Patent Description:
Physiologically active polypeptides are easily denatured due to low stability, degraded by proteases in the blood, and easily removed by the kidneys or liver. Thus, in order to maintain blood concentration and titer of a protein drug containing a physiologically active polypeptide as a pharmacological component, the protein drug needs to be frequently administered to a patient. However, since most protein drugs are administered to patients in the form of an injection, frequent administration via injection to maintain blood concentration of the physiologically active polypeptide causes severe pain to the patients and increases costs for treatment. To solve these problems, efforts have been made to maximize the efficacy of protein drugs by increasing blood stability of the protein drugs and maintaining the blood concentration thereof at a high level for a long period of time. However, these long-acting formulations of protein drugs should not induce immune responses in patients while increasing the stability of the protein drugs and maintaining the titer of the drug at a sufficiently high level.

As a method of stabilizing proteins, inhibiting contact with proteases, and suppressing renal clearance, a method of chemically adding a highly soluble polymer such as polyethylene glycol (hereinafter referred to as "PEG") to the surfaces of protein drugs has conventionally been used. However, while the method of using PEG may extend in vivo duration of the peptide drug by increasing a molecular weight of PEG, the titer of the peptide drug significantly decreases as the molecular weight increases, and a yield may decrease due to low reactivity with the peptide.

Therefore, as a method for increasing serum half-life, a conjugate of an immunoglobulin fragment and a physiologically active polypeptide has been used, and various studies have been conducted to improve preparation methods therefor (<CIT>). <CIT> discloses a method for the mass production of a monomeric or dimeric immunoglobulin Fc region, free of initial methionine residues, using a recombinant expression vector comprising a nucleotide sequence coding for a recombinant immunoglobulin Fc region comprising an immunoglobulin Fc region linked at the N-terminus thereof to an immunoglobulin Fc region via a peptide bond. <CIT> discloses a complex composition, of which positional isomers are minimized by using a N-terminus of an immunoglobulin Fc region as a binding site when the immunoglobulin Fc region is used as a carrier. Also provided are a protein complex which is prepared by N-terminal-specific binding of immunoglobulin Fc region, thereby prolonging blood half-life of the physiologically active polypeptide, maintaining in vivo potency at a high level, and having no risk of immune responses, a preparation method thereof, and a pharmaceutical composition including the same for improving in vivo duration and stability of the physiologically active polypeptide. The protein complex prepared by the present invention may be usefully applied to the development of long-acting formulations of various physiologically active polypeptide drugs.

In particular, there has been a steadily increasing need for the development of efficient processes for preparing a long-acting drug conjugate by simplifying the existing preparation process.

An object of the present invention is to provide a novel intermediate for preparing a long-acting drug conjugate.

Another object of the present invention is to provide a composition for preparing a long-acting drug conjugate including the intermediate.

Another object of the present invention is to provide a method of preparing a long-acting drug conjugate using the intermediate.

Another object of the present invention is to provide a long-acting drug conjugate prepared by the preparation method.

One aspect of the present invention provides a novel intermediate.

According to a first aspect, the present invention provides a compound having a structure of Formula <NUM> below or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:.

[Formula <NUM>]     X-L1-O(CH<NUM>CH<NUM>O)n-L2-R.

In the compound or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to the previous embodiment, wherein in Formula <NUM>,.

According to a second aspect, the present invention provides a compound having a structure of Formula <NUM> below or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:
<CHM>
wherein in Formula <NUM>, n is from <NUM> to <NUM>.

In the compound or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any of the previous embodiments, the immunoglobulin Fc region is derived from IgG, IgA, IgD, IgE, or IgM.

In the compound or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any of the previous embodiments, the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

In the compound or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any of the previous embodiments, the immunoglobulin Fc region is in a dimeric form.

In the compound or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any of the previous embodiments, the immunoglobulin Fc region comprises an amino acid sequence of SEQ ID NO: <NUM>.

In the compound or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof according to any of the previous embodiments, the compound has a size of <NUM> kDa to <NUM> kDa.

Another aspect of the present invention provides use of a composition in preparing a long-acting drug conjugate comprising a compound having a structure of Formula <NUM> below, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof,
wherein the drug is a physiologically active polypeptide:.

The use according to the previous embodiment,.

The use according to any of the previous embodiments, the physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-<NUM> (GLP-<NUM>), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, Gprotein-coupled receptors, interleukins, interleukin receptors, enzymes, interleukinbinding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, α-<NUM> antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activating factor, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, lenin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, atriopeptin, cartilage inducing factor, elcatonin, connective tissue activating 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, incretins, gastric inhibitory polypeptide (GIP), GLP-<NUM>/GIP dual agonist, GLP1/GIP/Glucagon trigonal agonist, cell surface antigens, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

The use according to any of the previous embodiments, R of the compound of the composition is linked to cysteine of the drug.

The use according to any of the previous embodiments,
wherein the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

The use according to any of the previous embodiments, wherein the composition is used to prepare a long-acting drug conjugate without performing ultrafiltration/diafiltration in preparation of the long-acting drug conjugate.

Another aspect of the present invention provides a method for preparing a long-acting conjugate of a physiologically active polypeptide.

In an embodiment, the preparation method comprises preparing a conjugate by linking a mono-PEGylated immunoglobulin Fc region, prepared by linking a linker of Formula <NUM> below to the N-terminus of an immunoglobulin Fc region comprising a hinge sequence that comprises an amino acid sequence of SEQ ID NO: <NUM> (Pro-Ser-Cys-Pro), to a physiologically active polypeptide:.

[Formula <NUM>]     CHO-L1-O(CH<NUM>CH<NUM>O)n-L2-R.

In the preparation method according to the previous embodiment, the mono-PEGylated immunoglobulin Fc region is prepared by linking the linker of Formula <NUM> above to the N-terminus of the immunoglobulin Fc region at a pH of <NUM> to <NUM> in the presence of a reducing agent.

In the preparation method according to any of the previous embodiments, the conjugate is prepared by linking the linker of the mono-PEGylated immunoglobulin Fc region to the physiologically active polypeptide at a pH of <NUM> to <NUM>.

In the preparation method according to any of the previous embodiments, the preparing of the conjugate is performed by reacting the mono-PEGylated immunoglobulin Fc region with the physiologically active polypeptide in a molar ratio of <NUM>:<NUM> to <NUM>:<NUM>.

In the preparation according to any of the previous embodiments, the method comprises preparing a mono-PEGylated immunoglobulin Fc region by linking a linker of Formula <NUM> to the N-terminus of the immunoglobulin Fc region; and preparing a conjugate by linking the linker of the mono-PEGylated immunoglobulin Fc region prepared in the previous step to a physiologically active polypeptide.

In the preparation according to any of the previous embodiments, the linker of the mono-PEGylated immunoglobulin Fc region is linked to a cysteine of the physiologically active polypeptide.

In the preparation according to any of the previous embodiments, the method comprises preparing a mono-PEGylated immunoglobulin Fc region by linking a linker of Formula <NUM> to the N-terminus of an immunoglobulin Fc region; purifying the monoPEGylated immunoglobulin Fc region prepared in the previous step by anionexchange chromatography in a buffer solution with a pH of <NUM> to <NUM>; and preparing a conjugate by linking the linker of the mono-PEGylated immunoglobulin Fc region purified in the previous step to a physiologically active polypeptide.

