JPS59144460A - Body fluids removing device - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (1) Background Technical Field of the Invention The present invention relates to a novel device for adsorbing and removing components in body fluids.

If there are pathogenic components in the body fluids of humans or animals, or if certain body fluid components are present in excess of normal values, a device is installed on the body fluid flow path of the extracorporeal circulation to adsorb and remove those body fluid components. Methods of treating diseases by removing or reducing the body fluid components are known. For example, a treatment method is known in which globulin present in blood in excess of a normal value during chronic infection or autoimmune disease is adsorbed and removed using the above-described method. Also, JP-A No. 55-141248
The issue describes a method for adsorbing and removing ferritin present in body fluids of cancer patients in excess of normal values.

The present invention thus relates to a device for adsorbing and removing components or pathogenic components present in body fluids in excess of normal values.

Problems with the Prior Art Conventional body fluid component removal devices have consisted of a support body on which an adsorbent for the component is fixed.

Therefore, in the unlikely event that even a portion of the fixed adsorbent is detached for some reason, the adsorbent may be mixed into body fluids.
There was a risk of serious injury to the human body.

Il. Object of the Invention The object of the present invention is to prevent the adsorbent fixed to the support from entering the human body in the above-mentioned body fluid component removal device even if it detaches from the support A. An object of the present invention is to provide a body fluid component removal device that can prevent the above.

111. DETAILED DESCRIPTION OF THE INVENTION In order to achieve the above objects, the present invention comprises the apparatuses described in the following sections.

(1) In a device that is placed on the body fluid flow path of extracorporeal circulation and that adsorbs and removes all the components in the body fluid using an adsorbent, the device has an adsorption main body that houses a support on which the adsorbent for the body fluid components is fixed. and an adsorption auxiliary part that accommodates a support on which the body fluid component is immobilized. 9. A body fluid component removal device, wherein the adsorption auxiliary part is disposed downstream of the body fluid flow from the adsorption main part.

(2) The body fluid component removal device according to item 1, wherein the adsorption main body portion and the adsorption auxiliary body portion are integrated.

(3) The body fluid component removal device according to item 1 or 2, wherein the component in the body fluid is human immunoglobulin G (still human-gamma globulin), and the adsorbent for the body fluid component is protein 8. .

(4) The support on which the adsorbent for the body fluid components is fixed has a pore size of 2
It has a large number of penetrating holes of 40-1600X and a surface area of 17-100 m2. The third layer is a porous bead with various
The body fluid component removal device described in Section 1.

(5) The body fluid component removal device according to item 4, wherein the porous beads have a particle size of 70 to 2 ooμ.

(6) The fourth porous bead has a pore diameter of 40'0 to 7oo' and a surface area of 17 to 100 m/9-.
The body fluid component removal device according to item 5.

(7) The body fluid component removal device according to any one of items 4 to 6, wherein the porous beads are glass beads, silica beads, or plastic beads.

(8) The support body on which the body fluid components are fixed has a pore diameter of 240 to 1
Has a large number of holes penetrating 600X, and has a surface area of 17~1
8. The body fluid component removal device according to items 3 to 7, which is a porous bead with a diameter of 00 m2/9.

(9) The body fluid component removal device according to item 8, wherein the porous beads have a particle size of 70 to 200 μm.

(Claim 8) or Item 9, wherein the porous beads have a pore size of 400 to 700X and a surface area of 17 to 100m2./W-;

(1υ The body fluid component removal device according to any one of items 8 to 10, wherein the porous beads are glass beads, silica beads, or plastic beads.

In the device of the present invention, the support on which the adsorbent for body fluid components is completely immobilized and the support on which the body fluid components are immobilized are prepared by methods known per se.

For example, when removing human)-IgG (-! or human-gamma globulin) from blood, prop-in A is used as an adsorbent.

Protein A is derived from the bacterium Staphylococcus aureus colawan l (5taphy)ococcus au.
It is a protein present in the cell wall of the human IgG (or human γ globulin) that is known to specifically adsorb human IgG (or human γ globulin).
Volume 124-127 (1970) and FEBS Letters, Volume 28, 73
-76 pages (1972)].

