JPS6159142B2 - - Google Patents

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, the method for manufacturing an immunoadsorbent device that can safely and efficiently adsorb and remove immunoglobulins and/or their complexes such as harmful autoantibodies found in the blood of patients with diseases related to abnormalities in the immune function of the living body. Regarding. As is well known, immunoglobulins such as autoantibodies and/or their complexes found in the blood are
It is believed that there is a close relationship with the causes and progression of conditions of autoimmune diseases such as cancer, rheumatoid arthritis, and systemic lupus erythematosus, as well as diseases that are deeply related to the immune system of the body, such as allergies and rejection reactions during organ transplants. It is considered. Recently, in order to remove malignant substances such as antigens, autoantibodies, and immune complexes in the patient's plasma, plasmapheresis therapy, in which the patient's plasma is replaced with other fresh frozen plasma or albumin preparations, has been performed, and symptoms have been significantly alleviated. , its progress prevention or curative effects have been confirmed. However, this plasmapheresis therapy has two drawbacks: (1) difficulty in obtaining large quantities of fresh frozen plasma or plasma components in a sustained manner to replenish the removed plasma;
(2) Because the plasma of another person is used, there is a high risk of infection with hepatitis viruses, etc., and this method cannot be widely used. Additionally, to remove malignant substances from patient plasma, there is a method using an ultrafiltration membrane, which has the advantage of not requiring supplementation of plasma components; however, (1) it cannot be separated cleanly due to molecular weight; (2) There is no selectivity for removal other than molecular weight, so useful substances in plasma are also removed. (2) Problems such as decreased overspeed due to membrane clogging and fluctuations in cut-off molecular weight. have. 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 are difficult because the immobilized substances are expensive and their activity is unstable.
It had the disadvantage that it was easily deactivated due to handling 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 it to 100°C or higher, but during this heating, the immobilized substance is deactivated and the bound substances such as autoantibodies are removed. The ability to bind to substances is lost. In order to overcome these problems, the present inventors
As a result of extensive research into an adsorbent that is simple and safe, can efficiently remove malignant substances such as autoantibodies from patients or plasma, and can be sterilized, we have found that 5 to 50% of crosslinking compounds can be added to linear polymer compounds. A crosslinked polymer compound having a hydroxyl group, obtained by using part by weight, with a water retention amount of 0.5 to 6 g/g
By covalently bonding an organic low-molecular compound capable of binding an adsorbed substance to a carrier in the range of 0.1 mg to 50 mg per ml of the carrier, it is possible to remove the target adsorbed substance and at the same time They discovered that the immobilized substance does not undergo denaturation and can be easily sterilized, leading to the completion of 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 provides a crosslinked polymer compound having a hydroxyl group, which is obtained by using 5 to 50 parts by weight of a crosslinkable compound to a linear polymer compound, and has a water retention amount of 0.5 to 6 g/g. After covalently bonding an organic low-molecular compound containing a functional site capable of binding with the adsorbed substance to the carrier in a range of 0.1 mg to 50 mg per 1 ml of the carrier, it is placed in a container through which the liquid to be treated can flow. This is a method for manufacturing an immunoadsorber, which is characterized by filling and further performing moist heat sterilization. The carrier used in the present invention can be used in any shape, such as granules, fibrous bodies, and sintered bodies;
A particulate carrier is the most preferable shape from the viewpoint of less damage during transportation. 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 6 g/g, more preferably 1.0 to 5.0 g/g. Those listed in can be suitably used. The water retention capacity is defined as the amount of saline that can be contained in the carrier per unit dry carrier when the carrier is equilibrated with physiological saline. Water retention capacity is 6
When it is larger than g/g, the mechanical strength of the carrier decreases, and the particles may be broken during manufacturing, sterilization, transportation, etc., which is not preferable. Water retention amount is 0.5g/
If it is smaller than g, the pore volume and surface area of the carrier particles will decrease, resulting in a decrease in adsorption capacity, which is not preferable. The carrier must have an average particle diameter of 25 to 2500μ, in order to be able to stably flow liquids with high viscosity and high solute concentration, such as body fluids such as blood and plasma, at high flow rates for long periods of time, and at the same time maintain high adsorption performance. More preferably 40
A diameter of ~1000μ is preferably used, but a diameter of 400μ or more is desirable in order to allow blood to circulate. The carrier used in the present invention is used to suppress interaction with plasma proteins, blood cell components, etc., and at the same time to retain a large amount of an organic compound having a site capable of binding to immunoglobulin and/or immunoglobulin complex. , a carrier having a hydroxyl group density of 5 meq/g 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. , a suitable carrier is obtained. When there are few crosslinking compounds,
This is not preferable because the physical strength of the carrier decreases and it becomes unable to withstand moist heat sterilization treatment. 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. Polymerization methods include condensation polymerization, radical polymerization, ionic polymerization,
