Activated branched polyethylene glycol derivative having active group at terminal and method for producing same

Abstract

This method for producing an activated branched polyethylene glycol derivative having an active group at a terminal has a step (a), a step (b), and a step (c). The step (a) is a reaction step in which a branched polyethylene glycol raw material and an activating agent are reacted to obtain an activated polyethylene glycol derivative, the step (b) is a purification step in which impurities in the activated polyethylene glycol derivative obtained in the reaction step (a) are removed, and the step (c) is a drying step in which the activated polyethylene glycol derivative obtained after the purification step (b) is dried using a dry gas to obtain an activated branched polyethylene glycol derivative having a moisture content of 0.001 mass% to 0.3 mass%.

Description

Activated branched polyethylene glycol derivative having an active group at the terminal and its method of production

[0001] The present invention relates to a branched activated polyethylene glycol derivative having an active group at its terminal and a method for producing the same.

[0002] In the pharmaceutical field, complexes formed by combining bioactive agents such as proteins with water-soluble compositions have improved efficacy compared to when bioactive agents are injected into the body, due to factors such as an increased half-life and the ability to evade the immune response system.

[0003] In addition, they are also effective in diagnostic materials and medical devices by stabilizing diagnostic results and evading the immune response system.

[0004] One example of a water-soluble composition is polyethylene glycol. Generally, bioactive agent-polyethylene glycol conjugates are formed by a reaction between the active agent and polyethylene glycol. To achieve this reaction, activated polyethylene glycol derivatives are used, in which the termini of polyethylene glycol are substituted with reactive functional groups. Examples of reactive functional groups include active groups that chemically bond with functional groups present on the surface of bioactive agents, such as amino groups, mercapto groups, carboxy groups, and unsaturated bonds. For example, when modifying amino groups, active groups such as formyl groups, epoxy groups, p-nitrophenyl ester groups, and N-hydroxysuccinimidyl groups are attached to the termini of the polyethylene glycol chain. For carboxy groups, active groups such as mercapto groups and amino groups are attached to the termini of the polyethylene glycol chain. For unsaturated bonds, active groups such as mercapto groups are attached to the termini of the polyethylene glycol chain.

[0005] Japanese Patent Application Publication No. 2013-227543

[0006] However, the inventors of the present invention have found that when an activated polyethylene glycol compound, which is a product of activation, is stored, the active group is deactivated and the terminal activation rate decreases. Specifically, in activated polyethylene glycol derivatives having an active ester, a carbonate ester, or the like at a terminal, hydrolysis of the active group at the terminal occurs during long-term storage, which can decrease the terminal activation rate.

[0007] An object of the present invention is to prevent the deactivation of the terminal active groups of a branched activated polyethylene glycol derivative during storage.

[0008] That is, the present invention is as follows: [1] A method for producing an activated branched polyethylene glycol derivative having an active group at its terminal, characterized by comprising the following steps (a), (b), and (c): Step (a): A reaction step of reacting a branched polyethylene glycol raw material with an activator to obtain an activated polyethylene glycol derivative; Step (b): A purification step of removing impurities from the activated polyethylene glycol derivative obtained in the reaction step (a); Step (c): A drying step of drying the activated polyethylene glycol derivative after the purification step (b) using a dry gas to obtain an activated branched polyethylene glycol derivative having a moisture content of 0.001% by mass or more and 0.3% by mass or less.

[0009] [2] The method for producing an activated branched polyethylene glycol derivative according to [1], wherein the dry gas in the step (c) is nitrogen, oxygen, carbon dioxide, argon, or a mixture thereof.

[0010] [3] A method for producing the activated branched polyethylene glycol derivative according to [1] or [2], wherein the activated branched polyethylene glycol derivative has a structure represented by the following formula (1) or (2):

[0011] (In formula (1), X is a branched skeleton containing at least a hydrocarbon group, PEG1 is a polyethylene glycol moiety, W is a hydrolyzable active group, A is a linker connecting the polyethylene glycol moiety PEG1 and the active group W, and m is an integer of 3 or more and 8 or less.) (In formula (2), X is a branched skeleton containing at least a hydrocarbon group; PEG1 is a polyethylene glycol moiety; W is a hydrolyzable active group; B is a linker connecting the branched skeleton X and the active group W; Y is a hydroxyl group or a protecting group for a hydroxyl group; and l and k are integers of 2 or more and 7 or less.)

[0012] [4] The method for producing an activated branched polyethylene glycol derivative according to [1] or [2], wherein the activated branched polyethylene glycol derivative has a number average molecular weight of 400 daltons or more and 100,000 daltons or less.

[0013] [5] The method for producing an activated branched polyethylene glycol derivative according to [1] or [2], wherein the activated branched polyethylene glycol derivative is a particulate solid, and the particulate solid has an average particle size of 0.1 μm or more and 10 mm or less.

[0014] [6] An activated branched polyethylene glycol derivative having an active group at its terminal, characterized in that it is a particulate solid having a moisture content of 0.001% by mass or more and 0.3% by mass or less, a terminal activation rate of 90% by mass or more, an average particle size of 0.1 μm or more and 10 mm or less, and a color difference of 0 or more and 10 or less.

[0015] The present inventors have investigated the deactivation of activated polyethylene glycol derivatives during storage and have come to the following findings.

