Method for producing activated polyethylene glycol derivatives

Abstract

Provided is a method for producing a polyethylene glycol derivative having a high activation purity, the method being capable of preventing an increase in polydispersity due to an increase in thermal history and being industrially feasible. A method for producing an activated polyethylene glycol derivative having, at the terminal, a functional group or a precursor of a functional group capable of reacting with a biologically relevant substance, comprising the following steps (A), (B) and (C). Step (A): A step for drying a powder of a polyethylene glycol compound having at least one group selected from the group consisting of a carboxyl group, a mercapto group, a hydroxyl group, and an amino group at a terminal in an atmosphere of 0-50 DEG C to obtain a dried powder having a water content of 0.10 mass% or less; a step (B) of adding an organic solvent having a water content of not more than 200 ppm to the dry powder obtained in the step (A) to dissolve the dry powder in the organic solvent to obtain a solution; step (C): a step for obtaining an activated polyethylene glycol derivative by reacting the polyethylene glycol compound contained in the solution obtained in step (B) with an activator.

Description

Technical Field

[0001] This invention relates to a method for producing activated polyethylene glycol derivatives. Background Technology

[0002] When drugs composed of biorelated substances such as hormones, cytokines, and enzymes are administered to living organisms, they are typically absorbed through glomerular filtration in the kidneys or by macrophages in organs such as the liver or spleen, and are then rapidly excreted from the body. Consequently, their short half-life in the blood makes it difficult to achieve sufficient pharmacological action. To address this issue, attempts have been made to chemically modify biorelated substances with water-soluble polymers such as polyethylene glycol (hereinafter sometimes referred to as "PEG"). This increases the molecular weight and forms a hydration layer, thus prolonging the half-life of biorelated substances in the blood. These modifications are known to produce effects such as reducing the toxicity and antigenicity of biorelated substances and improving their agglutination properties.

[0003] Activated polyethylene glycol (PEG) derivatives typically have active groups at the ends of the PEG chain that are chemically bonded to functional groups such as amino, thiol, carboxyl, or unsaturated bonds present on the surface of the protein to be modified. For example, in the case of amino modification, active groups such as formyl, epoxy, p-nitrophenyl ester, or N-hydroxysuccinimide ester are present at the ends of the PEG chain. In the case of thiol modification, active groups such as thiol, maleimide, substituted maleimide, allyl, or N-hydroxysuccinimide ester are present at the ends of the PEG chain. In the case of carboxyl modification, active groups such as thiol or amino are present at the ends of the PEG chain. In the case of unsaturated bond modification, active groups such as thiol are present at the ends of the PEG chain.

[0004] As activated polyethylene glycol derivatives used for drug modification, from the perspective of drug homogeneity, high-purity products with low polydispersity (weight-average molecular weight (Mw) / number-average molecular weight (Mn)) are required to prevent deviations in drug blood retention, immunogenicity, and efficacy. Furthermore, during the synthesis of activated polyethylene glycol derivatives, a high conversion rate to reactive functional groups is preferred to prevent byproducts such as PEG-based impurities and PEGylated formulations with different bonds.

[0005] By reacting polyethylene glycol compound raw materials with hydroxyl, amino, etc. at the ends with an activator that is a low molecular weight compound, activated polyethylene glycol derivatives with functional groups or precursors at the ends that can react with biologically related substances are obtained.

[0006] However, some activators are hydrolyzable and react sensitively with water in the reaction system, leading to deactivation. When the activator is deactivated, the purity of the activated polyethylene glycol derivative decreases, and when an excess of activator is added as expected for deactivation, the amount of impurities originating from the activator also increases. This increases the load on the purification step, making it necessary to minimize the amount of activator.

[0007] Polyethylene glycol (PEG) compounds with terminal carboxyl, thiol, hydroxyl, or amino groups exhibit a wide variety of properties, including hard wax forms, flakes obtained from coarse crushing, and granules. PEG compounds with terminal hydroxyl groups are obtained through anionic polymerization of active hydrogen compounds and ethylene oxide. The polymerization is carried out under virtually anhydrous conditions, but the compound absorbs moisture when removed from the thermostatic reactor in liquid form after polymerization and cooled and solidified in an atmospheric environment. Furthermore, PEG compounds after some reactions or purification can be granulated by solvent reprecipitation. The surface area of ​​granules is larger than that of waxy or flake solids, and therefore they are highly hygroscopic; even when dried during production, the moisture content may increase when handled in an atmospheric environment.

[0008] For the reasons mentioned above, polyethylene glycol powder typically contains 0.1% to 2% moisture before the activation reaction. Since this level affects the deactivation of the activator during the activation reaction, it is necessary to reduce the moisture content in the system by performing some drying steps before the reaction.

[0009] Patent document 1 describes mixing a polyethylene glycol compound with a solvent capable of forming an azeotropic mixture with water to obtain a mixture, subsequently drying the mixture by azeotropic distillation, and then activating the polyethylene glycol compound in the solution.

[0010] Existing technical documents

[0011] Patent documents

[0012] Patent Document 1: JP2013-227543A Summary of the Invention

[0013] The problem to be solved by the present invention

[0014] However, as described in Patent Document 1, in the method for producing activated polyethylene glycol derivatives by drying the polyethylene glycol compound in solution via azeotropic distillation and subsequently activating it, the quality of the activated polyethylene glycol derivative may decrease. This is because the continuous heating of the polyethylene glycol compound solution as a raw material at high temperatures leads to an increased thermal path and an increase in polydispersity due to the oxidative decomposition of the PEG chains. Generally, with larger production scales, the heating and cooling times are longer, making the increase in polydispersity due to the increased thermal path appear more significant.

[0015] As mentioned above, although activated polyethylene glycol derivatives are important materials in pharmaceutical applications, they are not easily obtained.

[0016] One object of the present invention is to provide a method for producing activated polyethylene glycol derivatives with high activation purity, which can prevent the increase in polydispersity due to the increase in thermal process, and is industrially feasible.

