WO2017141910A1 - Porous crosslinked cellulose gel and manufacturing method for same - Google Patents

Abstract

The problem of providing a porous crosslinked cellulose gel that is suitable as a filler for chromatography and combines high porosity and high mechanical strength, a manufacturing method for the porous crosslinked cellulose gel and an adsorbent for antibody purification, resulting from immobilizing an affinity ligand on the porous crosslinked cellulose gel, is resolved by providing a porous crosslinked cellulose gel having the two features of (1) a porosity of 90% or more and (2) a column pressure loss of not more than 0.4 MPa, a manufacturing method for the porous crosslinked cellulose gel and an adsorbent for antibody purification obtained by immobilizing an affinity ligand on the porous crosslinked cellulose gel.

Description

Porous crosslinked cellulose gel and method for producing the same

The present invention relates to a porous crosslinked cellulose gel useful as a chromatographic filler, particularly an adsorbent for purifying biopharmaceuticals such as antibody pharmaceuticals, and a method for producing the same. More specifically, the present invention relates to a porous crosslinked cellulose gel having both high porosity and mechanical strength, and a method for producing the same.

In recent years, biopharmaceuticals such as proteins and vaccines have been actively developed. In particular, the market for antibody drugs using antibodies as raw materials is growing rapidly. Generally, column chromatography using a porous carrier as a packing material is used in the purification process of biopharmaceutical production. For example, packing using an ion-exchange group or packing using an affinity ligand for the substance to be purified is used. The agent is being used. Particularly in antibody drug purification, affinity chromatography using a filler into which an affinity ligand for the antibody is introduced is often used because the antibody can be efficiently purified.

With the recent rapid growth of the biopharmaceutical market, there is a need for a chromatographic packing material that has a high adsorption capacity for the substance to be purified and an excellent mechanical strength that can be processed at a high flow rate for the purpose of improving productivity. . The high adsorption capacity of the filler can be achieved by a method of reducing the particle size of the porous carrier used as the filler or a method of increasing the porosity of the porous carrier. However, if the particle size of the porous carrier is reduced, the column pressure loss increases, making it difficult to increase the flow rate. If the porosity of the porous carrier is increased, the mechanical strength of the carrier is reduced. Processing may be difficult.

As a conventional porous carrier, for example, a silica gel carrier as disclosed in Patent Document 1, for example, a synthetic polymer carrier as disclosed in Patent Literature 2 is known. Furthermore, as a polysaccharide carrier using a polysaccharide which is a natural polymer as a raw material, for example, a porous crosslinked agarose gel as shown in Patent Document 3, for example, a porous cellulose gel as shown in Patent Document 4 such as Patent Document 5 As shown, polysaccharide composite particles using polysaccharides such as agarose, pullulan, dextran and cellulose as raw materials are known. Moreover, as a porous crosslinked cellulose gel whose mechanical strength has been increased by crosslinking treatment, for example, Patent Document 6 discloses a method for obtaining a porous crosslinked cellulose gel obtained by crosslinking a porous uncrosslinked cellulose gel with glycidyl ethers. Patent Document 7 discloses a method of obtaining a porous crosslinked cellulose gel obtained by crosslinking an uncrosslinked porous cellulose gel produced by the method described in Patent Document 4 with epichlorohydrin.

However, although the silica gel carrier has high mechanical strength and porosity, it tends to have a low adsorption capacity of the substance to be purified, and further has a weak alkali resistance, so that it is difficult to use or wash under alkaline conditions. Synthetic polymer carriers have good alkali resistance and mechanical strength, but tend to have low porosity and adsorption capacity of the substance to be purified. There is a strong tendency to adsorb non-specifically, and impurities may be mixed. The polysaccharide carrier has good alkali resistance, porosity, and adsorption capacity of the substance to be purified, but has insufficient mechanical strength for use as a packing material for chromatography capable of high flow rate treatment. Moreover, since the process which requires the high temperature of 100 degreeC or more is included in the manufacturing method of the porous cellulose gel of patent document 4 and patent document 6, when manufacturing on an industrial scale, a large installation and much In addition, the method for producing polysaccharide composite particles described in Patent Document 5 uses an expensive ionic liquid as a polysaccharide solvent. Therefore, these methods are expensive production methods. It is a problem.

JP-A-6-281638 JP 2009-244067 A Special Table 2000-508361 JP-A-10-195103 Japanese Patent Application Laid-Open No. 2012-126797 JP 2011-252929 A JP 2009-242770 A

An object of the present invention is a porous crosslinked cellulose that has solved the problems of the conventional porous carrier described above, such as having a high porosity and mechanical strength, and having a high adsorption capacity of a substance to be purified when an affinity ligand is introduced. It is in providing the manufacturing method of the gel, and the said porous crosslinked cellulose gel. More specifically, the object is to provide a chromatography filler and an antibody purification adsorbent using the porous crosslinked cellulose gel.

As a result of intensive studies to solve the above-mentioned problems, the present inventors have added a gelling agent to a cellulose dispersion having a temperature of 0 ° C. or more and 15 ° C. or less obtained by mixing a cellulose solution and an organic solvent. After obtaining an uncrosslinked cellulose gel, the porous uncrosslinked cellulose gel is subjected to crosslinking treatment using two types of polyfunctional crosslinking agents having different numbers of atoms between functional groups capable of reacting with the hydroxyl groups of cellulose. It was found that a porous crosslinked cellulose gel having both porosity and mechanical strength can be obtained. Further, it was found that the antibody purification adsorbent in which the affinity ligand for the antibody is immobilized on the porous crosslinked cellulose gel of the present invention has a high antibody adsorption amount and has excellent performance as an antibody purification adsorbent. It came to complete.

That is, the present invention provides the inventions described in the following (1) to (6).

(1) A porous crosslinked cellulose gel having the following characteristics (A) and (B).

(A) The porosity measured by the following operations (a) to (e) is 90% or more.

(A) The porous crosslinked cellulose gel is packed in a chromatography column.

(B) Using water as an eluent, measure the elution volume of blue dextran having a molecular weight of 2 million and sodium chloride from the column.

(C) The gel volume is calculated by subtracting the elution volume of blue dextran having a molecular weight of 2 million measured in operation (b) from the column volume of the column.

(D) The pore volume is calculated by subtracting the elution volume of blue dextran having a molecular weight of 2 million measured in operation (b) from the elution volume of sodium chloride measured in operation (b).

(E) The porosity is calculated by dividing the pore volume calculated in operation (d) by the gel volume calculated in operation (c).

(B) A chromatography column having an average particle diameter of 30 μm to 150 μm and an inner diameter of 6.6 mm is packed so as to have a height of 220 mm ± 5 mm, and water at 25 ° C. is poured into the column at a linear velocity of 1500 cm / hour. The column pressure loss under the flowed condition is 0.4 MPa or less.

(2) The method for producing a porous crosslinked cellulose gel according to (1), comprising the following steps (a) to (e):
(A) a step of obtaining a cellulose solution by dissolving cellulose in an alkaline aqueous solution;
(B) a step of obtaining a cellulose dispersion by mixing the cellulose solution obtained in step (a) with an organic solvent and an emulsifier;
(C) The step of obtaining a porous uncrosslinked cellulose gel by bringing the cellulose dispersion obtained in step (b) to 0 ° C. or more and 15 ° C. or less and adding a gelling agent;
(D) a step of obtaining a porous partially crosslinked cellulose gel by reacting the porous uncrosslinked cellulose gel obtained in step (c) with glycidyl ethers having at least two or more glycidyl groups,
(E) A step of obtaining a porous crosslinked cellulose gel by reacting the porous partially crosslinked cellulose gel obtained in the step (d) with a crosslinking agent having two or more functional groups capable of reacting with a hydroxyl group of cellulose.

(3) A chromatographic filler comprising the porous crosslinked cellulose gel described in (1) above.

(4) A protein purification method using the chromatography filler according to (3) above.

(5) An adsorbent for antibody purification, wherein an affinity ligand is immobilized on the porous crosslinked cellulose gel described in (1) above.

(6) An adsorbent for antibody purification, wherein the affinity ligand described in (5) above is protein A or Fc-binding protein.

Since the porous crosslinked cellulose gel obtained by the production method of the present invention has both high porosity and high mechanical strength, and has suitable pore characteristics and particle size as a packing material for chromatography, It is suitable as a packing material for chromatography used in the purification process of biopharmaceuticals. Moreover, the manufacturing method of the porous bridge | crosslinking cellulose gel of this invention is a manufacturing method which does not require a large facility, a great deal of energy, and an expensive raw material compared with the manufacturing method of the conventional porous cellulose gel. Furthermore, the adsorbent for antibody purification obtained by immobilizing the affinity ligand for the antibody on the porous crosslinked cellulose gel obtained by the production method of the present invention has a high adsorption capacity for the antibody, and further has a low column pressure loss and a high level. Since the flow rate treatment is possible, the productivity in the antibody drug purification process can be remarkably improved.

The flow rate and the column pressure loss of the porous crosslinked cellulose gels 1, 3, 5 produced in Examples 1, 2, and 3 and the commercially available porous crosslinked cellulose gel (Cellfine GCL-2000) evaluated in Comparative Example 4 are shown. It is a graph. It is the graph which showed the relationship between the flow rate of the porous bridge | crosslinking cellulose gel 10 manufactured in Example 7, and the porous bridge | crosslinking cellulose gels 12 and 13 manufactured in Comparative Examples 6 and 7, and column pressure loss.

Hereinafter, the present invention will be described in more detail.

The porous crosslinked cellulose gel of the present invention has (A) a porosity of 90% or more, (B) an average particle diameter of 30 μm or more and 150 μm or less, and is high in a chromatography column having an inner diameter of 6.6 mm. It is packed so that it becomes 220 mm ± 5 mm, and it has two characteristics that the column pressure loss is 0.4 MPa or less under the condition that 25 ° C. water is passed through the column at a linear velocity of 1500 cm / hour. Features.

The characteristic that the porosity (A) of the porous crosslinked cellulose gel of the present invention is 90% or more is measured by the operations described in the following (a) to (e). This measurement method is a general method for measuring the porosity of a porous carrier in the field of liquid chromatography.

(A) The porous crosslinked cellulose gel of the present invention is packed in a chromatography column.

(B) Using water as an eluent, measure the elution volume of blue dextran having a molecular weight of 2 million and sodium chloride from the column.

(C) The gel volume is calculated by subtracting the elution volume of blue dextran having a molecular weight of 2 million measured in operation (b) from the column volume of the column.

(D) The pore volume is calculated by subtracting the elution volume of blue dextran having a molecular weight of 2 million measured in operation (b) from the elution volume of sodium chloride measured in operation (b).

(E) The porosity is calculated by dividing the pore volume calculated in operation (d) by the gel volume calculated in operation (c).

That is, the porosity can be calculated from the following formula using the elution volume (Vn) of sodium chloride, the elution volume (Vo) of blue dextran having a molecular weight of 2 million, and the column volume (Vc). Rate (%) = ((Vn−Vo) / (Vc−Vo)) × 100.

When the porous crosslinked cellulose gel of the present invention, which is a porous carrier, is used as a chromatographic filler or an adsorbent for antibody purification, increasing the adsorption capacity of the substance to be purified is a method for increasing the porosity and particle size. However, if the porosity is increased too much, the mechanical strength decreases, and consolidation occurs as the linear velocity, which is the value obtained by dividing the flow rate by the column cross-sectional area, increases. Since the column pressure loss increases, high flow rate processing becomes difficult. Further, since the column pressure loss increases in inverse proportion to the square of the particle diameter, if the particle diameter is made too small, the column pressure loss similarly increases and high flow rate processing becomes difficult.

On the other hand, increasing the strength of the porous carrier can be achieved by increasing the concentration of the solid component constituting the carrier or increasing the amount of the crosslinking agent used. When the amount of the agent used is excessively large, the porosity of the porous carrier is lowered, so that the adsorption capacity for the substance to be purified is lowered.

Thus, in order to achieve both high adsorption capacity and high strength, a porous carrier having an appropriate porosity, mechanical strength, and particle size is necessary. Thus, a porous crosslinked cellulose gel having both a high adsorption capacity and a high mechanical strength can be produced.

The porosity of the porous crosslinked cellulose gel of the present invention needs to be 90% or more in terms of achieving both a high adsorption capacity for the substance to be purified and a high mechanical strength capable of a high flow rate treatment. % Or more and 95% or less is preferable. In addition, the mechanical strength of the porous crosslinked cellulose gel of the present invention, for the same reason, the column pressure loss measured under the above-mentioned conditions needs to be 0.4 MPa or less, and may be 0.3 MPa or less. preferable. Furthermore, the average particle size of the porous crosslinked cellulose gel of the present invention needs to be 30 μm or more and 150 μm or less for the same reason, and is 40 μm or more and 100 μm or less in that the adsorption capacity for the substance to be purified can be further increased. It is preferable. In addition, the exclusion molecular weight limit of polysaccharides such as dextran and pullulan of the porous crosslinked cellulose gel of the present invention is 500,000 to 5,000,000 in that a high adsorption capacity for a substance to be purified is achieved. Preferably, it is 1,000,000 to 3,000,000. The method for measuring the exclusion limit molecular weight of polysaccharides is not particularly limited as long as it is a general method for measuring the exclusion limit molecular weight of polysaccharides in the field of packing materials for chromatography. Examples of the method described in “Gel Filtration Method” Second Edition (Academic Publishing Center) and JP2012-141212 can be given.

The average particle diameter of the porous crosslinked cellulose gel of the present invention was obtained by taking an image of a slide glass with a scale of 10 to 200 μm using an optical microscope, and then taking an image of a plurality of particles to be measured at the same magnification. It can be obtained by measuring the major axis of a plurality of (for example, 100 or more) particles photographed using a ruler and calculating an average value thereof.

The particle size of the porous crosslinked cellulose gel can be adjusted to a desired range by a wet classification method using a JIS standard sieve made of stainless steel, for example, a sieve having an opening of 45 μm and a sieve having an opening of 90 μm. Depending on the wet classification used, a porous crosslinked cellulose gel of 45 μm or more and 90 μm or less can be obtained.

As shown by comparison between Examples 1 to 3 and Comparative Example 4 described later, the column pressure loss of the porous crosslinked cellulose gel of the present invention is the same as that of the porous crosslinked cellulose gel of the present invention. Cellulite GCL-2000 (manufactured by JNC, porous crosslinked cellulose gel) with a large particle size is lower than the column pressure loss when measured under the same conditions, and is generally used in biopharmaceutical purification processes Therefore, it is highly useful as a packing material for chromatography that can be processed at a high flow rate. The specific method for filling the porous crosslinked cellulose gel of the present invention into the column, the method for measuring the exclusion limit molecular weight of the polysaccharide, the method for measuring the porosity and the column pressure loss are as shown in the examples. .

The production method of the porous crosslinked cellulose gel in the present invention is as follows.
(A) a step of obtaining a cellulose solution by dissolving cellulose in an alkaline aqueous solution;
(B) a step of obtaining a cellulose dispersion by mixing the cellulose solution obtained in step (a) with a solution containing an organic solvent and an emulsifier soluble in the organic solvent,
(C) The step of obtaining a porous uncrosslinked cellulose gel by bringing the cellulose dispersion obtained in step (b) to 0 ° C. or more and 15 ° C. or less and adding a gelling agent;
(D) a step of obtaining a porous partially crosslinked cellulose gel by reacting the porous uncrosslinked cellulose gel obtained in step (c) with glycidyl ethers having at least two or more glycidyl groups,
(E) a step of obtaining a porous crosslinked cellulose gel by reacting the porous partially crosslinked cellulose gel obtained in the step (d) with a crosslinking agent having two or more functional groups capable of reacting with a hydroxyl group of cellulose;
It is characterized by including.

Details of each step from step (a) to step (e) will be described below.

(A) Step of obtaining a cellulose solution by dissolving cellulose in an alkaline aqueous solution The cellulose that can be used in the step (a) is not particularly limited as long as a cellulose solution can be obtained, but can be easily obtained. In addition, plant-derived cellulose such as wood pulp and microorganism-derived cellulose produced by microorganisms such as acetic acid bacteria are preferable, and plant-derived cellulose is more preferable in that it can be obtained at low cost. Moreover, cellulose derived from different plant species, such as cellulose derived from wood pulp, cellulose derived from cotton, and cellulose derived from hemp, can be used alone or as a mixture.

In Example 7 and Example 8 described later, when the porosity and mechanical strength of a porous crosslinked cellulose gel produced under the same conditions except for the origin of cellulose used as a raw material are compared, cotton-derived cellulose is used as a raw material. Thus, since a high porosity and mechanical strength can be achieved, it is preferable to use cotton-derived cellulose when celluloses having different origins are used alone.

The average degree of polymerization of the cellulose used in the step (a) is not particularly limited as long as a cellulose solution can be obtained, but when the cellulose having a low average degree of polymerization is used, the mechanical strength of the resulting porous cellulose gel is reduced. There is. On the other hand, when cellulose having a high average degree of polymerization is used, the solubility of cellulose is lowered, so that it may be difficult to completely dissolve the cellulose. Therefore, the average degree of polymerization of the cellulose used in the step (a) is preferably 100 or more and 1000 or less, more preferably 100 or more and 500 or less. The average polymerization degree of cellulose is, for example, the method described in “JEM1455: Method for measuring average polymerization degree of insulating paper for transformer”, that is, after dissolving cellulose in a copper / ethylenediamine solution, an Ostwald viscometer It can be determined by a viscosity measurement method using Cellulose having an average degree of polymerization of 100 or more and 500 or less may be a commercially available product. For example, as the wood pulp-derived cellulose, Asahi Kasei Theolas series, and as the cotton-derived cellulose, ADVANTEC filter paper powder series can be used. Among these, PH-101 is preferable for the Theolas series manufactured by Asahi Kasei, and filter paper powder C (300 mesh or more) is used for the filter paper powder series manufactured by ADVANTEC from the viewpoint of ease of preparation of the cellulose solution and degree of polymerization.

When the cellulose concentration in the cellulose solution obtained in the step (a) is decreased, the porosity of the porous cellulose gel obtained is increased, but the mechanical strength may be decreased, while the cellulose concentration is increased. Then, the porosity of porous cellulose gel may fall. Therefore, the cellulose concentration is preferably 2% by weight or more and 10% by weight or less, more preferably 4% by weight or more and 8% by weight or less.