[0016] That is, when an activated polyethylene glycol derivative is handled under atmospheric conditions, such as when it is stored in a can or divided into small portions, hydrolysis of the terminal active group occurs due to absorbed moisture, resulting in a deterioration in quality during storage. When activated polyethylene glycol derivatives exhibiting this phenomenon were investigated, it was found that in activated polyethylene glycol derivatives having a branched structure, deactivation of the terminal active group occurs during storage. In the case of linear activated polyethylene glycol derivatives, such deactivation of the terminal active group was not significant.

[0017] Upon investigating the reason for this, it was found that branched polyethylene glycol derivatives are prone to absorbing moisture during handling, which in turn makes the terminal hydrolyzable active groups prone to decomposition, resulting in a tendency for the terminal activation rate to decrease. Specifically, as shown in Figure 1, it was discovered that polyethylene glycol derivatives having a branched structure absorb significantly more moisture than polyethylene glycol derivatives having a linear structure under the same relative humidity environment.

[0018] Based on this finding, the present inventors have conceived of adjusting the moisture content to 0.001% by mass or more and 0.3% by mass or less by drying an activated branched polyethylene glycol using a dry gas after producing the activated branched polyethylene glycol. As a result, they have succeeded in suppressing deactivation of the terminal active groups during storage and obtaining an activated branched polyethylene glycol derivative of high purity that is industrially feasible, thereby arriving at the present invention.

[0019] 1 is a graph showing the amount of water adsorption (moisture value) at each relative humidity for monofunctional (linear), bifunctional (linear), and tetrafunctional (branched) polyethylene glycol (PEG) derivatives having active groups at their terminals. 2 is a graph showing the relationship between the rate of decrease in terminal activation rate of activated branched polyethylene glycol derivatives and storage period, showing the results for the cases where the drying step (c) was performed and the case where the drying step (c) was not performed.

[0020] [Activated Branched Polyethylene Glycol Derivative Having an Active Group at a Terminal] The activated branched polyethylene glycol derivative of the present invention is a particulate solid having a moisture content of 0.001% by mass or more and 0.3% by mass or less, a terminal activation rate of 90% by mass or more, an average particle size of 0.1 μm or more and 10 mm or less, and a color difference of 0 or more and 10 or less.

[0021] The present activated branched polyethylene glycol derivative is preferably represented by the following general formula (1) or (2):

[0022] (In formula (1), X represents a branched skeleton containing at least a hydrocarbon group; W represents a hydrolyzable active group; A represents a linker connecting the polyethylene glycol moiety PEG1 and the active group W; PEG1 represents a polyethylene glycol moiety; and m represents an integer of 3 or more and 8 or less.)

[0023] (In formula (2), X is a branched skeleton containing at least a hydrocarbon group; W is a hydrolyzable active group; B is a linker connecting the branched skeleton X and the active group W; Y is a hydroxyl group or a protecting group for a hydroxyl group; PEG1 is a polyethylene glycol moiety; and l and k are integers of 2 or more and 7 or less.)

[0024] Here, X is a branched skeleton containing at least a hydrocarbon group. Here, a branched skeleton means that X has multiple bonding moieties that bond to a polyethylene glycol chain or a linker. The number of bonding moieties of X is determined by the above-mentioned number m or the number l + k. Here, the hydrocarbon group of X may be an alkyl group or a hydrocarbon having an unsaturated double bond. Furthermore, the total number of carbon atoms in the hydrocarbon group of X is preferably 3 or more, more preferably 5 or more. Furthermore, the total number of carbon atoms in the hydrocarbon group of X is preferably 17 or less.

[0025] X may consist solely of a hydrocarbon group, or may have, in addition to a hydrocarbon group, one or more bonds selected from the group consisting of an amino bond, a urethane bond, an ether bond, an ester bond, and a carbonate bond.

[0026] Linker A may contain a hydrocarbon group and may have one or more bonds selected from the group consisting of an amide bond, a urea bond, a urethane bond, an ether bond, an ester bond, a carbonate bond, a thioether bond, and a secondary amine. The hydrocarbon group may or may not have an unsaturated bond. Linker A may not have a hydrocarbon group. The hydrocarbon group in linker A preferably has 1 to 10 carbon atoms, more preferably 1 to 6 carbon atoms.

[0027] Linker B may contain a hydrocarbon group and may contain one or more bonds selected from the group consisting of an amide bond, a urea bond, a urethane bond, an ether bond, an ester bond, a carbonate bond, a thioether bond, and a secondary amine. Furthermore, it may or may not contain a polyethylene glycol moiety. Here, the polyethylene glycol moiety contained in linker B is referred to as PEG2.

[0028] Here, W is a hydrolyzable active group. In a preferred embodiment, W is an active group such as an active ester or carbonate ester.

[0029] Exemplary structures of the activated ester include an N-hydroxysuccinimidyl (NHS) ester shown in formula (3), a thioester shown in formula (4), a sulfate ester shown in formula (5), and an aromatic ester shown in formula (6) in which any hydrogen atom on the aromatic ring is substituted with an electron-withdrawing group. In formula (4), R is a hydrocarbon group or any functional group with a hydrocarbon group as a linker; in formula (5), -O － Na + Salts with cations such as H + In formula (6), Q may be a bonded molecule. １ ~Q ５ is an electron-withdrawing group such as a hydrogen atom, a halogen atom, a nitro group, etc. Among formulas (3) to (6), formulas (3) and (6) are more preferred, and formula (3) is even more preferred.