In the preparation according to any of the previous embodiments, the method is performed without ultrafiltration/diafiltration after preparing the mono-PEGylated immunoglobulin Fc region.

In the preparation according to any of the previous embodiments, the method further comprises purifying the conjugate by hydrophobic interaction chromatography.

In the preparation according to any of the previous embodiments, in Formula <NUM>, L1 is a straight or branched-chain C<NUM>-C<NUM> alkylene; L2 is -a1-NHCO- or -a1-NHCO-a2-; in which a1 and a2 are each independently a straight or branched-chain C<NUM>-C<NUM> alkylene; n is from <NUM> to <NUM>; and R is maleimide.

In the preparation according to any of the previous embodiments, the linker has a structure of Formula <NUM> below:
<CHM>
wherein in Formula <NUM>, n is from <NUM> to <NUM>.

In the preparation according to any of the previous embodiments, the linker has a size of <NUM> kDa to <NUM> kDa.

In the preparation according to any of the previous embodiments, the physiologically active polypeptide is selected from the group consisting of glucagon-like peptide-<NUM> (GLP-<NUM>), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, Gprotein-coupled receptors, interleukins, interleukin receptors, enzymes, interleukinbinding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, α-<NUM> antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activating factor, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, lenin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, atriopeptin, cartilage inducing factor, elcatonin, connective tissue activating 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, incretins, gastric inhibitory polypeptide (GIP), GLP-<NUM>/GIP dual agonist, GLP1/GIP/Glucagon trigonal agonist, cell surface antigens, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

In the preparation according to any of the previous embodiments, the physiologically active polypeptide is a GLP-<NUM>/GIP/Glucagon trigonal agonist, glucagon,
or an analog thereof.

In the preparation according to any of the previous embodiments, the physiologically active polypeptide
includes one of amino acid sequences of SEQ ID NOS: <NUM> to <NUM>.

In the preparation according to any of the previous embodiments, the hinge sequence comprises an amino acid sequence of SEQ ID NO: <NUM> (Pro-Ser-Cys-Pro).

In the preparation according to any of the previous embodiments, the immunoglobulin Fc region is derived from IgG1, IgG2, IgG3, or IgG4.

Another aspect of the present invention provides use of a composition in preparing a long-acting drug conjugate comprising a compound having a structure of Formula <NUM> below or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof: wherein the drug is a physiologically active polypeptide:
<CHM>
<CHM>
wherein in Formula <NUM> above, n is from <NUM> to <NUM>. Another aspect of the present invention provides a method for preparing a long-acting conjugate of a physiologically active polypeptide, the method comprising:.

According to the method for preparing a long-acting drug conjugate using a novel intermediate according to the present invention, a long-acting drug conjugate may be prepared with a high yield although some of conventional purification processes are omitted, and thus productivity of the long-acting drug conjugate may be increased.

<FIG> shows results of analyzing a structure of a mono-PEGylated immunoglobulin Fc region by MALDI-TOF assay.

Throughout the specification, not only the conventional one-letter and three-letter codes for naturally occurring amino acids, but also those three-letter codes generally allowed for other amino acids, such as α-aminoisobutyric acid (Aib), Nmethylglycine (Sar), and α-methyl-glutamic acid are used. In addition, the amino acids mentioned herein are abbreviated according to the nomenclature rules of IUPAC-IUB as follows.

An aspect of the present invention provides a compound having a structure of Formula <NUM> below, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:.

In the present invention, the compound having a structure of Formula <NUM>, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof is a novel substance prepared for preparing a long-acting conjugate and may also be referred to as "intermediate" or "intermediate material" in the present application.

In a method of preparing a long-acting drug conjugate using the intermediate of the present invention, purification steps by ultrafiltration/diafiltration and hydrophobic interaction chromatography may be omitted, and effects on preparing the long-acting drug conjugate with a high yield may be obtained although the purification steps are omitted.

Specifically, the intermediate is in a form in which an immunoglobulin Fc region is linked to a linker. In the intermediate of Formula <NUM>, X is an immunoglobulin Fc region and comprises, at the N-terminus, a hinge sequence that comprises an amino acid sequence of SEQ ID NO: <NUM> (Pro-Ser-Cys-Pro), and L1-O(CH<NUM>CH<NUM>O)n-L2-R may be a linker. Specifically, in Formula <NUM> above, L1 is a straight or branched-chain C<NUM>-C<NUM> alkylene; L2 is -a1-NHCO- or -a1- NHCO-a2-; in which a1 and a2 are each independently a straight or branched-chain C<NUM>-C<NUM> alkylene; n is from <NUM> to <NUM>; and R is maleimide.

The L1 is a site binding to the immunoglobulin Fc region and may be a straight or branched-chain C<NUM>-C<NUM> alkylene. R is a site for linkage between the intermediate and a physiologically active polypeptide and, specifically, may include a reactive group (e.g., thiol, maleimide, aldehyde, and succinimidyl) capable of binding to a cysteine, or an amine group of the N-terminus, or a lysine residue of a physiologically active polypeptide.

In the present invention, the intermediate may have a structure of Formula <NUM> below:
<CHM>.

In Formula <NUM> above,
n is from <NUM> to <NUM>.

Specifically, the intermediate has a size
of <NUM> kDa to <NUM> kDa.

As used herein, the term "immunoglobulin Fc region" refers to a region including a heavy chain constant domain <NUM> (CH2) and/or a heavy chain constant domain <NUM> (CH3) excluding the heavy chain and light chain variable domains of the immunoglobulin. The immunoglobulin Fc region may be a component constituting a moiety of the long-acting drug conjugate of the present invention.

Specifically, X is an immunoglobulin Fc region derived from IgG, IgA, IgD, IgE, or IgM, more specifically, an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

In the present invention, the immunoglobulin Fc region may include a particular hinge sequence at the N-terminus.

As used herein, the term "hinge sequence" refers to a site located at a heavy chain and forming a dimer of the immunoglobulin Fc region via an inter disulfide bond.

As used herein, the term "N-terminus" refers to amino terminus of a protein or polypeptide and may include an amino acid residue located at the end of the amino terminus or <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> or more amino acids from the end of the amino terminus. The immunoglobulin Fc region of the present invention may include the hinge sequence at the N-terminus.

The hinge sequence of the present invention may consist of <NUM> to <NUM> amino acids including only one cysteine residue. More specifically, the hinge sequence of the present invention may have a sequence as follows: 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-Tyr-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-Pro-Ser-Cys-Pro, or Glu-Pro-Ser-Cys. More specifically, the hinge sequence comprises a sequence of SEQ ID NO: <NUM> (Pro-Ser-Cys-Pro).

X may be a dimer formed of two chain molecules of the immunoglobulin Fc region in the presence of the hinge sequence, and the intermediate of the present invention may be in a form in which one end of the linker is linked to one chain of the immunoglobulin Fc region in the dimeric form. In the present invention, the immunoglobulin Fc region is in a dimeric form.

In addition, X of the present invention may be an immunoglobulin Fc region comprising an amino acid sequence of SEQ ID NO: <NUM>.

