GB2465301A - Spherical sulphated cellulose - Google Patents

Spherical sulphated cellulose Download PDF

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Publication number
GB2465301A
GB2465301A GB1001425A GB201001425A GB2465301A GB 2465301 A GB2465301 A GB 2465301A GB 1001425 A GB1001425 A GB 1001425A GB 201001425 A GB201001425 A GB 201001425A GB 2465301 A GB2465301 A GB 2465301A
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spherical
cellulose
production process
subjected
treatment
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GB201001425D0 (en
GB2465301B (en
Inventor
Naoyuki Yoshida
Kazushi Ishida
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JNC Corp
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Chisso Corp
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Priority claimed from GB0616658A external-priority patent/GB2429708B/en
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 - B01D15/36
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/262—Synthetic macromolecular compounds obtained otherwise than by reactions only involving carbon to carbon unsaturated bonds, e.g. obtained by polycondensation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/26—Synthetic macromolecular compounds
    • B01J20/265—Synthetic macromolecular compounds modified or post-treated polymers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B01J20/28019—Spherical, ellipsoidal or cylindrical
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/281—Sorbents specially adapted for preparative, analytical or investigative chromatography
    • B01J20/286—Phases chemically bonded to a substrate, e.g. to silica or to polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B1/00—Preparatory treatment of cellulose for making derivatives thereof, e.g. pre-treatment, pre-soaking, activation
    • C08B1/02—Rendering cellulose suitable for esterification
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B5/00—Preparation of cellulose esters of inorganic acids, e.g. phosphates
    • C08B5/14—Cellulose sulfate
    • G01N30/48—

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Materials Engineering (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Medicinal Chemistry (AREA)
  • Polymers & Plastics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Polysaccharides And Polysaccharide Derivatives (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)

Abstract

A process for producing spherical sulphated cellulose comprises a step of subjecting spherical cellulose to sulphate esterification using a mixture of N,N-dimethylformamide and sulphuric anhydride. The spherical cellulose may be crosslinked and may be obtained by using crystalline cellulose as a starting material. The cellulose is typically subjected to an initial dehydration treatment carried out by liquid substitution using a water-soluble solvent, such as N,N-dimethylformamide. After sulphate esterification, the modified cellulose may be neutralized with an alkali. The sulphate esterification treatment may be conducted at 0-70°C.

Description

DESCRIFI1ON
SPHERICAL SULFATED CELLULOSE AND
PRODUCTION PROCESS FOR ThE SAME
TECHNICAL FIELD
The present invention relates to spherical sulfated cellulose, specifically to spherical sulfated cellulose which Is useful as a base material for affinity chromatography and a base material for medical use, and a production process for the same.
BACKGROUND ART
Spherical sulfated cellulose is available as a base material which has a group specificity and which is used for separating and refining biological materials such as proteins and virus. Spherical sulfated celluloac is a gelatinous matter obtained by subjecting solid granular particles of cross-linked or non-cross-linked cellulose to sulfate esterification while maintaining a solid granular state thereof and then neutralizing it with alkali. It is known that spherical sulfated cellulose has a biological affinity similar to that of heparin (refer to Japanese Patent Publication No. 23751/1992 and Japanese Patent Publication No. 23752.11992).
However, a sulfur content of spherical sulfated cellulose obtained.by conventional methods is as low as about 0.15 % (refer to Exainplel in Japanese Patent Publication No. 23751/1992), and an adsorbing ability for protein is not satlsfactoi.
DISCLOSURE OF INVENTION
In light of the circumstances described above, it would be desired to develop spheiical sulfated cellulose which has a high sulfur content and which is excellent in an ability for adsorbing proteins and a production process for the same are desired to be developed.
Intensive researches repeated by the present inventors in order to solve the problems described above have resulted in finding that a content of sulfur inoduced into spherical sulfated cellulose is enhanced by using a specific sulfate esterifying agent, and thus they have come to complete the present invention.
That is the present invenlion provides spherical sulfated cellulose a production process for the same and a protein adsorbing agent contalning the spherical sulfated cellulose and the like which are shown below.
(1) A production process for spherical sulfated ceLlulose, comprising a step in which spherical cellulose is subjected to sulfate esteiificalion treatment with a mixture of N,N.
diuiethylformamide and sulfuric anhydride.
(2) The production process of the above item (1), wherein the spherical cellulose is obtained by using crystalline cellulose as a starting material.
(3) The production process of the above item (1) or (2), wherein the spherical cellulose is cross-linked.
(4) The production process of any of the above items (1) to (3), wherein the sulfate io esterification treatment is ca3Tied out at 0 to 70C.
(5) The production process of any of the above items (I) to (4), comprising a step in which the spherical ccllulosc is subjected to dehydndion treatment.
(6) The production process of the above item (5), wherein the dehydration treatment is carried out by liquid substitution using a water-soluble solvcnt (7) The production process of the above item (6), wherein the water-soluble solvent contains NN-dimethyIfomiamide.
(8) The production process of any of the above items (I) to (7), comprising a step in which the spherical cellulose subjected to the sulfate esterification treatment is subjected to neutralization treatment with an alkali.
(9) Spherical sulfated ceUulosc having a sulfur content of 1 to 10 % by weight which can be produced by the production process of any of the above items (1) to (8).
(l0)A production process for spherical sulfated cellulose, comprising: (a) a step in which spherical cellulose is subjected to dehydration treatment by liquid substitution using a water-soluble solvent contalning N,N-diniethylfonnanide, (b) a step in which the spherical cellulose subjected to the dehydration treatment is sujected to sulfate esterification treatment with a mixture of NN-dimethythxmamide and sulfuric anhyciride and (C) a step in which the spherical cellulose subjected to the sulfate estetification treatment is neutralized with an alkali.
(11) The production process of the above item (10). wherein the spherical cellulose is obtained by using crystalline cellulose as a starting material.
(12) The production process of the above item (10) or (11), wherein the spherical cellulose is cross-linked.
(13) The production process of arty of the above items (10) to (12), wherein the sulfate esterification treatment is carried out at (I to 70'C.
(14) Spherical sulfated cellulose having a sulfi content of 11010 % by weight which can be produced by the production process of any of the above items (10) to (13).
(15) Spherical sulfated cellulose having a sulfur content of 11010 % by weight and a sphericily of 0.9 to 1.0.
