JP2008279366A - Porous carrier, adsorbents for purification using the same, manufacturing method thereof, and purification process using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a porous carrier which has strength to be capable of conducting the purification of antibody and the like with large scale and under high wire-speed without problems and which has a large amount of adsorption of the substance to be purified; to provide an adsorbent for purification using the same; to provide a manufacturing method thereof; and to simply and inexpensively provide the purification process using the same. <P>SOLUTION: The porous carrier is characterized in that: the compression stress at 5% compression is from 0.01 MPa or more to 1 MPa or less; the compression stress at 10% compression is from 0.03 MPa or more to 3 MPa or less; and the compression stress at 15% compression is from 0.06 MPa or more to 5 MPa or less. Also the adsorbent using the same, the manufacturing method thereof and the purification method using the same are provided. As a preferable embodiment, the strength of the porous carrier can be enlarged by causing a long-chain cross-linker with two or more functions to act. Thus, the purification of the antibody and the like can be performed with a large scale under a high speed and in a low cost. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an adsorbent for affinity chromatography, and more particularly to an adsorbent for purifying antibody drugs.

The porous carrier is widely used as an adsorbent for various chromatographies and an adsorbent for affinity chromatography. An adsorbent for affinity chromatography has been used as an adsorbent for purifying antibody drugs because it can efficiently purify a target substance or reduce the concentration of unwanted substances (see, for example, Non-Patent Document 1). In recent years, the antibody drug market has grown greatly, and in accordance with this, large-scale and high linear speeds of antibody drug purification have been actively carried out. With the increase in the scale of purification and the increase in linear velocity, it may be necessary to increase the strength of the adsorbent used for purification, that is, the porous carrier. When a porous carrier having a low strength is used at a large scale and under a high linear velocity, the porous carrier may be consolidated and problems such as a liquid not flowing may occur. Conventionally, as a gel with high strength, for example, a silica gel-based porous carrier as shown in Patent Document 1, for example, an agarose-based crosslinked porous carrier as shown in Patent Document 2, for example, a cellulose as shown in Patent Document 3 Known are porous porous carriers and crosslinked cellulose porous carriers.
JP-A-6-281638 Special table 2000-508361 JP-A-1-217041 "Affinity Chromatography", written by Seiichi Kasai et al., Tokyo Chemical Doujin, 1991

  However, silica gel-based porous carriers tend to have a small amount of adsorption of the purification target product, and are often difficult to use under alkaline conditions. In addition, conventional agarose-based crosslinked porous carriers often have a complicated crosslinking method and are often expensive. Cellulose-based porous carriers and cellulose-based crosslinked porous carriers have few disadvantages like glass-based porous carriers and agarose-based crosslinked porous carriers, but scales and lines that are becoming mainstream in recent antibody drug purification. At high speed, consolidation may occur.

  Therefore, the present invention has been made in view of the above-mentioned problems of the prior art, and has a strength capable of purifying antibodies and the like without causing compaction at a large scale and at a high linear velocity. It is an object of the present invention to provide a porous carrier having a large amount of adsorbed, an adsorbent for purification using the same, a production method thereof, and a purification method using them in a simple and inexpensive manner.

  In order to solve the above problems, the present inventors have provided the following invention as a result of intensive studies.

  That is, a porous stress having a compressive stress at 5% compression of 0.01 MPa to 1 MPa, a compressive stress of 10% compression of 0.03 MPa to 3 MPa, and a compressive stress of 15% compression of 0.06 MPa to 5 MPa. A quality carrier was provided.

  According to this, an adsorbent for purification having a strength capable of purifying antibodies and the like on a large scale and at a high linear velocity without problems and having a large amount of adsorption of the purification target product can be obtained.

  Further, the compression stress at 5% compression is 0.02 MPa to 1 MPa, the compression stress at 10% compression is 0.06 MPa to 3 MPa, and the compression stress at 15% compression is 0.1 MPa to 5 MPa, A porous support was provided.

  Moreover, the porous support | carrier bridge | crosslinked with the crosslinking agent whose functional group number is 2 or more and whose number of atoms between the most distant functional groups is 6 or more was provided.

  Moreover, the porous support | carrier characterized by the number of functional groups of a crosslinking agent being 3 or more was provided.

  Further, the present invention provides a porous carrier characterized in that a hydroxyl group exists between functional groups of the crosslinking agent.

  Moreover, the porous support | carrier characterized by the water content rate of a crosslinking agent being 50% or more was provided.

  Moreover, the porous support | carrier characterized by the viscosity of a crosslinking agent being 100 mPa * s or more and 50000 mPa * s or less was provided.

  Moreover, the porous support | carrier characterized by the functional group equivalent of a crosslinking agent being 100-600 is provided.

  Also provided is a porous carrier characterized in that the crosslinking agent is a glycidyl ether compound.

  In addition, a porous carrier is provided, wherein the amount of the crosslinking agent used is 0.01 to 10 times the volume of the carrier.

  Also provided is a porous carrier characterized in that the porous carrier contains a polysaccharide.

  Also provided is a porous carrier characterized in that the porous carrier contains cellulose or a cellulose derivative.

  Moreover, the porous support | carrier characterized by containing a formyl group was provided.

  Moreover, the porous support | carrier characterized by the volume average particle diameter being 20 micrometers or more and 300 micrometers or less was provided.

  Also provided is a method for producing a porous carrier.

  Also provided is an adsorbent for purification using a porous carrier.

  In addition, an adsorbent for purification is provided, which is characterized in that protein A is introduced as an affinity ligand.

  In addition, an adsorbent for purification is provided, wherein the amount of affinity ligand introduced is 1 mg or more and 1000 mg or less per mL of porous carrier.

  Further, the purification adsorbent is provided, wherein the amount of adsorption of the purification object is 1 mg or more per 1 mL of the purification adsorbent.

  Moreover, the manufacturing method of the adsorbent for refinement | purification was provided.

  Further, a purification method using the purification adsorbent was provided.

  Moreover, the purification method characterized by using a column 2 cm or more in diameter and 5 cm or more in height was provided.

  Moreover, the refinement | purification method characterized by having the process of letting liquid flow at a linear velocity of 300 cm / h or more was provided.

  According to the present invention, purification of antibody drugs and the like can be performed on a large scale, at high speed and at low cost.

  According to the present invention, antibody pharmaceuticals and the like can be purified on a large scale, at high speed and at low cost.

  In the present invention, the compression stress at 5% compression is 0.01 MPa to 1 MPa, the compression stress at 10% compression is 0.03 MPa to 3 MPa, and the compression stress at 15% compression is 0.06 MPa to 5 MPa. A porous support is provided.

  The present inventors have a compressive stress at 5% compression of 0.01 MPa to 1 MPa, a compressive stress of 10% compression of 0.03 MPa to 3 MPa, and a compressive stress of 15% compression of 0.06 MPa to 5 MPa. It has been found that a porous carrier that does not cause compaction even when purified at a large scale and at a high linear velocity can be obtained by the following porous carrier.

