JP2021161246A - Method for producing porous cellulose beads - Google Patents

Abstract

To provide a method for easily producing cellulose beads suitable for antibody adsorption without using an auxiliary raw material having high toxicity and corrosiveness and without going through a complicated process which is industrially disadvantageous.SOLUTION: There is provided a method for producing porous cross-linked cellulose beads including the steps of: (a) dissolving cellulose in a mixed solution of sodium hydroxide, lithium hydroxide, urea and water at a temperature of 5°C or lower and -20°C or higher to prepare a cellulose solution; (b) heating the cellulose solution to 10°C or higher and 40°C or lower; (c) dispersing the cellulose solution into a dispersion medium of 10°C or higher and 40°C or lower to prepare an emulsion; (d) bringing the emulsion into contact with a coagulation solvent to precipitate cellulose beads; and (e) cross-linking the precipitated cellulose beads, wherein a molar ratio of sodium hydroxide/lithium hydroxide in the step (a) is 0.3/0.7 to 0.8/0.2.SELECTED DRAWING: None

Description

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

Porous cellulose beads have the advantages of higher safety and less non-specific adsorption than when other synthetic polymers are used, and porous cellulose beads have various chromatographic adsorbents and affinity adsorbents. It is used as a base material for various adsorbents such as. In particular, affinity adsorbents have been used as medical adsorbents and adsorbents for purifying antibody drugs because they can efficiently purify the target substance and reduce the concentration of unnecessary substances. In particular, as a therapeutic adsorbent or a medical adsorbent for rheumatism, hemophilia, and dilated cardiomyopathy, an adsorbent in which protein A is immobilized on a porous carrier as an affinity ligand has been attracting attention (for example, Non-Patent Document 1). , Non-Patent Document 2).

Since it has been said that it is difficult to dissolve cellulose in the production of porous cellulose beads, many of them include complicated steps as compared with ordinary synthetic polymers. As one of them, a method of dissolving and coagulating in a solvent such as an aqueous solution of calcium thiocyanate, which is highly corrosive and toxic and increases the difficulty of equipment installation, is disclosed (for example, Patent Document 1). It is known that the cellulose solution used in this method behaves peculiarly, and that 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 this method are used as an adsorbent for an antibody or the like, high adsorption performance cannot be expected because the specific surface area is small. On the other hand, in order to increase the solubility of cellulose, a method of imparting a substituent to the hydroxyl group of cellulose, dissolving it in a general-purpose solvent for granulation, and removing the substituent after granulation to obtain a porous cellulosic carrier. (For example, Patent Document 2), but the process is complicated, the molecular weight decreases in the process of adding or removing a substituent, and it is used in high-speed processing and large scale, which have been demanded in recent years. It tends to be difficult to obtain appropriate mechanical strength to do so.

On the other hand, a method of dissolving cellulose in a low-temperature alkali / urea / water mixed solution has been reported (Non-Patent Document 4). Since this dissolution method has a low environmental load and the dissolution process is not complicated, various studies have been conducted.

Special Table 2009-242770 International release WO 2006/025371

Annals of the New York Academy of Sciences, Vol. 1051, (2005), p. 635-646 American Heart Journal, Vol. 152, Number 4, (2006), p. 712e1-712e6 Macromolecular Bioscience, vol. 5, (2005), p. 539-548 Journal of Chromatography, Vol. 316, (1984), p. 311-332

The present invention develops cellulose beads having a pore structure suitable for antibody adsorption simply and efficiently without using highly toxic and corrosive auxiliary materials and without going through complicated steps that are industrially disadvantageous. The purpose is.

As a result of diligent research to solve the above problems, the present inventors have obtained cellulose beads having an optimum structure for antibody adsorption by using a mixed solution of sodium hydroxide, lithium hydroxide, urea and water. We have found that it can be manufactured, and have completed the present invention.

Therefore, one aspect of the present invention is (a) a step of dissolving cellulose in a mixed solution of sodium hydroxide, lithium hydroxide, urea and water at −5 ° C. or lower and −20 ° C. or higher to prepare a cellulose solution. b) A step of heating the cellulose solution to 10 ° C. or higher and 40 ° C. or lower, (c) a step of dispersing the cellulose solution in a dispersion medium of 10 ° C. or higher and 40 ° C. or lower to prepare an emulsion, (d) the above. A method for producing porous crosslinked cellulose beads, which comprises a step of bringing the emulsion into contact with a coagulation solvent to precipitate cellulose beads and (e) a step of cross-linking the precipitated cellulose beads. The present invention relates to a method for producing porous crosslinked cellulose beads, which comprises a molar ratio of lithium oxide of 0.3 / 0.7 to 0.8 / 0.2.

