Method for analyzing branched-chain polyethylene glycol compound

Abstract

In the present invention, the purity of a branched-chain polyethylene glycol compound having a terminal functional group is analyzed. A labeled compound is obtained by labeling the terminal functional group with a labeling reagent having a UV absorption function. Then, the labeled compound is analyzed by reverse-phase liquid chromatography, and a chromatogram having a plurality of peaks is obtained by an ultraviolet absorbance detector. Then, the purity of the branched-chain polyethylene glycol compound is determined on the basis of the areas of the plurality of peaks.

Description

Analytical method for branched polyethylene glycol compounds

[0001] The present invention relates to a method for analyzing branched polyethylene glycol compounds.

[0002] Polyethylene glycol compounds are widely used as materials for drug delivery systems and medical hydrogels. For such medical applications, there is a demand for polyethylene glycol compounds with low levels of impurities.

[0003] For example, monomethoxypolyethylene glycol, a type of polyethylene glycol compound, is known to contain linear polyethylene glycol (hereinafter sometimes referred to as "diol") with hydroxyl groups at both ends, which is produced by the reaction of water molecules with ethylene oxide, as an impurity (Patent Document 1).

[0004] Diol compounds are formed during the production of polyethylene glycol compounds even in the presence of trace amounts of water in the system, making it difficult to completely suppress their formation. Therefore, there is a strong need for analytical methods to determine the amount of diol compounds in polyethylene glycol compounds.

[0005] Furthermore, during the polymerization reaction of polyethylene glycol compounds, polyethylene glycol impurities (hereinafter sometimes referred to as "polyethylene glycol impurities") with different skeletal structures or different numbers of hydroxyl groups from the target substance are generated through the decomposition of polymerization initiators, etc. Since such polyethylene glycol impurities are also highly likely to affect the safety of the drug, highly accurate purity analysis methods are required from the perspective of impurity control.

[0006] In this context, impurity control refers to the highly sensitive quantification of trace amounts of residual polyethylene glycol impurities during the derivatization process, confirming based on the analysis results that the content of derivatized polyethylene glycol impurities is within a range that does not affect quality characteristics such as the rate of active group introduction, completing predetermined processes such as reaction and purification, and moving on to the next process. If the content is within a range that affects quality characteristics, appropriate measures such as adding a purification process will be taken.

[0007] To date, gel permeation chromatography (hereinafter sometimes referred to as "GPC") has been used as an analytical method for detecting polyethylene glycol impurities in polyethylene glycol compounds, as shown in Patent Document 1 above. This method detects the target substance and impurities by sieving them apart using a column based on their molecular weight.

[0008] Japanese Patent Application Publication No. 11-335460

[0009] Incidentally, the GPC method described in Patent Document 1 separates and detects polyethylene glycol compounds of the target structure and polyethylene glycol impurities based on differences in molecular weight. However, it has become clear that with the GPC method described in Patent Document 1, separation and detection become more difficult as the difference in molecular weight between the target substance and the impurities decreases.

[0010] The present invention has been made in view of the above circumstances, and aims to provide an analytical method for separating and detecting a branched-chain polyethylene glycol compound, which is the target product, and polyethylene glycol impurities (for example, the diol compounds mentioned above) without depending on the difference in molecular weight.

[0011] As a result of diligent research, the inventors have discovered that by derivatizing the ends of a polyethylene glycol compound with a functional group having ultraviolet (UV) absorption, and using reverse-phase high-performance liquid chromatography (RP-HPLC), it is possible to quantify polyethylene glycol impurities in a mixture containing branched polyethylene glycol compounds and polyethylene glycol impurities, and to accurately measure the purity of the branched polyethylene glycol compound. Based on this finding, the present invention is as follows.

[0012] [1] A method for analyzing the purity of a branched polyethylene glycol compound having terminal functional groups, comprising the steps of: obtaining a labeled compound by labeling the terminal functional groups with a labeling reagent having UV absorption function; analyzing the labeled compound by reverse-phase liquid chromatography and obtaining a chromatogram having a plurality of peaks using an ultraviolet spectrophotometer; and determining the purity of the branched polyethylene glycol compound based on the area of ​​each of the plurality of peaks.

[0013] [2] The branched polyethylene glycol compound is of formula (1) The analytical method of [1], characterized in that the branched polyethylene glycol compound is represented by formula (1), where m is 0 or 1, when m is 0, s is 3 to 9, when m is 1, s is 2 to 8, s n are each independently 20 to 2000, Y and s X are each independently hydroxyl groups in which hydrogen atoms may be substituted with substituents, and E is a linker with an (s + m) valency.

[0014] [3] The analytical method of [2], characterized in that the substituent is a substituent selected from the group consisting of a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, an n-hexyl group, an isohexyl group, an n-heptyl group, an isoheptyl group, a phenyl group, a benzyl group, a trityl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, or a succimimidyl carbonate group.

