JPH10248927A - Adsorbent for purifying humor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable fast removal of desired substances by arranging a carrier to generate a flow of humor piercing the carrier when the flow of the humor exists on the perimeter thereof. SOLUTION: For example, carboxymethyl cellulose is mixed with about 6 normal of a NaOH aqueous solution and small particles of porous cellulose with an average particle size of about 25×10<-6> m are mingled into the mixture to agitate. The mixture thus obtained is injected into an ethanol solution together with a compressed nitrogen gas through a nozzle to obtain a carrier with the average particle size of 100×10<-6> m. A liquid in the carrier is replaced with ethanol to be freeze dried. This carrier is caused to react with epichlorohydrine at about 45 deg.C for two hour and then, with dexitran sulfate at about 40 deg.C for 24 hour to obtain an adsorbent with the dexitran sulfate immobilized thereon. The adsorption rate of the adsorbent is about 51%, about 0% and about 0% for low density lipoprotein-cholesterol, high density lipoprotein-cholesterol and albumin and has affinity to the low density lipoprotein.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a body fluid purifying adsorbent for removing a target substance at a high speed when a body fluid purifying method is carried out in the treatment of hyperlipidemia, autoimmune diseases, immune-related diseases and the like.

[0002]

2. Description of the Related Art As a method for removing and treating a substance present in a body fluid, a body fluid is passed through an adsorber filled with an adsorbent in which a substance having an affinity for a target substance in the body fluid is immobilized on a carrier. A body fluid purification method of adsorbing and removing a target substance has been used. For this purpose, a method was first used in which whole blood was passed through activated carbon, particularly coated activated carbon particles, to adsorb and remove the target substance. Further, with the development of plasma separation membranes, various adsorbers for adsorbing and removing a target substance from separated plasma have been developed. Patient Quality of Life (Qualit
From the viewpoint of y Of Life), it is desirable that the treatment time is short. For this purpose, there are several methods for shortening the treatment time by devising operation conditions and the like while keeping the same adsorbent.

First, it is conceivable to increase the flow rate of body fluid circulating extracorporeally to increase the amount of body fluid that comes into contact with the adsorbent per hour. However, extremely increasing the flow rate of the body fluid taken out of the patient and circulated extracorporeally impairs the quality of life of the patient. Conventionally, the flow rate of the body fluid circulating extracorporeally is 0.833 × 10 ^{−6 to} 3.33 × 1.
Is ^{0 -6 m 3 / s (50~200ml} / min). Therefore, there is a limit in increasing the flow rate of the body fluid circulating extracorporeally.

[0004] It is also conceivable to increase the adsorber volume to prolong the contact time of the bodily fluid with the adsorbent. But,
As the volume of the adsorber increases, the amount of body fluid existing outside the body during the treatment increases and the quality of life of the patient is impaired. Therefore, there is a limit in increasing the volume of the adsorber. Conventional adsorber volume for body fluid purification is at most 50x
It was 10 ^{-6 to} 500 × 10 ^-6 m ³ (50 to 500 ml).

[0005] Next, it is conceivable to shorten the treatment time by increasing the static adsorption performance of the adsorber.
The static adsorption performance is the magnitude of the saturated adsorption amount. As a method of increasing the static adsorption performance, it is conceivable to increase the adsorption amount per adsorbent. What influences the adsorption equilibrium is a substance having an affinity for the target substance and a contact area effective for adsorbing the target substance. However, substances having affinity for the target substance are limited to those having affinity for the target substance. In addition, for the purpose of purifying body fluids, it is limited to those having a small physiological effect. It is also conceivable to increase the effective contact area, but this contact surface requires at least a small hole for adsorbing the target substance. Therefore, the contact area of the porous body having such small holes has an upper limit depending on the diameter and number of the small holes.
As described above, there is a limit in improving the above-mentioned adsorption equilibrium relationship to increase the static adsorption performance.

As described above, it is difficult to shorten the treatment time while maintaining the adsorption amount by improving the volume of the adsorber, the flow rate of the body fluid, and the static adsorption performance due to restrictions based on the body fluid purification method. It was difficult.

Finally, it is conceivable to shorten the treatment time by increasing the dynamic adsorption performance of the adsorber. The dynamic adsorption performance is the magnitude of the adsorption speed. As a method of increasing the dynamic adsorption performance, it is conceivable to optimize the particle diameter of the adsorbent or the intraparticle diffusion coefficient of the target substance to increase the dynamic adsorption performance.