According to the present invention, cellulose beads suitable for antibody adsorption can be easily produced without using highly toxic and corrosive auxiliary raw materials and without going through complicated steps that are industrially disadvantageous.

An embodiment of the present invention will be described in detail below. Unless otherwise specified in the present specification, "A to B" representing a numerical range means "A or more and B or less". In addition, all of the documents described herein are incorporated herein by reference.

[1. Outline of the present invention]
The method for producing porous cellulose beads according to an embodiment of the present invention (hereinafter, referred to as “the present production method”) is (a) sodium hydroxide at −5 ° C. or lower and −20 ° C. or higher, lithium hydroxide, and the like. A step of dissolving cellulose in a mixed solution of urea and water to prepare a cellulose solution, (b) a step of heating the cellulose solution to 10 ° C. or higher and 40 ° C. or lower, and (c) a step of heating the cellulose solution to 10 ° C. or higher. It includes a step of dispersing in a dispersion medium at 40 ° C. or lower to prepare an emulsion, (d) a step of bringing the emulsion into contact with a coagulation solvent to precipitate cellulose beads, and (e) a step of cross-linking the precipitated cellulose beads. It has been developed by the present applicant that porous cellulose can be obtained by dispersing cellulose in a low-temperature aqueous sodium hydroxide solution and contacting it with a coagulation solvent (International Publication WO 2012/121258, etc.).

For unknown reasons, surprisingly, we found that instead of using a single alkaline substance, we used a combination of multiple alkaline substances to create cross-linked cellulose with pores suitable for antibody adsorption. For the first time, I found that I could make beads.

Hereinafter, the configuration of this manufacturing method will be described in detail.

[2. Method for manufacturing porous cellulose beads]
This manufacturing method is a method including the following steps (a) and (b) as essential steps.
-Step (a): A step of dissolving cellulose in a mixed solution of sodium hydroxide, lithium hydroxide, urea and water at -5 ° C or lower and -20 ° C or higher to prepare a cellulose solution.-Step (b): The step. Step / Step (c) of heating the cellulose solution prepared in (a) to 10 ° C. or higher and 40 ° C. or lower: Disperse the cellulose solution prepared in the step (b) in a dispersion medium of 10 ° C. or higher and 40 ° C. or lower. Step / Step (d): The emulsion prepared in the step (c) was brought into contact with a coagulation solvent to precipitate cellulose beads. Step / Step (e): Precipitated in the step (d). Steps for Crossing Cellulose Beads The method of the present invention will be described below for each step.

(Step (a))
In the step (a) of the present production method, cellulose is dissolved in a mixed solution of sodium hydroxide, lithium hydroxide, urea and water at −5 ° C. or lower and −20 ° C. or higher to prepare a cellulose solution.

Here, the molar ratio of sodium hydroxide / lithium hydroxide is preferably 0.3 / 0.7 to 0.8 / 0.2. It is more preferably 0.4 / 0.6 to 0.7 / 0.3, and more preferably 0.45 / 0.55 to 0.65 / 0.35. If the sodium hydroxide molar ratio is too high, the pore radius becomes too large for antibody adsorption, and if the sodium hydroxide molar ratio is too low, the pore radius becomes small for antibody adsorption.

In one embodiment of the present invention, the total concentration of sodium hydroxide and lithium hydroxide in the mixed solution of sodium hydroxide, lithium hydroxide, urea and water is preferably 5% by weight to 20% by weight. It is more preferably 6% by weight to 15% by weight, and more preferably 7% by weight to 12% by weight. Within the above concentration range, cellulose can be satisfactorily dissolved.

In one embodiment of the present invention, the urea concentration of the mixed solution of sodium hydroxide, lithium hydroxide, urea and water is preferably 10% by weight to 20% by weight. It is more preferably 11% by weight to 18% by weight, and even more preferably 11% by weight to 16% by weight. Within the above concentration range, cellulose can be satisfactorily dissolved.

In one embodiment of the present invention of the present invention, the cellulose concentration of the cellulose solution is preferably 3% by weight or more and 9% by weight or less. Within the above concentration range, cellulose can be satisfactorily dissolved.

In one embodiment of the present invention, the molecular weight of the cellulose raw material used is not particularly limited, but the degree of polymerization is preferably 100 to 1000. More preferably, it is 150 to 900, and more preferably 200 to 800. When the molecular weight of cellulose is in the above range, cellulose is easily dissolved.

