Formyl group-containing porous support, adsorbent using same, method for producing same, and method for producing the adsorbent

Abstract

The present invention relates to a method for producing a formyl group-containing porous base matrix, comprising the steps of introducing a spacer in a formyl group-containing porous particle; and then oxidizing the spacer with periodic acid and/or a periodate, to transform the part of the spacer into a formyl group; wherein the formyl group content in the porous particle after introduction of the spacer is not more than 3 mol per 1 mL of the porous particle. Also, the present invention relates to a method for producing an adsorbent, comprising the step of immobilizing an amino group-containing ligand on the formyl group-containing porous base matrix. According to the present invention, a formyl-group containing porous base matrix and an adsorbent produced from the porous base matrix of which adsorption amount is high and which is has high strength and of which ligand is difficult to be leaked are provided.

Claims

1. A method for producing a formyl group-containing porous base matrix, comprising the steps of: (a) introducing a spacer in a formyl group-containing porous particle by reacting formyl groups therein with functional groups in the spacer; subsequent to step (a), (b) treating formyl groups in said particle not used for introducing the spacer so that the formyl group content in the porous base matrix after introduction of the spacer is not more than 3 mol per 1 mL of the porous particle; and, subsequent to step (b), (c) oxidizing the spacer with periodic acid and/or a periodate, to transform a portion of the spacer into a formyl group.

2. The production method of a formyl group-containing porous base matrix according to claim 1, wherein the introduced spacer is a sugar or a sugar analog.

3. The production method of a formyl group-containing porous base matrix according to claim 1, wherein the introduced spacer is glucosamine.

4. The production method of a formyl group-containing porous base matrix according to claim 1, wherein the porous particle contains a polysaccharide.

5. The production method of a formyl group-containing porous base matrix according to claim 4, wherein the polysaccharide is cellulose and/or a cellulose derivative.

6. The production method of a formyl group-containing porous base matrix according to claim 1, wherein the porous particle is crosslinked.

7. A formyl group-containing porous base matrix, obtained by the production method of a formyl group-containing porous base matrix according to claim 1.

8. A method for producing an adsorbent comprising the step of immobilizing an amino group-containing ligand on the formyl group-containing base matrix according to claim 7.

9. An adsorbent, obtained by the production method according to claim 8.

10. A method for purification, wherein the adsorbent according to claim 9 is used.

Description

EXAMPLES

(1) Hereinafter, the examples of the present invention are described, but the present invention is in no way limited by the examples. The volume of the porous particle and the porous base matrix for reaction means the gravity settled volume unless otherwise specified. The gravity settled volume is obtained by placing a slurry of the porous base matrix and RO water in a weighing apparatus and allowing to stand for 2 hours in a vibration-free state until the volume could not be further reduced. In addition, unless otherwise specified, the volume of the porous base matrix when the functional group content is measured is the volume when a slurry of the porous base matrix and RO water is placed in a weighing apparatus and allowed to settle under vibration until the volume could not be further reduced.

(2) Measurement of Content of Formyl Group

(3) The content of formyl group was obtained by bringing 2 mL of a porous base matrix or porous particle substituted with a 0.1 M phosphate buffer having pH 8 and 2 mL of a 0.1 M phosphate buffer having pH 8 containing dissolved phenylhydrazine into contact with each other, stirring the resulting mixture at 40 C. for 1 hour, measuring the absorbance of the supernatant of the mixture at the maximum absorption around 278 nm by UV measurement, and estimating the quantity of phenylhydrazine to be adsorbed onto the porous base matrix or porous particle. At that time, the quantity by mole of phenylhydrazine to be loaded was controlled to be 3 times as much as the content of the assumed formyl group, and when the quantity of phenylhydrazine to be adsorbed onto the porous base matrix or porous particle was not more than 15% or not less than 45% relative to the quantity of phenylhydrazine to be loaded, the quantity of phenylhydrazine to be loaded was re-examined and the measurement was carried out again.

(4) Measurement of Compressive Stress

(5) A slurry containing 50 vol % of a porous particle or an adsorbent was poured in a measuring cylinder with an inner diameter of 15 mm and made of glass. While vibrating the measuring cylinder made of glass, the porous particle or adsorbent was precipitated and packed until the volume of the porous particle or adsorbent was not further decreased and the amount of the porous particle or adsorbent was adjusted to 4 mL by volume. The volume at that time was defined as the initial volume. A piston made of a metal which was processed so as not to cause friction with the inner wall of the measuring cylinder and as to prevent elution of the porous particle or adsorbent was attached to an auto-graph equipped with a load cell for 20 N (EZ-TEST, manufactured by SHIMADZU Corporation). The bottom face of the piston was adjusted at the position corresponding to 120 vol % of the porous particle or adsorbent. The piston was moved downward at a testing rate of 5 mm/minute while preventing bubble generation to compact the porous particle or adsorbent and decrease the volume, and the compressive stress at a desired point was measured.

(6) Quantitative Determination of Quantity of Glucosamine to be Immobilized

(7) Each porous base matrix in an amount of 1 mL was transferred into a glass filter (3G-2, manufactured by TOP), substituted 3 times with a 0.01 N sodium hydroxide solution in an amount of 3 times as much as that of the gel, and washed 6 times with RO water in an amount of 3 times as much as that of the gel. Then, the porous base matrix gel was suction-filtered for drying for 5 minutes, and substituted with acetic acid (manufactured by Wako Pure Chemical Industries, Ltd.). The obtained gel after the substitution was transferred into a 50 mL beaker and diluted up to 30 mL with acetic acid for non-aqueous titration (manufactured by Wako Pure Chemical Industries, Ltd.). The resulting gel was subjected to titration using a potentiometric automatic titration apparatus (AT-610, manufactured by KEM) with 0.004 N perchloric acid/acetic acid which was obtained by dilution of 0.1 M perchloric acid/acetic acid solvent with acetic acid for non-aqueous titration manufactured by Wako Pure Chemical Industries, Ltd., to measure the amount of glucosamine to be immobilized.

(8) Measurement of Dynamic Binding Capacity and Concentration of Leaching Ligand in Target Substance

(9) (1) Production of Solutions

(10) The respective solutions were produced, and deformed before use:

(11) A solution: phosphate buffer having pH 7.4 (manufactured by Sigma)1;

(12) B solution: 35 mM sodium acetate having pH 3.5, produced from acetic acid, sodium acetate and RO water manufactured by Wako Pure Chemical Industries, Ltd.;

(13) C solution: 1 M acetic acid, produced from acetic acid and RO water manufactured by Wako Pure Chemical Industries, Ltd.;

(14) D solution: a 1 mg/mL human polyclonal IgG solution, produced from Gammagard manufactured by Baxter and A solution;

(15) E solution: 6 M urea;

(16) F solution: a solution obtained by adding 0.2 vol % of a surfactant (polyoxyethylene(20) sorbitan monolaurate, manufactured by Wako Pure Chemical Industries, Ltd.) to A solution;

(17) a neutralization solution: 2 M tris(hydroxymethyl)aminomethane, produced from tris(hydroxymethyl)aminomethane manufactured by Sigma with RO water.

(18) (2) Packing and Preparation

(19) As an apparatus for column chromatography, AKTA explorer 100 (manufactured by GE Healthcare Biosciences) was used, and a 22 m mesh was attached to a column with a diameter of 0.5 cm and a height of 15 cm, and 3 mL of each adsorbent of the present invention was put in, and an aqueous 20% ethanol solution (produced from ethanol and RO water manufactured by Wako Pure Chemical Industries, Ltd.) was passed at a linear rate of 450 cm/h for 1 hour to pack the adsorbent in the column. A 15 mL tube for sampling was set in a fraction collector, and the neutralization solution was previously put in the sampling tube for collecting the eluate.

