JPH02177892A - Method for purifying human interferon beta - Google Patents

Abstract

PURPOSE:To purify the subject substance useful as an antiviral and antitumor agents by bringing a solution containing human interferon beta into contact with a carrier containing a specific ligand immobilized therein and then eluting the above-mentioned human interferon beta. CONSTITUTION:A solution containing human interferon beta (preferably genetic recombinant type) is brought into contact with a carrier containing a ligand, expressed by the formula and immobilized therein prefarably under neutral conditions. The above-mentioned human interferon beta is then eluted and purified with an acetic acid buffer solution (pH3.0-5.5), etc., containing 0.5-1M sodium chloride.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a method for purifying human interferon β.

[Prior art] Human interferon (hereinafter abbreviated as IFN) is a protein originally produced by human cells, and currently α
They are broadly classified into , β, and γ types, and some of them have already been used clinically as antiviral agents or antitumor agents because of their antiviral activity and cell proliferation inhibitory activity, respectively. In particular, with the practical application of so-called genetic recombination technology, it has become possible to produce IFN in large quantities using microorganisms and certain animal cells, and its clinical application has rapidly progressed. IFN used in actual clinical applications is used as a protein preparation with impurities removed to an insignificant level. In this case, how to efficiently remove impurities is the key to preparing safe and effective IFN preparations. For this reason, various ion exchange chromatography, hydrophobic chromatography, and affinity talomadography have been developed in order to efficiently purify IFN (for example, New Bioseparation, Vol. 2, 249-2).
(Page 61, published by CMC, 1988).

Among these, metal chelate chromatography, which utilizes the specific affinity between proteins and metals, was developed in 1970 by P.
It was proposed by Orath et al. (Nature, 258.598) and has also been used for purification of IFN. That is, it is used as a copper-gantt for the purification of IFN-α (Berg and Heron, J. Imm.
unol.

11, 489.1980), and zinc is said to be suitable for the purification of IFN-β (Pdy et al., J. Biol., C.
hc+n, 2525934, 1977), and nickel (Coppenhave
r, Methods in [Enzymol,,
119°199, 1986) and zinc (Geargiad
es, Mcjhods in Enzymol,
119.83.1986) is used as a ligand. In addition, as a carrier for immobilizing these metals,
Generally, agarose gel (product name: Chelating
5epharose 6B (Pharmacia/LKB), hydrophilic vinyl polymer gel (trade name CoAF-Chelate Toyopearl 650M, Tosoh), etc., both of which were described by Porath et al. (Nature, 258.598).
．． It is prepared as a carrier having a chelate group of the structural formula shown below, which is produced by bis-epoxide and iminodiacetic acid developed by (Bis-Epoxide method) (1975).
.

CH2C0OH Here, the metal chelate group is a biscarboxymethylimino group [N (CH2C00H)2, and the carrier and the chelate group are bonded by an alkyl chain called a spacer.

In addition to this bisepoxide method, the chelate group and spacer can be synthesized by a method using shrimp chlorohydrin (Hube
rt and Porath, J. Chromatogr.
198° 247, 1980), or tris-carboxymethyl ethylenediamine (Sulkovski,
Trends in Bi.

A method using the following methods is known: Technol., 3.1.1985). As mentioned above, it has been reported that metal chelate chromatography is useful for purifying IFH, but there have been few attempts to improve the spacer.

[Problems to be solved by the invention] As mentioned above, zinc chelate chromatography is useful for purifying IFN-β, but it cannot be purified by this method alone, and there is still room for further improvement. I had left it behind. In particular, in the case of clinical application of IFN-β produced by genetic recombination technology using non-human microorganisms or animal cells as hosts, it is necessary to completely remove contaminant proteins due to antigenicity to humans. .

In order to solve this problem, the present invention improves the chelate group of zinc chelate chromatography to produce highly purified IFN-
The purpose is to purify β.

