A kind of preparation method of the urokinase that can remove pyrogen and virus
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method of preparing urokinase fine work.
Background technology
Urokinase is a kind of serine protease that the uPA in human blood is discharged after Partial digestion from urine, for plasminogen activator, Profibrinolysin can be converted into plasmin, be mainly used in clinically treating the diseases such as Acute Myocardial Infarction, acute cerebral thrombosis formation, cerebral vessels embolism, thrombus occlusion disease, pulmonary infarction and central vein of retina thrombosis.
< < Chinese Pharmacopoeia > > 2005 editions and 2010 editions, clearly regulation " this product needs in process of production through 60 ℃ of heating 10 hours, so that inactivation of virus "; And quality standard requires the bacterial endotoxin of urokinase to be less than 1.0EU/ ten thousand units.
At present, domestic many experts have carried out deep experimental study for the preparation technology of urokinase, as a species specificity purified high-molecular urokinase chromatography media and preparation method and application (patent No. CN101693748A such as Yang Huaying), a kind of urokinase preparation method (patent No. CN101701215A such as Zhang Zhi's force) etc., all be combined with ion exchange chromatography, gel molecular sieve method, immunoaffinity chromatography, p-Aminobenzamidine-Sepharose affinity chromatography technology, purification refine obtains urokinase.Specific activity, polymer urokinase relative content and the bioactive rate of recovery of urokinase of product are improved constantly.
But for the removal research of virus and pyrogen in urokinase product, the document of delivering is less.
Bacterial endotoxin belongs to the one of pyrogen, and pyrogen means the pyrogenic substance that can cause the rising of warm blooded animal abnormal body temperature by microorganisms.It comprises bacillary pyrogen, endogenous polymer pyrogen, the low molecule pyrogen of endogenous and chemical pyrogen etc.Here " pyrogen " of indication, mainly refers to bacillary pyrogen, is meta-bolites, bacterium corpse and the intracellular toxin of some bacterium.What pyrogenicity ability was the strongest is the product of gram negative bacillus, is secondly gram-positive bacillus class, gram positive coccus a little less than, mould, yeast, even virus also can produce pyrogen.After the material that contains pyrogen enters human body, people know from experience produce feel cold, shiver, generate heat, perspire, feel sick, the symptom such as vomiting, body temperature can rise to more than 40 ℃ sometimes, severe patient is even gone into a coma, is collapsed, as rescue not in time, can threat to life.Therefore reducing phlegm and internal heat former is an indispensable step in this product production process.
Summary of the invention
The object of this invention is to provide a kind of urokinase preparation method that can remove virus and pyrogen.
Technical scheme of the present invention is: by urokinase crude product through DEAE-Mierocrystalline cellulose chromatography, CMC chromatography, affinity chromatography, reducing phlegm and internal heat after former obtains urokinase fine work, comprise the steps:
(1) crude product extracts: get urokinase crude product, add phosphate buffered saline buffer and dissolve, the ratio of urokinase crude product and phosphoric acid buffer is: 1g: 10~14ml; Stir; Filter, filter cake is added to phosphoric acid buffer again and dissolves, filter cake and phosphoric acid buffer in ratio be: 1g: 2~5ml; Stir, filter; Merge the filtrate of twice, regulate filtrate pH to 7.0~8.0;
(2) ultrafiltration: use ultra-filtration membrane ultrafiltration to 1/5 to 1/20 of original volume crude extract; Add water to after original volume, continue ultrafiltration to 1/5 to 1/15 of original volume; Add 0.025mol/L PB S balance liquid to original volume, continue ultrafiltration to 1/2 to 1/5 of original volume;
(3) DEAE-Mierocrystalline cellulose chromatography: the DEAE-Mierocrystalline cellulose of the 0.025mol/L PB S balance liquid balance of learning from else's experience, add in ultrafiltrated, stir, filter; On adding, once measure the DEAE-Mierocrystalline cellulose of 1/2 0.025mol/L PB S balance liquid balance in filtrate, stir, filter; Merge secondary filtrate;
(4) CMC chromatography: regulate filtrate pH to 3.0~5.0, adjust specific conductivity to 15~20 μ S/cH.CMC post is first used 0.05~0.2mol/L acetate buffer solution balance, then by upper filtrate CMC post, with 0.05%~0.2% ammonia soln wash-out, obtains elutriant.By elutriant 60 ℃ of water-bath sterilizations 10 hours;
(5) affinity chromatography: by Tryp sin-inhibitor-Sepharose dress post, by the 0.01mol/L PBS balance liquid washing balance containing 1mol/L NaCl; Sterilized elutriant is carried out to upper prop; With 1~3 bed volume of 0.01mol/LPBS balance liquid washing containing 1mol/L NaCl, then wash 0.1~1 bed volume with the 0.01mol/LPBS balance liquid containing 0.5mol/L NaCl; Finally use 0.01mol/LPBS elutriant wash-out, collect elutriant.
(6) precipitation, dry: regulate elutriant pH to 6.0~7.0, precipitate with ethanol, the volume ratio of elutriant and ethanol is 1: 3~8; By washing with alcohol precipitation, repeat 2~4 times again; Dry, obtain dry product.