(20) (3) Purification of IgG

(21) The A solution in an amount of 9 mL was passed at a linear rate of 300 cm/h and successively, while being monitored by UV, the D solution was passed at a linear rate of 300 cm/h until 10% of IgG was passed through. Then, 30 mL of the A solution was passed at a linear rate of 300 cm/h and 30 mL of the B solution was passed at a linear rate of 300 cm/h to elute IgG. Finally, 9 mL of the C solution was passed at a linear rate of 300 cm/h and 9 mL of the E solution was passed at a linear rate of 300 cm/h. The procedure after the completion of packing of the adsorbent was repeated further two times to measure the IgG quantity in the eluate and the concentration of the leaching ligand in IgG.

Production Example 1

(22) A porous cellulose particle having a volume average particle diameter of 92 m, a resin content of 6% and an exclusion limit molecular weight of 50,000,000 (CK-A, manufactured by Chisso Corporation) was classified in a wet condition for 2 hours using a mesh of 90 m (manufactured by NONAKA RIKAKI, wire diameter: 63 m) and a classifier (300-MM manufactured by TSUTSUI SCIENTIFIC INSTRUMENTS CO., LTD.) to obtain Porous particle A having a volume average particle diameter of 83 m.

(23) RO water was added to Porous particle A (2.3 L), to make the total volume 2.78 L, and the mixture was transferred into a separable flask. Into the flask, 0.246 L of 4N NaOH (prepared from NaOH manufactured by Wako Pure Chemical Industries, Ltd. and RO water) was added. In addition, sodium borohydride (3.3 g) was added, and the temperature of the mixture was increased to 40 C. in a water tank. A crosslinking agent containing glycerolpolyglycidyl ether (DENACOL EX314 manufactured by Nagase ChemteX Corporation) (1.64 L) was added thereto, and the mixture was stirred at 40 C. for 5 hours. After the reaction, the mixture was suction-filtered and washed with 20 times by volume of RO water on a glass filter (26G-2 manufactured by TOP) to obtain a crosslinked porous particle. The compressive stress of the obtained crosslinked porous particle was 0.020 MPa when compressed by 5%, 0.049 MPa when compressed by 10%, and 0.080 MPa when compressed by 15%.

(24) RO water was added to the obtained crosslinked porous particle, to make the total amount 2 times by volume of the amount of the crosslinked porous particle. The mixture was added into a glass beaker (1 L), and the beaker was sealed with two aluminium foils and heated at 120 C. for 40 minutes using an autoclave (Neoclave for high-pressure sterilization manufactured by SAKURA). After the temperature was cooled to room temperature, the mixture was suction-filtered and washed with RO water of which volume was 5 times relative to the porous particle of on a glass filter (26G-2 manufactured by TOP) to obtain a crosslinked porous particle in which epoxy group was transformed to glyceryl group.

(25) Then RO water was added to 2.3 L of the porous particle treated by autoclave, to make the total volume 2.78 L, and the mixture was transferred into a separable flask. Into the flask, 0.246 L of 4N NaOH (prepared from NaOH manufactured by Wako Pure Chemical Industries, Ltd. and RO water) was added. In addition, sodium borohydride (3.3 g) was added, and the temperature of the mixture was increased to 40 C. in a water tank. A crosslinking agent (DENACOL EX314 manufactured by Nagase ChemteX Corporation) (1.64 L) was added thereto, and the mixture was stirred at 40 C. for 5 hours. After the reaction, the mixture was suction-filtered and washed with RO water of which volume was 20 times relative to the porous particle on a glass filter (26G-2 manufactured by TOP) to obtain a crosslinked porous particle. The compressive stress of the obtained crosslinked porous particle was 0.020 MPa when compressed by 5%, 0.049 MPa when compressed by 10%, and 0.080 MPa when compressed by 15%.

(26) RO water was added to the obtained crosslinked porous particle, to make the total amount 2 times by volume of the amount of the crosslinked porous particle. The mixture was added into a glass beaker (1 L), and the beaker was sealed with two aluminium foils and heated at 120 C. for 40 minutes using an autoclave (Neoclave for high-pressure sterilization manufactured by SAKURA). After the temperature was cooled to room temperature, the mixture was washed with RO water of which volume was 5 times relative to the porous particle on a glass filter (26G-2 manufactured by TOP) to obtain Porous particle B.

Example 1

(27) To the porous Particle B obtained in Production Example 1 (523 mL), RO water was added to make the total volume 784.5 mL, and the mixture was transferred into a 2 L separable flask. The separable flask was immersed in a thermostat bash (Thermostatic water bath T-2S manufactured by THOMAS KAGAKU Co., Ltd.) at 25 C. A 11.5 mg/mL sodium periodate aqueous solution (523 mL) was prepared by dissolving sodium periodate (manufactured by Wako Pure Chemical Industries) in RO water. The solution was added into the separable flask, and the mixture was stirred with rotation number of 120 rpm at 25 C. for 1 hour. After the reaction, the porous particle was washed with RO water on a glass filter (26G-2 manufactured TOP) until the conductivity of the filtrate became not more than 5 S/cm. To the obtained porous particle, RO water was added to make the total volume 784.5 mL, and the mixture was transferred into a separable flask. The separable flask was immersed in a thermostat bash (Thermostatic water bath T-2S manufactured by THOMAS KAGAKU Co., Ltd.) at 25 C. A 11.5 mg/mL sodium periodate aqueous solution was prepared by dissolving sodium periodate (manufactured by Wako Pure Chemical Industries) in RO water. The solution (523 mL) was added into the separable flask, and the mixture was stirred using a stirrer (MAZELA Z) with rotation number of 120 rpm at 25 C. for 1 hour. After the reaction, the porous particle was washed with RO water on a glass filter (26G-2 manufactured TOP) until the conductivity of the filtrate became not more than 5 S/cm, to obtain Porous particle C. The electric conductivity of filtrate of washing was measured using an electric conductivity meter (ECTestr10 pure+ manufactured by EUTECH INSTRUMENTS). The content of formyl group of the obtained Porous particle C was measured by the above-described method; as a result, the content of formyl group was 68 mol per 1 mL of the Porous particle C.

(28) Then, a separable flask was marked up at the point of 1042 mL, and the obtained Porous particle C (521 mL) was added into the separable flask with RO water. The separable flask was immersed in a thermostat bash (Thermostatic water bath T-2S manufactured by THOMAS KAGAKU Co., Ltd.) with an input-cooler (ADVANTEC TBC120DA), and the reaction mixture was cooled to 15 C. After it was confirmed that the temperature of the reaction mixture became 15 C., fermentative glucosamine K (manufactured by Kyowa Hakko Co., Ltd.) of which volume was 10 times by mole relative to the content of formyl group of the Porous particle C was added, and the pH was adjusted to 11 by adding a sodium hydroxide aqueous solution. While the pH was fine-adjusted to 11 by adding a 4N sodium hydroxide aqueous solution, RO water was added to make the total volume of the reaction mixture 1042 mL. The mixture was stirred with rotation number of 120 rpm at 15 C. for 5 hours.

(29) Then, 2.96 g of sodium borohydride (manufactured by Wako Pure Chemical Industries) was added, and the mixture was stirred for 1 hour. After the reaction, the porous particle was washed on a glass filter (26G-2 manufactured by TOP) using RO water of which volume was 20 times by volume relative to the porous particle. The washed gel was added in a separable flask, and RO water was added to the mark of 1042 mL. Further sodium borohydride (2.96 g) was added, and the mixture was stirred at 25 C. for 1 hour. After the reaction, the porous particle was washed on a glass filter (26G-2 manufactured by TOP) using RO water of which volume was 20 times by volume relative to the gel. The reaction with sodium borohydride at 25 C. for 1 hour was repeated 2 times, and washing was carried out with RO water on a glass filter (26G-2 manufactured TOP) until the conductivity of the filtrate became not more than 5 S/cm, to obtain glucosaminized porous base matrix.

