JPH06104122B2 - β▲lower 2▼-microglobulin removal column - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION TECHNICAL FIELD The present invention relates to a column for removing β ₂ -microglobulin, and more particularly to a column for specifically adsorbing and removing β ₂ -microglobulin from blood.

BACKGROUND ART β ₂ -microglobulin is a histocompatibility antigen (in humans,
It is the light chain of the two-chain protein that constitutes the HLA class I. It is found on the surface of almost all cells and also in body fluids in a free state unbound to the heavy chain, but the physiological function of free _β2 -microglobulin is unknown. The entire amino acid sequence has already been elucidated for humans and various other animals, and the three-dimensional structure of the bovine protein has also been determined by X-ray crystallography. The results show that it is a simple protein with no sugar chains, with a molecular weight of approximately 12,000, and that it is structurally highly similar to the C domain (constant domain) of immunoglobulins. Furthermore, the amino acid sequence homology between animal species is quite high, at 60-80% (Proc. Natl.
Acad.Sci 257 , 2619 (1982), etc.

In patients undergoing long-term hemodialysis for kidney disease, the concentration of free _β2 -microglobulin in the blood is 10 to 100 times higher than in healthy individuals. This is thought to be because _β2 -microglobulin, which is broken down in the kidneys in healthy individuals, accumulates without being removed by hemodialysis.

The present inventors have detected and analyzed amyloid proteins deposited in the affected areas of patients with carpal tunnel syndrome, a condition that occurs at a high rate among dialysis patients, and have found that the majority of the amyloid proteins is _β2 -microglobulin.
It is believed that this is due to the deposition of _β2 -microglobulin in the affected area, which is accumulated in high concentrations in the blood during dialysis. It is expected that the onset of carpal tunnel syndrome can be suppressed by removing _β2 -microglobulin from the blood in parallel with dialysis. It is also possible that _β2 -microglobulin is involved in amyloid deposition in areas other than the carpal tunnel.

Until now, no disease thought to be caused by high concentrations of β ₂ -microglobulin in the blood has been known, and therefore no investigation has been made into its removal.

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a method for selectively removing β ₂ -microglobulin from blood.

The above object can be achieved by the present invention, which provides a column for adsorbing and removing β ₂ -microglobulin using an immobilized anti-β ₂ -microglobulin antibody.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows an example of a circuit for using a hemodialyzer and a β ₂ -microglobulin removal column in combination.
(a) shows the case where they are connected in series, and (b) shows the case where they are connected in parallel.

FIG. 2 shows the column fractions in Example 1.
- Schematic diagram of the results of polyacrylamide gel electrophoresis analysis.

FIG. 3 shows the total protein amount and β ₂ -microglobulin concentration of the column fractions in Example 2.

FIG. 4 shows the change over time in the amount of residual β ₂ -microglobulin in the blood in Example 4, where (a) shows an example in which a column immobilized with anti-β ₂ -microglobulin antibody was used, and (b) shows an example in which the column in (a) was reused.
(c) shows an example in which a column on which no anti-β ₂ -microglobulin antibody was immobilized was used.

BEST MODE FOR CARRYING OUT THE INVENTION The anti-β ₂ -microglobulin antibody used in the present invention is
Both polyclonal antibodies obtained by immunizing animals such as mice, rats, rabbits, goats, and sheep, and monoclonal antibodies obtained using cell fusion techniques, etc., can be used. The _β2 -microglobulin used for immunization can be of any animal species as long as the resulting antibody can bind to human _β2 -microglobulin. However, to increase the binding efficiency, antibodies derived from humans or monkeys are preferred, with human-derived antibodies being even more preferred. Peptide fragments or synthetic peptides with equivalent immunogenicity can also be used. In order to efficiently remove _β2 -microglobulin without affecting the function of blood cells, it is necessary to remove HL on the cell surface.
More preferable results are obtained by using a monoclonal antibody that does not react with the β ₂ -microglobulin that constitutes A and that binds only to free β ₂ -microglobulin.

The insoluble carriers used in the present invention include agarose,
Examples of suitable materials include natural and synthetic polymers such as cellulose, dextran, polyacrylamide, and polystyrene derivatives, but hydrophilic materials that minimize non-specific adsorption of blood components are preferred.
The beads may be in any form, such as beads, fibers, or films. When beads are used, the particle size is not important as long as the β ₂ -microglobulin-containing solution can be circulated through a column packed with the beads. However, in order to reduce the flow resistance, a diameter of 50 to 3,000 μm is preferred, and a diameter of 200 to 3,000 μm is preferred.
It is more preferable that the beads are physically strong and have small diameter changes due to pressure.

