JP2013088398A - Method for manufacturing porous crosslinked particle and separation agent, and separation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a surface-modified porous cross-linked particle and a separation agent suitable as a liquid chromatographic separation agent for a protein and capable of efficiently introducing a high molecule to the surface of a carrier having a large pore size.SOLUTION: A method for manufacturing a surface-modified porous cross-linked particle by fixing a high molecule having a primary hydroxyl group on a porous cross-linked particle having a functional group to be covalent-bound with a hydroxyl group on a surface by covalent binding is provided. A method for manufacturing a separation agent for introducing an interactive functional group into the surface-modified porous cross-linked particle is provided. A method for manufacturing a separation agent for fixing a high molecule having a primary hydroxyl group and an interactive functional group on the porous cross-linked particle having a functional group to be covalent-bound with a hydroxyl group on the surface by covalent binding of the primary hydroxyl group is provided.

Description

  The present invention relates to a method for producing surface-modified porous crosslinked particles and a separating agent, and a separating method. Specifically, the present invention relates to surface-modified porous crosslinked particles suitably used as a separation agent for liquid chromatography, a method for producing the separation agent, and a separation method using the separation agent. More specifically, the present invention relates to a method for producing surface-modified porous crosslinked particles and a separating agent suitably used as a separating agent for liquid chromatography suitable for protein separation, and a separation method using the separating agent.

  Carriers used in the separation agent for liquid chromatography include inorganic carriers such as silica gel and hydroxyapatite, natural polymer carriers such as agarose, dextran, cellulose and chitosan, polystyrene, poly (meth) acrylate, etc. These synthetic polymer carriers are known. These carriers are used as they are or with various functional groups added as necessary to enable use in various separation modes. Introduction of a functional group to the carrier is usually performed directly on the surface of the carrier or via a compound having a relatively small molecular weight called a spacer, regardless of the porosity of the carrier.

Regardless of the type of separating agent, it is desirable that the amount of adsorption of the separation object is large.
When the molecular weight of the separation target is small, the adsorption amount of the separation target of the obtained separation agent can be increased by increasing the specific surface area using the carrier as a porous structure.
However, when the molecular weight of the separation target is large, such as protein, the pore diameter is small in the carrier having a developed porous structure, so that the separation target cannot diffuse into the pores or the diffusion rate is low. For this reason, when the chromatography method is used, if the flow rate is increased, there is a problem that the amount of dynamic adsorption decreases because the diffusion of the separation target into the pores is not sufficiently performed.

  In order to increase the efficiency of diffusion of a carrier having a large molecular weight into the pores of the carrier, it is necessary to use a carrier having a large pore diameter. The amount of adsorption of objects will be reduced. For this reason, the conventional carrier in which a functional group is introduced directly on the surface of the carrier or via a compound having a relatively small molecular weight called a spacer has a high molecular weight such as protein for a separation target. There was no indication of the amount of adsorption.

  In order to solve this problem, various methods for introducing a polymer to the surface of a carrier having a large pore diameter have been studied.

  For example, Patent Document 1 discloses a separating agent in which a support having a hydroxyl group is used as a base and the surface thereof is coated with a polymer having a degree of polymerization of 2 to 100 having a covalent bond.

  Patent Document 2 discloses beads having an affinity structure including a basic matrix in which a water-soluble polymer called an extender is covalently bonded.

JP-A-1-310744 JP-T-2001-510397

According to the study by the present inventors, in the technique described in Patent Document 1, it is necessary to introduce a polymer having a degree of polymerization of 2 to 100 by graft polymerization, but on a production scale at an industrial level, It is considered that there is a problem that it is difficult to stably control the polymerization degree of the coating polymer on the substrate surface.

  In addition, Patent Document 2 describes that the extender has two or more structures having affinity for the substance to be adsorbed and is a non-polypeptide, but more detailed description of the extender. There is no. And as the Example 1, although the dextran solution is added to the basic | foundation matrix, since the usage-amount of the required dextran increases, there exists a problem that efficiency is low economically.