Meanwhile, the immunoglobulin Fc region of the present invention may be an extended Fc region including a part of or the entirety of a heavy chain constant domain <NUM> (CH1) and/or a light chain constant domain <NUM> (CL1) excluding the heavy chain and the light chain variable domains of the immunoglobulin, as long as the immunoglobulin Fc region has substantially identical or enhanced effects compared to the native type. Also, the immunoglobulin Fc region may be a region from which a considerably long part of the amino acid sequence corresponding to the CH2 and/or CH3 is removed.

For example, the immunoglobulin Fc region of the present invention may include <NUM>) CH1 domain, CH2 domain, CH3 domain and CH4 domain, <NUM>) CH1 domain and CH2 domain, <NUM>) CH1 domain and CH3 domain, <NUM>) CH2 domain and CH3 domain, <NUM>) a combination of one or more domains selected from CH1 domain, CH2 domain, CH3 domain, and CH4 domain and an immunoglobulin hinge region (or a part of the hinge region), or <NUM>) a dimer of each domain of the heavy chain constant domain and the light chain constant domain.

Also, the immunoglobulin Fc region of the present invention includes not only a naturally occurring amino acid sequence but also a sequence derivative thereof. The amino acid sequence derivative refers to a sequence different from the naturally occurring amino acid sequence due to a deletion, insertion, non-conservative or conservative substitution, or any combination of one or more amino acids of the naturally occurring amino acid sequence.

For example, in the case of IgG Fc, amino acid residues known to be important in linkage at positions <NUM> to <NUM>, <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM> may be used as a suitable target for modification.

Also, other various derivatives including those in which a site capable of forming a disulfide bond is deleted or certain amino acid residues are eliminated from the N-terminus of a native Fc form, and a methionine residue is added to the N-terminus of the native Fc form may be used. In addition, to remove effector functions, a complement binding site, such as a C1q binding site, may be deleted, and an antibody dependent cell mediated cytotoxicity (ADCC) site may be deleted. Techniques of preparing such sequence derivatives of the immunoglobulin Fc region are disclosed in International Patent Publication Nos. <CIT> and WO <NUM>/ <NUM>.

Amino acid exchanges in proteins and peptides, which do not generally alter the activity of molecules, are known in the art (<NPL>). The most commonly occurring exchanges of amino acid residues are exchanges between 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. If required, the Fc region may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, and amidation.

The above-described sequence derivatives of the Fc region are derivatives that have a biological activity equivalent to that of the immunoglobulin Fc region of the present invention or improved structural stability against heat, pH.

In addition, these immunoglobulin Fc regions may be obtained from native forms isolated from humans and other animals including cows, goats, swine, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or derivatives thereof, obtained from transformed animal cells or microorganisms. In this regard, they may be obtained from a native immunoglobulin by isolating whole immunoglobulins from living humans or animals and treating them with a protease. Papain digests the native immunoglobulin into Fab and Fc regions and pepsin digests the native immunoglobulin into pF'c and F(ab)<NUM> fragments. These fragments may be subjected to size-exclusion chromatography to isolate Fc or pF'c. In a more specific embodiment, a human-derived Fc region is a recombinant immunoglobulin Fc region obtained from a microorganism.

In addition, the immunoglobulin Fc region of the present invention may have natural glycans or increased or decreased glycans compared to the natural type, or be in a deglycosylated form. The increase, decrease, or removal of glycans of the immunoglobulin Fc may be achieved by any methods commonly used in the art such as a chemical method, an enzymatic method, and a genetic engineering method using a microorganism. In this regard, the immunoglobulin Fc region obtained by removing glycans shows a significant decrease in binding affinity to a complement c1q and a decrease in or loss of antibody-dependent cytotoxicity or complement-dependent cytotoxicity, and thus unnecessary immune responses are not induced thereby in living organisms. Based thereon, a deglycosylated or aglycosylated immunoglobulin Fc region may be more suitable as a drug carrier in view of the objects of the present invention.

As used herein, the term "deglycosylation" refers to a Fc region from which glycan is removed using an enzyme and the term "aglycosylation" refers to a Fc region that is not glycosylated and produced in prokaryotes, more specifically, E.

Meanwhile, the immunoglobulin Fc region may be derived from humans or animals such as cows, goats, swine, mice, rabbits, hamsters, rats, or guinea pigs. In a more specific embodiment, the immunoglobulin Fc region may be derived from humans.

In addition, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, or IgM, or any combination or hybrid thereof. In a more specific embodiment, the immunoglobulin Fc region is derived from IgG or IgM which are the most abundant proteins in human blood, and in an even more specific embodiment, it is derived from IgG known to enhance the half-lives of ligand-binding proteins. In a yet even more specific embodiment, the immunoglobulin Fc region is an IgG4 Fc region, and in the most specific embodiment, the immunoglobulin Fc region is an aglycosylated Fc region derived from human IgG4.

Meanwhile, as used herein, the term "combination" related to the immunoglobulin Fc region refers to formation of a linkage between a polypeptide encoding a single-chain immunoglobulin Fc region of the same origin and a singlechain polypeptide of a different origin when a dimer or a multimer is formed. That is, a dimer or multimer may be prepared using two or more Fc fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.

As used herein, the term "hybrid" means that sequences corresponding to two or more immunoglobulin Fc regions of different origins are present in a single-chain of an immunoglobulin constant domain. In the present invention, various hybrid forms are possible. That is, a domain hybrid may be composed of <NUM> to <NUM> domains selected from the group consisting of CH1, CH2, CH3, and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc, and IgD Fc and may further include a hinge region.

Meanwhile, IgG may also be classified into IgG1, IgG2, IgG3 and IgG4 subclasses, which may be combined or hybridized in the present invention. Preferred are IgG2 and IgG4 subclasses, and most preferred is the Fc fragment of IgG4 rarely having effector functions such as complement dependent cytotoxicity (CDC).

As used herein, the term "linker" refers to a moiety linking a drug (e.g., physiologically active polypeptide) to the immunoglobulin Fc region in the long-acting drug conjugate, and the linker may be a peptidyl linker or a non-peptidyl linker. Specifically, the linker may be represented by Formula <NUM> below:.

The linker may include polyethylene glycol and has particular chemical structures at both ends of polyethylene glycol.

Specifically, in Formula <NUM> above, L1 may be a straight or branched-chain C<NUM>-C<NUM> alkylene; L2 may be -a1-NHCO- or -a1-NHCO-a2-; in which a1 and a2 may be each independently a straight or branched-chain C<NUM>-C<NUM> alkylene; n may be from <NUM> to <NUM>; and R may be maleimide, and the linker may have a size of <NUM> kDa to <NUM> kDa, <NUM> kDa to <NUM> kDa, <NUM> kDa to <NUM> kDa, <NUM> kDa to <NUM> kDa, or <NUM> kDa to <NUM> kDa.

Also, the linker may have a structure of Formula <NUM> below:
<CHM>.

In Formula <NUM> above, n is from <NUM> to <NUM>.

One end of the linker may be linked to the immunoglobulin Fc region, specifically, the N-terminus of the immunoglobulin Fc region, more specifically, the hinge sequence located at the N-terminus of the immunoglobulin Fc region, even more specifically, a proline residue of the hinge sequence, to form the intermediate.

