Analysis method for reactive low molecular weight compounds contained in polyoxyethylene derivatives

Abstract

The present invention provides an analysis method in which a compound of formula (1) contained in a polyoxyethylene derivative is separated by means of reversed-phase chromatography, and the compound is subsequently detected by means of an ultraviolet-visible spectroscopic detector, the analysis method being characterized in that: ultraviolet light having a wavelength shorter than 200 nm is used as a detection wavelength; and an acidic substance that has a pKa of 4 or less and a maximum molar extinction coefficient of 20 M-1 cm-1 or less at 190 nm to 200 nm is used as an additive to a mobile phase for analysis.

Description

[Technical Field]

[0001] The present invention relates to a highly sensitive analytical method for low molecular weight compounds contained in polyoxyethylene derivatives, and more specifically to a highly sensitive analytical method for low molecular weight compounds contained in polyoxyethylene derivatives having at their terminals active groups used as polyoxyethylene modifying agents for polypeptides, physiologically active proteins, enzymes, etc. [Background technology]

[0002] In recent years, the development of polymeric compounds for pharmaceutical applications has been actively pursued, one example of which is terminally activated polyoxyethylene derivatives. Their main use is chemical modification of biologically relevant substances such as hormones, cytokines, antibodies, and enzymes, thereby increasing their molecular weight and forming a hydration layer, thereby extending the blood half-life of the biologically relevant substances. Modification with polyoxyethylene derivatives can also reduce the toxicity and antigenicity of biologically relevant substances and improve the solubility of poorly water-soluble drugs. The effectiveness of reducing the toxicity and antigenicity of biologically relevant substances varies depending on the molecular weight and amount of the polyoxyethylene derivative used for modification. For example, in the case of highly antigenic biologically relevant substances, the antigenicity of the biologically relevant substance can be reduced by modifying them with multiple polyoxyethylene derivatives.

[0003] The structure of polyoxyethylene derivatives usually has an active group at the end of the polyoxyethylene that can chemically bond with functional groups such as amino groups, mercapto groups, carboxyl groups, and unsaturated bonds present on the surface of bio-related substances such as proteins to be modified. For example, active groups that can be used to modify amino groups of bio-related substances include formyl groups, acetal groups, para-nitrophenyloxycarbonyl groups, and N-succinimidyloxycarbonyl groups. In particular, N-succinimidyloxycarbonyl groups are useful active groups for chemical modification of bio-related substances because they react under mild conditions and there are relatively many amino groups on the surface of bio-related substances that can react with N-succinimidyloxycarbonyl groups (Non-Patent Document 1).

[0004] Polyoxyethylene derivatives having an N-succinimidyloxycarbonyl group can be synthesized by reacting a polyoxyethylene derivative having a carboxylic acid at the terminal with N-hydroxysuccinimide in the presence of a condensing agent such as dicyclohexylcarbodiimide. During the condensation reaction, the reagents undergo a side reaction, resulting in the formation of the compound represented by formula (1).

[0005] [ka]

[0006] It is known that a reactive low molecular weight compound represented by the formula (N-succinimidyloxycarbonyl-β-alanine N-succinimidyl ester, β-Ala-NHS, CAS Registry Number: 21994-89-8) is produced as an impurity (Non-Patent Documents 2 and 3), and commercially available polyoxyethylene derivatives having an N-succinimidyloxycarbonyl group contain approximately 100 to 1000 ppm of the compound represented by formula (1).

[0007] The compound of formula (1) has an N-succinimidyloxycarbonylamino group and an N-succinimidyloxycarbonyl group at both ends. These functional groups are reactive with amino groups, and therefore its application as a bifunctional linker has been proposed (Patent Document 1, Non-Patent Document 3). However, when the compound of formula (1) is contained in a polyoxyethylene derivative, during chemical modification of biologically relevant substances such as proteins, the compound of formula (1) also reacts with the biologically relevant substance in the same manner as the polyoxyethylene derivative, resulting in impurities in the formulation. Therefore, highly sensitive analysis is required to control the content of the compound of formula (1), which is contained in small amounts in the polyoxyethylene derivative.