(7) the former I that reduces phlegm and internal heat: dry product is added to water, in the ratio of 1g: 60~75ml, dissolve, under agitation add the ethanol of equal-volume-20~0 ℃, in the ratio of overall solution volume 1ml: 5~10g, add ammonium acetate again, then slowly add 0.1~0.3mol/L sodium radio-phosphate,P-32 solution, the ratio of its consumption and dry product is: 1ml: 10~14g, stirs, finally add 0.1~0.5mol/L calcium acetate solution, the ratio of its consumption and dry product weight is: 1ml: 10~14g; Regulate pH to 8.5, stir, centrifugal, get supernatant liquor.
(8) the former II that reduces phlegm and internal heat: supernatant liquor is adjusted pH to 6.0~7.0, when stirring, adds ethanol to precipitate, supernatant liquor with add the volume ratio of ethanol to be: 1: 4~6, centrifugal.Use again washing with alcohol throw out, centrifugal, 2~4 times repeatedly; Then drying precipitate, obtains urokinase fine work.
Embodiment
Below in conjunction with specific embodiment, the invention will be further described.
Embodiment
Described utilize urokinase crude product through DEAE-Mierocrystalline cellulose chromatography, CMC chromatography, affinity chromatography, reducing phlegm and internal heat after former obtains urokinase fine work, comprise the steps:
1.DEAE-Mierocrystalline cellulose chromatography
1.1 crude products extract:
Get crude product 420g, be dissolved in 12 times of amount 1mol/L phosphoric acid buffers, stir and extract after 2 hours, filter to get filtrate.Filter cake is dissolved in 4 times of amount 1mol/L phosphoric acid buffers again, stirs, and filters to get filtrate.Merge secondary filtrate, with 5mol/LHCl tune pH to 7.5.
1.2 ultrafiltration:
By the crude extract of modulated pH to 7.5 with the ultra-filtration membrane ultrafiltration of Cut-off10000MW 1/12 o'clock to original volume, add water to original volume, continue ultrafiltration to 1/10 of original volume, then add 0.025mol/LPBS balance liquid to original volume, continue ultrafiltration to 1/3 of original volume.
1.3DEAE-Mierocrystalline cellulose chromatography
The DEAE-Mierocrystalline cellulose of the 0.025mol/LPB S balance liquid balance of 1.3.1 learning from else's experience, the ratio that adds a gram in 30 OD of ultrafiltrated (280nm) adds in ultrafiltrated, stirs, and filters to get filtrate.
1.3.2 with 0.025mol/LPBS balance liquid washing DEAE-Mierocrystalline cellulose.
1.3.3 in filtrate, add the DEAE-Mierocrystalline cellulose of half amount in 1.3.1, stir, filter to get filtrate.
1.3.4 merge secondary filtrate.
2.CMC chromatography
2.1 regulate pH to 4.2 by filtrate, and add sodium-chlor adjust filtrate electricity is led at 17 places.
The upper CMC post through 0.1mol/L acetate buffer solution equilibrate overnight of 2.2 filtrates, flow rate control is at 125ml/min; With 0.1% ammonia soln wash-out, obtain elutriant.
2.3 by elutriant 60 ℃ of water-bath sterilizations 10 hours.
3. affinity chromatography
3.1 fill post by Trypsin-inhibitor-Sepharose, with 25 times of bed volumes containing the 0.01mol/LPBS balance liquid washing balance of 1mol/LNaCl 12 hours.
3.2 by sterilized elutriant upper prop, and flow rate control is at 42ml/min.
After 3.3 upper props finish, with 2 bed volumes of 0.01mol/LPBS balance liquid washing containing 1mol/LNaCl, then wash 1 bed volume with the 0.01mol/LPBS balance liquid containing 0.5mol/L NaCl.
3.4 wash-outs:
With 0.01mol/LPBS elutriant wash-out, flow rate control, at 40ml/min, is collected effluent liquid.
3.5 precipitation
Regulate pH to 6.51 with NaOH solution, then precipitate with ethanol, the ethanol adding and the volume ratio of elutriant are 5: 1, and stirring, staticly settles 12 hours.
3.6 dry
After precipitation finishes, centrifugal, by washing with alcohol precipitation, repeat secondary; By drying precipitate, after being dried, obtain dry product 11.5g.
4. reduce phlegm and internal heat former
4.1 add 788ml water stirring and dissolving by above-mentioned dry product; Under agitation add equal-volume ethanol, and then add ammonium acetate, the consumption of ammonium acetate and the ratio of overall solution volume are:
8g: 1ml, solution clarification;
4.2 slowly add 0.2mol/L sodium radio-phosphate,P-32 solution 189ml again, stir after 15 minutes, add 0.3mol/L calcium acetate solution 189ml;
4.3 then, with 5mol/L NaOH adjusting pH to 8.5, stirs 1 hour, centrifugal, discards throw out;
The 4.4 last 5mol/L of using HCl regulate supernatant liquor pH to 6.68, under agitation, add 9860ml ethanol, and Precipitation, puts refrigerator standing 12 hours;
After 4.5 precipitations finish, centrifugal, with washing with alcohol precipitation, 2 times repeatedly;
4.6 is last, by drying precipitate, obtains dry product 29.6g, is finished product.
The above, be only preferred embodiment of the present invention, is not the restriction of the present invention being made to 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.