JP2007069193A - Temperature responsive chromatography carrier, manufacturing method and temperature responsive chromatography method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a temperature responsive chromatography carrier by the surface of which a polymer having hydration force changing within the temperature range of 0-80°C is fixed highly densely. <P>SOLUTION: A method for manufacturing the temperature responsive chromatography carrier comprises the steps of fixing an atom migrating radical polymerization initiator on the surface of a substrate and growing-and-reacting the polymer having hydration force changing within the temperature range of 0-80°C from the initiator by an atom migration radical method in the presence of a catalyst. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention is an external signal of temperature, and a carrier for liquid chromatography that is carried out by controlling the interaction of solid substances with useful substances such as pharmaceuticals, biological substances (proteins, DNA, glycolipids, etc.) and cells, The present invention relates to a production method and a temperature-responsive chromatography method using the same.

  High-performance liquid chromatography (HPLC) has a wide variety of combinations of mobile phase liquids and stationary phases, and can be selected variously according to the sample. Therefore, in recent years, it has been used for separation and purification of various substances. However, in the conventional chromatography, the surface structure of the stationary phase is not changed, and the interaction between the solute contained in the mobile phase and the stationary phase surface is mainly changed by changing the solvent of the mobile phase. Has been done. For example, in HPLC used in many fields, an organic solvent such as hexane, acetonitrile or chloroform is used as a mobile phase in a normal phase column using a carrier such as silica gel as a stationary phase, and an aqueous system. An organic solvent such as methanol or acetonitrile is used in the reverse phase column using the silica gel derivative separated in step 1 as a carrier.

  In ion exchange chromatography using an anion exchanger or a cation exchanger as a stationary phase, substance separation is performed by changing the external ion concentration or type. In recent years, due to rapid progress in genetic engineering and the like, bioactive peptides, proteins, DNA, and the like are expected to be widely used in various fields including pharmaceuticals, and their 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.

  However, since organic solvents, acids, alkalis, and surfactants used in conventional mobile phases impair the activity of physiologically active substances and at the same time become impurities, improvement of the system is expected. In addition, a separation / purification system that does not use these substances is required from the viewpoint of avoiding environmental pollution of such substances.

  Under such background, various studies have been made so far. Among them, the technology disclosed in Japanese Patent Publication No. 06-104061 particularly corresponds to those basic technologies. Here, cells are cultured at a temperature not higher than the upper critical lysis temperature or higher than the lower critical lysis temperature on a cell culture support in which the substrate surface is coated with a polymer having an upper or lower critical lysis temperature of 0 to 80 ° C. with respect to water. Thereafter, a technique is described in which the cultured cells are detached by setting the temperature to the upper critical solution temperature or lower or the lower critical solution temperature or lower. This is the first example of a temperature-responsive polymer used as a cell culture material in the biomedical field. In fact, when cells adhere to the surface of a substrate, the cells secrete adhesive proteins and attach through them. . Therefore, the phenomenon that cells peel from the surface of the substrate here also includes the separation of the adhesive protein secreted by the cells from the surface of the substrate. In fact, when cells obtained by this technique are replated or transplanted into a living tissue, the cells detached from the substrate adhere well to the substrate or tissue. This means that the detached cells retain the adhesive protein secreted during culture. That is, the technique here is the concept of the temperature-responsive chromatography technique in which the protein adsorbed by the temperature change in the present invention is desorbed.

  Under such circumstances, Japanese Patent Application Laid-Open No. 05-133947 has studied to immobilize on silica gel or polymer gel, which 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.

  The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a novel temperature-responsive chromatography carrier based on a completely different idea from the prior art. Another object of the present invention is to provide a method for producing such a carrier. It is another object of the present invention to provide a temperature-responsive chromatography method using the carrier.