(30) The amount of glucosamine to be introduced in the porous base matrix was 17 mol per 1 mL of the porous base matrix. The content of remaining formyl group which could not be treated with a reducing agent after the immobilization of glucosamine per 1 mL of the porous base matrix was 0 mol.

(31) To the obtained glucosaminized porous base matrix (109 mL), RO water was added to make the total volume 163.5 mL, and the mixture was transferred into a 500 mL separable flask. Into the flask, 11.5 mg/mL of sodium periodate aqueous solution (109 mL) was added, and the mixture was stirred with rotation number of 120 rpm at 25 C. for 1 hour. After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured by TOP) using RO water until the conductivity of the filtrate became not more than 5 S/cm, to obtain Formyl group-containing porous base matrix D. The compressive stress of the obtained crosslinked porous base matrix was 0.028 MPa when compressed by 5%, 0.067 MPa when compressed by 10%, and 0.109 MPa when compressed by 15%. The content of formyl group per 1 mL of the Porous base matrix D was 6.2 mol.

Example 2

(32) To the Porous particle B obtained in Production Example 1 (435 mL), RO water was added to make the total volume 652 mL, and the mixture was transferred into a 2 L separable flask. The separable flask was immersed in a thermostat bash (Thermostatic water bath T-2S manufactured by THOMAS KAGAKU Co., Ltd.) at 25 C. A 46.0 mg/mL sodium periodate aqueous solution (217.5 mL) was prepared by dissolving sodium periodate (manufactured by Wako Pure Chemical Industries) in RO water. The solution (217.5 mL) was added into the separable flask, and the mixture was stirred with rotation number of 120 rpm at 25 C. for 15 minutes. After the reaction, the porous particle was washed with RO water on a glass filter (26G-2 manufactured TOP) until the conductivity of the filtrate became not more than 5 S/cm, to obtain Porous particle E. The content of formyl group of the obtained Porous particle E was measured; as a result, the content of formyl group per 1 mL of the Porous particle E was 45.6 mol.

(33) Then, a separable flask was marked up at the point of 820 mL, and the obtained Porous particle E (410 mL) was added into the separable flask with RO water. The separable flask was immersed in a thermostat bash (Thermostatic water bath T-2S manufactured by THOMAS KAGAKU Co., Ltd.) with an input-cooler (ADVANTEC TBC120DA), and the reaction mixture was cooled to 15 C. After it was confirmed that the temperature of the reaction mixture became 15 C., fermentative glucosamine K (manufactured by Kyowa Hakko Co., Ltd.) of which amount was 10 times by mole relative to the content of formyl group of the Porous particle E was added, and the pH was adjusted to 11 by adding a 4N sodium hydroxide aqueous solution (prepared from sodium hydroxide manufactured by Wako Pure Chemical Industries and RO water). While the pH was fine-adjusted to 11 by adding a 4N sodium hydroxide aqueous solution, RO water was added to make the total volume of the reaction mixture 820 mL. The mixture was stirred with rotation number of 120 rpm at 15 C. for 5 hours.

(34) Then, 2.33 g of sodium borohydride (manufactured by Wako Pure Chemical Industries) was added, and the mixture was stirred for 1 hour. After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured by TOP) using RO water of which amount was 20 times by volume relative to the gel. The washed gel was added in a separable flask, and RO water was added up to the mark of 820 mL. Further sodium borohydride (2.33 g) was added, and the mixture was stirred at 25 C. for 1 hour. After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured by TOP) using RO water of which amount was 20 times by volume relative to the gel. The reaction with sodium borohydride at 25 C. for 1 hour was repeated 2 times, and washing procedure was finally carried out with RO water on a glass filter (26G-2 manufactured TOP) until the conductivity of the filtrate became not more than 5 S/cm, to obtain a glucosaminized porous base matrix.

(35) The amount of glucosamine to be introduced in the porous base matrix was 11.0 mol/mL per 1 mL of the porous base matrix. The content of remaining formyl group which could not be treated with a reducing agent after the immobilization of glucosamine per 1 mL of the porous base matrix was 0.4 mol.

(36) To the obtained glucosaminized porous base matrix (80 mL), RO water was added to make the total volume 120 mL, and the mixture was transferred into a separable flask. A 11.5 mg/mL sodium periodate aqueous solution was prepared. The solution (40 mL) was added, and the mixture was stirred using a stirrer (MAZELA Z) with rotation number of 150 rpm at 25 C. for 30 minutes. After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured by TOP) using RO water until the conductivity of the filtrate became not more than 5 S/cm, to obtain a formyl group-containing porous base matrix. The content of formyl group was 3.4 mol per 1 mL of the porous base matrix.

Example 3

(37) A glucosaminized porous base matrix was obtained and a formyl group-containing porous base matrix was subsequently obtained by the same method as Example 2 except that the reaction condition was at 15 C. for 1 hour once and at 37 C. for 1 hour twice instead of at 15 C. for 1 hour once and at 25 C. for 1 hour six times in the reductive reaction after glucosamine immobilization. The amount of glucosamine to be introduced was 9.6 mol/mL per 1 mL of the glucosaminized porous base matrix. The content of remaining formyl group which could not be treated with a reducing agent after the immobilization of glucosamine per 1 mL of the porous base matrix was 0.3 mol. The content of the remaining formyl group of the formyl group-containing porous base matrix was 4.3 mol per 1 mL of the porous base matrix.

Example 4

(38) A glucosaminized porous base matrix was obtained and a formyl group-containing porous base matrix was obtained by the same method as Example 2 except that the reaction condition was at 15 C. for 1 hour once and at 50 C. for 1 hour once instead of at 15 C. for 1 hour once and at 25 C. for 1 hour six times in the reductive reaction after glucosamine immobilization. The amount of glucosamine to be introduced was 8.7 mol/mL per 1 mL of the glucosaminized porous base matrix. The content of the remaining formyl group which could not be treated with a reducing agent after the immobilization of glucosamine was 0.4 mol per 1 mL of the porous base matrix. The content of formyl group of the formyl group-containing porous base matrix was 4.3 mol per 1 mL of the porous base matrix.

Example 5

(39) The formyl group-containing base matrix obtained in Example 1 (109 mL) was substituted with a buffer of which pH was 11 and contained 0.5 M sodium citrate (manufactured by Wako Pure Chemical Industries) and 0.15 M sodium chloride (manufactured by Wako Pure Chemical Industries) (327 mL) on a glass filter (26G-2 manufactured TOP). The substituted formyl group-containing porous base matrix was added into a separable flask marked at 197.6 mL with a buffer of which pH was 11 and contained 0.5 M sodium citrate and 0.15 M sodium chloride. A 52.85 mg/mL protein A solution (PNXL30 manufactured by KANEKA corporation) of which protein A was prepared by the method described in WO2006/004067 (16.5 mL) was added thereto, and the pH was adjusted to 11 using a 4 N NaOH (prepared from NaOH manufactured by Wako Pure Chemical Industries and RO water) and the total volume of the reaction mixture was adjusted to 197.6 mL. The mixture was stirred in a thermostat bash (Thermostatic water bath T-2S manufactured by THOMAS KAGAKU Co., Ltd.) with an input-cooler (ADVANTEC TBC120DA) using a stirrer (MAZELA Z) with rotation number of 150 rpm at 4 C. for 12 hours.

(40) After the reaction, the pH of the reaction mixture was adjusted to 6.8 using 4 M hydrochloric acid (prepared from hydrochloric acid manufactured by Wako Pure Chemical Industries and RO water). Then, 0.309 g of sodium borohydride (manufactured by Wako Pure Chemical Industries) was added thereto, and the mixture was gently stirred at 4 C. for 1 hour. After the reaction, the absorbance of maximum absorption around 277 nm of the reaction mixture was measured; as a result, the amount of protein A as affinity ligand to be introduced was 7.2 mg per 1 mL of the porous base matrix.

