JP5234727B2 - Method for producing temperature-responsive chromatographic carrier, chromatographic carrier obtained therefrom, and use thereof - Google Patents

Description

  The present invention can separate a high molecular weight physiologically active substance useful in the fields of biology, medicine, pharmacy and the like on a solid surface within a temperature range of 0 to 80 ° C. under specific conditions including an aqueous mobile phase. The present invention relates to a method for producing a carrier for liquid chromatography coated with a charged polymer that varies in sum.

  High-performance liquid chromatography (HPLC) techniques that are currently in widespread use various combinations of mobile phase liquids and carriers, and modes of interaction related to separation. For this reason, since it can select variously according to a sample, it is utilized for isolation | separation and refinement | purification of various substances in recent years. Due to such technological innovations, although there are limitations, it has become possible to separate and purify pharmaceuticals in the biochemical field and to separate peptides, proteins, nucleic acids and the like. Further, as the application to biopharmaceuticals such as recombinant proteins produced by genetic engineering techniques becomes more prominent, further innovations in these separation and purification technologies are becoming stronger.

  Chromatography conventionally used is performed by changing the solvent of the mobile phase, mainly for the interaction between the solute contained in the mobile phase and the surface of the support without changing the surface structure of the support. For example, reverse phase chromatography is a method comprising a hydrophobic carrier and a polar mobile phase. Solutes are separated by partitioning between the mobile phase and the carrier according to their hydrophobicity. In this case, the hydrophobicity of the solvent of the mobile phase is changed, and the partition between the mobile phase and the carrier is changed and eluted. In that case, since organic solvents, such as a methanol and acetonitrile, are used as a solvent of a mobile phase, there existed a problem which impaired the activity of the biological component used as separation object.

  In ion exchange chromatography, an anion exchanger or a cation exchanger is fixed on the surface of a carrier, and substance separation is performed by changing the concentration or type of counter ion present in the mobile phase. Silica, cellulose, dextran, a copolymer of styrene and divinylbenzene, and the like are often used as the holding carrier, and ion exchange groups such as sulfonic acid groups and quaternary ammonium groups are usually introduced into these carriers. The solute dissociates into cations, anions, and zwitterions according to the hydrogen ion concentration in the mobile phase, and when this solute is passed through the ion exchange column, it competes with the ions in the mobile phase for the reversely charged exchange groups on the surface of the carrier. It binds and distributes between the mobile phase and the support surface at a constant rate. Ion exchange chromatography is performed by utilizing the fact that the speed of movement in the column varies depending on the strength of the bond. This proportion of distribution can be varied in several ways. For example, a method of changing the concentration of competing ionic species in the mobile phase or a method of changing the hydrogen ion concentration of the mobile phase to change the degree of ionization of the ion exchange groups on the surface of the carrier. That is, in ion exchange chromatography, a method of separating by adjusting the ionic strength and hydrogen ion concentration of a mobile phase is generally performed. At that time, since an acid or a buffer solution having a high salt concentration is used as a mobile phase, there is a problem that the activity of a biological component to be separated is impaired.

  Separation of pharmaceuticals, peptides, proteins, nucleic acids and the like useful in the biochemical field is generally performed by ion exchange chromatography techniques or reverse phase chromatography techniques. However, in the current method, there is a problem that the activity of the separation target is impaired as described above, and bioactive peptides, proteins, DNA, etc. are widely used in various fields including pharmaceuticals due to rapid progress in genetic engineering in recent years. Its use is expected and its separation and purification are extremely important issues. In particular, there is an increasing need for a technique for separating and purifying a physiologically active substance without impairing its activity. Further, since organic solvents, acids, alkalis, and surfactants used in conventional mobile phases impair the activity of physiologically active substances and become contaminants, improvement of the system is expected. Therefore, a separation / purification system that does not use these substances is also required from the viewpoint of avoiding environmental pollution of such substances.

  Against this background, Japanese Patent Application Laid-Open No. 05-133947 has studied to immobilize a polymer on silica gel or polymer gel that is usually used as a chromatography carrier. However, as far as the examples are concerned, the result of separation of the solute when actually using the carrier (separation chart) is not shown, and what kind of substance can be separated using this carrier, and more specifically There was no indication of any particular issues, and the details were unknown.

On the other hand, Japanese Patent Application Laid-Open No. 07-318551 discloses an example of actually separating various steroids and further lymphocytes by immobilizing a temperature-responsive polymer on a silica gel surface and using the carrier. It is clearly shown that various steroids and lymphocytes can be separated due to the characteristics of the temperature-responsive polymer actually immobilized on the silica gel carrier surface. However, the temperature-responsive polymer immobilization method exemplified here can immobilize only up to about 0.7 mg / m ² on the substrate surface due to the bulk of the polymer, and the function of the chromatography carrier is limited. It was. Innovative techniques that improve the prior art by designing in detail the immobilization of temperature-responsive polymers to the substrate surface have been desired.

  Under such circumstances, in Japanese Patent Application No. 2005-291719, the temperature-responsive polymer is immobilized at a high density on the surface of the silica gel by using the atom transfer radical polymerization method, and the separation when actually using the carrier is performed. Examples are also illustrated. However, in any of the examples, the separation peak width is large, and further technical improvement has been demanded for use as a method for separating a useful physiologically active substance. Although the reason for this is not described at all in this publication, this is because the temperature-responsive polymer is immobilized at a high density, but N, N-dimethylformamide (DMF) is used as a reaction solvent. This is presumably because the reaction rate of the atom transfer radical polymerization method is too fast and the temperature-responsive polymer is sufficiently uniformly immobilized on the support surface.

