JP2003524680A - Affinity control type material using stimulus-responsive polymer and separation / purification method using the material - Google Patents

Abstract

  (57) [Summary] An affinity-controlling material is provided in which an affinity substance (ligand) having an affinity for a stimulus-responsive polymer and a target substance is independently bound to a support, preferably covalently. This material utilizes a support that suppresses nonspecific adsorption to proteins and has high separation performance, and uses at least one of conditions other than temperature (for example, pH in solution or concentration of organic solvent or salt). With a constant, a target substance such as a physiologically active substance can be separated and purified by physical stimulation to achieve excellent separation performance.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD OF THE INVENTION

The present invention provides (a) a stimulus-responsive polymer (stimulus-responsiv)
epolymer) and (b) affinity control type material (affinity con)
Affinity substance (ligand) having an affinity for a target substance present in a solution in contact with a controlling material (affinitive sub)
stance (ligand)). By subjecting the material to a stimulus, for example a physical stimulus, it is possible to change the chemical and / or physical environment provided around the ligand, thereby changing the affinity between the ligand and the target substance. it can. Environmental changes are typically associated with structural changes in stimuli-responsive macromolecules.

In the present invention, the term “solution” means various liquids, which typically contain dissolved buffer components and salts (ions). Typical solutions have solvents such as water, organic solvents and mixed solutions thereof as liquid components. Examples of the organic solvent include water-miscible solvents such as water-miscible alcohol, acetonitrile, tetrahydrofuran, and the like, and mixed solvents thereof. In the present invention, the terms "change in affinity", "changing affinity" and the like refer to apparent affinity, ie the affinity that can be measured under the conditions applied.

Affinity controlled materials are referred to below as separation media.
Alternatively, it is also referred to as a separation material. Furthermore, the present invention relates to affinity-controlled materials that remove or separate and / or purify desired target substances (metal ions, pharmaceuticals, biological components, etc.) from a mixture containing other substances.

Further, the present invention relates to a method for separating and purifying a target substance (metal ion, drug, biological component, etc.) using the affinity control type material. The method preferably includes keeping at least one of the conditions other than the temperature (for example, the pH in the solution or the concentration of the organic solvent or the salt) constant.

[0005]

[Prior art]

Affinity binding-based ion exchange chromatography, reverse phase chromatography, and other affinity chromatography and various batch protocols as a means for effectively separating and purifying biological components, pharmaceuticals, etc. from a mixture of substances. (Batch-wise proto
cols) etc. have been used so far. Further, in recent years, with the development of biotechnology, many new physiologically active substances such as recombinant proteins have been developed. At the same time, there is a need for an improved method of separating and purifying these substances without losing their physiological activity.

Separation and / or purification of target substances from a mixture of substances by affinity chromatography and other adsorption-based separation techniques typically involve a separation step (adsorption step) and a release step (desorption or elution step). Include. Compared to the binding process, the release process requires a change in the composition of the liquid in contact with the separation medium. Examples of making appropriate changes are the addition of organic solvents to the mobile phase, increasing the salt concentration of the mobile phase, or changing the pH value of the mobile phase. This is the turbulent liquid phase used in batch processing.
phase) or non-turbulent liquid phase
Phase) is also applied. These operations cause deactivation of the physiologically active substance. Further, even if a physiologically active substance can be separated and purified while retaining its activity by conventional chromatography, in many cases, the organic solvent, salt, etc. added to the mobile / liquid phase are at least purified or isolated. It is necessary to partially remove it from the target substance. This causes another problem that the activity and / or recovery / yield of the target substance is reduced.

In the last ten years, so-called chromatographic techniques and insoluble separation media of the kind used in chromatography
There is interest in combining stimuli-responsive macromolecules with other techniques based on binding or partitioning of the target substance to the N medium.

Recently, there has been a description of separation media containing ion-exchange groups covalently bound to stimuli-responsive polymers. For example, Japanese application 14 corresponding to WO99 / 61904
See 0722/98.

Galaef et al (J. Chromatog. A684 (1994) 3
7-43 and WO 94/154951 describe the temperature elution of target substances in a chromatography system in which a plurality of ligand groups are covalently bound to a base matrix. The temperature-responsive polymer is a multi-point bond (
It is indirectly attached to the ligand group by a multi-point attachment. That is, a thermo-responsive polymer (thermo-responsiv)
The binding of the e polymer) is the pre-binding of the ligand to the base matrix (
prior attachment). By varying the temperature, the ligand more or less affinity binds to its target substance.
bond).

Hofman et al. , (WO8706152) describe a separation method in which a ligand is bound to a temperature-responsive polymer. Binding and elution of the target substance occur on the same side of the critical solution temperature. The term critical solution temperature is further referred to in the description of thermoresponsive polymers.

