JPH0899999A - Production of alpha1 protease inhibitor - Google Patents

Abstract

PURPOSE: To obtain the subject substance capable of controlling the evolution of tissue inflammations without any danger of viral infection by developing a solution containing an α1-protease inhibitor on a cation exchange resin, adsorbing and removing contaminating impurity proteins and then recovering an unadsorbed fraction. CONSTITUTION: A fraction IV or a fraction IV-1 prepared by a Cohn ethanol separating method or its improved method is used as a plasma protein solution containing an α1 protease inhibitor (α1PI) and the resultant solution is treated by at least one means of dilution, dialysis or gel filtration chromatography, etc., to reduce the concentration of salts in the solution to <=about 0.02M and the pH of the solution is then regulated to 4.5-5.5. The regulated solution is subsequently brought into contact with a cation exchange resin having a selective affinity for contaminating impurity proteins other than the α1PI in the solution to adsorb the contaminating impurity proteins and the α1PI is separated into in an unadsorbed fraction to recover the α1PI. Thereby, the objective α1PI, useful for control and regulation of coagulation or complement systems, etc., control, etc., over the evolution of tissue inflammations, etc., and having high safety is obtained.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for producing an α1 protease inhibitor (hereinafter sometimes referred to as α1PI). More specifically, α1P having a reduced content of contaminant proteins from plasma or plasma fraction containing α1PI
It is intended to provide a manufacturing method capable of mass-producing I in a very simple manner, and a preparation having higher safety against the risk of viral infection.

[0002]

2. Description of the Related Art α1
PI is a glycoprotein having a molecular weight of 53,000, is contained in plasma in a large amount, and has a function as a serine protease inhibitor. Antithrombin III, heparin cofactor II, protein CI inhibitor, α1 antichymotrypsin, α2 plasmin inhibitor, plasminogen activator inhibitor, C1 inhibitor and other serine protease inhibitors (generally called serpins) Steal P
and Chothia C, J. Mol. Biol., 221 , p.615-621, (199
1)). Regarding the physiological function of α1PI, L in 1963.
aurell and Eriksson first reported that α1PI deficiency results in emphysema (Laurell CB and Eriksson, Scand.
J. Clin. Lab. Invest., 15 , p.132-140, (1963)). In addition, Travis et al. Demonstrated that α1PI has extremely high specificity for neutrophil elastase, and emphysema in α1PI deficiency cannot suppress neutrophil-derived elastase collected in alveoli due to α1PI deficiency. It was shown that the cause was the inability to suppress the collapse of elastin fibers in the alveolar wall (Keith B and Travis J, J. Biol. Chem., 255 , p.393.
1-3934 (1980)). Furthermore, α1PI has been found to be involved in the regulatory control of the coagulation system, kallikrein system, immune complement system, etc. due to its broad protease inhibition spectrum, and is also deeply involved in the biological defense reaction such as controlling the progression of tissue inflammation. (Koji Suzuki, Protein Nucleic Acid Enzyme, 34 ,
(8) (1989)).

Conventionally, the method for obtaining α1PI from plasma is as follows:
It was a complicated method combining anion exchange chromatography, affinity chromatography, gel filtration chromatography and the like. For example, after fractionating ammonium sulfate using plasma as a starting material, blue sepharose, D
A method of Travis et al. (Tra) in which column chromatography is carried out one after another with EAE-cellulose and Sephadex G-75.
vis J., Method in Enzymology, 80 , p.754-765), or after ammonium sulfate salting out, Zinc chelate, DE-5
The method of Kurecki combining each column chromatography of 2-cellulose is known (Kurecki T., Anal.
ytical Biochemistry 99 , p.415-420 (1979)). Several other methods have been reported, but all of them are similar to the above-mentioned method, and column chromatography is frequently used. Recently, Coan et al. Developed a purification method in which two-stage polyethylene glycol (PEG) fractionation was performed and a DEAE Sepharose column was further applied. However, as a process, a chromatography process was added to the fractionation process. It requires a lot of labor and time to separate and obtain proteins (Coan MM, Vox Sang, 48 , p.33.
3-342 (1985)).