[0015] [4] The analytical method of [1] or [2], characterized in that E is a residue obtained by removing all hydroxyl groups from glycerin, diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin, pentaerythritol, dipentaerythritol, tetrito, pentitol, or hexitol.

[0016] [5] The analytical method according to [1] or [2], characterized in that the number average molecular weight of the branched polyethylene glycol compound is 2,000 to 80,000.

[0017] [6] The analytical method according to [1] or [2], characterized in that the labeling reagent is a compound represented by the following formula (2): R-L-A (2) (wherein R represents an organic group having UV absorption function, L represents a linker, and A is a reactive group that can react with the terminal functional group of the branched polyethylene glycol compound.)

[0018] According to the present invention, even when branched polyethylene glycol compounds contain impurities of polyethylene glycol compounds with different structures or numbers of functional groups, separation from impurities can be improved compared to conventional methods, and the purity of branched polyethylene glycol compounds can be measured more accurately.

[0019] The UV chromatogram results obtained by reversed-phase liquid chromatography in Example 1 are shown. The UV chromatogram results obtained by reversed-phase liquid chromatography in Example 2 are shown. The UV chromatogram results obtained by reversed-phase liquid chromatography in Example 3 are shown. The UV chromatogram results obtained by reversed-phase liquid chromatography in Example 4 are shown. The UV chromatogram results obtained by reversed-phase liquid chromatography in Example 5 are shown. The UV chromatogram results obtained by reversed-phase liquid chromatography in Example 6 are shown. The ELSD chromatogram results obtained by reversed-phase liquid chromatography in Comparative Example 1 are shown.

[0020] The present invention will be described in order below. Unless otherwise clearly stated, the descriptions herein can be combined with each other.

[0021] The present invention relates to an analytical method for branched polyethylene glycol compounds. More specifically, the present invention relates to an analytical method for detecting a target product from a mixture containing a branched polyethylene glycol compound and polyethylene glycol impurities that differ from the target product in structure and number of functional groups.

[0022] In this specification, "branched polyethylene glycol compound" means a compound having two or more polyethylene glycol chains and having a branched structure.

[0023] In this specification, "polyethylene glycol impurity" means a compound having polyethylene glycol chains and having a different skeletal structure and number of functional groups than the target branched polyethylene glycol compound. The polyethylene glycol chains in the "polyethylene glycol impurity" may be one or two or more.

[0024] The number-average molecular weight (hereinafter sometimes abbreviated as "Mn") of the branched polyethylene glycol compound is preferably 500 or more, more preferably 1,000 or more, even more preferably 2,000 or more, even more preferably 90,000 or less, and particularly preferably 80,000 or less, from the viewpoint of separation between the branched polyethylene glycol compound and polyethylene glycol impurities (hereinafter sometimes abbreviated as "from the viewpoint of separation").

[0025] From the viewpoint of separation, the Mn content of polyethylene glycol impurities is preferably 500 or more, more preferably 2,000 or more, preferably 90,000 or less, and more preferably 80,000 or less.

[0026] The manganese (Mn) of polyethylene glycol compounds was measured by gel permeation chromatography (GPC) (reference: polyethylene glycol (HO-(C)). ２ H ４ It can be calculated using O)n-H).

[0027] A mixture containing branched polyethylene glycol compounds and linear polyethylene glycol compounds can be obtained by known methods for producing branched polyethylene glycol compounds.

[0028] Examples of methods for producing branched polyethylene glycol compounds include the following (i) and (ii).

[0029] (i) A method of adding ethylene oxide to a polyhydric alcohol having three or more hydroxyl groups as a starting material (see, for example, Japanese Patent Publication No. 2004-197077).

[0030] (ii) A method of reacting a linear polyethylene glycol compound having a hydroxyl group with a compound having three or more functional groups that can react with a hydroxyl group (see, for example, Japanese Patent Publication No. 9-504299)

[0031] In method (i) above, if water is present when ethylene oxide is added, linear polyethylene glycol with hydroxyl groups at both ends is formed as a polyethylene glycol impurity. In method (ii) above, the linear polyethylene glycol compound of the raw material may remain as a polyethylene glycol impurity.

[0032] The branched polyethylene glycol compound is preferably a branched compound represented by formula (1) (hereinafter sometimes abbreviated as "compound (1)"). (In formula (1), m is 0 or 1; when m is 0, s is 3 to 9; when m is 1, s is 2 to 8; s n are each independently 20 to 2000; Y and s X are each independently hydroxyl groups in which hydrogen atoms may be substituted with substituents; and E is a (s + m) valence linker.)

[0033] Here, "branched-chain compound" refers to a compound having a branched-chain structure. Therefore, compound (1) specifically refers to a compound represented by formula (1) that has a branched-chain structure.

