JP2016165677A - Adsorption material, and separation purification apparatus and separation purification method using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an adsorption material which is used when a target substance contained in a liquid to be treated is separated and purified and which can allow separation purification treatment to be performed appropriately, and to provide a separation purification apparatus and a separation purification method in each of which the adsorption material is used.SOLUTION: The adsorption material 1 includes: an adsorption site 10 comprising a functional molecule for adsorbing the target substance L to be freely desorbed; a temperature responsive site 20 comprising a temperature responsive molecule the phase of which is changed by a temperature change; and a carrier 30 for supporting the adsorption site 10 and the temperature responsive site 20. The temperature responsive site 20 has the operation of desorbing the target substance L to be adsorbed on the adsorption site 10 by a phase change followed by a temperature rise. The molecular weight of the adsorption site 10 is twice or less of that of the temperature responsive site 20. The separation purification apparatus includes an adsorption material 1-packed column and the separation purification method comprises a desorption step of desorbing the target substance L adsorbed on the adsorption material 1 by the phase change followed by the temperature rise of the temperature responsive site 20.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an adsorbent, a separation and purification apparatus using the same, and a separation and purification method.

  Affinity chromatography using an adsorbent having a specific affinity for a target substance is often used for separation and purification of biological substances such as proteins. In the separation and purification process using affinity chromatography, the target substance is selectively adsorbed on the adsorbent by contacting the target liquid containing the desired target substance, and the impurities contained in the target liquid are washed. Then, after removing the target substance, the target substance is separated and purified. In order to desorb the target substance adsorbed on the adsorbent, it is generally necessary to change the pH, salt concentration, etc. to reduce the affinity between the target substance and the adsorbent.

  However, if the target substance is desorbed by changing the pH to an acidic range or increasing the salt concentration, the target substance may be denatured and deteriorated. Further, after the separation and purification treatment, neutralization treatment of an acidic solution, dialysis treatment of a high salt concentration solution, and the like are required, and thus there are difficulties such as an increase in man-hours and waste and a reduction in recovery rate. In particular, in the field of handling biomolecules as pharmaceuticals and pharmaceutical raw materials, management operations that minimize the time for exposing biomolecules to a temperature condition of 42 ° C. or higher and an acidic condition of pH 5 or lower are desired because of quality control requirements. Yes. Therefore, as a means for desorbing the target substance adsorbed on the adsorbent, development of a method utilizing a phase change accompanying a temperature change of a temperature-responsive molecule has been advanced.

  For example, Patent Document 1 discloses an adsorbent for cell separation having a region on which a substance that specifically adsorbs a target cell is introduced and a region to which a stimulus-responsive polymer is bonded on the surface of the substrate. An adsorbent for cell separation, wherein a substance that specifically adsorbs cells is introduced to the substrate via a polymer having affinity for both the substance and the substrate but not for cells. Have been described. Specifically, it is described that poly (N-isopropylacrylamide) used as a temperature-responsive polymer is desorbed by lowering the temperature from 37 ° C. to 10 ° C. (See paragraphs 0061 and 0062).

  Patent Document 2 describes an affinity control-type material in which an affinity substance (ligand) having affinity for a stimulus-responsive polymer and a target substance is independently bonded to a support, preferably covalently bonded. Yes. Specifically, it is described that the target substance was desorbed by lowering the temperature of poly (N-isopropylacrylamide) used as the stimulus-responsive polymer from 40 ° C. to 20 ° C. (paragraph 0061). reference).

  Patent Document 3 includes a substance that specifically interacts with a stimulus-responsive polymer and a target substance, and the stimulus-responsive material polymer undergoes a structural change due to physical stimulation, thereby A separation material comprising a composite material in which the interaction of substances that specifically interact with each other is subjected to changes in the chemical or physical environment, and the interaction force with the target substance is reversibly changed by physical stimulation. In addition, a separation material is described in which a stimulus-responsive polymer has no affinity for a substance that specifically interacts with a target substance. Specifically, it is described that the target substance is desorbed by lowering the poly (N-isopropylacrylamide) used as the stimulus-responsive polymer from 40 ° C. to 2 ° C. or 10 ° C. (See Example 2 and Example 3).

JP 2006-223195 A Special table 2003-524680 International Publication No. 2001/74482

  As described in Patent Document 1, Patent Document 2, and Patent Document 3, when a separation and purification material (adsorbent) that desorbs a target substance by a phase change accompanying a temperature change of a temperature-responsive molecule is used, an acidic solution It is possible to avoid degradation of the target substance due to the above. However, these separation and purification materials (adsorbents) are intended to desorb the target substance due to a phase change accompanying the temperature drop of the temperature-responsive molecule. In general, biological substances handled as pharmaceuticals and pharmaceutical raw materials are stored in a solution at a low temperature of about 4 ° C. so that the activity and molecular structure are not impaired. In order to desorb the target substance by this, it is necessary to perform a process of raising the temperature of the liquid to be treated to the phase change temperature or higher prior to the separation and purification process.

  As the liquid to be treated for the separation and purification treatment, a culture solution that has been subjected to large-scale culture or a large amount of filtrate after the filtration treatment is often applied. And large capacity. Therefore, enormous heating costs and heating time are required to raise the temperature of the liquid to be treated containing the biological material to the phase change temperature or higher in advance. That is, with an adsorbent that has a mechanism for desorbing a target substance due to a phase change accompanying a temperature drop of a temperature-responsive molecule, deterioration of the target substance due to an acidic solution or the like can be reduced, but heating is increased as the liquid to be treated becomes larger. There is a problem that cost and heating time are required or the recovery rate is lowered, and the separation and purification processing efficiency of the liquid to be processed is easily impaired.

  Therefore, the present invention is an adsorbent used for the separation and purification treatment of the target substance contained in the liquid to be treated, the adsorbent capable of appropriately performing the separation and purification treatment, a separation and purification apparatus using the same, and An object is to provide a separation and purification method.

  In order to solve the above problems, an adsorbent according to the present invention includes an adsorption site composed of a functional molecule that adsorbs a target substance in a detachable manner, a temperature response site composed of a temperature-responsive molecule that changes phase according to a temperature change, and An adsorption site and a carrier supporting the temperature response site, wherein the temperature response site has an action of desorbing a target substance adsorbed on the adsorption site by a phase change accompanying a temperature rise, and the adsorption site The molecular weight of is characterized by being not more than twice the molecular weight of the temperature-responsive site.

  In addition, a separation and purification apparatus according to the present invention includes the adsorbent and a column packed with the adsorbent.

  The separation and purification method according to the present invention is a separation and purification method for separating and purifying a target substance contained in a liquid to be treated using a column filled with the adsorbent, wherein the adsorbent is An adsorption site composed of a functional molecule that adsorbs the target substance in a detachable manner, a temperature response site composed of a temperature-responsive molecule that changes phase according to a temperature change, and a carrier that immobilizes the adsorption site and the temperature response site. The temperature-responsive part has an action of desorbing the target substance adsorbed on the adsorption part by a phase change accompanying a temperature rise, and the molecular weight of the adsorption part is less than twice the molecular weight of the temperature-responsive part The separation and purification method is a separation and purification method for separating and purifying a target substance contained in a liquid to be treated using a column packed with the adsorbent, and the target substance is placed on the column. Contains An adsorption step of passing a working fluid and adsorbing the target substance to the adsorption site; a washing step of washing the adsorbent adsorbed by the target substance under conditions that do not desorb the target substance; The adsorbent is heated, and a desorption step of desorbing the target substance adsorbed on the adsorption site due to a phase change accompanying a temperature increase of the temperature response site, and the desorbed target substance from the column And an elution step for elution.

