WO2019220866A1 - Porous cellulose beads and method for producing adsorbent - Google Patents

Abstract

The present invention relates to a method for producing porous cellulose beads which comprises dispersing a phase, which is to be a dispersed phase and comprises either cellulose or a cellulose derivative, in a highly safe, continuous phase to obtain a W/O type emulsion and bringing the emulsion into contact with a coagulant. The method for producing porous cellulose beads is characterized by using a compound having an HLB value of 0.1 or higher but less than 1.8 as a surfactant. The problem was thus able to be solved.

Description

Method for producing porous cellulose beads and adsorbent

The present invention relates to a method for producing porous cellulose beads and an adsorbent.

As materials for adsorbents for purification of polymer drugs such as medical adsorbents and antibody drugs, polysaccharides such as agarose and cellulose with less nonspecific adsorption are preferred. Porous cellulose beads are difficult to crush, have relatively high mechanical strength, and contain many hydroxyl groups that can be used to introduce ligands that interact with target substances to be adsorbed. It is used as a base material for various adsorbents such as a body and an affinity adsorbent. In particular, affinity adsorbents have been used as medical adsorbents and pharmaceutical purifying adsorbents because they can efficiently purify target substances or reduce the concentration of unwanted substances. In particular, an adsorbent in which protein A is immobilized on a porous carrier using an affinity ligand is attracting attention as an adsorbent for treating rheumatism, hemophilia, dilated cardiomyopathy, or an adsorbent for purifying antibody drugs (eg, non-patent literature). 1, 2).

The production of porous cellulose beads often involves complicated steps compared to general synthetic polymer beads because it is difficult to dissolve cellulose in a general solvent. As one of them, a cellulose dope is prepared by dissolving cellulose at a high temperature of 100 ° C. or higher in a highly corrosive solvent such as a 60% high-concentration calcium thiocyanate aqueous solution, which increases the difficulty of installation. A method of forming the liquid droplets and solidifying them is disclosed (for example, Patent Document 1). It is known that the cellulose solution used in this method exhibits unique behavior, and the porous cellulose beads obtained by this method have considerably large pores and a wide pore size distribution ( For example, Non-Patent Document 3). Therefore, when the porous cellulose beads obtained by the method are used as an adsorbent such as an antibody, the specific surface area is expected to be small, and it cannot be expected to exhibit extremely high adsorption performance. Moreover, as a similar production method, a method including a step of dissolving cellulose in a lithium bromide aqueous solution having a high concentration of 60% at a high temperature of 100 ° C. or higher is disclosed, but it is also preferable from the viewpoint of environmental load. It is difficult (for example, Patent Document 2). On the other hand, in order to increase the solubility of cellulose, a method of giving a substituent to the hydroxyl group of cellulose, dissolving in a general-purpose solvent, granulating, and removing the substituent after granulation to obtain a porous cellulose carrier Is exemplified (for example, Patent Document 3), but the process is complicated.

As a method for producing a cellulose dope inexpensively, safely and simply, a method using a low-temperature alkaline aqueous solution as a solvent is known. However, in general, it is difficult for this method to completely dissolve cellulose, and special cellulose is often used. For example, Patent Document 4 discloses cellulose that is soluble in an alkaline solution. However, the cellulose requires a microfibril having a fiber diameter of 1 μm or less, and further subjected to special fine processing of 500 nm or less. Yes.

As shown in Patent Document 5, microbial cellulose is dissolved in an alkaline solution to prepare a cellulose solution. After adding a continuous phase solvent, the cellulose solution is made into droplets, and then the microbial cellulose particles are frozen and then washed. Although a method for obtaining cellulose beads is disclosed, energy is required to freeze the cellulose solution together with the continuous phase solvent. Moreover, microbial cellulose is a special raw material, and it is difficult at present to stably obtain a large amount thereof.

Very recently, Patent Document 6 reported that a cellulose dope can be produced at a relatively high temperature using an aqueous solution containing sodium hydroxide and urea, and that porous beads can be obtained from this cellulose dope. Yes. In Patent Document 7, a high-performance porous cellulose bead and an adsorbent using the same can be obtained by adding a certain additive to a cellulose dispersion obtained by treating a general-purpose cellulose raw material with a slightly low-temperature alkaline aqueous solution. It has been reported that it can be obtained. The porous beads obtained in Patent Documents 6 and 7 are capable of producing a cellulose dope by a method that is relatively simple and has few environmental burdens and product safety risks.

In Patent Documents 6 and 7, beads are obtained through a step of emulsifying cellulose dope in a continuous phase, and orthodichlorobenzene is used for the continuous phase of the emulsion. Orthodichlorobenzene has a low melting point and a high boiling point, has a high flash point, is hardly miscible with water, and is easily miscible with alcohol, and thus is a preferred continuous phase solvent in the production method of porous cellulose beads. According to Patent Document 7, a surfactant having an HLB (hydrophilic and hydrophobic balance) value of 4.3 is used in a production method using orthodichlorobenzene. As shown in Patent Document 8 and the like, usually, in a W / O type emulsion, an appropriate HLB value of about 3 to 6 is used for the surfactant to be used, and this also makes it easy to obtain spherical cellulose beads. You can see that However, orthodichlorobenzene may not be preferred from the viewpoints of product safety and environmental burden when used for medical purposes and pharmaceutical purification.

US Pat. No. 8,664,152 Japanese Patent Laying-Open No. 2015-187255 International Publication No. 2006/025371 JP-A-9-124702 JP 2010-236975 A JP 2011-231152 A International Publication No. 2016/167268 European Patent No. 2437723

Anals of the New York Academy of Sciences, 2005, Vol. 1051, p. 635-646 American Heart Journal, Vol. 152, Number 4, 2006, p. 712e1-712e6 Journal of Chromatography, 195 (1980) 221-230

The present invention has been made in view of the above-mentioned problems of the prior art, and is for obtaining high-performance spherical porous cellulose beads by a method with less risk of product safety and environmental burden.

In a production method in which a dispersed phase containing cellulose or a cellulose derivative is dispersed in a continuous phase to form a W / O emulsion and contacted with a coagulant to obtain porous cellulose beads, the HLB value is 0.1 or more and less than 1.8 By using this compound as a surfactant, it is possible to obtain porous cellulose beads with excellent sphericity even when using a solvent that is preferable in terms of product safety and environmental load, and the above problems can be solved. It was.
The present invention is shown below.

[1] A method for producing a porous cellulose bead,
A step of dispersing cellulose or a cellulose derivative in a solvent to obtain a dispersed phase;
Dispersing the dispersed phase in a continuous phase containing a surfactant to obtain a W / O type emulsion; and
A step of coagulating the cellulose or cellulose derivative by bringing the W / O emulsion into contact with a coagulant;
A method in which a surfactant is added to one or both of the solvent and the continuous phase, and the HLB value of the surfactant is 0.1 or more and less than 1.8.

[2] The method according to [1], wherein the flash point of the continuous phase is 73 ° C. or higher.

[3] The method according to [1] or [2], wherein the continuous phase has a boiling point of 181 ° C. or higher.

[4] The method according to any one of [1] to [3], wherein liquid paraffin having a kinematic viscosity of less than 74 mm ² / S is used as the continuous phase.

[5] The method according to any one of [1] to [4], wherein an HLB value of the surfactant is smaller than a value obtained by the following formula 1.
−0.0433 × kinematic viscosity of continuous phase [mm ² /S]+1.733 (Equation 1)

[6] The method according to any one of [1] to [5], wherein the concentration of water in the dispersed phase is 41% by mass or more.

[7] The liquid temperature at the time of production of the porous cellulose beads is less than the boiling point of the compound having the lowest boiling point among the compounds contained in the continuous phase, dispersed phase, surfactant and coagulant. The method according to any one of [6].

[8] The method according to any one of [1] to [7], wherein the coagulant is immiscible with the continuous phase.

[9] The method according to any one of [1] to [8], wherein the dispersed phase is prepared by mixing an alkaline aqueous solution and the cellulose or cellulose derivative powder.

[10] A step of producing porous cellulose beads by the method according to any one of [1] to [9], and
The manufacturing method of the crosslinked porous cellulose bead characterized by including the process of bridge | crosslinking the said porous cellulose bead using a crosslinking agent.