The first method, that is, a method of increasing the dynamic adsorption performance by reducing the particle diameter of the adsorbent to reduce the diffusion distance, is to reduce the particle diameter of the adsorbent to reduce body fluid. The flowing channel diameter becomes smaller,
In addition, since the pressure loss is increased, clogging may be caused. In consideration of a safe body fluid purification method, the particle diameter cannot be extremely reduced. In fact, the particle size of the conventional adsorbent is 50 × 10 ⁻⁶ m or more for plasma perfusion.
Less than 0 × 10 ⁻⁶ m, especially 100 × for direct blood perfusion
It was 10 ⁻⁶ m or more and less than 4000 × 10 ⁻⁶ m.

The second method, that is, a method of increasing the diffusion coefficient of the target substance in the adsorbent and rapidly increasing the dynamic adsorption performance in order to quickly move the target substance in the adsorbent, This method also has its limitations for the following reasons. In the case of the conventional body fluid purification adsorbent based on diffusion control, once the target substance is determined, the diffusion coefficient has a constant value depending on the structure of the adsorbent. Required. But,
No matter how much the structure is optimized, the diffusion coefficient of the target substance in the adsorbent does not exceed the diffusion coefficient in steric hindrance-free body fluids, and this method has its limitations.

Therefore, in the case of a conventional body fluid purifying adsorbent,
There is a limit to improving the dynamic adsorption performance by improving the particle size of the adsorbent and the intraparticle diffusion coefficient of the target substance, and it has been difficult to reduce the treatment time.

On the other hand, although it is difficult to use it as an adsorbent for purifying body fluid, it is expected that a carrier for chromatography will improve dynamic adsorption performance when a substance having an affinity for the target substance is immobilized. There is something you can do.

First, the principle of the dynamic adsorption performance will be described below. As a measure of the dynamic adsorption performance of the adsorbent, a breakthrough curve that represents the change over time in the concentration of the target substance at the outlet of the adsorber when a solution containing the target substance at a constant concentration is passed at a constant speed should be used. General. When estimating the dynamic adsorption performance of the adsorber under use conditions, it is preferable to keep the linear velocity in the adsorber constant, that is, to keep the flow state around the adsorbent constant. In this specification, the linear velocity in the adsorber refers to the moving velocity (m) of the mobile phase passing through the adsorber.
/ S).

On the other hand, the number of theoretical plates is generally used as an index indicating the performance of a column packed with a carrier on which no substance is adsorbed (carrier packed column). The number of theoretical plates means that when a solution containing the target substance is passed through a column packed with a carrier,
This is the number of stacks when it is considered that the minimum column height necessary for the target substance to reach the adsorption-desorption equilibrium is stacked in the carrier-packed column.

The relationship between the breakthrough curve, which indicates the dynamic adsorption performance of the adsorber, and the theoretical plate number, which is an index indicating the performance of the column packed with a carrier, is described by Kato et al. (Shigeo Kato et al., Journal of Fermentation and Biotechnology). Engineering, vol. 78, p. 246 (1994)), which are associated with the following three equations.

[0015]

(Equation 1)

[0016]

(Equation 2)

[0017]

(Equation 3)

Here, t is time [sec]. C is the eye
Concentration at the outlet of the adsorber [kg / m^Three] And time and
Both change. C₀Of the target substance flowing into the adsorber
Concentration [kg / m^Three] And is constant. V is the adsorber
Or the volume of a column packed with a carrier [m^Three] And constant
It is. q₀Is C₀Equilibrium adsorption amount [kg /
m ^Three], C₀Solution with a concentration of
After passing through the liquid and adsorbing it until the amount of adsorption no longer increases
Amount and constant. F is the flow rate of the solution [m^Three/ Sec]
However, it should be the same as the linear velocity in the adsorber during use.
Is set to be constant. N is the number of theoretical plates,
The same flow rate as when passing through the adsorber through the packed column
The value obtained for the target substance when the solution was passed through F
Constant. t^-Is the target substance in the column
Average residence time [seconds]. θ is t^-Division of t against
It is. α is a parameter that indicates the efficiency of the amount of adsorbent adsorbed.
It is a tar.

In order to show the effect of the number of theoretical plates on the breakthrough curve, FIG. In FIG. 1, the amount of adsorption q [kg / m ³ ] per unit volume of the adsorber adsorbed by each time t / t ⁻ is the area on the upper side of the breakthrough curve, that is, {1- (C / C ₀ )}. Is integrated over that time and divided by the adsorber volume. FIG. 2 shows the change over time of the adsorption amount q with respect to q ₀ by performing the above integration. From Fig. 2, it can be seen that the larger the number of theoretical plates in a column packed with a carrier, the larger the amount of adsorption that can be adsorbed in a certain period of time, and the shorter the time required to adsorb a certain amount, that is, the dynamic adsorption performance of the adsorber It can be seen that is improved. From this, it can be seen that in order to improve the dynamic adsorption performance of the adsorber, it is better to increase the number of theoretical plates in the carrier packed column.

