JPH1160601A - Cellulose-based carrier and its preparation - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a cellulose-based carrier with a large particle size which brings about a perfusion effect even on a liquid fed at a relatively low linear velocity by contacting a suspension of small porous cellulose-based particles in an alkaline solution with an acid solution. SOLUTION: A suspension is contacted with an acid solution to link together small cellulose-based particles so that a space is formed among the small cellulose-based particles thereby forming a cellulose-based carrier comprising cellulose-based particle aggregates. Preferably, the pH of an alkaline solution is 13 or higher, the concentration of the small cellulosebased particles in the suspension is 50-75 vol. % and the pH of the acid solution is 1 or below. The average diameter of the cellulose-based particle aggregates is preferably such that the ratio thereof to the average diameter of small cellulose-based particles is less than 50. The average diameter of the cellulose-based particle aggregates is preferably 20×10<-6> to 3×10<-3> m in usual.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a perfusion type cellulosic carrier and a method for producing the same.

[0002]

2. Description of the Related Art Particles having through-pores penetrating particles and sub-pores which are interconnected with the through-pores and have a smaller pore diameter than the through-pores are used as a carrier for chromatography. , A carrier for affinity chromatography,
When filled in a container as a carrier for enzyme immobilization and passed at an appropriate linear velocity, compared to a conventional carrier without through-pores,
It is known that the movement of the solute in the carrier becomes faster (perfusion effect) and achieves the respective target operations at a high speed (Japanese Unexamined Patent Publication No. 4-500726,
JP-T-Hei 6-507313). In the present specification, a carrier comprising the above-described particles having through pores and subpores is referred to as a perfusion-type carrier.

As a perfusion-type carrier, POROS (trade name), a chromatography carrier manufactured by Perceptive Biosystems, Inc. is on the market. This is a carrier in which small particles of a styrene-divinylbenzene copolymer are aggregated. Conventionally available perfusion type carriers have a small particle size (10 × 1
0 ^-6 to 50 × 10 ^-6 m), a perfusion effect is exerted when the liquid is passed at a high linear velocity of 2.8 × 10 ^-3 m / s or more. However, when a solution, a slurry solution, blood, or the like obtained from a culture tank is filled at a high linear velocity as a carrier in a container, clogging is likely to occur due to a small particle diameter. For example, when blood was passed, blood cells were clogged in the column or at the column entrance, causing hemolysis.
In addition, when the liquid passing speed is increased, clogging occurs in a short time.

However, heretofore, there has been no known perfusion type carrier in which a perfusion effect occurs when the liquid is passed at a large particle diameter and at a low linear velocity. Furthermore, a cellulose-based carrier was not known as a perfusion-type carrier.

[0005]

SUMMARY OF THE INVENTION In view of the above, the present invention provides a cellulosic carrier which can increase the particle diameter and produces a perfusion effect even when passed at a relatively low linear velocity, and a method for producing the same. The purpose is to provide.

[0006]

The present invention relates to a perfusion-type cellulosic carrier, in which porous cellulosic small particles are suspended in an alkaline solution to form a suspension. A cellulosic carrier comprising cellulosic particles in which the cellulosic small particles are connected to each other such that the cellulosic small particles are brought into contact with an acidic solution so as to form voids between the particles.

[0007] The present invention also provides a method for suspending porous cellulose-based small particles in an alkaline solution to form a suspension, and bringing the suspension into contact with an acidic solution to form particles of the cellulose-based small particles. This is a method for producing a perfusion-type cellulosic carrier in which the small cellulosic particles are connected to each other so as to provide voids therebetween. Hereinafter, the present invention will be described in detail.

[0008] The alkaline solution used in the present invention is not particularly limited.
Examples include aqueous lithium hydroxide, aqueous potassium hydroxide, aqueous cesium hydroxide, and aqueous rubidium hydroxide. Glycerin, a water-soluble polymer, or the like may be added to the alkaline solution for viscosity adjustment.