[11] A step of producing a crosslinked porous cellulose bead by the method according to [10], and
A method for producing an adsorbent comprising the step of immobilizing a ligand on the crosslinked porous cellulose beads.

[12] A method for purifying a target substance,
A step of producing an adsorbent by immobilizing a ligand that binds to the target substance on the crosslinked porous cellulose beads by the method described in [11], and
A method comprising the step of bringing a solution containing the target substance into contact with an adsorbent.

[13] The method according to [12], wherein the adsorbent is packed in a column and the solution is passed through the column.

[14] The method according to [13], wherein two or more columns are connected to flow the solution.

According to the present invention, high-performance spherical porous cellulose beads can be obtained by a method with less risk of product safety and environmental burden.

2 is a microscopic observation view of a porous body obtained in Granulation Comparative Example 1. FIG. 6 is a microscopic observation view of a porous body obtained in Granulation Comparative Example 2. FIG. 1 is a microscopic observation view of a porous body obtained in Granulation Example 1. FIG. 5 is a microscopic observation view of a porous body obtained in Granulation Example 2. FIG. It is a microscope observation figure of the porous body obtained in Granulation Example 3. It is a microscope observation figure of the porous body obtained in Granulation Example 4. It is a microscope observation figure of the porous body obtained in Granulation Example 5. It is a microscope observation figure of the porous body obtained in the granulation Example 6. 2 is a microscopic observation view of a porous body obtained in Granulation Reference Example 1. FIG. It is a graph of the pressure flow rate characteristic of the median particle diameter 60 micrometer goods which bridge | crosslinked the bead of granulation Example 3. FIG. 3 is a graph comparing the pressure flow rate of cross-linked porous beads according to the present invention and commercial PA resin.

Hereinafter, although this invention is demonstrated for every process, this invention is not limited to the specific example as described below.
1. Dispersed Phase Preparation Step In this step, cellulose or a cellulose derivative is dispersed in a solvent to obtain a dispersed phase. A dispersed phase containing cellulose or a cellulose derivative is also called a cellulose dope.

There is no limitation in particular in the kind of the said cellulose and a cellulose derivative. Cellulose is a polysaccharide in which glucose is linked by β-1,4-glucoside, and has the following structural formula.

The cellulose derivative refers to a compound in which one or more hydroxyl groups at the 2nd, 3rd and 6th positions of glucose constituting cellulose are modified. Examples of the cellulose derivative include, for example, a cellulose derivative in which one or more hydroxyl groups are modified with a hydroxy C _1-4 alkyl group such as hydroxypropyl cellulose; ethyl cellulose or the like, in which one or more hydroxyl groups are modified with a C _1-4 alkyl group. Cellulose derivatives such as hydroxypropylmethylcellulose, cellulose derivatives in which two or more hydroxyl groups are modified with a hydroxy C _1-4 alkyl group and a C _1-4 alkyl group; carboxymethyl cellulose and the like, wherein one or more hydroxyl groups are carboxy C _1-4 Cellulose derivatives modified with alkyl groups; cellulose derivatives in which one or more hydroxyl groups are modified with C _2-7 acyl groups, such as acetyl cellulose, propionyl cellulose, and butyryl cellulose. Hydroxypropylcellulose and carboxymethylcellulose may show water solubility depending on the degree of substitution. Ethylcellulose has improved affinity for organic solvents, depending on the degree of substitution. Hydroxypropyl methylcellulose also shows solubility in water or an aqueous alcohol solution and gels when the aqueous solution is heated. Further, the substituent may be finally eliminated. When the substituent is finally eliminated, the degree of substitution is preferably less than 2.0, more preferably less than 1.0, and even more preferably less than 0.5. In addition, a substitution degree means the average number of substituents per glucose unit which comprises a cellulose.

As shown in Patent Document 7, the present applicant has developed a method for obtaining porous cellulose beads without completely dissolving the cellulose, so that cellulose introduced with a substituent for increasing solubility is introduced. For example, it is not necessary to use a cellulose derivative, and it is preferable to use ordinary unsubstituted cellulose as a raw material. However, in order to efficiently disperse cellulose or cellulose derivatives in a solvent, it is preferable to use cellulose or cellulose derivative powder. The average particle size of the powder can be, for example, 10 μm or more and 250 μm or less. If the average particle diameter is 10 μm or more, energy for refining and secondary aggregation are more reliably suppressed, and if the powder is 250 μm or less, the dispersibility is excellent. The average particle size is preferably 100 μm or less, and more preferably 15 μm or more and 75 μm or less from the viewpoint of the handleability of the powder. The average particle diameter of the powder can be measured with a laser diffraction particle size distribution measuring device. The standard of the amount of particles when measuring the average particle size of the powder includes volume, area, length, and number. Generally, the volume is used as a reference. Hereinafter, the cellulose or cellulose derivative powder is abbreviated as “cellulose powder”.

The molecular weights of cellulose and cellulose derivatives as raw materials to be used are not particularly limited, but the degree of polymerization is preferably 1000 or less. If the degree of polymerization is 1000 or less, the dispersibility / swellability in a solvent is increased, which is preferable. Moreover, since the mechanical strength of the obtained porous cellulose bead will become large if a polymerization degree is 10 or more, it is preferable. A more preferable range of the degree of polymerization is 50 or more and 500 or less, further preferably 100 or more and 400 or less, particularly preferably 200 or more and 350 or less, and most preferably 250 or more and 350 or less.

The solvent for dispersing cellulose or the cellulose derivative is not particularly limited as long as it is a solvent that can favorably disperse cellulose or the cellulose derivative, and examples thereof include an aqueous solvent. The aqueous solvent refers to water and an aqueous solution. Examples of the aqueous solution include an aqueous alkaline solution, an aqueous thiocyanate solution, an aqueous lithium bromide solution, and an aqueous copper ammonia solution (Schweitzer solution). Although there is no limitation in particular about the density | concentration of the water in the disperse phase containing the cellulose or cellulose derivative regarding this invention, it is preferable that it is 41 mass% or more. If the said concentration is 41 mass% or more, since there are few adjuvants for producing a dispersed phase, a cellulose bead can be obtained with a method with little environmental impact. The concentration of water in the dispersed phase is more preferably 51% by mass or more, further preferably 61% by mass or more, and most preferably 71% by mass or more. The water concentration in the dispersed phase is preferably 97% by mass or less because cellulose beads having a good shape and mechanical strength are easily obtained, more preferably 90% by mass or less, and still more preferably 85% by mass. Or 80% by mass or less.

Other methods for preparing the dispersed phase are not particularly limited, but a method of mixing a low-temperature alkaline aqueous solution and cellulose powder is preferable because it is simple and has little environmental burden.

The alkali used in the present invention can be used without particular limitation as long as it shows alkalinity when it becomes an aqueous solution. Lithium hydroxide, sodium hydroxide and potassium hydroxide are preferable from the viewpoint of availability, and sodium hydroxide is most preferable from the viewpoint of product safety and price.

The alkali concentration of the aqueous alkali solution is not particularly limited, but is preferably 3% by mass or more and 20% by mass or less. If the alkali concentration is within this range, the dispersibility / swellability of cellulose or a cellulose derivative in an aqueous alkali solution is preferably increased. The concentration of alkali is more preferably 5% by mass or more and 15% by mass or less, and further preferably 7% by mass or more and 10% by mass or less.

It is also preferable to use an auxiliary agent in the dispersed phase of the present invention. In the present disclosure, “auxiliary agent” means an agent that promotes dispersion of cellulose or a cellulose derivative in a solvent or stabilizes a dispersed phase. The concentration of the auxiliary agent in the dispersed phase is preferably 3% by mass or more and 30% by mass or less. A concentration of the auxiliary agent within this range is preferable because the homogeneity of the dispersed phase is increased. The concentration of the auxiliary agent is more preferably 8% by mass or more and 30% by mass or less, further preferably 10% by mass or more and 20% by mass or less, and most preferably 12% by mass or more and 15% by mass or less. Examples of the auxiliary agent include inorganic salt auxiliary agents such as lithium bromide and zinc chloride; organic compound auxiliary agents such as urea and polyethylene glycol.