There are also numerous references describing chromatography based separation materials that include stimuli-responsive polymers but do not have a ligand covalently bound to a temperature-responsive polymer. Gewehr et al (Macromolecular Chemi
stry and Physics 193 (1992) 249-256)
Gel chromatography on porous silica beads coated with a temperature responsive polymer is described. Hosoya et al (Hosoya et al.) (Anal. Chem. 6).
7 (1995) 1907-1911); Yamamoto et al. (Yamamoto et al.) (Proc.114 ^th National Meeting of ^the
Pharmaceutical Society of Japan, Tok
yo (1994) 160; Kanazawa et al (Kanazawa et al.) (Pharmaceutical Journal 117 (10-11) (1997) 817-824); Kanazawa e.
t al (Kanazawa et al.) (Anal. Chem. 68 (1) (1996) 100-1.
05); Kanazawa et al (Kanazawa et al.) (Anal. Chem. 69).
(5) (1997) 823-830); Kanazawa et al (Kanazawa et al.) (J. Pharm. Biomed. Anal. 15 (1997) 1545-1).
550); Yakushiji et al (Yakushiji et al.) (Langmuir1)
4 (16) 1998) 4657-466268); Kanazawa et a.
l (Kanazawa et al.) (Trends Anal. Biochem. 17 (7) (19)
98) 435-440); Yakushiji et al (Yakushiji et al.) (An.
al. Chem. 71 (6) 1999) 1125-1130); Grace &
Co (EP534040); Okano (Okano) (JP-A-6-108643).
Describes reverse phase chromatography on a matrix coated with a thermoresponsive polymer used for the separation of biomolecules. This matrix may be porous. The hydrophobic groups utilized are naturally present in the polymer as such. No ligand is covalently attached to the macromolecule after polymerization.

(A) Stimuli-responsive polymer and affinity ligand (affinity liga)
nd) and a separation medium covalently functionalized with a conjugate.
b) Each aspect of the general idea of carrying out a chromatographic separation based on a separation medium functionalized by individual / independent binding of a stimuli-responsive macromolecule and an affinity ligand, 1999 respectively. March 2
It was announced at the National Convention of the Chemical Society of Japan on 8th and May 27, 1999 (SPS
J (Polymer Science Society of Japan), Annual Meeting, pp. 583).

Separation media are often in the form of particles that can be porous or non-porous. Typically, the particles will have a ligand directly or indirectly, for example,
It includes a base matrix that is bound through a spacer. The material of the particles may be synthetic macromolecules such as styrene crosslinked polymers, acrylate / methacrylate crosslinked polymers and crosslinked polymers of similar materials. However, polystyrene particles and polymethacrylate particles themselves are relatively hydrophobic and therefore often exhibit significant and non-specific adsorption of various substances that may be present with the target substance of interest. If such particles are to be used as supports in affinity-based separations, then it is necessary to make the particles sufficiently hydrophilic to minimize hydrophobic / non-specific adsorption. In order to improve the separation and purification ability of particles, particles of uniform particle size (monosized particles or monodispersed particles) are used.
It is often convenient to use particles).

Although hydrophilic synthetic polymer supports in the form of porous particles having a uniform particle size have been required for a long time, the number of manufacturing methods therefor is not so large.

[0015]

DISCLOSURE OF THE INVENTION

The object of the present invention is to provide a solution to the abovementioned problems, thereby enabling improved separation methods and materials. In view of the present situation, at least one of the conditions other than temperature (for example, pH in the solution or the concentration of the organic solvent or salt, etc.) is utilized by utilizing a support having high separation performance by suppressing non-specific adsorption with proteins.
It is intended to provide an affinity control type material for separating and purifying a target substance such as a physiologically active substance by a physical stimulus while keeping the two conditions constant, and a method for separating and purifying a target substance using the affinity control type material.

As a result of earnest studies, the present inventor has found that a stimuli-responsive polymer and an affinity substance (ligand) for a target substance are independently supported by a support, that is, a separated binding structure (sepa).
It was found that an affinity-controlled material can be synthesized by binding via rate linking structures. Further, the present inventor has made the target substance adsorbed by the affinity control type material under physical stimulation such as temperature change while keeping at least one condition other than physical stimulation constant. I found that it can be removed. For example, if this stimulus is a temperature change, the conditions to be kept constant can be selected from, for example, pH, the concentration of the organic solvent, the salt concentration and the like. Further, the present inventors have found that the binding ability between the ligand and the target substance depends on the length of the spacer that binds the ligand to the support / base matrix, or the size of the stimuli-responsive polymer that binds to the support. Found to depend on.

The present inventor may also use hydrophilic porous polymer particles, which may have a uniform particle size (monosized = evenly dispersed), as the support in the present invention. I found it. Thus, in this part of the invention, the inventor has found to use particles obtained by polymerisation of an emulsion / suspension of the monomers obtained by the membrane emulsification method. Also, in this embodiment, the particles are chemically treated with an acidic substance or a basic substance, and a hydrophilic group or / and a ligand and / or a stimuli-responsive polymer is covalently bonded, for example, a hydroxy group and / or an amino group is covalently bonded. It includes a group that enables This chemical treatment requires that the starting materials (monomers) used in the membrane emulsification process be able to react with the utilized acidic or basic substances. Typical reactive groups have hitherto been epoxy groups. The present invention has been completed based on these findings.

