Liquid preparations

Abstract

The present invention relates to a liquid formulation of a long-acting conjugate of a peptide active against a glucagon receptor and a GLP-1 receptor, and a method for producing the same.

Description

[Technical Field]

[0001] The present invention relates to a liquid formulation of a long-acting conjugate of a peptide active against a glucagon receptor and a GLP-1 receptor, and a method for producing the same. [Background technology]

[0002] Recent economic development and changes in dietary habits have led to a rapid increase in the incidence of metabolic syndrome-related diseases, including obesity, hyperlipidemia, hypertension, arteriosclerosis, hyperinsulinemia, diabetes, and liver disease. While these diseases may occur independently, they are generally closely related and often occur with various symptoms. According to the World Health Organization (WHO), more than one billion adults worldwide are overweight, of which at least three million are clinically obese. Overweight-related deaths occur annually in Europe, particularly in Europe, and more than 2.5 million worldwide. Obesity is a serious global disease that can cause various illnesses, yet many believe it can be overcome through individual self-help efforts. However, obesity is surprisingly difficult to treat due to its complex nature, which is linked to the mechanisms of appetite regulation and energy metabolism. Therefore, in order to treat obesity, not only the patient's own efforts but also methods to treat abnormal mechanisms of action related to appetite regulation and energy metabolism must be carried out at the same time, and therefore efforts are ongoing to develop medicines that can treat these abnormal mechanisms of action.

[0003] As a result of these efforts, obesity treatments such as rimonabant (Sanofi-Aventis), sibutramine (Abbott), Contrave (Takeda), and orlistat (Roche) have been developed. However, these drugs have drawbacks, such as fatal side effects and ineffective obesity treatment. Therefore, active research is underway to develop new drugs that can address the problems of existing obesity treatments. Recently, oxyntomodulin, which has activity at both GLP-1 and glucagon peptide receptors, has attracted attention. Oxyntomodulin is a peptide synthesized from preglucagon, the precursor of glucagon. It exhibits the food intake-inhibiting and satiety-promoting effects of GLP-1 and the lipolytic function of glucagon, raising its potential as an anti-obesity treatment. Based on the dual function of the oxyntomodulin peptide, active research is underway to develop drugs for the treatment of obesity. For example, Korean Patent Registration No. 925017 discloses a pharmaceutical composition for oral, parenteral, mucosal, rectal, subcutaneous, or transdermal administration for treating overweight in humans, which contains oxyntomodulin as an active ingredient. However, it has been reported that anti-obesity agents containing oxyntomodulin have a short in vivo half-life and exhibit low levels of therapeutic efficacy even when administered in high doses three times daily. Therefore, ongoing efforts are being made to modify oxyntomodulin to increase its in vivo half-life and enhance its therapeutic efficacy. However, previously developed oxyntomodulin and its derivatives still have two major disadvantages: they require daily administration due to their short half-life and low efficacy, and they require excessive drug administration. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Korean Patent Registration No. 925017 [Patent Document 2] Korean Patent No. 10-725315 [Patent Document 3] International Patent Publication No. WO97 / 34631 [Patent Document 4] International Patent Publication No. 96 / 32478 [Patent Document 5] International Publication No. WO2014-073842 [Non-patent literature]

[0005] [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]

[0006] There is an increasing need to develop a method for maintaining the in vivo efficacy of drugs even during long-term storage by adding a stabilizer to proteins and peptides in solution to suppress physicochemical changes in the drugs. [Means for solving the problem]

[0007] One object of the present invention is to provide a liquid formulation of a long-acting conjugate of a peptide active on the glucagon receptor and the GLP-1 (Glucagon-like peptide-1) receptor, in which the peptide and an immunoglobulin Fc segment are linked to each other.

[0008] Another object of the present invention is to provide a method for producing a liquid formulation of a long-acting conjugate of a peptide active against the glucagon receptor and the GLP-1 receptor. [Effects of the Invention]

[0009] The liquid formulation of the present invention, a long-acting conjugate of a peptide active against the glucagon receptor and the GLP-1 receptor, has a novel composition characterized by the presence of sugar but not sugar alcohol, and exhibits excellent long-term storage stability as well as excellent results in stress tests and / or accelerated tests for determining pharmaceutical stability, making it useful as a novel liquid formulation that can be stably stored without pharmaceutical degradation. Furthermore, the liquid formulation of the present invention has an osmotic pressure range similar to that of blood, which prevents pain upon administration to patients and improves convenience for patients. DETAILED DESCRIPTION OF THE INVENTION

[0010] One embodiment of the present invention is a liquid formulation of a long-acting conjugate of a peptide active on the glucagon receptor and GLP-1 (Glucagon-like peptide-1) receptor, in which the peptide and an immunoglobulin Fc segment are linked to each other. The long-acting conjugate can refer to a substance in which a peptide active on the glucagon receptor and GLP-1 receptor is covalently bound to an immunoglobulin Fc segment via a linker.

[0011] In one specific example, the liquid preparation is characterized by being a liquid preparation of a peptide conjugate comprising a peptide conjugate of the following chemical formula (1); a buffer substance; a sugar; and a nonionic surfactant:

[0012] TIFF0007723686000001.tif45129...(1) In the chemical formula (1), Q is a peptide represented by the following general formula 1: Z is human immunoglobulin Fc segment; n is a natural number, and the value of n is determined so that the average molecular weight, for example, the number average molecular weight, of the [OCHCH] moieties in the peptide bond is 10 kDa;

[0013] [General formula 1] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa12-Tyr-Leu-Asp-Xaa16-Lys-Arg-Ala-Xaa20-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys (SEQ ID NO: 1),

[0014] In this case, in general formula 1, Xaa2 is 2-aminoisobutyric acid, Xaa12 is lysine, Xaa16 is glutamic acid, and Xaa20 is lysine; A lactam ring may or may not be formed between residues Xaa12 and Xaa16 or Xaa16 and Xaa20.

[0015] A liquid formulation according to any one of the above specific examples, characterized in that the liquid formulation is a liquid formulation of a peptide conjugate comprising 18 to 940 nmol / mL of the peptide conjugate of chemical formula (1); a buffer substance in an amount sufficient to maintain the pH of the liquid formulation in the range of 4.5 to 7.5; 1 to 20% (w / v) of a sugar; and 0.001 to 0.2% (w / v) of a non-ionic surfactant.

[0016] A liquid formulation according to any one of the previous embodiments, wherein the peptide comprises the amino acid sequence of SEQ ID NO: 2, 3, or 4.

[0017] A liquid formulation according to any one of the previous embodiments, wherein the peptide, Q, comprises the amino acid sequence of SEQ ID NO:3.

[0018] A liquid formulation according to any one of the preceding embodiments, wherein Q is amidated at its C-terminus.

[0019] A liquid formulation according to any one of the previous embodiments, wherein Q is linked through the sulfur atom of a cysteine in the peptide.

[0020] A liquid formulation according to any one of the previous embodiments, characterized in that said immunoglobulin Fc fragment is derived from IgG4.

[0021] A liquid formulation according to any one of the preceding embodiments, wherein Z is a structure in which two polypeptide chains are linked by a disulfide bond, and are linked only through the nitrogen atom of one of the two chains.

[0022] A liquid formulation according to any one of the previous embodiments, characterized in that Z comprises a monomer having the amino acid sequence of SEQ ID NO:5.

[0023] A liquid formulation according to any one of the previous embodiments, characterized in that Z is linked through the nitrogen atom of its N-terminal proline.

[0024] A liquid formulation according to any one of the preceding embodiments, characterized in that the immunoglobulin Fc segment and Q are non-glycosylated.

[0025] A liquid formulation according to any one of the previous embodiments, characterized in that said liquid formulation does not contain a sugar alcohol.

[0026] A liquid formulation according to any one of the preceding embodiments, said liquid formulation may be free of or may include a tonicity agent.

[0027] A liquid formulation according to any one of the preceding embodiments, characterized in that the buffer substance is selected from the group consisting of citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof.

[0028] A liquid formulation according to any one of the previous embodiments, characterized in that the buffer substance is acetic acid and its salts.

[0029] A liquid formulation according to any one of the above specific examples, characterized in that the pH of said liquid formulation is 4.8 to 6.0.

[0030] A liquid formulation according to any one of the above embodiments, characterized in that the pH of said liquid formulation is 4.9 to 5.3.

[0031] The liquid formulation according to any one of the above-mentioned embodiments, characterized in that the concentration of the buffer substance is 5 to 100 mM to maintain the pH of the liquid formulation in the range of 4.5 to 7.5.

