JP6927259B2 - Adsorbents, columns, refiners and methods for manufacturing adsorbents - Google Patents

Description

The present invention relates to an adsorbent, a column, a purification apparatus, and a method for producing an adsorbent used for purifying a target substance.

Affinity purification using biomolecules such as proteins and antibodies as target substances involves adsorbing the target substance to an affinity adsorbent (hereinafter, simply referred to as "adsorbent"), washing non-adsorbed components, and targeting with an eluent. It is carried out after the substance is recovered from the adsorbent. The adsorbent is composed of immobilizing an adsorbent site that interacts with a target substance on the surface of a carrier made of a polysaccharide, a resin, an inorganic oxide, or the like. In particular, an adsorbent using a low molecular weight compound (low molecular weight compound) that specifically adsorbs to an antibody as an adsorbent site has a lower cost than the adsorbent using a protein such as protein A. It is attracting widespread attention because it has high chemical resistance and can reduce the deterioration of the adsorption site in processes such as strong alkaline cleaning.

Such adsorbents are described, for example, in Patent Document 1 and Non-Patent Documents 1 to 3.
Specifically, Patent Document 1 describes a sud bioaffinity chromatography adsorbent (corresponding to an adsorbent) used for selectively adsorbing immunoglobulin, (a) a solid support material, and (b) this solid. A sud bioaffinity chromatography adsorbent comprising a ligand (corresponding to an adsorption site) immobilized on the surface of a support material, wherein the ligand is a compound having a mercapto five-membered heterocycle having a predetermined chemical structure. Have been described.

Further, Non-Patent Documents 1 to 3 report on antibody purification by chromatography using a thiophilic ligand as an adsorbent.

Special Table No. 10-500615

E. Boschetti et al., J. Biochem. Biophys. Methods, 2001, 49, pp.361-389. A. Schwarz et al., React. Polym., 1995, 22, pp.259-266. P. Kumplume et al., J. Chromato. A, 2004, 1022, pp.41-50.

However, the function of an adsorbent using a small molecule compound as a ligand as described in Patent Document 1 and Non-Patent Documents 1 to 3 is greatly influenced by the linker molecule connecting the carrier and the small molecule compound. NS. Therefore, the small molecule compound has a problem that the carriers that can be used are limited. Further, an adsorbent using a small molecule compound as a ligand is greatly affected by the interfacial properties of the carrier, so that it is difficult to control the orientation of the ligand, and it is difficult to increase the adsorption capacity of a target substance such as an antibody. There is. In addition, in this specification, the interfacial property of a carrier means the surface property such as hydrophilicity and hydrophobicity.

The present invention has been made in view of such a situation, and a small molecule compound can be used as an adsorption site, more types of carriers can be used than before, and the adsorption capacity of the target substance can be used. It is an object of the present invention to provide an adsorbent, a column, a purification device, and a method for producing an adsorbent having a high content.

The present inventors have diligently researched and developed to solve the above problems, and found that the hydrophilicity and hydrophobicity of the entire stimulus-responsive polymer are hydrophilic and hydrophobic by controlling the amount of the adsorption site (low molecular weight compound) introduced into one polymer chain. We have found that the adsorption capacity of the target substance can be increased by immobilizing this on a carrier that satisfies a predetermined index while controlling the sexual balance, and have completed the present invention. That is, the present invention is an adsorption capable of adsorbing an extremely large amount of a target substance from a solution containing the target substance by combining a specific condition range of a stimulus-responsive polymer with a carrier having various interfacial properties. The material was found and the present invention was completed.

The adsorbent according to the present invention that solves the above-mentioned problems includes a carrier, an adsorption site for adsorbing a target substance, and an amino group into which the adsorption site is introduced , and a stimulus-responsive polymer immobilized on the carrier. When the carrier is dispersed in octanol and ethanol having different dielectric constants, the index A calculated from the evaluation value indicating the dispersibility of the carrier in each solvent is 0 or more and 1.0. or less, before Symbol stimuli-responsive polymer is partition coefficient CLogP is Ri der -0.46 to 1.3, the index a is to disperse the carrier to each of the solvent of the octanol and the ethanol The prepared dispersion was developed on a carrier and allowed to stand at room temperature for 10 minutes, and then subjected to a binarization treatment in which the aggregate portion in the image obtained by imaging with an optical microscope was treated to black and the others to white. The value obtained by dividing the length of the aggregate portion obtained in black by the diameter of one of the carriers is defined as φ, and the evaluation value 5: φ = 0.2 μm or more and less than 8.3 μm, the evaluation value 4 according to the value of φ. : Φ = 8.3 μm or more and less than 17 μm, evaluation value 3: φ = 17 μm or more and less than 27 μm, evaluation value 2: φ = 27 μm or more and less than 40 μm, evaluation value 1: φ = 40 μm or more, and this is evaluated per sample. The average value of the values obtained from the five images is adopted, and further, the average value obtained by repeating this twice for one sample when dispersed in the octanol. The index value is a value obtained by subtracting the evaluation value when dispersed in ethanol from the evaluation value, the carrier is formed of a synthetic resin, and the adsorption site is benzothiazole-2-acetic acid. , 4-Mercaptobenzothiazole-2-ethyl acetate, 5-Mercaptobenzothiazole-2-ethylacetate, 2-Mercaptothiazole, 3- (benzothiazole-2-ylthio) sodium propan-1-sulfonate, 2-mercapto- 6-Nitrobenzothiazole, 3- (2-benzothiazolylthio) propionic acid, 2-mercapto-1,3,4-thiasiazol, N-methyl-2-mercaptoimidazole, pyridylthioacetic acid, 2-benzothiazolylthioacetic acid It is assumed that at least one selected from the above is amide-bonded to the amino group. The stimulus-responsive polymer refers to a structure in which an adsorption site is introduced into a polymer chain.

According to the present invention, a small molecule compound can be used as an adsorption site, more types of carriers can be used than before, and an adsorbent, a column, a purification device and a purification device having a high adsorption capacity of a target substance can be used. A method for producing an adsorbent can be provided.
Issues, configurations and effects other than those described above will be clarified by the following description of the embodiments.

It is the schematic explaining the structure of the adsorbent which concerns on this embodiment. It is the schematic explaining the structure of the column which concerns on this embodiment. It is the schematic explaining the structure of the purification apparatus which concerns on this embodiment. It is a graph which shows the relationship between the index A and 10% DBC in each adsorbent which concerns on Examples 1-9 and Comparative Examples 1-3. The horizontal axis represents index A, and the vertical axis represents 10% DBC (mg / mL). The DBC refers to a dynamic adsorption capacity. In the figure, Examples are plotted with “■” and Comparative Examples are plotted with “○”.

Hereinafter, an embodiment of an adsorbent, a column, a purification apparatus, and a method for producing an adsorbent according to the present invention will be described in detail with reference to the drawings as appropriate.
In addition, "~" described in this specification is used in the meaning of having numerical values described before and after it as a lower limit value and an upper limit value. In the numerical range described stepwise in the present specification, the lower limit value or the upper limit value described in one numerical range may be replaced with the lower limit value or the upper limit value described in another stepwise. The lower or upper limit of the numerical range described herein may be replaced with the values shown in the examples.

For each element described in the present specification, a material selected from the material group constituting each element may be used alone or in combination of two or more. In addition, each element or material described in the present specification may be composed only of the material described in the present specification, and is not described in the present specification to the extent that the effect of the present invention is not impaired. You may have the material of.

<Adsorbent 1>
FIG. 1 is a schematic view illustrating the configuration of the adsorbent 1 according to the present embodiment.
As shown in FIG. 1, the adsorbent 1 includes a carrier 2 and an adsorption site 3 that adsorbs the target substance O, and also contains a stimulus-responsive polymer 4 immobilized on the carrier 2. The stimulus-responsive polymer 4 includes a polymer chain 5, and a plurality of adsorption sites 3 are introduced into the polymer chain 5 and the balance between hydrophilicity and hydrophobicity of the entire stimulus-responsive polymer 4 is controlled. The functional group R has been introduced.