  Macromolecules 38, 5937-5943 (2005) describes a technique in which atom transfer radical polymerization of N-isopropylacrylamide is performed in various solvents including isopropyl alcohol (IPA). A comparison of is also described. However, this technique relates to a polymerization reaction in a solution, and there is no description of immobilizing a temperature-responsive polymer on a solid chromatographic support, and there is no description that suggests that. . Furthermore, there was no description used for the atom transfer radical polymerization reaction in the copolymerization of the monomer as the raw material of the temperature-responsive polymer and the monomer having a charged functional group, and there was no description suggesting that. .

On the other hand, Japanese Patent Application No. 10-140722 proposes a technique for separation by chromatography using a carrier on which a temperature-responsive polymer having a charge is immobilized. According to this method, the polymer chain whose hydration power changes within the temperature range of 0 to 80 ° C. coated on the surface of the carrier contracts and swells due to the temperature change, but when the polymer chain contracts, When the ion exchange group is hidden and the polymer chain swells, the ion exchange group in the polymer chain is exposed. By controlling the temperature of the carrier, the charge amount on the carrier surface can be controlled. However, the method of immobilizing a charged polymer exemplified here can immobilize only up to about 0.7 mg / m ² on the substrate surface due to the bulk of the polymer, and the function of the chromatography carrier is limited. It was. There has been a demand for innovative technology that improves the prior art by designing in detail the immobilization of charged polymers on the surface of the carrier.

  The present invention has been made with the intention of solving the problems of the prior art as described above. That is, the present invention is a method for producing a temperature-responsive chromatography carrier capable of efficiently separating a physiologically active substance useful in the field of biochemistry from an idea completely different from the prior art, a chromatography carrier obtained therefrom, and a method for using the same. The purpose is to provide.

In order to solve the above problems, the present inventors have studied and developed from various angles. As a result, an atom transfer radical polymerization initiator was immobilized on the surface of the carrier, and charged with an isopropyl alcohol as a solvent by an atom transfer radical method under a polymerization catalyst from within the temperature range of 0 to 80 ° C. By carrying out the growth reaction of the polymer with varying hydration power, the charged polymer is uniformly immobilized on the surface of the carrier, and the effective binding capacity of the carrier with the protein is under the condition that the polymer chain swells. It was found that a chromatographic carrier having a molecular weight of 6000 to 80,000 μg / g per carrier weight was obtained. The technology shown in the present invention was completely unpredictable from the prior art, and is expected to develop into a new chromatography system that was never found in the prior art. The present invention has been completed based on such findings.

  That is, the present invention immobilizes an atom transfer radical polymerization initiator on the surface of a substrate, and has a hydration power within a temperature range of 0 to 80 ° C. by an atom transfer radical method performed in the isopropyl alcohol under the catalyst from the initiator. Provided is a method for producing a temperature-responsive chromatographic support characterized in that a polymer having a varying charge is grown and reacted. The present invention also provides that the charged polymer is uniformly immobilized on the surface of the carrier, and the effective binding capacity of the carrier with the protein is 6000 to 80000 μg / g per carrier weight under the condition that the polymer chain swells. A temperature-responsive chromatography carrier is provided. Furthermore, the present invention provides a method for separating, purifying and fractionating physiologically active substances such as albumin and globulin using the temperature-responsive chromatography carrier.

  A novel separation system is proposed by the method for producing a temperature-responsive chromatography carrier described in the present invention. If this system is used, useful physiologically active compounds such as albumin and globulin can be separated.

  As a result of various studies to satisfy the above-mentioned demands, the present inventors have dissolved the surface structure of the support in the mobile layer without changing the mobile phase by changing external conditions such as temperature, or The present invention has been completed by developing a technique for separating, purifying, and concentrating by changing the interaction between the dispersed specific substance and the support surface. The purpose of the present invention is to perform the atom transfer radical method under specific conditions. Provides a method for producing a temperature-responsive chromatographic carrier, which causes a growth reaction of a polymer having a charge and a change in hydration power within a temperature range of 0 to 80 ° C., a chromatographic carrier obtained therefrom, and a method for using the same Is.

  The present invention is a method for immobilizing a polymer having electric charge and changing hydration power within a temperature range of 0 to 80 ° C. on a chromatography carrier. In the case of immobilizing a temperature-responsive polymer on a chromatographic carrier, controlling the amount of immobilization is an extremely important technique because it affects the performance of the carrier. If the amount of immobilization is small, the temperature responsiveness is lost and the target compound is not sufficiently retained, which is not preferable. Further, it is desirable that the polymer having a uniform molecular weight is uniformly applied to the support surface. The shape of the carrier used in the present invention is not particularly limited, and examples thereof include particles, flat plates, and tubes. In particular, when the carrier of the present invention is used as a carrier for chromatography, the carrier is preferably silica gel or glass. At that time, the pore diameter is not particularly limited, but is preferably 50 to 5000 mm, preferably 100 to 1000 mm, and more preferably 120 to 500 mm. If the molecular weight is less than 50 kg, only the solute having a considerably low molecular weight is targeted, and if it is more than 5000 mm, the surface area of the carrier is reduced and the separation is remarkably deteriorated.

