Polyethylene glycol derivative, composition containing the same, and method for producing bioactive polypeptide conjugate using the same

Abstract

The polyethylene glycol derivative compound according to one embodiment is represented by Chemical Formula 1 in the specification. In Chemical Formula 1, n is a natural number from 30 to 115, and R 1 and R 2 are the same or different C1-C5 alkyl.

Description

Technical Field

[0001] The present invention relates to polyethylene glycol derivatives, compositions containing the same, and the production of bioactive polypeptide conjugates using such polyethylene glycol derivatives.

Background Art

[0002] Peptide pharmaceuticals are substances in which two or more amino acids are linked in a certain chemical bond form, and mainly refer to pharmaceuticals produced through chemical synthesis. Such peptide pharmaceuticals are known to have the characteristics of "biocompatibility" and "in vivo specificity", and side effects, even in small amounts, can exhibit strong pharmacological actions and activities. Therefore, such peptide pharmaceuticals are expected to complement the disadvantages of synthetic pharmaceuticals and protein pharmaceuticals.

[0003] However, peptide pharmaceuticals are easily decomposed into amino acids by protein enzymes in the digestive tract, making oral administration difficult. They must be transmitted through injection. Due to their short blood half-life, their bioavailability is low, and repeated administration is required. Also, due to their smaller size than protein pharmaceuticals, they cannot reach the target during drug administration and have the limitation of disappearing quickly in the body through the kidneys.

[0004] To solve such problems, methods such as increasing the biofilm permeability of peptide drugs and transmitting peptide drugs into the body by inhalation through the oral cavity or nasal cavity, changing specific amino acid sequences sensitive to protein hydrolases to suppress the degradation of peptides by protein hydrolases, producing fusion proteins of bioactive polypeptides and human serum albumin or human immunoglobulin fragments (Fc) using recombinant fusion technology, and chemically attaching highly soluble non-peptidic polymers to the peptide surface have been studied.

[0005] Polyethylene glycol (PEG), a non-peptidic polymer, is a polymer with non-ionic, non-toxic, biocompatible, and highly hydrophilic properties. Since the PEG can be easily chemically modified, it can be covalently attached to peptide or protein drugs, increasing the molecular weight of the drugs, thereby suppressing renal elimination and protecting the drugs from proteolytic enzymes. Since the PEG does not cause any special side effects, it has recently been actively studied as a method for extending the plasma half-life of peptide drugs.

[0006] PEG has been approved by the US Food and Drug Administration as a substance "generally recognized as safe (GRAS)". International Patent Publication WO06 / 076471 describes binding PEG to B-type natriuretic peptide (BNP), which is used as a therapeutic agent for congestive heart failure, to sustain its biological activity. US Patent No. 6,924,264 describes a method of binding PEG to lysine residues of exendin-4 to extend its in vivo duration.

[0007] The homopolymer of ethylene glycol, which is unmodified or unaltered PEG, has hydroxy groups at both or one of its ends. For use in binding to drugs, polyethylene glycol (PEG) derivatives can also be used. For example, polyethylene glycol (PEG) derivatives in which the hydroxy groups at one or both ends of the polyethylene glycol (PEG) chain are converted to highly reactive functional groups can also be used. For example, PEG-aldehyde, PEG-acetaldehyde, PEG-propionaldehyde, etc. can be used as the polyethylene glycol (PEG) derivatives. Such aldehyde polyethylene glycol (PEG) derivatives selectively react with one end of the amino acid of the protein with the aldehyde group present at the end, and form a chemical bond or are linked to the peptide drug.

[0008] However, when a polyethylene glycol (PEG) derivative in which both ends are aldehyde groups is used for drug conjugation, since both aldehyde groups can participate in the reaction, it leads to the formation of undesired adducts and excessive use of PEG, and as a result, it has an undesirable effect on the yield and purity. Summary of the Invention Problems to be Solved by the Invention

[0009] One object of the present invention is to provide a polyethylene glycol derivative that can be used in the production of peptide pharmaceuticals and a composition containing the same.

[0010] Another object of the present invention is to provide a method for producing a bioactive polypeptide conjugate using the aforementioned polyethylene glycol derivative. Means for Solving the Problems

[0011] The polyethylene glycol derivative compound according to one aspect is represented by the following Chemical Formula 1:

[0012] Chemical Formula 1

Chemical

[0013] The polyethylene glycol derivative composition according to one aspect is a polyethylene glycol derivative represented by the following chemical formula 1A, a polyethylene glycol derivative represented by the following chemical formula 2, and a polyethylene glycol derivative represented by the following chemical formula 3:

[0014] Chemical formula 1A [Chemical formula]

[0015] Chemical formula 2 [Chemical formula]

[0016] Chemical formula 3 [Chemical formula] n is also a natural number from 30 to 115.

[0017] In the composition, the content ratio of the polyethylene glycol derivative represented by the chemical formula 1A within the range of number average molecular weight of 2,950 to 3,650 is shown to be at least 70 area% based on high performance liquid chromatography (HPLC), the content ratio of the polyethylene glycol derivative represented by the chemical formula 2 within the range of number average molecular weight of 2,950 to 3,650 is shown to be 15 area% or less based on high performance liquid chromatography (HPLC), the content ratio of the polyethylene glycol derivative represented by the chemical formula 3 within the range of number average molecular weight of 2,950 to 3,650 is shown to be 10 area% or less based on high performance liquid chromatography (HPLC).

[0018] In the composition, the number average molecular weight of the polyethylene glycol derivative is in the range of 2,950 to 3,650 when measured by gel permeation chromatography. In the composition, the number average molecular weight of the polyethylene glycol derivative is in the range of 3,000 to 3,200 when measured by gel permeation chromatography.

[0019] A method for producing a bioactive polypeptide conjugate according to one aspect is (a) A pegylation step of reacting a polyethylene glycol derivative of the following Chemical Formula 1 with a bioactive polypeptide to generate a conjugate in which the bioactive polypeptide is covalently linked to the aldehyde carbon of the polyethylene glycol derivative of the following Chemical Formula 1, (b) A hydrolysis step of treating the conjugate under acidic aqueous conditions to generate a conjugate hydrolyzate, (c) A conjugation step of reacting the conjugate hydrolyzate with an immunoglobulin Fc fragment or a derivative thereof to generate a conjugate:

[0020] Chemical Formula 1

Chemical Formula

[0021] The reaction in step (a) is reductive amination, The linker is also a substance in which the N-terminal nitrogen of the bioactive polypeptide or the nitrogen atom of the ε-amino group of lysine is covalently linked to the aldehyde carbon atom of the compound of Chemical Formula 1. The PEGylation reaction in step (a) is also carried out in the pH range of 3.0 to 9.0. The hydrolysis in step (b) is also carried out in the pH range of 1.0 to 5.0. The conjugation reaction in step (c) is also reductive amination. The reductive amination is also carried out in the pH range of 5.0 to 8.5.