(41) The porous base matrix after the reaction was washed with RO water of which amount was 20 times by volume relative to the porous base matrix on a glass filter (26G-2 manufactured by TOP), and the washed base matrix was added into a separable flask. RO water was added up to the mark of 197.6 mL, and sodium borohydride (0.309 g) was added, and the mixture was stirred at 25 C. for 1 hour. After the reaction, washing procedure was carried out with RO water of which volume was 20 times. Then, substitutive procedure was carried out using 3 times volume of 0.01 M hydrochloric acid (prepared from hydrochloric acid manufactured by Wako Pure Chemical Industries and RO water). To the substituted porous base matrix, 0.01 M hydrochloric acid was added to make the total volume 220 mL, and the mixture was added into a separable flask and stirred at room temperature for 30 minutes for acid washing.

(42) After the acid washing, the porous base matrix was washed on a glass filter (26G-2 manufactured by TOP) using RO water of which amount was 20 times by volume relative to the porous base matrix, and then substitutive procedure was carried out with 3 times volume of an aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate (prepared from sodium hydroxide and sodium sulfate and RO water manufactured by Wako Pure Chemical Industries). To the substituted porous base matrix, an aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate to make the total volume 220 mL. The mixture was added into a separable flask, and stirred at room temperature for 20 minutes for alkali washing.

(43) After the alkali washing, the porous base matrix was washed on a glass filter (26G-2 manufactured by TOP) using RO water of which amount was 20 times by volume relative to the porous base matrix, and then substitutive procedure was carried out with 3 times volume of PBS (manufactured by SIGMA) of which pH was 7.4. Washing procedure was carried out until the conductivity of the filtrate became not more than 5 S/cm, to obtain the desired adsorbent immobilized with protein A. The electric conductivity of filtrate of washing was measured using an electric conductivity meter (ESTestr10 pure+ manufactured by EUTECH INSTRUMENTS).

(44) As the target substance for the obtained adsorbent, human polyclonal IgG (Gammagard manufactured by Baxter) was selected. The dynamic adsorbent amount and the amount of the ligand leaked into the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 37 mg at first time, 37 mg at second time and 38 mg at third time. The concentration of the leaching ligand in the purified IgG was 38 ppm at first time, 20 ppm at second time and 19 ppm at third time, relative to IgG.

Example 6

(45) An adsorbent immobilized with protein A was obtained by the same method as Example 5 using the formyl group-containing base matrix obtained in Example 2. The dynamic adsorbent amount and the amount of the ligand leaked into the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 37 mg at first time, 38 mg at second time and 39 mg at third time. The concentration of the leaching ligand in the purified IgG was 23 ppm at first time, 19 ppm at second time and 19 ppm at third time, relative to IgG.

Example 7

(46) An adsorbent immobilized with protein A was obtained by the same method as Example 5 using the formyl group-containing base matrix obtained in Example 3. The dynamic adsorbent amount and the amount of the ligand leaked into the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 34 mg at first time, 35 mg at second time and 36 mg at third time. The concentration of the leaching ligand in the purified IgG was 27 ppm at first time, 24 ppm at second time and 16 ppm at third time, relative to IgG.

Example 8

(47) An adsorbent immobilized with protein A was obtained by the same method as Example 5 using the formyl group-containing base matrix obtained in Example 4. The dynamic adsorbent amount and the amount of the ligand leaked into the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 31 mg at first time, 32 mg at second time and 32 mg at third time. The concentration of the leaching ligand in the purified IgG was 16 ppm at first time, 16 ppm at second time and 8 ppm at third time, relative to IgG.

Example 9

(48) An adsorbent was obtained by the same method as Example 7 except that the temperature for immobilizing protein A was changed from 4 C. to 21 C. The dynamic binding capacity and the amount of the ligand leaked into the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 34 mg.

Example 10

(49) An adsorbent was obtained by the same method as Example 7 except that a 17 mg/mL sodium hydroxide solution (12.4 mL) was prepared by dissolving the same amount of sodium hydroxide into RO water and the solution was added in 25 batches for 1 hour instead of adding sodium hydroxide as a powder after immobilizing protein A. The dynamic binding capacity was measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 39 mg for first time, 38 mg for second time and 39 mg for third time.

Example 11

(50) An adsorbent was obtained by the same method as Example 7 except that a 17 mg/mL sodium hydroxide solution (0.5 mL) was added instead of adding sodium hydroxide as a powder after immobilizing protein A. The dynamic binding capacity and the amount of the ligand leaked into the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 39 mg for second time and 39 mg for third time.

Example 12

(51) An adsorbent was obtained by the same method as Example 7 except that dimethylamineborane (manufactured by Wako Pure Chemical Industries) of the same amount as sodium hydroxide was added as a powder instead of adding sodium hydroxide for the first time after immobilizing protein A. The dynamic binding capacity was measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 36 mg for first time and 38 mg for third time.

Comparative Example 1

(52) A glucosaminized porous base matrix and Formyl group-containing porous base matrix F were obtained by the same method as Example 1 except that reductive reaction was not carried out after immobilizing glucosamine. The amount of glucosamine to be introduced in the porous base matrix was 20.2 mol per 1 mL of the porous base matrix. The content of the remaining formyl group which could not be treated with a reducing agent after the immobilization of glucosamine was 7 mol. The content of formyl group of the formyl group-containing base matrix was 14.5 mol per 1 mL of the Porous base matrix F. An adsorbent immobilized with protein A was obtained by the same method as Example 5 using the obtained formyl group-containing porous base matrix. The dynamic binding capacity and the amount of the ligand leaked into the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 37 mg at first time, 38 mg at second time and 38 mg at third time. The concentration of the leaching ligand in the purified IgG was 87 ppm at first time, 47 ppm at second time and 56 ppm at third time, relative to IgG.

Comparative Example 2

(53) To the Porous particle B obtained in Production Example 1 (64 mL), RO water was added to make the total volume 96 mL, and the mixture was transferred into a separable flask. The separable flask was immersed in a thermostat bash (Thermostatic water bath T-2S manufactured by THOMAS KAGAKU Co., Ltd.) at 25 C. A 1.44 mg/mL sodium periodate aqueous solution (64 mL) was prepared by dissolving sodium periodate (manufactured by Wako Pure Chemical Industries) in RO water. The solution was added into the separable flask, and the mixture was stirred using a stirrer (MAZELA Z) with rotation number of 120 rpm at 25 C. for 1 hour. After the reaction, the porous particle was washed with RO water on a glass filter (26G-2 manufactured TOP) until the conductivity of the filtrate became not more than 5 S/cm, to obtain Formyl group-containing porous base matrix G. The content of formyl group of the obtained Porous base matrix G was measured; as a result, the content of formyl group was 5.9 mol per 1 mL of the porous base matrix.

(54) The formyl group-containing base matrix (54.5 mL) was substituted with a buffer of which pH was 11 and contained 0.5 M phosphoric acid (manufactured by Wako Pure Chemical Industries) and 0.15 M sodium chloride (manufactured by Wako Pure Chemical Industries) (165 mL) on a glass filter (26G-2 manufactured TOP). To the substituted formyl group-containing porous base matrix, a buffer of which pH was 11 and contained 0.5 M phosphoric acid and 0.15 M sodium chloride was added to make the total amount 90.5 mL. The mixture was added into a separable flask. A 52.85 mg/mL protein A solution (PNXL30 manufactured by KANEKA corporation) of which protein A was prepared by the method described in WO2006/004067 (8.25 mL) was added thereto, and the mixture was stirred in a thermostat bash (Thermostatic water bath T-2S manufactured by THOMAS KAGAKU Co., Ltd.) with an input-cooler (ADVANTEC TBC120DA) using a stirrer (MAZELA Z) with rotation number of 150 rpm at 4 C. for 12 hours.