In one embodiment of the present invention, the temperature of the cellulose solution is preferably −5 ° C. to −20 ° C. More preferably, it is −8 ° C. to −19 ° C., and more preferably −10 ° C. to −17 ° C. At a temperature of −5 ° C. or higher, cellulose is difficult to dissolve, and at a temperature of −20 ° C. or lower, the cellulose solution freezes, resulting in poor operability.

(Step (b))
In the step (b) of the present production method, the cellulose solution prepared in the step (a) is heated to 10 ° C. to 40 ° C. More preferably, it is 15 ° C to 30 ° C, and more preferably 20 ° C to 28 ° C. When the heating temperature is within the above range, cellulose does not precipitate and the viscosity of the solution does not increase, so that the operation can be preferably performed.

(Step (c))
In the step (c) of this production method, the cellulose solution prepared in the step (b) is dispersed in a dispersion medium to prepare an emulsion.

In one embodiment of the present invention, the dispersion medium is not particularly limited as long as it is not mixed with the cellulose solution. Hydrocarbon-based solvents can be mentioned. Further, a surfactant such as a nonionic surfactant may be added to the dispersion medium.

Examples of the hydrocarbon solvent include hexane, decane, and liquid paraffin. Animal and vegetable oils include palm oil, shea oil, monkey oil, iripe oil, pork oil, beef oil, rapeseed oil, rice oil, peanut oil, olive oil, corn oil, soybean oil, perilla oil, cottonseed oil, sunflower oil, evening primrose oil, Examples include sesame oil, safflower oil, palm oil, cacao butter, palm kernel oil, fish oil, wakame oil, and comb oil. Examples of hydrogenated animal and vegetable fats and oils include palm hardened oil, palm extremely hardened oil, rapeseed hardened oil, rapeseed extremely hardened oil, soybean hardened oil, pork fat hardened oil, and fish oil hardened oil. The fatty acid glyceride may be any of tri-, di-, and mono-glycerides, and examples thereof include stearin glyceride, palmitic acid glyceride, and lauric glyceride. Examples of the aliphatic hydrocarbon solvent include beeswax, candelilla wax, rice bran wax and the like. Examples of the aromatic hydrocarbon solvent include benzene, toluene, chlorobenzene, dichlorobenzene and the like.

In order to prepare an emulsion, an appropriate amount of a surfactant may be further added to the dispersion medium. Examples of the surfactant include sorbitan fatty acid esters such as sorbitan laurate, sorbitan stearate, sorbitan oleate and sorbitan trioleate, and polyglycerin fatty acid esters such as polyglycerin polylysinolate, decaglycerin oleate and decaglycerin stearate. Can be done.

In one embodiment of the present invention, the amount of the dispersion medium used is not particularly limited, but may be an amount capable of sufficiently dispersing the cellulose solution prepared in the step (b). For example, it can be 1 to 10 times by volume with respect to the cellulose solution. It is more preferably 2 volume times to 8 volume times, and more preferably 4 volume times to 10 volume times. If the amount of the dispersion medium used is out of the above range, the amount of waste liquid may increase excessively.

The emulsion may be prepared by a conventional method. For example, it can be prepared by vigorously stirring the mixed solution containing the cellulose solution, the dispersion medium and the surfactant.

(Step (d))
In the step (d) of the present production method, the emulsion prepared in the step (c) is brought into contact with a coagulation solvent to prepare porous cellulose beads.

The coagulation solvent is not particularly limited as long as it has an affinity for the cellulose solution, and examples thereof include an alcohol solvent or a mixed solvent of water and an alcohol solvent. _{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 mixed solvent of water and alcohol solvent can be, for example, water: alcohol solvent = 80:20 to 1:99 in volume ratio.

In one embodiment of the present invention, the coagulation method is not particularly limited, but since the emulsion may be unstable, it is preferable to add the coagulation solvent in a state of vigorous stirring so that the droplets do not bond with each other.

After the coagulation solvent is added, the coagulated porous cellulose beads may be separated by filtration, centrifugation, or the like, and washed with water, alcohol, or the like. The obtained porous cellulose beads may be classified using a sieve or the like in order to make the particle size uniform.

(Step (e))
In the step (e) of the present production method, the cellulose beads produced in the step (d) are crosslinked with a cross-linking agent to produce porous crosslinked cellulose beads.

A cross-linking agent has two or more reactive groups capable of forming a covalent bond with a hydroxyl group on cellulose and can cross-link between cellulose molecules. The conditions for cross-linking the cross-linked porous cellulose particles and the cross-linking agent that can be used in the present invention are not particularly limited. For example, the method described in WO2008 / 146906 can be used. The entire contents of this international publication are incorporated herein by reference.