The alkaline aqueous solution that can be used in the step (a) is not particularly limited as long as a cellulose solution is obtained, but a sodium hydroxide aqueous solution or a mixed aqueous solution of sodium hydroxide and urea and / or thiourea is preferable. When the aqueous alkali solution is an aqueous sodium hydroxide solution, in order to obtain a cellulose solution, for example, the suspension obtained by adding cellulose to the aqueous sodium hydroxide solution at 20 to 30 ° C. is frozen, and then from 20 ° C. A general method involves an operation of freezing the cellulose solution, such as repeating the operation of dissolving the suspension at 30 ° C. On the other hand, in the case of a mixed aqueous solution of sodium hydroxide and urea and / or thiourea, by using a mixed aqueous solution of sodium hydroxide and urea and / or thiourea in the concentration range described later, from 20 ° C. without freezing. A cellulose solution can be obtained by cooling and stirring at 30 ° C. or, if necessary, between 0 ° C. and 20 ° C. Accordingly, a mixed aqueous solution of sodium hydroxide and urea and / or thiourea is more preferable in that cellulose can be easily dissolved. Further, in the step of obtaining a porous uncrosslinked cellulose gel described later (step (c)), the alkaline aqueous solution is water in that the formation of a porous uncrosslinked cellulose gel having a large particle size that is not used in step (d) can be suppressed. A mixed aqueous solution of sodium oxide and urea and thiourea, or a mixed aqueous solution of sodium hydroxide and thiourea is more preferable.

When the aqueous alkali solution used in step (a) is a mixed aqueous solution of sodium hydroxide and urea, dissolution of cellulose is difficult if the concentration of sodium hydroxide and urea in the mixed aqueous solution is low, while the concentration of sodium hydroxide and urea is low. If it is high, decomposition or modification of cellulose may occur. Accordingly, the sodium hydroxide concentration in the alkaline aqueous solution is preferably 5% by weight or more and 12% by weight or less, and the urea concentration is preferably 4% by weight or more and 30% by weight or less, and more preferably the sodium hydroxide concentration is 7% by weight or more and 10% by weight. Hereinafter, the urea concentration is 6 wt% or more and 28 wt% or less. The weight ratio of sodium hydroxide and urea is not particularly limited as long as a cellulose solution can be obtained, but in order to obtain a cellulose solution by stirring between 0 ° C. and 25 ° C., it is appropriate for the sodium hydroxide concentration. For example, when the sodium hydroxide concentration is 7% by weight, the urea concentration is 15 to 28% by weight. When the sodium hydroxide concentration is 8% by weight, the urea concentration is When the concentration is 10 wt% or more and 28 wt% or less and the sodium hydroxide concentration is 9 to 10 wt%, a cellulose solution can be obtained by setting the urea concentration to 6 wt% or more and 28 wt% or less.

When the alkaline aqueous solution used in the step (a) is a mixed aqueous solution of sodium hydroxide and thiourea, the sodium hydroxide concentration is 6% by weight to 12% by weight and the thiourea concentration is 3% by weight or more and 8% for the reasons described above. The sodium hydroxide concentration is preferably 8 wt% or more and 10 wt% or less, and the thiourea concentration is 4 wt% or more and 7 wt% or less. In the case of a mixed aqueous solution of sodium hydroxide and thiourea, the solubility of thiourea in water is low, so it is preferable to increase the sodium hydroxide concentration compared to the case of a mixed aqueous solution of sodium hydroxide and urea.

When the alkaline aqueous solution used in step (a) is a mixed aqueous solution of sodium hydroxide, urea and thiourea, the sodium hydroxide concentration is 6 wt% or more and 12 wt% or less, and the urea concentration is 2 wt% or more for the reasons described above. 20 wt% or less, thiourea concentration is preferably 1 wt% or more and 6 wt% or less, more preferably sodium hydroxide concentration is 8 wt% or more and 10 wt% or less, urea concentration is 3 wt% or more and 15 wt% or less, The thiourea concentration is 2% by weight or more and 5% by weight or less. The weight ratio of sodium hydroxide, urea and thiourea is not particularly limited as long as a cellulose solution is obtained. However, if the urea concentration is increased, thiourea may not be dissolved. For example, when the sodium hydroxide concentration is 9 to 10% by weight, the urea concentration is 3 to 10% by weight and the thiourea concentration is 2 to 4% by weight. A cellulose solution can be obtained.

The method for dissolving cellulose in an alkaline aqueous solution is to add cellulose to the above-mentioned mixed aqueous solution of sodium hydroxide and urea and / or thiourea, and then between 20 ° C. and 30 ° C., or between 0 ° C. and 20 ° C. as necessary. A method of cooling and stirring the solution is preferable. In general, it is known that cellulose easily dissolves at a temperature not lower than the freezing point of an alkaline aqueous solution and not higher than 20 ° C. However, when adding cellulose to a cooled alkaline aqueous solution, it takes time to dissolve unless the addition method is appropriate. There is a case. Therefore, the method of dissolving cellulose in an alkaline aqueous solution is more preferably a method of adding cellulose to an alkaline aqueous solution at 20 ° C. to 30 ° C. to disperse the cellulose, and then cooling and stirring between 0 ° C. and 20 ° C. . The dissolution of cellulose can be confirmed by visually confirming that the state of the alkaline aqueous solution to which cellulose has been added has become transparent from the suspended state.

According to the method of the present invention, cellulose is added at 0 ° C. to 20 ° C. to a mixed aqueous solution of sodium hydroxide and urea preliminarily cooled from −15 ° C. to −8 ° C. disclosed in JP-T-2008-542560. Compared with the method of dissolving, the cellulose solution can be obtained without cooling to 0 ° C. or lower. Further, compared with the method disclosed in JP 2011-231152 A, a wood pulp-derived cellulose as compared with a method in which wood pulp-derived cellulose is dissolved in a mixed aqueous solution of sodium hydroxide and urea and / or thiourea at 10 ° C. to 15 ° C. Cellulose solutions can also be obtained from other cotton-derived cellulose and microorganism-derived cellulose.

(B) Step of obtaining a cellulose dispersion by mixing the cellulose solution obtained in step (a) with an organic solvent and an emulsifier The organic solvent that can be used in step (b) is a stable cellulose dispersion. From the viewpoint of properties, an organic solvent having a specific gravity at 20 ° C. of 0.6 to 1.5 is preferable. Specifically, aliphatic hydrocarbons having 5 to 8 carbon atoms such as pentane, hexane, and octane, aromatic hydrocarbons having 6 to 10 carbon atoms such as toluene, xylene, and ethylbenzene, monochlorobenzene, o-dibenzene, and the like. Examples thereof include halogenated aromatic hydrocarbons such as chlorobenzene, ethers such as tert-butyl methyl ether, cyclopentyl methyl ether and anisole, and esters such as benzyl acetate and cyclohexyl acetate. Among these, ethers containing aromatic hydrocarbons such as toluene, xylene and ethylbenzene and aromatic hydrocarbons such as anisole are preferable, and aromatic hydrocarbons such as toluene, xylene and ethylbenzene are more preferable. Moreover, although there is no restriction | limiting in particular in the usage-amount of an organic solvent, From the point which improves productivity, it is preferable to use 1 to 5 times volume with respect to a cellulose solution, and it is more preferable to use 1 to 3 times volume.

The emulsifier that can be used in the step (b) is not particularly limited as long as it is an emulsifier that dissolves in the organic solvent described above, but cellulose derivatives such as methylcellulose, ethylcellulose, and cellulose acetate are high in the emulsifying action of the cellulose solution. Sorbitan fatty acid esters such as sorbitan monooleate and sorbitan trioleate are preferred, and among these, cellulose derivatives are more preferred. The amount of the emulsifier added to the organic solvent is preferably 0.1 to 3% by weight, more preferably 0.5 to 2% by weight of the emulsifier with respect to the organic solvent in terms of the emulsifying action of the cellulose solution. %.

The method of mixing the cellulose solution obtained in the step (a) and the solution containing the organic solvent and the emulsifier described above is not particularly limited as long as a cellulose dispersion is obtained, but it is easy to control the emulsified state of the cellulose solution. In this respect, a method using stirring using a stirring blade is preferred. The mixing temperature is not particularly limited as long as it is not higher than the boiling point of the organic solvent described above, but is preferably 0 ° C. or higher and 50 ° C. or lower, and preferably 0 ° C. or higher and 35 ° C. or lower in view of easy mixing operation. More preferred. Specifically, a cellulose dispersion is prepared by adding a cellulose solution having a temperature of 0 ° C. or more and 35 ° C. or less under a condition in which a solution containing an organic solvent at 0 ° C. or more and 35 ° C. or less and an emulsifier soluble in the organic solvent is stirred. Can be obtained. Moreover, if the stirring speed is adjusted to an appropriate stirring speed according to the size and shape of the container to be used, the stirring blade, and the emulsifier concentration so that the porous uncrosslinked cellulose gel obtained in the step (c) has a desired particle size. Good.

(C) A step of obtaining a porous uncrosslinked cellulose gel by adding a gelling agent to the cellulose dispersion of 0 ° C. or more and 15 ° C. or less obtained in step (b). Adding a gelling agent to the cellulose dispersion There are many known methods for obtaining porous uncrosslinked cellulose gel, and according to these methods, the cellulose gel can be obtained by adding a gelling agent to the cellulose dispersion regardless of the temperature of the dispersion. It is possible to get.

On the other hand, when the change of the transparency with the temperature of the cellulose solution obtained in the step (a) was observed, the cellulose solution that was transparent at 0 ° C. or more and 15 ° C. or less was dissolved with increasing temperature when the cellulose solution was heated to 15 ° C. or more. There is a case where the phenomenon that the transparency of the cellulose solution is lowered due to the lowering of the property is confirmed. Compared with the case where a gelling agent is added to a cellulose dispersion at 15 ° C. or higher where the transparency of the cellulose solution is lowered, as shown by comparison between Examples 1 to 3 and Comparative Examples 1 to 3 described later, cellulose Obtaining a porous cellulose gel having a high porosity and an adsorbent for antibody purification having a high amount of antibody adsorption by adding a gelling agent to a cellulose dispersion at 0 ° C. to 15 ° C. in a transparent state Can do. Therefore, the cellulose dispersion obtained in the step (b) needs to be in a state of 0 ° C. or higher and 15 ° C. or lower before adding the gelling agent, and is in a state of 0 ° C. or higher and 10 ° C. or lower. Is preferred.

The stirring of the cellulose dispersion is preferably continued for 15 minutes to 3 hours, and more preferably for 30 minutes to 2 hours after confirming that the temperature of the dispersion is in the range of 0 ° C. to 10 ° C. preferable. Moreover, although it is preferable to maintain the stirring speed of the above-mentioned process (b), you may adjust a stirring speed suitably according to the state of a dispersion liquid.

The gelling agent that can be used in step (c) is not particularly limited as long as it can gel the cellulose solution. Specifically, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2- Examples thereof include alcohols having 1 to 4 carbon atoms such as butanol, ketones such as acetone and methyl ethyl ketone, acidic aqueous solutions such as hydrochloric acid and dilute sulfuric acid, and aqueous inorganic salt solutions such as aqueous sodium sulfate and aqueous magnesium sulfate. These gelling agents may be added directly to the cellulose dispersion, but gelling agents that are insoluble in organic solvents such as acidic aqueous solutions and inorganic salt aqueous solutions are emulsified with an organic solvent containing an emulsifier that is soluble in the aforementioned organic solvents. You may add as a W / O type emulsion obtained by doing. Among these gelling agents, alcohols and ketones are preferable in view of being soluble in an organic solvent, alcohols are more preferable, and methanol is more preferable. Further, the addition amount of the gelling agent is preferably 0.2 to 1 times the volume of the cellulose dispersion, and 0.2 to 0.5 times the volume is preferably used in terms of surely gelling. More preferred.

The addition method of the gelling agent can be obtained by adding a whole amount of the gelling agent to the cellulose dispersion at one time to obtain a bulk cellulose gel in which the particulate cellulose gels are bonded together. A method of gradually adding the gelling agent to the cellulose dispersion is preferred, and a method of adding the gelling agent at a constant rate using a liquid feed pump is more preferred. The addition rate of the gelling agent is preferably 0.001 to 0.05 times the volume of the gelling agent per minute with respect to the volume of the cellulose dispersion in terms of increasing the yield of the resulting cellulose gel, More preferably, 0.005 to 0.03 times the volume of gelling agent is added per minute. In addition, the temperature of the gelling agent to be added is preferably in a state of 0 ° C. or higher and 15 ° C. or lower in advance, and is in a state of 0 ° C. or higher and 10 ° C. or lower in order to suppress the temperature increase of the cellulose dispersion. It is more preferable. After completion of the addition of the gelling agent to the cellulose dispersion, the cellulose gel suspension containing the cellulose gel can be obtained by continuing the stirring preferably for 5 minutes to 1 hour, more preferably for 10 minutes to 30 minutes.

The obtained cellulose gel suspension was prepared by removing the organic solvent and the emulsifier using a washing solvent, and then removing alkali components such as sodium hydroxide and urea and / or thiourea using water. A crosslinked cellulose gel can be obtained. Specifically, after adding a washing solvent to the cellulose gel suspension and repeating the method of filtration or decantation to wash the cellulose gel with the washing solvent, the pH of the washing solution is similarly adjusted using water. By washing the cellulose gel until it becomes porous, a porous uncrosslinked cellulose gel can be obtained.

The washing solvent that can be used in the step (c) is preferably soluble in the organic solvent and the emulsifier used in the step (b) and is water-soluble. Specifically, methanol, ethanol, 1 -Alcohols such as propanol, 2-propanol, ethylene glycol and glycerol can be exemplified. These alcohols can be used alone, but a mixture of several kinds of alcohols or a mixture of alcohols and water can also be used. Moreover, since the organic solvent and an emulsifier may remain | survive if washing | cleaning is inadequate, the usage-amount of the solvent for washing | cleaning uses 5 to 30 times volume with respect to the organic solvent used at the process (b). It is preferable to use 10 to 20 times the volume.

(D) A step of obtaining a porous partially crosslinked cellulose gel by reacting the porous uncrosslinked cellulose gel obtained in the step (c) with glycidyl ethers having at least two or more glycidyl groups. And step (e) is by cross-linking the porous uncrosslinked cellulose gel obtained in step (c) with two types of cross-linking agents having different numbers of atoms between functional groups capable of reacting with the hydroxyl groups of cellulose. This is a step of obtaining a porous crosslinked cellulose gel.

Step (d) is a reaction in which a glycidyl ether as a crosslinking agent is added to a suspension obtained by adding a solvent to the porous uncrosslinked cellulose gel obtained in step (c) and heated under stirring. This is a step of obtaining a porous partially cross-linked cellulose gel by adding a base that promotes water and a reducing agent that reduces cellulose, and further heating under stirring conditions.

The glycidyl ethers that can be used in the step (d) are preferably glycidyl ethers having at least two glycidyl groups in terms of increasing the mechanical strength of the resulting porous crosslinked cellulose gel. Specifically, ethylene glycol diglycidyl ether, glycerol diglycidyl ether, 1,4-butanediol diglycidyl ether, 1,6-hexanediol diglycidyl ether, diethylene glycol diglycidyl ether, tetraethylene glycol diglycidyl ether, resorcinol diglycidyl ether Triglycidyl ethers such as glycerol triglycidyl ether, erythritol triglycidyl ether, diglycerol triglycidyl ether Pentaerythritol tetraglycidyl ether, tetraglycidyl ethers such as pentaerythritol tetraglycidyl ether can be exemplified. Among these, glycerol diglycidyl ether, glycerol triglycidyl ether, erythritol triglycidyl ether, and erythritol tetraglycidyl ether are more preferable. These glycidyl ethers can be used alone, but several mixtures can also be used. Commercially available products may be used as the aforementioned glycidyl ethers, and for example, Denacol EX-313 (glycerol polyglycidyl ether) and Denacol EX-614B (sorbitol polyglycidyl ether) manufactured by Nagase ChemteX can be used.

The solvent that can be used in step (d) is not particularly limited as long as a porous uncrosslinked cellulose gel suspension can be obtained, and water, organic solvents, and mixtures thereof can be used. Examples of organic solvents include ketones such as acetone, methyl ethyl ketone, and methyl propyl ketone, ethers such as diethyl ether, 1,4-dioxane, and ethylene glycol dimethyl ether, nitrogen-containing solvents such as dimethylformamide, and sulfur-containing solvents such as dimethyl sulfoxide. It can be illustrated. Among these solvents, a mixed solvent of water and 1,4-dioxane, water and dimethylformamide, and water and dimethyl sulfoxide is preferable in that the dispersibility of the porous uncrosslinked cellulose gel and the solubility of glycidyl ethers are high.

Although there is no restriction | limiting in particular in the usage-amount of a solvent, In the point which improves the dispersibility of porous uncrosslinked cellulose gel, it should use 0.5 to 3 times the amount of solvent with respect to the moisture content of porous uncrosslinked cellulose gel. Is preferred. The mixing ratio of the organic solvent and water is not particularly limited, but the ratio of the organic solvent to the whole reaction solution is preferably 20 to 80% by weight.

When the amount of the crosslinking agent used is small, the mechanical strength of the resulting porous crosslinked cellulose gel is reduced. On the other hand, when the amount is large, the crosslinking agent reacts to solidify the entire reaction solution. Therefore, it is preferable to use 1 to 5 times the amount of the crosslinking agent relative to the dry cellulose weight contained in the porous uncrosslinked cellulose gel, more preferably 1.5 to 3 times the amount. The dry cellulose weight contained in the porous uncrosslinked cellulose gel should be measured by, for example, heating and drying the porous uncrosslinked cellulose gel in a water-containing state using a heat drying moisture meter manufactured by A & D. Can do.

The reaction temperature in step (d) is preferably 30 to 70 ° C, more preferably 40 to 60 ° C. The method of stirring the reaction solution is preferably a method using a stirring blade in terms of suppressing the destruction of the cellulose gel. The stirring speed is not particularly limited as long as the cellulose gel can be well dispersed in the suspension.

After adding the porous uncrosslinked cellulose gel obtained in the step (c) to the reaction vessel, the solvent and the glycidyl ether as the crosslinking agent, heating at the aforementioned temperature for 30 to 60 minutes under the stirring condition, the crosslinking reaction is performed. For the purpose of promoting, it is preferable to add a base to the reaction solution. Specifically, inorganic bases such as sodium hydroxide and potassium hydroxide and organic bases such as triethylamine and diisopropylethylamine can be exemplified. Among these, inorganic bases such as sodium hydroxide and potassium hydroxide are preferable, and sodium hydroxide is more preferable. Although there is no restriction | limiting in particular in the addition amount of a base, It is preferable that it is 0.5 to 2 times amount with respect to the dry cellulose weight contained in porous uncrosslinked cellulose gel.