[0017] Problem Solving Methods

[0018] As a result of in-depth research to achieve the above objectives, the inventors have discovered that by using polyethylene glycol compounds with specific properties as raw materials and performing an activation reaction after a specific drying step, it is possible to obtain activated polyethylene glycol derivatives with high activation purity and prevent quality degradation.

[0019] That is, the present invention is as follows.

[0020] [1] A method for producing an activated polyethylene glycol derivative, said activated polyethylene glycol derivative having at its terminal a functional group or a precursor of a functional group capable of reacting with a biologically relevant substance, said method comprising the following steps (A), (B) and (C):

[0021] Step (A): Drying polyethylene glycol compound powder in an atmosphere above 0°C and below 50°C to obtain dried powder with a moisture content of less than 0.10% by mass, wherein the polyethylene glycol compound has one or more groups selected from the group consisting of carboxyl, mercapto, hydroxyl and amino groups at its end;

[0022] Step (B): Add an organic solvent with a water content not exceeding 200 ppm to the dried powder obtained in step (A) to dissolve the dried powder in the organic solvent under a closed environment to obtain a solution; and

[0023] Step (C): The step of reacting the polyethylene glycol compound contained in the solution obtained in step (B) with an activator to obtain an activated polyethylene glycol derivative.

[0024] [2] The method for producing activated polyethylene glycol derivatives according to [1], wherein the organic solvent is an aprotic solvent.

[0025] [3] The method for producing activated polyethylene glycol derivatives according to [1] or [2], wherein the average particle size of the powder particles in step (A) is 0.1 μm or more and 10 mm or less.

[0026] [4] The method for producing activated polyethylene glycol derivatives according to [1] or [2], wherein the number average molecular weight of the polyethylene glycol compound in step (A) is above 2,000 Daltons and below 80,000 Daltons.

[0027] [5] The method for producing polyethylene glycol derivatives according to [1] or [2], wherein the activated polyethylene glycol derivative is represented by the following formula (1):

[0028] PEG-X···(1)

[0029] (in the above formula (1),

[0030] PEG represents a polyethylene glycol moiety having a linear or branched structure, and

[0031] X represents a functional group or a precursor of a functional group that can react with biologically relevant substances.

[0032] [6] A method for producing polyethylene glycol derivatives according to [1] or [2], wherein the activator is a compound selected from the group consisting of disuccinimide carbonate, p-nitrophenyl chloroformate, bis(1-benzotriazole) carbonate, trichlorophenyl chloroformate, p-nitrophenyl succinimide carbonate, p-nitrophenyl 1-benzotriazole carbonate, pentafluorophenyl chloroformate, 1,1'-carbonyldiimidazole, succinic anhydride, glutaric anhydride, halomethanesulfonyl, halotrifluoromethanesulfonyl, halotoluenesulfonyl, alkyl 6-halohexanoate, azodicarbonate, N,N'-dicyclohexylcarbodiimide and N-hydroxysuccinimide.

[0033] Invention Effects

[0034] According to the present invention, it is possible to achieve the effects of improving activation purity, preventing increased polydispersity due to increased thermal history, and producing industrially feasible activated polyethylene glycol derivatives. Attached Figure Description

[0035] Figure 1 This is a graph showing the relationship between temperature, relative humidity, and the rate of change of the mass of the measured sample during the moisture adsorption / desorption measurement in Experimental Example 1. Detailed Implementation

[0036] [Activated polyethylene glycol derivatives]

[0037] The activated polyethylene glycol derivatives according to the present invention have a functional group at the end that is capable of reacting with biologically relevant substances or a precursor of the above functional group.

[0038] In a preferred embodiment, the activated polyethylene glycol derivative has the following formula (2).

[0039] PEG-X···(2)

[0040] In formula (2), PEG represents a polyethylene glycol moiety having a straight-chain or branched structure, and X represents a functional group capable of reacting with biologically relevant substances or a precursor of the aforementioned functional group.

[0041] More specifically, an activated polyethylene glycol derivative represented by formula (3) can be exemplified.

[0042] [Chemical Formula 1]

[0043]

[0044] Here, Z is a residue obtained by removing active hydrogen groups from a compound having 2 to 8 active hydrogen groups (-GH) (Z(GH)n: n = 2 to 8). An active hydrogen group is a functional group having active hydrogen. Examples of active hydrogen groups (-GH) include hydroxyl, carboxyl, amino, secondary amino, and thiol groups. When the active hydrogen group (-GH) is hydroxyl or carboxyl, residue Z is a dehydroxylated residue; and when the active hydrogen group (-GH) is amino, secondary amino, or thiol, residue Z is a dehydrogenated residue.

[0045] Specific examples of compounds having 2 to 8 active hydrogen groups (-GH) (Z(GH)n: n = 2 to 8) include: polyols such as ethylene glycol, propylene glycol, trimethylene glycol, isopropylene glycol, butanediol, tetramethylene glycol, trimethylolpropane, glycerol, diglycerol, triglycerol, hexaglycerol, pentaerythritol, dipentaerythritol, and xylitol; amino acids or peptides having amino, carboxyl, or thiol groups, such as lysine and glutamic acid; or compounds such as organic amines and organic carboxylic acids.

[0046] a, b, c, and d are integers that satisfy "0≤a≤8, 0≤b≤8, 0≤c≤7, 0≤d≤7" and "1≤a+b≤8, 2≤a+b+c+d≤8".

[0047] Y1, Y2, Y3, and Y4 each independently represent an ether bond, amide bond, ester bond, carbamate bond, carbonate bond, secondary amino bond, thioether bond, disulfide bond, thioester bond, or an alkylene group that may contain the above groups. Preferred examples of alkylene groups include methylene, ethylene, propylene, isopropylene, butylene, isobutylene, pentylene, isopentylene, and hexylene, which may be branched.

[0048] Polymer1 and Polymer2 are straight or branched polyethylene glycol chains. A branched polyethylene glycol chain is a polyethylene glycol chain that branches into two or more chains via an intermediate linking group and may have multiple branch points. An example is a polyethylene glycol chain with two or more branches and a polyol such as glycerol as a branch point, as shown in formula (i) below.

