JP2006119119A - Polymer and ligand-immobilizing carrier - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high-thermostability, low-impurity and high-affinity polymer, wherein the bond between a ligand and a carrier is chemically stable, and to provide a ligand-immobilizing carrier that includes the polymer. <P>SOLUTION: This polymerized polymer, containing at least styrene-based monomer and t-butyl methacrylate, contains 30-90 mol.% styrene-based monomer. At least a part of the polymer is substituted with a functional group, containing a maleimidyl group represented by Formula (1) (wherein X is -O- or -NH-; and Q is a spacer group). <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a polymer that can be suitably used for diagnostic agents, test agents, and liquid chromatography, and particularly to a polymer that can be suitably used as a carrier for immobilizing a ligand. The ligand immobilization carrier is suitable for separating or purifying useful substances or pathogenic substances in a liquid such as an antibody using the principle of affinity chromatography.

A substance (ligand) having an affinity for a substance intended for separation or purification (target substance) is immobilized on an insoluble carrier, and the target substance is specifically adsorbed and recovered by this ligand. The technique to perform is known as affinity chromatography (for example, refer nonpatent literature 1). In addition, for the purpose of antibody purification, a method using an affinity gel in which a kappa light chain binding protein is bound to agarose as a ligand (for example, see Patent Document 1), or a hydrophilic spacer having an epoxy group at both ends is used. A carrier (for example, see Patent Document 2) in which a ligand (antibody) is immobilized on water-insoluble cellulose by an epoxy reaction is disclosed.
However, when a carrier gel derived from a natural product such as agarose is used, there is a drawback that the gel itself dissolves at 40 ° C. or higher and the stability to temperature is poor. In the case of cellulose, a highly toxic reagent such as epichlorohydrin must be used to synthesize epoxidized cellulose, and the epoxidized cellulose produced has a low bond selectivity with the functional group present in the ligand, As a result, the obtained immobilization carrier has a disadvantage that the affinity performance is low. Furthermore, carrier gels such as agarose or cellulose have the disadvantage of low purity because they are derived from natural products.

In addition, a ligand-immobilized carrier in which a ligand and a carrier are linked by a disulfide bond is disclosed (see, for example, Non-Patent Document 1 and Patent Document 3). However, the disulfide bond is chemically unstable and has a disadvantage that it is easily cleaved when a compound having another thiol group (for example, a peptide or protein having a cysteine residue) is present.
As an immobilization carrier, polystyrene, polymethacrylic acid and derivatives thereof having functional groups such as thiol group, amino group, hydroxyl group and carboxyl group introduced therein, or copolymers thereof, as well as polyvinyl alcohol and styrene-divinylbenzene. Synthetic polymer compounds such as copolymers are disclosed (see, for example, Patent Document 4 and Patent Document 5). However, in the case of the above-mentioned general synthetic polymer, for example, in the case of a ligand-immobilized carrier made of an aliphatic polymer such as polymethyl methacrylate or polymethacrylic acid, the antibody separability is remarkably lowered, and polystyrene, hydroxymethyl polystyrene It has been found that the aromatic polymers such as (1) have the disadvantage that non-specific binding occurs in which antibodies other than the target substance are bound, and the selectivity is low.
Japanese Patent Laid-Open No. 7-146280 JP-A-6-34633 Japanese Patent Laid-Open No. 2004-45120 JP-A-6-34633 JP 2004-45120 A "Immobilized Affinity Ligand Techniques" Academic Press 1992

  An object of the present invention is to provide a polymer having high thermal stability of the carrier, low impurities, chemically stable binding between the ligand and the carrier, high affinity, and a carrier for immobilizing a ligand containing the polymer. That is.

一般式（１）中、Ｘは−Ｏ−又は−ＮＨ−を表し、Ｑはスペーサー基を表す。 A polymer obtained by polymerizing at least a styrene monomer and t-butyl methacrylate, wherein the styrene monomer is 30 mol% or more and 90 mol% or less, and at least a part of the polymer is represented by the following general formula (1). The above-mentioned problems have been solved by a polymer that is substituted with a functional group containing a maleimidyl group represented by the formula:

In general formula (1), X represents —O— or —NH—, and Q represents a spacer group.

