JP2000103800A - Purification of immunoglobulin a - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a purification process that can efficiently purify serum Ig A in high yield in an industrial scale. SOLUTION: The purification process for serum Ig A comprises the treatment of a solution containing Ig A (serum immunoglobulin A) with metal chelate chromatography, the extraction with water, polyethylene glycol fractionation, treatment with an anionic ion-exchange resin and the treatment with hydrophobic chromatography. According to the present invention. as Ig A can be efficiently separated from other plasma proteins by subjecting them to the metal chelate chromatography, the serum Ig A can be efficiently and economically collected in an pinduntrical scale.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for purifying serum immunoglobulin A (IgA), and more particularly to a method for purifying serum IgA on an industrial scale with a high yield.

[0002]

2. Description of the Related Art IgA is classified into serum IgA and secretory IgA. Secretory IgA is a major fraction of the immunoglobulin composition in the exocrine fluid and is responsible for so-called local immunity on mucosal surfaces. On the other hand, serum IgA is 1 of serum Ig.
0-20% (concentration in normal human serum is 60-490 mg /
dl) and is the second most abundant after IgG.
Serum IgA is mainly a monomer molecule having a molecular weight of about 170,000, but about 15% exists as a polymer of (IgA) _2-4 (most of which are dimers, trimers and tetramers). Is present in only trace amounts).

[0003] Serum IgA has various biological defense effects, such as having an ability to promote bacterial aggregation and phagocytosis of neutrophils, and is used for the prevention and treatment of necrotizing colitis and the like. In addition, bacterial infection diarrhea, antibiotic resistant diarrhea, rotavirus infection,
Clinical applications to respiratory tract infection, allergic diseases and infections in immunodeficiency state, IgA deficiency, autoimmune diseases, and the like are also expected.

[0004] Serum IgA is separated and purified from serum or plasma, or a fraction thereof, as a raw material, and is formulated. As a method for purifying serum IgA, for example,
Methods using ion exchanger adsorption and immunoadsorbent adsorption (Litman et al., Biochim. Biophys. Acta, 263, 89, 1972).
), A method using the caprylic acid precipitation method and the DEAE cellulose adsorption method (Pejaudier et al., Vox Sang, 23, 165, 1972).
), A method using ammonium sulfate salting out method and rivanol precipitation method (Sch
wick et al., Vox Sang, 23, 1972), a method using a zinc acetate precipitation method and a polyethylene glycol fractionation method (US Pat. No. 3,808,189), and the like. Among them, the method of Litman et al. Is not suitable for purification on an industrial scale, and all other methods have problems in purity and yield. Thereafter, as a method for purifying serum IgA with high purity on an industrial scale, a sodium sulfate fractionation method (Japanese Patent Publication No.
8-43372), a method combining virus inactivation treatment and polyethylene glycol fractionation (Japanese Unexamined Patent Publication No.
No. 145031), and a method combining a virus inactivation treatment with a thiophilic matrix adsorption treatment or gel filtration (JP-A-8-59699) and the like have been newly reported.

[0005]

SUMMARY OF THE INVENTION The present invention relates to serum IgA
To provide a novel purification method that can purify the compound on an industrial scale with higher yield.

[0006]

Means for Solving the Problems The present inventors have made intensive studies to achieve the above object, and as a result, by subjecting an IgA-containing solution to metal chelate chromatography, fibrinogen, albumin, IgG, and complement were obtained. It has been found that various contaminating proteins such as components can be efficiently removed, and by combining a conventionally known purification step with metal chelate chromatography, high-purity serum Ig substantially free of contaminating proteins and degradation products of IgA.
A was successfully recovered in good yield, and the present invention was completed.

That is, the present invention comprises a method for purifying serum IgA including a step of subjecting a serum IgA-containing solution to metal chelate chromatography, and a combination of polyethylene glycol fractionation, anion exchanger treatment and hydrophobic chromatography. Any one of the above methods, wherein the metal chelate chromatography treatment is performed under conditions of pH 3 to 5.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION In the method of the present invention, the serum IgA-containing solution as a starting material is not particularly limited. For example, serum or plasma derived from humans, or fractions thereof are mentioned. In consideration of cost reduction, fibrinogen, serum albumin, I
It is particularly preferable to use a conventionally discarded fraction as a raw material after fractionating plasma proteins such as other immunoglobulin classes such as gG. Such fractions include corn II + III, III, and IV fractions obtained by the cold alcohol fractionation method of corn (J. Am. Chem.
Soc., 68, 459, 1946). These fractions are advantageous in that they have a high IgA content. In addition, a residue fraction obtained by fractionating other immunoglobulin classes such as IgG and other plasma proteins from these fractions can also be preferably used.