  The present invention provides a surface-modified porous crosslinked particle in which a polymer is efficiently introduced on the surface of a carrier, and a separating agent in which an interactive functional group is introduced into the surface-modified porous crosslinked particle, in particular, liquid chromatography of proteins. It is an object of the present invention to provide surface-modified porous cross-linked particles and a separating agent suitable for lithography.

  As a result of repeated investigations to solve the above problems, the present inventors have immobilized a polymer having a primary hydroxyl group to the porous crosslinked particle having a functional group capable of covalently bonding to a hydroxyl group by covalent bonding. As a result, the present inventors have found that the surface-modified porous crosslinked particles obtained by doing so are extremely effective for the above-mentioned problems, and have reached the present invention.

  That is, the gist of the present invention is as follows.

[1] A method for producing a surface-modified porous crosslinked particle, wherein a polymer having a primary hydroxyl group is immobilized by covalent bonding on a porous crosslinked particle having a functional group capable of covalently bonding with a hydroxyl group on the surface. .

[2] The method for producing surface-modified porous crosslinked particles according to [1], wherein the polymer is a synthetic polymer.

[3] A separation agent characterized by introducing an interactive functional group into the surface-modified porous crosslinked particles produced by the method for producing a surface-modified porous crosslinked particle according to [1] or [2] Production method.

[4] The method for producing a separating agent according to [3], wherein the interactive functional group is introduced by covalently fixing to the polymer having the primary hydroxyl group.

[5] The method for producing a separating agent according to any one of [3] or [4], wherein the interactive functional group is an ion exchange group.

[6] The method for producing a separating agent according to [3] or [4], wherein the interactive functional group is an affinity ligand.

[7] The method for producing a separating agent according to [3] or [4], wherein the interactive functional group includes any one of an alkyl group, a phenyl group, and a polyalkyl ether group.

[8] An adsorptive separation method using the separating agent produced by the method for producing a separating agent according to any one of [3] to [7].

[9] A chromatographic separation method using the separation agent produced by the method for producing a separation agent according to any one of [3] to [7].

[10] The adsorptive separation method according to [8], wherein the separation target is a protein.

[11] The chromatographic separation method according to [9], wherein the separation target is a protein.

ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method which can introduce | transduce a polymer | macromolecule efficiently on the surface can be provided by using porous bridge | crosslinking particle | grains with a large pore diameter as a support | carrier.
Moreover, according to this invention, the method of manufacturing the separating agent suitable for adsorption separation of high molecular weight separation objects, such as protein, can be provided.
Furthermore, according to the present invention, it is possible to provide a method for producing surface-modified porous crosslinked particles and a separating agent with good economic efficiency and stability even on an industrial scale.

  The interactive functional group introduced into the separating agent according to the present invention is preferably an ion exchange group; an affinity ligand; a hydrophobic group such as an alkyl group, a phenyl group and a polyalkyl ether group, and such an interactive functional group. In particular, the separating agent into which can be introduced can be effectively used as an adsorbent for high-molecular substances such as proteins, especially as a separating agent for chromatography.

  The present invention will be described in detail below, but the present invention is not limited to the following embodiments, and can be implemented with various modifications within the scope of the gist thereof. In addition, when using the expression “to” in the present specification, it is used as an expression including numerical values or physical property values before and after the expression.

In the present specification, “(meth) acryl” means one or both of “acryl” and “methacryl”, and “(co) polymerization” means one or both of “polymerization” and “copolymerization”. Mean both. "(Poly) alkylene ..." means one or both of "alkylene ..." and "polyalkylene ...". The same applies to "(poly) ethylene ...".
In the following, the surface-modified porous crosslinked particles produced by the production method of the present invention are referred to as “surface-modified porous crosslinked particles of the present invention”, and the separating agent produced by the production method of the present invention is referred to as “the present invention. Is called “separating agent”. In the present invention, the crosslinked particles mean those having a three-dimensional network molecular structure.

