JP2016142724A - Refiner and refining method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a refiner that has high capture amount of a target substance and that can be inhibited from capturing an impurity in a non-specific manner.SOLUTION: The refiner related to the present invention refines a target substance and comprises a column tube and a filler 12 filled into the column tube. The filler 12 is composed of a porous carrier having a plurality of pores 123 with a diameter of 1nm or more and 50 nm or less. The porous carrier includes an amphoteric ion substance 124 and a capture section 125 that is specifically combined with the target substance.SELECTED DRAWING: Figure 3

Description

  The present invention relates to a purification apparatus and a purification method.

  In producing useful substances, techniques for separating and purifying target substances from contaminants are widely used. Such techniques include a distillation method in which a liquid compound is heated, gasified and then condensed, a recrystallization method in which the compound is heated and dissolved in a solvent and cooled, and a solution and compound in which the compound is dissolved. Reprecipitation method that obtains the target compound as a precipitate by mixing a solvent that is not very soluble (poor solvent), sublimation method that makes a solid compound into a gas and then turns it into a solid, and uses the difference in affinity with silica, etc. The column method (chromatography method) is known.

  The column method is suitable for purification with high purity without degrading the target substance. Therefore, for example, an antibody protein that is an antibody drug is separated with high purity from a cell culture solution containing a large amount of contaminants using a column method without deterioration. On the other hand, the column method has a demerit that it is difficult to purify a large volume at a time as compared with other separation and purification techniques.

  Examples of the column method include affinity chromatography method, ion exchange chromatography method, hydrophobic interaction chromatography method, gel filtration chromatography method and the like. In the purification of antibody drugs, a combination of a plurality of methods is used, such as an affinity chromatography method in which protein A is immobilized by rough purification, and an ion exchange chromatography method is used in the subsequent step.

  Here, conventionally, a bead-like porous carrier (bead-like carrier) formed of agarose, silica gel or the like has been used as a packing material for a column used for chromatography. The bead-shaped carrier is a carrier having a structure having pores of different sizes inside a bead having a diameter of about 10 to 100 μm.

  A bead-like support (agarose beads) formed of agarose has an advantage that a non-specific trapping amount of impurities is low due to its high biocompatibility. Therefore, agarose beads are mainly used as a packing material for a protein purification column, but have a drawback of low mechanical strength against pressure loss. Further, the bead-shaped carrier has a drawback that when the carrier is packed in the column, a narrowed portion is generated between the carriers and the flow path is reduced. In this case, it has been pointed out that the flow path entering the constricted portion is greatly bent due to the influence of the opposing carrier, and the pressure loss increases (see Patent Document 1). For these reasons, when a bead-shaped carrier is used, the flow rate cannot generally be set high, and there is a disadvantage that it takes time for the treatment process.

  In order to shorten the processing time by increasing the flow rate, for example, Patent Document 1 proposes a monolithic porous carrier (monolithic carrier) instead of a bead-like carrier. The monolithic carrier refers to a single porous carrier having a skeleton and voids continuously (that is, having a three-dimensional network structure). The voids are often macropores having a pore diameter of about 50 nm or more, and mesopores having a pore diameter of about 2 to 50 nm or micropores having a pore diameter of about 2 nm or less are formed on the surface of the skeleton. There are many cases. When a monolith support is used as the column packing, the liquid phase passes through macropores that are continuously formed in the monolith support.

  Specifically, Patent Document 1 discloses a skeleton body made of silica gel or silica glass having a three-dimensional network structure, and pores penetrating from the surface of the skeleton body to the inside formed by being dispersed on the surface of the skeleton body. A column for purifying an antibody comprising an adsorbent formed by immobilizing protein A on the surface of a porous carrier having a pore diameter measured by a nitrogen adsorption method of 40 nm or more and 70 nm or less, The pre-purification solution is allowed to flow through the antibody purification column under a condition where the contact time between the pre-purification solution containing the antibody to be purified and the adsorbent is 40 seconds or less, and the antibody in the pre-purification solution An antibody purification method characterized by adsorbing to an adsorbent and purifying the antibody is disclosed.

  According to the invention described in Patent Document 1, by using silica monolith as a carrier and using a central pore diameter in an optimal range for antibody adsorption, a high dynamic adsorption capacity (dBC, It is also said that the antibody purification can be carried out with high efficiency and in a short time by reducing the time required for the adsorption step to 40 seconds or less.

Japanese Patent Laid-Open No. 2014-2008

  Since the silica monolith (monolith carrier) described in Patent Document 1 has a three-dimensional network structure, it is structurally less subject to pressure load than conventional bead-like carriers and can be produced relatively easily. There is an advantage.

Here, in general, when the macropores in the monolith support are increased, the flow path is secured and the pressure loss is reduced, but the surface area is reduced, so that the capture amount (adsorption amount) of the target substance is decreased. On the other hand, when the macropores in the monolith support are reduced, the surface area is increased, but the flow path is reduced, so that the pressure loss is increased and high flow rate processing becomes difficult.
In addition, under the low flow rate condition, there is sufficient time for the target substance to diffuse into the macropores, so that it can penetrate into the mesopores in the macropores. The entire area of the mesopore surface area cannot be used effectively because the chance of contact with the trapped portion inside the mesopore is reduced. Therefore, even if high flow rate processing becomes possible by using a monolithic carrier with low pressure loss, the capture efficiency (adsorption efficiency) decreases due to a decrease in the target substance that can enter the mesopores. And it is difficult to significantly reduce processing costs.

  That is, the invention described in Patent Document 1 has a problem that it is difficult to achieve both flow rate and capture efficiency when a treatment solution containing a target substance is passed through a column. In other words, the invention described in Patent Document 1 has a problem that it is difficult to increase the capture amount of the target substance. Further, the silica material mainly used in the monolithic carrier has a problem that non-specific trapping of impurities is large. In addition, when nonspecific trapping of impurities increases, there is a possibility that the purity of the collected target substance is lowered and the column life is shortened.

  This invention is made | formed in view of the said problem, and makes it a subject to provide the refinement | purification apparatus and purification method which can suppress the non-specific capture | acquisition of an impurity with the capture amount of a target substance being high.

  A purification apparatus according to the present invention that solves the above-described problems is a purification apparatus that purifies a target substance, and includes a column tube and a filler packed in the column tube, and the filler has a pore diameter of 1 nm. It is composed of a porous carrier having a plurality of pores having a diameter of 50 nm or less, and the porous carrier includes a zwitterionic substance and a capturing part that specifically binds to the target substance.

