KR950004014B1 - METHOD FOR PURIFICATION OF Ñß-INTERFERON - Google Patents

Abstract

The activation process of recombinant alpha interferon (I) consists of converting purified (I) to reducing form in a solution (II) containing 1-10 mm reduced glutathione and 0.1-1 mM oxidized glutathione. The ratio of the reduced thiol in (II) is needed to 5-10 fold.

Description

Improved Purification Method of Recombinant Alpha Interferon

1 shows the separation of alpha interferon using CM-Sepharose.

2 shows a non-reducing SDS-PAGE of the result of converting an interferon dimer into a monomer using an oxidation-reducing agent.

3 shows the results of C18 reverse phase high pressure chromatography of recombinant alpha interferon.

The present invention relates to a method for increasing the purification yield during the purification of recombinant alpha interferon, and more particularly, to convert the recombinant alpha interferon dimer by disulfide bonds generated during the purification process into monomers using an oxidation-reduction action. The present invention relates to a method for increasing the purification yield of a protein.

Interferon first became known in 1957 when Isaac and Lindenmann developed a factor that inhibited the reproduction of viruses when chickens were infected with the influenza A virus (Isaacs and Lindenmenn, J., Proc, R. Soc. Lond., B 147 , 258-267,1957).

Subsequently, numerous studies have shown that interferon acts as a regulator of not only the antiviral properties but also the expression, structure, and function of cell-to-cell genes, and interferon also has a direct anti-proliferative effect. This suggests a possibility as an anticancer agent.

Alpha-interferon is produced when white blood cells (B-cells, T-cells, macrophages) are stimulated by B-cell mitogens, foreign macromolecules, viruses or cancer cells. It is known to exist and consists mostly of 165 or 166 amino acids.

The alpha inferferon used in the first clinical trials was obtained from buffery leukocytes stimulated with Sendai Virus, at which time the protein purity was only about 1% (Cantell, K. and Hirvonen, Tex. Re. Biol. Med ., 35 , 138-144,1977).

Recently, clinical trials using genetically modified human alpha interferon, which has bioactivity, have been shown to be effective in the treatment of various solid cancers. In particular, bladder cancer (Torti, FM et al ., J. Clin . Onco ., 3 , 506-512, 1985), kidney cancer (Vugrin, D. et al . , Cancer treat.Rep., 69 , 817-820, . 1985), and said to be effective for, in recent years, such as such as (Davis, GG is known that the effect in the treatment of hepatitis C virus, N. En l, J. Med, 321, 1501-1506,1989; Bisceglie, AD et al., N. En gl. J. Med ., 321 , 1506-1510,1989).

As a method for obtaining a native alpha interferon, reverse-phase high performance liquid chromatography (Rubinstein, M. et al. , Arch Biochem. Biophys ., 210, 307-318, 1981) or single Techniques for obtaining relatively high purity proteins are known using antibody substitution columns (Berg, K. and Heron, I., Methods Enzymol ., 78 , 487-499,1981).

As a method for obtaining a recombinant human alpha interferon with activity, a method of selectively separating human alpha interferon from other contaminating proteins using a single antibody affinity column and then further purifying several times using several columns has been devised. (Tarnowski, SJ et al., Methods Enzymol ., 119 , 153-165, 1986; Pestka, S. and Tarnowski, SJ, Pharmac. Ther ., 29 , 299-319, 1985). However, this method has the disadvantage that the effect is good but the mass production of a single antibody selective against human alpha interferon. In addition, the purification of recombinant human alpha interferon has been performed by Cu-chelate columns (Thatcher, DR and Panayotatos, N., Methods Enzymol ., 119 , 166-177, 1986).

Alpha interferon (αA) has a disulfide group connected between the 1 and 98 cysteine groups, and another disulfide group is connected between the 29 and 138 cysteine groups. However, when recombinant alpha interferon is expressed in yeast or Escherichia coli, the two disulfide bonds in the interferon are not formed properly but remain in a reduced state, and the protein itself is maintained in the cell in a denatured form.