The number of theoretical plates of a column packed with a carrier is determined by the minimum column height (height equivalent to the theoretical plate) required for the target substance to reach the adsorption-desorption equilibrium and the height of the column packed with the carrier. It is represented by

[0021]

(Equation 4)

Here, L [m] is the height of the column packed with the carrier,
HETP [m] is a height equivalent to a theoretical plate. Since the column height is constant, increasing the number of theoretical plates of the carrier-packed column means reducing the height equivalent to the theoretical plate, which is a characteristic of the packed carrier. The adsorption performance can be improved. The theoretical plate number depends on factors such as the shape of the container, whereas the theoretical plate equivalent height is a characteristic that depends only on the properties of the carrier.In other words, when discussing the theoretical plate equivalent height, breakthrough is required. A carrier packed column having a shape different from that of the adsorber used for the curve measurement may be used. However, the linear velocities in the carrier packed column must be the same.

As a carrier having a height corresponding to a theoretical plate that is smaller than that of a conventional carrier in which mass transfer of a target substance is only diffusion, when a flow is present around carrier particles, a flow penetrating into the particles is generated. It is known that there is a carrier having a structure (NB Aphean et al., Journal of
Chromatography, volume 519, page 1 (1990
Year): Shigeo Kato et al., Journal of Fermentation and Bioengineering, 78, 2
46 pages (1994)). In other words, a flow that penetrates through the carrier particles reduces the height corresponding to the theoretical plate measured for the carrier. Therefore, an adsorber in which an adsorbent in which a substance having an affinity for a target substance is immobilized is filled into a carrier having a structure in which a flow penetrates the inside of the carrier particle when there is a flow around the particle is dynamic. The adsorption performance is improved.

As such a carrier having a structure in which a flow penetrates inside a carrier particle when there is a flow around the carrier particle, Poros (commercially available as a carrier for chromatography by Perceptive Biosystems Inc.) Name) (particle size 10 × 10 ⁻⁶ m, 20 × 10 ⁻⁶ m,
50 * 10 < ^-6 > m) (Tokuhyohei 4-500726) is known. However, since such a carrier is a carrier for chromatography, it needs to be in a range that does not hinder the operation of packing and liquid passing, and its particle size is small.

[0025]

The use of a chromatographic carrier such as the poros described above as a carrier for purifying body fluids has many disadvantages due to restrictions based on the method for purifying body fluids. It is difficult to do.
For example, when whole blood, which is a bodily fluid, is passed through a column filled with a carrier having a small particle size for chromatography at a conventional extracorporeal circulating fluid flow rate, blood cells are clogged in the column or at the column entrance. It causes hemolysis.

The present invention has been made in view of such circumstances, and in order to shorten the treatment time while maintaining the amount of adsorption, the adsorbent for body fluid purification capable of rapidly removing the target substance. It is intended to provide materials.

[0027]

Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present invention has been made. That is, the present invention is a bodily fluid purification adsorbent obtained by immobilizing a substance having an affinity for a target substance in a bodily fluid on a particulate, slab-like or irregular-shaped carrier, wherein the carrier has a bodily fluid around it. When there is a flow of the body fluid, a body fluid purifying adsorbent having a structure in which a body fluid flow passing through the inside of the carrier is generated, a body fluid purifying adsorber filled with the body fluid purifying adsorbent, and the body fluid purifying adsorber are used. This is a body fluid purifying method for purifying body fluids.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described specifically. The body fluid purifying adsorbent of the present invention is obtained by immobilizing a substance having an affinity for a target substance on a carrier. The body fluid purifying adsorbent of the present invention is obtained by immobilizing a substance having an affinity for a target substance on a carrier having a structure in which a flow that penetrates through the inside of the carrier particle occurs when there is a flow around the carrier particle. .

A carrier having a structure in which a flow penetrates through the inside of the particle when there is a flow around the carrier particle means that when a flow of a liquid such as a body fluid or the like occurs around the carrier particle, the pressure gradient is caused by the pressure gradient. A carrier having a structure in which a part of the flow flows through the inside of the carrier particles. The carrier needs a through hole having a sufficient diameter.

In the adsorbent of the present invention, a flow penetrating through the carrier particles is generated to reduce the height equivalent to the theoretical plate described in the prior art in this specification. The ratio of the average particle diameter of the carrier particles to the average diameter of the pores is preferably 70 or less, more preferably 50 or less.

