JP3941980B2 - Cellulosic carrier and method for producing the same - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a perfusion type cellulosic carrier and a method for producing the same.
[0002]
[Prior art]
Particles having through-pores that penetrate the particles and sub-pores that are interconnected to the through-pores and have a pore diameter smaller than that of the through-pores are used as a carrier for chromatography and for affinity chromatography. When filled in a container as a carrier or enzyme immobilization carrier and passed through at an appropriate linear velocity, the movement of solutes in the carrier is faster (perfusion effect) than conventional carriers that do not have through pores. It is known that each target work can be accomplished at high speed (Japanese Patent Publication No. 4-5000726 and Japanese Patent Publication No. 6-507313). In the present specification, a carrier composed of particles having the above-described through pores and subpores is referred to as a perfusion type carrier.
[0003]
As a perfusion type carrier, POROS (trade name), a chromatography carrier manufactured by Perceptive Biosystems, is marketed. This is a carrier in which small particles of a styrene-divinylbenzene copolymer are assembled.
Conventionally available perfusion-type carriers have a small particle size (10 × 10 ⁻⁶ to 50 × 10 ⁻⁶ m), and pass through at a high linear velocity of 2.8 × 10 ⁻³ m / s or more. Produces a perfusion effect. However, when a container filled as a carrier and a solution obtained from a culture tank, a slurry solution, blood, or the like is passed at a high linear velocity, clogging is likely to occur due to the small particle size. For example, when blood was passed, blood cells were clogged in the column or at the column inlet, causing hemolysis. Further, when the liquid passing speed is increased, clogging occurs in a short time.
[0004]
However, a perfusion-type carrier that produces a perfusion effect when the particle diameter is large and the liquid is passed at a low linear velocity has not been known so far. Furthermore, a cellulose-based carrier has not been known as a perfusion type carrier.
[0005]
[Problems to be solved by the invention]
In view of the above, an object of the present invention is to provide a cellulosic carrier capable of increasing the particle diameter and producing a perfusion effect even when liquid is passed at a relatively low linear velocity, and a method for producing the same.
[0006]
[Means for Solving the Problems]
The present invention is a perfusion-type cellulosic carrier, wherein porous cellulosic small particles are suspended in an alkaline solution to form a suspension, and the suspension is brought into contact with an acidic solution, It is a cellulose carrier made of a cellulosic particle body in which the cellulosic small particles are connected to each other so that voids are provided between the particles of the cellulosic small particles.
[0007]
Further, the present invention provides a suspension by suspending porous cellulose-based small particles in an alkaline solution, and contacting the suspension with an acidic solution so that voids are formed between the particles of the cellulose-based small particles. The perfusion type cellulosic carrier is produced by connecting the cellulosic small particles to each other.
The present invention is described in detail below.
[0008]
The alkaline solution used in the present invention is not particularly limited, and examples thereof include a sodium hydroxide aqueous solution, a lithium hydroxide aqueous solution, a potassium hydroxide aqueous solution, a cesium hydroxide aqueous solution, and a rubidium hydroxide aqueous solution.
To the alkaline solution, glycerin, a water-soluble polymer or the like may be added for viscosity adjustment.
[0009]
The alkaline solution preferably has a pH of 13 or more (solution concentration of 0.1 N or more). More preferably, the pH is 14.3 or more (solution concentration is 2N or more). When the pH is less than 13, when contacting the acidic solution as a suspension containing cellulosic small particles, the cellulosic small particles are dispersed with each other and cannot be connected. The value of pH is dissociation degree = 1 in the acid or an aqueous solution of alkali, [H ^+] × ^- was determined from the equation = 10 pH = -log ₁₀ as ^-14 [H ^+] [OH]. Therefore, for example, when the concentration of the alkaline solution is 0.1 N, the pH is 13.
[0010]
Cellulose small particles used in the present invention are conventionally used for applications such as gel filtration agents, cellulosic ion exchanger raw materials, affinity chromatography carriers, polymer carriers, body fluid purification carriers, cosmetic additives, etc. Can be used.