[0034] The symbols in equation (1) are explained below in order. When m is 0 in equation (1), it means that Y does not exist in equation (1). n in equation (1) is the number of repetitions of the oxyethylene unit. This number of repetitions is an average value and may be a decimal. In equation (2), when m is 0, s is an integer from 3 to 9, preferably from 3 to 5. In equation (1), when m is 1, s is an integer from 2 to 8, preferably from 2 to 4. Therefore, there are multiple n and multiple X in equation (1). The s n may be the same or different from each other. Similarly, the s X may be the same or different from each other.

[0035] In formula (1), the s n are each independently a number between 20 and 2000, preferably between 200 and 1000. The s n may be the same or different from each other.

[0036] In formula (1), Y and s X are each independently a hydroxyl group, in which the hydrogen atom may be substituted with a substituent. From the viewpoint of ease of analysis of compound (1), it is preferable that the s X are the same as each other.

[0037] In the present invention, "hydroxyl group in which a hydrogen atom may be substituted with a substituent" means a hydroxyl group (-OH) or a hydroxyl group in which a hydrogen atom is substituted with a substituent. "Hydroxyl group in which a hydrogen atom is substituted with a substituent" means a hydroxyl group (-OD) in which the hydrogen atom (H) of a hydroxyl group (-OH) is substituted with a substituent (D).

[0038] The substituent D of the hydroxyl group described above is preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, an n-hexyl group, an isohexyl group, an n-heptyl group, an isoheptyl group, a phenyl group, a benzyl group, a trityl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, or a succimidyl carbonate group, more preferably a benzyl group, a trityl group, or a succimidyl carbonate group, and even more preferably a succimidyl carbonate group.

[0039] The s X groups are each independently preferably a hydroxy group whose hydrogen atom may be substituted with a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, an n-hexyl group, an isohexyl group, an n-heptyl group, an isoheptyl group, a phenyl group, a benzyl group, a trityl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, or a succinimidyl carbonate group; more preferably each independently a hydroxy group whose hydrogen atom may be substituted with a benzyl group, a trityl group, or a succinimidyl carbonate group; and still more preferably both are a hydroxy group whose hydrogen atom is substituted with a succinimidyl carbonate group, or an unsubstituted hydroxy group.

[0040] Y is preferably a hydroxy group whose hydrogen atom may be substituted with a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, an n-hexyl group, an isohexyl group, an n-heptyl group, an isoheptyl group, a phenyl group, a benzyl group, a trityl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, or a succinimidyl carbonate group; more preferably a hydroxy group whose hydrogen atom may be substituted with a benzyl group, a trityl group, or a succinimidyl carbonate group; and still more preferably a hydroxy group whose hydrogen atom is substituted with a succinimidyl carbonate group.

[0041] E in formula (1) is an (s+m)-valent linker, that is, a trivalent to nonavalent linker. E is preferably a linker having a structure of a residue obtained by removing all hydroxy groups from glycerin, diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin, pentaerythritol, dipentaerythritol, tetritol, pentitol, or hexitol.

[0042] The aforementioned diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, and heptaglycerin are compounds generally collectively referred to as "polyglycerin" (also known as polyglycerol). The term "polyglycerin" refers to a compound having a structure in which a plurality of glycerin molecules (also known as glycerol) are linked via ether bonds. Specifically, "diglycerin" refers to a compound having a structure in which two glycerin molecules are linked via an ether bond, and "heptaglycerin" refers to a compound having a structure in which seven glycerin molecules are linked via an ether bond. "Triglycerin", "tetraglycerin", "pentaglycerin", and "hexaglycerin" have the same meanings as "diglycerin" and "heptaglycerin" respectively. Polyglycerin may have a structure in which a plurality of glycerin molecules are linked linearly, or may have a structure in which a plurality of glycerin molecules are linked in a branched chain form.

[0043] As used herein, the term "tetritol" refers to a compound of formula: C ４ H ６ (OH) ４ , which represents a tetravalent sugar alcohol, and examples thereof include erythritol.

[0044] As used herein, the term "pentitol" refers to a compound of formula: C ５ H ７ (OH) ５ , which represents a pentavalent sugar alcohol, and examples thereof include xylitol.

[0045] As used herein, the term "hexitol" refers to a compound of formula: C ６ H ８ (OH) ６ , which represents a hexavalent sugar alcohol, and examples thereof include mannitol.

[0046] In formula (1), when m is 0, s is an integer of 3 to 5; when m is 1, s is an integer of 2 to 4; and E is selected from formula (3a), (3b), (3c)

[0047] It is preferable that the linker be represented by one of the following formulas (3a), (3b), and (3c), where * indicates a bond position. Note that * indicates a bond position, not a carbon atom. For example, "*-" indicates a single bond.