Known polymerization methods such as ring-opening 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). For example, it can be made by copolymerizing a vinyl monomer or vinylene monomer with a vinyl or allyl crosslinking agent. Examples of the hydrophilic crosslinked polymer compound in this case include crosslinked vinyl polymers such as crosslinked polyvinyl alcohol, crosslinked 2-hydroxyethyl acrylate, and crosslinked 2-hydroxyethyl methacrylate. Examples of crosslinking agents include 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. Polyvinyl ethers such as glyoxal, polyallyl ethers such as diarylidene pentaerythrite and tetraallyloxyethane, and glycidyl acrylates such as glycidyl methacrylate can be used. In particular, triallylisocyanurate 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, triallyl of polyvinyl alcohol obtained by copolymerizing a vinyl ester of carboxylic acid and a vinyl compound having an isocyanurate ring (allyl compound) and hydrolyzing the copolymer. Isocyanurate crosslinkers provide particularly good supports in terms of mechanical strength, hardness, pore stability, and chemical properties. All known methods such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation insolubilization on the polymer surface can be used to bond the organic low-molecular compound to the carrier. It is preferable to use it by holding and insolubilizing it 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,
Examples include the halogenated triazine method, the bromoacetyl bromide method, the ethyl chloroformate method, and the 1,1'-carbonyldiimidazole method. The activation method of the present invention is limited to the above examples as long as it can perform a substitution and/or addition reaction with a nucleophilic reactive group having active hydrogen such as an amino group, a hydroxyl group, a carboxyl group, or a thiol group of the organic low-molecular compound. It's not something you can do. The organic low-molecular-weight compound having a functional site capable of binding to immunoglobulins such as autoantigens and autoantibodies or complexes thereof used in the present invention is one that does not change its molecular structure when heated during moist heat sterilization. Must-have. 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 at a high rate in the blood of chronic rheumatism patients. There are also heat-denatured deoxyribonucleic acids and purine 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 autoimmune patients such as systemic lupus erythematosus patients. There are compounds or derivatives thereof that contain bases or pyrimidine bases as constituents, and also sugars and/or oligos that have strong binding ability to sugar chains and glycoproteins present on the surface of cell membranes that can be detected in various autoimmune diseases. These include sugars and their derivatives. The organic low-molecular compound used in the present invention is preferably a compound that does not have antigenicity in the body when released from a carrier during clinical use, and has a molecular weight of 10,000 or less, particularly a polypeptide compound with a molecular weight of 1000 or less. It is preferable that In the present invention, the amount of the organic low molecular weight compound bound to the carrier is preferably in the range of 0.1 mg to 50 mg per ml of carrier. 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 preferable. The immunoadsorber of the present invention fills and holds the adsorbent as described above in a container through which a liquid to be treated can flow,
It was sterilized using moist heat. FIG. 1 shows an embodiment of the immunoadsorber of the present invention, in which a cap 5 having a fluid inlet 4 is screwed into the opening at one end of a cylinder 1 through a packing 3 with a filter 2 stretched inside. Fitted 6, cylinder 1
A cap 5' having a fluid outlet 4' is screwed into the opening at the other end through a packing 3' with a filter 2' stretched inside, and an adsorbent is inserted into the gap between the filters 2 and 2'. Fill and hold adsorbent layer 7
It is formed by forming. 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 appropriate volume of the adsorbent layer 7 is about 50 to 400 ml when used for extracorporeal circulation. The method of moist heat sterilization is to use an autoclave as stipulated in the Japanese Pharmacopoeia Law.
It is necessary to appropriately select sterilization conditions from among 30 minutes at 121°C, 20 minutes at 121°C, and 15 minutes at 131°C. It is necessary to select the materials of the immunoadsorber's container body and other members 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. For this purpose, it is important to heat-treat the adsorbent in an autoclave before filling it into the adsorber. It is also effective to use a rubber ball or the like having a buffering effect in order to compensate for the difference between the volume change of the container of the immunoadsorber and the volume change of the internal filling liquid. When using the adsorber of the present invention in extracorporeal circulation,
There are roughly two methods as follows. One method involves separating blood taken from the body into plasma components and blood cell components using a centrifuge or membrane plasma separator, and then passing the plasma components through the device of the present invention.
After purification, the blood is returned 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 adsorption device 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 2-hydroxy methacrylate 100 g, ethylene glycol dimethacrylate 25 g, glycidyl methacrylate 12 g, ethyl acetate 124 g, heptane 124 g, polyvinyl acetate (degree of polymerization 500) 3.1 g and 2,2'-azobisisobutyronitrile 3.1 g
A homogeneous mixed liquid consisting of polyvinyl alcohol 1
wt%, sodium dihydrogen phosphate dihydrate 0.05 wt% and disodium hydrogen phosphate dodecahydrate
Pour 400ml of water containing 1.5% by weight into a flask, stir well, and heat at 60°C for 18 hours.
Suspension polymerization was carried out by heating and stirring at °C for 5 hours to obtain a particulate copolymer. After washing with water, classify the
A carrier with an average particle size of 180μ was obtained. In addition, the water retention amount of the obtained particulate carrier was 4.5g/
g, and its specific surface area was 10 m ² /g. 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 hydrogen carbonate whose pH was adjusted to 9.5 using an aqueous sodium hydroxide solution, added to 10 ml of carrier, and
After shaking and reacting at ℃ for 16 hours and blocking unreacted activated functional groups with glycine,
Washing with physiological saline was repeated to obtain an immunoadsorbent material. The adsorbent was filled into a 4 ml container as shown in FIG. 1 to prepare an immunoadsorbent, and then sterilized in an autoclave at 121° C. 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 the sterilization treatment was observed. An adsorption experiment was conducted using the model experimental system shown in FIG. 2 using the immunoadsorber before and after sterilization. That is, 15 ml of systemic lupus erythematosus patient's plasma 9 is put into the container 8, and the pump 10 pumps the blood at 0.5 ml per minute.
ml flow rate and sent to the immunoadsorber 11, drip chamber 12 and sampling port 13.
tube 14 to be returned to container 8 through
was installed. After circulating 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.