That is, the present invention relates to affinity controlled materials / separation media of the type defined in the introduction. One of the main features is that the stimuli-responsive macromolecule and the ligand are bound to the base matrix by individual / independent binding. Varying the appropriate level and / or intensity of stimulus for the macromolecule from one side of the critical level / intensity to the other side causes reversible affinity between the ligand and its target substance. Changes are brought about. This stimulus may be a physical stimulus and the experimental part gives a temperature change as a typical example.

Further, the present invention is to change the chemical or physical environment of the stimuli-responsive polymer under physical stimulation while keeping at least one condition other than the changed condition constant. That is, the affinity control for reversibly changing the affinity of the target substance for the affinity substance by exposing the thermoresponsive polymer to the change of one physical stimulus while keeping at least one of the other stimuli constant. Regarding mold material. If the stimulus to be changed is temperature, then at least one condition / stimulus other than temperature (eg, pH, concentration of organic solvent or concentration of salt) is kept essentially constant.

Furthermore, the present invention provides that the affinity between the ligand and the target substance is (a) the length of the spacer that binds the ligand to the base matrix, or (b) the size of the stimuli-responsive polymer, eg, the molecular weight. By an affinity controlled material / separation medium as defined above depending on the size as reflected by

Furthermore, the invention provides that the support (base matrix) preferably has a uniform particle size and / or is produced by polymerizing a monomer emulsion / suspension obtained from the membrane emulsification process. It relates to an affinity-controlled material as defined above comprising hydrophilic porous polymer particles. Hydrophobic particles having a functional group reactive with an acidic substance or a basic substance can reduce the degree of hydrophobicity by reacting with a substance of this type. A typical example of a reactive group is an epoxy group. That is,
If the film emulsification method requires polymerization, the starting monomers contain epoxy groups.

Furthermore, the present invention relates to the use of such an affinity control type material as a packing material for chromatography. Another embodiment of the present invention is a method for separating one or more target substances from a liquid sample (solution) (liquid I). This embodiment comprises the steps of: (a) contacting a liquid sample containing the target substance (liquid (I)) with a separation medium (containing chromatographic packing material), which separation medium is functionalized with a ligand. This ligand has affinity binding with the target substance.
This contact is performed under conditions that allow the target substance to bind to the ligand; (b) the target substance from the ligand is free of this target substance. Liquid (I
Contacting said carrier with liquid (II) so as to release into I).

The liquid sample is preferably separated from the separation medium between steps (a) and (B). After step (b), liquid (II) may be separated from the separation medium. The target substance may be removed from the liquid (II) if so desired. The separation medium may be washed between steps (a) and (B).

Molecules that are biologically and / or physiologically active, such as nucleotide structures (including nucleic acids), polypeptide structures (including proteins), carbohydrate structures,
A biological organic molecule having a structure selected from steroid structures and the like (bio)
For target substances that are organic molecules, the liquids have typically been aqueous.

This embodiment of the invention is characterized by the following points: (i) The separation medium comprises a stimuli-responsive macromolecule and a ligand bound separately as defined elsewhere herein. A support / base matrix, and (ii) a stimuli-responsive polymer that enhances binding of the target substance to the ligand in step (a) and at least during binding of the target substance to the ligand. The stimuli-responsive macromolecules of the target substance are subjected to a conformational level / intensity of stimulation on the separation medium, and (iii) in step (b) and at least during the release of the target substance from the ligand. Subjecting the separation material to a level / strength stimulus that results in a conformation that prevents it from binding to the ligand.

It is preferable to apply the same kind of stimulation to the steps (a) and (B). Compared to step (a), the level / intensity of stimulation in step (b) depends on the stimulus-sensitive polymer used.
On the opposite side of the critical level / strength. If step a is repeated after step b, typically after individual washing / regeneration steps and equilibration steps, the process can be cycled.

The various embodiments of the process according to the invention can be carried out both batch-wise and by chromatographic methods. Chromatographic methods include, for example, subjecting the bed of the separation medium to appropriate stimulation with respect to the individual steps and the stimuli-responsive polymer bound to the base matrix,
It can be carried out by passing various liquids in a plug flow (mobile phase) through the bed. This floor is a porous monolith (po
It may be a loose monolith) or a bed of packed or fluidized particles.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION

Stimulus-Responsive Polymer The physical stimulus to be used in the present invention is, for example, temperature.

Other stimuli may be applied, such as light, magnetic fields, pH, etc., depending on the particular stimuli-responsive polymer used. Stimuli-responsive macromolecules are often referred to as "intelligent polymers."