In order to solve the above problems, the present inventors have studied and developed from various angles. As a result, surprisingly, the temperature responsiveness in which the polymer whose hydration power changes within the temperature range of 0 to 80 ° C. is immobilized on the substrate surface at a high density of 0.08 molecular chain / nm ² or more. It has been found that when a chromatography carrier is used, the amount of change with respect to temperature change is significantly improved as compared with the chromatography carrier obtained from the prior art. The technology shown in the present invention was completely unpredictable from the prior art, and is expected to develop into a new chromatographic system that was not present in the prior art. The present invention has been completed based on such findings.

That is, the present invention is characterized in that a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized on the substrate surface at a high density at a rate of 0.08 molecular chain / nm ² or more. A temperature-responsive chromatography carrier is provided.
In addition, the present invention immobilizes an atom transfer radical polymerization initiator on the substrate surface, and grows a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. by the atom transfer radical method under the catalyst from the initiator. There is provided a method for producing the above-described temperature-responsive chromatography carrier, characterized by reacting.
Furthermore, the present invention provides a temperature-responsive chromatography method using the temperature-responsive chromatography carrier.

  With the temperature-responsive chromatographic support described in the present invention, a novel separation system is proposed. Using this system, a wide range of peptides, proteins, and cells 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 stationary phase in the mobile layer without changing the mobile phase, for example, by changing external conditions such as temperature. Alternatively, 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 stationary phase surface. The purpose of the present invention is to change external conditions. The present invention provides a chromatographic method capable of reversibly changing the surface characteristics of a stationary phase, thereby allowing separation, purification and concentration by a single aqueous mobile phase, and a packing material as a stationary phase used in the chromatography. is there.

  The gist of the present invention is a chromatographic method characterized in that a specific substance is separated using a filler capable of changing the characteristics of the stationary phase surface with temperature while the mobile phase is fixed in an aqueous system. It is. The present invention also provides a method for producing the temperature-responsive chromatography carrier. Furthermore, the present invention shows a temperature-responsive chromatography method using the same. That is, by using the present invention, it is possible to separate biological elements such as peptides, proteins and cells by setting the external temperature to a critical temperature or higher. Therefore, at this time, since chemicals such as organic solvents, acids, alkalis and surfactants are not used at all, they can be prevented from becoming contaminants, and analysis while maintaining the functions of proteins and cells can be performed. The same can be used for separation.

  In the conventional chromatographic method, a sample containing various compounds in a single mobile phase, especially when separating and analyzing a plurality of samples having greatly different polarities, it is difficult to separate and the time required for the separation becomes very long. End up. Therefore, when handling such a sample, separation is performed by a solvent gradient method in which the amount and type of organic solvent are continuously changed over time or a step gradient method in which steps are changed step by step. However, the temperature gradient according to the present invention is used. In the method or step gradient method, it is possible to achieve the same separation by changing the column temperature continuously or stepwise with a single mobile phase instead of using an organic solvent. Thus, contamination of the above-mentioned contaminants can be prevented, separation can be performed while maintaining the functions of proteins and cells, and desired components can be separated in a short time by controlling the temperature.

The present invention is specifically shown below. The present invention relates to a temperature-responsive chromatographic support in which a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized at a high density at a rate of 0.08 molecular chain / nm ² or more on the substrate surface. It is. And the characteristic of the polymer fix | immobilized on the support | carrier surface by this high-density fixation expresses notably. The reason for this is not clear at the present time, but it is considered that the immobilized polymer is present at a high density on the surface of the carrier, and is therefore closely related to the nearby polymer chain. There is no restriction on the technology.

