JP2714817B2 - Method for producing urokinase precursor - Google Patents

Description

The present invention relates to a method for producing a urokinase precursor (hereinafter simply referred to as a precursor). More specifically, the present invention relates to a method for removing an antibody component from a precursor-containing composition for the purpose of removing contaminant proteins containing antibodies mixed in a precursor-containing fraction obtained by antibody affinity chromatography.

(Prior art)

The precursor is a kind of fibrinolytic enzyme existing in the living body, and its details are described in JP-A-60-62981. Although the precursor is inactive as it is, it is a so-called zymogen which expresses enzymatic activity by plasmin treatment, and can be produced in a serum-free medium of human kidney cells.

The precursor is a protein having a single chain structure composed of 411 amino acids and having a molecular weight of about 50,000 to 55,000. Although the precursor does not show enzymatic activity as described above, the plasminogen activator activity expressed by plasmin treatment is completely inhibited by the anti-urokinase antibody. Precursors have specific affinity for fibrin and have different thrombolytic properties than conventional urokinase, such as selectively degrading fibrin, the fiber that makes up the thrombus. Precursors having excellent properties different from urokinase are expected to be widely used clinically as fibrinolytic enzymes.

Precursor purification methods include CM-Sephadex treatment, affinity chromatography with an anti-precursor antibody (JP-A-60-62981), affinity chromatography with an anti-UK antibody, and ConA-Sepharose treatment (JP-A-61 -177987) and the like. In particular, a method using antibody affinity chromatography is a useful means for obtaining a high-purity precursor.

[Problems to be solved by the invention]

The use of the affinity chromatography makes it possible to easily and efficiently purify the target substance. On the other hand, antibodies used in the affinity chromatography include antibodies obtained by immunizing animals other than humans (IgG ), I.e., there are many heterologous proteins, and in this affinity chromatography, in general, they may be used under extreme conditions such as large fluctuations in pH, so they were immobilized as ligands on an insoluble carrier. There is a problem that antibodies, that is, heterologous proteins are detached to some extent.

[Means for solving the problem]

In view of the above circumstances, the present inventors have conducted various studies and found that the precursor-containing fraction was obtained by binding a compound selected from aminobenzamidine, aminophenylguanidine, and a basic amino acid to a water-insoluble carrier. By treating with an immobilized carrier, and collecting the non-adsorbed fraction,
Finding that contaminant proteins including antibody components can be removed,
The present invention has been completed.

That is, the present invention treats an aqueous solution containing a urokinase precursor using an immobilized carrier in which a compound selected from aminobenzamidine, aminophenylguanidine, and a basic amino acid is bound to a water-insoluble carrier, and the non-adsorbed fraction is treated. A method for removing an antibody component from a urokinase precursor-containing composition, which comprises recovering the composition.

The precursor used in the present invention has almost no fibrinolytic activity as it is, but is converted to double-chain urokinase by enzymatic treatment with plasmin or the like and exhibits fibrinolytic activity. It also shows some fibrinolytic activity even in the presence of fibrin.

The first representative example of the precursor used in the present invention has a molecular weight of 50,000 to 55,000 and has a single-chain peptide bond structure.

Such precursors include, for example, constituent amino acid 41
One (for the amino acid sequence, see the section of the preparation of the precursor in Examples and Experimental Examples below) (JP-A-60-62).
No. 981).

The origin of the precursor is not particularly limited, and examples include those prepared by a cell culture method, a genetic engineering method and the like. Cell culture method is described in JP-A-60-62981, etc.
The genetic engineering method is disclosed in, for example, Japanese Patent Application Laid-Open No. Sho 60-188051.

The term “precursor” used in the present invention is not limited to those described above, and is a concept that includes derivatives thereof. Such derivatives include protein molecules in which the entire region or part of the epidermal growth factor domain of the precursor has been deleted or the entire region or part thereof has been substituted with another amino acid residue, or a finger domain of the precursor. Or a protein molecule in which the entire region or a part thereof has been deleted, or the entire region or a part thereof has been substituted with another amino acid residue. Therefore, unless otherwise specified, the term “precursor” as used herein means the precursor itself and the precursor derivative as described above.

This precursor derivative usually has a molecular weight of about 40,000 to 50,000 and has a single-chain peptide bond structure like the precursor itself.
The expression mode of the fibrinolytic activity is the same as that of the precursor itself.

This derivative is prepared, for example, by a genetic engineering technique.

As the specific activity of the precursor, no activity was exhibited as it was when measured by the synthetic substrate method, and 100% in the presence of fibrin.
~ 1000UK unit / mg, 80,000 ~ 200,000U for plasmin treatment
K unit / mg is exemplified.

As a starting material of the present invention, a precursor-containing fraction (aqueous solution) obtained by performing antibody affinity chromatography is suitably used. Specifically, a precursor-containing fraction (aqueous solution) in which antibody components such as an anti-UK antibody, an anti-precursor antibody, and an anti-IgG antibody are mixed is preferably used as the contaminating protein.

The immobilization carrier used in the present invention is obtained by covalently binding aminobenzamidine, aminophenylguanidine or a basic amino acid to a water-insoluble carrier.

Examples of the basic amino acid used in the present invention include arginine and lysine.

As the water-insoluble carrier, those known per se may be used. For example, cellulose, agarose, dextran, polyacrylamide, amino acid copolymer and the like are preferably used.