Examples of the cross-linking agent include halohydrins such as epichlorohydrin, epibromohydrin, and dichlorohydrin; bifunctional dichloromethane (bisoxylan); and polyfunctional polyepoxide (polyoxylan). As the cross-linking agent, only one kind may be used alone, or two or more kinds may be used in combination.

The solvent for the reaction of cross-linking the porous cellulose beads with a cross-linking agent may be appropriately selected. For example, in addition to water, water miscibility with alcohol-based solvents such as methanol, ethanol and isopropanol, and nitrile-based solvents such as acetonitrile. Organic solvents can be mentioned. Further, the cross-linking reaction solvent may be used as a mixture of 2 or more.

The cross-linking reaction may be carried out a plurality of times, and the reaction solvent and the cross-linking agent may be changed each time. For example, the first cross-linking reaction may be carried out in a water-miscible organic solvent, and the final cross-linking reaction may be carried out in water. In this case, the solvent composition in the middle may be the same as or different from that of either the first round or the final round, or may be an intermediate composition between them. Furthermore, all times may be carried out in an aqueous solvent. The same applies to the cross-linking agent. When the cross-linking reaction is repeated a plurality of times, it is preferable to wash the cross-linked porous cellulose beads with water or the like to remove the cross-linking agent between the cross-linking reactions.

A base may be added to the reaction solution to promote the cross-linking reaction. Such bases include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal bicarbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; alkali metal carbonates such as sodium carbonate and potassium carbonate; triethylamine and pyridine. Organic bases such as.

After the cross-linking reaction, the porous cross-linked cellulose beads are insoluble and may be washed with a solvent such as water.

According to this production method, crosslinked porous cellulose beads having high productivity and excellent antibody adsorption can be obtained.

[3. Porous cross-linked cellulose beads]
The porous crosslinked cellulose beads (hereinafter referred to as “the present beads”) according to the embodiment of the present invention have an intraparticle pore ratio of 90% or more, a pore radius of 35 to 50 nm, and a volume median diameter. Is 45 to 70 μm.

Since the beads are produced by the production method, they have an advantage of being excellent in antibody adsorption ability.

The volume median diameter of the beads is not particularly limited, but is preferably 45 to 70 μm, more preferably 50 to 65 μm, still more preferably 55 to 63 μm from the viewpoint of achieving excellent dynamic antibody adsorption ability. The volume median diameter of the beads is measured by the method described in Examples.

The porosity in the particles of the beads is preferably 90% or more, more preferably 91% or more, still more preferably 92% or more, from the viewpoint of achieving excellent dynamic antibody adsorption ability. The intra-particle porosity of the beads is measured by the method described in Examples.

The pore radius of the beads is preferably 35 to 50 nm, more preferably 37 to 48 nm, and even more preferably 40 to 45 nm from the viewpoint of achieving excellent dynamic antibody adsorption ability. The average pore radius of the beads is measured by the method described in Examples.

In order to specifically adsorb the antibody, the beads can bind a protein A ligand, and the binding conditions and the protein A ligand are not particularly limited. For example, the method described in WO2012 / 133349 can be used. The entire contents of this international publication are incorporated herein by reference.

The present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the claims, and the embodiment obtained by appropriately combining the technical means disclosed in each of the different embodiments. Is also included in the technical scope of the present invention.

That is, one embodiment of the present invention is as follows.
<1> (a) A step of preparing a cellulose solution by dissolving cellulose in a mixed solution of sodium hydroxide, lithium hydroxide, urea and water at -5 ° C or lower and -20 ° C or higher.
(B) A step of heating the cellulose solution to 10 ° C. or higher and 40 ° C. or lower.
(C) A step of preparing an emulsion by dispersing the cellulose solution in a dispersion medium having a temperature of 10 ° C. or higher and 40 ° C. or lower.
(D) A step of bringing the emulsion into contact with a coagulation solvent to precipitate cellulose beads.
(E) A method for producing porous crosslinked cellulose beads, which comprises a step of crosslinking the precipitated cellulose beads.
A method for producing porous crosslinked cellulose beads, wherein the molar ratio of sodium hydroxide / lithium hydroxide in the step (a) is 0.3 / 0.7 to 0.8 / 0.2.
<2> A method for producing porous crosslinked cellulose beads, wherein the urea concentration of the mixed solution of sodium hydroxide, lithium hydroxide, urea and water in the step (a) is 10% by weight to 20% by weight.
<3> A method for producing porous crosslinked cellulose beads, which comprises a cellulose concentration of 3% by weight or more and 9% by weight or less in the early stage cellulose solution. <4> The coagulation solvent is an alcohol solvent or water and alcohol. A method for producing porous crosslinked cellulose beads, which is a mixed solvent with a system solvent.
<5> Porous crosslinked cellulose beads having a porosity in particles of 90% or more, a pore radius of 35 nm to 50 nm, and a volume median diameter of 45 μm to 70 μm.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to these Examples.