Examples of the target substance O include physiologically active substances and industrially useful proteins. Examples of the physiologically active substance include antibodies (antibody molecules / antibody proteins). The antibody in the present embodiment refers to gamma globulin and immunoglobulin, and in particular, includes either an antibody that can be administered to humans as a pharmaceutical product or an antibody that is not administered to humans as introduced into an analytical reagent. Is done. Antibodies applicable as pharmaceuticals are not limited to classes or subclasses. For example, there are five types of IgG, IgM, IgA, IgD, and IgE having different constant region structures, and any of the immunoglobulins may be used. Human antibodies have four subclasses of IgG1 to IgG4, and IgA also has two subclasses of IgA1 and IgA2, but the present embodiment is not particularly limited. Further, the antibody can be classified according to the origin and the production method, and may be either a natural human antibody, a recombinant human antibody produced by a gene recombination technique, or a monoclonal antibody or a polyclonal antibody. In particular, among these antibodies, application to human IgG, which is the most important as a pharmaceutical raw material, is meaningful. Further, the antibody may be a humanized antibody, a chimera with human IgG, or the like. The humanized antibody refers to a variable region in which the complementarity determining region is derived from an organism other than human and the other framework regions are derived from human. The chimera with human IgG means that the variable region is derived from a non-human organism such as a mouse, but the other constant region is replaced with a human-derived immunoglobulin. The bioactive substance is not limited to the antibody, and includes any substance alone or a group of compounds that exerts a physiological action or a pharmacological action on an organism. Examples of substances and compound groups that exert physiological and pharmacological actions on living organisms include alkaloids, cytokines, plant hormones, neurotransmitters, pheromones, hormones (animal hormones), growth factors, and growth regulators. Factors, growth inhibitory factors and the like can be mentioned. Examples of simple substances and compound groups that exert physiological and pharmacological actions on living organisms include biopharmacy such as tissue plasminogen activator and insulin used as thrombolytic agents. Examples of industrially useful proteins include oxidoreductases, transferases, hydrolases, depleting and adding enzymes, isomerases, and synthases. Further, the target substance O includes, for example, carotenoids such as β-carotene and astaxanthin, pigments such as chlorophyll and bacteriochlorophyll, phycobilin proteins such as phycocyanin used for coloring foods or cosmetics, and physiologically active substances such as fatty acids. Can be mentioned.

For antibodies such as pharmaceuticals, for example, a production step of producing an antibody as a raw material by cell culture, a purification step of selectively recovering the antibody from a plurality of impurities contained in the culture to increase the purity, and removing the virus. It is produced through a virus removal step, a concentration / liquid exchange step of concentrating a solution containing a virus-removed antibody, exchanging a buffer solution, a formulation step of formulating into an appropriate drug form, and the like. The flow is known as a general process and is not limited thereto.

In the above steps, the adsorbent 1 according to the present embodiment is particularly used in the purification step. The role of the adsorbent 1 is to separate the antibody and impurities contained in the solution and purify and recover the antibody. Examples of the purification step include, but are not limited to, initial purification using affinity chromatography, and then medium-term and late-stage purification steps using an ion exchanger. In the following description, the solution immediately before being put into the purification step (initial step) using the adsorbent 1 according to the present embodiment may be referred to as an antibody-containing solution.

The description of the adsorbent 1 will be continued.
The carrier 2 may be either porous or non-porous. Examples of the mode of the carrier 2 include a plate shape, a bead (particle) shape, a fibrous shape such as a non-woven fabric or a woven fabric, a film shape, a monolith shape, a hollow thread shape, and the like. Further, the carrier 2 may have a core-shell structure.

The carrier 2 may be formed of, for example, a material containing a polysaccharide, a synthetic resin, an inorganic compound, and a composite material thereof. The polysaccharide may be a crosslinked polysaccharide. Examples of the polysaccharide or the crosslinked polysaccharide include agarose, crosslinked agarose, hydrophobized agarose, cellulose and the like. Examples of the synthetic resin include acrylic resins (for example, polyalkyl acrylate, polyalkyl methacrylate, polyglycidyl acrylate, polyglycidyl methacrylate, etc.), polystyrene, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, polysiloxane, polyfluorinated ethylene, and the like. Be done. Examples of the inorganic compound include silica, metal oxides (for example, alumina, titania, zirconia, iron oxide, etc.), ferrite, hydroxyapatite, silicate and the like. The carrier 2 preferably has a polysaccharide, a crosslinked polysaccharide, a synthetic resin, silica or a metal oxide, and more preferably has a polysaccharide or a crosslinked polysaccharide. The carrier 2 may have these materials at least on the surface of the carrier 2. That is, the surface of the carrier 2 may have a core-shell structure having a polysaccharide, a crosslinked polysaccharide, a synthetic resin, silica or a metal oxide, preferably a polysaccharide or a crosslinked polysaccharide. Therefore, it is preferable that the surface of the carrier 2 or the carrier 2 has a polysaccharide, a crosslinked polysaccharide, a synthetic resin, silica or a metal oxide, and more preferably a polysaccharide or a crosslinked polysaccharide.

Further, the surface of the carrier 2 may be modified with a reactive group such as a carboxyl group, an amino group, a hydroxyl group, an epoxy group or an ester group. The surface of the carrier 2 is preferably modified with a carboxyl group from the viewpoint that a larger amount of the stimulus-responsive polymer 4 can be introduced, that is, a larger amount of the adsorption site 3 can be introduced.

The average particle size of the carrier 2 is preferably 3 μm or more and 100 μm or less. In this way, the surface area of the carrier 2 is sufficiently obtained, so that the dynamic adsorption capacity (DBC) is further increased. The DBC is an index showing how much the target substance O is adsorbed on the adsorbent 1 (bonding capacity of the adsorbent 1) while the target substance O is flowing, and the amount of adsorption per unit volume. It can be expressed as (for example, "mg / mL").

The average particle size can be measured, for example, by a method according to JIS Z 8815. Further, the average particle size can be measured by, for example, a vibration type sieving device, a laser diffraction type particle size distribution measuring device, or a photon correlation method particle size distribution measuring device. For example, when the carrier is polystyrene, it can be measured by a vibration type sieving device, when it is Sepharose (registered trademark), it can be measured by a laser diffraction type particle size distribution measurement, and when it is a magnetic fine particle, it can be measured by a photon correlation method particle size distribution measuring device. .. Further, those having an average particle size of 40 μm to several mm can be measured by a vibrating sieving device, and those having an average particle size of 100 to 1000 μm can be measured by a laser diffraction type particle size distribution measuring device.

The adsorption site 3 in the present embodiment is a site derived from a small molecule compound. The adsorption site 3 corresponds to, for example, a ligand when the target substance O is an antibody and the column for purifying the antibody is an affinity column.

The adsorption site 3 is introduced into the side chain of the polymer chain 5 via an arbitrary chemical bond such as an amide bond. The compound used for introducing the adsorption site 3 is not particularly limited as long as it has a portion capable of binding to the functional group R (for example, an amino group) of the polymer chain 5. Examples of such a compound include a compound having a carboxyl group, an azlactone group, an imidazole group, a cyano ester group, an epoxy group, an acetyl group, an acetoxy group, an acryloyl group, and an alkoxy group for forming an amide bond. Examples of such a compound include a carboxylic acid anhydride, a carboxylic acid halide, a compound having an isocyanate group for forming a urea bond, a compound having an isothiocyanate group for forming a thiourea bond, and a sulfone. Examples thereof include a compound having a sulfonyl chloride group for forming an amide bond, a compound having an oxazoline group for forming an amide ester bond, and the like.

Specific examples of the compound used for introducing the adsorption site 3 include mercaptoimidazole, mercaptobenzothiazole, mercaptotetrazole, and mercaptothiadiazole. More specifically, benzothiazole-2-acetic acid, 4-mercaptobenzothiazole-2-ethyl acetate, 5-mercaptobenzothiazole-2-ethylacetate, 2-mercaptothiazole, 3- (benzothiazole-2-ylthio) Sodium propan-1-sulfonate, 2-mercapto-6-nitrobenzothiazole, 3- (2-benzothiazolylthio) propionic acid, 2-mercapto-1,3,4-thiazylazole, N-methyl-2-mercapto Examples thereof include imidazole, pyridylthioacetic acid (for example, 2-pyridylthioacetic acid, 4-pyridylthioacetic acid), 2-benzothiazolylthioacetic acid and the like.

The amount of the adsorption site 3 introduced in the stimulus-responsive polymer 4 is sufficient if it is 5% or more of the number of functional groups contained in the polymer chain 5 having a functional group R such as a plurality of amino groups, and is maximum. About 95% can be introduced. From the viewpoint of the high antibody adsorption characteristics of the adsorbent 1, it is preferably 7% or more. When the monomer is polymerized to form the polymer chain 5, if the functional group R into which the adsorption site 3 of the monomer can be introduced is known, the number of functional groups in the polymer chain 5 can also be increased. It can be easily grasped. Therefore, for example, when one molecule of the polymer chain 5 has 30 functional groups R into which the adsorption site 3 can be introduced, the amount of the adsorption site 3 introduced is 5% to 95% (the number of functional groups). 1.5 to 28.5). That is, in the present embodiment, it is preferable that the stimulus-responsive polymer 4 has a plurality of adsorption sites 3 and a plurality of functional groups R. In this way, the balance between hydrophilicity and hydrophobicity of the entire stimulus-responsive polymer 4 can be more easily controlled, and the DBC can be further increased.

In the present embodiment, the amount of the adsorbed site 3 introduced into the adsorbent 1 is determined by ¹ H-NMR for determining the introduction rate of the adsorbed site 3 introduced into the stimulus-responsive polymer 4 per molecule, and then the carrier 2 It is determined by identifying the amount of stimulus-responsive polymer 4 immobilized on top. That is, the introduction amount of the adsorption site 3 in the adsorbent 1 is the adsorption site introduction rate (amount) per molecule and the adsorption amount of the stimulus-responsive polymer 4 immobilized on the carrier 2 on the carrier 2. It is determined by the product of the amount of the stimulus-responsive polymer 4.