[Method for producing surface-modified porous crosslinked particles]
The method for producing a surface-modified porous crosslinked particle of the present invention comprises a porous crosslinked particle having a functional group capable of covalently bonding with a hydroxyl group on the surface (hereinafter referred to as “porous crosslinked particle having a functional group capable of covalently bonding to a hydroxyl group on the surface”. ”Is sometimes referred to as“ porous crosslinked particle A ”), and a polymer having a primary hydroxyl group (hereinafter,“ polymer having a primary hydroxyl group ”may be referred to as“ polymer B ”). It is fixed by covalent bond.

<Porous crosslinked particle A>
The polymer substance constituting the porous crosslinked particle A as a carrier used in the present invention may be a natural polymer or a synthetic polymer as long as it has a functional group capable of covalently bonding with a hydroxyl group on its surface. Either can be used.

  Examples of natural polymers include agarose, dextran, cellulose, chitosan, glucomannan and the like. Since these natural polymers have a hydroxyl group on the surface, a porous structure is introduced into the hydroxyl group by introducing a crosslinked structure using a crosslinking agent such as epichlorohydrin, (poly) alkylene glycol diglycidyl ether, or alkylene diisocyanate. The crosslinked particles A can be obtained.

  Synthetic polymers include aromatic monovinyl compounds such as styrene monomers such as styrene, ethylstyrene, methylstyrene, hydroxystyrene, and chlorostyrene; methyl (meth) acrylate, ethyl (meth) acrylate, ( (Meth) acrylic acid such as hydroxyethyl methacrylate, hydroxypropyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, cyclohexyl (meth) acrylate, glycerol mono (meth) acrylate, etc. Esters; (meth) acrylamides such as (meth) acrylamide, dimethyl (meth) acrylamide, hydroxyethyl (meth) acrylamide; nitriles such as (meth) acrylonitrile; glycidyl (meth) acrylate, 4,5-epoxybuty Epoxy group-containing compounds such as (meth) acrylate and 9,10-epoxystearyl (meth) acrylate; after (co) polymerizing one or more monovinyl monomers such as other vinyl esters and vinyl ethers The resulting (co) polymer was introduced into a porous crosslinked particle by introducing a crosslinked structure using a crosslinking agent such as epichlorohydrin, (poly) alkylene glycol diglycidyl ether, alkylene diisocyanate, or divinylbenzene. , Aromatic polyvinyl compounds such as trivinylbenzene, poly (meth) acrylates such as (poly) ethylene glycol di (meth) acrylate, glycerol di (meth) acrylate, polycarboxylic acid polyvinyl esters, Polycarboxylic acid polyallyl esters, One or two or more of polyvinyl compounds such as riol polyvinyl ethers, polyol polyallyl ethers, butadiene, methylenebisacrylamide, triallyl isocyanurate, or one such polyvinyl compound Porous crosslinked particles obtained by copolymerizing two or more and one or more of the above-described monovinyl monomers can be used.

  In view of industrial productivity, as the synthetic polymer, one or two or more kinds of polyvinyl compounds and one or more kinds of monovinyl monomers are copolymerized as porous crosslinked particles A. preferable.

  In addition, as the shape of the porous crosslinked particles A used in the present invention, both an indefinite shape and a spherical shape are used, but considering the case of using for liquid chromatography separation, a spherical shape is preferable, and an average particle size thereof Is preferably in the range of 0.5 to 1000 μm, more preferably 1 to 200 μm, and still more preferably 2 to 100 μm. If the average particle size is smaller than this range, the pressure loss when the column is packed and passed through increases, so that the flow rate cannot be increased and the processing efficiency tends to decrease. On the other hand, if the average particle size is larger than this range, the amount of adsorption and separation performance tend to decrease.

  The average particle diameter of the porous crosslinked particles A can be measured by a known method. For example, an average particle diameter can be obtained by measuring 100 or more particle diameters with an optical microscope and calculating a volume median diameter from the distribution.

