JPH05271299A - Protein a-immobilized adsorbent - Google Patents

Abstract

PURPOSE:To obtain the subject adsorbent favorably maintaining specific adsorp tion power to antibody (immunoglobulin), thus capable of efficiently separating and purifying antibody, by reaction of an acid anhydride group-bearing com pound with fiber surface followed by reaction and immobilization of protein A. CONSTITUTION:Fibers such as nylon 6 is immersed in 3N HCl at 30 deg.C for 30min to hydrolyze part thereof and induce free amino group on the fiber surface followed by thoroughly washing with distilled water and then drying. The resulting fibers is then immersed in an acetone solution of an acid anhydride group-bearing compound such as 2% (W/V) maleic anhydride-methyl vinyl ether copolymer at room temperature for one hour and washed with acetone and then dried in a vacuum. The resultant fibers is immersed in a protein A-contg. 10mM acetic acid buffer solution (pH4.0) at room temperature for 2hr to immobilize the protein A on the fiber surface, thus obtaining the objective protein A-immobilized adsorbent having the above-mentioned advantages.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a protein A-immobilized adsorbent which is used for immobilizing protein A on the fiber surface and efficiently separating and purifying antibodies.

[0002]

2. Description of the Related Art Conventionally, polyester, polyamide,
It has been proposed to adsorb physiologically active substances such as antibodies using fibers such as polyacrylonitrile for separation and purification (see Japanese Patent Publication No. 48-23891). The performance required of the adsorbent used for such adsorption is that it has a large specific adsorptivity for a target substance such as an antibody, a large adsorption capacity per unit weight, and is sufficient when performing separation and purification operations. It has excellent mechanical strength and stability.

[0003]

However, none of the conventional adsorbents has sufficiently obtained these performances. In particular, since the adsorption capacity per unit weight is small, the apparatus required for adsorption becomes large, which is not efficient and economically disadvantageous.

[0004]

In view of this point, the present inventors have made various studies on protein A-immobilized adsorbents in order to develop an antibody-specific adsorbent having a large adsorption capacity per unit weight. The invention has been reached.

That is, the protein A-immobilized adsorbent of the present invention is characterized by immobilizing protein A by reacting a compound having an acid anhydride group on the fiber surface.

The present invention will be described in detail below. As the fiber used in the present invention, fibers such as polyamide such as nylon 4, nylon 6 and nylon 12 and polyurethane, as well as polyester such as polyethylene terephthalate, polyacrylonitrile, polyvinyl alcohol and regenerated cellulose can be used. it can. Also,
These fibers may be made of a copolymer. The single yarn fineness is preferably 10 to 0.001 denier, and particularly preferably 4 to 0.001 denier. Further, the fiber can be used as a fiber aggregate of various shapes such as spun yarn, cotton, non-woven fabric, paper, woven fabric and knitted fabric.

Examples of the compound having an acid anhydride group used in the present invention include polycarboxylic acid anhydrides such as maleic anhydride methyl vinyl ether copolymer, maleic anhydride ethylene copolymer and maleic anhydride styrene copolymer. , Maleic anhydride, phthalic anhydride and the like.

[0008] The protein A used in the present invention is a protein having a molecular weight of about 42,000 existing in the bacterial cell wall and specifically binds to immunoglobulin.

The protein A-immobilized adsorbent of the present invention is prepared by reacting a compound having an acid anhydride group on the fiber surface to introduce an acid anhydride group, and then reacting protein A with the above acid anhydride group. And the amino group of protein A are bound and immobilized.

In the present invention, in the case of a fiber having a reactive group capable of reacting with an acid anhydride group, for example, a fiber having an amino group, a compound having an acid anhydride group can be directly reacted with the fiber surface. When there is no reactive group capable of reacting with the acid anhydride group, first, amines such as ethylenediamine, trimethylenediamine, and tetramethylenediamine, and imines such as ethyleneimine, glutaraldehyde, hexamethylenediisocyanate, and the like. After introducing an amino group with a bifunctional reagent or a condensing agent such as cyclohexylmorpholinoethylcarbodiimidemethotoluenesulfonic acid or dicyclohexylcarbodiimide, a compound having an acid anhydride group can be reacted with the fiber surface. Further, maleic anhydride can be graft-polymerized on the fiber surface by γ-ray or electron beam to react with acid anhydride. The fiber reacted with the compound having an acid anhydride group has an active acid anhydride group on the surface of the fiber, so that a bond easily occurs with the amino group of protein A under mild conditions, and covalent bond It can be fixed. The protein A immobilization treatment can be achieved by adding fibers prepared by reacting a compound having an acid anhydride group to a solution of protein A dissolved in water or physiological saline and stirring the mixture at room temperature for several hours. An acid, base, salt or the like may be added to the protein A solution.