  Examples of the polymer whose hydration power changes within the temperature range of 0 to 80 ° C. used in the present invention include a polymer having a lower critical solution temperature (LCST) and a polymer having an upper critical solution temperature (UCST). It can be a polymer, copolymer, or mixture. Examples of such a polymer include polymers described in Japanese Patent Publication No. 06-104061. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, vinyl ether derivatives, and polyvinyl alcohol partially acetylated products. Of these, any two or more of them 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. At that time, since the substance to be separated is a biological substance, the separation is carried out in the range of 5 ° C. to 50 ° C., and therefore the polymer is poly-Nn-propylacrylamide (lower critical solution temperature of homopolymer). 21 ° C), poly-Nn-propylmethacrylamide (27 ° C), poly-N-isopropylacrylamide (32 ° C), poly-N-isopropylmethacrylamide (43 ° C), poly-N-cyclopropyl Acrylamide (at 45 ° C), poly-N-ethoxyethylacrylamide (at about 35 ° C), poly-N-ethoxyethylmethacrylamide (at about 45 ° C), poly-N-tetrahydrofurfurylacrylamide (at about 28 ° C) , Poly-N-tetrahydrofurfuryl methacrylamide (about 35 ° C.), poly-N, N-ethylmethylacrylamide ( 56 ° C.), poly -N, N-diethyl acrylamide (the 32 ° C.), and the like.

  The hydrophilic polymer used in the present invention may be either a homopolymer or a copolymer. For example, polyacrylamide, poly-N, N-diethylacrylamide, poly-N, N-dimethylacrylamide, polyethylene oxide, polyacrylic acid and its salts, polyhydroxyethyl methacrylate, polyhydroxyethyl acrylate, polyvinyl alcohol, polyvinyl pyrrolidone, cellulose Examples thereof include water-containing polymers such as carboxymethyl cellulose, but are not particularly limited.

In the present invention, the polymer is immobilized at a high density. 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 can be 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.

In the present invention, the above-described polymer is immobilized on a silica gel carrier. The immobilization method is not particularly limited, but, for example, an atom transfer radical polymerization initiator is immobilized on the surface of the substrate, and the temperature range of 0 to 80 ° C. by the atom transfer radical method under the catalyst from the initiator. Among them, there is a method of growing and reacting a polymer whose hydration power changes. 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. The solvent used in the polymerization is not particularly limited, and examples thereof include dimethylformaldehyde (DMF). Other initiator concentrations, copper halide concentrations, ligand complex concentrations, reaction temperatures, reaction times, and the like during polymerization are not particularly limited, and 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.

  The shape of the base material used in the present invention is not particularly limited, and examples thereof include a particle shape, a flat plate shape, and a tubular shape. In particular, when the carrier of the present invention is used as a carrier for chromatography, silica gel is preferable as the carrier. 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, the temperature-responsive chromatographic support thus obtained is packed in a column and attached to a normal liquid chromatography apparatus, and used as a liquid chromatography system. In that case, the separation of the present invention is affected by the temperature of the support 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.

  Although the separation method is not particularly limited, an example is a method of separating a solute in a column packed with a carrier at a constant temperature. The surface of the carrier of the present invention varies depending on the temperature. Depending on the substance to be separated, it may be separated only by setting an appropriate constant temperature.

  As an example of another separation method, the temperature at which the characteristics of the support surface change may be confirmed in advance, and the solute may be 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.

  When changing the temperature, the temperature change may be changed intermittently once or more times after starting to flow the solute, or may be changed continuously. Moreover, you may combine those methods. The temperature change at that time may be performed manually, or an apparatus capable of automatically controlling the temperature according to a program may be used.

  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.

  Furthermore, two or more types of temperature-responsive liquid chromatography carriers may be packed in the same column, and the solute may be separated while changing the temperature so as to sandwich the temperature at which the characteristics of the carrier surface change. In this case, for example, when two types of carriers are used, different temperature ranges are generated on the three surfaces of the carrier, and the temperature may be changed by the above-described method so as to sandwich these three temperatures. . This may be performed by packing two or more types of temperature-responsive chromatography carriers in two or more columns.

  As an example of another separation method, a temperature-responsive liquid chromatography carrier is used, the column inlet end temperature and the column outlet end temperature are set so as to sandwich the temperature at which the characteristics of the carrier surface change, and the temperature in the column is changed from the inlet end. A method of separating the solute by providing a temperature gradient to the outlet end can be mentioned. The method of changing the temperature step by step is not particularly limited. For example, the column inlet end temperature and the column outlet end temperature are monitored sufficiently to keep the entire column warm, and multiple aluminum blocks with different temperatures are connected to the column. The method of making it, etc. are mentioned.