[0022] The sequence of the Fc fragment or its derivative also has a proline at the N-terminus. In the conjugate, the nitrogen atom of the N-terminal proline of the Fc fragment or its derivative is also covalently linked to the carbon atom derived from the acetal carbon. The Fc fragment or its derivative may have the amino acid sequence of SEQ ID NO: 2. The R 1 and the R 2 are also ethyl. n is also a natural number from 67 to 83.

Advantages of the Invention

[0023] By binding a bioactive polypeptide and an Fc fragment or its derivative to a polyethylene glycol derivative having a reactive aldehyde group at one end and a non-reactive acetal group at the other end, a bioactive polypeptide conjugate can be produced in a high yield.

Brief Description of the Drawings

[0024]

Figure 1

Figure 2A

Figure 2B

Figure 3A

Figure 3B

Figure 3C

Figure 3D

Figure 4A

Figure 4B

Figure 5

Modes for Carrying Out the Invention

[0025] Hereinafter, the present invention will be described in detail. All technical terms used in the present invention are used in the meaning generally understood by those skilled in the relevant field of the present invention unless otherwise specifically defined. Also, although desirable methods and samples are described in this specification, those similar to or equivalent to them are also included in the scope of the present invention. Also, the numerical values described in this specification are considered to include the meaning of "about" even if not explicitly stated. The content of all publications described as references in this specification is incorporated herein by reference in its entirety.

[0026] A polyethylene glycol derivative according to one aspect of the present invention will be described. The polyethylene glycol derivative is a polyethylene glycol (PEG) derivative having an aldehyde group (ALD) at one end and an acetal group (ACT) at the other end. The polyethylene glycol derivative is also represented by the following formula 1:

[0027] <Formula 1> ALD-PEG-ACT In formula 1, ALD is an aldehyde functional group, PEG is a polyethylene glycol moiety, and ACT is an acetal group.

[0028] In formula 1, the polyethylene glycol (PEG) moiety is represented by (-O-C2H4)n-O-, where n is 30 to 115, or also 50 to 100. In a more specific example, n is also in the range of 67 to 83. The polyethylene glycol (PEG) can bind to a bioactive polypeptide and an Fc fragment or its derivative, increase the in vivo half-life of the bioactive polypeptide, and play a role in transporting the bioactive polypeptide in the body.

[0029] The aldehyde group (ALD) is a reactive functional group and is also an alkyl aldehyde, for example, a C2-C6 alkyl aldehyde. Specifically, the aldehyde group (ALD) is also a propionaldehyde group, a butyraldehyde group, etc., but is not particularly limited thereto. The aldehyde group (ALD) can react with a bioactive polypeptide to bind the bioactive polypeptide to polyethylene glycol (PEG). Alternatively, the aldehyde group (ALD) can react with an Fc fragment or its derivative to bind the Fc fragment or its derivative to polyethylene glycol (PEG).

[0030] The acetal group (ACT) is a non-reactive functional group and does not react with a bioactive polypeptide or an Fc fragment or its derivative. Therefore, while one end of polyethylene glycol (PEG) is bound to a bioactive polypeptide or an Fc fragment or its derivative via the reaction of an aldehyde group (ALD), the other end of polyethylene glycol (PEG) is also protected by the non-reactive acetal group (ACT).

[0031] The acetal group (ACT) is -R 1 CH(OR 2 )(OR 3 ) and is represented by R 1 to R 3 are each independently also a C2-C6 alkyl group. Specifically, the acetal group (ACT) may be, for example, a 1,1-diethoxypropyl group, a 1,1-diethoxybutyl group, etc., but is not particularly limited thereto.

[0032] On the other hand, the acetal group (ACT) can be hydrolyzed to an aldehyde group to form a second aldehyde group (ALD). The second aldehyde group (ALD) can react with a bioactive polypeptide or an Fc fragment or its derivative to bind the bioactive polypeptide or the Fc fragment or its derivative to the other end of polyethylene glycol (PEG).

[0033] A specific example of the polyethylene glycol derivative of Formula 1 is also represented by Chemical Formula 1 below:

[0034] Chemical Formula 1

Chemical Structure

[0035] n is also in the range of 30 to 115, for example, 50 to 100, for example, 67 to 83. The acetal group in Chemical Formula 1 is hydrolyzed in the process of forming a bioactive peptide conjugate and is also converted into a second aldehyde group.

[0036] Specifically, the polyethylene glycol derivative of Chemical Formula 1 is also represented by the following Chemical Formula 1A:

[0037] Chemical Formula 1A

Chemical Formula

[0038] A polyethylene glycol derivative composition according to another aspect of the present invention will be described. The aforementioned polyethylene glycol derivative can form a composition containing impurities. The impurities include a polyethylene glycol derivative in which both ends of the polyethylene glycol are acetal groups and / or a polyethylene glycol derivative in which both ends of the polyethylene glycol are aldehyde groups. Regarding the content ratio of the polyethylene glycol derivative having an aldehyde group at one end and an acetal group at the other end in the composition, it can be regarded as the purity of the polyethylene glycol derivative having an aldehyde group at one end and an acetal group at the other end. The content of such impurities is also calculated as the area percentage of the peak measured by high performance liquid chromatography (HPLC).

[0039] For example, the polyethylene glycol derivative composition a polyethylene glycol derivative represented by the following Chemical Formula 1A, a polyethylene glycol derivative represented by the following Chemical Formula 2, Those containing a polyethylene glycol derivative represented by the following Chemical Formula 3:

[0040] Chemical Formula 1A

Chem.

[0041] Chemical Formula 2

Chem.

[0042] Chemical Formula 3

Chem.

[0043] In the composition, The polyethylene glycol derivative represented by Chemical Formula 1A has a number average molecular weight in the range of 2,950 to 3,650, and may have a content ratio shown as at least 75 area % based on high performance liquid chromatography (HPLC). The polyethylene glycol derivative represented by Chemical Formula 2 has a number average molecular weight in the range of 2,950 to 3,650, and may have a content ratio shown as 15 area % or less based on high performance liquid chromatography (HPLC). The polyethylene glycol derivative represented by Chemical Formula 3 has a number average molecular weight in the range of 2,950 to 3,650, and may have a content ratio shown as 10 area % or less based on high performance liquid chromatography (HPLC).