[0004]

In view of the above-mentioned problems, the inventors of the present invention have earnestly studied a method for separating and obtaining α1PI and studied various methods. As a result, the α1PI-containing fraction obtained from plasma Spread the solution on the cation exchange resin,
The present invention has been completed which provides a novel preparation method characterized by adsorbing a contaminated impure protein and separating high-purity α1PI into a non-adsorbed fraction. When the preparation method is applied, α1PI can be prepared on an industrial scale extremely easily and efficiently as compared with the conventional method.

The starting material used in the present invention is a corn fraction IV (FIV) or a corn IV-prepared using the corn ethanol separation method or a modification thereof.
1 (FIV-1) is the most preferred embodiment, and further includes a rework of these fractions from which other proteins were first removed. In the method for separating and obtaining α1PI from the α1PI-containing fraction, the α1PI-containing solution has a salt concentration of 0.0
The pH is adjusted to 2M or less by using a method such as dilution by adding distilled water, dialysis or gel filtration, and further adjusted to about pH 4.5 to 5.5. If impure protein precipitates, it is filtered or centrifuged to obtain a clear solution. In any case, the α1PI solution preferably has a protein concentration of 1 to 5% and a pH of about 4.5 to 5.5. α1
PH 4.5 if the PI-containing fraction is solid or precipitated
Dissolve in a buffer such as phosphate buffer or acetate buffer having a salt concentration of 0.02 M or less at ˜5.5, and if impure protein precipitates, it is similarly treated by filtration or centrifugation to clarify. Use as a solution.

The prepared plasma protein solution containing α1PI is developed on a cation exchange resin which has been previously equilibrated with a buffer such as a phosphate or acetate buffer having a salt concentration of 0.02 M or less. The buffer to be used may have a salt concentration of 0.02 M or less, and is not particularly limited to the phosphate and acetate buffers. Further, there is no particular restriction on the mode of development, and either the column method or the batch method can be adopted. The cation exchange resin used may be a commercially available cation exchange resin such as CM Sepharose, SP-Sepharose, S-Sepharose, SP Toyopearl. The cation exchange resin is not particularly limited, but SP-Sepharose is a particularly preferable embodiment.

After development on chromatography, the non-adsorbed fraction is collected. The non-adsorbed fraction was subjected to gel filtration analysis and SDS.
-PAGE analysis shows that α1PI has a purity of 80% or more, and this fraction is concentrated to reduce the volume by membrane filtration or the like to obtain an α1PI concentrate. It is desirable that the α1PI concentrate is free of virus infectivity. The concentrate may be heated for a sufficient period of time, for example about 10 hours, to a temperature such as about 60 ° C. or higher to reduce viral infectivity. At this time, citrate ions coexist in the solution. For example, the protein concentration of α1PI concentrated solution is 20mg
/ Ml, the citrate ion of the solution is about 0.15-0.4.
Let M. The pH of the mixture during this heating step is about 6.0-
It is desirable to set it to 7.0.

In order to further enhance the stabilization of α1PI during heating, it is desirable to use carbohydrates as stabilizers with sodium citrate. For this purpose,
Mono-, di- and trisaccharides such as arabinose, glucose,
Galactose, maltose, fructose, fibose, mannose, ramose, sucrose and the like, and sugar alcohols such as sorbitol and mannitol and the like can be mentioned, and sucrose is a preferred embodiment. It may be added as a carbohydrate to the α1PI solution so that the final concentration will be 40 to 70%.

The present invention will be specifically described below with reference to test examples and examples, but the present invention is not limited thereto.

