JPS6222659A - Method and apparatus for regenerating low specific gravity lipoprotein adsorbing material - 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 (Field of Industrial Application) The present invention is a low-density lipoprotein that selectively adsorbs and removes low-density lipoproteins, which are thought to be closely related to various diseases caused by increased plasma lipids. The present invention relates to a method for regenerating a specific gravity lipoprotein adsorbent and an apparatus therefor.

As is well known, an increase in lipids in the blood, including low-density lipoprotein (%), which is a cause of arteriosclerosis, is thought to be closely related to the progression of arteriosclerosis. Myocardial infarction occurs when arteriosclerosis progresses,
The possibility of suffering from serious circulatory system symptoms such as cerebral infarction is extremely high, and the mortality rate is also high.

Therefore, by selectively adsorbing and removing low-density lipoproteins from body fluid components such as blood and plasma, SaraK is expected to prevent the progression of the above-mentioned diseases, alleviate symptoms, and speed up healing. was.

(Prior art) Existing technologies that can be used for the above purpose include adsorption (Lupie) using an adsorbent in which heharin is immobilized on agarose gel.
n, P-J, et, a4: A new a
The management
of family hypercholeste
-rolemia, Removal of plus
ma -cholesterol based on t
he principle of affinity
chromato-graphy, Lancet,
2: 1241-1264.1974. ) and chromatography using glass powder or glass beads (Carlson, L.A.: Chroma
tographic 5eparation of s
erum 1ipoprote-ins on gla
ss powder columns, Script
ion of the method and some
applications, Cl1n, Chim.

Acta, 5: 52B-558.1960. ).

In addition, recently, an improved invention suitable for the adsorption and removal of low-density lipoproteins by extracorporeal circulation (% 1986-10245)
6) and the invention of an adsorbent with a further improved adsorption capacity for low-density lipoproteins (Patent Application No. 80777-1989,
80778) is now available.

Technological progress has been made.

In addition, attempts have been made to treat body fluids with these adsorbents, adsorb and remove low-density lipoproteins, and then desorb the low-density lipoproteins from the adsorbents to regenerate and reuse the adsorbents. Ta.

For example, in the case of an adsorbent with immobilized heparin.

How to wash lipoproteins using 5-chloride sodium chloride (
Ddwin A, Burgstaler et a.
l, : Laboratory Study, Rem
oval of plasma
ns fromcirculating blood
with a Heparin-Agarosecol
umnlMayo C11n Proc 55:
180-184°1980), and when using glass, potassium bicarbona
te-carbonate buffer (pH 8,
8, 9, 6, 9, 8 and C01) (Carlson, L.A.: Chrom
atographic separation of s
erum 1ipoproteins on glass
sprayer column, Description
n of the method and some a
applications, Cl1n, Chim,
Acta, 5:528-538.1960. ).

(Problems to be Solved by the Invention) However, although these adsorbent regeneration methods are sufficient as regeneration means for conventional adsorbents whose adsorption performance is not very high, recent low-density lipoprotein adsorption Even when the above-described adsorbent regeneration method is applied to polyanionic adsorbents with improved performance, the adsorbents are not regenerated sufficiently, and improvements have been desired.

(Means for Solving the Problems) The present inventors have solved the above-mentioned problems and have created a simple and safe method.
As a result of intensive research to provide an efficient adsorbent regeneration method, we found that by using a liquid containing a substance that has the property of reacting with metal ions to form metal chelates as an eluent,
The inventors discovered that adsorbent material can be regenerated easily and safely with surprisingly high efficiency, leading to the completion of the present invention.

That is, the present invention provides low-density lipoprotein adsorption #1 having a polyanionic moiety on its surface, which adsorbs low-density lipoproteins and/or other cationic substances in body fluids, and a solution containing at least one type of chelating agent. A method for regenerating a low-density lipoprotein adsorbent, the method comprising: washing with a low-density lipoprotein adsorbent;
The device used directly for carrying out this method consists of a container with two liquid inlets and two liquid outlets containing a low-density lipoprotein adsorbent having polyanion moieties on five surfaces. This is a device for adsorption and regeneration of low-density lipoproteins.