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).
belongs to. When the pH is less than 13, when contacting the acidic solution as a suspension containing cellulosic small particles,
The cellulosic small particles are in a state of being mutually dispersed,
It cannot be linked. The value of pH is such that the degree of dissociation in an aqueous solution of acid or alkali = 1, [H ⁺ ]
× [OH ⁻ ] = 10 ⁻¹⁴ and pH = −log
₁₀ [H ⁺ ]. Therefore, for example, when the concentration of the alkaline solution is 0.1N, the pH is 13.

The small cellulosic particles used in the present invention include gel filtration agents, raw materials for cellulosic ion exchangers, affinity chromatography carriers, polymer carriers, body fluid purifying carriers, cosmetic additives and the like. Conventionally used ones can be used.

The above-mentioned small cellulose-based particles are composed of cellulose-based materials such as cellulose, cellulose derivatives, regenerated cellulose and the like. The cellulose is not particularly limited, and examples thereof include defatted cotton fibers, hemp, pulp obtained from wood, and purified cellulose obtained by purifying pulp.

The cellulose derivative is not particularly restricted but includes, for example, those in which a part of the hydroxyl groups of cellulose are esterified (ester derivatives); those in which the hydroxyl groups of cellulose are etherified (ether derivatives). it can. The ester derivative of cellulose is not particularly limited, for example, cellulose acetate, cellulose propionate, nitrocellulose, cellulose phosphate,
Examples thereof include cellulose acetate butyrate, cellulose nitrate, and dithiocarboxylic acid ester of cellulose (viscose rayon). The ether derivative of cellulose is not particularly limited, and includes, for example, methyl cellulose, ethyl cellulose, benzyl cellulose, trityl cellulose, cyanoethyl cellulose, carboxymethyl cellulose, carboxyethyl cellulose, aminoethyl cellulose, oxyethyl cellulose, and the like.

The above-mentioned regenerated cellulose is obtained by converting the above-mentioned cellulose once into a cellulose derivative which is easy to mold, and then converting the cellulose into cellulose again. Specifically, for example, cellulose such as cellulose acetate or cellulose propionate is used. Examples thereof include those prepared by hydrolyzing ester derivatives and the like.

The above-mentioned cellulose-based small particles are porous having a pore size according to the application. The porous cellulose-based small particles can be produced by a method disclosed in, for example, JP-A-63-90501, JP-A-63-92602, and the like. Specifically, for example, the above-mentioned small cellulosic particles can be produced by the following method or the like.

(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 the dispersion, or mix with the coagulant of cellulose xanthate,
The cellulose xanthate in the dispersion is coagulated as fine particles. At this time, since the fine particles of cellulose xanthate contain a water-soluble polymer compound, they are removed. Next, the cellulose fine particles of cellulose xanthate are neutralized with an acid to regenerate the cellulose, thereby obtaining the small cellulose-based particles.

The coagulation of the fine particles of cellulose xanthate can be carried out by adding an acid to the above-mentioned dispersion in addition to the above. In this case, after the water-soluble polymer compound is removed, the added acid is neutralized to regenerate the cellulose, thereby obtaining the small cellulose-based particles.

(2) Viscose, calcium carbonate and a water-soluble anionic polymer compound are mixed to form a fine particle dispersion of viscose containing calcium carbonate,
Viscose in the dispersion is coagulated by heating the dispersion or mixing a coagulant, and then neutralized with an acid to produce cellulose fine particles. Thereafter, the cellulose fine particles are separated from the dispersion liquid, and the calcium carbonate is removed by acid decomposition, followed by drying to obtain the cellulose-based small particles.

The cellulosic carrier of the present invention is formed by suspending the cellulosic small particles in the alkaline solution to form a suspension, and bringing the suspension into contact with an acidic solution.

The time for suspending the cellulosic small particles in the alkaline solution is preferably 1 minute or more. If it is less than 1 minute, it is difficult to sufficiently connect the above-mentioned small cellulose-based particles. More preferably, it is one hour or more.