[0049] [Chemical Formula 2]

[0050]

[0051] (Here, m1, m2, and m3 are integers, preferably 1 to 3636, more preferably 1 to 2728, and even more preferably 1 to 1818.)

[0052] The PEG in this invention is a polymer with a polydispersity of preferably 1.2 or less, more preferably 1.1 or less, and most preferably 1.07 or less, as measured by gel permeation chromatography (GPC).

[0053] The number-average molecular weight of the PEG portion in equation (2) is a value measured by GPC analysis and is calculated based on a calibration curve made using PEGs with various known molecular weights. There are no particular limitations on the number-average molecular weight of the PEG portion, but it is preferably 2,000 Daltons or more and 80,000 Daltons or less, more preferably 2,000 Daltons or more and 60,000 Daltons or less, and even more preferably 2,000 Daltons or more and 50,000 Daltons or less.

[0054] In formula (3), A1 and A2 each independently represent -L1-(CH2)l1-, -L1-(CH2)l1-L2-(CH2)l2 or a single bond, L1 represents an ether bond, amide bond, carbamate bond, secondary amino bond or a single bond, L2 represents an ether bond, amide bond, carbamate bond, single bond or thioether bond, and l1 and l2 each independently represent an integer from 0 to 5.

[0055] X1 and X2 are each independently a functional group capable of reacting with biologically relevant substances or a precursor of such functional group. The number of such functional groups or precursors of such functional groups in the activated polyethylene glycol derivative is not limited to one, and may be two or more.

[0056] R1 and R2 are each independently a structure that does not react with the activator described above in the reaction of this invention. This structure is not particularly limited and may have functional groups capable of reacting with biologically relevant substances, precursors of the aforementioned functional groups, or hydrocarbons. Examples of such structures are illustrated below.

[0057] Alkynyl, substituted alkynyl (e.g., alkynyl substituted with a hydrocarbon group having 1 to 5 carbon atoms), alkenyl (e.g., allyl or vinyl), alkyl, ester, carbonate (e.g., succinimide carbonate or p-nitrophenyl carbonate), formyl, isocyanate, isothiocyanate, epoxy, carboxyl, maleimide, substituted maleimide, dithiopyridyl, substituted sulfonyl (e.g., methanesulfonyl or toluenesulfonyl), vinylsulfonyl, iodoacetamide, alkylcarbonyl, azide, acryloyl, sulfonyloxy (e.g., alkylsulfonyloxy), and α-haloacetyl.

[0058] In this invention, examples of functional groups or precursors of such functional groups that are capable of reacting with biologically relevant substances and are contained in activated polyethylene glycol derivatives are given below.

[0059] Epoxy group, p-nitrophenyl ester group, N-hydroxysuccinimide ester group, mercapto group, maleimide group, substituted maleimide group, allyl group, azide group, biotin group, formyl group, amino group, methanesulfonyl group, toluenesulfonyl group, phthalimide group, carboxyl group, acetal-protected formyl group, 9-fluorenylmethoxycarbonyl-protected amino group, tert-butoxycarbonyl-protected amino group, and benzyloxycarbonyl-protected amino group.

[0060] The production method according to the present invention includes steps (A), (B), and (C). Each step will be described in sequence below.

[0061] Step (A): Drying polyethylene glycol compound powder in an atmosphere above 0°C and below 50°C to obtain dried powder with a moisture content of 0.10% by mass or less, wherein the polyethylene glycol compound has one or more terminal groups selected from the group consisting of carboxyl, mercapto, hydroxyl, and amino groups.

[0062] The raw material in step (A) is a polyethylene glycol compound having one or more groups selected from the group consisting of carboxyl, thiol, hydroxyl, and amino at the end. The polyethylene glycol compound is preferably PEG having the structure represented by formula (4).

[0063] [Chemical Formula 3]

[0064]

[0065] In equation (4), W1 and W2 independently represent carboxyl, mercapto, hydroxyl, and amino groups, respectively. Other symbols have the same definitions as in equation (2).

[0066] The polyethylene glycol compound is in the form of dried powder particles. There are no particular limitations on the drying method, examples of which include vacuum drying and drying using circulating drying gas. The average particle size of the powder particles is preferably 0.1 μm or more and 10 mm or less, and more preferably 0.1 μm or more and 1 mm or less, a value measured by sieving analysis.

[0067] The container used in the method according to the invention is a container capable of drying polyethylene glycol compound powder and capable of adding solvent to dried polyethylene glycol derivative powder without contact with the atmosphere. Here, "without contact with the atmosphere" means preventing contact with the atmosphere by sealing the container or filling the container with an inert gas to create a positive pressure in the container with an inert gas, or by housing the container in a sealed device and filling the device with an inert gas or filling the device with an inert gas to create a positive pressure in the device with an inert gas.

[0068] The moisture content of the dried powder particles is preferably below 0.10% by mass.

[0069] When drying powder particles by reduced pressure drying, the pressure is preferably -1.0 MPaG or more and -0.01 MPaG or less, and more preferably -1.0 MPaG or more and -0.1 MPaG or less.

[0070] There are no particular restrictions on the atmosphere during vacuum drying, but an inert gas, such as nitrogen or argon, is preferred.

[0071] When using a drying gas cycle to dry powder particles, there are no particular restrictions on the type of gas, but inert gases such as nitrogen or argon are preferred.

[0072] There are no particular limitations on the temperature during the drying of the powder particles, and any temperature equal to or lower than the melting point of the polyethylene glycol compound powder particles can be applied. The temperature is preferably above 0°C and below 50°C, and more preferably above 15°C and below 35°C.

[0073] Step (B): Add an organic solvent with a water content of less than 200 ppm to the dried powder obtained in step (A) to dissolve the dried powder in the organic solvent to obtain a solution.

