JPS5810055A - Production of immune adsorbing 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 The present invention relates to a method for manufacturing an immunoadsorbent that removes high molecular weight substances dissolved in blood and purifies blood. More specifically, we will introduce an immunoadsorber that can safely and efficiently adsorb and remove harmful immunoglobulins and/or their complexes, such as harmful autoantibodies, found in the blood of patients with diseases related to abnormalities in the body's immune function. Regarding the manufacturing method.

As is well known, immunoglobulins such as autoantibodies and/or their complexes found in the blood can cause autoimmune diseases such as cancer, rheumatoid arthritis, and systemic lupus erythematosus, as well as biological diseases such as allergies and rejection reactions during organ transplants. It is thought that there is a close relationship with the causes and progression of pathological conditions of diseases that are deeply related to immune function.

Recently, in order to remove malignant substances such as antigens, autoantibodies, and immune complexes in the patient's plasma, plasmapheresis therapy, which replaces the patient's plasma with other fresh frozen plasma or an albumin preparation, has been performed, which has significantly alleviated symptoms. , progress prevention, or curative effects have been confirmed.

However, this plasmapheresis therapy has two problems: (1) It is difficult to obtain a large amount of fresh frozen plasma or plasma components in a sustained manner to replenish the removed plasma, and (2) it uses someone else's plasma, so it is difficult to prevent hepatitis. There are nine drawbacks when there is a high risk of infection by viruses, etc., and it is not something that can be applied to the general public.

Additionally, to remove malignant substances from patient plasma, there is a method that uses an ultrathermal filtration membrane, which has the advantage of not requiring supplementation of plasma components; (2) There is no selectivity for removal other than molecular weight, so useful substances in the plasma are also removed. (2) Decrease in filtration rate due to the eyelid in II, fluctuation in cut-off molecular weight, etc. It has the following problems.

Furthermore, a method has been proposed in which biopolymers such as protein A, DNA, the first component of complement, denatured biopolymers, synthetic nucleic acid polymers, etc. are immobilized on a carrier to remove autoantibodies and their complexes. There is.

However, these methods have the disadvantage that the immobilized substance is expensive and its activity is unstable, so that it is easily deactivated due to handling in an O compost during immobilization, storage after immobilization, etc. A particularly critical problem is that, due to its instability, sterilization operations cannot be performed to achieve sterility, which is an essential requirement for a therapeutic device. One of the sterilization operations is moist heat sterilization using an autoclave, etc., and the purpose is achieved by heating to 100°C or higher, but during this heating, the immobilized substance is deactivated,
The ability to bind to bound substances such as autoantibodies is lost.

In order to overcome these problems, the present inventors have conducted intensive research into an adsorbent that is simple and safe, can efficiently remove malignant substances such as autoantibodies from patient blood or plasma, and can be sterilized. As a result, by bonding an organic low-molecular-weight compound that can bind to an adsorbed substance to an insoluble carrier having a hydroxyl group, it is possible to remove the target adsorbed substance, and at the same time, there is no denaturation of the immobilized substance, making it sterile. They discovered that the process can be easily performed and completed the present invention. This made it possible to perform extracorporeal circulation safely and overcome the difficulties in actual clinical application.

That is, the present invention involves bonding an organic low-molecular compound containing a functional site capable of binding to an adsorbed substance to an insoluble carrier having a hydroxyl group, and then filling the insoluble carrier into a container through which the liquid to be treated can flow. This is a method for manufacturing an immunoadsorber characterized by performing thermal sterilization treatment.

The carrier used in the present invention can be used in any shape, such as granules, fibrous bodies, and sintered bodies, but it is easy to manufacture, less likely to be damaged during transportation, etc. From this point of view, a particulate carrier is the most preferable shape.

The carrier used in the present invention is a crosslinked polymer compound that is insoluble in water and has a hydroxyl group, and has a water retention capacity of 0.5 to 69/9, more preferably 1.0 to 69/9.
Those within the range of 5.0979 can be suitably used. The water retention capacity is defined as the amount of physiological saline that can be contained in the carrier per unit dry carrier when the carrier is equilibrated with physiological saline. If the water retention amount is larger than 69%, the mechanical strength of the carrier decreases, and particles may break during manufacturing, sterilization, transportation, etc., which is undesirable. Water retention amount is 0.59/9
When the particle size becomes too small, the pore volume and surface area of the carrier particles decrease, which is undesirable because the adsorption capacity decreases.