[0011]
The said cellulose small particle is comprised from cellulose materials, such as a cellulose, a cellulose derivative, a regenerated cellulose, for example.
The cellulose is not particularly limited, and examples thereof include those obtained by degreasing cotton fibers, hemp, pulp obtained from wood, and purified cellulose obtained by purifying pulp.
[0012]
The cellulose derivative is not particularly limited, and examples thereof include those obtained by esterifying a part of the hydroxyl group of cellulose (ester derivative); those obtained by etherifying the hydroxyl group of cellulose (ether derivative).
The ester derivative of cellulose is not particularly limited, and examples thereof include cellulose acetate, cellulose propionate, nitrocellulose, cellulose phosphate, cellulose acetate butyrate, cellulose nitrate, and cellulose dithiocarboxylate (viscose rayon). Can do.
The ether derivative of cellulose is not particularly limited, and examples thereof include methyl cellulose, ethyl cellulose, benzyl cellulose, trityl cellulose, cyanoethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, aminoethyl cellulose, and oxyethyl cellulose.
[0013]
The regenerated cellulose is a cellulose derivative that is easy to mold once and then converted into cellulose again after being molded. Specifically, for example, cellulose ester derivatives such as cellulose acetate and cellulose propionate, etc. And the like prepared by hydrolysis.
[0014]
The cellulose-based small particles are porous having a pore size corresponding to the application. The porous cellulosic small particles can be produced, for example, by the methods disclosed in JP-A-63-90501, JP-A-63-92602, and the like. Specifically, for example, the cellulose-based small particles can be produced by the following method or the like.
[0015]
(1) An aqueous alkaline polymer solution containing cellulose xanthate and a water-soluble polymer compound and a water-soluble anionic polymer compound are mixed to prepare a fine particle dispersion of the aqueous alkaline polymer solution. Heat or mix with a cellulose xanthate coagulant to coagulate the cellulose xanthate in the dispersion as fine particles. At this time, since the cellulose xanthate fine particles contain a water-soluble polymer compound, they are removed. Subsequently, the cellulose xanthate fine particles are neutralized with an acid to regenerate the cellulose to obtain the cellulose-based small particles.
[0016]
In addition to the above, the cellulose xanthate fine particles can be solidified by adding an acid to the dispersion. In this case, after removing the water-soluble polymer compound, the added acid is neutralized to regenerate cellulose to obtain the cellulose-based small particles.
[0017]
(2) Viscose, calcium carbonate and a water-soluble anionic polymer compound are mixed to produce a fine particle dispersion of viscose containing calcium carbonate, and the dispersion is heated or mixed with a coagulant. The viscose in the dispersion is coagulated and then neutralized with an acid to produce cellulose fine particles. Then, the cellulose fine particles are obtained by separating the cellulose fine particles from the dispersion, removing calcium carbonate by acid decomposition, and drying.
[0018]
The cellulosic carrier of the present invention is formed by suspending the cellulosic small particles in the alkaline solution to form a suspension, and contacting the suspension with an acidic solution.
[0019]
The time for suspending the cellulose-based small particles in the alkaline solution is preferably 1 minute or longer. When it is less than 1 minute, it is difficult to sufficiently connect the cellulose-based small particles. More preferably, it is 1 hour or more.
[0020]
The suspension concentration of the cellulose-based small particles is preferably 50 to 75% by volume.
The suspension concentration is the ratio of the total volume of cellulosic small particles in the suspension to the volume of the suspension.
When the suspension concentration of the cellulose-based small particles is less than 50% by volume, when the droplets of the suspension are brought into contact with an acidic solution, a fragment-like cellulose-based molded body is obtained and the strength thereof is weak, 75 If the volume% is exceeded, droplets having a smooth surface cannot be obtained, and the shape of the cellulose-based molded body becomes a lump. More preferably, it is 60-70 volume%.
[0021]
The droplets preferably have an average diameter of 3 × 10 ⁻³ m or less. When the average diameter exceeds 3 × 10 ⁻³ m, the action exerted by the surface tension becomes small and it becomes difficult to form droplets.