  ADVANTAGE OF THE INVENTION According to this invention, it is an adsorption material used for the separation-purification process of the target substance contained in a to-be-processed liquid, Comprising: Adsorption material which can perform a separation-purification process appropriately, Separation-purification apparatus using the same, and Separation and purification methods can be provided.

It is a figure which shows typically the structure and effect | action of an adsorbent which concern on embodiment of this invention. (A) is a state before the target substance is adsorbed on the adsorbent, (b) is a state where the target substance is adsorbed on the adsorbent, (c) is a state where a phase change occurs in the adsorbent, (d) Indicates a state in which the target substance is desorbed from the adsorbent in which the phase change has occurred. It is a figure which shows an example of the manufacturing method of the adsorbent which concerns on embodiment of this invention. (A) shows a step of binding the temperature-responsive molecule to the carrier, and (b) shows a step of binding the functional molecule to the temperature-responsive molecule. It is a figure which shows an example of schematic structure of a manufacturing system provided with the isolation | separation refinement | purification apparatus to which the adsorbent which concerns on embodiment of this invention is applied. It is a figure which shows an example of schematic structure of a manufacturing system provided with the isolation | separation refinement | purification apparatus to which the adsorbent which concerns on a comparative example is applied. It is a figure which shows the correlation of the molecular weight ratio of an adsorption | suction site | part and a temperature response site | part with respect to the collection | recovery rate of a target substance.

  First, an adsorbent according to an embodiment of the present invention will be described. In addition, about the structure which is common in the following each figure, the same code | symbol is attached | subjected and the overlapping description is abbreviate | omitted.

  FIG. 1 is a diagram schematically showing the structure and operation of an adsorbent according to an embodiment of the present invention. (A) is a state before the target substance is adsorbed on the adsorbent, (b) is a state where the target substance is adsorbed on the adsorbent, (c) is a state where a phase change occurs in the adsorbent, (d) Indicates a state in which the target substance is desorbed from the adsorbent in which the phase change has occurred.

  As shown in FIG. 1A, the adsorbent 1 according to this embodiment includes an adsorption site 10, a temperature response site 20, and a carrier 30. The adsorbent 1 is a separation / purification material used for separation / purification processing of the target substance L contained in the liquid to be treated, and is a material suitable as a packing material for a separation / purification apparatus (column) for affinity chromatography, for example. is there.

  A solution containing the desired target substance L together with other impurities is applied to the adsorbent 1 as a liquid to be treated for separation and purification (see FIG. 1A). In FIG. 1, IgG is exemplified as the target substance L, but the target substance L is not limited to this. When the adsorbent 1 and the liquid to be treated are brought into contact with each other, as shown in FIG. 1 (b), the target substance L in the liquid to be treated is selectively selected to the adsorption site 10 of the adsorbent 1 with a relatively high affinity. Adsorb to. At this time, the impurities contained in the liquid to be treated have a relatively low affinity for the adsorption site 10 and are not adsorbed or weakly adsorbed, so that the target substance L is left by the washing treatment. Removed.

  The adsorbent 1 according to the present embodiment has an action of desorbing the target substance L by a phase change accompanying a temperature rise of the temperature-responsive molecule (temperature-responsive part 20). Therefore, when the temperature of the adsorbent 1 is increased, the temperature response portion 20 changes to a three-dimensional structure in an agglomerated state as shown in FIG. As a result, the affinity between the adsorption site 10 and the target substance L is reduced, and the target substance L adsorbed on the adsorption site 10 is desorbed (see FIG. 1D). In the adsorbent 1 according to the present embodiment, the separation and purification process of the desired target substance L contained in the liquid to be processed is thus realized.

  Specifically, the adsorbent 1 has a structure in which the branch structure schematically shown in FIG. 1 is repeated innumerably, and a large number of adsorption sites 10 and temperature response sites 20 are supported on a carrier 30. And such a support | carrier 30 comprises the isolation | separation refinement | purification material by single-piece | unit single-piece | unit or by a large collection.

  The liquid to be processed that is processed in the separation and purification process using the adsorbent 1 is an aqueous solution containing the desired target substance L that specifically adsorbs to the adsorption site 10. That is, a solution containing a desired target substance L having a high affinity with the adsorption site 10 and impurities or the like having a relatively low affinity with the adsorption site 10 is provided for the treatment. The aqueous solution may be either an aqueous solution using water as a solvent or a mixed solvent solution of water and an organic solvent. Such a solution includes a suspension or a dispersion.

  The adsorption site 10 is composed of a functional molecule that adsorbs the target substance L in a detachable manner. In the low temperature range lower than the phase change temperature of the temperature response site 20, the functional molecule exhibits a strong affinity for the target material L and specifically adsorbs the target material L, and is higher than the phase change temperature of the temperature response site 20. In the high temperature range, the affinity is weakened so that the target substance L can be detached.

  The adsorption of the target substance L to the adsorption site 10 may be either a chemical bond or a physical bond, or may be a multipoint bond. For example, hydrogen bond, hydrophobic interaction, π-π interaction, dipole-dipole interaction, charge-dipole interaction, charge-charge interaction, charge transfer interaction, van der Waals bond, anchor effect, etc. A reversible bond is formed by an appropriate interaction or a combination thereof.

  As the target substance L, an appropriate substance such as a biological substance, a cell, an organic compound, an inorganic compound, a complex, a metal ion, an inorganic fine particle, or a metal fine particle is applied as long as it is a substance that specifically binds to the adsorption site 10. Is possible. From the viewpoint of taking advantage of the characteristics of the adsorbent 1, a substance that is desired to be stored in a low temperature range of about 0 ° C. or higher and 20 ° C. or lower, or a substance that has low stability in an acidic range of about pH 5 or lower is preferable. Examples of such target substance L include biological substances, cells, acid-reactive substances that react intramolecularly with acids having a pH of about 5 or less, or intermolecular reactions with impurities, etc., and decompose by acids having a pH of about 5 or less. Examples include acid-decomposable substances that generate water.

  Examples of biological substances include proteins, peptides, amino acids, saccharides, nucleic acids, lipids, metabolites, and the like. Examples of the cells include human cells, animal cells, plant cells, and microbial cells including bacteria and fungi. As cells, blood cells, recombinant cells, fused cells, and the like can be applied. Examples of the acid-reactive substance and the acid-decomposable substance include various substances such as esters and amides.

  As the functional molecule constituting the adsorption site 10, any functional low molecule or functional polymer having an appropriate structure may be used as long as it is a molecule that specifically adsorbs the target substance to be separated and purified. it can. Examples of functional small molecules include 5-membered or 6-membered aromatic or heteroaromatic rings, sulfide groups, sulfonyl groups, amide groups, amino acids, nucleic acids, sugars, and hydrophobicity such as cyclophane and calixarene. A functional molecule having at least one molecular structure selected from the group consisting of cyclic compounds having a space, and a group consisting of a benzene skeleton, a pyridine skeleton, a pyrazine skeleton, a pyrrole skeleton, a triazine skeleton, a diazole skeleton, and a triazole skeleton It is preferable to use a functional molecule having at least one selected skeleton. When a functional molecule having such a skeleton is used, it is easy to form an interaction by multipoint bonding, particularly when a protein, nucleic acid, or the like is used as a target substance. In particular, the sulfide group is preferable in that it can form a functional molecule exhibiting antibody binding ability by thiophilic interaction. Moreover, as a functional polymer, it is preferable to use the functional molecule which consists of a peptide, protein, polysaccharide, polyacrylamide derivative, polymethacrylic acid derivative, polyfunctional polyamide, or polyfunctional polyester, for example. Such a water-soluble stable functional polymer facilitates the molecular design of the adsorption site 10.

