WO2015056680A1 - 多孔質セルロースビーズの製造方法 - Google Patents
多孔質セルロースビーズの製造方法 Download PDFInfo
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- WO2015056680A1 WO2015056680A1 PCT/JP2014/077361 JP2014077361W WO2015056680A1 WO 2015056680 A1 WO2015056680 A1 WO 2015056680A1 JP 2014077361 W JP2014077361 W JP 2014077361W WO 2015056680 A1 WO2015056680 A1 WO 2015056680A1
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- 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
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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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
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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
- B01D15/3828—Ligand exchange chromatography, e.g. complexation, chelation or metal interaction chromatography
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- 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
- B01J20/282—Porous sorbents
- B01J20/285—Porous sorbents based on polymers
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- 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
- B01J20/30—Processes for preparing, regenerating, or reactivating
- B01J20/3085—Chemical treatments not covered by groups B01J20/3007 - B01J20/3078
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/16—Extraction; Separation; Purification by chromatography
- C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
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- 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
- C08B1/003—Preparation of cellulose solutions, i.e. dopes, with different possible solvents, e.g. ionic liquids
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- 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
- C08B1/08—Alkali cellulose
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/16—Making expandable particles
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J9/00—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof
- C08J9/28—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a liquid phase from a macromolecular composition or article, e.g. drying of coagulum
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L1/00—Compositions of cellulose, modified cellulose or cellulose derivatives
- C08L1/02—Cellulose; Modified cellulose
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- 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
- B01J20/267—Cross-linked polymers
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- 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
- B01J20/28054—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 surface properties or porosity
- B01J20/28078—Pore diameter
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2201/00—Foams characterised by the foaming process
- C08J2201/04—Foams characterised by the foaming process characterised by the elimination of a liquid or solid component, e.g. precipitation, leaching out, evaporation
- C08J2201/054—Precipitating the polymer by adding a non-solvent or a different solvent
- C08J2201/0542—Precipitating the polymer by adding a non-solvent or a different solvent from an organic solvent-based polymer composition
- C08J2201/0543—Precipitating the polymer by adding a non-solvent or a different solvent from an organic solvent-based polymer composition the non-solvent being organic
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08J—WORKING-UP; GENERAL PROCESSES OF COMPOUNDING; AFTER-TREATMENT NOT COVERED BY SUBCLASSES C08B, C08C, C08F, C08G or C08H
- C08J2301/00—Characterised by the use of cellulose, modified cellulose or cellulose derivatives
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/50—Conditioning of the sorbent material or stationary liquid
- G01N30/52—Physical parameters
- G01N2030/524—Physical parameters structural properties
- G01N2030/525—Physical parameters structural properties surface properties, e.g. porosity
Definitions
- the present invention relates to a method for producing porous cellulose beads.
- Porous cellulose beads have the advantages of higher safety and less non-specific adsorption than when other synthetic polymers are used. In addition, there are advantages such as high mechanical strength and a large amount of hydroxyl groups that can be used to introduce a ligand that interacts with the target substance to be adsorbed. Accordingly, porous cellulose beads are used as a base material for various adsorbents such as various chromatographic adsorbents and affinity adsorbents. In particular, affinity adsorbents have been used as medical adsorbents and antibody drug purifier adsorbents because they can efficiently purify the target product and reduce the concentration of unwanted substances.
- Non-Patent Document 1 As an adsorbent for treating or treating rheumatism, hemophilia, dilated cardiomyopathy, an adsorbent in which protein A is immobilized on a porous carrier as an affinity ligand has attracted attention (for example, Non-Patent Document 1).
- Non-Patent Document 2 As an adsorbent for treating or treating rheumatism, hemophilia, dilated cardiomyopathy, an adsorbent in which protein A is immobilized on a porous carrier as an affinity ligand has attracted attention (for example, Non-Patent Document 1).
- Non-Patent Document 2 As an adsorbent for treating or treating rheumatism, hemophilia, dilated cardiomyopathy, an adsorbent in which protein A is immobilized on a porous carrier as an affinity ligand has attracted attention (for example, Non-Patent Document 1).
- Non-Patent Document 2 As an a
- an adsorbent in which protein A is immobilized on a porous carrier using an affinity ligand is attracting attention as an adsorbent for purifying antibody drugs that can specifically adsorb immunoglobulin (IgG).
- IgG immunoglobulin
- Patent Document 1 a method of dissolving and coagulating in a solvent such as an aqueous calcium thiocyanate solution that is highly corrosive and toxic and increases the difficulty of installation is disclosed. It is known that the cellulose solution used in this method exhibits unique behavior, and the porous cellulose beads obtained by this method have considerably large pores and a wide pore size distribution ( For example, Non-Patent Document 3).
- porous cellulose beads obtained by the method are used as an adsorbent such as an antibody, it cannot be expected to show a high adsorption performance because the specific surface area is small.
- a method of giving a substituent to the hydroxyl group of cellulose, dissolving in a general-purpose solvent, granulating, and removing the substituent after granulation to obtain a porous cellulose carrier (For example, Patent Document 2), however, the process is complicated, and the molecular weight is lowered in the process of adding or removing a substituent. There is a tendency that it is difficult to obtain an appropriate strength.
- Patent Documents 3 and 4 a method is disclosed in which cellulose can be dissolved in a low temperature sodium hydroxide aqueous solution (for example, Patent Documents 3 and 4).
- a mixture of cellulose and a hydrogen bond-cleaving solution is heated at 100 to 350 ° C. under pressure and then dissolved in an alkaline aqueous solution. Yes.
- Such a process is industrially disadvantageous.
- Patent Document 5 discloses cellulose that is soluble in an alkaline solution, but the cellulose has a microfibril diameter reduced to 1 ⁇ m or less and further refined to 500 nm or less. Such a refinement process is not suitable for industrial production.
- microbial cellulose is dissolved in an alkaline solution to prepare a cellulose solution, and after adding a dispersion solvent, particles are formed, and then the microbial cellulose particles are frozen and then washed.
- a method for obtaining cellulose beads is disclosed, the process is complicated and not suitable for an industrial production method.
- the present invention has a narrow pore size distribution and a pore shape suitable for an adsorbent simply and efficiently without using a complicated process that is industrially disadvantageous, without using highly toxic and corrosive auxiliary materials. It aims at providing the method of manufacturing the cellulose bead excellent in adsorption capacity.
- the present inventors added a water-soluble low molecular weight organic compound to a cellulose fine dispersion prepared by mixing a low-temperature alkaline aqueous solution and cellulose powder.
- the present invention was completed by finding that porous cellulose beads having a sharp pore size distribution and a higher adsorption ability when a ligand was immobilized can be produced satisfactorily.
- [1] a) A step of mixing a low-temperature alkaline aqueous solution and cellulose to prepare a cellulose fine dispersion, b) a step of adding a water-soluble low molecular weight organic compound to the cellulose fine dispersion to prepare a mixed solution; c) A step of producing an emulsion by dispersing the mixed liquid in a dispersion medium, d) A method for producing porous cellulose beads, comprising the step of bringing the emulsion into contact with a coagulation solvent.
- the water-soluble low molecular weight organic compound is one or more amino acids selected from the group consisting of glycine, alanine, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine and histidine.
- the dispersion medium is one or more oil-soluble solvents selected from the group consisting of animal and vegetable oils and fats, hydrogenated animal and vegetable oils and fats, fatty acid triglycerides, aliphatic hydrocarbon solvents, and aromatic hydrocarbon solvents.
- the method for producing porous cellulose beads according to any one of the above [1] to [4].
- porous cellulose bead according to any one of [1] to [5], wherein the coagulation solvent is an alcohol solvent or a mixed solvent of water and an alcohol solvent. Production method.
- Adsorption characterized by immobilizing a ligand that interacts with a target substance on the beads obtained by the method for producing porous cellulose beads according to any one of [1] to [6] above body.
- An adsorbent comprising porous cellulose beads produced by the method according to any one of [1] to [6] above, and a ligand that interacts with a target product.
- a narrow pore size distribution and a pore shape suitable for an adsorbent can be obtained simply and efficiently without using a complicated process that is industrially disadvantageous, without using highly toxic and corrosive auxiliary materials.
- Cellulose beads having excellent adsorptivity can be produced.
- FIG. 2 is a surface enlarged SEM observation image of beads obtained in Example 1.
- FIG. 2 is a surface enlarged SEM observation image of beads obtained in Comparative Example 1.
- 3 is a graph showing the pore size distribution of beads obtained in Examples 3 to 5 and Comparative Example 3.
- 4 is a graph showing the pore size distribution of beads obtained in Examples 3 and 6 and Comparative Example 3.