(16) The spherical sulfated cellulose of the above item (15), having a sulfur content oft to 6% by weight (17) A protein adsorbing agent comprising the spherical sulfated cellulose of any of the above items (9) and (14)to(16).
(18) A filler for affinity chromatography characterized by using the protein adsorbing agent of the above item (17).
According to the process 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, which is excellent in an adsorbing ability for proteins, can be provided. The spherical sulfated cellulose of the present invention is useful as a medical base material for separating and rrfining various viruses and protcins an adsorbing agent and a filler for chromatography, particularly a filler for affinity chromatography.
BRIFY DESCRIPTION OF DRAWINGS
Fig. I is a graph showing relation of a sulfur content of spherical sulfated cellulose to a lysozynie adsorbing amounL
BEST MODE FOR CARRYING OUT THE INVEN11ON
The spherical sulfated cellulose, the production process for the sanc the protein adsorbing agent and the like according to the present inVention shall be explained below in details.
A. Production process for spherical sulfated cellulose Firsi the production process for spherical sulfated cellulose of the present invention shall be explained.
The production process for spherical sulfated cellulose of the present invention is characterized by comprising a atep in which spherical cellulose is subjected to sulfate esterification treatment with a mixture of N,N-dimethylformamide and sulfuric anhydride.
In the process of the present invention, the mixture of NN-dlmethylfonnamide and sulfuric anhydride is used as a sulfate esterifying agent for spherical cellulose to t subject spherical cellulose to suffate esteriflcation treatment A combined matter or a complex which is formed by NN-dünethy1fonnsinide and sulfuric unhydride is contained in the above mixture (refer to the following formula), and it is considered that this works effectively on the sulfate estetification of the spherical cellulose.
S
\ ,CH3 ___,CH3 + $03 -N So / / H CH3 H CH3 The spherical cellulose used in the present invention shall not specifically be restricted, and conventional cross-linked or non-cross-linked spherical cellulose can be used. The spherical cellulose is a solid granular particle having a spherical form, and it is usually gelatinous. The spherical cellulose used in the present invention has prëlbrably a sphexicity of 0.8 to 1.0. In this case, the "spbericity" means minor diameter/major diameter of the spherical cellulose, The spherical cellulose described above can be obtained, for example, by dissolving crystalline cellulose or cellulose comprising a crystalline region or an is amorphous region end reproducing it A specific production process for spherical cellulose includes, for example, processes in which it goes througb acetic acid esters, described in Japanese Patent Publication No. 39565/1980 and Japanese Patent Publication No. 40618/1980, a process in which it is granulated from a solution using calcium thiocyanate, described in Japanese Patent Publication No. 62252/1988 and a process in which it is produced from a paraformaklehydedimncthylsulfoxide solution, described in Japanese Patent Publication No. 38203/1984. Further, given is a process in which it is molded fiom a cellulose solution obtained by dissolving cellulose in amide containing lithium chloride, described in Patent No. 3663666.
Spherical cellulose obtained by using crystalline cellulose as a starting material is preferably used as the spherical ccilulose used in the present invention since sulfur can be allowed to be contained at a higher concentration. It is because spherical cellulose originating in crystalline cellulose has a high strength and Is less liable to be dissolved in uatej, so that spherical sulfated cellulose is not softened too much if sulfur is allowed to be contained at a high concentration. Cross-linked spherical cellulose is preferred as the spherical cellulose used in the present Invention since it has a high physical stabilit In particular, cross-linked spherical cellulose obtained by cross-linking spherical cellulose originating in crystalline cellulose is more preferred since It is excellent in a physical stability. Spherical cellulose is increased in hydrophilicity as sulfate esteri&ation is advanced, so that a spherical foun is less liable to be maintained in a certain case. Howevgr use of spherical cellulose having a high physical stability maks it possible to produce spherical sulfated celMose which has a high strength aial islessliabletobedissolvedjuwatet Cross-linked spherical cellulose can be produced by subjecting non-avas-linked spherical cellulose to cross.lhiking treathient A cross-linking method shall not specifically be restricted as long as it is usually used for cross-linldng cellulose.
Multifbnctional epoxy compounds such as epichioruhydrin can be given as the example of a cross-linking agent.
CommerciaUy available products can also be used as the spherical cellulose used iii the present mvention. The non-cross-linked spherical cellulose includes, for example, Cellufine GC-15, 011-25, GC-l00 and OC-200 (brand names, rnanuturcd by Chisso Corporation) and AvIceI (brand nm"e, manufactured by Asahi Chemical Industry Co., Ltd.). The cress-linked spherical cellulose includes Cdllufbie GCL-25 GCL-90 and GCL-2000 (brand names, manufactured by Chisso Corporation). Further, capable of being used are Viscopcarl (brand name, manufactured by Rengo Co., Ltd.) which is commercially available as cellulose particles reproduced from viscose, Perloza MT series (brand name, manufactured by Iontsorb Co., Ltd.) and "Cellulose, Bcad (catalogue cord C8204, manufactured by Sigma Co., Ltd.).
An average particle diameter of the spherical cellulose used in the present invention is preferably I to 1,000 pm, mom preferably 100 to 500 pm and further preferably 250 to 300 pm. The average particle diameter can suitably be selected according to use applications. When the spherical sulfated cellulose of the present invention obtained using spherical cellulose es a starting material is used fo for example, a filler for chromatography, the void ratio is enhanced if the average paiticle diameter is too large, and it tends to be difficult to obtain a certain separative power.
On the other hand, if it is too small, pressure Is likely to be applied to bring about deformation. Accordingly, an average particle diameter of the spherical cellulose is piufezably5Oto25Opzn,znorepreferably5oto 125pm.
On the other hand, when the spherical sulfated cellulose of the present invention is used for medical applications, the too small average particle diameter allows blood platelets aixi blood cells contained in the blood to cause clogging in a void between the spherical celluloses in a certain case, and therefore an average particle diameter of the spherical cellulose is preferably 50 to 1000 pm, more preferably 300 to
S
700 jun.