[Human IgG (or human γ globulin G)] and protein A can be immobilized using various general protein immobilization methods. That is, the support is activated with a fixing agent, and then human-IgG (-d-human-gamma globulin) or protein A is reacted with it.

Conventionally known supports and fixing agents are used.

For example, cellulose, agarose, polyacrylamide, polyaminostyrene or porous glass are used as supports. In particular, porous glass is desirable because of its high mechanical strength, and typically aminopropylated glass is used. The fixing agent depends on the support used; cyanogen bromide or novinyl sulfone is used as a fixing agent when the support is cellulose or agarose, glutaraldehyde is used for polyacrylamide, and polyaminostyrene is used as the fixing agent. In the case of aminopropylated porous glass, gluturaldehyde or carbodiimide is used.

A combination of aminopropylated porous glass and glutaraldehyde is particularly desirable.

After the support is activated by treatment with the above-mentioned immobilizing agent, human IgG (or human γ globulin) is reacted with protein Af. Activation reactions and immobilization reactions are usually carried out in buffer, chamber, 'fFL. The form of the support is preferably non-woven fabric, woven fabric, cotton, thread, or beads for reasons such as flowability of body fluids.

Here, non-woven fabrics can also include paper-like ones.

It is preferable to use porous beads in one hole in the beads, and in order to immobilize as much protein A as possible, the surface area of the support is required to be large. It's hopeful. On the other hand, however, the pores of the beads are required to have a pore size such that protein A can easily enter and be immobilized therein, and that IgG can easily be adsorbed to the immobilized protein A.

Furthermore, since bodily fluids that have come into contact with the adsorbent are returned to the body, harmful substances must not be eluted from the adsorbent support. Furthermore, when the adsorbent is packed in a column and used, it is also required that the flow rate of liquid passing through the column is not reduced or clogging does not occur, and that it is easy to handle.

Therefore, porous beads having a large number of penetrating pores with a pore diameter of 240 to 1600× and a surface area of 917 to 100 m 2 per gram are preferred. The pore size of the bead hole is critical,
240 to 1600X is efficient, more preferably 400 to 700X, and the surface area is 17 to 10 per 11
It is 0m. It is necessary that these pores penetrate through the support particles, and a support having only closed pores has a low adsorption capacity. The particle size of the crushed beads is preferably 70 to 200 μm from the viewpoint of handling, flow rate, clogging, etc. when packed in a column. There are no particular restrictions on the material of the beads, but those that elute harmful substances and those that are weak and break easily should be avoided since harmful substances and broken pieces may enter the body along with body fluids. Examples of desirable particles include glass beads, silica beads, beads of plastics such as polyaminostyrene, and the like.

The same applies to the support on which IgGf is immobilized.

Methods known per se are used to immobilize protein A on these beads. That is, in the case of glass or phosphoric beads, the beads are aminopropylated and then activated by treatment with glutaraldehyde, followed by reaction with protein A. In the case of polyaminostyrene beads, they are activated with carbodiimide and then reacted with protein A. Activation and immobilization reactions are usually carried out in a buffer at room temperature or at 4°C overnight. -I will be heard.

Furthermore, a method known per se is used to immobilize IgG on the beads. That is, in the case of glass or soribeads, this is carried out by converting the beads into aminozorobyls, then activating them by treating them with glutaraldehyde, and then reacting them with IgG. In the case of polyaminostyrene beads, they are activated by carboimide and then reacted with IgG. Activation reactions and immobilization reactions are usually carried out in a buffer at room temperature.

When using the device of the present invention to remove components in body fluids, methods known per se are used.

That is, desired components in the body fluid can be removed by leading the patient's body fluid out through extracorporeal circulation, passing it through the device of the present invention, and then returning the body fluid back into the patient's body.