[0044] In the composition, the number average molecular weight of the polyethylene glycol derivative is also a value measured by gel permeation chromatography. In the composition, the number average molecular weight of the polyethylene glycol derivative is also in the range of 3,000 to 3,200 when measured by gel permeation chromatography. The number average molecular weight of a compound is also measured by gel permeation chromatography.

[0045] A method for producing a bioactive polypeptide conjugate according to another aspect of the present invention will be described. FIG. 1 is a flowchart for explaining a method for producing a bioactive polypeptide conjugate according to one aspect. Referring to FIG. 1, the method for producing the bioactive polypeptide conjugate first provides a polyethylene glycol (PEG) derivative containing a polyethylene glycol having an aldehyde functional group at one end and an acetal functional group at the other end (S110). The polyethylene glycol derivative is a compound represented by the aforementioned Formula 1, Chemical Formula 1 or Chemical Formula 1A. Refer to the description related to the compound of Formula 1, Chemical Formula 1 or Chemical Formula 1A above.

[0046] Next, the polyethylene glycol derivative is reacted with the bioactive polypeptide (S120). By this reaction, a conjugate in which the bioactive polypeptide is covalently linked to the aldehyde carbon of the polyethylene glycol derivative can be formed. Such a conjugate formation reaction is also a pegylation reaction. The pegylation reaction is also a reductive amination reaction. The pegylation reaction is carried out in the range of pH 3.0 to 9.0, for example, in the range of pH 5.0 to 9.0.

[0047] The bioactive polypeptide includes, for example, hormones, cytokines, enzymes, antibodies, growth factors, transcription regulators, blood coagulation factors, vaccines, structural proteins, ligand proteins, or receptors. For example, the bioactive polypeptide may be exendin-4 or imidazo-acetyl exendin-4. For example, the conjugate is also a substance in which the N-terminal nitrogen of the bioactive polypeptide or the nitrogen atom of the ε-amino group of lysine is covalently linked to the aldehyde carbon atom of the compound of Chemical Formula 1.

[0048] Next, the acetal group at the other end of the polyethylene glycol derivative, one end of which is bound to the bioactive polypeptide, can be converted into an aldehyde group (S130). For example, the acetal group can be converted into an aldehyde group by hydrolysis. At this time, the hydrolysis is carried out, for example, under acidic aqueous conditions. The hydrolysis is also carried out in the range of pH 1.0 to 5.0, for example, in the range of pH 2.0 to 5.0. The R of the acetal group of Chemical Formula 1 1 and / or R 2 When is a propyl group, the aldehyde group generated by hydrolysis is also a propionaldehyde group.

[0049] Next, the converted aldehyde group of the polyethylene glycol derivative can be reacted with an Fc fragment or a derivative thereof (S140). By this reaction, the other end of the polyethylene glycol derivative can be conjugated to the Fc fragment or a derivative thereof. At this time, the Fc fragment or a derivative thereof is also covalently linked to the carbon atom derived from the carbon of the acetal group.

[0050] For example, when the polyethylene glycol derivative is the polyethylene glycol derivative represented by the above Chemical Formula 1, the aldehyde group of the polyethylene glycol derivative can first be reacted with a bioactive polypeptide to bind the bioactive polypeptide to one end of the polyethylene glycol derivative. At this time, the bioactive polypeptide can be pegylated with the aldehyde group. For the pegylation reaction, for example, a reductive amination reaction can be used. Next, in the polyethylene glycol derivative-bioactive polypeptide conjugate thus obtained, the acetal group (ACT) of the functional group remaining without reaction can be hydrolyzed to be converted into a second aldehyde group having reactivity. The converted second aldehyde group of the polyethylene glycol derivative-bioactive polypeptide conjugate can be reacted with an Fc fragment or a derivative thereof. The reaction is a conjugation reaction, and for example, a reductive amination reaction can be used. The reductive amination is also carried out in the range of pH 5.0 to 8.5. By this reaction, an Fc fragment or a derivative thereof can be bound to the other end of the polyethylene glycol derivative-bioactive polypeptide conjugate. Through such a process, a bioactive polypeptide conjugate in which one end of the polyethylene glycol derivative is linked to a bioactive polypeptide and the other end is linked to an Fc fragment or a derivative thereof can be formed. The conjugate is also, for example, a conjugate in which the nitrogen atom of the N-terminal proline of the Fc fragment or a derivative thereof is covalently linked to a carbon atom derived from the acetal carbon of the polyethylene glycol derivative. The acetyl carbon is the carbon to which an -OR group is linked in the acetyl group.

[0051] The method for producing a bioactive polypeptide conjugate using a polyethylene glycol derivative according to an embodiment of the present invention has an advantage of high yield while suppressing the generation of impurities or related substances. In the step of linking a bioactive polypeptide to a polyethylene glycol derivative containing a functional group at the end (PEGylation), as a by-product, an analog intermediate in which two or more units of the polyethylene glycol derivative are linked to one unit of the bioactive polypeptide is inevitably generated. If the molecular weight of the polyethylene glycol derivative is not large, it is not easy to perfectly purify and filter out such an analog intermediate before entering the subsequent linking reaction step only by general chromatographic methods.

[0052] When using a prior art polyethylene glycol derivative having aldehyde functional groups at both ends, such remaining analog intermediates react with the Fc fragment in the subsequent linking step, generating impurities in the form of multimers (dimers, trimers, etc.). Also, in the production using a prior art polyethylene glycol derivative, in the PEGylation step, a polyethylene glycol conjugate having a sandwich structure such as a bioactive polypeptide - polyethylene glycol derivative - bioactive polypeptide is also generated, and there is a disadvantage that the yield is reduced due to such reaction by-products.

[0053] Even when using a prior art polyethylene glycol derivative having aldehyde functional groups at both ends, due to the high reactivity of the aldehyde groups, the aldehyde groups first react with substances other than the reaction target and are thus lost. Further, in the manufacturing process of the polyethylene glycol derivative, impurities with altered end groups are generated, and the polyethylene glycol derivative itself also contains a certain amount of impurities. Due to such inactivation and alteration of the aldehyde groups, one end aldehyde group is converted to a hydroxy group, an ether group, etc. When an analog having a single aldehyde group undergoes a pegylation reaction, a polyethylene glycol conjugate with a corresponding structure is generated. Such a polyethylene glycol conjugate does not have an aldehyde group capable of reacting with an Fc fragment or its derivative by a subsequent ligation reaction, and thus the final conjugate cannot be obtained. Therefore, the generation of such analogs having a single aldehyde group also causes impurities and reduces the yield in the prior art method for manufacturing conjugates.