  Furthermore, the purification method according to the present invention comprises a column tube and a filler packed in the column tube, wherein the filler is from a porous carrier having a plurality of pores having a pore diameter of 1 nm to 50 nm. The porous carrier is a purification method for purifying a target substance using a purification apparatus including a zwitterionic substance and a capture unit that specifically binds to the target substance, and is packed in the column tube An equilibration step for equilibrating the filler, and after the equilibration step, a processing solution containing the target substance and components other than the target substance is passed through the filler, and the target substance is captured by the capturing unit. A capture step, after the capture step, a washing solution is passed through the filler to wash away components other than the target substance captured by the capture unit, and after the wash step, the eluent is eluted into the filler. Let the liquid flow, The target substance is trapped in the serial capture portion eluted from the filler, characterized in that it comprises a elution step of recovering.

The purification apparatus according to the present invention has a high trapping amount of the target substance and can suppress nonspecific trapping of impurities.
Moreover, the purification method according to the present invention has a high trapping amount of the target substance and can suppress nonspecific trapping of impurities.

It is a schematic block diagram of the target substance manufacturing apparatus using the refiner | purifier which concerns on this invention. It is a schematic sectional drawing explaining the structure of the refiner | purifier which concerns on this invention. It is a principal part expanded sectional view explaining the filler used for the refiner | purifier which concerns on this invention. It is a schematic sectional drawing which shows other embodiment of the refiner | purifier which concerns on this invention. It is a schematic sectional drawing which shows other embodiment of the refiner | purifier which concerns on this invention. It is a schematic sectional drawing which shows other embodiment of the refiner | purifier which concerns on this invention. It is a flowchart explaining the content of the purification method which concerns on this invention. 6 is a graph showing the relationship between the amount of protein A immobilized and the amount of antibody captured relative to the organic monolith carrier according to Example 1 and Comparative Example 1 measured in [Measurement 2]. It is a graph which shows the comparison result of the antibody capture amount per protein A unit amount with respect to the cysteine concentration in Example 1 and Comparative Examples 2 and 3 measured in [Measurement 2] (relative amount based on the measurement result of Example 1). is there. It is a graph which shows the comparison result of the antibody capture | acquisition amount per unit amount of protein A (relative amount on the basis of the measurement result of the comparative example 1) in the organic monolith carrier which concerns on Example 1 and the comparative example 1 which were measured by [Measurement 2]. is there. 7 is a graph showing a comparison result of the amount of antibody captured per unit amount of protein A (relative amount based on the measurement result of Comparative Example 4) in the organic monolithic carrier according to Example 2 and Comparative Example 4 measured in [Measurement 2]. is there. The graph which shows the comparison result of the nonspecific adsorption amount (relative amount on the basis of the measurement result of the comparative example 1) of BSA using each organic monolith support | carrier which concerns on Example 1 and Comparative Example 1 measured by [Measurement 3] It is.

  DESCRIPTION OF EMBODIMENTS Hereinafter, an embodiment (embodiment) for carrying out a purification apparatus and a purification method according to the present invention will be described in detail with reference to the drawings as appropriate.

[Target substance production equipment]
First, with reference to FIG. 1, an example of the target substance manufacturing apparatus 100 in which the purification apparatus 10 according to the present invention is used will be briefly described. FIG. 1 is a schematic configuration diagram of a target substance manufacturing apparatus 100 using the purification apparatus 10 according to the present invention.

  As shown in FIG. 1, the purification apparatus 10 is used as a part of the target substance manufacturing apparatus 100. Specifically, the target substance manufacturing apparatus 100 includes a processing solution storage tank 110, a cleaning liquid storage tank 120, an eluent storage tank 130, a valve 140 connected to these tanks, a liquid passing pump 150, A purifier 10 and a target substance storage tank 160 are provided. In addition, when setting it as the aspect which performs post-cleaning, the refiner | purifier 10 may be equipped with the post-cleaning liquid storage tank, and the post-cleaning liquid storage tank may be connected with the valve | bulb 140 like other tanks. (Not shown).

  The processing solution storage tank 110 is a tank that stores a processing solution to be processed including a target substance. The cleaning liquid storage tank 120 is a tank for storing a cleaning liquid for cleaning the filler 12 (see FIG. 2) in the purification apparatus 10. The eluent storage tank 130 is a tank that stores an eluent for eluting the target substance captured by the filler 12 in the purification apparatus 10. These tanks should just be formed with the material which is not corroded by each solution. Examples of such a material include stainless steel and aluminum alloy. The treatment solution only needs to contain a target substance, and examples thereof include a culture solution in which cells are cultured, a crushing treatment solution in which cell membranes of cultured animal cells and cell walls of plant cells are crushed. The washing liquid and the eluent can be appropriately selected according to the target substance. The target substance will be described later.

  The valve 140 is connected to the processing solution storage tank 110, the cleaning liquid storage tank 120, and the eluent storage tank 130, and can freely switch the solution to be passed through the liquid passing pump 150 and the purification device 10. The liquid passing pump 150 can apply pressure to the solution switched by the valve 140 and pass it through the purifier 10.

The purification apparatus 10 includes a filler 12 described later, and purifies the target substance. Details of the purification apparatus 10 will be described later.
The target substance storage tank 160 is a tank that stores an eluent containing the target substance purified by the purification apparatus 10. The target substance storage tank 160 can be formed of stainless steel, aluminum alloy or the like, as with other tanks.

[Purification equipment]
Next, the purification apparatus 10 according to the present invention will be described with reference to FIGS. FIG. 2 is a schematic cross-sectional view illustrating the configuration of the purification apparatus 10 according to the present invention. FIG. 3 is an enlarged cross-sectional view of a main part for explaining the filler 12 used in the purification apparatus 10 according to the present invention.

  As described above, the purification apparatus 10 is used for purifying a target substance. Here, examples of the target substance include physiologically active substances and industrially useful proteins. Examples of physiologically active substances include antibodies (antibody molecules). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, humanized antibodies, human antibodies, immunoglobulins, and the like. The physiologically active substance is not limited to an antibody, and any substance or compound group that exhibits a physiological action or a pharmacological action on an organism is included. Substances and compounds that exhibit physiological and pharmacological effects on organisms include, for example, alkaloids, cytokines, plant hormones, neurotransmitters, pheromones, hormones (animal hormones), growth factors, and growth regulation. Factors, growth inhibitory factors, and the like. As an example of the substance simple substance and compound group which express a physiological action and a pharmacological action with respect to a living body, biopharmaceuticals, such as a tissue type plasminogen activator used as a thrombolytic agent, and insulin, are mentioned, for example. Examples of industrially useful proteins include oxidoreductases, transferases, hydrolases, removal / addition enzymes, isomerases, and synthetic enzymes.

