JPH0611330B2 - Lipoprotein removal device - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a device for removing harmful components in blood, and more particularly to lipoprotein, particularly low density lipoprotein (LDL) and / or very low density lipoprotein from plasma separated from blood. The present invention relates to a lipoprotein removal device that selectively removes (VLDL) by adsorption.

[0002]

2. Description of the Related Art Among the lipoproteins present in blood, LDL
Is rich in cholesterol and is known to cause arteriosclerosis. Particularly, in hypercholesterolemia such as familial hyperlipidemia, the LDL value is several times higher than the normal value, which causes hardening of the coronary arteries and the like. LD in blood for this treatment
Although drug therapy such as diet therapy, probucol, and cholestyramine is performed for the purpose of lowering L, the effects are limited and side effects are also a concern. Especially for familial hyperlipidemia, so-called plasma exchange therapy, in which the plasma of a patient is separated and then replaced with normal plasma or a replacement fluid containing albumin, etc., is almost the only effective treatment method at present. is there. However, as is well known, plasmapheresis therapy (1)
Need to use expensive fresh plasma or plasma products,
(2) There are drawbacks such that hepatitis virus may be infected, and (3) not only harmful components but also useful components are removed at the same time.

Attempts have been made to remove harmful components by means of a membrane for the purpose of eliminating these drawbacks, but none which is satisfactory in terms of selectivity has not yet been obtained. Further, for the same purpose, attempts have been made to use so-called immunoadsorbents on which antigens, antibodies, etc. are immobilized. Although this is almost satisfactory in terms of selectivity, it is difficult and expensive to obtain the antigens and antibodies to be used. Has a fatal drawback.

Further, an adsorbent based on the so-called affinity chromatograph, in which a compound (so-called ligand) having an affinity for harmful components is immobilized, has been tried. The ligand used for this is relatively inexpensive and has relatively good selectivity, which is convenient, but since a soft gel typified by agarose is used as a carrier, it is difficult to obtain a sufficient flow rate when packed in a column. It was In other words, if these adsorbents are to be used for blood and plasma perfusion therapy (so-called plasmapheresis, etc.) that uses an extracorporeal circulation circuit that has been developed in recent years, a special shape of the column is required to obtain a high flow rate. Also, there are many problems such as the need to prepare a spare column because it often causes clogging, and it has not reached a situation where stable treatment can be performed. It is obvious that a carrier having a high mechanical strength may be used to improve the flow characteristics of the adsorbent, but it is known that the use of these carriers lowers the adsorption ability as compared with a soft gel such as agarose. ing.

On the other hand, it is known that polyanion compounds such as sulfated polysaccharides have affinity with lipoproteins and form precipitates in the presence of metal ions [eg M. Burnstein an
d HRScholnic.Adv. in Lipid, Res., 11, 67 (1973)], and is used for clinical analysis and the like. However, if LDL is to be removed from the blood of a patient by this method, at least 0.05% of the polyanion compound and 0.02 M or more of metal ion must be added to the plasma to be treated, and Since it was necessary to separate the precipitate by a method such as centrifugation, the operation was complicated and dangerous, and it was practically impossible to apply.

[0006]

DISCLOSURE OF THE INVENTION The present invention provides a lipoprotein adsorbent which is inexpensive and has good flow characteristics, and which does not have a lower adsorptivity as compared with the case where a soft gel is used as a carrier and has an excellent removal capacity. The present invention provides a device for removing lipoprotein from plasma, which comprises a column packed with this.

[0007]

Means for Solving the Problems As a result of earnest research by the present inventors, the column was packed with an adsorbent obtained by using a specific porous polymer hard gel and immobilizing a polyanion compound having an affinity for lipoprotein on the gel. The inventors have found that the above-mentioned problems can be solved by the device, and have completed the present invention. That is, the present invention has a) plasma separation means, b) an inlet for plasma that is sent from the plasma separation means, and an outlet for plasma after lipoprotein adsorption and removal, and the exclusion limit molecular weight of globular proteins is 100. A gist is a device for removing lipoproteins from plasma, which comprises a column filled with an adsorbent comprising a polyanion compound having an affinity for lipoproteins immobilized on a porous polymer hard gel of 10,000 to 100 million.

