JP5332090B2 - Spherical sulfated cellulose and method for producing the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a spherical sulfated cellulose which has a high sulfur content and which is excellent in the adsorption of proteins, a production process for the same, a protein adsorbing agent containing the above spherical sulfated cellulose and a chromatography apparatus using the same for a filler. <P>SOLUTION: The process for producing spherical sulfated cellulose comprises a step of subjecting spherical cellulose to sulphate esterification using a mixture of N, N-dimethylformamide and sulphuric anhydride. <P>COPYRIGHT: (C)2007,JPO&amp;INPIT

Description

  The present invention relates to spherical sulfated cellulose, particularly to spherical sulfated cellulose useful as a substrate for affinity chromatography, a medical substrate, and the like, and a method for producing the same.

Spherical sulfated cellulose is used as a base material for separating and purifying biological substances such as proteins and viruses. Spherical sulfated cellulose is a gel-like product obtained by sulfate esterifying solid granular particles of crosslinked or non-crosslinked cellulose while maintaining the solid granular state, and then neutralizing with alkali. It is known to have a chemical affinity (see Japanese Patent Publication No. 4-23751 (Patent Document 1) and Japanese Patent Publication No. 4-23752 (Patent Document 2)).
However, the sulfur concentration of the spherical sulfated cellulose obtained by the conventional method is as low as about 0.15% (see Example 1 of Patent Document 1), and the protein adsorption capacity was not sufficient.
Japanese Patent Publication No.4-223751 Japanese Patent Publication No.4-223752

  In view of the above situation, development of spherical sulfated cellulose having a high sulfur concentration and excellent protein adsorption ability and a method for producing the same are desired.

  As a result of intensive studies to solve the above problems, the present inventors have found that the sulfur concentration introduced into the spherical cellulose can be increased by using a specific sulfate esterifying agent. It came to be completed.

  That is, the present invention provides a spherical sulfated cellulose as shown below, a production method thereof, a protein adsorbent containing the spherical sulfated cellulose, and the like.

(1) A method for producing spherical sulfated cellulose, comprising a step of subjecting spherical cellulose to a sulfate esterification treatment using a mixture of N, N-dimethylformamide and sulfuric anhydride.
(2) The production method according to (1) above, wherein the spherical cellulose is obtained using crystalline cellulose as a raw material.
(3) The production method according to (1) or (2) above, wherein the spherical cellulose is crosslinked.
(4) The production method according to any one of (1) to (3) above, wherein the sulfate esterification treatment is performed at 0 to 70 ° C.
(5) The production method according to any one of (1) to (4) above, which comprises a step of dehydrating spherical cellulose.
(6) The production method according to (5), wherein the dehydration treatment is performed by liquid replacement using a water-soluble solvent.
(7) The method according to (6) above, wherein the water-soluble solvent contains N, N-dimethylformamide.
(8) The production method according to any one of (1) to (7) above, comprising a step of neutralizing the spherical cellulose subjected to the sulfate esterification treatment with an alkali.

(9) Spherical sulfated cellulose having a sulfur concentration of 1 to 10% by weight that can be produced by the production method according to any one of (1) to (8) above.

(10) (a) a step of dehydrating spherical cellulose by liquid replacement using a water-soluble solvent containing N, N-dimethylformamide;
(B) a step of subjecting the dehydrated spherical cellulose to a sulfate esterification treatment using a mixture of N, N-dimethylformamide and sulfuric anhydride,
(C) a step of neutralizing the spherical cellulose treated with sulfate ester with an alkali;
A process for producing spherical sulfated cellulose, comprising:
(11) The production method according to (10) above, wherein the spherical cellulose is obtained using crystalline cellulose as a raw material.
(12) The production method according to (10) or (11) above, wherein the spherical cellulose is crosslinked.
(13) The production method according to any one of (10) to (12) above, wherein the sulfate esterification treatment is performed at 0 to 70 ° C.

(14) Spherical sulfated cellulose having a sulfur concentration of 1 to 10% by weight that can be produced by the production method according to any one of (10) to (13).

(15) A spherical sulfated cellulose having a sulfur concentration of 1 to 10% by weight and a sphericity of 0.9 to 1.0.
(16) The spherical sulfated cellulose as described in (15) above, wherein the sulfur concentration is 1 to 6% by weight.

(17) A protein adsorbent comprising the spherical sulfated cellulose according to any one of (9) and (14) to (16).

(18) A packing material for affinity chromatography using the protein adsorbent according to (17) above.

  According to the method of the present invention, spherical sulfated cellulose containing sulfur at a high concentration can be obtained. That is, according to the present invention, spherical sulfated cellulose excellent in protein adsorption ability can be provided. The spherical sulfated cellulose of the present invention is useful as a medical substrate, adsorbent, or chromatographic filler for separation and purification of various viruses and proteins, particularly an affinity chromatography filler.

  Hereinafter, the spherical sulfated cellulose of the present invention, the production method thereof, and the protein adsorbent containing the spherical sulfated cellulose will be described in detail.