[0054] When the polyethylene glycol derivative according to an embodiment of the present invention is used in the manufacture of a bioactive polypeptide conjugate, such impurity generation can be basically prevented. Different from the prior art polyethylene glycol derivative, the polyethylene glycol derivative according to an embodiment of the present invention contains an aldehyde group only at one end, and thus basically cannot form a multimer. Further, in the manufacturing method using the polyethylene glycol derivative according to an embodiment of the present invention, if the aldehyde group disappears or changes to an analog before pegylation, the bioactive polypeptide - polyethylene glycol conjugate, which is a pegylation product, cannot be generated from the beginning. Therefore, the generation of analogs can be suppressed, purification becomes simple, and this leads to an improvement in the yield at the pegylation stage. Furthermore, as will be described later, the efficiency of the ligation reaction stage after pegylation is also higher than that of the prior art, and the purity in the purification process is also improved.

[0055] Regarding the above advantages, the yields of the manufacturing method of the present invention and the prior art are generally compared. First, in a specific embodiment of the present invention, generally, the aldehyde activity (the degree to which aldehyde groups are maintained until immediately before the coupling reaction) of the bioactive polypeptide - polyethylene glycol conjugate, which is a pegylated product, is at the level of 80 - 95%. This is much higher compared to the aldehyde activity of 60 - 75% of the conjugate using polyethylene glycol derivatives of the prior art. Also, in the manufacturing method of the present invention, since the yield in the coupling step is also improved, as a specific embodiment of the present invention, the final yield of the bioactive polypeptide conjugate is generally improved to the level of about 1.2 times to about 1.7 times that of the manufacturing method of the prior art.

[0056] Hereinafter, the manufacturing method of the bioactive polypeptide conjugate of the present invention will be described with specific examples.

[0057] First, a polyethylene glycol derivative (ALD - PEG - DEP) in which both ends of the ethylene glycol repeating unit are modified with a propyl aldehyde group and a 3 - diethoxypropyl group, respectively, was produced.

[0058] ●Test Example 1: Production of ALD - PEG - DEP 1 (a) Production of PEG - Ms [Chemical formula]

[0059] In a reactor under a nitrogen atmosphere, a solution prepared by dissolving 100 g of polyethylene glycol (PEG) (number - average molecular weight 3,400) in 300 mL of dichloromethane (CH2Cl2) was cooled to 5°C. To this solution, 23.0 mL of triethylamine (TEA) was added, and while maintaining 5°C, 12.6 mL of methanesulfonyl chloride (MsCl) was added. After stirring this reaction solution at 5°C for 2.5 hours, 300 mL of distilled water was added, and after stirring for 10 minutes, the organic layer was separated.

[0060] 300 mL of dichloromethane was added to the aqueous layer, and after further extraction of the organic layer, it was combined with the already separated organic layer. After washing the combined organic layer with distilled water, it was dried over anhydrous magnesium sulfate and filtered. After concentrating the filtrate under reduced pressure, the concentrate was dissolved in 100 mL of dichloromethane and added dropwise to 1,500 mL of methyl t-butyl ether over 30 minutes. After stirring the reaction solution at room temperature for 1 hour, the solid was filtered, washed with methyl t-butyl ether, and dried under nitrogen at room temperature to obtain 97 g (yield: 92.6%) of the target compound PEG-Ms (mesylate).

[0061] (b) Production of PEG-DEP

Chemical formula

[0062] Under a nitrogen atmosphere, 10 g of PEG-Ms produced as described above and 40 mL of toluene were charged into the second reactor. The solution of the activated first reactor was gradually added dropwise thereto over 1 hour. After stirring at room temperature for 2 hours and completion of the reaction, a saturated aqueous ammonium chloride solution was added to the reaction product. After stirring the reaction product for 5 minutes, dichloromethane was added and the organic layer was extracted. Dichloromethane was added to the aqueous layer and the organic layer was further extracted. The organic layers were collected and concentrated under reduced pressure, completely dissolved in 10 mL of dichloromethane, and then methyl t-butyl ether was added dropwise for crystallization. After stirring at room temperature for 2 hours, the crystals were filtered and washed with methyl t-butyl ether. The crystals were dried under nitrogen at room temperature to obtain 9.3 g (yield: 90.0%) of the target compound PEG-diethyl acetal (PEG-DEP).

[0063] (c) Production of ALD-PEG-DEP

Chemical formula

[0064] ● Test Example 2: Production of ALD-PEG-DEP 2

Chemical formula

[0065] (b) Production of ALD-PEG-DEP Under a nitrogen atmosphere, 45 mL of 3,3-diethoxy-1-propanol and 400 mL of toluene were charged into the first reactor. 15.7 g of potassium t-pentoxide (t-PeOK) was added, the temperature was raised to 50 °C, and it was stirred for 1 hour. The reaction solution was cooled to room temperature. In a nitrogen atmosphere, 100 g of PEG-Ms produced as described above and 400 mL of toluene were charged into the second reactor. The solution of the activated first reactor was gradually added dropwise thereto over 1 hour. After stirring at room temperature for 2.5 hours until the reaction was completed, distilled water was added to the reaction product. After stirring the reaction product for 5 minutes, the aqueous layer was separated. Dichloromethane and toluene were added to the aqueous layer to extract the organic layer. Distilled water was added to the organic layer, and after stirring for 5 minutes, the aqueous layer was further separated.

[0066] 13 mL of acetic acid was added to the separated aqueous layer, and the mixture was stirred at room temperature for 2.5 hours. After adding a 5% aqueous sodium hydrogen carbonate solution thereto, the mixture was stirred for 5 minutes. Dichloromethane and hexane were mixed in the third reactor, and then the aforementioned reaction solution was added. After stirring the mixture for 10 minutes, the aqueous layer was separated. A 5% aqueous sodium hydrogen carbonate solution was added to the separated aqueous layer, and the mixture was stirred for 5 minutes. Dichloromethane and hexane were mixed in the third reactor, and then the aforementioned reaction solution was added. After stirring for 10 minutes, the organic layer was separated. Distilled water was added to the separated organic layer and stirred, and then the organic layer was separated. The organic layer was dried over magnesium sulfate, filtered, and then concentrated under reduced pressure. Dichloromethane was added to the concentrated solution to dissolve it, and then methyl t-butyl ether was added dropwise to cause crystallization. After stirring at room temperature for 30 minutes, the crystals were filtered and washed with methyl t-butyl ether. The crystals were dried under nitrogen at room temperature to obtain 10.1 g (yield: 10.1%) of 3.4 kDa ALD-PEG-DEP, which is the target compound. The number average molecular weight measured by gel permeation chromatography was 3,163.