[0008] A method using liquid chromatography is commonly used to analyze low molecular weight impurities such as the compound of formula (1), and highly sensitive detection methods include ultraviolet-visible spectroscopic detectors, fluorescence detectors, mass spectrometry detectors, etc. However, no analytical method for highly sensitive detection of the compound of formula (1) using these detectors has been reported to date. As described above, the compound of formula (1), which is produced as a by-product during the synthesis of polyoxyethylene derivatives having an N-succinimidyloxycarbonyl group at the terminal, can react with biologically relevant substances even in small amounts and cause the production of impurities as by-products. Therefore, from the perspective of pharmaceutical quality control, it is necessary to analyze the compound of formula (1) contained in polyoxyethylene derivatives with high sensitivity. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2010 / 107520 [Patent Document 2] International Publication No. 1997 / 003106 [Non-patent literature]

[0010] [Non-Patent Document 1] Bioconjugate Techniques 3rd edition, 2013 [Non-patent document 2] Tetrahedron 1968, 24, 6935-6939 [Non-patent document 3] Reactive & Functional Polymers 2007, 67, 529-539 Summary of the Invention [Problem to be solved by the invention]

[0011] Therefore, the problem to be solved by the present invention is to provide a compound represented by the formula (1) contained in a polyoxyethylene derivative.

[0012] [ka]

[0013] The object of the present invention is to provide a method for analyzing compounds with high sensitivity. [Means for solving the problem]

[0014] The present invention is characterized by a method for highly sensitively analyzing a polyoxyethylene derivative having an N-succinimidyloxycarbonyl group at its terminal that chemically bonds to a biologically relevant substance by reversed-phase liquid chromatography, and for analyzing the compound of formula (1) contained in an extremely small amount based on the area value of a chromatogram obtained with an ultraviolet-visible spectrophotometer.

[0015] That is, the present invention relates to the following (1) and (2). (1) Polyoxyethylene derivatives of formula (1)

[0016] [ka]

[0017] This is an analytical method in which the compounds are separated by reversed-phase chromatography and then detected using an ultraviolet-visible spectrophotometer. The detection wavelength is shorter than 200 nm, and an additive to the mobile phase for analysis is a compound with a maximum molar absorption coefficient of 20 M or less at 190-200 nm. -1 cm -1 An analytical method characterized by using an acidic substance having a pKa of 4 or less.

[0018] (2) The polyoxyethylene derivative is represented by the formula (2)

[0019] [ka]

[0020] (wherein Z is a residue of a compound having 2 to 8 hydroxyl groups and 2 to 21 carbon atoms, which may contain an oxygen atom and / or a nitrogen atom; OA 1 and O.A. 2 is an oxyethylene group, a and b are each independently 0 to 5,000 and a+b is 20 or more, and L 1 and L 2 are each independently an alkylene group which may have a bond selected from an ester bond, a urethane bond, an amide bond, a urea bond, an ether bond, and a thioether bond in the alkylene chain or at the terminal thereof; p and q are each independently 0 or 1; s is 0 to 8; t is 1 to 8, and 2≦s+t≦8; X 1 is an alkoxy group or an N-succinimidyloxycarbonyl group, and X 2 is an N-succinimidyloxycarbonyl group. a, b, OA 1 ,OA 2 , p, q, L 1 , L 2 and X 1 When a plurality of each of these is present in one molecule, they are the same or different. [Effects of the Invention]

[0021] The present invention enables the provision of polyoxyethylene derivatives containing a low content of the compound of formula (1) by analyzing with high sensitivity the compound of formula (1) contained in the polyoxyethylene derivative. Since even a small amount of the compound of formula (1) contained in the polyoxyethylene derivative used for modifying biologically relevant substances can cause impurities during the production of pharmaceuticals, the present invention is extremely useful in the quality control of pharmaceuticals. DETAILED DESCRIPTION OF THE INVENTION