  Regarding the particle size distribution of the porous crosslinked particles A, the uniformity coefficient, which is a value obtained by dividing the 90% particle size in the volume distribution by the 40% particle size, is 1.4 or less, preferably 1.3 or less, more preferably It is preferable that the particle size distribution is narrow as 1.2 or less. That is, a smaller uniformity coefficient is usually preferable because the pressure loss when the column is packed and passed is reduced. On the other hand, a uniformity coefficient of 1.0 means that the particle diameter is completely uniform, which is difficult in actual production. For this reason, the uniformity coefficient is usually greater than 1.0. Examples of a method for setting the uniformity coefficient below the upper limit include classification using a sieve, a water sieve using a water stream, and a wind sieve using an air stream.

The uniformity coefficient of the porous crosslinked particles A is “Diaion (registered trademark) Ion Exchange Resin / Synthetic Adsorbent Manual 1” Revised 4th Edition (October 10, 2008) issued by Ion Exchange Resin Division, Mitsubishi Chemical Corporation. ) It is a value calculated according to a known calculation method described on pages 140 to 141. More specifically, it can be measured by the method described below. First, a particle size distribution diagram is obtained from the average particle size obtained by the above method. From the particle size distribution chart, the 40% point is obtained as the “40% residual diameter” and the 90% point as the “effective diameter” from the particles having a large average particle diameter, and the uniformity coefficient is calculated by the following formula. To do.
(Uniformity coefficient) = (40% residual diameter (mm)) / (effective diameter (mm))

  Further, for the porous structure of the porous crosslinked particles A used in the present invention, the pore volume measured by a mercury porosimeter is usually 0.2 to 2.5 ml / g, particularly 0.2 to 2.0 ml / g. A range is preferred. The larger the pore volume, the larger the amount of adsorption, which is preferable. However, when the pore volume is excessively large, the mechanical strength of the particles tends to decrease.

  Further, the most frequent radius of the porous crosslinked particles A is usually in the range of 1 nm to 1 μm, particularly 2 to 500 nm, especially 10 to 300 nm. When the most frequent radius is smaller than the above range, it becomes difficult for the protein to be separated or the like to enter the pores of the particles, and as a result, the amount of adsorption tends to decrease. On the other hand, if the most frequent radius exceeds the above range, a space that does not contribute to adsorption is created inside the pores, and the amount of adsorption tends to decrease, and the mechanical strength of the particles also tends to decrease. is there.

Furthermore, the specific surface area of the porous crosslinked particles A measured by the BET method is usually in the range of ^{2 to} 1500 m ² / g, particularly 5 to 1000 m ² / g. When the specific surface area is smaller than the above range, the amount of adsorption tends to be low. When the specific surface area is large, the pore diameter decreases accordingly. The amount of adsorption tends to decrease.

  The porous crosslinked particle A used in the present invention has a functional group capable of covalently bonding with a hydroxyl group on the surface thereof. Examples of the functional group that can be covalently bonded to a hydroxyl group include a hydroxyl group, a carboxyl group, a sulfone group, an isocyanate group, a halogen group such as a chloro group, and an epoxy group, preferably a halogen group such as an isocyanate group and a chloro group, and an epoxy group. And particularly preferably an epoxy group. Here, in the present invention, the surface of the porous cross-linked particle A is used in a sense including not only the outer surface constituting the cross-linked particle but also the surface in the pores.

  The amount of the functional group that can be covalently bonded to the hydroxyl group of the porous crosslinked particle A is usually in the range of 0.001 to 10 meq / g and in the range of 0.01 to 5 meq / g. preferable. When the functional group capable of covalently bonding with the hydroxyl group of the porous crosslinked particle A is not less than the lower limit, it is preferable in terms of the efficiency of immobilizing the polymer B to the porous crosslinked particle A, and on the other hand, not more than the upper limit. And the number of components that maintain the mechanical strength of the porous crosslinked particles is increased.

  In order to obtain a porous crosslinked particle A having a functional group capable of being covalently bonded to a hydroxyl group, the above-mentioned hydroxyl group can be covalently bonded to the hydroxyl group as a polyvinyl compound and / or monovinyl monomer used in the (co) polymerization of the synthetic polymer. It is preferable to use one having a functional group that can be converted to a functional group that can be covalently bonded to the above hydroxyl group by a post-reaction, and then one that has been converted by a post-reaction. .