The concentration of cellulose or cellulose derivative in the dispersed phase is not particularly limited and may be appropriately adjusted. For example, it may be about 1% by mass or more and 20% by mass or less. The concentration is preferably 2% by mass or more, more preferably 3% by mass or more, particularly preferably 3.8% by mass or more, and most preferably 4.0% by mass or more from the viewpoint of the mechanical strength of the porous beads. It is. Further, from the viewpoint of adsorption performance and homogeneity of the dispersed phase, it is preferably 10% by mass or less, more preferably 8% by mass or less, particularly preferably 6% by mass or less, and most preferably 5% by mass or less.

The method for preparing the dispersed phase may be in accordance with a conventional method. For example, a mixture of a solvent and cellulose or a cellulose derivative may be stirred while maintaining a low temperature, and examples already known by many of the present applicants can be suitably used. A specific example of the low temperature is −22 ° C. or more and less than 15 ° C. However, immediately after the addition of an alkaline compound such as sodium hydroxide, the temperature rapidly rises and sometimes exceeds 15 ° C., but such temporary temperature rise is excluded. In addition, at −12 ° C., there seems to be some change point in the properties of the dispersed phase, and it may be difficult to obtain reproducibility. It is not preferable to keep the dispersed phase at −12 ° C. for a certain period of time. It is preferable to include a step of keeping cold at a low temperature. In particular, it is more preferable to include a step of keeping the cold at a temperature lower than −12 ° C. The cooling time is preferably from 20 minutes to 12 hours, more preferably from 30 minutes to 6 hours, and particularly preferably from 60 minutes to 3 hours. However, when the temperature is adjusted to a temperature lower than −12 ° C. from the time when the raw material is charged, the viscosity of the dispersed phase rises before the cellulose powder and auxiliary agent are homogeneously dispersed, and thus a homogeneously dispersed phase and thus porous cellulose beads having a homogeneous structure are obtained. For this reason, it is preferable that the temperature is higher than −12 ° C. when the raw material is charged. A more preferable raw material temperature is preferably 0 ° C. or higher from the viewpoint of preventing freezing, more preferably 4 ° C. or higher, and 10 ° C. or higher in order to suppress precipitation of alkaline compounds and auxiliaries. Is also preferable.

2. Step for preparing W / O type emulsion In this step, a dispersed phase is dispersed in a continuous phase to obtain a W / O type emulsion.

The present invention relates to a production method in which a dispersed phase containing cellulose or a cellulose derivative is dispersed in a continuous phase to form a W / O emulsion, and contacted with a coagulant to obtain porous cellulose beads. By using a compound of less than .8 as a surfactant, it is possible to provide a production method in consideration of product safety and environmental load reduction. As described above, ortho-dichlorobenzene or the like is used for the well-known high-performance porous cellulose beads in the continuous phase, which may not be appropriate from the viewpoint of product safety and environmental load. Therefore, in order to solve the above-mentioned problems, the present inventors have started an investigation using a continuous phase that is preferable in terms of product safety and environmental load compared to orthodichlorobenzene. However, as illustrated in Comparative Example 1 for granulation, spherical beads could not be obtained. As a result of intensive studies, the present inventors have used a compound having an HLB value of less than 1.8 as a surfactant, even when using a continuous phase that is preferable in terms of product safety and environmental burden than orthodichlorobenzene. It was clarified that porous cellulose beads with excellent sphericity can be obtained. According to Patent Document 7, a surfactant having an HLB value of 4.3 is used in a production method using orthodichlorobenzene. Usually, in the W / O type emulsion, the HLB value of the surfactant to be used is suitably 3 to 6, and the HLB value found by the present inventors is extremely unusual in view of the conventional technical knowledge, Such an example has not been found so far. The HLB value of the surfactant is preferably 1.0 or less because the sphericity of the beads is further improved, and when the viscosity of the continuous phase is large, it is preferably 0.5 or less. Moreover, if the HLB value of surfactant is 0.1 or more, it is preferable because the obtained porous cellulose beads are easy to wash with water, and if it is 0.3 or more, the beads can be more preferably washed.

In the present invention, it is preferable to add a surfactant to one or both of the solvent of the dispersed phase and the continuous phase of the W / O emulsion, and to add the surfactant to the continuous phase of the W / O emulsion. The type of surfactant is not particularly limited as long as it satisfies the HLB value, but compounds having fatty acid components such as lauric acid, palmitic acid, stearic acid, oleic acid, erucic acid, polyglycerin fatty acid, for example, Fatty acid esters and the like can be preferably used. Among these, polyglycerin polyricinoleate is preferable because it is easily available.

As described above, the present inventors also found that, in the course of the study, when the HLB value of the surfactant is selected in accordance with the viscosity of the continuous phase used, highly spherical cellulose beads are easily obtained. That is, it is preferable to use a surfactant having a small HLB value as the viscosity of the continuous phase increases. As used herein, the term “continuous phase” suitably includes not only the main solvent of the continuous phase but also additives such as thickeners. To be more specific, the HLB value of the surfactant is preferably smaller than the value obtained by Formula 1, more preferably smaller than the value obtained by Formula 2, and most preferably smaller than the value obtained by Formula 3.

Formula 1: -0.0433 × continuous phase kinematic viscosity [mm ² /S]+1.733
Formula 2: −0.0192 × Kinematic viscosity of continuous phase [mm ² /S]+1.0769
Formula 3: -0.0357 × kinematic viscosity of continuous phase [mm ² /S]+1.429

Although there is no limitation in particular in the usage-amount of surfactant, since it is easy to obtain a spherical bead if it is 0.1 mass part or more with respect to 100 mass parts of continuous phases in W / O emulsion, More preferably, it is 1. The amount is not less than 1.5 parts by mass, and more preferably not less than 1.5 parts by mass. Moreover, if it is less than 10 mass parts, it is preferable from a viewpoint of production cost, and it is still more preferable that it is less than 7 mass parts.

The continuous phase solvent is not particularly limited, but it is preferable to use other than chlorinated aromatic compounds from the viewpoint of product safety and environmental load. Specific examples of preferable continuous phase solvents include animal and vegetable oils and fats, hydrogenated animal and vegetable oils and fats, fatty acid glycerides, and aliphatic hydrocarbon solvents. As animal and vegetable oils and fats, palm oil, shea fat, monkey fat, iripe fat, pork fat, beef tallow, rapeseed oil, rice oil, peanut oil, olive oil, corn oil, soybean oil, perilla oil, cottonseed oil, sunflower oil, evening primrose oil, Sesame oil, safflower oil, coconut oil, cacao butter, palm kernel oil, fish oil, wakame oil, kombu oil and the like can be mentioned. As hydrogenated animal and vegetable oils and fats, mention may be made of hardened palm oil, hardened palm oil, hardened rapeseed oil, hardened rapeseed oil, hardened soybean oil, hardened tallow fat, hardened fish oil and the like. The fatty acid glyceride may be any of tri-, di-, and mono-glycerides, and examples thereof include stearic glyceride, palmitic glyceride, and lauric glyceride. Examples of the aliphatic hydrocarbon solvent include beeswax, candelilla wax, rice bran wax and the like. Moreover, when using a porous cellulose bead for pharmaceutical refinement | purification or medical use, it is also preferable to use a continuous phase with a high purity degree. In this case, the flash point of the continuous phase solvent is preferably 73 ° C. or higher from the viewpoint of safety during production, more preferably 81 ° C. or higher, and may be 100 ° C. or higher. Similarly, from the viewpoint of safety during production, the boiling point of the continuous phase solvent is preferably 181 ° C. or higher, more preferably 184 ° C. or higher, even more preferably 195 ° C. or higher, and the boiling point is so high that it cannot be measured. May be. Examples of the solvent having these preferable flash points and boiling points include liquid paraffin. The upper limits of the flash point and boiling point are not particularly limited, but for example, both are preferably 500 ° C. or lower.

When liquid paraffin is used as the continuous phase solvent in connection with the present invention, it is possible to select a grade exhibiting various viscosities, but this grade selection makes it easy to adjust the particle size distribution and is restricted by laws and regulations such as the Fire Service Act. Is preferable because it can be relaxed.

The amount of the continuous phase used may be an amount that can sufficiently disperse the droplets of the dispersed phase. For example, it can be 1 mass times or more with respect to the dispersed phase. On the other hand, if the amount of the continuous phase is too large, the amount of waste liquid may increase excessively, and the ratio is preferably 10 times by mass or less. Moreover, 7 mass times or less are more preferable, and 5 mass times or less are still more preferable. In addition, when the amount of the continuous phase is small relative to the dispersed phase, an O / W / O emulsion in which the continuous phase enters the droplets of the dispersed phase may result, and as a result, porous beads having a homogeneous structure may not be obtained. Therefore, the ratio is preferably 2 times or more, more preferably 3 times or more, and particularly preferably 4 times or more.