[0022]
The method of making the suspension into droplets is not particularly limited, and examples thereof include a method of discharging the suspension into a gas phase from a capillary and a method of using a sprayer. Among them, it is preferable to use a nebulizer or the like because fine droplets can be obtained.
[0023]
The type of the atomizer is not particularly limited as long as it is an apparatus that can subdivide the average diameter of the droplets to 3 × 10 ⁻³ m or less. For example, a rotating disk type, a pressure nozzle type, a two-fluid nozzle type Can be mentioned.
[0024]
The above rotating disk type sprayer is to spray a solution on a high-speed disk, shake off the solution by centrifugal force, and collide with a gas such as air to be atomized.
The pressure nozzle type sprayer discharges a high-pressure solution from a small hole and causes the solution to collide with a gas such as ambient air and spray the solution.
The above two-fluid nozzle type atomizer sprays the solution itself with a high-speed gas by a compressed gas even when the pressure is low.
[0025]
The acidic solution preferably has a pH of 1 or less (solution concentration of 0.1 N or more). More preferably, the pH is −0.3 (solution concentration of 2 N or more) or less. When pH exceeds 1, when making it contact with an acidic solution as a suspension containing a cellulose small particle, the said cellulose small particle will be in the state disperse | distributed mutually, and it is difficult to connect.
[0026]
It does not specifically limit as said acidic solution, For example, hydrochloric acid aqueous solution, sulfuric acid aqueous solution, nitric acid aqueous solution, phosphoric acid aqueous solution etc. can be mentioned.
In order to adjust the viscosity, glycerin, a water-soluble polymer, or the like may be added to the acidic solution.
[0027]
The method of bringing the droplets of the suspension into contact with the acidic solution is not particularly limited. For example, the method of immersing the droplets in the acidic solution; the acid solution is refined, for example, atomized. Examples thereof include a method of bringing a droplet into contact.
The time for bringing the droplets of the suspension into contact with the acidic solution is preferably 1 minute or longer. If it is less than 1 minute, the cellulose-based small particles cannot be sufficiently connected. More preferably, it is 1 hour or more.
[0028]
In the cellulose-based particle body, the mode in which the cellulose-based small particles are connected to each other does not necessarily have to be based on a covalent bond, and substantially maintains a bonded state between the particles. Any state that can do this is acceptable. For example, the connection of cellulose-based small particles includes connection by entanglement of cellulose molecules between particles, connection by chemical bonds such as hydrogen bonds, and the like.
[0029]
The average diameter of the cellulose-based particles is preferably such that the ratio of the average diameter of the cellulose-based particles to the average diameter of the cellulose-based small particles is less than 50. When the value of the ratio is 50 or more, the voids serving as through pores are reduced, and the perfusion effect is reduced.
The average diameter of the cellulose-based particle body is appropriately set according to the application. Usually, it is preferably 20 × 10 ^{−6 to} 3 × 10 ⁻³ m.
When filling the cellulosic particles into a container and passing a solvent that easily clogs, the average diameter of the cellulosic carrier to be used is preferably 100 × 10 ⁻⁶ m or more, In the range where clogging does not occur, 3 × 10 ⁻⁴ m / s or more is preferable. When the average diameter is less than 100 × 10 ⁻⁶ m, clogging is likely to occur, and when the liquid passing speed is less than 3 × 10 ⁻⁴ m / s, the perfusion effect is small and the object is achieved. Inefficient work per hour.
[0030]
The cellulosic particles preferably have a specific surface area of 2 × 10 ⁴ m ² / kg or more in the BET method when dried. If it is less than 2 × 10 ⁴ m ² / kg, the working area depending on the application becomes small. More preferably, it is 5 × 10 ⁴ m ² / kg or more.
[0031]
The cellulosic carrier of the present invention uses the cellulosic particle body itself described in detail above as the carrier itself. The cellulosic particles are composed of a plurality of cellulosic small particles interconnected with the cellulosic small particles so as to provide voids between the cellulosic small particles, and the voids between the particles become pores. The pores of the plurality of small cellulose particles after connection facing the through pores become subpores. The shape is usually spheroid or spherical.
