WO2014157670A1 - 液体クロマトグラフィー用カチオン交換体、その製造方法及びその用途 - Google Patents
液体クロマトグラフィー用カチオン交換体、その製造方法及びその用途 Download PDFInfo
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- WO2014157670A1 WO2014157670A1 PCT/JP2014/059296 JP2014059296W WO2014157670A1 WO 2014157670 A1 WO2014157670 A1 WO 2014157670A1 JP 2014059296 W JP2014059296 W JP 2014059296W WO 2014157670 A1 WO2014157670 A1 WO 2014157670A1
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- WIPO (PCT)
- Prior art keywords
- liquid chromatography
- cation exchanger
- particles
- polyacrylic acid
- acid
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Classifications
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/18—Ion-exchange chromatography
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation
Definitions
- the present invention relates to a cation exchanger for liquid chromatography having a high holding power, a sufficient adsorption capacity for a target, a high resolution and a mechanical strength for a target to be separated and analyzed, and a low operating pressure.
- a cation exchanger for liquid chromatography.
- a target substance such as protein (also simply referred to as a target).
- a target substance such as protein (also simply referred to as a target).
- It has a sufficient target adsorption capacity (adsorption capacity) to maintain the separation performance of trace components.
- High resolution and selectivity for separation / analysis accuracy improvement (separation and selectivity).
- It has a mechanical strength that can withstand a high operating flow rate in order to shorten the separation and analysis time (mechanical strength).
- Low operating pressure that does not cause a decrease in operating flow rate (operating pressure).
- the cation exchanger for liquid chromatography is obtained by introducing a cation exchange group (ligand) into particles constituting the exchanger.
- a cation exchange group ligand
- the particles for the purpose of improving the separation and selectivity in (3) above, use non-porous particles without intra-particle diffusion (see Non-Patent Document 1) or particles having an average particle size of about 10 ⁇ m. It is proposed to use. With regard to the average particle size, further micronization has progressed, and it has recently been proposed to use particles having an average particle size of 5 ⁇ m or less. In order to prevent such a long separation / analysis time due to the use of fine particles, it is necessary to apply a higher operating pressure, and the mechanical strength of (4) required for the particles is about 100 MPa. ing.
- a cation exchange group a sulfonic acid group, a carboxyl group, a phosphoric acid group, etc. are known conventionally. Among them, the dissociation degree of the carboxyl group is easily changed depending on the pH of the eluent, and exhibits a specific adsorption behavior such as formation of a hydrogen bond. Therefore, the selectivity easily changes depending on the elution conditions.
- a strong cation exchange type such as a sulfonic acid type is widely used for separation of protein isomers and the like because it can separate difficult samples.
- a method for introducing a carboxylic acid group into a particle a method in which a monomer such as acrylic acid or methacrylic acid and a crosslinkable monomer are copolymerized, or a method in which a halogenated alkylcarboxylic acid is desalted and condensed with an alkali on a crosslinked particle having a hydroxyl group.
- a method of introducing a compound having a polyfunctional carboxyl group such as polyacrylic acid using particles having a functional group capable of binding to a carboxylic acid such as an epoxy group, an amino group, or a hydroxyl group, and using a heating or condensing agent.
- Non-porous particles having a synthetic polymer as a skeleton can achieve the mechanical strength of (4) as well as the separation and selectivity of (3) above. Therefore, it is conceivable to apply a conventional cation exchange group introduction method to non-porous polymer particles.
- synthetic polymer particles generally comprise a co-polymer of a monomer having a functional group for later introducing a cation exchange group on the particle surface and a polyfunctional unsaturated crosslinking monomer for increasing the mechanical strength of the particle. It manufactures using a polymer.
- the amount of functional groups on the particle surface decreases, and a sufficient amount of cation exchange groups cannot be introduced on the particle surface.
- the cation exchange group density on the surface decreases.
- the retention force of the target becomes insufficient (the retention force of (1) above cannot be achieved), and due to the small surface area due to the non-porous property, in particular, biopolymers such as proteins and nucleic acids, Therefore, the adsorption capacity of (2) is impaired, and the target elution peak becomes broad.
- Patent Documents 1 to 4 A method of immobilizing a water-soluble polymer in the pores (see Patent Document 5) has been proposed.
- the polymer chain is fixed to the particle surface by grafting, the operating pressure is increased, and the operating flow velocity is reduced against the operating pressure of (5) above.
- the method of fixing the water-soluble polymer in the pores cannot be applied to non-porous particles.
- the object of the present invention can meet the improvement of the characteristics (1) to (5) described above, and in particular, the liquid chromatograph (3) with improved separation and selectivity. It is to provide a cation exchanger for lithography.
- the present inventor has found that a cation exchanger for liquid chromatography in which polyacrylic acid into which a dicarboxylic acid compound having an amino group is introduced is substantially fixed on the surface of non-porous particles. Have a high holding power for the object, a sufficient adsorption capacity of the object, and a sufficient mechanical strength.
- charge isomers such as antibodies
- the inventors have found that they exhibit high separation performance and selectivity, and have a low operating pressure, thereby completing the present invention. That is, the present invention has the following gist.
- a cation exchanger for liquid chromatography characterized in that polyacrylic acid in which a dicarboxylic acid compound having an amino group is introduced by an amide bond is fixed on the surface of non-porous particles.
- the non-porous particles are silica, zirconia or alumina inorganic base material, or a cross-linked polysaccharide or vinyl monomer cross-linked organic base material having a volume average particle diameter of 5 ⁇ m or less.
- Cation exchanger for lithography 3.
- amidation condensing agent is 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl morpholinium chloride
- a method for producing a cation exchanger is 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl morpholinium chloride
- a column for liquid chromatography comprising the cation exchanger for liquid chromatography according to any one of 1 to 6 above.
- 14 14.
- 15. 14 A method for measuring glycohemoglobin by ion exchange chromatography using the liquid chromatography column as described in 13 above.
- the cation exchanger of the present invention can improve the above characteristics (1) to (5) as compared with the conventional cation exchange pair, in particular, the adsorption capacity of (2) and the separation of (3), Selectivity can be greatly improved.