The method of the present invention is characterized in that serum IgA is purified on an industrial scale with good yield by appropriately combining a metal chelate chromatography treatment with a conventionally known serum IgA purification step. As the conventionally known method selected here, for example, salting out treatment, extraction treatment with an aqueous solvent or the like, fractionation treatment with polyethylene glycol or the like, anion exchanger treatment, cation exchanger treatment, hydrophobic chromatography treatment, Ultrafiltration membrane treatment, gel filtration treatment and the like can be mentioned.

In one embodiment of the present invention, an extraction treatment with an aqueous solvent, a polyethylene glycol fractionation treatment, an anion exchanger treatment and a hydrophobic chromatography treatment are used in combination with a metal chelate chromatography treatment.
These treatments may be performed in any order, but preferably a metal chelate chromatography treatment is incorporated at the end of the purification step. A preferred example is a method in which extraction treatment with an aqueous solvent, polyethylene glycol fractionation, anion exchanger treatment, hydrophobic chromatography treatment, and metal chelate chromatography treatment are performed in this order. Hereinafter, embodiments of each processing step will be described in more detail.

(1) Extraction treatment with aqueous solvent In this step, the fraction containing serum IgA is suspended in an appropriate aqueous solvent to extract and collect IgA. Processing conditions include pH
5 to 9, and a salt concentration of about 0.001 to 0.5M. The extraction ratio is, for example, at least 3 parts by volume, preferably about 5 to 20 parts by volume per 1 part by weight of the IgA-containing fraction. Further, the extraction treatment is preferably performed at about 0 to 5 ° C. Purified water or the like is preferably used as the aqueous solvent.

(2) Polyethylene glycol fractionation In this step, the supernatant is clarified by contacting and mixing the IgA-containing fraction with polyethylene glycol. The molecular weight of the polyethylene glycol used is 1000 to 10
000, preferably about 2000 to 6000. The processing conditions include a polyethylene glycol concentration of 0.5 to
5w / v%, pH 5-9, salt concentration 0.01-
About 0.5M is exemplified. The protein concentration of the IgA-containing fraction used in this step is, for example, 1 to 20 w / v%. As the solvent, a buffer such as a phosphate buffer or a carbonate buffer can be used. Further, the pH can be adjusted using hydrochloric acid, sodium hydroxide, or the like. The fractionation treatment is performed by stirring the solution at about 0 to 5 ° C. for about 30 minutes to 24 hours. Then, for example, centrifugation (5
000 to 10000 rpm for about 10 to 30 minutes) to collect the supernatant.

(3) Anion exchanger treatment In this step, the IgA-containing fraction is brought into contact with the anion exchanger to collect IgA in the adsorbed fraction. Examples of the anion exchanger include a diethylaminoethyl (DEAE) group type and a quaternary aminoethyl (QAE) group type. DEAE
As a base, DEAE-agarose (trade name: DEA
E-Sepharose, manufactured by Pharmacia), DEAE-polyvinyl (trade name: DEAE-Toyopearl, manufactured by Tosoh Corporation) and the like. The QAE base type includes QA
Examples include E-agarose (trade name: Q-Sepharose, manufactured by Pharmacia), QAE-polyvinyl (trade name: QAE-Toyopearl, manufactured by Tosoh Corporation) and the like. The contact and washing conditions are about pH 6 to 9, preferably 6.5 to 6.5.
For example, a salt concentration of about 8, a salt concentration of about 0.001 to 0.05M, and preferably about 0.01 to 0.025M. The elution conditions are a pH of about 6 to 9, preferably about 6.5 to 8, and a salt concentration of about 0.05 to 1M.