In the present invention, the term "pharmaceutically acceptable" refers to a substance that may be effectively used for the intended use within the scope of pharmaco-medical decision without inducing excessive toxicity, irritation, or allergic responses.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base. Examples of a suitable acid may 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, naphthalene2-sulfonic acid, and benzenesulfonic acid. Examples of the salt derived from a suitable base may include alkali metals such as sodium and potassium, alkali earth metals such as magnesium, and ammonium.

The present invention comprises not only the compound or a pharmaceutically acceptable salt thereof, but also a solvate prepared therefrom.

In addition, the compound may be present in the form of an enantiomer (R or S isomer), racemate, or diastereomer, or any mixture thereof in the case of having an asymmetric carbon center (absent carbon) in a substituent thereof. In addition, the compound may be present in the form of an exo or endo isomer in the case of having a bridged ring.

An aspect of the present invention provides a composition including a compound having a structure of Formula <NUM> below, or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof, wherein the drug is a physiologically active polypeptide:.

Specifically, in Formula <NUM> above, L1 is a straight or branched-chain C<NUM>-C<NUM> alkylene; L2 is -a1-NHCO- or -a1-NHCO-a2-; in which a1 and a2 are each independently a straight or branched-chain C<NUM>-C<NUM> alkylene; n is from <NUM> to <NUM>; and R is maleimide.

The composition of the present invention comprises the intermediate and has a use for preparing a long-acting drug conjugate.

Specifically, since the composition of the present invention comprises the intermediate in which the linker is linked to the immunoglobulin Fc region, the composition of the present invention may be reacted with a physiologically active polypeptide such that the linker of the intermediate is linked to the physiologically active polypeptide, thereby preparing a long-acting drug conjugate. More specifically, the long-acting drug conjugate may be prepared via linkage between R of Formula <NUM> corresponding to one end of the linker and a cysteine, or an amine group such as the N-terminus, or a lysine residue of the physiologically active polypeptide.

In the present invention, any physiologically active polypeptide of the longacting drug conjugate that may be prepared using the composition may fall within the scope of the present invention regardless of type, size, origin, as long as the physiologically active polypeptide has pharmacological effects on disease. Examples of the physiologically active polypeptide may include glucagon-like peptide1 (GLP-<NUM>), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, G-proteincoupled receptors, interleukins, interleukin receptors, enzymes, interleukin-binding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, α-<NUM> antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activating factor, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, lenin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, atriopeptin, cartilage inducing factor, elcatonin, connective tissue activating 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, incretins, gastric inhibitory polypeptide (GIP), GLP-<NUM>/GIP dual agonist, GLP1/GIP/Glucagon trigonal agonist, cell surface antigens, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments.

More specifically, the physiologically active polypeptide may be glucagon-like peptide-<NUM> (GLP-<NUM>), glucagon, insulin, enzyme, incretin, gastric inhibitory polypeptide (GIP), GLP-<NUM>/GIP dual agonist, or GLP-<NUM>/GIP/Glucagon triple agonist.

Because a method for preparing a long-acting drug conjugate using the intermediate or the composition including the same according to the present invention is performed by linking the intermediate, in which the linker is linked to the immunoglobulin Fc region, to the physiologically active polypeptide, any physiologically active polypeptide including an amino acid residue or a reactive group capable of binding to the intermediate may be used regardless of types thereof to prepare the long-acting drug conjugate using the intermediate or the composition including the same of the present invention.

In the present invention, the immunoglobulin Fc region is derived [<NUM>] from IgG1, IgG2,
IgG3, or IgG4.

In the case of using the composition of the present invention, the long-acting drug conjugate may be prepared without performing ultrafiltration/diafiltration and one cycle of hydrophobic interaction chromatography may be optionally omitted.

Specifically, when the long-acting drug conjugate is prepared by reacting the intermediate or the composition including the same according to the present invention with the drug, purity of the long-acting drug conjugate may be <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% or more. The purity may be measured by any method well known in the art, specifically, by SE-HPLC, RP-HPLC, and IE-HPLC, but any method available in the art. More specifically, the purity of the long-acting drug conjugate prepared using the composition of the present invention may be <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IE-HPLC.

Also, the composition of the present invention may further include a buffer, a stabilizer, a preservative, a salt, required to stabilize the intermediate and to prepare the long-acting drug conjugate.

Another aspect of the present invention provides a kit for preparing a long-acting drug conjugate including the composition. The kit may include a reagent, manual for preparing the long-acting drug conjugate.

Another aspect of the present invention provides a method for preparing a long-acting drug conjugate.

The preparation method of the present invention is a method for preparing a long-acting drug conjugate in which a drug is linked to an immunoglobulin Fc region via a linker, specifically, a method for preparing a long-acting drug conjugate by linking the intermediate to the drug.

Specifically, the preparation method is characterized by sequentially performing i) linking a linker including polyethylene glycol (PEG) to an immunoglobulin Fc region, and ii) linking the linker, which is linked to the immunoglobulin Fc region, to a drug (e.g., a physiologically active polypeptide or protein). That is, the preparation method is characterized by performing steps in a particular order, i.e., performing a first step of preparing the intermediate by linking the linker including PEG to the immunoglobulin Fc region, and then performing a second step of linking the drug to the intermediate. Alternatively, the preparation method of the present invention may also be performed only by the second step of preparing the long-acting drug conjugate via a reaction between the intermediate or the composition for preparing the long-acting drug conjugate including the same and the drug, without performing the first step. This preparation method may be referred to as "reverse order preparation method" in the present application.

In the present invention, in the case of the method for preparing the long-acting drug conjugate performed by preparing the intermediate first and then linking the intermediate to the drug, the purification processes by ultrafiltration/diafiltration and hydrophobic interaction chromatography may be omitted and it was confirmed that the long-acting drug conjugate may be prepared with a high yield although the purification processes are omitted.

According to the convention preparation method in which the linker is first linked to the physiologically active polypeptide and then linked to the immunoglobulin Fc region without forming the intermediate, when the physiologically active polypeptide-linked linker (e.g., polyethylene glycol) is linked to the immunoglobulin Fc region, ultrafiltration/diafiltration is required as a separate process after the linker is linked to the physiologically active polypeptide and before the linked product is linked to the immunoglobulin Fc region to reduce the risk of aggregation that may occur due to low pH conditions (pH of about <NUM>) of an equilibrium buffer and an elution buffer used for purification of the physiologically active polypeptide-linked linker and to adjust the pH conditions for reaction using an appropriate buffer. On the contrary, in the preparation method according to the present invention in which the intermediate is prepared by linking the linker to the immunoglobulin Fc region first, a pH of a buffer used in purification of the immunoglobulin Fc region-linked linker is relatively high, and thus the ultrafiltration/diafiltration process may be omitted and then a process of linking the immunoglobulin Fc region-linked linker to the physiologically active polypeptide may be performed.

Therefore, in the preparation method of the present invention, ultrafiltration/ diafiltration may not be performed after preparing a mono-PEGylated immunoglobulin Fc region. In the method for preparing a long-acting drug conjugate according to the present invention, a pH of a solution used to purify the mono-PEGylated immunoglobulin Fc region is not significantly different from a pH of a solution used for a subsequent reaction so that linkage to the drug may be performed without conducting the ultrafiltration/diafiltration. By omitting the ultrafiltration/diafiltration process, the risk of formation of aggregate impurities in a concentration step may be reduced and the preparation process may be simplified so that cost reduction effects may be expected in the case where the technology is commercialized.