- the method for producing porous cellulose beads according to the present invention includes: a) a step of mixing a low-temperature alkaline aqueous solution and cellulose to produce a cellulose fine dispersion; b) adding a water-soluble low-molecular organic compound to the cellulose fine dispersion.
- a step of preparing a mixed liquid c) a step of dispersing the mixed liquid in a dispersion medium to prepare an emulsion, and d) a step of contacting the emulsion with a coagulation solvent to obtain porous cellulose beads.
- porous cellulose can be obtained by dispersing cellulose in a low-temperature sodium hydroxide aqueous solution and bringing it into contact with a coagulation solvent.
- the present inventors have increased the amount of adsorption by adding a water-soluble low molecular organic compound to the cellulose fine dispersion compared with the case where no water-soluble low molecular organic compound is added. It has been found that larger adsorbers can be obtained. Perhaps the water-soluble low-molecular-weight organic compound is suitably dispersed in the cellulose fine dispersion to form a microscopic area, and the water-soluble low-molecular-weight organic compound migrates to the coagulation solvent or washing solvent, thereby forming pores that are advantageous for adsorption. I think that it may be formed.
- Step a Preparation Step of Cellulose Fine Dispersion a cellulose fine dispersion is prepared by mixing a low-temperature alkaline aqueous solution and cellulose.
- low temperature refers to a temperature lower than normal temperature. If the temperature is lower than normal temperature, there is no major problem, but if it is -20 ° C or higher, it is preferable because the temperature control equipment is simple and the running cost is low. Moreover, if it is 10 degrees C or less, since coloring of a cellulose dispersion liquid decreases and the dispersibility and swelling property of a cellulose become high, it is preferable.
- the temperature is preferably ⁇ 10 ° C. or higher and 20 ° C. or lower. If it is ⁇ 10 ° C. or higher, freezing of the alkaline aqueous solution can be suppressed.
- a cellulose dispersion liquid can be prepared efficiently and coloring of a cellulose dispersion liquid can be suppressed.
- the temperature is preferably ⁇ 5 ° C. or higher, more preferably ⁇ 2 ° C. or higher, particularly preferably ⁇ 1 ° C. or higher, and is 0 ° C. or higher in view of the handling of water used in the cellulose dispersion and ease of temperature adjustment. Most preferred. Especially, 15 degrees C or less is more preferable, 9 degrees C or less is more preferable, 5 degrees C or less is more preferable, 4 degrees C or less is more preferable, 1 degrees C or less is more preferable. Moreover, if the said temperature is 9 degrees C or less, since the sphericity of the obtained porous cellulose bead becomes high, it is preferable.
- Alkali can be used without particular limitation as long as it shows alkalinity when it becomes an aqueous solution.
- Lithium hydroxide, sodium hydroxide and potassium hydroxide are preferable from the viewpoint of availability, and sodium hydroxide is most preferable from the viewpoint of product safety and price.
- the alkali concentration of the alkaline aqueous solution is not particularly limited, but is preferably 3 to 20% by weight. If the alkali concentration is within this range, the dispersibility / swellability of cellulose in an alkaline aqueous solution is increased, which is preferable.
- the concentration of alkali is more preferably 5 to 15% by weight, further preferably 7 to 10% by weight, and most preferably 8 to 10% by weight.
- the type of cellulose is not particularly limited.
- substituted cellulose such as cellulose into which a substituent for increasing solubility is introduced, and ordinary non-substituted cellulose is used as a raw material. it can.
- cellulose powder in order to efficiently disperse the cellulose in the alkaline aqueous solution, it is preferable to use cellulose powder as the cellulose.
- the molecular weight of the cellulose raw material used is not particularly limited, but the degree of polymerization is preferably 1000 or less. When the degree of polymerization is 1000 or less, the dispersibility / swellability in an aqueous alkali solution is increased, which is preferable. Moreover, since the mechanical strength of the obtained porous cellulose bead will become large if a polymerization degree is 10 or more, it is preferable.
- a more preferable range of the polymerization degree is 50 or more and 500 or less, more preferably 100 or more and 400 or less, particularly preferably 200 or more and 350 or less, and most preferably 250 or more and 350 or less.
- the concentration of cellulose in the cellulose fine dispersion is not particularly limited and may be appropriately adjusted. For example, it may be about 1% by weight or more and 20% by weight or less.
- the concentration is more preferably 2% by weight or more, further preferably 5% by weight or more, more preferably 15% by weight or less, and further preferably 10% by weight or less.
- the method for preparing the cellulose fine dispersion may be in accordance with a conventional method. For example, what is necessary is just to stir violently, maintaining the mixture of aqueous alkali solution and a cellulose at low temperature.
- Step b Step for Producing Mixed Liquid Containing Cellulose and Water-Soluble Low-Molecular Organic Compound
- a water-soluble low-molecular organic compound is added to the cellulose fine dispersion to produce a mixed solution.
- the water-soluble low molecular weight organic compound that can be used in the present invention is not particularly limited.
- the term “low molecule” as used herein refers to an organic resin that is not polymerized by polymerization or the like, and preferably has a molecular weight of 1000 or less.
- water-soluble as used in the present invention means that it has a relatively high solubility as well as infinite solubility in water. Specifically, for example, an organic compound in which the amount of water required to dissolve 1 g or 1 mL at 20 ⁇ 5 ° C. is less than 30 mL can be used. The amount of water is more preferably less than 10 mL, and even more preferably less than 1 mL.
- Specific water-soluble low molecular weight organic compounds include alcohols such as butanol and propanol; polyols such as propylene glycol, glycerin, ethylene glycol and triethylene glycol; sugars such as sucrose and fructose; cellosolves such as methyl cellosolve and butyl cellosolve Carbitols such as diethylene glycol monomethyl ether and diethylene glycol diethyl ether; ethers such as tetraethylene glycol dimethyl ether and 1,4-dioxane; esters such as ethylene glycol monomethyl ether acetate can be used.
- alcohols such as butanol and propanol
- polyols such as propylene glycol, glycerin, ethylene glycol and triethylene glycol
- sugars such as sucrose and fructose
- cellosolves such as methyl cellosolve and butyl cellosolve
- Carbitols such as diethylene glycol mono
- the water-soluble low molecular weight organic compound that can be used in the present invention is preferably a solid at room temperature because the amount of adsorption becomes larger.
- alkyl sulfates such as alkyl sulfates and polyoxyethylene alkyl sulfates
- solid organic acids such as citric acid or salts thereof
- quaternary ammonium salts such as alkyltrimethylammonium chloride and dialkyldimethylammonium chloride
- glycine Amino acids such as alanine, serine, threonine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine or the like
- vitamins such as vitamin C and vitamin B.
- the water-soluble low-molecular organic compound is a compound having low toxicity from the viewpoint of product safety.
- water-soluble low molecular weight organic compounds with low toxicity include amino acids and vitamins.
- amino acids glycine, alanine, and lysine are preferable, and among vitamins, vitamin C and vitamin B are preferable from the viewpoint of being relatively inexpensive.
- the amount of the water-soluble low-molecular organic compound used is not particularly limited and may be adjusted as appropriate.
- the concentration of the water-soluble low-molecular organic compound in the mixed solution is about 0.1 wt% or more and about 5 wt% or less. It should just become.
- the method for adding the water-soluble low molecular weight organic compound to the cellulose dispersion is not particularly limited.
- a water-soluble low molecular organic compound may be added to the cellulose dispersion, or a water-soluble low molecular organic compound may be added during the preparation of the cellulose dispersion.
- the water-soluble low molecular weight organic compound is liquid or solid, it may be added as it is, dissolved in a solvent and added as a solution, or added as a dispersion or slurry.
- the solvent or dispersion medium there are no particular limitations on the solvent or dispersion medium in this case, and an organic solvent or water can be used.
- the temperature condition at the time of adding the water-soluble low molecular organic compound but it is preferably 25 ° C. or less from the viewpoint of preventing bead coloring.
- the water-soluble low molecular weight organic compound does not necessarily need to be uniformly dispersed or dissolved in the cellulose dispersion. However, when it is desired to uniformly disperse or dissolve the water-soluble low molecular organic compound, operations such as natural diffusion, stirring, and shaking can be performed. .
- Step c Emulsion Preparation Step
- an emulsion is prepared by dispersing the mixed liquid in a dispersion medium.
- Examples of the dispersion medium constituting the emulsion include animal and vegetable oils and fats, hydrogenated animal and vegetable oils and fats, fatty acid glycerides, aliphatic hydrocarbon solvents, and aromatic hydrocarbon solvents.
- a surfactant such as a nonionic surfactant may be used.
- palm oil, shea fat, monkey fat, iripe fat, pork fat, beef tallow, rapeseed oil, rice oil, peanut oil, olive oil, corn oil, soybean oil, perilla oil, cottonseed oil, sunflower oil, evening primrose oil, Sesame oil, safflower oil, coconut oil, cacao butter, palm kernel oil, fish oil, wakame oil, kombu oil and the like can be mentioned.