[0032] The liquid formulation according to any one of the previous embodiments, wherein the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof.

[0033] A liquid formulation according to any one of the previous embodiments, characterized in that the sugar is sucrose.

[0034] A liquid formulation according to any one of the previous embodiments, characterized in that the sugar is present in the liquid formulation at a concentration of 1-20% (w / v).

[0035] A liquid formulation according to any one of the previous embodiments, wherein the non-ionic surfactant is a poloxamer, a polysorbate, or a combination thereof.

[0036] A liquid formulation according to any one of the preceding embodiments, wherein the non-ionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

[0037] A liquid formulation according to any one of the previous embodiments, characterized in that the non-ionic surfactant is present in the liquid formulation at a concentration of 0.001% to 0.2% (w / v).

[0038] A liquid formulation according to any one of the above embodiments, characterized in that the peptide conjugate of formula (1) is present in the liquid formulation at a concentration of 18 to 940 nmol / ml.

[0039] A liquid formulation according to any one of the preceding embodiments, wherein the liquid formulation further comprises an amino acid selected from the group consisting of arginine, glycine, methionine, and combinations thereof as a stabilizer.

[0040] A liquid formulation according to any one of the above-mentioned embodiments, characterized in that the liquid formulation comprises a peptide conjugate of chemical formula (1); a buffer selected from the group consisting of citric acid and salts thereof, acetic acid and salts thereof, histidine and salts thereof, phosphoric acid and salts thereof, and combinations thereof; a sugar selected from the group consisting of glucose, fructose, galactose, lactose, maltose, sucrose, and combinations thereof; and a non-ionic surfactant selected from the group consisting of poloxamer, polysorbate, and combinations thereof, and has a pH of 4.8 to 6.0.

[0041] A liquid formulation according to any one of the above-mentioned embodiments, characterized in that the liquid formulation comprises: a peptide conjugate of chemical formula (1); 5 to 100 mM of a buffer substance selected from the group consisting of citric acid and salts thereof, acetic acid and salts thereof, histidine and salts thereof, phosphoric acid and salts thereof, and combinations thereof, for maintaining the pH of the liquid formulation in the range of 4.8 to 6.0; 1% to 20% (w / v) of a sugar selected from the group consisting of glucose, fructose, galactose, lactose, maltose, sucrose, and combinations thereof; and 0.001% to 0.2% (w / v) of a non-ionic surfactant selected from the group consisting of poloxamer, polysorbate, and combinations thereof.

[0042] A liquid formulation according to any one of the above-mentioned specific examples, characterized in that the liquid formulation comprises: a peptide conjugate of chemical formula (1); 5 to 100 mM acetic acid or a salt thereof for maintaining the pH of the liquid formulation in the range of 4.8 to 6.0; 1% to 20% (w / v) sucrose; and 0.001% to 0.2% (w / v) polysorbate.

[0043] A liquid formulation according to any one of the previous embodiments, characterized in that said immunoglobulin Fc fragment is an Fc fragment derived from IgG, IgA, IgD, IgE or IgM.

[0044] A liquid formulation according to any one of the preceding embodiments, wherein the immunoglobulin Fc fragment is selected from the group consisting of: (a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of one or more of 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; and (f) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0045] A liquid formulation according to any one of the preceding embodiments, wherein each domain of the immunoglobulin Fc fragment is a hybrid of domains of different origin derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0046] A liquid formulation according to any one of the preceding embodiments, characterized in that the immunoglobulin Fc fragment is in the form of a dimer or multimer, composed of single immunoglobulin chains consisting of domains of identical origin.

[0047] A liquid formulation according to any one of the previous embodiments, characterized in that said immunoglobulin Fc fragment is an IgG4 Fc fragment.

[0048] A liquid formulation according to any one of the previous embodiments, characterized in that said immunoglobulin Fc fragment is a human non-glycosylated IgG4 Fc fragment.

[0049] The liquid formulation according to any one of the above-mentioned embodiments, wherein the immunoglobulin Fc fragment is a derivative of native Fc, including a variant in which a site capable of forming a disulfide bond has been removed, a variant in which some amino acids have been removed from the N-terminus of native Fc, a variant in which a methionine residue has been added to the N-terminus of native Fc, a variant in which a complement binding site has been removed, or a variant in which an ADCC (antibody dependent cell mediated cytotoxicity) site has been removed, or a combination of the above variants.

[0050] A liquid formulation according to any one of the preceding embodiments, characterized in that the liquid formulation comprises: a peptide conjugate of chemical formula (1) comprising Q containing SEQ ID NO: 2, 3, or 4; 5 to 100 mM of a buffer substance selected from the group consisting of citric acid and salts thereof, acetic acid and salts thereof, histidine and salts thereof, phosphate, and combinations thereof, for maintaining the pH of the liquid formulation in the range of 4.8 to 6.0; 1% to 20% (w / v) of a sugar selected from the group consisting of glucose, fructose, galactose, lactose, maltose, sucrose, or combinations thereof; and 0.001% to 0.2% (w / v) of a non-ionic surfactant selected from the group consisting of poloxamer, polysorbate, and combinations thereof.

[0051] A liquid formulation according to any one of the preceding embodiments, characterized in that the liquid formulation comprises: a peptide conjugate of chemical formula (1) comprising Q containing SEQ ID NO: 2, 3, or 4; 5 to 100 mM of a buffer substance selected from the group consisting of citrate, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, for maintaining the pH of the liquid formulation in the range of 4.8 to 6.0; 1% to 20% (w / v) sucrose; and 0.001% to 0.2% (w / v) of a non-ionic surfactant selected from the group consisting of poloxamer, polysorbate, and combinations thereof.

[0052] A liquid formulation according to any one of the above-mentioned specific examples, characterized in that the liquid formulation comprises: 93 to 565 nmol / mL of a peptide conjugate of chemical formula (1) in which Q, amidated at the C-terminus, is linked through a cysteine sulfur atom; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 4.8 to 5.5; 4 to 10% (w / v) of a sugar; and 0.01 to 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof.

[0053] A liquid formulation according to any one of the above-mentioned specific examples, characterized in that the liquid formulation comprises: 93 to 565 nmol / mL of a peptide conjugate of chemical formula (1), in which Q, whose C-terminus is amidated, is linked through the sulfur atom of cysteine and Z is linked through the nitrogen atom of the N-terminal proline; 5 to 25 mM of a buffer substance selected from citric acid and salts thereof, acetic acid and salts thereof, histidine and salts thereof, phosphoric acid and salts thereof, and combinations thereof, so that the pH of the liquid formulation is 4.8 to 5.5; 4 to 10% (w / v) of a sugar; and 0.01 to 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof.

[0054] The liquid formulation according to any one of the above-mentioned specific examples, characterized in that the concentration of the peptide conjugate of formula (1) in the liquid formulation is 274 to 474 nmol / mL.

[0055] The liquid formulation according to any one of the above-mentioned embodiments, characterized in that the concentration of the peptide conjugate of formula (1) in the liquid formulation is 320-430 nmol / mL.

[0056] A liquid formulation according to any one of the above specific examples, characterized in that the liquid formulation remains transparent after storage for one week under harsh test conditions of 40±2°C and 75±5% relative humidity.

[0057] Another embodiment of the present invention is a method for preparing a liquid formulation of a peptide conjugate according to any one of the above-mentioned embodiments, comprising mixing (a) the peptide conjugate of formula (1) with (b) i) a buffer substance, ii) a sugar, and iii) a surfactant.

[0058] The specific contents for carrying out the present invention will be described as follows.

[0059] Note that each description and embodiment disclosed in this application also applies to other descriptions and embodiments. In other words, any combination of various elements disclosed in this application is included in the present invention. Furthermore, the present invention is not limited to the specific description below.

[0060] Additionally, those skilled 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.

[0061] 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), and the amino acids referred to herein as abbreviations are described according to the IUPAC-IUB nomenclature system.

[0062] 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

[0063] One embodiment of the present invention provides a liquid formulation of a long-acting conjugate of a peptide active on the glucagon receptor and GLP-1 (Glucagon-like peptide-1) receptor, in which the peptide and an immunoglobulin Fc segment are linked to each other.

[0064] Specifically, the present invention provides a liquid formulation comprising a peptide conjugate of the following chemical formula (1): a buffer substance; a sugar; and a non-ionic surfactant.