The cellulosic small particles have a suspension concentration of 5
It is preferably from 0 to 75% by volume. The suspension concentration is the ratio of the total volume of the small cellulosic particles in the suspension to the volume of the suspension. When the suspension concentration of the above-mentioned small cellulosic particles is less than 50% by volume, when the droplets of the suspension are brought into contact with an acidic solution, a fragmentary cellulose-based molded product is obtained, and its strength is low. When the content is more than the volume%, droplets having a smooth surface cannot be obtained, and the shape of the cellulose-based molded product becomes a lump. More preferably, it is 60 to 70% by volume.

The size of the droplet is as follows: the average diameter is 3 × 10
^-3 m or less. Average diameter 3 × 10 ^-3
If it exceeds m, the effect exerted by the surface tension becomes small, and it becomes difficult to form droplets.

The method for converting the suspension into droplets is not particularly limited, and includes, for example, a method of discharging the suspension into a gas phase from a capillary, a method of using a sprayer, and the like. Among them, it is preferable to use a sprayer or the like, since a fine droplet can be obtained.

The type of the above atomizer is not particularly limited as long as it is an apparatus capable of subdividing the average diameter of droplets to 3 × 10 ⁻³ m or less. For example, a rotary disk type, a pressure nozzle type, Fluid nozzle type and the like can be mentioned.

The above-mentioned rotary disk type sprayer is a device in which a solution is made to flow on a high-speed disk, the solution is shaken off by centrifugal force, and is made to collide with a gas such as air to atomize. The pressure nozzle type sprayer discharges a high-pressure solution from a small hole and collides the solution with a gas such as ambient air to spray the solution. In the two-fluid nozzle type sprayer, even when the solution itself is at a low pressure, the solution is sprayed with a compressed gas at a high speed to atomize the solution.

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 is 2N or more) or less. When the pH exceeds 1, when the suspension containing the cellulosic small particles is brought into contact with the acidic solution, the cellulosic small particles are in a state of being mutually dispersed, and it is difficult to connect them.

The acidic solution is not particularly restricted but includes, for example, aqueous hydrochloric acid, aqueous sulfuric acid, aqueous nitric acid and aqueous phosphoric acid. Glycerin, a water-soluble polymer or the like may be added to the acidic solution for adjusting the viscosity.

The method of bringing the droplets of the suspension into contact with the acidic solution is not particularly limited. For example, a method of immersing the droplets in the acidic solution; And contacting the droplet. The time for bringing the droplets of the suspension into contact with the acidic solution is preferably 1 minute or more. If the time is less than 1 minute, the small cellulosic particles cannot be sufficiently linked. More preferably, it is one hour or more.

In the above cellulose-based particles,
The mode in which the small cellulosic particles are connected to each other is not necessarily based on a covalent bond, but may be any state as long as the bonding state between the particles can be substantially maintained stably. For example, the connection of cellulosic small particles includes connection by entanglement of cellulose molecules between particles, connection by chemical bond such as hydrogen bond, and the like.

As for the average diameter of the above-mentioned cellulose-based particles, the value of the ratio of the average diameter of the cellulose-based particles to the average diameter of the above-mentioned small cellulose-based particles is preferably less than 50. When the value of the above ratio is 50 or more, the voids serving as through pores become small, and the perfusion effect becomes small. The average diameter of the above-mentioned cellulose-based particles is appropriately set according to the use. Usually 20 × 10 ^{-6 to} 3 × 10 ^-3
m is preferable. When filling the container with the cellulosic particles, and passing a solvent that is easily clogged,
The average diameter of the cellulosic carrier used is 100 ×
The flow rate is preferably 10 ⁻⁶ m / s or more, and the flow rate is preferably 3 × 10 ⁻⁴ m / s or more as long as clogging does not occur. When the average diameter is less than 100 × 10 ⁻⁶ m, clogging is liable to occur, and when the liquid passing speed is less than 3 × 10 ⁻⁴ m / s, the perfusion effect is small, so that the object is achieved. The efficiency of work per hour becomes poor.