Further, since the adsorbent of the present invention is used for purifying body fluid, there is a restriction on the linear velocity of the solution based on the method for purifying body fluid. Therefore, the carrier in the present invention is packed in a column, and a solution containing only the target substance is subjected to a linear velocity of 1 × 10 ^−4.
When the liquid is passed in the range of not less than 10 × 10 ⁻⁴ m / s and not more than 10 × 10 ⁻⁴ m / s, the height equivalent to the theoretical plate, which is the carrier characteristic, is 0.5%.
m, and more preferably 0.1 m or less.
It is as follows.

A typical method for measuring the height equivalent to a theoretical plate is as follows. The target substance is injected into the column filled with the carrier in a pulsed manner, and the elution curve is detected. When the number of theoretical plates is large as in chromatography, the shape of the elution curve has a Gaussian distribution, and the height equivalent to theoretical plates (HETP) is calculated using the following equation.

[0033]

(Equation 5)

Where L [m] is the height of the packed column, Tr
[Sec] is the holding time, and Wt [sec] is the half width. The retention time is the time when the peak apex of the elution curve is detected,
The half width is the time width of a half position of the peak apex (F. Gais, "Optimization of Liquid Chromatography", Kodansha, page 18, (1980)).

However, unlike the case of chromatography, the particle size of the adsorbent for body fluid purification is large and the length of the container is limited, and the target substance obtained under the conditions of the container filled with the adsorbent for body fluid purification is not limited. Elution curves are rarely Gaussian in their shape. In this case, the shape of the elution curve can be used as an index that qualitatively indicates the performance of the carrier.

When mass transfer is insufficient, which corresponds to the case where the height of the theoretical plate is large and the number of theoretical plates is small, many of the target substances elute with the flow of the solution filled in the container without sufficiently contacting the carrier. . Therefore, the peak top position exists immediately after the elution of the solution corresponding to the void volume between the particles of the carrier ends, and thereafter, the target substance gradually elutes as the elution time increases.

On the other hand, when the mass transfer is good, which corresponds to the case where the height corresponding to the theoretical plate is small and the number of theoretical plates is large, the number of times the target substance comes into contact with the carrier increases as the mass transfer becomes good. Therefore, the time during which the target substance is retained in the container is also increased, the peak immediately after the solution corresponding to the void volume between the carriers has been eluted is reduced, and the position of the peak top also moves backward. Further, the shape of the elution curve approaches a Gaussian distribution.

In the present invention, the shape of the through-holes of the carrier particles is not limited as long as a part of the flow in the vessel only flows through the inside of the carrier particles. . For example, the cross-sectional shape may be circular, polygonal or irregular, and the through-paths within the carrier particles may be straight or tortuous. In addition, it is preferable that a plurality of through-holes exist, and these through-holes are, for example, a configuration in which a plurality of through-holes of the same type or a plurality of through-holes of different types are arranged in parallel, or a random, not parallel, configuration. It may have a configuration or the like that is in a different direction.

The carrier only needs to have a structure in which a bodily fluid flow that passes through the inside of the carrier occurs when a bodily fluid flows around the carrier. In addition, the shape of the carrier may be any of a particle shape, a slab shape, and an irregular shape.
Particles are preferred from the viewpoint of ease of handling and the like.

The above-mentioned carrier is not particularly restricted but includes, for example, a porous body having through-holes, fine particles aggregated into particles, fibers aggregated into particles, and particles processed into through-holes. And the like. In addition, as the microparticles or fibers used in the microparticles obtained by assembling the microparticles, or in the microparticles or fibers obtained by assembling the fibers, those having small holes capable of adsorbing the target substance, Those having a large contact surface are preferable.
The one that was processed into the above particles to create a through hole,
It is preferable that the particles before processing have many small holes capable of adsorbing the target substance.

It is preferable that the carrier has a strength such that particles are deformed when the body fluid passes and consolidation that prevents passage of the body fluid does not occur.

Examples of the method for producing the carrier include a method of assembling particles to form through holes, a method of assembling fibers, and a method of forming processed holes. Examples of methods for assembling particles and fibers include methods of assembling during the polymerization reaction to produce particles or fibers, organic solvents, heating, adhesives only on surfaces adjacent to each other without destroying the structure of the particles or fibers. , An acid, an alkali solution or the like to make them adhere to each other. The space of the aggregated particles or fibers is a through hole. Examples of a method of forming a processing hole include a laser drilling technique and solvent leaching.

Examples of the material of the carrier include natural polymer compounds such as cellulose, chitin, chitosan and agarose; modified natural compounds such as acyl cellulose and acyl chitin; polystyrene, polymethacrylic acid and derivatives thereof, and copolymers thereof. Coalescence, furthermore polyvinyl alcohol,
Synthetic polymer compounds such as styrene divinylbenzene copolymer; inorganic substances such as glass, alumina, and ceramics are used.