(55) After the reaction, the pH of the reaction mixture was adjusted to 8 using 4M hydrochloric acid (prepared from hydrochloric acid manufactured by Wako Pure Chemical Industries and RO water). Then, 0.155 g of sodium borohydride (manufactured by Wako Pure Chemical Industries) was added thereto, and the mixture was gently stirred at 4 C. for 1 hour. After the reaction, the absorbance of maximum absorption around 276 nm of the reaction mixture was measured; as a result, the amount of protein A as affinity ligand to be introduced was 6.7 mg per 1 mL of the porous base matrix.

(56) The porous base matrix after the reaction was washed with RO water of which amount was 20 times by volume relative to the porous base matrix on a glass filter (26G-2 manufactured by TOP), and RO water was added to the washed base matrix to make the total volume 109 mL. The mixture was added into a separable flask. Sodium borohydride (0.155 g) was added thereto, and the mixture was stirred at 25 C. for 1 hour. After the reaction, washing procedure was carried out with 20 times by volume of RO water. Then substitutive procedure was carried out using 3 times volume of 0.01 M hydrochloric acid (prepared from hydrochloric acid manufactured by Wako Pure Chemical Industries and RO water). To the substituted porous base matrix, 0.01 M hydrochloric acid was added to make the total volume 109 mL, and the mixture was into a separable flask and stirred at room temperature for 30 minutes for acid washing.

(57) After the acid washing, the porous base matrix was washed on a glass filter (26G-2 manufactured by TOP) using RO water of which amount was 20 times by volume relative to the porous base matrix, and then substitutive procedure was carried out with 3 times volume of a aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate (prepared from sodium hydroxide and sodium sulfate and RO water manufactured by Wako Pure Chemical Industries). To the substituted porous base matrix, an aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate to make the total volume 109 mL. The mixture was added into a separable flask and stirred at room temperature for 20 minutes for alkali washing.

(58) After the alkali washing, the porous base matrix was washed on a glass filter (26G-2 manufactured by TOP) using RO water of which amount was 20 times by volume relative to the porous base matrix, and then substitutive procedure was carried out with 3 times volume of PBS (manufactured by SIGMA) relative to the gel and of which pH was 7.4. Washing procedure was carried out until the conductivity of the filtrate became not more than 5 S/cm, to obtain the desired adsorbent immobilized with protein A. The electric conductivity of filtrate of washing was measured using an electric conductivity meter (ESTestr10 pure+ manufactured by EUTECH INSTRUMENTS).

(59) The dynamic binding capacity and the amount of the ligand leaked into the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 35 mg at first time, 35 mg at second time and 35 mg at third time. The concentration of the leaching ligand in the purified IgG was >100 ppm at first time, >100 ppm at second time and >100 ppm at third time, relative to IgG.

Comparative Example 3

(60) A crosslinked porous particle was obtained by the same method as Production Example 1 except that CK-A manufactured by Chisso Corporation was not classified. Then, RO water was added to the crosslinked porous particle (11 mL) to make the total volume 12.6 mL, and the mixture was added into a centrifuge tube (50 mL, manufactured by IWAKI GARASU). To the centrifuge tube, 3.7 mL of 2 M sodium hydroxide aqueous solution (prepared from sodium hydroxide manufactured by Wako Pure Chemical Industries and RO water) was added. The mixture was heated to 40 C. for 30 minutes. After the temperature of the mixture became 40 C., 1.3 mL of epichlorohydrin (manufactured by Wako Pure Chemical Industries) was added thereto, and the mixture was shaked using a thermostat shaking apparatus (Thermostatic water bath T-25 manufactured by THOMAS KAGAKU Co., Ltd.) at 100 rotation/minute at 40 C. for 2 hours.

(61) After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured TOP) using RO water of which amount was 20 times by volume relative to the porous particle, to obtain epoxidized porous particle of which epoxy content was 5.7 mol per 1 mL of the porous particle. The epoxidized porous particle (9.5 mL) was substituted with a 0.5 M carbonate buffer of which pH was 10 (prepared from sodium hydrogencarbonate, sodium carbonate and RO water manufactured by Wako Pure Chemical Industries) (30 mL) on a glass filter (26G-2 manufactured TOP). To the epoxidized porous particle after substitution, a 0.5 M carbonate buffer of which pH was 10 was added to make the total volume 19 mL, and the mixture was added into a centrifuge tube (50 mL, manufactured by IWAKI GARASU). A hydrochloride salt of D(+)-glucosamine (manufactured by Wako Pure Chemical Industries) (0.18 g) was added thereto, and the mixture was shaked using a thermostat shaking apparatus (Thermostatic water bath T-25 manufactured by THOMAS KAGAKU Co., Ltd.) at 100 rotation/minute at 50 C. overnight.

(62) After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured TOP) using RO water of which amount was 20 times by volume relative to the porous base matrix, to obtain glucosaminized porous base matrix of which amount of glucosamine was 1.4 mol per 1 mL of the porous base matrix. To the obtained glucosaminized porous base matrix (10 mL), RO water was added to make the total amount 15 mL. The mixture was added into a centrifuge tube (50 mL, manufactured by IWAKI GARASU). Sodium periodate (manufactured by Wako Pure Chemical Industries) (115 mg) was dissolved in RO water (10 mL), and the sodium periodate aqueous solution was added to the centrifuge tube. The mixture was shaked using a mixrotor (Variable mixrotor VMR-5 manufactured IUCHISEIEIDO) in an incubator (Incubator LOW-TEMP ICB-151L manufactured by IWAKI GARASU) at 100 rotation/minute at 25 C. for 1 hour.

(63) After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured TOP) using RO water of which amount was 20 times by volume relative to the porous base matrix, to obtain a formyl group-containing porous base matrix. The content of formyl group of the obtained formyl group-containing porous base matrix was measured by the above-described method; as a result, the content of formyl group was 5.9 mol per 1 mL of the porous base matrix.

(64) The formyl group-containing base matrix (7.1 mL) was substituted with a buffer of which pH was 10 and contained 0.5 M phosphoric acid and 0.15 M sodium chloride (prepared from bissodium phosphorate, sodium chloride, sodium hydroxide and RO water manufactured by Wako Pure Chemical Industries) (30 mL) on a glass filter (26G-2 manufactured TOP). To the substituted formyl group-containing porous base matrix, a buffer of which pH was 10 and which contained 0.5 M phosphoric acid and 0.15 M sodium chloride was added to make the total amount 11.8 mL. The mixture was added into a centrifuge tube (50 mL, manufactured by IWAKI GARASU). A 52.6 mg/mL protein A solution (PNXL28 manufactured by KANEKA corporation) of which protein A was prepared by the method described in WO2006/004067 (1.08 mL) was added thereto, and the mixture was shaked using a mixrotor (Variable mixrotor VMR-5 manufactured IUCHISEIEIDO) in an incubator (Incubator LOW-TEMP ICB-151L manufactured by IWAKI GARASU) at 100 rotation/minute at 4 C. for 12 hours.

(65) After the pH of the reaction mixture was adjusted to 8 using 4 M hydrochloric acid (prepared from hydrochloric acid manufactured by Wako Pure Chemical Industries and RO water), sodium borohydride (0.02 g) was added thereto. The mixture was gently stirred at 4 C. for 1 hour. After the reaction, the absorbance of maximum absorption around 277 nm of the reaction mixture was measured; as a result, the amount of protein A as affinity ligand to be introduced was 4.7 mg per 1 mL of the porous base matrix.

(66) After the reaction, the porous base matrix was washed with RO water on a glass filter (26G-2 manufactured TOP) until the conductivity of the filtrate became not more than 5 S/cm, to obtain the desired adsorbent immobilized with protein A.

