JPH04247235A - Adsorbent for beta2-microglobulin - Google Patents

Abstract

PURPOSE:To efficiently and selectively adsorb and remove beta2-microglubulin which exists at high concn. in the blood of a patient under treatment with artificial hemodialysis. CONSTITUTION:A porous body containing polyamino acid as a structural component is prepared by controlling the micropores to such a range that beta2- miicroglobulin can be diffused but other coexisting proteins (albumin, globulin) in blood are hardly diffused (in the range from 10000 to 50000 in terms of dextran mol.wt.). Then the surface of this porous body is treated with formic acid to form a beta structure to the polyamino acid.

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 no sugar chains, similar to the C domain of immunoglobulins. By the way, in patients undergoing hemodialysis for a long period of time, free β2 in the blood
- 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 frequently occurs 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]Also, there is an example in which an inert carrier is supported with an anti-β2-microglobulin antibody (Japanese Patent Application Laid-Open No. 62-871597), but preparation of the adsorbent such as antibody production cloning takes time and cost. Not suitable for mass use. Examples of bonding and adsorption of strongly hydrophobic ligands to various porous carriers (JP-A-63-99875, JP-A-63-99875;
-240068) has a large adsorption amount of β2-microglobulin due to its strong hydrophobicity, but other proteins (especially highly hydrophobic proteins, such as immunoglobulins) that exist in large quantities in actual blood or plasma, It even adsorbs 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, since the pore diameter of the carriers disclosed in these prior art is in the range of 20 to 2000 angstroms, it is considered difficult to select between them and other proteins in terms of size. Furthermore, the adsorbents of the prior art are not biocompatible, and problems remain in terms of blood compatibility when directly applied to actual blood or plasma.

0004

Problems to be Solved by the Invention The adsorbent of the present invention overcomes the drawbacks of conventional adsorbents and selectively and efficiently collects β2-microglobulin from blood or plasma.
The object of the present invention is to provide an adsorbent that can be removed by adsorption.

[0005]

[Means for Solving the Problems] The present inventors have conventionally discovered that the surface structure of polyamino acids gives them selectivity in their interaction with protein molecules, and by controlling the pores of the porous surface, they can diffuse into the interior. It was discovered that protein molecules can be limited, and an application has already been filed under JP-A-2-209155. As a result of intensive research into adsorbents with even higher adsorption ability, the present inventors found that the above-mentioned polyamino acid porous material not only has high selectivity for protein adsorption by controlling the surface structure and pore diameter. The selectivity of diffusive permeation also increases, and the surface structure of the polyamino acid is such that the β structure changes to β2.
- It was discovered that the polyamino acid interacts specifically with microglobulin, and that treatment of a polyamino acid porous material with formic acid contributes to the generation of β structure, and based on these findings, the present invention was completed.

That is, the present invention provides a β2 material obtained by treating a porous material containing a polyamino acid as a constituent with formic acid.
- Provides an adsorbent for microglobulin. In the adsorbent, the pore diameter of the porous body containing polyamino acids as a constituent component is preferably in the range of 10,000 to 50,000 in terms of the molecular weight of dextran. In these adsorbents, preferably, the polyamino acid further has a β structure.

[0007] The polyamino acids in the present invention include alanine, cystine, glycine, proline, oxyproline,
Leucine, isoleucine, methionine, phenylalanine, serine, threonine, tryptophan, tyrosine,
Valine, arginine, lysine, histidine, glutamic acid, aspartic acid and one of the derivatives of these amino acids
A porous body containing a polyamino acid, which is a polymerization of one or more kinds of polyamino acids, may be one in which the above-mentioned polyamino acid itself is made into a porous membrane or into a porous fiber. Porous beads may also be used. Further, it may be made porous after being coated or chemically bonded onto various carriers, or 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. Furthermore, various shapes of these carriers can be used, such as powder, beads, membranes, fibers, and hollow fibers, and they can be selected depending on the module such as an adsorption column or an adsorption cartridge.

[0008] 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. As a method for making polyamino acids themselves porous, for example, the polyamino acids are dissolved in a suitable solvent, dispersed in a medium that is incompatible with the solvent, the solvent of the polyamino acids is removed by evaporation, and the polyamino acids are precipitated. (No. 62-1728) or a method of polymerizing the amino acid NCA (N-carboxy-β-amino acid anhydride) before polymerization in a solvent that is compatible with it but becomes incompatible with it after polymerization. There is. 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.

Polyamino acids that can be made porous by the former method include polyalanine, polyaspartic acid, polyglutamic acid, polyglycine, polyoxyproline, polyisoleucine, polyleucine, polyproline, polyserine, polythreonine, polyvaline, and These derivatives, copolymers of two or more types, etc. can be mentioned.

[0010] 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 material obtained by these methods can be changed arbitrarily, but when actually removing β2-microglobulin by passing blood or plasma through it, it is necessary to adjust the size to the size of the adsorption column, adsorption cartridge, etc. You can choose.

