JPH04256441A - Adsorbent for beta2-microglobulin - Google Patents

Abstract

PURPOSE:To make beta2-microglobulin selectively adsorbed and remove it from blood or blood plasma and to avoid other usual proteins from being adsorbed by using the porous body of polyamino acid having both of a hydrophobic group and a carboxyl group as the absorbent. CONSTITUTION:The polyamino acid formed by polymerizing hydrophobic deriv. of amino acids, such as glutamic acid and aspartic acid, is made into a porous film, porous fiber, etc., to form the porous body of the polyamino acid. A bifunctional regent, such as toluene diisocyanate, is brought into reaction therewith to incorporate both of the hydrophobic group and the carboxyl group therein. The pore size of the porous body is specified to a 10000 to 50000 range in terms of mol. wt. of dextran. The resulted absorbent selectively adsorbs away the beta2-microglobulin in the blood or blood plasma and does not adsorb the useful proteins exclusive of the beta2-microgrobulin.

Description

[Detailed description of the invention]

0001

[Field of Industrial Application] The present invention relates to an adsorbent for β2-microglobulin, and more particularly to an adsorbent for removing β2-microglobulin from blood or plasma. .

0002

BACKGROUND OF THE INVENTION Through analysis of its entire amino acid sequence, β2-microglobulin has been found to be a simple protein with a molecular weight of about 12,000 and without sugar chains, similar to the C domain of immunoglobulins. By the way, in patients undergoing hemodialysis for a long period of time, free β in the blood
2-Microglobulin concentration is 10 to 100 times higher than in healthy individuals, and β2-microglobulin is thought to be the causative agent of carpal tunnel syndrome (amyloidosis), which occurs at a high rate in dialysis patients. . Attempts have been made to remove β2-microglobulin from blood or plasma, but no practical removal method has yet been found. For example, separation using a dialysis membrane has the disadvantage that useful proteins other than β2-microglobulin are also removed, and the amount removed is small.

[0003] Furthermore, an example in which an anti-β2-microglobulin antibody was supported on an inert carrier (Japanese Patent Laid-Open No. 62-871597
However, it is not suitable for large-scale use because it takes time and cost to prepare adsorbents such as antibody production and cloning.

[0004] Also, examples of bonding and adsorbing highly hydrophobic ligands to various porous carriers (Japanese Patent Laid-Open No. 63-9987
No. 5, No. 62-240068) has a large adsorption amount of β2-microglobulin due to its strong hydrophobicity, but it also adsorbs other proteins (particularly highly hydrophobic proteins, e.g. It also adsorbs immunoglobulin, etc.), lipids, etc. Therefore, in actual blood or plasma, the adsorption amount and adsorption rate of β2-microglobulin will be small, making it difficult to use. Furthermore, the pore size of the carrier disclosed in these prior art, 2
In the range of 0 to 2000 angstroms, size selection with other proteins is considered difficult. In addition, conventional adsorbents are not biocompatible, so they can be used to directly absorb actual blood.
When applied to plasma, problems remain in terms of blood compatibility.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide an adsorbent that overcomes the drawbacks of conventional adsorbents and can selectively adsorb and remove β2-microglobulin from blood or plasma. With the goal.

[0006]

[Means for Solving the Problems] The present inventors have discovered that dextran has a pore size that allows β2-microglobulin to diffuse into it, while making it difficult for other proteins present in large amounts in blood (albumin, globulin) to diffuse. The molecular weight was found to be in the range of 10,000 to 50,000.

[0007] Furthermore, β2-microglobulin has a basic domain and can electrically interact with acidic groups such as carboxyl groups and sulfonic acid groups.
Among the amino acids that make up microglobulin, we found that the hydrophobic groups in the side chains of neutral amino acids interact with the hydrophobic groups of adsorbents, and we found that adsorbents with both acidic and hydrophobic groups have a synergistic effect. We have discovered that this phenomenon appears and have completed the present invention. That is, the present invention provides an adsorbent for β2-microglobulin made of a polyamino acid porous material characterized by having both a hydrophobic group and a carboxyl group as functional groups for adsorption. Preferably, the pore diameter is from 10,000 to 50,000 based on the molecular weight of dextran. The hydrophobic group in the present invention refers to an alkyl group such as a methyl group, an ethyl group, a propyl group, a hexyl group, and an aromatic hydrophobic group such as a phenyl group and a naphthyl group.