A. Method for producing spherical sulfated cellulose First, the method for producing spherical sulfated cellulose of the present invention will be described.
The method for producing spherical sulfated cellulose of the present invention is characterized by including a step of subjecting spherical cellulose to a sulfate esterification treatment using a mixture of N, N-dimethylformamide and sulfuric anhydride.

In the method of the present invention, the spherical cellulose is subjected to sulfate esterification using a mixture of N, N-dimethylformamide and sulfuric anhydride as the sulfate esterifying agent for spherical cellulose. This mixture contains a complex or complex formed by N, N-dimethylformamide and sulfuric anhydride (see the following formula), which effectively acts on the sulfate esterification of spherical cellulose. Conceivable.

  The spherical cellulose used in the present invention is not particularly limited, and ordinary crosslinked or non-crosslinked spherical cellulose can be used. Spherical cellulose is a solid granular particle having a spherical shape and is usually in a gel form, but the spherical cellulose used in the present invention preferably has a sphericity of 0.8 to 1.0. Here, “sphericity” means the short diameter / long diameter of the spherical cellulose.

  Such spherical cellulose can be obtained, for example, by dissolving and regenerating crystalline cellulose or cellulose composed of a crystalline region and an amorphous region. Specifically, as a method for producing spherical cellulose, for example, a method via an acetate described in JP-B-55-39565, JP-B-55-40618 and the like, and JP-B-63-62252 are described. And a method of granulating from a solution using calcium thiocyanate, and a method of producing from a paraformaldehyde / dimethyl sulfoxide solution described in JP-A-59-38203. In addition, a method described in Japanese Patent No. 3663666, in which cellulose is molded from a cellulose solution in which amide containing lithium chloride is dissolved, may be used.

  As the spherical cellulose used in the present invention, it is preferable to use spherical cellulose obtained from crystalline cellulose as a raw material because sulfur can be contained at a higher concentration. This is because spherical cellulose derived from crystalline cellulose has high strength and is hardly soluble in water, so that spherical sulfated cellulose does not become too soft even when sulfur is contained at a high concentration. The spherical cellulose used in the present invention is preferably a crosslinked spherical cellulose because of such high physical stability. In particular, a crosslinked spherical cellulose obtained by crosslinking spherical cellulose derived from crystalline cellulose is more preferable because it is excellent in physical stability. As the esterification progresses, the hydrophilicity of the spherical cellulose increases, and it may be difficult to keep the spherical shape. By using the spherical cellulose with high physical stability, it produces a high-strength spherical sulfated cellulose that is difficult to dissolve in water. can do.

  Cross-linked spherical cellulose can be produced by cross-linking non-cross-linked spherical cellulose. The crosslinking method is not particularly limited as long as it is a method usually used for crosslinking cellulose. As a crosslinking agent, polyfunctional epoxy compounds, such as epichlorohydrin, can be illustrated, for example.

  A commercial item can also be used as spherical cellulose used for this invention. For example, as non-cross-linked spherical cellulose, Cellufine GC-15, GH-25, GC-100, GC-200 (product name: Chisso Corporation), Avicel (product name: Asahi Kasei Kogyo Co., Ltd.) and the like can be mentioned. It is done. Examples of the crosslinked spherical cellulose include Cellufine GCL-25, GCL-90, GCL-2000 (product name: Chisso Corporation) and the like. In addition, Viscopar (product name: Rengo Co., Ltd.), Perloza MT series (product name: Iontsorb), “Cellulose, Beaded” (catalog code C8204, Sigma) commercially available as regenerated cellulose particles from viscose Etc. can also be used.

The average particle diameter of the spherical cellulose used in the present invention is preferably 1 μm to 1,000 μm, more preferably 100 μm to 500 μm, and further preferably 250 μm to 300 μm. The average particle size can be appropriately selected according to the intended use. For example, when the spherical sulfated cellulose of the present invention obtained from spherical cellulose as a raw material is used as a filler for chromatography, if the average particle size is too large, the porosity becomes high and it becomes difficult to obtain a certain resolution. On the contrary, if it is too small, there is a risk of pressure being applied and deformation. Therefore, the average particle size of the spherical cellulose is preferably 50 μm to 250 μm, and more preferably 50 μm to 125 μm.
On the other hand, when the spherical sulfated cellulose of the present invention is used for medical purposes, if the average particle size is too small, blood platelets and blood cell components may clog the voids between the spherical cellulose. The average particle size of cellulose is preferably 50 μm to 1000 μm, and more preferably 300 μm to 700 μm.

  In the present invention, the average particle diameter of the spherical cellulose can be calculated from the particle diameter (arithmetic diameter) measured using the electric resistance method. The electrical resistance method is a method that utilizes a change in electrical resistance between two electrodes that occurs when particles pass through a sensitive region. Since the electrical resistance is proportional to the volume of the particles, the particle diameter of the spherical cellulose can be measured by measuring the change in the electrical resistance and converting this to the particle diameter. All data of the “arithmetic diameter” values measured in this way are averaged to obtain an average particle diameter. As the measuring device, a precision particle size distribution measuring device (product name “Multisizer 3”) manufactured by Beckman Coulter, Inc. can be used.