In the present invention, an average particle diameter of the spherical cellulose can be calculated from particle diameters (arithmetic diameter) measured using an electric resistance method. The electric resistance method Is a method making use of a change in an electric resistance between two electrodes brought about when particles pass through a sensitive legiorL Since an electric resistance is proportional to a volume of a particle, a change In an electric resistance is measured, and this is converted to a particle diameter, whereby a particle diameter of the spherical cellulose can be measured. The whole data of the "arithmetic diameter" values thus measured is averaged to obtain the average particle diameter. A precision particle size distribution mcasuting apparatus (brand name Multlsizer 3) manufactured by Beckman Coulter, Inc. can be used as the measuring apparatus.
The spherical cellulose used in the present invention has preferably an exclusion limit molecular weight of 2,000 to 3,000,000. An exclusion limit molecular )5 weight of the spherical cellulose is varied to a large extent depending on a size of protein and vinrs which arc objects for absorption, and therefore it is suitably determined. In this case, the exclusion limit molecular weight can be determined by filling a colunm with the spherical cellulose end allowing substances having known molecular weights to flow through it to plot volumes (or elation time) of clution peaks of the respective substances versus the molecular weights. An exclusion limit molecular weight is a molecular weight in which the substances cannot get into pores of the spherical cellulose. An elution volume (or clution time) of a substance having a molecular weight, which is not smaller than the exclusion Limit molecular weight, is measured as the same value. Substances which can be used for measurement of an exclusion limit molecular weight as a substance having a known molecular weight include proteins, sugars and synthetic polymers such as polyethylene glycols and polyethylene oxides Dehydration treatment; Spherical cellulose usually containe moisture of about 90 % based on an own weight thereof even after centriftgnl dehydration. When spherical cellulose is subjected to sulfate esterificatlon in the State that a large amount of moisture is present as described above, a sulfate esterifying agent is deactivated by this moisture, and sulfate caterificafian does not proceed efficiently in a certain case. Accordingly, in the present invention, the spherical cellulose is preferably sujected to dehydration ueatrnent before carrying out the sulfate esterification treatment to remove moisture in advance as much as possible.
The dehydration eatment shall not specifically be restricted as long as moisture contained in the spherical cellulose can sufficiently be removed without damaging the form of the spherical cellulose. A dehydration treating method includes, for example, drying by beating and liquid substitution using a waler-soluble solvent.
Drying by heating can be carried out as well under a vacu condition.
Drying by heating is convenient as a method for removing moisture, but it foUows shrinkage or breakage of the spherical cellulose in a certain case. Accordingly, the liquid substitution in which such shrinkage or breakage are less liable to be brought about is preferably used in the proceas of the present invention. According to the liquidsubstitution, moisture can be removed in the state that the form of the spherical cellulose is held. In particular, spherical celluloac originating in crystalline cellulose has a low density and is liable to cause shrinkage or breakage by drying by heating, end therefore the liquid substitution is preferably used.
The liquid substitution can be carried out by sufficiently stirring spherical cellulose together with a waler-soluble solvent in a vessel, leaving it standing shill and then carrying out decantation of the supernatant. Repeating of the above operation mk jt possible to sumeicatly remove moishnc contained in the spherical cellulose.
The above operation is repeated until moisture contained in the supernatant reaches preferably 2 % by weight or less, more preferably 1 % by weight or less and particularly preferably 0.3 % by weight or less to remove the moisture, whereby subsequent sulfate esterification treatment can efficiently be carried out.
The water-soluble solvent used iii the present invention shall not spccificallybc restricted as long as it has an afni'y to water and does not rctzd pro'ess of the sulfate esterillcation treatment Aprotic organic solvents are preferred as the above water-soluble solvent The aprotic organic solvents include, fbr example, acetonihrile, diinethylsulfoxide, N,N-dimethylformer&de, N,N-dimethylacetaznide, N-methylpynolidone, tetrahydrofuran, dioxane, pyridine, lriethylmiiine and piperidine.
Among them, N)I.dimcthylforiianiide that is used as well for a sulfate esterifying agent is preferably contained as the water-soluble solvent front the viewpoint that the sulfate esterificalion treatment can more efficiently be carried out. NN- dimelhylformaxnide is preferably used alone or in a mixed solvent of N,N- dimethylformainicle with other water-soluble solvents. In particular, N,N-dirnethylformamide is preferably used alone.
Sulfate esterificatiori treatment: Ncxt the sulfate csterification treatment is carried out.
First, a mixture of N,N-dimethylfonnamide and sulfuric anhydride is prepared.
A mixing ratio of N,N-diniethylforinarnlde to sulfuric anhydride shall not specifically be resthcted as long as it falls in a range in which N,N-düncthylfonnwnide is excessive to sulfuric anhydride. A sulfuric anbychide concentration in the mixture described above �S preferably 10 to 30 % by weight. If the sulfuric anhydride concentration is within the above range, the sulfate estenfication reaction of the spherical cellulose may preceed to sufficiently, resulting in ease of sulfur content control. Particularly preferred concentration of sulfuric arthydride in the mixture isiS % by weight A concentration of sulfuric anhydrido can be measured, for example, by adding water to the mixture described above to turn sulfuric anhydride into sulfuric acid and then carrying out neutralization titration with sodium hydioxide. Sulfuric anhydride is reacted with water in a ratio 1:1, and therefore a concentration (mole concentration) of sulfuric acid measured by the neutralization titration described above means, as it is,a concentration (mole concentration) of sulibric anhythide.
Thc mixture of NN-dimcihylfonnantidc and sulfuric anbydride can be prepared by dropwise adding sulfuric anh)dride to NN-dunvthy1forznaniide and mixing them. Hetisgendinntixinginacincase,andthereforemixingispreferably carried out on a temperature condition of 5C or lower using an icc bath.
Next, the mixture of N,N-diznetbylformainide and sulfuric anbydride is dropwise added to a dispersion of the spherical cellulose and N,-diincthylfornzamide.
In the present invention, the mixture of N,N.dimethylformarnidc and sulfuric anhydride is preferably dropwise added to the dispersion of the spherical cellulose and N,N-dimctbylformanude in the state that the dispersion is held at a low temperature of 5'C or lower usmg for example, an ice bath in order to carry out homogeneously the reaction.
