the production method may include the steps of linking the linker of formula (3) to the N-terminus of an immunoglobulin Fc region to prepare a single PEGylated immunoglobulin Fc region; purifying the single PEGylated immunoglobulin Fc region prepared in the step by anion exchange chromatography in a buffer solution of pH 6.0 to 8.5, pH 6.0 to 8.0, pH 6.0 to 7.5, pH 6.0 to 7.0, pH 6.1 to 6.9, pH 6.2 to 6.8, or pH 6.3 to 6.7; and linking the linker of the single PEGylated immunoglobulin Fc region purified in the step to a physiologically active polypeptide to prepare a conjugate. and raw The method may include, but is not limited to, a step of linking the compound to a biologically active polypeptide to prepare a conjugate.

[0153] Further, in the production method of the present invention, (i) the single-pegylated immunoglobulin Fc region is produced by linking the linker of formula (3) to the N-terminus of the immunoglobulin Fc region in the presence of a reducing agent at pH 4.0 to 8.0, pH 4.5 to 7.5, pH 5.5 to 7.5, pH 5.6 to 7.4, pH 5.7 to 7.3, or pH 5.8 to 7.2; and / or (ii) The conjugate may be, but is not limited to, one produced by linking a linker of a single pegylated immunoglobulin Fc region to a physiologically active polypeptide at pH 5.5 to 8.0, pH 6.0 to 7.5, or pH 6.5 to 7.5.

[0154] Furthermore, the step of producing the conjugate in the production method of the present invention may involve reacting the same amount of physiologically active polypeptide as the single PEGylated immunoglobulin Fc region, or more specifically, the single PEGylated immunoglobulin Fc region:physiologically active polypeptide may be reacted at a molar ratio of 1:1 to 1:10, 1:1 to 1:7, 1:1 to 1:5, or 1:1 to 1:3, but is not limited thereto.

[0155] The term "single-pegylated immunoglobulin Fc region" as used herein refers to an intermediate substance produced during the production process of the long-acting drug conjugate according to the present invention, in which a linker containing one polyethylene glycol is linked to the immunoglobulin Fc region. That is, in the present invention, the term "single-pegylated immunoglobulin Fc region" can be used interchangeably with "intermediate" or "intermediate substance."

[0156] In the present invention, the immunoglobulin Fc region may be, but is not limited to, an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4. Furthermore, the immunoglobulin Fc region may be an immunoglobulin Fc region comprising a hinge sequence mutated to have only one cysteine ​​residue by deleting a portion of the amino acid sequence shown below. Specifically, the hinge sequence may comprise the amino acid sequence of SEQ ID NO: 8 (Ser-Cys-Pro) or SEQ ID NO: 9 (Pro-Ser-Cys-Pro), but is not limited thereto.

[0157] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

[0158] The "long-acting drug conjugate" of the present invention refers to a drug conjugate with an increased half-life, which has a structure in which a drug (biologically active polypeptide) that exhibits pharmacological activity in the body is linked to the Fc region of an immunoglobulin via a linker. For the purposes of the present invention, the long-acting drug conjugate may be, but is not limited to, an intermediate or a single-pegylated immunoglobulin Fc region to which a drug is conjugated.

[0159] Specifically, the drug is not limited to a specific substance, and may be a natural or non-natural protein, enzyme, antibody, compound, etc., as long as it exhibits the effect of preventing, treating, or improving a specific disease. More specifically, the drug may be a physiologically active polypeptide or protein, and more specifically, the physiologically active polypeptide may be glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, G protein-coupled receptor (G protein-coupled receptor), or the like. receptor), interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage-activating factors, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy-inhibitory factors, necrotic glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, metastatic growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, hyperglycosylated erythropoietic factors, angiopoietins, hemoglobin, thrombin, thrombin receptor-activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activators, fibrin-binding peptides, urokinase, streptokinase, hirudin, protein C, and C -reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, atriopeptin, chondroinductive factor, elcatonin, connective tissue active factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, incretin, GIP (Gastric Inhibitory Protein)The physiologically active polypeptide may be, but is not limited to, a GLP-1 / GIP dual-active polypeptide, a GLP-1 / GIP / glucagon triple-active polypeptide, a cell surface antigen, a virus-derived vaccine antigen, a monoclonal antibody, a polyclonal antibody, or an antibody fragment. More specifically, the physiologically active polypeptide may be, but is not limited to, a GLP-1 / GIP / glucagon triple-active polypeptide, glucagon, or an analog thereof. More specifically, the physiologically active polypeptide may comprise, essentially consist of, or consist of the amino acid sequence of any one of SEQ ID NOS: 1 to 6, but is not limited to these.

[0160] Also within the scope of the present invention are variants, derivatives, and fragments of said physiologically active polypeptides.

[0161] In the present invention, the term "variant" refers to a peptide that differs in one or more amino acid sequences from a naturally occurring physiologically active polypeptide and has the same function as the naturally occurring physiologically active polypeptide, and may be prepared by any one of substitution, addition, deletion, and modification of some amino acids in the naturally occurring sequence, or a combination of these methods.

[0162] In the present invention, the term "derivative" includes peptides, peptide derivatives, peptidomimetics, etc., which are obtained by modifying the naturally occurring physiologically active polypeptide by adding, deleting, or substituting some of the amino acids of the naturally occurring physiologically active polypeptide and which exhibit activity similar to that of the naturally occurring physiologically active polypeptide.

[0163] In the present invention, the term "fragment" refers to a form in which one or more amino acids have been added or deleted at the amino or carboxy terminus, and the added amino acids may be non-naturally occurring amino acids (e.g., D-amino acids).

[0164] The methods for producing biologically active polypeptide variants, derivatives, and fragments can be used independently or in combination, including, for example, biologically active polypeptides with one or more amino acid sequence differences and deamination of the N-terminal amino acid residue.