The target adsorbent of the present invention is low-density lipoprotein, and to explain in more detail, it has a molecular weight of 2.2
0' to 3,5 x 10", water density 1.003 to 1,034 (P/d), flotation coefficient (1,0+63)
is 0 to 20 X 1 o-“crrl” expression-! -ct
yh-sr-凰, diameter 20.0 to 30. Onm lipoprotein (5CANU.

A, M: plasma 1ipoproteins
: an 1ntroduction.

” The Biochemistry of Ath
erosclerosis”ed.

by 5CANU A≧y1. , j979. P, 5-8
．． ), and also with other cationic substances #-i
, polyvalent metal ions such as calcium and magnesium, and cationic proteins, and refers to substances that can be adsorbed by adsorbents having polyanion moieties on the surface.

Next, constituent elements of the adsorbent targeted by the present invention will be explained in detail.

In the present invention, the polyanion moiety of the low-density lipoprotein adsorbent refers to a functional group with a molecular weight of 600 or more and a negative charge in one molecule, that is, a carboxyl group (C0
OH, COO"-), sulfonic acid group (5ol) I a
80m-), etc., which have many functional groups that exhibit negative charges in plasma. Examples include vinyl synthetic polyanions such as polyacrylic acid, polyvinyl sulfonic acid, polyvinyl phosphoric acid, and polymethacrylic acid, polystyrene sulfonic acid, poly-α-methylstyrene sulfonic acid, polystyrene phosphoric acid-SO styrene polyanions, and vinyl anions. Copolymers of, for example, polyanions such as styrene-maleic acid copolymers, synthetic peptide polyanions such as polyglutamic acid, polyaspartic acid, etc.

Examples include synthetic nucleic acid polyanions such as poly U and poly A, and natural polyanions such as polyphosphoric acid, polyphosphate ester, alkinoic acid, and heparin.

Among them, synthetic polyanions have excellent chemical stability and
It is superior to natural products in that it is easy to obtain a product that is stable against high-pressure steam sterilization, gamma ray sterilization, ethylene oxide sterilization, etc., and the molecular weight can be adjusted relatively easily, so it is recommended. In addition, in the case of polyanions obtained by synthesis,
This is preferable because a polyanion that does not easily cause complement activation as seen in natural polysaccharides can be easily obtained. Furthermore, the advantage of being able to perform graft polymerization directly onto a carrier, such as with 1-vinyl anions, is that polyanions with large molecular weights can be immobilized in a high retention amount on the carrier.
] give favorable results.

In addition, since the low-density lipoprotein, which is the target substance to be adsorbed, is a huge lipoprotein with a diameter of about 20 OA, it is preferable that the structure of the polyanion part is a chain structure, and it is preferable that it extends long from the surface of the adsorbent. It's nice. Further, it is preferable that the polyanion moiety has at least one negative charge density per molecular weight of 300 equivalents. More preferably 5 molecular weight 2
There should be at least one Ic per 00, and preferably one per unit of molecular weight 70 to 150. The molecular weight mentioned here.

It also includes the molecular weight of functional groups that exhibit negative charges. The molecular weight of the polyanion moiety must be at least 600, since low density lipoproteins cannot be adsorbed to gold if it becomes small. A value of 2,500 or more is preferred, and a range of 10,000 to 5,000,000 is more preferred.

The polyanion moiety has a large number of negatively charged functional groups,
It is thought that by recognizing multiple points on low-density lipoproteins, it binds low-density lipoproteins with strong Coulomb force.

Among the functional groups showing negative charge, carboxyl group (C0O
H,COO-) gives favorable results in %. Since it is a weaker acid than the sulfonic acid group (5OsHe 50m ``''), it has low adsorption to useful proteins such as albumin. In addition, blood coagulation system proteins are less adsorbed and less likely to be activated. Furthermore, complement system proteins are also difficult to adsorb.

Among the above-mentioned polyanion moieties, polycarboxylic acids such as polyacrylic acid and polymethacrylic acid are particularly stable.
I can recommend it.

The density of negative charge is 1 ml from 1 μeq of adsorbent 1!
! The range of q has good adsorption performance for low-density lipoproteins, good adsorption selectivity, and ha that has little effect on the coagulation fibrinolytic system and the complement system.
i range. When the negative charge density is lower than 1 μeq/m, the adsorption ability of low-density lipoproteins falls short of practical performance, and when it exceeds 1 meq, non-selective adsorption increases, which adversely affects the coagulation fibrinolytic system and the complement system. The preferred range is 5μ.
eq/m to 700 μeq/ml, more preferably 10 μeq/− to 500 μeq/−.