[0048] The linker represented by formula (3a) is a linker having a structure obtained by removing a hydroxyl group from glycerol. The linker represented by formula (3b) is a linker having a structure obtained by removing a hydroxyl group from pentaerythritol. The linker represented by formula (3c) is a linker having a structure obtained by removing a hydroxyl group from pentitol (e.g., xylitol).

[0049] In equation (1), if m is 0, s is an integer of 3 or 4; if m is 1, s is an integer of 2 or 3; and it is more preferable that E is a linker represented by equation (3a) or equation (3b).

[0050] (Labeling reaction step) The analytical method of the present invention is a reaction step in which a labeling reagent having a reactive functional group that reacts with terminal functional groups and having UV absorption function is reacted with a branched polyethylene glycol compound and polyethylene glycol impurities to obtain a labeled reaction product in which terminal functional groups are labeled.

[0051] In a preferred embodiment, the labeling reagent has the structure of formula (2): R-L-A (2) (wherein R represents an organic group having UV absorption function, L represents a linker, and A is a reactive group that can react with the terminal functional group of a branched polyethylene glycol compound.)

[0052] In formula (2), L is the linker (bonding site) to the reactive group. This linking site preferably contains a saturated hydrocarbon group or an unsaturated hydrocarbon group having 1 to 24 carbon atoms, and more preferably contains a saturated hydrocarbon group or an unsaturated hydrocarbon group having 1 to 12 carbon atoms. By making the number of carbon atoms in these hydrocarbon groups 24 or less, and even more preferably 12 or less, the progress of the labeling reaction can be accelerated.

[0053] In formula (2), L may consist only of saturated or unsaturated hydrocarbon groups having 1 to 24 carbon atoms. Alternatively, L may have ester bonds, ether bonds, urethane bonds, amide bonds, etc., in addition to saturated or unsaturated hydrocarbon groups having 1 to 24 carbon atoms.

[0054] In formula (2), preferred forms of R are a naphthyl group, a dinitrobenzoyl group, and an indolyl group, with the latter being a dinitrobenzoyl group and an indolyl group.

[0055] In formula (2), A is a reactive group that can react with the terminal functional group of a branched polyethylene glycol compound, and preferred forms include an amino group, an acyl halogenated group, a succimidyl group, a carboxyl group, a maleimide group, a thiol group, a hydroxyl group, and an azide group, with amino groups, acyl halogenated groups, and carboxyl groups being preferred.

[0056] Regarding the labeling reagent of formula (2) having UV absorption function, specific examples include, when the terminal functional group is a hydroxyl group, benzyl chloride, indoleacetic acid, 3,5-dinitrobenzoyl chloride, etc. can be used, preferably indoleacetic acid or 3,5-dinitrobenzoyl chloride. When the terminal functional group is a succimidyl carbonate terminal group, tryptamine, etc., can be used.

[0057] The labeling reaction used in this invention must be quantitative in order to accurately measure the purity of the branched polyethylene glycol compound, and it is important that the reaction proceeds quantitatively without decomposition or side reactions, resulting in a stable labeled product. Furthermore, it is desirable that the procedure be as simple as possible. The conditions for the labeling reaction are described in detail below.

[0058] The amount of labeling reagent added is arbitrary as long as it is in excess of the terminal hydroxyl groups of the branched polyethylene glycol compound, in order to perform a quantitative labeling reaction, but is preferably 5 to 100 equivalents, more preferably 5 to 50 equivalents.

[0059] During the labeling reaction, base catalysts such as triethylamine, pyridine, and 4-dimethylaminopyridine, as well as condensing agents such as N,N'-dicyclohexylcarbodiimide and N,N'-diisopropylcarbodiimide, may be added as needed.

[0060] The reaction solvent for the labeling reaction can be any organic solvent except alcohol, depending on the solubility and reactivity of the labeling reagent. Any organic solvent in which the branched polyethylene glycol compound and the labeling reagent are soluble is acceptable, including acetonitrile, dioxane, methylene chloride, chloroform, benzene, and toluene.

[0061] The amount of reaction solvent used in the labeling reaction is arbitrary, but typically 1 to 100 parts by mass, preferably 2 to 50 parts by mass, of the branched polyethylene glycol compound is desirable. If the amount of reaction solvent used is less than 1 part by mass, the reaction rate may decrease due to an increase in viscosity. Also, if the amount of reaction solvent used is more than 100 parts by mass, the reaction rate may decrease.

[0062] The reaction temperature and reaction time for the labeling reaction are arbitrary, allowing for the determination of conditions suitable for the labeling reaction.

[0063] The labeled branched polyethylene glycol compound can be recovered from the resulting reaction solution by known means (e.g., solid-liquid separation by adding a poor solvent).