[Measurement and evaluation method]
Measurements and evaluations in Examples, Comparative Examples and Production Examples were performed by the following methods.

(Volume median diameter)
The volume median diameter of the beads was measured using a laser diffraction / scattering type particle size distribution measuring device LA-950 manufactured by HORIBA.

(Pore radius)
22.8 mL of the beads were dispersed in distilled water and degassed for 30 minutes. The degassed porous cellulose beads were packed in a column (“Tricorn 10/300” manufactured by GE Healthcare Japan Co., Ltd.). Shimadzu size exclusion chromatography system ("DGU-20A3", "RID-10A", "LC-20AD", "SIL-20AC", "CTO-20AC" included, "LCSolution" as software (Use) was used for measurement.

As a marker, dextran or glucose shown in Table 1 was dissolved in a 20 mM phosphate buffer (pH 7.5) containing 0.2 M NaCl and used.

While passing a 20 mM phosphate buffer (pH 7.5) containing 0.2 M NaCl in the column at a flow rate of 0.33 mL / min, first, in order to determine the volume other than the bead portion in the column, the molecular weight is 4 × 10 ^7. The solution of dextran was injected, and the flow rate from the injection to the peak observed by the RI monitor was determined. The concentration of the dextran solution having a molecular weight of 4 × 10 ⁷ was 10 mg / mL, and the injection amount was 40 μL. Next, the flow rate of each marker solution was determined in the same manner. The measured values were substituted into the following equation to calculate the value of K _{D.
K D = (V R -V 0} ) / (V t -V 0)
Wherein, V _R represents the liquid passing amount (mL) to peak from the injection of the marker solution is observed, V ₀ is injected peaks from observation dextran solution having a molecular weight of 4 × 10 ⁷ The amount of liquid to be passed (mL) is indicated, and V _t indicates the volume (mL) of the bead pores in the column. V _t is measured from a marker having a molecular weight of 180. ]
The porosity in the particles is represented by _{(V t −} V ₀ ) / V _0.

It was calculated pore radius by the following formula in Non-Patent Document 4 described from the value of K _D.

a r _p is the average pore radius, _{s p} represents the standard deviation.

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

(2) Filling and preparation Using AKTA Pure 150 (manufactured by GE Healthcare) as a column chromatography device, 3 mL of an adsorbent sample was placed in a column having an inner diameter of 0.5 cm, and the height of the adsorbent layer was set to 15 cm. A 0.2 M aqueous NaCl solution prepared from sodium chloride and distilled water was passed through the column at a flow rate of 3 mL / min for 10 minutes to fill the column with the adsorbent. A 15 mL collection tube was set in the fraction collector, and a neutralizing solution was previously put 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 to elute IgG. Next, 9 mL of C solution, 15 mL of A solution, 9 mL of E solution, and 15 mL of A solution 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 4 minutes was adjusted to 0.75 mL / min.

(4) Dynamic adsorbed amount The dynamic adsorbed amount of IgG was determined from the amount of IgG adsorbed on the adsorbent and the volume of the adsorbent before IgG broke by 5%. The amount of dynamic adsorption is called 5% DBC.

[Example 1]
(Preparation of cellulose solution)
48.0 wt% sodium hydroxide (manufactured by Nakaraitesk Co., Ltd.) 30.2 g, lithium hydroxide monohydrate (manufactured by Kishida Chemical Co., Ltd.) 15.4 g, urea (manufactured by Wako Pure Chemical Industries, Ltd.) 57.5 g, 200 g of pure water was mixed and dissolved and kept at 10 ° C. to prepare an alkaline aqueous solution. 17 g of powdered cellulose (Nippon Paper Industries, Ltd., W200G) was dispersed in 80 g of water, added to the alkaline aqueous solution, and then the temperature was lowered to -12 ° C. to dissolve the cellulose. The sodium hydroxide concentration of the solvent excluding the cellulose weight was 3.8% by weight, the lithium hydroxide concentration was 2.3% by weight, the urea concentration was 15% by weight, and the sodium hydroxide molar amount / lithium hydroxide molar amount was It is 0.5 / 0.5. The cellulose solution was held at −12 ° C. for 60 minutes, then heated to 40 ° C. and held for 30 minutes.