In the present invention, a temperature-responsive polymer whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized on the carrier. Examples of the immobilization method include a method in which an atom transfer radical polymerization initiator is immobilized on the surface of a substrate, and a temperature-responsive polymer is grown from the initiator by the atom transfer radical method in the presence of a catalyst. The initiator used in that case is not particularly limited, but when the substrate is silica or glass as in the present invention, for example, 1-trichlorosilyl-2- (m, p-chloromethylphenyl) ethane 2- (4-chlorosulfonylphenyl) ethyltrimethoxysilane, (3- (2-bromoisobutyryl) propyl) dimethylethoxysilane, and the like. In the present invention, polymer chains are grown from this initiator. The catalyst at that time is not particularly limited, but when an N-alkyl-substituted (meth) acrylamide derivative is selected as a polymer whose hydration power changes, Cu ^I Cl, Cu ^I as copper halide (Cu ^I X). Br and the like. The ligand complex for the copper halide is not particularly limited, but tris (2- (dimethylamino) ethyl) amine (Me ₆ TREN), N, N, N ″, N ″ -pentamethyl. Diethylenetriamine (PMDETA), 1,1,4,7,10,10-hexamethyltriethylenetetraamine (HMTETA), 1,4,8,11-tetramethyl 1,4,8,11-azacyclotetradecane (Me) ₄ Cyclam), bipyridine and the like.

  As the solvent used in the polymerization in the present invention, isopropyl alcohol (IPA) is suitable. The inventors conducted various studies. First, N-isopropylacrylamide was selected as a raw material for the temperature-responsive polymer, and when the atom transfer radical polymerization reaction was performed in a solution at room temperature, dimethylformaldehyde was used as a reaction solvent. It was found that the reaction rate was comparable even when any of (DMF), water, and IPA was selected. However, in the case of a solid-phase reaction in which N-isopropylacrylamide is immobilized on the surface of a solid chromatographic support as in the present invention, assuming that the reaction solvent is IPA, the other two are selected. It was found that the reaction rate was remarkably slower than that. In addition, t-butyl alcohol shown in the above Macromolecules 38, 5937-5943 (2005) may solidify at room temperature, and therefore the reaction temperature must be increased to room temperature or higher, resulting in an increase in the reaction rate. It was found that this was inappropriate for the present invention. The knowledge here is not known at all in the prior art, and according to the present invention, the molecular weight of the polymer chain gradually increases when IPA is selected as the reaction solvent for the immobilization polymerization on the carrier, The amount of polymer chains immobilized on the carrier surface will also gradually increase. Therefore, according to the method of the present invention, the polymer chain can be uniformly immobilized on the surface of the carrier. Furthermore, by stopping the reaction at a predetermined time, a carrier having an immobilized state at the time when the reaction is stopped can be produced with good reproducibility.

  The present invention immobilizes an atom transfer radical polymerization initiator on the surface of a carrier, has isopropyl alcohol as a solvent, has an electric charge by the atom transfer radical method from the initiator under a polymerization catalyst, and is within a temperature range of 0 to 80 ° C. It is a method of growing and reacting a polymer whose hydration power changes, but the initiator concentration, copper halide concentration, ligand complex concentration, reaction temperature, reaction time, etc. during other polymerization are not particularly limited, It may be changed according to the purpose. Furthermore, the state of the reaction solution may be allowed to stand or be stirred, but the latter is preferred in view of the uniform fixation on the surface of the carrier.

    In the present invention, the polymer having a charge that changes its hydration power within a temperature range of 0 to 80 ° C., which is immobilized on a chromatography carrier, is not particularly limited, but the temperature at which the hydration power changes within the temperature range. It is obtained by introducing a charged functional group into the responsive polymer. Examples of such a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer.

  Among these, since poly (N-isopropylacrylamide) has a lower critical temperature at 32 ° C., the surface of the carrier chemically modified with this polymer greatly changes the hydrophilic / hydrophobic surface properties at this critical temperature. Is used by grafting or coating on the surface of the chromatographic packing material, the retention force on the sample can be changed by temperature. As a result, the retention behavior can be controlled by temperature without changing the composition of the eluate. In order to set the lower critical temperature to 32 ° C. or higher, hydrophilic comonomers such as acrylamide, methacrylic acid, acrylic acid, dimethylacrylamide, and vinylpyrrolidone that are more hydrophilic than isopropylacrylamide are copolymerized with N-isopropylacrylamide. It is possible to make adjustments. Further, when the lower critical temperature is desired to be 32 ° C. or lower, it can be adjusted by copolymerization with a hydrophobic comonomer such as styrene, alkyl methacrylate, alkyl acrylate or the like which is a hydrophobic monomer.

  Further, the lower critical temperature of polydiethylacrylamide is about 30 ° C. to 32 ° C., and the surface physical property changes to hydrophilic / hydrophobic with this temperature as a boundary, as in the case of poly (N-isopropylacrylamide) described above. In addition, the holding force for the sample can be adjusted by the temperature. The novel chromatographic support utilized in the present invention is made by chemical modification or polymer coating.

  In the present invention, the polymer coated on the surface of the carrier causes hydration and dehydration by changing the temperature. The temperature range is 0 ° C. to 80 ° C., preferably 10 ° C. to 50 ° C., more preferably. It is 20 degreeC-45 degreeC. If the temperature exceeds 80 ° C., the mobile phase is water, which is not preferable because workability such as evaporation is deteriorated. On the other hand, if it is lower than 0 ° C., the mobile phase may freeze, which is not preferable.

  In the present invention, the polymer coated on the surface of the carrier is charged. The method for imparting the charge is not particularly limited. Usually, when synthesizing the temperature-responsive polymer chain coated on the surface of the carrier, a method of copolymerization including an ionic monomer having a functional group that generates a charge can be given. . Examples of the ionic monomer include dialkylaminoalkyl (meth) acrylamide, dialkylaminoalkyl (meth) acrylate, aminoalkyl (meth) acrylate, aminostyrene, aminoalkylstyrene, aminoalkyl (meth ) Acrylamide, alkyloxyalkyltrimethylammonium salt, (meth) acrylamide alkyltrimethylammonium salt and the like, and as a structural unit of a polymer having a carboxyl group, a structural unit of a polymer having acrylic acid, methacrylic acid, or sulfonic acid ( Examples thereof include, but are not limited to, meth) acrylamide alkyl sulfonic acid.