[0064] The poor solvent for solid-liquid separation is preferably at least one selected from the group consisting of n-hexane, diethyl ether, and methyl tert-butyl ether, and more preferably n-hexane. The amount of poor solvent used is preferably 100 to 200 parts by mass, more preferably 120 to 180 parts by mass, per 100 parts by mass of solvent in the solution containing the branched-chain PEG compound. The temperature when adding the poor solvent to the solution containing the branched-chain PEG compound is preferably 25 to 45°C, more preferably 30 to 40°C.

[0065] The solid containing the branched polyethylene glycol compound precipitated by the addition of a poor solvent can be recovered by known means (e.g., filtration or centrifugation).

[0066] (Analysis process) In this invention, the labeled reaction product is eluted by liquid chromatography using an ion exchange column, and a chromatogram having multiple peaks is obtained using an ultraviolet absorbance spectrophotometer.

[0067] In other words, the labeled reaction product obtained in the labeling reaction step can be used directly as a sample for reversed-phase liquid chromatography and analyzed quickly and easily. However, it is preferable to perform desalting treatment using a gel filtration column before reversed-phase liquid chromatography.

[0068] Any gel filtration column capable of separating low molecular weight substances with a molecular weight of 1000 or less is acceptable. Desalting is performed by the following procedure: First, the gel filtration column is equilibrated with the buffer solution to be used as the eluent in RP-HPLC analysis, and then the reaction solution containing the labeled compound is added. Further eluent is added to separate the high molecular weight fraction that elutes first, and this is used as the analytical sample.

[0069] Labeled branched polyethylene glycol compounds, which are given polarity differences and UV absorption properties through labeling, are then separated by high-performance liquid chromatography using a reversed-phase column and measured. The measurement conditions are described in detail below.

[0070] Reverse-phase columns typically use stainless steel columns with a length of 5 to 30 cm and an inner diameter of 2 to 10 mm. These columns can be used either packed with reverse-phase chromatography packing material or already packed with it.

[0071] The packing material for reverse-phase chromatography can have a propyl (3-C3 alkyl) group, a cyanopropyl (CN) group, an octyl (8-C3 alkyl) group, a phenyl (Ph) group, or an octadecyl (18-C3 alkyl) group, with octadecyl groups being preferred. As the substrate having these functional groups, a polymer gel or a silica gel can be used, with silica gels being preferred. The gel pore size is preferably 60 to 300 angstroms, and more preferably 300 angstroms.

[0072] The eluent used in reverse-phase chromatography can be any eluent suitable for the separation of the reverse-phase column used. Specifically, formic acid buffer, acetate buffer, phosphate buffer, carbonate buffer, borate buffer, glycine buffer, Tris-HCl buffer, monoethanolamine-HCl buffer, etc., can be selected arbitrarily.

[0073] Furthermore, a salt solution, an organic solvent, or both may be optionally mixed with the buffer solution. Suitable salt solutions include sodium chloride, potassium chloride, sodium sulfate, and potassium sulfate. Suitable organic solvents include methanol, ethanol, and acetonitrile, which are easily miscible with water, and their concentrations can be mixed in the range of 0 to 50% by volume.

[0074] The pH of the eluent is arbitrary within the applicable range of the column being used, and is usually used in the range of 2.0 to 12.0.

[0075] The gradient conditions for the eluent are determined by setting appropriate gradient conditions based on the structure and molecular weight of the branched polyethylene glycol compound. Generally, in reversed-phase columns, separation is achieved by the difference in the magnitude of hydrophobic interactions between substances. Furthermore, the degree of separation can be adjusted by the concentration of the buffer solution used as the eluent.

[0076] In the analytical method of the present invention, an ultraviolet absorbance spectrophotometer is used as the optical detector for reverse-phase chromatography. In the present invention, gradient elution is necessary to separate the derivatized polyethylene glycol compound by utilizing the difference in hydrophobicity. Generally, when performing analysis using gradient elution, the refractive index of the baseline changes, making it difficult to use a differential refractive index detector (RI). For this reason, it is preferable to use an ultraviolet absorbance spectrophotometer or the like.

[0077] (Purity Calculation Process) The resulting chromatogram contains peaks corresponding to the target branched polyethylene glycol compound and peaks corresponding to impurities with different branching structures and numbers of functional groups. The area of ​​each peak corresponds to the molar ratio of the derivatized functional group, but can be converted to the mass percentage of each compound by calculating using the following formula. Here, the mass percentage is expressed by the following formula: Mass percentage of each peak [%] = {(Area of ​​each compound peak / Number of functional groups of each compound) / (Sum of (Area of ​​each compound peak / Number of functional groups of each compound))} × 100

[0078] In other words, the percentage calculated using the above formula for the peak corresponding to the target product, the branched polyethylene glycol compound, indicates the mass ratio (purity) of the branched polyethylene glycol compound. Furthermore, the percentage calculated using the above formula for the peak corresponding to polyethylene glycol impurities with different branching structures or functional group numbers indicates the mass ratio of the impurities. This allows us to determine the purity of the branched polyethylene glycol compound.