Furthermore, in the general formula (1), it is preferable that the spacer group represented by Q contains a polyoxyethylene chain and the polymer is water-insoluble, or the polymer is crosslinked and has a particle shape. .
These polymers can be suitably used as a ligand immobilization carrier.

  According to the present invention, a polymer having high thermal stability of the carrier, low impurities, chemically stable binding between the ligand and the carrier, high affinity, and a ligand immobilization carrier containing the polymer are obtained. be able to.

  The polymer of the present invention is a polymer obtained by polymerization including at least a styrene monomer and t-butyl methacrylate, wherein the styrene monomer is 30 mol% or more and 90 mol% or less, and at least a part of the polymer is It is a polymer characterized by being substituted with a functional group containing a maleimidyl group represented by the general formula (1).

The present invention will be specifically described below.
The polymer of the present invention is a polymer obtained by polymerization including at least a styrene monomer and t-butyl methacrylate, wherein the styrene monomer is 30 mol% or more and 90 mol% or less, and at least a part of the polymer is It is a polymer characterized by being substituted with a functional group containing a maleimidyl group represented by the following general formula (1). Preferably, the styrenic monomer is 40 mol% or more and 80 mol% or less.

As a component other than the styrene monomer and t-butyl methacrylate, other polymer components can be contained within a range satisfying the above conditions. As other components, (meth) acrylic acid, hydroxyethyl (meth) acrylate, and divinylbenzene and ethylene glycol dimethacrylate in the case of crosslinking are preferable.
When the component styrene monomer is larger than 90 mol%, the selectivity of the ligand is lowered and physical adsorption other than the target substance is increased. On the other hand, when the component styrene monomer is less than 30 mol%, the affinity of the ligand is lowered and the adsorption of the target substance is remarkably lowered.

  A functional group containing a maleimidyl group represented by the following general formula (1) can be substituted with a carboxylic acid group portion of t-butyl methacrylate. The ratio of substitution is preferably 1% or more and 100% or less, and more preferably 10% or more and 90% or less with respect to the entire carboxylic acid group present.

The functional group containing a maleimidyl group represented by the general formula (1) will be described.

  Here, X represents -O- or -NH-. Q represents a spacer group, preferably a divalent linking group containing a polyoxyethylene chain, an alkylene chain, an amino chain, or a polyoxypropylene group, more preferably represented by the following (1) or (2). A divalent linking group.

  When the spacer group is (1) above, a represents an integer of 1 or more. The length of the polyoxyethylene chain is not particularly limited, but a molecular weight of 60 to 10,000 is preferable. More preferably, it is 100 to 1000 (the polyoxyethylene chain described herein includes a single ethylene oxide). b represents 0 or an integer of 1 or more, preferably 0 or an integer of 1 to 6, more preferably 0 to 2, and particularly preferably 0 or 1.

When the spacer group is (1) above, it can be obtained by bonding the terminal hydroxyl group of polyoxyethylene and the hydroxyl group-containing maleimidyl by an etherification reaction. When an alkylene group is interposed between the maleimidyl group and the hydroxyl group (when b is 1 or more), it can be obtained by using a compound such as hydroxyalkylmalemidyl.
When the spacer group is (1) and X is —O—, the hydroxyl group at one end of the polyoxyethylene when the terminal hydroxyl group of polyoxyethylene and the hydroxyl group-containing maleimidyl are bonded by an etherification reaction It is.
When the spacer group is (1) and X is -NH-, (i) the terminal hydroxyl group of polyoxyethylene and the hydroxyl group-containing maleimidyl are bonded by an etherification reaction, and the other terminal hydroxyl group of polyoxyethylene is bonded. (Ii) maleic anhydride is reacted with a compound in which both ends of the spacer in (1) are amines, and then dehydrated and cyclized. (Iii) one end of polyoxyethylene After converting the hydroxyl group to an amino group, the other terminal hydroxyl group of polyoxyethylene and the hydroxyl group-containing maleimidyl are subjected to an etherification reaction. (Iv) After converting the terminal hydroxyl group of polyoxyethylene to an amino group, N- Target substance by introducing maleimidyl group by reacting bifunctional reagent having hydroxysuccinimide ester group and maleimidyl group It is possible to obtain.