  The porous crosslinked particles A may be used alone or in combination of two or more.

<Polymer B>
In the present invention, the polymer B is immobilized to the porous crosslinked particles A by covalent bonds.

  As the polymer B used in the present invention, any of a natural polymer and a synthetic polymer can be used as long as it has a primary hydroxyl group. In the present invention, after immobilizing the polymer B to the porous crosslinked particles A, when the separating agent of the present invention is produced by introducing an interactive functional group described later, the polymer B is porous. In addition to the primary hydroxyl group that is covalently bonded to the crosslinked particle A, it is also preferable to have a functional group that can bind to an interactive functional group. Examples of functional groups that can be bonded to such interactive functional groups include secondary hydroxyl groups, tertiary hydroxyl groups, carboxyl groups, amino groups, sulfone groups, isocyanate groups, chloro groups and other halogen groups and epoxy groups. .

  The polymer B is preferably a hydrophilic polymer, more preferably a natural polymer or a synthetic polymer having a plurality of hydroxyl groups. Examples of the natural polymer include agarose, chitosan, glucomannan and the like, and modified cellulose such as hydroxypropylcellulose and carboxymethylcellulose. Synthetic polymers include polyether polyols such as polyglycerin, polyglycidol, allyl glycidyl ether-glycidol copolymer, hydroxyethyl (meth) acrylate, 2,3-dihydroxypropyl (meth) acrylate, etc. One or two monovinyl monomers containing a primary hydroxyl group such as hydroxyalkyl (meth) acrylates, hydroxyalkyl vinyl ethers such as hydroxyethyl vinyl ether and hydroxybutyl vinyl ether, alkylol acrylamides such as methylol acrylamide and hydroxyethyl acrylamide One or more kinds of (co) polymers or copolymers of one or more of these monovinyl monomers containing a primary hydroxyl group and one or more of monovinyl monomers containing other functional groups, etc. Can be mentionedIn addition, about the monovinyl monomer containing another functional group, what contains the functional group which can introduce | transduce the interactive functional group mentioned above or an interactive functional group by post-reaction is also preferable. As the polymer B, a synthetic polymer is more preferable. This is because the synthetic polymer is usually higher in purity than the natural polymer and the immobilization reaction to the porous crosslinked particles A is easy to control.

  Although there is no restriction | limiting in particular about the molecular weight of the polymer B, Usually, 100 or more, Preferably it is 200 or more, More preferably, it is 250 or more, On the other hand, Usually, 5,000,000 or less, Preferably it is 1,000,000 or less. More preferably, it is 600,000 or less. If the molecular weight of the polymer B is too small, the effect of improving the adsorption amount of the present invention by immobilizing the polymer B on the porous crosslinked particles A tends to be reduced. On the other hand, if the polymer B is too large, the immobilized polymer Since B occupies the majority of the spaces in the pores of the porous crosslinked particles A, there is less room for a high molecular weight separation target such as a protein to diffuse and penetrate into the spaces in the pores.

The amount of the polymer B immobilized on the porous crosslinked particles A is obtained by immobilizing the polymer B on the weight W ₀ of the porous crosslinked particles A before the polymer B is immobilized. As an immobilization rate calculated by the following formula from the weight W ₁ in which the surface-modified porous crosslinked particles of the present invention are made constant by a method such as a vacuum drying method, usually 0.1 to 30%, particularly 0.5 to 20%. It is preferable that
Immobilization rate = {(W ₁ −W ₀ ) / W ₀ } × 100

  If the immobilization rate is too small, the effect of improving the adsorption amount of the present invention by immobilizing the polymer B on the porous crosslinked particles A cannot be sufficiently obtained. By occupying the majority of the spaces in the pores of the porous crosslinked particles A, there is less room for a high molecular weight separation target such as protein to diffuse and penetrate into the spaces in the pores.