[0079] The present invention will be described in more detail by reference to examples and comparative examples. (Example 1) (Labeling reaction step) A PEG mixture containing a tetrafunctional polyethylene glycol compound represented by the following formula (4) (Mn = 18,091, 0.18 g) and a linear bifunctional polyethylene glycol impurity represented by the following formula (5) (Mn = 18,628, 0.02 g) was analyzed (linear bifunctional polyethylene glycol impurity content = 9.97% by mass, GPC main infraction purity = 97.6%, 0.2 g). This PEG mixture and 3,5-dinitrobenzoyl chloride (0.12 g) were weighed into a glass flask and dissolved in tetrahydrofuran (8.9 g). After dissolution, pyridine (0.079 g) was added and the solution was stirred at 40°C for 1 hour. The reaction solution was diluted with ethyl acetate (23 g) and insoluble matter was removed by filtration. n-hexane (8 g) was added to the obtained solution at 25°C to precipitate the labeled polyethylene glycol compound. The precipitated crystals were collected by filtration, washed with a mixed solvent of ethyl acetate (25 g) and n-hexane (8 g), and the crystals were collected by filtration. After repeating this crystal washing five times, the crystals were dissolved in ethyl acetate (25 g) at 40°C, and n-hexane (8 g) was added at 25°C to precipitate the crystals. The precipitated crystals were collected by filtration to obtain a sample for analysis.

[0080]

[0081] (Analysis Process) Next, RP-HPLC measurement was performed under the following conditions. (RP-HPLC Measurement Conditions) HPLC instrument: Alliance 6890 (Waters Co., Ltd.) Separation column: Zorbax 300SB-C18 (3.0 × 150 mm) (Agilent Technologies, Inc.) Mobile phase A: Water Mobile phase B: Acetonitrile flow rate: 0.6 mL / min Sample concentration: 25 mg / mL Injection volume: 10 μL Detector: Ultraviolet absorbance spectrophotometer (210 nm) Gradient conditions: As shown in Table 1.

[0082]

[0083] (Calculation Process) Figure 1 shows the UV chromatogram of the sample being measured. In Figure 1, peak 1 corresponds to the peak of compound (1). The mass percentage of the target substance was calculated from the area of ​​each peak and defined as the purity. As a result, the purity was 84.75%.

[0084] (Example 2) (Labeling reaction step) A PEG mixture containing a tetrafunctional polyethylene glycol compound represented by formula (4) (Mn = 1,833, 0.045 g) and a linear bifunctional polyethylene glycol impurity represented by formula (5) (Mn = 1,910, 0.005 g) (linear bifunctional polyethylene glycol impurity content = 10.0% by mass, GPC main infraction purity = 97.72%, 0.05 g) was weighed into a glass flask and dissolved in tetrahydrofuran (300 μL). After dissolution, pyridine (20 μL) was charged, and a solution (600 μL) of 3,5-dinitrobenzoyl chloride (0.4 g) separately dissolved in tetrahydrofuran (2 mL) was added, and the mixture was stirred at 40°C for 1 hour.

[0085] Next, the entire reaction solution was added to a gel filtration column (PD-10 (Amersham Biosciences)) equilibrated with the eluent used for RP-HPLC measurement. Further eluent was added, and the high molecular weight fraction that eluted first was collected in a vial for RP-HPLC measurement and used as the measurement sample (desalting step).

[0086] (Analysis and Calculation Process) RP-HPLC measurement was performed under the same conditions as in Example 1. The UV chromatogram of the sample is shown in Figure 2. In Figure 2, peak 1 corresponds to the peak of compound (1). The mass percentage of the target substance was calculated from the area value of each peak and defined as the purity. As a result, the purity was 95.04%.

[0087] (Example 3) (Labeling reaction step) A PEG mixture containing a tetrafunctional polyethylene glycol compound represented by formula (4) (Mn = 34,531, 0.045 g) and a linear monofunctional polyethylene glycol impurity represented by formula (6) below (Mn = 42,851, 0.005 g) (linear monofunctional polyethylene glycol impurity content = 10.0% by mass, GPC main infraction purity = 84.16%, 0.05 g) was weighed into a glass flask and dissolved in tetrahydrofuran (300 μL). After dissolution, pyridine (20 μL) was charged, and a solution (200 μL) of 3,5-dinitrobenzoyl chloride (0.2 g) separately dissolved in tetrahydrofuran (1 mL) was added, and the mixture was stirred at 40°C for 1 hour.

[0088] Next, the entire reaction solution was added to a gel filtration column (PD-10 (Amersham Biosciences)) equilibrated with the eluent used for RP-HPLC measurement. Further eluent was added, and the high molecular weight fraction that eluted first was collected in a vial for RP-HPLC measurement and used as the measurement sample (desalting step).