[0065] TIFF0007723686000002.tif47135...(1)

[0066] In the chemical formula (1), Q is a peptide represented by the following general formula 1: Z is human immunoglobulin Fc segment; n is a natural number, and the value of n is determined so that the average molecular weight, for example, the number average molecular weight, of the [OCHCH] moieties in the peptide bond is 10 kDa; [General formula 1] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa12-Tyr-Leu-Asp-Xaa16-Lys-Arg-Ala-Xaa20-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys (SEQ ID NO: 1),

[0067] In this case, in general formula 1, Xaa2 is 2-aminoisobutyric acid (Aib), Xaa12 is lysine (K), Xaa16 is glutamic acid (E), and Xaa20 is lysine (K); A lactam ring may or may not be formed between residues Xaa12 and Xaa16 or Xaa16 and Xaa20.

[0068] The peptide conjugate of formula (1) has a structure in which a peptide Q of SEQ ID NO: 1 and a human immunoglobulin Fc fragment Z are covalently linked via an ethylene glycol repeating moiety, where Q is linked to the succinimide ring of formula (1) and Z is linked to the oxypropylene group of formula (1).

[0069] Q of the peptide conjugate may be a peptide having activity against the glucagon receptor and the GLP-1 (Glucagon-like peptide-1) receptor.

[0070] In one embodiment, the point at which Q is attached to the succinimide ring of formula (1) is the sulfur atom of the C-terminal system of Q.

[0071] Z is a human immunoglobulin Fc fragment, and as used herein, the term "human immunoglobulin Fc fragment" encompasses not only the native sequence obtained by papain digestion of immunoglobulin, but also derivatives thereof, such as sequences that differ from the native sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, of one or more amino acid residues in the native sequence.

[0072] The site of Z linked to the oxypropylene group is not particularly limited. In one embodiment of the present invention, the site of Z linked to the oxypropylene group may be the N-terminal nitrogen or a nitrogen atom of an internal residue of Z (e.g., the epsilon nitrogen of lysine). In one specific embodiment of the present invention, the site of Z linked to the oxypropylene group of Chemical Formula (1) may be the N-terminal proline of Z.

[0073] Z may be a structure in which two polypeptide chains are linked by a disulfide bond, but is not limited to such a structure in which only one of the two chains is linked through a nitrogen atom. The linkage through the nitrogen atom can be via reductive amination to the epsilon-amino atom of lysine or the N-terminal amino group.

[0074] The reductive amination reaction refers to a reaction in which an amine group or amino group of a reactant reacts with an aldehyde (i.e., a functional group capable of reductive amination) of another reactant to generate an amine, and then an amine bond is formed by a reduction reaction, and is an organic synthesis reaction widely known in the art.

[0075] In one specific example, Z may be linked through the nitrogen atom of the N-terminal proline, but is not limited thereto.

[0076] In one specific example, the immunoglobulin Fc fragment and Q may be non-glycosylated, but are not limited thereto. In the present invention, the peptide conjugate of chemical formula (1) is referred to as a "long-acting conjugate." In the present invention, a long-acting conjugate may be referred to interchangeably as a "long-acting oxyntomodulin derivative conjugate" or a "peptide conjugate."

[0077] In the present invention, the term "liquid preparation" refers to a drug that has been formulated into a liquid form from a pharmaceutical product, and includes both liquid preparations for internal use and liquid preparations for external use.

[0078] The liquid formulation of the present invention comprises a pharmacologically effective peptide conjugate of chemical formula (1) and a substance that stably maintains and / or preserves the pharmacologically effective substance for a certain period of time when the substance is formulated into a liquid form.

[0079] In the liquid formulation of the peptide conjugate of formula (1) of the present invention, storage stability is important to ensure accurate dosing.

[0080] The liquid formulation may contain a buffer substance, a sugar, and a non-ionic surfactant. Such a liquid formulation may be a solution formulation that allows stable storage of a long-acting conjugate of a peptide active against a glucagon receptor and a GLP-1 receptor.

[0081] The liquid formulation may further comprise a stabilizer, which is an amino acid selected from the group consisting of arginine, glycine, methionine, and combinations thereof, but is not limited thereto.

[0082] For example, the liquid formulation may essentially contain a buffer, sugar, and nonionic surfactant in addition to the pharmacologically effective peptide conjugate of formula (1), or may essentially contain a buffer, sugar, and nonionic surfactant with an amino acid stabilizer as an additional component, but is not limited thereto. The inventors surprisingly discovered that, compared to existing formulations containing a buffer, sugar alcohol, nonionic surfactant, and amino acid, when sugar is substituted for sugar in the formulation, superior stability is achieved even without the amino acid stabilizer. Furthermore, while conventional formulations containing a buffer, sugar alcohol, nonionic surfactant, and amino acid have the problem of high osmotic pressure compared to blood at concentrations of components sufficient to ensure formulation stability, the formulations of the present invention not only reduce osmotic pressure to a level similar to that of blood, but also have the advantage of achieving stability at least equivalent to or improved from that of conventional formulations. Specifically, by substituting sugar alcohol for sugar and partially adjusting the pH in conventional formulations, the aforementioned stability and an osmotic pressure similar to that of blood can be achieved.

[0083] Generally, the osmotic pressure of blood is about 300 mOsm / kg. The osmotic pressure of the formulation of the present invention may be in a range similar to that of blood, for example, 300±50 mOsm / kg. Thus, the formulation of the present invention having an osmotic pressure similar to that of blood does not cause pain to patients upon administration and can improve the convenience of administration.

[0084] On the other hand, methionine, an amino acid that may be additionally contained in the formulation of the present invention, may be excluded because its presence or absence does not affect stability.

[0085] According to one specific embodiment of the present invention, the liquid formulation can stably maintain the long-acting conjugate for up to 12 months, and has excellent long-term stability, with a good survival rate for up to 6 months even under accelerated conditions, and maintains transparency for up to 4 weeks even under harsh conditions.

[0086] One embodiment of the liquid formulation of the present invention may be sugar alcohol-free.

[0087] A specific example of the liquid formulation of the present invention may or may not contain an isotonicity agent, for example, but is not limited to, the liquid formulation of the present invention may not contain sodium chloride, sodium sulfate, sodium citrate, or the like.

[0088] The tonicity adjusting agent is a substance that adjusts osmotic pressure and can play a role in maintaining an appropriate osmotic pressure when the liquid formulation according to the present invention is administered into the body.

[0089] Representative examples of such isotonicity agents include water-soluble inorganic salts such as sodium chloride, sodium sulfate, or sodium citrate, and specifically include sodium chloride, but are not particularly limited thereto.

[0090] In the formulation according to the present invention, the concentration of the isotonicity agent may be, but is not limited to, 0 to 200 mM, 0 to 150 mM, 0 to 100 mM, 10 to 200 mM, 10 to 150 mM, 10 to 100 mM, 10 to 50 mM, 20 to 100 mM, 20 to 80 mM, 20 to 50 mM, 20 to 30 mM, or 40 to 50 mM. Such an isotonicity agent may be, but is not limited to, an optional component additionally contained in the liquid formulation.

[0091] It is clear that the following applies to the type and concentration of each component constituting the liquid preparation, as well as to pH.

[0092] The buffer substance, which is one component contained in the liquid formulation of the present invention, can maintain the pH of the solution so that the pH of the liquid formulation does not change suddenly, thereby stabilizing the peptide conjugate of formula (1). The buffer substance is also called a buffer system, and the buffer substance or buffer system serves to maintain the pH of the liquid formulation. Any buffer substance that maintains a pH that can stabilize the peptide conjugate of formula (1), which is the target substance to be stabilized, can be used without limitation.

[0093] The buffer substance may be a pH buffer substance such as phosphoric acid and its conjugate base, an alkali salt (e.g., phosphate salts: sodium phosphate, potassium phosphate, or their hydrogen or dihydrogen salts), citric acid and its salts (e.g., sodium citrate), acetic acid and its salts (e.g., sodium acetate), histidine and its salts, or a mixture of these buffer substances may also be used, but is not limited to these.

[0094] The liquid formulation of the present invention may contain a buffer solution containing the buffer substance as a solvent for the liquid formulation. Specifically, the buffer solution may be selected from the group consisting of citrate buffer solutions (e.g., sodium citrate buffer solutions), acetate buffer solutions (e.g., sodium acetate buffer solutions), phosphate buffer solutions (e.g., sodium phosphate buffer solutions), histidine buffer solutions, and combinations thereof. The buffer solution or the buffer substance in the liquid formulation (citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, or combinations thereof) may be contained in a concentration sufficient to maintain the target pH of the liquid formulation.