The negative charge density can be measured according to a conventional method for measuring the ion exchange capacity of a cation exchange resin.

Examples of the method for producing the low-density lipoprotein adsorbent according to the present invention include a method of activating a carrier and covalently bonding a polyanion, and a method of graft polymerizing an anion monomer to the carrier to form a polyanion graft chain. It will be done.

The carrier only needs to be able to immobilize the polyanion, and both hydrophilic and hydrophobic carriers can be used. However, when using a hydrophobic carrier, non-specific adsorption of albumin to the carrier sometimes occurs, so it is preferable to use a hydrophilic carrier. gives a more favorable result.

The shape of the insoluble carrier can be any known shape such as particulate, fibrous, hollow fiber, or membrane. It is preferable that the shape is as follows.

The carrier may be made of any material as long as it can fix the polyanion. As carriers that can be used, organic or inorganic porous materials such as cellulose gel, dextran gel, agarose gel, polyacrylamide gel, porous glass, and polymer gel can be used. All carrier materials used in can be used, but the adsorbed substance, low-density lipoprotein, has a diameter of 200 to 300 particles and a molecular weight of d! 2,2X10-5,5X106, the exclusion limit molecular weight of the support is 2.2 million or more, and the pore diameter is 200,000.
Among the above-mentioned nine carriers, carriers made of cross-linked copolymers whose main constituent is vinyl alcohol units have higher adsorption efficiency due to their hydrophilicity. Interaction with solutes is small, reducing non-specific adsorption to a minimum. Furthermore, it has extremely excellent properties such as not interacting with the complement system and coagulation system in plasma.

In terms of physical properties, it exhibits an excellent pore size distribution, has heat resistance, enables heat sterilization, and has excellent physical and mechanical strength, which is a characteristic of synthetic polymers. When used as a carrier for adsorbent for whole blood, it also has extremely excellent properties such as having little interaction with blood cell components and minimizing thrombus formation, non-specific adhesion of blood cell components, residual blood, etc. .

A crosslinked polymer having vinyl alcohol units as a main component can be synthesized by polymerizing a monomer having a hydroxyl group or by introducing a hydroxyl group through a chemical reaction of the polymer. It is also possible to synthesize both in combination.

As the polymerization method, a radical polymerization method can be used. The crosslinking agent may be introduced by copolymerization during polymerization, or t
A: It may be introduced through a chemical reaction between polymers (between polymers, between a polymer and a crosslinking agent), or when both are used in combination, it is more effective.An example is the copolymerization of a vinyl monomer and a vinyl or allyl crosslinker. It can be made well. Examples of the vinyl monomer in this case include vinyl esters of carboxylic acids such as vinyl acetate and vinyl propionate, and vinyl ethers such as methyl vinyl ether and ethyl vinyl ether.

Triallylisocyanurate is used as a crosslinking agent.

Allyl compounds such as triallyl cyanurate, di(meth)acrylates such as ethylene glycol dimethacrylate and diethylene glycol dimethacrylate, polyvinyl ethers such as butanediol divinyl ether, diethylene glycol divinyl ether, and divinylglyoxal; Polyallyl ethers such as arylidene pentaerythritol and nato-2-allyloxyethane.

Glycidyl acrylates such as chrycidyl methacrylate can be used. Furthermore, copolymerized comonomers with other comonomers can also be used, if necessary.

In the case of a vinyl copolymer, a vinyl compound (vinyl ester of carboxylic acid and incyanuride) (
The triallylisocyanurate crosslinked product of polyvinyl alcohol obtained by copolymerizing allyl compound) and cross-decomposing the copolymer provides a good support for IWPK in terms of strength and chemical stability.

-1g- All known methods such as covalent bonding, ionic bonding, physical adsorption, embedding, or precipitation insolubilization on the polymer surface can be used to immobilize the polyanion on the surface of the insoluble carrier. Considering its elution properties, it is immobilized by covalent bonding.