  Specific examples of the combination of the functional molecule constituting the adsorption site 10 and the target substance L include antibodies, antigens, protein A, protein G, protein L, Fc receptors, peptide fragments thereof, or antibody adsorption capacity A combination of a synthetic low molecule or a synthetic polymer, a lectin and a sugar chain, a partial structure of a sugar chain, or a combination of these derivatives, a protein, a protein receptor, a partial structure of a protein receptor, Combinations of these derivatives, cofactors, or synthetic small molecules that interact specifically, such as dyes, nucleic acids (polynucleotides such as DNA and RNA), complementary nucleic acids, or complementary artificials A combination with a nucleic acid, a combination of biotin and avidin, or streptavidin can be exemplified.

  The adsorption site 10 is supported by the carrier 30. In FIG. 1, the adsorption site 10 is bonded to the side chain of the temperature response site 20, but it may be bonded to the end of the temperature response site 20, or only the carrier 30 instead of the temperature response site 20. It may be directly bonded, or may be directly bonded to both the temperature response site 20 and the carrier 30. Moreover, the adsorption | suction site | part 10 may be couple | bonded with the single temperature response site | part 20 or the support | carrier 30, and multiple may be couple | bonded. The adsorption site 10 may be bonded to the temperature response site 20 or the carrier 30 via a linker (spacer). The linker is preferably a molecule that does not irreversibly adsorb the target substance.

  The binding between the adsorption site 10 and the temperature responsive site 20 or the carrier 30 may be either a covalent bond or a non-covalent bond. For example, the bond between the adsorption site 10 and the temperature responsive site 20 or the carrier 30 is a covalent bond by an appropriate reaction such as a nucleophilic addition reaction, a nucleophilic substitution reaction, a radical addition reaction, a rearrangement reaction, a sigmatropy reaction, a condensation reaction, Formed by non-covalent bonds by multipoint adsorption, hydrogen bonding, hydrophobic interaction, π-π interaction, dipole-dipole interaction, charge-dipole interaction, charge-charge interaction, charge transfer interaction, etc. can do. More specifically, for example, epoxide and nucleophile, carboxylic acid or activated carboxylic acid and nucleophile, alkyl halide and nucleophile, aryl halide and nucleophile, unsaturated carbonyl and nucleophile, unsaturated Reactions such as nitrile and nucleophile, amine and aldehyde, silanol and silane coupling agent, and binding by biotin and avidin, biotin and streptavidin, etc. can be used.

  The molecular weight of the adsorption part 10 shall be 2 times or less the molecular weight of the temperature response part 20 described later. When the molecular weight of the adsorption site 10 is made smaller than the molecular weight of the temperature response site 20 in this way, the recovery rate of the target substance L in the separation and purification process is increased, and a recovery rate of about 30% or more can be realized. The ratio between the molecular weight of the adsorption site 10 and the temperature response site 20 can be changed to further improve the recovery rate. For example, when a recovery rate of 40% or more is required, the molecular weight of the adsorption site 10 is preferably 0.5 times or less than the molecular weight of the temperature response site 20, and a recovery rate of 70% or more is required. For this, the molecular weight of the adsorption site 10 is preferably 0.2 times or less than the molecular weight of the temperature response site 20. The molecular weight of the adsorption site 10 is preferably 40 or more. When the molecular weight of the adsorption site 10 is 40 or more, a predetermined target substance can be specifically adsorbed.

  The temperature responsive portion 20 is made of a temperature responsive molecule that changes phase with a temperature change. The temperature-responsive molecule indicates a lower critical solution temperature (LCST). That is, the temperature-responsive molecule is present in a solution to be treated in a low temperature region lower than the lower critical solution temperature, and is present in a phase-insoluble state in a high temperature region higher than the lower critical solution temperature. Therefore, the temperature-responsive part 20 changes phase with the lower critical solution temperature as a boundary and causes a remarkable change in the three-dimensional structure, thereby increasing the affinity between the adsorption part 10 and the target substance L bound to the adsorption part 10. Make it variable.

  The temperature response part 20 has an action of desorbing the target substance L adsorbed on the adsorption part 10 by a phase change accompanying a temperature rise. When the temperature response portion 20 is heated from a low temperature range lower than the lower critical solution temperature (phase change temperature) indicated by the temperature responsive molecule to a higher temperature range higher than the lower critical solution temperature (phase change temperature), the phase With the change in the three-dimensional structure due to the change, the affinity between the adsorption site 10 and the target substance L adsorbed on the adsorption site 10 is lower than that at the time of adsorption, and the target substance L is desorbed from the adsorbent 1. Become.

  As the temperature-responsive molecule constituting the temperature-responsive portion 20, a temperature-responsive polymer that exhibits a lower critical solution temperature can be used. The temperature-responsive polymer is composed of acrylamide derivatives, methacrylic acid derivatives such as methacrylic acid esters, acrylic acid derivatives such as acrylic acid esters, vinyl alcohol derivatives, N-modified ε-polylysine derivatives as monomers (monomer units). Ε-polylysine derivatives (lysine derivatives) such as γ-glutamic acid derivatives such as γ-glutamic acid amide (glutamic acid derivatives), δ-aspartic acid derivatives such as δ-aspartic acid amide derivatives (aspartic acid derivatives), lactic acid, diol derivatives, Caprolactams, caprolactones, sugars, siloxanes and the like can be included. The temperature-responsive molecule may be a polymer of any of these monomers or a copolymer of a combination of these monomers. The copolymer may be any of random type, block type and graft type. These monomers are, independently of each other, hydroxy group, carboxyl group, amino group, thiol group, epoxy group, nitrile group, halogen atom, methyl group, ethyl group, n-propyl group, iso-propyl group, n -Alkyl groups such as butyl group, sec-butyl group, iso-butyl group, tert-butyl group, n-pentyl group, n-hexyl group, cycloalkyl group optionally having these as a substituent, alkoxy It may be substituted with a substituent (functional group) such as a group, an aryl group, an aralkyl group, an acyl group, and an aminoacyl group. The temperature-responsive polymer is not limited to a linear polymer, and may be a branched polymer, a dendrimer, a gel, a crosslinked gel, or the like.

  Examples of the acrylamide derivative include N-isopropylacrylamide, N-diisopropylacrylamide, N-ethylacrylamide, N-diethylacrylamide, N-methylacrylamide, N-dimethylacrylamide, and other N-substituted derivatives. Examples of the methacrylic acid derivative include butyl methacrylate, propyl methacrylate, ethyl methacrylate, methyl methacrylate, and other O-substituted derivatives. Examples of the acrylic acid derivative include butyl acrylate, propyl acrylate, ethyl acrylate, methyl acrylate, and other O-substituted derivatives. Examples of the vinyl alcohol derivative include vinyl alcohol methyl ether, vinyl alcohol acetylate, and other O-substituted derivatives. Examples of the ε-polylysine derivative include lysine valeric acid amide, lysine butyric acid amide, lysine propionic acid amide, N-hydroxypentyl lysine, N-hydroxybutyl lysine, N-hydroxypropyl lysine, and other N-substituted derivatives. Can be mentioned. Examples of the γ-glutamic acid derivative include glutamic acid hydroxyhexylamide, glutamic acid hydroxypentylamide, glutamic acid hydroxybutylamide, glutamic acid hydroxypropylamide, and other O-substituted derivatives. Examples of the δ-aspartic acid derivative include aspartic acid hydroxyhexylamide, aspartic acid hydroxypentylamide, aspartic acid hydroxybutylamide, aspartic acid hydroxypropylamide, and other O-substituted derivatives. Examples of the diol derivative include polyethylene glycol and polypropylene glycol. Examples of the saccharide include hydroxypropyl cellulose.

  As the temperature responsive molecule constituting the temperature responsive portion 20, a temperature responsive low molecule showing the lower critical solution temperature can be used together with the temperature responsive polymer showing the lower critical solution temperature. As the temperature-responsive low molecule, for example, spiropyran, azobenzene, diarylethene, derivatives thereof, liquid crystal molecules, and the like that exhibit temperature responsiveness as well as photoresponsiveness can be used.

  The phase change temperature of the temperature response part 20 is preferably in the range of 0 ° C. or more and 60 ° C. or less, more preferably in the range of 0 ° C. or more and 40 ° C. or less, and in the range of 10 ° C. or more and 40 ° C. or less. Is more preferable. When the phase change temperature of the temperature responsive part 20 is in such a temperature range, the target substance L can be adsorbed and desorbed under mild temperature conditions, so that deterioration of the target substance L can be avoided. It is. The phase change temperature of the temperature responsive part 20 is affected by the solvent, impurities, pH, etc. of the liquid to be treated, but the molecular design is such that the temperature responsive part 20 changes phase under pH conditions of pH 6-8. Is preferred. Such molecular design of the temperature responsive portion 20 can be performed by introducing various functional groups into the temperature responsive molecule. For example, the balance between hydrophilic group and hydrophobic group, introduction of charge interaction, etc. Can be realized.

  The temperature response part 20 is supported by the carrier 30. In FIG. 1, the temperature responsive portion 20 is bonded to the carrier 30 via the side chain, but may be bonded to the carrier 30 via the end. In addition, a single temperature response site 20 may be bonded to a single carrier 30, or a plurality of temperature response sites 20 may be bonded. Further, the temperature responsive portion 20 may be bonded to the carrier 30 via a linker (spacer).

  The bond between the temperature responsive site 20 and the carrier 30 may be either a covalent bond or a non-covalent bond. For example, the bond between the temperature response site 20 and the carrier 30 can be formed as described above in the same manner as the bond between the adsorption site 10 and the temperature response site 20 or the carrier 30.

  The carrier 30 supports the adsorption site 10 and the temperature response site 20. The carrier 30 preferably has high chemical and physical stability in the liquid to be treated, and plays a role of imparting mechanical strength, formability, handleability, and the like to the adsorbent 1.

  The carrier 30 can have an appropriate shape, and may be either porous or non-porous. Specific examples of the shape of the carrier 30 include a plate shape, a bead shape, a fiber shape such as a nonwoven fabric and a woven fabric, a film shape, a monolith shape, and a hollow fiber shape.

  The material of the carrier 30 may be either an organic material or an inorganic material, or may be a composite material using these materials in combination. Specifically, for example, polysaccharides such as agarose, sepharose, cellulose, polystyrene, polyalkyl methacrylate, polyglycidyl methacrylate, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, polysiloxane, polyfluorinated ethylene, polyethylene, polypropylene, polyethylene terephthalate. Synthetic resins such as polytrimethylene terephthalate, polyvinyl chloride, polyvinyl acetate, polycarbonate, PA6, PA66, PA11, PA12, carbon materials such as graphite, carbon fiber, carbon nanotube, fullerene, silica, alumina, titania, Oxides such as zirconia and iron oxide, carbonates such as calcium carbonate, phosphates such as hydroxyapatite and aluminum phosphate, borosilicate glass, etc. Silicates such as oxalate, silicate, diatomaceous earth, sulfates such as aluminum sulfate and calcium sulfate, inorganic compounds such as silicon carbide and silicon nitride, ferrite, permalloy, chromium steel, iron-aluminum alloy, gold, silver, Metal materials such as platinum, palladium, and rhodium can be used. Among these, preferred materials are agarose, sepharose, cellulose, polystyrene, silica, iron oxide or ferrite useful as a magnetic carrier from the viewpoints of stability and ease of production.

  The carrier 30 may have an appropriate reactive group such as a carboxy group, an amino group, or a hydroxy group that forms a covalent bond with the adsorption site 10 or the temperature response site 20 on the surface. The carrier 30 may be surface-modified with molecules other than the adsorption site 10 and the temperature response site 20. For example, blocking that prevents non-specific adsorption of the target substance, surface modification that controls the orientation of the adsorption site 10 or the temperature response site 20, surface modification that modifies the dispersibility or adsorption of the carrier 30, etc. It may be done.

  The adsorbent 1 according to the present embodiment can be manufactured by using a conventionally known appropriate reaction. For example, the functional molecule constituting the adsorption site 10, the temperature responsive molecule constituting the temperature responsive site 20, the carrier 30 having a reactive group, etc. can be prepared using a known polymer reaction or the like, It can be obtained by using an activated surface treatment technique or by utilizing a separation / purification technique for natural products and biochemical products. Each bond of the functional molecule constituting the adsorption site 10, the temperature responsive molecule constituting the temperature response site 20, and the carrier 30 having a reactive group is, for example, the functional molecule constituting the adsorption site 10 and the temperature response. A method of binding the temperature-responsive molecule constituting the site 20 and then binding to the carrier 30; and the functionality of constituting the adsorption site 10 after binding the temperature-responsive molecule constituting the temperature-responsive site 20 to the carrier 30 A method of binding a molecule to a temperature-responsive molecule constituting the temperature-responsive site 20, and a method of binding the functional molecule constituting the adsorption site 10 and the temperature-responsive molecule constituting the temperature-responsive site 20 together with the carrier 30 Etc. can be taken.

  Drawing 2 is a figure showing an example of a manufacturing method of an adsorbent concerning an embodiment of the present invention. (A) shows a step of binding the temperature-responsive molecule to the carrier, and (b) shows a step of binding the functional molecule to the temperature-responsive molecule.

  In the method for manufacturing the adsorbent 1 shown in FIG. 2, the temperature responsive molecule constituting the temperature responsive portion 20 is bonded to the carrier 30, and then the functional molecule constituting the adsorbed portion 10 is constituted in the temperature responsive portion 20. The method of binding to a temperature-responsive molecule is illustrated. In this method, a carrier having a carboxy terminus is used as the carrier 30, and glutaraldehyde is used as a linker between the adsorption site 10 and the temperature response site 20.

  In the step of binding the temperature-responsive molecule to the carrier 30, as shown in FIG. 2 (a), first, the carrier 30 is activated by reacting the carboxy terminus of the carrier 30 with carbodiimide hydrochloride (EDC · HCl). Let This reaction may be performed, for example, at pH 5.8 and 37 ° C. for about 2 hours. Subsequently, after removing unreacted substances and by-products, the activated carrier 30 is reacted with the temperature response site 20. This reaction may be performed, for example, at pH 5.8 and 37 ° C. for about 2 hours. Through such a reaction, the temperature-responsive molecule 20 and the carrier 30 can be bound via the nucleophilic amino group of the temperature-responsive site 20 (see the right figure in FIG. 2A). ). According to such a reaction using carbodiimide as a dehydration condensing agent, the temperature-responsive molecule is bound to the carrier 30 under mild conditions such as a temperature condition of about 4 ° C. to 40 ° C. and a pH condition of about pH 4 to about 7 or less. It is also possible to make it.