Also, the preparation method of the present invention may further include purifying the conjugate by hydrophobic interaction chromatography.

Specifically, the hydrophobic interaction chromatography may be performed only once or more than once in accordance with properties of the drug of the long-acting drug conjugate and type and size of the linker.

According to the preparation method of the present invention, not only an amount of the expensive drug may be reduced but also an amount of unreacted immunoglobulin Fc regions may be reduced, so that the entire or a part of the purification process by hydrophobic interaction chromatography may be omitted to obtain effects on reducing raw materials required for preparation of the long-acting drug conjugate and costs therefor compared to the conventional method.

Meanwhile, although the ultrafiltration/diafiltration and hydrophobic interaction chromatography processes, which have been performed in the conventional method for preparing the long-acting drug conjugate, are omitted and only the final purification process (e.g., one cycle of hydrophobic interaction chromatography) is performed in the preparation method of the present invention, it is advantageous in that a purity of the final conjugate obtained by the present invention is maintained compared to that of the conventional preparation method. That is, according to the preparation method of the present invention, the final purity may be maintained although some of the purification processes are omitted so that productivity of the long-acting drug conjugate may be improved.

The purity of the long-acting drug conjugate according to the present invention may be measured by any method well known in the art and examples of the method may be SE-HPLC, RP-HPLC, and IE-HPLC.

According to the preparation method of the present invention, the final purity of the long-acting drug conjugate may be <NUM>% or more, specifically, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>% or more.

Meanwhile, in the preparation method of the present invention, the monoPEGylated immunoglobulin Fc region is prepared first and then linked to the physiologically active polypeptide, so that the long-acting drug conjugate may be prepared with a higher yield compared to the conventional method in terms of not only the physiologically active polypeptide but also the immunoglobulin Fc region.

In an embodiment of the present invention, it was confirmed that the yield of the long-acting drug conjugate obtained by the preparation method of the present invention was increased twice or more compared to the yield of the long-acting drug conjugate obtained by the conventional method.

Specifically, the preparation method of the present invention relates to a method for preparing a long-acting drug conjugate including preparing a conjugate by linking a mono-PEGylated immunoglobulin Fc region, which is prepared by linking a linker of Formula <NUM> below to the N-terminus of an immunoglobulin Fc region comprising a hinge sequence that comprises an amino acid sequence of SEQ ID NO: <NUM> (Pro-Ser-Cys-Pro), to a physiologically active polypeptide:.

In addition, in the preparation method of the present invention,.

In addition, the step of preparing the conjugate according to the preparation method of the present invention may be performed by reacting the physiologically active polypeptide in an amount equivalent to or more than an amount of the monoPEGylated immunoglobulin Fc region, and specifically, a molar ratio of monoPEGylated immunoglobulin Fc region : physiologically active polypeptide may be from <NUM>:<NUM> to <NUM>:<NUM>.

As used herein, the term "mono-PEGylated immunoglobulin Fc region" refers to an intermediate material that is produced in the middle of the method for preparing the long-acting drug conjugate according to the present invention in which one linker including one polyethylene glycol is linked to the immunoglobulin Fc region. That is, in the present invention, the "mono-PEGylated immunoglobulin Fc region" may be used interchangeably with "intermediate" or "intermediate material".

In the present invention, the immunoglobulin Fc region may be an immunoglobulin Fc region derived from IgG1, IgG2, IgG3, or IgG4.

In addition, the immunoglobulin Fc region is an immunoglobulin Fc region comprising a hinge sequence that comprises an amino acid sequence of
SEQ ID NO: <NUM> (Pro-Ser-Cys-Pro).

The "long-acting drug conjugate" of the present invention refers to a drug conjugate having a structure in which a drug (physiologically active polypeptide) having a pharmacological activity in the body is linked to an immunoglobulin Fc region via a linker and an increased half-life. In view of the objects of the present invention, the long-acting drug conjugate may be one in which the intermediate or monoPEGylated immunoglobulin Fc region is linked to the drug.

Specifically, the drug is not limited to particular substances as long as the drug has preventive, therapeutic, or alleviating effects on a certain disease and may be a natural or non-natural protein, enzyme, antibody, compound. More specifically, the drug may be a physiologically active polypeptide or protein, even more specifically, the physiologically active polypeptide may be glucagon-like peptide-<NUM> (GLP-<NUM>), granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), erythropoietin (EPO), glucagon, insulin, growth hormone releasing hormone, growth hormone releasing peptide, interferons, interferon receptors, G-proteincoupled receptors, interleukins, interleukin receptors, enzymes, interleukin-binding protein, cytokine-binding protein, macrophage activating factor, macrophage peptide, B cell factor, T cell factor, protein A, allergy inhibitor, cell necrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastasis growth factor, α-<NUM> antitrypsin, albumin, α-lactalbumin, apolipoprotein-E, highly glycosylated erythropoietin, angiopoietins, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factors VII, VIIa, VIII, IX, and XIII, plasminogen activating factor, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, lenin inhibitor, collagenase inhibitor, superoxide dismutase, leptin, platelet-derived growth factor, epithelial growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone stimulating protein, calcitonin, atriopeptin, cartilage inducing factor, elcatonin, connective tissue activating 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, incretins, gastric inhibitory polypeptide (GIP), GLP-<NUM>/GIP dual agonist, GLP1/GIP/Glucagon trigonal agonist, cell surface antigens, virus derived vaccine antigens, monoclonal antibodies, polyclonal antibodies, and antibody fragments. More specifically, the physiologically active polypeptide may be GLP1/GIP/Glucagon trigonal agonist, glucagon, or a analog thereof. Even more specifically, the physiologically active polypeptide may include, essentially consist of, or consist of one of amino acid sequences of SEQ ID NOS: <NUM> to <NUM>.

As used herein, the term "variant" refers to a peptide having an amino acid sequence in which one or more amino acids are different from those of a native physiologically active polypeptide while retaining the same functions as those of the native physiologically active polypeptide, and the variant may be prepared by substitution, addition, deletion, modification, or any combination of some amino acids of the amino acid sequence of the native physiologically active polypeptide.

As used herein, the term "derivative" refers to a peptide, a peptide analog, or a peptidomimetic obtained by modifying one or more amino acids of the native physiologically active polypeptide by addition, deletion, or substitution to have similar activity to that of the native physiologically active polypeptide.

As used herein, the term "fragment" refers to a form obtained by adding/ deleting one or more amino acids to/from the N-terminus or the C-terminus, and the added amino acid may be any amino acid that does not exist in nature (e.g., D-amino acid).

The methods for preparing the variant, derivative, and fragment of the physiologically active polypeptide may be used independently or in combination. For example, any physiologically active polypeptide having one or more different amino acids in the amino acid sequence and deamination of an amino acid residue at the N-terminus may be included therein.

