CN111748545B

CN111748545B - Method for adsorbing urokinase in urine by using agarose gel 4B

Info

Publication number: CN111748545B
Application number: CN202010656590.9A
Authority: CN
Inventors: 顾京; 李争; 刘艳红; 郭芬; 唐维; 魏飘飘
Original assignee: Jiangsu Youlika Biological Technology Co ltd
Current assignee: Henan Eureka Biotechnology Co ltd
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2022-09-30
Anticipated expiration: 2040-07-09
Also published as: CN111748545A

Abstract

The invention discloses a method for adsorbing urokinase in urine by using agarose gel 4B, and relates to the technical field of separation and extraction. The method comprises the following steps: (1) putting the agarose gel 4B affinity bag into a filter flask, and enabling urine to flow into the filter flask through the agarose gel 4B affinity bag; (2) taking out the agarose gel 4B affinity bag after the urine flow is finished, filling the agarose gel 4B affinity bag into a chromatographic column, washing the chromatographic column by using a buffer solution, then desorbing the affinity bag by using a desorption solution, and collecting the affinity bag when the color of the elution solution is changed, thus obtaining a urokinase solution; (3) and (3) carrying out freeze drying on the urokinase solution obtained in the step (2) to obtain urokinase powder. The method for adsorbing urokinase in urine provided by the invention does not need to treat urine, is simple to operate, and has good yield and high purity of the obtained urokinase.

Description

Method for adsorbing urokinase in urine by using agarose gel 4B

Technical Field

The invention relates to the technical field of separation and extraction, and particularly relates to a method for adsorbing urokinase in urine by using agarose gel 4B.

Background

Urokinase is a thrombolytic drug extracted from fresh human urine, is a serine protease produced by human renal tubular epithelial cells, is a basic protein, has an isoelectric point of about pH8.7, is white amorphous powder, and is easily soluble in water. The dilute solution has unstable property, needs to be prepared fresh, and cannot be diluted by an acid solution, and the freeze-dried state can be stable for years. Urokinase is a very specific proteolytic enzyme. The activity of the synthetic substrate is similar to that of trypsin and plasmin, and the synthetic substrate also has esterase activity, has no antigenicity and does not generate antibodies in vivo. The half-life period in vivo is (14 +/-6) min.

Urokinase activates plasminogen to active plasmin, which converts insoluble fibrin to soluble peptides, thereby dissolving the thrombus. Therefore, it is clinically used for treating thrombosis, thromboembolism and other diseases. When urokinase is combined with an anticancer agent, the urokinase can dissolve fibrin around cancer cells, so that the anticancer agent can penetrate into the cancer cells more effectively, thereby improving the capability of the anticancer agent in killing the cancer cells. Therefore, urokinase is also a good cancer adjuvant, and it has no problem of antigenicity and can be used for a long time.

As urokinase has important medical value, a plurality of special journals report methods for extracting crude urokinase, and because the urokinase content in human urine is very low, the key point of taking urokinase is how to enrich urokinase from a large amount of urine, and from the prior published documents, three methods are mainly provided: (1) and a foaming method: stirring at high speed to foam the enzyme solution, liquefying the foam, and adding ammonium sulfate to precipitate urokinase; (2) the precipitant method: adding a precipitating agent into urine to precipitate urokinase, wherein the urokinase is remained in the precipitate; (3) adsorbent, the most used in this method, and the selection of a suitable adsorbent for selective adsorption of urokinase.

Chinese patent application 201510549397.1 discloses a method for enriching urine protein by directly adsorbing urine protein in urine in a urinal or a urinal through a filter cloth bag containing modified silica gel, and transporting the filter cloth bag adsorbing urine protein to a processing point for subsequent treatment. The method utilizes the isoelectric point property of specific urine protein, and uses modified silica gel, macroporous resin, chitin, ion resin, etc.; directly and effectively adsorb urine proteins such as urine trypsin inhibitor, human urine kininogenase, urokinase and the like, and the step of collecting urine is avoided. The activity of the lyophilized powder obtained by the method is 4.12 × 10 ⁵ IU, which does not meet the requirements.

Chinese patent application 201510691424.1 provides a method for extracting urokinase and ulinastatin from urine, comprising: collecting urine and adjusting the pH value of the urine; adding powdered silica gel into urine and stirring to make urokinase protein in urine adsorbed by silica gel; obtaining silica gel by a filtering mode and adjusting the pH value of the filtered urine to obtain urokinase protein liquid from the silica gel; adding urokinase protein liquid into saturated water to extract urokinase, and adding ammonium sulfate into the ulinastatin protein liquid to extract ulinastatin. The invention can extract crude urokinase and crude ulinastatin from urine, thereby greatly improving the economic value of urine. However, urokinase is also eluted during the adsorption process, thereby causing a loss of urokinase, thereby decreasing the yield of urokinase.