[0011] Formic acid treatment in the present invention may include adding formic acid during the synthesis process of the polyamino acid porous material to promote the formation of β structure, or treatment with formic acid vapor or direct immersion in formic acid after making it porous. You may do so. That is, there is a method in which formic acid is added as part of the aforementioned poor solvent to generate a porous body. The amount of formic acid added can be arbitrarily selected within a range that allows generation of a porous body, but it is preferable that the pore diameter of the resulting porous body is in the range of 10,000 to 50,000 in terms of the molecular weight of dextran.

The pore diameter of the porous material can be evaluated by gel permeation chromatography (GPC) using dextran standard molecular weight substance. That is, a porous material containing a polyamino acid as a component 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. As for the size of the stainless steel column, a commercially available column (for example, 4.6 mmφ*150 mm manufactured by Nippon Seimitsu Co., Ltd.) is sufficient. The standard molecular weight substance used for GPC measurement is preferably one that has as little interaction as possible with the porous body;
Dextran standard molecular weight material (manufactured by Sigma Chemical Co.) 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. The measurement method may be normal 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. The porous material containing polyamino acids as a constituent component preferably has an exclusion limit molecular weight in the range of 10,000 to 50,000 in terms of the molecular weight of dextran when used as an adsorbent for β2-microglobulin. The β structure of the polyamino acid porous material can be identified by infrared absorption spectrum. That is,
In the absorption spectrum of the amide I band, the random coil is at 1655 cm, and the α-helix is at 1650 cm.
cm-1, β structure has absorption at 1630 and 1690 cm-1.

[0013]

[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, it is thought that the protein molecules that can diffuse into the adsorbent can be limited by controlling the pore size of the adsorbent, and that the β structure of the polyamino acid specifically adsorbs β2-microglobulin.

[0014]

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, and (2) adsorption amount and adsorption rate of β2-microglobulin. (3) Adsorptive property with a large value has excellent effects such as biocompatibility.

[0015]

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) Poly-γ-methyl-L with an average degree of polymerization of 500
- Add 4 ml of methyl laurate and 4 ml of formic acid to 50 g of a 2.5% dichloroethane solution of glutamate (hereinafter referred to as PMLG) 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 and formic acid. Furthermore, the internal methyl laurate is completely extracted and removed using methanol. This porous material was classified to 37 to 74
Take out the μm one and fill it into a stainless steel column.
GPC measurement was performed. As a result, the exclusion limit molecular weight was approximately 4
It was 10,000. The infrared absorption spectrum of this porous material was as shown in FIG. 1, and the formation of a β structure was observed.

(Production Example 2) A porous body was produced in exactly the same manner as in Production Example 1 except that the amount of methyl laurate was changed to 8 ml and formic acid was not added. This was immersed in formic acid at room temperature for 3 hours, washed with distilled water to completely remove the formic acid, and then GP
C measurement was performed. As a result, the exclusion limit molecular weight was approximately 30,000. Moreover, when the infrared absorption spectrum of this porous body was taken, it was similar to that shown in FIG. 1, and the formation of a β structure was observed.

(Comparative Example 1) A porous body was produced in exactly the same manner as in Production Example 1 except that formic acid was not added. Fill this into a stainless steel column and
PC measurement was performed. As a result, the exclusion limit molecular weight was approximately 40,000. Further, the infrared absorption spectrum of this porous body was as shown in FIG. 2, and no formation of β structure was observed. (Comparative Example 2) A porous material obtained in the same manner as in Production Example 2 except that the amount of methyl laurate was changed to 4 ml was packed into a stainless steel column, and GPC
Measurements were taken. As a result, the exclusion limit molecular weight was approximately 4,000.

(Example) Production Examples 1 and 2 and Comparative Examples 1 and 2
1 g of each of the adsorbents obtained in step 1 was immersed in 50 ml of plasma from a patient undergoing artificial dialysis, and the concentration of β2-microglobulin (β2), total protein concentration (TP), and albumin in the plasma was measured at 37°C while stirring. /globulin ratio (A/
G) Changes over time were measured. 0 hours 4
Time β2 T
P A/G β2 TP
A/G mg/L
g/dl - mg/L
g/dl - Production example 1 1
1.5 4.1 1.5
1.6 4.1 1.5
Production example 2 11.5 4.1
1.5 1.8
4.1 1.5 Comparative example 1 11
．． 5 4.1 1.5
5.2 4.1 1.5
Comparative example 2 11.5 4.1
1.5 8.9
4.1 1.5 In the adsorbent of the comparative example, a large amount of β2-microglobulin in plasma remains.

[Brief explanation of the drawing]

FIG. 1 is an infrared absorption spectrum of a polyamino acid-containing porous material treated with formic acid of the present invention.

FIG. 2 is an infrared absorption spectrum of a porous material similar to FIG. 1 but not treated with formic acid.

Claims (3)

[Claims] 1. An adsorbent for β2-microglobulin, which is obtained by treating a porous material containing a polyamino acid as a constituent with formic acid. 2. The adsorbent for β2-microglobulin according to claim 1, wherein the porous body containing a polyamino acid as a constituent has a pore diameter in the range of 10,000 to 50,000 in terms of the molecular weight of dextran. . 3. The adsorbent for β2-microglobulin according to claim 1 or 2, wherein the polyamino acid has a β structure.