The amount of the stimulus-responsive polymer 4 immobilized on the carrier 2 is identified as follows. First, a solution in which a condensing agent and a stimulus-responsive polymer 4 are dissolved is brought into contact with the carrier 2 to carry out an immobilization reaction. Then, the solution is washed with a solvent a plurality of times, and the solution containing the adsorption site 3 in the recovered supernatant solution is analyzed by high-speed liquid chromatography. The amount of the stimulus-responsive polymer 4 immobilized on the carrier 2 can be estimated by subtracting the amount of the adsorption site 3 eluted during washing from the stimulus-responsive polymer 4 at the time of charging.

Further, the stimulus-responsive polymer 4 includes a polymer chain 5 having a plurality of functional groups R and a compound for introducing the adsorption site 3, and a polymer chain 5: a compound for introducing the adsorption site 3. It is obtained by reacting with a molar ratio of 1: 0.01 to 10, preferably a molar ratio of 1: 0.1 to 5, and more preferably a molar ratio of 1: 0.1 to 2.

As the functional group R, one having a positive charge or one having a negative charge in an arbitrary aqueous solution or a buffer solution can be appropriately selected and used. The valence of the electric charge carried by the functional group R may be 1 to 3. That is, since the stimulus-responsive polymer 4 having such a functional group R is an ionic polymer, the target substance O can be recovered without using a strong acid or the like.

The functional group R is not particularly limited as long as it can control the hydrophilicity / hydrophobic balance of the entire stimulus-responsive polymer 4. Therefore, the functional group R includes, for example, an amino group, an imino group, various nitrogen-containing aromatic groups (pyrrole group, imidazolyl group, pyridyl group, pyrimidyl group, oxazolyl group, thiazolyl group, triazolyl group, etc.), guanidyl group, phenol. Sexual hydroxyl group, hydroxy group, aldehyde group, carbonyl group, carboxyl group, azo group, nitro group, nitroso group, thiol group, sulfo group, ether group, boronic acid group, fluoro group, chloro group, bromo group, iodo group, phosphoryl Examples thereof include a group, a phosphinyl group, a silicate group, a phosphate group and a group of a derivative thereof. As the functional group R, only one of these may be used, or a plurality of types may be used in combination. As the functional group R, an amino group can be preferably used among these. That is, a polyamine having a plurality of amino groups can be preferably used for the polymer chain 5.

Further, the functional group R may be one capable of forming a hydrogen bond with the target substance O. In this way, the selectivity of the target substance O can be improved, so that a high separation ability can be obtained. Examples of such a functional group R include a sulfo group, a carboxyl group, a hydroxyl group, an amino group and the like.

The polymer chain 5 may be a dendrimer. Examples of the polymer chain 5 include polylysine (α-polylysine, ε-polylysine), polyethyleneimine (linear polyethyleneimine, branched polyethyleneimine), polyallylamine, polyvinylamine, and derivatives and copolymers having them as a partial structure. However, ε-polylysine, polyethyleneimine, polyallysine or those containing them as a partial structure are preferable, and ε-polylysine is more preferable from the viewpoint that more adsorption sites 3 can be introduced.

The weight average molecular weight of the polymer chain 5 is preferably 1000 to 100,000, more preferably 1000 to 6000. The weight average molecular weight of the polymer chain 5 in the present embodiment means that measured by gel permeation chromatography.

As the combination of the polymer chain 5 and the adsorption site 3, each combination of ε-polylysine, polyethyleneimine or polyallylamine and the above-mentioned adsorption site 3 is preferable from the viewpoint of the high target substance adsorption characteristics of the adsorbent 1.

The adsorption site 3 may be introduced into the polymer chain 5 either before or after immobilizing the polymer chain 5 on the carrier 2. That is, the adsorption site 3 may be introduced into the polymer chain 5, and then the polymer chain 5 into which the adsorption site 3 has been introduced may be immobilized on the carrier 2, and the polymer chain 5 having no adsorption site 3 may be used as the carrier. After immobilization to 2, the adsorption site 3 may be introduced into the immobilized polymer chain 5.

Next, the conditions of the partition coefficient CLogP of the stimulus-responsive polymer 4 in which the increase in DBC is observed will be described. In the present embodiment, the partition coefficient CLogP is one of the predicted values of LogP and can be calculated by a computer. The partition coefficient CRogP can be calculated by using software having a function capable of calculating this. Examples of such software include chemical structural formula drawing software. And, as such chemical structural formula drawing software, ChemDraw is mentioned, for example. Further, the LogP described above refers to an index of a dimensionless number that defines the hydrophobicity and lipophilicity of the compound. LogP is an experimental value, and for example, a partition coefficient using n-octanol and water can be used.

In the present embodiment, as described above, the stimulus-responsive polymer 4 having the small molecule compound as the adsorption site 3 is used. In this embodiment, since the DBC is increased by using such a stimulus-responsive polymer 4, the partition coefficient CLogP indicating the degree of hydrophobicity of the stimulus-responsive polymer 4 is −0.46 or more and 1.3 or less. It takes that. In this way, when the stimulus-responsive polymer 4 having the partition coefficient CLogP within this numerical range is immobilized on the carrier 2, the stimulus-responsive polymer 4 satisfying the index A described later is more optimally high in stimulus responsiveness. The combination with the molecule 4 can be performed, and the DBC is greatly improved.

For the partition coefficient CLogP of the stimulus-responsive polymer 4 in the present embodiment, the respective partition coefficients CLogP are obtained from the chemical structural formulas of the polymer chain 5 and the adsorption site 3, and the amount of the adsorption site introduced per molecule of the polymer chain 5 is obtained. Is changed, and the average value is defined as the partition coefficient CLogP of the stimulus-responsive polymer 4. The larger the value of the partition coefficient CLogP, the more hydrophobic the stimulus-responsive polymer 4 shows, and the smaller the partition coefficient CRogP, the more hydrophilic the property.

Next, the conditions for the carrier 2 in which the increase in DBC is observed will be described. In this embodiment, a stimulus-responsive polymer 4 having a small molecule compound as an adsorption site 3 is used. Then, in this embodiment, in order to increase the DBC by using such a stimulus-responsive polymer 4, it is necessary that the index A described later, which indicates the dispersibility of the carrier 2 with respect to the solvent, is 1.0 or less. As described above, the combination of the carrier 2 having the index A of 1.0 or less and the stimulus-responsive polymer 4 having the partition coefficient CLogP of −0.46 or more and 1.3 or less significantly improves DBC. .. The calculation method of the index A is specifically described below.

First, ethanol (dielectric constant ε: 24.3 farads per meter (F / m)) and octanol (dielectric constant ε: 10.3 F / m) are prepared as organic solvent phases having different dielectric constants. The carrier 2 is dispersed in each solvent, and the mixture is stirred so that the carrier 2 and the solvent are mixed. At this time, care should be taken so that the carrier 2 is sufficiently dispersed in the solvent. Then, 3 mL of the dispersion liquid of various carriers 2 is developed on a glass petri dish (major axis = 35 mm) and allowed to stand at room temperature for 10 minutes. Then, focus on various carriers 2 with a lens having a magnification of 20 times with an optical microscope, and image at 5 points per sample. The aggregated portion of the carrier 2 observed in the field of view of the obtained image (20 mm × 27 mm) is binarized with image software (ImageJ). The aggregate portion of the image is treated black and the others are treated white, and the length of the aggregate portion obtained in black, particularly the length of the major axis, is the carrier diameter (diameter) of one carrier 2. The divided value is defined as φ. Then, according to the value of φ, the following evaluation values 1 to 5 are classified in 5 stages. However, in this embodiment, it is assumed that the carrier 2 has a carrier diameter of 3 to 200 μm. For the value of φ, the average value of the values obtained from the images taken at five points per sample is adopted. Further, the above experiment is repeated twice for one sample, and the average value of the two evaluation values obtained for each time is used as the evaluation value of the carrier 2. That is, a value such as 0.5 means an intermediate evaluation value, and for example, 3.5 is positioned as an intermediate evaluation value between the evaluation value 3 and the evaluation value 4.

Evaluation value 5: φ = 0.2 μm or more and less than 8.3 μm (Findings: Carrier 2 is stably and well dispersed in the solvent).
Evaluation value 4: φ = 8.3 μm or more and less than 17 μm (Findings: By mixing the carrier 2 well, a part of it is dispersed in the solvent, and both small aggregates and isolated particles are mixed).
Evaluation value 3: φ = 17 μm or more and less than 27 μm (Findings: A part of the carrier 2 is dispersed with respect to the solvent to form small aggregates).
Evaluation value 2: φ = 27 μm or more and less than 40 μm (Findings: Carrier 2 is hardly dispersed in the solvent, and large aggregates are formed.)
Evaluation value 1: φ = 40 μm or more (Findings: Carrier 2 is not dispersed in the solvent, forming large aggregates.)