- a vinyl monomer copolymer (crosslinked product) is used as the particles of the present invention, a mechanical strength that can withstand 100 MPa can be realized, and the mechanical strength of (4) can be improved.
- Separation, selectivity and (5) operating pressure can be improved.
- the method of introducing a dicarboxylic acid having an amino group after introducing polyacrylic acid improves the operating pressure of (5) because the operating pressure increase due to polymer introduction is small, and can be measured at a faster flow rate. 3) Separation, selectivity and (4) mechanical strength can be improved.
- Example 2 is a chromatogram of Examples 1 to 3 and Comparative Examples 1 to 4. It is a chromatogram of Example 2 and Comparative Example 4. It is a chromatogram of Example 1 and Comparative Example 4.
- the cation exchanger for liquid chromatography of the present invention is based on non-porous particles.
- non-porous in the present invention includes not only particles having no micropores, but also particles having pores of a size that cannot be separated by an object to be separated using a cation exchanger.
- the average diameter of the holes is preferably 20 mm or less, and more preferably 10 mm or less.
- Methods for controlling the size of the pores of the particles are known in the prior art, for example US Pat. No. 4,382,124.
- the non-porous particles preferably have a volume average particle diameter (D50) of 5 ⁇ m or less, more preferably 1.5 to 3.0 ⁇ m, particularly for the purpose of improving separation and selectivity in (3).
- the base material for the non-porous particles examples include an inorganic base material (for example, silica, zirconia, alumina, etc.), an organic base material (for example, a cross-linked polysaccharide, a vinyl monomer cross-linked body such as acrylamide, acrylic ester, styrene). If it is good.
- an inorganic base material a silica base material is preferable from a viewpoint of the cation exchange property which the base material has, and as an organic base material, a crosslinked hydrophilic (meth) acrylic acid ester is preferable from the viewpoint of hydrophilicity and micropores. .
- organic base particles include monofunctional vinyl monomers (glycidyl methacrylate, vinyl benzyl glycidyl ether, etc.) and polyfunctional vinyl monomers disclosed in, for example, Japanese Patent Publication No. 58-58026 and Japanese Unexamined Patent Publication No. 53-90991.
- Ethylene glycol dimethacrylate, polyethylene glycol dimethacrylate, glycene polymethacrylate, divinylbenzene, etc. a method by suspension polymerization of a mixed solution, or a seed combined with the monomer described in Japanese Patent Application Laid-Open No. 2001-2716 It can be produced by polymerization.
- a copolymer of a monofunctional vinyl monomer and a polyfunctional vinyl monomer is preferable.
- the ratio of the polyfunctional vinyl monomer is preferably 5% by weight or more, more preferably 15 to 30% by weight.
- a functional group for introducing a sufficient amount of carboxylic acid groups and fixing polyacrylic acid in which a dicarboxylic acid compound having an amino group is introduced particularly
- the ratio of the polyfunctional vinyl monomer is preferably 50% by weight or less, and more preferably 15 to 30% by weight.
- a copolymer of a monofunctional vinyl monomer having an epoxy group such as glycidyl methacrylate, and a polyfunctional vinyl monomer
- the epoxy group is directly used or the epoxy group is hydrolyzed to open the ring, Hydroxylation can be performed by hydrophilizing with a functional polyhydric alcohol or the like.
- an epoxy group may be introduced using a polyfunctional epoxy compound such as epichlorohydrin or ethylene glycol diglycidyl ether, and then reacted in the same manner as described above. Is possible.
- the cation exchanger of the present invention has a carboxylic acid group as a cation exchange group introduced on its surface and a polyacrylic acid in which a dicarboxylic acid compound having an amino group is introduced by an amide bond. is there.
- a polycarboxylic acid polymer such as polyacrylic acid is first introduced using an epoxy group on the surface, and a dicarboxylic acid having an amino group is introduced into the polycarboxylic acid polymer introduced on the surface using a condensing agent such as carbodiimide. It is obtained by doing.
- the polyacrylic acid introduced by the amide bond of the dicarboxylic acid compound having an amino group to the carboxyl group of the polyacrylic acid includes:
- the average molecular weight is preferably in the range of 5,000 to 20,000, more preferably 5,000 to 10,000. If the molecular weight is small, the effect of improving the characteristics of (1) to (3) is small, and if the molecular weight is too large, the operating pressure increases and the effect of improving the characteristic of (5) is small.
- the polyacrylic acid in which the dicarboxylic acid compound having an amino group is introduced by an amide bond can be introduced into the particles after previously introducing the dicarboxylic acid compound having an amino group into the polyacrylic acid.
- a method in which polyacrylic acid is introduced into the particle in advance and then a dicarboxylic acid compound having an amino group is introduced into the polyacrylic acid on the particle surface using an amidation condensing agent such as carbodiimide is preferable. is there. According to this method, an effect that the operation pressure is further reduced can be obtained.
- the dicarboxylic acid compound having an amino group it is preferable to use acidic amino acids such as aspartic acid and glutamic acid.
- the dicarboxylic acid compound having an amino group can be introduced into polyacrylic acid using an amidation condensation agent of carboxylic acid.
- an amide such as 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methyl morpholinium chloride
- the organic solvent include water-soluble alcohols such as methanol, ethanol, and propanol, dimethyl sulfoxide, dimethyl sulfoamide, and chloroform.
- dicarboxylic acid having an amino group is hardly soluble in an organic solvent
- a method using triazin-2-yl) -4-methyl morpholinium chloride is preferred.
- dicarboxylic acids having amino groups are generally poorly soluble in organic solvents
- 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride hereinafter referred to as “condensation in an aqueous solvent”
- a method using a water-soluble carbodiimide such as WSC) is also suitable.
- the carboxyl group of polyacrylic acid is changed to an active ester type such as N-hydroxysuccinimide, and then an active ester and a dicarboxylic acid having an amino group are used. It is further preferred to react with the compound.
- a functional group capable of reacting with the carboxyl group of polyacrylic acid is added to the particles and bonded.
- the functional group capable of reacting with a carboxyl group include an amino group, a hydroxyl group, and an epoxy group.