- hydrogenated animal and vegetable oils and fats mention may be made of hardened palm oil, hardened palm oil, hardened rapeseed oil, hardened rapeseed oil, hardened soybean oil, hardened tallow fat, hardened fish oil and the like.
- the fatty acid glyceride may be any of tri-, di-, and mono-glycerides, and examples thereof include stearic glyceride, palmitic glyceride, and lauric glyceride.
- examples of the aliphatic hydrocarbon solvent include beeswax, candelilla wax, rice bran wax and the like.
- examples of the aromatic hydrocarbon solvent include benzene, toluene, chlorobenzene, dichlorobenzene and the like.
- a surfactant In order to prepare an emulsion, an appropriate amount of a surfactant may be added.
- the surfactant include sorbitan fatty acid esters such as sorbitan laurate, sorbitan stearate, sorbitan oleate, and sorbitan trioleate.
- the amount of the dispersion medium used may be an amount that can sufficiently disperse the liquid mixture droplets. For example, it can be 1 mass times or more with respect to the said liquid mixture. On the other hand, if the amount of the dispersion medium is too large, the amount of waste liquid may increase excessively, and the ratio is preferably 10 times by mass or less. The ratio is more preferably 2 times by mass or more, more preferably 4 times by mass or more, more preferably 8 times by mass or less, still more preferably 7 times by mass or less, and particularly preferably 6 times by mass or less.
- the emulsion may be prepared by a conventional method. For example, it can be prepared by vigorously stirring the mixed solution containing the mixed solution, the dispersion medium and the surfactant.
- Step d Coagulation Step Next, the emulsion is brought into contact with a coagulation solvent to extract the solvent from the droplets of the cellulose fine dispersion to obtain porous cellulose beads.
- the coagulation solvent is not particularly limited as long as it has an affinity for the solvent of the fine cellulose dispersion, and examples thereof include alcohol solvents and mixed solvents of water and alcohol solvents.
- the alcohol solvent include C 1-4 alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, s-butanol, and t-butanol.
- the amount of the coagulation solvent used is not particularly limited and may be adjusted as appropriate.
- the amount of the coagulation solvent may be about 20 v / w% or more and 150 v / w% or less with respect to the used mixed solution.
- the coagulation method is not particularly limited, but since the emulsion may be unstable, it is preferable to add the coagulation solvent with vigorous stirring so that the droplets do not bind to each other.
- the coagulated porous cellulose beads may be separated by filtration or centrifugation, and washed with water or alcohol.
- the obtained porous cellulose beads may be classified using a sieve or the like in order to make the particle diameter uniform.
- Step e Crosslinking Step of Porous Cellulose Beads
- the porous cellulose beads obtained above are preferably crosslinked with a crosslinking agent to form crosslinked porous cellulose beads in order to increase the strength.
- the crosslinking agent refers to one having two or more reactive groups capable of forming a covalent bond with a hydroxyl group on cellulose and capable of crosslinking between cellulose molecules.
- crosslinking conditions or crosslinking agent of the crosslinked porous cellulose beads that can be used in the present invention.
- the method described in WO2008 / 146906 can be used.
- the entire contents of this international publication are incorporated herein by reference.
- crosslinking agent examples include halohydrins such as epichlorohydrin, epibromohydrin and dichlorohydrin; bifunctional bisepoxides (bisoxiranes); and polyfunctional polyepoxides (polyoxiranes).
- a crosslinking agent may be used individually by 1 type, and may use 2 or more types together.
- the solvent for the reaction for cross-linking the porous cellulose beads with a cross-linking agent may be selected as appropriate.
- water miscibility such as alcohol solvents such as methanol, ethanol and isopropanol, and nitrile solvents such as acetonitrile, etc. Mention may be made of organic solvents. Further, two or more crosslinking reaction solvents may be mixed and used.
- the crosslinking reaction may be performed a plurality of times, and the reaction solvent and the crosslinking agent may be changed each time.
- the first crosslinking reaction may be performed in a water-miscible organic solvent
- the final crosslinking reaction may be performed in water.
- the intermediate solvent composition may be the same as or different from either the first time or the last time, or may be an intermediate composition thereof.
- all rounds may be carried out in an aqueous solvent. The same applies to the crosslinking agent.
- a base may be added to the reaction solution.
- bases include alkali metal hydroxides such as sodium hydroxide and potassium hydroxide; alkali metal hydrogen carbonates such as sodium hydrogen carbonate and potassium hydrogen carbonate; alkali metal carbonates such as sodium carbonate and potassium carbonate; triethylamine and pyridine.
- organic bases such as
- the crosslinked porous cellulose beads are insoluble, and may be washed with a solvent such as water.
- Step f Ligand Immobilization Step
- the porous cellulose beads according to the present invention can be made into an adsorbent by immobilizing a ligand that interacts with a target product. Since the adsorbent that can be obtained in the present invention has the characteristic that there is little nonspecific adsorption, it is possible to provide highly safe drugs and treatments, and also save labor in the intermediate washing step during purification and treatment. Can be realized.
- the “ligand” refers to an affinity ligand that has a specific affinity for an object to be purified by adsorbing to an adsorbent and interacts with the object.
- the target substance is an antibody
- antigens, proteins, peptide fragments and the like that specifically interact with the antibody can be exemplified.
- the ligand that can be used for the adsorbent according to the present invention is not particularly limited as long as it has a specific affinity for an object to be purified using the adsorbent according to the present invention.
- the method for immobilizing the ligand on the porous cellulose beads according to the present invention is not particularly limited, and a conventional method can be used.
- a conventional method can be used.
- cyanogen bromide method, trichlorotriazine method, epoxy Method immobilization of amino group-containing ligands using methods such as tresyl chloride method, periodate oxidation method, divinyl sulfonic acid method, benzoquinone method, carbonyldiimidazole method, acyl azide method; epoxy method, diazo coupling method, etc.
- a method of immobilizing a hydroxyl group-containing ligand using a method a method of immobilizing a thiol group-containing ligand using an epoxy method, tresyl chloride method, divinyl sulfonic acid method, etc .; a carboxylic acid-containing ligand or formyl group on an amination carrier
- immobilization methods such as a method of immobilizing the contained ligand can be mentioned. The entire contents of this document are incorporated herein by reference.
- the adsorbent according to the present invention can be used as an adsorbent for purification, but can also be used as an adsorbent for purifying antibody drugs and a medical adsorbent that have attracted attention in recent years.
- the ligand when used in adsorbents for antibody drug purification, for example, antigens and proteins highly specific for antibodies, protein A, protein G, protein L and their variants, antibodies Examples thereof include amino group-containing ligands such as peptides having binding activity.
- an adsorbent capable of specifically adsorbing immunoglobulin (IgG) an adsorbent obtained by immobilizing protein A, protein G, or a variant thereof as a ligand on a porous carrier has attracted attention.
- the protein A or the like that can be used in the present invention is not particularly limited, and natural products and genetically modified products can be used without limitation.
- antibody binding domains, mutants thereof, those containing oligomers thereof, fusion proteins, and the like may be used.
- the number of polymerizations of the oligomer can be 2 or more and 10 or less.
- the adsorbent of the present invention in which protein A is immobilized can also be used as a therapeutic adsorbent that can be used for the treatment of dilated cardiomyopathy and the like.
- the adsorbent of the present invention in which dextran sulfate or the like is immobilized can be used as an adsorbent for treating hypercholesterolemia.
- the method for introducing the ligand into the porous cellulose beads can be selected from the various immobilization methods described above, but more preferably, the reaction between the formyl group contained in the porous particles and the amino group of the ligand is performed.
- JP-A-1-278534 The entire contents of this publication are incorporated herein by reference.
- the amount of ligand immobilized on the adsorbent of the present invention is not particularly limited, and can be, for example, 1 mg or more and 1000 mg or less per mL of porous cellulose beads. If the said ratio is 1 mg or more, since the adsorption amount with respect to a target object becomes large, it is preferable, since manufacturing cost can be suppressed if it is 1000 mg or less, it is preferable.
- the amount of ligand immobilized is preferably 2 mg or more, more preferably 4 mg or more, particularly preferably 5 mg or more, more preferably 500 mg or less, further preferably 250 mg or less, particularly preferably 200 mg or less per mL of porous cellulose beads. 100 mg or less is most preferable.
- the use of the adsorbent of the present invention is not particularly limited, but it is suitable for a medical adsorbent, especially a therapeutic adsorbent that adsorbs and removes large-sized pathogenic substances (such as LDL cholesterol) because the surface porosity can be improved. Can be used. Moreover, it can be used as various chromatographic carriers, especially industrial chromatographic carriers packed in large-diameter columns. In particular, when used as an adsorbent for antibody drug purification, which has been in great demand in recent years, the effect can be exhibited. From such a viewpoint, it can be suitably used as an adsorbent obtained by introducing protein A, protein G, or protein L into the porous beads of the present invention.