The derivative of the physiologically active polypeptide includes biosimilar and biobetter forms. For example, with respect to biosimilars, the biosimilar may be any biosimilar enzyme available in the long-acting drug conjugate of the present invention although there is a difference between a known enzyme and a host for its expression, a difference in glycosylation feature and the degree thereof, and a difference in the degree of substitution in a particular amino acid residue of the corresponding enzyme in light of the standard sequence where the degree of substitution is not <NUM>% substitution. The physiologically active polypeptide and the variant, derivative and fragment thereof may be produced from animal cells, E. coli, yeast, insect cells, plant cells, living animals, via genetic recombination, and any commercially available physiologically active polypeptides, and variants, derivatives, and fragments thereof may also be used.

In addition, the physiologically active polypeptide, and the variant, derivative and fragment thereof may include an amino acid sequence having a homology of at least <NUM>%, specifically, at least <NUM>%, more specifically, at least <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, <NUM>%, or <NUM>%, and the physiologically active polypeptide, and the variant, derivative, and fragment thereof may be obtained from microorganisms by genetic recombination technologies or commercially available.

As used herein, the term "homology" refers to the degree of similarity between amino acid sequences of a wild-type protein or nucleotide sequences encoding the same and includes a sequence identical to the amino acid sequence or nucleotide sequence of the present invention by the above-described percentage or more. The homology may be determined by comparing the sequences via visual observation but may also be determined using a bioinformatic algorithm, which provides analysis results of a degree of homology by aligning sequences to be compared. The homology between the two amino acid sequences may be indicated in percentage. Useful automated algorithms may be used in GAP, BESTFIT, FASTA, and TFASTA computer software modules of Wisconsin Genetics Software Package (Genetics Computer Group, Madison, WI, USA). The automated alignment algorithms in the modules include the Needleman & Wunsch algorithm, the Pearson & Lipman algorithm, and the Smith & Waterman sequence algorithm. Other useful algorithms and homology determinations on alignment are automated in software such as FASTP, BLAST, BLAST2, PSIBLAST, and CLUSTAL W.

Information on sequences of the physiologically active polypeptide, and the variant, derivative, and fragment thereof, and nucleotide sequences encoding the same may be obtained from known database of the NCBI GenBank.

The amino acids substituted or added may be not only <NUM> amino acids commonly found in human proteins but also atypical or non-naturally occurring amino acids. Commercial sources of atypical amino acids may include Sigma-Aldrich, ChemPep Inc. and Genzyme pharmaceuticals. The peptides including theses amino acids and typical peptide sequences may be synthesized and purchased from commercial suppliers, e.g., American Peptide Company, Bachem (USA) or Anygen (Korea).

In addition, the physiologically active polypeptide, and the variant, derivative, and fragment thereof according to the present invention may be in a varied form where the N-terminus and/or C-terminus is chemically modified or protected by organic groups, or amino acids may be added to the termini of the peptide, for protection from proteases in vivo while increasing stability thereof.

Particularly, since the N- and C-termini of chemically synthesized peptides are electrically charged, the N-terminus may be acetylated and/or the C-terminus may be amidated to remove the charges.

In addition, the peptide according to the present invention includes all of those in the form of the peptide itself, a salt thereof (e.g., a pharmaceutically acceptable salt of the peptide), or a solvate thereof. Also, the peptide may be in any pharmaceutically acceptable form.

The type of the salt is not particularly limited. However, the salt is preferably in a form safe and effective to an individual, e.g., a mammal.

As used herein, the term "solvate" refers to a complex of the peptide or a salt thereof according to the present invention and a solvent molecule.

The method for preparing the long-acting drug conjugate according to the present invention may be a method for preparing a conjugate in which a physiologically active polypeptide is linked to an immunoglobulin Fc region via a linker.

In the present invention, linkage between the linker and the immunoglobulin Fc region may be formed by a covalent bond or a non-covalent bond between one end of the linker and the N-terminus of the immunoglobulin Fc region, but binding sites or methods for the linkage are not particularly limited. Specifically, the mono-PEGylated immunoglobulin Fc region may be prepared by linking a proline at the N-terminus of the immunoglobulin Fc region to a -CHO group of the linker.

In the present invention, the linker may have a structure of Formula <NUM> below:
<CHM>.

In addition, the linker has a size of <NUM> kDa to <NUM> kDa.

The mono-PEGylated immunoglobulin Fc region may have a structure of Formula <NUM> below.

The preparation method of the present invention may be performed by linking the physiologically active polypeptide to one end of the mono-PEGylated immunoglobulin Fc region having the structure of Formula <NUM>.

In addition, the other end of the linker which is not linked to the immunoglobulin.

Fc region may be linked to the physiologically active polypeptide, specifically, a -SH group or an amino acid containing a -SH group, or a cysteine of the physiologically active polypeptide.

When the long-acting drug conjugate is prepared according to the preparation method of the present invention in which the mono-PEGylated immunoglobulin Fc region is prepared first and linked to the physiologically active polypeptide, it was confirmed that the purity of the final conjugate may be maintained with an increased yield compared to the conventional preparation method although the ultrafiltration/ diafiltration and hydrophobic interaction chromatography processes are omitted and only the final purification process (e.g., one cycle of hydrophobic interaction chromatography) is performed in the preparation method according to the present invention.

Another aspect of the present invention provides a long-acting drug conjugate prepared by the above-described method.

Because the long-acting drug conjugate prepared by the preparation of the present invention has an increased half-life compared to the physiologically active polypeptide that is not linked to the linker or the immunoglobulin Fc region, advantageous effects on preparation of drugs may be obtained.

The long-acting drug conjugate prepared by the preparation method of the present invention may be used in preparation of drugs or compositions for the purposes of prevention, treatment, and alleviation of diseases.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely presented to exemplify the present invention, and the scope of the present invention is not limited thereto.

A PEGylated physiologically active polypeptide was linked to an immunoglobulin Fc region to prepare a long-acting conjugate.

In order to PEGylate a physiologically active polypeptide (GLP1/GIP/Glucagon trigonal agonist analog <NUM>, SEQ ID NO: <NUM>) at a cysteine residue (-SH group), the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> was reacted with a linker containing PEG (maleimide-<NUM> kDa-PEG-aldehyde) (Formula <NUM>) for about <NUM> hour in a molar ratio of <NUM>:<NUM> to <NUM>:<NUM> with a GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> concentration of about <NUM>/L. Specifically, the reaction was performed in a <NUM> Tris buffer containing isopropanol (pH of <NUM>, <NUM> ± <NUM>). In order to obtain a monoPEGylated GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM>, the reaction solution was diluted with an equilibrium buffer including sodium citrate and ethanol to a total volume of <NUM> times and purified. In this regard, the mono-PEGylated GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> was purified using an SP High Performance column (GE Healthcare, cation-exchange chromatography) using a solution including sodium citrate and ethanol and a potassium chloride concentration gradient. After the purified solution of the PEGylated GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> was diluted with water, the buffer solution was replaced with a <NUM> potassium phosphate solution through ultrafiltration/diafiltration (UF/DF), followed by concentration to recover a resultant with a final concentration of about <NUM>/L or more.

The mono-PEGylated GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> prepared as described above was linked to an immunoglobulin Fc region to prepare a long-acting conjugate as follows.

In order to link an aldehyde group of PEG of the mono-PEGylated GLP1/GIP/Glucagon trigonal agonist analog <NUM> to the amino terminus of an immunoglobulin Fc region, the mono-PEGylated GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> was reacted with the immunoglobulin Fc region in a molar ratio of <NUM>:<NUM> at a temperature of <NUM> ± <NUM> for about <NUM> hours with a total protein concentration (GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> and immunoglobulin Fc region) of <NUM>/L.

