WO2018186222A1 - Billes de cellulose poreuses et adsorbant - Google Patents

Billes de cellulose poreuses et adsorbant Download PDF

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Publication number
WO2018186222A1
WO2018186222A1 PCT/JP2018/012221 JP2018012221W WO2018186222A1 WO 2018186222 A1 WO2018186222 A1 WO 2018186222A1 JP 2018012221 W JP2018012221 W JP 2018012221W WO 2018186222 A1 WO2018186222 A1 WO 2018186222A1
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cellulose
beads
dope
porous cellulose
adsorbent
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PCT/JP2018/012221
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English (en)
Japanese (ja)
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義和 河井
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株式会社カネカ
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Priority to JP2019511163A priority Critical patent/JPWO2018186222A1/ja
Publication of WO2018186222A1 publication Critical patent/WO2018186222A1/fr

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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
    • 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
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • 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
    • B01J20/282—Porous sorbents
    • 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
    • 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
    • C08B1/08—Alkali cellulose
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B15/00—Preparation of other cellulose derivatives or modified cellulose, e.g. complexes
    • C08B15/10—Crosslinking of cellulose
    • 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
    • C08J9/26—Working-up of macromolecular substances to porous or cellular articles or materials; After-treatment thereof by elimination of a solid phase from a macromolecular composition or article, e.g. leaching out

Definitions

  • the present invention relates to a porous cellulose bead and an adsorbent.
  • adsorbents for purification of polymer drugs such as medical adsorbents and antibody drugs
  • polysaccharides such as agarose and cellulose with less nonspecific adsorption are preferred.
  • Porous cellulose beads are difficult to be crushed due to their relatively high mechanical strength, and they contain various hydroxyl groups that can be used to introduce ligands that interact with the target substance to be adsorbed. It is used as a base material for various adsorbents such as adsorbents and affinity adsorbers.
  • affinity adsorbents have been used as adsorbents for medical treatment and pharmaceutical purification because they can efficiently purify target substances or reduce the concentration of unwanted substances.
  • an adsorbent in which protein A is immobilized on a porous carrier using an affinity ligand is attracting attention as an adsorbent for treating rheumatism, hemophilia, dilated cardiomyopathy, or an adsorbent for purifying antibody drugs (eg, non-patent literature). 1, 2).
  • porous cellulose beads often involves complicated steps compared to general synthetic polymer beads because it is difficult to dissolve cellulose in a general solvent.
  • a cellulose solution is prepared by dissolving it in a highly corrosive and highly difficult solvent, such as a 60% high-concentration calcium thiocyanate aqueous solution.
  • Patent Document 1 It is known that the cellulose solution used in this method exhibits unique behavior, and the porous cellulose beads obtained by this method have considerably large pores and a wide pore size distribution ( For example, Non-Patent Document 3).
  • porous cellulose beads obtained by the method are used as an adsorbent such as an antibody, the specific surface area is expected to be small, and high adsorption performance cannot be expected.
  • a method of giving a substituent to the hydroxyl group of cellulose, dissolving in a general-purpose solvent, granulating, and removing the substituent after granulation to obtain a porous cellulose carrier Is exemplified (for example, Patent Document 2), but the process is complicated.
  • Patent Document 3 discloses cellulose that is soluble in an alkaline solution.
  • the microfibril has a fiber diameter of 1 ⁇ m or less, further 500 nm or less, and is subjected to special fine processing. Yes.
  • microbial cellulose is dissolved in an alkaline solution to prepare a cellulose solution. After adding a continuous phase solvent, the cellulose solution is made into droplets, and then the microbial cellulose particles are frozen and then washed. Although a method for obtaining cellulose beads is disclosed, energy is required to freeze the cellulose solution together with the continuous phase solvent. Moreover, microbial cellulose is a special raw material, and it is difficult at present to stably obtain a large amount thereof.
  • Patent Document 5 reported that a cellulose dope can be produced at a relatively high temperature using an aqueous solution containing sodium hydroxide and urea, and that porous beads can be obtained from this cellulose dope. Yes.
  • Patent Document 6 a high-performance porous cellulose bead and an adsorbent using the same can be obtained by adding a certain additive to a cellulose dispersion obtained by treating a general-purpose cellulose raw material with a slightly low-temperature alkaline aqueous solution. It has been reported that it can be obtained.
  • Non-Patent Document 4 adsorption using a target substance that is fluorescently labeled is used.
  • a behavioral analysis method has been shown, and it has been reported that a site that is easily adsorbed in a certain adsorbent particle varies depending on ionic strength. In other words, it is possible to obtain the optimum ion intensity in advance by preliminary examination using such transmission observation, which is very significant.
  • Non-Patent Document 5 the time-dependent change in the adsorption behavior into the adsorbent particles is observed and it is shown that the adsorption proceeds to the center of the beads in about 40 minutes. That is, the optimum adsorption time can be obtained by preliminary examination using such transmission observation.
  • Non-Patent Document 6 reports a method for obtaining mass transfer characteristics by analyzing an experiment in which a fluorescently labeled antibody is adsorbed on a protein A-immobilized adsorbent using a fluorescence microscope. From this result, a precise purification method is reported. It is suggested that can be constructed.