Moreover, it is preferable to add a reducing agent such as sodium borohydride, sodium cyanoborohydride, dimethylamine borane to the reaction solution for the purpose of reducing the reducing end of cellulose. Among these, sodium borohydride is used. More preferred. There is no restriction | limiting in particular in the addition amount of a reducing agent, It is preferable that it is 0.01 to 0.05 time amount with respect to the dry cellulose weight contained in porous uncrosslinked cellulose gel.

After the addition of the base and the reducing agent, it is preferable to continue stirring for 8 to 24 hours while maintaining the reaction temperature at 30 to 70 ° C., preferably 40 to 60 ° C. After the reaction, the reaction solution is cooled to 40 ° C. or lower, and then washed with water using a glass filter or the like, whereby the desired porous partially crosslinked cellulose gel can be obtained.

(E) A step of obtaining a porous crosslinked cellulose gel by reacting the porous partially crosslinked cellulose gel obtained in step (d) with a crosslinking agent having two or more functional groups capable of reacting with the hydroxyl groups of cellulose. (E) can react with the reducing agent which reduces a cellulose and the hydroxyl group of a cellulose, after heating the suspension which added the solvent to the porous partial bridge | crosslinking cellulose gel obtained at the process (d) on stirring conditions. This is a step of obtaining a porous crosslinked cellulose gel by adding a crosslinking agent having two or more functional groups and a base for promoting a crosslinking reaction, and further heating under stirring conditions.

The solvent that can be used in the step (e) is not particularly limited as long as a porous partially crosslinked cellulose gel suspension can be obtained, and water, an inorganic salt aqueous solution, an organic solvent, and a mixture thereof can be used. it can. Examples of inorganic salts include sodium sulfate, magnesium sulfate, lithium chloride, sodium chloride, magnesium chloride, potassium chloride, sodium phosphate, sodium monohydrogen phosphate, sodium dihydrogen phosphate, and the like. Examples of the organic solvent include ketones such as acetone, methyl ethyl ketone, and methyl propyl ketone, ethers such as diethyl ether, dipropyl ether, 1,4-dioxane, and ethylene glycol dimethyl ether, nitrogen-containing solvents such as dimethylformamide, dimethyl sulfoxide, and the like. Examples thereof include sulfur-containing solvents such as Among these solvents, water or an aqueous solution of inorganic salt is preferable, and an aqueous solution of sodium sulfate is more preferable in terms of increasing the efficiency of the crosslinking reaction.

Although there is no restriction | limiting in particular in the usage-amount of a solvent, 0.5 to 3 times the amount of solvent should be used with respect to the moisture content of a porous partially crosslinked cellulose gel by the point which improves the dispersibility of a porous partially crosslinked cellulose gel. Is preferred. Also, the inorganic salt concentration in the inorganic salt aqueous solution is not particularly limited as long as the inorganic salt aqueous solution can be obtained. However, if the inorganic salt concentration is too high, inorganic salt crystals are formed when the reaction solution is cooled from 20 ° C to 25 ° C. For example, when the inorganic salt is sodium sulfate, it is preferably 15 to 25% by weight because it may be precipitated.

The reaction temperature in the step (e) is preferably 30 to 70 ° C, more preferably 40 to 60 ° C. The method of stirring the reaction solution is preferably a method using a stirring blade in terms of suppressing the destruction of the cellulose gel. The stirring speed is not particularly limited as long as the cellulose gel can be well dispersed in the suspension.

After heating the cellulosic suspension obtained by adding the porous partially crosslinked cellulose gel obtained in step (d) and the above solvent to the reaction vessel at the above temperature for 30 to 60 minutes under stirring conditions, It is preferable to add a reducing agent for the purpose of reducing the reducing end of cellulose. Specifically, sodium borohydride, sodium cyanoborohydride, and dimethylamine borane can be exemplified, and among these, sodium borohydride is more preferable. Although there is no restriction | limiting in particular in the addition amount of a reducing agent, It is preferable that it is 0.01 to 0.05 times amount with respect to the dry cellulose weight contained in porous partially crosslinked cellulose gel.

Next, crosslinking of the porous partially crosslinked cellulose gel is performed by adding a crosslinking agent having two or more functional groups capable of reacting with a hydroxyl group of cellulose and a base for promoting the crosslinking reaction to the cellulose suspension to which the reducing agent is added. Perform the reaction.

The crosslinking agent having two or more functional groups capable of reacting with the hydroxyl group of cellulose that can be used in the step (e) further increases the mechanical strength of the porous partially crosslinked cellulose gel obtained in the step (d). In this respect, it is preferable to use a crosslinking agent having a smaller number of atoms between functional groups capable of reacting with the hydroxyl group of cellulose than the glycidyl ethers used in step (d). Specifically, epihalohydrins such as epichlorohydrin and epibromohydrin, 1,2-dichloroethane, 1,2-dibromoethane, 1,2-iodoethane, 1,2-dichloropropane, 1,3-diphenyl Halogenated hydrocarbons such as chloropropane, 1,2-dibromopropane, 1,3-dibromopropane, 1,3-diiodopropane, 1-bromo-3-chloropropane, 1,3-dichloro-2-propanol, 2 And halogen-containing alcohols such as 2,3-dichloro-1-propanol and 2,3-dibromo-1-propanol. Among these, epihalohydrins such as epichlorohydrin and epibromohydrin are preferable, and epichlorohydrin is more preferable because of its high reactivity with the hydroxyl group of cellulose in an aqueous inorganic salt solution.

Although there is no restriction | limiting in particular in the usage-amount of a crosslinking agent, When the usage-amount is small, since the mechanical strength of the porous bridge | crosslinking cellulose gel obtained falls, the porous uncrosslinked cellulose gel obtained at the process (c) It is preferable to use 0.5 to 8 times the amount of the crosslinking agent, and more preferably 0.5 to 5 times the amount of the dry cellulose contained in. In addition, it is preferable to add the necessary amount of the crosslinking agent continuously or stepwise for 2 to 12 hours, more preferably 4 to 8 hours.

Examples of the base that can be used in the step (e) include inorganic bases such as sodium hydroxide and potassium hydroxide, and organic bases such as triethylamine and diisopropylethylamine. Among these, inorganic bases such as sodium hydroxide and potassium hydroxide are preferable, and sodium hydroxide is more preferable. Although there is no restriction | limiting in particular in the addition amount of a base, It is preferable that it is 0.5 to 2 times amount with respect to the dry cellulose weight contained in porous uncrosslinked cellulose gel. In addition, it is preferable to add the necessary amount continuously or stepwise for 2 to 12 hours, more preferably 4 to 8 hours, and it is more preferable to add the base continuously or stepwise simultaneously with the above-mentioned crosslinking agent. preferable.

After adding the crosslinking agent and the base, it is preferable to continue stirring for 8 to 24 hours while maintaining the temperature of the reaction solution at 30 to 70 ° C., preferably 40 to 60 ° C., and continue stirring for 12 to 24 hours. More preferably. After the reaction, the reaction solution is cooled to 40 ° C. or lower and then washed with water using a glass filter or the like to obtain the target porous crosslinked cellulose gel.

Furthermore, after the step (e), the porous crosslinked cellulose gel of the present invention having an average particle diameter of 30 μm or more and 150 μm or less can be obtained by performing classification according to the method described above.

Since the porous crosslinked cellulose gel of the present invention is porous, for example, substances to be measured having different molecular weights can be separated by the pore characteristics such as the pore diameter distribution of the gel. In the field of liquid chromatography, the gel distribution coefficient (Kav) is often used as an index indicating the pore characteristics of a porous carrier, and the pore characteristics can be determined by obtaining Kav for various substances with known molecular weights. I can grasp it.

Kav can be measured, for example, by the method described in Biochemical Experimental Method 11 “Gel Filtration Method” Second Edition (Academic Publishing Center). Specifically, using the elution volume (Ve) of the measurement target substance having a known molecular weight, the column volume (Vc), and the elution volume (Vo) of blue dextran having a molecular weight of 2 million, as in the above-described porosity calculation formula. It can be calculated from the calculation formula shown below.
Kav = (Ve−Vo) / (Vc−Vo).

The specific method for measuring Kav of the porous crosslinked cellulose gel of the present invention is as shown in the examples.

Further, the Kav of the porous crosslinked cellulose gel of the present invention is the concentration of each constituent component of cellulose, sodium hydroxide, urea and / or thiourea in the cellulose solution in the step (a) of the present invention, and the step (d). And it can adjust by adjusting bridge | crosslinking conditions, such as a crosslinking agent density | concentration and reaction temperature in a process (e). Among these variables capable of adjusting Kav, the cellulose concentration is the most important. In the porous crosslinked cellulose gel of the present invention, the cellulose concentration is 2 wt% or more and 10 wt% or less, more preferably 4 wt% or more and 8 wt%. By setting it as the following, as shown in the below-mentioned Example, Kav suitable as a packing material for chromatography can be obtained.

Since the porous crosslinked cellulose gel of the present invention has pore characteristics and particle sizes that can be used for separation and purification of biopharmaceuticals such as antibody drugs, affinity chromatography, ion exchange chromatography, hydrophobic interaction It can be suitably used as a packing material for various types of chromatography such as chromatography, size exclusion chromatography, reverse phase chromatography, covalent bond chromatography, and chelate chromatography. In addition, the porous crosslinked cellulose gel of the present invention has a high porosity that can be expected to have a high adsorption capacity for a substance to be purified and a high mechanical strength that enables a high flow rate treatment. Can be suitably used as a packing material for ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography frequently used in US Pat.

The protein purification method using the porous crosslinked cellulose gel in the present invention as a packing material for chromatography is not particularly limited as long as it is a method based on a general chromatography operation, but the porous crosslinked cellulose gel of the present invention is a machine. A method in which the chromatographic column is packed and used is preferable because of its high mechanical strength and high flow rate treatment. As for the size (inner diameter and length) of the column for chromatography, a column having an appropriate size may be used according to the throughput of the purification raw material such as a solution containing the substance to be purified.

When the porous cross-linked cellulose gel of the present invention is used as the above-mentioned chromatographic filler, various functional groups according to the application are introduced into the porous cross-linked cellulose gel by a general method in other chromatographic fillers. can do. For example, a filler for ion exchange chromatography is introduced by introducing a charged group into the porous crosslinked cellulose gel of the present invention, and a hydrophobic interaction chromatography is introduced by introducing a hydrophobic group such as an alkyl group or an aryl group. A filler can be provided. The method for introducing a charged group or a hydrophobic group into the porous crosslinked cellulose gel of the present invention is not particularly limited as long as it is a method for introducing a charged group or a hydrophobic group into a general polysaccharide porous carrier.

In addition, after introducing functional groups for immobilizing affinity ligands such as epoxy groups, formyl groups, amino groups, maleimide groups, carboxyl groups into the porous crosslinked cellulose gel of the present invention, affinity ligands for sugar chains such as concanavalin A, By immobilizing affinity ligands for antibodies such as protein A, protein G, and Fc binding protein, it is possible to provide a packing material for affinity chromatography for each substance to be purified.

As a method for introducing an epoxy group into the porous crosslinked cellulose gel of the present invention, for example, the porous crosslinked cellulose gel of the present invention is converted into epichlorohydrin, ethylene glycol diglycidyl ether, 1,4-butanediol diglycidyl ether. A method of reacting an epoxy group-containing compound such as the above under basic conditions can be exemplified. Examples of the method for introducing a formyl group into the porous crosslinked cellulose gel of the present invention include a method of reacting the porous crosslinked cellulose gel of the present invention with bifunctional aldehydes such as glutaraldehyde, and a porous crosslinked cellulose gel. A method of reacting with an oxidizing agent such as sodium periodate can be exemplified. In addition, the porous crosslinked cellulose gel introduced with an epoxy group by the above-described method and the porous group introduced with an adjacent hydroxyl group by reacting a compound such as D-glucamine, N-methyl-D-glucamine, and α-thioglycerol. A method of reacting the crosslinked cellulose gel with an oxidizing agent such as sodium periodate can be exemplified. As a method for introducing an amino group into the porous crosslinked cellulose gel of the present invention, for example, a porous crosslinked cellulose gel into which an epoxy group has been introduced by the above-described method is used, such as ethylenediamine, diethylenetriamine, tris (2-aminoethyl) amine and the like. The method of making it react with the compound which has an at least 2 or more amino group can be illustrated. As a method for introducing a maleimide group into the porous crosslinked cellulose gel of the present invention, for example, a porous crosslinked cellulose gel into which an amino group has been introduced by the above-described method is used as 3-maleimidopropionic acid N-succinimidyl, 4-maleimidobutyric acid N Examples thereof include a method of reacting with a compound such as -succinimidyl and 6-maleimidohexanoic acid N-succinimidyl. Examples of the method for introducing a carboxyl group into the porous crosslinked cellulose gel of the present invention include a method of reacting the porous crosslinked cellulose gel obtained according to the present invention with haloacetic acid such as monochloroacetic acid and monobromoacetic acid under basic conditions. In addition, porous crosslinked cellulose gel into which epoxy groups have been introduced by the above-described method is used for amino acids such as glycine, alanine, aspartic acid and glutamic acid, amino acids such as β-alanine, 4-aminobutyric acid and 6-aminohexanoic acid. Examples thereof include a method of reacting a group-containing carboxylic acid and a sulfur-containing carboxylic acid such as thioglycolic acid or thiomalic acid under basic conditions.

The method for immobilizing the affinity ligand on the porous crosslinked cellulose gel introduced with the functional group for immobilizing the affinity ligand is not particularly limited as long as it is a general affinity ligand immobilization method. For example, when the affinity ligand immobilization functional group is an epoxy group, the epoxy group introduced into the porous crosslinked cellulose gel of the present invention and the active functional group (lysine amino group or cysteine mercapto group) in the affinity ligand are bases. The method of making it react on sexual conditions can be illustrated. When the functional group for immobilization is a formyl group, the formyl group introduced into the porous crosslinked cellulose gel of the present invention and the amino group of lysine in the affinity ligand are reacted at 10 to 40 ° C. under conditions of pH 8 to 12. Thereafter, a method of adding a reducing agent such as sodium borohydride or dimethylamine borane and reacting at 10 to 40 ° C. can be exemplified. When the functional group for immobilization is an amino group or a carboxyl group, the amino group or carboxyl group introduced into the porous crosslinked cellulose gel of the present invention, N-hydroxysuccinimide, 1-ethyl-3- (3-dimethylaminopropyl) An example is a method of reacting the carboxyl group of aspartic acid and / or glutamic acid or the amino group of lysine in the affinity ligand in the presence of carbodiimide hydrochloride under conditions of pH 4 to 7 at 10 to 40 ° C. When the functional group for immobilization is a maleimide group, the maleimide group introduced into the porous crosslinked cellulose gel of the present invention and the mercapto group of cysteine in the affinity ligand are reacted at 10 to 40 ° C. under conditions of pH 6 to 8. A method can be illustrated.

Among these affinity ligand immobilization methods, the method of reacting the active functional group of the affinity ligand with the formyl group, carboxyl group, maleimide group introduced into the porous crosslinked cellulose gel is preferable because of the high immobilization yield. More preferred is a method of reacting a formyl group or maleimide group introduced into a porous crosslinked cellulose gel with an active functional group of an affinity ligand.

Furthermore, the affinity ligand is immobilized via a spacer atom (hereinafter referred to as spacer) introduced between the hydroxyl group of the porous crosslinked cellulose gel of the present invention and the active functional group of the affinity ligand in order to increase the adsorption capacity for the substance to be purified. It is more preferable to use the method. The number of atoms of the spacer is not particularly limited as long as the affinity ligand has the ability to adsorb to the substance to be purified. However, the number of atoms from 2 atoms to 50 in terms of increasing the contact efficiency between the affinity ligand and the substance to be purified to increase the adsorption capacity. An atom is preferable, and 3 to 30 atoms are more preferable.

The amount of affinity ligand immobilized on the porous crosslinked cellulose gel in which formyl group or maleimide group is introduced into the porous crosslinked cellulose gel of the present invention is 1 mL in terms of increasing the adsorption amount of the substance to be purified and reducing the production cost. It is preferably 1 mg or more and 100 mg or less, more preferably 5 mg or more and 50 mg or less, and even more preferably 5 mg or more and 30 mg or less per 1 porous crosslinked cellulose gel. The amount of affinity ligand immobilized on the porous crosslinked cellulose gel was determined by collecting the immobilized reaction solution and the gel washing solution after the reaction, and measuring the absorbance from the affinity ligand to determine the amount of unreacted affinity ligand. It can be calculated by subtracting the amount of unreacted affinity ligand from the amount of affinity ligand used in the oxidization reaction. The introduction amount of the above-mentioned various functional groups and affinity ligands into the porous crosslinked cellulose gel of the present invention may be determined according to the use of the packing material for chromatography, and the introduction amount of various functional groups changes the reaction conditions. Can be adjusted. Specific methods for introducing the functional group for immobilizing the affinity ligand into the porous crosslinked cellulose gel of the present invention and immobilizing the affinity ligand are as shown in the Examples.

According to the above-described method, an affinity ligand for an antibody such as protein A, protein G, or Fc-binding protein is introduced into the porous crosslinked cellulose gel in which the functional group for immobilizing affinity ligand is introduced into the porous crosslinked cellulose gel of the present invention. Thus, as shown in the Examples described later, it is possible to obtain a packing material for affinity chromatography having a high antibody adsorption capacity suitable for antibody purification, that is, an adsorbent for antibody purification.

As shown in the Examples below, the affinity ligand for the antibody used for the preparation of the adsorbent for antibody purification is protein A or Fc binding in that a high antibody adsorption amount can be realized with a low ligand immobilization amount. Sex protein is preferred, and protein A is more preferred.

Hereinafter, the present invention will be described in more detail with reference to Examples and Comparative Examples, but the present invention is not limited thereto.

In addition, in the following examples and comparative examples, when the cellulose gel is described by weight, unless otherwise specified, it is the wet weight measured after filtering the cellulose gel suspended in water with a glass filter or the like, Moreover, when it describes with "volume", unless there is particular description, the cellulose gel suspended in water is added to the container with a scale, and the sedimentation volume when it is left to stand for 12 hours or more is measured visually.