When the spacer group is (2) above, c represents an integer of 1 or more, preferably an integer of 1 to 12, and more preferably 1 to 6.
In the case where the spacer group is (2) and X is —O—, a compound having an alkylene group interposed between the maleimidyl group and the hydroxyl group, for example, a compound such as hydroxymethylmalemidyl is used as a target substance. Can be obtained.
When the spacer group is (2) and X is -NH-, the target substance can be obtained by the same method as in the case where the spacer group is (1).

  The spacer group represented by Q is preferably a spacer group having a polyoxyethylene chain represented by the above (1). In particular, it is preferable that the carrier for immobilizing a ligand has a particle shape. By introducing a polyoxyethylene chain, dispersibility in water and in a buffer solution is increased, and operability is facilitated. When the spacer group represented by Q is a linking group having a polyoxyethylene chain represented by (1) above, X is preferably —O—.

The method for introducing the maleimidyl group into the copolymer via Q and X may be any method and can be appropriately selected depending on the purpose.
For example, when X is —O—, a base such as a (meth) acrylic acid t-butyl copolymer is hydrolyzed with an acid or an alkali, and the ester part is once converted into a carboxyl group, The compound having a hydroxyl group at the end of X may be introduced by ester reaction, or may be introduced by direct ester exchange reaction with the substrate using an ester exchange catalyst. Or it can also obtain by copolymerizing the corresponding monomer which X and Q couple | bonded beforehand. In the case where Q has a polyoxyethylene chain as shown in (1) above, a method in which an ester reaction is carried out after hydrolysis to a carboxylic acid is preferred.

The amount of maleimidyl group contained in the polymer of the present invention is not particularly limited, but it is preferably 0.001 mmol / g or more, and from the viewpoint of increasing the reaction amount per unit mass as much as possible, 0.01 mmol / G or more and 1.0 mmol / g or less is more preferable, and 0.05 mmol / g or more and 0.5 mmol / g or less is particularly preferable. By setting it to 0.001 mmol / g or more, the SH group-containing substance can be immobilized as an affinity carrier, and by setting it to 0.05 mmol / g or more, the immobilization ability is particularly satisfied.
The amount of maleimidyl groups in the polymer is a value measured by the following method.

First, a certain amount of polymer particles are weighed into a screw-cap test tube, and a predetermined amount of 2-mercaptoethylamine (Tokyo Kasei Co., Ltd.) prepared in advance is added so that the amount of 2-mercaptoethylamine is excessive. And stirring reaction.
Next, after centrifuging the polymer particles, 2-mercaptoamine in the supernatant solution is reacted with 4,4′-dithiodipyridine, and 2 in the supernatant solution is obtained from the absorbance at 324 nm using a molar extinction coefficient ε = 19800. -Determine the amount of mercaptoethylamine B (mol).
As a blank, the amount of 2-mercaptoamine C (mol) in a system not containing a sample is measured in the same manner, and the amount of maleimidyl group M (mmol / g) is determined according to the following formula.
M = (C−B) / W × 1000

  In the present invention, the styrene monomer means an aromatic monomer copolymerizable with t-butyl methacrylate, and includes styrene, methylstyrene, aminostyrene, acetoxystyrene, t-butylstyrene, chlorostyrene, bromostyrene, fluorostyrene, Examples include chloromethyl styrene, cyanomethyl styrene, methoxy styrene, and phenyl styrene. Of these, styrene is particularly preferred from the viewpoint of polymerizability and price.