- the target product can be purified using the adsorbent according to the present invention.
- the adsorbent of the present invention may be brought into contact with a solution containing the target product.
- the contact method is not particularly limited, and the adsorbent according to the present invention may be added to a solution containing the target product.
- the column is packed with the adsorbent of the present invention to prepare a solution containing the target product.
- the target substance may be selectively adsorbed to the adsorbent of the present invention. Since the adsorbent according to the present invention has high strength, particularly when packed in a column, liquid can be passed at a high speed and the target product can be purified efficiently.
- the adsorbent of the present invention on which the target substance is selectively adsorbed is separated from the solution by filtration or centrifugation.
- the target product and other substances can be separated.
- the eluate is used to separate the object from the adsorbent of the present invention.
- an acidic buffer having a pH of about 2.5 or more and 4.5 or less can be used.
- Test Example 1 SEM Observation of Bead Surface
- the porous cellulose beads or adsorbents obtained in each production example and reference example were washed with 5 times volume of 30% ethanol, and 30 parts of the liquid contained in the porous cellulose beads were washed. Replaced with% ethanol. Subsequently, the porous cellulose beads were similarly treated using 50% ethanol, 70% ethanol, 90% ethanol, special grade ethanol, special grade ethanol, and special grade ethanol in this order, and the liquid portion was replaced with ethanol. Further, the porous cellulose beads were treated in the same manner using a mixed solution of t-butyl alcohol / ethanol 3/7.
- t-butyl alcohol / ethanol 5/5, 7/7, 9/1, 10/0, 10/0, 10/0, and the liquid portion was t.
- the porous cellulose beads that had been freeze-dried were subjected to vapor deposition treatment, and SEM images were taken.
- Test Example 2 Dynamic adsorption amount measurement in RT (Residence time) 3 minutes (1) Solution preparation The following solutions were prepared.
- Liquid A pH 7.4 phosphate buffer (manufactured by Sigma)
- Solution B 35 mM sodium acetate at pH 3.5M (prepared with acetic acid, sodium acetate, RO water manufactured by Nacalai Tesque)
- Liquid C 1M acetic acid (prepared with acetic acid and RO water manufactured by Nacalai Tesque)
- Solution D 1 mg / mL human polyclonal IgG solution (prepared with 1500 mg / 10 mL of “gamma globulin NICHIYAKU” manufactured by NICHIYAKU and solution A)
- Liquid E 6M urea (prepared with urea and RO water manufactured by Kanto Chemical Co., Inc.) Each solution was degassed before use.
- AKTAexplorer 100 manufactured by GE Healthcare Bioscience
- a 22 ⁇ m mesh is attached to a column having a diameter of 0.5 cm and a height of 15 cm, and the adsorbent of the present invention is attached.
- 3 mL each was added and filled with a 20% ethanol aqueous solution (prepared with ethanol and RO water manufactured by Wako Pure Chemical Industries, Ltd.) at a linear velocity of 450 cm / h for 1 hour.
- a 15 mL collection tube was set in the fraction collector.
- the eluate collection tube was previously filled with a neutralizing solution.
- Test Example 3 Determination of Epoxy Group Epoxidized porous cellulose beads were suction filtered (suction dry) for 15 minutes on a glass filter (“3G-2” manufactured by TOP), and 1.5 g of the porous carrier after suction drying was screwed A tube (manufactured by Maruemu) was weighed, and 4.5 mL of 1.3 M aqueous sodium thiosulfate solution (prepared with sodium thiosulfate and RO water manufactured by Wako Pure Chemical Industries, Ltd.) was added.
- Test Example 4 Measurement of LDL Cholesterol Level Anticoagulation treatment was performed by adding 500 ⁇ L of heparin to 100 mL of fresh blood of a healthy person and mixing gently. Healthy human fresh blood subjected to anticoagulation treatment was centrifuged at 3000 rpm for 15 minutes to obtain plasma having an LDL cholesterol concentration of 70 mg / dL. 3 mL of this plasma was added to 0.5 mL of the adsorbent washed with physiological saline, and shaken at 37 ° C. for 2 hours.
- the amount of LDL cholesterol in the supernatant after shaking was measured using an LDL cholesterol kit (“Coletest LDL” manufactured by Sekisui Medical), and the amount of LDL cholesterol adsorbed on the adsorbent was determined.
- LDL cholesterol kit (“Coletest LDL” manufactured by Sekisui Medical)
- the amount of LDL cholesterol adsorbed on the adsorbent was determined.
- the amount of LDL cholesterol in a solution subjected to the same operation as described above was determined except that the same amount of physiological saline was used instead of the adsorbent.
- Non-oriented protein A used in the present invention has an amino acid sequence represented by SEQ ID NO: 1. This is a part of the protein A derived from Staphylococcus aureus excluding the signal sequence (S domain) and cell wall binding domain (X domain), and is described as SPA ′ in WO2006 / 004067. The said protein A was prepared according to the method as described in the Example of WO2006 / 004067. The entire contents of this WO2006 / 004067 are incorporated herein by reference.
- Example 1 (1) Preparation of alkaline aqueous solution Using sodium hydroxide and distilled water manufactured by Wako Pure Chemical Industries, Ltd., a 33 wt% aqueous sodium hydroxide solution was prepared, and its temperature was adjusted to 4 ° C.
- the obtained crosslinked beads twice were transferred to a container, distilled water was added to make the total volume 10 times the volume of crosslinked porous cellulose beads, and the mixture was heated at 120 ° C. for 60 minutes using an autoclave. After allowing to cool to room temperature, it was washed with 5 times or more volume of distilled water of the beads to obtain autoclaved twice crosslinked beads.
- Adsorbent preparation An adsorbent with protein A immobilized thereon was prepared according to the following procedure. RO water is added to 11.0 mL of the crosslinked porous cellulose beads obtained in (5) above to make the total volume 17.0 mL, and the mixture is placed in a 50 mL centrifuge tube. This is mixed rotor at 25 ° C. After mounting on the mix rotor MR-3 "), the mixture was stirred. Next, 6.0 mL of an 8.64 mg / mL sodium periodate aqueous solution in which sodium periodate (manufactured by Wako Pure Chemical Industries, Ltd.) is dissolved in RO water is prepared, and the above-mentioned crosslinked porous cellulose beads are put therein.
- the mixture was stirred at 25 ° C. for 1 hour.
- the filtrate was washed with RO water on a glass filter (“11GP100” manufactured by Shibata Co., Ltd.) until the electric conductivity of the filtrate was 1 ⁇ S / cm or less to obtain formyl group-containing crosslinked porous cellulose beads.
- the electrical conductivity of the washing filtrate was measured with a conductivity meter (“ECTester 10 Pure +” manufactured by EUTECH INSTRUMENTS).
- the mixture was stirred using a rotor (“Mix Rotor MR-3” manufactured by ASONE). Subsequently, 0.39 mL of 5.5% aqueous dimethylamine borane (DMAB) solution (prepared with dimethylamine borane manufactured by Kishida Chemical Co., Ltd. and RO water) was added and stirred at 6 ° C. for 1 hour, and then the reaction temperature was raised to 25 ° C. The mixture was reacted at 25 ° C. for 18 hours with stirring using a mix rotor (“Mix Rotor MR-3” manufactured by ASONE). After the reaction, the UV absorbance at the absorption maximum near 278 nm of the reaction solution was measured to determine the amount of protein A introduced.
- DMAB dimethylamine borane
- the beads after the reaction were washed on a glass filter (“11GP100” manufactured by Shibata Co., Ltd.) with 3 times the volume of RO water of the beads.
- 3 times volume of 0.1N citric acid aqueous solution (prepared with citric acid monohydrate and RO water manufactured by Kanto Chemical Co., Ltd.) is added to the beads to make the total volume 30 mL or more, put in a centrifuge tube, and 25 ° C. Acid washing was performed with stirring for 30 minutes.
- the beads were washed on a glass filter (“11GP100” manufactured by Shibata) with 3 times the volume of RO water of the beads, and then 3 times the volume of 0.05 M sodium hydroxide + 1 M sodium sulfate aqueous solution ( Nacalai Tesque sodium hydroxide, Kanto Chemical sodium sulfate and RO water) were added.
- 0.05 M sodium hydroxide + 1 M sodium sulfate aqueous solution was added to the beads to make the total volume 30 mL or more, put into a centrifuge tube, and washed with alkali while stirring at room temperature for 30 minutes.