(67) As the target substance for the obtained adsorbent, human polyclonal IgG (Gammagard manufactured by Baxter) was selected. The dynamic binding capacity and the amount of the leaching ligand in the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 22 mg at first time, 23 mg at second time and 27 mg at third time. The concentration of the leaching ligand in the purified IgG was 297 ppm at first time, 214 ppm at second time and 198 ppm at third time, relative to IgG.

Production Example 2

(68) To the Porous particle B obtained by the same method as Production Example 1 (100 mL), RO water was added to make the total volume 150 mL, and the mixture was transferred into a separable flask (manufactured by TOP, 500 mL). Sodium periodate (manufactured by Wako Pure Chemical Industries) (2.30 g) was dissolved in RO water (50 mL), and the solution was added into the separable flask. The mixture was stirred with rotation number of 150 rpm at 25 C. for 15 minutes. After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured TOP) using RO water of which amount was 20 times by volume relative to the porous particle, to obtain Porous particle H. The content of formyl group of the obtained formyl group-containing porous particle was measured by the above-described method; as a result, the content of formyl group was 57 mol per 1 mL of the porous particle.

Example 13

(69) The Porous particle H prepared by the same method as Production Example 2 (99 mL) was mixed with RO water (99 mL) at 1:1, to obtain a slurry. After the temperature of the slurry was adjusted to 15 C., the slurry was suction-filtered for drying on a glass filter (26G-2 manufactured TOP) for 15 minutes. The obtained suction-dried Porous particle I was added into a glass separable flask (manufactured by TOP, 500 mL). Then, hydrochloride salt of glucosamine (glucosamine K manufactured by Kyowa Hakko Co.) (3.3 g) was added to the separable flask. Then, RO water of 15 C. was added while the hydrochloride salt of glucosamine was dissolved, and the total amount of the reaction mixture after dissolution was adjusted to 180 mL. After the temperature of the reaction mixture was adjusted to 15 C., the pH was adjusted to 10 and the total amount of the reaction mixture was adjusted to 198 mL using 4 N sodium hydroxide aqueous solution and RO water. Then, the mixture was stirred at 150 rotation/minute at 15 C. for 5 hours. Then, sodium borohydride (manufactured by Wako Pure Chemical Industries) (0.56 g) was added, and the mixture was stirred at 150 rotation/minute at 15 C. for 60 minutes. After the reaction, washing procedure was carried out with RO water of which amount was 40 times by mole relative to the porous base matrix on a glass filter (26G-2 manufactured TOP).

(70) Then, all of the washed porous base matrix was added into a glass separable flask (manufactured by TOP, 500 mL), and RO water was added to make the total volume 198 mL. Then, sodium borohydroxide (manufactured by Wako Pure Chemical Industries) (0.56 g) was added, and the mixture was stirred at 150 rotation/minute at 15 C. for 60 minutes. After the reaction, washing procedure was carried out on a glass filter (26G-2 manufactured by TOP) using RO water of which amount was 40 times by volume relative to the porous base matrix. The above-described procedure in was repeated two times, to obtain the desired porous base matrix. The amount of glucosamine to be immobilized in the porous base matrix was measured by non-water titration; as a result, the amount was 10 mol per 1 mL of the base matrix.

Example 14

(71) A porous base matrix was prepared by the same method as Example 13 except that pH was adjusted to 9 after the addition of the hydrochloride salt of glucosamine. The amount of glucosamine to be immobilized was 10 mol per 1 mL of the porous base matrix.

Example 15

(72) A porous base matrix was prepared by the same method as Example 13 except that pH was adjusted to 8 after the addition of the hydrochloride salt of glucosamine. The amount of glucosamine to be immobilized was 10 mol per 1 mL of the porous base matrix.

Example 16

(73) The porous base matrix prepared in Example 13 (94 mL) was substituted with a 0.01 M citrate buffer (prepared from trisodium citrate dihydrate, citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries) (280 mL), and the buffer was added to make the total volume 141 mL. The mixture was added into a separable flask (manufactured by TOP, 500 mL). Sodium periodate (manufactured by Wako Pure Chemical Industries) (0.54 g) was dissolved in RO water (94 mL), and the sodium periodate aqueous solution was added in the separable flask. The mixture was stirred at 150 rotation/minute at 5 C. for 40 minutes. After the reaction, washing procedure was carried out with RO water of which amount was 40 times by volume relative to the porous base matrix on a glass filter (26G-2 manufactured TOP), to obtain formyl group-containing porous base matrix. The content of formyl group of the obtained base matrix was measured by the above-described method; as a result, the content of formyl group was 4 mol per 1 mL of the porous base matrix.

(74) The formyl group-containing base matrix (92.8 mL) was substituted with a buffer containing 0.6 M trisodium citrate dihydrate and 0.2 M sodium chloride (prepared from trisodium citrate dihydrate, sodium chloride and RO water manufactured by Wako Pure Chemical Industries) (280 mL), and a buffer containing 0.6 M trisodium citrate and 0.2 M sodium chloride was added to make the total volume 132 mL. The mixture was added into a separable flask (manufactured by TOP, 500 mL). A 52.8 mg/mL protein A solution (PNXL35 manufactured by KANEKA corporation) of which protein A was prepared by the method described in WO2006/004067 (14.06 mL) was added thereto, and the pH was adjusted to 12 using a 0.08 N NaOH (prepared from NaOH manufactured by Wako Pure Chemical Industries and RO water), and the mixture was stirred at 150 rotation/minute at 4 C. for 4 hours.

(75) After the reaction, the pH of the reaction mixture was adjusted to 7 using 0.1 M citric acid (prepared from citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries). Then, dimethylaminoborane (408 mg) was added thereto, and the mixture was stirred at 150 rotation/minute at 4 C. for 1 hour and then at 150 rotation/minute at 25 C. for 8 hours. After the reaction, the absorbance of maximum absorption around 277 nm of the reaction mixture was measured; as a result, the amount of protein A as affinity ligand to be introduced was 7.6 mg per 1 mL of the porous base matrix.

(76) The porous base matrix after the reaction was washed with RO water of which amount was 10 times by volume relative to the porous base matrix on a glass filter (26G-2 manufactured by TOP), and substitutive procedure was carried out using 0.1 M citric acid (prepared from citric acid monohydrate manufactured by Wako Pure Chemical Industries and RO water). Then, 0.1 M citric acid was added to the substituted base matrix, to make the total volume 186 mL. The mixture was added into a separable flask (manufactured by TOP, 500 mL), and stirred at 150 rotation/minute at 25 C. for 30 minutes for acid washing.

(77) After the acid washing, the porous base matrix was substituted with an aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate (prepared from sodium hydroxide, sodium sulfate and RO water manufactured by Wako Pure Chemical Industries) of which volume was 3 times by volume on a glass filter (26G-2 manufactured by TOP). To the substituted porous base matrix, an aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate, to make the total volume 186 mL. The mixture was added into a separable flask and stirred at 25 C. for 20 minutes for alkali washing.

(78) After the alkali washing, the porous base matrix was substituted with a 0.5 M citrate buffer (prepared from trisodium citrate dihydrate, citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries) (278 mL) of which pH was 6. Then, washing procedure was carried out until the conductivity of the filtrate became not more than 5 S/cm.

(79) Subsequently, the porous base matrix was substituted with 20% aqueous ethanol (prepared from ethanol and RO water of Japanese Pharmacopoeia), and added into a polymer container (manufactured by SANPLATEC CO., LTD.), to obtain the desired adsorbent immobilized with protein A. The electric conductivity of filtrate of washing was measured using an electric conductivity meter (ESTestr10 pure+ manufactured by EUTECH INSTRUMENTS).