For collecting urokinase from human urine, how to improve the extraction rate of urine protein in urine is very important, and therefore, the development of a method for improving the yield of urokinase is urgently needed. In view of this, in order to solve the problems in the prior art, the present invention provides a method for adsorbing urokinase in urine by using agarose gel 4B, which can significantly increase the yield of urokinase and improve the purity of urokinase.

Disclosure of Invention

The invention aims to provide a method for adsorbing urokinase in urine by using agarose gel 4B, which can obviously improve the yield of urokinase and the purity of urokinase.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a method for adsorbing urokinase in urine by agarose gel 4B comprises the following steps:

(1) putting the agarose gel 4B affinity bag into a filter flask, and enabling the urine to flow into the filter flask through the agarose gel 4B affinity bag;

(2) taking out the agarose gel 4B affinity bag after the urine flow is finished, filling the agarose gel 4B affinity bag into a chromatographic column, washing the chromatographic column by using a buffer solution, then desorbing the affinity bag by using a desorption solution, and collecting the affinity bag when the color of the elution solution is changed, thus obtaining a urokinase solution;

(3) and (3) carrying out freeze drying on the urokinase solution obtained in the step (2) to obtain urokinase powder.

Preferably, in the step (1), the volume ratio of the urine to the agarose gel 4B is 400000-600000:15, and more preferably 500000: 15.

Preferably, in step (1), the flow rate of urine passing through the reaction system is 1-10mL/min, more preferably 4 mL/min.

Preferably, in the step (2), the chromatographic column is selected from any one of BPG/GE, Chromaflow/GE, Axichrom/GE, Quick Scale/Merck, SAC-BIO/Lisui technology, ABH-BIO/Lisui technology, EAC-BIO/Lisui technology, XK16/20/GE and GCC/Lisui technology, and is further preferably XK 16/20/GE.

Preferably, in the step (2), the volume ratio of the sepharose 4B to the chromatography column is 0.1-0.5: 1, more preferably 0.3: 1.

Preferably, in the step (2), the buffer is at least one selected from the group consisting of a phosphate buffer, a Tris-HCl buffer, an ammonium sulfate buffer, a citrate buffer, an acetate-Tris buffer, and a glycine-Tris buffer, preferably the phosphate buffer, and more preferably 0.02M phosphate buffer.

Preferably, in step (2), the buffer is used in an amount of 2 to 4 column volumes, preferably 3 column volumes.

Preferably, in the step (2), the desorption solution is a mixed solution containing glacial acetic acid and a sodium chloride solution; the volume ratio of the glacial acetic acid to the sodium chloride solution is 3:400-600, and is further preferably 3: 500; the concentration of the sodium chloride solution is 0.01 to 0.03g/mL, and more preferably 0.02 g/mL.

Preferably, in the step (2), the amount of the stripping solution is 1.5 to 2.5 column volumes, and more preferably 2 column volumes.

Preferably, in the step (2), the flow rate of the stripping solution is 1 to 4mL/min, and more preferably 2.5 mL/min.

Preferably, in the step (2), the color change means that the color changes from light gray to dark gray or from light yellow to dark yellow.

Preferably, in the step (3), the freeze-drying specifically means: putting the urokinase solution obtained in the step (2) into a freeze dryer, freezing for 2-4 hours at the temperature of 35-55 ℃, then heating to room temperature, and keeping for 1.5-2.5 hours; further preferably, the freeze-drying specifically means: and (3) putting the urokinase solution obtained in the step (2) into a freeze dryer, freezing the urokinase solution for 3 hours at minus 45 ℃, and then heating the solution to room temperature for 2 hours.

Compared with the prior art, the invention has the beneficial effects that:

(1) according to the method for adsorbing urokinase in urine, urine does not need to be processed, collected urine directly penetrates through the urine and the agarose gel 4B, then the urine and the agarose gel 4B are filled into a chromatographic column, and urokinase can be obtained through elution, so that the operation is simple, and the loss of urokinase is well reduced;

(2) the method of the invention can separate urokinase better, reduce the loss of urokinase, and obtain urokinase with good yield and high purity.

Drawings

FIG. 1 is a map of a urokinase solution obtained in example 1, in which 02 is a target peak;

FIG. 2 is a urokinase powder pattern obtained in example 1, in which 02 is the target peak.

Detailed Description

The present invention will be further explained with reference to specific embodiments in order to make the technical means, the original characteristics, the achieved objects and the effects of the present invention easy to understand, but the following embodiments are only preferred embodiments of the present invention, and not all embodiments are possible. Based on the embodiments in the implementation, other embodiments obtained by those skilled in the art without any creative efforts belong to the protection scope of the present invention.

The experimental methods in the following examples are conventional methods unless otherwise specified, and materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

In the following examples, Sepharose 4B affinity pack is an affinity chromatography gel prepared by this company using Sepharose 4B, Sepharose 4B was purchased from GE, cat # 17-0120-05.

Example 1

(1) Putting 15mL of sepharose 4B affinity bag into a filter bottle, taking 500L of urine, passing through the sepharose 4B affinity bag at the flow rate of 4mL/min, and flowing into the filter bottle, wherein the urine detection is 380 thousand iu in total;

(2) taking out the agarose gel 4B affinity bag after the urine flow is finished, loading the agarose gel 4B affinity bag into a chromatographic column (XK16/20/GE), pressing tightly, enabling the volume ratio of the agarose gel 4B to the chromatographic column to be 0.3:1, washing the agarose gel 4B and the chromatographic column by using a 3-fold volume of 0.02M phosphate buffer, then using 2-fold volume of stripping solution (1L of 0.02g/mL sodium chloride solution and 6mL of glacial acetic acid) to strip, enabling the flow rate of the stripping solution to be 2.5mL/min, and collecting the stripping solution when the color of the stripping solution is changed from light ash to dark ash to obtain a urokinase solution;

(3) and (3) putting the solution collected in the step (2) into a freeze dryer, freezing for 3 hours at minus 45 ℃, then heating to room temperature, carrying out vacuum freeze drying for 2 hours, and taking out to obtain urokinase powder.

Example 2

(1) Putting 15mL of the agarose gel 4B affinity bag into a filter bottle, taking 400L of urine, passing through the agarose gel 4B affinity bag at the flow rate of 1mL/min, and flowing into the filter bottle, wherein the urine detection is 302 thousand iu in total;

(2) after the urine flow is finished, taking out the sepharose 4B affinity bag, putting the sepharose 4B affinity bag into a chromatographic column (GCC/ear-benefiting technology), pressing tightly, wherein the volume ratio of the sepharose 4B to the chromatographic column is 0.1:1, washing the sepharose 4B and the chromatographic column by using 2 times of 0.02M phosphate buffer solution, then desorbing the sepharose 4B and the chromatographic column by using 1.5 times of desorption solution (0.8L of 0.02g/mL sodium chloride solution and 6mL of glacial acetic acid), wherein the flow rate of the desorption solution is 1mL/min, and collecting the desorption solution when the color of the desorption solution is changed from light ash to dark ash to obtain a urokinase solution;

(3) and (3) putting the solution collected in the step (2) into a freeze dryer, freezing for 4 hours at the temperature of minus 35 ℃, then heating to room temperature, carrying out vacuum freeze drying for 1.5 hours, and taking out to obtain urokinase powder.

Example 3

(1) Putting 15mL of the agarose gel 4B affinity bag into a filter bottle, taking 600L of urine, passing through the agarose gel 4B affinity bag at the flow rate of 10mL/min, flowing into the filter bottle, and detecting the urine by 458 thousand iu;

(2) taking out the sepharose 4B affinity bag after the urine flow is finished, loading the sepharose 4B affinity bag into a chromatographic column (XK16/20/GE), pressing tightly, enabling the volume ratio of the sepharose 4B to the chromatographic column to be 0.5:1, washing the sepharose 4B and the chromatographic column by using a 4-fold volume of 0.02M phosphate buffer, then using 2.5-fold volume of stripping liquid (1.2L of 0.02g/mL sodium chloride solution and 6mL of glacial acetic acid) to strip, enabling the flow rate of the stripping liquid to be 4mL/min, and collecting the stripping liquid when the color of the stripping liquid is changed from light ash to dark ash to obtain a urokinase solution;

(3) and (3) putting the solution collected in the step (2) into a freeze dryer, freezing for 2 hours at the temperature of minus 55 ℃, then heating to room temperature, carrying out vacuum freeze drying for 2.5 hours, and taking out to obtain urokinase powder.