[0032]
The cellulosic carrier of the present invention can be used for multiple purposes by setting the cellulosic small particles having a pore size according to the application and the above-mentioned diameter ratio. Examples thereof include gel filtration carriers, raw materials for cellulosic ion exchangers, carriers for affinity chromatography, carriers for adsorption of fragrances and chemicals, immobilized carriers for cells and enzymes, carriers for body fluid purification, and the like.
[0033]
In the production method of the present invention, the cellulose-based particles are suspended by suspending porous cellulose-based small particles in an alkaline solution, and the suspension is brought into contact with an acidic solution. The cellulosic small particles are produced by interconnecting the small cell particles so that voids are provided between the small particles.
[0034]
The method for producing a cellulose-based carrier of the present invention can easily connect the cellulose-based small particles and can form voids between the cellulose-based small particles. It is suitable for manufacturing. In addition, the method for producing a cellulose-based carrier of the present invention does not use an organic solvent in the production process, is easy to wash, and is very preferable for preventing environmental pollution.
[0035]
【Example】
Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.
[0036]
Example 1
Porous cellulose small particles (manufactured by Chisso Corporation) having an average diameter of 20 × 10 ⁻⁶ m were mixed in a 6N aqueous sodium hydroxide solution (pH = 14.8) so that the suspension concentration was 70% by volume. . After sufficiently stirring with a stirrer, a droplet of this suspension was brought into contact with a 5N aqueous hydrochloric acid solution (pH = −0.7) with a capillary having a hole diameter of 0.7 × 10 ⁻³ m. Obtained.
The particle size was about 2 × 10 ⁻³ m. The obtained cellulose carrier was washed with pure water.
The liquid in the obtained cellulosic carrier was replaced with ethanol, then with 2-methyl-2-propanol, freeze-dried using a freeze dryer (manufactured by Eiko Eng. CO Ltd.), and gold was added. After vapor deposition, when observed with a scanning electron microscope (manufactured by Topcon Corporation), as shown in FIG. 1, the obtained cellulose-based carrier had a spherical shape. As shown in FIG. 3, there were voids between the connected cellulose small particles. Moreover, as shown in FIG. 4, the pores of the porous cellulose small particles after connection were also observed.
[0037]
Comparative Example 1
Porous cellulose small particles having an average diameter of 20 × 10 ⁻⁶ m (manufactured by Chisso Corporation) were mixed in pure water so that the suspension concentration was 70% by volume. After sufficiently stirring with a stirrer, the droplets of this suspension were brought into contact with a 5N aqueous hydrochloric acid solution (pH = −0.7) with a capillary having a hole diameter of 0.7 × 10 ⁻³ m. Became dispersed.
[0038]
Comparative Example 2
Porous cellulose small particles (manufactured by Chisso Corporation) having an average diameter of 20 × 10 ⁻⁶ m were mixed in a 6N aqueous sodium hydroxide solution (pH = 14.8) so that the suspension concentration was 70% by volume. . After sufficiently stirring with a stirrer, the droplets of this suspension were brought into contact with pure water with a capillary having a hole diameter of 0.7 × 10 ⁻³ m, whereby a disk-shaped cellulose molded body was obtained. When shaken, the shape of the compact collapsed and the small cellulose particles were dispersed.
[0039]
Comparative Example 3
Porous cellulose small particles (manufactured by Chisso Corporation) having an average diameter of 20 × 10 ⁻⁶ m were mixed in a 6N aqueous sodium hydroxide solution (pH = 14.8) so that the suspension concentration was 40% by volume. . After sufficiently stirring with a stirrer, the droplets of this suspension were brought into contact with a 5N aqueous hydrochloric acid solution (pH = −0.7) with a capillary having a hole diameter of 0.7 × 10 ⁻³ m. A cellulose molded product was obtained. When shaken, the shape of the compact collapsed and the small cellulose particles were dispersed.