[0095] The pH of the liquid preparation may be, but is not limited to, about pH 4.5 to about pH 7.5, for example, about pH 4.5 to about pH 7.0, about pH 4.5 to about pH 6.5, about pH 4.5 to about pH 6.3, about pH 4.5 to about pH 6.0, about pH 4.5 to about pH 5.9, about pH 4.6 to about pH 5.8, about pH 4.7 to about pH 5.8, about pH 4.8 to about pH 6.0, about pH 4.8 to about pH 5.8, about pH 4.8 to about pH 5.7, about pH 4.8 to about pH 5.6, about pH 4.8 to about pH 5.5, about pH 4.8 to about pH 5.4, about pH 4.8 to about pH 5.3, about pH 4.9 to about pH 5.3, about pH 4.9 to about pH 5.2, about pH 5.0 to about pH 5.2, or about pH 5.1.

[0096] The concentration of the liquid preparation to achieve the target pH may be about 1 mM to about 200 mM, and more specifically may be about 5 mM to about 100 mM, about 5 mM to about 80 mM, about 5 mM to about 40 mM, about 8 mM to about 40 mM, about 5 mM to about 30 mM, or about 5 mM to about 25 mM, but is not particularly limited thereto.

[0097] The saccharide, a component of the liquid formulation of the present invention, refers to a monosaccharide, disaccharide, polysaccharide, oligosaccharide, etc., and can increase the stability of long-acting conjugates of peptides active against glucagon receptors and glucagon-like peptide-1 (GLP-1) receptors. Specific examples include, but are not limited to, monosaccharides such as mannose, glucose, fructose, galactose, fucose, and xylose; disaccharides such as lactose, maltose, and sucrose; and polysaccharides such as raffinose and dextran.

[0098] The sugar may be present at a concentration of about 1 to about 20% (w / v), about 1 to 15% (w / v), about 2 to about 15% (w / v), about 2 to about 12% (w / v), about 2 to about 12% (w / v), about 3 to about 10% (w / v), about 4 to about 10% (w / v), about 5 to about 10% (w / v), about 6 to about 10% (w / v), about 7 to about 10% (w / v), about 7 to about 9% (w / v), about 8 to about 9% (w / v), or about 8.5% (w / v) of the total solution of the liquid preparation, but is not particularly limited thereto.

[0099] Although not particularly limited thereto, a nonionic surfactant, which is one component contained in the liquid formulation, can reduce the surface tension of a protein solution and prevent the protein from adsorbing or aggregating on a hydrophobic surface.

[0100] Specific examples of nonionic surfactants used in the present invention include polysorbates (e.g., polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate; the number (20) after polyoxyethylene means the total number of oxyethylene groups (-(CH2CHO)-))), poloxamer (PEO-PPO-PEO copolymer; PEO: poly(ethylene oxide), PPO: poly(propylene oxide), polyethylene-polypropylene glycol, polyoxyethylene compounds (for example, polyoxyethylene-stearate, polyoxyethylene alkyl ether (alkyl: C1-C30), polyoxyethylene monoallyl ether, alkylphenyl polyoxyethylene copolymer (alkyl: C1-C30), etc.), sodium dodecyl sulfate (SDS), etc., or polysorbate or poloxamer may also be mentioned, and these may be used in the form of a combination of one or more thereof.

[0101] Specifically, the nonionic surfactant may be polysorbate 80, polysorbate 60, polysorbate 40, polysorbate 20, or poloxamer 188, or a combination thereof, but is not particularly limited thereto.

[0102] In the present invention, the nonionic surfactant is preferably not contained in a high concentration. Specifically, the nonionic surfactant may be contained in the formulation of the present invention at a concentration of about 0.2% (w / v) or less, for example, about 0.001 to about 0.2% (w / v), about 0.001 to about 0.1% (w / v), about 0.001 to about 0.05% (w / v), about 0.005 to about 0.08% (w / v), about 0.002 to about 0.05% (w / v), about 0.005 to about 0.05% (w / v), about 0.01 to about 0.05% (w / v), about 0.01 to about 0.04% (w / v), about 0.01 to about 0.03% (w / v), or about 0.02% (w / v), but is not particularly limited thereto.

[0103] The amino acid stabilizer that may be added as an optional component to the liquid formulation may be, but is not limited to, methionine, arginine, glycine, or a combination thereof, and may be in the L-form, but is not particularly limited thereto.

[0104] The amino acids can suppress the production of impurities that may occur due to oxidation reactions of proteins, but are not particularly limited to these.

[0105] The amino acid may be present in the formulation at a concentration of about 0.01 to about 1 mg / mL, about 0.01 to about 0.8 mg / mL, about 0.01 to about 0.5 mg / mL, about 0.02 to about 0.5 mg / mL, or about 0.02 to about 0.4 mg / mL, but is not particularly limited thereto.

[0106] Meanwhile, the liquid formulation of the present invention may optionally further contain, in addition to the essential components of the liquid formulation, sugar; buffer substance; and nonionic surfactant, an optional component, an amino acid as a stabilizer, and other components or substances known in the art within the scope that do not impair the effects of the present invention, but is not limited thereto.

[0107] The long-acting conjugate, which is the peptide conjugate of chemical formula (1), can be present in a pharmacologically effective amount in the liquid formulation, and examples of such amounts include those in which the concentration of the peptide conjugate of chemical formula (1) is about 18 to about 940 nmol / mL, about 18.7 to about 935 nmol / mL, about 18 to about 842 nmol / mL, about 18 to about 748 nmol / mL, about 18 to about 655 nmol / mL, about 18 to about 561 nmol / mL, about 18 to about 468 nmol / mL, about 18 to about 374 nmol / mL, about 93 to about 940 nmol / mL, about 93.5 to about 561 nmol / mL, about 93 to about 842 nmol / mL, about 93 to about 748 nmol / mL, about 93 to about 655 nmol / mL, about 93 to about 565 nmol / mL, and about 93 to about 561 nmol / mL. L, about 93 to about 468 nmol / mL, about 150 to about 468 nmol / mL, about 200 to about 468 nmol / mL, about 250 to about 468 nmol / mL, about 274 to about 474 nm ol / mL, about 280 to about 468 nmol / mL, about 300 to about 468 nmol / mL, about 300 to about 450 nmol / mL, about 320 to about 430 nmol / mL, about 320 to about The concentration may be, but is not limited to, 440 nmol / mL, about 340 to about 420 nmol / mL, about 340 to about 400 nmol / mL, about 350 to about 400 nmol / mL, about 360 to about 390 nmol / mL, about 365 to about 385 nmol / mL, about 370 to about 380 nmol / mL, about 93 to about 374 nmol / mL, or about 374 nmol / mL.

[0108] In the present invention, the term "about" refers to a range that includes, but is not limited to, ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, ±0.01, etc., and includes all numerical values in a range that is equal to or similar to the numerical value following the term "about."

[0109] As a specific example, the liquid preparation may contain a peptide conjugate of chemical formula (1); a buffer substance selected from the group consisting of citric acid and salts thereof, acetic acid and salts thereof, histidine and salts thereof, phosphoric acid and salts thereof, and combinations thereof; a sugar selected from the group consisting of glucose, fructose, galactose, lactose, maltose, sucrose, and combinations thereof; and a nonionic surfactant selected from the group consisting of poloxamer, polysorbate, and combinations thereof, and have a pH of 4.8 to 6.0.

[0110] As one specific example, the liquid formulation may contain 18 to 940 nmol / mL of the peptide conjugate of chemical formula (1); 5 to 100 mM of a buffer substance selected from the group consisting of citric acid and salts thereof, acetic acid and salts thereof, histidine and salts thereof, phosphoric acid and salts thereof, and combinations thereof, for maintaining the pH of the liquid formulation in the range of 4.8 to 6.0; 1% to 20% (w / v) of a sugar selected from the group consisting of glucose, fructose, galactose, lactose, maltose, sucrose, or combinations thereof; and 0.001% to 0.2% (w / v) of a nonionic surfactant selected from the group consisting of poloxamer, polysorbate, and combinations thereof.

[0111] As a specific example, the liquid formulation may contain, but is not limited to, 18 to 940 nmol / mL of the peptide conjugate of formula (1); 5 to 100 mM of acetic acid or a salt thereof to maintain the pH of the liquid formulation in the range of 4.8 to 6.0; 1% to 20% (w / v) sucrose; and 0.001% to 0.2% (w / v) polysorbate.