Then, the dispersibility of the carrier 2 when dispersed in the solvent according to the above criteria is evaluated as follows. As a result of the examination, the value obtained by subtracting the evaluation value when dispersed in ethanol from the evaluation value when dispersed in octanol is optimal as the value indicating the properties of the carrier 2 (index A described above). I understood. _{Therefore, the evaluation value (evaluation value ε-octanol} ) obtained by dispersing the carrier 2 in octanol (dielectric constant ε: 10.3 F / m) and ethanol (dielectric constant ε: 24.3 F / m) having different dielectric constant values. from to as evaluation value ε _ethanol), according to the following equation (1), defines the index a.
A = Evaluation value ε- _octanol -Evaluation value ε _Ethanol (1)

A stimulus-responsive polymer 4 having a partition coefficient of −0.46 or more and 1.3 or less is immobilized on a carrier 2 having an index A of 1.0 or less to produce an adsorbent 1. Then, as one embodiment, when the antibody which is the target substance O is purified, it is carried out using the column 20 (see FIG. 2) packed with the adsorbent 1. Specifically, the antibody-containing solution is passed through the adsorbent 1 packed inside the column 20, and if necessary, the non-specifically adsorbed component is washed, and then the adsorbent is used with an eluent. Purification can be performed by recovering the antibody adsorbed on 1. The antibody-containing solution to be passed is preferably having a pH in the range of 7 to 8. In the present embodiment, the index A indicating the dispersibility of the stimulus-responsive polymer 4 is controlled to be 1.0 or less, so that the hydrophilicity / hydrophobic balance of the entire stimulus-responsive polymer 4 is appropriately balanced. The adsorption site 3 is arranged so as to be separated from the surface of the carrier 2. Therefore, the target substance O is easily adsorbed on the adsorption site 3, and the DBC is improved. Therefore, when the adsorbent 1 is used, the productivity can be increased in an actual drug manufacturing process, an analytical reagent manufacturing process, or the like.

Next, the presumed adsorption / desorption mechanism of the target substance O for the adsorbent 1 according to the present embodiment will be described using an antibody as an example.
In the state where the stimulus-responsive polymer 4 has a large charge, the functional group R is in a more charged state in water, and the polymer chain 5 of the stimulus-responsive polymer 4 is caused by the charge repulsion between the charged sites. Is in a relatively elongated state. At this time, when the adsorption site 3 introduced into the stimulus-responsive polymer 4 is hydrophobic, the interaction between the hydrophilic stimulus-responsive polymer 4 and the hydrophobic adsorption site 3 is relatively weak, so that the adsorption site 3 is adsorbed. Site 3 adsorbs the antibody (target substance O) (see FIG. 1). Then, when the charge of the stimulus-responsive polymer 4 decreases due to a change in the external environment such as when an eluent is passed through the solution, the hydrophobicity of the polymer chain 5 increases. Therefore, when the adsorption site 3 is hydrophobic, the equilibrium shifts in the direction in which the interaction between the stimulus-responsive polymer 4 and the adsorption site 3 becomes stronger. As a result, the adsorption force at which the adsorption site 3 adsorbs the antibody is weakened, and the antibody is desorbed from the adsorption site 3. Under certain conditions, when the charge of the stimulus-responsive polymer 4 is small, the polymer chain 5 of the stimulus-responsive polymer 4 is in a relatively contracted state because the charge repulsion is small.

Changes in the external environment are not particularly limited as long as they can change the charge density of the stimulus-responsive polymer 4 and the interaction between the adsorption site 3 and the antibody. Changes in the external environment include, for example, changes in the salt concentration, dielectric constant, pH, temperature, and the like of the solution with which the adsorbent 1 is in contact. Changes in the external environment include, for example, changes in the electrostatic shielding effect when the salt concentration changes, changes in the permittivity due to changes in the type of solvent and solute, and equilibrium movement of charges when the pH changes. The charge density changes. The change in the external environment is preferably a change in the salt concentration, temperature, dielectric constant, and pH of the solution with which the adsorbent 1 is in contact, and a change in the salt concentration and a change in pH are more preferable. These changes in the external environment may be combined. Due to changes in the external environment, the charge density of the stimulus-responsive polymer 4 and the interaction between the adsorption site 3 and the antibody change, and the antibody is adsorbed or desorbed. Therefore, the adsorbent 1 according to the present embodiment is an adsorbent 1 that is responsive to changes in the external environment. For example, when the change in the external environment is a change in pH, the adsorbent 1 is a pH-responsive adsorbent. When the material is changed in the external environment such as salt concentration, dielectric constant, and temperature, it becomes a salt concentration-responsive adsorbent, a dielectric-responsive adsorbent, and a temperature-responsive adsorbent, respectively.

The conformational change caused by the change in the charge density of the stimulus-responsive polymer 4 due to the change in the external environment can change the strength of the interaction between the antibody and the adsorption site 3 to change the adsorption characteristics. It may be a change.
In particular, when the pH is raised from near neutral pH as an external stimulus, it is presumed that the conformation of the stimulus-responsive polymer 4 on the carrier 2 takes a gradually contracted form from an elongated form. This conformational change of the stimulus-responsive polymer 4 helps to physically desorb the antibody from the adsorbent 1. Further, as the pH changes, the charges of the stimulus-responsive polymer 4 and the antibody change. Therefore, it is considered that the antibody adsorbed on the stimulus-responsive polymer 4 due to the effect of electric charge is also desorbed from the stimulus-responsive polymer 4, that is, the adsorbent 1 by charge repulsion or charge neutralization. When the target substance O is other than an antibody, it is considered that it is adsorbed and desorbed by the same mechanism.

From the above, the amount of charge charged can be controlled by changing the amount of adsorption site introduced in the stimulus-responsive polymer 4, that is, the amount of functional groups, and the hydrophilicity and hydrophobicity of the stimulus-responsive polymer 4 can be controlled. can do. As described above, the index indicating the hydrophilicity / hydrophobicity of the stimulus-responsive polymer 4 is represented by the partition coefficient CLogP.

Generally, when the index A indicating the dispersity of the carrier 2 is high, an interaction occurs between the interface of the target substance O and the adsorption site 3, and the adsorption of the antibody is inhibited, so that the adsorption ability is significantly increased. It is known to reduce. However, the stimulus-responsive polymer 4 in the present embodiment applies the partition coefficient CLogP of the stimulus-responsive polymer 4 in the optimum range even when the interfacial property of the carrier 2, that is, the index A indicating the degree of dispersion is high. Therefore, the antibody can be adsorbed with high efficiency.

Specifically, when the index A of the carrier 2 is high, for example, when the index A of the carrier 2 is 0.5, the stimulus-responsive polymer 4 having a low partition coefficient CLogP of about 0.19 is combined. Therefore, the DBC of the antibody can be set to about 30 mg / mL. This is because the amount of functional groups in the stimulus-responsive polymer 4 is large, so that the charge repels between the molecules, and the adsorption site 3 is not on the interface of the carrier 2 but on the solution interface side due to the interaction beyond the influence of the interface of the carrier 2. Is obtained by exposing. As a result, the adsorption ability of the antibody is improved. Further, the surface properties of the carrier 2 are improved by immobilizing the stimulus-responsive polymer 4 having a large amount of functional groups such as amino groups on the carrier 2 having a high index A of the carrier 2, that is, having a hydrophobic interfacial property. It can be improved and the adsorption capacity of the antibody is improved.

On the other hand, when the index A of the carrier 2 is low, that is, when the carrier 2 is hydrophilic, the interaction between the interface of the target substance O and the adsorption site 3 does not occur or is unlikely to occur. Therefore, the stimulus-responsive polymer 4 can be selected and combined with a higher degree of freedom as compared with the case where the index A of the carrier 2 is high. For example, when the index A of the carrier 2 is -2.5 to -3.5, any stimulus-responsive polymer 4 having a partition coefficient CLogP of 1.3 or less can be used.

As another usage mode, the adsorbent 1 can be used in a suspension state. In the conventional temperature-responsive adsorbent that utilizes a change in solubility, the hydrophobicity increases and the dispersibility decreases due to the temperature change. Then, there is a high risk that the adsorbent in the suspended state aggregates, adheres to the wall surface, and is localized at the interface. However, even in such a usage mode, the adsorbent 1 according to the present embodiment does not utilize the change in solubility due to the temperature change for the adsorption or desorption of the antibody, so that there is almost no such risk. ..

<Column and purification equipment>
FIG. 2 is a schematic view illustrating the configuration of the column 20 according to the present embodiment. FIG. 3 is a schematic view illustrating the configuration of the purification apparatus 30 according to the present embodiment. In the description of the column 20 and the purification apparatus 30, the description of the elements already described in detail with respect to the adsorbent 1 will be omitted.

As shown in FIG. 2, the column 20 is formed by filling the bottomed tubular body 21 with the adsorbent 1. A discharge port (not shown) is provided at the bottom of the tubular body 21, and various solutions that have passed through the column 20 are discharged from this discharge port. The column 20 is a device for selectively recovering a target substance O such as an antibody from a plurality of impurities contained during culturing to increase the purity.