The carrier has an average particle size of 25 to 2, which allows liquids with high viscosity and high solute concentration, such as body fluids such as blood and plasma, to flow stably at high flow rates for long periods of time, and at the same time maintains high adsorption performance.
500μ, more preferably 40 to 1000μ, is preferably used, but in order to circulate blood, 400μ
The above is desirable.

The carrier used in the present invention is rounded to suppress interaction with plasma proteins, blood cell components, etc., and at the same time retains a large amount of organic compound having a site capable of binding to immunoglobulin and/or immunoglobulin complex. , 5m
A carrier having a hydroxyl group density of 0.01 or more is preferably used.

The carrier used in the present invention is composed of a linear polymer compound and a crosslinkable compound, and the crosslinkable compound is used in an amount of 5 parts by weight or more, more preferably 5 to 50 parts by weight based on the linear polymer compound. Thus, a suitable carrier can be obtained. If the amount of the crosslinking compound is too small, the physical strength of the carrier will decrease and it will not be able to withstand thermal sterilization, which is not preferable. Too much crosslinking compound is not preferred because the hydroxyl group density decreases and undesirable interactions increase.

A hydrophilic crosslinked polymer compound having a hydroxyl group can be synthesized by polymerizing a monomer having a hydroxyl group or introducing a hydroxyl group through a chemical reaction of a polymer. It is also possible to synthesize both in combination. As the polymerization method, known polymerization methods such as condensation polymerization, radical polymerization, ionic polymerization, and ring polymerization can be used.

The crosslinking agent is preferably introduced by copolymerization during polymerization. Alternatively, it may be introduced through a chemical reaction between polymers (between polymers, between a polymer and a crosslinking agent).

As an example, vinyl polymers can be made by copolymerizing a vinylene monomer and a vinyl or allyl crosslinking agent. In this case, the hydrophilic crosslinked polymer compound includes crosslinked polyvinyl A'7# Co-Jz, crosslinked 112
Examples include crosslinked vinyl polymers such as -hydroxyethyl acrylate and crosslinked 2-hydroxyethyl methacrylate.

As a crosslinking agent, allyl compounds such as triallyl isocyanurate and triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate, butanediol divinyl ether, diethylene glycol divinyl ether, and tetravinyl are used. Polyvinyl ethers such as glyoxal, polyallyl ethers such as diarylidene pentaerythritol and tetraallyl xyethane, and glycidyl acrylates such as glycidyl methacrylate can be used. In particular, triallyl in cyanurate units are preferred from the viewpoints of mechanical strength, hardness, micropore structure, and chemical properties.

If necessary, copolymerized comonomers such as vinyl esters and vinyl ethers may also be used.

In the case of vinyl or vinylene copolymers, polyvinyl 7 alcohol is obtained by copolymerizing a vinyl ester of a carboxylic acid and a vinyl compound having an incyanurate ring (allyl compound), and then hydrolyzing the copolymer. Triallylisocyanurate [111] provides a particularly good support in terms of mechanical strength, hardness, pore stability, and chemical properties.

Methods for binding organic low-molecular compounds to carriers include covalent bonding,
Any known method can be used, such as ionic bonding, physical adsorption, embedding, precipitation on the surface of the polymer, etc., but in view of the elution properties of bound substances, it is preferable to hold and insolubilize by covalent bonding. For this purpose, known methods for activating and binding carriers commonly used in immobilized enzymes, affinity chromatography, and affinity chromatography can be used.

Examples of activation methods include cyanogen halide method, epichlorohydrin method, bisepoxide method, halogenated triazine method, bromoacetyl bromide method, ethyl chloroformate method, 1,1'-carbonyldiimidazole method, etc. can. The activation method of the present invention involves substituting and/or substituting a nucleophilic reactive group having active hydrogen, such as an amino group, a hydroxyl group, a carboxyl group, or a thiol group, on the organic low-molecular compound.
It is sufficient that the addition reaction can be carried out, and the method is not limited to the above example.