In the present invention, the polymer is fixed with high density and uniformity. The degree of immobilization is preferably 0.08 molecular chain / nm ² or more, preferably 0.1 molecular chain / nm ² or more, more preferably 0.12 molecular chain in terms of the number of molecular chains per unit area. / Nm ² or more is preferable. If the degree of polymer immobilization on the surface of the substrate is 0.08 molecular chain / nm ² or less, the characteristics of individual polymer chains are expressed just like the polymer immobilization on the substrate surface by the conventional method. It is not preferred as the carrier of the invention. The calculation method of the numerical value indicating the degree of immobilization is not particularly limited, but for example, a molecular weight obtained by preparing a polymer that is not immobilized on the substrate surface under similar reaction conditions and analyzing the polymer chain And the amount of immobilized polymer obtained from elemental analysis of the carrier on which the polymer is immobilized.

  The molecular weight of the polymer to be coated is not particularly limited as long as the molecular weight is sufficiently large to cause a change in hydration power within a temperature range of 0 to 80 ° C., but the polymer molecular weight is preferably 1000 or more, preferably Is 2,000 or more, more preferably 5,000 or more. A molecular weight of 1000 or less is not preferable because the molecular weight is too low and a change in hydration power cannot be expressed. Further, if the molecular weight is 5000 or more, the molecular weight of the polymer is too high, so that the molecule itself becomes bulky and the temperature responsiveness decreases, which is not preferable.

Furthermore, immobilization of the polymer onto the substrate at which in this invention may have a range of 0.8~10.0mg / ^{m 2,} preferably from 0.9~8.0mg / ^{m 2,} further The range of 1.0 to 6.0 mg / m ² is preferable. If it is 0.8 mg / m ² or less, the temperature responsiveness is not recognized, and even if the value is higher than 10.0 mg / m ^{2, the} temperature responsiveness decreases due to the bulk of the polymer, which is not preferable. . The measurement of the amount of immobilization may be in accordance with a conventional method, for example, elemental analysis, the amount of ESCA may be mentioned, and any method may be used. The state of the polymer to be immobilized in the present invention is not particularly limited, and may be linear or cross-linked. In order to achieve immobilization, a straight-chain product of the whole company is preferable.

  Thus, according to the production method shown in the present invention, it is possible to obtain a high-performance liquid chromatography carrier that could not be obtained by the prior art. As one of its functions, in the analysis by the front end chromatography method used as a conventional method, the effective binding capacity of the protein per unit weight of the carrier is determined based on the condition that the temperature-responsive polymer chain on the surface of the carrier swells. 6,000 to 80,000 μg / g are obtained per unit. In the present invention, all the carriers obtained here can be used for liquid chromatography. However, the effective binding capacity of the protein per unit weight of the carrier is determined based on the condition that the temperature-responsive polymer chain on the surface of the carrier swells. In particular, a value of 7000 to 50000 μg / g is preferable, and a value of 8000 to 20000 μg / g is more preferable. When the effective binding capacity is 6000 μg / g or less, a large amount of carrier is required for analyzing and separating the physiologically active compound, which is not preferable as the carrier of the present invention. Further, if the effective binding capacity is 80000 μg / g or more, the charge density per carrier surface becomes high, and there is a concern about the denaturation of the physiologically active substance to be separated, and a countermeasure for that is required, which is not necessarily a suitable carrier. If it is the method shown by this invention, a thing suitable as a support | carrier for liquid chromatography can be provided now.

  Furthermore, the temperature-responsive polymer is immobilized on the surface of the carrier obtained by the present invention, and the temperature-responsive polymer chain contracts or swells by changing the external environment temperature. As a result, the effective binding capacity of the protein per carrier unit weight represented by the above-mentioned 6000-80000 μg / g varies depending on the state of the temperature-responsive polymer chain. At that time, if the effective binding capacity of the protein per weight of the carrier is selected under the condition that the temperature-responsive polymer chain on the surface of the support swells, the effective binding capacity with the protein changes the temperature. In the range of 500 to 80,000 μg / g per carrier weight. In the present invention, all of the carriers obtained here can be used for liquid chromatography, but the effective binding capacity of the protein per unit weight of the carrier is determined under the condition that the temperature-responsive polymer chain on the surface of the carrier is contracted. In particular, it is preferably 500 to 5000 μg / g, more preferably 1000 to 3000 μg / g. When the effective binding capacity is 5000 μg / g or more, the separation for analyzing and separating the physiologically active compound is deteriorated, which is not preferable as a simple substance of the present invention.

  The temperature-responsive chromatography carrier obtained by the present invention is packed in a column and attached to a normal liquid chromatography apparatus, and used as a liquid chromatography system. At that time, the separation performance is affected by the temperature of the carrier packed in the column. At that time, the method of applying the temperature to the carrier is not particularly limited. For example, it is possible to attach the whole or part of the column packed with the carrier to an aluminum block, water bath, air layer, jacket or the like having a predetermined temperature. Can be mentioned.

  The separation method is not particularly limited, and there is a method in which the temperature at which the characteristics of the support surface change is confirmed in advance, and the solute is separated while changing the temperature so as to sandwich the temperature. In this case, since the characteristics of the support surface are greatly changed only by the temperature change, it is expected that a large difference occurs in the time (holding time) in which the signal appears depending on the solute. In the case of the present invention, it is the most effective utilization method that the separation is performed so as to sandwich a temperature at which the characteristics of the carrier surface greatly change.