It is preferable to use it after insolubilization. For this purpose, an immobilized enzyme, a known carrier activation method used in affinity chromatography, a binding method with a ligand, and a graft polymerization method in which the carrier or activated carrier is used as a backbone polymer and polyanions are used as branches are used. be able to.

Examples of activation methods include halogenated cyanide 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 is not 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 amine group, hydroxyl group, carboxyl group, or thiol group of a ligand. However, in consideration of chemical stability, thermal stability, etc., the method of incorporating epoxide is preferable, and the epichlorohydrin method is particularly recommended.

For carriers with silanol groups such as 7'ch silica-based and glass-based carriers, 27% r-aminopropyltriethoxysilane in γ-crisitoxypropylenetrimethane,
Various silane coupling agents such as r-mercaptopropyltrimethoxine, vinyltrichlorosilane, etc. are preferably used.

Examples of craft polymerization methods include methods that utilize chain transfer reactions, methods that utilize reactions such as dehydrogenation and dehalogenation by K such as radiation and ultraviolet light, and methods that utilize the formation of peroxides. , a cerium salt, a carrier having a reducing group such as thiol, aldehyde, or amine.
A method in which anionic monomers are graft-polymerized using an iron salt or the like as an initiator is simple and recommended. In addition, a graft polymerization system is preferably used because it can fix b-polyanion having a relatively large molecular weight to the inside of the carrier.

Two or more types of polyanions may be bound to the carrier.

Above, as a method for manufacturing a low-density lipoprotein adsorbent, a method of activating a carrier and then binding a polyanion has been described, but the method is not limited to this.

A low-density lipoprotein adsorber is a device in which the above-described low-density lipoprotein adsorbent is filled and held in a container equipped with an inlet and outlet for body fluids.

In the present invention, the adsorbent is generally used while being filled in a container equipped with an inlet and outlet for body fluids.

In FIG. 1, numeral 1 indicates an example of a low-density lipoprotein adsorber containing a low-density lipoprotein adsorbent;
A gasket in which a cap having a body fluid inlet is screwed into an opening at one end of the cylinder 2 via a backing 4 with a filter 3 stretched inside, and a filter 3' is stretched inside the opening at the other end of the cylinder 2. A cap 8 having a body fluid lead-out lower part is screwed through the filter 4' to form a container, and a low-density lipoprotein adsorbent is filled and held in the gap between the filters 5 and 3' to form an adsorbent layer 9. That's what happens.

The adsorbent layer 9 may be filled with a low-density lipoprotein adsorbent alone, or may be mixed or layered with other adsorbents.
. Other adsorbents include, for example.

Something like activated carbon, which has a wide range of adsorption capacities, can be used. A wide range of clinical effects can be expected due to the synergistic effect of the adsorbent. The appropriate volume of the adsorbent layer 9 is about 50 to 400 when used for extracorporeal circulation. When the apparatus of the present invention is used for extracorporeal circulation, there are two methods: KFi and Ojiji.

One K is to separate blood taken from the body into plasma components and blood cell components using a centrifuge or am plasma separator, and then pass the plasma components through the device to purify them. One method is to return the blood to the body together with blood cell components, and the other method is to take blood from the body and pass it directly through the nuclear device.
It is a method of purification.

The method of passing body fluids depends on clinical needs.

Alternatively, depending on the condition of the equipment, the liquid may be passed continuously. Also, the liquid may be passed intermittently.

The chelating agent referred to in the present invention refers to a metal chelate (a complex in which one ligand coordinates with two or more central metal traps to form a cyclic structure containing a metal atom) by reacting with a metal ion. ), examples include malonic acid, oxalic acid, phthalic acid, succinic acid, maleic acid, citraconic acid, itaconic acid (dibasic acid),
Aliphatic amines such as ethylene diamine, diethylene triamine, propylene diamine, α, α′-dipyridyl.

Aromatic amines such as phenanthroline, natural amino acids and peptides such as alanine, aspartic acid, glycine, glycylglycylglycine, glutamic acid, β-a22-N, N-diacetic acid, aminobenzoic acid-N, N-diacetic acid , iminodiacetic acid, aniline silicoic acid, 1,5-diaminocyclohexane-N, N'-tetraacetic acid, ethylenediaminenathodiacetic acid, pentamethylenediaminetetodiacetic acid, etc., non-naturally occurring amino acids, citric acid, gluconic acid.