[0165] The physiologically active polypeptide derivatives include biosimilars and biobetters, and examples of biosimilars include those that differ from known enzymes in their expression hosts, in the mode and degree of glycosylation, and, if residues at specific positions are not 100% substituted in light of the reference sequence of the enzyme, those that differ in the degree of substitution also qualify as biosimilar enzymes that can be used in the long-acting drug conjugates of the present invention. The physiologically active polypeptides, variants, derivatives, and fragments can be produced by genetic recombination from animal cells, E. coli, yeast, insect cells, plant cells, living animals, etc., and the production method is not limited thereto, and commercially available physiologically active polypeptides, variants, derivatives, and fragments may also be used.

[0166] Furthermore, the physiologically active polypeptides, variants, derivatives and fragments may comprise an amino acid sequence having 80% or more homology, specifically 90% or more, more specifically 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more homology thereto, and the physiologically active polypeptides, variants, derivatives and fragments may be obtained from microorganisms by recombinant technology or may be commercially available, but are not limited thereto.

[0167] The term "homology" as used herein refers to the degree of similarity between the amino acid sequence of a wild-type protein or the nucleotide sequence encoding it, and includes sequences that share the above percentage or more of identity with the amino acid sequence or nucleotide sequence of the present invention. Such homology can be determined by visually comparing the two sequences or by using a bioinformatics algorithm that aligns the sequences to be compared and analyzes the degree of homology. The homology between the two amino acid sequences can be expressed as a percentage. Useful automated algorithms are available in the GAP, BESTFIT, FASTA, and TFASTA computer software modules of the Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WV, USA). Automated alignment algorithms in these modules include the Needleman & Wunsch, Pearson & Lipman, and Smith & Waterman alignment algorithms. Other useful alignment algorithms and homology determinations are automated in software including FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

[0168] Information on the sequences of the physiologically active polypeptides, variants, derivatives and fragments and the nucleotide sequences encoding them can be obtained from publicly known databases such as NCBI.

[0169] The substituted or added amino acids can be any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids include Sigma-Aldrich, ChemPep, and Genzyme Pharmaceuticals. Peptides containing such amino acids and typical peptide sequences can be synthesized and purchased through commercial peptide synthesis companies, such as American Peptide Company and Bachem in the United States, or Anygen in Korea.

[0170] Furthermore, the physiologically active polypeptides, variants, derivatives and fragments according to the present invention may be in a modified form, such as by chemically modifying the N-terminus and / or C-terminus, protecting them with an organic group, or adding an amino acid to the peptide terminus, in order to protect them from in vivo protease cleavage and increase their stability.

[0171] In particular, in the case of chemically synthesized peptides, the N-terminus and C-terminus are charged, and therefore, in order to remove such charges, the N-terminus can be acetylated and / or the C-terminus can be amidated, but this is not a limitation.

[0172] Furthermore, the peptide according to the present invention includes the peptide itself, a salt thereof (e.g., a pharmaceutically acceptable salt of the peptide), or a solvate thereof. The peptide may be in any pharmaceutically acceptable form.

[0173] The type of the salt is not particularly limited, but is preferably in a form that is safe and effective for individuals, for example, mammals, but is not particularly limited thereto.

[0174] The term "solvate" as used in the present invention refers to a complex of the peptide according to the present invention or a salt thereof with solvent molecules.

[0175] Furthermore, even if the present invention describes a "peptide consisting of a specific sequence number," this does not exclude meaningless additions of sequences before or after the amino acid sequence of the sequence number, or naturally occurring mutations, or silent mutations thereof, as long as the peptide has the same or corresponding activity as the peptide consisting of the amino acid sequence of the sequence number, and it is self-evident that even if the peptide has such additions or mutations of sequences, it falls within the scope of the present application.

[0176] The method for producing the long-acting drug conjugate of the present invention may be, but is not limited to, a method for producing a conjugate in which a physiologically active polypeptide and an immunoglobulin Fc region are linked via a linker.

[0177] In the present invention, the linker and the immunoglobulin Fc region may be linked by a covalent or non-covalent bond between one end of the linker and the N-terminus of the immunoglobulin Fc region, but the linkage site and method are not particularly limited. Specifically, a single-pegylated immunoglobulin Fc region may be produced by linking the N-terminal proline of the immunoglobulin Fc region to the -CHO group of the linker, but this is not limitative.

[0178] In the present invention, the linker may have a structure represented by the following chemical formula (4), but is not limited thereto.

[0179] JPEG0007752664000005.jpg34131...(4)

[0180] In the chemical formula (4), n is 200-250.

[0181] Furthermore, the linker may have a size of, but is not limited to, 1 to 200 kDa, 1 to 150 kDa, 1 to 100 kDa, 1 to 50 kDa, or 1 to 10 kDa.

[0182] The single pegylated immunoglobulin Fc region may have a structure represented by the following chemical formula (2), but is not limited thereto:

[0183] TIFF0007752664000006.tif43135...(2)

[0184] The production method of the present invention may include, but is not limited to, a step of linking a physiologically active polypeptide to one end of the Fc region of a single-pegylated immunoglobulin having the structure of chemical formula (2).

[0185] Furthermore, the other end of the linker that is not linked to the immunoglobulin Fc region may be linked to a physiologically active polypeptide, specifically, but not limited to, to an -SH group or an amino acid containing an -SH group, or cysteine ​​of the physiologically active polypeptide.

[0186] In the present invention, the long-acting drug conjugate may have a structure represented by the following chemical formula (5), but is not limited thereto.

[0187] JPEG0007752664000007.jpg49132··(5)

[0188] The chemical formula (5) may have a structure in which a physiologically active polypeptide, a linker, and an immunoglobulin Fc region are sequentially linked from the left, but is not limited thereto.

[0189] In the examples of the present invention, it was confirmed that when a long-acting drug conjugate is produced by the production method of the present invention, in which a single PEGylated immunoglobulin Fc region is first produced and then linked to a physiologically active polypeptide, the ultra / diafiltration and hydrophobic interaction chromatography steps are omitted and only the final purification step (e.g., one hydrophobic interaction chromatography) is performed, while the purity of the final conjugate is maintained and the yield is improved compared to conventional processes.