  In the present invention, as described above, the solute can be separated only by temperature while the moving bed is fixed. In that case, the mobile phase is preferably a 100% aqueous system, but in the case of the present invention, the composition of the mobile phase is not particularly limited because of the characteristics of the polymer immobilized on the surface of the carrier. For example, the mobile phase contains a solvent. Even if it changes, pH may be changed and the salt may be included. At that time, the carrier concentration of the present invention may be used in combination with a solvent gradient method by changing the solvent concentration. Further, the mobile phase may be 100% organic solvent.

  By using the temperature-responsive chromatography carrier of the present invention described above and the chromatography method using the same, it is possible to separate pharmaceuticals and their metabolites, agricultural chemicals, peptides, proteins and cells. In that case, separation can be achieved by simple operation only by changing the temperature in the column.

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

DMF in the presence of CuCl and Me ₆ TREN as catalysts was added to silica beads (average particle size 5 μm, pore size 300 mm) into which (m, p-chloromethylphenylethyl) ethyltrichlorosilane, an atom transfer radical polymerization (ATRP) initiator, was introduced. ATRP was performed in a poly (N-isopropylacrylamide) (PIPAAm) brush on the silica particle surface. At this time, ATRP was carried out for 1 to 20 hours, and the amount of PIPAAm grafting was controlled by the reaction time. A column (φ4.6 × 50 mm) packed with this was connected to an HPLC system, and steroid elution experiments were performed while controlling the column temperature in a thermostatic bath. Steroids eluting from the column were detected by absorption at 254 nm.

As a result, the amount of PIPAAm grafting increased with the ATRP reaction time, and a PIPAAm grafting amount of 1.80 to 4.40 mg / m ² was obtained, which was 20 to 80 times that of the carrier obtained by the conventional method. The obtained results are shown in FIG. Next, steroid elution experiments were performed at 30 ° C. using three types of silica beads with different amounts of PIPAAm grafting. It was found that the steroid retention time increased as the graft amount increased. The results are shown in FIG. From this, it was found that the hydrophobic interaction with the steroid can be increased by increasing the PIPAAm graft amount. Further, a carrier having an ATRP reaction time of 3 hours was packed in a column, a temperature change from 10 ° C. to 50 ° C. was given, and a steroid elution experiment was performed. As a result, as shown in FIG. 3, the peaks of each steroid that overlapped at 10 ° C. were separated at 50 ° C. This is presumed to be because the PIPAAm brush constructed on silica beads dehydrates as the temperature rises, and hydrophobically interacts with the steroid. In addition, separation was performed using this column while changing the temperature of the mixed solution of insulin chain A and insulin. The obtained results are shown in FIG. Overlapping peaks began to separate at 30 ° C at low temperatures, and separation of insulin chain A and insulin could be achieved at 40 ° C. From the above, it is considered that the silica beads having the PIPAAm brush structure of the present invention are chromatography carriers capable of controlling a large hydrophobic interaction by temperature with a small surface area.

Comparative Example 1

(A) Synthesis method of poly (N-isopropylacrylamide) having a carboxyl group at one end N-isopropylacrylamide 20.0 g, 3-mercaptopropionic acid 0.09 g, 2,2′-azobis (isobutyronitrile) 0 .21 g was added to each polymerization tube, and 50 ml of dry N, N-dimethylformamide was added and dissolved. Next, after freezing under liquid nitrogen, oxygen in the polymerization tube was degassed with a vacuum oil pump, and the polymerization tube was immersed in methanol in a reduced pressure state to remove dissolved oxygen in N, N-dimethylformamide. This freeze deaeration operation was repeated three times. When degassing was completed, the reaction was carried out in an incubator at 70 ± 1 ° C. for 20 hours. Next, when the temperature was lowered to room temperature, it was dropped into dry diethyl ether which was concentrated under reduced pressure to precipitate poly (N-isopropylacrylamide) having a carboxyl group at one end. This precipitate was collected by filtration with a PTFE (polytetrafluoroethylene) filter (pore size: 3.0 μm) and dried under reduced pressure in a desiccator containing silica gel to obtain 19.0 g of a crude product. This was dissolved in 30 ml of dry N, N′-dimethylformamide, dropped into dry diethyl ether, and the precipitate was collected by filtration with a Teflon filter. This was dried under reduced pressure in a desiccator to obtain purified poly (N-isopropylacrylamide). The obtained polymer was confirmed to have poly (N-isopropylacrylamide) having a molecular weight of 15,000 and having about 1 carboxyl group at the molecular end by gel filtration chromatography using tetrahydrofuran as a solvent and acid-base measurement. .