  In the step of binding the functional molecule to the temperature-responsive molecule, as shown in FIG. 2B, first, the amino group of lysine (Lys) at the temperature-responsive site 20 is reacted with glutaraldehyde and activated. This reaction may be performed, for example, at pH 9.0 and 37 ° C. for about 2 hours. Subsequently, unreacted substances are removed with pure water, and then glutaraldehyde bound to the temperature response site 20 is reacted with the amino group of lysine (Lys) at the adsorption site 10. This reaction may be performed at, for example, pH 7.4 and 37 ° C. for about 2 hours. By undergoing such a reaction, the temperature-responsive molecule 20 and the adsorption site 10 can be bound via glutaraldehyde. The reaction can be allowed to proceed even when the temperature condition is from room temperature to about 37 ° C., the pH condition is in the range of about pH 6 to 9, and even more neutral.

  According to such a production method, the adsorbent 1 can be produced under mild conditions of temperature and pH, so that denaturation of the temperature-responsive molecule 20 and the adsorption site 10 can be avoided, and the adsorbent 1 Performance can be easily secured, and generation of acid and alkali waste liquids can be reduced. In addition, in FIG. 2, cross-linking by glutaraldehyde is schematically shown. However, since cross-linking by multimers of glutaraldehyde can be formed in an actual reaction, steric hindrance and interaction between molecules are partially eliminated. As a result, it may be easy to ensure a certain level of performance for the adsorbent.

  Next, a separation / purification apparatus according to an embodiment of the present invention will be described.

  FIG. 3 is a diagram illustrating an example of a schematic configuration of a manufacturing system including a separation and purification apparatus to which the adsorbent according to the embodiment of the present invention is applied.

  For example, as shown in FIG. 3, the adsorbent 1 can be applied as a column packing material for a separation purification apparatus 70 for affinity chromatography provided in the manufacturing system 100. The manufacturing system 100 is a device that manufactures biological materials that are used as pharmaceuticals, pharmaceutical raw materials, and the like, and includes a culture tank 50, a storage tank 60, and a separation and purification apparatus 70. Further, the separation and purification device 70 is provided with a heating means 80. The separation and purification apparatus 70 to which the adsorbent 1 according to the present embodiment is applied plays a role of separating and purifying biomaterials produced biochemically by culture from impurities in such a manufacturing system 100.

  Specifically, the separation and purification device 70 includes the adsorbent 1 and a column packed with the adsorbent 1. The column is composed of a hollow container having an arbitrary shape, and the column is filled with a liquid to be treated containing the target substance L, a washing liquid for washing impurities, an eluent for desorbing the target substance L, and the like. A liquid inlet and a liquid outlet for passing through the adsorbent 1 are provided. The separation and purification device 70 is incorporated in the manufacturing system 100 by connecting pipes to the liquid inlet and the liquid outlet.

  The material of the column of the separation and purification apparatus 70 may be either an organic material or an inorganic material, or a composite material using these materials in combination. Specifically, for example, glass, stainless steel, aluminum alloy, polyethylene, polypropylene, polyethylene terephthalate, polytrimethylene terephthalate, polyvinyl chloride, polyvinyl acetate, polycarbonate, PA6, PA66, PA11, PA12 and the like can be used. .

  It is preferable that a heat insulating material is interposed in the column. The heat insulating material can be installed, for example, between the packed adsorbent 1 and the column inner wall or around the column outer wall. By adiabatic between the filler 1 and the outside air, heat loss due to the temperature rise of the adsorbent 1 is reduced, and an efficient temperature rise is enabled by heat conduction in the filler 1. Cost and heating time can be suppressed more effectively. As the heat insulating material, for example, a foam type heat insulating material such as urethane foam or phenol foam, a fiber type heat insulating material such as glass fiber, mineral fiber, or resin fiber, a vacuum heat insulating material, or a combination thereof may be used. it can.

  The heating means 80 has a function of increasing the temperature of the adsorbent 1 provided in the separation and purification apparatus 70 with the phase change temperature of the temperature-responsive molecule interposed therebetween. The temperature of the adsorbent is raised by the heating means 80 beyond the phase change temperature, so that the target substance L adsorbed on the adsorbent 1 is desorbed. The heating means 80 preferably has a function of adjusting the temperature of the adsorbent 1 in a range of at least 0 ° C. to 40 ° C., preferably in a range of 0 ° C. to 60 ° C.

  In FIG. 3, the heating means 80 is attached to the column of the separation and purification apparatus 70 and is configured to heat the adsorbent 1 packed in the column inside the column. It is good also as a form which installs independently and contacts the adsorbent 1 which the target substance L has adsorbed, after heating the desorption liquid (eluate) for desorbing a target substance in the exterior of a column. Further, when the heating means 80 is attached to the column, even if it is a type in which heat is transferred from the outside of the column, a heat transfer tube extending inside the column is provided to control the temperature inside the column. The format to be performed may be used. In addition, when the heating means 80 is installed independently from the column, the temperature is adjusted in an in-line configuration in which the temperature is adjusted in the pipe through which the desorbed liquid flows, and in the storage tank in which the desorbed liquid is temporarily stored. Any of the offline forms for performing

  In the manufacturing system 100 shown in FIG. 3, predetermined cells are cultured in the culture tank 50, and a desired biological material is produced biochemically by the cells. For example, antibody production or the like is performed by culturing genetically modified animal cells or the like into which an antibody gene has been introduced. Then, the produced solution containing the biological material is transferred to the storage tank 60 and temporarily stored under a low temperature condition of about 4 ° C., for example. Thereafter, the solution containing the biological material is transferred to the separation and purification device 70, and the separation and purification device 70 performs separation and purification processing using the biological material as the target material L.

  Next, a separation and purification method according to an embodiment of the present invention will be described.

  The separation and purification method according to this embodiment is a method for separating and purifying the target substance L contained in the liquid to be treated using the column (separation and purification apparatus 70) packed with the adsorbent 1. . This separation and purification method is a method that includes an adsorption step, a washing step, a desorption step, and an elution step sequentially.

  In the adsorption process, the liquid to be treated containing the target substance L is passed through the column filled with the adsorbent 1, and the target substance is adsorbed to the adsorption site 10 provided in the adsorbent 1. Examples of the liquid to be treated include a culture liquid obtained in the culture tank 50, a filtrate obtained by filtering the culture liquid, and a low-temperature solution. The temperature of the liquid to be treated is preferably 0 ° C. or higher and not higher than the phase change temperature of the temperature-responsive molecule, more specifically, preferably 0 ° C. or higher and 20 ° C. or lower, more preferably The range is from 0 ° C. to 15 ° C., more preferably from 0 ° C. to 10 ° C., for example, about 4 ° C. (4 ° C. ± 4 ° C.). Therefore, a more preferable form of the separation and purification method according to the present embodiment is a method including a storage step of storing the liquid to be processed containing the target substance L in such a low temperature region as a pre-step of the adsorption step.

  In the adsorption step, the temperature condition is set to a temperature lower than the phase change temperature of the adsorbent 1. Specifically, the adsorption temperature is preferably in the range of 0 ° C. to 20 ° C., more preferably in the range of 0 ° C. to 15 ° C., and in the range of 0 ° C. to 10 ° C. Further preferred. When the target substance is adsorbed at such a temperature, the target substance L can be adsorbed by allowing the liquid to be treated to pass through the column without adjusting the temperature in advance. Can be avoided. Moreover, about other conditions, such as pH conditions and salt concentration, you may make liquid flow constant. On the other hand, the column through which the liquid to be treated is passed is preferably preliminarily adjusted so that the internal temperature of the column is lower than the phase change temperature of the adsorbent 1.