  If the compressive stress at 5% compression of the porous carrier is 0.01 MPa or more, the compressive stress at 10% compression is 0.03 MPa or more, and the compressive stress at 15% compression is 0.06 MPa or more, a large scale, A porous carrier that does not cause compaction even when purified at a high linear velocity is obtained. Moreover, if the compressive stress at 5% compression of the porous carrier is 1 MPa or less, the compressive stress at 10% compression is 3 MPa or less, and the compressive stress at 15% compression is 5 MPa or less, the brittleness is improved and fine particles are generated. Can be suppressed.

Here, the compression stress at the time of 5% compression is the stress when the porous carrier is compressed and the volume is reduced by 5% from the initial volume, and the compression stress at the time of 10% compression is the compression of the porous carrier. The stress when the volume is reduced by 10% from the initial volume and the compressive stress at the time of 15% compression are stresses when the porous carrier is compressed and the volume is reduced by 15% from the initial volume. The initial volume is a volume in a state in which the slurry containing the porous carrier is allowed to settle and filled until the volume of the porous carrier does not decrease while applying vibration to the slurry. The compression stress at the time of compression can be measured by the following method.
1) A 50 vol% slurry of a porous carrier is put into a glass graduated cylinder having an inner diameter of 15 mm.
2) While applying vibration to the glass graduated cylinder, settle and fill until the porous carrier volume does not decrease, and adjust the amount of the porous carrier so that the porous carrier volume is about 4 mL. The volume at this time is defined as the initial volume.
3) A metal piston (processed so as not to cause friction with the inner wall of the graduated cylinder and to prevent the porous carrier from leaking) is attached to an autograph (EZ-TEST manufactured by SHIMADZU) equipped with a 20N load cell.
4) Align the bottom surface of the piston with the position corresponding to 120 vol% of the porous carrier.
5) The piston is lowered at a test speed of 5 mm / min so as to prevent bubbles from entering, and the volume is reduced by compressing the porous carrier.
6) Measure the compressive stress at any point.

  Further, the porous carrier of the present invention has a compression stress at 5% compression of 0.01 MPa to 1 MPa, a compression stress of 10% compression of 0.04 MPa to 3 MPa, and a compression stress of 15% compression of 0. More preferably, the pressure is 0.06 MPa or more and 5 MPa or less. Further, the compression stress at 5% compression is 0.01 MPa or more and 1 MPa or less, the compression stress at 10% compression is 0.06 MPa or more and 3 MPa or less, and 15% compression. The compressive stress is preferably 0.07 MPa or more and 5 MPa or less. In particular, the compressive stress at 5% compression of the porous carrier is 0.02 MPa to 1 MPa, and the compressive stress at 10% compression is 0.06 MPa to 3 MPa and 15% compression because it is suitable for repeated use. The compressive stress at the time is particularly preferably 0.09 MPa or more and 5 MPa or less, and most preferably, the compressive stress at 5% compression is 0.02 MPa or more and 1 MPa or less, and the compressive stress at 10% compression is 0.08 MPa or more and 3 MPa. The compressive stress at 15% compression is 0.11 MPa or more and 5 MPa or less. Further, from the viewpoint of cost, the compression stress at 5% compression is 0.5 MPa or less, the compression stress at 10% compression is 1.5 MPa or less, and the compression stress at 15% compression is 2.5 MPa or less. Preferably, the compression stress at 5% compression is 0.2 MPa or less, the compression stress at 10% compression is 0.7 MPa or less, and the compression stress at 15% compression is 2 MPa or less, and further at 5% compression It is particularly preferable that the compressive stress is 0.1 MPa or less, the compressive stress at 10% compression is 0.3 MPa or less, and the compressive stress at 15% compression is 2 MPa or less.

  The method for increasing the strength of the porous carrier is not particularly limited, and a method for increasing the matrix content (for example, resin content) of the porous carrier is preferable, but from the advantage that the pore diameter of the porous carrier is difficult to be reduced. More preferably, the strength of the porous carrier is increased by acting a crosslinking agent. There are no particular limitations on the cross-linking agent and the cross-linking reaction conditions, and it can be carried out using known techniques. Examples thereof include halohydrins such as epichlorohydrin, epibromohydrin, dichlorohydrin, bifunctional bisepoxides (bisoxiranes), and polyfunctional polyepoxides (polyoxiranes). In addition, the present inventors have refined a porous carrier cross-linked with a cross-linking agent having two or more functional groups and six or more atoms between the most distant functional groups at a large scale and high linear velocity. It has been found that it is preferable as a porous carrier that does not cause compaction even if it is carried out. Although the reason is not clear, the present inventors have surprisingly found that when the number of functional groups of the crosslinking agent is 2 or more, the strength tends to increase. Moreover, although the reason is not clear, the present inventors have found that when the number of atoms between the most distant functional groups is 6 or more, the strength tends to increase. There are no particular limitations on the cross-linking agent used in the present invention having 2 or more functional groups and 6 or more atoms between the most distant functional groups. For example, resorcinol diglycidyl ether, neopentyl glycol Diglycidyl ether, 1,6-hexanediol diglycidyl ether, hydrogenate bisphenol A diglycidyl ether, glycerol diglycidyl ether, trimethylolpropane diglycidyl ether, diglycidyl terephthalate, diglycidyl orthophthalate, ethylene glycol diglycidyl ether, Examples include diethylene glycol diglycidyl ether and propylene glycol diglycidyl ether.

  Further, the number of functional groups of the crosslinking agent used in the present invention is preferably 3 or more because the strength of the porous carrier tends to increase. The crosslinking agent having 3 or more functional groups is not particularly limited. For example, glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, diglycerol polyglycidyl ether, polyglycerol polyglycidyl ether, sorbitol poly A glycidyl ether etc. can be mentioned.

  Moreover, it is preferable that the crosslinking agent used for this invention has a hydroxyl group between functional groups. The presence of a hydroxyl group between the functional groups of the cross-linking agent is preferable because the strength of the porous carrier tends to increase. Although the reason is not clear, the present inventors have surprisingly found that the presence of a hydroxyl group between the functional groups of the crosslinking agent tends to increase the strength of the porous carrier. Furthermore, it is more preferable that a hydroxyl group exists between the most distant functional groups of the crosslinking agent. Although the reason is not clear, the present inventors have surprisingly found that the presence of a hydroxyl group between the most distant functional groups of the crosslinking agent tends to increase the strength further.

  The cross-linking agent having a hydroxyl group between functional groups is not particularly limited. For example, sorbitol polyglycidyl ether (Nagase Chemtex Denacol EX-611, EX-612, EX-614, EX-614B, EX-622, etc.), polyglycerol polyglycidyl ether (Denacol EX-512, EX-521, etc. manufactured by Nagase ChemteX), diglycerol polyglycidyl ether (Denacol EX-421, manufactured by Nagase ChemteX), glycerol polyglycidyl Examples include ethers (Denacol EX-313, EX-314, etc., manufactured by Nagase ChemteX Corporation).