The present invention will be described in detail below. The carrier of the adsorbent suitable for use in the present invention is required to have (1) pressure resistance and (2) have pores having a relatively large diameter, and the polymer hard gel is most suitable for the present invention. It is a carrier. The hard gel referred to here is a gel which is less swelled by a solvent than a soft gel such as dextran, agarose, acrylamide and the like, and is hard to be deformed by pressure. Hard gel and soft gel can be distinguished by the following method. That is, as shown in the reference example below, the gel is uniformly packed in a cylindrical column, and the relationship between the pressure loss and the flow rate when the aqueous liquid is flowed is almost linear in the hard gel, whereas there is pressure in the soft gel. If the point is exceeded, the gel will be deformed and consolidated and the flow rate will not increase. In the present invention, when the column shown in the reference example described later is used, one having the above linear relationship up to at least 0.3 kg / cm ² is called a hard gel.

The next required property is to have pores having a relatively large diameter. That is, LDL is a macromolecule which is said to have a molecular weight of at least 1,000,000 or more, and it is necessary for LDL to be able to penetrate into pores in order to adsorb and remove it. Next, if LDL can penetrate into the pores,
The performance as an adsorbent is low unless the probability of penetrating into the pores is large to some extent, that is, the larger the distribution ratio (concentration of stationary phase / concentration of mobile phase) between the mobile phase and stationary phase (in the pores), the better. it is conceivable that. Therefore, it seems that the larger the pore size, the more advantageous. There are various methods for measuring the pore size, and the mercury intrusion method is most often used, but it may not be applicable in the case of polymer hard gel. Therefore, it is appropriate to use the exclusion limit molecular weight as a measure of the pore size. Exclusion limit molecular weight is a molecule that cannot enter (exclude) in the pores of gel permeation chromatography as described in books such as Hiroyuki Hatano and Toshihiko Hanai, Experimental High Performance Liquid Chromatography, Kagaku Dojin. The molecular weight of the one with the smallest molecular weight.

Phenomenonally, molecules having an exclusion limit molecular weight or more are eluted near the mobile phase volume Vo, so that the exclusion limit molecular weight can be determined by investigating the relationship with the elution volume using compounds having various molecular weights. it can. It is known that the exclusion limit molecular weight varies depending on the type of target compound,
Generally, globular proteins, dextran, polyethylene glycol, etc. have been well investigated, but lipoproteins have hardly been investigated. Therefore, it is appropriate to use the values obtained with the most similar globular proteins (including viruses). As a result of examination using various carriers having different exclusion limits, it was unexpectedly expected that the exclusion limit molecular weight was smaller than that of LDL by about 1 million LD.
It has been demonstrated that L-adsorption capacity is exhibited, and that the larger the pore size, the larger the capacity is. Rather, proteins other than LDL are removed, so that the optimum pore size range exists. That is, when a carrier having an exclusion limit molecular weight of less than 1 million is used, the amount of LDL removed is small and cannot be practically used, but the exclusion limit molecular weight is 1 million to several million.
Even with a carrier having a molecular weight close to L, an adsorbent that can be put to practical use to some extent can be obtained.

On the other hand, exclusion limit molecular weight and LDL adsorption amount, and adsorption of proteins other than LDL (so-called non-specific adsorption)
The amount of LDL adsorbed increases as the exclusion limit molecular weight increases, but this increase reaches a peak when the exclusion limit exceeds 10 million, while the adsorption of proteins other than LDL, such as LGG and IGM. Was found to be noticeable. Further, when the exclusion limit molecular weight exceeds 100 million, the amount of immobilized ligand decreases, and consequently the amount of LDL adsorbed decreases, and nonspecific adsorption cannot be ignored. Therefore, the preferred exclusion limit molecular weight of the carrier used in the present invention is 1 million or more and 100 million or less, and most preferably 3 million or more and 70
It is, 000,000 or less.

Next, regarding the porous structure of the carrier, the total porosity is preferable to the surface porosity, and the pore volume is preferably 20% or more. As the shape of the carrier, any shape such as a granular shape, a fibrous shape, a film shape, and a hollow fiber shape can be selected. When a particulate carrier is used, its particle size is preferably 1 μ or more and 5000 μ or less. Furthermore, it is convenient that a functional group that can be used in the immobilization reaction or a functional group that can be easily activated is present on the surface of the carrier. Typical examples of these functional groups include an amino group, a carboxyl group,
Examples thereof include a hydroxyl group, a thiol group, an acid anhydride group, a succinylimide group, a chlorine group, an aldehyde group, an amide group and an epoxy group.