Example 1 Production of Porous Crosslinked Cellulose Gel 1 Example 1 is porous from a 6% cellulose solution prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 7% by weight sodium hydroxide and 22% by weight urea. The present invention relates to the production of a crosslinked cellulose gel and the evaluation of properties as a chromatographic filler.
(1) Production of porous crosslinked cellulose gel 1 (a) Step of obtaining cellulose solution 5M sodium hydroxide aqueous solution (Kanto Chemical, 140.6 g), urea (Kanto Chemical, 74.6 g) and water (123.8 g) ADVANTEC filter paper powder C (18.0 g, average degree of polymerization 176) was added at 25 ° C. to a 7 wt% sodium hydroxide-22 wt% urea mixed aqueous solution (300 mL) prepared by mixing Was stirred for 2 hours to obtain a transparent cellulose solution 1.
(B) Step of obtaining a cellulose dispersion After stirring the cellulose solution 1 obtained in step (a) at 25 ° C. for 1 hour, toluene (manufactured by Kanto Chemical, 45 cP, 3.60 g) containing toluene (manufactured by Kanto Chemical, 400 mL) at 25 ° C., and stirred for 10 minutes at 500 rpm using a stirring blade to obtain a cellulose dispersion 1.
(C) Step of obtaining porous uncrosslinked cellulose gel Under conditions where stirring at 500 rpm was continued at 25 ° C., the cellulose dispersion 1 obtained in step (b) was ice-cooled, and the temperature of the dispersion was 5 ° C. or lower. Then, stirring was continued for 1 hour under ice cooling. Next, 10 mL / min of ice-cooled methanol (200 mL, manufactured by Kanto Chemical Co., Ltd.) was added to the cellulose dispersion, which was continuously stirred at 500 rpm, under the condition that the temperature of the cellulose dispersion was in the range of 0 ° C. to 10 ° C. Then, the mixture was further stirred for 10 minutes under ice cooling to obtain a cellulose gel suspension. The obtained cellulose gel suspension was washed 5 times with 1.2 L of ethanol and 5 times with 3.5 L of water, and then classified using a sieve, so that porous uncrosslinked particles having a particle size of 150 μm or less were obtained. Cellulose gel 1 (350 mL) and porous uncrosslinked cellulose gel 1 (60 mL) having a particle diameter of 150 μm or more were obtained.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 1 having a particle diameter of 150 μm or less obtained in step (c) (220.3 g, moisture content 92.0%, dry cellulose 17.6 g) , 1,4-dioxane (manufactured by Kanto Kagaku, 264.0 g), water (61.3 g), Denacol EX-313 (manufactured by Nagase ChemteX, 35.2 g), and after stirring at 50 ° C. for 30 minutes, Sodium borohydride (manufactured by Kanto Chemical Co., 528 mg) and a 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., 17.6 g) were added, and stirring was continued at 50 ° C. for 16 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain porous partially crosslinked cellulose gel 1.
(E) Step of obtaining porous cross-linked cellulose gel 25 wt% aqueous solution of sodium sulfate prepared from the total amount of porous partially cross-linked cellulose gel 1 obtained in step (d), anhydrous sodium sulfate (manufactured by Wako Pure Chemical Industries) and water ( 442.5 g) and mixed at 50 ° C. for 30 minutes. Next, after adding sodium borohydride (manufactured by Kanto Chemical Co., 528 mg) to the reaction solution, epichlorohydrin (manufactured by Tokyo Kasei Co., Ltd., 4.41 g) and 48% water were used under the condition of continuing stirring at 50 ° C. A sodium oxide aqueous solution (manufactured by Kanto Chemical Co., Ltd., 4.41 g) was added 12 times at 30 minute intervals, and stirring was continued at 50 ° C. for 15 hours after the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution. After the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 1 having a particle size of 45 μm or more and 90 μm or less ( 200 mL) was obtained.
(2) Exclusion limit molecular weight measurement The measurement of the exclusion limit molecular weight of polysaccharides of the porous crosslinked cellulose gel 1 of 45 μm or more and 90 μm or less obtained in the above step (e) is carried out by the method described in JP2012-141212A. went. The porous crosslinked cellulose gel 1 was washed with a 0.5 M aqueous sodium chloride solution using a glass filter and then suction filtered to obtain a suction-dried porous crosslinked cellulose gel 1. Next, suction-dried porous crosslinked cellulose gel 1 (20.0 g) was dispersed in a 0.5 M aqueous sodium chloride solution (30.0 g), and a reservoir stainless steel column (inner diameter 10.7 mm, length 300 mm) and outlet were used. Pour into a connected stainless steel column (inner diameter 10.7 mm, length 150 mm, column volume 13.5 mL) equipped with a stainless steel sintered filter, connect to a tube pump and 0.5 M at a constant pressure of 0.15 MPa. A sodium chloride aqueous solution (100 mL) was fed. After stopping the liquid feeding, after confirming that the internal pressure of the column had decreased to atmospheric pressure, the reservoir and the column were separated, the porous crosslinked cellulose gel 1 on the upper surface of the column was scraped off, and a stainless sintered filter was attached.

The column packed with porous crosslinked cellulose gel 1 was connected to an HPLC system (quantitative pump: DP-8020 manufactured by Tosoh Corp., autosampler: AS-8020 manufactured by Tosoh Corp., differential refractometer: RI-8020 manufactured by Tosoh Corp.), and the differential refractometer was connected. It was connected to a data processing system (SC-8020 manufactured by Tosoh Corporation). Next, pure water was passed at a flow rate of 1 mL / min, and 40 μL of a 0.2% ethylene glycol aqueous solution was injected from the autosampler so that the asymmetry coefficient of the ethylene glycol peak was in the range of 0.8 to 1.2. I confirmed that there was. Next, at a flow rate of 0.48 mL / min, 0.2% dextran aqueous solution (manufactured by SIGMA, molecular weight 4 × 107), 0.2% eight kinds of pullulan aqueous solutions (Showex STANDARD P-82, Showa Denko, weight average) Molecular weights Mw are 78.7 × 104, 40.4 × 104, 21.2 × 104, 11.2 × 104, 4.73 × 104, 2.28 × 104, 1.18 × 104, 0.59 ×, respectively. 104 and pullulan having Mw / Mn values of 1.23, 1.13, 1.13, 1.12, 1.06, 1.07, 1.10, 1.09, respectively, and ethylene glycol. Each 40 μL was injected to measure the elution volume. The elution volume of each measurement sample is plotted on the x-axis, the molecular weight of each measurement sample is plotted on the y-axis, the elution volume of dextran is 0 as the distribution coefficient, the elution volume of ethylene glycol as the distribution coefficient 1, and the distribution coefficient is 0.5 or more. The exclusion limit molecular weight of porous crosslinked cellulose gel 1 was 1.14 million as a result of drawing a straight line from 3 to 5 points including 1 point and calculating the exclusion limit molecular weight from the section with the elution volume of dextran. .
(3) Porosity and Kav Measurement Kav and porosity of the porous crosslinked cellulose gel 1 were measured by the methods described below. First, the porous cross-linked cellulose gel 1 is suspended in water, and then water is passed through the reservoir column and pump, whereby an omnifit glass column (inner diameter 6.6 mm, length 220 mm, column volume) is obtained. 7.5 mL) was packed so as to be closest packed. Next, after the column filled with the porous crosslinked cellulose gel was connected to an HPLC system (AKTA explorer 10S, manufactured by GE Healthcare Bioscience), water was passed at 0.3 mL / min, and a 0.2% molecular weight of 2 million 10 μL each of a blue dextran aqueous solution (manufactured by Sigma), sodium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) and water and 10 μL of the aqueous solution was measured, and the elution volume from the column was measured. Using the values of column volume (Vc), elution volume (Vo) of blue dextran having a molecular weight of 2 million, and elution volume (Vn) of sodium chloride, the porosity of the porous crosslinked cellulose gel 1 from the above-described porosity calculation formula As a result, the porosity was 91.3%.

Next, 0.05 M Tris-HCl buffer (pH 7.5) prepared from tris (hydroxymethyl) aminomethane (manufactured by Wako Pure Chemicals, hereinafter referred to as Tris) and dilute hydrochloric acid at a flow rate of 0.5 mL / min. ), And the HPLC eluate prepared by adding potassium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) to a final concentration of 0.1M is passed through 40 mL or more, and then the aforementioned HPLC eluate is added at 0.3 mL / min. Passed through and 0.2% cytochrome C solution (manufactured by Sigma, molecular weight 12400), 0.5% albumin solution (manufactured by Sigma, molecular weight 66000), 0.5% apoferritin (manufactured by Sigma, molecular weight) 443000) and 0.6% thyroglobulin (manufactured by Sigma, molecular weight 669000), and 10 μL of each solution was injected to measure the elution volume from the column. In addition, the measuring object protein solution was prepared by melt | dissolving measuring object protein in the above-mentioned HPLC solution. Using the elution volume (Ve) of each protein to be measured, the column volume (Vc), and the elution volume (Vo) of blue dextran having a molecular weight of 2 million, the Kav of the porous crosslinked cellulose gel 1 is calculated from the aforementioned Kav calculation formula. As a result, it was 0.91 for cytochrome C, 0.78 for albumin, 0.66 for apoferritin, and 0.60 for thyroglobulin.
(4) Measurement of column pressure loss For the measurement of the column pressure loss of the porous crosslinked cellulose gel 1, the column packed in the above (2) and the HPLC system were used. The column pressure loss measurement was performed by equilibrating the inside of the column by passing at least 40 mL of water through the column at a flow rate of 0.5 mL / min (linear velocity: 88 cm / hr), and then at a flow rate of 0.5 mL / min. Pass water, read the pump pressure of the HPLC system after 1 minute, and increase the flow rate by 0.5 mL / min at 1-minute intervals to the maximum flow rate of the HPLC system, 10 mL / min (linear velocity: 1755 cm / hr). This was done by reading the pump pressure after 1 minute at each flow rate. The column pressure loss is the flow rate when water is passed with the column filled with water from the pump pressure of the HPLC system at each flow rate when water is passed with the column filled with packing material. It was calculated by subtracting the pump pressure of the HPLC system. As a result of measuring the column pressure loss of the porous crosslinked cellulose gel 1, the column pressure loss at a linear velocity of 1491 cm / hour (flow rate: 8.5 mL / min) was 0.26 MPa.
(5) Formylation of porous crosslinked cellulose gel Porous crosslinked cellulose gel 1 (3.0 g) having a particle diameter of 45 μm or more and 90 μm or less obtained in step (e), sodium periodate (manufactured by Kanto Chemical Co., Inc.) and water A sodium periodate aqueous solution (10 mg / mL, 1.5 mL) and water (1.5 mL) prepared from the above were mixed and stirred at 25 ° C. for 60 minutes to carry out a formylation reaction of the porous crosslinked cellulose gel 1. It was. After the reaction, the porous crosslinked cellulose gel 1F obtained by formylating the porous crosslinked cellulose gel 1 was obtained by washing with a large amount of water using a glass filter.
(6) Protein A immobilization on formylated porous cross-linked cellulose gel A 50 vol% suspension (100 μL) prepared by adding water to the porous cross-linked cellulose gel 1F obtained in (4) above. It was added to a container (manufactured by BIO-RAD, mini-biospin chromatography column, volume 1.2 mL) and washed 5 times with 0.2 M phosphate buffer (pH 11.0, 150 μL) containing 0.5 M sodium chloride. . The 50% by volume suspension of the porous cross-linked cellulose gel 1F was prepared by allowing the porous cross-linked cellulose gel 1F suspended in water to settle in a graduated cylinder and leaving it until the volume became constant by tapping occasionally. It was prepared by adding water so that the volume of the porous crosslinked cellulose gel 1F was 50%.

Next, in a container containing porous crosslinked cellulose gel 1F (50 μL), 0.2 M phosphate buffer (pH 11.0, 120 μL) containing 0.5 M sodium chloride, protein A (manufactured by Repligen) and D A protein A solution (30.5 mg / mL, 20 μL) prepared from PBS (−) (manufactured by Wako Pure Chemical Industries, Ltd.) was added and stirred at 30 ° C. for 1 hour. Next, sodium borohydride aqueous solution (15 mg / mL, 10 μL) prepared from sodium borohydride (manufactured by Kanto Chemical Co., Inc.) and water is added, and the protein A is made porous by stirring at 30 ° C. for 1 hour. Immobilized in a cross-linked cellulose gel 1F. After the immobilization reaction, porous crosslinking is performed by washing D-PBS (-) (manufactured by Wako Pure Chemical Industries, Ltd.) with a PBS solution adjusted to pH 7.0 with dilute hydrochloric acid (hereinafter referred to as PBS 7.0 solution). An adsorbent for antibody purification 1FP in which protein A was immobilized on cellulose gel 1F was obtained.

The reaction solution and the washing solution are collected, the amount of unreacted protein A is calculated by measuring the absorbance at 280 nm, and then the amount of unreacted protein A is subtracted from the amount of protein A used in the reaction to adsorb the antibody. As a result of calculating the amount of protein A immobilized on 1FP, the amount immobilized was 10.7 mg per mL of gel.
(7) Antibody adsorption amount measurement of protein A-immobilized porous crosslinked cellulose gel The antibody purification adsorbent 1FP (50 μL) prepared in the above (5) was washed 5 times with PBS 7.0 solution (200 μL), and then reacted. Purify antibody by adding PBS 7.0 solution (140 μL) and gamma globulin preparation with concentration of 150 mg / mL (70 μL, manufactured by Chemical and Serum Therapy Research Institute) to the container and stirring at 25 ° C. for 2 hours. The antibody was adsorbed on the adsorbent 1FP.

After completion of the stirring, the antibody purifying adsorbent 1FP adsorbed with the antibody was washed four times with a PBS 7.0 solution (150 μL), and the amount of unadsorbed antibody was calculated by measuring the absorbance of the collected washing solution at 280 nm. Next, the antibody was desorbed by washing the adsorbent 1FP for antibody purification adsorbed with the antibody four times with 0.1 M citrate buffer (pH 3.0, 150 μL). As a result of calculating the antibody adsorption amount by measuring the absorbance at 280 nm of the washing solution with the collected citrate buffer, the antibody adsorption amount of the antibody purification adsorbent 1FP was 65.2 mg per mL of gel.
(8) Measurement of antibody adsorption amount of porous cross-linked cellulose gel without immobilized protein A Instead of the adsorbent 1FP for antibody purification, porous cross-linked cellulose gel 1 without immobilized protein A was used. ), The static antibody adsorption amount of the porous crosslinked cellulose gel 1 was measured, and as a result, the static antibody adsorption amount of the porous crosslinked cellulose gel 1 was 0.7 mg per mL of the gel. Therefore, it was revealed that nonspecific adsorption of the antibody to the porous crosslinked cellulose gel 1 was low.

Comparative Example 1 Production of Porous Crosslinked Cellulose Gel 2 Comparative Example 1 was prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 7% by weight sodium hydroxide and 22% by weight urea as in Example 1. This relates to the production of a porous crosslinked cellulose gel and the evaluation of its properties as a chromatographic filler when a cellulose solution is used and the addition temperature of the gelling agent in step (c) of Example 1 is 25 ° C. .
(1) Production of porous crosslinked cellulose gel 2 (a) Step of obtaining cellulose solution 5M sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., 103.1 g), urea (manufactured by Kanto Chemical Co., Ltd., 54.7 g) and water (128) ADVANTEC filter paper powder C (13.2 g, average polymerization degree 176) was added at 25 ° C. to a 7 wt% sodium hydroxide-22 wt% urea mixed aqueous solution (220 mL) prepared by mixing A transparent cellulose solution 2 was obtained by stirring for 2 hours under ice cooling.
(B) Step of obtaining a cellulose dispersion After stirring the cellulose solution 2 obtained in the step (a) at 25 ° C. for 1 hour, toluene (Kanto Chemical Co., Inc., 45 cP, 4.68 g) manufactured by Kanto Chemical Co., Inc. Manufactured at 520 mL) and stirred at 500 rpm for 10 minutes using a stirring blade to obtain a cellulose dispersion 2.
(C) Step of obtaining porous uncrosslinked cellulose gel 25 ° C. methanol (180 mL, manufactured by Kanto Chemical Co., Inc.) was added to the cellulose dispersion 2 obtained in step (b) under the condition that stirring at 500 rpm was continued at 25 ° C. Was added at a rate of 10 mL per minute, and stirring was further continued at 25 ° C. for 30 minutes to obtain a cellulose gel suspension. The obtained cellulose gel suspension was washed 5 times with 1.2 L of ethanol and 5 times with 3.5 L of water, and then classified using a sieve, so that porous uncrosslinked particles having a particle size of 150 μm or less were obtained. Cellulose gel 2 (260 mL) and porous uncrosslinked cellulose gel 2 (35 mL) having a particle size of 150 μm or more were obtained.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 2 having a particle diameter of 150 μm or less obtained in step (c) (156.0 g, moisture content 91.8%, dry cellulose 12.8 g) 1,4-dioxane (manufactured by Kanto Chemical Co., 192.0 g), water (48.8 g), Denacol EX-313 (manufactured by Nagase ChemteX, 25.6 g) were mixed and stirred at 50 ° C. for 30 minutes. Thereafter, sodium borohydride (manufactured by Kanto Chemical Co., 384 mg) and a 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., Ltd., 12.8 g) were added, and stirring was continued at 50 ° C. for 16 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain porous partially crosslinked cellulose gel 2.
(E) Step of obtaining porous crosslinked cellulose gel 25 wt% aqueous sodium sulfate solution prepared from the total amount of porous partially crosslinked cellulose gel 2 obtained in step (d), anhydrous sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) and water. (442.5 g) was mixed and stirred at 50 ° C. for 30 minutes. Next, after adding sodium borohydride (manufactured by Kanto Chemical Co., Inc., 384 mg) to the reaction solution, epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 3.20 g), 48 % Sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Ltd., 3.20 g) was added 12 times at 30 minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution, stirring was continued at 50 ° C. for 15 hours. did. After the reaction, the reaction solution is cooled to 40 ° C. or lower, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 2 having a particle diameter of 45 μm or more and 90 μm or less ( 140 mL) was obtained.
(2) Porosity and Kav Measurement The porosity of the porous crosslinked cellulose gel 2 calculated by the method described in Example 1 was 89.0%. Therefore, it was clarified that the porosity of the porous crosslinked cellulose gel 2 is lower than that of the porous crosslinked cellulose gel 1 produced in Example 1. Similarly, the Kav of the porous crosslinked cellulose gel 2 calculated by the method described in Example 1 is 0.88 for cytochrome C, 0.76 for albumin, 0.63 for apoferritin, and thyroglobulin. On the other hand, it was 0.57.
(3) Column pressure loss measurement The column pressure loss of the porous crosslinked cellulose gel 2 measured by the method described in Example 1 was 0.20 MPa at a linear velocity of 1491 cm / hour (flow rate: 8.5 mL / min).
(4) Formylation of porous cross-linked cellulose gel, protein A immobilization and antibody adsorption amount measurement After obtaining porous cross-linked cellulose gel 2F by formylating porous cross-linked cellulose gel 2 by the method described in Example 1 Then, an adsorbent for antibody purification 2FP having protein A immobilized thereon was obtained. The amount of protein A immobilized on the antibody purification adsorbent 2FP calculated by the method described in Example 1 was 11.5 mg per mL of gel. Similarly, the antibody adsorption amount of the antibody purification adsorbent 2FP calculated by the method described in Example 1 was 55.9 mg per mL of gel. Therefore, it was revealed that the antibody adsorption amount of the antibody purification adsorbent 2FP was lower than that of the antibody purification adsorbent 1FP produced in Example 1.