  The spherical cellulose used in the present invention preferably has an exclusion limit molecular weight of 2,000 to 3,000,000. The exclusion limit molecular weight of the spherical cellulose greatly varies depending on the protein or virus size to be adsorbed, and may be determined as appropriate. Here, the exclusion limit molecular weight can be obtained from a plot of the volume (or elution time) and molecular weight of the elution peak of each substance after the column is filled with spherical cellulose and a substance having a known molecular weight is allowed to flow. The exclusion limit molecular weight is a molecular weight that cannot enter the pores in the spherical cellulose. The elution volume (or elution time) of a substance having a molecular weight higher than the exclusion limit molecular weight is measured as the same value. Examples of substances that have a known molecular weight that can be used for measuring the exclusion limit molecular weight include synthetic polymers such as proteins, saccharides, polyethylene glycol, and polyethylene oxide.

(Dehydration treatment)
Spherical cellulose usually contains about 90% of its own weight even after centrifugal dehydration. When the spherical cellulose is subjected to sulfate esterification in the presence of a large amount of water in this way, the sulfate esterification agent may be deactivated by the moisture and the sulfate esterification may not proceed efficiently. For this reason, in the present invention, it is preferable to dehydrate the spherical cellulose before the sulfate esterification treatment to remove water as much as possible.

  A dehydration process will not be specifically limited if the water | moisture content in a spherical cellulose can fully be removed, without impairing the shape of a spherical cellulose. Examples of the dehydration method include heat drying and liquid replacement using a water-soluble solvent. Heat drying can also be performed under vacuum conditions.

  Heat drying is a simple method for removing moisture, but may involve shrinkage or destruction of spherical cellulose. For this reason, in the method of the present invention, it is preferable to use liquid replacement that is unlikely to cause such shrinkage or destruction. According to liquid replacement, moisture can be removed while maintaining the shape of the spherical cellulose. In particular, since spherical cellulose derived from crystalline cellulose has a low density and tends to shrink or break due to heat drying, it is preferable to use liquid replacement.

  The liquid replacement can be performed by sufficiently stirring the spherical cellulose together with the water-soluble solvent in a container, allowing to stand, and decanting the supernatant. By repeating this operation, water in the spherical cellulose can be sufficiently removed. This operation is repeated until the water content in the supernatant is preferably 2% by weight or less, more preferably 1% by weight or less, and particularly preferably 0.3% by weight or less. The sulfuric esterification treatment can be performed more efficiently.

The water-soluble solvent used in the present invention has an affinity for water, and
There is no particular limitation as long as it does not hinder the progress of the sulfate esterification treatment. As such a water-soluble solvent, an aprotic organic solvent is preferable. Examples of the aprotic organic solvent include acetonitrile, dimethyl sulfoxide, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, tetrahydrofuran, dioxane, pyridine, triethylamine, piperidine and the like.

  Among these, the water-soluble solvent preferably includes N, N-dimethylformamide, which is also used as a sulfate esterifying agent, from the viewpoint that the sulfate esterification treatment can be performed more efficiently. It is preferable to use a mixture of N, N-dimethylformamide and other water-soluble solvent alone. In particular, it is preferable to use N, N-dimethylformamide alone.

(Sulfate esterification treatment)
Next, a sulfuric esterification treatment is performed.
First, a mixture of N, N-dimethylformamide and sulfuric anhydride is prepared. The mixing ratio of N, N-dimethylformamide and sulfuric anhydride is not particularly limited as long as N, N-dimethylformamide is in an excess amount relative to sulfuric anhydride. The concentration of sulfuric anhydride in the mixture is preferably 10 to 30% by weight. If the concentration of anhydrous sulfuric acid is within this range, the sulfuric esterification reaction proceeds sufficiently and the control of the sulfur concentration is easy. The concentration of sulfuric anhydride in the mixture is particularly preferably 18% by weight. The concentration of sulfuric anhydride can be measured, for example, by adding water to the above mixture to convert sulfuric anhydride to sulfuric acid and then neutralizing with caustic soda. Since sulfuric anhydride and water react 1: 1, the sulfuric acid concentration (molar concentration) measured by neutralization titration means the sulfuric acid anhydrous concentration (molar concentration) as it is.

  A mixture of N, N-dimethylformamide and sulfuric anhydride can be prepared by adding sulfuric anhydride dropwise to N, N-dimethylformamide and mixing them. In addition, since mixing may be accompanied by heat generation, the mixing is preferably performed using an ice bath or the like at a temperature condition of 5 ° C. or lower.

  Next, a mixture of N, N-dimethylformamide and sulfuric anhydride is added dropwise to the dispersion of spherical cellulose and N, N-dimethylformamide. In the present invention, in order to carry out the reaction uniformly, the dispersion of spherical cellulose and N, N-dimethylformamide is kept at a low temperature of 5 ° C. or lower using, for example, an ice bath or the like, and N, N-dimethylformamide is used. It is preferable to add a mixture of sulfuric acid and sulfuric acid dropwise.