For the polymerization of the polymer of the present invention, a known method can be used. For example, a suspension polymerization method, an emulsion polymerization method, a dispersion polymerization method, a seed polymerization method and the like are preferably used. Furthermore, suspension polymerization can be performed using an emulsification method known as a membrane emulsification method. If necessary, a polymerization initiation catalyst known to those skilled in the art can be used. Specifically, organic peroxides such as diacyl peroxide, ketone peroxide and alkyl hydroperoxide; inorganic peroxides such as hydrogen peroxide and ozone; and azobisisobutyronitrile (AIBN; Wako Pure) (Available from Yakuhin as V-60), 2,2′-azobis (2-methylbutyronitrile) (available as V-59 from Wako Pure Chemical Industries, Ltd.) and 2,2′-azobis (2,4- Oil-soluble azo organic compound such as dimethylvaleronitrile (available as V-65 from Wako Pure Chemical Industries); 2,2′-azobis (2-amidinopropane) dioic acid salt (V- 50)), 2,2′-azobis [2-methyl-N- (2-hydroxyethyl) propionamide] (available as VA-086 from Wako Pure Chemical Industries, Ltd.) And a water-soluble azo organic compound such as 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dioic acid salt (available as VA-044 from Wako Pure Chemical Industries, Ltd.). If polymerization initiators are used, they are used in an amount sufficient to initiate polymerization well. Such amounts are well known to those skilled in the art. In general, it is preferably used in an amount of 0.1 to 5.0% by mass.

The obtained polymer can be diluted and dispersed in a solvent such as methanol, filtered, and further washed with water and / or solvent, and then isolated as a powder by ordinary means such as spray drying, reduced pressure drying, freeze drying and the like. it can.
The polymer polymerized as described above can be solidified by heat melting or dissolving in a solvent to form a desired shape and then cooling or removing the solvent.
The polymer of the present invention is cross-linked as necessary, but may be cross-linked at the time of polymerization or may be cross-linked after polymerization. The method for crosslinking is not particularly limited, and examples thereof include addition of a crosslinking agent, irradiation with electromagnetic waves, irradiation with electron beams, and irradiation with particle beams. The type of the crosslinking agent is not particularly limited, and examples thereof include divinylbenzene, ethylene glycol dimethacrylate, urea resin, and melamine resin.

The polymer of the present invention can be suitably used as a ligand immobilization carrier.
As a method for immobilizing the ligand on the carrier, a reaction between the maleimidyl group introduced into the carrier and the mercapto group of the ligand is preferably used. When the ligand is a peptide, it can be immobilized on a carrier using the mercapto group of a cysteine residue in the peptide chain. When the ligand is an antibody, the antibody is digested with pepsin and only the variable region of the antibody is And can be immobilized on a carrier using a mercapto group generated as a ligand. Further, the antibody can be reduced and fragmented with a thiol compound such as aminoethanethiol and immobilized on a carrier using the generated mercapto group. The reaction between the maleimidyl group and the mercapto group is rapid, and the resulting bond has high chemical stability. For immobilization of the ligand on the carrier, the carrier and the ligand may be mixed and stirred in pure water or a buffer.

  The ligand defined here is not limited to a synthetic product or a natural product as long as it interacts with the target substance, but an antibody or a receptor can be preferably used. If antibodies are used, for example, anti-CD3 antibody, anti-CD4 antibody, anti-CD28 antibody, anti-CD34 antibody, anti-CD199 antibody, anti-CCR4 antibody, anti-low density lipoprotein (LDL) antibody, antioxidant LDL antibody, anti-β2 micro Globulin antibodies, anti-staphylococcus aureus toxin antibodies, and the like can be used, and various antibodies can be selected depending on the purpose.

  If receptors are used, for example, cytokine receptors such as CCR3 and CCR4, immunoglobulin receptors such as Fcγ and Fcε, scavenger receptors such as RAGE and LDL receptors, T cell receptors and major histocompatibility antigens, etc. Although a cell recognition receptor etc. can be used, it is not limited to these.

  The carrier of the present invention is preferably water-insoluble. The form of the carrier of the present invention is not particularly limited, but when used as a liquid treatment column, particles, fibers, hollow fibers, yarn bundles, yarns, nets, knitted fabrics, woven fabrics, etc. are used, but they have a large surface area and cells. Considering that the flow resistance is low without clogging even when flowing, particles, fibers, knitted fabrics, woven fabrics, hollow fibers, and porous membranes are preferably used. Particles are particularly preferable from the viewpoint of operability. Furthermore, a ligand-immobilized carrier may be formed as a spot on a plate or a film. In this case, the particulate ligand-immobilized carrier may be fixed on the plate or the film, or the ligand-immobilized site may be directly formed on the plate or the film.

In the case of particles, the particle size can be selected according to various applications, but generally the number average particle size in the dry state is 0.1 to 1000 μm, and from the viewpoint of ease of solid-liquid separation. 1 to 200 μm is preferable, and 5 to 100 μm is particularly preferable.
Here, the number average particle size is obtained by taking a photograph of dry particles with an optical microscope or an electron microscope, measuring the particle size of 100 to 200 particles randomly selected from them, and summing them. Is the value divided by the number.

  In the present invention, a known method can be used to make the polymer into particles, and for example, a suspension polymerization method, an emulsion polymerization method, a dispersion polymerization method, a seed polymerization method and the like are preferably used. Furthermore, suspension polymerization can be performed using an emulsification method known as a membrane emulsification method.

  Since the ligand-immobilizing carrier in the present invention does not have a special reactive functional group, there is an advantage that there is no or very little adverse effect on the particle physical properties due to the reaction with the additive and the solvent and the residual. In addition, since the ligand-immobilizing support in the present invention has high stability to heat and solvent, impurities can be reduced and purity can be increased by performing a purification treatment such as Soxhlet extraction for a long time.

  Furthermore, a known dye, pigment, carbon black, magnetic powder or the like can be added to the ligand-immobilizing carrier in the present invention for the purpose of coloring. Microcapsules can also be used, and porous particles can also be used.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples. In addition, in an Example, a part shows a mass part.

[Example 1]
(Synthesis of crosslinked polymer particles)
Divinylbenzene (purity 55% by mass) was used as a crosslinking agent, and as shown in Table 1, styrene (manufactured by Wako Pure Chemical Industries, Ltd.), methyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) and t-butyl methacrylate (Japanese Base particles with different copolymerization ratios with Koyo Pure Chemical Co., Ltd. were produced. As the granulation method, a suspension polymerization method was used, classification operation was performed, and the number average particle size was adjusted to 50 μm. The measurement of the number average particle diameter is as described above.
The obtained particles were washed with ion exchange water and a solvent, and then isolated and dried to obtain base particles.

(Introduction of polyoxyethylene chain)
Each 10 parts of the base particle obtained above is dispersed in 75 parts of hydrochloric acid (manufactured by Wako Pure Chemical Industries, Ltd.), 150 parts of dioxane (manufactured by Wako Pure Chemical Industries, Ltd.) is added, and the reaction is carried out at 80 ° C. for 6 hours. It was. The obtained particles were dispersed / washed in methanol, further washed with ion exchange water and a solvent, and then isolated and dried to obtain polymethacrylic acid-containing carrier particles. From the quantification of carboxylic acid, 98% of the t-butyl methacrylate component of the copolymer was converted to methacrylic acid.
150 parts of polyethylene glycol 200 (manufactured by Wako Pure Chemical Industries, Ltd.) and 15 parts of sulfuric acid (manufactured by Wako Pure Chemical Industries, Ltd.) are added to 10 parts of any of the polymethacrylic acid-containing carrier particles P1 to P7 thus obtained. The mixture was mixed and reacted at 120 ° C. for 5 hours. The obtained particles were washed with ion-exchanged water and a solvent, and then isolated and dried to obtain polyoxyethylene chain-introduced base particles PP-1 to PP-7 into which polyoxyethylene chains were introduced into carrier particles.