- particles having amino groups are used, a method using an amidation condensing agent such as carbodiimide, and when particles having hydroxyl groups are used, a method of dehydration condensation by heating, or particles having epoxy groups are used. In such a case, a method of bonding under an alkali catalyst can be exemplified.
- Examples of the amidation condensing agent in the case of using particles having an amino group include 4- (4,6-dimethoxy-1,3,5-triazin-2-yl) -4-methylmoriphonium chloride, WSC and the like. Can be mentioned. Furthermore, sodium hydroxide, pyridine, etc. are mentioned as an alkali catalyst at the time of using the particle
- the reaction can be carried out in a solvent capable of dissolving polyacrylic acid, but after dissolving polyacrylic acid in a volatilizable solvent and dispersing the particles, the solvent is distilled off with an evaporator, etc.
- the method of bonding the carboxylic acid of polyacrylic acid and the functional group of the particles by heating does not necessarily require a catalyst or the like, and can introduce polyacrylic acid, which is preferable in terms of simplicity and introduction efficiency It is.
- the functionalities possessed by the particles It is preferable to use a hydroxyl group or an epoxy group as the group because the reaction conditions are mild. Specifically, 2 to 10% by weight, preferably 5 to 10% by weight of the polyacrylic acid or diacrylic acid compound having a dicarboxylic acid compound having an amino group is dissolved in water. It is preferable to adjust the pH to 5 to 10, preferably 6 to 8, disperse a predetermined amount of particles, distill water off, and then heat.
- the heating temperature is preferably 160 to 200 ° C. when the functional group of the particle is a hydroxyl group, and preferably 110 to 150 ° C. when the functional group is an epoxy group, and is preferably heated for 1 hour or longer.
- the amount of polyacrylic acid in which polyacrylic acid or a dicarboxylic acid compound having an amino group is introduced to the non-porous particles is set so that the cation exchange capacity of the cation exchanger is 5 to 50 ⁇ m for the purpose of improving (5). It is preferable to adjust in the range of equivalents / mL (meq / L), preferably 20 to 40 equivalents / mL (meq / L). By controlling the amount of such immobilization, a cation exchanger having low operating pressure can be realized.
- the recovery rate of the measurement sample may be reduced if an epoxy group that has not been used for fixing polyacrylic acid remains.
- a compound capable of reacting with an epoxy group and convert the remaining epoxy group into a chemical structure that does not adversely affect ion exchange between the filler and the sample.
- the compound capable of reacting with the epoxy group include water, polyhydric alcohols having a hydroxyl group such as ethylene glycol, glycerin, glucose, etc., giving a betaine structure, glycine, alanine, aspartic acid, glutamic acid, lysine, arginine, taurine, etc.
- polyacrylic acid into which a dicarboxylic acid compound having an amino group is introduced is substantially fixed on the surface of non-porous particles.
- Non-porous particles were produced by the method described in Japanese Patent Publication No. 2001-2716 (seed polymerization method). 30 g of benzyl methacrylate and 1.5 g of 2-ethylhexyl mercaptoacetate were mixed in a 500 mL three-necked flask, and 300 g of ion-exchanged water was added. Next, a magnetic stir bar was placed in the flask, immersed in an oil bath set at 72 ° C., a nitrogen introduction tube was installed, and stirring was performed at 150 rpm.
- 2,3 epoxypropyl methacrylate 64 g, ethylene dimethacrylate 16 g, 2,2′-azobis (2,4-dimethylvaleronitrile) (trade name V-65, manufactured by Wako Pure Chemical Industries, Ltd.) 0.2 g and sodium dodecyl sulfate 0.2 g was weighed into a 300 mL flask, and a magnetic stir bar was added and mixed with a magnetic stirrer. Furthermore, 100 mL of ion exchange water was added and emulsified with an ultrasonic homogenizer while stirring with a magnetic stirrer.
- the obtained non-porous particles are put into a 500 mL separable flask, 300 mL of ion exchange water is added, immersed in an oil bath set at 90 ° C., and heated for 24 hours with stirring to hydrolyze the epoxy groups of the particles. Went.
- the non-porous particles after hydrolyzing the epoxy group were particles having a uniform volume average particle diameter of 2.3 ⁇ m as observed by electron microscope observation.
- non-porous particles 100 g water suction dry
- epichlorohydrin 103 g and ion-exchanged water 100 g were put into a 500 mL separable flask, immersed in a water bath set at 45 ° C. and stirred.
- 88 g of 48% by weight aqueous sodium hydroxide solution was weighed, placed in a disposable syringe, placed in a syringe pump, and charged into a separable flask with stirring at a rate of 0.5 mL / min.
- reaction product was filtered through a glass filter, washed with water and acetone in this order, dried by ventilation, and epoxidized non-porous particles (epoxy equivalent 840 ⁇ equivalent / dry gel (g)) )
- the pore diameter of these particles was 200 or less in terms of polyethylene glycol and 10 or less.
- Example 1 ⁇ Introduction of polyacrylic acid> 0.75 g of polyacrylic acid (weight average molecular weight 5000, manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 50 mL of ion-exchanged water, and adjusted to pH 6.8 using 2N sodium hydroxide aqueous solution. Next, 10 g of epoxidized non-porous particles were added and dispersed in an ultrasonic water bath, then placed in a 200 mL eggplant flask, and water was distilled off with an evaporator. Thereafter, the eggplant flask was placed in an oven set at 130 ° C.
- the particles dispersed in water were heated in an autoclave at 140 ° C. for 3 hours to add water to the remaining epoxy groups.
- the amount of epoxy groups by the dioxane-hydrochloric acid method was 20 ⁇ equivalent / dry gel (g) or less.
- the particles were washed with 0.1 mol / L hydrochloric acid and water in this order, and titrated with 0.01 N sodium hydroxide. As a result, an ion exchange capacity of 25 ⁇ eq / mL was confirmed.