Stimuli-responsive macromolecules are characterized by undergoing structural and reversible changes in their physicochemical properties when they are exposed to the right type and intensity or level of stimulus. It can be said that this change is a conversion from a marked hydrophobicity to a marked hydrophilicity or a conversion from a marked hydrophilicity to a marked hydrophobicity. The exact level / intensity of stimulation required for this conversion to occur is the critical level of stimulation.
It is called level or critical intensity and depends on the structure of the polymer and often on other conditions (solvent, solute such as salt, etc.). The most well known and most utilized macromolecules of this type are responsive to heat (thermoresponsive or temperature responsive macromolecules). Temperature responsive macromolecules have a sharp temperature limit at which they switch from a predominantly hydrophilic state to a predominantly hydrophobic state and from a predominantly hydrophobic state to a predominantly hydrophilic state.
limit). For temperature responsive polymers in solution, conformational / physicochemical property changes are referred to as the so-called critical solution temperature (cri
Tal solution temperature (CST).

For temperature-responsive polymers in aqueous media, low critical solution temperature (LCST)
Or there is a high critical solution temperature (UCST). Regarding the polymer having LCST,
The polymer changes from a hydrophilic conformation to a hydrophobic conformation as it passes through the LCST from a temperature below the LCST. UCST
The polymer having a temperature from lower than UCST to UC
It changes from a hydrophobic conformation to a hydrophilic conformation as it passes through the ST. The exact values of LCST and UCST depend on the macromolecule and other application conditions (solvent, other solutes, etc.).

As mentioned above, one of the features of the present invention when the temperature responsive polymer is used is
Binding to and release from the ligand occurs on the opposite side of the applied CST.

The stimuli-responsive polymer used in the present invention has an insignificant affinity for the target substance as compared with the affinity between the target substance and the ligand bound to the support. It is preferable. It is preferred that there is no significant affinity between the ligand and the stimuli-responsive macromolecule.

Examples of the stimuli-responsive polymer used in the present invention include poly (N-substituted acrylamides) including poly (N-isopropylacrylamide) and poly ((N-isopropylmethacrylamide)). N-substituted methacrylamide), poly (N, N-disubstituted acrylamide), poly (N, N-disubstituted methacrylamide), polymethyl vinyl ether, poly (ethylene oxide-propylene oxide) copolymer, partially saponified polyvinyl alcohol Examples thereof include polyvinyl alcohol derivatives such as the above, and cellulose derivatives such as methyl cellulose. Further, in order to covalently bond the stimuli-responsive polymer to the support / base matrix, it is possible to introduce a reactive functional group such as an amino group, a carboxyl group or a hydroxyl group into the stimulus-responsive polymer.

Ligands Ligands are affinity bonds or covalent bonds,
It is possible to bind to the base matrix, preferably by the latter. According to the inventive concept, it is not provided by a stimuli-responsive macromolecule.

A typical one of the ligands binds to the target substance by more or less pure ionic (electrostatic) interactions. Alternatively, this binding involves more complex interactions such as conventional affinity binding (affinity adsorption). With respect to ionic interactions, ligands include positively or negatively charged entities (ion exchange; immobilized materials include primary amines, secondary amines, tertiary amines and quaternary amines). Groups such as quaternary ammonium, sulfonate, sulfate, phosphonate, phosphate, carboxy). The more complex interactions are illustrated by the ligands, which are two individual affinity members, as shown below.

(A) antibodies and antigens / haptens (b) lectins and carbohydrate structures (c) IgG binding proteins and IgG (d) macromolecular chelators and chelates (e) complementary nucleic acids Affinity members also catalyze reactions (Entities) involved in, for example, enzymes, enzyme substrates, cofactors, cosubstrates
(states) etc. are also included. Synthetic members of cell-cell and cell-surface interaction and biologically produced affinity members.
mimetics of bioproduced affinity mem
bers) are also included. The term ligand also refers to a complex having complex biomolecules by affinity, such as oligo- or polypeptide structures (including proteins), oligo and polynucleotide structures (including nucleic acids), oligo- or polysaccharide structures, etc. It also includes some complex organic molecules that bind to various biomolecules.

Still another example of the ligand used in the present invention is Cibacrone Blue F3G-A (trade name; manufactured by Fluka).
And other complex dyes (complex dyes), iminodiacetic acid, sugar chains such as glucose, proteins such as heparin and lectin, biotin, benzamidine, lysine, arginine, peptides and DNA is mentioned. Further, according to the present invention, it is also possible to control the binding ability of the target substance by covalently binding the affinity substance of the target substance to the support through a spacer such as a divalent alkyl group or an ethylene oxide group. Is. Base Matrix (ie Chromatographic Packing) The separation medium used in the method of the present invention comprises a base matrix (support) which may be based on organic and / or inorganic materials. If the liquid used is aqueous, the base matrix is preferably hydrophilic. This applies in particular to target substances which are biomolecules of the type mentioned above.