The target substances include lipoproteins that cause arteriosclerosis, such as low-density lipoprotein and ultra-low-density lipoprotein; immunoglobulins (A, D, E,
G, M), autoantibodies such as anti-DNA antibodies, anti-acetylcholine receptor antibodies, anti-blood group antibodies, and anti-platelet antibodies and antigen-antibody complexes; and target substances such as endotoxins, rheumatoid factors, macrophages, and cancer tissue infiltrating T cells. Can be

The substance having an affinity for the target substance is not particularly limited as long as it adsorbs the target substance. The affinity between a substance having an affinity for the target substance and the target substance is classified into two categories, biological affinity and physicochemical affinity. Examples of the substance having an affinity for the target substance utilizing the biological interaction include a substance on which an antigen is immobilized,
Examples of the substance include a substance on which an antibody is immobilized, and a substance utilizing a biological interaction such as complement binding or Fc binding. Substances having an affinity for the target substance utilizing physical interaction include substances utilizing electrostatic interaction or hydrophobic interaction. Of these, considering the stability of the activity in securing raw materials, manufacturing, sterilizing, storing, and storing adsorbents and columns, and the side effects of contact with blood, etc., Substances with affinity are preferred.

When a low-density lipoprotein is to be adsorbed, a substance having a negative group can be used as a specific example of a substance having an affinity for a target substance utilizing physical interaction. Examples of the substance having the negative group include dextran sulfate, heparin, chondroitin sulfate, chondroitin polysulfate, heparitin sulfate, xylan sulfate, caronin sulfate, cellulose sulfate, chitin sulfate,
Sulfated polysaccharides such as chitosan sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenan sulfate, starch sulfate, polyglucose sulfate, laminaran sulfate, galactan sulfate, levan sulfate, mepesulfate, etc .; phosphotungstic acid , Polysulfated anethole, polyvinyl alcohol sulfuric acid, polyphosphoric acid, polyacrylic acid and the like. Among them, sulfated polysaccharide is particularly effective. Further, preferable examples in terms of clinical utility include heparin and dextran sulfate.

The above-mentioned substances having a negative group are examples of substances having an affinity for a target substance utilizing physical interaction, which are used when trying to adsorb low-density lipoprotein. If is changed, a substance having a positive group and a hydrophobic group and exhibiting physical interaction may be used. Further, a plurality of substances having an affinity for the target substance may be immobilized. In addition, a substance having an affinity for the low-density lipoprotein includes aniline and the like.

As a method for immobilizing a substance having an affinity for the above-mentioned target substance on a carrier, there are known methods such as covalent bond, ionic bond, physical adsorption, embedding, and precipitation insolubilization on the surface.
An appropriate method can be selected depending on the substance having an affinity for the target substance and the material of the carrier. Considering the elution of a substance having an affinity for the target substance during sterilization, a method using covalent bonding is preferred. If necessary, a spacer may be introduced between the carrier and a substance having an affinity for the target substance.

When a substance having an affinity for a target substance is immobilized on a carrier by covalent bonding, examples of a method for bringing the reactivity to the substance having an affinity for the target substance to the carrier include:
The cyanogen halide method, epichlorohydrin method, bisepoxide method, bromoacetyl bromide method and the like are known. Specific functional groups used in the above reactions include:
Examples include an amino group, a carboxyl group, a hydroxyl group, a thiol group, an acid anhydride group, a succinylimide group, a chlorine group, an aldehyde group, an epoxy group, and a tresyl group. Among them, an epoxy group derived by the epichlorohydrin method is particularly preferable from the viewpoint of stability during heat sterilization.

In a preferred embodiment of the body fluid purifying adsorbent of the present invention, the average particle diameter is 100 × 10 ⁻⁶ m or more.
When the liquid is passed at 10 ^-4 m / s or more, a flow penetrating through the carrier particles is generated.

When the body fluid purifying adsorbent of the present invention is in direct contact with whole blood, the particle diameter of the adsorbent particles should be 100 × 10 ⁻⁶ m or more in consideration of clogging of blood cells and dynamic adsorption performance.
It is preferably less than 000 × 10 ⁻⁶ m, more preferably 100 × 10 ⁻⁶ m or more and less than 600 × 10 ⁻⁶ m.

When processing whole blood as a body fluid, it is necessary to increase the particle diameter as compared with a case where a liquid component such as plasma is flowed in order to secure a flow path through which blood cells and the like pass. However, when a conventional carrier is used, the diffusion distance increases as the particle diameter increases, and the dynamic adsorption performance decreases. When a conventional carrier having a large particle size is used for whole blood treatment, the dynamic adsorption performance is particularly insufficient.