  The sulfuric esterification treatment of spherical cellulose is, for example, after gradually dropping a mixture of N, N-dimethylformamide and sulfuric anhydride into a dispersion of spherical cellulose and N, N-dimethylformamide prepared in a reaction vessel, Perform by thorough stirring. This sulfate esterification treatment is preferably performed at 0 to 70 ° C, more preferably 0 to 50 ° C, further preferably 0 to 35 ° C, and particularly preferably 0 to 30 ° C. When there is a concern about coloring the spherical sulfated cellulose with an amine solvent, the sulfate esterification reaction is preferably performed at 0 to 30 ° C. The stirring time is not particularly limited as long as the sulfate esterification reaction can sufficiently proceed, but is usually 1 to 10 hours, preferably 2 to 6 hours, and more preferably 2 to 4 hours.

  The method of mixing the mixture of the spherical cellulose, N, N-dimethylformamide and sulfuric anhydride is an example, and the method is not limited to the above method as long as the sulfuric esterification reaction can be performed uniformly. Further, the amount of the mixture of N, N-dimethylformamide and sulfuric anhydride used may be in a range necessary and sufficient for sulfating spherical cellulose, and those skilled in the art can appropriately determine the range. it can. For example, using the value obtained by dividing the mass of spherical cellulose by the molecular weight corresponding to glucose (unit structural substance) as the number of moles of spherical cellulose, adjusting the amount of the mixture used so as to contain 1 to 5 times moles of sulfuric anhydride. The amount of the mixture used is adjusted so as to contain 1-3 moles, more preferably 1-1.5 moles of sulfuric anhydride.

(Neutralization treatment)
The spherical cellulose subjected to the sulfate esterification treatment is preferably recovered by filtration separation and the like, washed with an alcohol solvent such as methanol and ethanol, and then neutralized with an alkali.

The neutralization treatment can be performed by a method usually used when producing spherical sulfated cellulose. For example, it can be carried out using an aqueous solution or alcohol solution in which an alkali such as sodium hydroxide, potassium hydroxide, magnesium hydroxide is dissolved.
After the neutralization treatment, the product can be washed with ion exchange water or the like to obtain the target spherical sulfated cellulose.

  According to the method of the present invention, spherical sulfated cellulose having a high sulfur concentration and excellent adsorption ability can be obtained by a simple method.

According to a preferred embodiment of the present invention, the method of the present invention comprises:
(A) dehydrating spherical cellulose by liquid replacement using a water-soluble solvent containing N, N-dimethylformamide;
(B) a step of subjecting the dehydrated spherical cellulose to a sulfate esterification treatment using a mixture of N, N-dimethylformamide and sulfuric anhydride,
(C) a step of neutralizing the spherical cellulose treated with sulfate ester with an alkali;
including. Note that details of each step are as described above, and thus description thereof will not be repeated here.

B. Spherical sulfated cellulose The spherical sulfated cellulose that can be produced in this manner can contain sulfur at a high concentration. The spherical sulfated cellulose of the present invention preferably has a sulfur concentration of 1 to 10% by weight, more preferably 1 to 6% by weight, and still more preferably 1 to 4% by weight. Here, the sulfur concentration of the spherical sulfated cellulose refers to the sulfur content per dry weight of the spherical sulfated cellulose, and can be measured by an inductively coupled plasma method. Specifically, an inductively coupled plasma optical emission spectrometer (model number IRIS-AP) manufactured by Nippon Jarrel Ash Co., Ltd. can be used, and the measurement wavelength of sulfur can be measured at 182.034 nm. Since spherical sulfated cellulose is usually in the form of a gel, a sample obtained by decomposing spherical sulfated cellulose in advance is used as a sample.
The spherical sulfated cellulose of the present invention is a solid granular particle capable of containing sulfur at such a high concentration while maintaining a spherical shape, and its sphericity is preferably 0.9 to 1.0. . The sphericity can be determined by microscopic observation.
The exclusion limit molecular weight and particle diameter of the spherical sulfated cellulose of the present invention are not particularly limited. What is necessary is just to determine suitably according to adsorption object, a use, etc.

C. Protein adsorbent and use thereof The protein adsorbent of the present invention is characterized by containing the above-described spherical sulfated cellulose. Since the protein adsorbing ability is enhanced by including the spherical sulfated cellulose described above, the protein adsorbent of the present invention is suitably used for separation and purification of various proteins and viruses.

  The protein adsorbent of the present invention is useful as a packing material for chromatography such as affinity chromatography, ion exchange chromatography, gel filtration chromatography and the like. In particular, the spherical sulfated cellulose of the present invention has a blood coagulation inhibiting action, a fat serum clearing action and the like similar to heparin. In addition, because it has a heparin-like group-specific adsorption ability, it can be used as a packing material for affinity chromatography to search for proteins for ligands and ligands for proteins, and to separate these ligands, proteins, and antibodies. Can be purified.