Further, as shown in FIG. 3, the purification apparatus 30 includes a column 20 filled with the adsorbent 1. For example, the purification apparatus 30 includes a culture tank 31, a column 20, and a recovery container 32, as shown in FIG.
The culture tank 31 is a device for producing the target substance O by cell culture. The recovery container 32 is a container for collecting the solution containing the target substance O purified in the column 20. Known culture tanks 31 and recovery containers 32 can be used.
In addition to these, the purification device 30 is provided with various devices necessary for purification according to the target substance O, but the description of these devices will be omitted.

<Manufacturing method of adsorbent>
The adsorbent 1 according to the present embodiment can be produced by immobilizing the stimulus-responsive polymer 4 on the carrier 2. The stimulus-responsive polymer 4 is preferably bound to the carrier 2 via a chemical bond. That is, in one embodiment, in the adsorbent 1, the stimulus-responsive polymer 4 and the carrier 2 are immobilized via chemical bonds. The adsorption site 3 may be introduced at any time before or after the polymer chain 5 having a plurality of functional groups R is immobilized on the carrier 2. That is, after introducing the adsorption site 3 into the polymer chain 5 having a plurality of functional groups R, the polymer chain 5 into which the adsorption site 3 is introduced may be immobilized on the carrier 2. Further, the adsorption site 3 may be introduced into the polymer chain 5 after the polymer chain 5 having a plurality of functional groups R having no adsorption site 3 is immobilized on the carrier 2. Further, a hydrophilic or hydrophobic substituent may be further introduced into the functional group R remaining in the polymer chain 5 after the adsorption site 3 is introduced.

The adsorbent 1, the column 20 and the purification apparatus 30 according to the present embodiment described above are evaluated to show the dispersibility of the carrier 2 in each solvent when the carrier 2 is dispersed in octanol and ethanol having different dielectric constants. The index A calculated from the value is 1.0 or less, and the stimulus-responsive polymer 4 has a partition coefficient CLogP of −0.46 or more and 1.3 or less. Since the adsorbent 1, the column 20, and the purification device 30 have such a configuration, the hydrophilicity and hydrophobicity of the entire stimulus-responsive polymer 4 to the carrier 2 having various interfacial properties such as hydrophilicity and hydrophobicity. It is possible to immobilize the adsorption site 3 while maintaining a high adsorption capacity while controlling the balance between the two. Therefore, the adsorbent 1, the column 20, and the purification apparatus 30 can use a small molecule compound as the adsorption site 3, can use more types of carriers 2 than before, and have an adsorption capacity of the target substance O. Can be raised. As a result, by using the adsorbent 1, the column 20, and the purification apparatus 30, for example, it is possible to reduce the production cost of the antibody drug or the analytical reagent, and it is also possible to improve the productivity.
In addition, the method for producing the adsorbent 1 according to the present embodiment can reliably produce the adsorbent 1 described above.

Hereinafter, the present invention will be specifically described with reference to examples, but the technical scope of the present invention is not limited thereto.

[1] Synthesis of stimulus-responsive polymer <No. Synthesis of stimulus-responsive polymer related to P1>
Ε-Polylysine (EPL) was used as a polymer having a plurality of amino groups. In addition, mercaptobenzothiazole acetic acid (MBTA, 2-benzothiazolylthioacetic acid) was used as an adsorption site to be introduced into the amino group. MBTA is a thiophylic compound having an antibody-adsorbing ability.

EPL (Ichimaru Falcos, Polylysine 10) in water / dimethylformamide (DMF) solution (water: DMF = 1: 2 (volume ratio)) with MBTA (0.7 equivalent) and dehydration condensing agent 4- (4, 6-Dimethoxy-1,3,5-triazine-2-yl) -4-methylmorpholinium chloride (DMT-MM) (1.2 equivalent) was added, and the mixture was stirred at room temperature for 5 hours with the amino group of EPL. MBTA was introduced into EPL by forming an amide bond by reacting with the carboxyl group of MBTA. Hydrochloric acid was added to the obtained reaction solution to make it acidic, dialyzed against tetrahydrofuran (THF), and the resulting precipitate was filtered off and then dried with an evaporator to obtain No. A stimulus-responsive polymer according to P1 (hereinafter, referred to as “stimulus-responsive polymer P1” or the like according to the sample number) was obtained as a solid.

<Synthesis of stimulus-responsive polymer P2>
MBTA (0.6 eq) and DMT-MM (1.2 eq) were added to a water / DMF solution of EPL in the same manner as for the stimulus-responsive polymer P1, and the mixture was stirred at room temperature for 5 hours. Then, the precipitate generated in the same procedure as the stimulus-responsive polymer P1 was filtered off, and this was dried with an evaporator to obtain a stimulus-responsive polymer P2.

<Synthesis of stimulus-responsive polymer P3>
MBTA (0.45 eq) and DMT-MM (1.2 eq) were added to the water / DMF solution of EPL in the same manner as for the stimulus-responsive polymer P1, and the mixture was stirred at room temperature for 5 hours. Then, the precipitate formed in the same procedure as the stimulus-responsive polymer P1 was filtered off, and this was dried with an evaporator to obtain a stimulus-responsive polymer P3.

<Synthesis of stimulus-responsive polymer P4>
MBTA (0.3 eq) and DMT-MM (1.2 eq) were added to the water / DMF solution of EPL in the same manner as for the stimulus-responsive polymer P1, and the mixture was stirred at room temperature for 5 hours. Then, the precipitate formed in the same procedure as the stimulus-responsive polymer P1 was filtered off, and this was dried with an evaporator to obtain a stimulus-responsive polymer P4.

<Synthesis of stimulus-responsive polymer P5>
MBTA (0.07 eq) and DMT-MM (1.2 eq) were added to a water / DMF solution of EPL in the same manner as for the stimulus-responsive polymer P1, and the mixture was stirred at room temperature for 5 hours. Then, the precipitate generated in the same procedure as the stimulus-responsive polymer P1 was filtered off, and this was dried with an evaporator to obtain a stimulus-responsive polymer P5.

<Synthesis of stimulus-responsive polymer P6>
MBTA (0.9 eq) and DMT-MM (1.2 eq) were added to the water / DMF solution of EPL in the same manner as for the stimulus-responsive polymer P1, and the mixture was stirred at room temperature for 5 hours. Then, the precipitate generated in the same procedure as the stimulus-responsive polymer P1 was filtered off, and this was dried with an evaporator to obtain a stimulus-responsive polymer P6.

<Synthesis of stimulus-responsive polymer P7>
MBTA (0.03 eq) and DMT-MM (1.2 eq) were added to a water / DMF solution of EPL in the same manner as for the stimulus-responsive polymer P1, and the mixture was stirred at room temperature for 5 hours. Then, the precipitate generated in the same procedure as the stimulus-responsive polymer P1 was filtered off, and this was dried with an evaporator to obtain a stimulus-responsive polymer P7.

<Chemical composition evaluation and partition coefficient of stimulus-responsive polymers P1-7>
Table 1 shows the composition of the stimulus-responsive polymers P1 to 7, the adsorption site introduction rate, the adsorption site introduction amount per molecule, and the partition coefficient CLogP. In Table 1, as for the composition, the raw material compounds used for introducing the adsorption site are listed. The adsorption site introduction rate indicates the ratio occupied by the adsorption site with respect to the amount of amino groups of the polymer having a plurality of amino groups. The adsorption site introduction rate was ^{determined by 1 1} H-NMR measurement. The amount of adsorption site introduced per molecule was determined by the product of the number of amino groups possessed by the EPL used and the adsorption site introduction rate. In addition, the partition coefficient CLogP of the stimulus-responsive polymers P1 to 7 was calculated using the chemical structural formula drawing software ChemDraw.

[2] Carrier No. Carriers related to C1 to 9 (hereinafter, referred to as "carrier C1" or the like according to the sample number) were prepared (see Table 2). As the carrier, polystyrene, acrylic, methacrylate-based resin carrier, polysaccharide Sepharose carrier, silica, magnetic inorganic oxide (magnetic fine particles) and the like were used. Further, octanol (dielectric constant ε: 10.3 F / m) and ethanol (dielectric constant ε: 24.3 F / m) were prepared as organic solvent phases having different dielectric constant values. Then, each of the various carriers was dispersed in these organic solvent phases, and the carrier and the solvent were mixed well. Then, 3 mL of the dispersion liquid of various carriers was developed on a glass petri dish (major axis = 35 mm) and allowed to stand at room temperature for 10 minutes. Then, the focus was on various carriers with a lens having a magnification of 20 times with an optical microscope, and five points were imaged per sample. The aggregate portion of the carrier observed in the field of view of the obtained image (20 mm × 27 mm) was binarized with image software (ImageJ). The aggregate portion of the image was treated black, the others were treated white, and the length of the aggregate portion obtained in black, particularly the length of the major axis, was divided by the carrier diameter (diameter) of one carrier. The value was defined as φ. Then, according to the value of φ, the following evaluation values 1 to 5 were classified in 5 stages. As the value of φ, the average value of the values obtained from the images taken at 5 points per sample was adopted. Further, the above experiment was repeated twice for one sample, and the average value of the two evaluation values obtained for each time was used as the evaluation value of the carrier.