In order to isolate and remove unreacted immunoglobulin Fc regions after the reaction for linkage, the reaction solution was purified using a Butyl <NUM> Fast Flow column (GE Healthcare, hydrophobic interaction chromatography). In this case, a Tris buffer and sodium chloride were added to the reaction solution, and the reaction solution was purified using a solution including a Bis-Tris and a sodium chloride concentration gradient.

Thereafter, using a Source 15ISO column (GE Healthcare), hydrophobic interaction chromatography was performed. By-products were eliminated by this process, and an immunoglobulin Fc region-PEG-containing linker-GLP1/GIP/Glucagon trigonal agonist analog <NUM> conjugate was obtained. In this case, purification was performed using a buffer including sodium citrate and an ammonium sulfate concentration gradient.

In order to PEGylate a physiologically active polypeptide (Glucagon analog <NUM>, SEQ ID NO: <NUM>) at a cysteine residue (-SH group), Glucagon analog <NUM> was reacted with a linker containing PEG (maleimide-<NUM> kDa-PEG-aldehyde) (Formula <NUM>) for about <NUM> hour in a molar ratio of <NUM>:<NUM> with a Glucagon analog <NUM> concentration of <NUM>/L. Specifically, the reaction was performed in a <NUM> Tris buffer containing isopropanol (pH of <NUM>). In order to obtain a mono-PEGylated Glucagon analog <NUM>, the reaction solution was diluted with an equilibrium buffer including sodium citrate and ethanol to a total volume of <NUM> times and purified. In this regard, the mono-PEGylated Glucagon analog <NUM> was purified using an SP High Performance column (GE Healthcare, cation exchange chromatography) using a solution including sodium citrate and ethanol and a potassium chloride concentration gradient. After the purified solution of the PEGylated Glucagon analog <NUM> was diluted with water, the buffer solution was replaced with a <NUM> potassium phosphate solution through ultrafiltration/diafiltration (UF/DF), followed by concentration to recover a resultant with a final concentration of <NUM>/L or more.

The mono-PEGylated Glucagon analog <NUM> prepared as described above was linked to an immunoglobulin Fc region to prepare a long-acting conjugate as follows.

In order to link an aldehyde group of PEG of the mono-PEGylated Glucagon analog <NUM> to the amino terminus of the immunoglobulin Fc region, the mono-PEGylated Glucagon analog <NUM> was reacted with the immunoglobulin Fc region in a molar ratio of <NUM>:<NUM> at a temperature of <NUM> ± <NUM> for about <NUM> hours with a total protein concentration (Glucagon analog <NUM> and immunoglobulin Fc region) of <NUM>/L.

In order to isolate and remove unreacted immunoglobulin Fc regions after the reaction for linkage, the reaction solution was purified using a Butyl <NUM> Fast Flow column (GE Healthcare, hydrophobic interaction chromatography). In this case, a Tris buffer and sodium chloride were added to the reaction solution, and the reaction solution was purified using a solution including Bis-Tris and a sodium chloride concentration gradient.

Thereafter, using a Source 15ISO column (GE Healthcare), hydrophobic interaction chromatography was performed. By-products were eliminated by this process, and an immunoglobulin Fc region-PEG-containing linker-Glucagon analog <NUM> conjugate was obtained. In this case, purification was performed using a buffer including sodium citrate and an ammonium sulfate concentration gradient.

The present inventors have developed a process capable of efficiently producing the conjugate with a high purity by omitting the membrane filtration process and the purification process (hydrophobic interaction chromatography, Butyl <NUM> Fast Flow) from the process of preparing the conjugate according to the above-described Comparative Examples <NUM> and <NUM> as follows.

In order to PEGylate the N-terminus of an immunoglobulin Fc region (<NUM> kDa) having a hinge region with a Pro-Ser-Cys-Pro sequence at the N-terminus, the immunoglobulin Fc region was reacted with a linker containing PEG (structure of Formula <NUM>, <NUM> kDa) in a molar ratio (immunoglobulin Fc region : PEG-containing linker) of <NUM>:<NUM> with an immunoglobulin Fc region concentration of <NUM>/L at <NUM> ± <NUM> for about <NUM> hours.

Specifically, the reaction was performed in a composition including a <NUM> Bis-Tris buffer (pH <NUM>) and potassium phosphate, and <NUM> NaCNBH<NUM> (sodium cyanoborohydride) was added thereto as a reducing agent. In order to obtain a mono-PEGylated immunoglobulin Fc region, the reaction solution was diluted with the Bis-Tris buffer and purified.

Unlike the preparation method of the above-described Comparative Examples in which the mono-PEGylated GLP-<NUM>/GIP/Glucagon trigonal agonist analog and glucagon analog were purified by cation-exchange chromatography, the monoPEGylated immunoglobulin Fc region was purified using a CaptoQ ImpRes column (GE Healthcare, anion-exchange chromatography) using a Bis-Tris buffer and a sodium chloride concentration gradient.

The mono-PEGylated immunoglobulin Fc region prepared in Example <NUM>-<NUM> was structurally analyzed by MALDI-TOF and Peptide mapping. As a result of MALDITOF, the resultant was identical to an expected molecular weight of the monoPEGylated immunoglobulin Fc region (<FIG>), and as a result of Peptide mapping, it was confirmed that over <NUM>% of PEG was PEGylated at the N-terminus of the immunoglobulin Fc region.

Meanwhile, as a result of analyzing the mono-PEGylated immunoglobulin Fc region (Formula <NUM>) prepared in Example <NUM>-<NUM> above using SE-HPLC, RP-HPLC, and IE-HPLC assays, the purity was confirmed to be <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IE-HPLC.

Long-acting conjugates were prepared as follows by linking the monoPEGylated immunoglobulin Fc region prepared in Example <NUM>-<NUM> to various physiologically active peptides.

Unlike the preparation method of the Comparative Examples where the PEGylated physiologically active polypeptide was purified by cation-exchange chromatography and then subjected to buffer exchange and concentration by ultrafiltration/diafiltration (UF/DF), the mono-PEGylated immunoglobulin Fc region was reacted with the physiologically active polypeptide via peptide conjugation without performing ultrafiltration/diafiltration. Long-acting conjugates prepared as described above had high purity, and thus one of the two cycles of hydrophobic interaction chromatography could be omitted unlike the preparation method according to the Comparative Examples.

A long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM>) was prepared via peptide conjugation of the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> (SEQ ID NO: <NUM>), after anionexchange chromatography of Example <NUM>-<NUM>, without performing ultrafiltration/ diafiltration.

In this regard, in order to link a maleimide reactive group at one terminus of PEG of the mono-PEGylated immunoglobulin Fc region to the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM>, the mono-PEGylated immunoglobulin Fc region was reacted with the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> in a molar ratio of <NUM>:<NUM> with a GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> concentration of <NUM>/L at <NUM> ± <NUM> for about <NUM> hours. The reaction was performed in a Tris-Cl buffer (<NUM> ± <NUM>) including isopropanol. As a result of analyzing the resultant after reaction using SE-HPLC, RP-HPLC, and IE-HPLC assays, the purity was confirmed to be <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobic interaction chromatography once using a Source 15ISO column (GE Healthcare). By-products were eliminated by this process, and an immunoglobulin Fc region-PEG containing linker-GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> conjugate was obtained. In this case, purification was performed using a buffer including sodium citrate and an ammonium sulfate concentration gradient. It was confirmed that a yield obtained herein was increased by about twice or more compared to a yield of Comparative Example <NUM> with the same amount of the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM>.