<Immobilization method by covalent bond>
As a method of immobilizing the polymer B by covalent bonding to the porous crosslinked particle A, for example, the polymer B is covalently bonded to the porous crosslinked particle A having a halogen group such as chloro group on the surface. In the case of immobilization, it is possible to use the Williamson ether synthesis method, and in the case of immobilizing the polymer B by covalent bonding to the porous crosslinked particles A having an epoxy group on the surface, The immobilization reaction can be performed using an alkali catalyst or an acidic catalyst, or by any method without a catalyst.

  Regarding the solvent used for the immobilization reaction, any of an organic solvent system, an organic solvent / water mixed solvent system and an aqueous system can be used as long as the polymer B can be dissolved. Although it can select suitably based on well-known reaction conditions, in the case of the porous bridge | crosslinking particle | grains which have an epoxy group on the surface, for example, reaction temperature is 0-200 degreeC normally, and reaction time is 1 minute-60 hours normally. .

  In addition, after immobilizing the polymer B on the porous crosslinked particle A, the functional group capable of covalently bonding to the hydroxyl group remaining on the surface of the porous crosslinked particle A can be bonded to the interactive functional group by hydrolysis or the like. It may be converted into a group.

[Method for producing separating agent]
The method for producing the separating agent of the present invention is a method in which an interactive functional group is further introduced into the surface-modified porous crosslinked particles of the present invention produced by the method for producing surface-modified porous crosslinked particles of the present invention. In addition, in the production of the surface-modified porous crosslinked particles of the present invention, a method using the polymer B into which an interactive functional group has been introduced in advance may be used.

<Interactive functional group>
The interactive functional group introduced into the separating agent of the present invention is appropriately determined according to the use of the separating agent of the present invention. For example, in the use as a separating agent for liquid chromatography, various ion exchange groups, It is appropriately selected from affinity ligands and hydrophobic interaction groups. When separating a protein using the separating agent of the present invention, the ability to adsorb proteins is particularly excellent by appropriately selecting these interactive functional groups according to the protein to be separated. .

  Examples of the ion exchange group include a carboxyl group such as a carboxymethyl group, a phosphonoalkyl group such as a phosphonoethyl group, a sulfoalkyl group such as a sulfoethyl group, a sulfopropyl group, and a 2-methylpropanesulfonic acid group, and an alkylamino group. Groups, various alkylamino groups such as a dialkylamino group and a trialkylammonium group, and a pyridine group.

Examples of the affinity ligand include protein A, protein G, protein L and functional mutants thereof, various antibodies, or pseudo peptide ligands thereof, various dyes, lectins, nucleic acids such as oligonucleic acid, and the like. The protein separation application is not particularly limited as long as it is a substance having biochemical activity having affinity for proteins and can be immobilized on the porous crosslinked particles A and / or the polymer B.
In particular, when the main purpose is separation of antibodies, a ligand that can specifically bind to a part of an immunoglobulin is preferable. Among them, protein A, protein G, protein L, and variants thereof are alternately separated. It is preferable because of its high selectivity when used in the above.

  Examples of the hydrophobic interaction group include an alkyl group having 1 to 40 carbon atoms, a phenyl group, and a polyalkyl ether group having 1 to 10 carbon atoms and a repeating number of 2 to 100, preferably carbon number. Examples thereof include 4 to 18 alkyl groups, phenyl groups, and polyalkyl ether groups having 2 to 20 carbon atoms and 2 to 20 repeating carbon atoms.

  The separating agent of the present invention may have only one kind of these interactive functional groups, or may have two or more kinds.

<Introduction of interactive functional group into surface-modified porous crosslinked particles>
As a method for further introducing an interactive functional group into the surface-modified porous crosslinked particle of the present invention, the above-mentioned compound having an interactive functional group may be reacted with the surface-modified porous crosslinked particle. Examples of the compound having an interactive functional group that reacts with the surface-modified porous crosslinked particles include halogen groups such as hydroxyl groups, carboxyl groups, amino groups, sulfone groups, isocyanate groups, and chloro groups, and epoxy groups of the surface-modified porous crosslinked particles. And the like are preferred.

  These may be used alone or in combination of two or more.