(Preparation of porous cellulose beads)
HYCOAL K-230 (liquid paraffin, manufactured by Kaneda Co., Ltd.) 234.5 g, HYCOAL K-290 (liquid paraffin, manufactured by Kaneda Co., Ltd.) 404.5 g, PR-100 (manufactured by RIKEN Vitamin) 9.8 g, inner diameter 85 mm An emulsion was prepared by placing the mixture in a cylindrical container, stirring at 300 rpm to 40 ° C., adding 161 g of the above cellulose solution, and stirring. Stirring uses a three-stage stirring blade, and from the top, a slanted paddle, a flat turbine, and a disc turbine. The mixture was stirred for 20 minutes, and 150 ml of a coagulation solvent having a methanol / pure water volume ratio of 70/30 was added to obtain cellulose beads. The obtained cellulose beads were washed with isopropanol and then with water.

(Classification of cellulose beads)
The remaining cellulose beads obtained were sieved using a mesh with a comb opening of 38 μm and a comb opening of 90 μm, and beads in the range of 38 μm to 90 μm were collected.

(Crosslinking of cellulose beads)
After replacing 100 mL of the liquid portion contained in the classified porous cellulose beads with ethanol, the mixture was transferred to a reaction vessel so that the total amount of cellulose particles and ethanol was 125 g, and 80 mL of epichlorohydrin was added thereto. The solution temperature was adjusted to 40 ° C., 96 mL of a 1.8 N NaOH aqueous solution (prepared with sodium hydroxide and distilled water manufactured by Nacalai Tesque) was added, and the crosslinking reaction was started. 9.6 mL of 17.0 N NaOH aqueous solution was added 1.5 hours after the start of the reaction, and 9.6 mL of 17.0 N NaOH aqueous solution was added 3 hours and 4.5 hours after the start of the reaction. Six hours after the start of the reaction, the gel was collected and washed with distilled water having a volume of 20 times or more the volume of the beads.

The crosslinked cellulose beads obtained by the above crosslinking reaction were transferred to a reaction vessel so that the total amount of the cellulose beads and distilled water was 116.7 g. After adding 37.8 g of sodium sulfate and dissolving it, 33 mL of epichlorohydrin was added and the temperature was maintained at 40 ° C. 21 mL of 17.0 N NaOH aqueous solution was added to initiate the cross-linking reaction, and 5 mL of 17.0 N NaOH aqueous solution was added 2.5 hours after the start of the reaction. After 5 hours from the start of the reaction, the particles were collected and washed with distilled water having a volume of 20 times or more the volume of the particles.

(Classification of porous crosslinked cellulose beads)
The remaining cellulose beads obtained were sieved using a mesh with a mesh opening of 38 μm and a mesh with a comb opening of 75 μm, and beads in the range of 38 μm to 75 μm were collected. The volume median diameter of the obtained crosslinked cellulose beads was 61 μm, the average pore radius was 45.0 nm, and the porosity in the particles was 98%.

[Example 2]
Crosslinked cellulose beads were obtained in the same manner as in Example 1 except that the heating temperature at the time of preparing the cellulose solution and the temperature for producing the porous cellulose beads were set to 25 ° C. The volume median diameter of the obtained crosslinked cellulose beads was 60 μm, the average pore radius was 38.4 nm, and the porosity in the particles was 97%.

[Example 3]
Crosslinked cellulose beads were obtained in the same manner as in Example 1 except that the urea concentration at the time of preparing the cellulose solution was 11.5% by weight. The volume median diameter of the obtained crosslinked cellulose beads was 60 μm, the average pore radius was 41.9 nm, and the porosity in the particles was 98%.

[Example 4]
45.3 g of a 48.0 wt% sodium hydroxide aqueous solution, 7.7 g of lithium hydroxide monohydrate, 57.5 g of urea, and 200 g of pure water were mixed and dissolved and kept at 10 ° C. to prepare an alkaline aqueous solution. 17 g of powdered cellulose was dispersed in 80 g of water, added to the alkaline aqueous solution, and then the temperature was lowered to -12 ° C. to dissolve the cellulose. The sodium hydroxide concentration of the solvent excluding the cellulose weight was 5.6% by weight, the lithium hydroxide concentration was 1.1% by weight, the urea concentration was 15% by weight, and the sodium hydroxide molar amount / lithium hydroxide molar amount was It is 0.25 / 0.75. The cellulose solution was held at −12 ° C. for 60 minutes, then heated to 40 ° C. and held for 30 minutes. Crosslinked cellulose beads were obtained in the same manner as in Example 1 except for the above. The volume median diameter of the obtained crosslinked cellulose beads was 60 μm, the average pore radius was 39.9 nm, and the porosity in the particles was 98%.