[0030] Exemplary structures of carbonate esters include carbonate NHS shown in formula (7), thiocarbonate ester shown in formula (8), and carbonate phenyl ester shown in formula (9). R and Q in Equation (8) and Equation (9) １ ~Q ５ is the same as each symbol in formulas (4) to (6). Among formulas (7) to (9), formulas (7) and (9) are more preferred, and formula (7) is even more preferred.

[0031] Here, Y is a hydroxyl group or a protecting group for a hydroxyl group, and exemplary protecting groups for a hydroxyl group are an acetal group, an acetyl group, an isobutyryl group, a trialkylsilyl group, a trityl group, a benzyl group, a benzoyl group, a methoxy group, and a p-methoxybenzyl group.

[0032] The polyethylene glycol moiety of PEG1 and PEG2 contained in linker B means a moiety containing a polyethylene glycol chain. The polyethylene glycol chains of PEG1 and PEG2 have structures shown in formula (10) and formula (11), respectively. In formula (10), n is an integer. In formula (11), o is an integer. Here, n and o may be the same or different. The values ​​of n and o are determined by the values ​​of m and l+k described above and the number-average molecular weight of the activated branched polyethylene glycol derivative. The number-average molecular weight of the activated branched polyethylene glycol derivative of the present invention (particularly the activated polyethylene glycol derivatives of formulas (1) and (2)) depends on the polyethylene glycol moiety (particularly PEG1 and PEG2).

[0033] The number average molecular weight of the activated branched polyethylene glycol derivative is preferably 400 daltons or more and 100,000 daltons or less, and more preferably 1,000 daltons or more and 80,000 daltons or less.

[0034] The moisture content of the activated branched polyethylene glycol derivative is preferably 0.001% by mass or more and 0.3% by mass or less, and more preferably 0.001% by mass or more and 0.2% by mass or less, as measured by a Karl Fischer moisture meter.

[0035] The terminal activation rate of the activated branched polyethylene glycol derivative is preferably 90% by mass or more, and more preferably 93% by mass or more. Here, the terminal activation rate is measured by proton nuclear magnetic resonance (NMR).

[0036] The activated branched polyethylene glycol derivative is preferably a particulate solid. The particulate solid preferably has an average particle size of 0.1 μm or more and 10 mm or less, more preferably 0.1 μm or more and 1 mm or less. Here, the average particle size is measured using a Sato sieve, and the mass percentage of each particle size range is calculated and confirmed.

[0037] The color difference of the activated branched polyethylene glycol derivative is preferably 0 or more and 10 or less, and more preferably 0 or more and 5 or less. Here, the color difference is measured with a colorimeter.

[0038] [Method for Producing Activated Branched Polyethylene Glycol Derivatives Having Terminal Active Groups] The production method of the present invention comprises the following steps (a), (b), and (c). Each step will be described in turn below.

[0039] Step (a): A reaction step of obtaining an activated polyethylene glycol derivative by reacting a branched polyethylene glycol raw material with an activator.

[0040] The raw material in step (a) is a branched polyethylene glycol raw material having one or both of a hydroxyl group and a carboxyl group at its terminal. This branched polyethylene glycol raw material has the above-mentioned polyethylene glycol moiety and one or both of a hydroxyl group and a carboxyl group provided at its terminal. The branched polyethylene glycol raw material preferably has a structure as shown in formulas (12) and (13).

[0041]

[0042] In formulas (12) and (13), Z represents one or both of a hydroxyl group and a carboxyl group. The other symbols (X, Y, PEG1, A, B, m, l, and k) are the same as those in formulas (1) and (2).

[0043] The activator reacts with the hydroxyl group or carboxyl group of the branched polyethylene glycol raw material, and provides a hydrolyzable functional group at the end of the polyethylene glycol derivative that can react with a biologically relevant substance.

[0044] Suitable examples of hydrolyzable activators include chlorosulfuric acid, disuccinimidyl carbonate, thiol compounds, p-nitrophenol, p-nitrophenyl formate, and N-hydroxysuccinimide.

[0045] In addition to the activator, a low molecular weight compound such as a condensing agent or a base may be further added as a reaction accelerator.

[0046] The condensing agent may be a carbodiimide-based condensing agent, such as N,N-dicyclohexylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, or N,N-diisopropylcarbodiimide.

[0047] The base may be either an inorganic base or an organic base. Exemplary inorganic bases are salts such as sodium carbonate, sodium bicarbonate, and sodium acetate. Exemplary organic bases are triethylamine, N-methylmorpholine, N-phenylmorpholine, N,N-diisopropylethylamine, pyridine, 2,6-lutidine, 4-dimethylaminopyridine, 1,4-diazabicyclo[2.2.2]octane, and diazabicycloundecene.

[0048] Step (b): A purification step for removing impurities from the activated polyethylene glycol derivative obtained in the reaction step (a).

[0049] In step (b), any purification method can be used as long as it can remove impurities (low-molecular-weight impurities derived from activators and additives, and high-molecular-weight impurities generated by side reactions of polyethylene glycol compounds). Exemplary purification methods include crystallization, organic solvent washing using a two-layer system, aqueous solution washing using a two-layer system, adsorption treatment, and drying. Each purification method is described below.