[0112] As a specific example, the liquid formulation may contain, but is not limited to, 93 to 565 nmol / mL of a peptide conjugate of formula (1) linked through the sulfur atom of the cysteine of Q, the C-terminus of which is amidated; 5 to 25 mM of a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, so that the pH of the liquid formulation is 4.8 to 5.5; 4 to 10% (w / v) of a sugar; and 0.01 to 0.1% (w / v) of a nonionic surfactant selected from poloxamer, polysorbate, or combinations thereof.

[0113] The liquid preparation may be transparent when stored for one week under the severe test conditions of 40±2°C and a relative humidity of 75±5%.

[0114] As one specific example, the liquid formulation may be transparent when stored under harsh test conditions of 40±2°C and a relative humidity of 75±5% for about 1 week or more, about 2 weeks or more, about 3 weeks or more, or about 4 weeks or more.

[0115] According to one embodiment of the present invention, a liquid formulation comprising a peptide conjugate of formula (1), a buffer, a sugar, and a non-ionic surfactant, was found to be more stable for up to 6 months under accelerated conditions of 25±2°C and 60±5% relative humidity than a liquid formulation comprising a buffer, a sugar alcohol, an amino acid, and a non-ionic surfactant. It was also found to be transparent after storage for 1 week under severe test conditions of 40±2°C and 75%±5% relative humidity, and remained transparent for up to 4 weeks (Tables 6 to 9).

[0116] In this invention, the term "accelerated testing" refers to a test designed to increase the chemical decomposition or physical changes of raw materials or finished drugs by adopting excessive storage conditions as part of the official stability test for drugs. In addition to long-term stability tests, accelerated testing data can be used to evaluate long-term chemical effects under non-accelerated conditions, and to evaluate the effects of short-term deviations from the labeled storage conditions, such as those that may occur during transportation. This test is designed to increase the rate of chemical denaturation and physical changes of raw materials or drugs under conditions of temperature and humidity that are higher than normal storage conditions.

[0117] In the present invention, the term "stress testing" refers to a test that determines the fundamental characteristics of a drug's stability. It is conducted during the drug development process under conditions that are more severe than accelerated testing, and is useful for determining the expected degradation products and physical changes of a drug.

[0118] Q of the peptide conjugate of formula (1) may be a peptide active against the glucagon receptor and the GLP-1 (Glucagon-like peptide-1) receptor. The "peptide active against the glucagon receptor and the GLP-1 (Glucagon-like peptide-1) receptor" includes various substances, for example, various peptides, that have significant levels of activity against the glucagon receptor and the GLP-1 receptor.

[0119] Although not particularly limited thereto, the peptide having a significant level of activity on the glucagon receptor and the GLP-1 receptor may be referred to herein interchangeably as a "GCG / GLP-1 receptor dual agonist," a "dual acting agent," or an "oxyntomodulin derivative."

[0120] More specifically, the peptide having activity on the glucagon receptor and GLP-1 (Glucagon-like peptide-1) receptor may be a peptide having activity on the glucagon receptor and GLP-1 (Glucagon-like peptide-1) receptor, which comprises a sequence represented by the following general formula 1: [General formula 1] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa12-Tyr-Leu-Asp-Xaa16-Lys-Arg-Ala-Xaa20-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys (SEQ ID NO: 1),

[0121] In this case, in general formula 1, Xaa2 is 2-aminoisobutyric acid (Aib), Xaa12 is lysine (K), Xaa16 is glutamic acid (E), and Xaa20 is lysine (K); A lactam ring may or may not be formed between residues Xaa12 and Xaa16 or Xaa16 and Xaa20.

[0122] In this specification, "Aib" may be used interchangeably with "2-aminoisobutyric acid" or "aminoisobutyric acid," and 2-aminoisobutyric acid and aminoisobutyric acid may be used interchangeably.

[0123] The peptide may include, but is not limited to, an amino acid sequence comprising SEQ ID NO: 2, 3, or 4, or an amino acid sequence (essentially) selected from the group consisting of SEQ ID NO: 2, 3, or 4.

[0124] On the other hand, even if the present application describes a "peptide consisting of a specific sequence number," if the peptide has the same or corresponding activity as a peptide consisting of the amino acid sequence of the 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, 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.

[0125] Alternatively, such peptides may be non-naturally occurring.

[0126] The C-terminus of the peptide may be amidated, or may have a free carboxyl group (—COOH), or may include a peptide with an unmodified C-terminus, but is not limited thereto.

[0127] As a specific example, the C-terminus of Q may be amidated, but is not limited to this.

[0128] In one specific example, Q may be, but is not limited to, non-glycosylated.

[0129] Furthermore, the peptides active against the glucagon receptor and the GLP-1 (Glucagon-like peptide-1) receptor may contain an intramolecular bridge (e.g., a covalent or non-covalent bridge), specifically, may be in a form containing a ring, such as a ring formed between the 12th and 16th amino acids or the 16th and 20th amino acids in the general formula, but are not limited thereto. A non-limiting example of the ring may be a lactam bridge (or lactam ring).

[0130] 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.

[0131] 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.

[0132] The term "pharmaceutically acceptable" means, within the scope of medical judgment, a substance that can be effectively used for the desired purpose without inducing excessive toxicity, irritation, allergic reaction, or the like.

[0133] 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.

[0134] The term "solvate" used in the present invention refers to a complex formed between the peptide according to the present invention or a salt thereof and a solvent molecule.

[0135] In the present invention, the term "peptide conjugate of Chemical Formula (1)" refers to an active ingredient contained in the liquid formulation of the present invention and may be contained in the liquid formulation in a pharmacologically effective amount. Specifically, the peptide active against the glucagon receptor and the GLP-1 receptor and the immunoglobulin Fc region are linked to each other via a linker, and the conjugate can exhibit increased durability of efficacy compared to a peptide active against the glucagon receptor and the GLP-1 receptor to which the immunoglobulin Fc region is not bound. In the present invention, such a conjugate is referred to as a "long-acting conjugate." In the present invention, the term "long-acting conjugate" is also used interchangeably with "long-acting oxyntomodulin derivative conjugate" or "peptide conjugate."

[0136] Alternatively, such conjugates may be non-naturally occurring.

[0137] Furthermore, in the peptide conjugate of chemical formula (1), the link between Q, a peptide active against glucagon receptors and GLP-1 receptors, and the immunoglobulin Fc fragment may be a physical or chemical bond, or a non-covalent or covalent bond, specifically, a covalent bond, but is not limited thereto.

[0138] Furthermore, in the peptide conjugate of chemical formula (1), the method for linking Q, a peptide active on glucagon receptors and GLP-1 receptors, to the immunoglobulin Fc fragment is not particularly limited, and the peptide active on glucagon receptors and GLP-1 receptors and the immunoglobulin Fc fragment may be linked to each other via a linker.

[0139] Specifically, the long-acting conjugate contained in the liquid preparation of the present application may be represented by the above chemical formula (1).

[0140] A method for preparing such peptide conjugates is described in Korean Patent Registration No. 10-725315, the entire specification of which is incorporated herein by reference.

[0141] In the present invention, the term "immunoglobulin Fc segment" refers to the heavy chain constant region of an immunoglobulin, excluding the heavy and light chain variable regions. Specifically, the immunoglobulin Fc segment may include a portion of heavy chain constant region 2 (CH2) and / or heavy chain constant region 3 (CH3), and more specifically, may further include a hinge region (meaning the entire hinge region or a portion thereof).

[0142] The immunoglobulin Fc fragment is a component that forms part of the peptide conjugate of the present invention represented by chemical formula (1), and specifically, can correspond to Z in chemical formula (1).

[0143] Such an immunoglobulin Fc segment may include, but is not limited to, a hinge portion in the heavy chain constant region.

[0144] In the present invention, the immunoglobulin Fc segment may comprise a specific hinge sequence at the N-terminus.

[0145] As used herein, the term "hinge sequence" refers to the site located in the heavy chain that forms a dimer of immunoglobulin Fc segments through inter-disulfide bonds.

[0146] In the present invention, the hinge sequence may be mutated to have only one cysteine residue by deleting a portion of the hinge sequence having the following amino acid sequence, but is not limited thereto:

[0147] Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Cys-Pro (SEQ ID NO: 6).