[0190] Another aspect of the present invention provides a long-acting drug conjugate produced by the above method. The long-acting drug conjugates produced by the production method of the present invention have an increased half-life compared to physiologically active polypeptides to which a linker or immunoglobulin Fc region is not attached, and therefore have advantageous effects in the production of pharmaceuticals.

[0191] The long-acting drug conjugates produced by the production method of the present invention can be used to produce drugs or compositions that have applications in the prevention, treatment, and amelioration of diseases.

[0192] The present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0193] Comparative Example: Preparation of a conjugate by linking a PEGylated biologically active polypeptide to an immunoglobulin Fc region PEGylated bioactive polypeptides were linked to the immunoglobulin Fc region to prepare long-acting conjugates.

[0194] Comparative Example 1: Preparation of a conjugate by linking PEGylated GLP-1 / GIP / Glucagon triple-active analogue 1 to an immunoglobulin Fc region To PEGylate the cysteine ​​(-SH group) of the bioactive polypeptide (GLP-1 / GIP / Glucagon triple-active analog 1 (SEQ ID NO: 1)), the GLP-1 / GIP / Glucagon triple-active analog 1 was reacted at a molar ratio of 1:1.0-1.3 with a linker (chemical formula (4)) containing PEG (maleimide-10 kDa-PEG-aldehyde) at a concentration of 3 g / L for approximately 1 hour. Specifically, the reaction was carried out in a 50 mM Tris buffer (pH 7.5 @ 6 ± 4 °C) containing isopropanol. To obtain the single PEGylated GLP-1 / GIP / Glucagon triple-active analog 1, the reaction mixture was diluted to a total volume of 20 times with an equilibration buffer containing sodium citrate and ethanol, and purified. This reaction was carried out using SP High Performance (GE) Single pegylated GLP-1 / GIP / Glucagon triple-active analog 1 was purified using a (Healthcare, cation exchange chromatography) column with a solution containing sodium citrate and ethanol and a potassium chloride gradient. The purified solution of pegylated GLP-1 / GIP / Glucagon triple-active analog 1 was diluted with water, and then buffer exchanged with 0.1 M potassium phosphate solution and concentrated using a separation membrane ultra / diafiltration (UF / DF) method until the final recovered concentration was 3 g / L or more.

[0195] The single PEGylated GLP-1 / GIP / Glucagon triple activity analog 1 prepared above was linked to the immunoglobulin Fc region to prepare a long-acting conjugate as follows.

[0196] At this time, the molar ratio of single PEGylated GLP-1 / GIP / Glucagon triple activity analog 1 to immunoglobulin Fc region was 1:2, and the total protein concentration (GLP-1 / GIP / Glucagon triple activity analog 1 and immunoglobulin Fc region) was 30 g / L, so that the aldehyde group of PEG in single PEGylated GLP-1 / GIP / Glucagon triple activity analog 1 would bind to the amino terminus of the immunoglobulin Fc region. The reaction was carried out at 6±4°C for approximately 12 hours.

[0197] After the binding reaction, unreacted immunoglobulin Fc regions were separated and removed by purification using a Butyl 4 Fast Flow (GE Healthcare, hydrophobic interaction chromatography) column. Tris and sodium chloride were added to the reaction mixture, and the mixture was purified using a solution containing Bis-Tris and a sodium chloride concentration gradient.

[0198] Hydrophobic interaction chromatography was then performed using a Source 15ISO (GE Healthcare) column to remove process by-products, yielding the immunoglobulin Fc region-PEG-containing linker-GLP-1 / GIP / Glucagon triple-active analog 1 conjugate, which was purified using a sodium citrate-containing buffer and an ammonium sulfate gradient.

[0199] Comparative Example 2: Preparation of a conjugate by linking PEGylated glucagon analogue 1 to an immunoglobulin Fc region To PEGylate the cysteine ​​(-SH group) of the physiologically active polypeptide (glucagon analog 1; SEQ ID NO: 4), the glucagon analog 1 was reacted for approximately 1 hour at a molar ratio of 1:1.3 with a linker (chemical formula (1)) containing PEG (maleimide-10 kDa-PEG-aldehyde) at a concentration of 3 g / L. Specifically, the reaction was carried out in 50 mM Tris (pH 7.3) buffer containing isopropanol. To obtain mono-PEGylated glucagon analog 1, the reaction mixture was diluted to a total volume of 20 times with an equilibration buffer containing sodium citrate and ethanol, and then purified. The mono-PEGylated glucagon analog 1 was purified using an SP High Performance (GE Healthcare, cation exchange chromatography) column with a solution containing sodium citrate and ethanol and a potassium chloride gradient. The purified solution of PEGylated glucagon analogue 1 was diluted with water, and then buffer exchanged and concentrated with 0.1 M potassium phosphate solution using a membrane ultra / diafiltration (UF / DF) method until the final recovered concentration was 3 g / L or more.

[0200] The single PEGylated glucagon analog 1 prepared above was linked to the immunoglobulin Fc region to prepare a long-acting conjugate as follows.

[0201] At this time, the molar ratio of single PEGylated glucagon analog 1 to immunoglobulin Fc region was 1:5, and the total protein concentration (glucagon analog 1 and immunoglobulin Fc region) was 20 g / L, so that the aldehyde group of PEG in single PEGylated glucagon analog 1 would bind to the amino terminus of the immunoglobulin Fc region, and the reaction was carried out at 6±4°C for approximately 12 hours. After the binding reaction, unreacted immunoglobulin Fc regions were separated and removed by purification using a Butyl 4 Fast Flow (GE Healthcare, hydrophobic interaction chromatography) column. Tris and sodium chloride were added to the reaction mixture, and the mixture was purified using a solution containing Bis-Tris and a sodium chloride concentration gradient.

[0202] Then, hydrophobic interaction chromatography was performed using a Source 15ISO (GE Healthcare) column to remove process by-products and obtain the immunoglobulin Fc region-PEG linker-glucagon analog 1 conjugate, which was purified using a sodium citrate-containing buffer and an ammonium sulfate gradient.

[0203] The present inventors have developed the following process, which allows efficient conjugate production while maintaining high purity of the conjugate produced by omitting the membrane permeation step and purification step (hydrophobic interaction chromatography, Butyl 4 Fast Flow) in the conjugate production process of Comparative Examples 1 and 2.