(Example 2) Production of porous crosslinked cellulose gel 3 Example 2 was prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 9% by weight sodium hydroxide, 4% by weight urea and 4% by weight thiourea. The present invention relates to the production of a porous crosslinked cellulose gel from a cellulose solution and the evaluation of properties as a chromatographic filler.
(1) Production of porous crosslinked cellulose gel 3 (a) Step of obtaining cellulose solution 5M aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., 177.6 g), urea (manufactured by Kanto Chemical Co., Ltd., 13.3 g) and thiourea ( A filter paper manufactured by ADVANTEC Co., Ltd. was added to a 9 wt% sodium hydroxide-4 wt% urea-4 wt% thiourea mixed aqueous solution (300 mL) prepared by mixing 13.3 g) and water (128.8 g) manufactured by Kanto Chemical Co., Ltd. Powder C (18.0 g, average degree of polymerization 176) was added at 25 ° C., and the mixture was stirred for 2 hours under ice cooling to obtain a transparent cellulose solution 3.
(B) The process of obtaining a cellulose dispersion liquid The cellulose dispersion liquid 3 was obtained by the method described in Example 1 using the cellulose solution 3 obtained at the process (a).
(C) Step for obtaining porous uncrosslinked cellulose gel Porous uncrosslinked cellulose gel 3 having a particle diameter of 150 μm or less by the method described in Example 1 using cellulose dispersion 3 obtained in step (b). 350 mL) and porous uncrosslinked cellulose gel 3 (25 mL) having a particle diameter of 150 μm or more were obtained. Therefore, by using a mixed aqueous solution of sodium hydroxide, urea, and thiourea in step (a), the porous uncrosslinked particles having a particle diameter of 150 μm or more are further obtained than in Example 1 using the mixed aqueous solution of sodium hydroxide and urea. Generation of cellulose gel can be suppressed.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 3 having a particle diameter of 150 μm or less obtained in step (c) (221.5 g, moisture content 92.0%, dry cellulose 17.7 g) 1,4-dioxane (manufactured by Kanto Chemical Co., Inc., 265.5 g), water (61.7 g), Denacol EX-313 (manufactured by Nagase ChemteX Corporation, 35.4 g) were mixed and stirred at 50 ° C. for 30 minutes. After that, sodium borohydride (manufactured by Kanto Chemical Co., Ltd., 531 mg) and a 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., Ltd., 17.7 g) were added, and stirring was continued at 50 ° C. for 16 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 3.
(E) Step of obtaining porous crosslinked cellulose gel 25 wt% aqueous sodium sulfate solution prepared from the total amount of porous partially crosslinked cellulose gel 3 obtained in step (d), anhydrous sodium sulfate (Wako Pure Chemical Industries, Ltd.) and water. (442.5 g) was mixed and stirred at 50 ° C. for 30 minutes. Next, after adding sodium borohydride (manufactured by Kanto Chemical Co., Inc., 531 mg) to the reaction solution, stirring was continued at 50 ° C., and epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 4.43 g) and 48 were used. % Aqueous sodium hydroxide solution (4.43 g, manufactured by Kanto Chemical Co., Inc.) was added 12 times at 30 minute intervals. After the addition of epichlorohydrin and 48% aqueous sodium hydroxide solution was completed, stirring was continued at 50 ° C. for 15 hours. did. After the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 3 having a particle size of 45 μm or more and 90 μm or less ( 235 mL).
(2) Exclusion limit molecular weight, porosity, and Kav measurement The exclusion limit molecular weight of the polysaccharide of the porous crosslinked cellulose gel 3 measured by the method described in Example 1 was 1.4 million. Similarly, the porosity of the porous crosslinked cellulose gel 3 calculated by the method described in Example 1 was 90.8%. Similarly, the Kav of the porous crosslinked cellulose gel 3 calculated by the method described in Example 1 is 0.91 for cytochrome C, 0.77 for albumin, 0.66 for apoferritin, and thyroglobulin. On the other hand, it was 0.59.
(3) Column pressure loss measurement The column pressure loss of the porous crosslinked cellulose gel 3 measured by the method described in Example 1 was 0.21 MPa at a linear velocity of 1491 cm / hour (flow rate: 8.5 mL / min).
(4) Formylation of porous cross-linked cellulose gel, protein A immobilization and antibody adsorption amount measurement After obtaining porous cross-linked cellulose gel 3F by formylating porous cross-linked cellulose gel 3 by the method described in Example 1 As a result, an adsorbent 3FP for antibody purification with protein A immobilized thereon was obtained. The amount of protein A immobilized on the antibody purification adsorbent 3FP calculated by the method described in Example 1 was 11.3 mg per mL of gel. Similarly, the antibody adsorption amount of the adsorbent 3FP for antibody purification calculated by the method described in Example 1 was 66.3 mg per mL of gel.
(5) Measurement of static antibody adsorption amount of porous crosslinked cellulose gel without immobilized protein A Static antibody adsorption of porous crosslinked cellulose gel 3 without immobilized protein A measured by the method described in Example 1 The amount was 0.6 mg per mL of gel. Therefore, it was revealed that nonspecific adsorption of the antibody to the porous crosslinked cellulose gel 3 was low.

Comparative Example 2 Production of Porous Crosslinked Cellulose Gel 4 Comparative Example 2 is similar to Example 2 except that cotton-derived cellulose is dissolved in a mixed aqueous solution of 9% by weight sodium hydroxide, 4% by weight urea and 4% by weight thiourea. As a filler for chromatography and the production of a porous crosslinked cellulose gel when the addition temperature of the gelling agent in the step (c) of Example 2 is 25 ° C. It relates to characterization. In Comparative Example 2, a porous crosslinked cellulose gel was produced under the same conditions as in Example 2 with the mixing ratio of the organic solvent and the cellulose solution.
(1) Production of porous crosslinked cellulose gel 4 (a) Step of obtaining cellulose solution By the method described in Example 2, a transparent cellulose solution 4 was obtained.
(B) The process of obtaining a cellulose dispersion liquid The cellulose dispersion liquid 4 was obtained by the method described in Example 2 using the cellulose solution 4 obtained at the process (a).
(C) Step of obtaining porous uncrosslinked cellulose gel 25 ° C. methanol (200 mL, manufactured by Kanto Chemical Co., Inc.) was added to the cellulose dispersion 4 obtained in step (b) under the condition that stirring at 500 rpm was continued at 25 ° C. Was added at a rate of 10 mL per minute, and further stirring was continued at 25 ° C. for 10 minutes to obtain a cellulose gel suspension. The obtained cellulose gel suspension was washed 5 times with 1.2 L of ethanol and 5 times with 3.5 L of water, and then classified using a sieve, so that porous uncrosslinked particles having a particle size of 150 μm or less were obtained. Cellulose gel 4 (200 mL) and porous uncrosslinked cellulose gel 4 (200 mL) having a particle size of 150 μm or more were obtained. Accordingly, in Comparative Example 2 in which the addition temperature of the gelling agent was 25 ° C., compared with Example 2 in which the addition temperature of the gelling agent was 10 ° C. or less, porous uncrosslinked cellulose having a particle size of 150 μm or less. It was found that the yield of gel was reduced.

Moreover, as a result of observing the shape of the porous uncrosslinked cellulose gel 4 with an optical microscope, the porous uncrosslinked cellulose gel 4 having a particle diameter of 150 μm or less was observed as a particulate cellulose gel in which one particle was present alone. However, the porous uncrosslinked cellulose gel 4 having a particle diameter of 150 μm or more was observed as a massive cellulose gel in which a large number of particulate cellulose gels adhered to each other.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 4 having a particle diameter of 150 μm or less obtained in step (c) (98.3 g, moisture content 92.0%, dry cellulose 7.9 g) 1,4-dioxane (manufactured by Kanto Chemical Co., Inc., 118.5 g), water (28.1 g), Denacol EX-313 (manufactured by Nagase ChemteX Corp., 15.8 g) were mixed and stirred at 50 ° C. for 30 minutes. Thereafter, sodium borohydride (manufactured by Kanto Chemical Co., Ltd., 237 mg) and a 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., Ltd., 7.9 g) were added, and stirring was continued at 50 ° C. for 16 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 4.
(E) Step of obtaining porous cross-linked cellulose gel 25 wt% aqueous sodium sulfate solution prepared from the total amount of porous partially cross-linked cellulose gel 4 obtained in step (d), anhydrous sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) and water. (442.5 g) was mixed and stirred at 50 ° C. for 30 minutes. Next, sodium borohydride (manufactured by Kanto Chemical Co., Ltd., 237 mg) was added to the reaction solution, and stirring was continued at 50 ° C., and then epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 1.98 g) and 48 were used. % Sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Ltd., 1.98 g) was added 12 times at 30-minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution, stirring was continued at 50 ° C. for 15 hours. did. After the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 4 having a particle diameter of 45 μm or more and 90 μm or less ( 85 mL) was obtained.
(2) Porosity and Kav Measurement The porosity of the porous crosslinked cellulose gel 4 calculated by the method described in Example 1 was 88.8%. Therefore, it became clear that the porosity of the porous crosslinked cellulose gel 4 was lower than that of the porous crosslinked cellulose gel 3 produced in Example 2.

Similarly, the Kav of the porous crosslinked cellulose gel 4 calculated by the method described in Example 1 was 0.87 for cytochrome C, 0.73 for albumin, 0.63 for apoferritin, and thyroglobulin. On the other hand, it was 0.55.
(3) Measurement of column pressure loss The porous crosslinked cellulose gel 4 measured by the method described in Example 1 was 0.18 MPa at a linear velocity of 1491 cm / hr (flow rate of 8.5 mL / min).
(4) Formylation of porous cross-linked cellulose gel, protein A immobilization, and antibody adsorption amount measurement After obtaining porous cross-linked cellulose gel 4F obtained by formylating porous cross-linked cellulose gel 4 by the method described in Example 1 As a result, an adsorbent 4FP for antibody purification with protein A immobilized thereon was obtained. The amount of protein A immobilized on the antibody purification adsorbent 4FP calculated by the method described in Example 1 was 11.5 mg per mL of gel. Similarly, the antibody adsorption amount of the adsorbent 4FP for antibody purification calculated by the method described in Example 1 was 54.5 mg per mL of gel. Therefore, it was revealed that the antibody adsorption amount of the antibody purification adsorbent 4FP was lower than that of the antibody purification adsorbent 3FP produced in Example 2.

Example 3 Production of Porous Crosslinked Cellulose Gel 5 Example 3 is a porous material from a 6% cellulose solution prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 10% by weight sodium hydroxide and 6% by weight thiourea. It is related with manufacture of a property crosslinkable cellulose gel, and the characteristic evaluation as a filler for chromatography.
(1) Production of porous cross-linked cellulose gel 5 (a) Step of obtaining a cellulose solution 5M sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Ltd., 197.3 g), thiourea (manufactured by Kanto Chemical Co., Ltd., 20.0 g) and water ( ADVANTEC filter paper powder C (18.0 g, average polymerization degree 176) was added at 25 ° C. to a 10 wt% sodium hydroxide-6 wt% thiourea mixed aqueous solution (300 mL) prepared by mixing 115.7 g). The mixture was stirred for 2 hours under ice cooling to obtain a transparent cellulose solution 5.
(B) Step of obtaining a cellulose dispersion After stirring the cellulose solution 5 obtained in step (a) at 25 ° C. for 1 hour, toluene (Kanto Chemical Co., Ltd.) containing ethyl cellulose (Kanto Chemical Co., Inc., 45 cP, 3.60 g) Manufactured at 400 ° C., and stirred at 450 rpm for 10 minutes using a stirring blade to obtain a cellulose dispersion 5.
(C) Step of obtaining a porous uncrosslinked cellulose gel The cellulose dispersion 5 obtained in step (b) was ice-cooled under the condition that stirring at 450 rpm was continued at 25 ° C., and the temperature of the dispersion was 5 ° C. or lower. Then, stirring was continued for 1 hour under ice cooling. Next, under cooling conditions such that the temperature of the cellulose dispersion is in the range of 0 ° C. to 10 ° C., ice-cooled methanol (200 mL, manufactured by Kanto Chemical Co., Ltd.) was added to the cellulose dispersion continuously stirred at 500 rpm per minute. After adding at a rate of 10 mL, the cellulose gel suspension was obtained by continuing stirring for 10 minutes under ice cooling. The obtained cellulose suspension was washed successively with 1.2 L of ethanol 5 times and with 3.5 L of water 5 times in order, and then classified using a sieve to give a porous uncrosslinked cellulose having a particle size of 150 μm or less. Gel 5 (300 mL) and porous uncrosslinked cellulose gel 5 (25 mL) having a particle size of 150 μm or more were obtained. Therefore, by using a mixed aqueous solution of sodium hydroxide and thiourea in the step (a), a porous uncrosslinked cellulose gel having a particle size of 150 μm or more than that of Example 1 using a mixed aqueous solution of sodium hydroxide and urea. Generation can be suppressed.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 5 having a particle diameter of 150 μm or less obtained in step (c) (227.1 g, moisture content 92.2%, dry cellulose 17.7 g) 1,4-dioxane (manufactured by Kanto Chemical Co., Inc., 265.5 g), water (56.1 g) and Denacol EX-313 (manufactured by Nagase ChemteX Corp., 35.4 g) were added to the reaction vessel and added at 30 ° C. for 30 minutes. After stirring for a minute, sodium borohydride (manufactured by Kanto Chemical Co., 531 mg) and 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., Ltd., 17.7 g) were added, and stirring was continued at 50 ° C. for 16 hours. After completion of the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 5.
(E) Step of obtaining porous cross-linked cellulose gel 25 wt% aqueous solution of sodium sulfate prepared from the total amount of porous partially cross-linked cellulose gel 5 obtained in step (d), anhydrous sodium sulfate (Wako Pure Chemical Industries, Ltd.) and water (442.5 g) was added to the reaction vessel and stirred at 50 ° C. for 30 minutes. Next, after adding sodium borohydride (manufactured by Kanto Chemical Co., Inc., 531 mg) to the reaction solution, stirring was continued at 50 ° C., and epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 4.43 g) and 48 were used. % Aqueous sodium hydroxide solution (4.43 g, manufactured by Kanto Chemical Co., Inc.) was added 12 times at 30 minute intervals. After the addition of epichlorohydrin and 48% aqueous sodium hydroxide solution was completed, stirring was continued at 50 ° C. for 15 hours. did. After completion of the reaction, the reaction solution is cooled to 40 ° C. or lower, washed with a large amount of water using a glass filter, and then classified using a sieve to obtain a porous crosslinked cellulose gel 5 having a particle size of 45 μm or more and 90 μm or less. (190 mL) was obtained.
(2) Exclusion limit molecular weight, porosity and Kav measurement The exclusion limit molecular weight of the polysaccharide of the porous crosslinked cellulose gel 5 measured by the method described in Example 1 was 1.5 million. Similarly, the porosity of the porous crosslinked cellulose gel 5 was calculated by the method described in Example 1. As a result, the porosity was 90.6%. Moreover, as a result of calculating Kav of porous crosslinked cellulose gel 5 by the method described in Example 1, 0.89 for cytochrome C, 0.76 for albumin, 0.68 for apoferritin, It was 0.59 with respect to thyroglobulin.
(3) Column pressure loss measurement As a result of measuring the column pressure loss of the porous crosslinked cellulose gel 5 by the method described in Example 1, the column pressure loss at a linear velocity of 1491 cm / hr (flow rate 8.5 mL / min) is It was 0.24 MPa.
(4) Formylation of porous cross-linked cellulose gel, protein A immobilization and antibody adsorption amount measurement After obtaining porous cross-linked cellulose gel 5F by formylating porous cross-linked cellulose gel 5 by the method described in Example 1 A porous crosslinked cellulose gel 5FP having protein A immobilized thereon was obtained.

Similarly, as a result of calculating the protein A immobilization amount of the porous crosslinked cellulose gel 5FP by the method described in Example 1, the immobilization amount was 11.5 mg per mL of the gel. Similarly, as a result of calculating the antibody adsorption amount of porous crosslinked cellulose gel 5FP, the antibody adsorption amount was 62.4 mg per mL of gel.
(5) Measurement of antibody adsorption amount of porous crosslinked cellulose gel not immobilized with protein A By the method described in Example 1, the amount of static antibody adsorption of porous crosslinked cellulose gel 5 not immobilized with protein A was measured. As a result, the static antibody adsorption amount was 0.6 mg per mL of gel. Therefore, it was revealed that nonspecific adsorption of the antibody to the porous crosslinked cellulose gel 5 was low.

