JPH0421647B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to stable plasminogen activator precursor (hereinafter referred to as zymogen) compositions. <State of the art> The zymogen in the present invention is a protein that can be recovered from a serum-free medium of human kidney cells and has a molecular weight of about 5.
It has the characteristics of 1,000,000 daltons, does not undergo low molecular weight even when treated with a reducing agent, and expresses enzyme activity (plasminogen activator activity) when treated with a proteolytic enzyme such as plasmin. Furthermore, it has a higher affinity for fibrin than known urokinase. Therefore, this zymogen is highly expected to have clinical effects as a medicine for thrombolytic therapy. However, this zymogen had the property of adsorbing to glass walls and was unstable to heat. Furthermore, since this zymogen lacks storage stability in solution, it is desirable to use a lyophilized product when used as a pharmaceutical product. However, it was found that the activity could be lost even during freeze-drying. Therefore, the present zymogen must be stabilized in some way. <Disclosure of the Invention> The present inventors have conducted intensive research on the stabilization of the present zymogen, and have found that the present zymogen in an aqueous solution is stabilized when a certain amount of albumin is allowed to coexist. The present invention was completed by discovering that zymogen is not inactivated and that storage stability as a freeze-dried preparation is improved. That is, the present invention is a zymogen composition containing albumin as a stabilizer, and the blending ratio of zymogen and albumin is such that the amount of albumin is at least 30 mg per 10,000 to 1,000,000 U of the zymogen. The present invention relates to a stable zymogen composition characterized by: Zymogen (plasminogen) in the present invention
activator precursor) has the following properties (a) to (d). (a) The cell culture supernatant of human kidney cells was adsorbed onto a cation exchanger adjusted to pH 4.5 to 6.5, and the pH was adjusted to pH 7.5 to 9.5.
The eluate was eluted with a buffer of pH 6 to 8.
It is a protein that can be obtained by adsorbing it onto an affinity chromatography antibody column equilibrated with 100% and eluting it with an aqueous solution with a pH of 2 to 4. (b) The molecular weight measured by SDS-polyacrylamide gel electrophoresis is approximately 50,000 Daltons. (c) Low molecular weight does not occur due to reducing agent treatment. (d) Expresses plasminogen activator activity upon treatment with plasmin. The preparation method (a) above is a means for specifying the zymogen in the present invention, and the zymogen in the present invention can be obtained by the preparation method if it has the characteristics (a) to (d) above. Not limited to things. The zymogen used in the present invention is one obtained by culturing human kidney cells in a serum-free medium, collecting the zymogen produced in the medium, and purifying it by an appropriate combination of centrifugation, salting out, and chromatography. , other genetic engineering techniques such as Escherichia coli, Bacillus subtilis,
It can be widely used regardless of its origin, such as those produced by yeast, animal cells, etc. The present zymogen can be recovered by the following method. [Preparation of raw materials] Human kidney cells are used as raw materials.
Cult ure or diploid cells are obtained, subcultured, and the zymogen-producing cells are separated, which is used. For example, cells are implanted at a number of 2 to 20 × 10 ⁴ cells/ml, cultured for about 3 days, and when the number of cells becomes about three times the number of implanted cells, trypsinization is applied.
The one obtained by adding an EDTA mixture and collecting a monolayer of immature cells is used. [Culture conditions] As a medium, for example, Waymouth's medium,
A Dulbec co'smodified MEM medium is used, and 5% heat-inactivated fetal bovine serum is added to the medium during pre-culture, and a serum-free medium, preferably a serum-free medium supplemented with human serum albumin, is used during the production of the zymogen. Culture using serum medium. Human or bovine albumin, lactalbumin hydrolyzate, transferrin, various amino acids, various fatty acids, hormones such as insulin, etc. may be added to the serum-free medium. The culture medium is replaced every 2 to 3 days. The zymogen of the present invention is produced in this medium. [Recovery of the zymogen of the present invention] The zymogen of the present invention can be recovered from the medium by, for example, centrifuging the medium, vacuum concentration, salting out fractionation, gel filtration, concentration, ion exchange chromatography, affinity chromatography, etc. This is done by appropriately combining them. More specifically, it can be recovered, for example, by the following method. That is, first, the medium is centrifuged and the supernatant is collected. This recovered solution is partially purified by ion exchange chromatography. As the carrier, a weakly acidic cation exchanger is most suitable, such as CM-exchanger or Duolite. After adjusting the carrier to a pH of 4.5 to 6.5, preferably 5 to 6, the recovered liquid is developed and adsorbed onto the carrier. After washing with the above buffer, PH7.5~9.5,
More preferably, the present zymogen is eluted with a buffer solution having a pH of 8 to 9. Examples of the buffer include phosphate buffer and the like. Furthermore, this eluate is highly purified by affinity chromatography. As the carrier, either a polyclonal antibody column or a monoclonal antibody column may be used. In the case of the polyclonal method, the anti-zymogen antibody is prepared by immunizing an animal with highly purified zymogen,
It is obtained by collecting and purifying the obtained serum. The antiserum may be produced by a known method; for example, a mixed emulsion of highly purified zymogen and Freund's complete adjuvant is prepared, injected intradermally into the animal 2 to 3 times, and blood collected several days after the final immunization. After solidifying at room temperature, leave it at 4℃ overnight.
The antiserum is obtained by centrifugation at 3000 rpm for 20 minutes. There is no need to choose a particular animal species as the animal used for immunization; for example, rats, mice, rabbits,
Examples include goats and horses. Purification of the antiserum is described, for example, in J.Am.Chem.Soc., 62 , 3386.
(1940), Fed. Proc., 17 , 1161 (1958). In the case of the monoclonal method, an anti-zymogen antibody is obtained by a cell fusion method. The cell fusion method is carried out by means known per se, and one example is to react proliferative cells with lymphocytes producing the target antibody in the presence of polyethylene glycol. This method produces cells that have the ability to produce antibodies at the same time, and the antibody produced by these cells is a single antibody that reacts only with one antigenic determinant. The present invention uses mouse myeloma cells as proliferative cells and mouse spleen cells (B cells) that have been immunized with the present zymogen as antibody-producing lymphocytes to fuse together, and then cells producing the desired antibody are fused. A monoclonal antibody against the zymogen is obtained by screening. Furthermore, as a method for immobilizing the anti-zymogen antibody thus obtained without losing its activity, the following insoluble matrix can be applied. Copolymers of amino acids (J.Biol.
Chem., 236 , 1970 (1961), cellulose (Nature, 189 , 576 (1961)), agarose or sepadex (Nature, 215 , 1491 (1967),
Natufe, 245 , 3059 (1970)), polyacrylamide (Biochem., 8 , 4074 (1966)). Anti-zymogen antibodies can be efficiently immobilized by these methods.
Furthermore, by using the adsorbent thus obtained, the present zymogen can be obtained in good yield and with high purity. Affinity chromatography of zymogen according to the present invention is as follows. The zymogen partially purified using a cation exchanger is brought into contact with and adsorbed on an anti-zymogen antibody column equilibrated with a pH 6-8 buffer. After washing the column, elute with an aqueous solution of pH 2-4. It should be noted that the above-mentioned recovery method is only an example of the motigen recovery method of the present invention, and of course other methods may be used for recovery. [Characteristics of the zymogen of the present invention] Molecular weight When the molecular weight of the zymogen of the present invention was measured using SDS-polyacrylamide gel electrophoresis (Nature, 227 , 680-685 (1970)), it was approximately 50,000 Daltons. Ta. The molecular weight was determined by comparison with a standard protein of known molecular weight, and the protein was pretreated with 1% SDS and 1% 2-mercaptoethanol for 2 hours at 37°C or 2 minutes at 100°C. Ta. Enzyme Sensitivity A sensitivity experiment to plasmin was conducted according to J. Biol. Chem., 257 , 3276-3283 (1980).