[0040]
Comparative Example 4
Porous cellulose small particles having an average diameter of 20 × 10 ⁻⁶ m (manufactured by Chisso Corporation) were mixed in a 6N aqueous sodium hydroxide solution (pH = 14.8) so that the suspension concentration was 80% by volume. . After sufficiently stirring with a stirrer, a droplet of this suspension was brought into contact with a 5N aqueous hydrochloric acid solution (pH = -0.7) with a capillary having a hole diameter of 0.7 × 10 ⁻³ m. A droplet having a smooth surface was not formed, and the shape of the obtained cellulose-based molded body was a lump.
[0041]
Example 2
Porous cellulose small particles having an average diameter of 20 × 10 ⁻⁶ m (manufactured by Chisso Corporation) were mixed in a 6N aqueous sodium hydroxide solution (pH = 14.8) so that the suspension concentration was 70% by volume. A suspension was prepared and sufficiently stirred with a stirrer. Compressed nitrogen gas was ejected from the outer nozzle of a two-fluid nozzle (having an inner nozzle and an outer nozzle on a concentric circle), and at the same time, the suspension was discharged from the inner nozzle. The nitrogen injection pressure was 5 × 10 ³ kg / m ² , and the suspension discharge speed was 5.19 × 10 ⁻⁴ m ³ / s. A two-fluid nozzle having an inner nozzle diameter of 2.6 × 10 ⁻³ m and an outer nozzle diameter of 4.4 × 10 ⁻³ m was used. The discharge height was 4 m. The cellulosic carrier of the present invention was obtained in an acidic solution. The average particle size was about 200 × 10 ⁻⁶ m.
[0042]
The liquid in the obtained cellulose-based carrier is substituted with ethanol, then substituted with 2-methyl-2-propanol, freeze-dried using a freeze dryer (manufactured by Eiko Eng. CO Ltd.), and gold is deposited. Then, as observed with a scanning electron microscope (manufactured by Topcon Corporation), as shown in FIG. 5, the obtained cellulose-based carrier had a spherical shape. As shown in FIG. 6, there were voids between the connected cellulose small particles. Moreover, as shown in FIG. 7, the pores of the cellulose-based small particles after connection could be observed.
[0043]
Comparative Example 5
Porous cellulose particles (average diameter 179 × 10 ⁻⁶ m) (manufactured by Chisso Corp.) having the same structure (pore size, etc.) as the cellulose small particles used in Examples 1 and 2 and Comparative Examples 1 to 4 but having different average diameters A 23.2 degree physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) was allowed to flow through the packed column (inner diameter 0.01 m, length 0.05 m) at a linear velocity of about 5 × 10 ⁻⁴ m / s. 100 × 10 ⁻⁹ m ³ of a solution obtained by diluting a protein (L-2139, manufactured by SIGMA) 5 times with physiological saline was injected in a pulsed manner. The time-dependent change in the concentration of the low density lipoprotein was measured using an absorbance meter (manufactured by ATTO) at a wavelength of 280 nm. As shown in FIG. 8, it was confirmed that the peak top position was immediately after starting to elute. The cellulose particles used have pores that allow low density lipoproteins to enter the particles. Therefore, the result of the elution curve is not because there is no pore that allows the low-density lipoprotein to enter the cellulose particles, but because the particle size is large, the mass transfer distance is long and the low-density lipoprotein is This is considered to be due to elution from the outlet of the column together with the flow between the cellulose particles packed in the column without sufficiently moving into the cellulose particles.