[0148] The hinge sequence may be one in which the 8th or 11th cysteine residue in the hinge sequence of SEQ ID NO: 6 is deleted and only one cysteine residue is contained. The hinge sequence of the present invention may be one composed of 3 to 12 amino acids and containing only one cysteine residue, but is not limited thereto. 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 (SEQ ID NO: 7), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser-Pro (SEQ ID NO: 8), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 9), Glu -Ser-Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Pro (SEQ ID NO: 10), Lys-Tyr-Gly-Pro-Pro-Cys-Pro-Ser (SEQ ID NO: 11), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 12), Glu-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 13), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 14) 4), Glu-Pro-Ser-Cys-Pro (SEQ ID NO: 15), Pro-Ser-Cys-Pro (SEQ ID NO: 16), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 17), Lys-Tyr-Gly-Pro-Pro-Pro-Ser-Cys-Pro (SEQ ID NO: 18), Glu-Ser-Lys-Tyr-Gly-Pro-Ser-Cys-Pro (SEQ ID NO: 19) ), Glu-Ser-Lys-Tyr-Gly-Pro-Pro-Cys (SEQ ID NO: 20), Lys-Tyr-Gly-Pro-Pro-Cys-Pro (SEQ ID NO: 21), Glu-Ser-Lys-Pro-Ser-Cys-Pro (SEQ ID NO: 22), Glu-Ser-Pro-Ser-Cys-Pro (SEQ ID NO: 23), Glu-Pro-Ser-Cys (SEQ ID NO: 24), Ser-Cys-Pro (SEQ ID NO: 25). More specifically, the hinge sequence may comprise the amino acid sequence of SEQ ID NO: 25 (Ser-Cys-Pro) or SEQ ID NO: 16 (Pro-Ser-Cys-Pro), but is not limited thereto.

[0149] The immunoglobulin Fc fragment of the present invention 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 peptide conjugate of chemical formula (1) of the present invention may be in the form in which one end of a linker is linked to one chain of the dimeric immunoglobulin Fc fragment, but is not limited thereto.

[0150] 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 segment of the present invention may also include, but is not limited to, a hinge sequence at the N-terminus.

[0151] Furthermore, the immunoglobulin Fc region of the present invention may be an extended Fc fragment that excludes only the heavy and light chain variable regions of the immunoglobulin and includes part or the entire heavy chain constant region 1 (CH1) and / or light chain constant region 1 (CL1), as long as it exhibits effects substantially equivalent to or improved from those of the native form. 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.

[0152] For example, the immunoglobulin Fc fragment 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 of the CH1 domain, the CH2 domain, the CH3 domain, and the CH4 domain with an immunoglobulin hinge region (or a portion of the hinge region), or 6) a dimer of each domain of a heavy chain constant region and a light chain constant region.

[0153] In one specific example, the immunoglobulin Fc fragment may be in a dimeric form, and one molecule of the peptide of general formula 1 may be covalently linked to one Fc region of the dimeric form, and in this case, the immunoglobulin Fc and the peptide of general formula 1 may be linked to each other via a polyethylene glycol linker. Alternatively, two molecules of the peptide of general formula 1 may be symmetrically linked to one Fc region of the dimeric form. In this case, the immunoglobulin Fc and the peptide of general formula 1 may be linked to each other via a polyethylene glycol linker. However, the present invention is not limited to the above example.

[0154] Furthermore, the immunoglobulin Fc fragments of the present invention include not only naturally occurring amino acid sequences but also sequence derivatives thereof, which means that one or more amino acid residues in the naturally occurring amino acid sequence differ from the sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

[0155] 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, can be used as suitable sites for modification.

[0156] 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, complement binding sites, such as Clq binding sites, may be removed to eliminate effector functions, and ADCC (antibody dependent cell-mediated cytotoxicity) sites may also be removed. Techniques for producing such sequence derivatives of immunoglobulin Fc fragments are disclosed in International Patent Publication Nos. WO 97 / 34631 and WO 96 / 32478, among others.

[0157] 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 can also be performed.

[0158] The Fc derivative may exhibit biological activity equivalent to that of the Fc fragment of the present invention, and may have increased structural stability of the Fc region against heat, pH, and the like.

[0159] Furthermore, such Fc fragments may be obtained from natural forms isolated from the living bodies of animals such as humans, cows, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, or may be recombinant forms or derivatives thereof obtained from transformed animal cells or microorganisms. Here, obtaining natural forms may involve isolating whole immunoglobulins from the living body of a human or animal and then treating them with a 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 fragments can be separated using size-exclusion chromatography or other methods. In a more specific embodiment, the human-derived Fc fragment is a human immunoglobulin Fc fragment obtained from a microorganism.

[0160] Furthermore, the immunoglobulin Fc fragment may have native glycosylation, increased glycosylation compared to the native form, decreased glycosylation compared to the native form, or a form in which the glycosylation has been removed. Such increase, decrease, or removal of immunoglobulin Fc glycosylation can be achieved by conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms. Here, an immunoglobulin Fc fragment from which the glycosylation has been removed from the Fc fragment 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 more suited to the original purpose as a drug carrier is an immunoglobulin Fc fragment from which the glycosylation has been removed or which has been deglycosylated.

[0161] In the present invention, "deglycosylation" refers to an Fc fragment from which sugar chains have been removed using an enzyme, and "non-glycosylated" refers to an Fc fragment that is produced in a prokaryote, and in a more specific embodiment, in Escherichia coli, and is not glycosylated.

[0162] On the other hand, the immunoglobulin Fc fragment may be of animal origin, such as human or bovine, goat, porcine, mouse, rabbit, hamster, rat, guinea pig, and in a more specific embodiment, human origin, but is not limited thereto.

[0163] The immunoglobulin Fc fragment may also be an Fc fragment derived from IgG, IgA, IgD, IgE, IgM, or a combination or hybrid thereof. In a more specific embodiment, the Fc fragment is derived from IgG or IgM, which are the most abundant fragments in human blood, and in an even more specific embodiment, the Fc fragment is derived from IgG, which is known to improve the half-life of ligand-binding proteins. In an even more specific embodiment, the immunoglobulin Fc fragment is an IgG4 Fc fragment, and in a most specific embodiment, the immunoglobulin Fc fragment is a non-glycosylated Fc fragment derived from human IgG4, but is not limited thereto.

[0164] In a specific embodiment, the immunoglobulin Fc fragment is a fragment of human IgG4 Fc and may be in the form of a homodimer in which two monomers are linked through a disulfide bond (inter-chain form) between the cysteines at the third amino acids of each monomer, and in this case, each monomer of the homodimer independently has / can have two internal disulfide bonds (intra-chain form), i.e., an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199. The number of amino acids in each monomer may be 221, and the total number of amino acids forming the homodimer may be, but is not limited to, 442. Specifically, the immunoglobulin Fc segment may be, but is not limited to, a homodimer formed by two monomers having the amino acid sequence of SEQ ID NO: 5 (composed of 221 amino acids) via a disulfide bond between the cysteines at the third amino acids of each monomer, and the monomers of the homodimer may independently form an internal disulfide bond between cysteines at positions 35 and 95 and an internal disulfide bond between cysteines at positions 141 and 199. As an example, the immunoglobulin Fc segment may be, but is not limited to, a homodimer comprising the amino acid sequence of SEQ ID NO: 26 (composed of 442 amino acids).

[0165] Meanwhile, in the present invention, the term "combination" refers to the formation of a dimer or multimer by a polypeptide encoding a single-chain immunoglobulin Fc fragment of the same origin binding with a single-chain polypeptide of a different origin. That is, a dimer or multimer can be prepared from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

[0166] In the present invention, the term "hybrid" refers to the presence of sequences corresponding to immunoglobulin Fc fragments of two or more different origins within a single-chain immunoglobulin constant region. In the present invention, various forms of hybrids are possible. That is, hybrids of domains consisting of one to four 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.

[0167] Meanwhile, IgG can also be divided into subclasses, IgG1, IgG2, IgG3, and IgG4, and the present invention also allows for combinations or hybridization of these subclasses. Specifically, these are the IgG2 and IgG4 subclasses, and most specifically, the Fc fragment of IgG4, which has almost no effector functions such as complement-dependent cytotoxicity (CDC).

[0168] Another embodiment of the present invention provides a method for preparing a liquid formulation of a peptide conjugate of formula (1), comprising the steps of mixing (a) the peptide conjugate of formula (1) with (b) i) a buffer substance, ii) a sugar, and iii) a non-ionic surfactant.

[0169] The conjugate, buffer, sugar, and non-ionic surfactant, and liquid formulation are as described above.

[0170] In another embodiment, the present invention provides a pharmaceutical composition for preventing or treating obesity or diabetes, comprising the liquid formulation.