The sulfate esteriflcation treatment is carried out, for example, by slowly dropwise adding the nuxture of N,N-dimethylforrnamide and sulfuric anhydride to the dispersion of the spherical cellulose and N,N-dimethylformamidc which is ready in a reaction vessel and then sufficiently stirring them. The above sulfate esterication treatment is carried out preferably at 0 to 70C, more preferably 0 to 50C, further preferably 0 to 35'C and particularly preferably 0 to 30C. When coloring of the spherical sulfated cellulose by an amine base solvent is concerned, the sulfate esterificalion reaction is earned out preferably at 0 to 30C. The stirring time shall not specifically be restricted as long as it falls in a range in which the sulfate esterification reaction can sufficiently proceed, and it is usually I to 10 hours, prcrably 2 to 6 hours and more preferably 2 to 4 hours.
The above mixing method of the spherical cellulose with the mixture of NN-dimcthylforniamide and sulfuric anhydridc is one example, and it shall not be restricted to the method described above as long us the sulfate cstcrification reaction can homogeneously be carried out. A use ainowit of the mixture of N-dimethylfonnamide and sulfuric anhydride is allowed to fall in a range which is necessary and enough for carrying out sulfation of the spherical cellulose, and the range 0 thereof can suitably be determined by pcrsoxn averagely skilled In the art For example, a value obtained by dividing a mass of the spherical cellulose by a molecular weight corresponding to glucose (unit structorul substance) is set as a mole nwnber, and a use amount of the above mixture is preferably controLled so that sulfuric anhydride which is 1 to 5 times as much as it is contained. Ause amount of the above mxture is is controlled so that sulfuric anhydrlde which is more preferably 1 to 3 times, further preferably Ito 1.5 tIme as much asit is contained.
Neutralization treatment; Thc spherical cellulose subjected to the sulfate esterification trvatzneut is collected by filtering separation1 washed with an alcohol base solvent such as methanol, ethanol and the like and then preferably subjected to ncutrali7ation treatment with an alkali.
The neutralization treatment can be carried out by a mcthod usually used in producing spherical sulfated cellulose It can be carried out, for exampi; using an aqueous solution or an alcohol solution in which an alkali such as &odhnn hydroxide, potassium hydroxide and magnesium hydroxide Is dissolved.
Alter the neutralization treatment, the product is washed with ion-exchanged water and the like, and the intended spherical sulfated cellulose can be obtained.
According to the process of the present invention, the spherical sulfated cellulose which has a high sulfur content and which is excellent in an adsorbing ability can be obtained by a simple method.
According to the preferred embodiment of the present Invention, the process of the present Invention comprises: (a) a step in which spherical cellulose is subjected to dehydration trealment by liquid substitution using a water-soluble solvent cont2inhig N,N-dirnethylfoimaxnide, (b) a step in which the spherical cdliulosc suljccted to the dehydration treatment is subjected to sulfate esterificatlon teatment with a mixtire of N,N-dimethylfonnainidc and sulfuric enhydride and (c) a step in which the spherical cellulose subjected to the sulfate csteriflcation treatment is subjected to neutraliafion ire'tuiein with an alkali. The respective steps have been described above in details, and thefore they shall not repeatedly be ained here.
B, Spherical sulfated cellulose The spherical sulfated cdllulosc, which can be produced in the manner described above, can contain sulfur at a high conceniration. The spherical sulfated cellulose of the present invention has a sulfur content of preferably 1 to 10 % by weight, more preferably I to 6 % by weight and further preferably 1 to 4 % by weight. In this case, a sulfur content of the spherical sulfated cellulose ms a sulfur content of the spherical sulfated cellulose per a dry weight thereof and it can be measured by means of an inductively coupled plasma method. To be specific, it can be measured by means J5 of an inductively coupled plasma emission spectrometer (model nwnber IRJS-AP) manufactured by Nippon Janel Ash Co., Ltd., wherein a measuring wavelength of sulfur is set to 182034 nra. The spherical sulfated ccUulose is usually gelatbious and therefore the spherical sulfated cellulose, which is subjected in advance to decomposition treatment, is used as a sample for measurement The spherical sulfated cellulose of the present invcntion is a solid granular particle, which can contain sulfur at such a high conceutrtion as described above while maintaining a spherical form, and the spherzcity thcreo1is preferably 0.9 to 1.0. The sphericity can be determined by observation under a rnicrpscope.
An exclusion limit molecular weight and a parlide diameter of the spherical sulfated cellulose of the present Invention shall not specifically be restricted. They are allowed to be suitably determined according to the absoition objects and the uses.
C. Protein-adsorbing agent and uses thereof The protein-adsorbing agent of the present mvention is characterized by containing the spherical sulfated cellulose described above. The protein-adsorbing power is enhanced by covtnning the spherical sulfated cellulose described above, and therefore the protein adsorbing agent of the present Invention is suitably used for separating and refining various proteins and viniscs.
Thepiotein-adsorbingagcntoftheprcsentinvendonis useful as aflllcrfor chromatography such a affinity chromatography, ion exchange chromatography and gel 3$ filtering chromatography. In particular, the spherical sulfated cellulose of the present invention has a blood coagulation inhibiting action and a lipcmia clearing action which are similar to those of heparin. It has a heparin-like gro-speciflc adsorbing power, and therefore use thereof as a filler for affinity chromatography makes it possible to search for proteins to Iigandc and ligands to proteins and makes it possible to separate and refine these ligfinds and proteins and antibodies.
The protein adsorbing agent of the present invention can also be used as a base material for a blood purifying system for removing vizus, low density Upopzutcin (LI)!..), inunune complex, bilirubin and endotoxur which are deemed to be disease agents conlaired in the blood or the blood plasma. When disease agents and the like are removed using the protein adsorbing agent of the yLC,Cflt invention, removing methods therefore are varied depending on the properties of the disease agents to be removed.
For example, endotoxin is not adsorbed on the protein-adsorbing agent of the present invention, and therefore the intended substance is allowed to be adsorbed on. the protein-adsorbing agent of the column, and then the column is washed well, whereby 13 enciotoxin can be removed from the intended substance.
Substances which can be separated and refined using the protein-adsorbing agent of the present invention include, for example, heparinase and heparitinase which are heparin degradative enzymes, glycosaminoglycan degradative enzymes, DNA binding proteins (HMO: high mobility group protein, clioromatin and histone), bacteriophage, viral vector, Adeno-associaled Virus (AAV), Sendal vims, Follistathi, Activin, basic fibrobtast growth factor, blood coagulation ctor, lipase and nucleic acid degradative enzymes.