  Alternatively, as an example of another separation method, the adsorbed solute is released by once adsorbing the solute on the obtained temperature-responsive liquid chromatography carrier and then changing the temperature and changing the characteristics of the surface of the carrier. The method of using based on the catch and release method is mentioned. In this case, the mass to be adsorbed may or may not exceed the amount that can be adsorbed on the carrier. In any case, the adsorption is performed once, and then the temperature is changed to change the characteristics of the surface of the carrier to release the adsorbed solute.

  The chromatography shown in the present invention may use a buffer as a mobile phase and does not require an organic solvent. Here, the buffer solution is an aqueous solution containing inorganic salts, and specifically includes a phosphate buffer solution, a tris buffer, an ammonium chloride buffer solution, etc. It is not restricted. The concentration of the inorganic salt is 5 to 50 mM, preferably 10 to 40 mM, and more preferably 15 to 35 mM. If the concentration of the inorganic salt in the mobile phase is lower than 5 mM, there is a problem of impairing the activity of the physiologically active substance that is a solute, and the degree of dissociation of ion exchange groups on the surface of the chromatographic support becomes high, so It is not preferable because it is adsorbed and it becomes difficult to remove the solute from the surface of the carrier in the subsequent operation. Conversely, when the concentration of the inorganic salt is higher than 50 mM, the degree of dissociation of ion exchange groups on the surface of the chromatographic support is lowered, it becomes difficult to hold the solute on the surface of the support, and it is difficult to finally separate the solute, which is preferable. Absent.

  Furthermore, the neutral buffer used in the present invention has a pH of 6.5 to 7.5, preferably 6.8 to 7.2, and more preferably 7.0. When the ion exchange group introduced into the support surface is cationic, if the pH of the buffer solution is lower than 6.5, the degree of dissociation of the ion exchange group increases and the solute is strongly adsorbed on the support surface. Then, it is difficult to remove the solute from the surface of the carrier in the subsequent operation, which is not preferable. On the other hand, if the pH is higher than 7.5, the degree of dissociation of ion exchange groups on the surface of the chromatographic support becomes low, making it difficult to maintain the solute on the surface of the support, and it becomes difficult to finally separate the solute, which is not preferable. . Similarly, when the ion exchange group introduced onto the support surface is anionic, the degree of dissociation of the ion exchange group increases when the pH of the buffer is higher than 7.5, and the solute is strongly adsorbed on the support surface. Therefore, it is difficult to remove the solute from the surface of the carrier in the subsequent operation, which is not preferable. On the other hand, if the pH is lower than 6.5, the degree of dissociation of ion exchange groups on the surface of the chromatographic support becomes low, making it difficult to maintain the solute on the surface of the support, and it becomes difficult to finally separate the solute, which is not preferable. . The target substance here is not particularly limited as long as it is a polymer substance having physiological activity, and examples thereof include peptides, proteins, nucleic acids and the like, and preferably a physiologically active polymer having a molecular weight of 30000 or more. Is mentioned. Among them, in particular, a protein is a polymer substance having a three-dimensional structure, and therefore is suitable as a target substance of the present invention. In the present invention, the protein is not particularly limited. However, since the present invention is a separation method using a charged carrier surface, an acidic protein or a basic protein is preferable. The acidic protein or basic protein shown here may be a general one. Specifically, the acidic protein includes albumin, vimentin, ankyrin 3, actin, myosin, etc. The basic protein includes lysozyme. , Hemoglobin β chain, catalase, annexin, ezrin and the like, but are not particularly limited.

  Furthermore, according to the present invention, when the above-described phosphate buffer having a pH of 6.5 or less is used as a mobile phase and a surface is a negatively charged temperature-responsive chromatography carrier, a plurality of physiologically active polymers are used. It has been found that globulins can be desorbed by selectively holding the globulins from the aqueous solution in which the substance is dissolved by contracting the polymer chains on the surface of the carrier and swelling the polymer chains. In this case, the negatively charged temperature-responsive chromatographic carrier is not particularly limited as long as it can be obtained by the above-mentioned method, and the globulin to be separated is also γ-globulin, α-globulin, β-globulin. Any of these may be used and is not particularly limited. If the carrier obtained by the present invention is used, a very useful protein called γ-globulin can be recovered in a low damage state without denaturation with an organic solvent.

  By using the temperature-responsive chromatography carrier in the present invention described above, it is possible to separate a very useful physiologically active substance that can be used for pharmaceuticals and the like. In that case, separation can be achieved only by changing the temperature in the column by a simple operation, and since an organic solvent is not required for separation, the separated physiologically active substance can be obtained without modification.

  Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.