(Introduction of maleimidyl group)
5 parts of hydroxymethylmalemidyl and 350 parts of toluene are placed in any 10 parts of the polyoxyethylene chain-introduced base particles PP-1 to PP-7, and the mixture is heated and stirred at 60 to 70 ° C. 0.5 part of monohydrate was added, and the temperature was raised and reacted under reflux for 6 hours. The obtained particles were dispersed / washed in methanol and further subjected to Soxhlet extraction with ethanol for 8 hours. After filtration, washing again with ion-exchanged water and a solvent, isolation and drying were performed to obtain the ligand-immobilizing carrier of the present invention and comparative carriers (samples 1 to 7). The amount of maleimidyl groups in the thus obtained maleimidyl group-containing polymer particles was measured by the following method.

(Method for quantifying maleimidyl groups in maleimidyl group-containing carrier particles)
A certain amount of particles are weighed into a screw-cap test tube, and a predetermined amount of 2-mercaptoethylamine (manufactured by Tokyo Kasei Co., Ltd.) is added to the reaction solution so that the amount of 2-mercaptoethylamine is excessive, followed by a stirring reaction. I let you.
After centrifuging the particles, the 2-mercaptoamine in the supernatant solution is reacted with 4,4′-dithiodipyridine, and the amount of 2-mercaptoethylamine B ( mol). As a blank, the amount of 2-mercaptoamine C (mol) in a system not containing a sample was measured in the same manner, and the amount of maleimidyl group M (mmol / g) was determined according to the following formula.
M = (C−B) / W × 1000

  The obtained maleimidyl group amount is shown in Table 2. When 1 part of this particle was placed in 10 parts of pure water, subjected to ultrasonic treatment for 30 seconds and observed with a microscope, it was confirmed that the particles other than Sample 5 were well dispersed.

(Evaluation of impurities)
The obtained carrier was immersed in 0.1 mol / l phosphoric acid / EDTA buffer (pH 7.2) at 30 ° C. for 3 hours and then centrifuged, and the absorbance at 280 nm of the supernatant was measured with a spectrophotometer (Hitachi, Ltd.). It was measured using U-3310). In samples Nos. 1 to 4, the absorbance change was almost unchanged between 0.005 and 0.01. As a comparison, activated thiol Sepharose 4B (manufactured by Amersham Bioscience) was measured in the same manner, and the absorbance increased by 0.2. The agarose-based ligand immobilization carrier derived from a natural product is rich in impurities.

(Evaluate affinity characteristics)
The affinity characteristics of each carrier prepared in Example 1 were evaluated by the following method.
1) Mouse IgG (manufactured by Wako Pure Chemical Industries, Ltd., product code 132-13726) is used as a primary antibody, and this antibody is reduced and fragmented and bound to particles. Subsequently, after anti-mouse IgG, rabbit, IgG (H + L) (manufactured by Wako Pure Chemical Industries, Ltd., product code 017-17601) as a secondary antibody, affinity coupling is performed, and then the secondary antibody is separated to perform SDS. -Check for secondary antibody recovery by PAGE.
2) The primary antibody is directly mixed with the particles without reducing fragmentation. After affinity coupling of the secondary antibody, the secondary antibody is separated, and the presence or absence of secondary antibody recovery is examined by SDS-PAGE.

When the separation of the secondary antibody was confirmed by the operation of 1), the evaluation was ○ and ◎, particularly when the separation was good and could be clearly confirmed, and 場合, and when it was not confirmed at all, the evaluation was ×.
On the other hand, according to the level at which the separation of the secondary antibody was not confirmed by the operation of 2), ◯ and ◎ were marked, 特 に when the separation could not be confirmed at all was marked ◎, and when it was clearly confirmed, ×.
The results are shown in Table 3 below.

[Example 2]
(Introduction of aminoethoxyethanol chain)
Copolymerization of styrene (Wako Pure Chemical Industries, Ltd.) and t-butyl methacrylate (Wako Pure Chemical Industries, Ltd.) using divinylbenzene (purity 55 mass%) as a crosslinking agent in the same manner as in Example 1. Base particles having a ratio of 50:50 were prepared. As the granulation method, a suspension polymerization method was used, classification operation was performed, and the number average particle size was adjusted to 50 μm. The obtained particles were washed with ion exchange water and a solvent, and then isolated and dried to obtain base particles. 10 parts of these particles are dispersed in 50 parts of 2- (2-aminoethoxy) ethanol (manufactured by Wako Pure Chemical Industries, Ltd.), 15 parts of mesitylene (manufactured by Wako Pure Chemical Industries, Ltd.) are added, and tetra-n is added under a nitrogen atmosphere. -0.2 part of propoxy titanium was dripped, and it was made to react under recirculation | reflux for 14 hours. The obtained particles were dispersed / washed in methanol, further washed with ion-exchange water and a solvent, and then isolated and dried to obtain hydroxyl group-introduced base particles PP-8.