The organic low-molecular-weight compound used in this WAK, which has a functional site capable of binding to immunoglobulins such as autoantigens and autoantibodies, or their complexes, changes its molecular structure by heat transfusion during heating. It must be something that will not allow you to do so. For example, hydrophobic organic small molecules containing aromatic rings, such as human aggregation globulin and tryptophan, have a strong binding ability to autoantibodies called rheumatoid factors and their complexes, which are detected in the blood of chronic rheumatism patients in high-speed cars. There are also heat-denatured deoxyribonucleic acids and compounds that have a strong binding ability to anti-nuclear antibodies, anti-DNA antibodies, and their complexes, which are frequently found in the blood of patients with autoimmune diseases such as systemic lupus erythematosus patients. There are compounds or derivatives thereof that contain purine bases or pyrimidine bases as constituents, and they also contain sugars and/or derivatives that have a strong binding ability to sugar chains and glycoproteins present on the surface of cell membranes that are detected in various autoimmune diseases. These include oligosaccharides and their derivatives.

The organic low-molecular-weight compound used in the present invention is preferably a compound that does not have antigenicity in the body when released from the carrier during clinical use, and has a molecular weight of 10,000 or less,
In particular, it is preferable for polypeptide compounds to have a molecular weight of 1000 or less. In the present invention, the amount of the organic low molecular compound to be bound to the carrier is 0.1-9 to 50 m
A range of 9 is desirable. If the retained amount is too low, the adsorption capacity for the substance to be adsorbed is too low to be practical, and if the retained amount is too large, the specificity of adsorption may decrease, which is not preferred.

The immunoadsorber of the present invention uses the above-mentioned adsorbent in the liquid to be treated.
It is filled and held in a container that can be distributed and sterilized by heat.

FIG. 1 shows one embodiment of the immunoadsorber of the present invention, which includes a cylinder 1 having a fluid inlet 4 at one end opening through a backing 3 with a filter 2 placed inside. Screw the cap 5', which has a fluid outlet 4' through the opening K at the other end of the cylinder 1 and the backing 3' with the filter 2' on the inside, screw 6'. The adsorbent layer 7 is formed by filling and retaining an adsorbent in the gap between and 2'.

The adsorbent layer 7 may be filled with the adsorbent of the present invention alone, or may be mixed or laminated with other adsorbents. The volume of the adsorbent layer 7 is suitable for extracorporeal circulation.As a thermal sterilization method, as specified in the Japanese Pharmacopoeia Act, an autoclave is used for 115°C for 30 minutes, 121°C for 20 minutes, or 131°C for 15 minutes. It is necessary to select appropriate sterilization conditions from among these.

It is necessary to select the materials of the container body and other members of the immunoadsorber so as to meet the above-mentioned temperature conditions for sterilization. In addition, it is necessary to sufficiently degas the immunoadsorbent material so that there is no rapid volume change in the internal packing due to heating.

To do this, before filling the nuclear adsorber with l* adsorbent,
It is also important to perform heat treatment in an autoclave in advance. In addition, changes in the volume of the immunoadsorber container,
In order to compensate for the difference in volume of the internal filling liquid, it is also effective to use a rubber ball or the like that has a buffering effect.

When using the adsorber of the present invention in extracorporeal circulation, there are two methods as shown below by Ohji. One method is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or membrane plasma separator, and then pass the plasma components through the device of the present invention for purification. One method is to return the blood to the body together with blood cell components, and the other method is to directly pass the blood taken out from the body through the absorber of the present invention for purification.

The method for passing body fluids may be continuous or intermittent depending on clinical needs or the installation status of the equipment.

As described above, the adsorbent of the present invention can remove immunoglobulins such as autoantigens and autoantibodies in body fluids or their complexes very efficiently and can be used clinically with safety.

Hereinafter, embodiments of the present invention will be explained in more detail with reference to Examples.