- the beads were washed with 20 times volume of RO water on the glass filter (“11GP100” manufactured by Shibata). Next, 3 times the amount of beads, 0.5N trisodium citrate aqueous solution (prepared by Kanto Chemical Co., Ltd. trisodium citrate dihydrate + RO water) was added, and the filtrate was confirmed to be neutral. Then, the washing
- the amount of protein A introduced 35 g / L (adsorbent volume), and 5% DBC at RT 3 minutes was 39 g / L (adsorbent filled volume).
- the SEM observation image of this bead surface was shown in FIG.
- Comparative Example 1 An adsorbent was prepared in the same manner as in Example 1 except that the aqueous glycine solution was not added as the water-soluble low-molecular organic compound when the cellulose dispersion was prepared.
- the amount of protein A introduced was 35 g / L (adsorbent volume), and 5% DBC at RT 3 minutes was 31 g / L (adsorbent filled volume).
- the SEM observation image of this bead surface was shown in FIG.
- Example 2 (1) Epoxidation Uncrosslinked cellulose beads produced in the same manner as in Example 1 (3) were wet classified using 38 ⁇ m and 150 ⁇ m sieves. 1 part by volume of RO water was added to 1 part by volume of the classified beads, and 1.06 part by volume of 2N sodium hydroxide aqueous solution was further added, followed by heating at 45 ° C. for 30 minutes. Next, 0.36 part by volume of epichlorohydrin was added and stirred at 45 ° C. for 2 hours. Filtration was performed on a glass filter, and the beads were washed with a large amount of RO water to obtain epoxy group-containing porous cellulose beads. The epoxy content was 34 ⁇ mol / g wet weight.
- the obtained adsorbent had an LDL cholesterol adsorption rate of 98.4% and an adsorption amount of 3.7 g / L (adsorbent volume).
- Comparative Example 2 An adsorbent was prepared in the same manner as in Example 2 except that the aqueous glycine solution was not added as the water-soluble low-molecular organic compound when preparing the cellulose dispersion.
- the adsorption rate of LDL cholesterol of the obtained adsorbent was 91.9%, and the adsorbed amount was 3.4 g / L (adsorbent volume).
- Reference example 1 An LDL cholesterol adsorption test was conducted in the same manner as in Example 3 except that the adsorbent filled in the adsorption plasma purifier “Liposorber LA-15” (manufactured by Kaneka) was used. As a result, the adsorption rate of LDL cholesterol was 93.5%, and the adsorption amount was 3.5 g / L (adsorbent volume).
- Example 3 Production of porous cellulose beads Porous cellulose beads were produced in the same manner as in Examples 1 (1) to (3) above. The obtained porous cellulose beads were sieved to collect porous cellulose beads having a particle diameter of 38 ⁇ m to 150 ⁇ m. The liquid portion contained in 100 mL of the collected porous cellulose beads was replaced with ethanol, and then transferred to a reaction vessel so that the total amount of cellulose beads and ethanol was 97 g. Thereto were added 28 g of distilled water and 80 mL of epichlorohydrin.
- the solution temperature was adjusted to 40 ° C., and 96 mL of a 1.8N NaOH aqueous solution (prepared with sodium hydroxide and distilled water manufactured by Nacalai Tesque) was added to initiate the crosslinking reaction.
- 9.6 mL of 17.0 N NaOH aqueous solution was added 1.5 hours after the start of the reaction, and 9.6 mL of 17.0 N NaOH aqueous solution was added 3 hours and 4.5 hours after the start of the reaction. After 6 hours from the start of the reaction, the gel was collected and washed with distilled water of 20 times volume by volume or more.
- the crosslinked cellulose beads obtained by the crosslinking reaction were transferred to a reaction vessel so that the total amount of cellulose beads and distilled water was 116.7 g. After 37.8 g of sodium sulfate was added and dissolved therein, 33 mL of epichlorohydrin was added and kept at 40 ° C. 21 mL of 17.0 N NaOH aqueous solution was added to start the crosslinking reaction, and 5 mL of 17.0 N NaOH aqueous solution was added 2.5 hours after the start of the reaction. After 5 hours from the start of the reaction, the gel was collected and washed with distilled water of 20 times volume or more of the beads.
- K av Measurement the porous cellulose beads 22.8mL of gel distribution coefficient was dispersed in distilled water, and degassed for 30 minutes.
- the column (“Tricorn 10/300” manufactured by GE Healthcare Japan) was packed with the degassed porous cellulose beads. Size exclusion chromatography system (“DGU-20A3”, “RID-10A”, “LC-20AD”, “SIL-20AC”, “CTO-20AC”) manufactured by Shimadzu Corporation, and “LCSolution” as software Measurement).
- the following dextran or glucose was used by dissolving in 50 mM phosphate buffer (pH 7.5) containing 1 M NaCl.
- V R represents the liquid passing amount (mL) to peak from the injection of the marker solution is observed
- V 0 is injected peaks from observation dextran solution having a molecular weight of 4 ⁇ 10 7
- the results are shown in Table 2.
- Comparative Example 3 Preparation Example of Crosslinked Porous Cellulose Beads Without Using Water-Soluble Low-Molecular Organic Compound (1)
- Preparation of Cellulose Dispersion 103 g of distilled water and 9.3 g of cellulose were put into a separable flask, and a two-stage disc The slurry was stirred at 150 to 200 rpm for 30 minutes until the temperature of the slurry reached 4 ° C. using a turbine blade. Subsequently, 41 g of 33 wt% sodium hydroxide aqueous solution cooled to 4 ° C. was added and held for 30 minutes while stirring at a speed of 500 rpm.
- Example 3 (3) Measurement of gel distribution coefficient and calculation of pore size distribution
- the obtained porous cellulose beads were crosslinked under the same conditions as in Example 3 (1), and then gel distribution was performed in the same manner as in Example 3 (2). The coefficient was measured, and the pore size distribution was calculated in the same manner as in Example 3 (3) above.
- Table 3 shows the measurement results of the gel partition coefficient Kav .
- FIG. 3 a graph of the pore size distribution is shown in FIG. 3 together with the results of Example 3 above.
- the crosslinked porous cellulose beads of Comparative Example 3 prepared without using a water-soluble low molecular organic compound have a broad pore size distribution, whereas they are prepared using a water-soluble low molecular organic compound. It was shown that the pore size distribution of the crosslinked porous cellulose beads of Example 3 was sharper.
- Example 4 Porous cellulose beads were prepared in the same manner as in Example 3 except that a 6 wt% alanine aqueous solution was used instead of the 6 wt% glycine aqueous solution.
- the gel distribution coefficient of the obtained porous cellulose beads was measured in the same manner as in Example 3 (2), and the pore size distribution was calculated in the same manner as in Example 3 (3).
- Table 4 shows the measurement results of the gel partition coefficient Kav .
- Example 3 a graph of the pore size distribution is shown in FIG. 3 together with the results of Example 3 and Comparative Example 3. As shown in FIG. 3, the pore size distribution of the crosslinked porous cellulose beads of Example 4 produced using alanine as the water-soluble low molecular organic compound was sharper than the pore size distribution of Example 3.
- Example 5 Porous cellulose beads were produced in the same manner as in Example 3 except that a 9 wt% glycine aqueous solution was used instead of the 6 wt% glycine aqueous solution.
- the gel distribution coefficient of the obtained porous cellulose beads was measured in the same manner as in Example 3 (2), and the pore size distribution was calculated in the same manner as in Example 3 (3).
- Table 5 shows the measurement results of the gel partition coefficient Kav .
- FIG. 3 A graph of the pore size distribution is shown in FIG. 3 together with the results of Example 3 and the like. As shown in FIG. 3, the pore size distribution of the crosslinked porous cellulose beads of Example 5 produced using a water-soluble low-molecular-weight organic compound at a higher concentration was even sharper.
- Example 6 Porous cellulose beads were produced in the same manner as in Example 3 except that the amount of cellulose used was changed from 9.4 g to 7.7 g.
- the gel distribution coefficient of the obtained porous cellulose beads was measured in the same manner as in Example 3 (2), and the pore size distribution was calculated in the same manner as in Example 3 (3).
- Table 6 shows the measurement results of the gel partition coefficient Kav .
- FIG. 4 a graph of pore size distribution is shown in FIG. 4 together with the results of Example 3 and Comparative Example 3. As shown in FIG. 4, the pore size distribution of the crosslinked porous cellulose beads became sharper by increasing the use ratio of the water-soluble low molecular weight organic compound to cellulose.