  In addition, the protein adsorbent of the present invention is a substrate for a blood purification system that removes viruses, low-density lipoprotein (LDL), immune complexes, bilirubin, endotoxin and the like that are considered to be pathogenic substances contained in blood or plasma. Can also be used. In addition, when removing a pathogenic substance etc. using the protein adsorbent of this invention, the removal method changes with properties of the pathogenic substance to remove. For example, since endotoxin does not adsorb to the protein adsorbent of the present invention, endotoxin can be removed from the target substance by adsorbing the target substance to the protein adsorbent and thoroughly washing the column.

  Examples of substances that can be separated and purified by the protein adsorbent of the present invention include heparin degrading enzymes (heparinase, heparitinase), glycosaminoglycan degrading enzymes, DNA-binding proteins (HMG: high mobility group protein, chromatin, histone). , Bacteriophage, viral vector, Adeno-associated Virus (AAV), Sendai virus, Follistatin, Activin, basic fibroblast growth factor, blood coagulation factor, lipase, nucleic acid Examples include degrading enzymes.

  EXAMPLES Hereinafter, although an Example and a comparative example demonstrate this invention, this invention is not limited to these Examples.

[Example 1]
As the starting spherical cellulose, Cellufine GH-25 (product name: Chisso Corporation) was used. The spherical cellulose had a particle size of 44 to 105 μm. The spherical cellulose had an average particle size of 67.05 μm and an exclusion limit molecular weight of 2500.
In order to remove water in the spherical cellulose, 10 g of water content was weighed in a 50 ml beaker, and 20 ml of N, N-dimethylformamide was added and stirred for 30 minutes. The mixture was allowed to stand after stirring, and the water content of the supernatant was measured by the Karl Fischer method. This operation was repeated until the water content in the supernatant liquid was in the range of 1 to 2% by weight. Since the water content in the supernatant finally became 1.05% by weight, the following sulfate esterification treatment was performed using the dehydrated spherical cellulose.
First, dehydrated spherical cellulose was dispersed in N, N-dimethylformamide maintained at a temperature of 5 ° C. or lower in an ice bath.
To this dispersion, 10.97 g of an 18 wt% anhydrous sulfuric acid-dimethylformamide solution cooled to 5 ° C. was gradually added dropwise and reacted for 4 hours while maintaining the reaction temperature at 30 ± 2 ° C. After completion of the reaction, the reaction completion solution was separated by filtration, and the filtrate was washed with methanol. The 18 wt% anhydrous sulfuric acid-dimethylformamide solution used was prepared by adding 278 g of anhydrous sulfuric acid dropwise with stirring to 2500 g of N, N-dimethylformamide cooled to 5 ° C. or less in an ice bath (hereinafter referred to as “below”). In the same way). At this time, the dropping speed was adjusted so that the temperature in the liquid did not exceed 5 ° C.
Subsequently, the filtrate was charged into ion-exchanged water cooled to 10 ° C. or less and neutralized with 1M NaOH. Then, after sufficiently washing with ion exchange water, spherical sulfated cellulose was obtained.

The sulfur concentration of the obtained spherical sulfated cellulose was measured by an inductively coupled plasma method. For the measurement, spherical sulfated cellulose pretreated by the following method was used.
First, 20 mg of spherical sulfated cellulose was weighed in a 100 ml beaker, 10 ml of nitric acid was added, and it was decomposed on a hot plate (250 ° C.) until the brown color became light. At this time, attention was paid not to dry. After allowing to cool, 5 ml of nitric acid and 2 ml of perchloric acid were further added and decomposed on a hot plate until white smoke appeared. Subsequently, after allowing to cool, 10 ml of a 1: 1 hydrochloric acid aqueous solution (volume ratio) was added and dissolved, and the volume was adjusted to 100 ml in a volumetric flask to obtain a measurement sample.

  The measurement was performed using an inductively coupled plasma optical emission spectrometer (model number IRIS-AP) manufactured by Nippon Jarrell-Ash, and the measurement wavelength of sulfur was 182.034 nm. As a result, the sulfur concentration of the spherical sulfated cellulose was 1.31% by weight.

[Example 2]
The same Cellufine GH-25 used in Example 1 was used as the starting material.
In order to remove the water in the spherical cellulose, 52.7 g of water content was weighed in a 200 ml beaker, and 100 ml of N, N-dimethylformamide was added and stirred for 30 minutes. The mixture was allowed to stand after stirring, and the dehydration treatment was repeated until the water content in the supernatant liquid was in the range of 1 to 2% by weight in the same manner as in Example 1. Finally, the water content in the supernatant liquid was 1.60% by weight, and the desulfurized spherical cellulose was used for the subsequent sulfate esterification treatment.
Next, dehydrated spherical cellulose was dispersed in N, N-dimethylformamide at a temperature of 5 ° C. or lower. To this dispersion, 54.99 g of an 18 wt% anhydrous sulfuric acid-dimethylformamide solution cooled to 5 ° C. was gradually added dropwise. The reaction was carried out for 4 hours while maintaining the reaction temperature at 30 ± 2 ° C. After completion of the reaction, the reaction solution was separated by filtration, and the filtrate was washed with methanol.
Subsequently, the filtrate was charged into ion-exchanged water cooled to 10 ° C. or less and neutralized with 1M NaOH. Then, after sufficiently washing with ion exchange water, spherical sulfated cellulose was obtained.
As a result of measuring the sulfur concentration of the spherical sulfated cellulose in the same manner as in Example 1, the sulfur concentration of the spherical sulfated cellulose was 1.81% by weight.