Typical examples of the polymer hard gel suitable for the present invention include styrene-divinylbenzene copolymer, crosslinked polyvinyl alcohol, crosslinked polyacrylate, crosslinked vinyl ether-maleic anhydride copolymer, and crosslinked styrene-. Examples thereof include, but are not limited to, maleic anhydride copolymers, hard porous materials of synthetic polymers such as crosslinked polyamide, and those whose surfaces are coated with polysaccharides, synthetic polymers and the like. These polymer hard gels may be used alone or in combination of two or more.

Typical examples of the polyanion compound having affinity with lipoprotein suitable for use in the present invention include dextran sulfate, chondroitin sulfate, chondroitin polysulfate, heparanic acid, keratan sulfate, heparitin sulfate, xylan sulfate, caronine sulfate, Cellulose sulfate, chitin sulfate, chitosan sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate,
Carrageenin sulfate, starch sulfate, polyglucose sulfate,
Examples thereof include sulfated polysaccharides such as laminarin sulfate, galactan sulfate, levan sulfate, mebesulfate, phosphotungstic acid, polysulfated anethole, polyvinyl alcohol sulfate, and polyphosphoric acid. The most preferable examples include dextran sulfate and chondroitin polysulfate.

As a method of immobilizing a compound (ligand) having an affinity for lipoprotein on a carrier, various known methods can be used. That is, a physical adsorption method, an ionic bonding method, a covalent bonding method and the like. The adsorbent used in the present invention is
Since it is important that the ligand is not detached during sterilization, a covalent bond method with a strong bond is preferable, and it is preferable that the ligand is covalently crosslinked even when the ionic bond method is used. If necessary, a spacer may be introduced between the carrier and the ligand. The amount of immobilized ligand varies depending on the nature and activity of the ligand, but 0.02 per 1 ml of column volume is required to obtain a significant amount of lipoprotein adsorption.
It is preferably at least mg, and preferably 100 mg or less in consideration of economic efficiency. More preferably, it is 0.5 mg or more and 20 mg or less per 1 ml of the column volume.

The adsorbent obtained as described above is packed in a column having an inlet for plasma sent from the plasma separation means on one side and an outlet for plasma after lipoprotein adsorption and removal on the other side. To be done. The plasma separation means is not particularly limited, and known separation means can be used. To remove lipoproteins using the device of the present invention, first, plasma is continuously separated from blood by plasma separation means, and then the plasma is introduced from an inlet into a column filled with an adsorbent, where The lipoprotein in plasma is selectively adsorbed and removed, and then discharged from the column through the outlet. When removing LDL using the apparatus of the present invention, it is possible to improve the removal efficiency and selectivity by adding polyvalent metal ions to the plasma to be treated.
The polyvalent metal ions used for this purpose include alkaline earth metal ions such as calcium, magnesium, barium and strontium, group II element ions such as aluminum, group VII element ions such as manganese, and group VIII such as cobalt.
Examples include genus element ions.

[0017]

The present invention will be described in more detail with reference to the following examples. Reference Example A glass cylindrical column (inner diameter 9 mm, column length 150 mm) equipped with filters with pore diameters of 15 μm on both ends, agarose gel (Biorad Biogel A5m, particle size 50-100 mesh) as a soft gel, and Tosoh as a polymer hard gel. Toyo Pearl HW65 (particle size 50-100μ
m) were uniformly filled, and water was caused to flow by a peristaltic pump to determine the relationship between the flow rate and the pressure loss. The results are shown in Fig. 1. FIG. 1 shows that in the case of polymer hard gel, the flow rate increases almost in proportion to the increase in pressure, whereas in the case of agarose gel, the flow rate does not increase even if pressure is increased by causing consolidation. ing.