As a result, the zymogen of the present invention itself did not exhibit plasminogen activator activity. However, the activity is expressed by plasmin treatment, and the degree of activity expression depends on the concentration of plasmin treatment (Table 1) and the treatment time (Table 2).
depended on. The activity measurement method is as described below. In the former experiment, the amount of this zymogen protein was 1.3 μ
g/ml was prepared and pretreated with plasmin at each concentration for about 60 minutes, and then the enzyme activity expressed was measured. In the latter experiment, 0.1 μg/ml of plasmin and 1.3 μg/ml of the present zymogen protein were prepared, and the effect of treatment with plasmin was measured over time. [Table] [Table] Reducing agent treatment 1% SDS, 1% 2-mercaptoethanol, 37
The resistance of the zymogen of the present invention to treatment at 100°C for 2 hours or 100°C for 2 minutes was investigated according to a molecular weight measurement method. As a result, the untreated zymogen and the treated zymogen showed the same electrophoretic pattern, confirming that this enzyme was a single chain. Activity measurement method Synthetic substrate method (Cleason et al. Haemostasis.
776 (1978)) or the flat plate method (Astratup et al.
Arch.Biochem.Biophys., 40 , 346-351,
(1952)). As fibrinogen, bovine, fibrinogen, and Ff.I (containing a trace amount of plasmin) from Miles were used. Regarding other properties Active center: When the zymogen of the present invention was brought into contact with Sepharose gel on which p-aminobenzamidine, which binds to the serine active site of urokinase, was immobilized, it was not adsorbed. From this, it is presumed that the serine active site of the zymogen of the present invention is located inside the molecule, and its higher-order structure is different from that of conventional urokinase. Fibrin affinity: The zymogen of the present invention has a strong affinity for fibrin and has properties similar to tissue plasminogen activator. Neutralization of enzyme activity by anti-urokinase antibody and anti-human melanoma Yuki-TPA antibody: The activity of the zymogen of the present invention was expressed using plasmin. Furthermore, add anti-urokinase antibody or anti-human melanoma-derived TPA antibody, and store at 37°C for 90
After standing for a minute, the remaining enzyme activity was measured by the synthetic substrate method or the plate method, and it was found that the enzyme activity of the zymogen of the present invention expressed by plasmin treatment was inhibited by the anti-urokinase antibody; It was not inhibited by TPA antibody. From the above, the zymogen of the present invention is
It is a precursor of urokinase and shows similar properties to TPA in terms of fibrin affinity, but TPA
and its precursors. [Preparation of Albumin] The albumin used in the present invention is preferably human-derived albumin from the viewpoint of antigenicity, and is not particularly limited as long as it is purified for medical use. Its purity is determined by electrophoretic analysis of 80%
% or more is preferably albumin. Methods for obtaining human-derived albumin include ethanol fractionation (Japanese Patent Publication No. 47-2869, Japanese Patent Publication No. 35-5297), heating in the presence of an organic acid (Japanese Patent Publication No. 43-1604,
Examples include Japanese Patent Publication No. 51-401321). Particularly preferably, albumin is heat treated (preferably at 60°C,
(about 10 hours) to inactivate hepatitis viruses, etc. [Albumin content] The blending ratio of this zymogen and albumin is at least 10,000 to 1,000,000 U of albumin.
The ratio is equivalent to 30 mg or more, preferably 10,000 to 1,000,000 U of this zymogen to albumin.
This ratio corresponds to 30mg to 50mg. In addition, when freeze-drying the present zymogen-containing aqueous solution, regardless of the content of the present zymogen, at least 3 mg/ml or more and preferably 5 mg/ml of the present zymogen should be added to the aqueous solution.
