CN104072637A - Preparation method for low-molecular-weight heparin calcium - Google Patents
Preparation method for low-molecular-weight heparin calcium Download PDFInfo
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- CN104072637A CN104072637A CN201410320475.9A CN201410320475A CN104072637A CN 104072637 A CN104072637 A CN 104072637A CN 201410320475 A CN201410320475 A CN 201410320475A CN 104072637 A CN104072637 A CN 104072637A
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Abstract
The invention relates to the field of medical synthesis, and discloses a preparation method for low-molecular-weight heparin calcium. The method disclosed by the invention comprises the following steps: preparing a low-molecular-weight heparin sodium solution by virtue of a sodium nitrite method at first, and then carrying out resin calcification on cation exchange resin by use of a calcium chloride solution, then carrying out calcium-sodium ion substitution on the calcified cation resin columns on the low-molecular-weight heparin sodium solution, and finally filtering, freeze-drying and crushing the substitution solution after the calcium-sodium ion substitution to obtain the low-molecular-weight heparin calcium. According to the preparation method disclosed by the invention, a step of preparing low-molecular-weight heparin with the extremely low pH value is avoided; the low-molecular-weight heparin calcium is obtained by resin one-time moderate substitution for the low-molecular-weight heparin sodium, so that the loss of anti-Xa factor titer in severe environments is avoided, the anti-Xa/anti-IIa ratio is increased, and the production steps are simplified.
Description
Technical field
The present invention relates to the synthetic field of medicine, be specifically related to a kind of preparation method of low molecular weight calcium heparin.
Background technology
Heparin is a kind of mixture being made up of the oligose such as uronic acid and glucosamine, has anticoagulating active, from finding the history of existing more than 80 years so far, still there is no a kind of product that can substitute it completely at present, is still important biochemical drug.Heparin is thrombosis and the venothrombotic choice drug for the treatment of after prevention operation.But a large amount of uses can cause bleeding and the side effect such as induced platelet minimizing, has limited its a large amount of use.
Low molecular weight heparin is the bglii fragment of the molecular weight that obtains of heparin fractionated or degraded, its weight-average molecular weight, at 3000-8000, have the anti-Xa factor effect suitable with heparin, and antithrombin (IIa) activity reduces greatly, its anti-bolt effect is less on blood coagulation system impact, bleeding tendency is less, also has molecule little, easily absorbs, bioavailability is high, the features such as Half-life in vivo is long, its validity and security, due to heparin, have become the focus of anti-freezing class drug research at present.
Low molecular weight calcium heparin is the calcium salt of low molecular weight heparin, compared with sodium salt, calcium salt is slowly diffusion after injection, can not reduce the calcium colloid of capillary vessel between local cells, can not change vascular permeability yet, the side effect of the local hemorrhage that almost sodium-free salt may cause through subcutaneous injection, therefore calcium salt is more suitable for clinical use.
The standard of weighing the quality of low molecular heparin calcium has two, and one is that anti-Xa factor is tired, and another is exactly the anti-II a ratio of anti-Xa/, and the value of these two standards is higher, and quality product is better.Publication number is that the Chinese patent of CN1554671A discloses " a kind of preparation method of low molecular heparin calcium of low nitrite content ", it changes low molecular weight heparin sodium into low molecular weight calcium heparin through acid-base reaction repeatedly based on nitrite method, although it has successfully reduced the content of nitrite, but in preparation process, pH value is lower, add the follow-up strong neutralization reaction of soda acid in calcium step that turns, the certain loss of having tired of antagonism Xa factor, cause anti-Xa factor to tire and the not high problem of the anti-II a ratio of anti-Xa/, affect quality product, this process operations is more loaded down with trivial details simultaneously, may introduce other reagent impurity.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of preparation method of low molecular weight calcium heparin, the anti-Xa factor that makes this preparation method can improve low molecular weight calcium heparin is tired and the anti-II a ratio of anti-Xa/;
A further object of the present invention is to provide a kind of preparation method of low molecular weight calcium heparin, this preparation method can be simplified and prepare low molecular weight calcium heparin processing step.
In existing low molecular weight calcium heparin preparation method (CN1554671A), anti-Xa factor tire and the anti-II a ratio of anti-Xa/ lower, affect quality product, the simultaneously more loaded down with trivial details problem of process operations, the invention provides following solution:
A preparation method for low molecular weight calcium heparin, comprises the following steps:
Step 1, utilize sodium nitrite method to prepare low molecular weight heparin sodium solution;
Step 2, Zeo-karb carry out resin calcification with calcium chloride solution, and the resin cation (R.C.) on the low molecular weight heparin sodium solution of then being prepared by step 1 after calcification carries out the displacement of calcium sodium ion;
Step 3, the displacement liquid filtration after step 2 calcium sodium ion displacement, freeze-drying, pulverizing are obtained to low molecular weight calcium heparin.