Evaluation value 5: φ = 0.2 μm or more and less than 8.3 μm (Findings: The carrier is stably and well dispersed in the solvent).
Evaluation value 4: φ = 8.3 μm or more and less than 17 μm (Findings: By mixing the carrier well, a part is dispersed in the solvent, and both small aggregates and isolated particles are mixed).
Evaluation value 3: φ = 17 μm or more and less than 27 μm (Findings: A part of the carrier is dispersed with respect to the solvent to form small aggregates)
Evaluation value 2: φ = 27 μm or more and less than 40 μm (Findings: The carrier is hardly dispersed in the solvent, and large aggregates are formed.)
Evaluation value 1: φ = 40 μm or more (Findings: The carrier is not dispersed in the solvent and large aggregates are formed.)

Then, the dispersibility of the carrier when dispersed in the solvent according to the above criteria was evaluated as follows. _{Then, the evaluation values (evaluation value ε-octanol} , which are obtained by dispersing the carriers in octanol (dielectric constant ε: 10.3 F / m) and ethanol (dielectric constant ε: 24.3 F / m) having different dielectric constant values, respectively. from the evaluation value ε _ethanol), according to the following equation (1), defines the index a.
A = Evaluation value ε- _octanol -Evaluation value ε _Ethanol (1)
Table 2 shows the evaluation results regarding the materials of the carriers C1 to 9, the average particle size (μm), and the index A.

Compared with carriers C1 to 4 and 9 (carrier A group), carriers C5 to 8 (carrier B group) dispersed well in ethanol, and aggregates were formed in octanol. Therefore, the carrier B group was found to be more hydrophilic.

[3] Preparation of Adsorbents and Evaluation of Antibody Adsorption Ability According to Examples 1 to 9 and Comparative Examples 1 to 3 Carriers C1 to 9 and stimulus-responsive polymers P1 to 7 were immobilized by the combination shown in Table 3 described later. The adsorption capacity of the antibody was evaluated. In order to immobilize the stimulus-responsive polymers P1 to 7 on the carriers C1 to 9, an epoxy group and a carboxyl group were used as the functional groups of the carriers C1 to 9. In addition, these functional groups were modified if necessary.

<Example 1>
<Preparation of an adsorbent in which a stimulus-responsive polymer P3 is immobilized on carrier C1 (carrier A group)>
A 1% by mass solution of the stimulus-responsive polymer P3 was prepared using a THF / water mixed solution (THF: water = 4: 1 (volume ratio)) as a solvent. Suspension obtained from a solution of this stimulus-responsive polymer P3, a polystyrene carrier having a carboxyl group on the surface and an average particle size of 100 μm (Polybeads polystyrene series manufactured by Technochemical Co., Ltd.), and DMT-MM (10 equivalents). The solution was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 1 in which the stimulus-responsive polymer P3 was immobilized on the carrier C1.

<Evaluation of antibody adsorption ability of adsorbent according to Example 1>
In order to evaluate the antibody adsorption ability of the adsorption site of the adsorbent according to Example 1, an experiment was conducted according to the following procedure. Specifically, the adsorbent according to Example 1 was filled in a column for 1 mL, and 5 mL of a phosphate buffer solution (PBS buffer solution) having a pH of 7.4 containing 1% by mass of a predetermined human IgG was passed through the column. Then, the human IgG concentration in the solution eluted from the column was monitored, and the antibody adsorption capacity was calculated from the amount of the solution in which the 10% antibody solution leaked from the column. The antibody adsorption capacity referred to here is defined as the dynamic antibody adsorption capacity (10% DBC) on the adsorbent. The results are shown in Table 3 and FIG. 4 below. Note that FIG. 4 is a graph showing the relationship between the index A and 10% DBC in each of the adsorbents according to Examples 1 to 9 and Comparative Examples 1 to 3.
As shown in Table 3 and FIG. 4, it was confirmed that 24 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 1. It was confirmed that the adsorbent according to Example 1 could adsorb the antibody with high efficiency because the combination of the carrier and the stimulus-responsive polymer was good.

From the above results, when the carrier C1 having an index A indicating the degree of dispersibility of 0 and the stimulus-responsive polymer P3 having a distribution coefficient CLogP of 0.62 are combined, the 10% DBC is 24 mg / mL, which is a commercially available product. It has been clarified that the minimum function of the adsorbent distributed as 20 mg / mL or more can be obtained. It was considered that this was because the combination of the carrier and the stimulus-responsive polymer was good.

<Example 2>
<Preparation of an adsorbent in which a stimulus-responsive polymer P4 is immobilized on carrier C2 (carrier A group)>
A 1% by mass solution of the stimulus-responsive polymer P4 was prepared using a THF / water mixed solution (THF: water = 2: 1 (volume ratio)) as a solvent. Suspension obtained from a solution of this stimulus-responsive polymer P4, a polystyrene carrier having a carboxyl group on the surface and an average particle size of 50 μm (Polybeads polystyrene series manufactured by Technochemical Co., Ltd.), and DMT-MM (10 equivalents). The solution was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 2 in which the stimulus-responsive polymer P4 was immobilized on the carrier C2.

<Evaluation of antibody adsorption ability of adsorbent according to Example 2>
In order to evaluate the antibody adsorbing ability of the adsorbent site of the adsorbent according to Example 2, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 30 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 2.

From the above results, when the carrier C2 having an index A indicating the degree of dispersion of 0.5 and the stimulus-responsive polymer P4 having a partition coefficient CLogP of 0.19 are combined, the 10% DBC is as high as 30 mg / mL. was gotten. It was considered that this was because the combination of the carrier and the stimulus-responsive polymer was good.

<Example 3>
<Preparation of an adsorbent in which a stimulus-responsive polymer P4 is immobilized on carrier C3 (carrier A group)>
A 1% by mass solution of the stimulus-responsive polymer P4 was prepared using a THF / water mixed solution (THF: water = 2: 1 (volume ratio)) as a solvent. A suspension obtained from a solution of this stimulus-responsive polymer P4, an acrylic carrier having a carboxyl group on the surface and an average particle size of 30 μm (MX series manufactured by Soken), and DMT-MM (10 equivalents). It was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 3 in which the stimulus-responsive polymer P4 was immobilized on the carrier C3.

<Evaluation of antibody adsorption ability of adsorbent according to Example 3>
In order to evaluate the antibody adsorbing ability of the adsorbing site of the adsorbent according to Example 3, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 32 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 3.

From the above results, when the carrier C3 having an index A indicating the degree of dispersion of 0.5 and the stimulus-responsive polymer P4 having a partition coefficient CLogP of 0.19 are combined, the value of 10% DBC is as high as 32 mg / mL. was gotten. It was considered that this was because the combination of the carrier and the stimulus-responsive polymer was good.

<Example 4>
<Preparation of an adsorbent in which a stimulus-responsive polymer P5 is immobilized on carrier C4 (carrier A group)>
A 1% by mass solution of the stimulus-responsive polymer P5 was prepared using a THF / water mixed solution (THF: water = 1: 1 (volume ratio)) as a solvent. A suspension obtained from a solution of this stimulus-responsive polymer P5, a methacrylate carrier having a carboxyl group on the surface and an average particle size of 100 μm (Tosoh, Toyoperal AF-Carboxy 650M), and DMT-MM (10 equivalents). The turbid solution was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 4 in which the stimulus-responsive polymer P5 was immobilized on the carrier C4.

<Evaluation of antibody adsorption ability of adsorbent according to Example 4>
In order to evaluate the antibody adsorbing ability of the adsorbing site of the adsorbent according to Example 4, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 23 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 4.

From the above results, when the carrier C4 having an index A indicating the degree of dispersion of 1.0 and the stimulus-responsive polymer P5 having a partition coefficient CLogP of −0.46 are combined, the 10% DBC is 23 mg / mL. , It was clarified that the minimum function of 20 mg / mL or more of the adsorbent distributed as a commercial product can be obtained. It was considered that this was because the combination of the carrier and the stimulus-responsive polymer was good.

<Example 5>
<Preparation of an adsorbent in which a stimulus-responsive polymer P1 is immobilized on carrier C5 (carrier B group)>
A 1% by mass solution of the stimulus-responsive polymer P1 was prepared using a THF / water mixed solution (THF: water = 5: 1 (volume ratio)) as a solvent. Obtained from this stimulus-responsive polymer P1 solution, a Sepharose carrier having a carboxyl group on the surface and an average particle size of 100 μm (CM Sepharose 4 Fast Flow manufactured by GE Healthcare), and DMT-MM (10 equivalents). The suspension was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 5 in which the stimulus-responsive polymer P1 was immobilized on the carrier C5.