Examples 1 to 6 and Comparative Example 1 (1) Production of Adsorbents A to G Adsorbent A: Toyopearl HW55 [cross-linked acrylate gel (totally porous hard gel)] Abbreviated as protein exclusion limit) 700,000, particle size 50-100 μm] 10 ml, saturated NaOH aqueous solution 6 m
l, add epichlorohydrin 15ml and stir 50
The reaction was carried out at 0 ° C for 2 hours to obtain an epoxidized gel. 20 ml of concentrated aqueous ammonia was added to this gel and stirred at 50 ° C. for 2 hours to introduce an amino group. 0.5 g in 2 ml of the epoxidized gel obtained
Add 2 ml of an aqueous solution containing dextran sodium sulfate
The pH was adjusted to 12. After keeping this mixed solution at 40 ° C. and reacting for 18 hours, the gel was separated by filtration, and a 2 molar saline solution,
It was washed with a 0.5 molar saline solution and then with water. Unreacted epoxy groups were blocked by reacting with monoethanolamine to obtain a gel with dextran sodium sulfate immobilized. The immobilized amount was 1.6 mg / ml.

Adsorbent B: Toyopearl HW60 (protein exclusion limit 1,000,000, particle size 50 to 100 μm), which is a crosslinked acrylate gel (totally porous hard gel), was added with saturated NaOH aqueous solution 6 ml and epichlorohydrin 15 ml at 50 ° C. with stirring. And reacted for 2 hours to obtain an epoxidized gel. To this gel, add 20 ml of concentrated aqueous ammonia, and then at 50 ° C for 2
After stirring for an hour, an amino group was introduced. To 2 ml of the obtained epoxidized gel was added 2 ml of an aqueous solution containing 0.5 g of sodium dextran sulfate to adjust the pH to 12. Add this mixture to 4
After keeping at 0 ° C. and reacting for 18 hours, the gel was separated by filtration and washed with a 2 molar saline solution, a 0.5 molar saline solution, and then with water. Unreacted epoxy groups were blocked by reacting with monoethanolamine to obtain a gel with dextran sodium sulfate immobilized. Immobilization amount is 1.0 mg /
It was ml.

Adsorbent C: Crosslinked acrylate gel (totally porous hard gel) Toyopearl HW65 (protein exclusion limit 5,000,000, particle size 50 to 100 μm) 10 ml, saturated aqueous NaOH solution 6 ml, epichlorohydrin 15 ml were added and stirred at 50 ° C. And reacted for 2 hours to obtain an epoxidized gel. To this gel, add 20 ml of concentrated aqueous ammonia, and then at 50 ° C for 2
After stirring for an hour, an amino group was introduced. Then, 200 mg of chondroitin polysulfate was dissolved in 10 ml of water to adjust the pH to 4.5, and then 3 ml of the above-mentioned amino group-containing gel was added thereto. 200 mg of 1-ethyl-3- (dimethylaminopropyl) -carbodiimide was added thereto while keeping the pH at 4.5, and the mixture was shaken at 4 ° C. for 24 hours. After completion of the reaction, the product was washed with a 2 molar saline solution, a 0.5 molar saline solution and then with water to obtain chondroitin polysulfate-immobilized Toyopearl gel HW65. The immobilized amount was 1.2 mg / ml.

Adsorbent D: Cellulofine A-3 (manufactured by Chisso Corporation, protein exclusion limit 50,000,000, particle size 45 to 105 μm), which is a fully porous cellulose hard gel, was sucked on a filter to reduce water content. After that, 10g
Take 20g of NaOH 4g and heptane 12g,
Further, one drop of nonionic surfactant TWEEN20 was added and stirred at 40 ° C. for 2 hours. Epichlorohydrin 5
g was added and the mixture was stirred at 40 ° C. for 2 hours. After completion of the reaction, the supernatant was discarded and the gel was washed with water to obtain an epoxidized gel. 2 ml of an aqueous solution containing 0.5 g of dextran sulfate sodium was added to 2 ml of the obtained gel to adjust the pH to 12. After keeping this mixed solution at 40 ° C. for 18 hours to react,
The gel was filtered off and washed with 2 molar saline solution, 0.5 molar saline solution and then water. Unreacted epoxy groups were blocked by reacting with monoethanolamine to obtain a gel with dextran sodium sulfate immobilized. The immobilized amount was 1.5 mg / ml.

