CN109880817A - A kind of purification process of urokinase fine work - Google Patents
A kind of purification process of urokinase fine work Download PDFInfo
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- CN109880817A CN109880817A CN201711274440.6A CN201711274440A CN109880817A CN 109880817 A CN109880817 A CN 109880817A CN 201711274440 A CN201711274440 A CN 201711274440A CN 109880817 A CN109880817 A CN 109880817A
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Abstract
The invention discloses the technical methods using filter element filtering technology purifying urokinase fine work.It is compared with the traditional method, this method have the advantage that: by filter element filtering method, significantly reduce impurity content；The urokinase fine work high income of purifying, higher than work, thermal stability is good, and the indexs such as appearance, physics and chemistry, microbial limit meet or exceed the related drug standards both at home and abroad；Method and process is simple, and production cost is low, and yield is high, can industrialized production.
Description
Technical field
The invention belongs to field of biological pharmacy.Specifically utilize the technology of filter element filtering technology purifying urokinase fine work
Method.
Background technique
Urokinase is a kind of alkali protease that human renal cell generates, and is the very strong proteolytic enzyme of specificity, blood fibre
Fibrillarin lyase original is unique natural protein substrates, it, which acts on arginine-valine key, has been converted into plasminogen
Active fibrinolysin, to play pharmacological action.
Clinical application: being mainly used for cerebral thrombosis, arteriovenous thrombus and central retina arteriovenous thrombus etc. around limbs.
Currently, more classical purifying process is ion-exchange-resin process purifying urokinase semifinished product, then with various purifying
Method is purified.
Summary of the invention
The invention discloses using filter element filtering technology purifying urokinase fine work technical method, it the following steps are included:
Dissolution adjusts pH, filtering, precipitating, centrifugal dehydration, drying.
1, it dissolves: dissolving crude urokinase with purified water, ratio is that 68-70L purified water is added in every kg crude urokinase, so
95% cold ethyl alcohol of isometric temperature≤- 5 DEG C is added under stiring afterwards, so that the concentration of cold alcohol solution is reached 47%, then by overall
Ammonium acetate is added in product, and ratio is that 0.08kg ammonium acetate is added in every L solution, adds 0.2mol/L sodium radio-phosphate,P-32 solution, ratio is every
Kg crude urokinase is added 16-18L 0.2mol/L sodium radio-phosphate,P-32 solution and adds 0.3mol/L calcium acetate after stirring 15 minutes
Solution, ratio are that 16-18L 0.3mol/L calcium acetate solution is added in every kg crude urokinase, obtain urokinase feed liquid.
2, it adjusts pH: adjusting pH to 8.0-8.5 with 5.0 mol/L NaOH solutions, continue stirring 1-1.5 hours after stablizing,
It is then transferred to refrigerated centrifuge to be centrifuged 30 minutes, centrifugal rotational speed 3700rpm discards sediment, obtains urokinase centrifugate.
3, it filters: with the filter element filtering centrifugate of 20nm, obtaining urokinase filtered fluid.
4, precipitate: urokinase filtered fluid adjusts pH to 6.5-7.0 with 5.0 mol/L HCl solutions, and temperature is added under stiring
95% cold ethyl alcohol of≤- 5 DEG C of degree, ratio are that the cold ethyl alcohol of 5L95% is added in every L urokinase filtered fluid, and it is small to be then allowed to stand precipitating 20-24
When.
5, centrifugal dehydration: supernatant is removed in siphon, and lower sediment thing is placed in refrigerated centrifuge, centrifugal rotational speed 3700rpm,
Centrifugation time 30 minutes, liquid is discarded supernatant, with dehydrated alcohol centrifugal dehydration 2 times, liquid is discarded supernatant, obtains sediment.
6, dry: sediment is placed in phosphorus pentoxide and does room temperature vacuum drying, vacuum degree in the vacuum oven of desiccant
For -0.1MPa, urokinase highly finished product are obtained after dry.
The urokinase fine work obtained: yield >=80% is deposited under the conditions of 25 DEG C of temperature than > 140000IU/mg albumen living
Putting 6 months reduces≤5% than living.
Compared with prior art, the advantages and positive effects of the present invention are:
Urokinase fine work is purified using filter element filtering technology, by filter element filtering, significantly reduces impurity content；The urine of purifying swashs
Enzyme fine work, yield >=80%, than > 140000IU/mg albumen living, storing under the conditions of 25 DEG C of temperature 6 months reduces≤5% than living,
The indexs such as appearance, physics and chemistry, microbial limit meet or exceed the related drug standards both at home and abroad；Method and process is simple, is produced into
This is low, and yield is high, can industrialized production.
Specific embodiment
The present invention will be further described combined with specific embodiments below.
Embodiment 1
(1) 6.8L purified water is added in 0.1kg crude urokinase, and 95% cold second of temperature≤- 5 DEG C 6.8L is then added under stiring
Alcohol makes the concentration of cold alcohol solution reach 47%, then 1.09kg ammonium acetate is added by total volume, adds 1.6L 0.2mol/L phosphorus
Acid sodium solution adds 1.6L 0.3mol/L calcium acetate solution, obtains urokinase feed liquid after stirring 15 minutes.
(2) 5.0 mol/L NaOH solutions are added and adjust pH to 8.0, continue stirring 1 hour after stablizing, be then transferred to
Refrigerated centrifuge is centrifuged 30 minutes, and centrifugal rotational speed 3700rpm discards sediment, obtains urokinase centrifugate.
(3) the filter element filtering centrifugate for using 20nm, obtains urokinase filtered fluid.
(4) urokinase filtered fluid with 5.0 mol/L HCl solutions adjust pH to 6.5, under stiring be added 85L temperature≤-
The cold ethyl alcohol of the 95% of 5 DEG C, is then allowed to stand precipitating 20 hours.
(5) supernatant is removed in siphon, and lower sediment thing is placed in refrigerated centrifuge, centrifugal rotational speed 3700rpm, when centrifugation
Between 30 minutes, discard supernatant liquid, with dehydrated alcohol centrifugal dehydration 2 times, discard supernatant liquid, obtain sediment.
(6) sediment, which is placed in phosphorus pentoxide and does in the vacuum oven of desiccant room temperature, is dried in vacuo, and vacuum degree is-
0.1MPa obtains urokinase highly finished product after dry.
Detect urokinase fine work obtained: yield 86% stores 6 under the conditions of 25 DEG C of temperature than 165200IU/mg albumen living
3% is reduced than living within a month.
Embodiment 2
(1) 7L purified water is added in 0.1kg crude urokinase, and 95% cold ethyl alcohol of temperature≤- 5 DEG C 7L is then added under stiring,
So that the concentration of cold alcohol solution is reached 47%, then 1.12kg ammonium acetate is added by total volume, adds 1.7L 0.2mol/L phosphoric acid
Sodium solution adds 1.7L 0.3mol/L calcium acetate solution, obtains urokinase feed liquid after stirring 15 minutes.
(2) 5.0 mol/L NaOH solutions are added and adjust pH to 8.2, continue stirring 1.5 hours after stablizing, then shift
It is centrifuged 30 minutes to refrigerated centrifuge, centrifugal rotational speed 3700rpm discards sediment, obtains urokinase centrifugate.
(3) the filter element filtering centrifugate for using 20nm, obtains urokinase filtered fluid.
(4) urokinase filtered fluid with 5.0 mol/L HCl solutions adjust pH to 6.8, under stiring be added 88L temperature≤-
The cold ethyl alcohol of the 95% of 5 DEG C, is then allowed to stand precipitating 22 hours.
(5) supernatant is removed in siphon, and lower sediment thing is placed in refrigerated centrifuge, centrifugal rotational speed 3700rpm, when centrifugation
Between 30 minutes, discard supernatant liquid, with dehydrated alcohol centrifugal dehydration 2 times, discard supernatant liquid, obtain sediment.
(6) sediment, which is placed in phosphorus pentoxide and does in the vacuum oven of desiccant room temperature, is dried in vacuo, and vacuum degree is-
0.1MPa obtains urokinase highly finished product after dry.
Detect urokinase fine work obtained: yield 89% stores 6 under the conditions of 25 DEG C of temperature than 167900IU/mg albumen living
2% is reduced than living within a month.
Embodiment 3
(1) 6.9L purified water is added in 0.1kg crude urokinase, and 95% cold second of temperature≤- 5 DEG C 6.9L is then added under stiring
Alcohol makes the concentration of cold alcohol solution reach 47%, then 1.1kg ammonium acetate is added by total volume, adds 1.8L 0.2mol/L phosphorus
Acid sodium solution adds 1.8L 0.3mol/L calcium acetate solution, obtains urokinase feed liquid after stirring 15 minutes.
(2) 5.0 mol/L NaOH solutions are added and adjust pH to 8.4, continue stirring 1.5 hours after stablizing, then shift
It is centrifuged 30 minutes to refrigerated centrifuge, centrifugal rotational speed 3700rpm discards sediment, obtains urokinase centrifugate.
(3) the filter element filtering centrifugate for using 20nm, obtains urokinase filtered fluid.
(4) urokinase filtered fluid with 5.0 mol/L HCl solutions adjust pH to 7.0, under stiring be added 87L temperature≤-
The cold ethyl alcohol of the 95% of 5 DEG C, is then allowed to stand precipitating 24 hours.
(5) supernatant is removed in siphon, and lower sediment thing is placed in refrigerated centrifuge, centrifugal rotational speed 3700rpm, when centrifugation
Between 30 minutes, discard supernatant liquid, with dehydrated alcohol centrifugal dehydration 2 times, discard supernatant liquid, obtain sediment.
(6) sediment, which is placed in phosphorus pentoxide and does in the vacuum oven of desiccant room temperature, is dried in vacuo, and vacuum degree is-
0.1MPa obtains urokinase highly finished product after dry.
Detect urokinase fine work obtained: yield 85% stores 6 under the conditions of 25 DEG C of temperature than 174700IU/mg albumen living
3% is reduced than living within a month.
The above described is only a preferred embodiment of the present invention, be not that the invention has other forms of limitations, it is any ripe
Know the equivalent reality that professional and technical personnel was changed or be modified as equivalent variations possibly also with the technology contents of the disclosure above
Apply example.But without departing from the technical solutions of the present invention, to the above embodiments according to the technical essence of the invention
Any simple modification, equivalent variations and remodeling, still fall within the protection scope of technical solution of the present invention.
Claims (8)
1. a kind of purification process of urokinase fine work, which comprises the following steps: dissolution, adjust pH, filtering, precipitating,
Centrifugal dehydration, drying.
2. the purification process of urokinase fine work according to claim 1, which is characterized in that the dissolving step is: using
Purified water dissolves crude urokinase, and ratio is that 68-70L purified water is added in every kg crude urokinase, be then added under stiring etc.
The cold ethyl alcohol of the 95% of bulk temperature≤- 5 DEG C makes the concentration of cold alcohol solution reach 47%, then ammonium acetate, ratio is added by total volume
0.08kg ammonium acetate is added for every liter of solution, adds 0.2mol/L sodium radio-phosphate,P-32 solution, ratio is the addition of every kg crude urokinase
16-18L 0.2mol/L sodium radio-phosphate,P-32 solution after stirring 15 minutes, adds 0.3mol/L calcium acetate solution, and ratio is every kg urine
16-18L 0.3mol/L calcium acetate solution is added in kinases crude product, obtains urokinase feed liquid.
3. the purification process of urokinase fine work according to claim 1, which is characterized in that the adjusting pH step is:
With 5.0 mol/L NaOH solutions adjust pH to 8.0-8.5, stablize after continue stirring 1-1.5 hour, be then transferred to freeze from
Scheming is centrifuged 30 minutes, and centrifugal rotational speed 3700rpm discards sediment, obtains urokinase centrifugate.
4. the purification process of urokinase fine work according to claim 1, which is characterized in that the filtration step is: using
The filter element filtering centrifugate of 20nm, obtains urokinase filtered fluid.
5. the purification process of urokinase fine work according to claim 1, which is characterized in that the settling step is: urine
Kinases filtered fluid adjusts pH to 6.5-7.0 with 5.0 mol/L HCl solutions, and 95% cold second of temperature≤- 5 DEG C is added under stiring
Alcohol, ratio are that the cold ethyl alcohol of 5L 95% is added in every liter of urokinase filtered fluid, are then allowed to stand precipitating 20-24 hours.
6. the purification process of urokinase fine work according to claim 1, which is characterized in that the centrifugal dehydration step
Be: siphon supernatant, lower sediment thing are placed in refrigerated centrifuge, centrifugal rotational speed 3700rpm, centrifugation time 30 minutes, are abandoned
Supernatant is removed, with dehydrated alcohol centrifugal dehydration 2 times, liquid is discarded supernatant, obtains sediment.
7. the purification process of urokinase fine work according to claim 1, which is characterized in that the drying steps are: heavy
Starch is placed in phosphorus pentoxide and does room temperature vacuum drying in the vacuum oven of desiccant, and vacuum degree is -0.1MPa, obtains after dry
Urokinase highly finished product.
8. the purification process of urokinase fine work according to claim 1, which is characterized in that the urokinase essence obtained
Product: yield >=80%, than > 140000IU/mg albumen living, storing under the conditions of 25 DEG C of temperature 6 months reduces≤5% than living.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711274440.6A CN109880817A (en) | 2017-12-06 | 2017-12-06 | A kind of purification process of urokinase fine work |
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| CN201711274440.6A CN109880817A (en) | 2017-12-06 | 2017-12-06 | A kind of purification process of urokinase fine work |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115386564A (en) * | 2022-09-20 | 2022-11-25 | 河南省尤里卡生物科技有限公司 | Method for purifying urokinase |
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2017
- 2017-12-06 CN CN201711274440.6A patent/CN109880817A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115386564A (en) * | 2022-09-20 | 2022-11-25 | 河南省尤里卡生物科技有限公司 | Method for purifying urokinase |
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