(80) As the target substance for the obtained adsorbent, human polyclonal IgG (Gammagard manufactured by Baxter) was selected. The dynamic binding capacity and the amount of the leaching ligand in the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 37 mg at first time per 1 mL of the porous base matrix. The concentration of the leaching ligand in the purified IgG was 24 ppm relative to IgG. The content of formyl group of the obtained adsorbent was measured by the above-described method; as a result, the content of formyl group of the obtained adsorbent was 0.2 mol per 1 mL of the porous base matrix.

Example 17

(81) The desired adsorbent was obtained by the same procedure of Example 16 except that the porous base matrix prepared in Example 14 was used. The dynamic binding capacity of IgG was 37 mg per 1 mL of the porous base matrix. The concentration of the leaching ligand in the purified IgG was 22 ppm relative to IgG.

Example 18

(82) The desired adsorbent was obtained by the same procedure of Example 16 except that the porous base matrix prepared in Example 15 was used. The dynamic binding capacity of IgG was 37 mg per 1 mL of the porous base matrix. The concentration of the leaching ligand in the purified IgG was 26 ppm relative to IgG.

Example 19

(83) An adsorbent was prepared by the same procedure of Example 17 except that a citrate buffer (prepared from trisodium citrate dihydrate, citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries) of which pH was 2 was used instead of a citrate buffer of which pH was 3. In the Example, the content of formyl group of the formyl group-containing base matrix was 6 mol per 1 mL of the porous base matrix, and the amount of protein A as an affinity ligand to be immobilized was 7.2 mg per 1 mL of the porous base matrix. The dynamic binding capacity of IgG was 35 mg per 1 mL of the adsorbent. The concentration of the leaching ligand in the purified IgG was 38 ppm.

Example 20

(84) An adsorbent was prepared by the same procedure of Example 17 except that a citrate buffer (prepared from trisodium citrate dihydrate, citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries) of which pH was 5 was used instead of a citrate buffer of which pH was 3. In the Example, the content of formyl group of the formyl group-containing base matrix was 4 mol per 1 mL of the porous base matrix, and the amount of protein A as an affinity ligand to be immobilized was 6.5 mg per 1 mL of the porous base matrix. The dynamic binding capacity of IgG was 35 mg. The concentration of the leaching ligand in the purified IgG was 42 ppm.

Example 21

(85) The formyl group-containing base matrix prepared by the same method as Example 1 (92.8 mL) was substituted with a buffer containing 0.5 M trisodium citrate and 0.15 M sodium chloride (prepared from trisodium citrate dihydrate, sodium chloride and RO water manufactured by Wako Pure Chemical Industries) (278 mL). To the substituted formyl group-containing porous base matrix, a buffer containing 0.5 M trisodium citrate and 0.15 M sodium chloride was added to make the total volume 130 mL. The mixture was added into a separable flask (manufactured by TOP, 500 mL). A 52.85 mg/mL protein A solution (PNXL30 manufactured by KANEKA corporation) of which protein A was prepared by the method described in WO2006/004067 (14.04 mL) was added thereto, and the pH was adjusted to 11 using a 4M NaOH (prepared from NaOH manufactured by Wako Pure Chemical Industries and RO water). Further a buffer containing 0.5 M trisodium citrate and 0.15 M sodium chloride (prepared from trisodium citrate dihydrate, sodium chloride and RO water manufactured by Wako Pure Chemical Industries) of which pH was 11 was added to make the total volume 168 mL, and then the mixture was stirred at 150 rotation/minute at 4 C. for 12 hours.

(86) After the reaction, the pH of the reaction mixture was adjusted to 6.8 using 4M hydrochloric acid (prepared from hydrochloric acid manufactured by Wako Pure Chemical Industries and RO water). Then, sodium borohydride (263 mg) was added thereto, and the mixture was stirred at 150 rotation/minute at 4 C. for 1 hour. After the reaction, the absorbance of maximum absorption around 277 nm of the reaction mixture was measured; as a result, the amount of protein A as affinity ligand to be introduced was 7.2 mg per 1 mL of the porous base matrix.

(87) The porous base matrix after the reaction was washed with RO water of which amount was 10 times by volume relative to the porous base matrix on a glass filter (26G-2 manufactured by TOP), and all of the washed base matrix was added into a separable flask (manufactured by TOP, 500 mL). RO water was added thereto to make the total volume 168 mL. Then, sodium borohydride (263 mg) was added, and the mixture was stirred at 150 rotation/minute at 25 C. for 1 hour. After the reaction, washing procedure was carried out with RO water of which volume was 10 times by volume relative to the porous base matrix on a glass filter (26G-2 manufactured by TOP), and substitutive procedure matrix was carried out using 0.01 M hydrochloric acid (prepared from hydrochloric acid and RO water manufactured by Wako Pure Chemical Industries and RO water) of which volume was 3 times by volume. To the substituted porous base matrix, 0.01 M hydrochloric acid was added to make the total volume 168 mL, and the mixture was into a separable flask (manufactured by TOP, 500 mL) and shaked at 150 rotation/minute at 25 C. for 30 minutes for acid washing.

(88) After the acid washing, the porous base matrix was washed on a glass filter (26G-2 manufactured by TOP) using RO water of which amount was 10 times by volume relative to the porous base matrix, and then substitutive procedure was carried out with 3 times volume of a aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate (prepared from sodium hydroxide and sodium sulfate and RO water manufactured by Wako Pure Chemical Industries). To the substituted porous base matrix, an aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate to make the total volume 168 mL. All of the mixture was added into a separable flask, and shaked at 150 rotation/minute at 25 C. for 20 minutes for alkali washing.

(89) After the alkali washing, substitutive procedure for the porous base matrix was carried out using a 0.01 M citrate buffer (trisodium citrate dihydrate, citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries and RO water) of which pH was 6 (278 mL), and the porous base matrix was washed using RO water until the conductivity of the filtrate became not more than 5 S/cm. Then substitutive procedure was carried out using 20% aqueous ethanol (prepared from ethanol of and RO water Japanese Pharmacopoeia), and the porous base matrix was added into a 250 mL polymer container (manufactured by SANPLATEC CO., LTD.) using 20% aqueous ethanol, to obtain the desired adsorbent immobilized with protein A. The electric conductivity of filtrate of washing was measured using an electric conductivity meter (ESTestr10 pure+ manufactured by EUTECH INSTRUMENTS).

(90) As the target substance for the obtained adsorbent, human polyclonal IgG (Gammagard manufactured by Baxter) was selected. The dynamic binding capacity and the amount of the leaching ligand in the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 36 mg at first time, 36 mg at second time and 37 mg at third time. The concentration of the leaching ligand in the purified IgG was 69 ppm at first time, 47 ppm at second time and 36 ppm at third time.

Example 22

(91) An adsorbent was prepared by the same procedure of Example 21 except that a buffer containing 0.025 M phosphoric acid and 1.5 M sodium sulfate (prepared from disodium hydrogenphosphate Duodecihydrate, sodium sulfate and RO water manufactured by Wako Pure Chemical Industries) was used instead of a buffer containing 0.5 M trisodium citrate and 0.15 M sodium chloride, and a buffer, containing 0.025 M phosphoric acid and 1.5 M sodium sulfate (prepared from disodium hydrogenphosphate Duodecihydrate, sodium sulfate and RO water manufactured by Wako Pure Chemical Industries) of which pH was 11 instead of a buffer containing 0.5 M trisodium citrate and 0.15 M sodium chloride of which pH was 11. The amount of protein A as affinity ligand to be introduced was measured similarly to Example 2; as a result, the amount was 7.4 mg per 1 mL of the porous base matrix.

Example 23

(92) An adsorbent was prepared by the same method of Example 21 except that the pH to be adjusted to 11 in Example 21 was changed to 12.5. The amount of protein A to be immobilized was 6.0 mg per 1 mL of the porous base matrix, and the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 33 mg for first time, 33 mg for second time and 33 mg for third time. The concentration of the leaching ligand in the purified IgG was 35 ppm for first time.