[Example 3]
Cellufine (product name, Chisso Corporation) was used as a starting material. The manufacturing process of Cellufine is as follows.
(I) 0.46 kg of the above crystalline cellulose is added to an aqueous solution containing 60% by weight of calcium thiocyanate (as an anhydride), and heated to 110 ° C. to dissolve. (Ii) A surfactant is added to this solution, and the solution is dropped into 30 l of o-dichlorobenzene heated in advance at 130 to 140 ° C. and dispersed by stirring. (Iii) Next, the dispersion is cooled to 40 ° C. or lower, and 13 l of methanol is poured to obtain particles. (Iv) The suspension is separated by filtration, and the particles are washed with 13 l of methanol and separated by filtration. This washing operation is performed several times. (V) The desired spherical cellulose particles are obtained after further washing with a large amount of water.
The particle size of the spherical cellulose was 44 to 300 μm. The average particle size was 250 μm and the exclusion limit molecular weight was 3,000,000.

In order to remove water in the spherical cellulose, 50 g of water content was weighed in a 200 ml beaker, and 100 ml of N, N-dimethylformamide was added and stirred for 30 minutes. After stirring, the mixture was allowed to stand, and the dehydration treatment was repeated in the same manner as in Example 1 until the water content in the supernatant liquid reached a level of 0.2% by weight. Since the water content in the supernatant finally became 0.23% by weight, the desulfurized spherical cellulose was used for the subsequent sulfate esterification treatment.
First, dehydrated spherical cellulose was dispersed in N, N-dimethylformamide maintained at a temperature of 5 ° C. or lower in an ice bath. To this, 15.10 g of an 18 wt% anhydrous sulfuric acid-dimethylformamide solution cooled to 5 ° C. was gradually added dropwise. The mixture was stirred for 4 hours while maintaining the reaction temperature at 30 ± 2 ° C. After completion of the reaction, the reaction solution was separated by filtration, and the filtrate was washed with methanol.
Next, the filtrate was neutralized with 1M NaOH by ion-exchanged water cooling to 10 ° C. or lower. Thereafter, after washing with ion exchange water, spherical sulfated cellulose was obtained.
As a result of measuring the sulfur concentration of the spherical sulfated cellulose in the same manner as in Example 1, the sulfur concentration of the spherical sulfated cellulose was 2.80% by weight.

[Example 4]
Removal of water in the spherical cellulose was carried out in the same manner as in Example 3 except that 2000 g of water content was weighed in a 5 l beaker and 4 l of N, N-dimethylformamide was used. % Spherical cellulose was obtained.
Using all of this, 375.0 g of 18 wt% anhydrous sulfuric acid-dimethylformamide solution was subjected to sulfate esterification treatment and neutralization treatment in the same manner as in Example 3 to obtain spherical sulfated cellulose.
In the same manner as in Example 1, the sulfur concentration of the spherical sulfated cellulose was measured. As a result, the sulfur concentration of the spherical sulfated cellulose was 2.20% by weight.

[Example 5]
The same spherical cellulose used as the starting material in Example 3 was used and crosslinked by the following method.
In a 2 l separable flask, 176 g of spherical cellulose was weighed, heptane and a cationic surfactant (Japan Benzalkonium Chloride) were added, and the mixture was stirred for about 30 minutes. Thereafter, the temperature was raised to 30 ° C., 0.18 g of sodium borohydride, 420 ml of an aqueous sodium hydroxide solution adjusted to 5% by weight were added, and the mixture was stirred for 2 hours.
Next, the temperature was raised to 40 ° C., 60.0 g of epichlorohydrin was added, and this was further raised to 50 ± 1 ° C. and stirred for 4 hours. Thereafter, the mixture was cooled to about 35 ° C., neutralized with acetic acid, and methanol was added. After the reaction solution was separated by filtration, the filtrate was washed with methanol and water in this order to obtain crosslinked spherical cellulose.
The water content of the crosslinked spherical cellulose was removed in the same manner as in Example 3 except that 150 g of water content was weighed in a 500 ml beaker and 300 ml of N, N-dimethylformamide was used, and the water content in the supernatant liquid was 0.23. Dehydration treatment was performed until the weight was reached.
Sulfate esterification treatment and neutralization treatment were carried out in the same manner as in Example 3 except that the dehydrated crosslinked spherical cellulose was used in its entirety and 47.0 g of 18 wt% anhydrous sulfuric acid-dimethylformamide solution was used as the sulfate esterification agent. Spherical sulfated cellulose was obtained.
In the same manner as in Example 1, the sulfur concentration of the spherical sulfated cellulose was measured. As a result, the sulfur concentration of the spherical sulfated cellulose was 4.70% by weight.

