JPH0362433B2 - - Google Patents

Description

Detailed Description of the Invention (Industrial Application Field) The present invention provides a method for producing plasma from which harmful components in blood have been removed, and more specifically, a method for selectively removing lipoproteins, particularly low-density lipoproteins (LDL). The present invention relates to a method for producing adsorbed plasma. (Prior Art and Problems) Among the lipoproteins present in the blood, LDL contains a large amount of cholesterol and is known to cause arteriosclerosis. In particular, in cases of hypercholesterolemia such as familial hyperlipidemia, the LDL value is several times higher than the normal value, leading to hardening of the coronary arteries. For this treatment, dietary therapy, aimed at lowering blood LDL,
Drug treatments such as probucol and cholestyramine have been used, but their effectiveness is limited and there are concerns about side effects. Particularly for familial hyperlipidemia, so-called plasma exchange therapy, in which the patient's plasma is separated and replaced with normal plasma or a replacement fluid containing albumin, etc., is currently almost the only effective treatment. be. However, as is well known, plasma exchange therapy (1) requires the use of expensive fresh plasma or plasma preparations, (2) there is a risk of infection with hepatitis viruses, etc., and (3) it contains not only harmful components but also useful components at the same time. It has disadvantages such as being removed. In order to overcome these drawbacks, attempts have been made to remove harmful components using membranes, but no membranes have been found that are satisfactory in terms of selectivity. For the same purpose, attempts have been made to use so-called immunoadsorbents on which antigens, antibodies, etc. are immobilized.Although these are mostly satisfactory in terms of selectivity, they have the disadvantage that the antigens and antibodies used are difficult and expensive to obtain. It has some disadvantages. Furthermore, adsorbents based on the principle of so-called affinity chromatography, in which compounds having an affinity for harmful components (so-called ligands) are immobilized, have also been attempted. The ligands used for this are relatively inexpensive and have relatively good selectivity, but because the carrier is a soft gel such as agarose, it is difficult to obtain a sufficient flow rate when packed in a column. It was hot. In other words, if these adsorbents are to be used in blood or plasma perfusion therapy (so-called plasmapheresis, etc.) using the recently developed extracorporeal circulation circuit, special ingenuity is required in the column shape in order to obtain a high flow rate. However, there are many problems, such as the need to prepare spare columns because they often clog, and the situation has not been reached in which stable treatment can be performed. Although it is obvious that a carrier with high mechanical strength can be used to improve the flow characteristics of an adsorbent, it is known that the use of these carriers lowers the adsorption capacity compared to soft gels such as agarose. ing. On the other hand, it is known that polyanionic compounds such as sulfated polysaccharides have an affinity for lipoproteins and form precipitates in the presence of metal ions [for example, M.
Burnstein and HRScholnick.Adv.in Lipid,
Res., 11 , 67 (1973)] and is used for clinical analysis, etc. However, with this method, the
In order to remove LDL, it is necessary to add at least 0.05% of a polyanionic compound and 0.02M or more of metal ions to the plasma to be processed, and the resulting precipitate is separated by a method such as centrifugation. It is necessary, the operation is complicated, and the risk is high.
It was virtually impossible to apply. (Means for Solving the Problems) As a result of intensive research, the present inventors have found that by using a specific porous polymer hard gel and fixing a polyanionic compound that has an affinity for lipoproteins, the gel can be inexpensive and have good flow characteristics. The present invention has been achieved by obtaining a lipoprotein adsorbent with excellent removal ability, and with no decrease in adsorption ability compared to when a soft gel is used as a carrier, and by using this to produce plasma from which lipoproteins have been removed. . That is, in the present invention, a column is filled with a lipoprotein adsorbent made by immobilizing a polyanion compound having an affinity for lipoproteins on a porous polymer hard gel having an exclusion limit molecular weight of 1 million to 100 million for globular proteins. The gist of the present invention is a method for producing plasma from which lipoproteins have been removed, which is characterized by passing the separated plasma through the column. The present invention will be explained in detail below. The adsorbent carrier suitable for use in the present invention needs to (1) be pressure resistant and (2) have pores with a relatively large diameter, and polymer hard gel is most suitable for the present invention. It is a carrier. The term "hard gel" as used herein refers to a gel that swells less with solvents than soft gels such as dextran, agarose, acrylamide, etc., and is less likely to deform under pressure. Hard gels and soft gels can be distinguished by the following method. In other words, as shown in the reference example below, when gel is uniformly packed into a cylindrical column and an aqueous liquid is flowed, the relationship between pressure loss and flow rate is almost a straight line for hard gel, but for soft gel there is a pressure drop. Beyond this point, the gel deforms and becomes compacted, and the flow rate no longer increases. In the present