- MES 20 mmol / L morpholinoethane sulfonic acid
- the obtained cation exchanger was slurry-filled in a 4.6 mm inner diameter ⁇ 100 mm length liquid chromatography column using 10% ion exchange water as a dispersion. Using the obtained column, liquid chromatography was performed under the following conditions to measure the separation performance of the monoclonal antibody.
- the operating pressure of the cation exchanger was the operating pressure when the eluent A was fed at a flow rate of 0.6 mL / min.
- liquid chromatography was performed under the following conditions to measure the separation performance of glycated hemoglobin.
- Liquid chromatography conditions Eluent A: 20 mmol / L sodium phosphate buffer (pH 6.3)
- Eluent B 20 mmol / L sodium phosphate buffer (pH 6.3) containing 0.20 mol / L NaClO4
- Gradient 0-100% eluent B linear gradient, 10 minutes
- Flow rate 0.6 mL / min
- Detection UV 415 nm
- Target HbA1c calibrator 2 (manufactured by Tosoh Corporation) 5 ⁇ L
- the chromatogram was as shown in FIG. 3, and it was found that the separation of isomers was superior compared to the comparative example.
- Example 2 After fixing polyacrylic acid in the same manner as in ⁇ fixing polyacrylic acid> in Example 1, aspartic acid was introduced by the following method.
- the particles washed by centrifugal filtration three times with 0.5N sodium hydroxide and three times with 0.5N hydrochloric acid were collected as an ion-exchanged water suction dry gel.
- the ion exchange capacity of the obtained particles was 32 ⁇ eq / mL.
- the obtained cation exchanger was slurry-packed in a liquid chromatography column having a diameter of 4.6 mm and a length of 100 mm using 10% ion-exchanged water as a dispersion. Using the obtained column, liquid chromatography was performed under the same conditions as in Example 1 to measure the separation performance of the monoclonal antibody.
- the operating pressure of the cation exchanger was the operating pressure when the eluent A was fed at a flow rate of 0.6 mL / min.
- the chromatograms are shown in FIG. 1 and FIG. 2 (enlarged comparative diagram with elution time corrected together with Comparative Example 4). It was found that the retention was stronger than in the comparative example and the separation performance between isomers was excellent.
- the elution time difference from the acidic elution peak 1 to the basic elution peak 2 was 20.3 minutes, indicating a wide selectivity.
- the operating pressure was as low as 17.0 MPa.
- Example 3 After introducing polyacrylic acid by the same method as in ⁇ fixing polyacrylic acid> in Example 1, glutamic acid was introduced by the following method.
- the particles washed by centrifugal filtration three times with 0.5N sodium hydroxide and three times with 0.5N hydrochloric acid were collected as an ion-exchanged water suction dry gel.
- the obtained particles had an ion exchange capacity of 29 ⁇ eq / mL.
- the obtained cation exchanger was slurry-packed in a liquid chromatography column having a diameter of 4.6 mm and a length of 100 mm using 10% ion-exchanged water as a dispersion. Using the obtained column, liquid chromatography was performed under the same conditions as in Example 1 to measure the separation performance of the monoclonal antibody.
- the operating pressure of the cation exchanger was the operating pressure when the eluent A was fed at a flow rate of 0.6 mL / min.
- the chromatogram is as shown in FIG. 1, and it was found that the retention was stronger than that of the comparative example and the separation performance between isomers was excellent.
- the elution time difference from acidic elution peak 1 to basic elution peak 2 was 25.9 minutes, indicating a wide selectivity.
- the operating pressure was as low as 19.0 MPa.
- Comparative Example 1 The epoxidized non-porous particles obtained in the preparation of the non-porous substrate were dispersed in water, and the epoxy groups were converted to diol groups by heating in an autoclave at 140 ° C. for 3 hours. 4.5 g of suction-dried particles were dispersed in 8 g of ion-exchanged water, and 3.0 g of sodium ⁇ -chloroacetate was added to dissolve and disperse. Further, 2.7 g of 48% by weight sodium hydroxide was added, and the mixture was shaken in a water bath set at 55 ° C. for 2 hours.
- the obtained particles were washed with a glass filter in the order of 0.1 mol / L phosphoric acid, 0.1 mol / L sodium hydroxide and ion-exchanged water.
- the obtained particles had an ion exchange capacity of 36 ⁇ eq / mL.
- a liquid chromatography column was packed in the same manner as in Example 1, the measurement conditions were the same as in Example 1, and the separation performance of monoclonal antibodies was compared.
- the chromatogram was as shown in FIG. 1. The retention was weak, the peak was broad, and the separation performance between isomers was poor.
- the elution time difference from the acidic elution peak 1 to the basic elution peak 2 was 5.2 minutes, which was lower than that of Example 1.
- the operating pressure was 15.0 MPa and a low operating pressure.
- Comparative Example 2 Using the cation exchanger particles produced in Comparative Example 1, the same operation as in ⁇ Introduction of Aspartic Acid> in Example 1 was performed to obtain a cation exchanger into which aspartic acid was introduced. The obtained particles had an ion exchange capacity of 34 ⁇ eq / mL. A liquid chromatography column was packed in the same manner as in Example 1, the measurement conditions were the same as in Example 1, and the separation performance of monoclonal antibodies was compared. The chromatogram was as shown in FIG. 1. The retention was weak and the separation performance between isomers was not sufficient. The elution time difference from the acidic elution peak 1 to the basic elution peak 2 was 4.4 minutes, which was lower than that of Example 1. The operating pressure was 18.0 MPa, which was a relatively low operating pressure.
- Comparative Example 3 9 g of aspartic acid was added to an aqueous solution adjusted to pH 12.8 using an aqueous 0.2 mol / L sodium hydroxide solution, and 3 g of the epoxidized particles obtained in the preparation of the non-porous substrate were added, and the mixture was heated at 50 ° C. for 2 hours. Then, the mixture was heated in a water bath to obtain a cation exchanger in which aspartic acid was introduced into the epoxy group. The obtained particles had an ion exchange capacity of 42 ⁇ eq / mL.
- a liquid chromatography column was packed in the same manner as in Example 1, the measurement conditions were the same as in Example 1, and the separation performance of monoclonal antibodies was compared.