The base matrix is preferably based on a polymer, the polymer is preferably insoluble and swells slightly in water, and a gel is preferred. Derivatized (d
Included in this definition are hydrophobic macromolecules that have been made erivatized).
Suitable macromolecules are polyhydroxy macromolecules such as agarose, dextran,
Polysaccharide-based polyhydroxy polymers such as cellulose, starch, pullulan, etc., as well as polyacrylic acid amides, polymethacrylic acid amides, poly (hydroxyalkyl vinyl ethers), poly (hydroxyalkyl acrylates) and polymethacrylates (eg polyglycidyl). Methacrylate). Completely synthesized polymers such as polyvinyl alcohol and polymers based on styrene and divinylbenzene, and copolymers containing two or more of the monomers corresponding to said polymers. The water-soluble polymer is derivatized to become insoluble by binding to an insoluble substance through, for example, adsorption or covalent bond. Hydrophilic groups can be treated as hydrophobic polymers by polymerization of monomers that exhibit groups that can be converted to OH or by final hydrophilization of the polymers, for example by adsorption of suitable compounds such as hydrophilic polymers. It can be introduced on top (for example on a copolymer of monovinylbenzene and divinylbenzene).

Suitable inorganic materials used in the base matrix are silica, zirconium oxide, graphite, tantalum oxide and the like. Preferred base matrices lack hydrolytically labile groups, such as silane groups, ester groups, amide groups and groups present as such in silica. Preferred base matrices contain functional groups that can be used to covalently attach stimuli-responsive macromolecules and / or ligands. Examples of this type of functional group include a hydroxy group, a carboxy group, an amino group and the like.

The matrix may be porous or non-porous. This means that the matrix may be fully or partially permeable (porous) or completely impermeable (non-porous) to the compound to be removed. .

The pores may have a size ≧ 0.1 μm, for example ≧ 0.5 μm, which means spheres (≧ 0.5 μm in diameter each ≧ 0.5 μm).
a sphere ≧ 0.1 μm reactive ≧ 0.5 μm in d
i.e.) can be passed through. The liquid to be applied is of this type of pore system (conductive pore system).
It can be said that it is possible to flow through the system. When the support matrix is in the form of beads packed in the bed, the ratio of the pore size of the transmissive pore system to the diameter of the particles is typically in the range 0.01-0.3, preferably 0. .0
The section is 5-0.2. Pores of size ≧ 0.1 μm, eg ≧ 0.5 μm are often referred to as macropores.

The base matrix may also have pores with a size ≦ 0.5 μm, eg ≦ 0.1 μm, which means a diameter ≦ 0.5 μm, eg ≦ 0.1 μm.
This means that only m spheres can pass. Pore sizes <0.5 μm, eg ≦ 0.1 μm, are often micropores.
Called. In one embodiment of the invention, the base matrix is 1-1000μ.
m, preferably high performance applications
in the range of 5-50 μm, for preliminary purposes 50-
It has an irregular or spherical morphology with a size in the range of 300 μm. The particles used may be monodisperse or polydisperse.
size). The term monodispersed particles refers to a particle population (partially greater than 95% of particles having a size within ± 5% of their average diameter).
cle populations, which for the purposes of the present invention contemplate uniform sized expression particles. Polydispersed particles include other particle populations.

The base matrix may also be in the form of a monolith having at least macropores as defined above. Alternate geometric form
A geometric form is an inner wall of a pipe or the like.

The stimuli-responsive macromolecules and ligands as defined above are capable of binding to the outer and / or inner surface (macropore and / or micropore surface) of the base matrix. As mentioned above, the stimuli-responsive macromolecule and the ligand are bound to the base matrix by physical adsorption and / or covalent bonds, preferably the latter.

It is particularly preferred to use hydrophilic porous polymer particles having a uniform particle size as the support / base matrix, which particles, as mentioned above, depend on the membrane emulsification process and subsequent acidic or basic substances. Manufactured by chemical treatment.

The film emulsification method used in the present invention means that the first liquid permeates a glass film (preferably made of glass) and is sent to a second liquid immiscible with the first liquid, and thus in the second liquid. And forming substantially the same size of droplets. Note that this method is described, for example, in S. Omi, K .; Katami, A .; Yama
motor, and M.M. Iso, J .; Appl. Polym. Sci. , 51 (
1994) 1-11. If the first liquid contains a polymerizable monomer and the droplets undergo polymerization, particles will form in the second liquid.