  The treatment solution contains substances other than the target substance, that is, impurities. The impurity in the present invention refers to a molecule that is a substance other than the target substance that is specifically recognized by the trap part 125 (see FIG. 3) but traps (adsorbs) on the surface of the carrier part 121 (see FIG. 3). For example, in the treatment solution to be treated in the purification process of antibody pharmaceuticals, in addition to the target substance antibody, proteins secreted by cells by cell culture, proteins or DNA, RNA, lipids that constitute cultured cells, or for cell culture These media contain serum-derived proteins and other nutrient components that are contained in advance, and these substances become impurities. Impurities cause a decrease in purity during antibody recovery and deterioration of the column (purification device 10).

  Returning to FIG. 2, the description of the purification apparatus 10 will be continued. As shown in FIG. 2, the purification apparatus 10 includes a column tube 11 and a filler 12 filled in the column tube 11. At both ends of the column tube 11, there are provided liquid passing plugs 13, 13 that allow the inside and the outside of the column tube 11 to communicate with each other and allow a solvent or the like to pass therethrough.

(Column tube, liquid plug)
As long as the column tube 11 and the plug 13 for liquid passage are formed using a metal or a resin that does not react with the solution used for purification of the target substance and does not deform by the pressure applied to the liquid phase. Good. Examples of such a metal include stainless steel and aluminum alloy. Examples of the resin include polyethylene, polypropylene, polyvinyl chloride, polystyrene, polyvinyl acetate, polyurethane, polytetrafluoroethylene, acrylonitrile butadiene styrene resin, acrylonitrile styrene resin, acrylic resin, and rubber. The shape of the column tube 11 can be, for example, a cylindrical shape.

(filler)
As shown in the enlarged view of the main part of FIG. 3, the filler 12 includes a carrier part 121, a through-hole part 122, a hole part 123, a zwitterionic substance 124, and a capturing part 125. Note that the through-hole portion 122 corresponds to the above-described macropore, and the pore portion 123 corresponds to the above-described mesopore and micropore. That is, the filler 12 is composed of a porous carrier having a plurality of through-hole portions 122 having a pore diameter of about 50 nm or more and pore portions 123 having a pore diameter of 1 nm to 50 nm. In addition, the pore diameter of the hole part 123 should just be 1 nm or more and 30 nm or less. If the pore diameter of the pore 123 is 1 nm or more and 30 nm or less, the target substance can be reliably captured. The pore diameter of the through-hole part 122 and the pore diameter of the hole part 123 can be calculated by obtaining the pore diameter distribution using a specific surface area measuring device by a gas adsorption method.

The aforementioned porous carrier is preferably a monolith structure. When the porous carrier is a monolith structure, since it is a lump structure having a skeleton and voids continuously (that is, having a three-dimensional network structure), the flow rate can be increased to shorten the processing time, In addition, high capture efficiency can be maintained. In the conventional bead-like carrier (for example, agarose beads), the column height can only be increased to about several tens of centimeters due to the problem of pressure loss. Therefore, in order to increase the column capacity using conventional agarose beads or the like, in the case of a cylindrical column, it has to be expanded in the diameter direction. In addition, due to such circumstances, a column using conventional agarose beads or the like could only be a vertical purification apparatus that allows the solution to flow in the vertical direction.
However, as described above, when the porous carrier is a monolith structure, pressure loss can be reduced, so that the column can be expanded in the flow path direction, and the degree of freedom of the device structure can be increased. That is, a horizontal purification apparatus that allows the solution to flow in the horizontal direction can be obtained. In addition, if the porous body is a monolith structure, pressure loss can be reduced, so that the length in the flow path direction can be increased. Therefore, since the contact time of the target substance with the carrier can be increased, the amount of the target substance captured can be increased with the same carrier volume as compared with the agarose beads. Furthermore, the installation space for the apparatus can be reduced as compared with the conventional case.

  In the porous carrier described above, the zwitterionic substance 124 and the trap part 125 are fixed to at least a part of the surface, preferably the inner surface 123a of the hole 123 as shown in FIG. Of course, the zwitterionic substance 124 and the capturing part 125 may be fixed to a part other than the inner surface 123 a of the hole part 123, for example, the outer surface 121 a of the carrier part 121.

  The main flow path of the solution in the filler 12 is the through-hole portion 122. Various solutions flow, for example, in the direction of arrow A in FIG. 3 due to the pressure applied by the pump 150 for liquid flow. As described above, since the plurality of pores 123 exist on the surface of the porous carrier, the treatment solution and the target substance contained in the treatment solution are transferred from the through-hole portion 122 into the pore portion 123. Get in.

  At that time, the filler 12 used in the present invention is wet because the zwitterionic substance 124 is fixed to at least a part of the surface of the porous carrier (carrier part 121) (for example, the inner surface 123a of the hole 123). The property (hydrophilicity) is high. For this reason, the filler 12 has a treatment solution that easily enters the inside of the hole 123 as compared with a conventional filler in which the zwitterionic substance 124 is not fixed (for example, an adsorbent described in Patent Document 1). It has become. Therefore, by using the filler 12, at least a part of the surface of the carrier 121 (for example, the capturing part 125 fixed to the inner surface 123a of the hole 123), the target substance contained in the processing solution, and , And the capture unit 125 can easily capture the target substance. Therefore, even when the treatment solution is passed under a high flow rate condition that cannot be treated with a conventional filler, the target substance can enter the hole 123 and is fixed to the hole 123. Can be captured by the capturing unit 125. Such a filler 12 can improve the capture rate of the target substance by about 10 to 50% or more as compared with the conventional filler. In addition, the processing time for purifying the target substance can be significantly shortened compared to the conventional method.

  In addition, the filler 12 is neutral in charge and highly hydrophilic by fixing the zwitterionic substance 124 on the surface of the carrier portion 121. Therefore, non-specific interaction of impurities such as proteins contained in the eluent, that is, capture can be suppressed. In addition, protein has a complicated three-dimensional structure of polyamino acids, and has a hydrophobic region and an ionic region. Therefore, it is said that non-specific capture is caused by the hydrophobic interaction and electrostatic interaction of these regions with the surface of the carrier portion 121. Therefore, since these interactions can be avoided by the action of the zwitterionic substance 124 described above, nonspecific capture of impurities such as proteins can be suppressed as described above.