invention, when a column shown in the reference example below is used, a gel having the above linear relationship up to at least 0.3 Kg/cm ² is referred to as a hard gel. The next required property is to have pores with a relatively large diameter. That is, LDL is a large molecule with a molecular weight of at least 1 million or more, and in order to adsorb and remove it, it is necessary for LDL to be able to enter the pores. Next, even if LDL can enter the pores, its performance as an adsorbent will be low unless the probability of LDL entering the pores is high to some extent.In other words, the distribution ratio between the mobile phase and the stationary phase (inside the pores) is It is considered that a higher ratio (phase concentration/mobile phase concentration) is more preferable. Therefore, it seems that the larger the pore diameter is, the more advantageous it is. There are various methods for measuring pore diameter, and mercury intrusion method is the most commonly used, but it may not be applicable to polymer hard gels. Therefore, it is appropriate to use the exclusion limit molecular weight as a measure of the pore diameter. What is the exclusion limit molecular weight?As stated in books (for example, Hiroyuki Hatano and Toshihiko Hanai, Experimental High Performance Liquid Chromatography, Kagaku Doujin), the molecular weight cannot enter the pores (is excluded) in gel permeation chromatography. It refers to the molecular weight of the one with the smallest molecular weight among molecules. Phenomenologically, molecules with a molecular weight above the exclusion limit are eluted near the mobile phase volume Vo, so the exclusion limit molecular weight can be determined by examining the relationship with the elution volume using compounds of various molecular weights. It is known that the exclusion limit molecular weight varies depending on the type of target compound, and in general, it has been well investigated for globular proteins, dextran, polyethylene glycol, etc., but it has hardly been investigated for lipoproteins. Therefore, it is appropriate to use the values obtained using the most similar globular proteins (including viruses). As a result of studies using various carriers with different exclusion limits, we found that, contrary to expectations, the exclusion limit molecular weight was about 1 million, which is smaller than the molecular weight of LDL.
It has become clear that there is an optimal pore size range because it shows adsorption ability, and it is not that the larger the pore size, the greater the ability, but rather that proteins other than LDL are removed. In other words, if a carrier with an exclusion limit molecular weight of less than 1 million is used, LDL
Although the removal amount of LDL is too small to be practical, even a support with an exclusion limit molecular weight of 1 million to several million, which is close to the molecular weight of LDL, can provide an adsorbent that can be used practically to some extent. On the other hand, when examining the relationship between the exclusion limit molecular weight, the adsorption amount of LDL, and the adsorption of proteins other than LDL (so-called non-specific adsorption), we find that as the exclusion limit molecular weight increases, the adsorption amount of LDL increases, but this increase is excluded. It was found that when the limit exceeds 10 million, it reaches a plateau, and on the other hand, adsorption of proteins other than LDL, such as LGG and IGM, becomes noticeable. Furthermore, when the exclusion limit molecular weight exceeds 100 million, the amount of immobilized ligand decreases, resulting in a decrease in the amount of LDL adsorbed.
Non-specific adsorption can no longer be ignored. Therefore, the exclusion limit molecular weight of the carrier used in the present invention is preferably 1 million or more and 100 million or less, most preferably 3 million or more.
More than 70 million. Next, regarding the porous structure of the carrier, total porosity is preferable to surface porosity, and it is preferable that the pore volume is 20% or more. The shape of the carrier can be selected from any shape such as granules, fibers, membranes, and hollow fibers. When using a particulate carrier, the particle size is desirably 1 μm or more and 5000 μm or less. Furthermore, it is advantageous if a functional group that can be used in an immobilization reaction or a functional group that can be easily activated is present on the surface of the carrier. Representative examples of these functional groups include amino groups, carboxyl groups, hydroxyl groups, thiol groups, acid anhydride groups, succinylimide groups, chlorine groups, aldehyde groups, amide groups, and epoxy groups. Typical examples of polymer hard gels suitable for the present invention include styrene-divinylbenzene copolymer,
Hard porous bodies of synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked vinyl ether-maleic anhydride copolymer, cross-linked styrene-maleic anhydride copolymer, cross-linked polyamide, etc., and porous materials on their surfaces. Examples include those coated with sugars, synthetic polymers, etc.
It is not limited to these. These polymer hard gels may be used alone or in combination of two or more. Representative examples of polyanionic compounds with affinity for lipoproteins suitable for use in the present invention include heparin, 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, carrageenan sulfate, starch sulfate, Examples include sulfated polysaccharides such as polyglucose sulfate, laminarin sulfate, galactan sulfate, levan sulfate, and mepesulfate, phosphotungstic acid, polysulfated anethole, polyvinyl alcohol sulfate, and polyphosphoric acid. The most preferred examples include heparin, dextran sulfate, and chondroitin polysulfate. Compounds (ligands) that have affinity for lipoproteins
Various known methods can be used for immobilizing on a carrier. i.e. physical adsorption method,
These include ionic bonding, covalent bonding, etc. Since it is important that the adsorbent used in the present invention does not detach from the ligand during sterilization, it is preferable to use a strong covalent bonding method. Even if an ionic bonding method is used, the ligand is not covalently cross-linked. It is desirable to leave it there.
Furthermore, a spacer may be introduced between the carrier and the ligand if necessary. The amount of immobilized ligand varies depending on the properties and activity of the ligand, but is preferably 0.02 mg or more per ml of column volume in order to obtain a significant amount of lipoprotein adsorption, and desirably 100 mg or less in consideration of economic efficiency. More preferably, per ml of column volume
The amount is 0.5mg or more and 20mg or less. In the present invention, in order to produce plasma from which lipoproteins have been removed, plasma is first separated from blood, and then
It is better to pass the plasma through the column. When removing LDL using an adsorbent as in the present invention, it is possible to improve the removal efficiency and selectivity by adding polyvalent metal ions to the plasma to be treated. Examples of polyvalent metal ions used for this purpose include alkaline earth metal ions such as calcium, magnesium, barium, and strotium, ions of genus elements such as aluminum, ions of genus elements such as manganese, and element ions such as cobalt. (Example) The present invention will be explained in more detail with reference to Examples below. Reference example: In a glass cylindrical column (inner diameter 9 mm, column length 150 mm) equipped with a filter with a pore size of 15 μm at both ends,
Agarose gel (manufactured by Biorad) as a soft gel
Biogel A5m, particle size 50-100 mesh), Toyo Pearl manufactured by Toyo Soda Kogyo Co., Ltd. as a polymer hard gel
HW65 (particle size 50 to 100 μm) was uniformly filled, and water was poured using a peristaltic pump.
The relationship between flow rate and pressure loss was determined. The results are shown in Figure 1. According to the results, the flow rate of polymer hard gel increases almost in proportion to the increase in pressure, whereas the flow rate of agarose gel does not increase even if the pressure is increased due to compaction. Example 1 Cross-linked acrylate gel (fully porous hard gel) Toyopearl HW55 [molecular weight of globular protein exclusion limit (hereinafter abbreviated as protein exclusion limit) 700,000, particle size 50 to 100 μm], Toyopearl HW60 (protein exclusion limit) Limit 1000000, particle size 50-100μm), same HW65
(Protein exclusion limit 5000000, particle size 50-100μm),
Same HW75 (protein exclusion limit 50000000, particle size 50~
100 μm) Add 6 ml of saturated NaOH aqueous solution and 15 ml of epichlorohydrin to each 10 ml, and heat at 50°C while stirring.
After a time reaction, an epoxidized gel was obtained. 20 ml of concentrated ammonia water was added to this gel and stirred at 50°C for 2 hours to introduce amino groups. Next, 200 mg of heparin was dissolved in 10 ml of water and the pH was adjusted to 4.5, and then 3 ml of the above amino group-containing gel was added thereto. Add 200 mg of 1-ethyl-3-(dimethylaminopropyl)-carbodiimide to this and adjust the pH to 4.5.
The mixture was added while maintaining the temperature at 4°C, and the mixture was shaken at 4°C for 24 hours. After the reaction, 2 molar salt solution, 0.5 molar salt solution,
A heparin-immobilized gel was obtained by washing with water. The amount of immobilized heparin was 2.2 mg/ml and 1.8 mg/ml, respectively.
ml, 1.4 mg/ml, and 0.8 mg/ml. Example 2 Chondroitin polysulfate-immobilized Toyopearl gel HW65 was obtained in the same manner as in Example 1 except that chondroitin polysulfate was used instead of heparin. The amount of immobilization is
It was 1.2 mg/ml. Example 3 1 ml of each of the adsorbents synthesized in Examples 1 and 2 was placed in a test tube, and 3 ml of human plasma (containing 0.02M CaCl ₂ ) was added to this.
After stirring and leaving at 20℃ for 15 minutes, the cholesterol concentration and LDL (β-lipoprotein) in the supernatant were determined.
The amount was measured. The results are shown in Table 1. As can be seen from Table 1, when the adsorbent used in the present invention is used, lipoprotein adsorption is good;
Therefore, the supernatant cholesterol concentration and LDL
(β-lipoprotein) is decreased compared to the comparative example. 【table】

[Brief explanation of drawings]

FIG. 1 is a graph showing the relationship between flow rate and pressure loss using various reference example gels.

Claims (1)

[Claims] 1. Exclusion limit molecular weight of globular protein is 1 million or more. 1
A lipoprotein adsorbent made by fixing a polyanion compound having an affinity for lipoproteins to a porous polymer hard gel of 100 million yen or less is packed in a column, and plasma separated from blood is passed through the column. Method for producing removed plasma. 2. The method for producing plasma from which lipoproteins have been removed according to claim 1, wherein the porous polymer hard gel is made of a synthetic polymer. 3. The method for producing lipoprotein-free plasma according to claim 1, wherein the polyanion compound is a sulfated polysaccharide. 4. The method for producing lipoprotein-free plasma according to claim 3, wherein the sulfated polysaccharide is at least one selected from heparin, dextran sulfate, and chondroitin polysulfate. 5. The method for producing lipoprotein-free plasma according to claim 1, wherein the amount of the polyanion compound immobilized is 0.02 mg or more and 100 mg or less per ml of column volume.