Antibodies can be bound to insoluble carriers by chemical covalent bonding using coupling agents such as cyanogen bromide and carbodiimide, or cross-linking agents such as glutaraldehyde. Alternatively, anti-human _β2 -microglobulin antibodies can be bound via protein A that has been immobilized on an insoluble carrier in advance, thereby increasing the antigen adsorption capacity per antibody amount. In this case, however, it is necessary to chemically cross-link the protein A and the antibody to prevent antibody detachment.

The amount of antibody bound and the size of the column are not particularly limited, but in order to improve the therapeutic effect, 50 mg per column is recommended.
Since the amount of antigen β ₂ -microglobulin adsorbed per 1 g of antibody is about 50 mg to 150 mg, it is desirable that one column can adsorb 300 mg or more of β ₂ -microglobulin.
A binding amount of 000 mg or more of antibody is required. However, if two or more columns are used in one treatment, it is possible to reduce the amount of antibody per column.

The antibody-immobilized insoluble carrier thus obtained is packed into an appropriate column, and by passing blood through the column, β ₂ -microglobulin in the blood can be adsorbed and removed very selectively and efficiently.

During treatment, the _β2 -microglobulin removal column may be used alone, but considering that the main target patients are those undergoing dialysis, it is preferable to connect it in series or parallel to a hemodialyzer to circulate blood simultaneously, from the standpoint of ease of operation.

An example of connecting the column of the present invention to a hemodialyzer will be explained with reference to Fig. 1. An example of connecting them in series is shown in Fig. 1(a).
As shown in Fig. 1(a), blood taken out of the patient's body passes through a blood pump 1 and enters a hemodialyzer 2, where it is subjected to normal dialysis treatment with a dialysate 4, after which _β2 -microglobulin is removed in a _β2 -microglobulin removal column 3 and the blood is returned to the body. In Fig. 1(a), an example is shown in which the _β2 -microglobulin removal column 3 is connected after the hemodialyzer 2, but it is also possible to connect the β2-microglobulin removal column 3 before the hemodialyzer 2, i.e., before the blood pump 1.
The β 2 -microglobulin removal column 3 may be connected at any position before or after the β 2 -microglobulin removal column 3. An example of a parallel connection will be explained with reference to Figure 1(b). Blood taken out of the patient's body passes through the blood pump 1 and is separated into two directions. One enters the hemodialyzer 2 and undergoes normal dialysis treatment with the dialysate 4. The other enters the hemodialyzer 2, where the flow rate is adjusted by the auxiliary pump 5, and the β ₂ -microglobulin removal column 3 is removed. After that, the β 2 -microglobulin removal column 3 is combined with the blood coming out of the hemodialyzer 2 and returned to the body. Even in the case of a parallel connection, the β ₂ -microglobulin removal column 3 may be connected at any position in the circuit. In the case of a parallel connection, an auxiliary pump 5 may be used to keep the bypass blood flow constant, as shown in Figure 1(b). However, it is also possible to adjust the internal diameter of the circuit without using the auxiliary pump 5. The dialysis membrane of the hemodialyzer connected to the column of the present invention is made of a material selected from cellulose, cellulose acetate, polymethyl methacrylate,
Although there is no particular limitation, polyacrylonitrile, polysulfone, polyamide, polyester, polyvinyl alcohol, vinyl alcohol copolymers, etc. are used, in order to further increase the amount of _β2 -microglobulin removed, a dialysis membrane with a permeability of 2% or more for proteins with a molecular weight of 10,000 is desirable.

Alternatively, whole blood may be passed through the _β2 -microglobulin removal column of the present invention. However, although the procedure becomes more complicated, the same effect can be obtained by passing plasma from which blood cell components have been removed using a commonly used plasma separator instead of circulating whole blood.

Furthermore, the column used for adsorption can be regenerated and reused by passing an acidic solution of about pH 2 through it.

The column of the present invention selectively adsorbs _β2 -microglobulin, allowing for simple and efficient removal of _β2 -microglobulin from blood. Furthermore, the column of the present invention has the advantage that it can be reused repeatedly by eluting the adsorbed _β2 -microglobulin with an eluent.

The present invention will be described in more detail below with reference to examples.

Example 1 Agarose gel ("Affigel 10", BioRad) to which N-hydroxysuccinimide ester groups were introduced via a 10-atom long spacer ( _-OCH2CONH ( _CH2 )NHCO( _CH2 ) _2- ) was prepared.
1.46 mg of a commercially available anti-human β ₂ -microglobulin monoclonal antibody (Olac "MCA06") was added to 1 ml of the antibody solution (Olac "MCA06") in 0.1 MHE.
Add 1 ml of PES-NaOH buffer (pH 7.5) to the solution.
The mixture was stirred gently at 4°C overnight.

0.1 ml of 1 M ethanolamine-HCl (pH 8.0) was added and the mixture was reacted at room temperature for 1.5 hours to block the unreacted N-hydroxysuccinimide ester groups.
1 ml of 0.1 M acetic acid containing Cl-NaOH (pH 4.0) and 0.1 M carbonate
The gel was washed three times with 1 ml of NaOH (pH 9.0) and finally equilibrated with PBS. The amount of protein remaining in the solution after immobilization was 0.2 mg, which means that 1.44 mg of antibody was immobilized per 1 g of gel.

