CN106701722A

CN106701722A - Method for increasing purity of urokinase

Info

Publication number: CN106701722A
Application number: CN201611197215.2A
Authority: CN
Inventors: 刘乃山; 王小凤; 迟培升
Original assignee: Qingdao Jiulong Biological Medicine Group Co Ltd
Current assignee: Qingdao Jiulong Biological Medicine Group Co Ltd
Priority date: 2016-12-22
Filing date: 2016-12-22
Publication date: 2017-05-24

Abstract

The invention discloses a method for increasing the purity of urokinase. The method is characterized in that the urokinase is prepared by combining a D160 cationic resin exchange method, an affinity membrane chromatography method and a chromatography method and taking a crude urokinase product as a raw material. The quality of the obtained urokinase is better than the existing domestic level, the specific activity of the urokinase can reach 150,000 IU/mg.pr or above, the relative content of the macromolecular urokinase can reach 90% or above, and the bioactivity recovery rate of the urokinase can reach 65%. According to the method, the problems that an existing urokinase preparation method is tedious in operation, poor in product quality, low in yield, high in production cost and the like are solved.

Description

A kind of method for improving urokinase purity

Technical field

The present invention relates to a kind of method for improving urokinase purity, more particularly to a kind of affinity film chromatography technology and affine layer The method that analysis technology improves urokinase purity.

Background technology

Urokinase, abbreviation UK is a kind of alkaline serine protease of human renal cell generation, and serine and histidine are The essential amino acid of enzyme active center.It is primarily present in the urine of people and mammal, and human urine source urokinase mainly has naturally Two kinds of high molecular weight urokinase (54000Dal) and LMW-UK (33000Dal), high molecular weight urokinase thrombus Clinical effectiveness it is about higher 3 times than the urokinase of low-molecular-weight, in the world using the relative molecular weight of urokinase as quality standard One of important indicator.Clinically it is mainly used in treatment acute myocardial infarction, acute cerebral thrombosis formation, cerebral vessels embolism, thrombotic Obliteran, pulmonary embolism and central vein of retina thrombosis.Urokinase preparation method mainly has ion exchange at present Chromatography, gel molecular sieve method, immunoaffinity chromatography and p-Aminobenzamidine-Sepharose affinity chromatographys etc..Such as：Chen Xiangsheng Refine urokinase from crude urokinase using benzamidine sepharose gel affinity chromatography etc. reporting, than 27938IU/mg living Pr, activity recovery 62.96%, 4.03 times of purification；Zhu Yifeng etc. report using SephadexG-50 gel filtrations, CM-Sephadex C-50, ammonium sulfate precipitation and Bio-Gel P-30 gel filtrations etc. prepare urokinase fine work, than work 34080IU/mgpr, activity recovery 60% or so, 50 times of purification.Shen Jinyu etc. is reported and is used immunoaffinity chromatography Urokinase is refined from crude urokinase.Ion exchange and gel-filtration purified inefficiency, it is difficult to obtain the urokinase of high-purity Fine work；Immunoaffinity chromatography purifying urokinase can obtain the urokinase fine work of higher degree, but because immunoaffinity chromatography need to make Biological aglucon is used, it is expensive, it is difficult to obtain the aglucon that industrialized production is used；P-Aminobenzamidine-Sepharose is used as one Preferable urokinase fine work process for purification is planted, but must be also used together with other method and it is necessary to be many in extraction process Secondary use could obtain high-quality urokinase, be used for multiple times and cause that production cost is high, yield is low, and the production cycle is long.