[0204] Example 1: Preparation of mono-PEGylated immunoglobulin Fc regions Example 1-1. Preparation of a single PEGylated immunoglobulin Fc region To PEGylate the N-terminus of an immunoglobulin Fc region (49.8 kDa) having a hinge region with the Pro-Ser-Cys-Pro sequence at its N-terminus, the immunoglobulin Fc region was reacted at a molar ratio of 1:1 with a PEG-containing linker (structure of chemical formula (4), 10 kDa) at a concentration of 50 g / L at 6±4°C for approximately 4 hours.

[0205] JPEG0007752664000008.jpg34131...(4)

[0206] Specifically, the reaction was carried out in a solution containing 5 mM Bis Tris (pH 6.5) and potassium phosphate, and 10 mM NaCNBH3 (sodium cyanoborohydride) reducing agent was added. To obtain a single PEGylated immunoglobulin Fc region, the reaction solution was diluted with Bis Tris buffer and purified.

[0207] At this time, single PEGylated GLP-1 / GIP / Glucagon triple-active analogueUnlike the comparative manufacturing method in which the above was purified, the single PEGylated immunoglobulin Fc region was purified using a Capto Q ImpRes (GE Healthcare, anion exchange chromatography) column with a buffer containing BisTris and a sodium chloride gradient.

[0208] Example 1-2. Structural analysis of a single PEGylated immunoglobulin Fc region The structure of the mono-PEGylated immunoglobulin Fc region prepared by the method of Example 1-1 was analyzed by MALDI-TOF and peptide mapping. The MALDI-TOF analysis results showed that the molecular weight of the mono-PEGylated immunoglobulin Fc region was consistent with the predicted molecular weight ( FIG. 1 ), and the peptide mapping analysis results confirmed that PEG was PEGylated to the N-terminus of the immunoglobulin Fc region by 90% or more.

[0209] Meanwhile, the mono-PEGylated immunoglobulin Fc region (Chemical Formula (2)) prepared by the method of Example 1-1 was analyzed using SE-HPLC, RP-HPLC, and IE-HPLC. As a result, it was confirmed that the purity was 90% or more by SE-HPLC, 90% or more by RP-HPLC, and 80% or more by IE-HPLC.

[0210] TIFF0007752664000009.tif43136··(2)

[0211] Example 2: Preparation of a conjugate by linking a pegylated immunoglobulin Fc region to a biologically active polypeptide The single-pegylated immunoglobulin Fc region prepared in Example 1-1 was linked to various physiologically active peptides to prepare long-acting conjugates as follows.

[0212] Unlike the comparative manufacturing method in which the PEGylated bioactive polypeptide was purified by cation chromatography and then buffer exchanged and concentrated by membrane ultra / diafiltration (UF / DF), a single PEGylated immunoglobulin Fc region was reacted with the bioactive polypeptide by peptide conjugation without UF / DF. The long-acting conjugate thus produced was highly pure, and unlike the comparative manufacturing method, one of the two hydrophobic interaction chromatography steps could be omitted.

[0213] Example 2-1. Preparation of GLP-1 / GIP / Glucagon triple-active analog 1 conjugate After the anion chromatography of Example 1-1, peptide conjugation of GLP-1 / GIP / glucagon tri-active analog 1 (SEQ ID NO: 1) was carried out without ultra / diafiltration to prepare a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-GLP-1 / GIP / glucagon tri-active analog 1).

[0214] [SEQ ID NO: 1] JPEG0007752664000010.jpg29162

[0215] The reaction was carried out at a molar ratio of 1:1 between the mono-PEGylated immunoglobulin Fc region and GLP-1 / GIP / Glucagon tri-active analog 1, with a GLP-1 / GIP / Glucagon tri-active analog 1 protein concentration of 0.2 g / L, at 6±4°C for approximately 2 hours, so that the maleimide reactive group at one end of the PEG of the mono-PEGylated immunoglobulin Fc region was bound to GLP-1 / GIP / Glucagon tri-active analog 1. The reaction mixture was then incubated in Tris-Cl buffer (6±4°C) containing isopropanol. The reaction mixture was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC, and the conversion was confirmed to be greater than 90% by SE-HPLC, greater than 80% by RP-HPLC, and greater than 70% by IE-HPLC.

[0216] The reaction mixture was then subjected to a single round of hydrophobic interaction chromatography using a Source 15ISO (GE Healthcare) column. This allowed for the removal of reaction by-products, yielding a conjugate of immunoglobulin Fc region-PEG-containing linker-GLP-1 / GIP / Glucagon tri-active analog 1. This was purified using a sodium citrate-containing buffer and an ammonium sulfate gradient. The specific yield of the input GLP-1 / GIP / Glucagon tri-active analog 1 was confirmed to be more than two-fold higher than the yield in Comparative Example 1.

[0217] The eluted immunoglobulin Fc-PEG-containing linker-GLP-1 / GIP / glucagon triple-active analog 1 conjugate was analyzed by SE-HPLC, RP-HPLC, IE-HPLC, and SDS-PAGE. It was confirmed to have a high purity of over 90% by SE-HPLC, over 90% by RP-HPLC, and over 90% by IE-HPLC.

[0218] Example 2-2. Preparation of GLP-1 / GIP / Glucagon triple-active analogue 2 conjugate After the anion chromatography of Example 1-1, a peptide conjugation reaction of GLP-1 / GIP / Glucagon tri-active analog 2 (SEQ ID NO: 2) was carried out without ultra / diafiltration to prepare a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-GLP-1 / GIP / Glucagon tri-active analog 2).