The above-mentioned cellulosic particles preferably have a specific surface area when dried of at least 2 × 10 ⁴ m ² / kg by the BET method. When it is less than 2 × 10 ⁴ m ² / kg, the working area according to the use becomes small. More preferably, it is at least 5 × 10 ⁴ m ² / kg.

The cellulosic carrier of the present invention uses the cellulosic particle itself described in detail above as the carrier itself. In order to provide voids between the cellulose-based small particles in the cellulose-based particles, the cellulose-based small particles are composed of a plurality of cellulosic small particles interconnected with each other, and the voids between the particles serve as pores. The pores of the plurality of connected small cellulosic particles facing the through pores become subpores. Its shape is usually spheroidal or spherical.

The cellulosic carrier of the present invention can be used for many purposes by setting the cellulosic small particles having a pore size according to the application and the above-mentioned diameter ratio.
Examples of the use include a carrier for gel filtration, a raw material of a cellulosic ion exchanger, a carrier for affinity chromatography, a carrier for adsorption of fragrances and chemicals, a carrier for immobilizing bacteria and enzymes, and a carrier for body fluid purification.

In the production method of the present invention, the above-mentioned cellulosic particles are made into a suspension by suspending porous cellulosic small particles in an alkaline solution, and the above suspension is brought into contact with an acidic solution. The cellulosic small particles are produced by interconnecting the small cellulosic particles so as to provide voids between the small cellulosic particles.

The method for producing the cellulosic carrier of the present invention comprises:
Cellulose-based small particles can be easily connected and voids can be formed between the above-mentioned cellulosic small particles, and thus are suitable for producing the cellulosic carrier of the present invention. Further, the method for producing a cellulosic carrier of the present invention does not use an organic solvent in the production process, is easy to wash, and is very preferable in preventing environmental pollution.

[0035]

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.

Example 1 Porous small cellulose particles (manufactured by Chisso Corporation) having an average diameter of 20 × 10 ⁻⁶ m were mixed with a 6N aqueous sodium hydroxide solution (pH = 14) so that the suspension concentration was 70% by volume. .8). After sufficiently stirring with a stirrer, the hole diameter is 0.7 ×
Droplets of this suspension were contacted with a 5 N aqueous hydrochloric acid solution (pH = -0.7) using a 10 ^-3 m capillary to obtain a cellulose-based carrier. The particle size was about 2 × 10 ⁻³ m. The obtained cellulosic carrier was washed with pure water. The liquid in the obtained cellulosic carrier was replaced with ethanol, then replaced with 2-methyl-2-propanol, and lyophilized using a freeze dryer (manufactured by Eiko Eng. CO Ltd.) to remove gold. After vapor deposition, observation with a scanning electron microscope (manufactured by Topcon Corporation) revealed that the shape of the obtained cellulose-based carrier was spherical, as shown in FIG.
As shown in FIG. 3, there were voids between the connected cellulose small particles. In addition, as shown in FIG. 4, pores of the connected porous cellulose small particles were also observed.

Comparative Example 1 Porous small cellulose particles (manufactured by Chisso Corporation) having an average diameter of 20 × 10 ⁻⁶ m were mixed in pure water so as to have a suspension concentration of 70% by volume. After sufficiently stirring with a stirrer, 5 drops of the suspension were added to a capillary having a hole diameter of 0.7 × 10 ⁻³ m.
Upon contact with a specified aqueous hydrochloric acid solution (pH = -0.7), the cellulose small particles were in a dispersed state.

Comparative Example 2 Porous cellulose small particles (manufactured by Chisso Corporation) having an average diameter of 20 × 10 ⁻⁶ m were mixed with a 6N aqueous sodium hydroxide solution (pH = 14) so that the suspension concentration was 70% by volume. .8). After sufficiently stirring with a stirrer, the hole diameter is 0.7 ×
When a droplet of the present suspension was brought into contact with pure water using a 10 ^-3 m capillary, a disk-shaped cellulose molded product was obtained.
Upon shaking, the shape of the molded article collapsed, and the cellulose small particles became dispersed.