Therefore, the present inventors dissolve the recombinant alpha interferon using a protein denaturant containing a reducing agent such as β-mercaptoethanol or dithiothritol in the "recombinant human alpha inderferon purification method" Korean Patent Application No. 90-13450, The method has been filed by refolding the protein and subsequently purifying it using liquid column chromatography. The recombinant alpha interferon obtained by this method was a suitable material for pharmaceutical preparations having high purity and high titer.

Using cation exchange chromatography, only the alpha interferon retaining two disulfide bonds of the natural form and the disulfide bonds of Nos. 29 and 138 are connected, and the cysteine groups of Nos. 1 and 98 are reduced partially oxidized interferons. And, interferon dimers (dimers) connected to each other by disulfide groups can be efficiently separated from each other, and in particular, dimers can separate alpha interferons and alpha interferons of monomers using gel filtration chromatography. However, alpha interferon, which is preferred as a pharmaceutical formulation, must maintain the shape of a natural form with a disilide group maintained between 1-98 and 29-138, so that partial interoxidation and dimer alpha interferon are removed during purification. do. Therefore, the purification yield of recombinant alpha interferon tends to be inevitably lowered.

Accordingly, the present inventors have diligently studied to solve the above problems, and have developed a method of increasing the purification yield by converting an interferon dimer obtained after cation exchange chromatography into an interferon monomer using an appropriate redox agent. It was.

It is generally known to promote the disulfide formation of reduced proteins using oxidation reactions consisting of low molecular weight disulfide and thiol groups. It has been reported that the use of this academy-reducing agent increases the rate and yield of reconstitution l reoxidation by reshuffling proteins that are not bound correctly (Scheele, G. and Jacoby, R., J.). . Biol, Chem., 257, 12277-12282, 1982).

Based on the above theoretical basis, the present inventors have found that when an appropriate amount of oxidized thiol and reduced thiol groups are used for the alpha interferon dimer linked by disulfide bond, the unsafe alpha interferon dimer is separated and stable natural form It was shown that the monomer is composed of protein. Therefore, according to the present invention, it is possible to considerably increase the purification yield in purifying recombinant alpha interferon which is generally used.

Hereinafter, the present invention will be described in detail as follows.

The present invention provides a method for purifying recombinant alpha interferon, which comprises: (a) dissolving the recombinant alpha interferon using a protein denaturant, and then refolidng the protein; (B) performing anion exchange chromatography; (C) performing cation exchange chromatography; (D) The alpha interferon obtained by cation exchange chromatography is characterized in that the dimer is converted into an alpha interferon monomer using an oxidation-reducing agent to increase the purification yield of the total alpha interferon. The alpha interferon obtained from the dimer did not find any difference when comparing the physical and biological properties of the initial interferon monomer obtained from cation exchange chromatography.

The yeast cells used in the present invention are Luck-α-IFN-1Y containing the gene of alpha interferon cloned in the yeast expression vector and dated April 17, 1991 to KFCC, Accession No. KFCC-10715. Was deposited.

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

(Example 1)

Alpha interferon-expressed yeast ( Saccharomyces cerevisiae ) lkg was suspended in a buffer solution (20mM Tris-HCl, 1M urea, 1mM EDTA, 1mM PMSF, pH 8.0) with a homogenizer (Beckman) and 0.5-0.75mm of glass It was pulverized at a rate of 750 ml l in a dynomill with beads. The ground yeast solution obtained here was centrifuged at 12,000 rpm (4 ° C., Beckman JA-14 rotor) for 50 minutes to discard the supernatant and recover the precipitate. The collected precipitate was added again with 2 L of buffer solution (20 mM Tris-HCl, lM urea, 1 mM EDTA, 0.1% Triton X-100, pH 8.0) and suspended with a homogenizer (Beckman), followed by 12,000 rpm (Beckman JA- Sediment was recovered by centrifugation for 50 minutes). The recovered precipitate was further suspended with a homogenizer in 2 L of 1M guanidine solution (Guanidine HCl, Amresco) and centrifuged at 12,000 rpm for 30 minutes to recover the precipitate. At this time, the mass of the recovered precipitate was about 600g.