(B) Active esterification of poly (N-isopropylacrylamide) having a carboxyl group at one end. 11.35 g of purified poly (N-isopropylacrylamide) was dissolved in 100 ml of dry ethyl acetate, and 1.23 g of dicyclohexylcarbodiimide and N- Hydroxysuccinimide 0.69g was added and it was made to react at 0 degreeC for 2 hours, and room temperature (20-25 degreeC) for 12 hours, stirring well. Next, N, N'-dicyclohexylurea, which is a by-product, was filtered off with a PTFE filter, and the filtrate was concentrated under reduced pressure, then dropped into dry diethyl ether and precipitated, and filtered through a Teflon filter, and then cooled at room temperature. From which the solvent was distilled off, active esterified poly (N-isopropylacrylamide) was obtained.

(C) Binding of active esterified poly (N-isopropylacrylamide) and amino group carrier 2.0 g of active esterified poly (N-isopropylacrylamide) was dissolved in 50 ml of pure water, and 6.0 g of aminopropyl silica gel was added. After vigorous shaking at room temperature, the reaction was washed with 500 ml of cold water, and 2.0 g of active esterified poly (N-isopropylacrylamide) was again added to a solution of 50 ml of pure water and shaken vigorously at room temperature for 12 hours. That ’s it. This operation was repeated three times, washed with 500 ml of cold water, then washed with 100 ml of methanol and dried. Dissolve 3.0 g of active esterified poly (N-isopropylacrylamide) in 6 ml of N, N-dimethylformamide and add 1 ml of polystyrene fine particle suspension with a primary amino group on the surface (diameter: 1.0 ± 0.03 μm) , Stock solution concentration: 5 × 10 ¹¹ / ml) was added to a solution diluted with 24 ml of pure water at intervals of 30 minutes every 1 ml, and slowly mixed by inversion. After the entire amount was added, it was mixed by inverting at 4 ° C. or lower for 16 hours. After completion of the reaction, collection by centrifugation and washing by cold purification were repeated twice, followed by dilution with Hanks balanced salt solution (pH 7.4) (6 × 10 ⁹ , 6 × 10 ¹⁰ / ml). The polymer immobilization amount of the obtained carrier was 0.78 mg / m ² . It has been found that the method of the present invention is useful for high density immobilization of polymers.

  According to the present invention, it is possible to obtain a temperature-responsive chromatographic support in which a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized at a high density on the substrate surface. When this carrier is used, the amount of change of the substrate surface with respect to temperature change is significantly improved. As a result, the separation operation is simplified and the efficiency of the separation work is improved. As this separation object, utilization to a wide range of peptides, proteins and cells is strongly expected. Therefore, the present invention is extremely useful in the fields of medicine and biology.

  It is the figure which showed the amount of PIPAAm grafting when the reaction time shown in Example 1 was changed.   It is the figure which isolate | separated steroid, changing the kind of support | carrier shown in Example 1. FIG.   It is the figure which isolate | separated the steroid, changing the separation temperature shown in Example 1. FIG.   It is the figure which isolate | separated the insulin chain A and insulin, changing the isolation | separation temperature shown in Example 1. FIG.