The present application claims the priority of ispnese Patent Application 254121/2005 flIed on September 1, 2005, and the content of the same specification shall be incorporated into the present specification by reference.
The present invention shall be explained below with reference to examples and comparative examples, but the present invention shall not be restricted to these cxarnples.
IExanp1e 11 Celluflne OR-25 (brand came, manufactured by Chisso Corporation) was used as spherical cellulose which was a starting material. This spherical cellulose bad a particle diameter of 44 to 105 pin. The above spherical cellulose had an average particle diameter of 67.05 pin and an exchiaion limit molecular weight of 2500.
In order to remove moisture contained in the spherical cellulose, 10 g thereof in terms of a inoisture weight was weighed in a 50 ml beaker, and 20 ml of N,N-dimethytformamide was added thereto and stined for 30 inirnifes. The mixture was left st2uding still after stining, and a moisture content of the supeiuasaut was measured bya Karl Fischer's method. This operation was repeated until a moisture content of the supematant fell in a range of I to 2 % by weight. Finally, a moisture content of the supemalant reached 1.05 % by weight and therefore the above spherical cellulose subjected to the dehydration treatment was used to carry out subsequent sulfate esterificatiop teeattnent.
First, the spherical cellulose subjected to the dehydration tteaUnnt was dispersed in N,N-dinielhylfoinrarnide held at 5C or lower on en ice bath.
*A 18 weight % sulfuric anhydride-dirnethylformainide solution 10.97 g cooled to 5C was slowly added to the above dispersed solution to carry out reaction for 4 hours while maintinirg the reaction temperature at 30 � 2'C. After finishing the reaction, the reaction solution was separated by filtering, and a cake was washed with tuethanol. Used was the 18 weight % sulfuric anhydridc-dirneth)4fortnamide sohition prepared by dropwise zdding 278 g of sulfuric anbydiide to 2500 g of NN-diniethylfo main de cooled at 5'C or 1oon n icebath while stirring (hereinafter the same shall apply). In this case, the dropping speed was controlled so that temperature of the solution did not exceed 5C.
Next, the cake was put into ionexchanged water cooled at 1OC or lower and neutralized by IM-NaOH Then, it was sufficiently washed with ion.cxchanged water to obtain spherical sulfated cellulose.
A sulfur content of the spherical sulfated celMose thus obtained was measured by an inductively coupled plasma method. The spherical sulfated cellulose, which was subjectcd to pretreatment by the following method, was ised for measurement.
First, 20 mg of the spherical sulfated cellulose was weighed in a 100 ml beaker, and 10 ml of nitric acid was added into the bcake4 and then the spherical sulfated cellulose was decomposed en a hot plate (250C) until the brownish-red color became light. In this case, attention was paid so that it was not dried up. Alter left cooling down, 5 nil of nitric acid and 2 ml of pcrchloric acid were further added thereto, and it was decomposed on the hot plate until white smoke was emitted. Subsequently, after left cooling down, 10 ml of a!: 1 (volume ratio) hydrochloric acid aqueous solution was added to dissolve the ridue, and the volume was adjusted to 100 ml by a It measuring flask to prepare a sample for measurement.
An inductively coupled placimi emission spectrometer (model number IRIS-AP) manufactured by Nippon Janel Ash Co., Ltd. was used for measuremens, wherein a measuring wavelength of sulfur was set to 182.034 urn. As a result thereof, a sulfur content of the spherical sulfated cellulose was 131 % by weight.
xample 2J Cellufine GH-25 which was the same as used in Example 1 was used as a starting materi In order to remove moisture contained in the spherical cc1lulose 52.7 g thereof in teims of a moisture weight was weighed in a 200 ml beaker, and 100 ml of N,N-dirnethylfomnianjide was added thereto and stiued for 30 minutes The mixture was left srfinding still after stirring and dehydration treatment was repeated by the same method as in Example 1 until a moisture content of the supernatant fell in a range of 1 to 2 % by weight. Finally, a moisture content of the supernalant reached 1.60 % by weight, and therefore the above spbaical cellulose subjected to the dehydration treatment was used to carry out subsequent sulfate csteriflcation Uatmnert.
Next, the spherical cellulose subjected to the dehydration treatment was dispersed in thylfomiamide at a temperature of 5'C or lower. A 18 weight % ulfwic anhydride-dimethylfounamnide solution 54.99 g cooled to S'C was slowly added to the above dispersed solution. The reaction was carried out for 4 hours while xnaintRinin8 the reaction temperature at 30 � 2'C. After finishing the reaction, the reaction solution was separated by filtering, and a cake was washed with methanoL Then, the cake was put into ion-exchanged water cooled at b.C or lower and neutralized by IM-NaOH, Thereafter, it was sufficiently washed with ion-exchanged water to obtain spherical sulfated cellulose.
A sulfur content of the spherical sulfated cellulose was measured in the same manner as in Example 1 to result in finding that a sulfur content of the spherical sulfated cellulose was 1.81 %by weight lEumple 3J Cellufinc (brand name, manufactured by Cthso Corporation) was used as a starting material. Cdlluflzw was produced the following production steps.
(i) The ystalline cellulose 0.46 kg is added to an aqueous solution con1n 60 % by weight of calcium thiocyanate (as an anhydxide) and dissolved by heating at 110C. (ii) A surfactant is added to the above solution, and the solution is dropwise added to 301 of o-dichlorobenzenc heated in advance at 130 to 140C and dispersed by stirring. (iii) Then, the dispersed solution described above is cooled down to 40C or lower, and 13 1 of methanol is poured thereinto to obtain particles. (iv) This S suspension is separated by filtering, and the particles are *ashed with 131 of methanol and separated by filtering, This washing operation is carried out several times. (v) The particles are washed with a large amount of waler, and then intended spherical ceUulose particles are obtained.
The above spherical cellulose had a particle diameter of 44 to 300 sam. It bad an average particle diameter of 20 pm and an cXclualon limit molecular weight of 3,000,000.
In onlerto rmvc moistre contained in the spherical cellulose, $Og thereof in terms of a moisture weight was weighed in a 200 ml beaker, and 100 nil of N,N dirnethylformmiide was Rdd"d thereto and stirred for 30 minutes. The mixture was left standing still after stirring, and dehydration treatment was repeated by the same method as in Emnp!e I until a rnoistme content of the supernatant fell in a level of 0.2 % by weight Finally, a moisture content of the supernatant reached 0.23 % by weight, and therefore the above spherical cellulose subjected to the dehydration treatment was used to carry out subsequent sulfate esterification treathient.