A temperature-responsive positively charged carrier surface was prepared through the following two steps.
1-a) The following compounds were used to introduce an initiator-immobilized surface to silica gel.
m, p-Chloromethyltrichlorosilane 3.72 ml (MW: 288.1) and toluene 318 ml were mixed, and 16.0 g of silica gel containing water vapor was dispersed. The reaction was allowed to proceed for 18 hours at room temperature. After the reaction, it was filtered, washed with toluene and acetone, and dried at 110 degrees. This introduced an atom transfer radical polymerization (ATRP) initiator on the silica gel surface.
1-b) The following compounds were used for the formation of temperature responsive positively charged immobilized surfaces.
Initiator-immobilized silica gel 1.0 g prepared in 1-a)
IPAAm 3.89g
DMAPAAm (dimethylaminopropylacrylamide) 0.971 g
tBAAm (tert-butylacrylamide) 0.547g
CuCl 0.847g
CuCl ₂ 0.0115 g
Me ₆ TREN (Tris (2-dimethylaminoethyl) amine) 20.221 g
43 ml of isopropanol
IPAAm, DMAPAAm, and tBAAm were dissolved in 2-propanol and bubbled with nitrogen for 30 minutes. Thereafter, CuCl, CuCl ₂ and Me ₆ TREN were added to the solution under a nitrogen atmosphere and stirred for 30 minutes to form a CuCl / CuCl ₂ / Me ₆ TREN catalyst. This reaction solution was reacted with initiator-introduced silica beads in a glass container, and ATRP was performed at room temperature for 16 hours. After the reaction, the monomer, polymer and copper catalyst were washed with acetone and EDTA solution, and dried under reduced pressure at 50 ° C. for 3 hours. When the obtained carrier was analyzed by a conventional front end chromatography method, the effective binding capacity of the protein per unit weight of the carrier was 15000 μg per weight of the carrier under the condition that the temperature-responsive polymer chain on the surface of the carrier swells. / G was found to be obtained. Next, the polymer-modified carrier was packed in a stainless steel column, and the following analysis was performed.

Separation of globulin and albumin using a positively charged copolymer (poly (IPAAm-co-DMAPAAm-co-tBAAm)) brush-modified surface was performed as follows.
(Separation conditions) Poly (IPAAm-co-DMAPAAm-co-tBAAm) brush-modified silica beads as a column, Na ₂ PO ₃ / NaHPO ₃ buffer pH = 7.0, ionic strength = 16.7 mM as a mobile phase Was used.
(Result) At 10 ° C., both globulin and albumin are eluted without being adsorbed. At 40 ° C., the globulin was eluted, and only albumin was adsorbed on the copolymer surface and did not elute. The obtained results are shown in FIG. In addition, as shown in FIG. 2, it was shown from the peak area of the obtained chromatogram that although globulin continues to elute as the temperature increases, albumin causes adsorption. From this, albumin can be eluted by eluting only globulin at a column temperature of 40 ° C. and then lowering the temperature to 10 ° C. Thus, it can be seen that two kinds of blood proteins, globulin and albumin, can be separated by changing the temperature and changing the state of the copolymer on the silica bead surface.

Separation of globulin and albumin using a positively charged copolymer (poly (IPAAm-co-DMAPAAm-co-tBAAm)) brush-modified surface was performed as follows.
(Separation conditions) (Poly (IPAAm-co-DMAPAAm-co-tBAAm)) brush-modified silica beads as a column, Na ₂ PO ₃ / NaHPO ₃ buffer pH = 7.0, ionic strength = 16 as a mobile phase .7 mM was used.
(Result) At 10 ° C., both globulin and albumin are eluted without being adsorbed. At 40 ° C., globulin elutes and only albumin is adsorbed on the copolymer surface. Using this property, albumin could be eluted by eluting only globulin at a column temperature of 40 ° C. and then lowering the column temperature to 10 ° C. The results are shown in FIGS. By changing the state of the copolymer on the silica bead surface by changing the temperature in this way, it is possible to separate two types of blood proteins, globulin and albumin. It can be seen that a small amount of albumin present in γ globulin can be removed.

The surface of the temperature-responsive negatively charged carrier was produced through the following two steps.
4-a) The initiator was introduced into the silica gel surface in the same manner as in 1-a).
4-b) The following compounds were used for the formation of high density graft surfaces of temperature responsive negatively charged copolymers.
Initiator-immobilized silica gel 1.0 g prepared in 4-a)
IPAAm 3.89g
tBA (tert-butyl acrylate) 0.551 g
tBAAm (tert-butylacrylamide) 0.547g
CuCl 0.847g
CuCl ₂ 0.115 g
Me ₆ TREN (Tris (2-dimethylaminoethyl) amine) 20.221 g
43 ml of isopropanol
IPAAm, tBA, and tBAAm were dissolved in 2-propanol, and nitrogen bubbling was performed for 30 minutes. Thereafter, CuCl, CuCl ₂ and Me ₆ TREN were added to the solution under a nitrogen atmosphere and stirred for 30 minutes to form a CuCl / CuCl ₂ / Me ₆ TREN catalyst. This reaction solution was reacted with initiator-introduced silica beads in a glass container, and ATRP was performed at room temperature for 16 hours. After the reaction, the monomer, free polymer, and copper catalyst were washed with acetone and EDTA solution, and dried under reduced pressure at 50 ° C. for 3 hours. The recovered poly (IPAAm-co-tBA-co-tBAAm) brush-modified surface is dispersed in chloroform and hydrolyzed with methanesulfonic acid to hydrolyze the tert-butyl group of tBA, thereby poly (IPAAm-co-AAc). By using (acrylic acid) -co-tBAAm), a temperature-responsive negatively charged copolymer high-density graft silica gel having a large amount of anionic ion-exchange groups could be prepared. The obtained carrier was analyzed by a conventional front end chromatography method. As a result, the effective binding capacity of the protein per unit weight of the carrier was 13000 μg per weight of the carrier under the condition that the temperature-responsive polymer chain on the surface of the carrier swells. / G was found to be obtained. Next, the polymer-modified carrier was packed in a stainless steel column, and the following analysis was performed.