[0008] The polyamino acid in the present invention is a polymer of one or more of glutamic acid, aspartic acid, and hydrophobic ester derivatives of these amino acids, and any polyamino acid containing these amino acids or amino acid derivatives It may also be a copolymer with other amino acids. Examples of amino acids that can be copolymerized include alanine, glycine, oxyproline, isoleucine, leucine, proline, serine, threonine, valine, and derivatives thereof. One kind or two or more kinds of these may be used.

[0009] The polyamino acid porous material may be formed by forming the above-mentioned polyamino acid itself into a porous membrane, a porous fiber, or a porous bead. Furthermore, it may be coated or chemically bonded onto various carriers and then made porous, or it may be coated or chemically bonded onto a porous carrier. The carrier used for these may be an inorganic substance such as glass, metal salts, or carbon, or an organic substance such as a polymer. In addition, various shapes of these carriers can be used, such as powder, beads, membranes, fibers, and hollow fibers, and they can be selected according to the module such as the adsorption column or adsorption cartridge. .

[0010] The chemical bonding method utilizes the functional groups of the carrier and uses a suitable bonding reagent. For example, aminopropyl silica gel is reacted with a bifunctional reagent such as toluene diisocyanate or terephthalic acid chloride; Examples include a method of bonding amino groups, carboxyl groups, hydroxyl groups, etc. of the polyamino acid having both hydrophobic groups and carboxyl groups. As a method for making the polyamino acid itself porous, for example, the polyamino acid is dissolved in a suitable solvent, dispersed in a medium incompatible with the solvent, the solvent of the polyamino acid is removed by evaporation, and the polyamino acid is precipitated ( Japanese Unexamined Patent Publication 1986
-1728) and amino acid NCA (N-
There is a method in which the polymerization is carried out in a medium which is compatible with the carboxy-α-amino acid anhydride, but which becomes incompatible upon polymerization. In the former method, a poor solvent for the polyamino acid is added, and after precipitation, it can be removed by extraction or other methods to make it porous.The pore size can be adjusted depending on the type of poor solvent and the amount added. can be controlled.

[0011] In the latter method, basically any combination of amino acids and amino acid derivatives may be used, but the pore size can be controlled by appropriately combining the type of solvent, polymerization temperature, polymerization catalyst, and polymerization time. is necessary. The size of the porous body obtained by these methods can be changed arbitrarily, but when actually removing β2-microglobulin by passing blood or plasma through it, the size of the porous body obtained by these methods must be adjusted according to the module such as the adsorption column or adsorption cartridge. You can choose.

[0012] The pore diameter of the porous material in the present invention is preferably in the range of 10,000 to 50,000 in terms of the molecular weight of dextran. The pore diameter of the porous material was determined by gel permeation chromatography using a standard molecular weight dextran substance (
GPC).

That is, a porous material containing a polyamino acid as a constituent is sieved by an appropriate method and packed into a stainless steel column for liquid chromatography. The beads for pore size evaluation are preferably 100 μm or less, and are preferably as uniform in size as possible. The size of the stainless steel column is usually commercially available (for example, 4 made by Nippon Seimitsu Co., Ltd.).
．． A column (6mmφ*150mm) is sufficient. GPC
The standard molecular weight substance to be measured is preferably one that has as little interaction as possible with the porous body, and dextran standard molecular weight substance (manufactured by Sigma Chemical Co., Ltd.) is suitable. Other standard molecular weight substances, such as globular proteins and polyethylene glycol, are not suitable because they do not necessarily elute in the order of molecular weight or are adsorbed.

[0014] The measuring method may be an ordinary aqueous GPC measurement using standard molecular weight dextran. That is, the elution capacity of dextran of various molecular weights is measured using a liquid chromatography device using distilled water as an eluent.

