KR20020007231A - The purification of streptokinase - Google Patents

Abstract

PURPOSE: A process for purifying streptokinase is provided, thereby producing the high purity of streptokinase without streptolysin, endotoxin or pyrogen. CONSTITUTION: The process for purifying streptokinase comprises the steps of: cultivating Streptococcus, removing the cell and concentrating the culture; passing it through the column packed with cation or anion exchange resins to remove impurities and streptodornase; and treating the concentrate with cholesterol to remove streptolysin, in which the concentration of cholesterol ranges from 0.0050.05%, its temperature is maintained to 35 to 47 deg.C for 10 to 20 minutes after treating the concentrate with cholesterol, and 0.05 o 0.15 mole of phosphate buffer adjusted to pH 6.0 to 8.0 is used for removing streptodornase.

Description

Purification method of streptokinase {The purification of streptokinase}

The present invention relates to a method for purifying streptokinase derived from Streptococcus microorganisms. More specifically, the final product that is finally purified for use as a human injectable agent has adverse effects on not only streptodonase but also various impurities, in particular, the human body. The streptocin (hemotoxin, streptolysin 0), endotoxin, pyrogen, etc. should be removed and only streptokinase should be present in high concentration. The present invention effectively removes these substances and purifies only streptokinase with high purity. To provide a way.

Streptokinase is a protein produced by the microorganisms of the hemolytic Streptococcus , a polypeptide with a molecular weight of 46,000 to 50,000, and has the ability to activate plasminogen, a blood component, by plasmin. It acts as a thrombolytic factor. For this reason, Streptokinase was approved by the FDA for intravenous injection in 1987 and used as a thrombolytic drug for acute myocardial infarction, acute pulmonary embolism, and acute arterial occlusion in the US, Europe, and South America. In recent years, the production and development of streptokinase complexes with excellent clinical effects and resistance have been achieved, and thus the use of streptokinase raw materials has become important.

Existing methods for purifying streptokinase are methods using solubility, electrical properties, differences in molecular size and shape, and nonspecific physical interactions (US Pat. No. 3,098,015 / US Pat. No. 3,226,304). Purification method developed on the basis of the absence of cystine residues (EP 365,278 A2, 1989) or purification of streptokinase using a gel consisting of a matrix comprising a hydrophobic ligand such as phenylsepharose (EP 362,696 A1, 1990 ) Is known.

However, these known methods have disadvantages that are difficult to use for clinical purposes because they do not effectively remove excessive loss of streptokinase and more importantly, impurities and harmful substances such as streptolysin and endotoxin.

Under these circumstances, the present inventors studied methods for removing such harmful substances (streptolysin, endotoxin, pyrogen, etc.) while devising the following methods to enable the use of injectables. . First, a process step of removing cholesterol, which has been treated to remove streptodonase, impurities, and streptocin by using ion chromatography using cation and anion exchange resin, and second, cholesterol to concentrate in order to remove streptocin. Process step of adding and applying heat treatment, and third, devising and inventing a process step for removing endotoxin and exothermic material using an ultrafiltration membrane having a difference in membrane molecular weight limit, thereby preparing a thrombolytic agent containing streptokinase for injection. It is.

In particular, when cholesterol is added to remove streptocin, streptokinase is hardly lost and streptocin is 0.05 IU-0.15 IU, which is significantly lower than the level required by the European Pharmacopoeia (streptocin 1 IU or less, streptokinase 1,000,000 IU). Show the level.

It is an object of the present invention to provide a method for purifying streptokinase with high purity and effectively removing streptolysin, endotoxins, and pyrogens.

Other objects and applications of the present invention will become apparent to those skilled in the art from the following detailed description of the invention.

This will be described in more detail the present invention. The process steps described below are practical steps that can be applied at actual production levels.

When the microorganism of Streptococcus genus is cultured, the streptokinase does not exist in the cells but is secreted into the culture medium. Therefore, in order to recover and purify it, centrifugation is performed for about 8-10 hours at a speed of 8,000 rpm to remove the fungicide. At this time, the temperature is maintained at 4 ℃ to eliminate the risk of lowering the activity of the enzyme due to the centrifugation rate and the temperature rise with time. Cells and cell debris that could not be effectively removed during centrifugation can be effectively removed by using a membrane filtration device having a pore size of 0.22 μm.