  In the washing step, the adsorbent 1 on which the target substance L is adsorbed is washed under conditions that the target substance L is not desorbed. By washing the adsorbent 1 with the washing liquid, impurities or the like that are not adsorbed or weakly adsorbed due to low affinity for the adsorption site 10 and the unadsorbed target substance L are removed.

  In the cleaning step, the temperature condition is set lower than the phase change temperature of the adsorbent 1 so that the target substance L is not desorbed by the cleaning. Specifically, the cleaning temperature is preferably in the range of 0 ° C. to 20 ° C., more preferably in the range of 0 ° C. to 15 ° C., and in the range of 0 ° C. to 10 ° C. Further preferred. Other conditions such as pH and salt concentration may be constant. As the washing liquid, an appropriate buffer such as PBS can be used.

  In the desorption step, the temperature of the cleaned adsorbent 1 is increased, and the target substance L adsorbed on the adsorption site 10 is desorbed by a phase change accompanying the temperature increase of the temperature response site 20 provided in the adsorbent 1. . The adsorbent 1 preferably has a phase change temperature of the temperature response portion 20 in the range of 0 ° C. or higher and 40 ° C. or lower, and more preferably in the vicinity of 20 ° C. (10 ° C. or higher and 30 ° C. or lower).

  The desorption step is performed by raising the temperature condition to a temperature higher than the phase change temperature of the adsorbent 1. Specifically, the desorption temperature is preferably in the range of 21 ° C. to 40 ° C., more preferably in the range of 25 ° C. to 40 ° C., and in the range of 30 ° C. to 40 ° C. Is more preferable. If it is such temperature, the heating cost required for temperature rising of the adsorbent 1 will not become excessive, and thermal denaturation of the target substance L can also be avoided. The temperature of the adsorbent 1 can be increased by passing the heated desorption liquid or by heating the adsorbent 1 and the cleaning liquid passed through the adsorbent 1. As the desorbing solution, an appropriate buffer solution such as PBS can be used.

  In the elution step, the desorbed target substance L is eluted from the column. By passing the eluate through the adsorbent 1, the target substance L desorbed from the adsorption site 10 is eluted and recovered from the column, and the separation and purification process is completed. The elution step may be carried out integrally with the desorption step by passing the desorption solution (eluate) and then collecting it at the liquid outlet of the column.

  The elution step can be performed at the same temperature as the desorption temperature or at a temperature lower than the desorption temperature. From the viewpoint of reducing the deterioration of the target substance, it is preferable to pass an eluate having a low temperature of about 10 ° C. or lower. As the eluate, an appropriate buffer such as PBS can be used.

  According to such a separation and purification method according to the present embodiment, in the desorption step, the desorption of the target substance L adsorbed on the adsorbent 1 is heated or adsorbed on the desorbed liquid that is passed through the column. This can be achieved by either heating the column filled with the material 1 and the cleaning liquid passed through the adsorbent 1, and the total heat capacity of the object to be heated is relatively low.

  FIG. 4 is a diagram illustrating an example of a schematic configuration of a manufacturing system including a separation and purification apparatus to which an adsorbent according to a comparative example is applied.

  When a conventional adsorbent that desorbs a target substance due to a phase change accompanying a temperature drop of a temperature-responsive molecule (adsorbent according to a comparative example) is applied as a column packing material for affinity chromatography, the phase of the temperature-responsive molecule The target substance L must be adsorbed in a high temperature range higher than the change temperature, and the adsorbed target substance L must be desorbed by lowering the temperature to a low temperature range lower than the phase change temperature after the washing step. Therefore, in the manufacturing system 100C including the separation and purification device 70C to which the adsorbent according to the comparative example is applied, the heating unit 80 is provided in the liquid inlet side (upstream side) pipe of the separation and purification device 70C, as shown in FIG. Prior to the separation / purification process in the separation / purification apparatus 70C, a process of raising the temperature of the liquid to be treated containing the biological material to the phase change temperature or higher is required.

  In the manufacturing system 100C, after culturing in the culture tank 50, it is necessary to preheat the whole of the large-volume culture solution stored in the storage tank 60 under low temperature conditions. It takes time. On the other hand, according to the separation and purification apparatus and the separation and purification method to which the adsorbent 1 according to the present embodiment is applied, the target substance L adsorbed on the adsorbent 1 is changed in phase with the temperature rise of the temperature response portion 20. Therefore, it is not necessary to raise the temperature of a large volume of liquid to be processed in advance. That is, in the separation and purification apparatus and the separation and purification method according to the present embodiment, the column contents (adsorbent 1 and adsorbent 1 having a smaller total heat capacity than the culture solution or the like, or a desorbed solution that is smaller than the culture solution or the like. The target substance L can be desorbed by heating any one of the cleaning liquids that are passed through the liquid. Therefore, the heating cost and the heating time are suppressed, the deterioration of the target substance in the process is reduced, the process efficiency is hardly impaired, and the separation and purification process can be appropriately performed. Furthermore, the effect of improving the treatment efficiency can be made more advantageous as the volume of the liquid to be treated and the amount of adsorbent adsorbed are larger, and as the concentration of the target substance contained in the liquid to be treated is lower.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using the Example of this invention, the technical scope of this invention is not limited to this.

  As the adsorbent according to the example of the present invention, the adsorbent according to Examples 1 to 3 is manufactured, and the correlation of the molecular weight ratio between the adsorption site 10 and the temperature response site 20 with respect to the recovery rate of the target substance L is evaluated. Went. In addition, as a control for the adsorbent according to the example of the present invention, the adsorbents according to Comparative Examples 1 to 3 were manufactured and evaluated in the same manner.

  In the produced adsorbents according to Examples 1 to 3 and the adsorbents according to Comparative Examples 1 to 3, an ε-polylysine derivative represented by the following chemical formula was used as the temperature responsive portion 20. In the formula, X is an n-butoxycarbonyl group (-nBuCo), m is an integer of 1 or more, n is an integer greater than m, and the copolymerization format is arbitrary. The lower critical solution temperature of this ε-polylysine derivative is 28 ° C. Further, as the carrier 30, a polystyrene microplate having a carboxy terminus was used.

<Example 1>
As the adsorbent according to Example 1, an adsorbent in which the adsorption site 10 is (3-thia-5-pyridylpentyl) sulfonylethylamine represented by the following chemical formula was produced as follows. This adsorption site is a functional molecule having antibody adsorption ability by thiophilic interaction.

  First, 3 equivalents of N-hydroxysuccinimide ester of valeric acid was added to the ε-polylysine derivative aqueous solution and reacted at room temperature for 5 hours. Subsequently, dialysis was performed to remove low molecules, and an ε-polylysine derivative (EPL-V80) in which 80% of the side chains were converted to valeric acid amide was obtained. In addition, (3-thia-5-pyridylpentyl) vinylsulfone was prepared by reacting divinylsulfone and 2-pyridylethanethiol hydrochloride in the presence of a base.

  Then, the obtained ε-polylysine derivative (EPL-V80) was reacted with 1 equivalent of (3-thia-5-pyridylpentyl) vinylsulfone, so that 80% of the side chain was converted to valeric acid amide, An ε-polylysine derivative (EPL-V80-S5) in which 5% of the chain was converted to (3-thia-5-pyridylpentyl) sulfonylethylamine was obtained.

  Subsequently, the PBS solution (pH 5.8) of the obtained ε-polylysine derivative (EPL-V80-S5) is reacted with the carrier 30 whose carboxy terminus is activated with water-soluble carbodiimide (WSC). Thus, an adsorbent according to Example 1 was obtained.