  • the present invention has been made in view of the above-mentioned problems of the prior art, and does not use a corrosive or highly toxic auxiliary material, and does not go through complicated steps that are industrially disadvantageous. In order to obtain high-performance porous cellulose beads that form a homogeneous transmission observation image at low cost.
  • Porous cellulose comprising a step of cooling a cellulose dope prepared by mixing an alkaline aqueous solution and raw material cellulose powder to a temperature lower than ⁇ 12 ° C., and a step of adjusting the cellulose dope to a temperature higher than 15 ° C.
  • the manufacturing method of the porous cellulose bead characterized by including.
  • a method for purifying a target compound comprising: A step of producing an adsorbent by immobilizing a ligand that binds to a target substance on crosslinked porous cellulose beads by the production method according to [9] above; and A method comprising a step of bringing a solution containing a target substance into contact with an adsorbent.
  • FIG. 6 is a transmission observation image of beads obtained in Comparative Example 4.
  • 18 is a transmission observation image of beads obtained in Comparative Example 14.
  • 3 is a transmission observation image of beads obtained in Example 2.
  • FIG. It is a related figure of the cellulose concentration in dope in each granulation example, and solid content after granulation.
  • 10 is a transmission observation image of beads obtained in Comparative Example 21.
  • 2 is a pressure flow rate characteristic diagram of a crosslinked porous cellulose bead having a median particle diameter of 90 ⁇ m and a 20% compression stress of 0.11 MPa and an adsorbent of Reference Example 1.
  • FIG. 2 is a K av measurement result of the crosslinked porous cellulose beads and the adsorbent of Reference Example 1.
  • a cellulose dope prepared by mixing an alkaline aqueous solution and raw material cellulose powder is cooled to a temperature lower than ⁇ 12 ° C., and the cellulose dope is adjusted to a temperature higher than 15 ° C. after the cooling step.
  • It is a manufacturing method of the porous cellulose bead characterized by including a process.
  • Patent Document 6 and the like for producing a cellulose bead by dispersing cellulose in a low temperature sodium hydroxide aqueous solution and making it porous, but a specific example having the above-mentioned characteristics has not yet been seen. It has not been issued.
  • the present inventor has started development of porous cellulose beads having a homogeneous transmission observation image with an optical microscope in a method using a low-temperature alkaline aqueous solution.
  • the homogeneity of the transmission observation image means that, when observed at an appropriate magnification, an average of two or less portions that look like foreign matters having a length of 10 ⁇ m or more are in the beads.
  • the present inventor produced porous cellulose beads by lowering the temperature at which the dope was cooled than in the example of Patent Document 6. As a result, it was found that the transmission observation image of the dope by the optical microscope tends to be homogenized as the temperature for cooling the dope is lowered.
  • a method for producing porous cellulose beads comprising a step of cooling a cellulose dope produced by mixing an alkaline aqueous solution and raw material cellulose powder to a temperature lower than ⁇ 12 ° C. After cooling, it discovered that said subject could be solved by including the process of adjusting a cellulose dope to temperature higher than 15 degreeC before emulsification and porosification.
  • cellulose is dispersed in a low-temperature alkaline aqueous solution to obtain a cellulose dope. Then, by adjusting the temperature of the cellulose dope to above 15 ° C., porous cellulose beads exhibiting high adsorption performance It has also been found that can be obtained.
  • the present inventor can use an optical microscope as long as the value of [solid content of porous cellulose beads after granulation] / [solid content other than auxiliary materials in dope] is less than 1.35. It has been found that porous cellulose beads having a uniform transmission observation image and exhibiting high adsorption performance can be easily obtained.
  • the value of [Solid content of porous cellulose beads after granulation] / [Solid content other than auxiliary materials in dope] is more preferably 0.7 or more and less than 1.35. When the value is 0.7 or more, porous cellulose beads having excellent mechanical strength and a large specific surface area can be easily obtained.
  • the present inventors have found that when the value is less than 1.35, it is easy to obtain porous cellulose beads having a narrow pore size distribution.
  • the value is more preferably 0.8 or more and less than 1.31, and most preferably 0.9 or more and less than 1.28.
  • “granulation” means emulsification of cellulose dope and obtaining cellulose cellulose porous beads by contacting cellulose dope droplets in the emulsion with a coagulation solvent
  • “after granulation” refers to the situation in which no special operation has been performed after the step of making porous, which involves a change in the mass of the solid content of the bead. Refers to the dry weight per volume completely settled.
  • Solid content in dope refers to a component that is solid at room temperature alone before the dope preparation, and among these, “sub-material” refers to a porous process, a washing process, a crosslinking process, a ligand after the dope preparation. It refers to what is removed in the immobilization process, purification process, etc. For example, sodium hydroxide is considered a secondary material.
  • the temperature for cooling the cellulose dope is more preferably ⁇ 15 ° C. or lower. Further, if the temperature for cooling the cellulose dope is ⁇ 20 ° C. or higher, it is preferable from the viewpoint of energy cost required for cooling and equipment cost required for stirring.