[0022] The present invention will be described in detail below. Methods for detecting low-molecular-weight compounds with high sensitivity generally include UV-visible spectroscopic detectors, fluorescence detectors, and mass spectrometry detectors. However, fluorescence detectors can only detect compounds that exhibit fluorescence, and therefore cannot detect the compound of formula (1). Mass spectrometry detectors are also very expensive and have many limitations, such as the inability to use nonvolatile eluents and the risk of detector contamination when measuring high-concentration samples. On the other hand, UV-visible spectroscopic detectors are inexpensive, can be used with a variety of eluents, both volatile and nonvolatile, and are not contaminated by high-concentration samples. Therefore, they are widely used as versatile detectors. Therefore, it is preferable to use a UV-visible spectroscopic detector as the detector in the analytical method of the present invention. The wavelength used for detection (detection wavelength) is ultraviolet light with a wavelength shorter than 200 nm, preferably ultraviolet light in the 190-200 nm range, and particularly preferably ultraviolet light in the 194 nm range.

[0023] The separation mode used in the analytical method of the present invention is preferably reversed-phase chromatography, more preferably using a column packed with silica-based particles modified with octadecyl groups (C18), octyl groups (C8), phenyl groups (Ph), or triacontyl groups (C30) or similarly modified polymer-based particles. Reverse-phase chromatography using a column packed with silica-based particles modified with octadecyl groups is particularly preferred. The column used for analysis can be a stainless steel column with a length of 5 to 25 cm and an internal diameter of 1 to 6 mm, preferably a column length of 15 to 25 cm and an internal diameter of 2 to 5 mm. The particle size of the packing material is preferably 2 to 10 μm, more preferably 2 to 5 μm. The packing material is preferably fully porous or superficially porous particles, with a pore size of 5 to 50 nm, more preferably 7 to 30 nm. The column temperature is preferably 5 to 60°C, more preferably 20 to 40°C. The eluent (analytical mobile phase) used in the analysis can be a mixture of water and an organic solvent. Examples of suitable organic solvents include polar solvents miscible with water, such as acetonitrile, methanol, and 2-propanol, with acetonitrile being preferred. The organic solvent can be mixed in a ratio of 10 to 80% by volume, preferably 20 to 50%. Water and organic solvents can be premixed or mixed continuously using gradient analysis; using a premixed eluent is particularly preferred. Since the compound of formula (1) is known to react with water under neutral to basic conditions and decompose, the eluent must be acidic. An additive can be added to the eluent, preferably 0.01 to 1% of an acidic substance or its salt. If the added acidic substance has a large molar absorption coefficient in ultraviolet light at wavelengths of 190 to 200 nm, the chromatogram baseline and the peak of the compound of formula (1) may overlap, resulting in reduced peak detection sensitivity and poor analytical reproducibility. In addition, from the viewpoint of suppressing decomposition of the compound of formula (1), it is preferable that the pKa is 4 or less. The acidic substance to be added has a maximum molar absorption coefficient of 20 M or less in ultraviolet light having a wavelength of 190 to 200 nm. -1 cm -1Acidic substances with a pKa of 4 or less are preferred, and the maximum molar extinction coefficient is 5M -1 cm -1 More preferred are acidic substances with a pH of 10 or less and a pKa of 3 or less, and more preferred are phosphoric acid and perchloric acid, with phosphoric acid being the most preferred. The flow rate of the eluent is preferably 0.1 to 4 mL / min, and particularly preferably 0.2 to 1.5 mL / min.

[0024] The analytical sample of the present invention is a polyoxyethylene derivative containing a compound of formula (1). In this analytical sample preparation method, since the compound of formula (1) reacts with water and decomposes, as described above, the polyoxyethylene derivative is preferably dissolved in an aprotic organic solvent and a solvent miscible with the eluent used for analysis. Furthermore, in general, in liquid chromatography, ghost peaks that interfere with analysis may appear when the eluent and the dissolution solution of the analytical sample are different, so it is preferable to use the organic solvent used for the eluent. Furthermore, to suppress hydrolysis of the compound of formula (1), an acidic substance can be added as an additive to the dissolution solution of the analytical sample. As the acidic substance, acetic acid, formic acid, or trifluoroacetic acid (hereinafter also referred to as TFA) is preferably used, with trifluoroacetic acid being particularly preferred.