On the other hand, in the body fluid purifying adsorbent of the present invention, since the carrier generates a flow of the body fluid passing through the inside of the carrier particles with the flow of the body fluid around the carrier particles,
The mass transfer of the target substance is better than that of a conventional carrier in which the mass transfer is only diffusion. Therefore, when the particle size is 10
The adsorbent for body fluid purification of the present invention, which is at least 0 × 10 ⁻⁶ m and less than 4000 × 10 ⁻⁶ m, more preferably at least 100 × 10 ⁻⁶ m and less than 600 × 10 ⁻⁶ m, can directly contact whole blood. In the case of contact, dynamic adsorption performance is particularly improved.

The present invention also includes a body fluid purifying adsorber in which a container is filled with the above-described body fluid purifying adsorbent of the present invention. The method of using the adsorber is the same as that of the body fluid purifying adsorber used in the conventional plasma perfusion method and the direct blood perfusion method. An anticoagulant can be used in accordance with a normal body fluid purification method such as continuously injecting an anticoagulant into the body fluid circuit so as not to coagulate, or providing a pressure gauge for detecting the occurrence of clogging or the like.

[0055]

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.

Production Example 1 Preparation of Carrier Particles Carboxymethylcellulose (manufactured by Wako Pure Chemical Industries, Ltd.) and 6
By mixing with a specified aqueous NaOH solution, a solution containing 2.9% by weight of carboxymethylcellulose was prepared. The porous cellulose small particles having an average particle diameter of 25 × 10 ⁻⁶ m (manufactured by Chisso Corporation) were mixed with the above-mentioned NaOH of carboxymethyl cellulose.
Mixed in an aqueous solution (proportion of the total volume of the small cellulose particles in the suspension to the volume of the suspension: 65% by volume, ratio of the weight of carboxymethyl cellulose to the weight of the suspension: 1.0
% By weight) with stirring for 5 hours. Then 2
A compressed nitrogen gas is ejected from an outer nozzle of a fluid nozzle (having an inner nozzle and an outer nozzle on concentric circles), and at the same time, the above-mentioned suspension is discharged from the inner nozzle into a 99.5% ethanol solution as a coagulating liquid to form a coagulating liquid. Collected inside. The nitrogen gas ejection speed was 3.3 × 10 ⁻⁴ m ³ / s, and the suspension ejection speed was 1.2 × 10 ⁻⁷ m ³ / s. The diameter of the inner nozzle is 2.6 × 10 ⁻³ m, and the diameter of the outer nozzle is 4.4 × 10 ⁻³ m
Was used. The discharge height was 4 m.
The obtained carrier is washed with pure water, and the openings are 180 × 10 ^−6.
m and 355 × 10 ^-6 m sieve,
A carrier having an average particle size of 256 × 10 ⁻⁶ m was obtained.

The liquid in the obtained carrier was replaced with ethanol, then replaced with 2-methyl-2-propanol, and lyophilized (manufactured by Eiko Eng. Co. Ltd.).
Scanning electron microscope (manufactured by Topcon) after depositing gold
As shown in FIGS. 3 and 5, there are voids (through holes) between the connected cellulose small particles on the carrier surface and cross section, respectively, as shown in FIGS. 3 and 5, and as shown in FIGS. 4 and 6, Small holes (pores that can be adsorbed) were also observed on the carrier surface and cross section, respectively. The obtained carrier was a particle having a through hole and a small hole capable of being adsorbed, and had a structure in which a flow penetrating inside the particle occurred when there was a flow around the particle.

Reference Example 1 Measurement of Upper Limit Linear Velocity The carrier obtained in Production Example 1 (average particle size: 256 × 10
⁻⁶ m) was packed in a column having an inner diameter of 10 × 10 ⁻³ m and a length of 110 × 10 ⁻³ m, and fresh blood of cattle to which a citrate anticoagulant was added was kept warm at 37 ° C. and passed. Flow at a constant linear velocity,
When the pressure loss became constant, by switching to a higher linear velocity, the upper limit linear velocity at which a constant pressure loss could be maintained was measured. As a result, the upper limit linear velocity is 7.32
× 10 ⁻⁴ m / s.

Comparative Reference Example 1 Measurement of Upper Limit Linear Velocity Using a commercially available poros (trade name) (manufactured by Perceptive Biosystems, average particle size about 50 × 10 ⁻⁶ m), the same as in Reference Example 1, The upper limit linear velocity at which a constant pressure loss was maintained during the passage of fresh cow blood was measured. As a result, the linear velocity was the initial value of 0.75 × 10 ^−4.
Even at m / s, the pressure drop did not reach a constant value and continued to rise, and the packed column was clogged with blood and the experiment was stopped.