The eluted immunoglobulin Fc region-PEG-containing linker-GLP1/GIP/Glucagon trigonal agonist analog <NUM> conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity was confirmed since the purity was <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IE-HPLC.

In this regard, in order to link a maleimide reactive group at one terminus of PEG of the mono-PEGylated immunoglobulin Fc region to the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM>, the mono-PEGylated immunoglobulin Fc region was reacted with the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> in a molar ratio of <NUM>:<NUM> with a GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> concentration of <NUM>/L at <NUM> ± <NUM> for about <NUM> hours. The reaction was performed in a Tris-Cl buffer (<NUM> ± <NUM>) including isopropanol. As a result of analyzing the resultant after reaction using SE-HPLC, RP-HPLC, and IE-HPLC assays, the purity of the long-acting conjugate including the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> was confirmed to be <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IE-HPLC.

A long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM>) was prepared via peptide conjugation of the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> (SEQ ID NO: <NUM>), after anion exchange chromatography of Example <NUM>-<NUM>, without performing ultrafiltration/ diafiltration.

In this regard, in order to link a maleimide reactive group at one terminus of PEG of the mono-PEGylated immunoglobulin Fc region to cysteine of the GLP1/GIP/Glucagon trigonal agonist analog <NUM>, the mono-PEGylated immunoglobulin Fc region was reacted with the GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> in a molar ratio of <NUM>:<NUM> with a GLP-<NUM>/GIP/Glucagon trigonal agonist analog <NUM> concentration of <NUM>/L at <NUM> ± <NUM> for about <NUM> hours. The reaction was performed in a Tris-Cl buffer (<NUM> ± <NUM>) including isopropanol. As a result of analyzing the resultant after reaction using SE-HPLC, RP-HPLC, and IE-HPLC assays, the purity was confirmed to be <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IEHPLC.

A long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-Glucagon analog <NUM>) was prepared via peptide conjugation of Glucagon analog <NUM> (SEQ ID NO: <NUM>), after anion-exchange chromatography of Example <NUM>-<NUM>, without performing ultrafiltration/diafiltration.

In this regard, in order to link a maleimide reactive group at one terminus of PEG of the mono-PEGylated immunoglobulin Fc region to Glucagon analog <NUM>, the mono-PEGylated immunoglobulin Fc region was reacted with Glucagon analog <NUM> in a molar ratio of <NUM>:<NUM> with a Glucagon analog <NUM> concentration of <NUM>/L at <NUM> ± <NUM> for about <NUM> hours. The reaction was performed in a Tris-Cl buffer (<NUM> ± <NUM>) including isopropanol. As a result of analyzing the resultant after reaction using SE-HPLC, RP-HPLC, and IE-HPLC assays, the purity of the immunoglobulin Fc region-PEG containing linker-Glucagon analog <NUM> was confirmed to be <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobic interaction chromatography once using a Source 15ISO column (GE Healthcare). By-products were eliminated by this process, and an immunoglobulin Fc region-PEG containing linker-Glucagon analog <NUM> conjugate was obtained. In this case, purification was performed using a buffer including sodium citrate and an ammonium sulfate concentration gradient. It was confirmed that a yield obtained herein was increased by about <NUM> times or more compared to a yield of Comparative Example <NUM> with the same amount of Glucagon analog <NUM>.

The eluted immunoglobulin Fc region-PEG-containing linker-Glucagon analog <NUM> conjugate was analyzed by SE-HPLC, RP-HPLC, and IE-HPLC assays, and high purity was confirmed since the purity was <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IE-HPLC.

A long-acting conjugate (immunoglobulin Fc region-PEG-containing linker-Glucagon analog <NUM>) was prepared via peptide conjugation of Glucagon analog <NUM> (SEQ ID NO: <NUM>), after anion-exchange chromatography of Example <NUM>-<NUM>, without performing ultrafiltration/diafiltration.

[SEQ ID NO: <NUM>]
YXQGTFTSDYSKYLDSRRAQDFVQWLMNTC.

In this regard, in order to link a maleimide reactive group at one terminus of PEG of the mono-PEGylated immunoglobulin Fc region to cysteine of Glucagon analog <NUM>, the mono-PEGylated immunoglobulin Fc region was reacted with Glucagon analog <NUM> in a molar ratio of <NUM>:<NUM> with a Glucagon analog <NUM> concentration of <NUM>/L at <NUM> ± <NUM> for about <NUM> hours. The reaction was performed in a Tris-Cl buffer (<NUM> ± <NUM>) including isopropanol. As a result of analyzing the resultant after reaction using SE-HPLC, RP-HPLC, and IE-HPLC assays, the purity of the immunoglobulin Fc region-PEG-containing linker-Glucagon analog <NUM> was confirmed to be <NUM>% or more in SE-HPLC, <NUM>% or more in RP-HPLC, and <NUM>% or more in IE-HPLC.

Thereafter, the resultant of the reaction was subjected to hydrophobic interaction chromatography once using a Source 15ISO column (GE Healthcare). By-products were eliminated by this process, and an immunoglobulin Fc region-PEG containing linker-Glucagon analog <NUM> conjugate was obtained. In this case, purification was performed using a buffer including sodium citrate and an ammonium sulfate concentration gradient. It was confirmed that a yield obtained herein was increased by about <NUM> times or more compared to the existing yield with the same amount of Glucagon analog <NUM>.

Tables <NUM> and <NUM> show comparison results between the methods the comparative examples and examples.

Claim 1:
A compound having a structure of Formula <NUM> below or a stereoisomer, a solvate, or a pharmaceutically acceptable salt thereof:

        [Formula <NUM>]     X-L1-O(CH<NUM>CH<NUM>O)n-L2-R

wherein in Formula <NUM> above,
X is an immunoglobulin Fc region and comprises, at the N-terminus, a hinge sequence that comprises an amino acid sequence of SEQ ID NO: <NUM> (Pro-Ser-Cys-Pro);
L1 is a straight or branched-chain C<NUM>-C<NUM> alkylene and is linked to the N-terminal proline residue (Pro) of SEQ ID NO: <NUM>;
L2 is -a1-CONH-, -a1-NHCO-, -a1-NHCO-a2-, -COO-, -b1-COO-, -COO-b2-, or -b1-COO-b2-, in which a1, a2, b1, and b2 are each independently a straight or branched-chain C<NUM>-C<NUM> alkylene;
n is from <NUM> to <NUM>,<NUM>; and;
R is any one selected from the group consisting of <NUM>,<NUM>-dioxopyrrolidinyl, <NUM>,<NUM>-dioxopyrrolyl, aldehyde, maleimide, C<NUM>-C<NUM> aryl disulfide, C<NUM>-C<NUM> heteroaryl disulfide, vinyl sulfone, thiol, halogenated acetamide, succinimide, p-nitrophenyl carbonate, and thioester.