- the chromatogram was as shown in FIG. 1. The retention was weak and the separation performance between isomers was not sufficient.
- the elution time difference from the acidic elution peak 1 to the basic elution peak 2 was 5.4 minutes, which was lower than that of Example 1.
- the operating pressure was 14.0 MPa, which was a relatively low operating pressure.
- Comparative Example 4 Using the cation exchanger in which only the operation of ⁇ fixing polyacrylic acid> in Example 1 was performed, the column for liquid chromatography was packed in the same manner as in Example 1, and the measurement conditions were the same as in Example 1. The separation performance of monoclonal antibodies was compared. The chromatogram is as shown in FIG. 1 and FIG. 2 (enlarged comparative diagram with elution time corrected together with Example 2). Compared with Comparative Examples 1 and 2, the retention was stronger and the separation performance between isomers was also improved. However, the elution time difference from the acidic elution peak 1 to the basic elution peak 2 was 13.6 minutes, which was lower than in Examples 1 and 2. The operating pressure was as high as 30.0 MPa. Moreover, the separation performance of glycohemoglobin was compared as in Example 1. The chromatogram is as shown in FIG. 3, and the separation performance of isomers was insufficient.
- the cation exchanger of the present invention is useful as a cation exchanger for liquid chromatography in a wide range of fields, and is particularly used for separation and purification of biological samples such as various proteins including antibodies and glycohemoglobin. It should be noted that the entire content of the specification, claims, drawings and abstract of Japanese Patent Application No. 2013-0775020 filed on March 29, 2013 is cited herein as the disclosure of the specification of the present invention. Incorporated.
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Abstract
Description
(1)試料中に含まれる塩濃度の影響を受けにくくするために、タンパク質等の対象物質(単に、対象ともいう)に対して高い保持力を有する(保持力)。
(2)微量成分の分離性能を保つために十分な対象の吸着容量を有する(吸着容量)。
(3)分離・分析精度向上のために高い分離能、選択能を有する(分離、選択能)。
(4)分離・分析時間の短縮のために速い操作流速に耐える機械強度を有する(機械的強度)。
(5)操作流速の低下を招かない低操作圧である(操作圧)。
平均粒子径については更なる微粒子化が進み、平均粒子径5μm以下の粒子を使用することが近年では提案されている。このような微粒子の使用に伴う分離・分析時間の長時間化を防止するために、より高い操作圧を負荷する必要が生じ、粒子に求められる上記(4)の機械的強度は100MPa程度になっている。
スルホン酸型の様な強カチオン交換型では、分離困難な試料の分離を成しうることから、タンパク質異性体の分離等に広く用いられている。
カルボン酸基の粒子への導入方法としては、アクリル酸、メタクリル酸等のモノマーと架橋性モノマーを共重合させる方法や、水酸基を有する架橋粒子にハロゲン化アルキルカルボン酸をアルカリにより脱塩縮合させる方法、ポリアクリル酸などの多官能カルボキシル基を有する化合物を、エポキシ基、アミノ基、水酸基等のカルボン酸と結合可能である官能基を有する粒子と、加熱又は縮合剤等を用いて導入する方法が知られている。
上記(2)の吸着容量及び(5)の操作圧の改善を目的として、カルボン酸基を有する共重合ポリマーと、カルボン酸基と反応可能な官能基を有するポリマーを、粒子の表面に部分的架橋によって固定する方法も提案されている(特許文献6参照)。
すなわち、本発明は以下の要旨を有するものである。
2.前記非多孔性粒子が、体積平均粒子径5μm以下を有する、シリカ、ジルコニア若しくはアルミナの無機基材、又は架橋多糖若しくはビニル系モノマー架橋重合体の有機基材である、上記1に記載の液体クロマトグラフィー用カチオン交換体。
3.前記有機基材が、単官能性ビニルモノマーと多官能性ビニルモノマーの共重合体である、上記2に記載の液体クロマトグラフィー用カチオン交換体。
4.前記ポリアクリル酸は、分子量が5、000以上20、000以下である、上記1に記載の液体クロマトグラフィー用カチオン交換体。