When introducing body fluids to the removal device, ascites, blood, etc. may be directly introduced, or the blood may be separated into blood cells and plasma using a filtration device or a centrifugal device in advance, and only the plasma may be introduced to the removal device.

In the above example, protein A was used as the adsorbent in the apparatus of the present invention, but depending on the body fluid component to be removed, an antibody for the component may also be used as the adsorbent.

Next, a device for removing components from body fluids incorporating the device of the present invention will be described with reference to the accompanying drawings.

FIG. 1 is a diagram showing an example of the configuration of a body fluid component removal device. As can be seen in FIG. 1, the device comprises a separator 1, a flow rate controller 2 for the blood supplied to the separator, an end at the plasma outlet of the separator, and a blood flow path between the separator and the blood flow. It consists of a plasma channel 3 having an end downstream of the plasma flow channel -L, an adsorption main body section 4 arranged in the plasma flow channel -L, an adsorption auxiliary body section 5, and a quantitative liquid supply controller 6 for plasma. The blood is limited by the flow rate controller 2 and supplied to the separator 1, and a part of the plasma is separated from the blood by a filtration membrane built into the separator 1 for separating blood cells and plasma. The separated plasma enters the adsorption body 4 through the plasma path 3 of the separator 1, where desired liquid components are removed by an adsorbent fixed to a support.

Next, the plasma enters the adsorption auxiliary resting section 5, and if the desorbed adsorbent is mixed in the plasma, it is adsorbed and captured here by the body fluid component which is regulated at 12.1. blood! Around this time, the liquid was fed through the plasma flow path 3 by the quantitative liquid feed controller 6 at T(
It joins the blood.

FIG. 2 is a sectional view showing an example of the adsorption main body and the adsorption auxiliary body of the apparatus of the present invention. This main body part' or auxiliary body part consists of a container 9 having an inlet 17 and an outlet 8 at both ends, a support 10 containing an adsorbent or a body fluid component contained therein, and a support 10 having an inlet lower and an outlet 8. It consists of a filter 11 provided with a support 10 in between. The filter 11 is provided to prevent fragments of the support 10 from entering the bloodstream and flowing into the body.

FIG. 3 is a sectional view showing an example of the apparatus of the present invention in which the suction main body 1ηIs 4 and the auxiliary body portion 5 are integrated.

The adsorption main body part 4 and the auxiliary body part 5 can be constructed integrally since they have the same structure except for the components to be fixed. That is, the present device includes a cylindrical container 9 defined into two chambers by three filters 11 and 14 placed apart from each other.
A support 12 on which an adsorbent is immobilized and a support 13 on which body fluid components are immobilized are respectively housed inside, and plasma introduced from the entrance lower side contacts the support 12 and then contacts the support 13, and the blood plasma enters the outlet. 8.1].

Next, the present invention will be explained in more detail with reference to Examples.

Example (1) Preparation of adsorbent Aminopropyl porous glass beads (120-200 mesh, Pierce AMI)-CPG) and other specified beads each in 1 g of phosphate buffered saline with pT+ 7.2 25% glutaraldehyde 10 in water (PBS)
Me was added, dephosphorized, and stirred at room temperature for 1 hour. Wash with water and PBS. Next, 2mLj/me protein A (
(manufactured by Pharmacia) 15d" (Add and react overnight at 4°C. After removing the supernatant, add 15M) Risparanor (pH 7.6) 50
Add mA and leave at room temperature for 2 hours. Then 0,5MNaCt
0.1 M acetate buffer (p” 4.0) containing
．． 5M NaCtf: am0. IM carbonate buffer (
! Wash well alternately with i, (pH 8, 0), and finally with 1
) Wash with BS.

By the above operation, protein A of glass beads having an average pore diameter of 500×if 30 m9 is immobilized.

(2) Preparation of adsorption main body Porous glass beads with immobilized protein A 1y-t
Prepare the adsorption main body by filling the column.

Nylon mesocy was used for the filter, and polycarbonate was used for the column.