  In addition, the water content of the crosslinking agent used in the present invention is preferably 50% or more. It is preferable that the water content of the crosslinking agent is 50% or more because the strength of the porous carrier is further increased. In particular, when water is used as the solvent for the crosslinking reaction, it is preferable that the water content of the crosslinking agent is 50% or more because strength tends to increase. Although the reason is not clear, the present inventors have surprisingly found that the strength of the porous carrier tends to be increased when the water solubility of the crosslinking agent is 50% or more. Further, the water content of the crosslinking agent is particularly preferably 60% or more and 100% or less. Here, the water solubility refers to the ratio of the crosslinking agent actually dissolved in water when an operation of dissolving 10 parts of the crosslinking agent in 90 parts of water at room temperature is performed. The crosslinking agent having a water solubility of 50% or more is not particularly limited. For example, glycerol polyglycidyl ether (Nagase Chemtex Denacol EX-313, EX-314, etc.), diglycerol polyglycidyl ether (Nagase Chemtex) Denacol EX-421, etc.), polyglycerol polyglycidyl ether (Nagase ChemteX Denacol EX-512, EX-521, etc.), sorbitol polyglycidyl ether (Nagase Chemtex Denacol EX-614, EX-614B, etc.) , Ethylene glycol diglycidyl ether (Nagase ChemteX Denacol EX-810, EX-811 etc.), diethylene glycol diglycidyl ether (Nagase Chemtex Denacol EX-850, EX-851 etc.), PO Ethylene glycol diglycidyl ether (Denacol EX-821, EX-830, EX-832, EX-841, EX-861, EX-911, EX-941, EX-920, EX-931, etc. manufactured by Nagase ChemteX Corporation) Can be mentioned.

  Moreover, it is preferable that the viscosity of the crosslinking agent used for this invention is 100 mPa * s or more and 50000 mPa * s or less. When the viscosity of the cross-linking agent is within this range, the strength of the porous carrier is increased, and when used for the purification adsorbent, the amount of adsorption of the purification target product is increased, which is preferable. Although the reason is not clear, the present inventors have surprisingly discovered that the strength of the porous carrier tends to increase when the viscosity of the crosslinking agent is 100 mPa · s or more. Also, the reason is not clear, but surprisingly, if the viscosity of the cross-linking agent is 50000 mPa · s or less, the amount of the purification target adsorbed when the porous carrier is used as the adsorbent tends to increase. The present inventors have discovered. Moreover, the more preferable range of the viscosity is 100 mPa · s or more and 30000 mPa · s or less, more preferably 150 mPa · s or more and 25000 mPa · s or less, and particularly preferably 150 mPa · s or more and 5500 mPa · s or less. The viscosity can be measured with a Heppler type or B-type viscometer. The crosslinking agent having a viscosity of 100 mPa · s or more and 50000 mPa · s or less is not particularly limited. For example, resorcinol diglycidyl ether (such as Denacol EX-201 manufactured by Nagase ChemteX), neopentyl glycol diglycidyl ether (Nagase) Chematex Denacol EX-211, etc.), 1,6-hexanediol diglycidyl ether (Nagase ChemteX Denacol EX-212 etc.), hydrogenate bisphenol A diglycidyl ether (Nagase Chemtex Denacol EX-) 252), glycerol polyglycidyl ether (Nagase ChemteX Denacol EX-313, EX-314, etc.), trimethylolpropane polyglycidyl ether (Nagase Chemtex Denacol EX-321, etc.) , Pentaerythritol polyglycidyl ether (Danacol EX-411, etc., manufactured by Nagase ChemteX), diglycerol polyglycidyl ether (Denacol EX-421, manufactured by Nagase ChemteX), polyglycerol polyglycidyl ether (Denacol EX, manufactured by Nagase ChemteX) -512, EX-521, etc.), sorbitol polyglycidyl ether (Denacol EX-611, EX-612, EX-614, EX-614B, EX-622, etc., manufactured by Nagase ChemteX Corporation), diglycidyl terephthalate (Nagase ChemteX Corporation) Denacol EX-711, etc.), diglycidyl ortho-phthalate (Nagase ChemteX Denacol EX-721, etc.), ethylene glycol diglycidyl ether (Nagase Chemtex Denacol) X-810, EX-811, etc.), diethylene glycol diglycidyl ether (Nagase ChemteX Denacol EX-850, EX-851 etc.), polyethylene glycol diglycidyl ether (Nagase Chemtex Denacol EX-821, EX-830) EX-832, EX-841, EX-861, EX-911, EX-941, EX-920, EX-931, and the like.

  Moreover, it is preferable that the functional group equivalent of the crosslinking agent used for this invention is 100-600. When the functional group equivalent of the cross-linking agent is within this range, it is preferable because the strength of the porous carrier is increased and the amount of adsorption of the purification target product when used in the adsorbent is increased. The reason is not clear, but surprisingly, when the functional group equivalent is 100 or more and 600 or less, the present inventors have found that the strength of the porous carrier increases and the amount of adsorption of the purification target product tends to increase. Discovered. The more preferable range of the functional group equivalent of the crosslinking agent used in the present invention is 100 or more and 500 or less, and the more preferable range is 100 or more and 300 or less, and particularly preferably 130 or more and 190 or less. Here, the functional group equivalent is a weight per functional group equivalent, and can be determined by the molecular weight of the cross-linking agent divided by the number of functional groups. The cross-linking agent having a functional group equivalent of 100 or more and 500 or less is not particularly limited. For example, resorcinol diglycidyl ether (Nagase Chemtex Denacol EX-201 etc.), neopentyl glycol diglycidyl ether (Nagase Chemtex) Denacol EX-211, etc., manufactured by Co., Ltd.), 1,6-hexanediol diglycidyl ether (Denacol EX-212, manufactured by Nagase ChemteX Corporation), hydrogenated bisphenol A diglycidyl ether (Denacol EX-252, produced by Nagase ChemteX Corporation, etc.) ), Glycerol polyglycidyl ether (Denacol EX-313, EX-314, etc. manufactured by Nagase ChemteX), trimethylolpropane polyglycidyl ether (Denacol EX-321, manufactured by Nagase ChemteX), pentaerythri Polyglycidyl ether (Denacol EX-411, etc., manufactured by Nagase ChemteX), Diglycerol polyglycidyl ether (Denacol EX-421, manufactured by Nagase ChemteX), polyglycerol polyglycidyl ether (Denacol EX-, manufactured by Nagase ChemteX) 512, EX-521, etc.), sorbitol polyglycidyl ether (Nagase ChemteX Denacol EX-611, EX-612, EX-614, EX-614B, EX-622, etc.), diglycidyl terephthalate (manufactured by Nagase ChemteX) Denacol EX-711 etc.), diglycidyl ortho-phthalate (Nagase ChemteX Denacol EX-721 etc.), ethylene glycol diglycidyl ether (Nagase Chemtex Denacol EX-810, EX) 811 etc.), diethylene glycol diglycidyl ether (Nagase ChemteX Denacol EX-850, EX-851 etc.), polyethylene glycol diglycidyl ether (Nagase ChemteX Denacol EX-821, EX-830, EX-832, EX) -841, EX-861, EX-911, EX-941, EX-920, EX-931, etc.).

  Moreover, it is preferable that the crosslinking agent of this invention is a glycidyl ether type compound. Glycidyl ethers are preferred because they can be easily obtained industrially and the crosslinking reaction proceeds efficiently. The crosslinking agent containing a glycidyl ether compound is not particularly limited. For example, resorcinol diglycidyl ether (Nagase ChemteX Denacol EX-201 etc.), neopentyl glycol diglycidyl ether (Nagase Chemtex Denacol) EX-211, etc.), 1,6-hexanediol diglycidyl ether (Nagase ChemteX Denacol EX-212 etc.), hydrogenate bisphenol A diglycidyl ether (Nagase Chemtex Denacol EX-252 etc.), glycerol Polyglycidyl ether (Nagase ChemteX Denacol EX-313, EX-314, etc.), trimethylolpropane polyglycidyl ether (Nagase Chemtex Denacol EX-321, etc.), pentaerythrito Polyglycidyl ether (Denacol EX-411, manufactured by Nagase ChemteX), diglycerol polyglycidyl ether (Denacol EX-421, manufactured by Nagase ChemteX), polyglycerol polyglycidyl ether (Denacol EX-512, manufactured by Nagase ChemteX) , EX-521, etc.), sorbitol polyglycidyl ether (Nagase ChemteX Denacol EX-611, EX-612, EX-614, EX-614B, EX-622 etc.), diglycidyl terephthalate (Nagase Chemtex Denacol) EX-711), diglycidyl ortho-phthalate (Nagase ChemteX Denacol EX-721, etc.), ethylene glycol diglycidyl ether (Nagase Chemtex Denacol EX-810, EX- 11), diethylene glycol diglycidyl ether (Nagase ChemteX Denacol EX-850, EX-851 etc.), polyethylene glycol diglycidyl ether (Nagase Chemtex Denacol EX-821, EX-830, EX-832, EX) -841, EX-861, EX-911, EX-941, EX-920, EX-931, etc.).

  Moreover, it is preferable that the usage-amount of the crosslinking agent of this invention is 0.01 to 10 times the volume of a porous support | carrier. If the amount of the crosslinking agent used is 0.01 times or more of the volume of the carrier, it is preferable because the strength tends to increase due to the crosslinking reaction, and if it is 10 times or less, cost and unexpected radical reaction can be suppressed, Further, when a porous carrier is used for the adsorbent, the amount of the purification object to be purified tends to increase, which is preferable. Further, the more preferable range of the amount of the crosslinking agent used is 0.05 to 5 times the volume of the porous carrier, more preferably 0.1 to 3 times, particularly preferably 0.3 to 2 times. Yes, most preferably 0.3 times or more and 1 time or less.

  The method for increasing the strength of the porous carrier using these crosslinking agents is not particularly limited, but it is preferable to cause these crosslinking agents to act on the carrier under alkaline conditions from the viewpoint of reaction efficiency. There is no particular limitation on the method of charging the crosslinking agent, and the total amount used may be charged from the beginning of the reaction, the reaction may be repeated in multiple times, or the crosslinking agent may be added little by little using a dropping funnel or the like. Or a porous carrier may be charged into a reaction vessel charged with a crosslinking agent. The solvent for the crosslinking reaction is not particularly limited, but general-purpose organic solvents such as water, heptane, dimethyl sulfoxide, dimethylformamide, and dioxolane, alcohols such as ethanol, methanol, and propanol, and a mixed solvent of two or more of these are used. be able to. In order to increase the reaction efficiency, it is more preferable that a reducing agent such as sodium borohydride coexists. There is no particular limitation on the temperature during the crosslinking reaction, but it is preferably 0 ° C. or higher because it is advantageous in terms of reaction rate, and / or 100 ° C. or lower from the viewpoint of safety and damage to the porous carrier. Preferably, the temperature is 70 ° C. or lower because the functional group is difficult to deactivate. The cross-linking reaction is preferably performed with stirring or shaking, and the number of rotations or number of rotations per minute is 50 to 1000 for the reason that uniform stirring is possible and physical damage is not added to the porous carrier. Is preferably 100 times to 500 times, but is particularly preferably adjusted in accordance with the difference in specific gravity of each raw material and the strength of the porous carrier. The crosslinking reaction time is not particularly limited. However, when halohydrin is used, the bisepoxide (bisoxirane) or the polyepoxide (bisepoxide (bisepoxirane) or polyepoxide (bisepoxylan) is used when halohydrin is used because the functional group is deactivated or the carrier is less damaged. In the case of using (polyoxirane), it is preferably 2 hours or more and less than 15 hours, and more preferably adjusted in accordance with the reactivity of the crosslinking agent, the strength of the alkali, and the reaction temperature.

  The material of the porous carrier of the present invention is not particularly limited. For example, polysaccharide, polystyrene, styrene-divinylbenzene copolymer, polyacrylamide, polyacrylic acid, polymethacrylic acid, polyacrylic acid ester, polymethacrylic acid ester , Polyvinyl alcohol, and derivatives thereof. These have a coating layer such as a polymer material having a hydroxy group such as hydroxyethyl methacrylate or a graft copolymer such as a copolymer of a monomer having a polyethylene oxide chain and another polymerizable monomer. It may be. Among these, polysaccharides, polyvinyl alcohol, and the like can be preferably used because they easily introduce an active group on the surface of the carrier.

  Among these, it is more preferable that the porous carrier of the present invention contains a polysaccharide. Polysaccharides are preferred because they can be easily obtained industrially and have high safety to living bodies. The polysaccharide that can be used in the porous carrier of the present invention is not particularly limited, and examples thereof include agarose, cellulose, dextrin, chitosan, chitin, and derivatives thereof.

  The porous carrier of the present invention more preferably contains cellulose or a cellulose derivative. A porous carrier containing cellulose or a cellulose derivative has relatively high mechanical strength and is tough so that it is less likely to be broken or produce fine particles. When the column is packed, the liquid flows at a high linear velocity. Is also preferable because it is relatively difficult to consolidate. From the viewpoint of strength and cost, cellulose is most preferable as the material for the porous carrier of the present invention.

  The porous carrier of the present invention can be used as an adsorbent for purification. In order to use as an adsorbent for purification, an affinity ligand is often immobilized on a porous carrier. There are various immobilization methods such as cyanogen bromide method, trichlorotriazine method, epoxy method and tresyl chloride method shown in Tables 8 and 1 of FIGS. You can choose from the law. In particular, when immobilizing an affinity ligand having an amino group, the carrier formyl is used because of its safety, ease of immobilization, and use of a protein ligand produced by a relatively easy method. It is industrially preferable to use a reaction between the group and the amino group of the affinity ligand for immobilization. From these viewpoints, the porous carrier of the present invention preferably contains a formyl group. As a method for introducing a formyl group into a carrier, for example, there is a method in which a polysaccharide having a vicinal hydroxyl group is oxidized by a periodate oxidation method to form a formyl group on a sugar chain (hereinafter abbreviated as a sugar chain cleavage type). Can be mentioned. The adsorbent obtained through this method is preferred because of less leakage of the ligand. Further, as shown in FIGS. 8 and 15 of Non-Patent Document 1, various methods obtained by a method of causing glutaraldehyde to act, a method of causing periodate to act on a glyceryl group obtained by ring opening of an epoxy group, etc. And a method of introducing a formyl group via a spacer (hereinafter abbreviated as a spacer type). The adsorbent using the spacer type formyl group-containing carrier is preferable because the amount of the target substance to be purified tends to be relatively large. In addition, according to the invention that the present inventors have recently found uniquely, an amino sugar typified by glucosamine or the like was introduced as a spacer, and then a porous group containing a formyl group on the spacer by a periodate oxidation method was introduced. It has been found that by using a porous carrier, it is easy to obtain an adsorbent in which the leakage of the ligand is small and the adsorption amount of the purification object is large. Therefore, it is particularly preferable to apply this recent invention of the present inventors to a porous carrier having a high strength according to the present invention to obtain a porous carrier containing a formyl group and having a high strength. The formyl group content of these porous carriers is preferably 1 μmol or more and 100 μmol or less per mL of the porous carrier. If the formyl group content is 1 μmol or more per mL of porous carrier, the affinity ligand can be immobilized efficiently, and when used as a purification adsorbent, the amount of adsorption of the purification target is increased, which is preferable. Moreover, although the reason is not clear, the present inventors have surprisingly found that when the formyl group content is 100 μmol or less per mL of the porous support, the adsorption amount of the purification target product tends to increase. A more preferable range of the formyl group content is 2 μmol or more and 50 μmol or less per mL of the porous carrier, more preferably 2 μmol or more and 40 μmol or less, particularly preferably 4 μmol or more and 30 μmol or less, and most preferably 5 μmol or more and 25 μmol or less. . The formyl group content was determined by contacting 2 mL of a porous carrier substituted with 0.1 M phosphate buffer at pH 8 and 2 mL of 0.1 M phosphate buffer solution at pH 8 in which phenylhydrazine was dissolved, and stirring at 40 ° C. for 1 hour. The absorbance at the absorption maximum near 278 nm of the supernatant of the reaction solution is measured by UV measurement, and the amount of phenylhydrazine thus obtained is estimated as an adsorption amount to the porous carrier. At this time, the amount of phenylhydrazine input was 2 times the expected formyl group content, and when the amount of adsorption to the porous carrier was 25% or less or 75% or more with respect to the amount of phenylhydrazine input, The input amount of phenylhydrazine is reviewed and the measurement is performed again.

  The porous carrier of the present invention preferably has a volume average particle size of 20 μm or more and 300 μm or less. If the volume average particle diameter of the porous carrier is 20 μm or more, compaction is less likely to occur, and if it is 300 μm or less, the amount of adsorption of the purification target when used in the purification adsorbent is increased. A more preferable range of the volume average particle size of the porous carrier is 40 μm or more and 200 μm or less, more preferably 60 μm or more and 150 μm or less, particularly preferably 75 μm or more and 100 μm or less, and most preferably 80 μm or more and 95 μm or less. . The volume average particle diameter can be obtained by measuring the particle diameters of 100 randomly selected porous carriers. The particle size of each porous carrier is measured by taking a microphotograph of each porous carrier, storing it as electronic data, and using particle size measurement software (Image Pro Plus, manufactured by Media Cybernetics). be able to.

  The present inventors also provide an adsorbent for purification using the porous carrier of the present invention described above. The porous carrier of the present invention can be used as an adsorbent for purification. The adsorbent for purification that can use the porous carrier of the present invention is not particularly limited. For example, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, hydroxyapatite chromatography, etc. Examples of the adsorbent for various types of chromatography. In order to use the porous carrier of the present invention as an adsorbent for purification, an affinity ligand is often immobilized on the porous carrier. There are no particular limitations on the affinity ligand that can be immobilized, and various affinity ligands can be immobilized using a porous support activated to immobilize the desired affinity ligand. For example, as shown in Tables 8 and 1, Tables 8 and 2, and FIGS. 8 and 15 of Non-Patent Document 1, cyanogen bromide method, trichlorotriazine method, epoxy method, tresyl chloride method, periodate oxidation method, divinyl Method of immobilizing amino group-containing ligands using sulfonic acid method, benzoquinone method, carbonyldiimidazole method, acyl azide method, etc., method of immobilizing hydroxyl group-containing ligands using epoxy method, diazo coupling method, etc., epoxy method Various immobilization methods such as a method for immobilizing a thiol group-containing ligand using a tresyl chloride method, a divinyl sulfonic acid method, a method for immobilizing a carboxylic acid-containing ligand or a formyl group-containing ligand on an amination carrier Can be mentioned.

  There are no particular limitations on the affinity ligand when used in adsorbents for antibody drug purification, for example, antigens and proteins highly specific for antibodies, proteins G and L, variants thereof, and antibody binding activity A peptide etc. can be mentioned. In particular, as an adsorbent capable of specifically adsorbing and eluting immunoglobulin (IgG), an adsorbent in which protein A is immobilized on a carrier using an affinity ligand has attracted attention. In the field of antibody drug purification, large scale, high speed, and low cost are desired. From such a viewpoint, the adsorbent of the present invention is preferably a purification adsorbent into which protein A is introduced as an affinity ligand. There is no limitation in particular in the protein A which can be used for this invention, A natural product, a gene recombinant, etc. can be used without a restriction | limiting. Further, it may be an antibody binding domain and a variant thereof, a fusion protein or the like. In addition, molecular weight fractionation and fractionation using various chromatographic and membrane separation techniques such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and hydroxyapatite chromatography from bacterial cell extracts or culture supernatants. Protein A produced by combining and / or repeating purification methods selected from techniques such as fractional precipitation can also be used. In particular, protein A obtained by the method described in International Patent Publication WO2006 / 004067 and US Patent Publication US515151350 is preferable.

  The method for introducing protein A as an affinity ligand into the porous carrier can be selected from the various immobilization methods described above, but more preferably a formyl group contained in the porous carrier and an amino group of protein A. In particular, an amino sugar typified by glucosamine or the like is introduced as a spacer, and then a formyl group is contained on the spacer by a periodate oxidation method. In this method, an adsorbent for purification into which protein A is introduced as an affinity ligand is obtained using a porous support.

