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Method for separating urokinase from human urinary trypsin inhibitor

A trypsin inhibition and separation method technology, applied in the separation field of urokinase and human urinary trypsin inhibitor, can solve the problems of long cycle, decreased product quality, large activity loss, etc., and achieves short process time, low cost, improved live effect

Active Publication Date: 2013-05-01
YANGZHOU AIDEA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Some people try to recover UK and UTI by re-dissolving, and then processing with silica gel and other adsorbents, but the cycle is long and the activity loss is very large, reaching more than 30%. The quality of the product has seriously declined

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Mix 10 grams of UK crude product (31,500 U / g) and 10 grams of UTI crude product (23,300 U / g), add 100ml of deionized water, stir to dissolve, centrifuge to collect 92ml of supernatant, and use 1N NaOH solution to adjust the pH to 8.4; slowly add 16% (w / v) ammonium sulfate powder, while adding ammonium sulfate powder, stir to dissolve it, and protein precipitation appears in the solution, then add 4.6g ammonium sulfate powder, stir After dissolving, adjust the pH to 8.4 with 1N NaOH solution, and let it stand for 2 hours; centrifuge at 3000rpm for 10 minutes, collect the precipitate 1, continue to add ammonium sulfate powder to the supernatant to 60% (w / v), let it stand for 2 hours, 3000rpm Centrifuge for 10 minutes to collect pellet 2.

[0028] The activities of UK and UTI in precipitation 1 and precipitation 2 were determined, see Table 1.

[0029] Table 1:

[0030] UK total activity (u) UTI total activity (u) start 3.15×10 5 2.33×10 5 Pr...

Embodiment 2

[0033] Mix 10 grams of UK crude product (37,200 U / g) and 10 grams of UTI crude product (19,500 U / g), add 100ml of deionized water, stir to dissolve, centrifuge to collect 93ml of supernatant, and use 1N Adjust the pH to 9.6 with NaOH solution; slowly add 30% (w / v) ammonium sulfate powder, while adding ammonium sulfate powder, stir to dissolve it, then adjust the pH to 9.6 with 1N NaOH solution, and let it stand for 6 hours; 3000rpm Centrifuge for 10 minutes to collect the precipitate 1, continue to add ammonium sulfate powder to the supernatant to 55% (w / v), let stand for 2 hours, then centrifuge at 3000rpm for 10 minutes, and collect the precipitate 2.

[0034] The activities of UK and UTI in precipitation 1 and precipitation 2 were determined, see Table 2.

[0035] Table 2:

[0036] UK total activity (u) UTI total activity (u) start 3.72×10 5 1.95×10 5 Precipitation 1 3.59×10 5 2.63×10 4 Precipitation 2 not detected 1.57×10 5

...

Embodiment 3

[0039] Add 10.2kg of UK crude product mixed with UTI, add 50kg of deionized water, stir to dissolve, centrifuge to collect 46L of supernatant, adjust the pH of the supernatant to 9.0 with 1N NaOH solution; slowly add saturated ammonium sulfate solution, Stir while adding, until obvious protein precipitation appears in the solution, then add 3.5L saturated ammonium sulfate solution, adjust the pH to 9.0 with 1N NaOH solution, and let it stand for 2 hours; add diatomaceous earth for plate and frame filtration, and collect the precipitate 1 , add ammonium sulfate powder to the supernatant to 60% (w / v), let it stand for 6 hours, add diatomaceous earth for plate and frame filtration, and collect the precipitate 2. Ammonia solution can also be used to adjust the pH.

[0040] The activities of UK and UTI in precipitation 1 and precipitation 2 were determined, see Table 3.

[0041] table 3:

[0042] UK total activity (u) UTI total activity (u) start There is in...

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Abstract

The invention discloses a method for separating urokinase from a human urinary trypsin inhibitor. The method comprises the following steps: regulating pH of a solution containing the urokinase and the human urinary trypsin inhibitor; slowly adding ammonium sulfate powder while stirring until the solution generates obvious protein precipitation; regulating the pH to 9.0 plus or minus 0.6; after standing, centrifuging or adding diatomite for filtering, collecting the precipitation rich in the urokinase, and obtaining the urokinase; continuing to adding ammonium sulfate into supernatant while stirring to dissolve the ammonium sulfate; and after standing, centrifuging or adding diatomite for filtering, collecting the precipitation 2 rich in the human urinary trypsin inhibitor, and obtaining the human urinary trypsin inhibitor. The method is simple, convenient and feasible and has a short period, the UK and UTI activity is effectively protected, the UK degradation is reduced, the UK quality is increased, special equipment is not needed, and the invested cost of the equipment is reduced.

Description

technical field [0001] The invention relates to the technical field of purification process of urokinase and human urinary trypsin inhibitor and production of crude product, in particular to a separation method of urokinase and human urinary trypsin inhibitor. Background technique [0002] Male urine is an important source of human protein. At present, the two largest medicinal protein products in the market extracted from male urine are urokinase and human urinary trypsin inhibitor, which have important clinical therapeutic value and have received a wide range of applications. [0003] Urokinase (Urokinase, abbreviated as UK) is a clinically important thrombolytic drug, which is widely used in the treatment of thrombotic diseases in cardiovascular, cerebrovascular and peripheral blood vessels. It is mainly extracted from human urine. There are two forms of UK in human urine, high molecular weight (HUK) and low molecular weight (LUK). Since LUK is likely to cause side effec...

Claims

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Application Information

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IPC IPC(8): C12N9/72C07K14/81C07K1/30
Inventor 苗丕渠苏古方许冬至沈小宁
Owner YANGZHOU AIDEA BIOTECH
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