Comparative Reference Example 2 Measurement of upper limit linear velocity Small cellulose particles (average particle diameter 25) used in Production Example 1
× 10 ⁻⁶ m), a reference example using a porous cellulose carrier (manufactured by Chisso Corporation, average particle size 220 × 10 ⁻⁶ m) having the same structure as the pore size of the small pores and having a larger average particle size. As in 1, the upper limit linear velocity at which a constant pressure loss was maintained during the passage of fresh bovine blood was measured. as a result,
The upper limit linear velocity was 5.78 × 10 ⁻⁴ m / s.

As can be seen from Comparative Reference Example 1, Poros (trade name) which has a structure in which a flow penetrates through the inside of a carrier particle when a flow is present around the particle is available as a carrier for chromatography. In (2), direct blood perfusion was difficult due to small particle size. On the other hand, the carrier obtained in Production Example 1 had a large upper limit linear velocity at which a constant pressure loss could be maintained, as can be seen from Reference Example 1. In addition,
Columns used in ordinary body fluid purification methods (volume 40
0 × 10 ^-6 m ^3, to 110 × 10 ^-3 m) in length, the flow rate in passing blood in 7.32 × 10 ^-4 m / s is 2.6
6 × 10 ⁻⁶ m ³ / s (159 ml / min), within the range of the treatment condition (0.833 × 10 ^{−6 to} 3.33 × 10 3).
⁻⁶ m ³ / s (50 to 200 ml / min)).

Reference Example 2 Measurement of Elution Curve The carrier obtained in Production Example 1 (average particle size 256 × 10
^-6 m, a column packed with an inner diameter of 0.01 (the ratio of the average diameter of the carrier to the average particle diameter of the small cellulose particles = 10).
m, 0.20 m in length), a 23.2 degree physiological saline solution (manufactured by Otsuka Pharmaceutical Co., Ltd.) was flowed at a linear velocity of about 4.6 × 10 ⁻⁴ m / s,
Low density lipoprotein reagent (manufactured by SIGMA: L213)
A solution obtained by diluting 9) 5 times with a physiological saline solution 100 × 10 ⁻⁹
m ³ was injected in a pulsed manner. The change with time of the concentration of the low-density lipoprotein in the eluate was measured at a wavelength of 280 nm using an absorbance meter (manufactured by ATTO). FIG. 7 shows this result as an elution curve. The abscissa sita indicates the ratio of the elution amount to the void volume between the carriers, and the ordinate E indicates the solute concentration value converted so that the total cumulative area of the elution curve becomes 1. In FIG. 7, there are two peaks, and the position of the first peak top was small immediately after the solution corresponding to the void volume between the carriers was completely eluted (sita = 1), and the peak height was small.

When albumin (molecular weight: 66,000) was injected under the same conditions as in Reference Example 2, the position of the peak top was sita = about 1.8. Since the peak in the elution curve in the absence of adsorption comes first from a substance having a large molecular weight, in consideration of the above results, a low-density lipoprotein having a large molecular weight (molecular weight of 3,000,000 to 500
10,000) is the first peak.

Comparative Reference Example 3 Measurement of Elution Curve The carrier of Comparative Reference Example 2 (manufactured by Chisso, average particle size 220 ×
Using 10 ⁻⁶ m), an elution curve of low density lipoprotein was measured under the same conditions as in Reference Example 2. FIG. 8 shows this result as an elution curve. The position of the first peak top was immediately after the solution corresponding to the void volume between the carriers was completely eluted, and the peak height was large.

From the results of Reference Example 2 and Comparative Reference Example 3, the peak shape of Reference Example 2 was smaller than that of Comparative Reference Example 3 and the first peak height was shifted rearward as a whole.
Therefore, the carrier of Production Example 1 (average particle size 256 × 10
^-6 m) is the carrier of Comparative Reference Example 3 (average particle size 220 × 1
0 ^-6 m) from the mass transfer is good, it has been found that the performance is good. Although the carrier of Production Example 1 has a through hole even though the average particle diameter is larger than the carrier of Comparative Reference Example 3, when there is a flow around the carrier particle, a flow penetrating through the particle occurs. It can be estimated that mass transfer of the low density lipoprotein in the carrier can be accelerated.

In the elution curves of Reference Example 2 and Comparative Reference Example 3, the peak of the low-density lipoprotein appears immediately after the elution of the solution corresponding to the void volume between the carriers is due to the inside of the carrier. Not because there are no small holes that can enter, but because the particle size of the carrier is large, the mass transfer distance is long, and the low-density lipoprotein can not sufficiently contact the carrier particles, and the space between the carriers packed in the column This elutes from the column outlet together with the flow flowing through the void.
The cellulose small particles (average particle diameter 25 × 10 ⁻⁶ m) constituting the carrier used in Reference Example 2 and the carrier (average particle diameter 220 × 10 ⁻⁶ m) of Comparative Reference Example 3 have small pores and the like. It has the same structure and allows low-density lipoproteins to enter the pores. The insertion of the low-density lipoprotein into the pores of this structure was confirmed by adsorbing the low-density lipoprotein in Examples 1 and 2 using the carrier of Production Example 1.