  The amount of the affinity ligand introduced into the purification adsorbent of the present invention is preferably 1 mg or more and 1000 mg or less per mL of the porous carrier. If the amount of the affinity ligand introduced is 1 mg or more per mL of the porous carrier, the amount adsorbed on the purification target is preferably increased, and if it is 1000 mg or less, the production cost can be suppressed. A more preferable affinity ligand introduction amount is 2 mg or more and 120 mg or less, more preferably 3 mg or more and 60 mg or less, particularly preferably 4 mg or more and 30 mg or less, and most preferably 5 mg or more and 15 mg or less per mL of the porous carrier. . The introduction amount of the affinity ligand can be determined by measuring the absorbance derived from the affinity ligand in the supernatant of the reaction solution after the immobilization reaction. In addition, the amount of affinity ligand introduced can be determined using elemental analysis. For example, in the case of an amino group-containing affinity ligand, the introduction amount of the affinity ligand can be measured by performing N content analysis of the adsorbent for purification.

  Moreover, it is preferable that the adsorption amount of the purification object of the purification adsorbent of the present invention is 1 mg or more per 1 mL of the purification adsorbent. It is preferable that the amount of the purification object to be adsorbed is 1 mg or more per 1 mL of the purification adsorbent, because the purification can be performed efficiently. Moreover, if the amount of adsorption of the purification object is 1000 mg or less per 1 mL of the purification adsorbent, it is preferable because the adsorbed purification object is easily eluted from the purification adsorbent. More preferably, the adsorption amount of the purification object of the purification adsorbent is 5 mg or more and 500 mg or less, more preferably 10 mg or more and 250 mg or less, particularly preferably 20 mg or more and 150 mg or less. Preferably they are 30 mg or more and 80 mg or less. The adsorption amount of the purification target product was obtained by adding 70 mg of the purification target product to 35 mL of pH 7.4 phosphate buffer (Sigma) with respect to 0.5 mL of the purification adsorbent substituted with pH 7.4 phosphate buffer (manufactured by Sigma). The solution can be determined by measuring the amount of reduction of the purified product in the supernatant after contacting with the solution dissolved in the company) and stirring at 25 ° C. for 2 hours.

  As described above, the adsorbent for purification of the present invention can be produced by selecting an appropriate one each time from various affinity ligand immobilization reactions. When using a protein as the affinity ligand, the reaction temperature is preferably from the melting point to 100 ° C., more preferably from the melting point to 70 ° C., and even more preferably from the melting point to 50 ° C., from the viewpoint that the protein is difficult to deactivate. Particularly preferably, the melting point is not lower than 30 ° C. and most preferably not lower than 15 ° C. In addition, the pH of the reaction solution in the case of using a protein as an affinity ligand is preferably from pH 2 to less than 13, more preferably from pH 3 to less than 12, and still more preferably pH 4 from the viewpoint that the protein is hardly deactivated. It is 11 or less, particularly preferably pH 5 or more and 11 or less, and most preferably pH 6 or more and 11 or less. On the other hand, the pH of the reaction solution is preferably 7 or more and 11 or less, more preferably 8 or more and 11 or less, and even more preferably 9 or more pH, considering that the amount of affinity ligand immobilized tends to increase as the pH increases. 11 or less, particularly preferably pH 10 or more and 11 or less. The method for charging the affinity ligand is not particularly limited, and the entire amount used may be charged from the beginning of the reaction, the reaction may be repeated in multiple times, or the affinity ligand may be added little by little using a dropping funnel or the like. Or a porous carrier may be charged into a reaction vessel charged with an affinity ligand. There is no particular limitation on the solvent for the affinity ligand immobilization reaction, but general-purpose organic solvents such as water, heptane, dimethyl sulfoxide, dimethylformamide, dioxolane, alcohols such as ethanol, methanol, and propanol, and mixtures of two or more of these. A solvent can be used. It is also preferable to add at least one of carbonates, citrates, acetates, phosphates and the like for the purpose of increasing reaction efficiency and suppressing fluctuations in pH. The preferred concentration of these salts in the reaction solution is 0.001M to 10M, more preferably 0.01M to 5M, still more preferably 0.05M to 1M, and particularly preferably 0.1M to 0.5M. Moreover, when using high molecular compounds, such as protein, as an affinity ligand, it is also preferable to add salts, such as salt, in order to suppress the multipoint coupling | bonding between a porous support | carrier and an affinity ligand. The preferred concentration of these salts such as sodium chloride in the reaction solution is 0.001M to 10M, more preferably 0.01M to 5M, still more preferably 0.05M to 1M, and particularly preferably 0.1M to 0.5M. It is. In addition, when using a formyl group-containing porous carrier, a reducing agent such as sodium borohydride, trimethylamine borane, dimethylamine borane, picoline borane, pyridine borane, sodium cyanoborohydride, or sodium triacetoxyborohydride is immobilized on an affinity ligand. It is preferable to stabilize the immobilization by adding after completion or coexisting in the immobilization reaction. It is also preferable to act a sealant for the purpose of inactivating the active groups remaining on the porous carrier after the affinity ligand is immobilized. The sealant is not particularly limited, but is preferably a low molecular compound containing a functional group that reacts with an active group on the porous carrier. For example, the active group on the porous carrier is an epoxy group or a formyl group. In this case, it is preferable to use a low molecular compound containing an amino group, and examples thereof include glycine, monoethanolamine, and tris. The affinity ligand immobilization reaction and the reaction related thereto (for example, reduction reaction or sealing reaction) are preferably carried out with stirring or shaking, and the number of rotations or frequency per minute can be uniformly stirred. In addition, it is preferably from 1 to 1000 times, more preferably from 10 to 500 times, more preferably from 25 to 300 times, particularly preferably from the reason that physical damage is not applied to the porous carrier. Is 50 times or more and 150 times or less, but it is particularly preferable to adjust according to the difference in specific gravity of each raw material or the strength of the porous carrier. The affinity ligand immobilization reaction time is not particularly limited, but is preferably 0.1 hours or more and 1000 hours or less, more preferably 0.1 hours or less, because the affinity ligand is deactivated or the carrier is less damaged. 5 hours or more and 100 hours or less, more preferably 1 hour or more and 50 hours or less, particularly preferably 1.5 hours or more and 24 hours or less, and most preferably 3 hours or more and 12 hours or less, but the reactivity, pH, reaction It is more preferable to adjust according to temperature.

  The purification adsorbent of the present invention can be used for purification of various purification objects using affinity chromatography and various purification methods as shown in Non-Patent Document 1. Furthermore, the adsorbent for purification of the present invention enables purification of the purification object at a large scale, at high speed and at low cost.

  Therefore, the purification method using the purification adsorbent of the present invention preferably uses a column having a diameter of 2 cm or more and a height of 5 cm or more. If the diameter is 2 cm or more and the height is 5 cm or more, purification can be performed efficiently. From the viewpoint of purification accuracy and efficiency, the column size is preferably 2000 cm or less in diameter and 5000 cm or less in height. More preferably, the column has a diameter of 4 cm to 200 cm and a height of 7 cm to 300 cm, more preferably a diameter of 8 cm to 100 cm and a height of 9 cm to 150 cm, particularly preferably a diameter of 16 cm to 85 cm and a high height. It is 12 cm or more and 85 cm or less, most preferably 25 cm or more and 85 cm or less in diameter and 14 cm or more and 35 cm or less in height.