0.3 ml of the antibody-immobilized gel thus obtained was packed into a commercially available small column (φ=8 mm), and 2.4 ml of a model solution prepared by dissolving 0.1 mg/ml bovine serum albumin (BSA) and 0.1 mg/ml human β ₂ -microglobulin in PBS was added to the column at room temperature.
The flow rate was 1/h. 0.63 ml of each fraction was collected using a fraction collector.
20 μl was analyzed by SDS-polyacrylamide gel electrophoresis.

The results are shown in Figure 2. Figure 2 is a schematic diagram of the results of electrophoresis. Lane 1 shows the results of electrophoresis of the model protein solution before passing it through the column, and lanes 2 to 5 show the results of electrophoresis of column fractions 2 to 5, respectively.

The arrows indicate the migration positions of standard samples of β ₂ -microglobulin (β ₂ m) and BSA.

In all analyzed fractions 2 to 5, β
The ratio of the amount of _{β 2} -microglobulin to BSA was smaller than that in the solution before being applied to the column, indicating that only β ₂ -microglobulin was selectively adsorbed to the column.

After washing away the remaining proteins in the column with PBS, the antigen bound to the antibody was eluted with 50 mM glycine-HCl buffer (pH 2.4), and only _β2 -microglobulin was eluted. The results of electrophoresis of 20 μl of the eluate are shown in lane 6 of Figure 2.

Example 2: A column prepared in the same manner as in Example 1 was loaded with _β2 at a high level.
- Serum containing microglobulin from dialysis patients is circulated,
From the beginning of the flow, 0.32 ml fractions were collected using a fraction collector.

The total protein amount (expressed as absorbance at 280 nm) of each fraction and the concentration of β ₂ -microglobulin (β ₂ m) quantified by immunoassay are shown in Figure 3. In fractions 1 to 10, the ratio of β ₂ -microglobulin to total protein was significantly lower than that of the serum before loading onto the column, indicating that it had been adsorbed and removed by the antibody. (The absorbance at 280 nm of the serum before loading onto the column was 7.
2.1, the _β2 -microglobulin concentration was 40.5 mg/ml.) From the results in Figure 3, the total amount of _β2 -microglobulin adsorbed to the column was 0.049 mg, which means that 0.11 mg of _β2 -microglobulin was adsorbed per 1 mg of immobilized antibody.

Example 3: 9-atom long spacer ( _-OCH2CH (OH) _CH2NH ( _CH2 ) _4- )
2.8 ml of cellulose beads ("Formyl Cellulofine," manufactured by Chisso Corporation) to which formyl groups had been introduced via HCl and 2 mg of the commercially available anti-human _β2 -microglobulin monoclonal antibody used in Examples 1 and 2 were mixed in 6 ml of potassium phosphate buffer (pH 7.0), and after reacting for 2 hours at 4°C, the mixture was reduced with dimethylamineborane and further reacted overnight to prepare beads on which 0.54 mg of antibody was immobilized per ml of carrier. Unreacted formyl groups were blocked by reacting with the amino groups of Tris.

A small column was filled with 2.1 ml of these beads (1.1 mg of antibody), and 10 ml of healthy blood containing _β2 -microglobulin was circulated at a flow rate of 1 ml/min for 2 hours. Small amounts of blood were withdrawn at appropriate intervals, and the amount of _β2 -microglobulin ( _β2m ) in the blood was measured. The results are shown in Figure 4(a). Adsorption was complete within 10 minutes after the start of circulation, and the amount of adsorbed was 100 μg, approximately 1/10 of the amount of antibody used.

After washing the column with 1 M glycine-HCl buffer (pH 2.8), the same circulation experiment was carried out again.
As shown in Figure 4(b), adsorption was equal to or greater than that of the first experiment, and the column could be regenerated. In a control experiment using cellulose beads with no antibody immobilized on them (Figure 4(c)), almost no adsorption was observed.

Industrial Applicability As described above, the column of the present invention can specifically adsorb and remove _β2 -microglobulin from blood, and is therefore extremely useful for preventing and treating complications such as amyloidosis, including carpal tunnel syndrome, and bone disorders seen in dialysis patients.

Claims (3)

[Claims] 1. A β ₂ -microglobulin removal column comprising an anti-β ₂ -microglobulin antibody immobilized on an insoluble carrier. 2. The column according to claim 1, wherein the antibody is a monoclonal antibody. 3. A hemodialysis system comprising a hemodialyzer and a β ₂ -microglobulin removal column in which anti-β ₂ -microglobulin antibodies are immobilized on an insoluble carrier, connected in series or in parallel.