The material of the filler 12 is not particularly limited, and may be either an inorganic or organic material as a main component.
As the inorganic material, for example, at least one of silicon, titanium, zinc, and aluminum, or any one oxide selected from these, at least one of silicon, gallium, aluminum, and indium, or Any one of the nitrides selected from these can be used alone or as a composite.

  Examples of organic materials include polysaccharides such as agarose, cellulose, chitin, chitosan, amylose, heparin, hyaluronic acid, pectin, polyacrylamide, polyacrylic acid, polystyrene, polyvinyl alcohol, polymethacrylic acid, polyacrylic acid. Polymethacrylic acid ester, polyacrylic acid ester, and derivatives thereof can be used.

  When the porous carrier is a monolith structure body using an organic material, it is preferably formed using an organic polymer. Moreover, it is preferable that the said organic polymer is formed using the monomer containing an activation site | part, and it is preferable that an activation site | part is a glycidyl group (epoxy group). Examples of such a monomer include glycidyl methacrylate. That is, it is preferable that the organic polymer is crosslinked using glycidyl methacrylate. A method for producing the filler 12 formed by crosslinking using glycidyl methacrylate will be described later.

The porous carrier may be one synthesized by a general method for producing a synthetic polymer. As a general method for producing a synthetic polymer, for example, a radical polymerization reaction may be mentioned. Specifically, for example, a polymer can be formed by reacting in a solvent using a vinyl monomer, a divinyl compound as a crosslinking agent, and a radical initiator.
As the crosslinking agent in this case, for example, ethylene glycol dimethacrylate, polypropylene glycol dimethacrylate, polyethylene glycol divinyl ether, divinylbenzene, diallyl phthalate, methylene bisacrylamide, polyethylene glycol diacrylate, and the like can be used. By using such a crosslinking agent, the strength of the carrier portion 121 can be increased.
Examples of the radical initiator include azo compounds such as azobisisobutyronitrile and organic peroxides such as benzoyl peroxide and methyl ethyl ketone peroxide. In addition, hydrogen peroxide and iron (II ) What is used in combination with oxidizing agents, such as salt, and a reducing agent can also be used.

(Zwitterionic substance)
As the zwitterionic substance 124, a substance having a thiol group and fixed to the porous carrier (carrier 121) via the thiol group can be used. As such a zwitterionic substance 124, for example, at least one of an amino acid having a thiol group and an amino acid having a thiol group introduced therein can be used. More specifically, examples of amino acids having a thiol group include cysteine and cysteine derivatives. Introduction of a thiol group into an amino acid can be performed by a known method. In addition, the derivative | guide_body of cysteine means what substituted at least 1 (for example, hydroxyl group) among the functional groups which cysteine has with the other functional group (however, the thing which has the property of zwitterion is preferable).
Moreover, as the zwitterionic substance 124, for example, sulfobetaine 3-undecanethiol (N- (11-Mercaptoundecyl) -N, N-dimethyl-3-ammonio-1-propanesulfonate) can be used.
Further, as the zwitterionic substance 124, for example, zwitterionic groups such as phosphorylcholine groups, polyethylene glycol, and the like are functional groups that are neutral as charges and highly hydrophilic.
That is, as the zwitterionic substance 124, specifically, at least one of cysteine, a derivative of cysteine, sulfobetaine 3-undecanethiol, a phosphorylcholine group, and polyethylene glycol can be used.

(Capture part)
The capturing unit 125 can be appropriately set according to the target substance to be captured. The capturing unit 125 is preferably capable of specifically capturing a target substance. For example, when the target substance is an antibody, it is preferable to use at least one of protein A, protein G, and protein L as the capture unit 125. Since these all have immunoglobulin binding ability, they can be specifically captured and purified with high efficiency. If the target substance is an alkaloid as described above, a substance having a high affinity with it can be used as the capturing unit 125. If the target substance is a protein such as an enzyme, an antibody that specifically binds to the enzyme can be used as the capture unit 125.

[Method for producing filler]
Next, the manufacturing method of a filler is demonstrated.
The method for producing a filler includes a porous carrier production process and an immobilization process, and at least these processes are performed in the order described above.

(Porous carrier manufacturing process)
The porous carrier production process is a process for producing a porous carrier. The porous carrier can be produced by a known method (for example, Reference 1: Y. Ueki et al., Anal. Chem., December 2004, p. 7007 to 7012).

(Immobilization process)
The immobilization step is a step of immobilizing the zwitterionic substance 124 and the trapping portion 125 on at least a part of the surface of the porous carrier, preferably the inner surface 123a of the hole portion 123 as shown in FIG.

  Fixation of the zwitterionic substance 124 and the capturing part 125 to the surface of the porous carrier can be performed by a known method (for example, refer to Reference 2: JP 2011-252929 A). That is, it has an amino group using an epoxy method, a cyanogen bromide method, a trichlorotriazine method, a tresyl chloride method, a periodate oxidation method, a divinyl sulfonic acid method, a benzoquinone method, a carbonidyl imidazole method, an acyl azide method, etc. The capture unit 125 and the zwitterionic substance 124 are immobilized, the capture unit 125 having a hydroxyl group and the zwitterionic substance 124 are immobilized using an epoxy method, a diazo coupling method, etc., an epoxy method, a tresyl chloride method, a divinyl sulfone. Using an acid method or the like, the capture unit 125 having a thiol group and the zwitterionic substance 124 are immobilized, and further, the capture of having a carboxylic acid or a formyl group on the aminated carrier in which a part of the surface of the porous carrier is aminated. By immobilizing the portion 125 and the zwitterionic substance 124. As mentioned above, it is possible to fix the capturing part 125 and the zwitterionic material 124 to the surface of the porous support.

In addition, the trapping portion 125 and the zwitterionic substance 124 can be easily fixed to the inorganic porous carrier by using a silane coupling agent. In addition, a desired functional group can be added to the surface of the porous carrier. Examples of the functional group that can be added include a vinyl group, an epoxy group, a styryl group, a methacryl group, an acrylic group, an amino group, a mercapto group, and a sulfide group. Examples of silane coupling agents include vinyltrimethoxysilane, 2- (3,4-epoxycyclohexyl) ethyltrimethoxysilane, 3-glycidoxypropylmethyldimethoxysilane, p-styryltrimethoxysilane, and 3-methacryloxy. Examples include propylmethyldimethoxysilane, 3-acryloxypropyltrimethoxysilane, N-2- (aminoethyl) -3-aminopropylmethyldimethoxysilane, and 3-aminopropyltriethoxysilane.
After adding the functional group to the surface of the porous carrier using the silane coupling agent described above, for example, the epoxy method, cyanogen bromide method, trichlorotriazine method, tresyl chloride described in Reference 2 above. The functional group is activated by an activation method such as a method, periodate oxidation method, divinyl sulfonic acid method, benzoquinone method, carbonidyl imidazole method, acyl azide method, and the like. Can be immobilized.