[0067] ● Analytical Example: Comparison of the Characteristics of ALD-PEG-DEP and ALD-PEG-ALD The characteristics of ALD-PEG-DEP, a polyethylene glycol derivative produced by the method according to an embodiment of the present invention, and ALD-PEG-ALD, a commercial polyethylene glycol derivative (manufactured by Hanmi Fine Chemical Co., Ltd., Korea) with both ends modified with propyl aldehyde groups (the chemical formula weight of the ethylene glycol repeating unit is 3.4 kDa), were measured and compared. For this purpose, MALDI-TOF mass spectrometry, nuclear magnetic resonance analysis (1 1H NMR, 13 13C NMR) and Fourier transform infrared spectroscopy (FT-IR) were performed.

[0068] Figures 2A and 2B are MALDI-TOF mass spectrometry graphs of ALD-PEG-DEP produced in Test Example 2 and commercial ALD-PEG-ALD, respectively. The following Table 1 is a table comparing the molecular weights of the obtained ALD-PEG-DEP produced in Test Example 2 and commercial ALD-PEG-ALD.

[0069]

Table 1

[0070] Referring to Figures 2A, 2B and Table 1, it can be seen that there is no significant difference in molecular weight between the ALD-PEG-DEP of Test Example 2 and the commercially available ALD-PEG-ALD. Figures 3A and 3B are graphs of nuclear magnetic resonance ( 1 1H NMR) spectra of the ALD-PEG-DEP of Test Example 2 and commercial ALD-PEG-ALD, respectively. In the 1 1H NMR spectra of Figures 3A and 3B, through the peaks corresponding to the diethoxy group (the triplet near 4.6 ppm (b) and near 1.2 ppm (i), and the multiplet near 1.8 ppm (h)), it can be confirmed that the ALD-PEG-DEP of the test example has an acetal end group. In addition, in the 1 1H NMR spectrum of Figure 2B, no peak corresponding to the diethoxy group is shown.

[0071] Figures 3C and 3D are graphs of nuclear magnetic resonance ( 13 13C NMR) spectra of the ALD-PEG-DEP of Test Example 2 and commercial ALD-PEG-ALD, respectively. In the 13In the 13C NMR spectrum, peaks corresponding to an ethyl group were observed at δ 15 ppm (i) and 62 ppm (f), and a peak of a secondary carbon was observed at δ 101 ppm (b). In the graph of FIG. 3C, peaks of ALD-PEG-DEP having an asymmetric structure in terms of molecular structure were observed. In contrast, in the graph of FIG. 3D, peaks of ALD-PEG-ALD having a symmetric structure in terms of molecular structure were observed (the (e), (f) peaks of ALD-PEG-DEP vs. the (c) peak of ALD-PEG-ALD, the (h), (i) peaks of ALD-PEG-DEP vs. the (d) peak of ALD-PEG-ALD).

[0072] FIGS. 4A and 4B are the FT-IR spectra of ALD-PEG-DEP of Test Example 2 and commercial ALD-PEG-ALD, respectively. Since the FT-IR spectra of FIGS. 4A and 4B have similar peak patterns, it can be seen that ALD-PEG-DEP of Test Example 2 and commercial ALD-PEG-ALD have similar polymer backbones.

[0073] To determine the purity of the polyethylene glycol derivative composition containing ALD-PEG-DEP, it was analyzed by high performance liquid chromatography (HPLC). As the column, a column filled with octylsilylated silica gel (inner diameter 4.6 mm × length 250 mm (5.0 μm)) was used, and purified water and acetonitrile were used as the mobile phase, and analysis was performed using a reverse phase chromatography method. The content ratio of the polyethylene glycol derivative was calculated as the ratio of the areas of the peaks measured by high performance liquid chromatography.

[0074] ● Example 1: Production of CA GLP-2(RK)-PEG-Immunoglobulin Fc Conjugate Using ALD-PEG-DEP

Chemical Structure

[0075] As a bioactive polypeptide, the polypeptide CA GLP-2(RK) of SEQ ID NO:1, which is a derivative of human glucagon-like peptide-2 (GLP-2), was used to produce a bioactive polypeptide conjugate. The amino acid sequence of the polypeptide CA GLP-2(RK) of SEQ ID NO:1 is as follows: (4-imidazoacetyl-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Arg-Ile-Thr-Asp-Lys)

[0076] (1) Production of CA GLP-2(RK)-PEG-DEP conjugate The 34th lysine residue of CA GLP-2(RK) was linked to the aldehyde terminus of 3.4 kDa ALD-PEG-DEP to produce CA GLP-2(RK)-PEG-DEP. At this time, the molar ratio of CA GLP-2(RK) to ALD-PEG-ALD was set to 1:8, the concentration of the CA GLP-2(RK) peptide was set to 10 mg / mL, and the reaction was carried out at room temperature for about 25 hours. At this time, as the reaction solution, a solution containing 100 mM sodium cyanoborohydride (NaBH3CN) as a reducing agent in 50 mM triethanolamine (pH 8.0) was used. After completion of the reaction, the reaction mixture was purified using 20 mM Bis-Tris buffer (pH 6.2) and anion exchange chromatography using an NaCl concentration gradient to separate CA GLP-2(RK)-PEG-DEP. The purification yield was 59.6%, and the purity of the analysis results by SE-HPLC and RP-HPLC was 99% and 99%, respectively.

[0077] (2) Production of CA GLP-2(RK)-PEG-ALD conjugate To hydrolyze the diethoxy functional group (DEP) of the purified CA GLP-2(RK)-PEG-DEP and convert it to an aldehyde group (ALD), 20 mM sodium citrate (pH 2.0) buffer was used to lower the pH. Then, for the next step, 20 mM bis-tris (pH 6.5) buffer was used to exchange the buffer and obtain CA GLP-2(RK)-PEG-ALD.