Thus, according to the inventor's novel discoveries, a preferred support material (base matrix) is prepared as follows: From monomers and diluents etc. useful as starting materials for polymeric particles. A liquid mixture (first liquid) is prepared. Next, one side of the porous glass membrane is filled with this liquid mixture and the other side is filled with a second liquid immiscible with the first liquid. Pressure is applied to the liquid mixture to pass through the membrane to form droplets in the second liquid. For example, the first liquid may be immiscible with water and the second liquid may be aqueous and may contain an emulsion stabilizer or the like, resulting in a substantially uniform size. An emulsion consisting of small droplets having Subsequently, for example, by heating, latex particles having a uniform particle size are obtained, whereby the polymerization is carried out. If the monomer contains groups which are reactive with acidic or basic substances, the latex particles are stirred in a solution containing this type of substance to give hydrophilic porous polymer particles. During this post-processing,
Reactive functional groups such as amino groups can be introduced into the support. By using these reactive functional groups, the stimuli-responsive polymer or ligand can be covalently attached to the support.

Examples of the monomer used for producing the hydrophilic porous polymer particles having a uniform particle diameter include glycidyl acrylate, glycidyl methacrylate, diacrylate ester (eg ethylene diacrylate), dimethacrylic acid. Esters (eg, ethylene dimethacrylate), glycidyl vinyl benzyl ether, divinyl benzene and the like can be mentioned. Further, these monomers can be combined arbitrarily. Emulsion The diluent used in the production of the hydrophilic porous polymer particles having a uniform particle size of the present invention may be any compound that does not polymerize with the monomer used. Examples of such diluents include aromatic solvents / compounds such as toluene, aliphatic compounds such as dodecane, and the like.

Examples of the acidic substance or basic substance used for producing the hydrophilic porous polymer particles having a uniform particle diameter of the present invention include sulfuric acid, hydrochloric acid, nitric acid, acetic acid, sodium hydroxide, ammonia, 1,6 And aliphatic diamines such as diaminohexane.

Production of Separation Material of the Present Invention The affinity control material / separation material of the present invention can be produced, for example, as follows. (A) A method comprising covalently immobilizing a stimuli-responsive polymer or a copolymer thereof on a support, and then covalently immobilizing an affinity substance of a target substance on the support; or (b) A method comprising covalently immobilizing an affinity substance of a target substance to a support, and then immobilizing a stimuli-responsive polymer or a copolymer thereof to the support covalently; or (c) the target substance A method comprising simultaneously covalently immobilizing an affinity substance and a stimuli-responsive polymer or a copolymer thereof on a support.

[0052]

【Example】

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

[0053]

Example 1 1. Synthesis of stimuli-responsive polymer N-isopropylacrylamide (20 g), 3-mercaptopropionic acid (
0.18 g), and 2,2'-azobis (4-cyanovaleric acid) (0.27 g)
Was dissolved in tetrahydrofuran (200 ml) and placed in a polymerization tube. Oxygen in the solution was removed by the freeze-thaw degassing method. Polymerization was performed at 60 ° C. for 2 hours, and poly (N-isopropylacrylamide) having a carboxyl group at one end was synthesized by using diethyl ether as a reprecipitation solvent.

The molecular weight was measured by GPC (gel permeation chromatography) and end group quantification method. In GPC, dimethylformamide containing 10 mM lithium bromide as a mobile phase, α-3000 (manufactured by Tosoh Corporation) as a column, and polystyrene as a standard substance were used, and a number average molecular weight of about 4500 and a weight average molecular weight of about 10,000 ( N-isopropylacrylamide). In the end group determination method, 0.0
When the carboxyl group of poly (N-isopropylacrylamide) synthesized using a 1N aqueous sodium hydroxide solution was determined, the number average molecular weight was about 5,000.
Met. It was confirmed that the number average molecular weights of the synthesized poly (N-isopropylacrylamide) were almost the same by the GPC and the end group quantification method.

The synthesized poly (N-isopropylacrylamide) (10 g), N-hydroxysuccinimide (0.25 g) and N, N′-dicyclohexylcarbodiimide (0.45 g) were dissolved in tetrahydrofuran (60 ml) at room temperature. It was stirred for 12 hours. After the precipitate was collected by filtration, diethyl ether was used as a reprecipitation solvent, and the carboxyl group at one end was esterified with N-hydroxysuccinimide poly (
N-isopropylacrylamide) was synthesized. 2. Synthesis of hydrophilic porous polymer particles having uniform particle size Glycidyl methacrylate (3.1 ml), ethylene dimethacrylate (1.9 m
l), toluene (7.1 ml) and dodecane (0.4 ml) and 2,2'-
50 mg of azobis (2,4-dimethyl-valeronitrile) was used as a raw material, and pressure was applied to 2 through an MPG (microporous glass) pipe having an average pore diameter of 1.95 μm.
An O / W emulsion was prepared by extruding raw materials into a wt% polyvinyl alcohol aqueous solution. The polymerization was carried out at 70 ° C. for 6 hours to synthesize latex particles having a uniform particle size in high yield. The average particle size of these latex particles is 12.5 μm, and CV (
The coefficient of variation (coefficient of variation) is 12.
It was 4%, and it was confirmed that the particle diameters were uniform. Furthermore, the synthesized latex particles (3.5 g) were added to an aqueous solution (1) containing 1,6-hexyldiamine (1.8 g).
60 ml) and the mixture was stirred at 30 ° C. for 2 hours.