As preferably, step 1 is:
In step 1.1, the water-soluble solution of taking heparin sodium, to 2-4, add Sodium Nitrite reaction 4-6h to obtain degradation solution with vinegar acid for adjusting pH value;
Step 1.2, to adding 30% superoxol oxidation 15min in described degradation solution, then add in 25% sodium hydroxide solution and pH value to 6.5-7.0, obtain neutralizer;
Step 1.3, in described neutralizer, add 95% ethanol to carry out alcohol precipitation and separate alcohol hypostasis, water for injection dissolves and obtains low molecular weight heparin sodium solution for gained alcohol hypostasis.
Wherein, the ratio of described heparin sodium, Sodium Nitrite and 30% hydrogen peroxide is preferably 2.5kg:70g-90g:660mL, more preferably 2.5kg:80g-85g:660mL; Described each pH value all can be got a point value from appointing in the scope limiting, be not limited to the endpoint value of scope, and can with other parameter arbitrary combination, be not limited to above-mentioned preferred step 1 concrete scheme, for example, described in vinegar acid for adjusting pH value to 2-4, described pH value can be got within the scope of this such as 2.01,2.02......3.98,3.99 or 4.00 any one as pH value, and can with the concentration of hydrogen peroxide, sodium hydroxide and reaction times, oxidization time parameter arbitrary combination, other pH values are with reference to this explanation.
As preferably, described Zeo-karb is strongly acidic macroporous cation exchange resin, more preferably D001 type strongly acidic macroporous cation exchange resin or JZD001X7-II type large hole strong acid styrene system cation exchange resin.
As preferably, the weight-average molecular weight of described low molecular weight heparin sodium is 3000-4500D.
As preferably, the weight-average molecular weight of described low molecular weight calcium heparin is 3000-4500D.
As preferably, described resin calcification step is:
Calcium chloride solution on Zeo-karb, replaces to lower column liquid pH value and is after neutrality, and water for injection rinses Zeo-karb and completes resin calcification.
Wherein, described calcium chloride solution concentration is preferably 0.5mol/L.
It is the low molecular weight heparin step below 2 that the present invention avoids pH value in preparation process, reduce the decomposition of low molecular weight heparin under utmost point low ph environment, replacing by low molecular weight heparin sodium and Zeo-karb gentleness simultaneously, avoid adopting the acid-base neutralisation thermopositive reaction of calcium hydroxide and low molecular weight heparin, ensure the stable of low molecular weight calcium heparin, the loss of having avoided the anti-Xa factor under intense environment to tire.Analyze through detecting, the anti-Xa factor of low molecular weight calcium heparin prepared by the present invention is tired between 105-120IU/mg, apparently higher than the high resistance Xa factor of recording in the CN1554671A 102IU/mg (embodiment 1) that tires, the highest ratio of the anti-II a of anti-Xa/ reaches more than 4.4 simultaneously, the highest ratio 4.2 of recording higher than CN1554671A.
In addition, the present invention has simplified production stage, once replace by resin, improved production efficiency, adopt Zeo-karb, calcium ion in first selective adsorption raw material, then replace with low molecular weight heparin sodium, reduce the introducing of impurity, the low molecular weight calcium heparin producing of replacing, major part is combined calcium, ensures the reliable and stable of calcium contents.
Embodiment
The invention discloses a kind of preparation method of low molecular weight calcium heparin, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Preparation method of the present invention is described by preferred embodiment, related personnel obviously can change methods and applications as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
Be described further with regard to the preparation method of a kind of low molecular weight calcium heparin provided by the present invention below.
Embodiment 1: prepare low molecular weight calcium heparin
Degraded: get in the water-soluble solution of refined heparin sodium 2.5kg, to 2-4, add Sodium Nitrite 80.00g reaction 4-6h with vinegar acid for adjusting pH value.
Oxidation: the degradation solution that upper step makes, adds 30% (mass percent, lower same) superoxol 660mL to be oxidized 15min.
Neutralization: add in 25% sodium hydroxide solution and pH value to 6.5-7.0.
Alcohol precipitation: neutralizer adds 95% ethanol alcohol precipitation, alcohol hypostasis is low molecular weight heparin sodium solution after dissolving with water for injection.
Calcification resin: the upper 0.5mol/L calcium chloride solution of JZD001X7-II type large hole strong acid styrene system cation exchange resin, replace to lower column liquid pH value and be after neutrality, water for injection flushing ion exchange resin.
Calcium displacement: cationic exchange coloum on low molecular weight heparin sodium solution, carries out the displacement of sodium calcium.
Refining: displacement liquid, after 0.22um filters, obtains fine work solution.
Freeze-drying: fine work solution is placed in freeze drier freeze-drying, after pulverizing, obtains low molecular weight calcium heparin finished product 1858g.