The emulsion may be prepared by a conventional method. For example, it can prepare by stirring the liquid mixture containing the said dispersed phase, a continuous phase, and surfactant.

Generally, a dispersed phase containing cellulose or a cellulose derivative tends to have a higher viscosity at the same polymer concentration than a dispersed phase containing another polymer. Therefore, it is preferable to use a stirring blade used for emulsification that can easily obtain a large shearing force. Examples of agitation blades with high shear include turbine blades such as Rushton Turbine blades, Flat Blade Turbine blades, inclined paddle blades, propeller blades, blue margin blades, Dispersing Homizing blades, and the like. Among these, turbine blades are preferable from the viewpoint of shearing force and mixing properties.

When a stirring blade having a large shearing force is used, the entire mixing property tends to be lowered. Therefore, it is also preferable to use two or more stirring blades. In order to ensure the whole mixability, it is preferable that kinematic viscosity at 37.8 ° C. is used a continuous phase solvent is less than 74 mm ² / S, more preferably not more than 38mm ² / S, 16mm ² / More preferably, it is S or less. When the particle diameter of the obtained porous cellulose beads is desired to be larger than 30 μm, the kinematic viscosity at 37.8 ° C. of the continuous phase is preferably 4 mm ² / S or more.

3. Coagulation step In this step, porous cellulose beads are obtained by bringing the W / O emulsion into contact with a coagulant and coagulating the cellulose or cellulose derivative in the dispersed phase.

In the present invention, the coagulant for forming droplets of the dispersed phase is not particularly limited, but those showing affinity for the solvent of the dispersed phase are preferable. For example, an alcohol solvent and a mixed solvent of water and an alcohol solvent can be given. Examples of the alcohol solvent include C _1-4 alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, s-butanol, and t-butanol. The ratio of water and alcohol solvent in the aqueous alcohol solution can be, for example, water: alcohol solvent = 80: 20 to 5:95 in a volume ratio before mixing. In addition, acetic acid, citric acid, tartaric acid, formic acid or an aqueous solution thereof, hydrochloric acid, sulfuric acid and the like can also be used.

In the present invention, porous cellulose beads having high sphericity can be obtained even if the coagulant used is not miscible with the continuous phase. The term “not miscible” means that when the coagulant and the continuous phase are mixed at a volume of 1: 1 at room temperature, the dissolution rate is 20% or less. For example, methanol, which is a coagulant used in the examples of Patent Document 7, is well compatible with orthodichlorobenzene as a continuous phase solvent, so that excellent porous cellulose beads can be obtained if environmental concerns are excluded. ing. On the other hand, in the present invention, for example, when methanol is used as a coagulant and liquid paraffin is used as the continuous phase, both are hardly miscible, but excellent porous cellulose beads can be obtained. Although the reason is not clear, it is thought that the knowledge about the HLB value found by the present inventors is effective.

The amount of the coagulant used is not particularly limited and may be adjusted as appropriate. For example, it may be about 20 v / w% or more and 150 v / w% or less with respect to the dispersed phase.

After adding the coagulation solvent, the coagulated porous cellulose beads may be separated by filtration, centrifugation, oil-water separation, etc., and washed with water or alcohol. The obtained porous cellulose beads may be classified using a sieve or the like in order to make the particle diameter uniform.

In order to contribute the present invention to the industry promptly, the present inventors can manufacture at a liquid temperature lower than the boiling point of the compound having the lowest boiling point among the compounds contained in the continuous phase, dispersed phase, surfactant and coagulant. Attention has been made to improve manufacturing safety. This condition can be preferably used in the present invention.

4). Crosslinking step In this step, in order to increase the strength of the porous cellulose beads, crosslinked porous cellulose beads are obtained by crosslinking the porous cellulose beads with a crosslinking agent.

It is preferable that the porous cellulose beads of the present invention are crosslinked porous cellulose beads obtained by allowing a crosslinking agent to act because it is easy to provide an adsorbent suitable for high-speed purification. There are no particular limitations on the crosslinking conditions and the crosslinking agent. For example, the method described in WO2008 / 146906 can be used. For example, a cross-linking step may be performed by adding a cross-linking agent to the dispersed phase, or cross-linking may be performed by causing a cross-linking agent to act on the porous cellulose beads.

Examples of the crosslinking agent include halohydrins such as epichlorohydrin, epibromohydrin and dichlorohydrin; bifunctional bisepoxides (bisoxiranes); and polyfunctional polyepoxides (polyoxiranes). A crosslinking agent may be used individually by 1 type, and may use 2 or more types together.

The solvent for the reaction for cross-linking the porous cellulose beads with a cross-linking agent may be selected as appropriate. For example, in addition to water, water miscibility such as alcohol solvents such as methanol, ethanol and isopropanol, and nitrile solvents such as acetonitrile, etc. Mention may be made of organic solvents. Further, two or more crosslinking reaction solvents may be mixed and used.

The crosslinking reaction may be performed a plurality of times, and the reaction solvent and the crosslinking agent may be changed each time. For example, the first crosslinking reaction may be performed in a water-miscible organic solvent, and the final crosslinking reaction may be performed in water. In this case, the intermediate solvent composition may be the same as or different from either the first time or the last time, or may be an intermediate composition thereof. Furthermore, all rounds may be carried out in an aqueous solvent. The same applies to the crosslinking agent. When the crosslinking reaction is repeated a plurality of times, it is preferable to remove the crosslinking agent by washing the crosslinked porous cellulose with water or the like between the crosslinking reactions.

In order to promote the crosslinking reaction, a base may be added to the reaction solution. Such bases include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; alkali metal carbonates such as sodium carbonate and potassium carbonate; triethylamine and pyridine. And organic bases such as

After the crosslinking reaction, the crosslinked porous cellulose beads are insoluble, and thus separated from the reaction solution and then washed with a solvent such as water.

5. Ligand immobilization step In this step, an adsorbent for adsorbing a target substance having affinity for the ligand is obtained by immobilizing the ligand to the crosslinked porous cellulose beads. The porous cellulose beads and the crosslinked porous cellulose beads according to the present invention can be used as an adsorbent by immobilizing a ligand that interacts with a target substance. Since the adsorbent that can be obtained in the present invention has the characteristic that there is little nonspecific adsorption, it is possible to provide highly safe drugs and treatments, and also save labor in the intermediate washing step during purification and treatment. Can be realized. Moreover, since the porous cellulose beads and crosslinked porous cellulose beads of the present invention have high alkali resistance, an adsorbent capable of alkali cleaning can be obtained by immobilizing an alkali resistant ligand.

The “ligand” in the present invention refers to an affinity ligand that has a specific affinity for a target substance to be purified by adsorbing to an adsorbent and interacts with the target substance. For example, when the target substance is an antibody, an antigen, protein, or peptide fragment that specifically interacts with the antibody; when the target substance is an enzyme ligand, an enzyme using the ligand as a substrate; the target substance is an antigen In some cases, an antibody against the target antigen can be mentioned. The ligand that can be used for the adsorbent according to the present invention is not particularly limited as long as it has a specific affinity for the target substance to be purified using the adsorbent according to the present invention.

The method for immobilizing the ligand on the porous cellulose beads and the crosslinked porous cellulose beads according to the present invention is not particularly limited, and a conventional method can be used. For example, as shown in Seiichi Kasai et al., “Affinity Chromatography”, Tokyo Chemical Dojin, 1991, Tables 8 and 8, and FIGS. 8 and 15, cyanogen bromide method, trichlorotriazine method, epoxy Method, immobilization of amino group-containing ligands using methods such as tresyl chloride method, periodate oxidation method, divinyl sulfonic acid method, benzoquinone method, carbonyldiimidazole method, acyl azide method; epoxy method, diazo coupling method, etc. A method of immobilizing a hydroxyl group-containing ligand using a method; a method of immobilizing a thiol group-containing ligand using an epoxy method, a tresyl chloride method, a divinyl sulfonic acid method, etc .; a carboxylic acid-containing ligand or a formyl group on an amination carrier Various immobilization methods such as a method of immobilizing the contained ligand can be mentioned. The entire contents of this document are incorporated herein by reference.

The adsorbent according to the present invention can be used as an adsorbent for purification, but can also be used as an adsorbent for purifying antibody drugs and a medical adsorbent that have attracted attention in recent years. There are no particular limitations on the ligand when used in adsorbents for antibody drug purification, for example, antigens and proteins highly specific for antibodies, protein A, protein G, protein L and their variants, antibodies Examples thereof include amino group-containing ligands such as peptides having binding activity.

Particularly, as an adsorbent capable of specifically adsorbing immunoglobulin (IgG), an adsorbent obtained by immobilizing protein A, protein G, protein L, or a variant thereof as a ligand on a porous carrier has attracted attention. The protein A or the like that can be used in the present invention is not particularly limited, and natural products and genetically modified products can be used without limitation. In addition, antibody binding domains, mutants thereof, those containing oligomers thereof, fusion proteins, and the like may be used. The number of polymerizations of the oligomer can be 2 or more and 10 or less. In addition, molecular weight fractionation and fractionation using various chromatographic and membrane separation techniques such as ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, and hydroxyapatite chromatography from bacterial cell extracts or culture supernatants. Protein A or the like produced by combining and / or repeating purification methods selected from techniques such as fractional precipitation can also be used. In particular, protein A obtained by the method described in International Patent Publication WO2006 / 004067, US Patent Publication US515151350, WO2003 / 080655, JP2006-304633, WO2010 / 110288, and WO2012 / 133349 is preferable. The entire contents of these publications are incorporated herein by reference. The adsorbent of the present invention in which protein A is immobilized can also be used as a therapeutic adsorbent that can be used for the treatment of dilated cardiomyopathy and the like. Moreover, the adsorbent of the present invention in which dextran sulfate or the like is immobilized can be used as an adsorbent for treating hypercholesterolemia.

The method for introducing the ligand into the porous cellulose beads can be selected from the various immobilization methods described above, but more preferably, the reaction between the formyl group contained in the porous particles and the amino group of the ligand is performed. This is a method for immobilization. For example, there is a method described in WO2010 / 064437. The entire contents of this publication are incorporated herein by reference.

The amount of ligand immobilized on the adsorbent of the present invention is not particularly limited, and can be, for example, 1 mg or more and 300 mg or less per mL of porous cellulose beads or crosslinked porous cellulose beads. If the said ratio is 1 mg or more, since the adsorption amount with respect to a target substance becomes large, it is preferable since it can suppress manufacturing cost if it is 300 mg or less. The amount of ligand immobilized is preferably 2 mg or more, more preferably 4 mg or more, particularly preferably 5 mg or more, more preferably 100 mg or less, and further preferably 50 mg or less, per mL of porous cellulose beads or crosslinked porous cellulose beads. Preferably, 30 mg or less is particularly preferable, and 20 mg or less is most preferable.

The use of the adsorbent of the present invention is not particularly limited, but it is suitable for a medical adsorbent, especially a therapeutic adsorbent that adsorbs and removes large-sized pathogenic substances (such as LDL cholesterol) because the surface porosity can be improved. Can be used. Moreover, it can be used as various chromatographic carriers, especially industrial chromatographic carriers packed in large-diameter columns. In particular, when used as an adsorbent for antibody drug purification, which has been in great demand in recent years, the effect can be exhibited. From such a viewpoint, it can be suitably used as an adsorbent obtained by introducing protein A, protein G, or protein L into the porous beads of the present invention.

In recent years, for continuous chromatographic systems, relatively small porous beads having a median particle size of 36 μm or more and 64 μm or less and a column filled with these with a relatively low column height are desired. Since the present invention can provide a porous cellulose bead that can be easily adjusted in particle size, has good sphericity, and exhibits an appropriate compressive stress, it can accurately meet the need for column production that is relatively difficult to produce. I can respond. Here, the appropriate compressive stress is not particularly limited as long as it can be appropriately filled and used according to the application, but the stress when the settled beads are compressed by 20% is 0.01 MPa or more. If it is less than 1.0 MPa, it is preferable because good adsorption performance can be imparted. More preferably 0.04 MPa or more and 0.5 MPa or less, further preferably 0.06 MPa or more and 0.25 MPa or less, particularly preferably 0.09 MPa or more and 0.2 MPa or less, most preferably 0.10 MPa or more, 0.16 MPa. It is as follows. Examples of the method for adjusting the compressive stress include a method of adjusting the cellulose concentration in the dispersed phase and the degree of crosslinking.

6). Method for Purifying Target Substance In this method, a target substance is purified by bringing a solution containing the target substance into contact with the adsorbent and selectively adsorbing the target substance to the adsorbent. The target substance can be purified using the adsorbent according to the present invention. Specifically, the adsorbent of the present invention may be brought into contact with a solution containing the target substance. The contact method is not particularly limited, and the adsorbent according to the present invention may be added to a solution containing the target substance, or the column is filled with the adsorbent of the present invention and the solution containing the target substance is passed through. Thus, the target substance may be selectively adsorbed on the adsorbent of the present invention. Since the adsorbent according to the present invention has high strength, particularly when packed in a column, liquid can be passed at a high speed, and the target substance can be purified efficiently.

Next, the adsorbent of the present invention on which the target substance is selectively adsorbed is separated from the solution by filtration or centrifugation. When a column is used, the target substance is selectively adsorbed on the adsorbent and the remaining solution is discharged from the column, so that the adsorbent and the solution can be easily separated. By this step, the target substance and other substances can be separated. Furthermore, the target substance is separated from the adsorbent of the present invention using the eluate. As the eluate, for example, an acidic buffer having a pH of about 2.5 or more and 4.5 or less can be used. In addition, in adsorbents with large nonspecific adsorption, a long intermediate washing step may be required as a pre-elution step, but the porous cellulose beads of the present invention do not necessarily require such an intermediate washing step. do not do. Further, since the porous cellulose beads of the present invention have high alkali resistance, they can be washed with an alkaline washing liquid that can be prepared inexpensively and easily. When sodium hydroxide is used, it can be washed without problems even if its concentration is 0.1 N, and it can be used even if it is 0.5 N or more if the alkali resistance of the ligand is high. In the purification method according to the present invention, it is also preferable that two or more columns are connected and passed through.

This application claims the benefit of priority based on Japanese Patent Application No. 2018-96140 filed on May 18, 2018. The entire contents of Japanese Patent Application No. 2018-96140 filed on May 18, 2018 are hereby incorporated by reference.

Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to these examples. First, a method for testing physical properties of the produced porous cellulose beads will be described.

Test Example 1: Measurement of IgG adsorption characteristics (1) Solution preparation The following A to E solutions and neutralization solutions were prepared and degassed before use.
Solution A: A PBS buffer solution having a pH of 7.4 was prepared using “Phosphor buffered saline” manufactured by Sigma and distilled water.
Liquid B: A 35 mM aqueous sodium acetate solution having a pH of 3.5 was prepared using acetic acid, sodium acetate, and distilled water.
C liquid: 1M acetic acid aqueous solution was prepared using acetic acid and distilled water.
Solution D: An aqueous IgG solution having a concentration of 3 mg / mL was prepared using a polyclonal antibody (“Gamma Guard” manufactured by Baxter) and the solution A.
Liquid E: An aqueous solution in which the concentrations of sodium hydroxide and sodium chloride by Wako Pure Chemical Industries, Ltd. were 0.1 N sodium hydroxide and 1 M sodium chloride, respectively, was prepared and used as an alkaline cleaning liquid.
Neutralization solution: A 2M tris (hydroxymethyl) aminomethane aqueous solution was prepared with tris (hydroxymethyl) aminomethane and ultrapure water.

(2) Packing and preparation AKTA Pure 150 (manufactured by GE Healthcare) was used as an apparatus for column chromatography, and 3 mL of the adsorbent sample was placed in a column having an inner diameter of 0.5 cm, and the adsorbent layer height was set to 15 cm. A 0.2 M NaCl aqueous solution prepared from sodium chloride and distilled water was passed for 10 minutes at a flow rate of 3 mL / min, and the adsorbent was packed in the column. A 15 mL collection tube was set in the fraction collector, and a neutralization solution was put in advance in the eluate collection tube.

(3) IgG purification 15 mL of solution A was passed through the column, and then the required amount of solution D was passed. Next, 12 mL of solution A was passed through, and then 12 mL of solution B was passed through to elute IgG. Next, 9 mL of liquid C, 15 mL of liquid A, 9 mL of liquid E, and 15 mL of liquid A were passed. The flow rate was 1 mL / min except for the D solution, and the flow rate of the D solution was adjusted to a predetermined residence time (RT). For example, the flow rate for RT 6 minutes was adjusted to 0.5 mL / min.

(4) Dynamic adsorption amount The dynamic adsorption amount of IgG was determined from the amount of IgG adsorbed on the adsorbent and the adsorbent volume until IgG broke through 5%. The dynamic adsorption amount is referred to as 5% DBC.

(5) Static adsorption amount The static adsorption amount of IgG was calculated | required from IgG loading amount and adsorbent volume when each breakthrough curve of IgG of RT3min and RT6min crossed. The static adsorption amount is referred to as SBC.

(6) Operating binding capacity (OBC)
X. Gjoka et al. , J .; Chromatogr. A, 1416 (2015), 38-46, OBC was determined. The OBC represents the amount of antibody bound to the first column when the antibody solution starts to leak from the second column when two columns are connected and an antibody solution is loaded. In this example, for example, when obtaining OBC for RT 1 min, the loading time at 0.5% DBC when the antibody solution is loaded at a linear speed equivalent to RT 2 min is obtained, and then the antibody solution is added at the linear speed equivalent to RT 1 min. Obtained with time load.

Test Example 2: Measurement of 20% compressive stress (1) Sample preparation Pure water was added to the sample beads to prepare a slurry having a concentration of about 50% by volume. A homogenization / defoaming operation consisting of homogenization by stirring the slurry and subsequent defoaming by depressurization for 30 minutes or more was repeated three times to obtain a defoamed slurry. Separately from this operation, the object to be treated was changed to pure water, and the above homogenous / demethod operation was performed for 90 minutes or more to obtain defoamed water.

(2) Preparation of bead-filled syringe A hydrophilic disposable filter (pore size: 5.0 μm) was attached to the tip of a 2.5 mL disposable syringe (trade name “NORM-JECT” HANKE SASS WOLF). The piston of the syringe was removed, about 2 mL of defoamed water was added from the rear end side of the syringe, and defoamed slurry was added before the defoamed water was below the 0 mL mark. An aspirator was connected to the secondary side of the disposable filter, and the defoamed slurry was sucked while taking care that the liquid level did not fall below the bead surface. Suction was stopped when the liquid level dropped to about 0.5 mL above the bead surface. Subsequent operations were performed while appropriately adding the defoamed water so that the liquid level did not fall below the bead surface. The defoamed slurry was added or the beads were removed while applying vibration, and the bead surface was adjusted to a 1.5 mL mark, and it was confirmed that the bead surface did not drop even when vibration was applied. Slowly add defoamed water until it overflows from the syringe so that the beads do not flutter, and insert the piston with care to avoid bubbles. Hereinafter, this syringe is referred to as a “bead-filled syringe”.

(3) Measurement A load cell of 10K was attached to a FUDOH RHEO METER manufactured by Rheotech, the displacement speed dial was set to 2 cm / min, the bead-filled syringe was set, and displacement of the piston was started. The relationship between displacement and stress was recorded, and 20% compressive stress was determined based on the following formula.

20% compressive stress = [stress when the filled beads are compressed by 20%]-[stress when water is flowing before the piston pushes the beads]

Test Example 3: Measurement of solid content About 5 mL of sample beads were placed in a 15 mL centrifuge tube, and vibration was applied until the sample bead volume was not reduced any more, and the volume at that time was accurately measured. Hereinafter, such a volume is referred to as a “sedimentation volume”. Next, the beads in the centrifuge tube were transferred to a 3G glass filter and filtered. Note that the weight of the 3G glass filter was measured in advance by drying overnight in a 122 ° C. oven. Then, it was dried overnight in an oven at 122 ° C. and weighed. The solid content of the bead sample was calculated by dividing the weight by the volume.

Test Example 4: Measurement of pressure flow rate characteristics Beads having the same column volume and settling volume were prepared, and water was added thereto to prepare a 50% slurry. This 50% slurry was put into a column, and in the case of a column capable of passing water from the top at a linear speed of 60 cm / h or axial packing, the head was lowered at a linear speed of 60 cm / h. After the bead surface was stabilized, the head was lowered until a predetermined column volume was reached. The measurement was performed using AK Health Pure AKTA Pure 150 or AKTA Pilot, and the relationship between the linear velocity and the column differential pressure was examined.

Test Example 5: Measurement of median particle size The median particle size of beads and adsorbents was determined using a laser diffraction / scattering particle size distribution analyzer ("Partica LA950" manufactured by Horiba, Ltd.).

Granulation Comparative Example 1: Production of Porous Cellulose Beads (1) Compound Used As the cellulose, crystalline cellulose “PH-F20JP” or “PH-101” manufactured by Asahi Kasei Chemicals Corporation was used. Urea manufactured by Wako Pure Chemical Industries, Ltd. was used. The alkaline aqueous solution was prepared using sodium hydroxide and distilled water manufactured by Wako Pure Chemical Industries. Epichlorohydrin manufactured by Wako Pure Chemical Industries, Ltd. was used as the crosslinking agent after granulation. Other reagents were used without purification unless otherwise specified.

(2) Preparation of alkaline aqueous solution A sodium hydroxide aqueous solution was prepared using 27 g of sodium hydroxide and 32.4 g of pure water manufactured by Wako Pure Chemical Industries, Ltd., and the temperature was adjusted to 4 ° C.

(3) Preparation of dispersed phase 278 g of distilled water, 16 g of cellulose (PH-F20JP) and 46 g of urea are charged into a separable flask, and the temperature of the slurry is 4 ° C. using a disc turbine (rushton turbine) blade. Until 30 minutes at 150 to 200 rpm. In addition, the moisture content of the cellulose raw material was measured and the usage-amount of the raw material and the auxiliary material was adjusted in consideration of the water | moisture content. Next, the aqueous sodium hydroxide solution cooled to 4 ° C. was added, and the mixture was held for 30 minutes while stirring at a speed of 300 rpm. Thereafter, as a cooling step, the temperature was set to −15 ° C. and stirred at −15 ° C. for 60 minutes. Subsequently, this was adjusted to 25 degreeC, stirring. The concentration of water in the obtained dispersed phase was 77.7% by mass.

(4) Emulsification and porous formation 9.8 g of sorbitan monooleate (HLB value: 4.3) in 750 mL of continuous phase solvent (liquid paraffin, Kaneda K-140N, kinematic viscosity 4.6 mm ² / S, The flash point was 142 ° C. and the boiling point was so high that it could not be measured), and the mixture was stirred and mixed. While this was stirred at 750 rpm at 25 ° C., 156 g of the dispersed phase was added, and the dispersed phase was dispersed by stirring at 25 ° C. for 15 minutes. 87 mL of methanol was added as a coagulant and stirred at 750 rpm for 20 minutes. Thereafter, 14 g of acetic acid was added and neutralized by stirring at 750 rpm for 10 minutes. The solution was filtered through a glass filter “26G-3” manufactured by TOP, and then washed with isopropyl alcohol and water to collect porous cellulose beads.

(5) Classification of cellulose beads Wet classification was performed for 60 minutes using a 38 μm to 90 μm sieve.

(6) Observation with a microscope FIG. 1 illustrates a microscopic observation image of the porous body obtained. Only 23% of the total number of spherical beads was obtained.

Granulation Comparative Example 2
Preparation of beads was attempted in the same manner as in Comparative Example 1 except that the surfactant was changed to HLB1.8 sorbitan trioleate. FIG. 2 illustrates a microscopic image of the porous body obtained. Only 31% of the total number of spherical beads was obtained.

Granulation Example 1
Preparation of beads was attempted in the same manner as in Comparative Example 1 except that the surfactant was changed to polyglycerin ester of HLB 0.9 (manufactured by Riken Vitamin Co., Ltd .: PR-100) and the coagulant was changed to ethanol. FIG. 3 illustrates a microscopic observation image of the porous body obtained. 90% of the total number was obtained.

Granulation Example 2
(1) Compound to be used, (2) Preparation of aqueous alkali solution was carried out in the same manner as in Comparative Example 1 for granulation.

(3) Preparation of dispersed phase 15.25 kg of pure water, 0.919 kg of cellulose (PH-F20JP) and 2.49 kg of urea were charged into a cylindrical stainless steel container, and a two-stage disc turbine (ruston turbine) blade was used. The slurry was stirred at 283 rpm for 30 minutes until the temperature of the slurry reached 4 ° C. In addition, the moisture content of the cellulose raw material was measured, and the usage amount of the raw material and the auxiliary raw material was adjusted in consideration of the water content. Next, 2.884 kg of 48% sodium hydroxide manufactured by Kanto Chemical Co., Ltd. cooled to 4 ° C. at a rotation speed of 756 rpm was added and stirred for 30 minutes. Thereafter, it was cooled to −15 ° C. When the liquid temperature reached −9 ° C., the rotation speed was 57 rpm. After reaching −15 ° C., the mixture was stirred for 60 minutes. Subsequently, the rotational speed was set to 756 rpm and adjusted to 25 ° C. The concentration of water in the obtained dispersed phase was 77.8% by mass.

(4) Emulsification and porousization 1.225 kg of HLB value: 0.9 surfactant (PR-100 manufactured by Riken Vitamin Co., Ltd.) and 78.9 kg of continuous phase solvent (liquid paraffin, Kaneda K-140N, Kinematic viscosity 4.6 mm ² / S, flash point 142 ° C., boiling point is so high that it cannot be measured) and stirred in a cylindrical stainless steel container. While stirring this at 25 ° C. at 165 rpm, 19.55 kg of the dispersed phase was added, and the dispersed phase was dispersed by stirring at 25 ° C. for 15 minutes. 6.5 kg of methanol was added as a coagulant and stirred at 165 rpm for 20 minutes. Thereafter, 2.625 kg of acetic acid was added and neutralized by stirring at 165 rpm for 10 minutes. Stirring was stopped and the mixture was allowed to stand for 1 hour to separate the oil phase containing the continuous phase and the aqueous phase containing the beads, and the aqueous phase was separated from the bottom valve of the cylindrical stainless steel container. A required amount of the aqueous phase was filtered through a glass filter “26G-3” manufactured by TOP, and then washed with isopropyl alcohol and water to collect porous cellulose beads.

(5) Classification of cellulose beads Wet classification was performed for 60 minutes using a sieve of 38 μm and 75 μm or 90 μm.

(6) Observation with a microscope FIG. 4 illustrates a microscopic observation image of the porous body obtained. Of the total number, 94% of spherical beads were obtained.

Granulation Example 3
Beads were prepared in the same manner as granulation example 2 except that the amount of surfactant used was increased 3.5 times. FIG. 5 illustrates a microscope observation image of the porous body obtained. 95% of spherical beads were obtained out of the total number.

Granulation Example 4
The kinematic viscosity of the continuous phase solvent used is 13.6 mm ² / s (liquid paraffin, Kaneda K-230, flash point 176 ° C., boiling point is so high that the boiling point cannot be measured), the rotation speed after emulsification is 432 rpm, and solidification Beads were prepared in the same manner as granulation example 1 except that the amount of the agent was 43.5 mL. FIG. 6 illustrates a microscope observation image of the porous body obtained. 67% of spherical beads were obtained out of the total number.

Granulation Example 5
Beads were prepared in the same manner as granulation example 4 except that a surfactant having an HLB value of 0.4 (PR-300 manufactured by Riken Vitamin Co., Ltd.) was used. FIG. 7 illustrates a microscopic observation image of the porous body obtained. 83% of spherical beads were obtained out of the total number.

Granulation Example 6
The amount of cellulose in the dispersed phase is 14.7 g, the amount of water is increased by 1 g, the concentration of water in the resulting dispersed phase is 78.4% by mass, and the kinematic viscosity of the continuous phase solvent used is 35.3 mm ^2. / S (liquid paraffin, Kaneda K-290, flash point 206 ° C., boiling point is so high that the boiling point cannot be measured), the amount of the continuous phase solvent used is 488 mL, the rotation speed after emulsification is 300 rpm, and the coagulant used A bead was prepared in the same manner as in Granulation Example 1, except that the amount of was 65 mL. FIG. 8 illustrates a microscopic image of the obtained porous body. 62% of spherical beads were obtained out of the total number.

Granulation Example 7
Beads were prepared in the same manner as in Granulation Example 6 except that a surfactant having an HLB value of 0.4 (PR-300 manufactured by Riken Vitamin Co., Ltd.) was used. FIG. 9 illustrates a microscopic image of the obtained porous body. Of the total number, 91% of spherical beads were obtained.

Granulation Reference Example 1
The kinematic viscosity of the continuous phase solvent used is 77.6 mm ² / s (liquid paraffin, Kaneda K-350, flash point 254 ° C., boiling point is so high that the boiling point cannot be measured), and the rotational speed after emulsification is 750 rpm. Produced beads in the same manner as in Example 6 of granulation. FIG. 10 illustrates a microscopic observation image of the obtained porous body. Almost no spherical beads were obtained.

Table 1 shows the relationship between the number% of spherical beads obtained in the above granulation examples, the HLB value of the surfactant, and the continuous phase kinematic viscosity.

Example 8: Crosslinking of cellulose beads (1) First crosslinking step 96 mL of washed cellulose beads obtained in the above granulation example were prepared. When there were not enough beads, the procedure up to the above classification was repeated. The prepared beads were placed on a glass filter, ethanol was added, and the mixture was stirred well so that the bead slurry became uniform, and then the solvent substitution operation for removing the ethanol by suction was performed four times. The amount of ethanol was set to 233 mL for the first to third solvent replacement operations and 167 mL for the fourth solvent replacement operation. After the solvent replacement operation, ethanol was added to the whole so as to be 97 g while being transferred to a 500 mL separable flask, and then 28 g of water was added. Further, 80 mL of epichlorohydrin was added and stirred for 30 minutes at 200 rpm. Next, a mixed liquid composed of 10 mL of 17 M NaOH aqueous solution and 86 mL of water was added, and the porous cellulose beads were crosslinked by stirring for 1 hour and 30 minutes at a rotation speed of 350 rpm while maintaining the temperature at 40 ° C. Further, 9.6 mL of 17 M NaOH aqueous solution was added, and an additional treatment of stirring for 1.5 hours at a rotation speed of 350 rpm was carried out three times, followed by filtration and subsequent washing with water to obtain intermediate crosslinked beads.

(2) Second cross-linking step Water was added to the total amount of the obtained intermediate cross-linked beads to adjust the total volume to 117 mL, and the temperature was heated to 40 ° C. After adding 38 g of sodium sulfate and stirring for 10 minutes at a rotational speed of 150 rpm, 33 mL of epichlorohydrin was added and stirred for 10 minutes at a rotational speed of 250 rpm. Next, 21 mL of 17 M NaOH aqueous solution was added and stirred at 300 rpm for 2.5 hours. Finally, 5.1 mL of 17 M NaOH aqueous solution was added and further stirred for 2.5 hours. The reaction product was filtered, and the filtrate was washed with water to obtain crosslinked beads.

(3) Epoxy ring-opening treatment The obtained crosslinked beads and 50% slurry of water were heated in an autoclave at 121 ° C. for 60 minutes to open the epoxy group to form a diol group. The disappearance of the epoxy group was confirmed with a phenolphthalein indicator.

(4) Reclassification Wet classification was performed for 60 minutes using a 38 μm and 75 μm or 90 μm sieve. The obtained beads having each particle size were mixed to obtain crosslinked cellulose beads having a predetermined particle size.

The above-mentioned crosslinked cellulose beads were added according to the required amount, or obtained by increasing the scale. Table 2 shows the physical properties of the porous cellulose beads and the crosslinked porous cellulose beads.

As shown in Patent Document 7 and the like, mechanical strength that can be suitably used for industrial use is imparted if the 20% compressive stress is 0.11 MPa or more.

FIG. 10 shows the pressure flow rate characteristics of the crosslinked porous beads having a median particle size of 60 μm obtained by crosslinking the cellulose beads obtained in Granulation Example 3. The column used was AxiChrom 70 manufactured by GE Healthcare, the packing rate was 105% with respect to the tapping volume, and the column height was 20 cm. The mobile phase under test was pure water, but a comparison was also made between the case where the mobile phase during packing was pure water and the case where 20% ethanol with 0.4 M sodium chloride was used. When the mobile phase at the time of packing was water, axial packing was performed at a linear speed of 60 cm / h. When the mobile phase at the time of packing is 20% ethanol with 0.4M sodium chloride, bead slurry substituted with about 50% of the same mobile phase is put into the column, and the mobile phase is down-flowed at a linear velocity of 200 cm / h. The beads were allowed to settle while flowing, and axial packing was performed at a linear speed of 100 cm / h. In the pressure flow characteristics test, the pressure was read while gradually increasing the linear velocity, and the measurement was stopped when the bed height decreased by 1 mm or more. That is, in FIG. 10, the bed height is slightly lowered in the plot on the highest linear velocity side. Moreover, the result of having carried out the pressure flow rate test only with water without filling beads in the column is drawn as a blank.

Generally, in the case of 20 cm in height with an AxiChrom column, the ability to pass liquid at a linear velocity of 300 cm / h at 0.3 MPa or less is a criterion for judging whether or not it can be used for industrial use. As shown in FIG. 10, it was found that the crosslinked porous cellulose beads obtained according to the present invention can be passed at a linear velocity higher than 300 cm / h and the pressure is low.

FIG. 11 shows a comparison of pressure flow characteristics between the crosslinked porous beads used in the test of FIG. 10 and a commercially available PA resin (MabSelect SuRe pcc manufactured by GE Healthcare) that is considered suitable for a continuous column chromatography system. . The column used was GE Healthcare's AxiChrom 70, the packing rate was 105% of the tapping volume, and the column height was 4 cm. The mobile phase under test and the mobile phase during packing were pure water, and the axial packing was performed at a linear velocity of 60 cm / h. In the pressure flow characteristics test, the pressure was read while gradually increasing the linear velocity, and the measurement was stopped when the bed height decreased by 1 mm or more. That is, in FIG. 11, the bed height is slightly lowered in the plot on the highest linear velocity side. In addition, the beads are not packed in the column, and the result of the pressure flow rate test using only water is not drawn as a blank. As shown in FIG. 11, it was found that the crosslinked porous cellulose beads obtained by the present invention exhibited sufficient pressure flow characteristics even for a continuous column chromatography system.

Example 9: Preparation of adsorbent with immobilized ligand (1) Aldehydation reaction (1-1) Preparation of buffer 0.165 g of citric acid monohydrate and 0.0646 g of trisodium citrate dihydrate and water Was added to make 100 mL, and a pH 3.4 buffer was prepared.

(1-2) Reaction Using 4 times or more of the above-obtained cellulose beads after crosslinking, the liquid part was replaced with the above buffer with the above buffer, and the buffer was further added to bring the total amount to 6.0 mL. did. By adding 2.23 mL of 46.4 mg / mL sodium periodate aqueous solution and stirring at 25 ° C. for 35 minutes, aldehyde group-containing beads were obtained.

(2) Protein A Immobilization Reaction (2-1) Preparation of Protein A A protein A mutant was prepared according to the method described in Example 1 of WO2017 / 022672. Hereinafter, the obtained protein A mutant is abbreviated as “PA”. A solution containing this PA (protein A concentration 55 mg / mL) was prepared.

(2-2) Imination reaction-When the PA charge is 20 mg / mL Immediately after the aldehyde reaction, the mixture is filtered through a # 3 glass filter, and 0.9M dipotassium hydrogen phosphate aqueous solution is 3 times the beads. The liquid part was substituted by using the volume amount or more, and 0.9 M dipotassium hydrogen phosphate aqueous solution was added to make the total volume 6.0 mL. 1.48g of protein aqueous solution was added to this, and it stirred at 6 degreeC for 60 minutes. After stirring for 60 minutes, 0.75 mL of 2N aqueous sodium hydroxide solution was added to adjust the pH to 11 units. The mixture was stirred overnight at 6 ° C.

(2-3) Neutralization and reduction reaction The imination reaction solution after the overnight reaction was centrifuged, and the supernatant was extracted to adjust the liquid volume to 6.25 mL. The UV of the extracted reaction solution was measured to determine the amount of PA immobilized. 1.2 mL of an ethanol solution containing 1.1 w / v% picoline borane was added to the reaction vessel and stirred at 6 ° C. for 1 hour. Next, a 2.4 M aqueous citric acid solution was added to adjust the pH to 8, followed by stirring at 25 ° C. for 7 hours. Next, 2.4 mL of an aqueous solution containing 11 w / v% dimethylamine borane was added and stirred overnight at 25 ° C. The beads after the reaction were washed on a # 3 glass filter with 3 times the volume of water of the beads.

(2-4) Washing 3 mL of 0.1 M citric acid (hereinafter abbreviated as “acid buffer”) was passed through 1 mL of PA-immobilized beads on a # 3 glass filter, and the liquid part in the beads was removed. Replacement with acid buffer. The substituted PA-immobilized beads were transferred to a container, acid buffer was added to make the total volume 2 mL or more, and the mixture was stirred at 25 ° C. for 30 minutes for acid washing.

Next, alkali cleaning was performed in the same manner except that 0.05N sodium hydroxide + 1M sodium sulfate aqueous solution was used instead of the acid buffer.

Next, neutral washing was performed in the same manner except that a solution in which 0.1 M citric acid and 0.1 M sodium citrate were mixed to adjust the pH to 5.9 was used instead of the acid buffer. . The beads after neutral washing were washed with distilled water until the electric conductivity of the washing filtrate became 10 μs / cm or less to obtain an adsorbent on which protein A was immobilized.

Table 3 shows the IgG adsorption characteristics of each PA resin when the median particle size is 60 μm. The reference in the table is a commercially available PA resin that is said to be suitable for a continuous column chromatography system. Normally, the smaller the particle size, the higher the DBC and OBC. However, as shown in Table 3, the PA resin obtained in the present invention has a median particle size of 60 μm, even when the median particle size is adjusted to 60 μm. It was found to show high adsorption performance.

Claims (14)

A method for producing a porous cellulose bead comprising:
A step of dispersing cellulose or a cellulose derivative in a solvent to obtain a dispersed phase;
Dispersing the dispersed phase in a continuous phase containing a surfactant to obtain a W / O type emulsion; and
A step of coagulating the cellulose or cellulose derivative by bringing the W / O emulsion into contact with a coagulant;
A method in which a surfactant is added to one or both of the solvent and the continuous phase, and the HLB value of the surfactant is 0.1 or more and less than 1.8. The method according to claim 1, wherein the flash point of the continuous phase is 73 ° C or higher. The method according to claim 1 or 2, wherein the boiling point of the continuous phase is 181 ° C or higher. The method according to any one of claims 1 to 3, wherein liquid paraffin having a kinematic viscosity of less than 74 mm ² / S is used as the continuous phase. The method according to any one of claims 1 to 4, wherein an HLB value of the surfactant is smaller than a value obtained by the following formula 1.
-0.0433 × kinematic viscosity of continuous phase [mm ² /S]+1.733
... (Formula 1) The method according to any one of claims 1 to 5, wherein the concentration of water in the dispersed phase is 41% by mass or more. The liquid temperature during the production of the porous cellulose beads is less than the boiling point of the compound having the lowest boiling point among the compounds contained in the continuous phase, dispersed phase, surfactant and coagulant. The method according to one item. The method according to any one of claims 1 to 7, wherein the coagulant is immiscible with the continuous phase. The method according to any one of claims 1 to 8, wherein the dispersed phase is prepared by mixing an alkaline aqueous solution and the cellulose or cellulose derivative powder. Producing porous cellulose beads by the method according to any one of claims 1 to 9, and
The manufacturing method of the crosslinked porous cellulose bead characterized by including the process of bridge | crosslinking the said porous cellulose bead using a crosslinking agent. Producing cross-linked porous cellulose beads by the method of claim 10, and
A method for producing an adsorbent comprising the step of immobilizing a ligand on the crosslinked porous cellulose beads. A method for purifying a target substance,
A step of producing an adsorbent by immobilizing a ligand that binds to the target substance on the crosslinked porous cellulose beads by the method according to claim 11, and
A method comprising the step of bringing a solution containing the target substance into contact with an adsorbent. The method according to claim 12, wherein the adsorbent is packed in a column and the solution is passed through the column. The method according to claim 13, wherein two or more columns are connected to pass the solution.

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