  • “cellulose dope” refers to a dispersion in which fine cellulose particles or cellulose fibers are dispersed in an alkaline aqueous solution. The length of fine cellulose particles or cellulose fibers in the cellulose dope is ideally 10 ⁇ m or less.
  • the time for cooling the cellulose dope at a temperature lower than ⁇ 12 ° C. is not particularly limited, but if it is 30 minutes or longer, there is a tendency to obtain a more homogeneous porous cellulose bead with a transmission observation image obtained by an optical microscope. preferable. Moreover, if it is 60 minutes or more, a more preferable porous cellulose bead can be obtained. In addition, if the cooling is continued for a considerably long time, the cellulose dope tends to discolor, the viscosity increases, or gelation tends to occur. Therefore, the cooling time is preferably 48 hours or less, and 16 hours.
  • the above time can be defined as the time after the temperature of the cellulose dope reaches a predetermined temperature of ⁇ 12 ° C. or lower.
  • the cellulose dope temperature before the cooling step is preferably 25 ° C. or lower. If the said temperature is 25 degrees C or less, coloring of a cellulose dope will decrease and defects, such as generation
  • the temperature before the cooling step is more preferably ⁇ 5 ° C. or higher, more preferably ⁇ 2 ° C. or higher, particularly preferably ⁇ 1 ° C. or higher. Most preferably. Especially, 15 degrees C or less is more preferable, and 10 degrees C or less is still more preferable.
  • the alkali used in the present invention can be used without particular limitation as long as it shows alkalinity when it becomes an aqueous solution.
  • Lithium hydroxide, sodium hydroxide and potassium hydroxide are preferable from the viewpoint of availability, and sodium hydroxide is most preferable from the viewpoint of product safety and price.
  • the alkali concentration of the aqueous alkali solution is not particularly limited, but is preferably 3% by mass or more and 20% by mass or less. If the alkali concentration is within this range, the dispersibility / swellability of cellulose in an alkaline aqueous solution is increased, which is preferable.
  • the concentration of alkali is more preferably 5% by mass or more and 15% by mass or less, and further preferably 7% by mass or more and 10% by mass or less.
  • the “auxiliary agent” refers to an agent that promotes dispersion of cellulose in an alkaline aqueous solution or stabilizes the cellulose dope.
  • the concentration of the auxiliary agent in the dope is preferably 3% by mass or more and 30% by weight or less. If the concentration of the auxiliary agent is within this range, the homogeneity of the dope is preferably increased.
  • the concentration of the auxiliary agent is more preferably 8% by mass or more and 30% by mass or less, further preferably 10% by mass or more and 20% by mass or less, and most preferably 12% by mass or more and 15% by mass or less.
  • the type of cellulose is not particularly limited. Since the present applicant has developed a method for obtaining porous cellulose beads without completely dissolving cellulose as shown in Patent Document 6, cellulose introduced with a substituent for increasing solubility is disclosed. For example, it is not necessary to use substituted cellulose, and ordinary unsubstituted cellulose is used as a raw material. However, 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 degree of polymerization is 50 or more and 500 or less, further preferably 100 or more and 400 or less, particularly preferably 200 or more and 350 or less, and most preferably 250 or more and 350 or less.
  • the concentration of cellulose in the cellulose dope is not particularly limited and may be appropriately adjusted.
  • the concentration may be about 1% by mass or more and 20% by mass or less.
  • the concentration is preferably 2% by mass or more, more preferably 3% by mass or more, particularly preferably 3.8% by mass or more, and most preferably 4.0% by mass or more from the viewpoint of the mechanical strength of the porous beads. It is. Further, from the viewpoint of adsorption performance and dope homogeneity, it is preferably 10% by mass or less, more preferably 8% by mass or less, particularly preferably 6% by mass or less, and most preferably 5% by mass or less.
  • the method for preparing the cellulose dope may be according to a conventional method. For example, what is necessary is just to stir, maintaining the mixture of aqueous alkali solution and a cellulose at low temperature.
  • the cellulose dope according to the present invention it is preferable to filter the cellulose dope according to the present invention because the homogeneity of the dope is further improved and porous cellulose beads showing a more uniform transmission observation image tend to be obtained. From the viewpoint of quality control in the medical and pharmaceutical fields, it is preferable to perform filtration because a step of filtering the dope may be required.
  • the cellulose dope of Patent Document 6 is quite difficult to filter, and the state of the obtained porous cellulose beads is greatly different depending on the presence or absence of filtration.
  • the cellulose dope concerning this invention can implement filtration with a very simple instrument. Even if the cellulose concentration in the dope is a relatively high concentration such as 4% by weight or 5% by weight, the filtration can be carried out easily.
  • the filtration method and filter medium that can be used in the present invention, and an appropriate method / apparatus may be used.
  • the pore diameter of the filter medium There is no particular limitation on the pore diameter of the filter medium.
  • beads for affinity chromatography for industrial use are often 50 ⁇ m or more, and if foreign matter larger than this is mixed, removal after granulation becomes difficult. Therefore, the pore size of the filter medium is preferably 50 ⁇ m or less. . In small scale / high speed refining, a small particle size is often used. In this case, a filter medium having a pore size of 40 ⁇ m or less can be used. When it is desired to widen the particle size distribution, smaller beads may be included, and the pore size of the filter medium is preferably 30 ⁇ m or less.