[0089] (Analysis and Calculation Process) RP-HPLC measurement was performed under the same conditions as in Example 1. The UV chromatogram of the sample is shown in Figure 3. In Figure 3, peak 1 corresponds to the peak of the compound of formula (4). The mass percentage of the target substance was calculated from the area of ​​each peak and defined as the purity. As a result, the purity was 68.04%.

[0090]

[0091] (Example 4) (Labeling reaction step) A PEG mixture containing a branched tetrafunctional polyethylene glycol compound represented by formula (7) below (Mn = 66,762, 0.045 g) and a branched bifunctional polyethylene glycol impurity represented by formula (8) below (Mn = 76,804, 0.005 g) (branched bifunctional polyethylene glycol impurity = 10.0% by mass, GPC main infraction purity = 90.48%, 0.05 g) was weighed into a glass flask and dissolved in tetrahydrofuran (300 μL). After dissolution, pyridine (20 μL) was charged, and a solution (100 μL) of 3,5-dinitrobenzoyl chloride (0.1 g) separately dissolved in tetrahydrofuran (500 μL) was added, and the mixture was stirred at 40°C for 1 hour.

[0092] Next, the entire reaction solution was added to a gel filtration column (PD-10 (Amersham Biosciences)) equilibrated with the eluent used for RP-HPLC measurement. Further eluent was added, and the high molecular weight fraction that eluted first was collected in a vial for RP-HPLC measurement and used as the measurement sample (desalting step).

[0093] (Analysis and Calculation Process) RP-HPLC measurement was performed under the same conditions as in Example 1. The UV chromatogram of the sample is shown in Figure 4. In Figure 4, peak 1 corresponds to the peak of the compound of formula (7). The mass percentage of the target substance was calculated from the area of ​​each peak and defined as the purity. As a result, the purity was 81.54%.

[0094]

[0095] (Example 5) (Labeling reaction step) A polyethylene glycol compound represented by the following formula (9) (Mn = 42,794, 1.0 g), along with 3-indoleacetic acid (0.4 g) and 4-dimethylaminopyridine (0.02 g), were weighed into a glass flask and dissolved in acetonitrile (16 g). After dissolution, N,N'-diisopropylcarbodiimide (0.033 g) was added, and the solution was stirred at 15°C for 5 hours. The reaction solution was filtered to remove insoluble matter and diluted with ethyl acetate (72 g). n-hexane (79 g) was added to the obtained solution at 25°C to precipitate the labeled polyethylene glycol compound. The precipitated crystals were recovered by filtration and dissolved in ethyl acetate (90 g) at 40°C, and then n-hexane (33 g) was added at 25°C to precipitate the labeled polyethylene glycol compound. The precipitated crystals were recovered by filtration to obtain a sample for analysis.

[0096]

[0097] (Analysis Process) Next, RP-HPLC measurement was performed under the following conditions. (RP-HPLC Measurement Conditions) HPLC instrument: Alliance 6890 (Waters Co., Ltd.) Separation column: ACQUITY UPLC PST C18 (2.1 x 100 mm, 1.7 μm, Waters Co., Ltd.) Mobile phase A: Water Mobile phase B: Acetonitrile flow rate: 0.2 mL / min Sample concentration: 20 mg / mL Injection volume: 3 μL Detector: Ultraviolet absorbance spectrophotometer (254 nm) Gradient conditions: As shown in Table 2.

[0098]

[0099] The UV chromatogram of the sample being measured is shown in Figure 5. In Figure 5, peak 1 corresponds to the peak of the compound in equation (9). The mass percentage of the target substance was calculated from the area of ​​each peak and defined as the purity. As a result, the purity was 98.23%.

[0100] (Example 6) (Labeling reaction step) Tryptamine (0.1 g) was weighed into a glass vial, acetonitrile (9 g) was added and dissolved to prepare a tryptamine solution. Next, a polyethylene glycol compound represented by the following formula (10) (Mn = 42,794, 0.05 g) was weighed into another glass vial, and the previously prepared tryptamine solution (1 mL) was added. The solution was stirred at 25°C for 2 hours.

[0101] Next, the entire reaction solution was added to a gel filtration column (PD-10 (Amersham Biosciences)) equilibrated with the eluent used for RP-HPLC measurement. Further eluent was added, and the high molecular weight fraction that eluted first was collected in a vial for RP-HPLC measurement and used as the measurement sample (desalting step).