The content of the invention

It is an object of the invention to provide a kind of method for preparing urokinase as raw material with crude urokinase, the method production work The skill time is short, it is easy to which industrialized production, product quality is high, low production cost.The present invention is achieved like this, first urokinase Crude product through dissolving, centrifugation or filter, when volume is larger be concentrated by ultrafiltration etc. step obtain settled solution, adjust electrical conductivity, make its with The equilibrium liquid electrical conductivity that D160 resin cation chromatographic columns are used is consistent, then upper equipped with the absorption of D160 resin cations chromatographic column, Wash again, elute, affinity membrane purification column on the eluent for obtaining, affinity column on the scrubbed, eluent that affords, warp The filtrate wash, elute, being filtrated to get carries out freeze-drying, finally obtains urokinase.Its specific preparation method is as follows：(1) take A certain amount of crude urokinase (30000~50000IU/g of biological value, than 300~600IU/gpr living), with 8~10 times of urine The pH6.0 of kinases crude product amount (volume and weight ratio), 0.1mol/L phosphate buffers point add dissolving 2~3 times, stir every time molten Solution 0.5~1.0 hour, is then centrifuged for or filters, and merges supernatant or filtrate, is concentrated by ultrafiltration when volume is larger, and what is obtained is clear Clear solution is standby；(2) the settled solution regulation electrical conductivity for obtaining step (1), makes it make with D160 resin cation chromatographic columns Equilibrium liquid electrical conductivity is consistent, then upper with the pH6.0 containing 0.15~0.30mol/L sodium chloride, 0.1mol/L phosphate buffers The D160 resin cation chromatographic columns of balance, then with the pH6.0 containing 0.15~0.30mol/L sodium chloride, 0.1mol/L phosphoric acid Buffer solution is washed；Finally use pH10,1~3% ammoniacal liquor to be eluted, collect activity eluted peak；(3) step (2) is collected and is obtained Activity eluted peak with acetic acid adjust pH7.2, affinity membrane purification column is then gone up, with containing 0.45~0.60mol/L sodium chloride PH7.2,0.1mol/L phosphate buffer wash, then with contain 0.25~0.35mol/L sodium chloride pH3.5~4.5,0.1mol/L Acetate buffer solution is eluted, and collects activity eluted peak；(4) step (3) is collected the activity eluted peak acetic acid for obtaining and adjusts pH7.2, Then affinity column is gone up, with the pH7.2 containing 1.0~2.0mol/L sodium chloride, the washing of 0.1mol/L phosphate buffers, then with containing PH3.5~4.5 of 0.35~0.45mol/L sodium chloride, 0.1mol/L acetate buffer solutions wash-out, collect activity eluted peak, filter, Freeze-drying is finally carried out, urokinase is obtained.The present invention utilize D160 resin cation absorption methods, affinity film chromatography method with it is affine Chromatography etc. is used in combination, and urokinase is prepared by raw material of crude urokinase, and resulting urokinase quality is better than the existing country Level, urokinase can reach 150 than work, and more than 000IU/mgpr, macromolecule urokinase relative amount can reach more than 96%, The urokinase bioactivity rate of recovery can reach 65%.Operation present method solves existing urokinase preparation method generally existing is numerous Trivial, poor product quality, the problems such as yield is low and production cost is high.

Specific embodiment

Further illustrate technical solution of the invention with reference to specific embodiment, these embodiments it is not intended that It is the limitation to technical scheme.

Embodiment 1：

(1) 100g crude urokinases are taken, with pH6.0,0.1mol/L phosphate buffers 400ml dissolvings are stirred 50 minutes, centrifugation, Precipitation uses pH6.0 again, and 0.1mol/L phosphate buffers 400ml dissolvings are stirred 30 minutes, and centrifugation merges supernatant twice, obtains 820ml settled solutions, sampling detection, the unit of total biological value 0.042 hundred million.

(2) the 820ml settled solutions regulation electrical conductivity for obtaining step (1), makes it with D160 resin cation chromatographic columns The equilibrium liquid electrical conductivity for using is consistent, then upper with the pH6.0 containing 0.15mol/L sodium chloride, 0.1mol/L phosphate buffers balance D160 resin cation chromatographic columns, then with containing 0.15mol/L sodium chloride pH6.0,0.1mol/L phosphate buffers washing； PH10,1% ammoniacal liquor is finally used to be eluted, collection obtains the activity eluted peaks of 190ml, the unit of total biological value 0.039 hundred million.

(3) step (2) is collected the activity eluted peak acetic acid of 190ml for obtaining and adjusts pH7.2, then go up affinity membrane purifying Post, with the pH7.2 containing 0.45mol/L sodium chloride, the washing of 0.1mol/L phosphate buffers, then with containing 0.25mol/L sodium chloride PH3.5,0.1mol/L acetate buffer solution are eluted, and collection obtains the activity eluted peaks of 90ml, the unit of total biological value 0.035 hundred million.

(4) step (3) is collected the activity eluted peak acetic acid of 90ml for obtaining and adjusts pH7.2, then go up affinity column, used PH7.2 containing 1.0mol/L sodium chloride, 0.1mol/L phosphate buffer wash, then with contain 0.35mol/L sodium chloride pH3.5, 0.1mol/L acetate buffer solutions are eluted, and collect activity eluted peak, and filtering obtains 85ml filtrates, carries out freeze-drying, obtains 34.1mg urokinases, the unit of total biological value 0.028 hundred million, than 167000IU/mg.pr living, macromolecule urokinase relative amount 97.0%, active overall recovery 66.7%.

Embodiment 2：

(1) 200g crude urokinases are taken, with pH6.0,0.1mol/L phosphate buffers 1000ml dissolvings are stirred 60 minutes, centrifugation, Precipitation uses pH6.0 again, and 0.1mol/L phosphate buffers 1000ml dissolvings are stirred 30 minutes, and filtering merges supernatant, ultrafiltration twice Concentration, obtains 320ml concentrates, sampling detection, the unit of total biological value 0.080 hundred million.

(2) the 320ml concentrates regulation electrical conductivity for obtaining step (1), makes it make with D160 resin cation chromatographic columns Equilibrium liquid electrical conductivity is consistent, then above uses the pH6.0 containing 0.20mol/L sodium chloride, 0.1mol/L phosphate buffers balance D160 resin cation chromatographic columns, then with the pH6.0 containing 0.20mol/L sodium chloride, the washing of 0.1mol/L phosphate buffers；Most PH10,2% ammoniacal liquor is used to be eluted afterwards, collection obtains the activity eluted peaks of 260ml, the unit of total biological value 0.074 hundred million.

(3) step (2) is collected the activity eluted peak acetic acid of 260ml for obtaining and adjusts pH7.2, then go up affinity membrane purifying Post, with the pH7.2 containing 0.50mol/L sodium chloride, the washing of 0.1mol/L phosphate buffers, then with containing 0.30mol/L sodium chloride PH4.0,0.1mol/L acetate buffer solution are eluted, and collection obtains the activity eluted peaks of 100ml, the unit of total biological value 0.068 hundred million.

(4) step (3) is collected the activity eluted peak acetic acid of 100ml for obtaining and adjusts pH7.2, then go up affinity column, With the pH7.2 containing 0.50mol/L sodium chloride, the washing of 0.1mol/L phosphate buffers, then with containing 0.40mol/L sodium chloride PH4.0,0.1mol/L acetate buffer solution are eluted, and collect activity eluted peak, and filtering obtains 105ml filtrates, carries out freeze-drying, Obtain 62.8mg urokinases, the unit of total biological value 0.054 hundred million, than 176000IU/mgpr living, macromolecule urokinase is relative to be contained Amount 97.2%, active overall recovery 67.5%.

Claims

1. it is a kind of improve urokinase purity method, it is characterised in that the method in turn includes the following steps：

(1) a certain amount of crude urokinase is taken, with 8~12 times of pH6.0 of crude urokinase amount, 0.1mol/L phosphate buffers Divide 2~3 times and add dissolving, each stirring and dissolving 0.5~1.0 hour is then centrifuged for or filters, merge supernatant or filtrate, volume It is concentrated by ultrafiltration when larger, the settled solution for obtaining is standby；(2) settled solution for obtaining step (1) adjusts electrical conductivity, makes It is consistent with the equilibrium liquid electrical conductivity that D160 resin cation chromatographic columns are used, then upper with the sodium chloride containing 0.15~0.30mol/L PH6.0,0.1mol/L phosphate buffers balance D160 resin cation chromatographic columns, then with contain 0.15~0.30mol/L The pH6.0 of sodium chloride, 0.1mol/L phosphate buffer are washed；Finally use pH10,1~3% ammoniacal liquor to be eluted, collect wash-out and live Property peak；(3) step (2) is collected into the activity eluted peak acetic acid that obtains and adjusts pH7.2, then go up affinity membrane purification column, with containing The pH7.2 of 0.45~0.60mol/L sodium chloride, 0.1mol/L phosphate buffer are washed, then with the chlorination containing 0.25~0.35mol/L PH3.5~4.5 of sodium, 0.1mol/L acetate buffer solutions wash-out, collect activity eluted peak；(4) step (3) is collected what is obtained PH7.2 is adjusted in activity eluted peak with acetic acid, then goes up affinity column, with the pH7.2 containing 1.0~2.0mol/L sodium chloride, 0.1mol/L phosphate buffers are washed, then with pH3.5~4.5 of 0.35~0.45mol/L sodium chloride are contained, 0.1mol/L acetic acid delays Fliud flushing is eluted, and collects activity eluted peak, and filtering finally carries out freeze-drying, obtains urokinase.

2. the method for preparing urokinase according to claim 1, it is characterised in that the affinity membrane purifying that step (3) is used is situated between Matter is that, with polysulfones as carrier, p-Aminobenzamidine is affinity ligand, using dry-wet spinning masking technique be prepared into it is hollow Fiber affinity membrane；The affinity chromatography medium that step (4) is used is the p-Aminobenzamidine affinity ligand coupling with agarose as carrier Prepare.