[0010]

EXAMPLES Prior to Examples, α1PI in the present invention
The quantity test was performed using the following method. The ability of α1PI to inhibit elastase was evaluated by using a chromogenic substrate for elastase. Hydrolysis of N-succinyl-L-alanyl-L-alanyl-L-alanyl-p-nitroanilide by elastase increases the absorption at 450 nm.
This increase is continuously measured at 37 ° C to calculate the amount of hydrolysis per unit time. The linear change in absorption with and without α1PI is compared over time. Then, the amount of α1PI as an inhibitor was evaluated based on the fact that elastase and α1PI react stoichiometrically in a ratio of 1: 1 and the known amount of elastase (Travis J, Method in Enzymol.
ogy, 80 , p.754-765).

Test Example 1 (Effect of pH on cation chromatography) SP
-Chromatography was carried out using Sepharose (First Flow: Pharmacia), the pH of the developing solution and the buffer of the chromatography used at that time were changed, and changes in the α1PI recovery rate and purification coefficient were evaluated. Corn fraction FIV-
1.8 l of 15 mM phosphate / acetate buffer having a pH of 5.2 was added to 100 g of the precipitate of 1 and stirred and dissolved at 4 ° C. for 12 hours.
The insoluble fraction of this solution was filtered through a filter H050 manufactured by Pall Co. to obtain a clear fraction. FIV prepared by the above method
-1 The pH of 100 ml of the solution is changed to the pH shown in the following table with dilute acetic acid or dilute NaOH solution, and when precipitation occurs due to the change of pH, filtration is performed, and SP-Sepharose (First Flow: Pharmacia) 20 ml Then, the gel was washed with the same amount of the above buffer solution as the used gel amount, and the developing solution and the washing solution were collected and fractionated as a non-adsorbed fraction. Α1 at that time
The amount of PI was measured to calculate the recovery rate, and the protein amount was measured as A280 to calculate the purification coefficient. The recovery rate of the stock solution developed for chromatography is 100% and the purification coefficient is 1, and the results are shown in Table 1.

[0012]

[Table 1] * 1 Concentration was expressed as mg / ml by measuring the amount of α1PI having the ability to inhibit elastase. * 2 Specific activity was expressed as the value obtained by dividing the amount of α1PI (mg / ml) by A280. * 3 The refining factor is expressed as the value obtained by dividing the specific activity of the raw material liquid by the specific activity of other processes.

In this test example, the influence of pH on the recovery of α1PI and the increase in the degree of purification when it was applied to cation exchange chromatography was investigated, and the pH when it was applied to chromatography was 4. When the pH is 5 or less, the recovery rate and the degree of purification decrease, and when the pH is 5.5 or more, the recovery rate increases but the degree of purification decreases. Therefore, the pH of the developing solution at the time of developing to cation exchange chromatography needs to be in the range of pH 4.5 to pH 5.5, and preferably pH 5.2.

Test Example 2 (Effect of salt concentration on cation chromatography) S
Chromatography was carried out using P-Sepharose (First Flow: Pharmacia), and the salt concentration of the developing solution and the buffer used for the chromatography at that time were changed to evaluate the α1PI recovery rate and the purification coefficient. 10 g of FIV-1 precipitate was dissolved in 180 ml of various buffers having a pH of 5.2 shown in the table below, and 100 ml of the solution was equilibrated with the same buffer used for dissolution in advance. SP-Sepharose (First Flow: Pharmacia) It was developed to 20 ml and the non-adsorbed fraction was collected.
The recovery rate was calculated by measuring the amount of α1PI at that time, and the purification factor was calculated by measuring the protein amount as A280. The recovery rate of the undiluted solution applied to chromatography was 10
The results are shown in Table 2 with 0% and a purification factor of 1.

[0015]

[Table 2] * 1 Concentration was expressed as mg / ml by measuring the amount of α1PI having the ability to inhibit elastase. * 2 Specific activity was expressed as the value obtained by dividing the amount of α1PI (mg / ml) by A280. * 3 The refining factor is expressed as the value obtained by dividing the other specific activities by the specific activity of the raw material liquid.