  • the pore diameter of the filter medium is 0.1 ⁇ m or more, it is preferable because liquid feeding can be performed smoothly, more preferably 1 ⁇ m or more, still more preferably 5 ⁇ m or more, and most preferably 10 ⁇ m or more.
  • the pore diameter of the filter medium refers to the average pore diameter.
  • the material and properties of the filter medium can be used without any particular limitation.
  • the said filtration may be performed with respect to the cellulose dope after the said cooling process, and may be performed with respect to the cellulose dope after the temperature adjustment process of a postscript.
  • the temperature of the cellulose dope is adjusted to be higher than 15 ° C.
  • this process may be abbreviated as “temperature adjustment process”. If the said temperature is higher than 15 degreeC, there will be no limitation in particular. If the temperature is higher than 15 ° C., the value of [solid content of porous cellulose beads after granulation] / [solid content other than auxiliary materials in dope] tends to be smaller. Further, when the temperature is increased, the value of [solid content of the porous cellulose beads after granulation] / [solid content other than the auxiliary material in the dope] is further decreased. Preferably, it is more preferably 25 ° C or higher.
  • the temperature of the step of adjusting the cellulose dope to a temperature higher than 15 ° C is preferably higher than 25 ° C, more preferably 30 ° C or higher, further preferably 35 ° C or higher, and 40 ° C or higher. It is particularly preferred.
  • the said temperature is less than 60 degreeC, it becomes easy to maintain the sphericity of a porous cellulose, and since the mechanical strength of a bead is hard to fall, it is preferable. Moreover, it is still more preferable that it is 50 degrees C or less.
  • the temperature at a temperature just before the start of the porosification is water-based, if alcohol or the like is used for making the pores, heat is generated during mixing and the temperature rises by several degrees C., but the temperature described above is the temperature before this rise. It is convenient and preferable to manage.
  • the time of the temperature adjustment step is not particularly limited and may be adjusted as appropriate. For example, after adjusting the temperature of the cellulose dope to a predetermined temperature of 15 ° C. or higher, stirring may be continued for 1 minute or longer. Although the upper limit of the said time is not specifically limited, For example, it can be 60 minutes or less. From the viewpoint of the homogeneity and storage stability of the cellulose dope, the time is preferably 5 minutes or more, more preferably 10 minutes or more, still more preferably 15 minutes or more, more preferably 45 minutes or less, and more preferably 30 minutes or less. Preferably, 25 minutes or less is even more preferable.
  • the method for obtaining the porous cellulose beads from the cellulose dope according to the present invention is not particularly limited, and a conventionally known method can be used. Especially, the manufacturing method including the emulsification process which throws a cellulose dope into a continuous phase solvent and makes it a droplet is preferable from the point of the simplicity of an installation.
  • Examples of the continuous phase solvent 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.
  • animal and vegetable oils and fats palm oil, shea fat, monkey fat, iripe fat, pork fat, beef tallow, rapeseed oil, rice oil, peanut oil, olive oil, corn oil, soybean oil, perilla oil, cottonseed oil, sunflower oil, evening primrose oil, Sesame oil, safflower oil, coconut oil, cacao butter, palm kernel oil, fish oil, wakame oil, kombu oil and the like can be mentioned.
  • the fatty acid glyceride may be any of tri-, di-, and mono-glycerides, and examples thereof include stearic glyceride, palmitic glyceride, and lauric glyceride.
  • the aliphatic hydrocarbon solvent include beeswax, candelilla wax, rice bran wax and the like.
  • 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 continuous phase solvent used may be an amount capable of sufficiently dispersing the cellulose dope droplets. For example, it can be 1 mass times or more with respect to the cellulose dope. On the other hand, if the amount of the continuous phase solvent is too large, the amount of waste liquid may increase excessively, and the ratio is preferably 10 times by mass or less. Moreover, 7 mass times or less are more preferable, and 5 mass times or less are still more preferable. In addition, when the amount of the continuous phase solvent is small relative to the cellulose dope, it becomes an O / W / O emulsion in which the continuous phase solvent enters the droplets of the cellulose dope, and as a result, porous beads having a homogeneous structure cannot be obtained. Therefore, the ratio is preferably 2 times or more, more preferably 3 times or more, and particularly preferably 4 or more.
  • the emulsion may be prepared by a conventional method. For example, it can prepare by stirring the liquid mixture containing the said cellulose dope, a continuous phase solvent, and surfactant.
  • Porous cellulose beads having a value of the solid content of the porous cellulose beads] / [the solid content other than the auxiliary material in the dope] of less than 1.35 are preferred because they tend to be easily obtained.
  • Porous cellulose beads are obtained by adding a coagulation solvent to the emulsion and extracting the solvent in the cellulose dope droplets.
  • the coagulation solvent is not particularly limited as long as it has an affinity for the solvent of the cellulose dope, 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. For example, it can be about 20 v / w% or more and 150 v / w% or less with respect to the cellulose dope.
  • the coagulation method is not particularly limited, but when a step of emulsification is included, it is preferable to add the coagulation solvent in a state of being appropriately stirred so that the droplets are not bonded 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.