[0025] Increasing the concentration of the polyoxyethylene derivative dissolved in the solvent increases the concentration of the compound of formula (1) contained therein, improving analytical sensitivity; however, excessively high concentrations are known to result in poor peak separation. Therefore, the concentration of the polyoxyethylene derivative is preferably 50 to 300 mg / mL, with 50 to 200 mg / mL being particularly preferred. Increasing the sample injection volume during analysis increases the amount of polyoxyethylene derivative and the compound of formula (1) introduced into the column, improving analytical sensitivity. However, when the analytical sample of the present invention is dissolved in an aprotic organic solvent, increasing the sample injection volume is known to result in poor peak separation. Furthermore, a small sample injection volume results in poor reproducibility of the injection volume and poor reproducibility of the analytical results. Therefore, the sample injection volume (volume) during analysis is preferably 0.2% to 0.5% of the column volume, with 0.2% to 0.3% being particularly preferred.

[0026] When the sample concentration used in analysis is C (mg / mL), the sample injection amount I (mL), and the column volume V (mL), the sample amount per column volume is expressed as "C × I ÷ V". If this is small, sensitivity will decrease, and if it is large, peak separation will decrease. Therefore, it is preferably 0.1 to 1.5, and particularly preferably 0.1 to 0.6.

[0027] The signal-to-noise ratio (S / N ratio) can be used as an indicator of the detection sensitivity of the analytical method of the present invention. When a polyoxyethylene derivative containing 5 ppm of the compound of formula (1) is analyzed, the S / N ratio of the peak derived from the compound of formula (1) is preferably 10 or more, and more preferably 30 or more.

[0028] In the definition of formula (2), a and b are the average number of moles of oxyethylene groups added, each of which is 0 to 5,000, preferably 0 to 1,000, and a+b is 20 or more.

[0029] Examples of the compound having 2 to 8 hydroxyl groups and 2 to 21 carbon atoms, which may contain an oxygen atom and / or a nitrogen atom, in the definition of Z in formula (2) include ethylene glycol, triethanolamine, glycerin, diglycerin, tetrakis(2-hydroxyethyl)ethylenediamine, xylitol, pentaerythritol, dipentaerythritol, tripentaerythritol, hexaglycerin, etc., with ethylene glycol, glycerin, and pentaerythritol being preferred, and ethylene glycol being more preferred.

[0030] L in equation (2) 1 and L 2 The alkylene group in the definition of (1) includes, for example, a linear or branched alkylene group having 1 to 6 carbon atoms, and is preferably a methylene group, an ethylene group, a propylene group, a butylene group, or a pentylene group, and more preferably a methylene group, an ethylene group, or a pentylene group.

[0031] X in equation (2) 1 The alkoxy group in the definition of X includes, for example, a straight-chain or branched alkoxy group having 1 to 6 carbon atoms, and is preferably a methoxy group, an ethoxy group, a propoxy group, or an isopropyloxy group, and more preferably a methoxy group. 2 is an N-succinimidyloxycarbonyl group.

[0032] In the following formula (3), which is one of the preferred embodiments of the polyoxyethylene derivative of formula (2), Z in formula (2) is an ethylene glycol residue.

[0033] [ka]

[0034] In the following formula (4), which is one of the preferred embodiments of the polyoxyethylene derivative of formula (2), Z in formula (2) is a glycerin residue.

[0035] [ka]

[0036] In the following formula (5), which is one of the preferred embodiments of the polyoxyethylene derivative of formula (2), Z in formula (2) is a xylitol residue.

[0037] [ka]

[0038] In the following formula (6), which is one of the preferred embodiments of the polyoxyethylene derivative of formula (2), Z in formula (2) is a pentaerythritol residue.