[0078] (3) Production of CA GLP-2(RK)-PEG-immunoglobulin Fc conjugate Next, the molar ratio of the thus-obtained CA GLP-2(RK)-PEG-ALD to the immunoglobulin Fc fragment (SEQ ID NO: 2) was set to 1:2, the concentration of the total protein (CA GLP-2(RK) and immunoglobulin Fc fragment) was set to 30 mg / mL, and the conjugation reaction was allowed to proceed at room temperature for 14 to 16 hours. At this time, 20 mM bis-tris (pH 6.2), ethanol, and 30 mM NaBH3CN as a reducing agent were added to the reaction solution. After the reaction was terminated, CA GLP-2(RK)-PEG-immunoglobulin Fc was purified from the reaction mixture using hydrophobic interaction chromatography (HIC) and anion exchange chromatography. The purification yield was 70.2%, and the purity analyzed by SE-HPLC and RP-HPLC was 98.1% and 97%, respectively.

[0079] The amino acid sequence of the immunoglobulin Fc of SEQ ID NO: 2 is as follows: (Pro-Ser-Cys-Pro-Ala-Pro-Glu-Phe-Leu-Gly-Gly-Pro-Ser-Val-Phe-Leu-Phe-Pro-Pro-Lys-Pro-Lys-Asp-Thr-Leu-Met-Ile-Ser-Arg-Thr-Pro-Glu-Val-Thr-Cys-Val-Val-Val-Asp-Val-Ser-Gln-Glu-Asp-Pro-Glu-Val-Gln-Phe-Asn-Trp-Tyr-Val-Asp-Gly-Val-Glu-Val-His-Asn-Ala-Lys-Thr-Lys-Pro-Arg-Glu-Glu-Gln-Phe-Asn-Ser-Thr-Tyr-Arg-Val-Val-Ser-Val-Leu-Thr-Val-Leu-His-Gln-Asp-Trp-Leu-Asn-Gly-Lys-Glu-Tyr-Lys-Cys-Lys-Val-Ser-Asn-Lys-Gly-Leu-Pro-Ser-Ser-Ile-Glu-Lys-Thr-Ile-Ser-Lys-Ala-Lys-Gly-Gln-Pro-Arg-Glu-Pro-Gln-Val-Tyr-Thr-Leu-Pro-Pro-Ser-Gln-Glu-Glu-Met-Thr-Lys-Asn-Gln-Val-Ser-Leu-Thr-Cys-Leu-Val-Lys-Gly-Phe-Tyr-Pro-Ser-Asp-Ile-Ala-Val-Glu-Trp-Glu-Ser-Asn-Gly-Gln-Pro-Glu-Asn-Asn-Tyr-Lys-Thr-Thr-Pro-Pro-Val-Leu-Asp-Ser-Asp-Gly-Ser-Phe-Phe-Leu-Tyr-Ser-Arg-Leu-Thr-Val-Asp-Lys-Ser-Arg-Trp-Gln-Glu-Gly-Asn-Val-Phe-Ser-Cys-Ser-Val-Met-His-Glu-Ala-Leu-His-Asn-His-Tyr-Thr-Gln-Lys-Ser-Leu-Ser-Leu-Ser-Leu-Gly-Lys)

[0080] ● Comparative Example 1: Production of CA GLP-2(RK)-PEG-Immunoglobulin Fc conjugate using ALD-PEG-ALD

Chem.

[0081] (2) Preparation of CA GLP-2(RK)-PEG-immunoglobulin Fc conjugate Next, the molar ratio of CA GLP-2(RK)-PEG-ALD to immunoglobulin Fc fragment (SEQ ID NO: 2) was set to 1:2, the concentration of immunoglobulin Fc fragment was set to 30 mg / mL, and the reaction was carried out at room temperature for 14 to 16 hours. At this time, a solution containing 20 mM 2-picolyl borane complex was used as a reducing agent in 20 mM bis-tris (pH 6.2) and isopropanol as the reaction solution. After the reaction was completed, CA GLP-2(RK)-PEG-immunoglobulin Fc was purified from the reaction mixture using hydrophobic interaction chromatography (HIC) and anion exchange chromatography. The purification yield was 67.7%, and the purity analyzed by SE-HPLC and RP-HPLC was 98.6% and 96.1% respectively.

[0082] ● Analytical Example: Aldehyde Group Activity of Polyethylene Glycol Derivative Oils In the method for producing a physiologically active polypeptide conjugate, regarding the degree (activity level) of aldehyde groups remaining immediately before the ligation reaction step with the Fc fragment, an example using the polyethylene glycol derivative of the present invention was compared with a comparative example using a polyethylene glycol (PEG) derivative having aldehyde groups at both ends in the prior art.

[0083] Regarding the GLP-2 derivative, which is a physiologically active polypeptide used in the conjugate production process of Example 1, the steps of pegylation, chromatographic purification, and hydrolysis described in Example 1 were carried out until a conjugate hydrolyzate was obtained. For the same physiologically active polypeptide, pegylation and chromatographic purification described in Comparative Example 1 were carried out until the corresponding conjugate was obtained.

[0084] Next, the conjugate hydrolyzate and the conjugate were reacted with a reagent to form a derivative, and the degree of derivative formation was quantified by ultraviolet-visible spectrophotometry to measure the activity level of the aldehyde group, which is shown in FIG. 5. Referring to FIG. 5, the activity level of the aldehyde group of the conjugate hydrolyzate obtained using ALD-PEG-DEP according to the present invention was 87.2%, which was confirmed to maintain the aldehyde group at a considerably higher ratio compared to the 69.8% of the aldehyde group activity level of the conjugate obtained using conventional ALD-PEG-ALD.

[0085] Thus, in the method for producing a physiologically active polypeptide conjugate using the polyethylene glycol derivative compound according to the examples of the present invention, the activity of the aldehyde terminus can be maintained even higher. Furthermore, in subsequent steps, even when using the same purification process as in the prior art manufacturing method, the production yield of the conjugation reaction step in the manufacturing method of the present invention was improved compared to the prior art. In the case of the example of the present invention, the conjugation reaction production yield was 50.8%, which was increased by 25% or more compared to 40.5% when using conventional ALD-PEG-ALD. In the production of the CA GLP-2(RK)-PEG-immunoglobulin Fc conjugate, the overall yield in Example 1 of the present invention was 30.3%, showing an approximately 35% improvement compared to 22.4% in Comparative Example 1 of the prior art.

[0086] ● Example 2: Production of the CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate using ALD-PEG-DEP

Chemical formula

[0087] To pegylate 3.4 kDa ALD-PEG-DEP produced in Test Example 1 at the lysine 27 (Lys27) position of CA EXD4, the molar ratio of CA EXD4:ALD-PEG-DEP was set to 1:3, the concentration of CA EXD4 was set to 12 g / L, and the reaction was carried out at 8 ± 2 °C for about 16 hours. Specifically, the reaction was carried out under 0.1 M bis-tris (pH 7.9) and 45% (v / v) isopropanol, and sodium cyanoborohydride (SCB: sodium cyanoborohydride (NaCNBH3)) was added to a concentration of 50 mM as a reducing agent and reacted.