  The order of immobilizing the supplement part 125 and the zwitterionic substance 124 on the porous carrier used in the present invention is not particularly limited. In the present invention, the zwitterionic substance 124 may be immobilized after the supplemental portion 125 is first immobilized on the produced porous carrier, or after the zwitterionic substance 124 is first immobilized on the produced porous carrier. The supplementary part 125 may be immobilized by the above-mentioned method, or the supplementary part 125 and the zwitterionic substance 124 may be immobilized simultaneously on the produced porous carrier. Alternatively, the supplementary part 125 may be immobilized after the porous carrier is produced by polymerizing in advance using a monomer having the zwitterionic substance 124 in the side chain. Examples of the monomer having a zwitterionic group in the side chain include a zwitterionic vinyl monomer. When a zwitterionic vinyl monomer and a crosslinking agent having a vinyl group such as divinylbenzene are used in combination for radical polymerization, a porous carrier having the zwitterionic substance 124 can be obtained.

[Preferred Embodiment of Filler Production Method]
As described above, it is preferable that the filler 12 is formed of an organic polymer obtained by crosslinking using glycidyl methacrylate. Therefore, a method for producing the filler 12 formed of an organic polymer that is crosslinked using glycidyl methacrylate will be described.

  The filler 12 is produced by first using glycidyl methacrylate as a main component, using a divinyl compound such as ethylene glycol dimethacrylate or methylene bisacrylamide as a crosslinking agent, and azobisisobutyronitrile or benzoyl peroxide as an initiator. A porous carrier is obtained by radical polymerization of these using, for example.

  Next, by bringing the zwitterionic substance 124 into contact with the obtained porous carrier, it can be easily immobilized under mild conditions. Examples of the zwitterionic substance 124 include cysteine, a derivative of cysteine, and sulfobetaine 3-undecanethiol as described above. In this embodiment, it is preferable to use cysteine. In order to fix cysteine to the porous carrier, for example, the obtained porous carrier and a solution containing 0.01% by mass of cysteine are brought into contact under neutral conditions and kept at 40 ° C. overnight. . Only by doing this, the thiol group of cysteine can selectively react with the glycidyl group of the porous carrier to form a covalent bond, and the immobilization of cysteine to the porous carrier is achieved. According to this method, the zwitterionic substance 124 can be easily immobilized on the porous carrier by a one-step reaction. Further, since an inexpensive reagent called cysteine is used, the cost can be reduced.

  In the present embodiment, it is also possible to control the immobilization distribution of the capturing part 125 and the zwitterionic substance 124 by using a porous carrier having two different types of functional groups. Control of the immobilization distribution of the capture unit 125 and the zwitterionic substance 124 is performed, for example, by producing a porous carrier having a glycidyl group (epoxy group) and a carboxyl group, and via the amino group of the glycidyl group and the zwitterionic substance 124. After the reaction and fixation, the carboxyl group can be arbitrarily adjusted by activating the carboxyl group using an activating agent such as carbodiimide, and reacting the activated carboxyl group with the amino group of the capturing unit 125. That is, it is possible to control the immobilization distribution of the trapping portion 125 and the zwitterionic substance 124 by arbitrarily adjusting the distribution of the glycidyl group and the carboxyl group introduced into the porous carrier.

[Other configurations of purification equipment]
As mentioned above, although the preferable embodiment of the refiner | purifier 10 which concerns on this embodiment, and the filler 12 used for the said refiner | purifier 10 was demonstrated, the refiner | purifier can also be set as the other structures as follows.
For example, the monolith structure is preferably used as the porous carrier. However, the porous carrier of the monolith structure may be formed into a bead shape. Even with such a porous carrier, the pressure loss can be reduced, so that the column can be expanded in the direction of the flow path, and the degree of freedom of the device structure can be expanded. That is, a horizontal purification apparatus that allows the solution to flow in the horizontal direction can be obtained. In addition, if the porous body is a monolith structure, pressure loss can be reduced, so that the length in the flow path direction can be increased. Therefore, since the contact time of the target substance with the carrier can be increased, the amount of the target substance captured can be increased with the same carrier volume as compared with the agarose beads. Furthermore, the installation space for the apparatus can be reduced as compared with the conventional case.

Moreover, a refiner | purifier can be made into an aspect as shown to FIGS. 4 to 6 are schematic cross-sectional views showing other embodiments of the purification apparatus according to the present invention.
A purification device 10A shown in FIG. 4 is a device in which the purification device 10 shown in FIG. 2 is designed to be long along the solution flow direction. Moreover, the refiner | purifier 10B shown in FIG. 5 arrange | positions the refiner | purifier 10 shown in FIG. 2 in multiple numbers in series. And the refiner | purifier 10C shown in FIG. 6 arrange | positions the refiner | purifier 10 shown in FIG. 2 in multiple numbers in parallel. In any of the embodiments of the purification apparatuses 10A to 10C shown in FIGS. 4 to 6, since the column tube 11 is filled with the packing material 12 described above, the processing solution is used under a high flow rate condition that cannot be processed with the conventional packing material. Even when the liquid is passed, the target substance can enter the hole 123 and can be captured by the capturing part 125 fixed to the hole 123. Such a filler 12 can improve the capture efficiency of the target substance as compared with the conventional filler. In addition, the processing time for purifying the target substance can be significantly shortened compared to the conventional method.

  According to the purification apparatus described above, since the zwitterionic substance 124 is included, the amount of the target substance captured can be increased as compared with the conventional filler. Therefore, even when processing is performed at the same flow rate as before, processing time can be shortened. Moreover, even if the amount of immobilization of the capturing part 125 is reduced, the capturing efficiency equivalent to that of the conventional filler can be maintained. For example, in the purification of antibody drugs, protein A is often used as the capture unit 125. However, according to the present invention, since the zwitterionic substance 124 is included, the amount of antibody captured per unit amount of protein A can be reduced. Can be increased. Therefore, even if the amount of protein A immobilized on the porous carrier is reduced, an antibody capture amount equivalent to that of the conventional filler can be exhibited. In addition, since protein A is a protein mainly produced by the transformant of E. coli and is an expensive material, reducing the amount of protein A used is advantageous in terms of cost reduction.