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Abstract
Description
b)前記セルロース微分散液に水溶性低分子有機化合物を加え混合液を作製する工程、
c)前記混合液を分散媒に分散させてエマルションを作製する工程、
d)前記エマルションを凝固溶媒に接触させる工程を含むことを特徴とする、多孔質セルロースビーズの製造方法。
本工程では、低温のアルカリ水溶液とセルロースとを混合してセルロース微分散液を作製する。
次に、前記セルロース微分散液に水溶性低分子有機化合物を加え混合液を作製する。
本工程では、前記混合液を分散媒に分散させてエマルションを作製する。
次に、前記エマルションを凝固溶媒に接触させることによりセルロース微分散液の液滴から溶媒を抽出し、多孔質セルロースビーズを得る。
以上で得られた多孔質セルロースビーズは、強度を高めるために、架橋剤により架橋して架橋多孔質セルロースビーズとすることが好ましい。
本発明に係る多孔質セルロースビーズは、目的物と相互作用するリガンドを固定化することにより、吸着体とすることができる。本発明で得ることができる吸着体は非特異吸着が少ないといった特性を有していることから、安全性が高い薬や治療の提供が可能で、さらには精製や治療時に中間洗浄工程等を省力化することが可能となる。
各製造例、参考例で得られた多孔質セルロースビーズ、または吸着体を5倍体積量の30%エタノールで洗浄し、多孔質セルロースビーズに含まれる液体部分を30%エタノールで置換した。次いで、50%エタノール、70%エタノール、90%エタノール、特級エタノール、特級エタノール、特級エタノールを順に用いて多孔質セルロースビーズを同様に処理し、液体部分をエタノールで置換した。さらにt-ブチルアルコール/エタノールが3/7の混合液を用いて多孔質セルロースビーズを同様に処理した。次いで、t-ブチルアルコール/エタノール=5/5、7/7、9/1、10/0、10/0、10/0の混合液を用いて多孔質セルロースビーズを処理し、液体部分をt-ブチルアルコールで置換した後、凍結乾燥した。凍結乾燥を行なった多孔質セルロースビーズに蒸着処理を行い、SEM像を撮影した。
(1) 溶液調製
以下の溶液を調製した。
B液:pH3.5Mの35mM酢酸ナトリウム(ナカライテスク社製の酢酸、酢酸ナトリウム、RO水で調製)
C液:1M酢酸(ナカライテスク社製の酢酸とRO水で調製)
D液:1mg/mLのヒトポリクローナルIgG溶液(ニチヤク社製「ガンマグロブリンニチヤク」1500mg/10mLとA液で調製)
E液:6M尿素(関東化学社製の尿素とRO水で調製)
各溶液は、使用前に脱気した。
カラムクロマトグラフィー用装置として、AKTAexplorer 100(GEヘルスケアバイオサイエンス社製)を用い、直径0.5cm、高さ15cmのカラムに22μmのメッシュを取り付け、本発明の吸着体をそれぞれ3mL入れ、線速450cm/hで20%エタノール水溶液(和光純薬工業社製エタノールとRO水で調製)を1時間通液して充填した。フラクションコレクターに15mLの採取用チューブをセットした。この溶出液の採取用チューブについては、あらかじめ中和液を入れておいた。
A液を線速300cm/hで9mL通液し、次いでD液を、UVをモニターしながら、IgGが10%破過するまで線速300cm/hで通液した。ここで、5%破過した時のIgG負荷量をRT3分での5%DBCとした。次いで、A液を線速300cm/hで30mL通液し、B液を線速300cm/hで30mL通液してIgGを溶出させた。次にC液を線速300cm/hで9mL、E液を線速300cm/hで9mL通液し、再生処理を行った。
エポキシ化多孔質セルロースビーズを、グラスフィルター(TOP社製「3G-2」)上で15分間吸引ろ過(サクションドライ)し、サクションドライ後の多孔質担体1.5gをスクリュー管(マルエム社製)に秤量し、1.3Mチオ硫酸ナトリウム水溶液(和光純薬工業社製チオ硫酸ナトリウムとRO水で調製)4.5mLを加えた。45℃で30分間加温した後、RO水を加えて液量を50mLとし、100mLのガラス製ビーカーに移し、1%フェノールフタレイン溶液(和光純薬工業社製フェノールフタレインとエタノールで調製)を数滴添加した。0.01N塩酸(和光純薬工業社製、容量分析用)で滴定し、エポキシ基含量を求めた。
健常人新鮮血100mLにヘパリン500μLを添加し、穏やかに混和することにより、抗凝固処理を行った。抗凝固処理した健常人新鮮血を3000rpmで15分間遠心処理を行い、LDLコレステロール濃度が70mg/dLの血漿を得た。この血漿3mLを生理食塩水で洗浄した吸着体0.5mLに加えて、37℃で2時間振盪した。振盪後の上清のLDLコレステロール量をLDLコレステロールキット(積水メディカル社製「コレステストLDL」)を用いて測定し、吸着体に吸着されたLDLコレステロール量を求めた。また比較のため、吸着体の代わりに生理食塩水を同量用いた以外、前記と同様の操作を行った溶液のLDLコレステロール量を求めた。
[式中、C0は生理食塩水を用いた吸着操作におけるLDLコレステロールの濃度を示し、C1は吸着体を用いた吸着操作におけるLDLコレステロールの濃度を示す]
製造例1 プロテインAの調製
本発明で使用した無配向型プロテインAは、配列番号1で示されるアミノ酸配列を有する。これは、Staphylococcus aureus由来プロテインAからシグナルシーケンス(Sドメイン)及び細胞壁結合ドメイン(Xドメイン)を除いた部分にあたり、WO2006/004067においてSPA’として記載されているものである。当該プロテインAを、WO2006/004067の実施例に記載の方法に準じて調製した。なおこのWO2006/004067の全内容が、本願に参考のため援用される。
(1) アルカリ水溶液の作製
和光純薬社製の水酸化ナトリウムと蒸留水を用いて、33wt%の水酸化ナトリウム水溶液を作製し、その温度を4℃に調整した。
セパラブルフラスコに70gの蒸留水と9.3gのセルロースを投入し、二段ディスクタービン(rushton turbine)翼を用いてスラリーの温度が4℃になるまで、150~200rpmで30分間攪拌した。次いで、4℃に冷却した33wt%の水酸化ナトリウム水溶液を41g添加し、500rpmの速度で攪拌しながら30分間保持した。その後、作製したセルロース分散液に水溶性低分子有機化合物として6wt%グリシン水溶液33gを添加し、600rpmの速度で15分間撹拌を行った。
上記セルロース分散液に、1wt%のソルビタンモノオレエートが溶解した788gのo-ジクロロベンゼン溶液を投入し、4℃で600rpm、15分間撹拌することでセルロース液滴を分散させた。凝固溶剤としてメタノールを74mL添加し、4℃で600rpm、30分間攪拌した。その後、ガラスフィルター(TOP社製「26G-3」)で溶液を濾過し、次いで5倍体積量のメタノール、5倍体積量の蒸留水の順に洗浄を行ない、セルロースビーズを回収した。
得られた多孔質セルロースビーズを、38μmと90μmの篩を用いて湿式分級した。
分級後の上記多孔質セルロースビーズ20mLに蒸留水を加えて30mLとし、反応容器に移した。ここに架橋剤としてグリセロールポリグリシジルエーテルを含有するデナコールEX-314(ナガセケムテックス社製)を2.3g投入し、40℃に調整しながら攪拌を続けた。40℃に調整後、30分間攪拌した。次いで、2N NaOH水溶液(ナカライテスク社製水酸化ナトリウムと蒸留水で調製)7.1mLを用意し、1時間に1/4ずつ加えた。この間、温度を40℃に維持し、攪拌も継続した。最後の1/4量を添加後、同温度で1時間攪拌した。反応終了後、吸引濾過をしながら、ビーズの20倍体積量以上の蒸留水で洗浄し、架橋1回ビーズを得た。さらに同じ架橋反応をもう1回実施し、架橋2回ビーズを得た。
下記手順に従って、プロテインAを固定化した吸着体を作製した。上記(5)で得られた架橋多孔質セルロースビーズ11.0mLに、RO水を加えて全量を17.0mLとし、50mLの遠沈管に入れ、これを25℃にてミックスローター(アズワン社製「ミックスローターMR-3」)上に取り付けた後、攪拌した。次に、過ヨウ素酸ナトリウム(和光純薬工業社製)をRO水に溶解させた、8.64mg/mLの過ヨウ素酸ナトリウム水溶液を6.0mL調製し、先程の架橋多孔質セルロースビーズを入れた遠沈管に加え、25℃で1時間攪拌した。反応後、グラスフィルター(シバタ社製「11GP100」)上で、濾液の電気伝導度が1μS/cm以下となるまでRO水で洗浄し、ホルミル基含有架橋多孔質セルロースビーズを得た。洗浄濾液の電気伝導度は、導電率計(EUTECH INSTRUMENTS社製「ECTester10 Pure+」)で測定した。得られたホルミル基含有架橋多孔質セルロースビーズ9.0mLをグラスフィルター(シバタ社製「11GP100」)上で0.5Mクエン酸三ナトリウム二水和物(関東化学社製)+0.15M塩化ナトリウム(関東化学社製)バッファー30mLで置換した。pH8の0.5Mクエン酸三ナトリウム二水和物+0.15M塩化ナトリウムバッファーを用い、置換後のホルミル基含有架橋多孔質セルロースビーズを、遠沈管に入れ、総体積量14.0mLとなるように液量を調整した。
セルロース分散液作製時に水溶性低分子有機化合物としてグリシン水溶液を添加しないこと以外は、実施例1と同様に吸着体を作製した。得られた吸着体の物性評価を行った結果、プロテインA導入量:35g/L(吸着体体積)で、RT3分での5%DBCが31g/L(吸着体充填体積)であった。また、このビーズ表面のSEM観察像を図2に示した。
(1)エポキシ化
実施例1の(3)と同様に作製した未架橋セルロースビーズを、38μmと150μmの篩を用いて湿式分級した。この分級後のビーズ1体積部にRO水1体積部を加え、さらに2N水酸化ナトリウム水溶液を1.06体積部加えて45℃で30分間加温した。次にエピクロロヒドリンを0.36体積部加えて45℃2時間攪拌した。グラスフィルター上で濾過を行い、大量のRO水でビーズを洗浄し、エポキシ基含有多孔質セルロースビーズを得た。エポキシ含有量は湿潤重量1gあたり34μmolであった。