[0088] After completion of the reaction, a buffer solution containing sodium citrate and ethanol and a KCl linear concentration gradient were applied, and a SOURCE 15S column (Cytiva) was used to separate and purify the CA EXD4(Lys27)-PEG-DEP conjugate from the reaction solution. The yield of CA EXD4(Lys27)-PEG-DEP was confirmed to be about 50% compared to the input CA EXD4.

[0089] (2) Production of CA EXD4-PEG-ALD conjugate To hydrolyze the diethoxypropyl (DEP) functional group of the CA EXD4(Lys27)-PEG-DEP conjugate to a propyl aldehyde group (ALD), buffer exchange was performed with an acidic solution. Specifically, after diluting CA EXD4(Lys27)-PEG-DEP in water, buffer exchange and concentration were carried out with 25 mM hydrochloric acid via a separation membrane ultrafiltration / diafiltration (UF / DF) method, and the CA EXD4(Lys27)-PEG-ALD conjugate was separated so that the final recovered concentration was 0.8 g / L or more. As a result of analyzing the degree of conversion of the terminal functional group using RP-HPLC analysis, it was confirmed that the diethoxypropyl group was converted to the propyl aldehyde group by 95% or more.

[0090] (3) Production of CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate To produce the CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate, the CA EXD4(Lys27)-PEG-ALD conjugate obtained in (2) was ligated with immunoglobulin Fc (SEQ ID NO: 2). At this time, the molar ratio of the CA EXD4(Lys27)-PEG-ALD conjugate to immunoglobulin Fc was made 1:2, the concentration of the total protein (CA EXD4 and immunoglobulin Fc) was made 10 g / L, and the reaction was carried out at room temperature for 2 hours. At this time, as the reaction solution, a solution containing bis-tris and SCB, a reducing agent, was used.

[0091] After the reaction was completed, the CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate was obtained from the reaction mixture by using hydrophobic interaction chromatography and anion exchange chromatography. The yield of the CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate was confirmed to be about 50% to 60% compared with the input CA EXD4 bioactive polypeptide.

[0092] ● Comparative Example 2: Production of CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate using ALD-PEG-ALD

Chemical formula

[0093] After the reaction was completed, a buffer solution containing sodium citrate and ethanol and a KCl linear concentration gradient were applied, and a SOURCE 15S column (Cytiva) was used to separate and purify the CA EXD4(Lys27)-PEG-ALD conjugate from the reaction solution. Thereafter, the purified solution of the CA EXD4(Lys27)-PEG-ALD conjugate was diluted with water, and then buffer exchange and concentration were carried out with a 10 mM potassium phosphate solution through a separation membrane ultrafiltration / diafiltration (UF / DF) method so that the final recovery concentration was about 0.6 g / L or more.

[0094] The yield of the CA EXD4(Lys27)-PEG-ALD conjugate was confirmed to be 35% to 43% compared with the input CA EXD4 bioactive polypeptide.

[0095] (2) Production of CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate To produce the CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate, the CA EXD4(Lys27)-PEG-ALD conjugate obtained in (1) was ligated with immunoglobulin Fc (SEQ ID NO: 2). At this time, the molar ratio of the CA EXD4(Lys27)-PEG-ALD conjugate to immunoglobulin Fc was made 1:2, the concentration of the total protein (CA EXD4 and immunoglobulin Fc) was made 10 g / L, and the reaction was carried out at room temperature for 16 hours. At this time, as the reaction solution, a solution containing HEPES and SCB as an ethanol reducing agent was used. After the reaction was completed, the CA EXD4(Lys27)-PEG-immunoglobulin Fc conjugate was obtained from the reaction mixture by using hydrophobic interaction chromatography and anion exchange chromatography. The yield was confirmed to be 35% to 40% compared with the input bioactive polypeptide. When comparing the overall yields of CA-EXD4-PEG-immunoglobulin Fc production, the yield of Example 2 showed a result of being improved compared with the yield of Comparative Example 2.

[0096] ● Example 3: Production of GCSF Derivative-PEG-Immunoglobulin Fc Conjugate Using ALD-PEG-DEP

Chemical formula

[0097] After the binding reaction, GCSF derivative-PEG-DEP was purified using an SP-HP column (cation exchange chromatography) (Cytiva). At this time, a sodium acetate buffer solution was used and purification was carried out using a sodium chloride concentration gradient. The yield of GCSF-PEG-DEP was confirmed to be 61.2% compared to the input GCSF derivative.

[0098] (2) Hydrolysis of the DEP Functional Group To hydrolyze the diethoxypropyl (DEP) functional group of GCSF derivative-PEG-DEP to a propyl aldehyde group (ALD), buffer exchange was performed with an acidic solution. Specifically, the purified GCSF-PEG-DEP was subjected to buffer exchange via a separation membrane ultrafiltration / diafiltration (UF / DF) method to lower the pH of the buffer to 2.0, followed by storage at room temperature for 16 hours.

[0099] (3) Production of GCSF derivative-PEG-immunoglobulin Fc conjugate To produce the GCSF-PEG-immunoglobulin Fc conjugate, the GCSF derivative-PEG-ALD obtained in (2) was ligated with immunoglobulin Fc (SEQ ID NO: 2). At this time, the molar ratio of the GCSF derivative-PEG-ALD conjugate to immunoglobulin Fc was set to 1:4, the concentration of the total protein (GCSF and immunoglobulin Fc) was set to 50 g / L, and the reaction was carried out at 6 ± 4°C for 16 hours. Specifically, the reaction was carried out under 0.1 M potassium phosphate (pH 6.0), and SCB was added to a concentration of 20 mM as a reducing agent for the reaction. After the reaction was completed, the GCSF derivative-PEG-immunoglobulin Fc conjugate was obtained from the reaction mixture using hydrophobic interaction chromatography and anion exchange chromatography. The yield of the GCSF derivative-PEG-immunoglobulin Fc conjugate was confirmed to be 50.2% compared to the input GCSF derivative-PEG-DEP.

[0100] ● Comparative Example 3: Production of GCSF derivative-PEG-immunoglobulin Fc conjugate using ALD-PEG-ALD

Chemical formula

[0101] (2) Production of GCSF derivative-PEG-immunoglobulin Fc conjugate To produce the GCSF derivative-PEG-immunoglobulin Fc conjugate, the GCSF derivative-PEG-ALD and immunoglobulin Fc (SEQ ID NO: 2) were conjugated. At this time, the molar ratio of the GCSF-PEG-ALD conjugate to immunoglobulin Fc was set to 1:4, the concentration of the total protein (GCSF derivative and immunoglobulin Fc) was set to 50 g / L, and the reaction was carried out at 6 ± 4 °C for 16 hours. Specifically, the reaction was carried out under 0.1 M potassium phosphate (pH 6.0), and SCB was added as a reducing agent to a concentration of 20 mM for the reaction. After the reaction was completed, the GCSF derivative-PEG-immunoglobulin Fc conjugate was obtained from the reaction mixture using hydrophobic interaction chromatography and anion exchange chromatography. The yield of the GCSF derivative-PEG-DEP conjugate was confirmed to be 39.9% compared to the input GCSF derivative. When comparing the overall yields of the production of the GCSF derivative-PEG-immunoglobulin Fc conjugate, Example 3 according to the production method of the present invention showed a result that was 1.36 times higher than Comparative Example 3 of the prior art.

Claims

1. A polyethylene glycol derivative compound represented by the following Chemical Formula 1: Chemical Formula 1 【Chemical 1】 In Chemical Formula 1, n is a natural number from 30 to 115, R １ and R ２ are the same as or different from each other and are C １ -C ５ alkyl.

2. Said C １ -C ５ The polyethylene glycol derivative compound according to claim 1, wherein the alkyl is methyl, ethyl, propyl or butyl.

3. Said R １ and said R ２ are ethyl, and the polyethylene glycol derivative compound according to claim 1.

4. The polyethylene glycol derivative compound according to Claim 1, wherein the polyethylene glycol derivative compound is an isolated polyethylene glycol derivative compound.

5. The polyethylene glycol derivative compound according to any one of Claims 1 to 4, wherein n is a natural number from 50 to 100.

6. The polyethylene glycol derivative compound according to Claim 5, wherein n is a natural number from 67 to 83.

7. A polyethylene glycol derivative composition, comprising a polyethylene glycol derivative represented by the following Chemical Formula 1A, a polyethylene glycol derivative represented by the following Chemical Formula 2, and a polyethylene glycol derivative represented by the following Chemical Formula 3, Chemical Formula 1A [Chemical 2] Chemical Formula 2 【Chemical Formula 3】 Chemical Formula 3 【Chemical Formula 4】 In the composition, the content ratio of the polyethylene glycol derivative represented by Chemical Formula 1A within the range of a number average molecular weight of 2,950 to 3,650 is shown to be at least 70 area % based on high performance liquid chromatography (HPLC), the content ratio of the polyethylene glycol derivative represented by Chemical Formula 2 within the range of a number average molecular weight range of 2,950 to 3,650 is shown to be 15 area % or less based on high performance liquid chromatography (HPLC), the content ratio of the polyethylene glycol derivative represented by Chemical Formula 3 within the range of a number average molecular weight range of 2,950 to 3,650 is shown to be 10 area % or less based on high performance liquid chromatography (HPLC). A polyethylene glycol derivative composition.

8. The polyethylene glycol derivative composition according to Claim 7, wherein the number average molecular weight of the polyethylene glycol derivative is in the range of 2,950 to 3,650 when measured by gel permeation chromatography.

9. The polyethylene glycol derivative composition according to Claim 8, wherein the number average molecular weight of the polyethylene glycol derivative is in the range of 3,000 to 3,200 when measured by gel permeation chromatography.

10. (a) Reacting a polyethylene glycol derivative of the following Chemical Formula 1 with a bioactive polypeptide to produce a conjugate in which the bioactive polypeptide is covalently linked to the aldehyde carbon of the polyethylene glycol derivative of the following Chemical Formula 1, a pegylation step; (b) A hydrolysis step of treating the conjugate under acidic aqueous conditions to produce a conjugate hydrolyzate; (c) A conjugation step of reacting the conjugate hydrolyzate with an immunoglobulin Fc fragment or a derivative thereof to produce a conjugate, The method for producing a bioactive polypeptide conjugate, wherein the conjugate has a structure in which an Fc fragment or a derivative thereof is covalently linked to a carbon atom derived from the acetal carbon of the polyethylene glycol derivative of the following Chemical Formula 1 in the conjugate hydrolyzate: Chemical Formula 1 【Chemical Formula 5】 In Chemical Formula 1, n is a natural number from 50 to 100, R １ and R ２ are the same as or different from each other, and are C １ -C ５ alkyl.

11. The bioactive polypeptide is selected from the group consisting of hormones, cytokines, enzymes, antibodies, growth factors, transcription regulators, blood coagulation factors, vaccines, structural proteins, ligand proteins, or receptors. The method for producing a bioactive polypeptide conjugate according to Claim 10.

12. The reaction in step (a) is reductive amination, The conjugate is a substance in which the N-terminal nitrogen of the bioactive polypeptide or the ε-amino group nitrogen atom of lysine is covalently linked to the aldehyde carbon atom of the compound of Chemical Formula 1. The method for producing a bioactive polypeptide conjugate according to Claim 10.

13. The pegylation reaction in step (a) is carried out in the range of pH 3.0 to 9.

0. The method for producing a bioactive polypeptide conjugate according to Claim 12.

14. The hydrolysis in step (b) is carried out in the range of pH 1.0 to 5.

0. The method for producing a bioactive polypeptide conjugate according to Claim 12.

15. The conjugation reaction in step (c) is reductive amination. The method for producing a bioactive polypeptide conjugate according to Claim 10.

16. The reductive amination is carried out in the range of pH 5.0 to 8.

5. The method for producing a bioactive polypeptide conjugate according to Claim 15.

17. The sequence of the Fc fragment or its derivative has a proline at the N-terminus. The method for producing a bioactive polypeptide conjugate according to Claim 10.

18. The method for producing a bioactive polypeptide conjugate according to claim 17, wherein the nitrogen atom of the N-terminal proline of the Fc fragment or its derivative is covalently linked to a carbon atom derived from the acetal carbon.

19. The method for producing a bioactive polypeptide conjugate according to claim 18, wherein the Fc fragment or its derivative is SEQ ID NO:

2.

20. R １ and R ２ is ethyl, a method for producing a bioactive polypeptide according to claim 10.

21. The method for producing a bioactive polypeptide according to claim 10, wherein n is a natural number from 67 to 83.

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