<Example 2>
As the adsorbent according to Example 2, an adsorbent in which the adsorption site 10 is amino (4-hydroxyphenylethylamino) (phenylamino) triazine represented by the following chemical formula was produced as follows. The adsorption site 10 is a functional molecule having an antibody adsorption ability synthesized by imitating protein A.

  First, 1 equivalent of N-hydroxysuccinimide ester of valeric acid was added to the ε-polylysine derivative aqueous solution and reacted at room temperature for 5 hours. Subsequently, dialysis was performed to remove low molecules, and an ε-polylysine derivative (EPL-V57) in which 57% of the side chains were converted to valeric acid amide was obtained.

  The obtained ε-polylysine derivative (EPL-V57) was reacted with 1 equivalent of chloro (4-hydroxyphenylethylamino) (phenylamino) triazine, and 57% of the side chain was converted to valeric acid amide. An ε-polylysine derivative (EPL-V57-T10) in which 10% of the side chain was converted to chloro (4-hydroxyphenylethylamino) (phenylamino) triazine was obtained.

  Subsequently, the PBS solution (pH 5.8) of the obtained ε-polylysine derivative (EPL-V57-T10) was reacted with the carrier 30 whose carboxy terminus was activated by water-soluble carbodiimide (WSC). Thus, an adsorbent according to Example 2 was obtained.

<Example 3>
As the adsorbent according to Example 3, an adsorbent in which the adsorption site 10 is an extracellular domain having protein A antibody binding ability was produced as follows.

  First, in the same manner as in Example 1, an ε-polylysine derivative (EPL-V80) in which 80% of the side chains were converted to valeric acid amide was obtained. Protein A was cleaved with endoprotease “Glu-C”, and the resulting digest was fractionated by gel chromatography to recover a fragment of protein A corresponding to a molecular weight of about 5000.

  Subsequently, the PBS solution (pH 5.8) of the obtained ε-polylysine derivative (EPL-V80) was reacted with the carrier 30 activated with water-soluble carbodiimide (WSC) to produce a temperature response site. A carrier 30 having 20 bound thereto was obtained.

  Then, the carrier 30 to which the temperature response site 20 was bound was activated with glutaraldehyde, and the collected protein A fragment PBS solution (pH 7.4) was reacted to obtain the adsorbent according to Example 3.

<Comparative Example 1>
As the adsorbent according to Comparative Example 1, an adsorbent in which the adsorption site 10 is an extracellular domain having protein A antibody binding ability was produced as follows.

  First, in the same manner as in Example 1, an ε-polylysine derivative (EPL-V80) in which 80% of the side chains were converted to valeric acid amide was obtained. Protein A was cleaved with endoprotease “Glu-C”, and the resulting digest was fractionated by gel chromatography to recover a protein A fragment corresponding to a molecular weight of about 24,000.

  Subsequently, the PBS solution (pH 5.8) of the obtained ε-polylysine derivative (EPL-V80) was reacted with the carrier 30 activated with water-soluble carbodiimide (WSC) to produce a temperature response site. A carrier 30 having 20 bound thereto was obtained.

  The adsorbent according to Comparative Example 1 was prepared by activating the carrier 30 to which the temperature responsive site 20 was bound with glutaraldehyde and reacting the recovered protein A fragment with a PBS solution (pH 7.4).

<Comparative example 2>
As an adsorbent according to Comparative Example 2, an adsorbent in which the adsorption site 10 is protein A was produced as follows.

  First, in the same manner as in Example 1, an ε-polylysine derivative (EPL-V80) in which 80% of the side chains were converted to valeric acid amide was obtained.

  Subsequently, the PBS solution (pH 5.8) of the obtained ε-polylysine derivative (EPL-V80) was reacted with the carrier 30 activated with water-soluble carbodiimide (WSC) to produce a temperature response site. A carrier 30 having 20 bound thereto was obtained.

  The adsorbent according to Comparative Example 2 was prepared by activating the carrier 30 to which the temperature response site 20 was bound with glutaraldehyde and reacting with a PBS solution of protein A (pH 5.8).

<Comparative Example 3>
As an adsorbent according to Comparative Example 3, an adsorbent without the adsorption site 10 was manufactured as follows.

  First, in the same manner as in Example 1, an ε-polylysine derivative (EPL-V80) in which 80% of the side chains were converted to valeric acid amide was obtained.

  Subsequently, the PBS solution (pH 5.8) of the obtained ε-polylysine derivative (EPL-V80) was reacted with the carrier 30 activated with water-soluble carbodiimide (WSC), and Comparative Example 3 It was set as the adsorbent concerning.

  Next, using the adsorbents according to Examples 1 to 3 and the adsorbents according to Comparative Examples 1 to 3, the molecular weight ratio between the adsorption site 10 and the temperature response site 20 with respect to the recovery rate of the target substance L. The correlation was evaluated. In addition, as the target substance L, biotinylated IgG was used.

  First, a PBS solution (pH 7.4) of biotinylated IgG was added to a plurality of (six) wells of the carrier 30 (polystyrene microplate) of each adsorbent and allowed to stand at 4 ° C. for 30 minutes. Biotinylated IgG was adsorbed on the adsorbent.

  Subsequently, each well of the adsorbent carrier 30 (polystyrene microplate) was washed with a PBS solution (pH 7.4) to remove unadsorbed biotinylated IgG.

  A part (three) of the wells was heated from 4 ° C. to 37 ° C. and then allowed to stand for 1 hour to desorb the adsorbed biotinylated IgG. During this time, the remaining (three) wells were stored at 4 ° C. without being heated.

  Thereafter, labeled streptavidin is added to each well from which the biotinylated IgG has been desorbed and the remaining well, and the biotinylated IgG adsorbed by the ELISA method is quantified. The recovery rate by elution of was calculated. In the adsorbents according to Examples 1 to 3 and the adsorbents according to Comparative Examples 1 to 3, the recovery rate (%), the molecular weight (kDa) of each adsorption site 10 and the temperature response site 20, and The molecular weight ratio between the adsorption site 10 and the temperature response site 20 is shown in the following table.

  FIG. 5 is a diagram showing the correlation of the molecular weight ratio between the adsorption site and the temperature response site with respect to the recovery rate of the target substance.

  In FIG. 5, the vertical axis represents the recovery rate of biotinylated IgG (elution amount / total adsorption amount), and the horizontal axis represents the molecular weight ratio between the adsorption site and the temperature response site (molecular weight of the adsorption site / molecular weight of the temperature response site). Represents. Moreover, the plot of ● is the value in the adsorbent according to Examples 1 to 3, the plot of ■ is the value in the adsorbent according to Comparative Examples 1 to 2, and the plot of Δ is the temperature-responsive molecule. The value (refer patent document 3: International Publication 2001/74482) in the conventional adsorbent (control group) which desorbs a target substance using the phase change accompanying temperature fall is shown.