エポキシ基含有多孔質セルロースビーズ0.7体積部に26wt/vol%のデキストラン硫酸(硫黄含量約18%、分子量約4000)水溶液を添加して、液量を1.0体積部とした。次いで2N水酸化ナトリウム水溶液を添加し、pHを9.5に調整した。その後、40℃16時間攪拌した。グラスフィルター上で濾過を行い、大量のRO水でビーズを洗浄し、デキストラン硫酸が固定化されたビーズを得た。
デキストラン硫酸固定化ビーズ1体積部にRO水1体積部とモノエタノールアミン0.25体積部を添加し、45℃で2時間攪拌し、残存エポキシ基の封止反応を行った。グラスフィルター上で濾過を行い、大量のRO水でビーズを洗浄し、目的とする吸着体を得た。
セルロース分散液作製時に水溶性低分子有機化合物としてグリシン水溶液を添加しないこと以外は、実施例2と同様に吸着体を作製した。得られた吸着体のLDLコレステロールの吸着率は91.9%で、吸着量は3.4g/L(吸着体体積)であった。
吸着型血漿浄化器「リポソーバー LA-15」(カネカ社製)に充填されている吸着体を用いた以外は、実施例3と同様にLDLコレステロール吸着試験を行った。その結果、LDLコレステロールの吸着率は93.5%で、吸着量は3.5g/L(吸着体体積)であった。
(1) 多孔質セルロースビーズの作製
上記実施例1(1)~(3)と同様にして、多孔質セルロースビーズを作製した。得られた多孔質セルロースビーズを篩い分けし、粒子径38μmから150μmの多孔質セルロースビーズを集めた。集めた多孔質セルロースビーズ100mLに含まれる液体部分をエタノールで置換した後、反応容器に移し、セルロースビーズとエタノールの合計量が97gとなるようにした。そこに蒸留水28gとエピクロロヒドリン80mLを添加した。溶液温度を40℃に調整し、1.8N NaOH水溶液(ナカライテスク社製水酸化ナトリウムと蒸留水で調製)を96mL添加し、架橋反応を開始させた。反応開始から1.5時間後に17.0N NaOH水溶液を9.6mL添加し、反応開始から3時間後と4.5時間後にも17.0N NaOH水溶液を9.6mL添加した。反応開始から6時間後にゲルを回収し、ビーズの20倍体積量以上の蒸留水で洗浄した。
上記多孔質セルロースビーズ22.8mLを蒸留水に分散させ、30分間脱気した。脱気した多孔質セルロースビーズをカラム(GEヘルスケア・ジャパン社製「Tricorn 10/300」)に充填した。島津製作所社製のサイズ排除クロマトグラフィーシステム(「DGU-20A3」、「RID-10A」、「LC-20AD」、「SIL-20AC」、「CTO-20AC」を含み、ソフトウェアとしては「LCSolution」を使用)を用いて測定を行った。
[式中、VRは各マーカー溶液を注入してからピークが観測されるまでの通液量(mL)を示し、V0は分子量4×107のデキストラン溶液を注入してからピークが観測されるまでの通液量(mL)を示し、Vtはカラム内のビーズの体積(mL)を示す]
結果を表2に示す。
[式中、rmは各マーカーの粘度半径(nm)を示し、rpは各マーカーが多孔質セルロースビーズに侵入する細孔の半径(nm)を示す]
算出された多孔質セルロースビーズの細孔半径を横軸に、分子量180のマーカーがビーズ内に侵入した細孔体積(VR-V0)を100%とした場合の細孔径分布を縦軸にプロットしたグラフを図3に示す。
(1) セルロース分散液の作製
セパラブルフラスコに103gの蒸留水と9.3gのセルロースを投入し、二段ディスクタービン(rushton turbine)翼を用いてスラリーの温度が4℃になるまで、150~200rpmで30分間攪拌した。次いで、4℃に冷却した33wt%の水酸化ナトリウム水溶液を41g添加し、500rpmの速度で攪拌しながら30分間保持した。
上記セルロース分散液に、1wt%のソルビタンモノオレエートが溶解した788gのo-ジクロロベンゼン溶液を投入し、4℃で600rpm、15分間撹拌することでセルロース液滴を分散させた。凝固溶剤としてメタノールを74mL添加し、4℃で600rpm、30分間攪拌した。その後、ガラスフィルター(TOP社製「26G-3」)で溶液を濾過し、次いで5倍体積量のメタノール、5倍体積量の蒸留水の順に洗浄を行ない、セルロースビーズを回収した。
得られた多孔質セルロースビーズを上記実施例3(1)と同様の条件で架橋した後、上記実施例3(2)と同様にゲル分配係数を測定し、また、上記実施例3(3)と同様に細孔径分布を計算した。ゲル分配係数Kavの測定結果を表3に示す。
上記実施例3において、6wt%グリシン水溶液の代わりに6wt%アラニン水溶液を用いた以外は同様にして、多孔質セルロースビーズを作製した。
上記実施例3において、6wt%グリシン水溶液の代わりに9wt%グリシン水溶液を用いた以外は同様にして、多孔質セルロースビーズを作製した。
上記実施例3において、セルロースの使用量を9.4gから7.7gに変更した以外は同様にして、多孔質セルロースビーズを作製した。
Claims (10)
- a)低温のアルカリ水溶液とセルロースとを混合してセルロース微分散液を作製する工程、
b)前記セルロース微分散液に水溶性低分子有機化合物を加え混合液を作製する工程、
c)前記混合液を分散媒に分散させてエマルションを作製する工程、
d)前記エマルションを凝固溶媒に接触させる工程を含むことを特徴とする、多孔質セルロースビーズの製造方法。 - 前記a)の工程における前記アルカリ水溶液の液温が0~25℃であることを特徴とする請求項1に記載の方法。
- 前記水溶性低分子有機化合物が、アミノ酸であることを特徴とする請求項1または2に記載の多孔質セルロースビーズの製造方法。
- 前記水溶性低分子有機化合物が、グリシン、アラニン、セリン、トレオニン、アスパラギン、グルタミン、アスパラギン酸、グルタミン酸、リシン、アルギニンおよびヒスチジンからなる群から選択される1以上のアミノ酸であることを特徴とする請求項1または2に記載の多孔質セルロースビーズの製造方法。
- 前記分散媒が、動植物油脂、水素添加動植物油脂、脂肪酸トリグリセリド、脂肪族炭化水素系溶媒および芳香族炭化水素系溶媒からなる群から選択される1以上の油溶性溶媒であることを特徴とする請求項1~4のいずれか一項に記載の多孔質セルロースビーズの製造方法。
- 前記凝固溶媒が、アルコール系溶媒、または水とアルコール系溶媒との混合溶媒であることを特徴とする請求項1~5のいずれか一項に記載の多孔質セルロースビーズの製造方法。
- 請求項1~6のいずれか一項に記載の多孔質セルロースビーズの製造方法で得られたビーズに、目的物と相互作用するリガンドを固定化したことを特徴とする吸着体。
- 請求項1~6のいずれか一項に記載の方法で製造された多孔質セルロースビーズ、および、目的物と相互作用するリガンドを含むことを特徴とする吸着体。
- 請求項1~6のいずれか一項に記載の方法で製造された多孔質セルロースビーズに、目的物と相互作用するリガンドを固定化することにより吸着体を得る工程を含むことを特徴とする吸着体の製造方法。
- 請求項7または8に記載の吸着体を用いることを特徴とする精製方法。
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Publication number | Priority date | Publication date | Assignee | Title |
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WO2018008608A1 (ja) * | 2016-07-04 | 2018-01-11 | 日本たばこ産業株式会社 | 吸着剤、その吸着剤を有する喫煙物品用フィルター、およびその喫煙物品用フィルターを有する喫煙物品 |
CN114405483A (zh) * | 2021-12-13 | 2022-04-29 | 健帆生物科技集团股份有限公司 | 具有核壳结构的多孔纤维素微球吸附剂及制备方法和应用 |
Citations (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4634470A (en) | 1983-12-26 | 1987-01-06 | Asahi Kasei Kogyo Kabushiki Kaisha | Cellulose dope, process for preparation and method for application thereof |
US5151350A (en) | 1982-10-27 | 1992-09-29 | Repligen Corporation | Cloned genes encoding recombinant protein a |
US5410034A (en) | 1994-02-24 | 1995-04-25 | The United States Of America As Represented By The Secretary Of Agriculture | Alkaline method for dissolving cellulose |
JPH09124702A (ja) | 1995-11-02 | 1997-05-13 | Nisshinbo Ind Inc | アルカリに溶解するセルロースの製造法 |
JPH09132601A (ja) * | 1995-09-06 | 1997-05-20 | Bio Polymer Res:Kk | 多孔性セルロース粒子の製造方法 |
WO2003080655A1 (en) | 2002-03-25 | 2003-10-02 | Amersham Biosciences Ab | A mutated immunoglobulin-binding protein |
WO2006004067A1 (ja) | 2004-07-06 | 2006-01-12 | Kaneka Corporation | ブレビバチルス属細菌を用いたプロテインa様蛋白質の生産方法 |
WO2006025371A1 (ja) | 2004-08-30 | 2006-03-09 | Kaneka Corporation | 顆粒球吸着材 |