(Example 2)

1.8 g of 6M guanidine solution (6M guanidine solution HCl, 20 mM Tris-HCl, 100 mM β-mercaptoethane, pH 8.0) was added to 600 g of the recovered precipitate, and the mixture was dissolved at 4 ° C. overnight. The next day, the dissolved solution was centrifuged at 12,000 rpm (4 ° C, Beckman JA-14 rotor) for 30 minutes to settle and remove the undissolved precipitate and collect only the supernatant. At this time, the collected supernatant was dark brown and became about 2.1 L. Here, 6 L of complete solution (20mM Tris-HCl, 0.lmM PMSF, pH 8.0) was added well, and diluted 4 times, followed by centrifugation to obtain a supernatant. The supernatant was concentrated to 2 L using an ultrafiltration membrane and dialyzed three times in 50 L buffer (20 mM Tris-HCl, 0.12 M NaC1, 0.1 mM PMSF, pH 8.0) at 4 ° C.

(Example 3)

The dialysis solution was centrifuged at 12,000 rpm (4 ° C, Beckman JA-14 rotor) for 30 minutes to allow the precipitate to settle down, and then remove only the supernatant and collect the same supernatant (20 mM Tris-HCl, 0.12). m NaCl, pH 8.0) was passed through a DEAE-cellulose (Whatman) column at 10 cm / hour elution rate to collect the protein solution flowing out of the gel without sticking to 50 L of buffer solution (50 mM Na-acetate, pH 4.5). 3 times).

The dialysis solution was centrifuged at 12,000 rpm (4 ° C, Beckman's JA-14 rotor) for 30 minutes, and the resulting precipitate was allowed to settle down to remove the supernatant.

(Example 4)

The protein solution dialyzed at 50 mM sodium acetate, pH 4.5 was administered to the CM-Sepharose column equilibrated with the same buffer at a rate of 15 cm / hour and then washed with the same buffer until the peak of the protein was released without sticking to the gel. gave. Alpha interferon was eluted by continued flow of 50 mM sodium acetate, 0.12 M sodium chloride, pH 4.5 buffer at the same rate. When the peaks ran out, 50 mM sodium acetate and 0.5 M sodium chloride pH 4.5 were passed through the buffer to elute the protein again.

The chromatogram of the alpha interferon separation by CM-Sepharose column measured by absorbance at 280 mm is shown in FIG. In FIG. 1, the black bar portion (a) is the fraction containing partially oxidized alpha interferon, the black bar (b) is the fraction mainly containing alpha interferon homogeneous with the natural form, and the black bar (c) is the dimer. The main component is fraction. In FIG. 1, the partially oxidized alpha interferon is eluted from the column first, the main protein, which is the same alpha interferon as the native form, is subsequently eluted, and finally the alpha interferon dimer is subjected to the column under conditions of 0.5 M sodium chloride. Eluted from (c). In order to remove a small amount of partially oxidized alpha interferon contained in the same type of alpha interferon structure, re-separation was performed using a CM-Sepharose column to obtain a high purity alpha interferon.

(Example 5)

15% non-reduced S. D. Interferon dimers identified in S-polyacrylamide gel electrophoresis (non-reducing SDS-PAGE) (C in FIG. 1) were collected and dialyzed in 20 mM Tris-HCl (pH 8.0) buffer.

After the dialysis solution, cysteine (Sigma) and cysteine (cystine, Sigma) were added so that the concentrations were 5 mM and 0.5 mM, respectively, and the mixture was left to stand at 4 ° C or room temperature for at least 5 hours. S.D. After analysis by S-polyacrylamide ge1-electrophoresis (SDS-polyacrylamide ge1-electrophoresis) to confirm that the dimer was converted to the monomer, the sample was dialyzed in 50mM sodium acetate pH 4.5 buffer solution. Figure 2 shows the results of the conversion of alpha interferon dimer (A) to alpha interferon monomer (B) using the above method on a 15% non-reducing SDS-PAGE. The same results were obtained using oxidized glutathione and reduced glutathione.