[Purification method]
Next, the purification method according to the present invention will be described.
The purification method according to the present invention is a purification method for purifying a target substance using the above-described purification apparatus 10 (10A to 10C) according to the present invention. FIG. 7 is a flowchart for explaining the contents of the purification method according to the present invention.
As shown in FIG. 7, the purification method according to the present invention includes an equilibration step S1, a capture step S2, a washing step S3, and an elution step S4. These steps are performed in this order. is there. This purification method can be applied when the purification apparatus 10 is used as a disposable one.
On the other hand, when the purification apparatus 10 is used as a non-disposable type, that is, when it is used repeatedly a plurality of times, the post-cleaning step S5 is performed after the elution step S4, and the equilibration step S1 is completed after the post-cleaning step S5. Each process can be performed in the order described above.
Hereinafter, each step will be described. In addition, since it has already explained in full detail about the refiner | purifier 10 used with the refinement | purification method which concerns on this invention, description is abbreviate | omitted and suppose that the same code | symbol is attached | subjected about the same component.

(Equilibration process)
The equilibration step S1 is a step of equilibrating the packing material 12 packed in the column tube 11. The equilibration of the filler 12 can be performed, for example, by using a buffer solution. As the buffer solution, for example, acetate buffer solution, phosphate buffer solution, citrate buffer solution, borate buffer solution, tartaric acid buffer solution, Tris buffer solution, phosphate buffered saline and the like can be used.

(Capture process)
The capturing step S2 is a step in which, after the equilibration step S1, a processing solution containing the target substance and components other than the target substance is passed through the filler 12 in the column tube 11 and the target substance is captured by the capturing unit 125. is there. Examples of the treatment solution include a culture supernatant of serum containing Chinese hamster ovary (CHO) cells containing antibodies and serum.

(Washing process)
The washing step S3 is a step in which after the trapping step S2, the cleaning liquid is passed through the filler 12 in the column tube 11 to wash away components other than the target substance trapped by the trapping portion 125, that is, impurities. As the washing solution, for example, acetate buffer solution, phosphate buffer solution, citrate buffer solution, borate buffer solution, tartaric acid buffer solution, Tris buffer solution, phosphate buffered saline and the like can be used.

(Elution process)
The elution step S4 is a step in which, after the washing step S3, the eluent is passed through the packing material 12 in the column tube 11, and the target substance captured by the capturing unit 125 is eluted from the packing material 12 and collected. As the eluent, for example, an acidic solution such as sodium citrate buffer can be used.

(Post-cleaning process)
The post-cleaning step S5 is a step in which after the elution step S4, the post-cleaning liquid is passed through the filler 12 in the column tube 11 to wash away components (impurities) other than the target substance remaining on the porous carrier. As the post-cleaning liquid, for example, an alkaline solution such as an aqueous sodium hydroxide solution can be used.

  According to the purification method described above, since the filler 12 contains the zwitterionic substance 124, the amount of the target substance captured can be increased as compared with the conventional filler. Therefore, even when processing is performed at the same flow rate as before, processing time can be shortened. Moreover, even if the amount of immobilization of the capturing part 125 is reduced, the capturing efficiency equivalent to that of the conventional filler can be maintained. For example, protein A is often used as the capture unit 125 in the purification of antibody drugs. However, according to the present invention, since the filler 12 contains the zwitterionic substance 124, The amount of antibody captured can be increased. Therefore, even if the amount of protein A immobilized on the porous carrier is reduced, an antibody capture amount equivalent to that of the conventional filler can be exhibited. In addition, since protein A is a protein mainly produced by the transformant of E. coli and is an expensive material, reducing the amount of protein A used is advantageous in terms of cost reduction.

Next, the present invention will be described in more detail with reference to examples in which the effects of the present invention have been confirmed.
Examples 1 and 2 and Comparative Examples 1 to 4 were produced as follows.

[Example 1]
[1] Production of Organic Monolithic Carrier A porous carrier as a monolithic structure was prepared according to Reference 1 (Y. Ueki et al., Anal. Chem., December 2004, p.7007 to 7012). The resulting porous carrier was washed by shaking overnight in ethanol and air-dried. The pore diameter of the mesopores of the porous carrier thus obtained was 30 nm or less as shown in Table 1 below. Hereinafter, the porous carrier is referred to as an organic monolith carrier.

[2] Surface modification of organic monolith carrier After washing the organic monolith carrier obtained in [1] above with a phosphate buffer (PBS, pH 7.4), cysteine is contacted under a concentration of 0.01% by mass. And fixed at 40 ° C. overnight. The organic monolith carrier was washed again with PBS. Thereafter, the organic monolith carrier and protein A (1 mg / mL) are contacted at room temperature for 1 hour to immobilize protein A on the surface of the organic monolith carrier, and the organic monolith carrier is washed with PBS. The surface modification of was finished (Example 1).

  Non-immobilized protein A was measured on the organic monolith carrier according to Example 1 using a 660 nm protein assay kit (Thermo Scientific), and the amount of protein A immobilized on the organic monolith carrier was estimated. Thereafter, blocking was performed by injecting bovine serum albumin (BSA) into an organic monolith carrier in order to prevent the target substance antibody from being adsorbed to other than protein A immobilized on the carrier.

[Comparative Example 1]
An organic monolith support according to Comparative Example 1 was prepared in the same manner as in Example 1 except that cysteine was not immobilized and only protein A was immobilized.
Non-immobilized protein A was measured using a 660 nm protein assay kit (manufactured by Thermo Scientific) on the organic monolith carrier according to Comparative Example 1, and the amount of protein A immobilized on the organic monolith carrier was estimated. Thereafter, as in Example 1, blocking was performed by injecting bovine serum albumin (BSA) into an organic monolith carrier.

[Comparative Example 2]
An organic monolith support according to Comparative Example 2 was produced in the same manner as in Example 1 except that cysteine was contacted and immobilized under a concentration condition of 1% by mass.

[Comparative Example 3]
An organic monolith support according to Comparative Example 3 was prepared in the same manner as in Example 1 except that cysteine was contacted and immobilized under a concentration condition of 0.1% by mass.

[Example 2]
As in Example 1, according to Reference Document 1 (Y. Ueki et al., Anal. Chem., December 2004, p. 7007 to 7012), ethylene glycol dimethacrylate (EDMA), glycidyl methacrylate (GMA), azobisiso An organic monolith support according to Example 2 was prepared by changing the mixing ratio of butyronitrile (AIBN), n-propanol, 1,4-butanediol, and water. The pore diameter of the mesopores of the porous carrier thus obtained was 10 to 50 nm as shown in Table 1 below.