After removing the cells, the supernatant is concentrated and purified. First, the membrane is subjected to concentration using an ultrafiltration membrane with a molecular weight limit of about 20,000, thereby discarding substances having a molecular weight lower than that. The concentrated solution was passed through an anion exchange resin (DEAE, D i e thyl a mino e thyl) separates the Streptomyces D'xylanase and streptomycin kinase. A characteristic of Streptococcus microorganisms producing Streptokinase is that it secretes not only Streptokinase but also Streptodonase in culture. Therefore, in order to obtain high-purity streptokinase of thrombolytics, streptodonase must be removed, which is performed in this process step. When the concentrate is passed through an anion exchange resin, streptokinase and streptodonase are adsorbed on the resin surface, and these two substances are effectively separated by the following method. First, when passed through 0.12 mole of phosphate buffer solution adjusted to pH = 7.2, only streptodonase elutes. It can be discarded or stored separately and used in the manufacture of anti-inflammatory agents. Then clean the column by passing 0.03-0.05 mole of phosphate buffer adjusted to pH = 7.2, and then pass 0.15 mole of phosphate buffer adjusted to pH = 7.2 to elute streptokinase. Comes out. The eluted liquor is reconcentrated using an ultrafiltration membrane with a molecular weight limit of 10,000. This concentrate is passed through a cation exchange resin (CM, carboxy methyl) to adsorb and remove impurities contained in the concentrate. The buffer should be replaced with 0.03 mole phosphate solution with pH = 5.5. This is because CM (carboxy methyl) resins function in the pH range of 5.5. The membrane is then concentrated again using an ultrafiltration membrane with a molecular weight limit of around 10,000. The concentrate is then treated with cholesterol to remove the hemolytic toxin, streptolysin. The treatment concentration of cholesterol is treated at a concentration of about 2-5 grams in a 20 liter concentrate volume. At this time, cholesterol is not soluble in water, so dissolve in ethanol and treat. After this treatment, streptocin is removed by maintaining the temperature of the concentrate at 42 ° C. for 15 minutes. In this way, the yield of streptokinase is about 99% higher than before treatment, and the level of 0.1 IU (per 1,000,000 IU of streptokinase) has established a very effective process step. After 15 minutes, the temperature is cooled again. Next is passed through the anion exchange resin (DEAE, D i e thyl a mino e thyl) for the removal of cholesterol was used for Streptomyces lysine removed.

The eluate is passed through two kinds of ultrafiltration membranes with molecular weight limits of 10,000 and 100,000, which removes impurities (endotoxins, pyrogens, etc.) and concentrates streptokinase. That is, the molecular weight of endotoxins and pyrogens is usually 100,000 or more, and the molecular weight of streptokinase is usually about 46,000-50,000, so when passing through ultrafiltration membranes with a membrane molecular weight limit of 100,000, impurities are not passed through the membrane and the streptokinase is eluted. Comes out. When these eluents are passed through an ultrafiltration membrane with a membrane molecular weight limit of 10,000, the streptokinase is concentrated through the membrane. The buffer used in this process is 0.06 mole of phosphate buffer adjusted to pH = 7.2. This impurity removal and streptokinase concentration process is repeated until the concentrate specification meets the following conditions. In other words, less than 1 IU of streptodonase / 10,000 IU of streptokinase, less than 1 IU of streptocin / 1,000,000 IU of streptokinase, less than 8 IL of bacterial endotoxin / 1,000,000 IU of streptokinase, must meet the conditions for the detection of pyrogen . This concentrated solution will usually contain more than 4,000,000 IU of streptokinase / 1 ml of concentrate and its activity specificity is more than 120,000 IU / 1 mg protein.

A simple flow chart of the streptokinase production process above is as follows and it will be clear that this process step is the right of the skilled person.

Fermentation

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Centrifuge [Cell Removal Process]

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MF (membrane filtration) [Cellulum and cell debris removal process]

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UF (ultrafiltration) [membrane molecular weight limit 20,000 / concentration process]

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DEAE (Anion Exchange Resin) [Streptoronase Removal Process]

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UF [Membrane Molecular Weight Limit 10,000 / Concentration]

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Buffer exchange (phosphate buffer solution with pH 7.2 adjusted to 5.5)

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CM (Cation Exchange Resin) [Impuration Process]

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UF [Membrane Molecular Weight Limit 10,000 / Concentration]

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Cholesterol Treatment [Streptoricin Removal Process]

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DEAE [cholesterol removal process]

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UF [Membrane molecular weight limit 100,000 / endotoxin and pyrogen removal process]

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UF [Membrane Molecular Weight Limit 10,000 / Concentration]

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UF [Membrane molecular weight limit 100,000 / endotoxin and pyrogen removal process]

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UF [Membrane molecular weight limit 100,000 / endotoxin and pyrogen removal process]

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UF [Membrane Molecular Weight Limit 10,000 / Concentration]

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Claims (4)

A method for purifying streptokinase, comprising culturing Streptococcus strains, removing the cells and concentrating them, passing through a cation and an anion exchange resin to remove impurities and streptodonase, and removing streptocin by purifying the concentrate with cholesterol. . The method of claim 1, wherein the concentration of cholesterol to be treated for removal of streptolysin is in the range of 0.005-0.05%. The method of claim 1, wherein the concentration of the concentrate is maintained in the range of 35-47 ° C. for 10-20 minutes after cholesterol treatment of the concentrate for removal of streptolysin. Characterized in that the use of phosphate buffer solution adjusted to a concentration range of 0.05-0.15 mole and a pH adjusted to a range of 6.0-8.0 for removal thereof.