Comparative Example 3 Production of Porous Crosslinked Cellulose Gel 6 Comparative Example 3 was prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 10 wt% sodium hydroxide and 6 wt% thiourea in the same manner as in Example 6. This is related to the production of a porous crosslinked cellulose gel and the property evaluation as a packing material for chromatography when the addition temperature of the gelling agent in step (c) of Example 3 is 25 ° C. using a% cellulose solution. is there.
(1) Production of Porous Crosslinked Cellulose Gel 6 (a) Step of Obtaining Cellulose Solution 5M aqueous sodium hydroxide (Kanto Chemical Co., 144.7 g), thiourea (Kanto Chemical Co., 14.7 g) and water ( ADVANTEC filter paper powder C (13.2 g, average polymerization degree 176) was added at 25 ° C. to 10 wt% sodium hydroxide-6 wt% thiourea mixed aqueous solution (220 mL) prepared by mixing 84.9 g). Then, a transparent cellulose solution 6 was obtained by stirring for 2 hours under ice cooling.
(B) Step of obtaining a cellulose dispersion The cellulose dispersion 6 was obtained by the method described in Comparative Example 1 using the cellulose solution 6 obtained in the step (a).
(C) Step for obtaining porous uncrosslinked cellulose gel Porous uncrosslinked cellulose gel 6 having a particle diameter of 150 μm or less by the method described in Comparative Example 1 using cellulose dispersion 6 obtained in step (b). 220 mL) and porous uncrosslinked cellulose gel 6 (30 mL) having a particle diameter of 150 μm or more were obtained.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 6 having a particle diameter of 150 μm or less obtained in step (c) (125.2 g, moisture content 89.8%, dry cellulose 12.8 g) 1,4-dioxane (manufactured by Kanto Chemical Co., 192.0 g), water (79.6 g), Denacol EX-313 (manufactured by Nagase ChemteX Corp., 25.6 g) were added to the reaction vessel and added at 30 ° C. for 30 minutes. After stirring for a minute, sodium borohydride (Kanto Chemical Co., 384 mg) and 48% aqueous sodium hydroxide (Kanto Chemical Co., 12.8 g) were added, and stirring was continued at 50 ° C. for 16 hours. After completion of the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 6.
(E) Step of obtaining porous cross-linked cellulose gel 25 wt% aqueous sodium sulfate solution prepared from the total amount of porous partially cross-linked cellulose gel 6 obtained in step (d), anhydrous sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) and water. (442.5 g) was added to the reaction vessel and stirred at 50 ° C. for 30 minutes. Next, after adding sodium borohydride (manufactured by Kanto Chemical Co., Inc., 384 mg) to the reaction solution, epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 3.20 g), 48 % Sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Ltd., 3.20 g) was added 12 times at 30 minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution, stirring was continued at 50 ° C. for 15 hours. did. After completion of the reaction, the reaction solution is cooled to 40 ° C. or lower, washed with a large amount of water using a glass filter, and then classified using a sieve to obtain a porous crosslinked cellulose gel 6 having a particle size of 45 μm to 90 μm. (115 mL) was obtained.
(2) Porosity and Kav Measurement As a result of calculating the porosity of the porous crosslinked cellulose gel 6 by the method described in Example 1, the porosity was 86.5%. Therefore, it became clear that the porosity of the porous crosslinked cellulose gel 6 was lower than that of the porous crosslinked cellulose gel 5 produced in Example 3.

Moreover, as a result of calculating Kav of porous crosslinked cellulose gel 6 by the method described in Example 1, 0.84 for cytochrome C, 0.70 for albumin, 0.58 for apoferritin, It was 0.52 with respect to thyroglobulin.
(3) Column pressure loss measurement As a result of measuring the column pressure loss of the porous crosslinked cellulose gel 6 by the method described in Example 1, the column pressure loss at a linear velocity of 1491 cm / hr (flow rate 8.5 mL / min) is It was 0.16 MPa.
(4) Formylation of porous crosslinked cellulose gel, protein A immobilization, and antibody adsorption amount measurement After obtaining porous crosslinked cellulose gel 6F by formylating porous crosslinked cellulose gel 6 by the method described in Example 1 A porous crosslinked cellulose gel 6FP having protein A immobilized thereon was obtained.

Similarly, as a result of calculating the protein A immobilization amount of the porous crosslinked cellulose gel 6FP by the method described in Example 1, the immobilization amount was 11.5 mg per mL of the gel. Similarly, as a result of calculating the antibody adsorption amount of porous crosslinked cellulose gel 6FP, the antibody adsorption amount was 53.1 mg per mL of gel. Therefore, it was revealed that the antibody adsorption amount of the porous crosslinked cellulose gel 6FP was lower than that of the porous crosslinked cellulose gel 5FP produced in Example 3.

Table 1 summarizes the results of Examples 1 to 3 and Comparative Examples 1 to 3. The porous cross-linked cellulose gels 1, 3, and 5 produced in step (c) at a temperature during addition of the gelling agent at 0 to 10 ° C had a porosity of 90% or more, whereas the gelling agent The porosity of the porous crosslinked cellulose gels 2, 4, and 6 produced at a temperature of 25 ° C. at the time of addition was 90% or less. Furthermore, the amount of antibody adsorbed by the adsorbents 1FP, 3FP, and 5FP for antibody purification in which protein A is immobilized on each of the porous crosslinked cellulose gels 1, 3, and 5 is as follows. It was revealed that the antibody was higher than the adsorbents 2FP, 4FP, and 6FP for purifying antibodies.

Comparative Example 4 Characteristic Evaluation of Commercially Available Porous Crosslinked Polysaccharide Gel In Comparative Example 4, Cellufine GCL-2000 (manufactured by JNC, porous crosslinked cellulose gel, particle size 40) -130 μm), and the characteristics as a packing material for chromatography were evaluated by the method described in Example 1.
(1) Kav and porosity measurement The porosity of Cellufine GCL-2000 calculated by the method described in Example 1 was 89.6%. Similarly, Kav of Cellufine GCL-2000 calculated by the method described in Example 1 was 0.66 for cytochrome C, 0.42 for albumin, 0.26 for apoferritin, and for thyroglobulin. 0.19.
(2) Column pressure loss measurement The column pressure loss of Cellufine GCL-2000 measured by the method described in Example 1 was 0.25 MPa at a flow rate of 4.0 mL / min (linear velocity of 702 cm / hour). When the flow rate was increased to 4.5 mL / min, the pressure of the pump reached the limit of the HPLC system, and the liquid could not be passed.

A graph showing the relationship between the flow velocity and the column pressure loss in Examples 1 and 2 and Comparative Example 3 is shown in FIG. As is clear from FIG. 1, the column pressure loss of the porous crosslinked cellulose gels 1, 3, and 5 produced in Examples 1, 2, and 3 was found to be lower than that of Cellufine GCL-2000.
(3) Formylation, protein A immobilization and measurement of antibody adsorption amount of commercially available porous crosslinked polysaccharide gel After formylating Cellufine GCL-2000 from Cellufine GCL-2000 by the method described in Example 1, Protein A-immobilized Cellufine GCL-2000 was obtained. The amount of protein A immobilized on Protein A-immobilized Cellufine GCL-2000 calculated by the method described in Example 1 was 10.9 mg per mL of gel. Similarly, the antibody adsorption amount of protein A-immobilized Cellufine GCL-2000 calculated by the method described in Example 1 was 31.8 mg per mL of gel. Therefore, the adsorbents for antibody purification 1FP, 3FP, and 5FP in which protein A is immobilized on each of the porous crosslinked cellulose gels 1, 3, and 5 are compared with Cellufine GCL-2000 in which protein A is immobilized by the same method. It was revealed that the amount of antibody adsorption was high.

Table 2 shows the results of Examples 1 to 3 and Comparative Example 4.

Example 4 Production of Porous Crosslinked Cellulose Gel 7 Example 4 is porous from a 5% cellulose solution prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 7% by weight sodium hydroxide and 22% by weight urea. The present invention relates to the production of a crosslinked cellulose gel and the evaluation of properties as a chromatographic filler.
(1) Manufacture of porous crosslinked cellulose gel 7 (a) Step of obtaining cellulose solution 5M sodium hydroxide aqueous solution (Kanto Chemical Co., 140.6 g), urea (Kanto Chemical Co., 74.6 g) and water (123 ADVANTEC filter paper powder C (15.0 g, average polymerization degree 176) was added at 25 ° C. to 7 wt% sodium hydroxide-22 wt% urea mixed aqueous solution (300 mL) prepared by mixing 8 g). A transparent cellulose solution 7 was obtained by stirring for 2 hours under ice cooling.
(B) Step of obtaining a cellulose dispersion After stirring the cellulose solution 7 obtained in step (a) at 25 ° C. for 1 hour, toluene (Kanto Chemical Co., Inc., 45 cP, 3.40 g) manufactured by Kanto Chemical Co., Inc. Product, 400 mL) at 25 ° C., and stirred for 10 minutes at 450 rpm using a stirring blade to obtain a cellulose dispersion 7.
(C) Step of obtaining porous uncrosslinked cellulose gel The cellulose dispersion 7 obtained in step (b) was ice-cooled under the condition that stirring at 450 rpm was continued at 25 ° C., and the temperature of the dispersion was 5 ° C. or lower. Then, stirring was continued for 1 hour under ice cooling. Next, on the condition that the temperature of the cellulose dispersion was cooled to 0 ° C. to 10 ° C., ice-cooled methanol (200 mL, manufactured by Kanto Chemical Co., Ltd.) was added to the cellulose dispersion continuously stirred at 450 rpm per minute. After adding at a rate of 10 mL, the cellulose gel suspension was obtained by continuing stirring for 10 minutes under ice cooling. The obtained cellulose gel suspension was washed 5 times with 1.2 L of ethanol and 5 times with 3.5 L of water, and then classified using a sieve, so that porous uncrosslinked particles having a particle size of 150 μm or less were obtained. Cellulose gel 7 (350 mL) and porous uncrosslinked cellulose gel 7 (45 mL) having a particle diameter of 150 μm or more were obtained.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 7 having a particle diameter of 150 μm or less obtained in step (c) (231.3 g, moisture content 93.8%, dry cellulose 14.3 g) 1,4-dioxane (manufactured by Kanto Chemical Co., Inc., 286.0 g), water (69.0 g), Denacol EX-313 (manufactured by Nagase ChemteX Corp., 28.6 g) were mixed and stirred at 50 ° C. for 30 minutes. After that, sodium borohydride (manufactured by Kanto Chemical Co., 429 mg) and 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., 14.3 g) were added, and stirring was continued at 50 ° C. for 16 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 7.
(E) Step of obtaining porous cross-linked cellulose gel 25 wt% aqueous solution of sodium sulfate prepared from the total amount of porous partially cross-linked cellulose gel 7 obtained in step (d), anhydrous sodium sulfate (Wako Pure Chemical Industries, Ltd.) and water. (357.5 g) was mixed and stirred at 50 ° C. for 30 minutes. Next, sodium borohydride (manufactured by Kanto Chemical Co., Inc., 429 mg) was added to the reaction solution, and then stirring was continued at 50 ° C., and epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 4.78 g) and 48 were used. % Sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Ltd., 4.78 g) was added 12 times at 30 minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution, stirring was continued at 50 ° C. for 15 hours. did. After the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 7 having a particle diameter of 45 μm or more and 90 μm or less ( 195 mL).
(2) Exclusion Limit Molecular Weight, Porosity and Kav Measurement The exclusion limit molecular weight of the polysaccharide of the porous crosslinked cellulose gel 7 measured by the method described in Example 1 was 1.94 million. Similarly, the porosity of the porous crosslinked cellulose gel 7 calculated by the method described in Example 1 was 92.6%. Similarly, the Kav of the porous crosslinked cellulose gel 7 calculated by the method described in Example 1 was 0.92 for cytochrome C, 0.80 for albumin, 0.70 for apoferritin, and thyroglobulin. On the other hand, it was 0.63.
(3) Column pressure loss measurement The column pressure loss of the porous crosslinked cellulose gel 7 measured by the method described in Example 1 was 0.26 MPa at a linear velocity of 1491 cm / hour (flow rate 8.5 mL / min).
(4) Formylation of porous cross-linked cellulose gel, protein A immobilization, and antibody adsorption amount measurement After obtaining porous cross-linked cellulose gel 7F obtained by formylating porous cross-linked cellulose gel 7 by the method described in Example 1 Then, an adsorbent for antibody purification 7FP with protein A immobilized thereon was obtained. The amount of protein A immobilized on the antibody purification adsorbent 7FP calculated by the method described in Example 1 was 10.8 mg per mL of gel. Similarly, the antibody adsorption amount of the antibody purification adsorbent 7FP calculated by the method described in Example 1 was 64.9 mg per mL of gel.

Example 5 Production of Porous Crosslinked Cellulose Gel 8 Example 5 was prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 9% by weight sodium hydroxide, 4% by weight urea and 4% by weight thiourea. The present invention relates to the production of a porous crosslinked cellulose gel from a cellulose solution and the evaluation of properties as a chromatographic filler.
(1) Production of porous crosslinked cellulose gel 8 (a) Step of obtaining cellulose solution 5M aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., 177.6 g), urea (manufactured by Kanto Chemical Co., Ltd., 13.3 g) and thiourea ( A filter paper manufactured by ADVANTEC Co., Ltd. was added to a 9 wt% sodium hydroxide-4 wt% urea-4 wt% thiourea mixed aqueous solution (300 mL) prepared by mixing 13.3 g) and water (128.8 g) manufactured by Kanto Chemical Co., Ltd. Powder C (15.0 g, average polymerization degree 176) was added at 25 ° C., and the mixture was stirred for 2 hours under ice cooling to obtain a transparent cellulose solution 8.
(B) Step of obtaining a cellulose dispersion After stirring the cellulose solution 8 obtained in step (a) at 25 ° C. for 1 hour, toluene (Kanto Chemical Co., Inc., 45 cP, 3.40 g) manufactured by Kanto Chemical Co., Inc. Manufactured at 400 ° C., and stirred at 500 rpm for 10 minutes using a stirring blade to obtain a cellulose dispersion 8.
(C) Step of obtaining porous uncrosslinked cellulose gel Porous uncrosslinked cellulose gel 8 having a particle diameter of 150 μm or less by the method described in Example 1 using cellulose dispersion 8 obtained in step (b). 380 mL) and porous uncrosslinked cellulose gel 8 (40 mL) having a particle diameter of 150 μm or more were obtained.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 8 having a particle diameter of 150 μm or less obtained in step (c) (210.7 g, moisture content 93.0%, dry cellulose 14.7 g) 1,4-dioxane (Kanto Chemical Co., Ltd., 294.0 g), water (98.0 g), Denacol EX-313 (manufactured by Nagase ChemteX Corp., 29.4 g) were mixed and stirred at 50 ° C. for 30 minutes. After that, sodium borohydride (manufactured by Kanto Chemical Co., 441 mg) and a 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., Ltd., 14.7 g) were added, and stirring was continued at 50 ° C. for 16 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 8.
(E) Step of obtaining porous cross-linked cellulose gel 25 wt% aqueous solution of sodium sulfate prepared from the total amount of porous partially cross-linked cellulose gel 8 obtained in step (d), anhydrous sodium sulfate (Wako Pure Chemical Industries, Ltd.) and water. (367.5 g) was mixed and stirred at 50 ° C. for 30 minutes. Next, after adding sodium borohydride (manufactured by Kanto Chemical Co., Inc., 441 mg) to the reaction solution, epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 4.92 g) and 48 were used under the condition that stirring at 50 ° C. was continued. % Sodium hydroxide aqueous solution (4.92 g, manufactured by Kanto Chemical Co., Inc.) was added 12 times at 30-minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution, stirring was continued at 50 ° C. for 15 hours. did. After the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 8 having a particle diameter of 45 μm or more and 90 μm or less ( 180 mL) was obtained.
(2) Exclusion limit molecular weight, porosity, and Kav measurement The exclusion limit molecular weight of the polysaccharide of the porous crosslinked cellulose gel 8 measured by the method described in Example 1 was 2.04 million. Similarly, the porosity of the porous crosslinked cellulose gel 8 calculated by the method described in Example 1 was 91.4%. Similarly, the Kav of the porous crosslinked cellulose gel 8 calculated by the method described in Example 1 is 0.92 for cytochrome C, 0.80 for albumin, 0.70 for apoferritin, and thyroglobulin. On the other hand, it was 0.63.
(3) Column pressure loss measurement The column pressure loss of the porous crosslinked cellulose gel 8 measured by the method described in Example 1 was 0.28 MPa at a linear velocity of 1491 cm / hour (flow rate: 8.5 mL / min).
(4) Formylation of porous crosslinked cellulose gel, protein A immobilization and antibody adsorption amount measurement After obtaining porous crosslinked cellulose gel 8F by formylating porous crosslinked cellulose gel 8 by the method described in Example 1 Thus, an adsorbent for antibody purification 8FP with protein A immobilized thereon was obtained. The protein A immobilization amount of the antibody purification adsorbent 8FP calculated by the method described in Example 1 was 11.1 mg per mL of gel. Similarly, the antibody adsorption amount of the antibody purification adsorbent 8FP calculated by the method described in Example 1 was 59.4 mg per mL of gel.

Example 6 Production of Porous Crosslinked Cellulose Gel 9 Example 6 is a porous material from a 5% cellulose solution prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 10% by weight sodium hydroxide and 6% by weight thiourea. It is related with manufacture of a property crosslinkable cellulose gel, and the characteristic evaluation as a filler for chromatography.
(1) Production of porous crosslinked cellulose gel 9 (a) Step of obtaining a cellulose solution 5M sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., 197.3 g), thiourea (manufactured by Kanto Chemical Co., Ltd., 20.0 g) and water ( ADVANTEC filter paper powder C (15.0 g, average polymerization degree 176) was added at 25 ° C. to a 10 wt% sodium hydroxide-6 wt% thiourea mixed aqueous solution (300 mL) prepared by mixing 115.7 g). The mixture was stirred for 2 hours under ice cooling to obtain a transparent cellulose solution 9.
(B) The process of obtaining a cellulose dispersion liquid The cellulose dispersion liquid 9 was obtained by the method described in Example 4 using the cellulose solution 9 obtained at the process (a).
(C) Step for obtaining porous uncrosslinked cellulose gel Porous uncrosslinked cellulose gel 9 having a particle diameter of 150 μm or less (by the method described in Example 4) using cellulose dispersion 9 obtained in step (b). 350 mL) and porous uncrosslinked cellulose gel 9 (25 mL) having a particle diameter of 150 μm or more were obtained.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 9 having a particle diameter of 150 μm or less obtained in step (c) (194.1 g, moisture content 92.5%, dry cellulose 14.6 g) 1,4-dioxane (manufactured by Kanto Chemical Co., Inc., 292.0 g), water (112.5 g), Denacol EX-313 (manufactured by Nagase ChemteX Co., Ltd., 29.2 g) were added to the reaction vessel and added at After stirring for a minute, sodium borohydride (Kanto Chemical Co., 438 mg) and 48% aqueous sodium hydroxide (Kanto Chemical Co., 14.6 g) were added, and stirring was continued at 50 ° C. for 16 hours. After completion of the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 9.
(E) Step of obtaining porous crosslinked cellulose gel 25 wt% aqueous sodium sulfate solution prepared from the whole amount of porous partially crosslinked cellulose gel 9 obtained in step (d), anhydrous sodium sulfate (Wako Pure Chemical Industries, Ltd.) and water. (365.0 g) was added to the reaction vessel and stirred at 50 ° C. for 30 minutes. Next, sodium borohydride (manufactured by Kanto Chemical Co., Inc., 438 mg) was added to the reaction solution, and stirring was continued at 50 ° C., followed by epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 4.85 g) and 48. % Sodium hydroxide aqueous solution (4.85 g, manufactured by Kanto Chemical Co., Inc.) was added 12 times at 30 minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution was completed, stirring was continued at 50 ° C. for 15 hours. did. After completion of the reaction, the reaction solution is cooled to 40 ° C. or lower, washed with a large amount of water using a glass filter, and then classified using a sieve to obtain a porous crosslinked cellulose gel 9 having a particle size of 45 μm or more and 90 μm or less. (210 mL) was obtained.
(2) Exclusion limit molecular weight, porosity, and Kav measurement The exclusion limit molecular weight of the polysaccharide of the porous crosslinked cellulose gel 9 measured by the method described in Example 1 was 2.1 million. Similarly, the porosity of the porous crosslinked cellulose gel 9 was calculated by the method described in Example 1. As a result, the porosity was 91.2%. Moreover, as a result of calculating Kav of porous crosslinked cellulose gel 9 by the method described in Example 1, 0.92 for cytochrome C, 0.81 for albumin, 0.73 for apoferritin, 0.65 relative to thyroglobulin.
(3) Column pressure loss measurement As a result of measuring the column pressure loss of the porous crosslinked cellulose gel 9 by the method described in Example 1, the column pressure loss at a linear velocity of 1491 cm / hour (flow rate 8.5 mL / min) is It was 0.26 MPa.
(4) Formylation of porous crosslinked cellulose gel, protein A immobilization, and antibody adsorption amount measurement After obtaining porous crosslinked cellulose gel 9F by formylating porous crosslinked cellulose gel 9 by the method described in Example 1 A porous crosslinked cellulose gel 9FP having protein A immobilized thereon was obtained.