First, the spherical cdilulose subjected to the dehydration treatment was dispersed in NN-dimcthytforinamide held at a temperature of 5C or lower on n ice bath. A 18 weIght % sulfuric anhydride-dinwihylformanilde solution 15.10 g cooled to 5'C was slowly added thereto. The solulion was stirred for 4 hours while maintaining the reaction temperature at 30 � 2C. After fnihii the reaction, the s reaction solution was separated by filtering, and a cake was washed with methanol.
Then, the cake was neuliulized by IM-NaOR in ion-exchanged water cooled to lO'C or bwei Thereafter, it was washed with ion-exchanged water to obtain spherical sulfated cellulose.
A sulfur content of the spherical sulfated cellulose was measured in the same maimer as in Example Ito result in finding that a sulfur content of the spherical sulfated cellulose was 2.80 % by weight
tExample 41
Spherical cellulose particles in which a moisture content in the supernalant was 021 % by weight was obtained by the same method as in Example 3, except that in order to remove moisture contained in the spherical cellulose, 2000 g thereof in terms of a moisture weight was weighed in a 51 beaker and that 41 of N,N-dimetbylformamidc wes used.
The whole amount thereof was used, and a 18 weight % sulfuric azthydridc dimethylformarnide solution 375.0 g was used to CelTy out sulfate esterlfication beaünent and neutralization treatment in the same manners as in Example 3, whereby spherical sulfated cellulose was obtained.
A sulfur content of the spherical sulfated cellulose was measured in the same manner as in Example I. As a result thereof, a sulfur content of the spbcncal sulfated cellulose was 2.20% by weight (lmple5I The same spherical cellulose as that used as a starting material in Example 3 was used and cross-linked by the tbliowing method.
The spherical cellulose 176 g was weighed in a separable flask having a volume of 2 1, and heptane and a catlimic surfactant (Japanese Pharmacopoeia benzalkonjum chloride) were added thereto and stirred for about 30 minutes. Then, the temperature was raised up to 30'C, and 0.18 g of sodium boron hydride and 420 nil of a sodium hydroxide aqueous solution a4justed loS % by weight were added thereto and stirred for 2 hours.
Then, the temperature was raised up to 40'C, and 60.0 g of epich]orohydrin was added thereto. The temperature was further raised up to 50 � IC, and the solution was stirred for 4 hours. Then, the solution was cooled down to about 35C and neutralized with acetic acid, and then methanol was added thereto. The reaction solution was separated by filtering, and then a cake was washed in order with methanol and water to obtain cross-linked spherical cellulose.
Moisture contained in the cross-linked spherical cellulose was removed by the same method as in Example 3, and dehydration treatment was carried out until a moisture content of the supeniatant reached 013 % by weiglit, except that 150 g thereof in terms of a moisture weight was weighed in a 500 ml beaker and that 300 ml of NN-dimethylformaniidc was used.
Sulfate esterification Izealment and neutralization treatment were earned out in the same manners as in Example 3, except that the whole owit of the eross-linked spherical cellulose subjected to the dehydration treatment was used and that a 18 weight % sulfuric anhydridedimethylforrnamide solution 47.0 g was used, whereby is splicrical sulfated cellulose was obtained.
A sulfur content of the spherical sulfated cellulose was measured in the same manner as in Example I. As a result thereo4 a sulfur content ofthespherical sulfated cellulose was 4.70 % by weight Each 1000 particles of the spherical sulfated celluloses obtained in Examples I to 5 were observed under a microscope sd measured for a major diameter (RI) and a minor diameter (R2) to determine a sphericity (R7JR1) to find that a spbeiicity of the respective particles was 0.9 or more, and it was confirmed that a spherical font was held.
Nexi, in Examples 6 to 10, the spherical sulfated celluloses obtained in Examples 1 to 5 were measured for a lysozyme adsorbing amount
[Example 6J
First, the spherical sulfated cellulose obtained in P-nmple 1 was measured for a lysozyme adsorbing amount by the following method.
The spherical sulfated cellulose 3g which was stored in methanol and obtained by filtering under reduced pressure was put in a beaker, and 100 nil of ion-exchanged water was added thereto. The mixture was stirred for 10 minutes by means of a magnetic stirrer and then separated by filtering. Treatment in which the above sPhcncal sulfated cellulose separated by filtering was returned again to the beaker and in which 100 ml of iou-exchanged water was added thereto to stir and separate the mixture by filtering was repeated further three times.
The above spherical sulfated cellulose was put in a beaker, and 100 tnt of adsorbing buftbr was added thereto and stirred for one hour. A 0.OIM phosphoric acid (Na-Na) buffer solution (pH 7.0) conmining 0,lSM sodium chloride was used for the adsorbing buflhr.
Then, 2 ml of the spherical sulfated cellulose suspended in the adsorbing buffer was filled In a column, and the adsorbing buffer was allowed to flow at a flow velocity of 40 nd/hour to equilibrate Ike colunim Subsequently, 30 ml of a prepared solution obtained by adding the adsorbing buffer to 333 rag of a lysozyme solution (brand name "Lysozyme", manufactured by Wako Pure Chemical Industries, Ltd.) to adjust the volumeto lOOnil was weighedand circulatedataflowvelocityof 60 mI/hour for one bout Subsequently, 20 nil of the adsorbing buffer was allowed to flow at a flow velocity of 50 mI/hour to remove non-adsorbed lysozyme. Then, 50 ml of eluting buffer was allowed to flow at a flow velocity of 50 nd/hour, and 45 ml was collected.
The cluting buffer S ml was added to the above collected solution to make the volume ml, and this solution was set as an eluted solution. A0.OIM phosphoric acid (Na-Na) buffer solution (pH 7.0) containing 0.6M sodium chloride was used for the eluting buft.
A solution obtained by tIrin I ml of the lysozynie solution and adding the cluting buffer to it to make the volume tO ml was prepared1 and this solution was set as a standard solution.
The absorbances of the cluted solution and the standard solution were measured with the eluting buffer set as a coutnil. Measurement was carried out at a wavelength o1280 nm using a quartz cell.