[0050] The solvent used in the crystallization is preferably an organic solvent, more preferably an aprotic solvent, and most preferably acetonitrile, chloroform, ethyl acetate, dichloromethane, toluene, hexane, methyl tert-butyl ether, or 4-methyltetrahydropyran. A single solvent or a combination of two or more solvents may be used. The temperature during the crystallization is preferably 0°C or higher and 40°C or lower, more preferably 5°C or higher and 30°C or lower.

[0051] The solvents used in the organic solvent washing using a two-layer system must be a combination of organic solvents that are immiscible with each other, and are preferably aprotic solvents. The temperature during the organic washing may be any temperature up to the boiling point of the solvent, but is preferably 10°C or higher and 40°C or lower.

[0052] When washing with an aqueous solution using a two-layer system, the activated polyethylene glycol derivative is preferably dissolved in an aprotic solvent. The aqueous solution may be acidic, neutral, or basic. Examples of solutes in the aqueous solution include inorganic salts such as sodium chloride and sodium dihydrogen phosphate, water-soluble gases such as hydrogen chloride, organic bases such as ethylenediamine, and organic acids such as citric acid.

[0053] During adsorption, the activated polyethylene glycol derivative is preferably dissolved in a solvent, more preferably an aprotic solvent. Exemplary aprotic solvents include acetonitrile, chloroform, ethyl acetate, dichloromethane, toluene, and 4-methyltetrahydropyran. Examples of adsorbents include adsorbents of multiple metal solid solutions, silica-based adsorbents, and ion exchange resins.

[0054] After the above purification, drying under reduced pressure may be carried out.

[0055] Step (c): A drying step of drying the activated polyethylene glycol derivative after the purification step (b) using a dry gas to obtain an activated branched polyethylene glycol derivative having a moisture content of 0.001% by mass or more and 0.3% by mass or less.

[0056] Even when drying under reduced pressure is carried out in step (b), it is important in step (c) to blow a dry gas onto the activated branched polyethylene glycol derivative to reduce the moisture content, separately from drying under reduced pressure.

[0057] The dry gas used in step (c) is preferably nitrogen, oxygen, carbon dioxide, argon, or a mixture thereof, more preferably an inert gas such as nitrogen or argon. The temperature of the dry gas is preferably −15° C. or higher and 100° C. or lower, more preferably 0° C. or higher and 50° C. or lower, and even more preferably 15° C. or higher and 35° C. or lower.

[0058] In step (c), the moisture content is preferably 0.001% by mass or more and 0.3% by mass or less, and more preferably 0.001% by mass or more and 0.2% by mass or less.

[0059] The activated branched polyethylene glycol derivative having an active group at its terminal, shown in formula (6), obtained through steps (a), (b), and (c), showed a decrease in terminal activation rate of about 3% by mass when stored at 25°C for 3 months from immediately after production, as shown in Figure 2. On the other hand, the activated branched polyethylene glycol derivative obtained through steps (a) and (b) without performing step (c) showed a decrease in terminal activation rate of about 7% by mass when stored at 25°C for 3 months from immediately after production. Thus, it was found that performing step (c) inhibits the decrease in terminal activation rate of the activated branched polyethylene glycol derivative during storage, improving its stability.

[0060] The present invention will be explained in more detail below with reference to examples. The moisture content of activated branched polyethylene glycol derivatives was measured using analytical method A shown below. The terminal activation rate of activated branched polyethylene glycol derivatives was measured using analytical method B shown below. The average particle size of activated branched polyethylene glycol derivatives was measured using analytical method C shown below. The color difference of activated branched polyethylene glycol derivatives was measured using analytical method D shown below.

[0061] <Method for analyzing moisture value> The moisture value was measured using a Karl Fischer moisture meter. (Analysis method A) Karl Fischer moisture meter: MKC-501 manufactured by Kyoto Electronics Manufacturing Co., Ltd. Karl Fischer reagent: HYDRANAL COULOMAT AG (anode) manufactured by Honeywell, HYDRANAL COULOMAT CG (cathode) manufactured by Honeywell Measurement temperature: 20 to 25°C Measurement amount: 1 g

[0062] <Method for Analyzing Terminal Activation Rate> The terminal activation rate was analyzed by proton nuclear magnetic resonance (NMR). (Analysis Method B) NMR apparatus: JNM-ECA600 manufactured by JEOL Measurement temperature: 25°C Measurement solvent: deuterated chloroform manufactured by Kanto Chemical Co., Inc. Sample concentration: 20 to 30 mg / mL Measurement type: 1 H Number of accumulations: 64 to 1024 Data analysis: ALICE2 manufactured by JEOL

[0063] <Method for analyzing average particle size> The average particle size is a value calculated as a volume average based on the particle size distribution analyzed using a sieve shaker. (Analysis Method C) Sieve shaker: manufactured by Retsch Co. Test sieves: openings 200, 300, 450, 800, 1000 μm, diameter 10 cm

[0064] <Method for analyzing color difference> Color difference was analyzed using a colorimeter. (Analysis Method D) Colorimeter: ZE-2000 manufactured by Nippon Denshoku Industries Co., Ltd. Square cell: 10 x 36 x 45 mm Dissolving solution: sorbitol sodium acetate buffer solution (pH 4.0) Sample concentration: 1.0 g / mL

[0065] (Hygroscopicity of Linear and Branched Polyethylene Glycol Derivatives Having an Active Group at an End) The hygroscopicity of a tetrafunctional branched polyethylene glycol derivative (PEG) having a molecular weight of 10,000 and represented by formula (14), a bifunctional linear polyethylene glycol derivative (PEG) having a molecular weight of 10,000 and represented by formula (15), and a monofunctional linear polyethylene glycol derivative (PEG) having a molecular weight of 10,000 and represented by formula (16) was measured, and the results are shown in FIG. 1.