[0039] [ka]

[0040] In the following formula (7), which is one of the preferred embodiments of the polyoxyethylene derivative of formula (2), Z in formula (2) is a hexaglycerin residue.

[0041] [ka]

[0042] L in equation (2) 1 and L 2 are each independently a divalent spacer, and there are no particular limitations on these spacers as long as they are groups capable of forming a covalent bond, but they are preferably ester bonds, amide bonds, ether bonds, thioether bonds, urethane bonds, urea bonds, or alkylene groups which may contain these bonds, and more preferably ester bonds, amide bonds, ether bonds, urethane bonds, or alkylene groups which may contain these bonds, and particularly preferred embodiments are those shown in group (I) below. Two to five spacers of group (I) may also be combined.

[0043] Group (I)

[0044] [ka]

[0045] In (z1) to (z10), r in the formulas represents an integer of 0 to 10 (however, r is not 0 in (z1)), preferably represents an integer of 0 to 6, and more preferably represents an integer of 0 to 5. In (z2) to (z10), each r in the formulas may be the same or different.

[0046] L in equation (2) 1 is preferably (z1), (z2), (z3), (z4), (z5), (z6), (z7), (z8), (z9), or (z10) in group (I), and more preferably (z1), (z2), (z6), or (z9).

[0047] L in equation (2) 2 is preferably (z2), (z3), (z4), (z5), (z6), (z7), (z8), (z9), or (z10) in group (I), and more preferably (z2), (z6), or (z9).

[0048] In formula (2), p is preferably 0.

[0049] Variations The present invention is not limited to the configurations described in the above embodiments, and the following configurations, for example, are also included in the present invention. For example, the skeleton of the compound of formula (4) may be the skeleton of the following formula (11), and the skeleton of the compound of formula (5) may be the skeleton of the following formula (12).The compounds of formula (11) and formula (12) can also achieve the same effects as those of the above embodiment.

[0050] [ka]

[0051] [ka] [Example]

[0052] The present invention will be described in more detail with reference to examples and comparative examples, but the present invention is not limited to the following examples.

[0053] Example 1 10.0 mg of the compound of formula (1) (β-Ala-NHS) was weighed into a 50 mL volumetric flask, dissolved in acetonitrile, and then diluted to a final volume of 0.2 mg / mL. 0.5 mL of this solution was accurately weighed into a 100 mL volumetric flask, and diluted to a final volume with acetonitrile solution containing 0.1% TFA to prepare an additive solution (0.001 mg / mL). Next, a compound of formula (8) (NOF Corporation, SUNBRIGHT ME-200HS) was added to a 1 mL volumetric flask.

[0054] [ka]

[0055] 100 mg of the compound was weighed out, and 0.5 mL of the additive solution was accurately weighed and added thereto. The solution was then diluted with acetonitrile containing 0.1% TFA to prepare an analytical sample solution (100 mg / mL, 5 ppm of β-Ala-NHS was added to the compound of formula (8)). The analytical sample solution was subjected to HPLC measurement under the following conditions. The peak height of β-Ala-NHS in the analytical sample was 2325, and the S / N ratio was 56. In Example 1, the sample concentration C (mg / mL) was 100 mg / mL, the sample injection amount I (mL) was 0.01 mL, and the column volume V (mL) was 4.15 mL, so C×I÷V=100×0.01÷4.15=0.24.

[0056] (HPLC measurement conditions) HPLC equipment: Alliance2695 (Nihon Waters Co., Ltd.) Column: Inetsil ODS-3 (inner diameter 4.6 mm, length 250 mm, particle size 5 μm) Flow rate: 1.0mL / min Analysis time: 20 minutes Column temperature: 40℃ Eluent: Water / acetonitrile = 7 / 3, containing 0.02% phosphoric acid Injection volume: 10μL Detector: UV-visible spectroscopic detector (194 nm) (Nihon Waters Co., Ltd.)