Stabilization of the zymogen can be achieved by adding albumin to a concentration of ml or more. Note that in the present invention, it is of course possible to add other stabilizers in addition to albumin. For example, addition of inorganic salts or organic salts is suitable. [Freeze-drying treatment] In the case of freeze-drying treatment using albumin, it is carried out, for example, as follows. That is, an aqueous solution containing the purified zymogen is adjusted to pH 5 to 9, albumin is added in the above-mentioned stabilizing amount, the aqueous solution is sterilized and filtered, and then dispensed and frozen using a conventional method. This is done by subjecting it to drying. [Effects] The composition of the present invention thus provided is suitable as a pharmaceutical product with no loss of zymogen during the formulation process and excellent stability during storage. The present invention will be specifically explained below with reference to Examples, Experimental Examples, and Reference Examples, but the present invention is not limited by these in any way. <Example> After cultured human kidney cells were cultured in a serum-free medium for several days, the culture solution was centrifuged. The obtained supernatant was purified by various chromatographies to obtain the present zymogen with a specific activity of 56,000 U or more. Thereafter, 24,000 U/ml of this solution containing the present zymogen was adjusted to pH 7 with a phosphate buffer, and then human serum albumin was added in an amount of 35 mg/ml. This solution was sterilized and filtered, dispensed in 2 ml portions into 10 ml tube bottles, and freeze-dried under drying conditions at a final temperature of 25°C. The moisture content of the obtained dry product was determined based on biological product standards.
When tested according to the general test method, it was approximately 0.2%. When 2 ml of distilled water for injection was added to each dried product, it immediately dissolved, and the solution was clear and colorless. When the residual rates of the zymogen were determined for these solutions, there was no change in any of them compared to before freeze-drying. Also, during the formulation process,
There was no loss of this zymogen due to adsorption to the glass wall. <Experimental Example> An experiment was conducted to confirm the stabilizing effect of the present invention. Purified zymogen-containing solution (1×10 ⁴ U/ml,
5×10 ⁴ U/ml, 5×10 ⁵ U/ml) were added with various concentrations of human serum albumin (1-100 mg/ml),
Next, freeze-drying was performed. The titer of the lyophilized product was measured immediately after lyophilization and after storage at 50° C. for 3 months, and the residual activity rate relative to the titer immediately after addition of human serum albumin is shown in Table 3. [Table] <Reference example: Production example> Cultured human kidney cells were cultured for 3 days in a serum-free culture medium supplemented with 0.1% human serum albumin, the culture medium was centrifuged, and the supernatant was frozen and stored. After adjusting the pooled culture supernatant to pH5.5, CM-Sephadex C
-50 was touched. 0.16M phosphate buffer (PH5.5)
After washing the column with 0.16M phosphate buffer (PH
8.5) The adsorbed zymogen was eluted. On the other hand, mice previously immunized with this zymogen
Among the hybridomas obtained by fusing BALB/c spleen cells and mouse myeloma cells with polyethylene glycol, clones with high antibody production against this zymogen were selected. Anti-zymogen monoclonal antibodies were recovered from the culture medium of this fused cell. This monoclonal antibody activates BrCN
It was fixed on Sepharose 4B (Pharmacia). This monoclonal antibody column was equilibrated with 0.1M phosphate buffer (PH7.0) containing 0.4M NaCl, and the eluate containing the present motigen was contacted therewith. 0.1M phosphate buffer containing 0.4M NaCl (PH7.0)
After washing the column with 0.5M NaCl-containing 0.2M glycine-HCl aqueous solution (PH
2.5). After sterilizing and filtering the eluate,
The highly purified zymogen with a specific activity of at least 80,000 U/mg was obtained by freeze-drying. In addition, this purified product showed one band with a molecular weight of 50,000 by SDS-polyacrylamide gel electrophoresis.

Claims (1)

[Scope of Claims] 1. A plasminogen activator comprising a plasminogen activator precursor having the following characteristics (a) to (d) as a main component and containing at least albumin as a stabilizer. Beta precursor composition. (a) The cell culture supernatant of human kidney cells is adsorbed onto a cation exchanger adjusted to pH 4.5-6.5 and eluted with a buffer solution of pH 7.5-9.5, and the resulting eluate is adjusted to pH 6-8. It is a protein that can be obtained by adsorbing it to an equilibrated affinity chromatography antibody column and eluting with an aqueous solution of pH 2 to 4, and (b) has a molecular weight of about 5 as measured by SDS-polyacrylamide gel electrophoresis. 1,000,000 daltons, (c) no molecular weight reduction occurs when treated with a reducing agent, and (d) plasminogen activator activity is expressed when treated with plasmin.