  There is no particular limitation on the reaction system for introducing the interactive functional group by reacting the compound having the interactive functional group with the surface-modified porous crosslinked particles of the present invention. The surface-modified porous crosslinked particles of the present invention are added to a reaction solution in which the compound having an interactive functional group is dissolved in a solvent capable of dissolving the group-containing compound, and the reaction is performed by heating at a predetermined temperature for a predetermined time. You can do it.

<Introduced amount of interactive functional group>
The amount of the interactive functional group introduced into the separation agent of the present invention thus produced is, for example, 0.001 to 4 equivalent / L as the ion exchange capacity when the interactive functional group is an ion exchange group. -Separating agent particles are preferred, especially 0.01-2 equivalent / L-separating agent particles.

  When the interactive functional group is an affinity ligand, 1 micro equivalent / L-separator particle to 2 equivalent / L-separator particle is preferable.

  When the interactive functional group is a hydrophobic group, 0.001 equivalent / L-separator particle to 2 equivalent / L-separator particle is preferable.

[Separation object and separation method]
The separation agent of the present invention is suitably used for adsorption / desorption using a protein, particularly an antibody, as a separation target.
Particularly preferable separation targets include immunoglobulins or fusion proteins containing at least part of the Fc region of immunoglobulins or chemically modified products thereof. Among these, immunoglobulins are preferably monoclonal antibodies or polyclonal antibodies.
The adsorption / desorption treatment of these separation objects is preferably performed through the following steps.

(A) a step of bringing the solution containing the separation object into contact with the separation agent of the present invention and causing the separation object to be adsorbed on the separation agent (b) a step of eluting the separation object from the separation agent adsorbing the separation object

By such a method, it is possible to separate the various proteins as described above with high selectivity using the separating agent of the present invention.
In such adsorption / desorption treatment, a liquid chromatography column packed with the separation agent of the present invention is preferably used.

EXAMPLES Hereinafter, although this invention is demonstrated further more concretely using an Example, this invention is not limited to description of a following example, unless it deviates from the summary. In addition, the value of various manufacturing conditions and evaluation results in the following examples has a meaning as a preferable value of the upper limit or the lower limit in the embodiment of the present invention, and the preferable range is the above-described upper limit or lower limit value. A range defined by a combination of values of the following examples or values of the examples may be used.

[Example 1]
<Synthesis of surface-modified porous crosslinked particles>
Porous crosslinked particles were synthesized by suspension polymerization of glycidyl methacrylate having an epoxy group as a functional group capable of covalently bonding with a hydroxyl group and ethylene glycol dimethacrylate at a weight ratio of 70:30. With respect to the porous crosslinked particles, the amount of epoxy groups determined by the hydrochloric acid-dioxane method was 4.38 meq / g. This was sieved with a stainless mesh sieve so that the average particle size was 60 μm and the uniformity coefficient was 1.4 or less (the pore volume of this was 0.94 ml / g, the most frequent radius was 26.1 nm, BET specific surface area is 51 m ² / g.) 50 parts by weight of polyglycerol (“PG310” manufactured by Sakamoto Yakuhin Kogyo Co., Ltd.) as a polymer having a primary hydroxyl group and 50 parts by weight of dimethyl sulfoxide as a solvent with respect to 10 parts by weight. In addition, surface-modified porous crosslinked particles were synthesized by reacting at 120 ° C. for 6 hours. After washing the particles after the reaction with water, 50 parts by weight of 0.1% by weight sulfuric acid aqueous solution was added,
The remaining epoxy group was hydrolyzed by reacting at 50 ° C. for 3 hours. The weight increase rate (polyglycerin immobilization rate) of the surface-modified porous crosslinked particles obtained by washing the particles after the reaction with water and then drying under reduced pressure was 3.7%.

<Manufacture of separating agent>
To 10 parts by weight of the surface-modified porous crosslinked particles obtained above, 12.5 parts by weight of propane sultone as a compound having an interactive functional group and 12.5 parts by weight of tetrahydrofuran as a solvent are added. The separation agent was obtained by introducing a sulfopropyl group as an interactive functional group by reacting for 6 hours.