Oxyacids such as glyceric acid, glycolic acid, malic acid, 5-sulfosalicylic acid, tartaric acid, condensed phosphoric acids such as pyrophosphoric acid, trimetaphosphoric acid, triphosphoric acid, nitroacetic acid, 0-
Nitrocarboxylic acids such as nitrobenzoic acid, oxalacetic acid,
Acids such as pyruvic acid, salicylaldehyde and its derivatives such as salicylaldehyde, 3-chlorsalicylaldehyde, 5-sulfosalicylaldehyde, 2-oxy-1-
Oxidialdehydes such as nathaldehyde and 2-oxy-3-naphthaldehyde, β-diketones such as acetylacetone, benzoylacetone, 2-70ylbenzoylmethane, and trifluoroacetylacetone, 8-oxyquinoline, and catechol-3,5-disulfone. Examples include phenol fermentation conductors such as acids, 0,O'-dioxyazo dyes such as Eriochrome Punzku T1 and Eriochrome Plublak R, aminobenzenethiol, 0-aminophenol, and ethyl acetoacetate. Also usable are polyacrylic acids, polycarboxylic acids such as polymethacrylic acid, and polysaccharides such as heparin and alginic acid.

The presence of a high concentration of chelating agent shifts the adsorption equilibrium of cationic substances such as low-density lipoproteins, calcium, and magnesium, which are adsorbed on the adsorbent through interaction with the polyanion moiety of the adsorbent, causing them to be desorbed from the adsorbent. It is thought that it will be released.

Among chelating agents, considering that the adsorbent is reused for extracorporeal circulation treatment, sodium citrate, heharinna), IJum, etc. are highly recommended in terms of safety.

Next, a method for regenerating a low-density lipoprotein adsorbent will be explained using an example.

As mentioned above, the low-density lipoprotein adsorbent is usually filled into a container and used as a lipoprotein adsorber as shown in FIG. Body fluid is poured into this low-density lipoprotein adsorption device, and the low-density lipoprotein in the body fluid is adsorbed. After use, regeneration begins.5 Usually, in order to wash out the body fluid remaining in the low-density lipoprotein adsorbent, a physiological solution with a volume of 1 to 10 times the adsorbent capacity i is added. (e.g., physiological saline). This operation may be omitted if the regenerating liquid used thereafter does not have an adverse effect on body fluids, such as denaturation or aggregation.

Next, the regeneration solution of the adsorbent is passed through a low-density lipoprotein adsorber. Since the desorption efficiency is very high, almost complete desorption can be achieved by flowing 2 to 5 times the capacity of the adsorber. The flow rate can be arbitrarily selected as long as it is not extremely fast, and the temperature can also be arbitrarily selected.

Finally, considering the one-time use, it is replaced with a physiological solution or a preservation solution such as a disinfectant for preservation. If necessary, replace with physiological solution, then
Even if sterilization operations such as moist heat sterilization and R-ray sterilization are performed, the low-density lipoprotein adsorbent is regenerated.

Furthermore, if necessary, the low-density lipoprotein adsorbent can be regenerated even during the adsorption and removal of low-density lipoproteins in blood through extracorporeal circulation. In this case, use two or more low-density lipoprotein adsorbers, divide them into two systems, one for adsorbing low-density lipoproteins, and the other for regeneration, and operate them aseptically. is preferred. However, it is also possible to perform one operation intermittently.

In carrying out the method for regenerating the low-density lipoprotein adsorbent of the present invention, the low-density lipoprotein adsorbent is regenerated while the low-density lipoprotein in the blood is being adsorbed and removed by vortexing, especially by extracorporeal circulation. In this case, as a device for adsorption and regeneration of low-density lipoproteins, a container K having two systems of liquid inlets and two systems of liquid outlets, and a "low-density lipoprotein adsorbent having a polyanion portion on the surface" are stored. By using this material, the adsorption and regeneration operation of low-density lipoproteins is extremely simple, and sterile handling is also easy.

That is, one of the liquid inlets and one of the liquid outlets is used for adsorbing low-density lipoproteins, and the other liquid inlet and the other liquid outlet are used for regenerating the low-density lipoprotein adsorbent. Therefore, adsorption and regeneration of the low-density lipoprotein adsorbent can be easily and sterilely performed, which is very convenient.