(4) Hydrophobic Chromatographic Treatment In this step, the IgA-containing fraction is brought into contact with the hydrophobic chromatographic carrier to collect IgA into an adsorbed fraction or a non-adsorbed fraction. As a carrier for hydrophobic chromatography, a carrier having 4 to 1 carbon atoms is used.
And an alkyl group type (butyl group type, octyl group type, octyl decyl group type, etc.) and a phenyl group type. As the butyl group type, butyl-agarose, butyl-polyvinyl (trade name: butyl-Toyopearl, manufactured by Tosoh Corporation), as the octyl group type, octyl-agarose, as the octyldecyl group type, as octyldecyl-agarose, phenyl group type Is phenyl-cellulose (trade name: Phenylcellulofine, manufactured by Seikagaku Corporation) and the like.
When collecting IgA in the non-adsorbed fraction, the contact condition is pH6.
~ 9, preferably about 7 ~ 8, salt concentration 0.1 ~ 1M
Degree, preferably about 0.25 to 0.75M. When IgA is recovered in the adsorbed fraction, the contact conditions include a pH of about 6 to 9, preferably about 7 to 8, and a salt concentration of 1 to 8.
About 3M, preferably about 2-3M. The elution conditions are about pH 6 to 9, preferably about 7 to 8, and a salt concentration of about 0.1 to 1M, preferably 0.25 to 0.75.
It is about M.

(5) Metal Chelate Chromatography Treatment In this step, by bringing the IgA-containing fraction into contact with the metal chelate adsorbent, contaminating proteins in the fraction are efficiently separated and removed. Examples of the type of metal ion used include transition metals, for example, divalent ions of copper, nickel, zinc, and iron, and trivalent ions of iron and aluminum. Preferred are divalent ions of copper and nickel.
The metal chelate adsorbent is a metal ion-containing solution such as C
It is prepared by contacting a solution containing a u ²⁺ salt (for example, a CuSO ₄ solution) with a matrix to which a ligand is attached, and binding the ligand. The matrix portion of the chelate adsorbent is not particularly limited as long as it is a usual insoluble carrier. For example, a hydrophilic vinyl polymer (trade name: Toyopearl),
Crosslinked dextran (trade name: Sephadex), agarose (trade name: Sepharose), cellulose (trade name:
Cellulofine) and the like. On the other hand, examples of the ligand portion include an iminodiacetic acid group, a nitrilotriacetic acid group, and a tris (carboxymethyl) ethylenediamine group. The chelate adsorbent can be used as long as it is commercially available for metal chelate chromatography, for example, Chelating Sepharose 6B,
ChelatingSepharose FF (Pharmacia)
AF-Chelate Toyopearl 650 (manufactured by Tosoh Corporation), Chelate Cellulofine (manufactured by Seikagaku Corporation) and the like are preferably used.

The mixing ratio of the IgA and the metal chelate adsorbent is 0.001 to 10 m
l, preferably 0.01 to 1 ml.

In this step, IgA can be recovered in an adsorbed fraction or a non-adsorbed fraction. When eluting after adsorbing IgA, first, a salt concentration of about 0.01 to 1 M,
The metal chelate adsorbent is equilibrated using a buffer having a pH of about 5 to 8, specifically 10 to 100 mM acetic acid, phosphate or Tris-HCl buffer (pH 5 to 8) containing 1 M or less of sodium chloride.・ After washing, equilibrated pH
The adjusted IgA-containing fraction was brought into contact with the metal chelate adsorbent, and the adsorbed fraction was then subjected to a salt concentration of 0.01 to 1 M, pH
Elution is carried out using about 3 to 5 buffers described above.

On the other hand, when IgA is recovered in the non-adsorbed fraction, first, a buffer having a salt concentration of about 0.01 to 1 M and a pH of about 3 to 5, specifically 10 to 100 mM containing 1 M or less of sodium chloride is used. After equilibrating and washing the metal chelate adsorbent using acetic acid, phosphoric acid, or Tris-hydrochloric acid buffer (pH 3 to 5), the Ig adjusted to the equilibrated pH is used.
A fraction containing A is brought into contact with the metal chelate adsorbent to collect a non-adsorbed fraction.

In each case, the IgA after the treatment
In order to prevent metal ions from contaminating the containing fraction, a column comprising a metal-unbound chelate adsorbent equilibrated with a buffer having a salt concentration of about 0.01 to 1 M and a pH of about 3 to 5 is subjected to metal-bound chelate adsorption. It is preferable to perform this step using a system connected below a column made of a body (separation column).

The IgA obtained by the above purification step is
If necessary, further purification can be performed by a known method such as ultrafiltration membrane treatment (the molecular weight cut off is about 10,000 or less, or about 100,000 or less) or gel filtration HPLC.

The IgA obtained by the above purification step is
It consists essentially of monomers and dimers and is substantially free of degradation products. Here, “substantially free” means that the corresponding band has been purified to such an extent that it cannot be detected by gel electrophoresis under reducing and non-reducing conditions.
Also, contaminating plasma proteins, for example, IgG, serum albumin, prealbumin, ceruloplasmin, haptoglobin, fibrinogen, fibronectin, α1-antitrypsin, inter-α-trypsin inhibitor, α2-macroglobulin, C1-inhibitor, C1
3C, C4C, etc. are also sufficiently removed. Here, "sufficiently removed" means that the protein is purified to such an extent that the corresponding sedimentary gland is not detected in the octalony analysis.