The measured numerical values were substituted for the following calculating equation to calculate a lysozyme adsorbing amount. As a result thereof, the adsorbing ambunt was 18 mg/mI.
Msorbing amount (mg/mi) 0.333 x 25 x (absorbance of the cluted solution/absorbance of the standard solution) fump1e 71 A lysonyme adsorbing amount of the spherical sulfated cellulose obtained in Example 2 was measured hi the same mminr as in Example 6 to result in fin4ing that the adsorbing amount was 24 mg/mI.
lExampJegJ A lysozyme adsorbing amount of the spherical suiflited cellulose obtained in Example 3 was measured in the same manner as in Example 6, except that after renioving non-adsorbed Lysozyme., 150 ml of the cluting buffer was allowed to flow to collect 145 ml and that 5 ml of the duting buffer was added to the collected solution to make the volume 150 ml to prepare an cluted solution. As a result thereof, the adsorbing amount was 39.06 mgIml.
r21t1ple9J A lysozyxne adsorbing amount of the spherical sulfated cellulose obtained in Example 4 was measured in the same In as in Example 8. As a result thereot the adsorbing amount was 26.41 mg/mI.
I
[PTvnple 10) A lysozyn3c adsorbing amount of the spherical sulfated cellulose obtained in Example 5 was measured in the' same manner as in Example 8. As a result thereof, the S adsorbing amount was 40.33 mg/mI.
[Comparathe Example 1J Cellufinc Sulfate (brand me, manufactured by Chisso Corporation) was used as a sample to measure a lysozyme adsorbing amount in the same manner as in Example 6. As a result thereof, the adsorbing amount was 5 mg/mL A sulfur content of the Cdllufine Sulfate was measured in the same manner as in Example I to find that the su1t'ir content was 0.16 % by weight Shown In Fig. I is a relation of the sulfur contents of the spherical sulfated celkilosca obtained in Examples 6 to 10 and Comparative Example I to the lysozyinc adsorbing amounts thereoL in Fig. 1, the results obtained in Exaniples6 to 10 were shown by, and the result obtained in Comparative Example I was shown by A. It was found that the spherical sulfated ceiluloscspguduced by the process of the present invention had a three times or more larger lysoiymc adsorbing capacity than those of conventional preducts.
NexI in Example 11, compared ware the adsorbing amounts in the blood coagulation Vii! factor activity of the spherical sulfatcd cellulose obtained in Example 3 and Ceflufine Sulfate (brand name, manufactured by Chlsso Corporation).
Fwther, in Example 12, compared were the adsorbing amounts of the blood coagulation XII factor activity of the spherical sulfated cellulose obtained in Example 3 and Heparin Sephamsc 6 Fast Flow (brand name,, manufactured by Amersham Biosciences K. K.).
lExample 111
Human plasma (pool) (anticoagulant: sodium citrate, manufactured by Cosino Bio Co., Ltd.) 6 ml was weighed and put in 5pitz tubes. 2 ml of the spherical sulfated cellulose obtained in Example 3, which was washed with a physiological salt solution, was added into one of the spdz tubes and 2 ml of CeThflne Sulfate (brand name, manufactured by Chisso Corporation), which was washed with a physiological salt solution saddedintotheoberspiibcandeachspibewasstindat25Cfor $ 2 hours by shaking. Then, both wcte subjected to centdfagal separation, and each supematant was sampled to measure an adsorbing amount of the blood coagulation VIII factor activity. Plasma to which the gel was not added was used as a blank Each blood coagulation Viii factor activity was measured by Mitsubishi Chemical BCL according to an AP'lT (activated prothronibin time) method.
The measured value was shown by a relative value, wherein the measured value of standard human plasma was set to 100 36 The adsorbing amount was calculated according to the following equation.. The results thereof arc shown in Table Adsorbing amount (%/mI-gel) ((blank measuring value x volume) (sample measuring value x volume)) 12
Table 1
VIII factor activity adsorbing amount Sample %lmll Cellufain Sulfate 35 Spherical sulfated cellulose of' 191 Exarnple3 __________________________ It was found that the spherical sulfated cellulose prepared In Example 3 bad an about 5.5 times larger adsorbing capacity than that of Cellufine Sulfate which wa a commercially available product.
[Example 12J
The same operation as in Example t 1 was carried out to compare an adsorbing amount of the blood coagulation VII factor activity, except that Heparin Sepharose 6 Fast Flow (brand name, ra ufactomed by Amersham flioscicncesl( L) was used as comparison in place of Cellufine Sulfate (brand name, manufactured by Chisso Ccaporation).
Thc blood coagulation Xli factor activity was measured by Mitsubishi Chemical BCL according to the APTI' (activated pivthronibtn time) method.
The measured value was shown by a relative value, wherein the measured value of standard human pbLclnn was set to 100 36 The adsorbing amount was calculated according to the same equation as in Example Ii. The results thereof are
shown in Table 2.
Table 2
S
Xli factor activity adsorbing amount Sample ___________________ %ml-gel Heparin Sephamse 6 Fast Flow V 86 Spherical sulfated cellulose of 244 V Example3 __________________________V It was found that the spherical sulfated cellulose prepared in Example 3 had a 2.8 times or more larger adsorbing capacity than that of Heparin Sepharose 6 Fast Flow which was a commercially available product.
V
Next, in Example 13, compared were the adsorbing amounts y-globulia of the spherical sulfated cellulose prepared in Example 2 or 3, Cellufine Sulfate (brand name, manufactured by Chisso Corporation) and Heparin Sepharose 6 Fast Flow (brand name, manufactured by Azneisham Biosciences K. K.).
lExample 13J
Each 4 ml of the spherical sulfated cellulose prepared hi Example 2 or 3, CcUuline Sulfate (brand name, manufactured by Chisso Corporation) and Heparin Sepharose 6 Fast Flow (brand name, manufactured by Amersham Biosciences K. K.) was filled hr separate columns (1 rnm4 x 100 nun), and adsorbing buffer was allowed to pass at a flow velocity of 20 nil/hour to equilibrate each column. Hereinafter eachsolution was allowed to pass at a flow velocity of 20 ml/bow A 0.0814 sodium acetate-hydrochloric acid buffer solidion (pH 3.5) was used for the adsorbing buffeL A y-globulin solution (solution prepared by dissolving Bovine Olobu3ins (brand name, manufactured by Biochemical Industry Co., Ltd.) in adsorbing buffer) 20 nil was allowed to pass through each of the above columns. Then, the adsorbing buffer was allowed to pass to remove non-adsorbed matters.