  In addition, about each 1000 pieces of spherical sulfated cellulose obtained in Examples 1-5, it observes under a microscope and measures the long diameter (R1) and short diameter (R2) of each particle | grain, and sphericity (R2 / R1) ), The sphericity of each particle was 0.9 or more, and it was confirmed that the spherical shape was maintained.

  Next, in Examples 6 to 10, the lysozyme adsorption amount of the spherical sulfated cellulose obtained in Examples 1 to 5 was measured.

[Example 6]
First, the lysozyme adsorption amount of the spherical sulfated cellulose obtained in Example 1 was measured by the following method.
About 3 g of spherical sulfated cellulose stored in methanol, which was filtered under reduced pressure, was placed in a beaker, 100 ml of ion-exchanged water was added, and the mixture was stirred with a magnetic stirrer for 10 minutes, followed by filtration. The filtered sulfated spherical sulfated cellulose was returned to the beaker again, and the process of adding 100 ml of ion-exchanged water, stirring, and filtering and separating was repeated three more times.
The spherical sulfated cellulose was put in a beaker, 100 ml of adsorption buffer was added, and the mixture was stirred for 1 hour. As the adsorption buffer, 0.01 M phosphate (Na-Na) buffer solution (pH 7.0) containing 0.15 M sodium chloride was used.
Thereafter, 2 ml of spherical sulfated cellulose suspended in the adsorption buffer was packed in the column, and equilibrated by flowing 40 ml of the adsorption buffer at a flow rate of 40 ml / hour. Subsequently, 30 ml of a lysozyme solution (Wako Pure Chemical Industries, Ltd., product name “lysozyme”) 333 mg was added with adsorption buffer to a constant volume of 100 ml, and circulated for 1 hour at a flow rate of 60 ml / hour.

Subsequently, 20 ml of adsorption buffer was flowed at a flow rate of 50 ml / hour to remove unadsorbed lysozyme. Subsequently, 50 ml of elution buffer was flowed at a flow rate of 50 ml / hour, and 45 ml was collected. 5 ml of elution buffer was added to this collected solution to make 50 ml, and this solution was used as an eluate. In addition, 0.01M phosphoric acid (Na-Na) buffer solution (pH 7.0) containing 0.6M sodium chloride was used as the elution buffer.
Also, 1 ml of lysozyme solution was taken, and elution buffer was added thereto to prepare a 10 ml solution. This solution was used as a standard solution.

Using the elution buffer as a control, the absorbance of the eluate and standard solution was measured. The measurement was performed using a quartz cell at a wavelength of 280 nm.
The measurement value was substituted into the following calculation formula to calculate the lysozyme adsorption amount. As a result, the adsorption amount was 18 mg / ml.
Adsorption amount (mg / ml) = 0.333 × 25 × (absorbance of eluate / absorbance of standard solution)

[Example 7]
In the same manner as in Example 6, the amount of lysozyme adsorbed on the spherical sulfated cellulose obtained in Example 2 was measured. As a result, the amount adsorbed was 24 mg / ml.

[Example 8]
After removing unadsorbed lysozyme, 145 ml was collected by flowing 150 ml of elution buffer, and the sphere obtained in Example 3 was prepared in the same manner as in Example 6 except that 5 ml of elution Buffer was added to the collected liquid to prepare 150 ml. The lysozyme adsorption amount of sulfated cellulose was measured. As a result, the adsorption amount was 39.06 mg / ml.

[Example 9]
In the same manner as in Example 8, the lysozyme adsorption amount of the spherical sulfated cellulose obtained in Example 4 was measured. As a result, the adsorption amount was 26.41 mg / ml.

[Example 10]
In the same manner as in Example 8, the lysozyme adsorption amount of the spherical sulfated cellulose obtained in Example 5 was measured. As a result, the adsorption amount was 40.33 mg / ml.

[Comparative Example 1]
Lysozyme adsorption amount was measured in the same manner as in Example 6 using sulfated cellufine (product name: Chisso Corporation) as a sample. As a result, the adsorption amount was 5 mg / ml. In addition, when the sulfur concentration of sulfated cellufine was measured in the same manner as in Example 1, the sulfur concentration was 0.16% by weight.

  The relationship between the sulfur concentration of the spherical sulfated cellulose obtained in Examples 6 to 10 and Comparative Example 1 and the lysozyme adsorption amount is shown in FIG. In FIG. 1, the results of Examples 6 to 10 are indicated by ▪, and the result of Comparative Example 1 is indicated by Δ. It was found that the spherical sulfated cellulose produced by the method of the present invention has a lysozyme adsorption capacity that is 3 times or more that of the conventional product.

Next, in Example 11, the adsorption amount of blood coagulation factor VIII activity of the spherical sulfated cellulose of Example 3 and sulfated Cellufine (product name: Chisso Corporation) was compared.
In Example 12, the amount of adsorbed blood coagulation factor XII of the spherical sulfated cellulose of Example 3 and Heparin Sepharose 6 Fast Flow (product name: Amersham Biosciences) was compared.