In this test example, the influence of the salt concentration on the increase in the yield and the degree of purification of α1PI when it was applied to cation chromatography was investigated. When the salt concentration at the time of development into cation chromatography was 0.02 M (20 mM) or less, the degree of purification markedly increased.

Test Example 3 (Study of the Present Invention Using Various Cation Exchangers) α1PI was purified using various cation exchangers, and the recovery rate and the degree of purification at that time were evaluated. The evaluation method is FIV-1 solution 10 prepared by the method described in Test Example 1.
0 ml was developed on 20 ml of various cation exchangers previously equilibrated with 15 ml phosphate / acetic acid buffer of pH 5.2, and further washed with the same amount of the above-mentioned buffer as the gel amount used. The collected non-adsorbed fraction was collected. Α at that time
The recovery rate was calculated by measuring the amount of 1 PI, and the purification coefficient was calculated by measuring A280 as the protein amount. The results are summarized in Table 3.

[0018]

[Table 3] * 1 Concentration was expressed as mg / ml by measuring the amount of α1PI having the ability to inhibit elastase. * 2 Specific activity was expressed as the value obtained by dividing the amount of α1PI (mg / ml) by A280. * 3 The refining factor is expressed as the value obtained by dividing the specific activity of the raw material liquid by the specific activity of other processes.

In this example, the application of the present invention to the purification of α1PI using various cation exchangers was examined, and the results obtained depend on the type of cation exchanger in the present invention. It is shown that various cation exchangers on the market can be applied without particular restrictions.

Test Example 4 (Effect of sucrose on heat treatment) Based on the results obtained in Test Examples 1 and 2, corn fraction FIV-1 was used as a raw material and SP-Sepharose was added with 15 mM phosphoric acid at pH 5.2.
The mixture was developed using an acetate buffer, the non-adsorbed fraction containing α1PI was collected, and the pH was adjusted to 7.0. Next, use an ultrafiltration membrane to concentrate the protein to 2%,
An α1PI concentrate was prepared. Sucrose was added to the obtained α1PI concentrated solution so as to have the concentrations shown in the table below and completely dissolved by stirring, and then heat treatment was performed at 60 ° C. for 10 hours. As a result, sucrose was found to be useful as a stabilizer for heat treatment.

[0021]

[Table 4]

Test Example 5 (Effect of citrate ion on heat treatment) Sucrose was added at a concentration of 40% (W / V) to an α1PI concentrate prepared in the same manner as in Test Example 4, and further Trisodium citrate was added to give the concentrations given in the table below. After completely stirring and dissolving, the pH was adjusted to 7.0 and heat treatment was carried out at 60 ° C. for 10 hours. The amount of α1PI before and after heating was measured to calculate the recovery rate. The results are shown in Table 5.

[0023]

[Table 5]

This test example evaluates the effect of citrate as a stabilizer in the presence of a low concentration of sucrose, and shows that the optimum concentration is 0.15 to 0.3M.

Test Example 6 (Effect of pH on heat treatment) 40% (W / V) sucrose and 0% trisodium citrate were added to the α1PI concentrated solution prepared according to the methods of Test Examples 4 and 5. It was added to a concentration of 0.2M. After completely dissolving with stirring, the pH was adjusted to the values shown in the following table, and heat treatment was performed at 60 ° C. for 10 hours. The amount of α1PI before and after heating was measured to calculate the recovery rate. The results are shown in Table 6.

[0026]

[Table 6]

This test example shows that the optimum pH for stabilizing α1PI in the heat treatment is pH 6.0 to 7.0.