[0066] Specifically, each polyethylene glycol derivative was stored for 1 hour at room temperature of 20°C under each relative humidity (0, 10, 20, 30%) shown in Figure 1, and the amount of moisture increase after storage was measured. In Figure 1, the horizontal axis represents relative humidity, and the vertical axis represents the amount of moisture increase after storage.

[0067] As a result, it was discovered that the hygroscopicity of branched polyethylene glycol derivatives is significantly higher than that of linear polyethylene glycol derivatives. It is believed that this high hygroscopicity is the reason why the terminal activation rate of activated branched polyethylene glycol derivatives decreases significantly during storage.

[0068] Example 1 (Step (a)) Disuccinimidyl carbonate (Tokyo Chemical Industry Co., Ltd., 26 g: activator) and pyridine (Kanto Chemical Industry Co., Ltd., 12 g) were added to a dichloromethane (Kanto Chemical Industry Co., Ltd., 1050 g) solution of a branched polyethylene glycol raw material represented by formula (17) (molecular weight: 30,000, NOF Corp., 150 g) and stirred to obtain an activated branched polyethylene glycol derivative of formula (18).

[0069]

[0070]

[0071] Here, the compound of formula (18) belongs to formula (2), where Y is a protecting group for a hydroxyl group (methoxy group), and PEG1 is (CH ２ CH ２ O) ｎ where B is a linker containing PEG2, W is a succinimidyl carbonate group, and X is a branched skeleton consisting of a central hydrocarbon group and a urethane bond. Both k and l are 2.

[0072] (Purification step (b)) The reaction solution (dichloromethane solution) obtained in step (a) was filtered, diluted with dichloromethane (150 g), and washed four times with brine (437 g) containing 6N hydrochloric acid. The dichloromethane solution was then concentrated, and the dichloromethane was distilled off. Ethyl acetate (Kanto Chemical, 1500 g) was then added and dissolved, and magnesium sulfate (Kanto Chemical, 7.5 g) was added and dehydrated, after which the magnesium sulfate was removed by filtration. Hexane (Kanto Chemical, 900 g) was added to this solution to precipitate a solid, which was then filtered off. A mixed solution of ethyl acetate (1500 g) and hexane (600 g) was added to the recovered solid, and the mixture was stirred. The solid was then filtered off. This operation was repeated three times. Ethyl acetate (1350 g) was added to the resulting solid, and the solid was dissolved. Then, Kyoward 700 (registered trademark) (Kyowa Chemical Industry Co., Ltd., 30 g) was used for adsorption treatment, and Kyoward 700 (registered trademark) was removed by filtration. Hexane (600 g) was added and the slurry of the solid and hexane was stirred. The solid was separated by filtration and then dried under reduced pressure to obtain a particulate solid.

[0073] (Drying Step (c)) The particulate solid obtained in the purification step (b) was spread in a dry box under a nitrogen atmosphere and dried at 25°C for 2.5 hours by flowing dry nitrogen.

[0074] The particulate solid obtained above was analyzed, and the following measured values ​​were obtained: Moisture value: 0.03% Terminal activation rate: 97% by mass Average particle size: 869 μm Color difference: 4.8

[0075] Example 2 (Reaction step (a)) To a toluene solution of a branched polyethylene glycol raw material represented by formula (19) (molecular weight: 10,000, product of NOF Corp., 25 kg), N-hydrosuccinimide (manufactured by Midori Chemical Industry Co., Ltd., 3.5 kg: activator) and N,N-dicyclohexylcarbodiimide (manufactured by Osaka Organic Synthetic Chemical Industry Co., Ltd., 1.1 kg) were added, and the mixture was stirred at 40°C for 3 hours to obtain a polyethylene glycol derivative having an active group represented by formula (14).

[0076]

[0077]

[0078] Here, the compound of formula (14) belongs to formula (1), where PEG1 is (CH ２ CH ２ O) ｎ A is a propyl group and an ester bond (linker) adjacent to the propyl group, W is a succinimidyl ester, and X is a central hydrocarbon group C ５ H ８ It is a branched skeleton consisting of the following. m is 4.

[0079] (Purification step (b)) After filtering the reaction solution obtained in the reaction step (a), ethyl acetate (50 kg) and hexane (44 kg) were added to precipitate a solid, which was then filtered off. The obtained solid was dissolved in ethyl acetate (90 kg), and hexane (25 kg) was added to precipitate a solid, which was then filtered off. This operation was repeated three times, and the obtained solid was then dissolved in toluene (100 kg), and then added to hexane (150 kg) to precipitate a solid, which was then filtered off. Hexane (100 kg) was further added, and the slurry of the solid and hexane was stirred. The solid was then filtered off, and dried under reduced pressure to obtain a particulate solid.