Flask, Peristales? The pump and the adsorption main body are connected to each other with a tube, and the IgG solution contained in the flask is circulated and passed through the adsorption main body. The amount of IgG adsorbed by the adsorbent is calculated from the difference between the amount of IgG before the liquid is passed through the adsorption main body and the amount of IgG in the flask after the liquid is circulated for 30 minutes. Protein A immobilized on each bead 0.5J
The amount and amount of IgG adsorbed are shown in Table 1.

From the results shown in Table 1, it is clear that beads having pores with a pore diameter of 240 to 1600X specifically adsorb a large amount of IgG. On the other hand, beads with pores of 200X or less have a large amount of protein A immobilized, but IgG
In addition, beads having pores with a pore diameter of 2200× or more have a small amount of immobilized protein A and a small amount of IgG adsorbed.

(3) Fixation of body fluid components 1) Aminopropylated porous glass beads o17 (1)
) in the same manner as (except for protein A)'
-using IgG) to produce IgG-immobilized porous glass beads. As mentioned above, the porous beads have a pore size of 240
~1600X pores are preferred.

Approximately 3.5 mg of IgG was immobilized.

11) Human-γ globulin G (γ
G Nimidori Juji Co., Ltd.) was processed, γG was approximately 38 m.
g was fixed.

111) Add 100 mA of dimethyl sulfate to the nylon fiber IP and treat it over boiling water for 2.0 minutes. It is then cooled on ice, then immersed in cold methanol and left at room temperature for several hours. Next, wash with cold water and then wash with PBS.

The sulfuric acid-treated nylon yarn 1 was placed in PBS 2 Fl me containing 0.25 coefficient γG and left overnight at 4°C. After washing with PBS, 0.5 M glycine was added, and the mixture was left at room temperature for an hour. Wash with PBS.

Approximately 32 rng of human γG was immobilized by this operation.

(4) Preparation of adsorption auxiliary body part
Gt or human rGf: An adsorption auxiliary portion is prepared by filling a column with the immobilized support in the same manner as in (2) above.

(5) Descaling 11 and capture test 1) Flask, pelister 7]? Connect the tube and the adsorption main body with a tube, and place the PBS in the tube.
10 J, was circulated and the liquid was passed through the adsorption main body for 1 hour. The adsorption main body is incompletely washed and contains unfixed protein A. r'Bs', jr
After adding liquid J, determine the amount of protein A in the PBS in the flask.

Quantification of prodin A was performed using a 96-well flat microplate coated with IgG1 from rabbits immunized with protein A.
Protein A sample, alkaline phosphatase-sensitized IgG
The enzyme immunoassay method was used.

As a result, protein A was 5 mg/m! , it was found that there was some confusion.

11) Connect the peristaltic pump 0 and the adsorption main body to the flask with all tubes, and further attach the adsorption auxiliary body to the downstream of the adsorption main body. PBS was circulated in the same manner as in 1) above, and protein 8 in the PBS in the flask was quantified. As a result, protein A was not detected when any of the adsorption auxiliary parts prepared in (3) 1) to 111) above were used.

From the results of this test, it is clear that even if protein A is detached from the adsorption main body, the protein A is captured in the adsorption auxiliary body.

■1 Effects of the Invention According to the present invention, firstly, there is provided a body fluid component removal device that can prevent the adsorbent from mixing into the body fluid even when the adsorbent for the body fluid component is detached from the support. provided.

For example, when using the device of the present invention to remove human IgG in plasma with protein A, the
Even if lotiin A is desorbed, protein A can be prevented from entering the human body because it is adsorbed by the adsorption auxiliary body. Since rotiine A is a protein present in the cell walls of pathogenic bacteria and is a foreign protein to the human body, there is a risk that its entry into the human body may cause a more serious situation. Therefore, the body fluid component removal device of the present invention, which can prevent such dangers, is a medically extremely useful device.