  As shown by a broken line in FIG. 5, in the conventional adsorbent (control group), the recovery rate and the molecular weight ratio show a positive correlation, the molecular weight ratio increases, and the adsorption site becomes relatively larger, The recovery rate is improved. In contrast, in the adsorbents according to Examples 1 to 3 and Comparative Examples 1 to 2 in which the target substance is desorbed using the phase change accompanying the temperature rise of the temperature-responsive molecule, FIG. As indicated by the solid line, the recovery rate and the molecular weight ratio show a negative correlation, and the recovery rate of the target substance L is improved as the molecular weight ratio decreases and the adsorption site 10 becomes relatively small. I understand. In the adsorbents according to Comparative Examples 1 and 2, a sufficient recovery rate is not obtained, whereas the molecular weight ratio between the adsorption site 10 and the temperature response site 20 is small. In the adsorbent according to the present invention, a good recovery rate is realized. Further, it was confirmed that the adsorption of the target substance does not occur in the adsorbent according to Comparative Example 3 (not shown). As described above, in each adsorbent according to the embodiment of the present invention, the adsorption is based on a tendency different from that of the conventional adsorbent that desorbs the target substance by using the phase change accompanying the temperature decrease of the temperature-responsive molecule. It can be seen that a high recovery rate can be achieved by designing the molecular weight ratio between the site 10 and the temperature-responsive site 20.

  Next, the separation and purification apparatus according to the example was manufactured, and the separation and purification treatment of IgG was performed using the separation and purification apparatus.

  First, the ε-polylysine derivative (EPL-V57-T10) obtained in the same manner as in Example 2 was reacted with a surface-activated bead carrier so that the adsorption site 10 was amino (4-hydroxyphenylethylamino) (phenyl). An adsorbent was obtained which was amino) triazine, the temperature response site 20 was ε-polylysine, and the carrier 30 was a surface activated bead carrier. Next, the PBS solution (pH 7.4) of the obtained adsorbent was filled in a hollow cylindrical column having a diameter of 10 cm so as to have a height of 20 cm, and a heat insulating material was wound on the peripheral wall of the column. The liquid inlet and the liquid outlet of the column were respectively connected to a liquid feed pump and a heating device by pipes to obtain a separation and purification device according to the example.

  Subsequently, 10 L of a culture solution (treatment solution) containing 10 g of IgG (target substance L) and impurities and stored at 4 ° C. was introduced into the column at a flow rate of 200 cm / h to adsorb IgG. Adsorbed to the material.

  Next, 1.6 L of PBS solution (pH 7.4) was introduced into the column at a flow rate of 200 cm / h and washed to remove impurities.

  Then, after introducing a 37 ° C PBS solution (pH 7.4) into the column at a flow rate of 200 cm / h, the feeding is stopped, and the adsorbent is heated and adsorbed by holding it at 37 ° C for 30 minutes. The IgG that had been released was released.

  Thereafter, a 1.6 L PBS solution (pH 7.4) at 37 ° C. was again introduced into the column at a flow rate of 200 cm / h, and the desorbed IgG was recovered.

  As a result, the recovery rate by elution of the target substance L was about 80%. In this separation and purification treatment, 10 L of culture solution was used as the solution to be treated, whereas desorption of the target substance L adsorbed on the adsorption site 10 of the adsorbent was about 4 L of solution (PBS solution). This could be achieved only by heating to raise the temperature. Thus, according to the form in which the target substance L is desorbed using the phase change accompanying the temperature rise of the temperature-responsive molecule, the processing efficiency of the liquid to be processed is hardly impaired, and the heating cost and the heating time are reduced. I understand that

1 Adsorbent 1
10 Adsorption site 20 Temperature response site 30 Carrier 50 Culture tank 60 Storage tank 70 Separation and purification device 80 Heating means 100 Manufacturing system

Claims (14)

An adsorption site consisting of a functional molecule that adsorbs the target substance removably,
A temperature-responsive region composed of a temperature-responsive molecule that undergoes a phase change due to a temperature change;
A carrier that supports the adsorption site and the temperature response site;
The temperature response site has an action of desorbing the target substance adsorbed on the adsorption site by a phase change accompanying a temperature rise,
The adsorbent characterized in that the molecular weight of the adsorption site is not more than twice the molecular weight of the temperature-responsive site.   The adsorbent according to claim 1, wherein a phase change temperature of the temperature-responsive molecule is in a range of 0 ° C. or more and 60 ° C. or less.   The adsorbent according to claim 1, wherein the target substance is a biological substance, a cell, an acid-reactive substance that reacts with an acid, or an acid-decomposable substance that decomposes with an acid.   The adsorbent according to claim 3, wherein the biological substance is a protein, peptide, amino acid, saccharide, nucleic acid, lipid, or metabolite.   The adsorbent according to claim 1, wherein the adsorption site has a molecular weight of 40 or more.   The adsorption site is selected from the group consisting of 5-membered or 6-membered aromatic or heteroaromatic rings, sulfide groups, sulfonyl groups, amide groups, amino acids, nucleic acids, sugars, and cyclic compounds having a hydrophobic space. The adsorbent according to claim 1, wherein the adsorbent is a functional molecule having at least one molecular structure.   The adsorption site is a functional molecule having at least one skeleton selected from the group consisting of a benzene skeleton, a pyridine skeleton, a pyrazine skeleton, a pyrrole skeleton, a triazine skeleton, a diazole skeleton, and a triazole skeleton. The adsorbent according to claim 1.   The said adsorption site is a functional molecule consisting of a peptide, protein, polysaccharide, polyacrylamide derivative, polymethacrylic acid derivative, polyfunctional polyamide, or polyfunctional polyester. Adsorbent.   The temperature response site is a monomer, such as acrylamide derivatives, methacrylic acid derivatives such as methacrylic acid esters, acrylic acid derivatives such as acrylic acid esters, vinyl alcohol derivatives, lysine derivatives, glutamic acid derivatives, aspartic acid derivatives, lactic acid, diol derivatives. The adsorbent according to claim 1, comprising at least one selected from the group consisting of: caprolactams, caprolactones, saccharides, and siloxanes. A separation and purification device having an adsorbent and a column packed with the adsorbent,
The adsorbent is
An adsorption site consisting of a functional molecule that adsorbs the target substance removably,
A temperature-responsive region composed of a temperature-responsive molecule that undergoes a phase change due to a temperature change;
A carrier for immobilizing the adsorption site and the temperature response site,
The temperature responsive part has an action of desorbing the target substance adsorbed on the adsorption part by a phase change accompanying a temperature rise,
The separation and purification apparatus, wherein the molecular weight of the adsorption site is not more than twice the molecular weight of the temperature-responsive site.   The separation / purification apparatus according to claim 10, further comprising a heating unit that raises the temperature of the adsorbent.   The separation and purification device according to claim 10 or 11, wherein a heat insulating material is interposed in the column. A separation and purification method for separating and purifying a target substance contained in a liquid to be treated using a column packed with an adsorbent,
The adsorbent immobilizes the adsorption site composed of a functional molecule that adsorbs the target substance in a detachable manner, the temperature response site composed of a temperature-responsive molecule that changes phase according to temperature change, and the adsorption site and the temperature response site. The temperature responsive portion has a function of desorbing the target substance adsorbed on the adsorption site by a phase change accompanying a temperature rise, and the molecular weight of the adsorption site is the molecular weight of the temperature responsive site. Less than twice,
The separation and purification method includes:
An adsorption step of passing a liquid to be treated containing a target substance through the column and adsorbing the target substance to the adsorption site;
A cleaning step of cleaning the adsorbent on which the target substance is adsorbed under conditions where the target substance is not desorbed;
A desorption step of raising the temperature of the washed adsorbent and desorbing the target substance adsorbed on the adsorption site by a phase change accompanying a temperature increase of the temperature response site;
An elution step of eluting the desorbed target substance from the column. The phase change temperature of the temperature-responsive molecule is in the range of 0 ° C. or higher and 40 ° C. or lower;
The separation and purification method according to claim 13, wherein the temperature of the liquid to be treated is 0 ° C. or more and not more than the phase change temperature of the temperature-responsive molecule.

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