Similarly, as a result of calculating the protein A immobilization amount of the porous crosslinked cellulose gel 9FP by the method described in Example 1, the immobilization amount was 11.8 mg per mL of the gel. Similarly, as a result of calculating the antibody adsorption amount of the porous crosslinked cellulose gel 9FP, the antibody adsorption amount was 58.9 mg per mL of the gel.

The results of Examples 4 to 6 are shown in Table 3 together with the results of Examples 1 to 3.

Example 7 Production of Porous Crosslinked Cellulose Gel 10 Example 7 is porous from a 5% cellulose solution prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 8% by weight sodium hydroxide and 17% by weight urea. The present invention relates to production of a crosslinked cellulose gel and evaluation of properties as a chromatographic filler.
(1) Production of porous crosslinked cellulose gel 10 (a) Step of obtaining cellulose solution 5M aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., 160.7 g), urea (manufactured by Kanto Chemical Co., Ltd., 57.6 g) and water (120 ADVANTEC filter paper powder C (15.0 g, average polymerization degree 176) was added at 25 ° C. to 8 wt% sodium hydroxide-17 wt% urea mixed aqueous solution (300 mL) prepared by mixing 7 g). A transparent cellulose solution 10 was obtained by stirring for 2 hours under ice cooling.
(B) The process of obtaining a cellulose dispersion liquid The cellulose dispersion liquid 10 was obtained by the method described in Example 3 using the cellulose solution 10 obtained at the process (a).
(C) Step for obtaining porous uncrosslinked cellulose gel Porous uncrosslinked cellulose gel 10 having a particle diameter of 150 μm or less by the method described in Example 3 using cellulose dispersion 10 obtained in step (b). 350 mL) and porous uncrosslinked cellulose gel 10 (40 mL) having a particle diameter of 150 μm or more were obtained.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 10 having a particle diameter of 150 μm or less obtained in step (c) (227.6 g, moisture content 93.8%, dry cellulose 14.1 g) 1,4-dioxane (manufactured by Kanto Chemical Co., Inc., 282.0 g), water (68.5 g), Denacol EX-313 (manufactured by Nagase ChemteX Corp., 28.2 g) were mixed and stirred at 50 ° C. for 30 minutes. After that, sodium borohydride (manufactured by Kanto Chemical Co., Ltd., 423 mg) and a 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., Ltd., 14.1 g) were added, and stirring was continued at 50 ° C. for 16 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 10.
(E) Step of obtaining porous cross-linked cellulose gel 25 wt% aqueous solution of sodium sulfate prepared from the total amount of porous partially cross-linked cellulose gel 10 obtained in step (d), anhydrous sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) and water. (352.5 g) was mixed and stirred at 50 ° C. for 30 minutes. Next, sodium borohydride (manufactured by Kanto Chemical Co., Inc., 423 mg) was added to the reaction solution, and stirring was continued at 50 ° C., and epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 4.70 g) and 48 were used. % Sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Ltd., 4.70 g) was added 12 times at 30 minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution, stirring was continued at 50 ° C. for 15 hours. did. After the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 10 having a particle size of 45 μm or more and 90 μm or less ( 185 mL).
(2) Porosity and Kav Measurement The porosity of the porous crosslinked cellulose gel 10 calculated by the method described in Example 1 was 91.4%. Similarly, the Kav of the porous crosslinked cellulose gel 10 calculated by the method described in Example 1 is 0.92 for cytochrome C, 0.79 for albumin, 0.67 for apoferritin, and thyroglobulin. On the other hand, it was 0.61.
(3) Column pressure loss measurement The column pressure loss of the porous crosslinked cellulose gel 10 measured by the method described in Example 1 is 0.23 MPa at a linear velocity of 1491 cm / hour (flow rate of 8.5 mL / min). Met.
(4) Formylation of porous crosslinked cellulose gel, protein A immobilization, and antibody adsorption amount measurement After obtaining porous crosslinked cellulose gel 10F obtained by formylating porous crosslinked cellulose gel 10 according to the method described in Example 1. Thus, an adsorbent for antibody purification 10FP with protein A immobilized thereon was obtained. The amount of protein A immobilized on the antibody purification adsorbent 10FP calculated by the method described in Example 1 was 11.0 mg per mL of gel. Similarly, the antibody adsorption amount of the antibody purification adsorbent 10FP calculated by the method described in Example 1 was 62.5 mg per mL of gel.

Comparative Example 5 Production of Porous Crosslinked Cellulose Gel 11 Comparative Example 5 was prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 8 wt% sodium hydroxide and 17 wt% urea as in Example 7. After obtaining a porous uncrosslinked cellulose gel from a% cellulose solution, the step (e) in Example 7 was omitted to produce a porous crosslinked cellulose gel.
(1) Production of porous crosslinked cellulose gel 11 Porous uncrosslinked cellulose gel 11 having a particle diameter of 150 μm or less is obtained by carrying out the steps from step (a) to step (c) by the method described in Example 7. It was.
(D) Step of obtaining porous crosslinked cellulose gel Porous uncrosslinked cellulose gel 11 having a particle size of 150 μm or less (29.1 g, water content 91.4%, dry cellulose 2.5 g), 1,4-dioxane (Kanto Chemical) 50.0 g), water (23.4 g), Denacol EX-313 (manufactured by Nagase ChemteX Corporation, 5.0 g) were mixed and stirred at 50 ° C. for 30 minutes, and then sodium borohydride (Kanto Chemical) (75 mg) and 48% aqueous sodium hydroxide (manufactured by Kanto Chemical Co., 2.5 g) were added, and stirring was continued at 50 ° C. for 16 hours. After the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 11 having a particle diameter of 45 μm or more and 90 μm or less ( 28 mL) was obtained.
(2) Porosity and Kav measurement When the column was filled with porous crosslinked cellulose gel 11 having a particle size of 45 μm or more and 90 μm or less by the method described in Example 1, water was passed through at 0.3 mL / min. When the column pressure loss exceeded 1 MPa and the flow rate was further increased to 0.5 mL / min, the pressure of the pump reached the limit of the HPLC system, and liquid passage became impossible. Therefore, Kav, porosity, and column pressure loss measurement of the porous crosslinked cellulose gel 11 were not performed.

Comparative Example 6 Production of Porous Crosslinked Cellulose Gel 12 In Comparative Example 6, 5% prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 8 wt% sodium hydroxide and 17 wt% urea as in Example 7. After obtaining a porous uncrosslinked cellulose gel from a cellulose solution, the step (d) in Example 7 was performed according to the method described in Examples 1, 4 and 7 of Patent Document 6, and the step (e) was omitted. Thus, a porous crosslinked cellulose gel was produced.
(1) Production of porous crosslinked cellulose gel 12 By performing the steps from step (a) to step (c) by the method described in Example 7, porous uncrosslinked cellulose gel 12 having a particle diameter of 150 μm or less is obtained. It was.
(D) Step of obtaining porous crosslinked cellulose gel Porous uncrosslinked cellulose gel 12 (30.0 g, water content 91.5%, dry cellulose 2.6 g) having a particle size of 150 μm or less, sodium hydroxide (Wako Pure Chemical Industries, Ltd.) And 0.6M aqueous sodium hydroxide solution (30.0 mL) prepared from water was mixed and stirred at 40 ° C. for 30 minutes. Next, sodium borohydride (manufactured by Kanto Chemical Co., Ltd., 60 mg) and Denacol EX-313 (manufactured by Nagase ChemteX Corp., 30.0 mL) were added, and stirring was continued at 50 ° C. for 5 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain porous crosslinked cellulose gel 12A.

The total amount of the obtained porous crosslinked cellulose gel 12A was added to a heat-resistant glass container and heated at 121 ° C. for 40 minutes using an autoclave (SS-245 manufactured by Tommy Seiko Co., Ltd.). After the container was cooled to 40 ° C. or lower, it was washed with a large amount of water using a glass filter to obtain porous crosslinked cellulose gel 12B.

Next, the total amount of the obtained porous crosslinked cellulose gel 12B, sodium hydroxide (manufactured by Wako Pure Chemical Industries, Ltd.) and 0.6M aqueous sodium hydroxide solution (30.0 mL) prepared from water were mixed and mixed at 40 ° C. for 30. Stir for minutes. Furthermore, sodium borohydride (manufactured by Kanto Chemical Co., 60 mg) and Denacol EX-313 (manufactured by Nagase ChemteX, 30.0 mL) were added to the reaction solution, and stirring was continued at 50 ° C. for 5 hours. After the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain porous crosslinked cellulose gel 12C.

The total amount of the resulting porous crosslinked cellulose gel 12C was added to a heat-resistant glass container, and heated at 121 ° C. for 40 minutes using an autoclave (SS-245 manufactured by Tommy Seiko Co., Ltd.). The container is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and classified using a sieve to obtain porous crosslinked cellulose gel 12 (40 mL) having a particle size of 45 μm to 90 μm. It was.
(2) Porosity and Kav Measurement The porosity of the porous crosslinked cellulose gel 12 calculated by the method described in Example 1 was 86.7%. Similarly, the Kav of the porous crosslinked cellulose gel 12 calculated by the method described in Example 1 is 0.80 for cytochrome C, 0.70 for albumin, 0.62 for apoferritin, and thyroglobulin. On the other hand, it was 0.58.
(3) Column pressure loss measurement The column pressure loss of the porous crosslinked cellulose gel 12 measured by the method described in Example 1 was 1.56 MPa at a flow rate of 2.0 mL / min (linear velocity of 351 cm / hour). When the pressure was increased to 2.5 mL / min, the pressure of the pump reached the limit of the HPLC system and the liquid could not be passed.
(4) Formylation of porous crosslinked cellulose gel, protein A immobilization and antibody adsorption amount measurement After obtaining porous crosslinked cellulose gel 12F obtained by formylating porous crosslinked cellulose gel 12 by the method described in Example 1 As a result, an adsorbent 12FP for antibody purification with protein A immobilized thereon was obtained. The amount of protein A immobilized on the antibody purification adsorbent 12FP calculated by the method described in Example 1 was 11.3 mg per mL of gel. Similarly, the antibody adsorption amount of the adsorbent 12FP for antibody purification calculated by the method described in Example 1 was 44.1 mg per mL of gel. Therefore, it was revealed that the antibody adsorption amount of the antibody purification adsorbent 12FP was lower than that of the antibody purification adsorbent 10FP produced in Example 7.

Comparative Example 7 Production of Porous Crosslinked Cellulose Gel 13 In Comparative Example 7, 5% prepared by dissolving cotton-derived cellulose in a mixed aqueous solution of 8 wt% sodium hydroxide and 17 wt% urea in the same manner as in Example 7. After obtaining a porous uncrosslinked cellulose gel from the cellulose solution, the step (d) in Example 7 was omitted to produce a porous crosslinked cellulose gel.
(1) Production of porous crosslinked cellulose gel 13 By performing the steps from step (a) to step (c) by the method described in Example 7, porous uncrosslinked cellulose gel 13 having a particle diameter of 150 μm or less is obtained. It was.
(E) Step of obtaining porous crosslinked cellulose gel Porous uncrosslinked cellulose gel 13 (87.3 g, water content 91.5%, dry cellulose 7.4 g) having a particle size of 150 μm or less, and anhydrous sodium sulfate (Wako Pure Chemical Industries, Ltd.) And 25 wt% aqueous sodium sulfate solution (185.0 g) prepared from water were mixed and stirred at 50 ° C. for 30 minutes. Next, after adding sodium borohydride (manufactured by Kanto Chemical Co., Inc., 222 mg) to the reaction solution, stirring was continued at 50 ° C., and epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 2.47 g) and 48 were used. % Sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Inc., 2.47 g) was added 12 times at 30-minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution, stirring was continued at 50 ° C. for 15 hours. did. After the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 13 having a particle diameter of 45 μm or more and 90 μm or less ( 105 mL) was obtained.
(2) Porosity and Kav Measurement The porosity of the porous crosslinked cellulose gel 13 calculated by the method described in Example 1 was 89.8%. Similarly, the Kav of the porous crosslinked cellulose gel 13 calculated by the method described in Example 1 is 0.90 for cytochrome C, 0.75 for albumin, 0.67 for apoferritin, and thyroglobulin. On the other hand, it was 0.63.
(3) Column pressure loss measurement The column pressure loss of the porous crosslinked cellulose gel 13 measured by the method described in Example 1 was 1.44 MPa at a flow rate of 4.5 mL / min (linear velocity of 790 cm / hr). When the flow rate was increased to 5.0 mL / min, the pressure of the pump reached the limit of the HPLC system, and the liquid could not be passed.
(4) Formylation of porous crosslinked cellulose gel, protein A immobilization and antibody adsorption amount measurement After obtaining porous crosslinked cellulose gel 13F obtained by formylating porous crosslinked cellulose gel 13 by the method described in Example 1 As a result, an adsorbent for antibody purification 13FP with protein A immobilized thereon was obtained. The amount of protein A immobilized on the antibody purification adsorbent 13FP calculated by the method described in Example 1 was 11.5 mg per mL of gel. Similarly, the antibody adsorption amount of the adsorbent 13FP for antibody purification calculated by the method described in Example 1 was 55.4 mg per mL of gel. Therefore, it was revealed that the antibody adsorption amount of the antibody purification adsorbent 13FP was lower than that of the antibody purification adsorbent 10FP produced in Example 7.

FIG. 2 is a graph showing the relationship between the flow rate and the column pressure loss of the porous crosslinked cellulose gel 10 produced in Example 7 and the porous crosslinked cellulose gels 12 and 13 produced in Comparative Examples 6 and 7. As is clear from FIG. 2, the column pressure loss of the porous crosslinked cellulose gel 10 produced in Example 7 is lower than that of the porous crosslinked cellulose gels 12 and 13 produced in Comparative Examples 6 and 7. It was.

Table 4 summarizes the results of Example 7 and Comparative Examples 6 and 7.

Example 8 Production of Porous Crosslinked Cellulose Gel 14 Example 8 is a porous material from a 5% cellulose solution prepared by dissolving wood pulp-derived cellulose in a mixed aqueous solution of 8% by weight sodium hydroxide and 17% by weight urea. It is related with manufacture of a property crosslinkable cellulose gel, and the characteristic evaluation as a chromatography filler.
(1) Manufacture of porous crosslinked cellulose gel 14 (a) Step of obtaining cellulose solution Except for using filter paper powder C manufactured by ADVANTEC, Theolas PH-101 (15.0 g, average polymerization degree 173) manufactured by Asahi Kasei A transparent cellulose solution 14 was obtained by the method described in Example 7.
(B) Step of obtaining a cellulose dispersion After stirring the cellulose solution 14 obtained in step (a) for 1 hour at 25 ° C., toluene (Kanto Chemical Co., Inc., 45 cP, 3.60 g) manufactured by Kanto Chemical Co., Inc. Manufactured at 400 ° C. and stirred at 500 rpm for 10 minutes using a stirring blade to obtain a cellulose dispersion 14.
(C) Step of obtaining porous uncrosslinked cellulose gel Porous uncrosslinked cellulose gel 14 having a particle diameter of 150 μm or less by the method described in Example 1 using the cellulose dispersion 14 obtained in step (b). 350 mL) and porous uncrosslinked cellulose gel 14 (50 mL) having a particle diameter of 140 μm or more were obtained.
(D) Step of obtaining porous partially crosslinked cellulose gel Porous uncrosslinked cellulose gel 14 having a particle diameter of 140 μm or less obtained in step (c) (178.8 g, moisture content 92.1%, dry cellulose 14.1 g) 1,4-dioxane (manufactured by Kanto Chemical Co., Inc., 282.0 g), water (68.5 g), Denacol EX-313 (manufactured by Nagase ChemteX Corp., 28.2 g) were added to the reaction vessel, and the mixture was stirred at 50 ° C. for 30 minutes. After stirring for minutes, sodium borohydride (manufactured by Kanto Chemical Co., Ltd., 423 mg) and 48% aqueous sodium hydroxide (manufactured by Kanto Chemical Co., Ltd., 14.1 g) were added, and stirring was continued at 50 ° C. for 16 hours. After completion of the reaction, the reaction solution was cooled to 40 ° C. or lower, and then washed with a large amount of water using a glass filter to obtain a porous partially crosslinked cellulose gel 14.
(E) Step of obtaining porous cross-linked cellulose gel 25 wt% aqueous sodium sulfate solution prepared from the total amount of porous partially cross-linked cellulose gel 14 obtained in step (d), anhydrous sodium sulfate (manufactured by Wako Pure Chemical Industries, Ltd.) and water. (352.5 g) was added to the reaction vessel and stirred at 50 ° C. for 30 minutes. Next, sodium borohydride (manufactured by Kanto Chemical Co., Inc., 423 mg) was added to the reaction solution, and stirring was continued at 50 ° C., and epichlorohydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 4.70 g) and 48 were used. % Sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Ltd., 4.70 g) was added 12 times at 30 minute intervals. After the addition of epichlorohydrin and 48% sodium hydroxide aqueous solution, stirring was continued at 50 ° C. for 14 hours. did. After the completion of the reaction, the reaction solution is cooled to 40 ° C. or less, washed with a large amount of water using a glass filter, and then classified using a sieve to thereby obtain a porous crosslinked cellulose gel 14 having a particle size of 45 μm or more and 90 μm or less. (165 mL) was obtained.
(2) Porosity and Kav Measurement As a result of calculating the porosity of the porous crosslinked cellulose gel 14 by the method described in Example 1, the porosity was 90.5%. Moreover, as a result of calculating Kav of porous crosslinked cellulose gel 14 by the method described in Example 1, 0.91 for cytochrome C, 0.78 for albumin, 0.69 for apoferritin, It was 0.64 for thyroglobulin.
(3) Column pressure loss measurement As a result of measuring the column pressure loss of the porous crosslinked cellulose gel 14 by the method described in Example 1, the column pressure loss at a linear velocity of 1491 cm / hr (flow rate 8.5 mL / min) is It was 0.27 MPa.
(4) Formylation of porous cross-linked cellulose gel, protein A immobilization and antibody adsorption amount measurement After obtaining porous cross-linked cellulose gel 14F obtained by formylating porous cross-linked cellulose gel 14 by the method described in Example 1. A porous crosslinked cellulose gel 14FP having protein A immobilized thereon was obtained.