[0219] [SEQ ID NO: 2] JPEG0007752664000011.jpg20141

[0220] The mono-PEGylated immunoglobulin Fc region and GLP-1 / GIP / Glucagon tri-active analog 2 were reacted at a molar ratio of 1:1 and a protein concentration of 0.2 g / L at 6±4°C for approximately 2 hours, so that the maleimide reactive group at one end of the PEG of the mono-PEGylated immunoglobulin Fc region was conjugated to the cysteine ​​of GLP-1 / GIP / Glucagon tri-active analog 2. The reaction mixture was carried out in Tris-Cl buffer (6±4°C) containing isopropanol. The reaction product was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC. The purity of the long-acting conjugate containing GLP-1 / GIP / Glucagon tri-active analog 2 was confirmed to be 90% or higher by SE-HPLC, 80% or higher by RP-HPLC, and 70% or higher by IE-HPLC.

[0221] The reaction product was then subjected to a single round of hydrophobic interaction chromatography using a Source 15ISO (GE Healthcare) column. This allowed for the removal of reaction by-products, yielding immunoglobulin Fc region-PEG-containing linker-triple activated analog 2. Purification was performed using a sodium citrate-containing buffer and a gradient of ammonium sulfate. The specific yield of input triple activated analog 2 was confirmed to be approximately two-fold higher than that of Comparative Example 1.

[0222] The eluted immunoglobulin Fc region-PEG-containing linker-GLP-1 / GIP / Glucagon triple activity analog 2 conjugate was analyzed by SE-HPLC, RP-HPLC, IE-HPLC, and SDS-PAGE, and it was confirmed that it had been produced with a high purity of 90% or more by SE-HPLC, 90% or more by RP-HPLC, and 80% or more by IE-HPLC.

[0223] Example 2-3. Preparation of GLP-1 / GIP / Glucagon triple-active analog 3 conjugate After the anion chromatography of Example 1-1, a peptide conjugation reaction of GLP-1 / GIP / Glucagon tri-active analog 3 (SEQ ID NO: 3) was carried out without ultra / diafiltration to prepare a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-GLP-1 / GIP / Glucagon tri-active analog 3).

[0224] [SEQ ID NO: 3] JPEG0007752664000012.jpg21144

[0225] The reaction was carried out at a molar ratio of 1:1 between the mono-PEGylated immunoglobulin Fc region and the GLP-1 / GIP / Glucagon tri-active analog 3, with a GLP-1 / GIP / Glucagon tri-active analog 3 protein concentration of 0.2 g / L, at 6 ± 4°C for approximately 2 hours, so that the maleimide reactive group at one end of the PEG of the mono-PEGylated immunoglobulin Fc region would be conjugated to the cysteine ​​of the GLP-1 / GIP / Glucagon tri-active analog 3. The reaction mixture was carried out in Tris-Cl buffer (6 ± 4°C) containing isopropanol. The reaction product was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC, and was found to be >90% purified by SE-HPLC, >80% purified by RP-HPLC, and >70% purified by IE-HPLC.

[0226] The reaction product was then subjected to a single round of hydrophobic interaction chromatography using a Source 15ISO (GE Healthcare) column. This allowed for the removal of reaction by-products, yielding a conjugate of immunoglobulin Fc region-PEG-containing linker-GLP-1 / GIP / Glucagon triple-active analog 3. This was purified using a buffer containing sodium citrate and a gradient of ammonium sulfate. The specific yield of the input triple-active analog 3 was confirmed to be approximately two-fold higher than that of Comparative Example 1.

[0227] The eluted immunoglobulin Fc region-PEG-containing linker-GLP-1 / GIP / glucagon triple activity analog 3 conjugate was analyzed using SE-HPLC, RP-HPLC, and IE-HPLC, and was confirmed to have a high purity of 90% or more by SE-HPLC, 90% or more by RP-HPLC, and 90% or more by IE-HPLC.

[0228] Examples 2-4. Preparation of conjugates of glucagon analogue 1 After the anion chromatography of Example 1-1, a peptide conjugation reaction of glucagon analogue 1 (SEQ ID NO: 4) was carried out without ultra / diafiltration to prepare a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-glucagon analogue 1).

[0229] [SEQ ID NO: 4] JPEG0007752664000013.jpg28135

[0230] The reaction was carried out at a molar ratio of 1:1 between the mono-PEGylated immunoglobulin Fc region and glucagon analogue 1, with a glucagon analogue 1 protein concentration of 0.2 g / L, at 6±4°C for approximately 2 hours, so that the maleimide reactive group at one end of the PEG of the mono-PEGylated immunoglobulin Fc region was bound to glucagon analogue 1. The reaction mixture was carried out in Tris-Cl (6±4°C) buffer containing isopropanol. The reaction product was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC. The purity of the immunoglobulin Fc region-PEG-containing linker-glucagon analogue 1 was confirmed to be 90% or more by SE-HPLC, 70% or more by RP-HPLC, and 70% or more by IE-HPLC.

[0231] The reaction product was then subjected to a single round of hydrophobic interaction chromatography using a Source 15ISO (GE Healthcare) column. This allowed for the removal of reaction by-products, yielding a conjugate of immunoglobulin Fc region-PEG-containing linker-glucagon analog 1. The conjugate was purified using a sodium citrate-containing buffer and a gradient of ammonium sulfate. The specific yield of glucagon analog 1 input was confirmed to be 1.5-fold higher than that of Comparative Example 2.

[0232] The eluted immunoglobulin Fc-PEG-containing linker-glucagon analogue 1 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC, and it was confirmed that it had been produced with high purity of 90% or more by SE-HPLC, 90% or more by RP-HPLC, and 90% or more by IE-HPLC.

[0233] Example 2-5. Preparation of glucagon analogue 2 conjugate After the anion chromatography of Example 1-1, peptide conjugation reaction of glucagon analogue 2 (SEQ ID NO: 5) was carried out without ultra / diafiltration to prepare a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-glucagon analogue 2).

[0234] [SEQ ID NO: 5] JPEG0007752664000014.jpg20125

[0235] The reaction was carried out at a molar ratio of 1:1 between the mono-PEGylated immunoglobulin Fc region and glucagon analogue 2, with a glucagon analogue 2 protein concentration of 0.2 g / L, at 6±4°C for approximately 2 hours, so that the maleimide reactive group at one end of the PEG of the mono-PEGylated immunoglobulin Fc region was bound to glucagon analogue 2. The reaction mixture was carried out in Tris-Cl (6±4°C) buffer containing isopropanol. The reaction product was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC. The purity of the immunoglobulin Fc region-PEG-containing linker-glucagon analogue 2 conjugate was confirmed to be 90% or more by SE-HPLC, 70% or more by RP-HPLC, and 70% or more by IE-HPLC.