Immobilization may be performed according to a known method. For example,
The disclosure is disclosed in Japanese Patent Publication No. 57-13266. It is also possible to use a crosslinking agent and a condensing agent for covalent bonding. As an example, after agarose activated with bromide cyanide is combined with ε-aminocaproic acid (crosslinking agent), the agarose is reacted in the presence of 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (condensing agent). , Aminobenzamidine, aminophenylguanidine or a basic amino acid to obtain an immobilized carrier. Examples of such commercial products include benzamidine-Sepharose 6B (registered trademark, manufactured by Pharmacia) and the like.

Either a column method or a batch method may be used as the treatment method of the present invention.

In the case of the batch method, a buffer solution having a pH of 5 to 9 and an ionic strength of about 0.1 to 1 M [(for example, 0.1 to 1 M sodium chloride containing 0.01
-0.2 M phosphate buffer (pH 6-7) etc.] (protein concentration is preferably 0.5-10%)
Is contacted with the immobilized carrier equilibrated with the same buffer. The conditions are as follows: 1 to 100 ml of solution per 1 ml of immobilized carrier.
And preferably at 4 ± 2 ° C. for 30 minutes to 2 hours. Thereafter, centrifugation is performed to collect only the supernatant.

In the case of the column method, a buffer solution of pH 5 to 9 (same as above)
Is developed on an immobilized carrier (column) equilibrated with the same buffer to collect a non-adsorbed permeate fraction.

The precursor thus obtained is further purified, if necessary, and then formulated by a known method.

〔effect〕

According to the method of the present invention, a precursor can be produced in a simple processing step, and is suitable for a large-scale treatment, so that it is extremely useful as an industrial production method.

In addition, since the mixed antibody component or contaminant proteins such as UK can be removed, the obtained precursor has a further improved purity and can provide a preparation excellent in safety.

[Examples and experimental examples]

EXAMPLES The present invention is described in more detail with reference to Examples and Experimental Examples, but the present invention is not limited thereto.

Example 1 Cultured human kidney cells were cultured in a serum-free culture medium supplemented with 0.1% human serum albumin for 3 days, the culture medium was centrifuged, and the supernatant was frozen and stored. After adjusting the pH of the pooled culture supernatant to 5.5, it was brought into contact with CM-Sephadex C-50. After washing the column with a 0.16 M phosphate buffer (pH 5.5), the adsorbed precursor was eluted with a 0.16 M phosphate buffer (pH 8.5).

On the other hand, antiserum obtained from horses previously immunized with the precursor was purified to recover the anti-precursor antibody. This horse-derived anti-precursor antibody was immobilized on BrCN-activated agarose (Sepharose 4B, manufactured by Pharmacia).

This antibody column was equilibrated with a 0.1 M phosphate buffer (pH 6.5) containing 0.5 M sodium chloride, and the eluate containing the precursor was contacted with the equilibrium. 0.1M containing 0.5M sodium chloride
After washing the column with a phosphate buffer (pH 6.5), the adsorbed precursor was replaced with 0.2 M glycine-containing 0.5 M sodium chloride.
Elution was carried out with an aqueous hydrochloric acid solution (pH 2.5).

The eluate was concentrated and dialyzed against a 0.1 M phosphate buffer (pH 6.5) containing 0.5 M sodium chloride.

Agarose (Sepharose 4B, Pharmacia) immobilized with a rabbit-derived anti-horse IgG antibody (purified antiserum obtained by immunizing a rabbit with horse IgG) was equilibrated with the above buffer solution. Contacting the body-containing fraction,
The non-adsorbed fraction was collected.

Further, this fraction was contacted with para-aminobenzamidine-agarose (Benzamidine-Sepharose 6B, manufactured by Pharmacia) equilibrated with the above buffer, and the non-adsorbed fraction was collected.

 The fraction was sterilized and filtered, and then freeze-dried.

 The properties of the obtained precursor are as follows.

The molecular weight was about 54000 by SDS-polyacrylamide gel electrophoresis (under reduction) and showed a single band. The amino acid sequences of the amino acids are shown in Table 1.

The specific activity was 100 UK units / mg protein without plasmin treatment and 140,000 UK units / mg protein without plasmin treatment (activity was measured by a synthetic substrate method).

As the contaminating proteins, both the horse antibody and the rabbit antibody were below the detection limit.

Experimental Example 1 The removal effect of a rabbit antibody was mainly examined by the method of the present invention. The measurement was based on the RPHA method. The results are shown in Table 2.

As shown in the above table, it was found that by incorporating benzamidine treatment, heterologous proteins can be efficiently removed. Numerically, the removal rate of 88.3% or more for No. 1 and 88.7% for No. 2
Although the removal rate was as above, this is a numerical value at the detection limit of the RPHA method, and it is considered that almost 100% was actually removed.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Kazuo Morimoto 2-11 Nagatano-cho, Fukuchiyama-shi, Kyoto Inside the Midori Cross Osadano Plant (72) Inventor Hirofumi Arimura 2-1180-1 Invitation Otani, Hirakata-shi, Osaka Inside Midori Cross Research Laboratory, Ltd. (56) References JP-A-60-62981 (JP, A)

Claims (3)

(57) [Claims] An urokinase precursor-containing aqueous solution is treated with an immobilized carrier in which a compound selected from aminobenzamidine, aminophenylguanidine, and a basic amino acid is bound to a water-insoluble carrier, and the non-adsorbed fraction is treated. A method for removing an antibody component from a urokinase precursor-containing composition, which comprises recovering the composition. 2. The method according to claim 1, wherein the urokinase precursor-containing aqueous solution is obtained by antibody affinity chromatography. 3. A urokinase precursor-containing composition obtained by substantially removing contaminating antibody components.