Claims (21)

A temperature-responsive chromatography characterized in that a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. is immobilized at a high density at a rate of 0.08 molecular chain / nm ² or more on the substrate surface. Graphy carrier. Polymer immobilization of the substrate surface is 0.8~10.0mg / ^{m 2,} claim 1 temperature responsive chromatography carrier according. The temperature-responsive chromatography carrier according to any one of claims 1 and 2, wherein the polymer molecular chain is non-crosslinked. The polymer is composed of any one or two or more of poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives, copolymers thereof, polyvinyl methyl ether, and polyvinyl alcohol partially acetylated products. The temperature-responsive chromatography carrier according to any one of 1 to 3. The temperature-responsive chromatography carrier according to any one of claims 1 to 4, wherein the polymer is poly-N-isopropylacrylamide. The polymer is a copolymer in which hydrophilic molecules, hydrophobic molecules, and ionic molecules are included in the polymer molecular chain as long as the property of changing hydration power within the temperature range of 0 to 80 ° C. is not lost. The temperature-responsive chromatography carrier according to any one of claims 4 and 5. The temperature-responsive chromatography carrier according to any one of claims 1 to 6, wherein the substrate has a particle shape, a flat plate shape, or a tubular shape. It is characterized in that an atom transfer radical polymerization initiator is immobilized on the surface of a substrate, and a polymer whose hydration power changes within a temperature range of 0 to 80 ° C. by the atom transfer radical method from the initiator under a catalyst. A method for producing a temperature-responsive chromatography carrier. The method for producing a temperature-responsive chromatography carrier according to claim 8, wherein the atom transfer radical polymerization initiator is 2- (m, p-chloromethylphenylethyl) ethyltrichlorosilane. 10. The temperature-responsive chromatography carrier production according to claim 8, 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. Method. The temperature-responsive chromatography carrier production method according to any one of claims 8 to 10, wherein the polymerization catalyst is copper chloride as copper halide and tris (2- (dimethylamino) ethyl) amine as a ligand complex. Polymer immobilization of the substrate surface is 0.8~10.0mg / ^{m 2,} claim 8 to 11 temperature responsive chromatography carrier manufacturing method according to any one. The method for producing a temperature-responsive chromatography carrier according to any one of claims 8 to 12, wherein the polymer molecular chain is non-crosslinked. The polymer is composed of any one or two or more of poly-N-substituted acrylamide derivatives, poly-N-substituted methacrylamide derivatives, copolymers thereof, polyvinyl methyl ether, and polyvinyl alcohol partially acetylated products. The method for producing a temperature-responsive chromatography carrier according to any one of 8 to 13. The temperature-responsive chromatography carrier production method according to any one of claims 8 to 14, wherein the substrate is silica gel particles, glass plates, or glass particles. A temperature-responsive chromatography method comprising separating or concentrating a specific substance while changing the temperature so as to sandwich the temperature at which the surface characteristics of the temperature-responsive chromatography carrier according to claim 1 change. 18. The specific substance is adsorbed on the temperature-responsive chromatography carrier according to claim 1 and then the adsorbed specific substance is liberated by changing the temperature and changing the characteristics of the surface of the carrier. The temperature-responsive chromatography method described. A specific substance is separated while changing the temperature so that two or more types of temperature-responsive chromatographic supports according to claims 1 to 7 are packed in the same column and the temperature at which the characteristics of the support surface change is sandwiched. The temperature-responsive chromatography method according to any one of claims 16 and 17. Using the temperature-responsive chromatography carrier according to claim 1, the column inlet end temperature and the column outlet end temperature are set so as to sandwich the temperature at which the characteristics of the carrier surface change, and the temperature gradient in the column from the inlet end to the outlet end The temperature-responsive chromatographic method according to any one of claims 16 to 18, wherein the specific substance is separated by attaching. The temperature-responsive chromatography method according to any one of claims 16 to 19, wherein the mobile phase is an aqueous system. The temperature-responsive chromatography method according to claim 16 to 20, wherein the specific substance is a pharmaceutical product, a metabolite thereof, an agrochemical, a peptide, a protein, or a cell.

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