[Means for Solving the Problems] The present invention provides a method for producing IFN-β, which is characterized in that a solution containing IFN-β is brought into contact with a carrier on which a ligand consisting of OH is immobilized, and then the IFN-β is eluted. It is a purification method.

IFN-β here refers to IFN produced by stimulating cultured human cells with viruses, synthetic nucleic acids, etc.
-β, and IFN-β produced by microorganisms such as Escherichia coli and yeast that have incorporated the IFN-β structural gene using genetic recombination technology, and animal cells such as mice, hamsters, and monkeys, but preferably the gene Recombinant IFN
- refers to β.

In the purification of recombinant IFN-β, it is especially important to remove contaminant proteins derived from host cells, so the present inventors have completed the present invention after intensive study of the spacer for the ligand in zinc chelate chromatography. did.

That is, the zinc chelate carrier used in the present invention is characterized by having a spacer that is shorter than the spacer obtained by the bisepoxide method and can be synthesized, for example, by the shrimp chlorohydrin method.

The zinc chelate carrier used in the present invention can be produced, for example, by the following method.

A spacer having the structure shown below, synthesized from shrimp chlorohydrin and iminodiacetic acid, and a 0H CH2COO- chelate group are introduced into an insoluble gel carrier such as an agarose gel or a hydrophilic vinyl polymer gel. This gel carrier is brought into contact with a solution containing 1% zinc chloride in 50 mM acetate buffer (pH 5,0) to chelate 20 to 30 μM zinc per 1 ml gel.

The chromatography operation in the present invention is carried out as follows.

A solution containing IFN-β from which free amino acids, ammonium acid, metal chelating agents, etc. have been removed is brought into contact with the zinc chelate carrier prepared by the above method, preferably under neutral conditions.

After contacting the zinc chelate support with the IFN-β solution, the support is washed with a weakly acidic buffer of high ionic strength, such as an acetate buffer (pH 5,6-8.0) containing 0°5-IM sodium chloride. By doing so, almost all contaminant proteins other than IFN-β can be removed. IFN
For elution of -β, an acetate buffer containing 0.5-IM sodium chloride (pH 3, 0-5.5) or a neutral buffer such as 0.1H ethylenediaminetetraacetic acid, iminodiacetic acid, or histidine is used. It will be done. The concentration and volume of the buffer used here are not particularly limited, and the concentration and amount necessary to maintain the pH and the amount of adsorbed I
The amount necessary to substantially recover FN-β is used.

Furthermore, the method of the present invention may be used in combination with other methods such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, etc. In particular, it is preferable to use a method in combination with chromatography using a carrier bound with a blue dye, a silica bead column, or the like.

[Examples] Hereinafter, the present invention will be explained in more detail with reference to Examples.

IFN-β activity in the examples was measured by a cytopathic effect (CPE) inhibition method using human amnion-derived FL cells and vesicular stomatitis virus (VSV).
It is converted into international units (IU) by

Example 1 A carrier having a metal chelate group was synthesized by the epichlorohydrin method as follows.

That is, after washing 500 ml of commercially available 5epharose 4B (Pharmacia/LKB) with 1 g of distilled water, 75 ml of 4M sodium hydroxide and 20 ml of
After adding shrimp chlorohydrin, the mixture was stirred at 30°C for 6 hours. After the reaction, pass the gel through a glass filter.
After washing with 2M sodium carbonate solution of Ω, iminodiacetic acid solution (0.2 g/m
The gel was added to 350ml.

After reacting at 65°C for 24 hours, filter the glass filter.
In the second step, the sample was washed with 2 g of distilled water.

A 1% zinc chloride solution dissolved in 50mM acetate buffer (pH 4,5) was added to 5ml of this gel into which chelate groups were introduced.
0 ml was poured to chelate zinc to form a zinc chelate carrier.

