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A kind of separation and purification method of urokinase
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A technology for separation and purification and urokinase, which is applied in the field of urokinase purification, can solve problems such as inability to meet clinical applications, and achieve the effect of reducing environmental pollution
Active Publication Date: 2018-04-17
JIANGSU YOULIKA BIOTECHNOLOYG CO LTD
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Problems solved by technology
This method avoids the use of strong acid and strong alkali, can effectively separate endotoxin, and guarantees that urokinase is not destroyed simultaneously, and separation effect can reach pharmacopoeia standard requirement, and yield is more than 97%, but this method can only remove endotoxin in the crude product, but The ratio of large and small molecule urokinase is less than 60%, which cannot meet the needs of clinical application
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Embodiment 1
[0041] A method for separating and purifying urokinase, comprising the steps of:
[0042] 1) Preparation of crude urokinase
[0043] a) Male urine, adjust the pH to 8.5 at a temperature below 10°C, let it stand for 2 hours, discard the precipitate, and obtain the supernatant;
[0044] b) Take the supernatant, adjust the pH to 5.0-5.3 for acidification, and obtain acidified urine, which is adsorbed through a diatomite column;
[0045] c) washing the column with cold water at 5°C, and eluting with 0.02% ammonia water and 0.2M sodiumchloride solution to obtain eluent 1;
[0046] d) Eluent 1 is applied to a CM-C column under the condition of pH 4-4.2, and then eluted with ammonia water and 0.3M sodiumchloride solution of pH 11.5-11.8 to obtain eluent 2, which is frozen Dry to obtain crude urokinase.
[0047] 2) Purification of urokinase
[0048] 21) The crude product of urokinase was dissolved in pyrogen-free water with pH 6.5 to obtain a concentration of 2×10 5 IU / L clarif...
Embodiment 2
[0053] A method for separating and purifying urokinase, comprising the steps of:
[0054] The only difference from Example 1 is that in step 23), the clarified solution in step 21) is passed through the CM-agarosemicrosphere column at a speed of 80 mL / min.
[0055] Through measurement and calculation, the specific activity of the obtained purified urokinase is 23,000 IU / mg protein, the yield reaches 87%, and the proportion of large and small molecule urokinase is 86%.
Embodiment 3
[0057] A method for separating and purifying urokinase, comprising the steps of:
[0058] The only difference from Example 1 is that in step 23), the clarified solution in step 21) is passed through the CM-dextran microsphere column and the CM-agarose microsphere column at a speed of 80 mL / min in sequence.
[0059] Through measurement and calculation, the specific activity of the obtained purified urokinase is 22,000 IU / mg protein, the yield reaches 84%, and the ratio of large and small molecule urokinase is 90%.
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Abstract
The application belongs to the technical field of urokinase purification and specifically relates to a separation and purification method of urokinase. The method comprises the following steps: 1) preparation of a urokinase crude product; and 2) purification of urokinase. The step 2) comprises the following step: clear liquor obtained after the prepared urokinase crude product is dissolved is purified through a dextranmicrosphere column and / or an agarosemicrosphere column. By the separation and purification method provided by the application, specific activity of urokinase can be increased to 20,000 IU / mg proteins and above, yield reaches 75-87%, and the proportion of macromolecular and micromolecular urokinases is greater than 85%. As the use of strong acid and strong base is avoided during the separation and purification process of urokinases, environmental pollution is reduced.
Description
technical field [0001] The invention belongs to the technical field of urokinase purification, and in particular relates to a method for separating and purifying urokinase. Background technique [0002] Urokinase (UK for short) is an alkaline protease produced by the kidneys and mainly exists in human and mammalian urine, with an average human urine content of 5-6iu / ml. It is a serineprotease that can specifically recognize plasminogen and catalyze its transformation into plasmin. This enzyme breaks down insoluble fibrin into small soluble peptides, thereby dissolving thrombus. Therefore, clinically, UK is often used to dissolve thrombus, prevent and control myocardial infarction, hypertension, arteriosclerosis, etc. Urokinase has a variety of molecular weight forms, mainly in two forms, one is high molecular weight (54700, HUK) urokinase, and the other is low molecular weight (31300, LUK) urokinase. The two molecular weight forms of UK have the same mechanism of action ...
Claims
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Application Information
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