(Introduction of maleimidyl group)
In 10 parts of the above base particle PP-8, 5 parts of hydroxymethylmalemidyl and 350 parts of toluene are added and stirred at 60 to 70 ° C., and 0.5 part of p-toluenesulfonic acid monohydrate as a catalyst is added. The temperature was raised and reacted under reflux for 6 hours. The obtained particles were dispersed / washed in methanol and further subjected to Soxhlet extraction with ethanol for 8 hours. After filtration, it was washed again with ion-exchanged water and a solvent, and then isolated and dried to obtain the ligand-immobilizing carrier of the present invention (sample 8). The maleimidyl group content of the thus obtained maleimidyl group-containing polymer particles was measured by the same method as in (Samples 1 to 7) and found to be 0.1 mmol / g.

[Example 3]
(Introduction of maleimidyl group)
In 10 parts of the base particle P-2 produced in Example 1, 300 parts of thionyl chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was added and dispersed, and then heated and stirred at 75 to 80 ° C. and refluxed for 5 hours. Reacted. The resulting particles were dispersed / washed in toluene and isolated and dried. After 90 parts of dry toluene was added to 10 parts of these particles and redispersed, 3 parts of pyridine (manufactured by Wako Pure Chemical Industries, Ltd.) was added thereto, and 5 parts of hydroxymethylmalemidyl was added and reacted at room temperature for 12 hours. I let you. The obtained particles were repeatedly washed with ion-exchanged water and a solvent, and then isolated and dried to obtain a ligand-immobilizing carrier (sample 9) of the present invention. The maleimidyl group content of the thus obtained maleimidyl group-containing polymer particles was measured by the same method as in Samples 1 to 7, and found to be 0.2 mmol / g.

Impurities and affinity characteristics of Sample No. 8 and Sample No. 9 of the present invention obtained in Example 2 and Example 3 were evaluated by the same measurement method as in Example 1.
In the evaluation of impurities, in samples of sample number 8 and sample number 9, the change in absorbance hardly changed between 0.005 and 0.01.
The results of the affinity characteristics are shown in Table 3.

  As shown in Table 3, it was found that when the styrene content exceeds 90 mol%, physical adsorption increases and the selectivity decreases, and when the styrene content falls below 30 mol%, the affinity characteristic itself decreases. In particular, the affinity characteristics were good when the styrene content was 40 mol% or more and 80 mol% or less.

  Thus, it was found that the balance between the hydrophilicity and the hydrophobicity of the base particles is important for the expression of affinity characteristics. If the styrene content is less than 30 mol%, the hydrophilicity becomes too strong and the affinity with the antibody decreases, and if it exceeds 90 mol%, the hydrophobicity becomes too strong and physical adsorption increases, and the water dispersibility decreases. It is considered a thing. In particular, by setting the styrene content to 40 to 80 mol%, the balance between hydrophilicity and hydrophobicity becomes more preferable, and good affinity characteristics can be obtained.

Claims (4)

A polymer obtained by polymerizing at least a styrenic monomer and t-butyl methacrylate,
The styrenic monomer is 30 mol% or more and 90 mol% or less, and at least a part of the polymer is substituted with a functional group containing a maleimidyl group represented by the following general formula (1).

(In the formula, X represents —O— or —NH—, and Q represents a spacer group.)   The polymer of claim 1, wherein the polymer is water insoluble and the spacer group comprises a polyoxyethylene chain.   The polymer according to claim 1, wherein the polymer is crosslinked and has a particle shape.   A carrier for immobilizing a ligand comprising the polymer according to any one of claims 1 to 3.