Second, according to the present invention, there is provided a body fluid component removal device in which the plasma circuit is purified and the amount of plasma filled in the plasma circuit is reduced. That is, since the adsorption body containing the support body on which the adsorbent for the body fluid component is fixed and the adsorption auxiliary body containing the support body on which the body fluid component is fixed are integrated, the structure of the apparatus 1 is simplified. This has the advantage that only a small amount of plasma needs to be filled in the extracorporeal circulation circuit.

Thirdly, according to the present invention, a body fluid component removal device with excellent body fluid component adsorption ability is provided.

If the rgG adsorption capacity is high, a small column can be used when making an extracorporeal circulation column using it, and therefore, the volume of zoliming can be reduced, and costs can also be reduced.

Fourthly, according to the present invention, there is provided a body fluid component removal device that allows rapid liquid passage through the column, is less clogged, and is easy to handle.

The porous beads used in the present invention have sufficient strength so that body fluids can be passed through the column under pressure, and the liquid passing rate I
W can be further increased.

Fifth, the present invention provides a safe body fluid component removal device that does not elute harmful proboscis or release bead fragments when it comes into contact with body fluids.

[Brief explanation of the drawing]

FIG. 1 shows an example of the configuration of a device that removes body fluid components from a patient's body by fully incorporating the body fluid component removal device of the present invention, and FIG. 2 shows the dimensions of the adsorbed complement part of the device of the present invention. It is a sectional view showing an example of an adsorption auxiliary body part. FIG. 3 is a cross-sectional view showing an example of an apparatus of the present invention in which the suction main body part and the suction auxiliary body part are integrated. 1・Preparator, 2・Flow rate controller, 3・Plasma flow path, 4・・
Adsorption body part, 5... Adsorption auxiliary body part, 6... Plasma liquid supply controller, 7-- Inlet, 8-- Output [ ], 9-- Container,
10... Body fluid component adsorbent or support with body fluid components fixed, 1 and 14 - Noilku, 12... Support with body fluid component adsorbent fixed, 13... Support with body fluid components fixed

Claims (9)

[Claims] (1) In a device that is placed on the body fluid flow path of extracorporeal circulation and that adsorbs and removes components in the body fluid using an adsorbent, the device includes an adsorption body that houses a support on which an adsorbent for the body fluid component is fixed. and an adsorption auxiliary body part containing a support on which the body fluid component is immobilized, and the adsorption auxiliary body part is arranged at a position downstream of the body fluid flow from the adsorption main body part. Device. (2) The body fluid component removal device according to claim 1, wherein the adsorption main body portion and the adsorption auxiliary body portion are integrated. (3) Claim 1 or 2, wherein the component in the body fluid is human immunoglobulin G (or human-gamma globulin), and the adsorbent for the body fluid component is protein A.
The body fluid component removal device described in Section 1. (4) The support on which the adsorbent for the body fluid components is fixed has a pore size of 2
It has many penetrating holes of 40~1600X and has a surface area of 17~100 m2/f! - The body fluid component removal device according to claim 3, which is a porous bead. (5) The body fluid component removal device according to claim 4, wherein the porous beads have a particle size of 70 to 200 microns. (6) The body fluid component removal device according to claim 4 or 5, wherein the porous beads have a pore diameter of 4.00 to 700X and a surface area of 17 to 100 m2/¥. (7) The body fluid component removal device according to any one of claims 4 to 6, wherein the porous beads are glass beads, silica beads, plasti, or cube beads. (8) The support body on which the body fluid components are fixed has a pore diameter of 240 to L.
Has a large number of holes penetrating 600X, and has a surface area of 17~1
00m2/f! - The body fluid component removal device according to claims 3 to 7, which is a porous bead. (9) The body fluid component removal device according to claim 8, wherein the porous beads have a particle size of 70 to 200 microns. OQ The body fluid component removal device according to claim 8 or 9, wherein the porous beads have a pore diameter of 400 to 7007° and a surface area of 17 to 100 m27g. αυ The body fluid component removal device according to any one of claims 8 to 10, wherein the porous beads are glass beads, silica beads, or glass beads.