[Comparative Example 4]
An organic monolith support according to Comparative Example 4 was produced in the same manner as in Example 2 except that cysteine was not immobilized and only protein A was immobilized.

  Using the produced organic monolith carriers according to Examples 1 and 2 and Comparative Examples 1 to 4, the following [Measurement 1] to [Measurement 3] were appropriately measured.

[Measurement 1] Measurement of pore size distribution With respect to the organic monolith carriers according to Examples 1 and 2, the pore size distribution was measured using a specific surface area measurement device by a gas adsorption method. The results are shown in Table 1.

As shown in Table 1, the organic monolith support according to Example 1 and the organic monolith support according to Example 2 are both mesopores having a pore size distribution of several tens of nanometers and macropores having a pore size of several hundreds of nanometers. Polarization was confirmed. As shown in Table 1, the mesopore size in the region of the pore volume of 0.1 cm ³ / g · nm or more in the organic monolith support according to Example 1 was 30 nm or less. In addition, as shown in Table 1, the mesopore size in the region of the pore volume of 0.1 cm ³ / g · nm or more in the organic monolith support according to Example 2 was 10 to 50 nm.
From the above results, it can be said that Comparative Examples 1 to 3 produced under the same conditions as in Example 1 also have the same mesopore size and macropore size as in Example 1. Moreover, it can be said that the comparative example 4 produced on the conditions similar to Example 2 also has the same mesopore size and macropore size as Example 2. FIG.

[Measurement 2] Antibody capture amount per unit amount of protein A of organic monolith carrier Using each organic monolith carrier according to Examples 1 and 2 and Comparative Examples 1 to 4, the relative amount of antibody capture per unit amount of protein A , Sometimes simply referred to as “antibody capture amount”).
First, the antibody capture amount of each organic monolith carrier according to Examples 1 and 2 and Comparative Examples 1 to 4 was determined as follows, and as described later, this antibody capture amount was used to determine the amount of protein A per unit amount. The relative amount of antibody capture was calculated.

Each organic monolith carrier according to Examples 1 and 2 and Comparative Examples 1 to 4 was connected to a chromatography system (manufactured by GE Healthcare), and the solution on the downstream side of the carrier was used as an eluent while monitoring the absorbance at a wavelength of 280 nm. The inside of the carrier was washed by passing (pH 7.4) at a flow rate of 0.5 mL / min. After confirming that the baseline of the chromatograph was stable, the valve was switched and a 1 mg / mL antibody protein (IgG) solution as a treatment solution was continuously passed at a flow rate of 0.5 mL / min. The treatment solution was continued until the absorbance of the antibody that passed through the carrier was saturated. And the antibody capture | acquisition amount of each organic monolith carrier which concerns on Examples 1, 2 and Comparative Examples 1-4 was calculated | required by following formula (1).

Antibody capture amount = added antibody amount at the time when 5% leakage of the added antibody was observed Formula (1)

And the antibody capture relative amount per protein A unit amount of each organic monolith carrier according to Examples 1 and 2 and Comparative Examples 1 to 4 using the antibody capture amount determined by Formula (1) is expressed by the following formula (2). Calculated by However, in the following formula (2), X is the antibody capture amount (mg), and Y is the protein A immobilized amount (mg).

Relative amount of antibody captured per unit amount of protein A = X / Y (2)

[Measurement 3] Measurement of Nonspecific Adsorption Using each organic monolith support according to Example 1 and Comparative Example 1, nonspecific adsorption was measured as follows. For the measurement of non-specific adsorption, each organic monolith carrier according to Example 1 and Comparative Example 1 was washed with PBS (pH 7.4), contacted with cysteine (0.01% by mass), and kept at 40 ° C. overnight. Immobilization was performed. Thereafter, the carrier was connected to a chromatography system (manufactured by GE Healthcare), and PBS (pH 7.4) was passed through the solution on the downstream side of the carrier as an eluent while monitoring the absorbance at a wavelength of 280 nm at a flow rate of 0.5 mL / min. The inside of the carrier was washed with liquid. After confirming that the baseline of the chromatographic chart was stable, 100 μL of 1 mg / mL BSA was added to confirm the peak waveform when passing through the carrier. The peak area was determined, and the amount of BSA adsorbed nonspecifically to the carrier was calculated from a calibration curve prepared in advance.

[Test results and discussion]
[1] Comparison of antibody capture amount in the presence or absence of zwitterionic substance (cysteine) FIG. 8 shows relative antibody capture relative to the protein A fixed amount of Example 1 and Comparative Example 1 measured in [Measurement 2]. The quantity results are shown.
As shown in FIG. 8, in the organic monolith carrier according to Example 1, cysteine was immobilized on the carrier simultaneously with protein A. Therefore, compared with the organic monolith carrier according to Comparative Example 1, the antibody capture relative amount (comparison) It was confirmed that the relative amount based on the antibody capture amount of Example 1) increased.

Usually, under continuous liquid passage conditions, the smaller the mesopore (pore 125) pore diameter, the more difficult the antibody will enter, so a sufficient amount of antibody is captured against the immobilized protein A. I can't.
However, in the organic monolithic carrier according to Example 1, since cysteine was immobilized on the carrier surface as a zwitterionic substance, the hydrophilicity of the mesopores was increased and the infiltration of the solution was facilitated. Therefore, it is considered that the opportunity for the antibody to contact protein A immobilized in the mesopores has increased.

From the above viewpoint, as well as cysteine as a zwitterionic substance, a derivative of cysteine in which at least one of the functional groups of cysteine (for example, a hydroxyl group) is substituted with another functional group, and sulfobetaine 3-undecane It was speculated that thiols could also be used. In addition, as zwitterionic substances, zwitterionic groups such as phosphorylcholine groups, polyethylene glycol, and the like are functional groups that are neutral as charges and highly hydrophilic, so that instead of cysteine or together with cysteine, phosphorylcholine groups and It was speculated that at least one of the polyethylene glycols could be used.
Furthermore, the present invention also allows the cysteine thiol group to be selectively covalently bonded to the glycidyl group under neutral mild conditions simply by contacting the glycidyl group of the porous carrier with cysteine at a temperature of 40 ° C. It was confirmed that this is an advantage of the immobilization method.