Similarly, as a result of calculating the protein A immobilization amount of the porous crosslinked cellulose gel 14FP by the method described in Example 1, the immobilization amount was 11.1 mg per mL of the gel. Similarly, as a result of calculating the antibody adsorption amount of the porous crosslinked cellulose gel 14FP, the antibody adsorption amount was 62.6 mg per mL of the gel.

Table 5 summarizes the results of Examples 4, 7, and 8.

Example 9 Production of Antibody Purifying Adsorbent Immobilizing Protein A via a Spacer-1
In Example 9, an adsorbent for antibody purification in which protein A was immobilized on a porous crosslinked cellulose gel via a spacer (6 atoms) was produced, and the amount of antibody adsorption was measured.
(1) Introduction of spacer into porous crosslinked cellulose gel and formylation Porous crosslinked cellulose gel 1 (3.0 g), water (3.0 g), dimethyl sulfoxide (3.0 g), and epichloro produced in Example 1 Hydrin (manufactured by Tokyo Chemical Industry Co., Ltd., 0.6 g) is mixed and stirred at 30 ° C. for 30 minutes, and then a 48% aqueous sodium hydroxide solution (manufactured by Kanto Chemical Co., Inc., 1.64 mL) is added. Epoxidation reaction was performed by stirring for a period of time. After the reaction, an epoxidized porous crosslinked cellulose gel was obtained by washing with a large amount of water until the filtrate became neutral using a glass filter.

Next, an epoxidized porous crosslinked cellulose gel (3.0 g), D-glucamine (manufactured by Tokyo Chemical Industry Co., Ltd.) and water prepared from water (90 mg / mL, 3.0 mL), water (3.0 mL) Were mixed and stirred at 40 ° C. for 16 hours to carry out an amination reaction. After the reaction, the aminated porous crosslinked cellulose gel was obtained by washing with a large amount of water until the filtrate became neutral on the glass filter.

Aminated porous crosslinked cellulose gel (3.0 g), sodium periodate aqueous solution (10 mg / mL, 1.5 mL), water (1.5 mL) prepared from sodium periodate (manufactured by Kanto Chemical Co., Inc.) and water Were mixed and stirred at 25 ° C. for 60 minutes to carry out a formylation reaction. After the reaction, by washing with a large amount of water using a glass filter, a spacer was introduced into the porous crosslinked cellulose gel 1, and then a porous crosslinked cellulose gel 1S having a formyl group introduced therein was obtained.

In the same manner, from the porous crosslinked cellulose gel 3 produced in Example 2 and the porous crosslinked cellulose gel 5 produced in Example 3, a porous crosslinked cellulose gel 3S and a porous crosslinked cellulose gel 5S into which formyl groups have been introduced. Got.
(2) Immobilization of protein A on formylated porous crosslinked cellulose gel By the method described in Example 1, an adsorbent 1SP for antibody purification in which protein A was immobilized on porous crosslinked cellulose gel 1S was obtained. Similarly, as a result of calculating the protein A immobilization amount of the antibody purification adsorbent 1SP by the method described in Example 1, the immobilization amount was 11.0 mg per mL of gel.

By the same method, adsorbents 3SP and 5SP for antibody purification were obtained from porous crosslinked cellulose gels 3S and 5S. As a result of calculating the protein A immobilization amount of the antibody purification adsorbents 3SP and 5SP by the above-mentioned method, the immobilization amount of the antibody purification adsorbent 3SP was 10.9 mg per mL of gel and the antibody purification adsorbent 5SP was immobilized. The amount was 11.2 mg per mL of gel.
(3) Antibody adsorption amount measurement of protein A-immobilized antibody purification adsorbent As a result of calculating the antibody adsorption amount of the antibody purification adsorbent 1SP by the method described in Example 1, the antibody adsorption amount was 78. 5 mg. As a result of measuring the amount of antibody adsorption of the adsorbent 3SP for antibody purification and the adsorbent 5SP for antibody purification by the same technique, the adsorbed amount of the adsorbent 3SP for antibody purification was 80.7 mg per mL of gel, and the adsorbent 5SP for antibody purification. The amount of antibody adsorbed was 73.7 mg per mL of gel. The structures of the adsorbents 1SP, 3SP, and 5SP for antibody purification in which protein A is immobilized through a spacer (6 atoms) are shown below.

Example 10 Production of Adsorbent for Purifying Antibody with Protein A Immobilized through a Spacer-2
In Example 10, an adsorbent for antibody purification in which protein A was immobilized via a spacer (15 atoms) longer than that in Example 9 was produced, and the amount of adsorbed antibody was measured.
(1) Introduction of spacer into porous crosslinked cellulose gel and formylation Porous crosslinked cellulose gel 1 (3.0 g) produced in Example 1, 1,4-butanediol diglycidyl ether (Aldrich, 0.6 g) ), 0.2M aqueous sodium hydroxide solution (1.5 mL) and water (1.5 g) were mixed and stirred at 50 ° C. for 8 hours to carry out an epoxidation reaction. After the reaction, an epoxidized porous crosslinked cellulose gel was obtained by washing with a large amount of water until the filtrate became neutral using a glass filter.

Next, an epoxidized porous crosslinked cellulose gel (3.0 g), D-glucamine (manufactured by Tokyo Chemical Industry Co., Ltd.) and water prepared from water (90 mg / mL, 3.0 mL), water (3.0 mL) Were mixed and stirred at 40 ° C. for 16 hours to carry out an amination reaction. After the reaction, the aminated porous crosslinked cellulose gel was obtained by washing with a large amount of water until the filtrate became neutral on the glass filter.

Aminated porous crosslinked cellulose gel (3.0 g), sodium periodate aqueous solution (10 mg / mL, 1.5 mL), water (1.5 mL) prepared from sodium periodate (manufactured by Kanto Chemical Co., Inc.) and water Were mixed and stirred at 25 ° C. for 60 minutes to carry out a formylation reaction. After the reaction, by washing with a large amount of water using a glass filter, a spacer was introduced into the porous crosslinked cellulose gel 1, and then a porous crosslinked cellulose gel 1L into which a formyl group had been introduced was obtained.

By the same method, from the porous crosslinked cellulose gel 3 produced in Example 2 and the porous crosslinked cellulose gel 5 produced in Example 3, a porous crosslinked cellulose gel 3L and a porous crosslinked cellulose gel 5L into which formyl groups were introduced. Got.
(2) Protein A Immobilization on Formylated Porous Crosslinked Cellulose Gel By the method described in Example 1, an adsorbent 1LP for antibody purification in which protein A was immobilized on 1 L of porous crosslinked cellulose gel was obtained. Similarly, as a result of calculating the protein A immobilization amount of the adsorbent 1LP for antibody purification by the method described in Example 1, the immobilization amount was 11.5 mg per mL of gel.

In the same manner, adsorbents 3LP and 5LP for antibody purification were obtained from porous crosslinked cellulose gels 3L and 5L. As a result of calculating the protein A immobilization amount of the adsorbents 3LP and 5LP for antibody purification by the method described above, the immobilization amount of the adsorbent 3LP for antibody purification was 11.1 mg per mL of gel, and the adsorbent 5LP for antibody purification was immobilized. The amount was 10.9 mg per mL of gel.
(3) Antibody adsorption amount measurement of protein A-immobilized antibody purification adsorbent As a result of calculating the antibody adsorption amount of the antibody purification adsorbent 1LP by the method described in Example 1, the antibody adsorption amount was 83. 7 mg. As a result of measuring the antibody adsorption amount of the adsorbent 3LP for antibody purification and the adsorbent 5LP for antibody purification by the same technique, the adsorbed amount of the adsorbent 3LP for antibody purification was 84.6 mg per mL of gel, and the adsorbent 5LP for antibody purification. The antibody adsorption amount was 76.7 mg per mL of gel. The structures of the adsorbents 1LP, 3LP, and 5LP for antibody purification in which protein A is immobilized through a 15-atom spacer are shown below.

Table 6 summarizes the results of Examples 9 and 10 together with the results of Examples 1 to 3. The amount of antibody adsorption of the adsorbents 1SP, 3SP and 5SP for antibody purification introduced with a short spacer (6 atoms) is higher than that of adsorbents 1FP, 3FP and 5FP for antibody purification without introduction of a spacer, It is clear that the amount of antibody adsorbed by the adsorbents 1LP, 3LP and 5LP for antibody purification introduced with a long spacer (15 atoms) is higher than the adsorbents 1SP, 3SP and 5SP for antibody purification introduced with a short spacer. It became.

(Example 11) Production of adsorbent for antibody purification in which a spacer was introduced to immobilize an Fc binding protein In Example 11, after introducing a spacer into a porous crosslinked cellulose gel, maleimidation and Fc binding protein immobilization Fc-binding protein-immobilized porous cross-linked cellulose gel was prepared by sequentially performing the steps, and the amount of antibody adsorption was measured.
(1) Spacer introduction and maleimidation into porous crosslinked cellulose gel Porous crosslinked cellulose gel 1 (2.0 g) produced in Example 1, 1,4-butanediol diglycidyl ether (Aldrich, 1.0 g) ), 48% sodium hydroxide aqueous solution (manufactured by Kanto Chemical Co., Inc., 31 mg) and water (3.0 g) were mixed and stirred at 50 ° C. for 8 hours to carry out an epoxidation reaction. After the reaction, an epoxidized porous crosslinked cellulose gel was obtained by washing with a large amount of water until the filtrate became neutral using a glass filter.

Next, epoxidized porous crosslinked cellulose gel (2.0 g), ethylenediamine (manufactured by Tokyo Chemical Industry Co., Ltd., 1.0 g) and water (4.0 g) are mixed and stirred at 50 ° C. for 4 hours to carry out the amination reaction. I did it. After the reaction, the aminated porous crosslinked cellulose gel was obtained by washing with a large amount of water until the filtrate became neutral on the glass filter.

The aminated porous crosslinked cellulose gel (1.0 g), N-succinimidyl 3-maleimidopropionate (Tokyo Kasei Co., Ltd., 8.0 mg), 1,4-dioxane (Kanto Chemical Co., 2.0 mL) ) Were mixed and stirred at 35 ° C. for 4 hours to carry out a maleimidation reaction. After the reaction, by washing with a large amount of water using a glass filter, a spacer was introduced into the porous crosslinked cellulose gel 1, and then a porous crosslinked cellulose gel 1M having a maleimide group introduced therein was obtained. In the same manner, from the porous crosslinked cellulose gel 3 produced in Example 2 and the porous crosslinked cellulose gel 5 produced in Example 3, a porous crosslinked cellulose gel 3M and a porous crosslinked cellulose gel 5M into which formyl groups have been introduced. Got.
(2) Immobilization of Fc-binding protein on maleimidated porous cross-linked cellulose gel For immobilization of Fc-binding protein on maleimidized porous cross-linked cellulose gel, a method disclosed in JP 2014-187993 A is used. I did it.

A 50% by volume suspension (100 μL) prepared by adding water to the porous crosslinked cellulose gel 1M described above was added to a reaction vessel (BIO-RAD, mini-biospin chromatography column), and 50 mM phosphorus was added. Washed 5 times with acid buffer (pH 7.0, 150 μL). In addition, 50 volume% suspension of maleimide-ized porous crosslinked cellulose gel 1M causes maleimide-modified porous crosslinked cellulose gel 1M suspended in water to settle in a graduated cylinder, and is tapped occasionally to make the volume constant. Until the volume of the maleimidated porous crosslinked cellulose gel 1M was 50%, water was added.

Next, an Fc-binding protein solution (FcRm68-CG, FcRm68-CG, prepared by the method described in JP-A-2014-187993 is placed in a reaction vessel containing porous crosslinked cellulose gel 1M (50 μL) washed with a phosphate buffer. Concentration 9.2 mg / mL, 150 μL) and 1 M Tris-HCl buffer (pH 8.5, 7.5 μL) were added, and the mixture was stirred at 35 ° C. for 3 hours to thereby convert the Fc-binding protein into porous crosslinked cellulose gel 1M. Immobilized to. After the immobilization reaction, the antibody purification adsorbent 1MF having the Fc-binding protein immobilized thereon was obtained by washing with PBS 7.0 solution and 50 mM citrate buffer (pH 3.0).

Collect the reaction solution and washing solution, determine the amount of unreacted Fc binding protein by measuring the absorbance at 280 nm, and then subtract the amount of unreacted Fc binding protein from the amount of Fc binding protein used in the reaction. As a result of calculating the amount of Fc-binding protein immobilized on the antibody purification adsorbent 1MF, the amount immobilized was 22.5 mg per mL of gel. By the same method, adsorbents 3MF and 5MF for antibody purification were obtained from porous crosslinked cellulose gels 3M and 5M. Moreover, as a result of calculating the amount of FMF binding protein immobilized on the antibody purification adsorbent 3MF and 5MF by the method described above, the amount of antibody purification adsorbent 3MF immobilized was 20.8 mg per mL of gel, and the antibody purification adsorbent The amount of 5MF immobilized was 19.2 mg per mL of gel.
(3) Antibody adsorption amount measurement of Fc-binding protein-immobilized porous cross-linked cellulose gel The antibody adsorption amounts of the antibody purification adsorbents 1MF, 3MF, and 5MF calculated by the method described in Example 1 are the antibody purification adsorbents. The 1MF was 68.3 mg per mL of gel, the antibody purification adsorbent 3MF was 66.9 mg per mL of gel, and the antibody purification adsorbent 5MF was 62.3 mg per mL of gel.

As described above, the porous crosslinked cellulose gel obtained by the production method of the present invention has both high porosity and high mechanical strength, and has suitable pore characteristics and particle size as a chromatographic filler. ing. Therefore, the porous crosslinked cellulose gel of the present invention is particularly suitable as a chromatography filler used in a biopharmaceutical purification process.

Further, the method for producing a porous crosslinked cellulose gel of the present invention is a production method that does not require large equipment, a large amount of energy, and expensive raw materials, as compared with the conventional method for producing a porous cellulose gel.

Furthermore, the adsorbent for antibody purification obtained by immobilizing the affinity ligand for the antibody on the porous crosslinked cellulose gel obtained by the production method of the present invention has a high adsorption capacity for the antibody, and further has a low column pressure loss and a high level. Flow rate processing is possible. Therefore, the adsorbent for antibody purification of the present invention can remarkably improve the productivity in the purification process of antibody drugs.

Therefore, the present invention is particularly useful in the production of biopharmaceuticals such as antibody drugs.

Claims (6)

A porous crosslinked cellulose gel having the following features (1) and (2).
(1) The porosity measured by the following operations (a) to (e) is 90% or more.
(A) The porous crosslinked cellulose gel is packed in a chromatography column.
(B) Using water as an eluent, measure the elution volume of blue dextran having a molecular weight of 2 million and sodium chloride from the column.
(C) The gel volume is calculated by subtracting the elution volume of blue dextran having a molecular weight of 2 million measured in operation (b) from the column volume of the column.
(D) The pore volume is calculated by subtracting the elution volume of blue dextran having a molecular weight of 2 million measured in operation (b) from the elution volume of sodium chloride measured in operation (b).
(E) The porosity is calculated by dividing the pore volume calculated in operation (d) by the gel volume calculated in operation (c).
(2) A chromatography column having an average particle diameter of 30 μm to 150 μm and an inner diameter of 6.6 mm is packed so as to have a height of 220 mm ± 5 mm, and water at 25 ° C. is poured into the column at a linear velocity of 1500 cm / hour. The column pressure loss under the flowed condition is 0.4 MPa or less. The method for producing a porous crosslinked cellulose gel according to claim 1, comprising the following steps (a) to (e):
(A) a step of obtaining a cellulose solution by dissolving cellulose in an alkaline aqueous solution;
(B) a step of obtaining a cellulose dispersion by mixing the cellulose solution obtained in step (a) with an organic solvent and an emulsifier;
(C) The step of obtaining a porous uncrosslinked cellulose gel by bringing the cellulose dispersion obtained in step (b) to 0 ° C. or more and 15 ° C. or less and adding a gelling agent;
(D) a step of obtaining a porous partially crosslinked cellulose gel by reacting the porous uncrosslinked cellulose gel obtained in step (c) with glycidyl ethers having at least two or more glycidyl groups,
(E) A step of obtaining a porous crosslinked cellulose gel by reacting the porous partially crosslinked cellulose gel obtained in the step (d) with a crosslinking agent having two or more functional groups capable of reacting with a hydroxyl group of cellulose. A chromatographic filler comprising the porous crosslinked cellulose gel according to claim 1. A method for purifying a protein using the chromatography filler according to claim 3. An adsorbent for antibody purification, wherein an affinity ligand is immobilized on the porous crosslinked cellulose gel according to claim 1. An adsorbent for antibody purification, wherein the affinity ligand according to claim 5 is protein A or Fc-binding protein.

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