[0011]

In the present invention, a compound having an acid anhydride group is reacted on the surface of the fiber to introduce an active acid anhydride group on the fiber, and then protein A is reacted therewith to thereby activate the protein A. Can be fixed on the fiber without degrading. Since this protein A retains its excellent activity, it retains good specific adsorptivity with the antibody (immunoglobulin) peculiar to protein A, and the adsorptivity is utilized to efficiently separate the antibodies. , Can be purified.

[0012]

EXAMPLES The present invention will be described in more detail below with reference to examples. Example 1 A fiber (1 denier) made of nylon 6 was immersed in 3N hydrochloric acid at 30 ° C. for 30 minutes and then washed with distilled water. After washing and drying, it was immersed in a dehydrated acetone solution of a 2% (w / v) maleic anhydride-methyl vinyl ether copolymer at room temperature for 1 hour, washed with acetone, and vacuum dried. The protein A was immobilized by immersing the obtained fiber in a 10 mM acetic acid (pH 4.0) buffer containing protein A (manufactured by SIGMA) at room temperature for 2 hours.

1 g of the protein A-immobilized nylon fiber thus obtained was packed in a column (0.5 × 15 cm), and a 10 mM phosphate buffer solution (pH 7.2, hereinafter PBS buffer) containing 0.9% sodium chloride was packed. Abbreviated as liquid) 0.1%
(w / v) Equilibrated with a solution containing sodium azide and used for the subsequent reaction.

Serum obtained from rabbit blood is equivalent to PB
The sample diluted with S buffer was used. This sample was passed through a column packed with nylon fibers on which protein A was immobilized at a flow rate of 1 ml / 3 min, and the passing liquid was collected. Further, while confirming the presence or absence of protein in the passing solution by measuring the absorbance at 280 nm, the PBS buffer solution was passed through until the absorption of protein at this wavelength disappeared. Then, IgG (immunoglobulin G) adsorbed on the column was eluted with 100 mM citrate buffer (pH 4.0), and 1 M PBS was immediately added to the eluate.
Buffer was added.

The proteins contained in the thus-obtained fractions of the flow-through solution of the column and the fractions adsorbed to the column were respectively identified by SDS-polyacrylamide gel electrophoresis (Laemmli 1970). As a result, a single band corresponding to IgG was confirmed at a molecular weight of around 150,000 in the fraction adsorbed on the protein A-immobilized nylon fiber column, while other plasma proteins were confirmed in the flow-through fraction. , No band was detected at the position corresponding to IgG,
It was found that IgG was well adsorbed on the protein A-immobilized nylon fiber column.

Example 2 A fiber made of polyethylene terephthalate (1 denier) was immersed in 1N sodium hydroxide at 70 ° C. for 1 hour, washed with distilled water, and then immersed in 0.1N hydrochloric acid at 70 ° C. for 1 hour. did. After washing with distilled water and drying, 10% (w /
v) Polyethyleneimine aqueous solution and methanol 1:
The mixture was immersed in 5 liquid mixture for 30 minutes at room temperature, 5% (w / v) dicyclohexylcarbodiimide in methanol was added, and the mixture was subsequently immersed at room temperature for 2 hours. After being washed with methanol and dried, it was immersed in a dehydrated acetone solution of 2% (w / v) maleic anhydride-methyl vinyl ether copolymer at room temperature for 1 hour,
After washing with acetone, it was vacuum dried. The obtained fiber was mixed with 10 mM acetic acid (pH: Protein A (manufactured by SIGMA)
4.0) Immobilization of protein A was carried out by immersing in buffer for 2 hours at room temperature.

1 g of the thus-obtained Protein A-immobilized polyester fiber was packed in a column (0.5 × 15 cm) and added to a 10 mM phosphate buffer solution (pH 7.2) containing 0.9% sodium chloride at a pH of 0.2. The solution was equilibrated with a solution containing 1% (w / v) sodium azide and used for the subsequent reaction.