<Evaluation of antibody adsorption ability of adsorbent according to Example 5>
In order to evaluate the antibody adsorbing ability of the adsorbent site of the adsorbent according to Example 5, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 38 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 5.

From the above results, when the carrier C5 having an index A indicating the degree of dispersion of -2.5 and the stimulus-responsive polymer P1 having a partition coefficient CLogP of 1.3 are combined, the 10% DBC is as high as 38 mg / mL. It was confirmed that the antibody was adsorbed efficiently. It was considered that this was because the combination of the carrier and the stimulus-responsive polymer was good.

<Example 6>
<Preparation of an adsorbent in which a stimulus-responsive polymer P3 is immobilized on carrier C6 (carrier B group)>
A 1% by mass solution of the stimulus-responsive polymer P3 was prepared using a THF / water mixed solution (THF: water = 4: 1 (volume ratio)) as a solvent. A solution of this stimulus-responsive polymer P3, a silica carrier having a carboxyl group on the surface and an average particle size of 100 μm (made by AGC Si-Tech, MS GEL, D grade), and DMT-MM (10 equivalents). The suspension obtained from was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 6 in which the stimulus-responsive polymer P3 was immobilized on the carrier C6.

<Evaluation of antibody adsorption ability of adsorbent according to Example 6>
In order to evaluate the antibody adsorbing ability of the adsorbent site of the adsorbent according to Example 6, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 37 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 6.

From the above results, when the carrier C6 having an index A indicating the degree of dispersion of -3.0 and the stimulus-responsive polymer P3 having a partition coefficient CLogP of 0.62 are combined, the 10% DBC is as high as 37 mg / mL. It was confirmed that the antibody was adsorbed efficiently. It was considered that this was because the combination of the carrier and the stimulus-responsive polymer was good.

<Example 7>
<Preparation of an adsorbent in which a stimulus-responsive polymer P1 is immobilized on carrier C7 (carrier B group)>
A 1% by mass solution of the stimulus-responsive polymer P1 was prepared using a THF / water mixed solution (THF: water = 5: 1 (volume ratio)) as a solvent. A solution of this stimulus-responsive polymer P1, a silica carrier having a carboxyl group on the surface and an average particle size of 50 μm (manufactured by AGC Si-Tech, MSGEL, EP-DM grade), and DMT-MM (10 equivalents). ) And the suspension obtained from was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 7 in which the stimulus-responsive polymer P1 was immobilized on the carrier C7.

<Evaluation of antibody adsorption ability of adsorbent according to Example 7>
In order to evaluate the antibody adsorbing ability of the adsorbent site of the adsorbent according to Example 7, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 48 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 7.

From the above results, when the carrier C7 having an index A indicating the degree of dispersion of -3.5 and the stimulus-responsive polymer P1 having a partition coefficient CLogP of 1.3 are combined, the 10% DBC is as high as 48 mg / mL. It was confirmed that the antibody was adsorbed efficiently. It is considered that this is because the combination of the carrier which is hydrophilic and has a small average particle size and the stimulus-responsive polymer which is hydrophobic but introduces a large amount of the small molecule compound (MBTA) introduced as an adsorption site is good. rice field.

<Example 8>
<Preparation of an adsorbent in which a stimulus-responsive polymer P2 is immobilized on carrier C8 (carrier B group)>
A 1% by mass solution of the stimulus-responsive polymer P2 was prepared using a THF / water mixed solution (THF: water = 5: 1 (volume ratio)) as a solvent. A suspension obtained from a solution of this stimulus-responsive polymer P2, a magnetic carrier having a carboxyl group on the surface and an average particle size of 3 μm (Magnospere MS300 / Carboxyl manufactured by Takara Bio Inc.), and DMT-MM (10 equivalents). The turbid solution was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 8 in which the stimulus-responsive polymer P2 was immobilized on the carrier C8.

<Evaluation of antibody adsorption ability of adsorbent according to Example 8>
In order to evaluate the antibody adsorbing ability of the adsorbing site of the adsorbent according to Example 8, the same experiment was carried out in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 27 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 8.

From the above results, when the carrier C8 having an index A indicating the degree of dispersion of -1.0 and the stimulus-responsive polymer P2 having a partition coefficient CLogP of 1.1 are combined, the 10% DBC is 27 mg / mL. , It was confirmed that the antibody can be adsorbed at the same level as the commercially available product.

<Example 9>
<Preparation of an adsorbent in which a stimulus-responsive polymer P2 is immobilized on carrier C5 (carrier B group)>
A 1% by mass solution of the stimulus-responsive polymer P2 was prepared using a THF / water mixed solution (THF: water = 5: 1 (volume ratio)) as a solvent. Obtained from this stimulus-responsive polymer P2 solution, a Sepharose carrier having a carboxyl group on the surface and an average particle size of 100 μm (CM Sepharose 4 Fast Flow manufactured by GE Healthcare), and DMT-MM (10 equivalents). The suspension was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Example 9 in which the stimulus-responsive polymer P2 was immobilized on the carrier C5.

<Evaluation of antibody adsorption ability of adsorbent according to Example 9>
In order to evaluate the antibody adsorbing ability of the adsorbing site of the adsorbent according to Example 9, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 26 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Example 9.

From the above results, when the carrier C5 having an index A indicating the degree of dispersion of -2.5 and the stimulus-responsive polymer P2 having a partition coefficient CLogP of 1.1 are combined, the 10% DBC is 26 mg / mL. It was confirmed that an antibody adsorption capacity equivalent to that of a commercially available product can be obtained.

Comparing the adsorbent according to Example 9 with the adsorbent according to Example 5, 10% DBC adsorbs according to Example 5 even though the compositions of the carrier and the stimulus-responsive polymer are similar. The material was better. From this, it was considered that immobilization of a stimulus-responsive polymer having a more optimal partition coefficient CLogP with respect to the carrier could significantly improve DBC by 10%.

<Comparative example 1>
<Preparation of an adsorbent in which a stimulus-responsive polymer P6 is immobilized on carrier C3 (carrier A group)>
A 1% by mass solution of the stimulus-responsive polymer P6 was prepared using a THF / water mixed solution (THF: water = 5: 1 (volume ratio)) as a solvent. A suspension obtained from a solution of this stimulus-responsive polymer P6, an acrylic carrier having a carboxyl group on the surface and an average particle size of 30 μm (MX series manufactured by Soken), and DMT-MM (10 equivalents). It was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Comparative Example 1 in which the stimulus-responsive polymer P6 was immobilized on the carrier C3.

<Evaluation of antibody adsorption ability of adsorbent according to Comparative Example 1>
In order to evaluate the antibody adsorbing ability of the adsorbent site of the adsorbent according to Comparative Example 1, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 4 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Comparative Example 1.

From the above results, when the carrier C3 having an index A indicating the degree of dispersion of 0.5 and the stimulus-responsive polymer P6 having a partition coefficient CLogP of 1.9 are combined, the 10% DBC has a low value of 4 mg / mL. Indicated. From this result, it is considered that in the combination of the hydrophobic carrier and the hydrophobic stimulus-responsive polymer, the interaction between the carrier and the stimulus-responsive polymer was enhanced, and a sufficient amount of antibody adsorption ability could not be obtained. rice field.

<Comparative example 2>
<Preparation of an adsorbent in which a stimulus-responsive polymer P7 is immobilized on carrier C5 (carrier B group)>
A 1% by mass solution of the stimulus-responsive polymer P7 was prepared using a THF / water mixed solution (THF: water = 1: 1 (volume ratio)) as a solvent. Obtained from this solution of the stimulus-responsive polymer P7, a Sepharose carrier having a carboxyl group on the surface and an average particle size of 100 μm (CM Sepharose 4 Fast Flow manufactured by GE Healthcare), and DMT-MM (10 equivalents). The suspension was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Comparative Example 2 in which the stimulus-responsive polymer P7 was immobilized on the carrier C5.

<Evaluation of antibody adsorption ability of adsorbent according to Comparative Example 2>
In order to evaluate the antibody adsorbing ability of the adsorbing site of the adsorbent according to Comparative Example 2, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 6 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Comparative Example 2.

From the above results, when the carrier C5 having an index A indicating the degree of dispersion of -2.5 and the stimulus-responsive polymer P7 having a partition coefficient CLogP of -0.69 are combined, the 10% DBC is 6 mg / mL. It showed a low value. From this, the amount of the small molecule compound (MBTA) introduced as the adsorption site is small, and the combination of the hydrophilic carrier and the more hydrophilic stimulus-responsive polymer cannot adsorb a sufficient amount of antibody. confirmed.

<Comparative example 3>
<Preparation of an adsorbent in which a stimulus-responsive polymer P3 is immobilized on carrier C9 (carrier A group)>
A 1% by mass solution of the stimulus-responsive polymer P3 was prepared using a THF / water mixed solution (THF: water = 4: 1 (volume ratio)) as a solvent. Obtained from this stimulus-responsive polymer P3 solution, a methacrylate carrier having a carboxyl group on the surface and an average particle size of 45 μm (Techpolymer, MBX-40, manufactured by Sekisui Kasei Co., Ltd.), and DMT-MM (10 equivalents). The suspension was shaken overnight at room temperature. Then, it was washed with a mixed solvent of THF / water to obtain the adsorbent according to Comparative Example 3 in which the stimulus-responsive polymer P3 was immobilized on the carrier C9.