  The ion exchange capacity of the obtained separating agent particles was determined to be 0.10 equivalent per 1 L of particles by titration.

<Evaluation of dynamic adsorption capacity of separation agent protein>
A mouse IgG (20 mM sodium citrate + 15 mM sodium chloride) solution (pH 5.) filled with a column having an inner diameter of 6.4 mm and a length of 30 mm and having a concentration of 1 mg / ml at a flow rate of 1.0 ml / min. 2) The dynamic adsorption capacity determined by subtracting the amount of mouse IgG in the effluent from the column from the amount of mouse IgG determined from the flow rate through the column and passing through the column was 51.6 g per liter of particles. I was asked.

[Comparative Example 1]
The porous crosslinked particles having an epoxy group used in Example 1 were sieved so that the average particle diameter was 60 μm in the same manner as in Example 1, and 50 wt. The epoxy group was hydrolyzed by adding 3 parts and reacting at 50 ° C. for 3 hours.
3 parts by weight of propane sultone and 100 parts by weight of dimethyl sulfoxide are added to 10 parts by weight of the particles obtained by washing the particles after the reaction with water and dried under reduced pressure, and then 10% by weight of a 20% by weight aqueous sodium hydroxide solution as a catalyst. A sulfopropyl group was introduced by reacting at 30 ° C. for 7 hours.

  The ion exchange capacity of the obtained particles was determined to be 0.08 equivalent per 1 L of particles by titration.

  When this particle was packed in a 1 ml column and the dynamic adsorption capacity of mouse IgG was measured in the same manner as in Example 1, it was 35.0 g per liter of particle.

[Evaluation of results]
As is clear from the above results, after fixing polyglycerin, which is a polymer having primary hydroxyl groups, to porous crosslinked particles having epoxy groups, which are functional groups that can be covalently bonded to hydroxyl groups, on the surface, The separating agent particles of Example 1 in which a sulfopropyl group was introduced as an active functional group did not immobilize a polymer having a primary hydroxyl group on the porous crosslinked particles, but had a direct interactive functional group introduced in Comparative Example 1. Compared to particles, the difference in adsorption capacity is larger than the difference in ion exchange capacity. From this, it can be seen that the adsorption capacity was remarkably improved by introducing the polymer into the porous crosslinked particles.
In addition, according to the method of the present invention, a polymer having a primary hydroxyl group is immobilized on a porous crosslinked particle having a functional group capable of covalently bonding with a hydroxyl group by covalent bonding, whereby the porous crosslinked particle is immobilized. It can be seen that the polymer can be introduced efficiently.

Claims (11)

  A method for producing surface-modified porous crosslinked particles, wherein a polymer having a primary hydroxyl group is immobilized by covalent bonding to porous crosslinked particles having a functional group capable of covalently bonding with a hydroxyl group on the surface.   The method for producing surface-modified porous crosslinked particles according to claim 1, wherein the polymer is a synthetic polymer.   A method for producing a separating agent, wherein an interactive functional group is introduced into the surface-modified porous crosslinked particles produced by the method for producing surface-modified porous crosslinked particles according to claim 1.   The method for producing a separating agent according to claim 3, wherein the interactive functional group is introduced by covalently fixing to the polymer having the primary hydroxyl group.   The method for producing a separating agent according to claim 3, wherein the interactive functional group is an ion exchange group.   The method for producing a separating agent according to claim 3 or 4, wherein the interactive functional group is an affinity ligand.   The method for producing a separating agent according to claim 3 or 4, wherein the interactive functional group contains any of an alkyl group, a phenyl group, and a polyalkyl ether group.   An adsorption separation method using the separation agent produced by the method for producing a separation agent according to any one of claims 3 to 7.   A chromatographic separation method using the separation agent produced by the method for producing a separation agent according to any one of claims 3 to 7.   The adsorption separation method according to claim 8, wherein the separation object is a protein.   The chromatographic separation method according to claim 9, wherein the separation target is a protein.