[0171] In the present invention, the term "prevention" refers to any action that inhibits or delays the onset of a target disease. In the present invention, "prevention" refers to the inhibition or delay of the onset of diabetic symptoms such as abnormal blood glucose and abnormal insulin secretion regulation, or obesity symptoms such as weight gain and increased body fat percentage, by administering the conjugate of the present invention.

[0172] In the present invention, the term "treatment" refers to any action that alleviates, improves, or alleviates the symptoms of an existing disease. In the present invention, "treatment" refers to the administration of the conjugate of the present invention, which results in the alleviation, improvement, or alleviation of the diabetic symptoms or obesity symptoms, and the normalization of blood glucose, normalization of insulin secretion, weight loss, or reduction in body fat percentage.

[0173] In the present invention, the term "obesity" refers to a state in which there is an excess of adipose tissue in the body, and is defined as a body mass index (body weight (kg) divided by the square of height (m)) of 25 or more. Obesity is usually caused by an energy imbalance resulting from a long-term excessive intake of nutrients compared to energy expenditure. Obesity is a metabolic disease that affects the entire body, and increases the likelihood of developing diabetes and hyperlipidemia, sexual dysfunction, arthritis, and cardiovascular disease, and in some cases is associated with the development of cancer.

[0174] In the present invention, the term "diabetes" refers to a type of metabolic disease characterized by insufficient insulin secretion or inability to achieve normal function, characterized by hyperglycemia, which is an increase in blood glucose concentration, causing various symptoms and resulting in the excretion of glucose in the urine.

[0175] The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier, excipient, or diluent. In the present invention, the term "pharmaceutically acceptable" means a sufficient amount to exhibit a therapeutic effect and not cause side effects, and can be easily determined by a person skilled in the art depending on factors well known in the medical field, such as the type of disease, the patient's age, weight, health, sex, sensitivity of the patient to the drug, administration route, administration method, administration frequency, treatment period, and drugs used in combination or concomitantly.

[0176] In another embodiment, the present invention provides a method for preventing or treating obesity or diabetes, comprising administering the liquid formulation to an individual.

[0177] The liquid formulation, obesity and diabetes are as described above.

[0178] The individual is an individual suspected of having obesity or diabetes, and refers to a mammal, including humans, rats, livestock, etc., who has or may have the disease, but includes, without limitation, individuals who can be treated with the liquid formulation of the present invention.

[0179] The therapeutic methods of the present invention may involve administering a pharmaceutically effective amount of a pharmaceutical composition, including a liquid formulation. The appropriate total daily dose will be determined by the treating physician within the scope of sound medical judgment and may be administered in single or divided doses. However, for purposes of the present invention, the specific therapeutically effective amount for a particular patient will vary depending on a variety of factors, including the type and degree of response to be achieved, the specific composition, including whether other formulations are used, the patient's age, weight, general health, sex, and diet, the time and route of administration, the excretion rate of the composition, the duration of treatment, and drugs used in conjunction with or concurrently with the specific composition, as well as similar factors well known in the pharmaceutical arts.

[0180] The present invention will be described in more detail below with reference to the following examples, but the following examples are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0181] Example 1: Preparation of a GLP-1 / Glucagon Receptor Dual Agonist To determine the stability of a GLP-1 / Glucagon receptor dual agonist active at both the GLP-1 receptor and the glucagon receptor in the liquid formulation of the present invention, a GLP-1 / Glucagon receptor dual agonist having the amino acid sequence shown below was synthesized (Table 1). In the present application, the GLP-1 / Glucagon receptor dual agonist may be used in combination with an oxyntomodulin derivative.

[0182] [Table 1]

[0183] In Table 1, the amino acids shown in bold indicate that the amino acids shown in bold form a ring with each other. Also, Aib indicates aminoisobutyric acid, a non-natural amino acid.

[0184] Example 2: Preparation of mono-PEGylated immunoglobulin Fc fragments Example 2-1. Preparation of a monopegylated immunoglobulin Fc fragment To prepare a mono-pegylated immunoglobulin Fc fragment in which only one of the two N-termini of the immunoglobulin Fc fragment is PEGylated, an immunoglobulin Fc fragment (49.8 kDa, homodimer of SEQ ID NO: 5) having a hinge region with the sequence Pro-Ser-Cys-Pro at the N-terminus was reacted with a PEG linker of formula (2) (number-average molecular weight 10 kDa) at a molar ratio of immunoglobulin Fc:PEG linker = 1:1 at a concentration of 50 g / L at 6 ± 4°C for approximately 4 hours.

[0185] TIFF0007723686000004.tif36135...(2)

[0186] 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) as a reducing agent. To obtain a monopegylated immunoglobulin Fc fragment, the reaction mixture was diluted with Bis Tris buffer and purified.

[0187] At this time, the monopegylated immunoglobulin Fc fragment was purified using a CaptoQ ImpRes (GE Healthcare, anion exchange chromatography) column with a buffer containing BisTris and a sodium chloride gradient.

[0188] Example 2-2. Structural analysis of a monopegylated immunoglobulin Fc fragment The structure of the mono-pegylated immunoglobulin Fc fragment prepared by the method of Example 2-1 was analyzed by MALDI-TOF and peptide mapping. The MALDI-TOF analysis showed that the molecular weight was consistent with the predicted molecular weight of the mono-pegylated immunoglobulin Fc fragment, and the peptide mapping analysis confirmed that PEG was pegylated to more than 90% of the N-terminus of the immunoglobulin Fc fragment.

[0189] Meanwhile, the mono-PEGylated immunoglobulin Fc fragment (Chemical Formula (3)) prepared by the method of Example 2-1 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.

[0190] TIFF0007723686000005.tif48134...(3)

[0191] Example 3: Conjugate production by linking a pegylated immunoglobulin Fc fragment with an oxyntomodulin derivative The single-pegylated immunoglobulin Fc fragment prepared in Example 2-1 was linked to the oxyntomodulin derivative of SEQ ID NO: 3 (C-terminal amidated), selected as a representative oxyntomodulin derivative, to prepare a long-acting conjugate as follows.

[0192] A single pegylated immunoglobulin Fc segment was reacted with an oxyntomodulin derivative through peptide conjugation without ultra / diafiltration. Specifically, after the anion exchange chromatography of Example 2-1, a peptide conjugation reaction of the oxyntomodulin derivative (SEQ ID NO: 3), a GLP-1 / glucagon receptor dual agonist, was carried out without ultra / diafiltration to prepare a long-acting conjugate (immunoglobulin Fc segment-PEG-containing linker-oxyntomodulin derivative). In this application, the term "long-acting conjugate" may be used interchangeably with the term "long-acting oxyntomodulin derivative conjugate."

[0193] The mono-pegylated immunoglobulin Fc fragment and the oxyntomodulin derivative were reacted at a molar ratio of 1:1, with the oxyntomodulin derivative at 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 fragment would be conjugated to the cysteine of the oxyntomodulin derivative. 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 fragment-PEG-containing linker-oxyntomodulin derivative conjugate (Chemical Formula (4)) was confirmed to be over 90% by SE-HPLC, over 80% by RP-HPLC, and over 80% by IE-HPLC.

[0194] 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 fragment-PEG-containing linker-oxyntomodulin derivative. The conjugate was purified using a gradient of sodium citrate-containing buffer and ammonium sulfate.

[0195] The eluted immunoglobulin Fc fragment-PEG-containing linker-oxyntomodulin derivative conjugate (chemical formula (4)) was analyzed using MALDI-TOF, SE-HPLC, RP-HPLC, and IE-HPLC analytical methods. MALDI-TOF analysis showed that the molecular weight of the immunoglobulin Fc fragment-PEG-containing linker-oxyntomodulin derivative conjugate was consistent with the predicted molecular weight, and that it had been produced with high purity of over 90% by SE-HPLC, over 90% by RP-HPLC, and over 90% by IE-HPLC.

[0196] TIFF0007723686000006.tif60134...(4)

[0197] Example 4: Preparation of liquid formulations and evaluation of osmolality To confirm the stability of the newly created liquid formulation of the long-acting oxyntomodulin derivative conjugate prepared in Example 3, a liquid formulation with a composition publicly known from International Publication WO2014-073842 (Comparative Example) and a liquid formulation with a new composition according to the present invention (Example) were prepared with the compositions shown in Table 2 below. The formulations in the Examples are representative examples of the buffer, sugar, and nonionic surfactant components of the stabilizer of the present invention, with acetic acid and sodium acetate selected as the buffer, sucrose as the sugar, and polysorbate 20 as the nonionic surfactant.