Embodiment 2: prepare low molecular weight calcium heparin
Degraded: get in the water-soluble solution of refined heparin sodium 2.5kg, to 2-4, add Sodium Nitrite 81.25g reaction 4-6h with vinegar acid for adjusting pH value.
Oxidation: the degradation solution that upper step makes, adds 30% superoxol 660mL oxidation 15min.
Neutralization: add in 25% sodium hydroxide solution and pH value to 6.5-7.0.
Alcohol precipitation: neutralizer adds 95% ethanol alcohol precipitation, alcohol hypostasis is low molecular weight heparin sodium solution after dissolving with water for injection.
Calcification resin: the upper 0.5mol/L calcium chloride solution of JZD001X7-II type large hole strong acid styrene system cation exchange resin, replace to lower column liquid pH value and be after neutrality, water for injection flushing ion exchange resin.
Calcium displacement: cationic exchange coloum on low molecular weight heparin sodium solution, carries out the displacement of sodium calcium.
Refining: displacement liquid, after 0.22um filters, obtains fine work solution.
Freeze-drying: fine work solution is placed in freeze drier freeze-drying, after pulverizing, obtains low molecular weight calcium heparin finished product 1932g.
Embodiment 3: prepare low molecular weight calcium heparin
Degraded: get in the water-soluble solution of refined heparin sodium 2.5kg, to 2-4, add Sodium Nitrite 82.5g reaction 4-6h with vinegar acid for adjusting pH value.
Oxidation: the degradation solution that upper step makes, adds 30% superoxol 660mL oxidation 15min.
Neutralization: add in 25% sodium hydroxide solution and pH value to 6.5-7.0.
Alcohol precipitation: neutralizer adds 95% ethanol alcohol precipitation, alcohol hypostasis is low molecular weight heparin sodium solution after dissolving with water for injection.
Calcification resin: 0.5mol/L calcium chloride solution on D001 type strongly acidic macroporous cation exchange resin, to replace to lower column liquid pH value and be after neutrality, water for injection rinses ion exchange resin.
Calcium displacement: cationic exchange coloum on low molecular weight heparin sodium solution, carries out the displacement of sodium calcium.
Refining: displacement liquid, after 0.22um filters, obtains fine work solution.
Freeze-drying: fine work solution is placed in freeze drier freeze-drying, after pulverizing, obtains low molecular weight calcium heparin finished product 2047g.
Embodiment 4: prepare low molecular weight calcium heparin
Degraded: get in the water-soluble solution of refined heparin sodium 2.5kg, to 2-4, add Sodium Nitrite 83.75g reaction 4-6h with vinegar acid for adjusting pH value.
Oxidation: the degradation solution that upper step makes, adds 30% superoxol 660mL oxidation 15min.
Neutralization: add in 25% sodium hydroxide solution and pH value to 6.5-7.0.
Alcohol precipitation: neutralizer adds 95% ethanol alcohol precipitation, alcohol hypostasis is low molecular weight heparin sodium solution after dissolving with water for injection.
Calcification resin: 0.5mol/L calcium chloride solution on D001 type strongly acidic macroporous cation exchange resin, to replace to lower column liquid pH value and be after neutrality, water for injection rinses ion exchange resin.
Calcium displacement: cationic exchange coloum on low molecular weight heparin sodium solution, carries out the displacement of sodium calcium.
Refining: displacement liquid, after 0.22um filters, obtains fine work solution.
Freeze-drying: fine work solution is placed in freeze drier freeze-drying, after pulverizing, obtains low molecular weight calcium heparin finished product 2195g.
Embodiment 5: product detects
Low molecular weight calcium heparin prepared by embodiment 1-embodiment 4 detects, and contrast product is product prepared by the method that adopts CN1554671A embodiment 1 to record, and detected result is as following table:
Table 1 product detected result
As shown in Table 1, product prepared by the present invention meets every quality standard of low molecular weight calcium heparin, and tires at anti-Xa factor, is significantly better than CN1554671A aspect the anti-II a ratio of anti-Xa/ and weight loss on drying ratio.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. a preparation method for low molecular weight calcium heparin, is characterized in that, comprises the following steps:
Step 1, utilize sodium nitrite method to prepare low molecular weight heparin sodium solution;
Step 2, Zeo-karb carry out resin calcification with calcium chloride solution, and the cationic resin column on the low molecular weight heparin sodium solution of then being prepared by step 1 after calcification is carried out the displacement of calcium sodium ion;
Step 3, by displacement liquid filtration, freeze-drying, pulverizing after step 2 calcium sodium ion displacement, obtain low molecular weight calcium heparin.