The interaction between negatively charged copolymer (poly (IPAAm-co-AAc-co-tBAAm)) brush-modified silica, globulin and albumin was measured as follows.
(Separation conditions) Poly (IPAAm-co-AAc-co-tBAAm) brush-modified silica beads as the column, Na ₂ PO ₃ / NaHPO ₃ buffer pH = 5.8, ionic strength = 16.7 mM as the mobile phase Was used.
(Results) When the chromatogram was measured by changing the temperature from 10 ° C. to 50 ° C., the peak of γ globulin was small at a low temperature, but the peak gradually increased and eluted as the temperature was increased. On the other hand, albumin showed a behavior of continuing to elute regardless of the temperature change. The obtained results are shown in FIG. Moreover, as shown in FIG. 6, from the peak area of the obtained chromatogram, it was shown that the globulin tends to be adsorbed at a low temperature and eluted as the temperature is increased. On the other hand, albumin continued to elute even when the temperature was changed. Therefore, negatively charged copolymer (poly (IPAAm-co-AAc-co-tBAAm)) has a large difference in interaction with globulin and albumin on the brush-modified surface. It was found that by changing the state, only γ globulin can be selectively adsorbed, and separation by selective adsorption of γ globulin from blood and concentration of γ globulin are possible.

Comparative Example 1

The polymerization rate of the temperature-responsive polymer in isopropanol was measured as follows.
IPAAm 3g
CuCl 0.013g
Me ₆ TREN 0.031g
3.83 ml of isopropanol
IPAAm was dissolved in isopropanol and nitrogen bubbled for 30 minutes. Thereafter, CuCl and Me ₆ TREN were added to the solution under a nitrogen atmosphere, and stirred for 30 minutes to form a CuCl / Me ₆ TREN catalyst. This reaction solution was put in a glass container, 0.0176 ml of p-chloro-xylene as an initiator was added, and ATRP was performed at room temperature for 0.5 to 6 hours. After the reaction, the solution was dialyzed, and the polymer and the copper catalyst removed were lyophilized to obtain a polymer. The obtained PIPAAm molecular weight was measured by gel permeation chromatography.

Comparative Example 2

The polymerization rate of the temperature-responsive polymer in DMF was measured as follows.
IPAAm 3g
CuCl 0.013g
Me ₆ TREN 0.031g
DMF 3.83ml
IPAAm was dissolved in DMF and bubbled with nitrogen for 30 minutes. Thereafter, CuCl and Me ₆ TREN were added to the solution under a nitrogen atmosphere, and stirred for 30 minutes to form a CuCl / Me ₆ TREN catalyst. This reaction solution was put into a glass container, 0.0176 ml of p-chloro-xylene as an initiator was added, and ATRP was performed at room temperature for 0.5 to 6 hours. After the reaction, the solution was dialyzed, and the polymer and the copper catalyst removed were lyophilized to obtain a polymer. The obtained PIPAAm molecular weight was measured by gel permeation chromatography.

The results obtained in Comparative Example 1 and Comparative Example 2 are shown in FIG. As a result, polymerization rates of isopropanol ( Comparative Example 1 ) and DMF ( Comparative Example 2 ) were comparable. Thereby, when PIPAAm was prepared by ATRP in a solution, it turned out that there is no difference between both.

A temperature responsive polymer immobilization surface was made in isopropanol.
Initiator-immobilized silica gel 1.0 g prepared in 1-a)
IPAAm 4.86g
CuCl 0.847g
CuCl ₂ 0.0115 g
Me ₆ TREN 20.221g
43 ml of isopropanol
IPAAm was dissolved in isopropanol and nitrogen bubbled for 30 minutes. Thereafter, CuCl, CuCl ₂ and Me ₆ TREN were added to the solution under a nitrogen atmosphere and stirred for 30 minutes to form a CuCl / CuCl ₂ / Me ₆ TREN catalyst. This reaction solution was reacted with the initiator-introduced silica beads in a glass container, and ATRP was performed at room temperature for 0.5 to 15 hours. After the reaction, the monomer, free polymer, and copper catalyst were washed with acetone and EDTA solution, and dried under reduced pressure at 50 ° C. for 3 hours. The amount of polymer graft was calculated by CHN elemental analysis.

Comparative Example 3

A temperature responsive polymer immobilization surface was made in DMF.
Initiator-immobilized silica gel 1.0 g prepared in 1-a)
IPAAm 4.86g
CuCl 0.847g
CuCl ₂ 0.0115 g
Me ₆ TREN 20.221g
43 ml of DMF
IPAAm was dissolved in DMF and bubbled with nitrogen for 30 minutes. Thereafter, CuCl, CuCl ₂ and Me ₆ TREN were added to the solution under a nitrogen atmosphere and stirred for 30 minutes to form a CuCl / CuCl ₂ / Me ₆ TREN catalyst. This reaction solution was reacted with the initiator-introduced silica beads in a glass container, and ATRP was performed at room temperature for 0.5 to 15 hours. After the reaction, the monomer, free polymer, and copper catalyst were washed with acetone and EDTA solution, and dried under reduced pressure at 50 ° C. for 3 hours. The amount of polymer graft was calculated by CHN elemental analysis.

  The results obtained in Example 6 and Comparative Example 3 are shown in FIG. As a result, when the polymerization rate was estimated from the amount of immobilized PIPAAm, it was found that the polymerization rate was significantly slower when isopropanol (Example 6) was used than with DMF (Comparative Example 3). As a result, when PIPAAm is immobilized on the support by ATRP, it is possible to control the PIPAAm immobilization state on the support surface by using isopropanol as the reaction solvent, and the amount of PIPAAm grafting can be adjusted by the reaction time. I found it possible.