  In addition, the purification method using the purification adsorbent of the present invention preferably includes a step of passing liquid at a linear speed of 300 cm / h or more. It is preferable to have a step of passing liquid at a linear velocity of 300 cm / h or more because purification can be performed efficiently. Further, from the viewpoint of purification accuracy and the durability of the purification apparatus, the purification using the purification adsorbent of the present invention is preferably performed at a linear velocity of 10,000 cm / h or less. More preferably, the linear velocity of purification is 400 cm / h or more and 5000 cm / h or less, more preferably 500 cm / h or more and 2500 cm / h or less, particularly preferably 600 cm / h or more and 1500 cm / h or less, and most preferably 700 cm / h or more and 1200 cm / h or less. It is as follows.

  The porous carrier of the present invention, the adsorbent for purification using the same, the production method thereof, and the purification method using the same have a scale, speed and cost of purification as compared with the case of not using the present invention. It can be remarkably improved.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

Example 1
A 1: 1 slurry of a porous cellulose carrier (CK-A made by Chisso) having a volume average particle size of 92 μm, a resin content of 6%, and an exclusion limit molecular weight of 50 million and RO water was added to a glass filter (TOP 17G- 2) Suction filtration (suction dry) was performed for 15 minutes above. 27.4 g of the obtained porous cellulose carrier that has been suction-dried is put into a plastic container (100 mL, manufactured by Sampratec), and 0.6 M aqueous sodium hydroxide solution (sodium hydroxide and RO water manufactured by Wako Pure Chemical Industries, Ltd.) is added thereto. 27.4 mL was added and heated at 40 ° C. for 30 minutes. After the liquid temperature was heated to 40 ° C., 54.8 mg of sodium borohydride (manufactured by Wako Pure Chemical Industries, Ltd.) and Denacol EX-314 (manufactured by Nagase ChemteX) containing glycerol polyglycidyl ether as a crosslinking agent It added in order of 27.4 mL, and it was made to react, shaking at 100 times / minute for 5 hours at 40 degreeC using the constant temperature shaker (Thermostatic water bath T-25 by Thomas Science company). After completion of the reaction, the target porous carrier was obtained by washing with 20 times the volume of RO water of the porous carrier while suction filtration on a glass filter (17G-2 manufactured by TOP).

(Example 2)
A target porous carrier was obtained in the same manner as in Example 1 except that Denacol EX-321 (manufactured by Nagase ChemteX Corporation) containing trimethylolpropane polyglycidyl ether as a crosslinking agent was used.
(Example 3)
A target porous carrier was obtained in the same manner as in Example 1 except that Denacol EX-421 (manufactured by Nagase ChemteX Corporation) containing diglycerol polyglycidyl ether as a crosslinking agent was used.

Example 4
The porous carrier produced in Example 1 was heated at 120 ° C. for 40 minutes using an autoclave (manufactured by Sakura High Pressure Sterilizer Neoclave), allowed to cool to room temperature, and then glass filter (17G-2, TOP). Above, it was washed with 5 times the volume of RO water of the porous carrier. The washed porous carrier was further subjected to a crosslinking reaction in the same manner as in Example 1 to obtain the intended porous carrier.

(Example 5)
The porous carrier produced in Example 2 was heated at 120 ° C. for 40 minutes using an autoclave (Sakura High Pressure Sterilizer Neoclave), allowed to cool to room temperature, and then a glass filter (17G-2 from TOP). Above, it was washed with 5 times the volume of RO water of the porous carrier. The washed porous carrier was further subjected to a crosslinking reaction in the same manner as in Example 2 to obtain the target porous carrier.

(Example 6)
The porous carrier prepared in Example 3 was heated at 120 ° C. for 40 minutes using an autoclave (Sakura High Pressure Sterilizer Neoclave), allowed to cool to room temperature, and then a glass filter (TOP G17G-2). Above, it was washed with 5 times the volume of RO water of the porous carrier. The washed porous carrier was further subjected to a crosslinking reaction in the same manner as in Example 3 to obtain the intended porous carrier.

(Example 7)
The porous carrier prepared in Example 4 was heated at 120 ° C. for 40 minutes using an autoclave (Sakura High Pressure Sterilizer Neoclave), allowed to cool to room temperature, and then glass filter (TOP G17G-2). Above, it was washed with 5 times the volume of RO water of the porous carrier. 2. 10 mL of washed porous carrier and 2.6 mL of RO water are placed in a centrifuge tube (50 mL manufactured by Iwaki Glass Co., Ltd.), and further 2M sodium hydroxide aqueous solution (adjusted with sodium hydroxide and RO water manufactured by Wako Pure Chemical Industries, Ltd.) 7 mL was added thereto and warmed at 40 ° C. for 30 minutes. After the liquid temperature is heated to 40 ° C., 1.3 mL of epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.) is added, and a constant temperature shaker (thermostatic water bath T-25, manufactured by Thomas Scientific Co., Ltd.) is used. The reaction was carried out at 40 ° C. for 2 hours with shaking at 100 times / minute. After completion of the reaction, it was washed with 20 times volume of RO water on the glass filter (17G-2 manufactured by TOP) to obtain an epoxidized porous carrier. 30 mL of this epoxidized porous carrier was placed on a glass filter (17G-2 manufactured by TOP Co., Ltd.) and adjusted to 0.5 mL carbonate buffer (adjusted with sodium bicarbonate, sodium carbonate and RO water manufactured by Wako Pure Chemical Industries, Ltd.) at a pH of 10 Substituted. 10 mL of the epoxidized porous carrier after replacement and 10 mL of 0.5 M carbonate buffer having a pH of 10 are placed in a centrifuge tube (50 mL manufactured by Iwaki Glass Co., Ltd.), and 95 mg of D (+)-glucosamine hydrochloride (manufactured by Wako Pure Chemical Industries, Ltd.) is added. In addition, using a constant temperature shaker (Thermostatic Water Bath T-25, manufactured by Thomas Science Co., Ltd.), the mixture was shaken at 50 ° C. for 4 hours at 100 times / minute, and then allowed to stand at room temperature for 12 hours for reaction. . After the reaction, it was washed with 20 times volume of RO water on the glass filter (TOP 17G-2) to obtain a glucosamined porous carrier. 8.5 mL of the obtained glucosamined porous carrier and 4.3 mL of RO water were placed in a centrifuge tube (50 mL manufactured by Iwaki Glass Co., Ltd.). Next, 98 mg of sodium periodate (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in 8.5 mL of RO water, this sodium periodate aqueous solution was added to the centrifuge tube, and an incubator (incubator LOW-TEMP manufactured by Iwaki Glass Co., Ltd.) was added. ICB-151L), using a mix rotor (Variable Mix Rotor VMR-5 manufactured by Inoue Seieido Co., Ltd.), the reaction was carried out at 25 ° C. for 1 hour while shaking at 100 times / minute. After the reaction, on a glass filter (17G-2 manufactured by TOP), it was washed with 20 times the volume of RO water of the porous carrier to obtain a formyl group-containing porous carrier. As a result of measuring the formyl group content of the obtained formyl group-containing porous carrier by the above-mentioned method, the formyl group content was 56 μmol per mL of the porous carrier. 7.4 mL of this formyl group-containing porous carrier was placed on a glass filter (17G-2, manufactured by TOP), 0.5 M phosphoric acid + 0.15 M saline buffer (pH 10), disodium hydrogen phosphate manufactured by Wako Pure Chemical Industries, Ltd., chloride (Adjusted with sodium, sodium hydroxide and RO water). 7.4 mL of a formyl group-containing porous carrier after replacement and 4.9 mL of 0.5 M phosphoric acid + 0.15 M saline buffer having a pH of 10 are placed in a centrifuge tube (50 mL manufactured by Iwaki Glass Co., Ltd.) and described in International Patent Publication No. WO2006 / 004067 1.13 mL of a protein A-containing solution (PNXL28 manufactured by Kaneka Corporation) having a protein A concentration of 52.6 mg / mL prepared in the above method was added, and in an incubator (incubator LOW-TEMP ICB-151L manufactured by Iwaki Glass Co., Ltd.). Using a mix rotor (Variable mix rotor VMR-5 manufactured by Inoue Seieido Co., Ltd.), the reaction was carried out with shaking at 100 times / minute at 4 ° C. for 12 hours. After adjusting using 4M hydrochloric acid (adjusted with hydrochloric acid and RO water manufactured by Wako Pure Chemical Industries, Ltd.) so that the pH of the reaction solution after the reaction is 8, 21 mg of sodium borohydride was added, and then at 4 ° C. for 1 hour. , Reacted while gently shaking. After the reaction, the absorbance at the absorption maximum near 277 nm of the reaction solution was measured. As a result, it was found that the introduction amount of protein A as an affinity ligand was 8 mg per 1 mL of the porous carrier. The porous support after the reaction was washed on a glass filter (17G-2 manufactured by TOP) with 50 times the volume of RO water of the porous support to obtain the target protein A-immobilized purification adsorbent. As a result of obtaining the adsorption amount of IgG, which is the purification target product of the obtained adsorbent for purification, by the above-mentioned method, it was found that it was 26 mg per 1 mL of the adsorbent for purification.