Comparative Example 3 Porous small cellulose particles (manufactured by Chisso Corporation) having an average diameter of 20 × 10 ⁻⁶ m were mixed with a 6N aqueous sodium hydroxide solution (pH = 14) so that the suspension concentration was 40% by volume. .8). After sufficiently stirring with a stirrer, the hole diameter is 0.7 ×
When a droplet of the present suspension was brought into contact with a 5 N aqueous hydrochloric acid solution (pH = -0.7) using a 10 ^-3 m capillary, a cellulose molded article in a fragmented state was obtained. Upon shaking, the shape of the molded article collapsed, and the cellulose small particles became dispersed.

Comparative Example 4 Porous small cellulose particles (manufactured by Chisso Corporation) having an average diameter of 20 × 10 ⁻⁶ m were mixed with a 6N aqueous sodium hydroxide solution (pH = 14) so that the suspension concentration was 80% by volume. .8). After sufficiently stirring with a stirrer, the hole diameter is 0.7 ×
When a droplet of the present suspension was brought into contact with a 5N aqueous hydrochloric acid solution (pH = −0.7) using a 10 ⁻³ m capillary, a droplet having a smooth surface was not formed, and the resulting suspension was obtained. The shape of the cellulose-based molded article was lump.

Example 2 Porous small cellulose particles (manufactured by Chisso) having an average diameter of 20 × 10 ⁻⁶ m were mixed with a 6N aqueous sodium hydroxide solution (pH = 14) so that the suspension concentration was 70% by volume. .8) to prepare a suspension, which was sufficiently stirred with a stirrer.
The 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. Nitrogen injection pressure is 5 × 1
At 0 ³ kg / m ² , the discharge speed of the suspension was 5.19 × 10
^-4 m ³ / s. The diameter of the inner nozzle is 2.6 × 10 ^-3
m, a two-fluid nozzle having an outer nozzle diameter of 4.4 × 10 ⁻³ m was used. The discharge height was 4 m. The cellulose-based carrier of the present invention was obtained in an acidic solution. Average particle size is about 200
× 10 ^-6 m.

The liquid in the obtained cellulosic carrier was replaced with ethanol, and then replaced with 2-methyl-2-propanol, followed by freeze-drying (Eiko Eng. CO Lt.).
d. Lyophilized using gold, and after gold deposition,
When observed with a scanning electron microscope (manufactured by Topcon Corporation),
As shown in FIG. 5, the shape of the obtained cellulosic carrier was spherical. As shown in FIG. 6, there were voids between the connected cellulose small particles. Also, as shown in FIG.
The pores of the cellulosic small particles after the connection were also observed.

Comparative Example 5 Porous cellulose particles (average diameter 179 × 10 ⁻⁶ m) having the same structure (pore size, etc.) as the small cellulose particles used in Examples 1 and 2 and Comparative Examples 1 to 4 and having a different average diameter. Column (manufactured by Chisso) (inner diameter 0.01 m, length 0.0
5m), 23.2 degree physiological saline (manufactured by Otsuka Pharmaceutical Co., Ltd.)
At a linear velocity of about 5 × 10 ⁻⁴ m / s, and a 100 × 10 ⁻⁹ m ³ solution obtained by diluting low-density lipoprotein (L-2139, manufactured by SIGMA) five-fold with physiological saline was pulsed. Was injected. Time-dependent change in low-density lipoprotein concentration
The measurement was performed at a wavelength of 80 nm using an absorbance meter (manufactured by ATTO). As shown in FIG. 8, it was confirmed that the position of the peak top was immediately after the start of elution. In addition,
The cellulose particles used have pores that allow the low density lipoprotein to enter the particles. Therefore, the result of the elution curve is not due to the lack of pores that allow the low density lipoprotein to enter the cellulose particles, but due to the large particle size, the long mass transfer distance, the low density lipoprotein 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 being able to move sufficiently into the above-mentioned cellulose particles.