Example 1 The carrier obtained in Production Example 1 was reacted with epichlorohydrin at 45 ° C. for 2 hours, and then reacted with dextran sulfate at 40 ° C. for 24 hours to obtain an adsorbent on which dextran sulfate was immobilized.

The above-mentioned adsorbent was added so that the ratio of sedimentation volume to one volume of 6 volumes of fresh human serum was 1 volume,
After shaking at 37 degrees for 10 hours, the concentration of the supernatant was measured to determine the adsorption rate. Adsorption rate [%] = (stock solution concentration-supernatant solution concentration) / stock solution concentration x
100 The adsorption rates for low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, and albumin were 51%, 0%, and 0%, respectively, indicating an affinity for low-density lipoprotein.

Example 2 The carrier obtained in Production Example 1 was reacted with epichlorohydrin at 45 ° C. for 2 hours and then with aniline at 50 ° C. for 6 hours to obtain an adsorbent on which aniline was immobilized.

The adsorption rate of the adsorbent was determined under the same conditions as in Example 1. The adsorption rates for low-density lipoprotein-cholesterol, high-density lipoprotein-cholesterol, and albumin are 55% and 0%, respectively.
And 0%, indicating an affinity for low density lipoprotein.

According to Examples 1 and 2, the carrier obtained in Production Example 1 in which a substance having an affinity for the target substance was immobilized,
It was found that it could be applied as an adsorbent.

[0072]

Since the body fluid purifying adsorbent of the present invention has the above-mentioned structure, it can be expected that the dynamic adsorbing performance is large and the quality of life of the patient can be improved by shortening the treatment time.

[Brief description of the drawings]

FIG. 1 is a diagram showing the influence of the number of theoretical stages on a breakthrough curve.

FIG. 2 is a diagram showing a change with time in the amount of adsorption with respect to q ₀ .

FIG. 3 is a photograph of the surface of the carrier obtained in Production Example 1 enlarged 200 times.

FIG. 4 is a photograph of the surface of the carrier obtained in Production Example 1 magnified 5000 times.

FIG. 5 is a photograph of a cross section of the carrier obtained in Production Example 1 enlarged 200 times.

FIG. 6 is a photograph of a cross section of the carrier obtained in Production Example 1 magnified 5000 times.

FIG. 7 is a diagram showing an elution curve of a low-density lipoprotein based on Reference Example 2.

FIG. 8 is a diagram showing an elution curve of low-density lipoprotein based on Comparative Reference Example 3.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code FI G01N 30/00 G01N 1/28 J

Claims (8)

[Claims] 1. A body fluid purifying adsorbent comprising a substance having an affinity for a target substance in a body fluid immobilized on a particulate, slab-like or irregular-shaped carrier, wherein the carrier has a body fluid surrounding it. The body fluid purifying adsorbent has a structure in which a body fluid flow penetrating through the carrier is generated when there is a flow of the body fluid. 2. The body fluid purifying apparatus according to claim 1, wherein a substance having an affinity for the target substance is immobilized on a carrier having a structure in which a flow is generated through the inside of the carrier particles when there is a flow around the particles. Adsorbent. 3. A flow penetrating through the carrier particles when the average particle diameter is 100 × 10 ⁻⁶ m or more and the liquid is passed through the container at a linear velocity of 3 × 10 ⁻⁴ m / s or more. The adsorbent for purifying body fluid according to claim 1 or 2, which is produced. 4. A particle diameter of 100 × 10 ⁻⁶ m or more, 400
4. The body fluid purifying adsorbent according to claim 1, wherein the adsorbent is less than 0 × 10 ⁻⁶ m and is in direct contact with whole blood. 5. The carrier particles have through holes, and the ratio of the average particle diameter of the carrier particles to the average diameter of the through holes is 70 or less.
Or the adsorbent for purifying body fluid according to 4. 6. The carrier is packed in a column, and a solution containing only the target substance is subjected to a linear velocity of 1 × 10 ⁻⁴ m / s or more and 10 × 1
6. The adsorbent for body fluid purification according to claim 1, wherein the height equivalent to a theoretical plate is 0.5 m or less when the liquid is passed in a range of 0 ^-4 m / s or less. 7. A body fluid purifying adsorber filled with the bodily fluid purifying adsorbent according to claim 1, 2, 3, 4, 5, or 6. 8. A method for purifying a body fluid, comprising purifying a body fluid using the adsorber for purifying a body fluid according to claim 7.