(Effects of the Invention) As described above, according to the method for regenerating a low-density lipoprotein adsorbent of the present invention, it is possible to completely regenerate a polyanionic adsorbent that exhibits extremely high adsorption capacity and high selectivity. ,
It has become possible to perform this process easily and safely, making it possible to reuse the low-density lipoprotein adsorbent and adsorbing a large amount of low-density lipoprotein with a single low-density lipoprotein adsorber. It became possible to do so.

Since a chelating agent is used as a regenerating agent, regeneration efficiency is high, and by using a chelating agent that is safe even if it enters the living body, regeneration during extracorporeal circulation treatment is also possible.

The present invention is effective in regenerating a low-density lipoprotein adsorbent for treatment of familial hypercholesterolemia, has unprecedented complete regeneration ability, and is safe.

(Example) Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 Using the low-density lipoprotein adsorption device shown in FIG. 1, experiments on adsorption, regeneration, and re-adsorption of low-density lipoproteins were conducted.

Low-density lipoprotein adsorption device has an inner diameter of 0.8 crn5 and a length of 2.
．． 0 (Wb) A container having an internal volume of 1.0- and filled with a low-density lipoprotein adsorbent of 1.0- was used.

The low-density lipoprotein adsorbent was manufactured by the method described below.

Vinyl acetate 1000F, ) realyl isocyanurate 4
14f, ethyl acetate 1000r% to butane 1000f%
Polyvinyl acetate (degree of polymerization 500) 70f and 2.2'
-7 A homogeneous mixture of zobisisobutylonitrile 361p, 1% by weight of polyvinyl alcohol, 0.05% by weight of sodium dihydrogen phosphate dihydrate, and 1.5% by weight of disodium hydrogen phosphate dodecahydrate. Add 4 tons of water in which % by weight was dissolved and stir well, then heat at 65C for 18 hours.

Further, suspension polymerization was carried out by heating and stirring at 75'C for 5 hours.
A granular copolymer was obtained. During the polymerization, the stirring speed was controlled to keep the particle size small. After filtration, washing with water, and extraction with acetone, the copolymer was transesterified in a solution consisting of 465 t of caustic soda and 20 t of methanol at 40 C for 18 hours. The average particle diameter of the obtained particles was 1j40 μm.

This gel was filled into a stainless steel tube with an inner diameter of 7,511 m and a length of 120 m, and aqueous solutions of dextran and polyethylene glycol with various molecular weights, albumin, immunoglobulin G1, immunoglobulin M, β-lipoprotein, tobacco, and mosaic were added. When the virus was measured in a phosphate buffered salt solution, each virus was eluted in descending order of molecular weight.

The exclusion limit molecular weight for dextran was approximately 5XIQ'', and the exclusion limit molecular weight for protein was approximately 2X10'.

9. A solution of human γ-globulin and human fulbumin was run through an aqueous solution containing 0.5 M sodium chloride and 0.1 M sodium phosphate as a solvent. It was collected in

The nonspecific adsorption of the gel was very low. All sample measurements were performed at a flow rate of 1 m/mjR.

Next, the transesterified gel was thoroughly washed with water, and the dried gel was suspended in a solution consisting of 150 tk dimethyl sulfoxide 1800 tj and epichlorohydrin 1200 mj, and 50 ml of an aqueous sodium thihydroxide solution was added.
The reaction was carried out under stirring at saC for 5 hours. After the reaction was completed, the glass filter was washed with dimethyl sulfoxide of FA compound and then with 20 L of water to obtain an epoxy group-bonded gel.

The amount of epoxy group bonded in this gel is 1mK @ 0.11mm
It was OT. Using the epoxy group-bonded gel, polyacrylic acid (weight average molecular weight 9×104, manufactured by Aldrich, USA) was bound as a ligand to prepare an adsorbent.

4.5ft of polyacrylic acid - 50 Owt K with distilled water
Lb This was used as the ligand solution. Add 100 d'i of the epoxy group-bound gel to this ligand solution, and
The ligand binding reaction was carried out while shaking for hours.

After this, the polyacrylic acid was thoroughly washed with water, dehydrated by suction, and then immersed in a 0.1 N hydroxide solution for 30 minutes to make the polyacrylic acid into a sodium form. It was used as a lipoprotein adsorbent.