[0015]

[Operation] According to the present invention, a technique for selectively adsorbing β2-microglobulin from blood or plasma has been established. Although the mechanism is not clear, for example, controlling the pore size of the adsorbent can limit the protein molecules that can diffuse into the interior, and it is not because the hydrophobic groups and carboxyl groups of polyamino acids specifically adsorb β2-microglobulin. I think so.

[0016]

Effects of the Invention The adsorbent for β2-microglobulin of the present invention has (1) high selectivity without adsorbing useful proteins other than β2-microglobulin; (2) β2-microglobulin adsorbent has high selectivity;
-High adsorption amount and speed of microglobulin (3
) The adsorbent has excellent effects such as biocompatibility.

[0017]

EXAMPLES The present invention will be explained in more detail with reference to Examples below, but the present invention is not limited to these Examples. Note that the β2-microglobulin adsorption test will be described later. Production Example 1 2.5% dichloroethane solution of poly-γ-methyl-L-glutamate (hereinafter referred to as PMLG) with an average degree of polymerization of 500
Add 4 ml of methyl laurate to the mixture and stir well. Dichloroethane was removed by evaporation at 50° C. while stirring and dispersing this in 500 ml of a 2.5% aqueous solution of partially saponified polyvinyl alcohol. The obtained polyamino acid porous material is filtered and thoroughly washed with warm water to completely remove polyvinyl alcohol. Furthermore, the internal methyl laurate is completely extracted and removed using methanol. This porous material was classified, and those having a diameter of 37 to 74 μm were taken out, filled in a stainless steel column, and subjected to GPC measurement. As a result, the exclusion limit molecular weight was approximately 40,000. Further, the porous polyamino acid particles were left in a 1N aqueous sodium hydroxide solution at 30° C. for 1 hour, and then the caustic soda was completely washed away with distilled water. When a part of the particles was dried and the carboxyl groups were quantified by titration, it was found that about 45% of the methyl ester moieties were converted to carboxyl groups by hydrolysis.

Production Example 2 Production Example 1 except that poly-β-benzyl-L-aspartate with an average degree of polymerization of 450 was used instead of poly-γ-methyl-L-glutamate with an average degree of polymerization of 500. A porous body was produced in exactly the same manner as in Example 1. The exclusion limit molecular weight of the obtained particles was approximately 50,000. This was treated with caustic soda in the same manner as in Production Example 1, and the carboxyl groups were quantified, and it was found that about 65% of the benzyl ester moieties had become carboxyl groups due to hydrolysis.

Comparative Example 1 A porous body was produced in exactly the same manner as in Production Example 1, except that the caustic soda treatment was not performed.

Comparative Example 2 In Production Example 2, a porous body was produced in exactly the same manner as in Production Example 1, except that the caustic soda treatment was not performed.

Example 1 g of each of the adsorbents obtained in Production Examples 1 and 2 and Comparative Examples 1 and 2 was immersed in 50 ml of plasma from an artificial dialysis patient.
β2-microglobulin concentration (β2), total protein concentration (TP),
Changes in albumin/globulin ratio (A/G) over time were measured. 0 hours
4 hours
β2 TP A/G
β2 TP A/G
mg/L g/dl −
mg/L g/dl - Production example 1
13.5 4.2 1.5 2.6
4.2 1.5 Production example 2 13.5 4.2
1.5 2.3 4.2 1.5 Comparative Example 1 13.5 4.2 1.5
4.5 4.2 1.5 Comparative example 2 13.5
4.2 1.5 6.4 4.2 1
．． 5. As is clear from the above, β2 in Production Examples 1 and 2 is approximately half or less than that in Comparative Examples 1 and 2 after 4 hours.

Claims (2)

[Claims] 1. An adsorbent for β2-microglobulin, which is made of a porous polyamino acid, and is characterized by having both a hydrophobic group and a carboxy group as functional groups for adsorption. 2. The adsorbent for β2-microglobulin according to claim 1, wherein the pore diameter is from 10,000 to 50,000 based on the molecular weight of dextran.