Hydrochloric acid-calcium chloride method (Nakamura et al., Collection of Polymers,
37 (7) (1981) 485-491) describes that epoxy groups were present at 3.1 mmol / g on the surface of latex particles before treatment with 1,6-hexyldiamine. . In addition, the assay method using titration
0 on the surface of the hydrophilic porous polymer particles having a uniform particle size treated with hexyldiamine
． It was revealed that 36 mmol / g of amino group was introduced.

Further, the hydrophilic polymer particles having a uniform particle size and treated with 1,6-hexyldiamine (3.5 g) were stirred in 10 ml of acetic anhydride to acetylate the amino groups on the surface to form amidated particles. Synthesized. The amidated particles did not adsorb bovine serum albumin (BSA) in the mobile phase of 20 mM phosphate buffer (pH 7.0). From this, it was revealed that the hydrophilic porous polymer particles having a uniform particle size have hydrophilicity useful as a chromatography carrier (base matrix) for separating proteins such as affinity chromatography. 3. Immobilization of poly (N-isopropylacrylamide) onto a support 1,6-hexyldiamine-treated hydrophilic porous polymer particles having a uniform particle size (
4.5 g), poly (N-isopropylacrylamide) (4.5 g) in which the carboxyl group at one end was esterified with N-hydroxysuccinimide, and acetonitrile (75 ml) were stirred at room temperature for 12 hours. After washing with acetonitrile, tetrahydrofuran, methanol, and acetone, it was dried at room temperature. According to elemental analysis, 3.4 wt% of poly (
It was confirmed that N-isopropylacrylamide) was immobilized. Further, in order to compare the affinity control ability with temperature, a filler (noCB) in which the amino group remaining on the support was acetylated with acetic anhydride was also synthesized. 4. Immobilization of Cibacron Blue F3G-A on a support Poly (N-isopropylacrylamide) -immobilized support (0.70 g)
(Obtained from 3 above), 1,3-butadiene diepoxide (0.09 ml)
) Or ethylene glycol diglycidyl ether (0.21 g) and acetonitrile (10 ml) at 30 ° C. for 1 hour with stirring, whereby the amino group remaining on the support and one epoxy group of the diepoxide compound serving as a spacer are removed. It was made to react. Acetic anhydride (0.11 ml) was added to the suspension and the mixture was stirred at 30 ° C. for 1 hour to acetylate the unreacted amino group of the support. The support on which poly (N-isopropylacrylamide) and the spacer were immobilized was washed with acetonitrile and acetone, and then dried at room temperature.

A mixture of supports (0.61 g) each having poly (N-isopropylacrylamide) and a spacer immobilized thereon, aminohexylated cibacron blue F3G
-A (0.89 g) and water (10 ml) were added, pH was adjusted to 11 with sodium hydroxide, and the mixture was stirred at 25 ° C for 3 hours to give poly (N-isopropylacrylamide).
And Cibacron Blue F3G-A were respectively immobilized on a support to synthesize a filler. The amount of Cibacron Blue F3G-A immobilized was determined by titration, and the amount of the filler (CB-4) using 1,3-butadiene diepoxide as a spacer was 21 μmol /
g, ethylene glycol diglycidyl ether (CB-10) as a spacer
The filler used was 12 μmol / g.

[0059]

Example 2 1. Filling of fillers NoCB, CB-4 and CB-10 were wet-filled with water and had an inner diameter of 4.
Each of them was packed in a stainless steel column having a length of 6 mm and a length of 30 mm. 2. Measurement of Adsorption Amount for BSA Adsorbed by Filler Using a citrate buffer solution (I = 0.01) of pH 5 as a mobile phase, the adsorption curve of each filler at 40 ° C. with reference to 20 ° C. I asked for. The results are shown in Table 1.

[0060]

[Table 1]