<Evaluation of antibody adsorption ability of adsorbent according to Comparative Example 3>
In order to evaluate the antibody adsorption ability of the adsorbent site of the adsorbent according to Comparative Example 3, the same experiment was conducted in the same procedure as that of the adsorbent according to Example 1. The results are shown in Table 3 and FIG. 4 below.
As shown in Table 3 and FIG. 4, it was confirmed that 15 mg of the antibody was adsorbed on 1 mL of the adsorbent according to Comparative Example 3.

From the above results, when the carrier C9 having an index A indicating the degree of dispersion of 1.5 and the stimulus-responsive polymer P3 having a partition coefficient CLogP of 0.62 are combined, the 10% DBC is 15 mg / mL. It was slightly inferior to the standard of 20 mg / mL for commercial products. From this, even if it is a stimulus-responsive polymer that has been confirmed to have adsorptive ability, the adsorptive ability of the antibody may be increased depending on the combination with a hydrophobic carrier having an index A of 1.5 or more. It was found to be slightly reduced. Comparing the adsorbent according to Comparative Example 3 with the adsorbent according to Example 4, it was considered that the index A of the carrier capable of achieving high-efficiency adsorption of the antibody should be 1.0 or less for the adsorbent. ..

From the above results, even when a carrier of the hydrophobic carrier group A, which has been difficult to use in the past, is used, a stimulus-responsive polymer having a partition coefficient CLogP of −0.46 or more and 1.3 or less. It was found that the DBC can be increased by 10% by combining the above. When a carrier belonging to the hydrophilic carrier B group is used, a high DBC can be obtained by producing an adsorbent using a stimulus-responsive polymer having a partition coefficient CLogP of −0.46 or more and 1.3 or less. It turned out to be obtained. That is, according to the present invention, the optimum combination is such that the index A of the carrier is 1.0 or less and the partition coefficient CRogP of the stimulus-responsive polymer is in the range of −0.46 or more and 1.3 or less. Therefore, a low-molecular-weight compound can be used as an adsorption site, more types of carriers can be used than before, and an adsorbent having a high adsorption capacity of a target substance, and a column and a purification device using the adsorbent. Turned out to be able to provide.

From this, it is possible to provide an adsorbent having excellent antibody productivity for producing an antibody for a pharmaceutical product or an analytical reagent, and a column and a purification apparatus using the same. Then, according to the present invention, it is possible to purify an antibody with an adsorbent using a small molecule compound that can improve the adsorption efficiency of the antibody. Therefore, according to the present invention, it is possible to realize low-cost production and productivity improvement of antibody drugs, analytical reagents and the like. Therefore, according to the present invention, not only therapeutic antibodies used for rheumatism and cancer treatment, which have been difficult to treat in the past, but also antibodies in biochemical reagents and clinical test reagents used in analysis and testing are included with high efficiency. Since it can be produced and provided, it can greatly contribute to the progress of medical technology.

Although the adsorbent, the column, the purification apparatus, and the method for producing the adsorbent according to the present invention have been described in detail with reference to the embodiments and examples, the gist of the present invention is not limited to this, and various modifications are made. Is included. For example, the above-described embodiment has been described in detail in order to explain the present invention in an easy-to-understand manner, and is not necessarily limited to the one including all the described configurations. Further, it is possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. Further, it is possible to add / delete / replace other configurations with respect to a part of the configurations of each embodiment.

1 Adsorbent 2 Carrier 3 Adsorption site 4 Stimulus-responsive polymer 5 Polymer chain 20 Column 30 Purifier O Target substance R Functional group

Claims (7)

With the carrier
A stimulus-responsive polymer that contains an adsorption site that adsorbs a target substance and an amino group into which the adsorption site is introduced and is immobilized on the carrier.
Is an adsorbent containing
When the carrier is dispersed in octanol and ethanol having different dielectric constants, the index A calculated from the evaluation value indicating the dispersibility of the carrier in each solvent is 0 or more and 1.0 or less .
Before SL stimuli-responsive polymer is partition coefficient CLogP is Ri der -0.46 to 1.3,
For the index A, a dispersion liquid in which the carrier is dispersed in each solvent of the octanol and the ethanol is developed on a chalet, allowed to stand at room temperature for 10 minutes, and then coagulated in an image obtained by imaging with an optical microscope. A binarization process is performed in which the aggregated portion is treated to black and the others are treated to white, and the value obtained by dividing the length of the aggregate portion obtained in black by the diameter of one carrier is defined as φ, and the value of φ is defined as φ. According to, evaluation value 5: φ = 0.2 μm or more and less than 8.3 μm, evaluation value 4: φ = 8.3 μm or more and less than 17 μm, evaluation value 3: φ = 17 μm or more and less than 27 μm, evaluation value 2: φ = 27 μm or more and 40 μm Less than, evaluation value 1: φ = 40 μm or more was evaluated, and the average value of the values obtained from the above images at five locations per sample was adopted, and this was further applied to one sample. It is an average value obtained by repeating it twice, and the value obtained by subtracting the evaluation value when dispersed in ethanol from the evaluation value when dispersed in octanol is used as an index value.
The carrier is made of a synthetic resin and
The adsorption sites are benzothiazole-2-acetic acid, 4-mercaptobenzothiazole-2-ethyl acetate, 5-mercaptobenzothiazole-2-ethylacetate, 2-mercaptothiazole, 3- (benzothiazole-2-ylthio) propane. Sodium -1-sulfonic acid, 2-mercapto-6-nitrobenzothiazole, 3- (2-benzothiazolylthio) propionic acid, 2-mercapto-1,3,4-thiazylazole, N-methyl-2-mercaptoimidazole , Pyridylthioacetic acid, 2-benzothiazolylthioacetic acid, an adsorbent characterized in that at least one selected is amide-bonded to the amino group. In the adsorbent according to claim 1,
Wherein said carrier, Po Li methacrylate, polystyrene, polyacrylamide, adsorbent, characterized in that at least one member selected from the group of acrylic Le. In the adsorbent according to claim 1,
An adsorbent, wherein the stimulus-responsive polymer has a plurality of the adsorption sites and a plurality of amino groups. In the adsorbent according to claim 1,
An adsorbent having an average particle size of 3 μm or more and 100 μm or less of the carrier. A column characterized in that the adsorbent according to any one of claims 1 to 4 is filled. A purification apparatus comprising a column filled with the adsorbent according to any one of claims 1 to 4. A manufacturing method for manufacturing the adsorbent according to any one of claims 1 to 4.
When dispersed in octanol and ethanol having different dielectric constants , a carrier having an index A of 0 or more and 1.0 or less calculated from an evaluation value indicating dispersibility in each solvent has a partition coefficient of -0.46 or more. It fixes a stimulus-responsive polymer that is 1.3 or less .
The stimulus-responsive polymer contains an adsorption site for adsorbing a target substance and an amino group into which the adsorption site is introduced, and is immobilized on the carrier.
For the index A, a dispersion liquid in which the carrier is dispersed in each solvent of the octanol and the ethanol is developed on a chalet, allowed to stand at room temperature for 10 minutes, and then coagulated in an image obtained by imaging with an optical microscope. A binarization process is performed in which the aggregated portion is treated to black and the others are treated to white, and the value obtained by dividing the length of the aggregate portion obtained in black by the diameter of one carrier is defined as φ, and the value of φ is defined as φ. According to, evaluation value 5: φ = 0.2 μm or more and less than 8.3 μm, evaluation value 4: φ = 8.3 μm or more and less than 17 μm, evaluation value 3: φ = 17 μm or more and less than 27 μm, evaluation value 2: φ = 27 μm or more and 40 μm Less than, evaluation value 1: φ = 40 μm or more was evaluated, and the average value of the values obtained from the above images at five locations per sample was adopted, and this was further applied to one sample. It is an average value obtained by repeating it twice, and the value obtained by subtracting the evaluation value when dispersed in ethanol from the evaluation value when dispersed in octanol is used as an index value.
The carrier is made of a synthetic resin and
The adsorption sites are benzothiazole-2-acetic acid, 4-mercaptobenzothiazole-2-ethyl acetate, 5-mercaptobenzothiazole-2-ethylacetate, 2-mercaptothiazole, 3- (benzothiazole-2-ylthio) propane. Sodium -1-sulfonic acid, 2-mercapto-6-nitrobenzothiazole, 3- (2-benzothiazolylthio) propionic acid, 2-mercapto-1,3,4-thiazylazole, N-methyl-2-mercaptoimidazole , A method for producing an adsorbent , wherein at least one selected from pyridylthioacetic acid and 2-benzothiazolylthioacetic acid is amide-bonded to the amino group.

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