JP2006304633A (ja) | 2005-04-26 | 2006-11-09 | Apro Life Science Institute Inc | イムノグロブリン結合タンパク質 |
WO2008146906A1 (ja) | 2007-05-30 | 2008-12-04 | Kaneka Corporation | ホルミル基含有多孔質担体、それを用いた吸着体、およびそれらの製造方法 |
JP2009242770A (ja) | 2007-08-31 | 2009-10-22 | Chisso Corp | 多孔性セルロースゲル、その製造方法及びその用途 |
WO2010110288A1 (ja) | 2009-03-24 | 2010-09-30 | 株式会社カネカ | 免疫グロブリンに親和性を有するタンパク質および免疫グロブリン結合性アフィニティーリガンド |
JP2010236975A (ja) | 2009-03-31 | 2010-10-21 | Tosoh Corp | 細孔を有する微生物セルロース粒子の製造方法 |
WO2012033223A1 (ja) * | 2010-09-10 | 2012-03-15 | 株式会社カネカ | 多孔質粒子の製造方法、多孔質粒子、吸着体、およびタンパク質の精製方法 |
WO2012121258A1 (ja) | 2011-03-08 | 2012-09-13 | 株式会社カネカ | 多孔質セルロースビーズの製造方法 |
JP2013211520A (ja) | 2012-01-27 | 2013-10-10 | Rohm Co Ltd | チップ抵抗器の製造方法 |
JP2013215120A (ja) | 2012-04-06 | 2013-10-24 | Shimano Inc | クーラーボックス |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP3601229B2 (ja) * | 1997-01-14 | 2004-12-15 | チッソ株式会社 | 多孔性球状セルロース粒子 |
CN101274985B (zh) * | 2008-05-12 | 2011-04-20 | 武汉大学 | 一种磁性纤维素微球及其制备方法和用途 |
JP5691233B2 (ja) * | 2010-04-23 | 2015-04-01 | Jnc株式会社 | 結晶性セルロースの溶解方法及び多孔性セルロースの製造方法 |
-
2014
- 2014-10-14 US US15/028,632 patent/US20160236171A1/en not_active Abandoned
- 2014-10-14 WO PCT/JP2014/077361 patent/WO2015056680A1/ja active Application Filing
- 2014-10-14 EP EP14854425.7A patent/EP3059251B1/en not_active Not-in-force
- 2014-10-14 JP JP2015542619A patent/JP6442409B2/ja active Active
Patent Citations (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5151350A (en) | 1982-10-27 | 1992-09-29 | Repligen Corporation | Cloned genes encoding recombinant protein a |
US4634470A (en) | 1983-12-26 | 1987-01-06 | Asahi Kasei Kogyo Kabushiki Kaisha | Cellulose dope, process for preparation and method for application thereof |
US5410034A (en) | 1994-02-24 | 1995-04-25 | The United States Of America As Represented By The Secretary Of Agriculture | Alkaline method for dissolving cellulose |
JPH09132601A (ja) * | 1995-09-06 | 1997-05-20 | Bio Polymer Res:Kk | 多孔性セルロース粒子の製造方法 |
JPH09124702A (ja) | 1995-11-02 | 1997-05-13 | Nisshinbo Ind Inc | アルカリに溶解するセルロースの製造法 |
WO2003080655A1 (en) | 2002-03-25 | 2003-10-02 | Amersham Biosciences Ab | A mutated immunoglobulin-binding protein |
WO2006004067A1 (ja) | 2004-07-06 | 2006-01-12 | Kaneka Corporation | ブレビバチルス属細菌を用いたプロテインa様蛋白質の生産方法 |
WO2006025371A1 (ja) | 2004-08-30 | 2006-03-09 | Kaneka Corporation | 顆粒球吸着材 |
JP2006304633A (ja) | 2005-04-26 | 2006-11-09 | Apro Life Science Institute Inc | イムノグロブリン結合タンパク質 |
WO2008146906A1 (ja) | 2007-05-30 | 2008-12-04 | Kaneka Corporation | ホルミル基含有多孔質担体、それを用いた吸着体、およびそれらの製造方法 |
JP2009242770A (ja) | 2007-08-31 | 2009-10-22 | Chisso Corp | 多孔性セルロースゲル、その製造方法及びその用途 |
WO2010110288A1 (ja) | 2009-03-24 | 2010-09-30 | 株式会社カネカ | 免疫グロブリンに親和性を有するタンパク質および免疫グロブリン結合性アフィニティーリガンド |
JP2010236975A (ja) | 2009-03-31 | 2010-10-21 | Tosoh Corp | 細孔を有する微生物セルロース粒子の製造方法 |
WO2012033223A1 (ja) * | 2010-09-10 | 2012-03-15 | 株式会社カネカ | 多孔質粒子の製造方法、多孔質粒子、吸着体、およびタンパク質の精製方法 |
WO2012121258A1 (ja) | 2011-03-08 | 2012-09-13 | 株式会社カネカ | 多孔質セルロースビーズの製造方法 |
JP2013211520A (ja) | 2012-01-27 | 2013-10-10 | Rohm Co Ltd | チップ抵抗器の製造方法 |
JP2013215120A (ja) | 2012-04-06 | 2013-10-24 | Shimano Inc | クーラーボックス |
Non-Patent Citations (5)
Title |
---|
AMERICAN HEART JOURNAL, vol. 152, no. 4, 2006, pages 712EL - 712E6 |
ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, vol. 1051, 2005, pages 635 - 646 |
JOURNAL OF CHROMATOGRAPHY, vol. 195, 1980, pages 221 - 230 |
KENICHI KASAI ET AL.: "Affinity chromatography", 1991, TOKYO KAGAKUDOUJIN |
See also references of EP3059251A4 * |
Cited By (5)
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WO2018008608A1 (ja) * | 2016-07-04 | 2018-01-11 | 日本たばこ産業株式会社 | 吸着剤、その吸着剤を有する喫煙物品用フィルター、およびその喫煙物品用フィルターを有する喫煙物品 |
EA037985B1 (ru) * | 2016-07-04 | 2021-06-21 | Джапан Тобакко Инк. | Адсорбент, фильтр для курительного изделия, снабженный указанным адсорбентом, и курительное изделие, снабженное указанным фильтром для курительных изделий |
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CN114405483A (zh) * | 2021-12-13 | 2022-04-29 | 健帆生物科技集团股份有限公司 | 具有核壳结构的多孔纤维素微球吸附剂及制备方法和应用 |
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JPWO2015056680A1 (ja) | 2017-03-09 |
EP3059251B1 (en) | 2019-04-03 |
EP3059251A4 (en) | 2017-07-19 |
US20160236171A1 (en) | 2016-08-18 |
JP6442409B2 (ja) | 2018-12-19 |
EP3059251A1 (en) | 2016-08-24 |
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