5.前記アミノ基を有するジカルボン酸化合物が、アスパラギン酸又はグルタミン酸である上記1に記載の液体クロマトグラフィー用カチオン交換体。
6.前記カチオン交換体のイオン交換容量が、10~50μ当量/mLである上記1に記載の液体クロマトグラフィー用カチオン交換体。
8.前記非多孔性粒子が、平均粒子径が5μm以下の、シリカ、ジルコニア若しくはアルミナの無機基材、又は架橋多糖若しくはビニル系モノマー架橋体の有機基材である、上記(7)に記載の液体クロマトグラフィー用カチオン交換体を製造する方法。
9.溶媒として水を使用する、上記7又は8に記載の液体クロマトグラフィー用カチオン交換体を製造する方法。
11.前記ポリアクリル酸が導入された粒子と、アミノ基を有するジカルボン酸化合物とを反応させる触媒として、さらにアミド化縮合剤を用いる、上記7乃至10のいずれかに記載の液体クロマトグラフィー用カチオン交換体を製造する方法。
12.前記アミド化縮合剤が、4-(4,6-ジメトキシ-1,3,5-トリアジン-2-イル)-4-メチルモリホニウム クロリドである、上記(11)に記載の液体クロマトグラフィー用カチオン交換体を製造する方法。
14.上記13に記載の液体クロマトグラフィー用カラムを用い、イオン交換クロマトグラフィーにより抗体の電荷異性体を測定する方法。
15.上記13に記載の液体クロマトグラフィー用カラムを用い、イオン交換クロマトグラフィーによりグリコヘモグロビンを測定する方法。
本発明の粒子として、ビニルモノマー共重合体(架橋体)を使用する場合、100MPaに耐えうる機械強度が実現でき、(4)の機械的強度の改善が可能であり、また、(3)の分離、選択能及び(5)の操作圧が改善し得る。特にポリアクリル酸を導入した後、アミノ基を有するジカルボン酸を導入する手法は、ポリマー導入による操作圧増大が小さいために(5)の操作圧が改善され、より速い流速で測定できることから、(3)の分離、選択能及び(4)の機械的強度の改善ができる。
非多孔性粒子は、特に(3)の分離、選択能の改善を目的として、体積平均粒子径(D50)を5μm以下とすることが好ましく、1.5~3.0μmがより好ましい。
特に、有機基材の粒子は、例えば日本特公昭58-58026号や日本特開昭53-90991号に開示の、単官能ビニルモノマー(グリシジルメタクリレート、ビニルベンジルグリシジルエーテル等)と、多官能ビニルモノマー(エチレングリコールジメタクリレート、ポリエチレングリコールジメタクリレート、グリセンポリメタクリレート、ジビニルベンゼン等)を組み合わせた混合液の懸濁重合による方法、あるいは日本特開2001-2716号に記載の、前記モノマーを組み合わせたシード重合による方法で製造できる。
エポキシ基を有していない共重合体であれば、例えばエピクロロヒドリン、エチレングリコールジグリシジルエーテル等の多官能エポキシ化合物等を用いてエポキシ基を導入した後、上記と同様に反応させることが可能である。
上記(1)~(3)及び(5)の特性を改善するために、アミノ基を有するジカルボン酸化合物がポリアクリル酸のカルボキシル基とアミド結合することにより導入されたポリアクリル酸としては、重量平均分子量が5、000~20、000の範囲のものが好ましく、5、000~10,000以下がより好ましい。分子量が小さいと(1)~(3)の特性の改善効果が小さく、分子量が大すぎると、操作圧が上昇して、(5)の特性の改善効果が小さくなる。
アミノ基を有するジカルボン酸化合物としては、好ましくはアスパラギン酸、グルタミン酸等の酸性アミノ酸を用いることが好適である。
有機溶媒としては、メタノール、エタノール、プロパノール等の水溶性アルコール類、ジメチルスルホキシド、ジメチルスルホアミド、クロロホルム等が挙げられる。
また、アミノ基を有するジカルボン酸は一般的に有機溶媒へ難溶であるため、水溶媒中での縮合が可能である、1-エチル-3-(3-ジメチルアミノプロピル)カルボジイミド塩酸塩(以下、WSCともいう。)等の水溶性カルボジイミドを用いる方法も好適である。カルボジイミドを用いる場合は、アミノ基を有するジカルボン酸化合物の重合を禁止するため、ポリアクリル酸のカルボキシル基をN-ヒドロキシスクシンイミド等の活性エステル型とした後、活性エステルと、アミノ基を有するジカルボン酸化合物とを反応させることがさらに好適である。
カルボキシル基と反応可能である官能基としては、アミノ基、水酸基、エポキシ基等を挙げることができる。
アミノ基を有する粒子を用いた場合には、前記カルボジイミド等のアミド化縮合剤を用いる方法、水酸基を有する粒子を用いた場合には、加熱により脱水縮合する方法、またエポキシ基を有する粒子を用いた場合にはアルカリ触媒下で結合させる方法を例示できる。
アミノ基を有する粒子を用いた場合のアミド化縮合剤としては、4-(4,6-ジメトキシ-1,3,5-トリアジン-2-イル)-4-メチルモリホニウム クロリド、WSC等が挙げられる。さらに、エポキシ基を有する粒子を用いた場合のアルカリ触媒としては、水酸化ナトリウム、ピリジン等が挙げられる。
具体的には、粒子に対して2~10重量%、好ましくは5~10重量%のポリアクリル酸もしくはアミノ基を有するジカルボン酸化合物が導入されたポリアクリル酸を水に溶解した後、溶液のpHを5~10、好ましくは6~8に調製し、所定量の粒子を分散後、水を留去した後に加熱するのが好ましい。加熱温度は、粒子の官能基が水酸基の場合、160~200℃が好ましく、官能基がエポキシ基の場合、110~150℃が好ましく、1時間以上加熱するのが好ましい。
反応形態としては、残存するエポキシ基が試料と相互作用ができなくなれば良く、大過剰のエポキシ基に導入する化合物を用い、一般的な反応条件下で、必要に応じて触媒を用いることができる。
日本特許公開2001-2716号に記載された方法(シード重合法)により、非多孔性粒子を製造した。
ベンジルメタクリレート30g及びメルカプト酢酸2-エチルヘキシル1.5gを500mL三つ口フラスコに入れて混合し、イオン交換水を300g投入した。次いで、フラスコ内にマグネティック撹拌子を入れ、72℃に設定したオイルバスに浸し、窒素導入管を設置して、150rpmで撹拌した。これとは別に、50mL容器に過硫酸カリウム0.9g及びイオン交換水30gを計り取り溶解した。30分経過後、三つ口フラスコに設置したゴム栓から、過硫酸カリウム水溶液を注射器で投入した。回転数を300rpmとしてソープフリー乳化重合を実施した。2時間重合を継続した後、凝集分を取り除いてシード溶液を回収した。シード溶液の固形分含有率は、5.16重量%であり、体積平均粒子径(D50)は電子顕微鏡による測定で0.48μmであった。
<ポリアクリル酸の導入>