Example 1 100 g of a corn fraction FIV-1 precipitate was added with 15 of pH 5.2.
1.8 L of mM phosphate / acetate buffer was added, and the mixture was stirred and dissolved at 4 ° C. for 12 hours. The insoluble fraction of this solution was filtered through a filter H050 manufactured by Pall Co. to obtain a clear fraction. The clarified fraction was developed on SP-Sepharose (First Flow 400: Pharmacia) which had been equilibrated with 15 mM phosphate / acetate buffer in advance, and the buffer was used in the same amount as the gel amount in the column chromatography used. It was washed with and the non-adsorbed fraction was collected. Adjust the pH of the collected non-adsorbed fractions to 6.5, use an ultrafiltration membrane,
Concentrate so that the protein concentration becomes 2%, and α1PI
A concentrated solution was prepared. Trisodium citrate and sucrose were added to the α1PI concentrate to a final concentration of 0.2M and 40M, respectively.
% (W / V), and dissolved by stirring. After confirming that the added stabilizer is completely dissolved,
Heat treatment was performed for 0 hours. The results obtained are shown in Table 7. Further, when the purity of the α1PI concentrate obtained in this Example was checked by SDS gel electrophoresis, a purity of about 80% was obtained, and it was purified to a purity that does not hinder practical use. Was found (see Figure 1).

[0029]

[Table 7] * 1 Concentration was expressed as mg / ml by measuring the amount of α1PI having the ability to inhibit elastase. * 2 Specific activity was expressed as the value obtained by dividing the amount of α1PI (mg / ml) by A280. * 3 The refining factor is expressed as the value obtained by dividing the specific activity of the raw material liquid by the specific activity of other processes.

In this example, the FIV-1 solution was used as a raw material, and the solution was directly applied to cation exchange chromatography to evaluate the increase in the α1PI recovery rate and the degree of purification. The results show that the cation exchange chromatography alone can increase the degree of purification without lowering the recovery rate, and that heat treatment can be suitably performed.

Example 2 100 g of a corn fraction FIV precipitate was added to 15 mM of pH 6.5.
1.8 l of a phosphate buffer was added and dissolved by stirring at 4 ° C for 12 hours. Acetic acid was added to adjust the pH of the solution to 5.2, and the insoluble fraction was centrifuged at 5,000 g for 30 minutes to obtain a supernatant. Further, this supernatant was filtered through a filter H050 manufactured by Pall Co. to obtain a clear fraction. The clarified fraction was developed on SP-Sepharose (First Flow 400: Pharmacia), which had been equilibrated with 15 mM phosphate / acetate buffer in advance, and further,
The column was washed with the same amount of the column chromatography gel used as the above buffer, and the non-adsorbed fraction was collected. The pH of the collected non-adsorbed fraction was adjusted to 6.5, and concentrated using an ultrafiltration membrane so that the protein concentration was 2% to prepare an α1PI concentrated liquid. Trisodium citrate and sucrose were added to the α1PI concentrate at final concentrations of 0.2M and 40% (W /
V), and dissolved by stirring. After confirming that the added stabilizer was completely dissolved, heat treatment was carried out at 60 ° C. for 10 hours. The results obtained are shown in Table 8. In addition, the α1PI concentrate obtained in this Example was SD
When the purity was checked by S-gel electrophoresis, a purity of about 80% was obtained, and it was found that the product was purified to a purity that would not hinder practical use (see FIG. 2).

[0032]

[Table 8] * 1 Concentration was expressed as mg / ml by measuring the amount of α1PI having the ability to inhibit elastase. * 2 Specific activity was expressed as the value obtained by dividing the amount of α1PI (mg / ml) by A280. * 3 The refining factor is expressed as the value obtained by dividing the specific activity of the raw material liquid by the specific activity of other processes.

In this example, a FIV solution was used as a raw material,
The method was directly applied to cation exchange chromatography, and the increase in the recovery rate and the degree of purification of α1PI at that time was evaluated. The results show that the cation exchange chromatography alone can increase the degree of purification without lowering the recovery rate as in Example 1, and that the heat treatment can be suitably performed.