[0236] The reaction product was then subjected to a single round of hydrophobic interaction chromatography using a Source 15ISO (GE Healthcare) column. This allowed for the removal of reaction by-products, yielding a conjugate of immunoglobulin Fc region-PEG-containing linker-glucagon analogue 2. The conjugate was purified using a sodium citrate-containing buffer and a gradient of ammonium sulfate. The specific yield of the input glucagon analogue 2 was confirmed to be approximately 1.5-fold higher than that of Comparative Example 2.

[0237] The eluted immunoglobulin Fc-PEG-containing linker-glucagon analogue 2 conjugate was analyzed by SE-HPLC, RP-HPLC and IE-HPLC analytical methods, and it was confirmed that it had been produced with high purity of 90% or more by SE-HPLC, 90% or more by RP-HPLC and 90% or more by IE-HPLC.

[0238] Example 2-6. Preparation of glucagon analogue 3 conjugate After the anion chromatography of Example 1-1, peptide conjugation reaction of glucagon analogue 3 (SEQ ID NO: 6) was carried out without ultra / diafiltration to prepare a long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-glucagon analogue 3).

[0239] [SEQ ID NO: 6] JPEG0007752664000015.jpg8107

[0240] The reaction was carried out at a molar ratio of 1:1 between the mono-PEGylated immunoglobulin Fc region and glucagon analogue 3, with a protein concentration of 0.2 g / L of glucagon analogue 3, at 6 ± 4°C for approximately 2 hours, so that the maleimide reactive group at one end of the PEG of the mono-PEGylated immunoglobulin Fc region would bind to the cysteine ​​of glucagon analogue 3. The reaction mixture was then carried out in a Tris-Cl (6 ± 4°C) buffer containing isopropanol. The reaction mixture was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC. The purity of the immunoglobulin Fc region-PEG-containing linker-glucagon analogue 3 conjugate was confirmed to be 90% or more by SE-HPLC, 70% or more by RP-HPLC, and 70% or more by IE-HPLC.

[0241] The reaction product was then subjected to a single round of hydrophobic interaction chromatography using a Source 15ISO (GE Healthcare) column. This allowed for the removal of reaction by-products, yielding a conjugate of immunoglobulin Fc region-PEG-containing linker-glucagon analogue 3. This was purified using a sodium citrate-containing buffer and an ammonium sulfate gradient. The specific yield of input glucagon analogue 3 was confirmed to be approximately 1.5-fold higher than the conventional yield.

[0242] The eluted immunoglobulin Fc region-PEG-containing linker-glucagon analogue 3 conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC, and it was confirmed that it had been produced with high purity of 90% or more by SE-HPLC, 90% or more by RP-HPLC, and 90% or more by IE-HPLC.

[0243] Tables 1 and 2 below are tables comparing the methods of the comparative examples and examples of the present invention.

[0244] [Table 1]

[0245] [Table 2]

[0246] From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are merely illustrative and not limiting. The scope of the present invention should be interpreted as including within the meaning and scope of the claims below, and all modifications and variations derived from the equivalent concepts thereof, rather than the above detailed description. Another aspect of the present invention may be as follows. [1] A compound having the structure of the following chemical formula (1), its stereoisomer, solvate, or pharmaceutically acceptable salt thereof: TIFF0007752664000018.tif6169 In the chemical formula (1), X is an immunoglobulin Fc region; L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 10 to 2400; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof. [2] In the chemical formula (1), L1 is a straight or branched C1-C6 alkylene; L2 is -α1-NHCO- or -α1-NHCO-α2-; a1 and a2 are each independently a C1 to C6 straight or branched chain alkylene; n is 200-250; The compound according to [1] above, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein R is maleimide. [3] The compound according to [1] above, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein X is an immunoglobulin Fc region comprising a hinge sequence at the N-terminus. [4] The compound according to [3] above, its stereoisomer, solvate, or pharmaceutically acceptable salt thereof, wherein the hinge sequence is an immunoglobulin Fc region comprising a mutated hinge sequence in which the following amino acids are partially deleted to have only one cysteine ​​residue: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7). [5] The compound according to [4], its stereoisomer, solvate, or pharmaceutically acceptable salt thereof, wherein the hinge sequence comprises the amino acid sequence of SEQ ID NO: 8 (Ser-Cys-Pro) or SEQ ID NO: 9 (Pro-Ser-Cys-Pro). [6] The compound according to [4], its stereoisomer, solvate, or pharmaceutically acceptable salt thereof, wherein L1 is bonded to an amine or thiol reactive group located at the end of X. [7] The compound according to [1] above, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a structure represented by the following chemical formula (2): TIFF0007752664000019.tif43135 ··(2) In the chemical formula (2), n is 200-250. [8] The compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof according to [1], wherein X is an immunoglobulin Fc region derived from IgG, IgA, IgD, IgE, or IgM. [9] The compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof according to [8] above, wherein X is an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[10] The compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof according to [1], wherein X is a dimeric immunoglobulin Fc region.

[11] The compound according to [1], its stereoisomer, solvate, or pharmaceutically acceptable salt thereof, wherein X is an immunoglobulin Fc region comprising the amino acid sequence of SEQ ID NO: 10.

[12] The compound according to [1] above, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein the compound has a size of 40 to 250 kDa.

[13] A composition for producing a long-acting drug conjugate, comprising a compound having a structure represented by the following chemical formula (1), a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof: a composition wherein the drug is a biologically active polypeptide; TIFF0007752664000020.tif6169 In the chemical formula (1), X is an immunoglobulin Fc region; L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 10 to 2400; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof.

[14] In the chemical formula (1), L1 is a straight or branched C1-C6 alkylene; L2 is -α1-NHCO- or -α1-NHCO-α2-; a1 and a2 are each independently a C1 to C6 straight or branched chain alkylene; n is 200-250; The composition according to

[13] above, wherein R is maleimide.