  • the porous cellulose beads of the present invention are preferably crosslinked porous cellulose beads obtained by allowing a crosslinking agent to act, because it is easy to provide an adsorbent suitable for high-speed purification.
  • a crosslinking agent for example, the method described in WO2008 / 146906 can be used.
  • the crosslinking step may be performed by adding a crosslinking agent to the cellulose dope following the temperature adjustment step, or the porous cellulose beads may be crosslinked by acting a crosslinking agent.
  • 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.
  • the porous cellulose beads according to the present invention can be used as an adsorbent by immobilizing a ligand that interacts with a target substance. Since the adsorbent that can be obtained in the present invention has the characteristic that there is little nonspecific adsorption, it is possible to provide highly safe drugs and treatments, and also save labor in the intermediate washing step during purification and treatment. Can be realized. In addition, since the porous cellulose beads of the present invention have high alkali resistance, an adsorbent capable of alkali cleaning can be obtained by immobilizing an alkali resistant ligand.
  • the “ligand” in the present invention refers to an affinity ligand that has a specific affinity for a target substance to be purified by adsorbing to an adsorbent and interacts with the target substance.
  • the target substance is an antibody, an antigen, protein, or peptide fragment that specifically interacts with the antibody
  • the target compound is a ligand of the enzyme, an enzyme using the ligand as a substrate
  • the target compound is the antigen
  • an antibody against the target antigen can be mentioned.
  • the ligand that can be used for the adsorbent according to the present invention is not particularly limited as long as it has a specific affinity for the target substance to be purified using the adsorbent according to the present invention.
  • the method for immobilizing the ligand on the porous cellulose beads 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, a tresyl chloride method, a divinyl sulfonic acid method, etc .; a carboxylic acid-containing ligand or a 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.
  • the amount of ligand immobilized on the adsorbent of the present invention is not particularly limited, and can be, for example, 1 mg or more and 300 mg or less per mL of porous cellulose beads. If the said ratio is 1 mg or more, since the adsorption amount with respect to a target substance becomes large, it is preferable since it can suppress manufacturing cost if it is 300 mg or less.
  • the amount of ligand immobilized is preferably 2 mg or more, more preferably 4 mg or more, particularly preferably 5 mg or more, more preferably 100 mg or less, further preferably 50 mg or less, particularly preferably 30 mg or less, per mL of porous cellulose beads. 20 mg or less is most preferable.
  • the use of the adsorbent of the present invention is not particularly limited, but it is suitable for a medical adsorbent, especially a therapeutic adsorbent that adsorbs and removes large-sized pathogenic substances (such as LDL cholesterol) because the surface porosity can be improved. Can be used. Moreover, it can be used as various chromatographic carriers, especially industrial chromatographic carriers packed in large-diameter columns. In particular, when used as an adsorbent for antibody drug purification, which has been in great demand in recent years, the effect can be exhibited. From such a viewpoint, it can be suitably used as an adsorbent obtained by introducing protein A, protein G, or protein L into the porous beads of the present invention.
  • the present invention can provide a porous cellulose bead that can be easily adjusted in particle size, has good trueness, and exhibits an appropriate compressive stress, and therefore accurately meets the need for column fabrication that is relatively difficult to fabricate.
  • the appropriate compressive stress is not particularly limited as long as it can be appropriately filled and used according to the application, but the stress when the settled beads are compressed by 20% is 0.01 MPa or more. If it is less than 1.0 MPa, it is preferable because good adsorption performance can be imparted.
  • a method for adjusting the compressive stress include a method of adjusting the cellulose concentration in the cellulose dope and the degree of crosslinking.
  • the target substance 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 substance.
  • the contact method is not particularly limited, and the adsorbent according to the present invention may be added to a solution containing the target substance.
  • the column is filled with the adsorbent of the present invention, and the solution containing the target substance is prepared.
  • the target substance may be selectively adsorbed to the adsorbent of the present invention by passing the liquid. Since the adsorbent according to the present invention has high strength, particularly when packed in a column, liquid can be passed at a high speed, and the target substance can be purified efficiently.
  • the adsorbent of the present invention on which the target substance is selectively adsorbed is separated from the solution by filtration or centrifugation.
  • the adsorbent and the solution are easily separated.
  • the target substance and other substances can be separated.
  • the target substance is separated from the adsorbent of the present invention using the eluate.
  • the eluate for example, an acidic buffer having a pH of about 2.5 or more and 4.5 or less can be used.
  • a long intermediate washing step may be required as a pre-elution step, but the porous cellulose beads of the present invention do not necessarily require such an intermediate washing step. do not do.
  • porous cellulose beads of the present invention have high alkali resistance, they can be washed with an alkaline washing liquid that can be prepared inexpensively and easily.
  • sodium hydroxide When sodium hydroxide is used, it can be washed without problems even if its concentration is 0.1 N, and it can be used even if it is 0.5 N or more if the alkali resistance of the ligand is high.
  • Test Example 1 Measurement of dynamic adsorption amount (1) Solution preparation The following A to E solutions and neutralization solutions were prepared and defoamed before use.
  • Solution A A PBS buffer solution having a pH of 7.4 was prepared using “Phosphor buffered saline” manufactured by Sigma and distilled water.