[0198] The existing comparative formulation had a higher osmotic pressure than blood, and a new formulation was created to further improve stability. To improve stability, the pH of the existing formulation was lowered and mannitol was replaced with sucrose. This confirmed that the osmotic pressure also decreased to a level similar to that of blood, and the addition of methionine did not affect stability, so it was removed in the examples of the present invention. Osmotic pressure measurements showed that the comparative formulation was 494 mOsm / kg and the example formulation was 305 mOsm / kg, similar to the osmotic pressure of blood (~300 mOsm / kg).

[0199] [Table 2]

[0200] Example 5: Evaluation of long-term stability of the liquid formulations of the examples To confirm the long-term stability of the liquid formulation of the long-acting oxyntomodulin derivative conjugate prepared in Example 4 (Example), it was stored at 5±3°C for 0 to 12 months and then analyzed using ion exchange-high performance liquid chromatography (IE-HPLC), size exclusion-high performance liquid chromatography (SE-HPLC), and reverse phase-high performance liquid chromatography (RP-HPLC). In Tables 3, 4, and 5, IE-HPLC (%), RP-HPLC (%), and SE-HPLC (%) indicate the area% of the peak corresponding to the long-acting oxyntomodulin derivative conjugate at each analysis time point.

[0201] [Table 3]

[0202] [Table 4]

[0203] [Table 5]

[0204] As can be seen from the above results, the composition of the liquid preparation of the present invention was confirmed to be highly stable for up to 12 months, and was found to be the optimal composition for long-term storage.

[0205] Example 6: Evaluation of accelerated stability To confirm the accelerated stability of the liquid formulations of the Example and Comparative Examples prepared in Example 4, they were stored for 0-6 months at 25±2°C and 60±5% relative humidity, and then analyzed using IE-HPLC, RP-HPLC, and SE-HPLC. Accelerated stability testing is a test designed to increase chemical decomposition or physical changes in raw or finished pharmaceuticals by employing excessive storage conditions. In addition to long-term stability testing, accelerated stability evaluation can evaluate long-term chemical effects under non-accelerated conditions and the effects of short-term deviations from the labeled storage conditions, such as those that may occur during transportation. In Tables 6, 7, and 8, IE-HPLC (%), RP-HPLC (%), and SE-HPLC (%) indicate the area% of the peak corresponding to the sustained-acting oxyntomodulin derivative conjugate at each analysis time point.

[0206] [Table 6]

[0207] [Table 7]

[0208] [Table 8]

[0209] As can be seen from the results in Tables 6, 7, and 8, the liquid formulation of the present invention, which does not contain sugar alcohol but contains sucrose, was found to be more stable for up to 6 months under accelerated conditions than the comparative example. These results suggest that the liquid formulation of the present invention has high stability.

[0210] Example 7: Evaluation of Severe Stability To confirm the harsh stability of the liquid formulation of the example of the long-acting oxyntomodulin derivative conjugate prepared in Example 4, it was stored separately from the liquid formulation of the comparative example at 40±2°C and 75±5% relative humidity for 0-4 weeks, and then its properties were analyzed. The harsh stability test is a test that determines the effect of changes in conditions on a product when the product is exposed to conditions other than those specified for storage, and assesses the decomposition patterns and physicochemical stability of the raw drug substance and / or finished drug product. Table 9 shows the properties of the liquid formulations of the comparative example and example under these harsh conditions.

[0211] [Table 9]

[0212] As can be seen from the results in Table 9, under harsh conditions, the liquid formulation composition of the comparative example caused the long-acting oxyntomodulin derivative conjugate to become opaque after one week and precipitated by the third week. In contrast, the liquid formulation composition of the present invention, which does not contain a sugar alcohol but contains the sugar sucrose, maintained its transparency for up to four weeks, confirming that the long-acting oxyntomodulin derivative conjugate, which is the target of stabilization, was stable without precipitation. These results suggest that the liquid formulation composition of the present invention is more stable than known compositions and can therefore be provided as a liquid pharmaceutical formulation.

[0213] 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.

Claims

1. A liquid formulation of a peptide conjugate, said liquid formulation comprising 18 to 940 nmol / mL of a peptide conjugate of the following chemical formula (1): a buffer substance in an amount to maintain the pH of the liquid formulation in the range of 4.5 to 7.5; 1-20% (w / v) sugar; and A liquid formulation comprising: 0.001-0.2% (w / v) of a non-ionic surfactant; 【Chemical 1】 ・・・（１） In the chemical formula (1), Q is a peptide of the following general formula 1: Z is a human immunoglobulin Fc fragment; n is a natural number, and the value of n is the [OCH 2 CH 2 ] The average molecular weight of the n moiety is determined to be 10 kDa, [General formula 1] His-Xaa2-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Xaa12-Tyr-Leu-Asp-Xaa1 6-Lys-Arg-Ala-Xaa20-Glu-Phe-Val-Gln-Trp-Leu-Met-Asn-Thr-Cys (SEQ ID NO: 1), In this case, in general formula 1, Xaa2 is 2-aminoisobutyric acid (Aib), Xaa12 is lysine (K), Xaa16 is glutamic acid (E), and Xaa20 is lysine (K); A lactam ring may or may not be formed between residues Xaa12 and Xaa16 or between residues Xaa16 and Xaa20.

2. The liquid formulation according to claim 1, wherein the peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 2 to 4.

3. 2. The liquid formulation of claim 1, wherein Q is the amino acid sequence of SEQ ID NO:

3.

4. The liquid formulation according to claim 1 , wherein Q is amidated at its C-terminus.

5. 2. The liquid formulation of claim 1, wherein Q is linked through the sulfur atom of the cysteine.

6. The liquid formulation of claim 1 , wherein the immunoglobulin Fc fragment is derived from IgG4.

7. The liquid formulation according to claim 1, wherein Z is a structure in which two polypeptide chains are linked by a disulfide bond, and the two chains are linked only through a nitrogen atom of one of the chains.

8. The liquid formulation of any one of claims 1 to 7, wherein Z comprises a monomer having the amino acid sequence of SEQ ID NO:

5.

9. 9. The liquid formulation of claim 8, wherein Z is linked through the nitrogen atom of its N-terminal proline.

10. The liquid formulation of claim 1 , wherein the immunoglobulin Fc segment and Q are non-glycosylated.

11. 2. The liquid formulation of claim 1, wherein the buffering substance is selected from the group consisting of citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof.

12. 12. The liquid formulation of claim 11, wherein the buffer substance is acetic acid and its salts.

13. 2. The liquid formulation according to claim 1, wherein the pH of the liquid formulation is 4.8 to 6.

0.

14. 14. The liquid formulation of claim 13, wherein the pH of the liquid formulation is 4.9 to 5.

3.

15. The liquid formulation of claim 1 , wherein the liquid formulation does not contain a sugar alcohol.

16. 10. The liquid formulation of claim 1, wherein the sugar is glucose, fructose, galactose, lactose, maltose, sucrose, or a combination thereof.

17. 17. The liquid formulation of claim 16, wherein the sugar is sucrose.

18. 10. The liquid formulation of claim 1, wherein the non-ionic surfactant is a poloxamer, a polysorbate, or a combination thereof.

19. 19. The liquid formulation of claim 18, wherein the non-ionic surfactant is selected from the group consisting of poloxamer 188, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, and combinations thereof.

20. 10. The liquid formulation of claim 1, wherein the liquid formulation further comprises a stabilizer selected from the group consisting of arginine, glycine, methionine, and combinations thereof.

21. The liquid formulation 93 to 565 nmol / mL of a peptide conjugate of formula (1) linked through the sulfur atom of cysteine of Q, the C-terminus of which is amidated; a buffer substance selected from citric acid and its salts, acetic acid and its salts, histidine and its salts, phosphoric acid and its salts, and combinations thereof, in an amount of 5 to 25 mM, so that the pH of the liquid formulation is 4.8 to 5.5; 4-10% (w / v) sugar; and 10. The liquid formulation of claim 9, comprising 0.01 to 0.1% (w / v) of a non-ionic surfactant selected from poloxamer, polysorbate, or a combination thereof.

22. The liquid formulation according to claim 21, wherein the concentration of the peptide conjugate of formula (1) is 274 to 474 nmol / mL.

23. 22. The liquid formulation according to claim 21, wherein the liquid formulation remains transparent after storage for one week under severe test conditions of 40±2°C and 75±5% relative humidity.

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