[15] The physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), granulocyte colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, and G protein-coupled receptor. receptor), interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage-activating factors, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy-inhibitory factors, necrotic glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, metastatic growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, hyperglycosylated erythropoietic factors, angiopoietins, hemoglobin, thrombin, thrombin receptor-activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activators, fibrin-binding peptides, urokinase, streptokinase, hirudin, protein C, and C -reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, atriopeptin, chondroinductive factor, elcatonin, connective tissue active factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, incretin, GIP (Gastric The composition according to

[13] , wherein the antibody is selected from the group consisting of a GLP-1 / GIP dual inhibitory polypeptide, a GLP-1 / GIP / glucagon triple inhibitory polypeptide, a cell surface antigen, a virus-derived vaccine antigen, a monoclonal antibody, a polyclonal antibody, and an antibody fragment.

[16] The composition according to

[13] , wherein R of the compound of the composition binds to cysteine ​​of the drug.

[17] The composition according to

[13] , wherein X is an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

[18] The composition described in

[13] , which enables the production of a long-acting drug conjugate without ultrafiltration / diafiltration.

[19] A method for producing a long-acting physiologically active polypeptide conjugate, comprising the step of linking a single-pegylated immunoglobulin Fc region, which is produced by linking a linker represented by the following chemical formula (3) to the N-terminus of an immunoglobulin Fc region comprising a hinge sequence, to a physiologically active polypeptide to produce a conjugate: TIFF0007752664000021.tif5161 In the chemical formula (3), L1 is a straight or branched C1-C6 alkylene; L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, where a1, a2, b1, and b2 are each independently a C1 to C6 straight or branched alkylene; n is 10 to 2400; R is any one selected from the group consisting of 2,5-dioxopyrrolidinyl, 2,5-dioxopyrrolyl, aldehyde, maleimide, C6-C20 aryl disulfide, C5-C20 heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, thioester, and derivatives thereof.

[20] The method according to

[19] above, wherein the single-pegylated immunoglobulin Fc region is produced by linking the linker of chemical formula (3) to the N-terminus of the immunoglobulin Fc region in the presence of a reducing agent at pH 4.0 to 8.0.

[21] The method according to

[19] above, wherein the conjugate is produced by linking a linker of a single PEGylated immunoglobulin Fc region to a physiologically active polypeptide at pH 5.5 to 8.0.

[22] The method according to

[19] , wherein the step of producing the conjugate comprises reacting a single PEGylated immunoglobulin Fc region and a physiologically active polypeptide at a molar ratio of 1:1 to 1:3.

[23] The method according to

[19] , comprising the steps of: linking the linker of formula (3) to the N-terminus of the immunoglobulin Fc region to produce a single-pegylated immunoglobulin Fc region; and linking the linker of the single-pegylated immunoglobulin Fc region produced in the previous step to a physiologically active polypeptide to produce a conjugate.

[24] The production method described in

[23] above, wherein the linker of the single pegylated immunoglobulin Fc region is linked to a cysteine ​​of a physiologically active polypeptide.

[25] The production method according to

[23] above, comprising the steps of: linking a linker represented by chemical formula (3) to the N-terminus of the immunoglobulin Fc region to produce a single-pegylated immunoglobulin Fc region; purifying the single-pegylated immunoglobulin Fc region produced in the step by anion exchange chromatography in a buffer solution of pH 6.0 to 8.5; and linking the linker of the single-pegylated immunoglobulin Fc region purified in the step to a physiologically active polypeptide to produce a conjugate.

[26] The production method described in

[23] above, characterized in that ultrafiltration / diafiltration is not performed after the production step of the single PEGylated immunoglobulin Fc region.

[27] The method of producing according to

[19] above, further comprising purifying the conjugate by hydrophobic interaction chromatography.

[28] The production method according to

[19] above, wherein in the chemical formula (3), L1 is a linear or branched C1-C6 alkylene; L2 is -a1-NHCO- or -a1-NHCO-a2-; a1 and a2 each independently represent a C1-C6 linear or branched alkylene; n is 200 to 250; and R is maleimide.

[29] The method for producing according to

[19] above, wherein the linker has a structure represented by the following chemical formula (4): TIFF0007752664000022.tif34131 ···(4) In the chemical formula (4), n is 200-250.

[30] The method for producing according to

[19] above, wherein the linker has a size of 1 to 100 kDa.

[31] The physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, and G protein-coupled receptor. receptor), interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage-activating factor, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy-inhibitory factors, necrotic glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, metastatic growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, hyperglycosylated erythropoietic factors, angiopoietins, hemoglobin, thrombin, thrombin receptor-activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein Protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, atriopeptin, chondroinductive factor, elcatonin, connective tissue active factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, incretin, GLP-1 / GIP dual activator, GIP (Gastric The method for producing the antibody according to

[19] , wherein the antibody is selected from the group consisting of a GLP-1 / GIP / glucagon triple inhibitory polypeptide, a GLP-1 / GIP / glucagon triple inhibitory polypeptide, a cell surface antigen, a virus-derived vaccine antigen, a monoclonal antibody, a polyclonal antibody, and an antibody fragment.

[32] The method for production according to

[31] , wherein the physiologically active polypeptide is a GLP-1 / GIP / glucagon triple active substance, glucagon, or an analog thereof.

[33] The method of producing according to

[32] above, wherein the physiologically active polypeptide comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 6.

[34] The manufacturing method described in

[19] , wherein the hinge sequence is a mutated hinge sequence having the following amino acid sequence, in which a portion of the hinge sequence is deleted to have only one cysteine ​​residue: Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 7).

[35] The manufacturing method described in

[34] , wherein the hinge sequence comprises the amino acid sequence of SEQ ID NO: 8 (Ser-Cys-Pro) or SEQ ID NO: 9 (Pro-Ser-Cys-Pro).

[36] The production method described in

[19] , wherein the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

[37] A long-acting drug conjugate produced by the composition described in any one of

[13] to

[18] above or the method described in any one of

[19] to

[36] above.