[Example 11]
Two Spitz tubes were prepared, and 6 ml of human plasma (anticoagulant: sodium citrate, Cosmo Bio Inc.) was weighed into each Spitz tube. Next, 2 ml of spherical sulfated cellulose of Example 3 washed with physiological saline was added to one Spitz tube, and 2 ml of sulfated cellufine (product name, Chisso) was added to the other Spitz tube, Each was stirred for 2 hours at 25 ° C. Thereafter, each supernatant was sampled by centrifugation, and the adsorption amount of blood coagulation factor VIII activity was measured. Plasma with no gel added was used as a blank.
The measurement of blood coagulation factor VIII activity was performed by Mitsubishi Chemical BSC, Inc., and the APTT method (Activated Prothrombin Time method).
The measured value was expressed as a relative value with standard human plasma as 100%. The amount of adsorption was calculated by the following formula. The results are shown in Table 1.
Adsorption amount (% / ml-gel) = ((blank measurement value × volume) − (sample measurement value × volume)) / 2

  The spherical sulfated cellulose of Example 3 was found to have an adsorption capacity about 5.5 times that of commercially available sulfated cellulose.

[Example 12]
For comparison, the same operation as in Example 11 was performed except that Heparin Sepharose 6 Fast Flow (product name: Amersham Biosciences) was used instead of sulfated cellufine (product name: Chisso). The adsorption amount of blood coagulation factor XII was compared.
The measurement of blood coagulation factor XII activity was performed by Mitsubishi Chemical BLC, Inc., and the APTT method (Activated Prothrombin Time method).
The measured value was expressed as a relative value with standard human plasma as 100%. The adsorption amount was calculated by the same calculation formula as in Example 11. The results are shown in Table 2.

  The spherical sulfated cellulose of Example 3 was found to have an adsorption capacity of 2.8 times or more of the commercially available Heparin Sepharose 6 Fast Flow.

  Next, in Example 13, the spherical sulfated cellulose, sulfated Cellufine (product name: Chisso Corp.) and Heparin Sepharose 6 Fast Flow (product name: Amersham Bioscience Corp.) of Example 2 or 3 were used. The amount of γ-globulin adsorbed was compared.

[Example 13]
Separately, 4 ml each of the spherical sulfated cellulose, sulfated Cellufine (product name: Chisso) or Heparin Sepharose 6 Fast Flow (product name: Amersham Biosciences) obtained in Example 2 or 3 Column (7 mmφ × 100 mm) and equilibrated through the adsorption buffer at a flow rate of 20 ml / hr. (Hereinafter, the flow rate of each solution was 20 ml / hr.) As the adsorption buffer, 0.08M sodium acetate-hydrochloric acid buffer (pH 3.5) was used.
20 ml of γ-globulin solution (Bovine γ-Globulins (solution obtained by dissolving Seikagaku Corporation) with adsorption buffer) was passed through each column, and then the adsorption buffer was passed through to remove unadsorbed components. did.
Subsequently, elution was carried out with an elution buffer, and this solution was used as an eluate. The elution buffer used was 0.08 M sodium acetate-hydrochloric acid buffer (pH 3.5) containing 0.6 M NaCl.
Using the elution buffer as a control, the absorbance of the eluate and standard solution (prepared solution of 1 ml of γ-globulin solution and added to adsorption buffer to make 50 ml) was measured. The measurement was performed using a quartz cell at a wavelength of 280 nm.
The measured numerical value was substituted into the following formula to calculate the amount of γ-globulin adsorbed. The results are shown in Table 3.
Adsorption amount (mg / ml) = 100 × (absorbance of eluate / absorbance of standard solution)

  The spherical sulfated cellulose of the present invention is useful as a medical substrate, an adsorbent, or a chromatographic filler for separating and purifying various viruses and proteins.

It is the graph which showed the relationship between the sulfur concentration of spherical sulfated cellulose, and lysozyme adsorption amount.

Claims (6)

(A) a step of obtaining spherical cellulose from crystalline cellulose as a raw material ;
(B) a step of crosslinking the spherical cellulose to obtain a crosslinked spherical cellulose ;
(C) dehydrating the crosslinked spherical cellulose by liquid replacement using a water-soluble solvent containing N, N-dimethylformamide;
(D) a step of subjecting the dehydrated crosslinked spherical cellulose to a sulfate esterification treatment using a mixture of N, N-dimethylformamide and sulfuric anhydride,
(E) The manufacturing method of the crosslinked spherical sulfated cellulose characterized by including the process of neutralizing the crosslinked spherical cellulose which carried out the sulfate esterification process with the alkali. The production method according to claim 1 , wherein the sulfuric esterification treatment is performed at 0 to 70 ° C. Cross-linked spherical sulfated cellulose having a sulfur concentration of 1 to 6 wt%, which can be produced by the production method according to claim 1 or 2 . A crosslinked spherical sulfuric acid having a sulfur concentration of 1 to 6% by weight and having a sphericity of 0.9 to 1.0, which can be produced by the production method according to claim 1 or 2. Cellulose. A protein adsorbent comprising the crosslinked spherical sulfated cellulose according to claim 3 or 4 . A packing material for affinity chromatography using the protein adsorbent according to claim 5 .

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