[Example 5]
Crosslinked cellulose beads were obtained in the same manner as in Example 4 except that 150 ml of a coagulation solvent having a methanol / pure water volume ratio of 45/55 was added at the time of producing the porous cellulose beads. The volume median diameter of the obtained crosslinked cellulose beads was 62 μm, the average pore radius was 40.7 nm, and the porosity in the particles was 94%.

[Example 6]
Crosslinked cellulose beads were obtained in the same manner as in Example 1 except that the amount of cellulose was 14 g at the time of producing the porous cellulose beads. The volume median diameter of the obtained crosslinked cellulose beads was 58 μm, the average pore radius was 44.9 nm, and the porosity in the particles was 97%.

[Comparative Example 1]
52.5 g of a 48.0 wt% sodium hydroxide aqueous solution and 46.0 g of urea were mixed and dissolved and kept at 10 ° C. to prepare an alkaline aqueous solution. 17 g of powdered cellulose was dispersed in 283.5 g of water, added to the alkaline aqueous solution, and then the temperature was lowered to -12 ° C. to dissolve the cellulose. The sodium hydroxide concentration of the solvent excluding the cellulose weight is 6.7% by weight, and the urea concentration is 12% by weight. The cellulose solution was held at −12 ° C. for 60 minutes, then heated to 40 ° C. and held for 30 minutes. Crosslinked cellulose beads were obtained in the same manner as in Example 1 except for the above. The volume median diameter of the obtained crosslinked cellulose beads was 60 μm, the average pore radius was 51.2 nm, and the porosity in the particles was 90%.

[Comparative Example 2]
31.0 g of lithium hydroxide monohydrate, 57.5 g of urea, and 210 g of pure water were mixed and dissolved and kept at 10 ° C. to prepare an alkaline aqueous solution. 17 g of powdered cellulose was dispersed in 80 g of water, added to the alkaline aqueous solution, and then the temperature was lowered to -12 ° C. to dissolve the cellulose. The concentration of lithium hydroxide in the solvent excluding the weight of cellulose is 4.7% by weight, and the concentration of urea is 15.2% by weight. Crosslinked cellulose beads were obtained in the same manner as in Example 2 except for the above. The volume median diameter of the obtained crosslinked cellulose beads was 58 μm, the average pore radius was 28.0 nm, and the porosity in the particles was 95%.

[Comparative Example 3]
31.5 g of lithium hydroxide monohydrate, 46.0 g of urea, and 137.3 g of pure water were mixed and dissolved, and kept at 10 ° C. to prepare an alkaline aqueous solution. 17 g of powdered cellulose was dispersed in 170 g of water, added to the alkaline aqueous solution, and then the temperature was lowered to -12 ° C. to dissolve the cellulose. The concentration of lithium hydroxide in the solvent excluding the weight of cellulose is 4.7% by weight, and the concentration of urea is 12.0% by weight. The cellulose solution was held at −12 ° C. for 60 minutes, then heated to 40 ° C. and held for 30 minutes. Crosslinked cellulose beads were obtained in the same manner as in Example 1 except for the above. The volume median diameter of the obtained crosslinked cellulose beads was 60 μm, the average pore radius was 33.8 nm, and the porosity in the particles was 98%.

[Test example]
The porous crosslinked cellulose beads obtained in Examples 1 to 5 and Comparative Examples 1 and 2 were bound with a protein A ligand by the following method.

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

(Aldehyde reaction)
Water was added to 0.165 g of citric acid monohydrate and 0.0646 g of trisodium citrate dihydrate to make 100 mL, and a buffer having a pH of 3.4 was prepared.

The liquid portion was replaced with the above buffer by using 3 times or more the amount of the above buffer with respect to 3.5 mL of the cellulose beads after cross-linking, and the above buffer was further added to bring the total volume to 6.0 mL. 2.01 mL of 11.2 mg / mL sodium periodate aqueous solution was added, and the mixture was stirred at 25 ° C. for 35 minutes. Then, the mixture was filtered through a glass filter of # 3, and washed with distilled water until the electric conductivity of the filtrate became 1 μS / cm or less to obtain aldehyde group-containing beads. The electrical conductivity of the washing filtrate was measured with a conductivity meter (“ECTester10 Pure +” manufactured by EUTECH INSTRUMENTS). Under these reaction conditions, about 30 μmol of aldehyde group is introduced per 1 mL of crosslinked porous cellulose beads.

(Protein A immobilization reaction)
<Preparation of orientation-controlled alkali-resistant protein A>
With reference to WO2012 / 133349, the modified C domain 5 conjugate described in WO2012 / 133349 was prepared as an orientation controlled alkali resistant protein A. The orientation-controlled alkali-resistant protein A has the amino acid sequence shown in SEQ ID NO: 2. The entire contents of WO2012 / 133349 are incorporated herein by reference. Hereinafter, "protein A" is abbreviated as "PA".