Adsorbent E: Cellulofine A-3, which is a fully porous cellulose hard gel (protein exclusion limit of 50,00)
(000, particle size 45-105 μm,) is sucked on the filter to reduce water content, and then 10 g is taken and
4 g of 0% NaOH, 12 g of heptane, and 1 drop of the nonionic surfactant TWEEN20 were added, and the mixture was stirred at 40 ° C. for 2 hours. 5 g of epichlorohydrin was added thereto, and the mixture was stirred at 40 ° C. for 2 hours. After completion of the reaction, the supernatant was discarded and the gel was washed with water to obtain an epoxidized gel. To the obtained epoxidized gel, 15 ml of concentrated aqueous ammonia was added, and the mixture was stirred at 40 ° C. for 1.5 hours, then the gel was filtered off and washed with water to obtain a gel having an amino group introduced therein. Then, 200 mg of polyvinyl alcohol sulfuric acid was dissolved in 10 ml of water to adjust the pH to 4.5, and then 3 ml of the amino group-containing gel was added thereto. To this was added 200 mg of 1-ethyl-3- (dimethylaminopropyl) -carbodiimide while maintaining the pH at 4.5.
Shake at 24 ° C. for 24 hours. After completion of the reaction, the mixture was washed with a 2 molar saline solution, a 0.5 molar saline solution, and then with water to obtain a polyvinyl alcohol sulfate-immobilized gel. Immobilization amount is 1.5
It was mg / ml.

Adsorbent F: 5 g of Cellulofine A-3 sucked on the filter to reduce the water content was weighed, and 1,4-butanediol diglycidyl ether 2.5
Further, 7.5 ml of 0.1N NaOH aqueous solution was added, and the mixture was reacted at room temperature for 18 hours with stirring to introduce an epoxy group into the gel. To 2 ml of the obtained epoxidized gel was added 2 ml of an aqueous solution containing 0.5 g of sodium dextran sulfate to adjust the pH to 12. After keeping this mixed solution at 40 ° C. and reacting for 18 hours, the gel was filtered off, and a 2 molar saline solution, 0.5
It was washed with a molar saline solution and then with water. Unreacted epoxy groups were blocked by reacting with monoethanolamine to obtain a gel with dextran sodium sulfate immobilized. The immobilized amount was 1.8 mg / ml.

Adsorbent G: Toyopearl HW65 10 ml saturated N
6 ml of an aOH aqueous solution and 15 ml of epichlorohydrin were added and reacted at 50 ° C. for 2 hours while stirring to obtain an epoxidized gel. To 2 ml of the obtained epoxidized gel was added 2 ml of an aqueous solution containing 0.5 g of sodium dextran sulfate to adjust the pH to 12. The mixture was kept at 40 ° C. and reacted for 18 hours, then the gel was separated by filtration and washed with a 2 molar saline solution, a 0.5 molar saline solution and then with water. Unreacted epoxy groups were blocked by reacting with monoethanolamine to obtain a gel with dextran sodium sulfate immobilized. The immobilized amount was 1.2 mg / ml.

(2) Preparation of device and removal of lipoproteins Adsorbents A to G obtained in (1) above were each 9 m in inside diameter.
A column with m and a length of 47 mm was uniformly packed, and a plasma separation means was connected to one plasma inlet of the column to prepare a lipoprotein removal device. Using these devices, 18 ml of human plasma was flown at a rate of 0.3 ml / min to adsorb and remove lipoproteins in plasma. The lipoprotein concentration and protein concentration in the plasma that flowed out from the column were measured, and the removal rate of each was determined by comparison with the original plasma concentration. The results are shown in Table 1. The removal rates of proteins in the table are shown excluding the decrease in protein concentration due to removal of lipoproteins.

[0026]

[Table 1] Note) VLDL: Very low density lipoprotein LDL: Low density lipoprotein HDL: High density lipoprotein

[0027]

INDUSTRIAL APPLICABILITY As described above, the device of the present invention is inexpensive and has good flow characteristics, and is superior in lipoprotein adsorption / removal ability as compared with the case where a soft gel is used as a carrier.

[Brief description of drawings]

FIG. 1 is a graph showing a relationship between a flow velocity and a pressure loss when a soft gel “Biogel A5m” and a polymer hard gel “Toyopearl HW65” of Reference Example are used.

Claims (1)

[Claims] 1. An a) plasma separation means, b) an inflow port for plasma sent from the plasma separation means, and an outflow port for plasma after lipoprotein adsorption / removal, and the exclusion limit molecular weight of globular proteins is 100. An apparatus for removing lipoproteins from plasma, which comprises a column filled with an adsorbent comprising a polyanion compound having an affinity for lipoproteins immobilized on a porous polymer hard gel of 10,000 to 100 million.