  • Liquid B A 35 mM aqueous sodium acetate solution having a pH of 3.5 was prepared using acetic acid, sodium acetate, and distilled water.
  • C liquid 1M acetic acid aqueous solution was prepared using acetic acid and distilled water.
  • Solution D An aqueous IgG solution having a concentration of 3 mg / mL was prepared using a polyclonal antibody (“Gamma Guard” manufactured by Baxter) and the solution A.
  • Solution E An aqueous solution in which the concentrations of sodium hydroxide and sodium chloride manufactured by Wako Pure Chemical Industries, Ltd. were 0.1N sodium hydroxide and 1M sodium chloride, respectively, was prepared and used as an alkaline cleaning solution.
  • Neutralization solution A 2M tris (hydroxymethyl) aminomethane aqueous solution was prepared with tris (hydroxymethyl) aminomethane and ultrapure water.
  • the dynamic adsorption amount of IgG was determined from the amount of IgG adsorbed on the adsorbent and the adsorbent volume until IgG broke through 5%.
  • the dynamic adsorption amount is referred to as 5% DBC.
  • Static adsorption amount The static adsorption amount of IgG was calculated
  • the static adsorption amount is referred to as SBC.
  • Test Example 2 Measurement of 20% compressive stress (1) Sample preparation Pure water was added to sample beads to prepare a slurry having a concentration of about 50% by volume. A homogenization / defoaming operation consisting of homogenization by stirring the slurry and subsequent defoaming by depressurization for 30 minutes or more was repeated three times to obtain a defoamed slurry. Separately from this operation, the object to be treated was changed to pure water, and the above homogenous / demethod operation was performed for 90 minutes or more to obtain defoamed water.
  • Test Example 3 K av: Measurement porous cellulose beads 22.8mL of gel phase 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
  • Test Example 4 Measurement of solid content About 5 mL of sample beads were placed in a 15 mL centrifuge tube, and vibration was applied until the sample bead volume was not reduced any more, and the volume at that time was accurately measured. Hereinafter, such a volume is referred to as a “sedimentation volume”. Next, the beads in the centrifuge tube were transferred to a 3G glass filter and filtered. Note that the weight of the 3G glass filter was measured in advance by drying overnight in a 122 ° C. oven. Then, it was dried overnight in an oven at 122 ° C. and weighed. The solid content of the bead sample was calculated by dividing the weight by the volume.
  • Test Example 5 Measurement of pressure flow rate characteristics Beads having a predetermined column volume and the same sedimentation volume were prepared, and water was added thereto to prepare a 50% slurry. This 50% slurry was put into a column and allowed to stand for 2 hours or more. Next, water was passed from the top, and the flow rate was increased until the bead surface reached a predetermined column volume. The measurement was performed using AK Health Pure AKTA Pure 150 or AKTA Pilot, and the relationship between the linear velocity and the column differential pressure was examined.
  • Test Example 6 Measurement of median particle size The median particle size of beads and adsorbents was determined using a laser diffraction / scattering particle size distribution analyzer ("Partica LA950" manufactured by HORIBA, Ltd.).
  • Example 1 Production of Porous Cellulose Beads (1) Compound to be used As cellulose, crystalline cellulose “PH-F20JP” manufactured by Asahi Kasei Chemicals Corporation was used. Urea manufactured by Wako Pure Chemical Industries, Ltd. was used. The alkaline aqueous solution was prepared using sodium hydroxide and distilled water manufactured by Wako Pure Chemical Industries. As a dispersion medium, o-dichlorobenzene manufactured by Wako Pure Chemical Industries, Ltd. was used. As a surfactant, sorbitan monooleate (span 80 equivalent) manufactured by Wako Pure Chemical Industries, Ltd. was used.
  • Denacol EX321 manufactured by Nagase ChemteX Corporation was used as a water-soluble crosslinking agent to be added to the dope.
  • As the crosslinking agent after granulation epichlorohydrin manufactured by Wako Pure Chemical Industries, Ltd. was used. Other reagents were used without purification unless otherwise specified.
  • the cellulose dope was charged into 979.8 g of o-dichlorobenzene solution in which 9.8 g of sorbitan monooleate was dissolved, and after cooling at 600 rpm for 15 minutes, as shown in Table 2
  • the cellulose droplets were dispersed by stirring at the adjusted temperature.
  • As a solidifying solvent 87 mL of methanol adjusted to the same temperature as the adjusted temperature after cooling shown in Table 2 was added, and stirred at 600 rpm for 20 minutes. Thereafter, 14 g of acetic acid was added and neutralized by stirring at 600 rpm for 10 minutes.
  • the solution was filtered through a glass filter “26G-3” manufactured by TOP, and then washed with ethanol and water to collect porous cellulose beads.
  • Epoxy ring-opening treatment The resulting crosslinked beads and 50% slurry of water were heated in an autoclave at 121 ° C. for 60 minutes to open the epoxy groups to form diol groups. The disappearance of the epoxy group can be confirmed with a phenolphthalein indicator.
  • Example 2 Preparation of adsorbent with immobilized ligand (1) Aldehydation reaction (1-1) Preparation of buffer 0.165 g of citric acid monohydrate and 0.0646 g of trisodium citrate dihydrate and water was added to make 100 mL, and a pH 3.4 buffer was prepared.