The adsorption amount of BSA is larger in CB-10 than in CB-4. This indicates that the spacer length between the support and Cibacron Blue F3G-A affects the adsorption amount of BSA. It is also shown that poly (N-isopropylacrylamide) bound to the support does not significantly affect the adsorption of BSA. 3. Affinity Control by Temperature Change of Packing Material in Affinity Chromatography Using CB-10, after adsorbing BSA to Cibacron Blue F3G-A which is a ligand of the packing material CB-10 at 40 ° C, the temperature was raised to 20 ° C. The structure of the stimuli-responsive polymer was changed by changing it. Due to the structural change of the polymer
It was confirmed that SA was desorbed and eluted in the mobile phase. The result is shown in FIG. A citrate buffer solution (I = 0.01) having a pH of 5 was used as a mobile phase, and BSA
111 μg) was loaded onto the column of CB-10. Micro BCA ^TM PROTE
The amount of BSA in the eluate was measured using IN ASSAY REAGENT KIT (manufactured by Pierce). Excess BSA was eluted with an eluate volume of 1-4 ml. After elution of BSA was not confirmed by flowing the mobile phase (column) at 40 ° C until the amount of the eluate was 6 ml, the flow of the mobile phase was stopped and the mixture was cooled at 20 ° C for 20 minutes. 20 ° C
When the mobile phase was allowed to flow again as it was, BSA adsorbed to the ligand was desorbed at 40 ° C. and was eluted from the column due to the structural change of poly (N-isopropylacrylamide) bound to the support (eluate volume 7 ~ 9 ml). The amount of BSA eluted by this temperature change was 90% of the amount of CB-10 adsorbed on BSA. These results show that the target substance is removed or separated using a material containing a support / base matrix in which a stimuli-responsive polymer and a ligand having an affinity for the target substance are covalently immobilized through separate bonds. It has been clarified that it can be purified. Moreover, this result shows that the affinity between the target substance and the ligand can be controlled by a physical stimulus such as temperature.

[0062]

【The invention's effect】

  The affinity control type material in the present invention has the following advantages. 1) There is no need to use chemically harsh elution conditions for separation and purification of target substances     Therefore, the activity or recovery rate of physiologically active substances, etc. is greater than that of conventional separation and purification methods.     Can be improved. 2) The affinity substance of the target substance and the stimuli-responsive polymer are covalently bound to the support.     Therefore, they do not peel off and interfere with separation and purification. 3) Regeneration of packing material when used as packing material for affinity chromatography     Can be carried out more quickly than with conventional carriers. 4) Many types not possible with conventional affinity chromatography packing materials     It becomes possible to separate and purify the target substance.

[Brief description of drawings]

FIG. 1 is a chromatogram when BC-10 was used to control the affinity of BSA by changing the temperature.

[Procedure for Amendment] Submission for translation of Article 34 Amendment of Patent Cooperation Treaty

[Submission date] June 15, 2001 (2001.6.15)

[Procedure Amendment 1]

[Document name to be amended] Statement

[Correction target item name] 0010

[Correction method] Change

[Contents of correction]

Ohnishi et al (Japanese Patent Laid-Open No. 9-49830), Ohnish
i (JP-A-8-103653), Ohnishi (JP-A-7-13650)
5) and Ohnishi (JP-A-7-135957) disclose a separation material containing a stimuli-responsive polymer and a substance having a specific affinity for the surface of a support matrix. However, these publications report elution of a target substance by temperature change based on a separation system that immobilizes a copolymer (complex) obtained by binding a ligand to a temperature-responsive polymer on a base matrix. Only. When such a complex is used, the elution temperature of the target substance varies depending on the type and property of the ligand, and therefore the temperature must be controlled by changing the type of the complex depending on the type of the ligand. Hofman et al. , (WO8706152) describe a separation method in which a ligand is bound to a temperature-responsive polymer. Binding and elution of the target substance occur on the same side of the critical solution temperature. The term critical solution temperature is further referred to in the description of thermoresponsive polymers.

─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 4D017 AA03 BA07 CA20 DA03 EB07                 4G066 AC22C AE01B AE02B AE14B                       CA07 CA08 CA20 DA07 DA11                       EA02                 4H045 AA20 CA42 GA26                 4J031 AA03 AA13 AA15 AA16 AA20                       AA22 AA53 AF08

Claims (9)

[Claims] 1. An affinity control type material in which a stimuli-responsive polymer and an affinity substance (ligand) having an affinity for a target substance are independently bound to a support, preferably covalently bound. 2. The chemical or physical environment around the affinity substance provided by the polymer is changed by subjecting the mixture of the affinity control type material and the affinity substance in the solution to physical stimulation. The affinity control material according to claim 1, which can change the affinity between the affinity substance and the target substance. 3. The affinity of the target substance for the affinity substance is reversibly changed by physical stimulation while keeping at least one of the conditions other than temperature constant.
Alternatively, the affinity control type material according to item 2. 4. The affinity control type material according to claim 1, 2 or 3, wherein the physical stimulus is a temperature change. 5. The affinity control type material according to claim 1, wherein the affinity substance of the target substance does not interact with the stimuli-responsive polymer. 6. The binding ability of a target substance is controlled by the length of a spacer for binding an affinity substance of the target substance to a support or the size of a stimuli-responsive polymer. The affinity control type material according to any one of items. 7. The support contains hydrophilic porous polymer particles having a uniform particle size, which are produced by a film emulsification method using a monomer having an epoxy group in a side chain as a raw material and a chemical treatment with an acidic substance or a basic substance. 7. The affinity control type material according to any one of claims 1 to 6. 8. The affinity control type material according to any one of claims 1 to 7, which is used as a packing material for chromatography. 9. A method for separating and purifying a target substance using the affinity control type material according to claim 1.