ポリアクリル酸(重量平均分子量5000、和光純薬工業社製)0.75gをイオン交換水50mLに溶解し、2N-水酸化ナトリウム水溶液を用いてpH6.8に調整した。次いで、エポキシ化非多孔性粒子10gを投入し、超音波水浴で分散した後、200mLナスフラスコに入れ、エバポレータ―にて、水分を留去した。その後、130℃に設定したオーブンにナスフラスコを入れ、3時間加熱した後、ナスフラスコ中に50mLのイオン交換水を加えて粒子を分散した。ガラスフィルターを用いてろ過した後、0.1mol/Lリン酸、0.1mol/L水酸化ナトリウム及びイオン交換水の順で粒子を洗浄した。ジオキサン-塩酸法により、未反応エポキシ基が550μ当
量/ドライゲル(g)確認された。
粒子を0.1mol/L塩酸、水の順で洗浄し、0.01N水酸化ナトリムを用いて滴定したところ、25μ当量/mLのイオン交換容量が確認された。
上記粒子のイオン交換水サクションドライを4.5g計量し、40mLの遠心管に投入し、20mmol/L モリホリノエタンスルホン酸(以下、MESと記載する)(pH5.5)を用いて、3回遠心ろ過により溶媒置換した。さらに10mLの20mmol/LのMES(pH5.5)を加え粒子を分散した。さらに、N-ヒドロキシスクシンイミド540mgを加え、分散した。WSC(ペプチド研社製)224mgを5mLの20mmol/LのMES(pH5.5)に溶解し、投入後素早く撹拌した。1時間静置後、20mmol/L MES(pH5.5)で5回遠心ろ過により洗浄した。その後、14mmol/L濃度、pH6.5に調整したアスパラギン酸溶液20mLを投入し、分散した後、1晩静置した。得られた粒子のイオン交換容量は27μ当量/mLであった。
溶離液A:20mmol/L MES (pH5.5)
溶離液B:0.3mol/L NaCl in 20mmol/L MES (pH5.5)
グラジエント:37~57%溶離液B直線グラジエント、60分
流速:0.6mL/分
検出:UV230nm
対象:モノクローナル抗体 1/10溶離液A希釈液 10μL
クロマトグラムは図1に示したとおりであり、比較例と比較して保持が強く、異性体間の分離性能にも優れていることが分かった。酸性側溶出ピーク1から塩基性側溶出ピーク2までの溶出時間差は17.1分であり、広い選択性を示した。操作圧は13.0MPaと低操作圧であった。
液体クロマトグラフィー条件
溶離液A:20mmol/L リン酸ナトリウム緩衝液(pH6.3)
溶離液B:0.20mol/LのNaClO4を含む20mmol/L リン酸ナトリウム緩衝液(pH6.3)
グラジエント:0~100%溶離液B直線グラジエント、10分
流速:0.6mL/分
検出:UV415nm
対象:HbA1cキャリブレータ2(東ソー社製) 5μL
クロマトグラムは図3に示したとおりであり、比較例と比較し異性体の分離に優れていることが分かった。
実施例1の<ポリアクリル酸の固定>と同様な方法で、ポリアクリル酸を固定した後、下記の方法でアスパラギン酸を導入した。
粒子のイオン交換水サクションドライを4.5g計量し、20mLのエタノール水を加え粒子を分散した。さらに14mmol/L濃度、pH7.0に調整したアスパラギン酸水溶液20mL、4-(4,6-ジメトキシー1,3,5―トリアジンー2-イル)-4-メチルモルホリニウム=クロリドn水和物を60mg加え、分散した。室温にて1時間30分攪拌した後、イオン交換水で3回遠心ろ過により洗浄した。さらに0.5N水酸化ナトリウムで3回、0.5N塩酸で3回遠心ろ過により洗浄した粒子をイオン交換水サクションドライゲルとして回収した。得られた粒子のイオン交換容量は32μ当量/mLであった。
クロマトグラムは図1、図2(比較例4とあわせて、溶出時間を補正した拡大比較図)に示した。比較例と比較して保持が強く、異性体間の分離性能にも優れていることが分かった。酸性側溶出ピーク1から塩基性側溶出ピーク2までの溶出時間差は20.3分であり、広い選択性を示した。操作圧は17.0MPaと低かった。
実施例1の<ポリアクリル酸の固定>と同様な方法で、ポリアクリル酸を導入後、下記の方法でグルタミン酸を導入した。
粒子のイオン交換水サクションドライを4.5g計量し、20mLのエタノール水を加え粒子を分散した。さらに14mmol/L濃度、pH7.0に調整したグルタミン酸水溶液20mL、4-(4,6-ジメトキシー1,3,5―トリアジンー2-イル)-4-メチルモルホリニウム=クロリドn水和物を60mg加え、分散した。室温にて1時間30分攪拌した後、イオン交換水で3回遠心ろ過により洗浄した。さらに0.5N水酸化ナトリウムで3回、0.5N塩酸で3回遠心ろ過により洗浄した粒子をイオン交換水サクションドライゲルとして回収した。得られた粒子のイオン交換容量は29μ当量/mLであった。
クロマトグラムは図1に示したとおりであり、比較例と比較して保持が強く、異性体間の分離性能にも優れていることが分かった。酸性側溶出ピーク1から塩基性側溶出ピーク2までの溶出時間差は25.9分であり、広い選択性を示した。操作圧は19.0MPaと低操作圧であった。
非多孔性基材の作製で得られたエポキシ化非多孔性粒子を水に分散し、オートクレープ中で140℃、3時間加熱することによりエポキシ基をジオール基に変換した。サクションドライの粒子4.5gを8gのイオン交換水に分散し、3.0gのα―クロロ酢酸ナトリウムを加え溶解、分散した。さらに48重量%水酸化ナトリウムを2.7g加え、55℃に設定したウォーターバス中で2時間振盪した。得られた粒子をガラスフィルターにて0.1mol/Lリン酸、0.1mol/L水酸化ナトリウム及びイオン交換水の順で粒子を洗浄した。得られた粒子のイオン交換容量は36μ当量/mLであった。実施例1と同様に液体クロマトグラフィー用カラムに充填し、測定条件を実施例1と同様に実施し、モノクローナル抗体の分離性能を比較した。
クロマトグラムは図1に示したとおりであり、保持が弱く、ブロードなピークとなり異性体間の分離性能が悪いものであった。酸性側溶出ピーク1から塩基性側溶出ピーク2までの溶出時間差は5.2分であり、実施例1よりも選択性の低いものであった。操作圧は15.0MPaと低操作圧であった。
比較例1で作製したカチオン交換体粒子を用い、実施例1の<アスパラギン酸の導入>と同じ操作を行い、アスパラギン酸が導入されたカチオン交換体を得た。得られた粒子のイオン交換容量は34μ当量/mLであった。実施例1と同様に液体クロマトグラフィー用カラムに充填し、測定条件を実施例1と同様に実施し、モノクローナル抗体の分離性能を比較した。
クロマトグラムは図1に示したとおりであり、保持が弱く、異性体間の分離性能も十分ではないものであった。酸性側溶出ピーク1から塩基性側溶出ピーク2までの溶出時間差は4.4分であり、実施例1よりも選択性の低いものであった。操作圧は18.0MPaと比較的低操作圧であった。
9gのアスパラギン酸を0.2mol/L水酸化ナトリウム水溶液を用い、pH12.8に調整した水溶液に、非多孔性基材の作製で得られたエポキシ化粒子3gを投入し、50℃で2時間、水浴中で加熱し、エポキシ基にアスパラギン酸が導入されたカチオン交換体を得た。得られた粒子のイオン交換容量は42μ当量/mLであった。実施例1と同様に液体クロマトグラフィー用カラムに充填し、測定条件を実施例1と同様に実施し、モノクローナル抗体の分離性能を比較した。