[0044]
Example 3
Column (inner diameter 0.01 m, length 0) packed with the carrier (average particle size: about 200 × 10 ⁻⁶ m, ratio of average diameter of cellulose carrier to average diameter of cellulose small particles = 10) obtained in Example 2 .05m) 23.2 degree physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) was allowed to flow at a linear velocity of about 5 × 10 ⁻⁴ m / s, and low density lipoprotein (L-2139, manufactured by SIGMA) was physiological saline. The solution 100 × 10 ⁻⁹ m ³ diluted 5 times with was pulse-injected. The time-dependent change in the concentration of the low density lipoprotein was measured using an absorbance meter (manufactured by ATTO) at a wavelength of 280 nm. As shown in FIG. 9, the peak top position was confirmed to be slower than that of Comparative Example 5. The carrier used in this example was a small cellulose particle (particle size: about 20 × 10 ⁻⁶ ) having the same pores as the cellulose particles (particle size: 179 × 10 ⁻⁶ m) used in Comparative Example 5. m), a perfusion type carrier (particle diameter: about 200 × 10 ⁻⁶ m). Therefore, even if the particle size of the carrier is large, the result of the above elution curve is that the mass transfer of the low density lipoprotein in the carrier is accelerated by making the structure a perfusion type, and the low density lipoprotein is in the carrier. It is thought that it was because it was able to move enough.
[0045]
【The invention's effect】
Since the cellulosic carrier of the present invention has the above-described configuration, it is suitable for adsorption of gel filtration carrier, cellulosic ion exchanger raw material, affinity chromatography carrier, fragrance, medicine, etc., depending on its size and internal structure. It can be suitably used for applications such as a carrier, a microbial cell / enzyme immobilization carrier, and a body fluid purification carrier. Moreover, since the manufacturing method of the cellulose carrier of this invention is as above-mentioned, the cellulose carrier of this invention can be manufactured easily.
[Brief description of the drawings]
1 is a photograph of the surface of a cellulosic carrier of Example 1 magnified 40 times. FIG.
FIG. 2 is a photograph in which the particle cross section of the cellulose-based carrier of Example 1 is enlarged 40 times.
FIG. 3 is a photograph in which the cross section of the cellulosic carrier of Example 1 is magnified 500 times.
4 is a photograph of a cross section of the cellulose carrier of Example 1 magnified 5000 times. FIG.
5 is a photograph in which the particle surface of the cellulosic carrier of Example 2 is magnified 200 times. FIG.
6 is a photograph in which the particle surface of the cellulosic carrier of Example 2 is magnified 1000 times. FIG.
7 is a photograph in which the particle surface of the cellulosic carrier of Example 2 is magnified 5000 times. FIG.
FIG. 8 shows an elution curve of low density lipoprotein based on Comparative Example 5.
FIG. 9 shows an elution curve of low-density lipoprotein based on Example 3.

Claims (6)

A perfusion-type cellulosic carrier, in which porous cellulose-based small particles are suspended in an alkaline solution having a pH of 13 or more, and the suspension concentration of the cellulose-based small particles is 50 to 75% by volume and, the suspension is contacted to a pH of 1 or less of the acid solution, so as to provide a gap between the particles of the cellulose-based fine particles, formed by mutually by connecting the cellulose-based fine particle, the particle size A perfusion-type cellulosic carrier comprising a cellulosic particle having a particle size of 20 × 10 ^{−6 to} 3 × 10 ⁻³ m . The average diameter of the cellulosic particle body, cellulosic average diameter ratio of the value of claims 1 Symbol placement of perfusion type is less than 50 cellulosic carrier cellulosic particle body to the mean diameter of small particles. The average diameter of the cellulosic particles is 100 × 10 ⁻⁶ m or more, and a perfusion effect occurs when the particles are filled in a container and passed through at a linear velocity of 3 × 10 ⁻⁴ m / s or more. perfusion type cellulosic carrier of claim 1 or 2 wherein. A through-pore passing through the cellulosic particle body and a pore interconnected with the through-pore, the sub-pore having a smaller pore diameter than the through-pore, The perfusion-type cellulosic carrier described. pH cellulosic porous by suspending small particles suspended concentration of cellulosic small particles and 50 to 75 volume% of the suspension to 13 or more alkaline solution, the suspension, the pH is 1 Production of a perfusion-type cellulosic carrier characterized in that the cellulosic small particles are connected to each other so as to provide a void between the cellulosic small particles by contacting with the following acidic solution. Method. 6. The perfusion according to claim 5, wherein the perforations have through-pores penetrating through the cellulosic particles and sub-pores interconnected to the through-pores and having a pore diameter smaller than that of the through-pores. Method for producing a cellulosic carrier of the mold.

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