Next each was chited with cluting buffer, and each solution was set as an eluted solution. A 0.OSM sodium acetate-hydrochloric acid buffer solution (pH 3.5) continiiig 0.6M-NaCI was used for the duting buffer.
The absorbances of each ehited solution and the standard solution (a prepared solution obtained by adding the adsorbing buffer to I nil of the y-globulin solution to make the volume 50 ml) were measured with the eluting buffer set as a control.
Measurement was canled out at a wavelength of 280 nm using a quartz celL The measured numencal values were substituted for the following calculating equation to calculate the y-globulin adsorbing mounts. The results thereof are shown
in Table 3,
Adsorbing amount (mgml) = 100 x (absorbance of the eluted solution / tO absorbance of the standard solution)
Table 3
i-Globulin adsorbing amount Sample (_) Spherical sulfated cellulose of 02 Example 2 ____________________________ Spherical sulfated cellulose of 51 58 Exaxnple3 _____________________ cellufine Sulfate 0.10 1eparin Sepharose 6 Fast Flow ___________________ rwusrRLALAPPUcABILnY The spherical sulfated cellulose of the present inventioc is useful as a medical base material for separating and ieluing various viruses and proteins, an adsorbing agent and a filler!br chromatography

Claims (9)

  1. CLAIMS1. A production process for spherical sulfated ce1luio, a p in which spherical cellulose is sutjected to sulfate esterification treatment with a mixture of NN-dimethylfonnamide and sultric anhydiidc.
  2. 2. The production process according to claim 1, wherein the spherical cellulose is obtained by using crystalline cellulose as a starting materiaL 3. The production process according to claim I cr2, wherein the spherical cellulose is cross-linked.4. The production process according to any of claims I to 3, wherein the sulfate caterification treatment is carried out at 0 to 70C.5. The production process according to any of cLaims 1 to 4, comprising a step in which the spherical cellulose is subjected to dehydration treatment.6. The production process according to claim 5, wherein the dehydration treatment is carricd out by liquid substitution using a water-soluble solvent.7. The production process according to claim 6, wherein the water-soluble solvent contains NN-dimcthyiforrxiantide.8. The production process according to any of cLaims Ito?, comprising a step in which the spherical cellulose subjected to the sulfate esterification treatment is subjected to neuiralirtion treatment with an alkali.9. A production process for spherical sulfated cellulose, comprising: (a) a step in which spherical cellulose is subjected to dehydration treatment by liquid substitution using a water-soluble solvent containing N,N-dlm hytforinaznide, (b) a step in which the spherical cellulose subjected to the dehydration treatment is subjected to sulfate esterificazion trestment with a mixtwe of NN-dintethylformamide and sulfuric anhydride and (c) a step in winch the spherical cellulose subjected to the sulfate csteiification treatment is neutralized with an alkali.10. The produciion process according to claim 9, wherein the spherical cellulose is obtained by using crystalline cellulose as a starting material.11. The production process according to claim 9 or 10, wherein the spherical ceo5ciscrass.1jnked.12. The production process according to any of claims 9 to 11 wherein the sulfate esterification binici is carried out at 0 to 70'C.Amendments to the claims have been filed as followsCLAIMS1. A production process for spherical sulfated cellulose, comprising a step in which spherical cellulose is subjected to sulfate esterification treatment with a mixture of N,N-dimethylformamide and sulfuric anhydride.2. The production process according to claim 1, wherein the spherical cellulose is obtained by using crystalline cellulose as a starting material.
  3. 3. The production process according to claim 1 or 2, wherein the spherical cellulose is cross-linked.
  4. 4. The production process according to any of claims 1 to 3, wherein the sulfate esterification treatment is carried out at 0 to 70°C.* . e. 15
  5. 5. The production process according to any of claims I to 4, comprising a step in which the spherical cellulose is subjected to dehydration treatment.*** ..* * S
  6. 6. The production process according to claim 5, wherein the dehydration treatment is carried out by liquid substitution using a water-soluble solvent.
  7. 7. The production process according to claim 6, wherein the water-soluble solvent r* :* contains N,N-dimethylfonnamide.
  8. 8. The production process according to any of claims 1 to 7, comprising a step in which the spherical cellulose subjected to the sulfate esterification treatment is subjected to neutralization treatment with an alkali.
  9. 9. A production process according to claim I, comprising: (a) a step in which spherical cellulose is subjected to dehydration treatment by liquid substitution using a water-soluble solvent containing N,N-dimethylformamide, (b) a step in which the spherical cellulose subjected to the dehydration treatment is subjected to sulfate esterification treatment with a mixture of N,N-.dimethylformamide and sulfuric anhydride and (c) a step in which the spherical cellulose subjected to the sulfate esterification treatment is neutralized with an alkali.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1110335A (en) * 1965-07-15 1968-04-18 Kelco Co Process of preparing a gellable colloidal cellulose sulfate and product
JPS6154450A (en) * 1984-08-24 1986-03-18 Chemo Sero Therapeut Res Inst Gel for affinity chromatography having group specificity and its production
JPS6154451A (en) * 1984-08-24 1986-03-18 Chemo Sero Therapeut Res Inst Gel for affinity chromatography having group specificity and its production
EP1698641A1 (en) * 2005-03-01 2006-09-06 Chisso Corporation Compound selected from sulfated cellulose and salts thereof and dermatitis therapeutic agent

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1110335A (en) * 1965-07-15 1968-04-18 Kelco Co Process of preparing a gellable colloidal cellulose sulfate and product
JPS6154450A (en) * 1984-08-24 1986-03-18 Chemo Sero Therapeut Res Inst Gel for affinity chromatography having group specificity and its production
JPS6154451A (en) * 1984-08-24 1986-03-18 Chemo Sero Therapeut Res Inst Gel for affinity chromatography having group specificity and its production
EP1698641A1 (en) * 2005-03-01 2006-09-06 Chisso Corporation Compound selected from sulfated cellulose and salts thereof and dermatitis therapeutic agent

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