クロマトグラムは図1に示したとおりであり、保持が弱く、異性体間の分離性能も十分ではなかった。酸性側溶出ピーク1から塩基性側溶出ピーク2までの溶出時間差は5.4分であり、実施例1よりも選択性の低いものであった。操作圧は14.0MPaと比較的低操作圧であった。
実施例1の<ポリアクリル酸の固定>の操作のみを行ったカチオン交換体を用いて、実施例1と同様に液体クロマトグラフィー用カラムに充填し、測定条件を実施例1と同様にして、モノクローナル抗体の分離性能を比較した。クロマトグラムは図1及び図2(実施例2とあわせて、溶出時間を補正した拡大比較図)に示したとおりである。比較例1、2と比較して、保持は強くなり、異性体間の分離性能も改善されていた。しかし酸性側溶出ピーク1から塩基性側溶出ピーク2までの溶出時間差は13.6分であり、実施例1及び2よりも選択性の低いものであった。操作圧は30.0MPaと高い操作圧となった。
また、実施例1と同様にグリコヘモグロビンの分離性能を比較した。クロマトグラムは図3に示したとおりであり、異性体の分離性能は不十分であった。
なお、2013年3月29日に出願された日本特許出願2013-075020号の明細書、特許請求の範囲、図面及び要約書の全内容をここに引用し、本発明の明細書の開示として、取り入れるものである。
Claims (11)
- 非多孔性粒子の表面に、アミノ基を有するジカルボン酸化合物がアミド結合により導入されたポリアクリル酸が固定されていることを特徴とする液体クロマトグラフィー用カチオン交換体。
- 前記非多孔性粒子が、体積平均粒子径5μm以下を有する、シリカ、ジルコニア若しくはアルミナの無機基材、又は架橋多糖若しくはビニル系モノマー架橋重合体の有機基材である請求項1に記載の液体クロマトグラフィー用カチオン交換体。
- 前記有機基材が、単官能性ビニルモノマーと多官能性ビニルモノマーの架橋重合体である、請求項2に記載の液体クロマトグラフィー用カチオン交換体。
- 前記ポリアクリル酸は、重量平均分子量が5、000以上20、000以下である、請求項1乃至3のいずれかに記載の液体クロマトグラフィー用カチオン交換体。
- 前記アミノ基を有するジカルボン酸化合物が、アスパラギン酸又はグルタミン酸である請求項1乃至4のいずれかに記載の液体クロマトグラフィー用カチオン交換体。
- イオン交換容量が、10~50μ当量/mLである請求項1乃至5のいずれかに記載の液体クロマトグラフィー用カチオン交換体。
- 請求項1乃至6のいずれかに記載の液体クロマトグラフィー用カチオン交換体の製造方法であり、非多孔性粒子が有する官能基と、ポリアクリル酸に含まれるカルボン酸とを反応させて、非多孔性粒子の表面にポリアクリル酸を固定し、かかる固定されたポリアクリル酸とアミノ基を有するジカルボン酸化合物とを反応させることを特徴とする製造方法。
- 請求項1乃至6のいずれかに記載の液体クロマトグラフィー用カチオン交換体の製造方法であり、非多孔性粒子が有する官能基と、アミノ基を有するジカルボン酸化合物が導入されたポリアクリル酸とを反応させて、非多孔性粒子の表面に上記ポリアクリル酸を固定することを特徴とする製造方法。
- 請求項1乃至6のいずれかに記載の液体クロマトグラフィー用カチオン交換体を充填して成る液体クロマトグラフィー用カラム。
- 請求項9に記載の液体クロマトグラフィー用カラムを用い、イオン交換クロマトグラフィーにより抗体の電荷異性体を測定する方法。
- 請求項9に記載の液体クロマトグラフィー用カラムを用い、イオン交換クロマトグラフィーによりグリコヘモグロビンを測定する方法。
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JP2015508789A JP6332267B2 (ja) | 2013-03-29 | 2014-03-28 | 液体クロマトグラフィー用カチオン交換体、その製造方法及びその用途 |
EP14773104.6A EP2980582A4 (en) | 2013-03-29 | 2014-03-28 | Cation exchangers for liquid chromatography, process for the preparation thereof and use |
US14/779,771 US20160084804A1 (en) | 2013-03-29 | 2014-03-28 | Cation exchanger for liquid chromatography, process for producing same, and use thereof |
CN201480011528.7A CN105190305A (zh) | 2013-03-29 | 2014-03-28 | 液相色谱用阳离子交换体、其制造方法及其用途 |
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- 2014-03-28 US US14/779,771 patent/US20160084804A1/en not_active Abandoned
- 2014-03-28 WO PCT/JP2014/059296 patent/WO2014157670A1/ja active Application Filing
- 2014-03-28 JP JP2015508789A patent/JP6332267B2/ja not_active Expired - Fee Related
- 2014-03-28 CN CN201480011528.7A patent/CN105190305A/zh active Pending
- 2014-03-28 EP EP14773104.6A patent/EP2980582A4/en not_active Ceased
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See also references of EP2980582A4 |
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EP2980582A4 (en) | 2016-12-07 |
JPWO2014157670A1 (ja) | 2017-02-16 |
EP2980582A1 (en) | 2016-02-03 |
CN105190305A (zh) | 2015-12-23 |
US20160084804A1 (en) | 2016-03-24 |
JP6332267B2 (ja) | 2018-05-30 |
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