[0080] (Drying Step (c)) The particulate solid obtained in the purification step (b) was spread in a dry box under a nitrogen atmosphere and dried at 25°C for 5 hours by flowing dry nitrogen.

[0081] The particulate solid obtained above was analyzed, and the following measured values ​​were obtained: Moisture value: 0.02% Terminal activation rate: 93% by mass Average particle size: 517 μm Color difference: 0.6

[0082] Example 3 (Reaction step (a)) N-hydrosuccinimide (1 g: activator) and N,N-dicyclohexylcarbodiimide (2 g) were added to a toluene solution of a branched polyethylene glycol raw material represented by formula (20) (molecular weight: 40,000, product of NOF Corp., 25 g), and the mixture was stirred at 40°C for 3 hours to obtain an activated branched polyethylene glycol derivative having an active group at its terminal, represented by formula (21).

[0083]

[0084]

[0085] Here, the compound of formula (21) belongs to formula (1), where PEG1 is (CH ２ CH ２ O) ｎ and A is CH ２ (linker), W is a succinimidyl ester, X is an atomic group consisting of a central hydrocarbon group and an ether bond (branched skeleton), and m is 8.

[0086] (Purification step (b)) The reaction solution obtained in the reaction step (a) was filtered, diluted with ethyl acetate (75 g), and hexane (75 g) was added to precipitate a solid, which was then filtered off. The resulting solid was then dissolved in ethyl acetate (200 g), and the solid was precipitated with hexane (75 g) and filtered off. This procedure was repeated five times. Hexane (100 g) was added to the solid, and the slurry of the solid and hexane was stirred. The solid was filtered off, and then dried under reduced pressure to obtain a particulate solid.

[0087] (Drying Step (c)) The particulate solid obtained in step (b) was spread in a dry box under a nitrogen atmosphere, and dried at 25° C. for 5 hours by flowing dry nitrogen.

[0088] The solid obtained above was analyzed, and the following measured values ​​were obtained: Moisture value: 0.03% Terminal activation rate: 93% by mass Average particle size: 570 μm Color difference: 1.6

[0089] Comparative Example 1 The reaction step (a) and the purification step (b) were carried out in the same manner as in Example 1 to obtain an activated branched polyethylene glycol derivative of formula (18). However, unlike Example 1, the drying step (c) was not carried out.

[0090] Comparative Example 2 The reaction step (a) and the purification step (b) were carried out in the same manner as in Example 2 to obtain an activated branched polyethylene glycol derivative of formula (14). However, unlike Example 2, the drying step (c) was not carried out.

[0091] Comparative Example 3 The reaction step (a) and purification step (b) were carried out in the same manner as in Example 3 to obtain an activated branched polyethylene glycol derivative of formula (21). However, unlike Example 3, the drying step (c) was not carried out. The measured values ​​of the physical properties of each of the obtained activated branched polyethylene glycol derivatives are shown in Tables 1 and 2.

[0092]

[0093]

[0094] Example 4 and Comparative Example 4 An activated branched polyethylene glycol derivative represented by formula (14) was produced, and its storage stability at 25°C was confirmed. However, in Example 4, an activated branched polyethylene glycol derivative represented by formula (14) having a moisture content of 0.15% was obtained by carrying out the reaction step (a), purification step (b), and drying step (c). The resulting activated branched polyethylene glycol derivative was stored at 25°C in an environment of 11.5% relative humidity for three months immediately after production, and the rate of decrease Δ in the terminal activation rate was measured. As a result, the rate of decrease was 1% by mass after one month, and 3% by mass after three months. The results are shown in Table 3 and FIG. 2.

[0095] In Comparative Example 4, the reaction step (a) and the purification step (b) were performed, but the drying step (c) was not performed. This resulted in an activated branched polyethylene glycol derivative of formula (14) having a moisture content of 0.32%. The activated branched polyethylene glycol derivative thus obtained was stored at 25°C in an environment of 11.5% relative humidity for three months immediately after production, and the rate of decrease Δ in the terminal activation rate was measured. As a result, the rate of decrease was 3% by mass after one month, and 7% by mass after three months.

[0096]

[0097] (Comparative Example 5) (Step (a)) N-hydroxysuccinimide (activator) and N,N-dicyclohexylcarbodiimide were added to a toluene solution of a linear polyethylene glycol raw material of formula (22) (molecular weight: 4,500, manufactured by NOF Corporation), and the mixture was stirred at 40°C for 2 hours to obtain a polyethylene glycol derivative having an active group of formula (23).

[0098] (Purification step (b)) After filtering the reaction solution obtained in the reaction step (a), hexane was added to precipitate a solid, which was then filtered off. Thereafter, the obtained solid was dissolved in a mixed solvent of acetonitrile and ethyl acetate, and the solid was precipitated with hexane and filtered off. This procedure was repeated twice. Hexane was added to the solid, and the slurry of the solid and hexane was stirred. The solid was filtered off, and then dried under reduced pressure to obtain a particulate solid.

[0099] (Drying Step (c)) The particulate solid obtained in the purification step (b) was spread in a dry box under a nitrogen atmosphere and dried at 25°C for 8 hours by flowing dry nitrogen.