Serum Ig obtained by the purification method of the present invention
A is a virus inactivation treatment, addition of a stabilizer, sterilization filtration,
Formulations can be made by known techniques such as subdivision / dispensing and freeze-drying. The IgA preparation thus prepared is
It can be used clinically as injections, oral preparations, sprays, nasal drops and the like.

[0023]

The present invention will be described in more detail with reference to the following examples, which are merely illustrative and do not limit the scope of the present invention in any way.

Example 1 54 g of an extraction residue obtained by extracting and separating IgG from a corn II + III fraction using a 10-fold amount of purified water was suspended in a 10-fold (540 ml) amount of purified water at 4 ° C. at pH 7.0. After stirring at 5 overnight, the supernatant was collected by centrifugation. Sodium phosphate (25 mM) and polyethylene glycol (average molecular weight: 4,000) were added to a concentration of 1 w / v%, and the mixture was allowed to stand under ice-cooling overnight, and then centrifuged to obtain a supernatant. 25
DEAE equilibrated with mM phosphate buffer (pH 7.5)
-Hydrophilic polyvinyl (trade name: DEAE-Toyopearl 6)
The supernatant was applied to a column (50M, manufactured by Tosoh Corporation) and washed with the same buffer solution.
Elution was performed using a phosphate buffer (pH 7.5), and the adsorbed fraction was collected. Then, the fraction (180 ml) was
Butyl-hydrophilic polyvinyl equilibrated with 25 mM phosphate buffer (pH 7.5) containing 0.75 M sodium chloride (trade name: butyl-Toyopearl 650M, manufactured by Tosoh Corporation)
The mixture was applied to a column, and a non-adsorbed fraction was collected. The fraction (90 ml) was subjected to 25 mM sodium chloride containing 25 mM
Copper chelate-hydrophilic polyvinyl (trade name: copper chelate-Toyopearl 650) equilibrated with acetate buffer (pH 4)
M, manufactured by Tosoh Corporation) column, and a non-adsorbed fraction was collected. The fraction was further concentrated using a 100 kDa ultrafiltration membrane (Macrocep, manufactured by Filtron). 4
The average recovery of IgA (value before and after copper chelate chromatography) in the experiments was about 54%. Table 1 shows the results of examining contaminating proteins in the non-adsorbed fraction obtained by copper chelate chromatography. It was found that all the plasma proteins examined were sufficiently removed.

[0025]

[Table 1]

The purity of the final purified product and the properties of IgA were as follows. 1) IgA purity: 90% or more 2) SDS-PAGE (reducing conditions): IgA H chain (α
Chain) and a band corresponding to the L chain. 3) SDS-PAGE (non-reducing conditions): a band corresponding to a monomer or dimer of IgA was detected. 4) Gel filtration HPLC: peaks corresponding to monomers, dimers and higher polymers, and minor peaks of lower molecules were detected.

[0027]

According to the present invention, serum IgA can be efficiently and economically purified on an industrial scale. In particular, I
It is extremely useful for separating and purifying IgA and IgG from starting materials that contaminate gG.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 37/00 A61K 31/00 637 A61K 39/395 39/395 X C07K 1/18 C07K 1/18 1 / 20 1/20 1/22 1/22 (72) Inventor Katsuhiro Uryu 2-25-1, Shodai Otani, Hirakata City, Osaka Yoshitomi Pharmaceutical Co., Ltd.Osaka Research Laboratory (72) Inventor Yahiro Uemura Chuo-ku, Osaka-shi, Osaka 2-6-9 Hiranocho Yoshitomi Pharmaceutical Co., Ltd. F-term (reference) 4C085 AA33 BB33 CC22 DD32 DD33 DD34 DD36 DD40 EE01 4H045 AA20 DA75 GA21 GA23 GA25

Claims (3)

[Claims] 1. A method for purifying serum immunoglobulin A, comprising a step of subjecting a solution containing serum immunoglobulin A to metal chelate chromatography. 2. The method according to claim 1, further comprising a combination of polyethylene glycol fractionation treatment, anion exchanger treatment and hydrophobic chromatography treatment. 3. The method according to claim 1, wherein the metal chelate chromatography is carried out under a condition of pH 3-5.