[0074] There are no particular limitations on the method of adding an organic solvent with a water content of less than 200 ppm to dry powder particles and dissolving the powder particles to obtain a solution. The organic solvent can be added to the container without exposing the polyethylene glycol derivative powder particles to outside air. Examples of methods for adding the organic solvent include pump transfer, pressurized transfer, and depressurized transfer.

[0075] The water content in the organic solvent used in steps (B) and (C) is less than 200 ppm, preferably less than 150 ppm, and more preferably less than 100 ppm. There is no particular limitation on the lower limit of the water content, and it can be 0 ppm.

[0076] The organic solvents used in this invention are preferably aprotic solvents that do not react with the activator. Examples of solvents include toluene, benzene, ethyl acetate, dimethylformamide, dimethyl sulfoxide, diethyl ether, methyl tert-butyl ether, tetrahydrofuran, acetonitrile, acetone, chloroform, and dichloromethane. Essential conditions for the solvent in this invention are that the PEG dissolves, the reaction proceeds, the activator is not deactivated, and the functional groups do not decompose after the reaction. From the viewpoint that the activator or the functional groups after the reaction are likely to decompose, lower alcohols such as methanol or ethanol, which are commonly used as reaction solvents, are not preferred.

[0077] Step (C): Reacting the polyethylene glycol compound contained in the solution obtained in step (B) with an activator to obtain an activated polyethylene glycol derivative.

[0078] An activator reacts with one or more groups selected from the group consisting of carboxyl, thiol, hydroxyl, and amino groups in a polyethylene glycol compound to provide an activated polyethylene glycol derivative having a functional group at its terminal that is capable of reacting with biologically relevant substances or a precursor of such functional group.

[0079] Preferred examples of activators include the following.

[0080] One or more compounds selected from the group consisting of disuccinimide carbonate, p-nitrophenyl chloroformate, bis(1-benzotriazolyl) carbonate, trichlorophenyl chloroformate, p-nitrophenyl succinimide carbonate, p-nitrophenyl 1-benzotriazolyl carbonate, pentafluorophenyl chloroformate, 1,1'-carbonyldiimidazole, succinic anhydride, glutaric anhydride, halomethanesulfonyl, halotrifluoromethanesulfonyl, halotoluenesulfonyl, alkyl 6-halohexanoate, azodicarbonate, N,N'-dicyclohexylcarbodiimide, and N-hydroxysuccinimide.

[0081] In the reaction of step (C), a catalyst such as a base can be used as a reaction promoter, and the base can be an inorganic base or an organic base.

[0082] Examples of the base include salts of alkali metals and alkaline earth metals or amine compounds, such as hydroxides, carbonates, bicarbonates, acetates, citrates, phosphates, borates, citrates, phthalates, tartrates or lactates. Exemplary salts include sodium carbonate, sodium bicarbonate and sodium acetate. As amine compounds, primary amines and secondary amines may react with the activator or the functional group after the reaction, and thus tertiary amines are more preferred. Examples of the base as a tertiary amine include triethylamine, N-methylmorpholine, N-phenylmorpholine, N,N-diisopropylethylamine, pyridine, 2,6-dimethylpyridine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane and diazabicycloundecene. In the case of using a salt, since the salt may contain moisture, the salt can be added together with the powder of the polyethylene glycol compound in step (A) and dried.

[0083] Examples

[0084] Hereinafter, the present invention will be described in more detail with reference to examples.

[0085] The GPC polydispersity of the polyethylene glycol derivative was measured by the following analysis method A. The activation rate of the polyethylene glycol derivative was measured by the following analysis method B. The moisture content of the polyethylene glycol derivative was measured by the following analysis method C.

[0086] <Analysis method for GPC polydispersity>

[0087] (Analysis method A)

[0088] GPC system: Prominence manufactured by Shimadzu Corporation

[0089] Detector: Differential refractometer RID-20A manufactured by Shimadzu Corporation

[0090] GPC column: Two PL gel MIXED-D connected in series manufactured by Agilent Technologies

[0091] Column oven temperature: 65 °C

[0092] Eluent: N,N-dimethylformamide containing 10 mmol / L lithium bromide

[0093] Flow rate: 0.7 mL / min

[0094] Sample concentration: 1 mg / mL

[0095] Injection volume: 0.1 mL

[0096] Standards for calibration curves: GPC polymer standards of PEG with molecular weights from 600 to 70,000, manufactured by Agilent Technologies.

[0097] Data Analysis: Lab Solution manufactured by Shimadzu Corporation

[0098] <Methods for analyzing activation rate>

[0099] Activation rate was analyzed by proton nuclear magnetic resonance (1H-NMR).

[0100] (Analysis Method B)

[0101] 1H-NMR apparatus: JNM-ECA600 manufactured by JEOL.

[0102] Temperature measured: 25℃

[0103] Measurement solvent: deuterated chloroform manufactured by KANTO KAGAKU

[0104] Sample concentration: 25 mg / mL

[0105] Measurement of nuclei: 1H

[0106] Total number of times: 64 to 256

[0107] Data Analysis: ALICE2 manufactured by JEOL

[0108] <Methods for analyzing moisture levels>

[0109] The moisture level was measured using a Karl Fischer hygrometer.

[0110] (Analysis Method C)

[0111] Karl Fischer hygrometer: MKC-501 manufactured by KYOTO ELECTRONICS MANUFACTURING CO.,LTD.

[0112] Karl Fischer reagents: Honeywell-manufactured HYDRANAL COULOMAT AG (anode) and Honeywell-manufactured HYDRANAL COULOMAT CG (cathode).

[0113] Temperature measured: 25℃

[0114] Measurement quantity: 1g

[0115] <Experimental Example 1>

[0116] The following study was conducted to examine the hygroscopicity of polyethylene glycol compound powder particles.

[0117] Moisture adsorption / desorption measurements were performed on powder particles (Mw: 2000 Daltons, reactive functional group: one hydroxyl group, 27.8 mg) of the raw material (polyethylene glycol compound) represented by formula (5) using an IGA sorp manufactured by Hiden Isochema. As a pretreatment, the polyethylene glycol compound powder particles were placed in an SUS mesh and dried at 25°C and 0% RH for 5 hours to obtain dried powder particles.