[0100] Comparative Example 6 The reaction step (a) and the purification step (b) were carried out in the same manner as in Comparative Example 5 to obtain an activated branched polyethylene glycol derivative of formula (23). However, unlike Comparative Example 5, the drying step (c) was not carried out.

[0101] The activated branched polyethylene glycol derivatives of Comparative Examples 5 and 6 were stored at 25°C under a relative humidity of 11.5% for 3 months from immediately after production, and the rate of decrease Δ in the terminal activation rate was measured. The results are shown in Table 4.

[0102]

[0103] The rate of decrease in the terminal activation rate was small in both Comparative Examples 5 and 6, in which activated linear polyethylene glycol derivatives were produced. At the same time, the difference in the rate of decrease in the terminal activation rate was small between Comparative Example 5, which included the drying step (c), and Comparative Example 6, which did not include the drying step (c).

[0104] This indicates that in the case of activated branched polyethylene glycol derivatives as in the present invention, the drying step (c) has a significant effect.

[0105] Example 5 (Reaction step a) To a toluene solution of a branched polyethylene glycol raw material represented by formula (24) (molecular weight: 60,000, product of NOF Corp., 15 g), N-hydrosuccinimide (manufactured by Midori Chemical Industry Co., Ltd., 691 mg: activator) and N,N-dicyclohexylcarbodiimide (manufactured by Osaka Organic Synthetic Chemical Industry Co., Ltd., 825 mg) were added, and the mixture was stirred at 40°C for 3 hours to obtain a polyethylene glycol derivative having an active group represented by formula (25).

[0106]

[0107]

[0108] Here, the compound of formula (25) belongs to formula (2), where Y is a protecting group for a hydroxyl group (methoxy group), and PEG1 is (CH ２ CH ２ O) ｎ where B is a linker containing an ether bond, a propyl group, and an amide bond, W is a succinimidyl ester, and X is a central hydrocarbon group C ３ H ５ It is a branched skeleton consisting of the following. k is 1 and l is 2.

[0109] (Purification step b) Toluene (75 g) was added to the reaction solution obtained in the reaction step (a), and the mixture was filtered. Then, hexane (75 g) was added to precipitate a solid, which was then filtered off. The resulting solid was then washed with a mixed solution of ethyl acetate (150 g) and hexane (75 g), and the operation of filtering off the solid was repeated three times. The solid was dissolved in 150 g of ethyl acetate, and then the solid was precipitated with hexane (75 g) and filtered off. Hexane (120 g) was added to the solid, and the slurry of the solid and hexane was stirred. The solid was filtered off, and then dried under reduced pressure to obtain a particulate solid.

[0110] (Drying Step (c)) The particulate solid obtained in step (b) was spread in a dry box under a nitrogen atmosphere, and dried at 25° C. for 1 hour by flowing dry nitrogen.

[0111] The solid obtained above was analyzed, and the following measured values ​​were obtained: Moisture value: 0.2% Terminal activation rate: 92% by mass Average particle size: 1014 μm Color difference: 4.1

[0112] Example 6 (Reaction step a) To a toluene solution of a branched polyethylene glycol raw material represented by formula (26) (molecular weight: 40,000, product of NOF Corp., 50 g), N-hydrosuccinimide (manufactured by Midori Chemical Industry Co., Ltd., 4 g: activator) and N,N-dicyclohexylcarbodiimide (manufactured by Osaka Organic Synthetic Chemical Industry Co., Ltd., 4 g) were added, and the mixture was stirred at 40°C for 3 hours to obtain a polyethylene glycol derivative having an active group represented by formula (27).

[0113]

[0114]

[0115] Here, the compound of formula (27) belongs to formula (1), where PEG1 is (CH ２ CH ２ O) ｎ A is a linker consisting of a propyl group and an ester bond adjacent to the propyl group, W is a succinimidyl ester, and X is an atomic group (branched skeleton) consisting of a central hydrocarbon group and an ether bond. m is 8.

[0116] (Purification step b) Toluene (100 g) was added to the reaction solution obtained in reaction step (a), and the mixture was filtered. The mixture was then diluted with ethyl acetate (150 g). 200 g of hexane was added to precipitate a solid, which was then filtered off. The resulting solid was then washed with a mixed solution of ethyl acetate (398 g), acetonitrile (3 g), and hexane (200 g) containing 2,6-di-tert-butyl-p-cresol (Kanto Chemical, 80 mg), and the solid was filtered off. This procedure was repeated five times. The solid was dissolved in a mixed solvent of ethyl acetate (398 g) and acetonitrile (3 g) containing 2,6-di-tert-butyl-p-cresol (80 mg), and then precipitated with hexane (200 g) and filtered off. 200 g of hexane was added to the solid, and the resulting slurry of the solid and hexane was stirred. The solid was then filtered off and dried under reduced pressure to obtain a particulate solid.

[0117] (Drying Step (c)) The particulate solid obtained in step (b) was spread in a dry box under a nitrogen atmosphere, and dried at 25° C. for 1 hour by flowing dry nitrogen.