[2] Optimization of Zwitterionic Substance (Cysteine) Immobilization Amount Also, FIG. 9 shows the concentration at the time of cysteine immobilization (cysteine concentration) in Example 1 and Comparative Examples 2 and 3 measured in [Measurement 2] above. The comparison result of the amount of antibody captured per unit amount of protein A (based on the measurement result of Example 1) is shown.
As shown in FIG. 9, when the concentration at the time of cysteine immobilization was 0.1% by mass or more, it was confirmed that protein A hardly captured the antibody. From this result, it is inferred that when the zwitterionic substance is excessively present on the surface of the porous carrier, the antibody is prevented from approaching the vicinity of the carrier surface and from entering the mesopores of the carrier. Accordingly, the conditions for immobilizing cysteine as a zwitterionic substance on a carrier are preferably a concentration lower than 0.1% by mass, and more preferably 0.01% by mass.

[3] Comparison of effects in different pore diameters As shown in Table 1, the mesopores of the organic monolith support according to Example 1 have a pore diameter of 30 nm or less, and according to Comparative Example 1 manufactured under the same conditions. It can be said that the mesopore diameter of the organic monolith support is 30 nm or less.
FIG. 10 shows a comparison of the amount of antibody captured per unit amount of protein A (relative amount based on the measurement result of Comparative Example 1) in the organic monolithic carrier according to Example 1 and Comparative Example 1 measured in [Measurement 2]. Results are shown.
As shown in FIG. 10, since the organic monolith carrier according to Example 1 had cysteine immobilized thereon, the amount of antibody captured was 1. compared to the organic monolith carrier according to Comparative Example 1 to which cysteine was not immobilized. It increased 5 times.

In addition, as shown in Table 1, the mesopore diameter of the organic monolith support according to Example 2 was 10 to 50 nm, and the mesopores of the organic monolith support according to Comparative Example 4 manufactured under the same conditions were used. It can be said that the pore diameter is 10 to 50 nm.
FIG. 11 shows a comparison of the amount of antibody captured per unit amount of protein A (relative amount based on the measurement result of Comparative Example 4) in the organic monolithic carrier according to Example 2 and Comparative Example 4 measured in [Measurement 2]. Results are shown.
As shown in FIG. 11, since the cysteine was immobilized on the organic monolith support according to Example 2, the amount of antibody captured was 1. compared to the organic monolith support according to Comparative Example 4 on which cysteine was not immobilized. It increased by a factor of 1.

  The following can be said with reference to FIGS. 10 and 11 and Table 1 described above. That is, the group of organic monolith carriers according to Example 1 and Comparative Example 1 (a group having a small mesopore size) and the group of organic monolith carriers according to Example 2 and a Comparative Example 4 (a group having a large mesopore size) In comparison, it was found that the group having a smaller mesopore size has a higher effect of immobilizing cysteine. It was also found that the size of mesopores in the carrier can be set within the range of 1 nm to 50 nm, more preferably within the range of 1 nm to 30 nm.

[4] Comparison of non-specific adsorption amount with and without zwitterionic substance (cysteine) FIG. 12 shows the non-adsorption of BSA using each organic monolith carrier according to Example 1 and Comparative Example 1 measured in [Measurement 3]. The comparison result of specific adsorption amount (relative amount on the basis of the measurement result of the comparative example 1) is shown.
As shown in FIG. 12, each organic monolith carrier according to Example 1 had cysteine immobilized on the carrier. Therefore, compared to the organic monolith carrier according to Comparative Example 1 in which cysteine was not immobilized on the carrier, BSA The amount of nonspecific adsorption of (impurities) could be reduced by 10 times or more.
From the above results, as well as cysteine as a zwitterionic substance, a derivative of cysteine in which at least one of the functional groups of cysteine (for example, hydroxyl group) is substituted with another functional group, and sulfobetaine 3-undecanethiol It was speculated that can also be used. In addition, as zwitterionic substances, zwitterionic groups such as phosphorylcholine groups, polyethylene glycol, and the like are functional groups that are neutral as charges and highly hydrophilic, so that instead of cysteine or together with cysteine, phosphorylcholine groups and It was speculated that at least one of the polyethylene glycols could be used.

DESCRIPTION OF SYMBOLS 10, 10A, 10B, 10C Purification apparatus 11 Column tube 12 Filler 121 Carrier part 122 Through-hole part 123 Hole part 124 Zwitterionic substance 125 Capture part 13 Plug for liquid passage 100 Target substance manufacturing apparatus 110 Processing solution storage tank 120 Cleaning liquid storage Tank 130 Eluent storage tank 140 Valve 150 Flowing pump 160 Target substance storage tank

Claims (13)

A purification device for purifying the target substance,
A column tube and a filler filled in the column tube,
The filler comprises a porous carrier having a plurality of pores having a pore diameter of 1 nm or more and 50 nm or less,
The purification apparatus, wherein the porous carrier includes a zwitterionic substance and a capture unit that specifically binds to the target substance.   The purification apparatus according to claim 1, wherein the porous carrier is a monolith structure.   The purification apparatus according to claim 2, wherein the monolith structure is formed using an organic polymer.   4. The purification apparatus according to claim 3, wherein the organic polymer is formed using a monomer including an activation site.   The purification apparatus according to claim 4, wherein the activation site is a glycidyl group.   The purification apparatus according to claim 1, wherein the zwitterionic substance has a thiol group and is fixed to the porous carrier via the thiol group.   The purification apparatus according to claim 1, wherein the zwitterionic substance is at least one of an amino acid having a thiol group and an amino acid having a thiol group introduced therein.   The purification apparatus according to claim 1, wherein the zwitterionic substance is at least one of cysteine, a derivative of cysteine, sulfobetaine 3-undecanethiol, a phosphorylcholine group, and polyethylene glycol.   The purification apparatus according to claim 1, wherein the target substance is an antibody.   The purification apparatus according to claim 1, wherein the capturing unit is at least one of protein A, protein G, and protein L.   The purification apparatus according to claim 1, wherein a pore diameter of the pore portion is 1 nm or more and 30 nm or less. A column tube and a filler filled in the column tube,
The filler comprises a porous carrier having a plurality of pores having a pore diameter of 1 nm or more and 50 nm or less,
The porous carrier is a purification method for purifying a target substance using a purification apparatus including a zwitterionic substance and a capture unit that specifically binds to the target substance.
An equilibration step of equilibrating the packing material packed in the column tube;
After the equilibration step, a capturing step in which a processing solution containing the target substance and components other than the target substance is passed through the filler, and the target substance is captured by the capturing unit;
After the capturing step, a cleaning step of passing a cleaning liquid through the filler and washing away components other than the target substance captured by the capturing unit;
An elution step in which, after the washing step, an eluent is passed through the filler, and the target substance captured by the capturing part is eluted from the filler and recovered. .   13. A post-cleaning step is included, wherein after the elution step, a post-cleaning liquid is passed through the filler to wash away components other than the target substance remaining on the porous carrier. Purification method.