Example 1 100 g of 2-hydroxy methacrylate, 25 g of ethylene glycol dimethacrylate, 12 g of glycidyl methacrylate, 124 g of ethyl acetate, 1249 g of heptane,
Polyvinyl acetate (degree of polymerization 500) 3.19 and 2,2
A homogeneous liquid mixture consisting of 3.19% of polyvinyl alcohol, 0.05% of sodium dihydrogen phosphate dihydrate, and 12% of sodium hydrogen phosphate (12) Add 400ml of water in which 1.5% by weight of hydrate has been dissolved into a flask, stir well, and add 60ml of water.
C. for 18 hours and then heated and stirred at 75.degree. C. for 5 hours to perform suspension polymerization to obtain a particulate copolymer. After O filtering and washing with water, classification was performed to obtain a carrier having an average particle size of 180 .mu.m.

Further, the water retention amount of the obtained particulate carrier was 4.59/g, and its specific surface area was a 10 d/9. The exclusion limit molecular weight of the standard globular protein measured using phosphate buffered saline was approximately 1.5 million.

The organic low-molecular-weight compounds shown in Table 1 were dissolved in 0.1 M sodium bicarbonate whose pH was adjusted to 9.5 using an aqueous sodium hydroxide solution, added to carrier 10-, and added to the solution at 25°C.
After shaking and reacting for 6 hours and blocking unreacted activated functional groups with glycine, F separation and washing with physiological saline were repeated to obtain an immunoadsorbent.

The adsorbent was filled into a 4- container as shown in Fig. 1 to form an immunoadsorbent, and then heated at 121°C in an autoclave.
Sterilization was performed for 20 minutes.

From the observation results of the adsorbent and the content liquid in the adsorber using an optical microscope, no destruction such as rupture or cracking of the adsorbent due to sterilization was observed.

Adsorption experiments were conducted using the model experimental system shown in FIG. 2, using immunoadsorbers before and after sterilization.

That is, 15 d of systemic lupus erythematosus patient's plasma (9) is put into the container (8), and the pump (10) is pumped at 0.00 m/min.
It is pumped out at a flow rate of 5- and sent to the immunoadsorber (11), and then connected to the drip chamber (12) and the sampling port (13).
) to be returned to the container (8).
4) was installed.

After circulating the plasma for 3 hours using the above experimental system, the plasma was sampled, and anti-DNA antibodies, which are autoantibodies, in the plasma were measured by a hemagglutination method, and anti-nuclear antibodies were measured by an enzyme-linked antibody method.

Immune complexes were determined by measuring complement consumption in polyethylene glycol precipitates.

The results are shown in Table 1.

Around V-4cIi's Crying Watch
Example 2 A carrier was prepared in the same manner as in Example 1, and various organic low-molecular compounds shown in Table 2 were retained on the carrier to serve as an immunomodulating material. An adsorption experiment similar to that in Example 1 was conducted using chronic rheumatism patient plasma.

Rheumatoid factor, which is an autoantibody, was measured by the latex agglutination method and Waller-Rose method, and 0 immune complexes were measured in the same manner as in Example 1.

The results are shown in Table 2.

ku- ~ -moan &0 no to ■ -
A carrier with an average particle size of 450 μm prepared in the same manner as in Example 1 was coated with 28 μmo L-) lipdoan methyl ester.
Using a sterilized immunoadsorbent filled with 4Ad-bonded adsorbent, blood 15- from a patient suffering from chronic rheumatoid arthritis was recirculated for 3 hours in the experimental system shown in Figure 2. The rate of decrease in white blood cells was less than 5X, and the rate of decrease in platelets was less than 40X. Rheumatoid factor (latex agglutination) was reduced by one-eighth.

[Brief explanation of the drawing]

FIG. 1 is a sectional view showing one example of the immunoadsorber of the present invention, and FIG.
The figure is an explanatory diagram of a model experiment using the immunoadsorber of the present invention. ″ 1...Cylinder, 2.2'...Filter, 3.3'...
...Backing, 4,4'...Body fluid inlet/outlet 1.5,
5'...Cap, 6.6'...Screw, 7...Adsorbent. Figure 1 2vA

Claims (1)

[Claims] After bonding an organic low-molecular compound containing a functional site capable of bonding with an adsorbed substance to an insoluble carrier having a hydroxyl group, it is filled into a container through which the liquid to be treated can flow, and further subjected to moist heat sterilization. A method for manufacturing an immunoadsorber characterized by: