CN103740687A

CN103740687A - Method for preparing crude urokinase

Info

Publication number: CN103740687A
Application number: CN201310643507.4A
Authority: CN
Inventors: 刘乃山; 张晓涵; 安迪; 张元泽
Original assignee: QINGDAO KANGYUAN PHARMACEUTICAL CO Ltd
Current assignee: QINGDAO KANGYUAN PHARMACEUTICAL CO Ltd
Priority date: 2013-11-30
Filing date: 2013-11-30
Publication date: 2014-04-23
Anticipated expiration: 2033-11-30
Also published as: CN103740687B

Abstract

The invention discloses a method for preparing crude urokinase. The technical proposal of the method is as follows: using an adsorption process, a desorption process, a salting out process to prepare the crude urokinase from qualified urine. The method has the advantages of simple operation and high yield.

Description

A kind of method of preparing urokinase crude product

Technical field

The present invention relates to biological technical field, relate in particular to the method for preparing urokinase crude product from qualified urine.

Background technology

Urokinase is a kind of Sumizyme MP, by kidney, is produced, and is mainly present in human body and mammiferous urine; the normal function of human body during blood coagulation; but under some pathological state, by forming thrombus block blood when circulation in blood vessel, will cause serious consequence.Urokinase is thrombolytic agent, but itself is not directly used in clot itself, but first swashs intravital Profibrinolysin, becomes activated plasmin, and plasmin acts on Taka-proteinase grumeleuse, makes it resolve into the many carbon of solubility.

Urokinase has various molecular weights form, the Urokinase clot lysis thrombus clinical effectiveness of high molecular approximately doubles than low-molecular-weight, and therefore, the kinase whose molecular weight of helping a small child urinate by holding his legs apart is in the world as one of inspection item of quality standard, for reducing low-molecular-weight generation, in production process, pH value is unsuitable too low.

Because urokinase has important pharmaceutical value, so in succession there are a lot of monographs, reported the extracting method of urokinase crude product, because of urokinase content in human urine very low, the key of getting urokinase is exactly enrichment urokinase from a large amount of urines how, from the monograph of delivering at present, mainly contain three class methods: (1), foaming: with high-speed stirring, make the foaming of enzyme liquid, then make foam liquefaction, add ammonium sulfate to make urokinase precipitation.(2), precipitation agent method: add precipitation agent in urine, make urokinase precipitation, urokinase is retained in precipitation.(3), sorbent material method: this method application at most, is selected suitable sorbent material, selectively adsorbing urine kinases.

This technique adopts ion exchange resin as sorbent material exactly, produces urokinase crude product.

Summary of the invention

The object of the invention is to extract urokinase crude product from qualified urine.。

Technical scheme of the present invention is: utilize absorption process, desorption operation, salting-out procedures from qualified urine, to prepare the method for urokinase crude product.The present invention is simple to operate and have a higher yield.Comprise the steps:

(1) absorption process: get two layers of filtered through gauze for qualified urine, filter in stirred pot; When constantly stirring, with speed slowly, add D-160A ion exchange resin, and measure with precision instrument, regulate between urine pH to 4.5～6.5; Whip attachment 1～2 hour, discards urine, and wash resin with water and remove residual urine and impurity, then till being washed till clarification with phosphoric acid buffer;

(2) desorption operation: stir wash-out 0.5～1.5 hour with the ammoniacal liquor of sodium chloride-containing, suction filtration, collects elutriant, and resin is used same eluant solution 20～50 minutes again, collects elutriant, merges secondary elutriant;

(3) salting-out procedures: calculate and add solid ammonium sulfate by the volume of elutriant, stirring and dissolving, reach 50%～70% saturation ratio, in-4～0 ℃ of supernatant liquor, with acetic acid, adjust pH to 4.5～6.5, add 1～3 times of amount acetone, after slightly stirring, place precipitation 12～16 hours, centrifugal, the urokinase salt analysis of products of collecting precipitation;

(4) analysis of products of urokinase salt being placed to shady and cool place preserves;

Embodiment

Below in conjunction with specific embodiment, the invention will be further described.

Embodiment 1

Described utilize absorption process, desorption operation, salting-out procedures are prepared the method for urokinase crude product from qualified urine, comprise the steps:

1. absorption process:

1.1 get 500 kilograms of qualified urines, by two layers of filtered through gauze, filter in stirred pot;

1.2 when constantly stirring, and adds D-160A ion exchange resin, and measure with precision instrument with speed slowly, regulates urine pH to 6.0;

1.3 whip attachment 1.5 hours, discard urine, and wash resin with water and remove residual urine and impurity, then till being washed till clarification with phosphoric acid buffer; ;

2. desorption operation:

The ammoniacal liquor of 2.1 use sodium chloride-containing stirs wash-out 1 hour, and suction filtration is collected elutriant,

2.2 resins are used same eluant solution 30 minutes again, collect elutriant, merge secondary elutriant;

3. salting-out procedures:

3.1 volumes by elutriant calculate and add solid ammonium sulfate, and stirring and dissolving, reaches 65% saturation ratio;

3.2 adjust pH to 5.0 in-4～0 ℃ of supernatant liquor with acetic acid;

3.3 add 2 times of amount acetone, after slightly stirring, place precipitation 14 hours, centrifugal, the urokinase salt analysis of products of collecting precipitation;

4. the analysis of products of urokinase salt being placed to shady and cool place preserves;

The above, be only preferred embodiment of the present invention, is not the present invention to be done to the restriction of other form, and any those skilled in the art may utilize the technology contents of above-mentioned announcement to be changed or be modified as the equivalent embodiment of equivalent variations.But every technical solution of the present invention content that do not depart from, any simple modification, equivalent variations and the remodeling above embodiment done according to technical spirit of the present invention, still belong to the protection domain of technical solution of the present invention.

Claims

1. a method of preparing urokinase crude product, is characterized in that, the method comprises the steps:

(1) absorption process: get two layers of filtered through gauze for qualified urine, filter in stirred pot; When constantly stirring, slowly add ion exchange resin, regulate pH, whip attachment, washing resin is to clarification;

(2) desorption operation: stir wash-out, suction filtration, collects elutriant, and wash-out, collects elutriant again, merges twice elutriant;

(3) salting-out procedures: add solid ammonium sulfate, stirring and dissolving, regulates pH value, adds acetone, stirs, and places precipitation, centrifugal, obtains the analysis of products of urokinase salt, i.e. urokinase crude product.

2. method according to claim 1, is characterized in that: in step (1), regulate pH value to 4.5～6.5 of urine; The time of whip attachment is: 1～2 hour; During washing resin, first wash residual urine and impurity with water, then with phosphoric acid buffer, be washed till clarification.

3. method according to claim 1, is characterized in that: in step (2), and the ammoniacal liquor that elutriant used is sodium chloride-containing; Stirring for the first time elution time is 0.5～1.5 hour; Stirring for the second time elution time is 20～50 minutes.

4. method according to claim 1, is characterized in that: in step (3), according to the volume of elutriant, add solid ammonium sulfate, stirring and dissolving, makes solution reach 50%～70% saturation ratio; At-4～0 ℃ with acetic acid regulator solution supernatant liquor pH value to 4.5～6.5; With acetone precipitation, sedimentation time is 12～16 hours; The weight that adds solid ammonium sulfate in every 1000ml solution is 300～450g.

5. method according to claim 1, is characterized in that: the urokinase crude product of gained should be preserved in the cool.