  • FIG. 1 shows a transmission observation image of the beads obtained in Comparative Example 4.
  • This granulation example is particularly deeply related to the example of Patent Document 6 that is considered to have high adsorption performance, but as described above, a homogeneous transmission observation image cannot be obtained, and the length in the bead is 10 ⁇ m.
  • the above-mentioned average number of foreign matters is also over 2, and there is a possibility that the analysis as in Non-Patent Document 6 becomes difficult.
  • the transmission observation images had the same tendency.
  • FIG. 2 shows a transmission observation image of the beads obtained in Comparative Example 14 of the granulation example. Although the average number of foreign substances was reduced, an image was obtained as if there was a structure with low permeability in the beads. In addition, the number of foreign matters could not be measured due to the structure having a low transmittance. Similar observation images were also obtained in Comparative Examples 12, 13, and 15-19. As shown in Table 2, Comparative Examples 12 to 15 were those in which the dope cooling temperature was ⁇ 15 ° C. in order to reduce the number of foreign matters, but all were [solid content of porous cellulose beads after granulation] / The value of [Solid content other than auxiliary materials in dope] was large.
  • cellulose is dispersed in a low-temperature alkaline aqueous solution. Therefore, the present inventor considered that the lower the cooling process temperature, the more inhomogeneous observation images due to foreign matters in the beads are eliminated and the analysis as in Non-Patent Document 6 becomes possible. As shown in FIG. 2, an unfavorable observation image different from inhomogeneity due to foreign matter or the like was obtained.
  • FIG. 3 shows a transmission observation image of the beads obtained in Example 2.
  • the dope cooling temperature was set to ⁇ 15 ° C., and then the dope temperature was raised to 25 ° C., but as shown in FIG. 3, the transmission observation images were good.
  • the value of [Solid content of porous cellulose beads after granulation] / [Solid content other than auxiliary materials in dope] was good. It is believed that some of the condensation effects described above may have been mitigated.
  • Example 2 is similar to Comparative Example 4 and [Solid content of porous cellulose beads after granulation] / [Solid content other than auxiliary material in dope, which is closely related to Patent Document 6 which is considered to have high adsorption performance.
  • FIG. 4 shows the relationship between the cellulose concentration in the dope and the solid content after granulation in each granulation example. It is proved that a series of main granulation examples have found an example in which the value of [solid content of porous cellulose beads after granulation] / [solid content other than auxiliary materials in dope] can be skillfully controlled. It was done.
  • Test Example 7 Observation of dope A small amount of the cellulose dope being produced was extracted in the middle of the cooling step in the same manner as in Example 2 and observed under a microscope, and the number of foreign matters was measured. The results are shown in Table 3.
  • Comparative Example 20 A cellulose dope was prepared in the same manner as in Comparative Example 4, and the dope was subjected to centrifugal filtration at 2500 ° C. at 4 ° C. The dope supernatant was used to emulsify and make porous as in Comparative Example 4 to obtain porous cellulose beads. As a result of permeation observation of the obtained porous cellulose beads, an observation image similar to that in FIG. 1 was obtained. The solid content of the beads was 3.8 w / v%.
  • Test Example 8 Filtration Example of Cellulose Dope A cellulose dope was prepared in the same manner as in Example 2, and a glass filter (“26G-3” manufactured by TOP, pore size: 16 to 40 ⁇ m) and an aspirator (“AS-01” manufactured by ASONE, Inc.) And suction filtration was carried out in a 25 ° C. atmosphere. Filtration was performed smoothly.
  • Example 5 Porous cellulose beads were obtained in the same manner as in Example 2 except that the cellulose dope filtered in Test Example 8 was used. The physical properties of the obtained porous cellulose beads were also the same as in Example 2. It can be said that beads were obtained through a production method preferable from the viewpoint of removing foreign substances required for adsorbents for pharmaceutical purification and adsorbents for medical use. Further, when applied to the analysis as in Non-Patent Document 6, it can be expected that a more precise test can be performed.
  • the dope supernatant obtained in the same manner as in Comparative Example 7 was filtered in the same manner as in Comparative Example 1 to obtain porous cellulose beads by the same method as in Comparative Example 20.
  • the solid content of the beads was 3.1 w / v%, which was smaller than those of Comparative Example 4 and Comparative Example 20, an observation image with extremely low transmittance was obtained as shown in FIG.
  • the cellulose dope produced by a conventionally known method is difficult to filter. It can also be seen that the effect of achieving the object of the present invention cannot be obtained even if the centrifugation of the foreign matter is attempted. Furthermore, when the supernatant obtained by centrifuging a conventional cellulose dope was filtered, it was found that the observed image was further deteriorated.
  • Test Example 9 Pressure Flow Rate Characteristics of Crosslinked Porous Cellulose Beads
  • Crosslinked porous cellulose beads having a median particle size of 90 ⁇ m were obtained from Comparative Example 4, Crosslinking Examples, and Classification Examples.
  • the 20% compressive stress of this crosslinked porous cellulose bead was 0.11 MPa.