(Example 6)

The dialysis sample was again isolated from the monomer alpha interferon by the method of Example 4. In this case, the biological titers of the alpha interferon separated by Example 4 and the alpha interferon monomer obtained from the dimer were compared.

(Example 7)

When the alpha interferon partial oxidation type (A in FIG. 1) and the dimer (C in FIG. 1) collected in Example 4 were collected and tested by the method of Example 5, an increased result in terms of purification yield was obtained. .

(Example 8)

The concentrated protein solution was passed through S-300 (Pharmacia, Material: Cross-linked Agarose) at 3 cm / hour in a pre-equilibrated buffer (20 mM NH ₄ -acetate, 0.12 M NaC1, pH 4.5). Gel filtration chromatography. The fractions obtained here were non-reducing SDS-PAGE to collect fractions containing only monomeric alpha interferons containing almost no oligomeric alpha interferon and dialyzed three times in 20 L of PBS (phosphate buffered saline) buffer solution. I was.

Determination of Biological Activity of Purified Alpha-Interferon

Antiviral titers using the cytopathic effect were determined to measure the physiological activity of the recombinant alpha interferon finally purified by the above method. Male calf kidney cell suspension (MDBK, 3.5 × 10 ⁵ cells / ml) incubated in a T 75 flask was placed in a 96-well plate in 100 μl and incubated for 18 to 24 hours in a 37 ° C., 5% CO ₂ incubator. .

After washing the whole hole with phosphate buffer (pH 7.0), inoculate 100ml of 1 × 10 ^{5 ~} 4 × 10 ⁵ pfu / ml of vesicular stomatitis virus (VSV) at 37 ℃, 5% CO ₂ incubator. After culturing for 24 hours to form a cell monolayer, the test solution alpha interferon was diluted 2 times and left for 48 hours in 37 ℃, 5% CO ₂ incubator, stained with a crystal biot and the absorbance was measured at 600nm.

Protein concentration of recombinant human alpha interferon was measured using the Lowly method after TCA precipitation. The alpha interferon standard (Gxa-01-901-535) was obtained from the National Institutes of Health (NIH) and its specific activity was 2.0 × 10 ⁸ IU / mg.

The titer of the monomer alpha interferon obtained in Example 4 and the monomer alpha interferon obtained in Examples 5 and 6 both showed inactivation of 1.8 × 10 ⁸ IU / mg or more, and there was a statistically significant difference. Did not find. Thus, monomer alpha interferon obtained by reprocessing dimer alpha interferon remained homogeneous with alpha-interferon monomer obtained early in CM-Sepharose chromatography in terms of biological titer.

Reverse Phase High Performance Chromatography

Reverse-phase HPLC was performed to determine the homogeneity of the alpha interferon obtained in Examples 5 and 6, starting from the fully oxidized alpha-interferon monomer and dimer obtained at the time of CM-sepharose separation. It was.

Inject the protein into a C18 column (μ-Bondapak, Waters) equilibrated with 15% (v / v) acetonitrile (15% acetonitrile / 0.05% trifluoro acetic acid / H ₂ O) and then 25 over 5 minutes. When rapidly linearly gradient with% (v / v) acetonitrile and then slowly linearly gradient with 56% (v / v) acetonitrile over 35 minutes, both types of proteins appear at the same position as shown in FIG. Eluted from the column. Therefore, it could be seen that the alpha interferon monomers obtained by the two methods maintain homogeneity in terms of physical and chemical aspects.

Claims (5)

In purifying recombinant alpha interferon, dimers and alpha-oxidized interferons in alpha intiferon obtained by cationic liquid column chromatography were used as redox agents having thiol groups. A method for purifying recombinant alpha interferon comprising converting into monomeric alpha-interferon. The purification method according to claim 1, wherein cysteine and cystine are used as redox agents. The purification method according to claim 1, wherein oxidized glutathione and reduced glutathione are used as oxidation-reducing agents. The method of claim 1, wherein the concentration of reducing agent thiol is 1 mM to 10 mM and the oxidant thiol concentration is 0.1 mM to 1 mM. The method of claim 1, wherein the concentration of reducing agent thiol is 5 to 10 times greater than the concentration of oxidant thiol.