(Example 8)
Using the porous carrier prepared in Example 6, a formyl group-containing porous carrier having a formyl group content of 58 μmol per mL of porous carrier was obtained in the same manner as in Example 7. Next, in the same manner as in Example 7, the amount of protein A introduced as an affinity ligand was 8 mg per mL of the porous carrier, and the amount of adsorption of IgG as the purification target was 25 mg per mL of the adsorbent for purification. Got.

(Comparative Example 1)
A porous cellulose carrier (CK-A manufactured by Chisso) having a volume average particle size of 92 μm, a resin content of 6%, and an exclusion limit molecular weight of 50 million was used as a comparison of the present invention.

(Comparative Example 2)
A porous cellulose carrier (CK-C manufactured by Chisso) showing a resin content about 1.7 times that of CK-A was used as a comparison of the present invention.

(Comparative Example 3)
A porous carrier was obtained in the same manner as in Example 1 except that epichlorohydrin (manufactured by Wako Pure Chemical Industries, Ltd.) was used as the crosslinking agent.

(Reference Example 1)
RProtein A Sepharose Fast Flow (manufactured by GE Healthcare BioSciences AB) in which protein A was introduced as an affinity ligand into a porous agarose carrier was used as a reference.

(Reference example 2)
MabSelect (manufactured by GE Healthcare BioSciences AB), in which protein A was introduced as an affinity ligand in a porous agarose cross-linked carrier, was used as a reference.

(Compressive stress measurement)
The compressive stress of each porous carrier or purification adsorbent was measured by the method described above. The results are shown in Table 1 and FIG.

  The present invention can be used in the field of affinity chromatography, particularly in the field of antibody drug purification.

Examples of the present invention, comparative examples, and reference examples are shown.

Claims (23)

  A porous carrier having a compressive stress of 0.01 MPa to 1 MPa at 10% compression and a compressive stress of 0.03 MPa to 3 MPa at 10% compression, and a compressive stress of 0.06 MPa to 5 MPa at 15% compression .   The compressive stress at 5% compression is 0.02 MPa to 1 MPa, the compressive stress at 10% compression is 0.06 MPa to 3 MPa, and the compressive stress at 15% compression is 0.1 MPa to 5 MPa. 2. The porous carrier according to 1.   The porous carrier according to any one of claims 1 and 2, wherein the porous carrier is crosslinked by a crosslinking agent having two or more functional groups and having six or more atoms between the most distant functional groups.   The porous carrier according to any one of claims 1 to 3, wherein the porous carrier is crosslinked with a crosslinking agent having 3 or more functional groups.   The porous carrier according to any one of claims 1 to 4, wherein the porous carrier is crosslinked by a crosslinking agent having a hydroxyl group between functional groups.   The porous carrier according to any one of claims 1 to 5, wherein the porous carrier is crosslinked with a crosslinking agent having a water solubility of 50% or more.   The porous carrier according to any one of claims 1 to 6, wherein the porous carrier is crosslinked with a crosslinking agent having a viscosity of 100 mPa · s or more and 50000 mPa · s or less.   The porous carrier according to any one of claims 1 to 7, wherein the porous carrier is crosslinked with a crosslinking agent having a functional group equivalent of 100 or more and 600 or less.   The porous carrier according to any one of claims 1 to 8, wherein the porous carrier is crosslinked with a crosslinking agent which is a glycidyl ether compound.   The porous carrier crosslinked with a crosslinking agent, wherein the amount of the crosslinking agent used is 0.01 to 10 times the volume of the carrier. Porous support.   The porous carrier according to claim 1, wherein the porous carrier contains a polysaccharide.   The porous carrier according to any one of claims 1 to 11, wherein the porous carrier contains cellulose or a cellulose derivative.   A porous carrier according to any one of claims 1 to 12, wherein the porous carrier contains a formyl group.   14. The porous carrier according to claim 1, wherein the volume average particle diameter is 20 μm or more and 300 μm or less.   The manufacturing method of the porous support | carrier as described in any one of Claims 1-14.   An adsorbent for purification using the porous carrier according to any one of claims 1 to 15.   The adsorbent for purification according to claim 16, wherein protein A is introduced as an affinity ligand.   18. The purification adsorbent according to claim 16, wherein the amount of the affinity ligand introduced is 1 mg or more and 1000 mg or less per mL of the porous carrier.   The adsorbent for purification according to any one of claims 16 to 18, wherein the amount of adsorption of the purification object is 1 mg or more per 1 mL of the adsorbent for purification.   The method for producing an adsorbent for purification according to any one of claims 16 to 19.   A purification method using the purification adsorbent according to any one of claims 16 to 19.   The purification method according to claim 21, wherein a column having a diameter of 2 cm or more and a height of 5 cm or more is used.   The purification method according to any one of claims 21 and 22, further comprising a step of passing liquid at a linear velocity of 300 cm / h or more.