[0057] (Comparative Example 1) The analytical sample solution of Example 1 was used, and the additive in the HPLC eluent was 0.02% phosphoric acid (maximum molar extinction coefficient ε of 190-200 nm = 1.2 M -1 cm -1 , pKa=2.1) to 0.1% acetic acid (maximum molar extinction coefficient ε=38 M at 190-200 nm). -1 cm -1 , pKa=4.8), and HPLC measurement was performed under the same conditions as in Example 1 except for the additives. The peak height of β-Ala-NHS in the analytical sample solution was 769, and the S / N ratio was 2.

[0058] (Comparative Example 2) The analytical sample solution of Example 1 was used, and the additive in the HPLC eluent was 0.02% phosphoric acid (maximum molar absorption coefficient ε of 190-200 nm = 1.2 M -1 cm -1 , pKa=2.1) to 0.1% TFA (maximum molar extinction coefficient ε=216 M at 190-200 nm) -1 cm -1 , pKa=0.5), and HPLC measurement was performed under the same conditions as in Example 1 except for the additive. The peak height of β-Ala-NHS in the analytical sample solution was 540, and the S / N ratio was 2.

[0059] Example 2 When preparing the analytical sample solution, the amount of the compound of formula (8) weighed out was changed from 100 mg to 30 mg, and the amount of the additive solution was changed from 0.5 mL to 0.15 mL, thereby changing the sample concentration prepared from 100 mg / mL (β-Ala-NHS added at 5 ppm relative to the compound of formula (8)) to 30 mg / mL (β-Ala-NHS added at 5 ppm relative to the compound of formula (8)), and HPLC measurement was performed under the same conditions as in Example 1. The peak height of β-Ala-NHS in the analytical sample at this time was 698, and the S / N ratio was 11. In Example 2, the sample concentration C (mg / mL) was 30 mg / mL, the sample injection amount I (mL) was 0.01 mL, and the column volume V (mL) was 4.15 mL, so C×I÷V=30×0.01÷4.15=0.07.

[0060] [Table 1]

[0061] Example 3 Compounds (8) to (9) of Example 1

[0062] [ka]

[0063] The peak height of β-Ala-NHS in the sample was 2520 and the S / N ratio was 57.

[0064] Example 4 Compounds (8) to (10) of Example 1

[0065] [ka]

[0066] The peak height of β-Ala-NHS in the sample was 2210 and the S / N ratio was 54. [Industrial Applicability]

[0067] The present invention provides a highly sensitive analytical method for low molecular weight compounds contained in polyoxyethylene derivatives.

[0068] This application is based on patent application No. 2021-115693 filed in Japan (filing date: July 13, 2021), the contents of which are incorporated in their entirety into this specification.

Claims

1. Polyoxyethylene derivatives include those represented by formula (1) 【Chemistry 1】 This is an analytical method in which the compound is separated by reversed-phase chromatography and then detected by an ultraviolet-visible spectrophotometer. The detection wavelength is shorter than 200 nm, and an additive to the mobile phase for analysis is a compound having a maximum molar absorption coefficient of 20 M or less at 190-200 nm. －１ cm －１ An analytical method characterized by using an acidic substance having a pH of 0.5 or less and a pKa of 4 or less.

2. The polyoxyethylene derivative is represented by the formula (2) 【Chemistry 2】 (wherein Z is a residue of a compound having 2 to 8 hydroxyl groups and 2 to 21 carbon atoms, which may contain oxygen and / or nitrogen atoms; OA １ and O.A. ２ is an oxyethylene group, a and b are each independently 0 to 5,000 and a+b is 20 or more, and L １ and L ２ are each independently an alkylene group which may have a bond selected from an ester bond, a urethane bond, an amide bond, a urea bond, an ether bond, and a thioether bond in the alkylene chain or at the terminal thereof; p and q are each independently 0 or 1; s is 0 to 8; t is 1 to 8, and 2≦s+t≦8; X １ is an alkoxy group or an N-succinimidyloxycarbonyl group, and X ２ is an N-succinimidyloxycarbonyl group. a, b, OA １ , O.A. ２ , p, q, L １ , L ２ and X １ 2. The analytical method according to claim 1, wherein, when a plurality of each of the above is present in one molecule, they are the same or different.

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