Serum obtained from rabbit blood is equivalent to PB
The sample diluted with S buffer was used. This sample was passed through a protein A-immobilized polyester fiber column at a flow rate of 1 ml / 3 min, and the passing liquid was collected. Furthermore, 280 nm
While confirming the presence or absence of protein in the passage solution by measuring the absorbance at, the PBS buffer solution was passed through until the absorption of protein at this wavelength disappeared. Next, IgG adsorbed on the column was eluted with 100 mM citrate buffer (pH 4.0), and 1 M PBS buffer was immediately added to the eluate.

The thus-obtained fraction of the liquid passing through the column and the fraction adsorbed to the column were respectively subjected to SDS-polyacrylamide gel electrophoresis (Laemmli 1970) to identify the proteins contained therein. As a result, a single band corresponding to IgG was confirmed at a molecular weight of about 150,000 in the fraction adsorbed on the protein A-immobilized polyester fiber column, while other plasma proteins were confirmed in the flow-through fraction. No band was detected at the position corresponding to IgG.

Example 3 Polyurethane fiber (5 denier) was immersed in distilled water and subjected to hot water treatment at 80 ° C. for 1 hour. After immersing the polyurethane fiber which was washed with distilled water after hot water treatment and dried in an acetone solution in which the total monomer concentration was 3 mol / ml and the monomer composition of maleic anhydride and 2-hydroxyethyl methacrylate was 1: 3, ⁶⁰ COー Gamma ray dose rate 10kGy / h,
The graft polymerization reaction was carried out by simultaneously irradiating for 1 hour under a nitrogen atmosphere at room temperature and atmospheric pressure. After the irradiation, the fiber was taken out, washed well with acetone, and then dried. The protein A was immobilized by immersing the obtained fiber in a 10 mM acetic acid (pH 4.0) buffer containing protein A (manufactured by SIGMA) at room temperature for 2 hours.

1 g of the thus obtained protein A-immobilized polyurethane fiber was packed in a column (0.5 × 15 cm), and then added to a 10 mM phosphate buffer solution (pH 7.2) containing 0.9% sodium chloride at a pH of 0.2. The solution was equilibrated with a solution containing 1% (w / v) sodium azide and used for the subsequent reaction.

Serum obtained from rabbit blood is equivalent to PB
The sample diluted with S buffer was used. This sample was passed through a protein A-immobilized polyurethane fiber column at a flow rate of 1 ml / 3 min, and the passing liquid was collected. Furthermore, 280 nm
While confirming the presence or absence of protein in the passage solution by measuring the absorbance at, the PBS buffer solution was passed through until the absorption of protein at this wavelength disappeared. Next, IgG adsorbed on the column was eluted with 100 mM citrate buffer (pH 4.0), and 1 M PBS buffer was immediately added to the eluate.

The protein contained in each of the thus obtained fractions of the liquid passing through the column and the fractions adsorbed to the column was identified by using SDS-polyacrylamide gel electrophoresis (Laemmli 1970). As a result, a single band corresponding to IgG was confirmed at a molecular weight of around 150,000 in the fraction adsorbed on the protein A-immobilized polyurethane fiber column, while other plasma proteins were confirmed in the flow-through fraction. No band was detected at the position corresponding to IgG.

Comparative Example 1 Protein A-Sepharose CL-4B (Pharmacia)
After swelling 1.5 g of a solution of 0.1% (w / v) sodium azide in a 10 mM phosphate buffer (pH 8.0) containing 0.9% sodium chloride, the column (0.5 X15
cm) and equilibrated with the same solution.

Protein A-Sepharos thus obtained
The rabbit serum was passed through the e column in the same manner as in Example 1, and the fraction adsorbed on the column and the passed fraction were respectively subjected to SDS-
IgG by polyacrylamide gel electrophoresis
Was confirmed to be purified. As a result, in the fraction adsorbed on the column, a small amount of plasma protein band was observed in addition to the band at the position corresponding to IgG, while in the fraction of the flow-through, in addition to plasma protein, a small amount was found in IgG. Corresponding bands were also observed, indicating that the selective adsorption of IgG on the column was not good.

[0026]

EFFECTS OF THE INVENTION According to the adsorbent on which protein A is immobilized according to the present invention, the protein A is immobilized on the fiber without lowering the activity thereof, and therefore the protein A is specifically adsorbed with an antibody (immunoglobulin) specific to protein A. Keeps good
By selectively adsorbing the antibody, the antibody can be separated and purified very efficiently, which is economically advantageous.

Claims (1)

[Claims] 1. A protein A-immobilized adsorbent characterized by immobilizing protein A by reacting a compound having an acid anhydride group on the fiber surface.