[Table] Example 2 Various organic low-molecular compounds shown in Table 2 were retained on a carrier prepared in the same manner as in Example 1 to prepare an immunoadsorbent. An adsorption experiment similar to that in Example 1 was conducted using chronic rheumatism patient plasma. Rheumatoid factor, an autoantibody, was measured by latex agglutination method and Waller-Rose method.
Immune complexes were measured in the same manner as in Example 1. The results are shown in Table 2.

[Table] Example 3 Average particle size 450 μ produced in the same manner as Example 1
L-tryptophan methyl ester is added to the carrier.
In the experimental system shown in Figure 2, 15 ml of blood from a chronic rheumatoid patient was recirculated for 3 hours using a sterilized immunoadsorbent filled with adsorbent bound to 28 μmol/ml. The rate of decrease in red blood cells and white blood cells was less than 5%, and the rate of decrease in platelets was less than 40%. Rheumatoid factor (latex agglutination) was reduced by one-eighth.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional view showing an example of the immunoadsorbent of the present invention, and FIG. 2 is an explanatory diagram of a model experiment using the immunoadsorbent of the present invention. 1...Cylinder, 2, 2'...Filter, 3,
3'...Packing, 4,4'...Body fluid inlet/outlet,
5, 5'... Cap, 6, 6'... Screw, 7...
adsorbent.

Claims (1)

[Claims] 1 Add 5 to 50% crosslinkable compound to linear polymer compound
A crosslinked polymer compound having a hydroxyl group, obtained by using 50 parts by weight, with a water retention capacity of 0.5
After covalently bonding an organic low-molecular compound containing a functional site capable of bonding with an adsorbed substance to a carrier of ~6 g/g in the range of 0.1 mg to 50 mg per 1 ml of the carrier, the solution to be treated is distributed. 1. A method for producing an immunoadsorber, which comprises filling the immunoadsorber into a container capable of sterilizing the immunoadsorber, and further performing a moist heat sterilization process.