<Iminization reaction-when the amount of PA charged is 20 mg / mL>
Water was added to 2.941 g of trisodium citrate dihydrate to make 100 mL, and a buffer having a pH of 8 was prepared. The liquid portion in the beads was replaced with the above buffer by passing 3 times the amount of the buffer with respect to the total amount (3.5 mL) of the aldehyde group-containing beads on the glass filter of # 3. The replaced aldehyde group-containing beads were placed in a reaction vessel, and PA-immobilized buffer was added so that the total volume was 7.5 mL. 20 mg (net amount) of protein A was added to 1 mL of beads. After adjusting the temperature to 6 ° C., the pH was adjusted to 12 with a 0.08 N aqueous sodium hydroxide solution, and the mixture was stirred overnight at 6 ° C.

<Neutralization and reduction reaction>
The iminization reaction solution after the overnight reaction was filtered, and the filtrate was measured by UV to determine the amount of PA immobilized. A 0.1 M aqueous solution of citric acid was added to the Na buffer of citric acid having a pH of 8.0 to prepare a buffer having a pH adjusted to 5.0, and the buffer was used in an amount three times as much as that of the filtered beads. The liquid portion in the beads was replaced. The mixture was transferred to a reactor while adjusting the total volume to 7.0 mL with a buffer of p5.0, and stirred at 6 ° C. for 4 hours while maintaining neutralization.

Then, after adding 1.93 mL of a 5.5% aqueous solution of dimethylamine borane, the reaction temperature was raised to 25 ° C., and the mixture was stirred at 25 ° C. overnight. The beads after the reaction were washed on a # 3 glass filter with 3 times the volume of water as the beads.

<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 glass filter of # 3, and the liquid portion in the beads was replaced with an acid buffer. The replaced PA-immobilized beads were transferred to a container, acid buffer was added to bring the total volume to 2 mL or more, and the mixture was stirred at 25 ° C. for 30 minutes to carry out acid washing.

Then, alkaline washing was carried out 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 carried out in the same manner except that a solution prepared by mixing 0.1 M citric acid and 0.1 M sodium citrate to adjust the pH to 5.9 was used instead of the above acid buffer. ..

The beads after neutral washing were washed with distilled water until the conductivity of the washing filtrate became 10 μs / cm or less to obtain an adsorbent on which protein A was immobilized.

Table 2 shows the RT 5/4% DBC of the obtained adsorbent.

〔result〕
From Table 2, it was shown that the examples had higher antibody adsorption performance as compared with the comparative examples. It was also shown that the molar amount of sodium hydroxide / molar amount of lithium hydroxide in the cellulose solution has a pore radius suitable for antibody adsorption only in a specific range.

From the above, it was found that according to this production method, porous crosslinked cellulose beads having a high antibody adsorption capacity can be produced.

Claims (5)

(A) A step of preparing a cellulose solution by dissolving cellulose in a mixed solution of sodium hydroxide, lithium hydroxide, urea and water at −5 ° C. or lower and −20 ° C. or higher.
(B) A step of heating the cellulose solution to 10 ° C. or higher and 40 ° C. or lower.
(C) A step of preparing an emulsion by dispersing the cellulose solution in a dispersion medium having a temperature of 10 ° C. or higher and 40 ° C. or lower.
(D) A step of bringing the emulsion into contact with a coagulation solvent to precipitate cellulose beads.
(E) A method for producing porous crosslinked cellulose beads, which comprises a step of crosslinking the precipitated cellulose beads.
A method for producing porous crosslinked cellulose beads, wherein the molar ratio of sodium hydroxide / lithium hydroxide in the step (a) is 0.3 / 0.7 to 0.8 / 0.2. The porous crosslinked cellulose beads according to claim 1, wherein the urea concentration of the mixed solution of sodium hydroxide, lithium hydroxide, urea and water in the step (a) is 10% by weight to 20% by weight. Production method. The method for producing porous crosslinked cellulose beads according to claim 1, wherein the cellulose concentration of the cellulose solution in the first half is 3% by weight or more and 9% by weight or less. The method for producing a porous crosslinked cellulose bead according to any one of claims 1 to 3, wherein the coagulation solvent is an alcohol solvent or a mixed solvent of water and an alcohol solvent. Porous crosslinked cellulose beads having a porosity in particles of 90% or more, an average pore radius of 35 nm to 50 nm, and a volume median diameter of 45 μm to 70 μm.