Example 24

(93) An adsorbent was prepared by the same method of Example 21 except that the pH to be adjusted to 11 in Example 21 was changed to 13. The amount of protein A to be immobilized was 4.0 mg per 1 mL of the porous base matrix, and the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 28 mg for first time, 33 mg for second time and 33 mg for third time. The concentration of the leaching ligand in the purified IgG was 23 ppm for first time.

Example 25

(94) An adsorbent was prepared by the same method of Example 21 except that the pH to be adjusted to 11 in Example 21 was changed to 10. The amount of protein A to be immobilized was 4.0 mg per 1 mL of the porous base matrix, and the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 29 mg.

Example 26

(95) The formyl group-containing base matrix prepared by the same method as Example 1 (92.8 mL) was substituted with a buffer containing 0.6 M trisodium citrate and 0.2 M sodium chloride (prepared from trisodium citrate dihydrate, sodium chloride and RO water manufactured by Wako Pure Chemical Industries) (280 mL). To the substituted Porous base matrix F, a buffer containing 0.6 M trisodium citrate and 0.2 M sodium chloride was added to make the total volume 132 mL. The mixture was added into a separable flask (manufactured by TOP, 500 mL). A 52.8 mg/mL protein A solution (PNXL35 manufactured by KANEKA corporation) of which protein A was prepared by the method described in WO2006/004067 (14.06 mL) was added thereto, and the pH was adjusted to 12 using a 0.08 N NaOH aqueous solution (prepared from NaOH manufactured by Wako Pure Chemical Industries and RO water) and the mixture was stirred at 150 rotation/minute at 4 C. for 4 hours. The pH of the reaction mixture after the reaction was adjusted to 7 using 0.1 M citric acid (prepared from citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries). The mixture was stirred at the same temperature for 1 hour, and then dimethylamineborane (408 mg) was added. The mixture was stirred at 150 rotation/minute at 4 C. for 1 hour and at 150 rotation/minute at 25 C. for 5 hours.

(96) The porous base matrix after the reaction was washed with RO water of which amount was 10 times by volume relative to the porous base matrix on a glass filter (26G-2 manufactured by TOP), and substitutive procedure was carried out using 3 times by volume of 0.1 M citric acid (prepared by citric acid monohydrate manufactured by Wako Pure Chemical Industries and RO water). To the substituted porous base matrix, 0.1 M citric acid was added to make the total volume 186 mL, and the mixture was added into a separable flask (manufactured by TOP, 500 mL) and stirred at 150 rotation/minute at 25 C. for 30 minutes for acid washing.

(97) After the acid washing for the porous base matrix, substitutive procedure was carried out using an aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate (prepared from sodium hydroxide and sodium sulfate and RO water manufactured by Wako Pure Chemical Industries) on a glass filter (26G-2 manufactured by TOP). To the substituted porous base matrix, an aqueous solution of 0.05 M sodium hydroxide and 1 M sodium sulfate to make the total volume 186 mL. All of the mixture was added into a separable flask and stirred at 150 rotation/minute at 25 C. for 20 minutes for alkali washing.

(98) After the alkali washing, substitutive procedure for the porous base matrix was carried out using a 0.5 M citrate buffer of which pH was 6 (prepared from trisodium citrate dihydrate, citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries) (278 mL) on a glass filter (26G-2 manufactured by TOP). The porous base matrix was washed using RO water until the conductivity of the filtrate became not more than 5 S/cm. Then, substitutive procedure was carried out using 20% aqueous ethanol (prepared from ethanol of and RO water Japanese Pharmacopoeia), and the porous base matrix was added into a 250 mL polymer container (manufactured by SANPLATEC CO., LTD.) using 20% aqueous ethanol, to obtain the desired adsorbent immobilized with protein A. The porous base matrix was washed using RO water until the conductivity of the filtrate became not more than 5 S/cm. Then, substitutive procedure was carried out using 20% aqueous ethanol (prepared from ethanol of and RO water Japanese Pharmacopoeia), and the porous base matrix was added into a 250 mL polymer container (manufactured by SANPLATEC CO., LTD.) using 20% aqueous ethanol, to obtain the desired adsorbent immobilized with protein A. The electric conductivity of filtrate of washing was measured using an electric conductivity meter (ESTestr10 pure+ manufactured by EUTECH INSTRUMENTS).

(99) As the target substance for the obtained adsorbent, human polyclonal IgG (Gammagard manufactured by Baxter) was selected. The dynamic binding capacity and the amount of the leaching ligand in the target substance were measured; as a result, the dynamic binding capacity of IgG, i.e. 5% dynamic binding capacity, was 34 mg per 1 mL of the porous base matrix. The concentration of the leaching ligand in the purified IgG was 30 ppm.

Example 27

(100) An adsorbent was prepared by the same method of Example 26 except that the duration time for stirring at the same temperature after the pH was adjusted to 7 using 0.1 M citric acid was 2 hours. The dynamic binding capacity of IgG was 36 mg per 1 mL of the porous base matrix.

Example 28

(101) An adsorbent was prepared by the same method of Example 26 except that the duration time for stirring at the same temperature after the pH was adjusted to 7 using 0.1 M citric acid was 4 hours. The dynamic binding capacity of IgG was 37 mg per 1 mL of the porous base matrix, and the concentration of the leaching ligand in the purified IgG was 22 ppm.

Example 29

(102) An adsorbent was prepared by the same method of Example 26 except that the duration time for stirring at the same temperature after the pH was adjusted to 7 using 0.1 M citric acid was 6 hours. The dynamic binding capacity of IgG was 36 mg per 1 mL of the porous base matrix, and the concentration of the leaching ligand in the purified IgG was 45 ppm.

Example 30

(103) An adsorbent was prepared by the same method of Example 26 except that the duration time for stirring at the same temperature after the pH was adjusted to 7 using 0.1 M citric acid was 15 hours. The dynamic binding capacity of IgG was 37 mg per 1 mL of the porous base matrix

Example 31

(104) An adsorbent was prepared by the same method of Example 26 except that the pH was adjusted to 3 using 1.6 M citric acid after protein A was reacted at pH 12 and the duration time of subsequent stirring at the same temperature was 4 hours. The dynamic binding capacity of IgG was 36 mg per 1 mL of the porous base matrix, and the concentration of the leaching ligand in the purified IgG was 30 ppm.

Example 32

(105) An adsorbent was prepared by the same method of Example 26 except that dimethylamineborane was added immediately after the pH was adjusted to 7 using 0.1 M citric acid. The dynamic binding capacity of IgG was 31 mg per 1 mL of the porous base matrix, and the concentration of the leaching ligand in the purified IgG was 22 ppm.

Example 33

(106) An adsorbent was prepared by the same method of Example 33 except that substitutive procedure for Porous particle B prepared by Production Example 1 was carried out using a citrate buffer (prepared from trisodium citrate dihydrate, citric acid monohydrate and RO water manufactured by Wako Pure Chemical Industries) (282 mL), and the amount of the mixture was adjusted using the buffer, and 0.16 g of sodium periodate was used. The content of formyl group of the Porous base matrix E was 7 mol per 1 mL of the porous base matrix, and the amount of protein A as an affinity ligand to be immobilized was 5.0 mg per 1 mL of the porous base matrix. The dynamic binding capacity of IgG was 31 mg, and the concentration of the leaching ligand in the purified IgG was 36 ppm. The content of formyl group of the obtained adsorbent was 0.2 mol per 1 mL of the porous base matrix.

Comparative Example 4

(107) An adsorbent was prepared by the same method of Example 16 except that dimethylamineborane was not used. The content of formyl group of the obtained adsorbent was 2 mol per 1 mL of the porous base matrix. The dynamic binding capacity of IgG was 27 mg per 1 mL of the adsorbent, and the concentration of the leaching ligand in the purified IgG was 615 ppm.