Example 3 500 g of a precipitate of corn fraction FIV-1 was added to a pH of 8.8 at 0.8.
1M Tris-HCl / 0.02M Sodium Chloride Buffer 9.0
It was dissolved in 1. 50% polyethylene glycol 4,000 solution was added to this solution to make 15%, and then p
Adjust H to 6.5 and stir at 4 ° C for 1 hour,
Then, it was left to stand for 2 hours. Then it was centrifuged and the supernatant was collected. Polyethylene glycol 4,000 was added to this supernatant to a final concentration of 30%.
Stir at ℃ for 3 hours, then centrifuge and precipitate fraction 200g
Got The obtained precipitate fraction was dissolved by adding 800 ml of a 15 mM phosphate / acetate buffer solution having a pH of 5.2 so that the protein concentration was 2%. The lysate was developed on SP-Sepharose (First Flow 300: Pharmacia), which had been equilibrated with the above buffer,
After collecting the non-adsorbed fraction, the pH of the solution was adjusted to 7.0. Then, using an ultrafiltration membrane, concentration was carried out to a protein concentration of 2% to prepare an α1PI concentrated liquid. Trisodium citrate and sucrose were added to the α1PI concentrated solution so that the final concentrations were 0.3 M and 40% (W / V), and dissolved with stirring. After confirming that the added stabilizer was completely dissolved, heat treatment was carried out at 60 ° C. for 10 hours. The results obtained are shown in Table 9. Further, when the purity of the α1PI concentrated solution obtained in this Example was checked by SDS gel electrophoresis, a purity of about 80% was obtained, and the α1PI concentrated solution was purified to a purity that does not hinder practical use. Was found (see Figure 3).

[0035]

[Table 9] * 1 Concentration was expressed as mg / ml by measuring the amount of α1PI having the ability to inhibit elastase. * 2 Specific activity was expressed as the value obtained by dividing the amount of α1PI (mg / ml) by A280. * 3 The refining factor is expressed as the value obtained by dividing the specific activity of the raw material liquid by the specific activity of other processes.

In this example, the FIV-1 solution was used as a raw material in the form of P.
Crude fractionation was performed using EG, and the precipitated fraction after fractionation was applied to cation exchange chromatography.
The recovery rate and the degree of purification were evaluated. The results show that, as in the case of Examples 1 and 2, the α1PI-containing fraction can increase the degree of purification by cation exchange chromatography alone.

[0037]

[Brief description of drawings]

FIG. 1 is an electrophoretic diagram (drawing-substituting photograph) showing the purity of an α1PI concentrated liquid obtained according to the present invention.

FIG. 2 is an electrophoretic diagram (drawing-substituting photograph) showing the purity of the α1PI concentrated solution obtained according to the present invention.

FIG. 3 is an electrophoretogram (drawing-substituting photograph) showing the purity of an α1PI concentrated solution obtained according to the present invention.

Claims (5)

[Claims] 1. A solution containing an α1 protease inhibitor (hereinafter sometimes referred to as α1PI) is developed on a cation exchange resin to adsorb a contaminated protein.
A method for producing an α1 protease inhibitor, which comprises separating α1PI into a non-adsorbed fraction and collecting the fraction. 2. A plasma protein solution containing (a) α1PI is treated by at least one means selected from dilution, dialysis and gel filtration chromatography to reduce the concentration of salts in the solution to about 0.02 M or less. (B) Next, the pH of the solution is adjusted to 4.5 to 5.5, and (c) the above solution is
2. A step of contacting with a cation exchange resin having a selective affinity for a contaminated impure protein other than α1PI in the solution, separating α1PI into a non-adsorbed fraction, and collecting the fraction.
A method for producing the described α1 protease inhibitor. 3. The method for producing an α1 protease inhibitor according to claim 1 or 2, wherein the plasma protein solution containing α1PI is Fraction IV or Fraction IV-1 prepared by the Cohn's ethanol separation method or a modification thereof. . 4. The α according to claim 1 or 2, further comprising a step of performing a heat treatment for inactivating infectious contaminant viruses in the presence of sucrose and citrate ions.
1. A method for producing a protease inhibitor. 5. The method for producing an α1 protease inhibitor according to claim 4, wherein the α1PI solution is heated at a temperature of 60 ° C. or higher for 10 hours under the condition of pH 6.0 to 7.0.