[0118] The solid obtained above was analyzed, and the following measured values ​​were obtained: Moisture value: 0.3% Terminal activation rate: 93% by mass Average particle size: 1064 μm Color difference: 1.9

[0119] Comparative Example 7 The reaction step (a) and the purification step (b) were carried out in the same manner as in Example 5 to obtain an activated branched polyethylene glycol derivative of formula (25). However, unlike Example 5, the drying step (c) was not carried out.

[0120] Comparative Example 8 The reaction step (a) and purification step (b) were carried out in the same manner as in Example 6 to obtain an activated branched polyethylene glycol derivative of formula (27). However, unlike Example 6, the drying step (c) was not carried out. The measured values ​​of the physical properties of the activated branched polyethylene glycol derivatives obtained in Examples 5 and 6 and Comparative Examples 7 and 8 are shown in Tables 5 and 6.

[0121]

[0122]

[0123] Example 7 and Comparative Example 9 The compounds of Example 1 and Comparative Example 1 were each stored at 25° C. for 4 weeks, and the rate of decrease Δ in the terminal activation rate was measured. As a result, the compound of Example 1 had a decrease of 1% by mass after 2 weeks and 2% by mass after 4 weeks, while the compound of Comparative Example 1 had a decrease of 11% by mass after 2 weeks and 15% by mass after 4 weeks.

[0124] Example 8 and Comparative Example 10 The compounds of Example 5 and Comparative Example 7 were each stored at 25° C. for 4 weeks, and the rate of decrease Δ in the terminal activation rate was measured. As a result, the compound of Example 5 had decreased by 10% by mass after 2 weeks and 14% by mass after 4 weeks, while the compound of Comparative Example 7 had decreased by 24% by mass after 2 weeks and 29% by mass after 4 weeks.

[0125] Example 9 and Comparative Example 11 The compounds of Example 6 and Comparative Example 8 were each stored at 25°C for 4 weeks, and the rate of decrease Δ in the terminal activation rate was measured. As a result, the compound of Example 6 decreased by 9% by mass after 2 weeks and 12% by mass after 4 weeks, while the compound of Comparative Example 8 decreased by 12% by mass after 2 weeks and 21% by mass after 4 weeks. The rate of decrease Δ in the terminal activation rate for Examples 7 to 10 and Comparative Examples 9 to 11 is shown in Table 7.

[0126]

[0127] It was revealed that the rate of decrease in the terminal activation rate was suppressed by performing step (c) for all compounds. From the above, the results in Table 7 reinforce the fact that the drying step (c) is highly effective in activated branched polyethylene glycol derivatives.

[0128] The production method of the present invention can be used as a production method for branched polyethylene glycol derivatives containing terminally hydrolyzable active groups. Furthermore, the branched activated polyethylene glycols having terminally active groups produced by the present invention are useful as raw materials for pharmaceuticals, diagnostic reagents, and medical devices.

[0129] Although the present invention has been described in detail and with reference to specific embodiments, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the spirit and scope of the present invention. This application is based on a Japanese patent application (Patent Application No. 2024-128941) filed on August 5, 2024, the contents of which are incorporated herein by reference.

Claims

1. A method for producing an activated branched polyethylene glycol derivative having an active group at its terminal, characterized by comprising the following steps (a), (b), and (c): Step (a): A reaction step of reacting a branched polyethylene glycol raw material with an activator to obtain an activated polyethylene glycol derivative; Step (b): A purification step of removing impurities from the activated polyethylene glycol derivative obtained in the reaction step (a); Step (c): A drying step of drying the activated polyethylene glycol derivative after the purification step (b) using a dry gas to obtain an activated branched polyethylene glycol derivative having a moisture content of 0.001% by mass or more and 0.3% by mass or less.

2. The method for producing an activated branched polyethylene glycol derivative according to claim 1, wherein the dry gas in step (c) is nitrogen, oxygen, carbon dioxide, argon, or a mixture thereof.

3. A method for producing an activated branched polyethylene glycol derivative according to claim 1 or 2, wherein the activated branched polyethylene glycol derivative has a structure represented by the following formula (1) or (2): (In formula (1), X is a branched skeleton containing at least a hydrocarbon group, PEG1 is a polyethylene glycol moiety, W is a hydrolyzable active group, A is a linker connecting the polyethylene glycol moiety PEG1 and the active group W, and m is an integer of 3 or more and 8 or less.) (In formula (2), X is a branched skeleton containing at least a hydrocarbon group; PEG1 is a polyethylene glycol moiety; W is a hydrolyzable active group; B is a linker connecting the branched skeleton X and the active group W; Y is a hydroxyl group or a protecting group for a hydroxyl group; and l and k are integers of 2 or more and 7 or less.) 4. A method for producing an activated branched polyethylene glycol derivative according to claim 1 or 2, characterized in that the number average molecular weight of the activated branched polyethylene glycol derivative is 400 daltons or more and 100,000 daltons or less.

5. A method for producing an activated branched polyethylene glycol derivative according to claim 1 or 2, characterized in that the activated branched polyethylene glycol derivative is a particulate solid, and the average particle size of this particulate solid is 0.1 μm or more and 10 mm or less.

6. An activated branched polyethylene glycol derivative having an active group at its terminal, characterized in that it is a particulate solid having a moisture content of 0.001% by mass or more and 0.3% by mass or less, a terminal activation rate of 90% by mass, an average particle size of 0.1 μm or more and 10 mm or less, and a color difference of 0 or more and 10 or less.

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