Example 2 After sufficiently culturing and propagating Escherichia coli into which the IFN-β structural gene had been incorporated, the bacterial cells were crushed by high pressure.

Furthermore, after adding 0.5% polyethyleneimine to remove nucleic acids, 70% saturated ammonium sulfate was added to salt out proteins, and 20mM phosphate buffer containing 1M sodium chloride (p
It was redissolved with H7,4) to obtain a purified stock solution. The protein concentration of the purified stock solution is 30 mg/ml, and the IFN-β activity is 6X.
1061U/m! (Specific activity 2 x 1051U/mg
)Met.

130ml of this purified stock solution was added to 80ml (2,6,x 1
5cm) Pour onto a silica bead column and add 20mM phosphate buffer (pH 7.4) containing 1M sodium chloride 80
After washing with 0ml, 50% ethylene glycol and 1M
20mM phosphate buffer (pH 7,
4) IFN-β was eluted with 160 ml. This IFN-
The recovery rate of IFN-β in β solution was 90%, and the specific activity was 2.8.
It was X1061υ/mg.

The eluate from this silica bead column was transferred to a 5 ml (1°6×2.
5 cm), followed by 50% ethylene glycol, 150 ml of 20 mM phosphate buffer (pH 7,4) containing 1 M sodium chloride, and 150 ml of 0.1 M acetate buffer (pH 5,6) containing 1 M sodium chloride.
The column was washed with Furthermore, 0.1M acetate buffer containing 0.5M sodium chloride (pH 5.0 and
4,0) IFN-β was eluted with 240 ml and used as a purified fraction.

The IFN-β recovery rate of this purified fraction was 24%, and the specific activity was 1. It was 6 x 10811 J/mg. Furthermore, the amount of contaminating E. coli-derived protein contained in this purified fraction was determined to be 0.54% by enzyme immunoassay.

Comparative Example 1 160 ml of the silica bead column purified fraction of IFNβ obtained by the same method as shown in Example 2 was mixed with zinc chelate gel (trade name: "Chelating 5epharose 6B") into which a spacer was introduced by the bisepoxide method.
5 ml (1.6 x 2.5 cm column) of zinc chelate (Pharmana/LKB). Chromatography was then performed under the same conditions as in Example 2 to purify IFN-β with zinc chelate column. A fraction was obtained. The recovery rate of IFN-β in this purified fraction was 43%.
Specific activity is 1. It was 1×10B1U/mg. Furthermore, the amount of contaminating E. coli-derived protein contained in this purified fraction was determined to be 1.36% by enzyme immunoassay.

[Effects of the invention] As already mentioned, IFN-β, which is currently used clinically in humans as an antiviral agent and an antitumor agent, requires the removal of contaminant proteins to a level that does not cause problems during its production. is required. To this end, it is desired that various chromatography techniques, which have been considered useful for the purification of IFN-β, be improved to have higher performance. In particular, large amounts of
Now that it has become possible to produce FN-β, the need for it has become even greater.

IFN-β purified by the method of the present invention has a higher concentration than IFN-β purified by the conventional bis-epoxy method and a spacer synthesized with iminodiacetic acid and a zinc chelate column in which zinc is chelated to the chelate group. It is highly pure, and the amount of contaminant proteins that may be mixed into the purified sample is further reduced.

That is, instead of the conventionally used zinc chelate gel with a spacer synthesized by the bis-epoxide method, the zinc chelate gel with the spacer and Gant shown in the present invention was used to perform zinc chelate chromatography of IFN-β. By carrying out this method, it is possible to obtain a higher quality IFN-β purified sample containing less contaminant proteins than before.

Claims (2)

[Claims] (1) A solution containing human interferon β is brought into contact with a carrier on which a ligand represented by the following formula (numerical formula, chemical formula, table, etc.) is immobilized, and then the human interferon β is eluted. Purification method for human interferon β. (2) The purification method according to claim (1), wherein the human interferon β is a genetically recombinant type.