[0102]

[0103] (Analysis Process) Next, RP-HPLC measurement was performed under the following conditions. (RP-HPLC Measurement Conditions) HPLC instrument: Alliance 6890 (Waters Co., Ltd.) Separation column: apHera C4 (4.6 mm × 15 cm, 5 μm Sigma-Aldrich) Mobile phase A: 1 mmol / L hydrochloric acid / acetonitrile (2 / 1) Mobile phase B: 1 mmol / L hydrochloric acid / acetonitrile (1 / 1) Flow rate: 1.0 mL / min Injection volume: 50 μL Detector: UV absorbance spectrophotometer (223 nm) Gradient conditions: As shown in Table 3.

[0104]

[0105] (Calculation Process) The UV chromatogram of the sample to be measured is shown in Figure 6. In Figure 6, peak 1 corresponds to the peak of the compound of formula (10). The mass percentage of the target substance was calculated from the area of ​​each peak and was used as the purity. As a result, the purity was 98.52%.

[0106] (Comparative Example 1) A PEG mixture (1.0 g) containing a tetrafunctional polyethylene glycol compound represented by formula (4) (Mn = 18,091, 0.90 g) and a linear bifunctional polyethylene glycol impurity represented by formula (5) (Mn = 18,628, 0.10 g) was weighed into a glass flask.

[0107] Next, RP-HPLC measurements were performed under the following conditions: (RP-HPLC measurement conditions) HPLC instrument: Alliance 6890 (Waters Co., Ltd.) Separation column: Zorbax 300SB-C18 (3.0 × 150 mm) (Agilent Technologies, Inc.) Mobile phase A: Water Mobile phase B: Acetonitrile flow rate: 0.6 mL / min Sample concentration: 25 mg / mL Injection volume: 10 μL Detector: ELSD Gradient conditions: As shown in Table 4.

[0108]

[0109] The UV chromatogram of the measured sample is shown in Figure 7. In Figure 7, peak 1 corresponds to the peak of the mixture of the compound of formula (4) and the compound of formula (5).

[0110] In Comparative Example 1, a tetrafunctional polyethylene glycol compound represented by formula (4) was mixed with a linear bifunctional polyethylene glycol compound represented by formula (5), and RP-HPLC measurement was performed without a labeling reaction step. Since no labeling reaction was performed, detection was performed by ELSD. As a result, the peaks of the two compounds did not separate, and it was not possible to calculate the purity of the tetrafunctional polyethylene glycol compound. From this, it can be seen that the purity of the target polyethylene glycol compound cannot be calculated by RP-HPLC measurement without derivatization.

[0111] According to the present invention, even when branched polyethylene glycol compounds contain impurities of polyethylene glycol compounds with different structures or numbers of functional groups, separation from impurities can be improved compared to conventional methods, and the purity of branched polyethylene glycol compounds can be measured more accurately.

[0112] 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 invention. This application is based on Japanese Patent Application No. 2025-049259 filed on 25 March 2025, the contents of which are incorporated herein by reference.

Claims

1. A method for analyzing the purity of a branched polyethylene glycol compound having terminal functional groups, comprising the steps of: obtaining a labeled compound by labeling the terminal functional groups with a labeling reagent having UV absorption function; analyzing the labeled compound by reverse-phase liquid chromatography and obtaining a chromatogram having multiple peaks using an ultraviolet spectrophotometer; and determining the purity of the branched polyethylene glycol compound based on the area of ​​each of the multiple peaks.

2. The branched polyethylene glycol compound is of formula (1) The analytical method according to claim 1, characterized in that the branched polyethylene glycol compound is represented by formula (1), where m is 0 or 1, when m is 0, s is 3 to 9, when m is 1, s is 2 to 8, s n are each independently 20 to 2000, Y and s X are each independently hydroxyl groups in which hydrogen atoms may be substituted with substituents, and E is a linker with an (s + m) valency.

3. The analytical method according to claim 2, characterized in that the substituent is a substituent selected from the group consisting of a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a sec-butyl group, a tert-butyl group, an n-pentyl group, an isopentyl group, an n-hexyl group, an isohexyl group, an n-heptyl group, an isoheptyl group, a phenyl group, a benzyl group, a trityl group, a tert-butyldimethylsilyl group, a tert-butyldiphenylsilyl group, or a succimimidyl carbonate group.

4. The analytical method according to claim 1 or 2, characterized in that E is a residue obtained by removing all hydroxyl groups from glycerin, diglycerin, triglycerin, tetraglycerin, pentaglycerin, hexaglycerin, heptaglycerin, pentaerythritol, dipentaerythritol, tetriitol, pentitol, or hexitol.

5. The analytical method according to claim 1 or 2, characterized in that the number average molecular weight of the branched polyethylene glycol compound is 2,000 to 80,000.

6. The analytical method according to claim 1 or 2, characterized in that the labeling reagent is a compound represented by the following formula (2): R-L-A (2) (wherein R represents an organic group having UV absorption function, L represents a linker, and A is a reactive group that can react with the terminal functional group of the branched polyethylene glycol compound.)

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