Adsorption experiments were performed using plasma from patients with familial hypercholesterolemia.

First, the low-density lipoprotein adsorbent was brimmed with physiological saline, and then familial hypercholesterolemia patient plasma was flowed through the low-density lipoprotein adsorbent at a flow rate of 0.17 d/-. The first physiological saline flowing out of the adsorber 1
- was discarded, and the remaining plasma was collected one by one into a test tube.

After plasma t-12-flow, a 29.4 f/dl solution of trisodium citrate dihydrate (adjusted to pH 7 with hydrochloric acid) was used as a regenerating solution, and the flow rate was the same as that for plasma.
- It was washed away. Thereafter, after washing the regenerating solution with physiological saline 5-, familial hypercholesterolemia patient plasma was flowed again at the same flow rate to perform a re-adsorption test.

For all effluents during adsorption, regeneration and re-adsorption tests,
Figure 2 shows the results of measuring the cholesterol concentration. The horizontal axis is the amount of effluent, and the vertical axis is the concentration of cholesterol in each effluent.

First, in the first adsorption test, the adsorption amount of cholesterol (since it is plasma from a patient with familial hypercholesterolemia, it is mostly low-density lipoprotein cholesterol) is shown in the shaded area in Figure 2. It is expressed in area and calculated as 2
It becomes 5In9. Next, the amount of cholesterol leaked out by the regeneration solution is expressed by the area of the shaded area in Figure 2. When calculated, it matches the area of the shaded area, indicating that all of the adsorbed cholesterol is desorbed by the regeneration solution. , it can be seen that it leaked out of the adsorber. Next, the amount of cholesterol adsorbed in the second adsorption test is expressed by the area of the shaded area ■ in Figure 2, and since this also matches the area of the shaded area, the low-density lipoprotein adsorbent is You can see that it has been completely regenerated.

Comparative Example 1 Everything was carried out in the same manner as in Example 1 except that 5% saline was used as the regenerating solution. The cholesterol desorbed by the regenerating solution was 7% of the amount of cholesterol adsorbed in the first time.
The amount of cholesterol adsorbed in the second test was also 68% of that in the first test.

That is, the low-density lipoprotein adsorbent could not be completely regenerated with the 5T saline solution.

Example 2 A 4.52 f/dt solution of tetrasodium ethylenediaminetetraacetic acid tetrahydrate (pH-7 with hydrochloric acid, O
The procedure was carried out in the same manner as in Example 1, except that K adjustment) was used, and the amount of cholesterol adsorbed in the first time, the amount of cholesterol desorbed by the regenerating solution, and the amount of cholesterol adsorbed in the second time were all the same. That is, the low-density lipoprotein adsorbent was completely regenerated.

291 Example 3 As a regenerating liquid, 0.0% of polyacrylic acid with a molecular weight of 2000 was used.
72 f/di solution (pH 7.5 with sodium hydroxide)
The procedure was carried out in the same manner as in Example 1, except that (adjusted to 0) was used, and the amount of cholesterol adsorbed in the first time, the amount of cholesterol desorbed by the regenerating solution, and the amount of cholesterol adsorbed in the second time completely matched. It was found that the low-density lipoprotein adsorbent was completely regenerated by the regenerating solution.

[Brief explanation of the drawing]

FIG. 1 is a cross-sectional schematic diagram showing a low-density lipoprotein adsorbent filled with a low-density lipoprotein adsorbent having a polyanion portion on the surface, and FIG. 2 is a graph showing the experimental results of Example 1. . 1...Low-density lipoprotein adsorber 2...
...Cylinder 313'...Filter 4...Puts Φ ring 5...Body fluid inlet 6,8...・・・・・・
... Cap 9 ... Adsorbent layer

Claims (2)

[Claims] (1) Washing a low-density lipoprotein adsorbent having a polyanionic moiety on its surface, which has adsorbed low-density lipoproteins and/or other cationic substances in body fluids, with a solution containing at least one type of chelating agent. A method for regenerating a low-density lipoprotein adsorbent, characterized by: (2) Adsorption and regeneration of low-density lipoproteins, characterized in that a low-density lipoprotein adsorbent having a polyanion portion on the surface is housed in a container having two systems of liquid inlets and two systems of liquid outlets. equipment.