[0118] The dried powder (27.46 mg) was allowed to stand for 1 hour in an environment controlled at 25°C and any relative humidity, and the mass after standing was measured. The measurement results are shown in Table 1 and... Figure 1 middle.

[0119] The results revealed that the dried polyethylene glycol powder absorbs moisture and increases in mass with increasing relative humidity. Furthermore, it was found that the polyethylene glycol powder absorbs moisture immediately upon increasing relative humidity and is strongly influenced by ambient temperature and humidity.

[0120] Therefore, when polyethylene glycol powder reacts with an activator to obtain a target activated polyethylene glycol derivative, the reaction process may be hindered when polyethylene glycol powder with absorbed moisture is used as a raw material.

[0121] [Chemical Formula 4]

[0122] H——(OCH2CH2) n ——OCH3 (5)

[0123] (n = approximately 45)

[0124] [Table 1]

[0125] Relative humidity (%RH) Rate of change in mass (mass%) (initial) 0 10 0.08 20 0.18 30 0.31 40 0.47 50 0.67 60 0.96 70 1.56 80 5.27 85 16.38

[0126] <Experimental Example 2>

[0127] To examine the mass change of polyethylene glycol (PEG) compounds while performing azeotropic distillation to dry the granular PEG compounds, the following study was conducted.

[0128] A 300 mL four-necked round-bottom flask equipped with a mechanical stirrer, a Dimroth cooling tube, a moisture metering receiver, a thermometer, and a nitrogen injection tube was filled with a powder of polyethylene glycol compound represented by formula (6) (Mw: 40,000 Daltons, reactive functional groups: two hydroxyl groups, 10 g, moisture content: 1.1% by mass) and toluene (70 g, moisture content: 68 ppm), and dissolved in a jacketed heater at 50 °C while stirring under nitrogen. Azeotropic distillation was then performed at 115 °C using a jacketed heater. Table 2 shows the azeotropic distillation time and the GPC polydispersity (Mp) in Analytical Method A. w The relationship between / Mn).

[0129] The results revealed that the polyethylene glycol compound deteriorated and its polydispersity improved with increasing azeotropic distillation time. It was found that, when drying was carried out via azeotropic distillation, as production scale increased, the heating time required to reach the temperature suitable for azeotropic distillation was prolonged, thereby promoting the deterioration of the polyethylene glycol compound and improving its polydispersity.

[0130] [Chemical Formula 5]

[0131] H——(OCH2CH2) n —OH formula (6)

[0132] (n = approximately 909)

[0133] [Table 2]

[0134] Azeotropic distillation time (hours) GPC polydispersity (Mw / Mn) (initial) 1.11 1 1.11 3 1.12 6 1.13 9 1.15 12 1.16 18 1.19 24 1.25

[0135] <Example 1>

[0136] Powdered granules of polyethylene glycol compound (Mw: 40,000, reactive functional groups: two hydroxyl groups, 10 g, 250 μmol, moisture content: 1.1 wt%, GPC polydispersity: 1.11) represented by formula (6) were loaded into a 300 mL four-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, a three-way stopcock connected to a nitrogen line, and a glass stopper. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 3 hours to obtain dried powdered granules with a moisture content of 0.01 wt% (step (A)).

[0137] After step (A), the pressure was restored with nitrogen, and toluene (70 g, moisture value: 129 ppm) was injected using a glass syringe and needle by keeping the dried powder particles in a nitrogen atmosphere without contact with the outside air. The solution was then heated to 40°C and dissolved by stirring (step (B)).

[0138] Triethylamine (TEA, 58 mg, 1150 mmol) as a base catalyst and methanesulfonyl chloride (57 mg, 1000 mmol) as an activator were added to the resulting solution, and the mixture was then reacted under nitrogen at 40 °C for 3 hours. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the polyethylene glycol derivative represented by formula (7) (step (C)).

[0139] The polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 256 measurements, and an activation rate of 85% was found. GPC measurements were performed, and the polydispersity was found to be 1.10. Moisture content was measured, and the moisture content was found to be 0.2% by mass.

[0140] [Chemical Formula 6]

[0141]

[0142] <Example 2>

[0143] Powdered granules of polyethylene glycol compound (Mw: 43,000 Daltons, reactive functional group: one hydroxyl group, 10 g, 233 μmol, moisture value: 0.1% by mass, GPC polydispersity: 1.04) represented by formula (8) were loaded into a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 1 hour to obtain dried powdered granules with a moisture value of 0.02% by mass (step (A)).

[0144] After step (A), the pressure is restored with nitrogen, and dichloromethane (70 g, moisture value: 42 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then dissolved by stirring at 25°C to obtain a solution (step (B)).

[0145] Disuccinimidyl carbonate (DSC, 918 mg, 3584 μmol) as an activator and pyridine (434 μL, 5233 μmol) as a base catalyst were added to the solution obtained in step (B), and the mixture was then reacted under nitrogen at 27 °C for 15 hours. Ethyl acetate (500 g) was added to the reacted solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the activated polyethylene glycol derivative represented by formula (9) (step (C)).

[0146] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 256 measurements, and the activation rate was found to be 98%. GPC measurements were performed, and the polydispersity was found to be 1.04. Moisture content was measured, and the moisture content was found to be 0.3% by mass.

[0147] [Chemical Formula 7]

[0148]

[0149] [Chemical Formula 8]

[0150]

[0151] <Example 3>

[0152] A 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line was filled with a powder of polyethylene glycol compound represented by formula (5) (Mw: 2,000 Daltons, reactive functional group: one hydroxyl group, 5 g, 2.5 mmol, moisture value: 0.5% by mass, GPC polydispersity: 1.06) and sodium acetate (15 mg) as an alkaline catalyst. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 1 hour to obtain a dried powder with a moisture value of 0.08% by mass (step (A)).

[0153] After step (A), the pressure is restored with nitrogen, and toluene (5 g, moisture value: 67 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then heated to 40°C and dissolved by stirring to obtain a solution (step (B)).

[0154] Succinic anhydride (263 mg, 2.63 mmol) was added as an activator to the resulting solution, and the mixture was then reacted under nitrogen at 70 °C for 12 hours. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the activated polyethylene glycol derivative represented by formula (10) (step (C)).

[0155] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 64 measurements, and the activation rate was found to be 91%. GPC measurements were performed, and the polydispersity was found to be 1.06. Moisture content was measured, and the moisture content was found to be 0.3% by mass.

[0156] [Chemical Formula 9]

[0157]

[0158] <Example 4>

[0159] Powdered granules of polyethylene glycol compound (Mw: 10,000 Daltons, reactive functional groups: four amino groups, 5 g, 500 μmol, moisture content: 0.5% by mass, GPC polydispersity: 1.06) and sodium acetate (15 mg) as an alkaline catalyst were added to a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 2 hours to obtain dried powdered granules with a moisture content of 0.07% by mass (step (A)).

[0160] After step (A), the pressure is restored with nitrogen, and toluene (5 g, moisture value: 92 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then heated to 40°C and dissolved by stirring to obtain a solution (step (B)).

[0161] Succinic anhydride (210 mg, 525 μmol) was added as an activator to the solution in step (B), and the mixture was then reacted at 70 °C for 12 hours under nitrogen. Ethyl acetate (500 g) was added to the reacted solution and mixed at 40 °C. Hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the activated polyethylene glycol derivative represented by formula (12) (step (C)).

[0162] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 128 measurements, and the activation rate was found to be 95%. GPC measurements were performed, and the polydispersity was found to be 1.05. Moisture content was measured, and the moisture content was found to be 0.5% by mass.

[0163] [Chemical Formula 10]

[0164]

[0165] [Chemical Formula 11]

[0166]

[0167] <Example 5>

[0168] The polyethylene glycol compound represented by formula (5) (Mw: 2,000 Daltons, reactive functional group: one hydroxyl group, 1 g, 500 μmol, moisture value: 0.5% by mass, GPC polydispersity: 1.03) powder was packed into a 20 mL screw cap glass bottle, which was then placed in a glove box and dried by stirring with a scraper for 1 hour while circulating drying nitrogen to obtain dried powder with a moisture value of 0.08% by mass (step (A)).

[0169] After step (A), place the stir bar into the screw-cap glass bottle in the glove box, add toluene (2g, moisture value: 62ppm) and chloroform (18g, moisture value: 68ppm), close the cap, then remove the screw-cap glass bottle from the glove box and stir the mixture with a magnetic stirrer at 25°C to obtain a solution (step (B)).

[0170] Phthalimide (129 mg, 875 μmol), triphenylphosphine (TPP, 230 mg, 875 μmol), and diisopropyl azodicarboxylate (DIAD, 152 mg, 750 mol) were added to the obtained solution, and the mixture was then reacted under nitrogen at 25 °C for 2 hours and 30 minutes. After the reaction, ethyl acetate (100 g) was added to the solution and mixed at 40 °C, and hexane (100 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (100 g) to the obtained crystals to dissolve at 40 °C and adding hexane (100 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the activated polyethylene glycol derivative represented by formula (13) (step (C)).

[0171] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 64 measurements, and the activation rate was found to be 88%. GPC measurements were performed, and the polydispersity was found to be 1.03. Moisture content was measured, and the moisture content was found to be 0.5% by mass.

[0172] [Chemical Formula 12]

[0173]

[0174] <Example 6>

[0175] Powdered granules of polyethylene glycol compound (Mw: 40,000 Daltons, reactive functional groups: two hydroxyl groups, 10 g, 250 μmol, moisture content: 1.1% by mass, GPC polydispersity: 1.11) represented by formula (6) were loaded into a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 1 hour to obtain dried powdered granules with a moisture content of 0.03% by mass (step (A)).

[0176] After step (A), the pressure is restored with nitrogen, and dichloromethane (70 g, moisture value: 51 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then dissolved by stirring at 25°C to obtain a solution (step (B)).

[0177] Disuccinimidyl carbonate (DSC, 643 mg, 2510 μmol) as an activator and pyridine (303 μL, 3750 μmol) as a base catalyst were added to the solution obtained in step (B), and the mixture was then reacted under nitrogen at 25 °C for 16 hours. Ethyl acetate (500 g) was added to the reacted solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the activated polyethylene glycol derivative represented by formula (14) (step (C)).

[0178] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 256 measurements, and the activation rate was found to be 100%. GPC measurements were performed, and the polydispersity was found to be 1.11. Moisture content was measured, and the moisture value was found to be 0.3% by mass.

[0179] [Chemical Formula 13]

[0180]

[0181] <Example 7>

[0182] Powdered granules of polyethylene glycol compound (Mw: 80,000 Daltons, reactive functional group: one hydroxyl group, 2 g, 25 μmol, moisture value: 1.0 wt%, GPC polydispersity: 1.06) represented by formula (15) were loaded into a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 3 hours to obtain dried powdered granules with a moisture value of 0.08 wt% (step (A)).

[0183] After step (A), the pressure is restored with nitrogen, and dichloromethane (10 g, moisture value: 106 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then dissolved by stirring at 25°C to obtain a solution (step (B)).

[0184] Disuccinimidyl carbonate (DSC, 96 mg, 375 μmol) as an activator and pyridine (46 μL, 569 μmol) as a base catalyst were added to the solution obtained in step (B), and the mixture was then reacted under nitrogen at 25 °C for hours. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the activated polyethylene glycol derivative represented by formula (16) (step (C)).

[0185] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 256 measurements, and the activation rate was found to be 100%. GPC measurements were performed, and the polydispersity was found to be 1.06. Moisture content was measured, and the moisture value was found to be 0.3% by mass.

[0186] [Chemical Formula 14]

[0187]

[0188] [Chemical Formula 15]

[0189]

[0190] <Example 8>

[0191] Powdered granules of polyethylene glycol compound (Mw: 40,000 Daltons, reactive functional groups: eight hydroxyl groups, 2 g, 50 μmol, moisture content: 0.8% by mass, GPC polydispersity: 1.07) represented by formula (17) were loaded into a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 3 hours to obtain dried powdered granules with a moisture content of 0.09% by mass (step (A)).

[0192] After step (A), the pressure is restored with nitrogen, and dichloromethane (10 g, moisture value: 104 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then dissolved by stirring at 25°C to obtain a solution (step (B)).

[0193] Disuccinimidyl carbonate (DSC, 206 mg, 804 μmol) as an activator and pyridine (97 μL, 1199 μmol) as a base catalyst were added to the solution obtained in step (B), and the mixture was then reacted under nitrogen at 25 °C for hours. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the activated polyethylene glycol derivative represented by formula (18) (step (C)).

[0194] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 256 measurements, and the activation rate was found to be 99%. GPC measurements were performed, and the polydispersity was found to be 1.07. Moisture content was measured, and the moisture content was found to be 0.3% by mass.

[0195] [Chemical Formula 16]

[0196]

[0197] [Chemical Formula 17]

[0198]

[0199] <Example 9>

[0200] A 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line was filled with a powder of polyethylene glycol compound represented by formula (5) (Mw: 2,000 Daltons, reactive functional group: one hydroxyl group, 10 g, 5.0 mmol, moisture value: 0.5% by mass, GPC polydispersity: 1.03) and sodium acetate (3 mg) as an alkaline catalyst. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 3 hours to obtain a dried powder with a moisture value of 0.03% by mass (step (A)).

[0201] After step (A), the pressure is restored with nitrogen, and 4-methyltetrahydropyran (MTHP, 10 g, moisture value: 94 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then heated to 40 °C and dissolved by stirring to obtain a solution (step (B)).

[0202] Succinic anhydride (528 mg, 5.28 mmol) was added as an activator to the resulting solution, and the mixture was then reacted under nitrogen at 70 °C for 12 hours. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, followed by vacuum drying to obtain the activated polyethylene glycol derivative represented by formula (10) (step (C)).

[0203] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 64 measurements, and the activation rate was found to be 85%. GPC measurements were performed, and the polydispersity was found to be 1.03. Moisture content was measured, and the moisture content was found to be 0.4% by mass.

[0204] <Example 10>

[0205] Powdered granules of polyethylene glycol compound represented by formula (19) (Mw: 5,000 Daltons, reactive functional group: one carbonyl group, 2 g, 400 μmol, moisture value: 0.9% by mass, GPC polydispersity: 1.03) were loaded into a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 3 hours to obtain dried powdered granules with a moisture value of 0.02% by mass (step (A)).

[0206] After step (A), the pressure is restored with nitrogen, and toluene (10 g, moisture value: 83 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then heated to 40°C and dissolved by stirring to obtain a solution (step (B)).

[0207] N,N'-dicyclohexylcarbodiimide (DCC, 165 mg, 800 μmol) and N-hydroxysuccinimide (NHS, 101 mg, 878 μmol) were added as activators to the resulting solution, and the mixture was then reacted under nitrogen at 40 °C for 3 hours. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the filtrate obtained by vacuum filtration to crystallize at 25 °C, followed by vacuum filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, and then dried under reduced pressure to obtain the activated polyethylene glycol derivative represented by formula (20) (step (C)).

[0208] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 128 measurements, and the activation rate was found to be 99%. GPC measurements were performed, and the polydispersity was found to be 1.03. Moisture content was measured, and the moisture content was found to be 0.2% by mass.

[0209] [Chemical Formula 18]

[0210]

[0211] [Chemical Formula 19]

[0212]

[0213] <Example 11>

[0214] Powdered granules of polyethylene glycol compound represented by formula (5) (Mw: 2,000 Daltons, reactive functional group: one hydroxyl group, 10 g, 5.0 mmol, moisture value: 0.5% by mass, GPC polydispersity: 1.03) were loaded into a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, an L-shaped tube connected to a vacuum pump, a check valve, and a three-way stopcock connected to a nitrogen line. The pressure was reduced to below -0.1 MPaG using a vacuum pump, and the result was dried for 3 hours to obtain dried powdered granules with a moisture value of 0.01% by mass (step (A)).

[0215] After step (A), the pressure is restored with nitrogen, and toluene (11 g, moisture value: 93 ppm) is injected into the dry powder particles using a glass syringe and needle under a nitrogen atmosphere without contact with the atmosphere. The powder particles are then heated to 40°C and dissolved by stirring to obtain a solution (step (B)).

[0216] The resulting solution was added with p-nitrobenzene chloroformate (1.11 g, 5.5 mmol) as an activator and triethylamine (TEA, 837 μL, 6.0 μmol) as a base catalyst, and the mixture was then reacted under nitrogen at 60 °C for 2 h. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, and then dried under reduced pressure to obtain the activated polyethylene glycol derivative represented by formula (21) (step (C)).

[0217] The activated polyethylene glycol derivative obtained in step (C) was subjected to 1H-NMR measurements, with a cumulative total of 64 measurements, and the activation rate was found to be 91%. GPC measurements were performed, and the polydispersity was found to be 1.03. Moisture content was measured, and the moisture content was found to be 0.3% by mass.

[0218] [Chemical Formula 20]

[0219]

[0220] To confirm the effect of moisture absorption by polyethylene glycol powder on the reaction, the following study was conducted.

[0221] <Comparative Example 1>

[0222] Powdered polyethylene glycol compound (Mw: 40,000 Daltons, reactive functional groups: two hydroxyl groups, 10 g, 250 μmol, moisture content: 1.1 wt%, GPC polydispersity: 1.11) represented by formula (6) was added to a 300 mL four-necked round-bottom flask equipped with a mechanical stirrer, Dimroth cooling tube, thermometer and nitrogen injection tube. Toluene (70 g, moisture content: 41 ppm) was added to the mixture, and then the mixture was heated to 40 °C in a water bath and dissolved by stirring to obtain a solution.

[0223] Triethylamine (TEA, 58 mg, 1150 μmol) and methanesulfonyl chloride (MsCl, 57 mg, 1000 μmol) were added to the resulting solution, and the mixture was then reacted under nitrogen at 40 °C for 3 hours. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, and then drying under reduced pressure to obtain the activated polyethylene glycol derivative represented by formula (7).

[0224] The activated polyethylene glycol derivative was subjected to 1H-NMR measurements, with a cumulative total of 256 measurements, revealing an activation rate as low as 25%. GPC measurements showed a polydispersity of 1.11. Moisture content was measured to be 0.2% by mass.

[0225] <Comparative Example 2>

[0226] Powdered polyethylene glycol compound (Mw: 40,000 Daltons, reactive functional groups: two hydroxyl groups, 10 g, 250 μmol, moisture content: 0.8% by mass, GPC polydispersity: 1.06) represented by formula (6) was added to a 100 mL three-necked round-bottom flask equipped with a mechanical stirrer, Dimroth cooling tube, thermometer and nitrogen injection tube. Dichloromethane (72 g, moisture content: 104 ppm) was added to the mixture, and the mixture was then dissolved by stirring at 25 °C to obtain a solution.

[0227] Disuccinimidyl carbonate (DSC, 643 mg, 2510 μmol) as an activator and pyridine (303 μL, 3750 μmol) as a base catalyst were added to the resulting solution, and the mixture was then reacted under nitrogen at 25 °C for 18 hours. After the reaction, ethyl acetate (500 g) was added to the solution and mixed at 40 °C, and hexane (500 g) was added to the solution to crystallize at 25 °C, followed by filtration to obtain crystals. The process of adding ethyl acetate (500 g) to the obtained crystals to dissolve at 40 °C and adding hexane (500 g) to crystallize at 25 °C was repeated to remove low molecular weight impurities, and then drying under reduced pressure to obtain the activated polyethylene glycol derivative represented by formula (14).

[0228] The activated polyethylene glycol derivative was subjected to 1H-NMR measurements, with a cumulative total of 256 measurements, revealing an activation rate as low as 61%. GPC measurements showed a polydispersity of 1.06. Moisture content was measured to be 0.3% by mass.

[0229] Table 3 shows the results for Example 1 and Comparative Example 1, as well as Example 6 and Comparative Example 2. As shown in Table 3, it was found that the activation rate of the activated polyethylene glycol derivative was significantly reduced in Comparative Examples 1 and 2.

[0230] [Table 3]

[0231]

[0232] Table 4 shows the results of Examples 1 to 11. As shown in Table 4, it was found that, according to the present invention, even under the reaction conditions, it is possible to produce polyethylene glycol derivatives with high activation purity without increasing polydispersity.

[0233] [Table 4]

[0234]

[0235] Industrial application

[0236] According to the present invention, it is possible to achieve the effects of improving activation purity, preventing increased polydispersity due to increased thermal history, and producing industrially feasible activated polyethylene glycol derivatives.

[0237] Although the invention has been described in detail with reference to specific embodiments thereof, it will be apparent to those skilled in the art that various changes or modifications can be made to the invention without departing from its spirit and scope.

[0238] This application is based on Japanese Patent Application No. 2023-051234, filed on March 28, 2023, the contents of which are incorporated herein by reference.

Claims

1. A method for producing an activated polyethylene glycol derivative, said activated polyethylene glycol derivative having at its terminal a functional group capable of reacting with a biorelevant substance or a precursor of said functional group, said method comprising the following steps (A), (B) and (C): Step (A): Drying polyethylene glycol compound powder in an atmosphere above 0°C and below 50°C to obtain dried powder with a moisture content of less than 0.10% by mass, wherein the polyethylene glycol compound has one or more groups at the end selected from the group consisting of carboxyl, mercapto, hydroxyl and amino groups. Step (B): Add an organic solvent with a water content of less than 200 ppm to the dried powder obtained in step (A) to dissolve the dried powder in the organic solvent to obtain a solution; and Step (C): The step of reacting the polyethylene glycol compound contained in the solution obtained in step (B) with an activator to obtain an activated polyethylene glycol derivative.

2. The method for producing activated polyethylene glycol derivatives according to claim 1, wherein, The organic solvent is an aprotic solvent.

3. The method for producing activated polyethylene glycol derivatives according to claim 1 or 2, wherein, The average particle size of the powder particles in step (A) is greater than 0.1 μm and less than 10 mm.

4. The method for producing activated polyethylene glycol derivatives according to claim 1 or 2, wherein, The number-average molecular weight of the polyethylene glycol compound in step (A) is above 2,000 Daltons and below 80,000 Daltons.

5. The method for producing polyethylene glycol derivatives according to claim 1 or 2, wherein the activated polyethylene glycol derivative is represented by the following formula (1): PEG-X…(1) In the above formula (1), PEG represents a polyethylene glycol moiety having a linear or branched structure, and X represents a functional group or a precursor of a functional group that can react with biologically relevant substances.

6. The method for producing polyethylene glycol derivatives according to claim 1 or 2, wherein, The activator is one or more compounds selected from the group consisting of disuccinimide carbonate, p-nitrophenyl chloroformate, bis(1-benzotriazolyl) carbonate, trichlorophenyl chloroformate, p-nitrophenyl succinimide carbonate, p-nitrophenyl 1-benzotriazolyl carbonate, pentafluorophenyl chloroformate, 1,1'-carbonyldiimidazole, succinic anhydride, glutaric anhydride, halomethanesulfonyl, halotrifluoromethanesulfonyl, halotoluenesulfonyl, alkyl 6-halohexanoate, azodicarbonate, N,N'-dicyclohexylcarbodiimide, and N-hydroxysuccinimide.

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