Comparative example 1

In contrast to example 1, agarose gel 4B was replaced with the same amount of silica gel, and the rest was the same.

Comparative example 2

In contrast to example 1, the same amount of chitin was used in place of Sepharose 4B, and the rest was the same.

Comparative example 3

Unlike example 1, the amount of the solution used was 4 column volumes, and the rest was the same.

Comparative example 4

Unlike example 1, the urine flow rate was 0.6mL/min, and the rest was the same.

Test example 1

The urokinase powder obtained in the examples and the comparative examples was diluted by 10mg according to the 2020 edition [ Chinese pharmacopoeia ] test method, and the activity content was measured, and the yield was calculated according to the following formula:

the yield ═ yield (enzyme solution/powder total active content ÷ urine total active content) × 100%

The test results of the examples and comparative examples are shown in table 1:

table 1.

As can be seen from Table 1, the yield of the embodiment of the invention is high, wherein the yield of the urokinase solution activity in the embodiment 1 reaches 72%, and the yield of the urokinase powder activity in the embodiment 1 reaches 71%. From comparative examples 3 and 4, it can be seen that the yield is not significantly changed by increasing the amount of the stripping solution or decreasing the flow rate of the urine, and the amount and flow rate in example 1 are preferred from the viewpoint of cost and time.

Test example 2

Referring to example 1, the spectrum obtained by high performance liquid chromatography according to standard method of the pharmacopoeia of 2015 edition is shown in figure 1 and figure 2.

The spectra of FIG. 1 were analyzed as shown in Table 2:

table 2.

Peak number	Name of peak	Retention time	Peak height	Peak area	Content (wt.)
						1	-	21.484	113535.961	13281305.000	23.8550
2	-	28.338	96910.352	11423309.000	20.5178
						3	02	33.173	184770.750	27426234.000	49.2613
4	-	39.777	11329.014	927477.750	1.6659
						5	-	41.444	9600.839	1409818.875	2.5322
6	-	45.313	5178.754	531627.375	0.9549
						7	-	47.447	7329.811	675278.313	1.2129
Total of	-	-	428655.480	55675050.313	100.0000

As shown in Table 2, the retention time of the target peak 02 was 33.173, and the integrated relative content of the chromatogram was 49.26%, which shows that the urokinase adsorbed by the present invention has a greatly improved purity as compared with silica gel (purity of 10% or less).

The spectra of FIG. 2 were analyzed as shown in Table 3:

table 3.

Peak number	Name of peak	Retention time	Peak height	Peak area	Content (wt.)
						1	-	21.542	99471.438	10422151.000	24.9912
2	-	28.504	89094.477	9665433.000	23.1767
						3	02	33.273	158751.172	20800140.000	49.8766
4	-	40.010	5182.828	745367.688	1.7873
						5	-	45.213	1431.538	70125.602	0.1682
Total of	-	-	353931.452	41703217.289	100.0000

As can be seen from Table 3, the data repeatability is completely consistent with that of Table II, which proves that the repeatability of the invention is good and can be used for scale-up production.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

1. A method for adsorbing urokinase in urine by using agarose gel 4B is characterized by comprising the following steps:

(1) putting the agarose gel 4B affinity bag into a filter flask, and enabling the urine to flow into the filter flask through the agarose gel 4B affinity bag; the volume ratio of the urine to the agarose gel 4B is 400000-600000:15, and the flow rate of the urine passing through is 1-10 mL/min;

(2) taking out the agarose gel 4B affinity bag after the urine flow is finished, filling the agarose gel 4B affinity bag into a chromatographic column, washing the chromatographic column by using a buffer solution, then desorbing the affinity bag by using a desorption solution, and collecting the affinity bag when the color of the elution solution is changed, thus obtaining a urokinase solution; the desorption solution is a mixed solution containing glacial acetic acid and a sodium chloride solution, the volume ratio of the glacial acetic acid to the sodium chloride solution is 3: 400-; the dosage of the buffer solution is 2-4 times of the column volume; the concentration of the sodium chloride solution is 0.01-0.03 g/mL; the dosage of the stripping solution is 1.5 to 2.5 times of the column volume; the flow rate of the stripping solution is 1-4 mL/min;

2. The method according to claim 1, wherein in the step (2), the buffer is a phosphate buffer.

3. The method according to claim 1, wherein in step (3), the freeze-drying specifically comprises: and (3) putting the urokinase solution obtained in the step (2) into a freeze dryer, freezing for 2-4 hours at the temperature of 35-55 ℃, and then heating to room temperature for 1.5-2.5 hours.