Example 3 The carrier obtained in Example 2 (average particle size: about 200 × 10
^-6 m, a ratio of the average diameter of the cellulose carrier to the average diameter of the cellulose small particles = 10) packed in a column (0.01 m in inner diameter, 0.05 m in length) filled with 23.2 ° physiological saline (Otsuka Pharmaceutical Co., Ltd.) Manufactured at a linear velocity of about 5 × 10 ⁻⁴ m / s and a low-density lipoprotein (L-2139, SIGMA
100 × 10 5 solution diluted 5 times with physiological saline
The ^-9 m ³ were pulsed injected. The time-dependent change in the concentration of the low-density lipoprotein is measured at an absorbance meter (A
(Manufactured by TTO). As shown in FIG. 9, it was confirmed that the position of the peak top was later than that of Comparative Example 5. The carrier used in this example was a cellulose small particle (particle diameter: about 20) having the same pores as the cellulose particles (particle diameter: 179 × 10 ⁻⁶ m) used in Comparative Example 5.
× 10 ^-6 m) (particle diameter: about 200 × 10 ^-6 m). Therefore, the results of the above elution curves show that even if the particle size of the carrier is large, mass transfer of the low-density lipoprotein in the carrier becomes faster by making the structure a perfusion type, It is considered that this was due to sufficient movement.

[0045]

The cellulosic carrier of the present invention has the above-mentioned constitution, and therefore, depending on its size and internal structure, a carrier for gel filtration, a raw material for a cellulosic ion exchanger, a carrier for affinity chromatography, a fragrance, It can be suitably used for applications such as a carrier for adsorbing chemicals, a carrier for immobilizing cells and enzymes, and a carrier for purifying body fluids. Further, since the method for producing the cellulosic carrier of the present invention is as described above, the cellulosic carrier of the present invention can be easily produced.

[Brief description of the drawings]

FIG. 1 shows that the particle surface of the cellulosic carrier of Example 1 was 40
It is a photograph magnified twice.

FIG. 2 shows a cross section of particles of the cellulosic carrier of Example 1 of 40.
It is a photograph magnified twice.

FIG. 3 shows a cross section of particles of the cellulosic carrier of Example 1 of 50.
It is a photograph magnified 0 times.

FIG. 4 shows the cross section of the particles of the cellulosic carrier of Example 1 as 50
It is a photograph magnified by 00 times.

FIG. 5 shows that the particle surface of the cellulosic carrier of Example 2 was 20
It is a photograph magnified 0 times.

FIG. 6 shows that the particle surface of the cellulose-based carrier of Example 2 was 10
It is a photograph magnified by 00 times.

FIG. 7 shows that the particle surface of the cellulose-based carrier of Example 2 was 50
It is a photograph magnified by 00 times.

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)

[Claims] 1. A perfusion-type cellulosic carrier, in which porous cellulosic small particles are suspended in an alkaline solution to form a suspension, and the suspension is contacted with an acidic solution. A cellulosic carrier comprising a cellulosic particle obtained by connecting the cellulosic small particles to each other so as to provide a gap between the cellulosic small particles. 2. The alkaline solution has a pH of 13 or more, and the suspension concentration of the cellulosic small particles is 50 to 7
The cellulosic carrier according to claim 1, wherein the content is 5% by volume, and the acidic solution has a pH of 1 or less. 3. The cellulose-based carrier according to claim 1, wherein the average diameter of the cellulose-based particles is 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. 4. An average diameter of the cellulosic particles is 10%.
4. The perfusion effect according to claim 1, wherein the particle is 0 × 10 ⁻⁶ m or more, and the perfusion effect occurs when the particles are filled in a container and passed at a linear velocity of 3 × 10 ⁻⁴ m / s or more. Cellulosic carriers. 5. A method in which porous cellulose-based particles are suspended in an alkaline solution to form a suspension. The suspension is brought into contact with an acidic solution to form voids between the cellulose-based particles. A method for producing a perfusion-type cellulosic carrier, wherein the small cellulosic particles are connected to each other so as to be provided. 6. The alkaline solution has a pH of 13 or more, and the suspension concentration of the cellulosic small particles is 50 to 7
The method for producing a cellulosic carrier according to claim 5, wherein the acidic solution has a pH of 1 or less at 5% by volume.