  According to the present invention, there can be obtained a temperature-responsive chromatographic support in which a polymer having a charge whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized on the support surface at a high density. When this carrier is used, the interaction of the physiologically active substance on the surface of the carrier with respect to temperature changes is remarkably changed. As this separation object, for example, the use for separation, purification, and separation of proteins and peptides is strongly expected. Therefore, the present invention is extremely useful in the fields of medicine and biology.

  It is a chromatogram of HSA (human serum albumin) and a globulin using the poly (IPAAm-co-DMAPAAm-co-tBAAm) brush modification silica support | carrier when changing analysis temperature shown in Example 2. FIG.   It is a figure which shows the relationship between the peak area of the chromatogram of HSA and a globulin using the poly (IPAAm-co-DMAPAAm-co-tBAAm) brush modification silica support | carrier shown in Example 2, and analysis temperature.   It is the figure which showed isolation | separation of the globulin and HSA using the poly (IPAAm-co-DMAPAAm-co-tBAAm) brush modification support | carrier shown in Example 3. FIG.   FIG. 4 is a blot diagram of globulin and HSA obtained by separation using a poly (IPAAm-co-DMAPAAm-co-tBAAm) brush-modified carrier shown in Example 3.   It is the chromatogram of HSA and a globulin using the poly (IPAAm-co-AAc-co-tBAAm) brush modification silica support | carrier when changing analysis temperature shown in Example 5. FIG.   It is the figure which showed the relationship between the peak area of the chromatogram of HSA and a globulin using the poly (IPAAm-co-AAc-co-tBAAm) brush modification silica support | carrier shown in Example 5, and temperature.   It is a figure which shows the relationship between the reaction time by the difference in a solvent shown in the comparative example 1 and the comparative example 2, and the molecular weight of PNIPAAm.   It is a figure which shows the relationship between the reaction time by the difference in a solvent shown in Example 6 and Comparative Example 3, and the weight of the PNIPAAm fixed to the silica gel.

Claims (16)

An atom transfer radical polymerization initiator is immobilized on the surface of the carrier, and isopropyl alcohol is used as a solvent. The initiator is charged by an atom transfer radical polymerization method under a polymerization catalyst and hydrated within a temperature range of 0 to 80 ° C. force a method for producing a temperature-responsive chromatographic support for growing reaction, a polymer changes have the charge, polymer hydration force varies in the temperature range of 0 to 80 ° C., the coating on the carrier surface The method is characterized in that when the temperature-responsive polymer chain is synthesized, it is obtained by copolymerization with an ionic monomer having at least a functional group that generates a charge .   The method for producing a temperature-responsive chromatography carrier according to claim 1, wherein the charged polymer is a copolymer of polyalkylacrylamide.   The method for producing a temperature-responsive chromatography carrier according to claim 2, wherein the polyalkylacrylamide is poly- (N-isopropylacrylamide) and / or poly- (N, N-diethylacrylamide).   The temperature-responsive chromatography carrier production method according to any one of claims 1 to 3, wherein the carrier is silica gel particles, glass plates, or glass particles.   The method for producing a temperature-responsive chromatography carrier according to any one of claims 1 to 4, wherein the atom transfer radical polymerization initiator is 2- (m, p-chloromethylphenylethyl) ethyltrichlorosilane. The temperature-responsive chromatography carrier according to any one of claims 1 to 5, wherein the atom transfer radical polymerization initiator is immobilized at a high density at a rate of 0.08 molecular chain / nm ² or more. Production method.   The temperature-responsive chromatography carrier production method according to any one of claims 1 to 6, wherein the polymerization catalyst is copper chloride as a copper halide and tris (2- (dimethylamino) ethyl) amine as a ligand complex. The method for producing a temperature-responsive chromatography carrier according to any one of claims 1 to 7, wherein the amount of immobilized polymer having charge on the surface of the carrier is 0.8 to 10.0 mg / m ² .   The charged polymer is uniformly immobilized on the surface of the carrier, and the effective binding capacity of the carrier to the protein is 6000 to 80,000 μg / g per weight of the carrier under the condition that the polymer chain swells. A temperature-responsive chromatography carrier obtained from the method for producing a temperature-responsive chromatography carrier according to any one of 1 to 8.   The temperature-responsive chromatography carrier according to claim 9, wherein the effective binding capacity of the protein on the surface of the carrier is changed in the range of 500 to 80,000 µg / g per weight of the carrier by changing the temperature. A plurality of physiologically active polymer substances were dissolved using a neutral 5-50 mM phosphate buffer as a mobile phase and the temperature-responsive chromatography carrier according to any one of claims 9 and 10 as a carrier. A method for separating a physiologically active substance, comprising separating a physiologically active polymer substance having a molecular weight of 30000 or more from an aqueous solution.   The carrier surface is positively charged, and albumin is retained from the aqueous solution in which the bioactive polymer is dissolved by contracting the polymer chain on the surface of the carrier, and the albumin is desorbed by swelling the polymer chain. The method for separating a physiologically active substance according to claim 11, wherein the polymer substance and albumin are separated. A 5-50 mM phosphate buffer having a pH of 6.5 or lower is used as a mobile phase, and a carrier surface according to any one of claims 9 and 10 is a negatively charged thermoresponsive chromatographic carrier. The globulin is retained by shrinking the polymer chain on the surface of the carrier from the aqueous solution in which the bioactive polymer is dissolved, and the globulin is desorbed by swelling the polymer chain to separate the globulin from the bioactive polymer. A method for separating a physiologically active substance.   The method for separating a physiologically active substance according to claim 13, wherein the globulin is γ-globulin.   A method for purifying a physiologically active substance using the method for separating a physiologically active substance according to any one of claims 11 to 14.   The separation method of the bioactive substance using the separation method of the bioactive substance of any one of Claims 11-14.

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