  • Reference Example 1 MabSelect SuRe LX manufactured by GE Healthcare, which is well known as a high-adsorption type agarose protein A resin for large columns, was used.
  • FIG. 6 shows a comparison of the pressure flow characteristics of the two.
  • the column used had an inner diameter of 5 cm, a packed bed height of 5 cm, and the amount of packed beads was 98.1 mL in terms of sedimentation volume. As shown in FIG.
  • the crosslinked porous cellulose beads according to the present granulation example, the crosslinking example, and the classification example are equal to or more than the reference example 1 if the median particle size is 90 ⁇ m or more and the 20% compression stress is 0.11 MPa or more. It can be seen that the pressure flow rate characteristic is shown.
  • Test Example 10 Adsorption performance of adsorbent Table 4 shows the adsorption performance and 20% compressive stress value of the adsorbent using crosslinked porous cellulose beads having a particle size adjusted to 90 ⁇ m.
  • the adsorbent having a median particle size of 90 ⁇ m obtained from Example 2 relating to the present invention has a 20% compressive stress of 0.11 MPa, which is equivalent to Reference Example 1 known for large columns. It is suggested to show pressure flow characteristics. Also, the adsorption performance was inferior to that of Comparative Example 4 reported as a highly adsorbent cellulose protein A resin. Moreover, although this adsorption
  • the porous cellulose beads of the present invention since the porous cellulose beads of the present invention have a good permeation observation image, it can contribute to labor saving in the optimization study of the purification method. Also, from the results of the adsorbent obtained from a series of granulation examples, if the value of [solid content of porous cellulose beads after granulation] / [solid content other than auxiliary materials in dope] is too large, It was also found that the adsorption performance, particularly SBC, tends to be low.
  • Test Example 11 Adsorption performance of adsorbent Table 5 shows the adsorption performance of an adsorbent using crosslinked porous cellulose beads having a particle size adjusted to 50 ⁇ m.
  • Reference Example 2 shows the results of TOYOPEARL AF-rProtein A HC-650F manufactured by Tosoh Corporation, which is attracting attention as a small particle size protein A resin for a continuous chromatographic system.
  • the adsorbent having a median particle size of 50 ⁇ m obtained from Example 2 relating to the present invention has an adsorption characteristic equal to or higher than that of the protein A resin of Reference Example 2 which is attracting attention for use in a continuous chromatographic system.
  • the present invention an excellent porous cellulose bead that can provide an adsorbent for a continuous chromatographic purification system can be provided.
  • Test Example 12 K av inverse of the crosslinked porous beads obtained in the measurement 7 in each granulation example plot of K av measurements of the adsorbent of the method and the cross-linking porous cellulose beads crosslinked with Reference Example 1
  • the curve of size exclusion chromatography (ISEC) is shown. If the value of K av is large, the pore volume in the marker molecule is large. When the slope of this curve is steep, the pore size distribution is relatively sharp, and when the slope is gentle, the pore size distribution is broad. In general, the former tends to have a larger particle specific surface area and saturated adsorption amount.
  • the sufficiently small marker molecule K av can be regarded as the intraparticle porosity of the bead.

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Abstract

La présente invention aborde le problème consistant à fournir des billes de cellulose poreuses à faible adsorption non spécifique présentant des caractéristiques de pores permettant une bonne image d'observation par transmission et un niveau d'adsorption élevé par un procédé simple peu toxique et faiblement corrosif ; et à fournir, à l'aide desdites billes, des billes de cellulose poreuses réticulées, ainsi qu'un adsorbant, une colonne et un procédé de purification les utilisant. Le problème a pu être résolu en inventant un procédé de production de billes de cellulose poreuses caractérisé en ce qu'il comprend une étape de refroidissement d'une solution de cellulose à filer préparée par mélange d'une solution aqueuse alcaline et d'une matière première à base de poudre de cellulose à une température inférieure à -12 °C et une étape d'ajustement de la température de la solution de cellulose à filer à une température supérieure à 15 °C.
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02235944A (ja) * 1989-03-09 1990-09-18 Nisshinbo Ind Inc 多孔性セルロース及びセルロース誘導体粒子の製造方法
JPH02235902A (ja) * 1989-03-09 1990-09-18 Nisshinbo Ind Inc ザンタイト架橋を有するセルロース及びセルロース誘導体粒子およびその製造方法
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
JP2011231152A (ja) * 2010-04-23 2011-11-17 Jnc Corp 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
JP2015199868A (ja) * 2014-04-09 2015-11-12 東ソー株式会社 多孔性架橋セルロースゲル、その製造方法及びその用途

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02235944A (ja) * 1989-03-09 1990-09-18 Nisshinbo Ind Inc 多孔性セルロース及びセルロース誘導体粒子の製造方法
JPH02235902A (ja) * 1989-03-09 1990-09-18 Nisshinbo Ind Inc ザンタイト架橋を有するセルロース及びセルロース誘導体粒子およびその製造方法
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
JP2011231152A (ja) * 2010-04-23 2011-11-17 Jnc Corp 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
JP2015199868A (ja) * 2014-04-09 2015-11-12 東ソー株式会社 多孔性架橋セルロースゲル、その製造方法及びその用途

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