Claims

1. A compound having the structure of the following chemical formula (2), wherein the immunoglobulin Fc region comprises a hinge sequence consisting of the amino acid sequence of SEQ ID NO: 9 (Pro-Ser-Cys-Pro) at the N-terminus, a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof, wherein the compound is linked to a physiologically active polypeptide: ・・（２） In the chemical formula (2), n is 200 to 250.

2. The physiologically active polypeptides include glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, G protein-coupled receptor (G-CRR), receptor), interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage-activating factors, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy-inhibitory factors, cytotoxic glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, metastatic growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, hyperglycosylated erythropoietic factors, angiopoietins, hemoglobin, thrombin, thrombin receptor-activating peptides, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activators, fibrin-binding peptides, urokinase, streptokinase, hirudin, protein C, C -reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, atriopeptin, chondrogenic factor, elcatonin, connective tissue active factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, incretin, GIP (Gastric Inhibitory Protein)2. The compound according to claim 1, its stereoisomer, solvate, or pharmaceutically acceptable salt thereof, which is selected from the group consisting of a GLP-1 / GIP dual activator, a GLP-1 / GIP / glucagon triple activator, a cell surface antigen, a virus-derived vaccine antigen, a monoclonal antibody, a polyclonal antibody, and an antibody fragment.

3. 2. The compound of claim 1, its stereoisomer, solvate, or pharmaceutically acceptable salt thereof, wherein the immunoglobulin Fc region is an immunoglobulin Fc region derived from IgG, IgA, IgD, IgE, or IgM.

4. 4. The compound of claim 3, its stereoisomer, solvate, or pharmaceutically acceptable salt thereof, wherein the immunoglobulin Fc region is an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

5. 2. The compound of claim 1, its stereoisomer, solvate or pharmaceutically acceptable salt thereof, wherein the immunoglobulin Fc region is a dimeric immunoglobulin Fc region.

6. 2. The compound of claim 1, its stereoisomer, solvate or pharmaceutically acceptable salt thereof, wherein the immunoglobulin Fc region is an immunoglobulin Fc region comprising the amino acid sequence of SEQ ID NO:

10.

7. The compound, stereoisomer, solvate, or pharmaceutically acceptable salt thereof according to claim 1, wherein the physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, interleukin, enzymes, gastric inhibitory polypeptide (GIP), GLP-1 / GIP dual active substance, and GLP-1 / GIP / glucagon triple active substance.

8. A method for producing a long-acting physiologically active polypeptide conjugate, comprising the step of linking a compound having a structure represented by the following chemical formula (2), a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof to a physiologically active polypeptide to produce a conjugate: The physiologically active polypeptides include glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone-releasing hormone, growth hormone-releasing peptide, interferon, interferon receptor, G protein-coupled receptor (G-CRR), receptor), interleukins, interleukin receptors, enzymes, interleukin-binding proteins, cytokine-binding proteins, macrophage-activating factors, macrophage peptides, B-cell factors, T-cell factors, protein A, allergy-inhibitory factors, cytotoxic glycoproteins, immunotoxins, lymphotoxins, tumor necrosis factors, tumor suppressors, metastatic growth factors, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein E, hyperglycosylated erythropoietic factors, angiopoietins, hemoglobin, thrombin, thrombin receptor-activating peptides, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activators, fibrin-binding peptides, urokinase, streptokinase, hirudin, protein C, C -reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, osteogenic growth factor, osteogenic protein, calcitonin, atriopeptin, chondroinductive factor, elcatonin, connective tissue active factor, tissue factor pathway inhibitor, follicle-stimulating hormone, luteinizing hormone, luteinizing hormone-releasing hormone, nerve growth factor, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, adrenocortical hormone, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin-releasing factor, thyroid-stimulating hormone, autotaxin, lactoferrin, myostatin, incretin, GIP (Gastric Integrative Metabolite) a GLP-1 / GIP dual activator, a GLP-1 / GIP / glucagon triple activator, a cell surface antigen, a virus-derived vaccine antigen, a monoclonal antibody, a polyclonal antibody, and an antibody fragment, ・・（２） In the chemical formula (2), n is 200 to 250.

9. The method according to claim 8, wherein the step of preparing the conjugate comprises reacting a compound having a structure of chemical formula (2), a stereoisomer thereof, a solvate thereof, or a pharmaceutically acceptable salt thereof with a physiologically active polypeptide in a molar ratio of 1:1 to 1:

3.

10. The method according to claim 8, wherein the maleimide of the compound having the structure of chemical formula (2), its stereoisomer, solvate, or a pharmaceutically acceptable salt thereof is linked to a cysteine ​​residue of a physiologically active polypeptide.

11. The method according to claim 8, wherein ultrafiltration / diafiltration is not performed after the preparation step of the compound having the structure of chemical formula (2), its stereoisomer, solvate, or a pharmaceutically acceptable salt thereof.

12. The method of claim 8, further comprising purifying the conjugate by hydrophobic interaction chromatography.

13. 9. The method according to claim 8, wherein the physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-1 (GLP-1), neutrophil colony-stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, interleukin, enzymes, gastric inhibitory polypeptide (GIP), GLP-1 / GIP dual active substance, and GLP-1 / GIP / glucagon triple active substance.

14. The method according to claim 8, wherein the physiologically active polypeptide comprises any one of the amino acid sequences set forth in SEQ ID NOs: 1 to 6.

15. The method of claim 8 , wherein the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

16. The method according to claim 8, wherein the compound having the structure of chemical formula (2), its stereoisomer, solvate or pharmaceutically acceptable salt thereof and the physiologically active polypeptide are linked via a linker having the structure of the following chemical formula (4): ・・・（４） In the chemical formula (4), n is 200 to 250.

17. The method of claim 16, wherein the linker has a size of 1 to 100 kDa.

18. The method of claim 8 , wherein the immunoglobulin Fc region comprises a hinge sequence at the N-terminus, the hinge sequence comprising the amino acid sequence of SEQ ID NO: 9 (Pro-Ser-Cys-Pro).

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