2. preparation method according to claim 1, is characterized in that, step 1 is:
In step 1.1, the water-soluble solution of taking heparin sodium, to 2-4, add Sodium Nitrite reaction 4-6h to obtain degradation solution with vinegar acid for adjusting pH value;
Step 1.2, to adding 30% superoxol oxidation 15min in described degradation solution, then add in 25% sodium hydroxide solution and pH value to 6.5-7.0, obtain neutralizer;
Step 1.3, in described neutralizer, add 95% ethanol to carry out alcohol precipitation and separate alcohol hypostasis, gained alcohol hypostasis dissolves with water for injection, obtains low molecular weight heparin sodium solution.
3. preparation method according to claim 2, is characterized in that, the ratio of described heparin sodium, Sodium Nitrite and 30% hydrogen peroxide is 2.5kg:70g-90g:660mL.
4. preparation method according to claim 1, is characterized in that, described Zeo-karb is strongly acidic macroporous cation exchange resin.
5. preparation method according to claim 4, is characterized in that, described strongly acidic macroporous cation exchange resin is D001 type strongly acidic macroporous cation exchange resin or JZD001X7-II type large hole strong acid styrene system cation exchange resin.
6. preparation method according to claim 1, is characterized in that, the weight-average molecular weight of described low molecular weight heparin sodium is 3000-4500D.
7. preparation method according to claim 1, is characterized in that, the weight-average molecular weight of described low molecular weight calcium heparin is 3000-4500D.
8. preparation method according to claim 1, is characterized in that, described resin calcification step is:
Calcium chloride solution on Zeo-karb, replaces to lower column liquid pH value and is after neutrality, and water for injection rinses Zeo-karb and completes resin calcification.
9. preparation method according to claim 8, is characterized in that, described calcium chloride solution concentration is 0.5mol/L.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262510A (en) * | 2014-10-11 | 2015-01-07 | 枣庄赛诺康生化股份有限公司 | Preparation method for low molecular weight heparin sodium with ultralow free sulfate group content |
| CN106986954A (en) * | 2017-04-19 | 2017-07-28 | 烟台东诚药业集团股份有限公司 | A kind of Dalteparin Sodium and Nadroparin Calcium combined preparation process |
| CN108409890A (en) * | 2018-03-16 | 2018-08-17 | 湖北亿诺瑞生物制药有限公司 | A method of producing calciparine from heparin sodium crude |
| CN109575156A (en) * | 2018-11-05 | 2019-04-05 | 上海宝维医药技术有限公司 | A kind of purification process of low molecular weight heparin |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2047978C (en) * | 1990-07-27 | 2001-02-27 | Jose-Luis Orozco | Process for preparing heparin calcium |
| CN1554671A (en) * | 2003-12-24 | 2004-12-15 | 合肥兆峰科大药业有限公司 | Process for preparing low moledule heparin calcium of low nitrite content |
| CN101519459A (en) * | 2008-02-26 | 2009-09-02 | 苏州法思特生物制药科技有限公司 | Technique for producing ultra-low molecular heparin sodium (calcium) |
| CN103315951A (en) * | 2013-07-17 | 2013-09-25 | 海南通用同盟药业有限公司 | Low-molecular-weight heparin calcium injection |
| CN103382232A (en) * | 2012-05-04 | 2013-11-06 | 常州泰康制药有限公司 | Preparation and purification process for nadroparin calcium |
| CN103408676A (en) * | 2013-07-15 | 2013-11-27 | 河北常山生化药业股份有限公司 | Nadroparin calcium preparation technology |
-
2014
- 2014-07-07 CN CN201410320475.9A patent/CN104072637B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2047978C (en) * | 1990-07-27 | 2001-02-27 | Jose-Luis Orozco | Process for preparing heparin calcium |
| CN1554671A (en) * | 2003-12-24 | 2004-12-15 | 合肥兆峰科大药业有限公司 | Process for preparing low moledule heparin calcium of low nitrite content |
| CN101519459A (en) * | 2008-02-26 | 2009-09-02 | 苏州法思特生物制药科技有限公司 | Technique for producing ultra-low molecular heparin sodium (calcium) |
| CN103382232A (en) * | 2012-05-04 | 2013-11-06 | 常州泰康制药有限公司 | Preparation and purification process for nadroparin calcium |
| CN103408676A (en) * | 2013-07-15 | 2013-11-27 | 河北常山生化药业股份有限公司 | Nadroparin calcium preparation technology |
| CN103315951A (en) * | 2013-07-17 | 2013-09-25 | 海南通用同盟药业有限公司 | Low-molecular-weight heparin calcium injection |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104262510A (en) * | 2014-10-11 | 2015-01-07 | 枣庄赛诺康生化股份有限公司 | Preparation method for low molecular weight heparin sodium with ultralow free sulfate group content |
| CN104262510B (en) * | 2014-10-11 | 2016-08-17 | 山东万邦赛诺康生化制药股份有限公司 | A kind of preparation method of the low molecular weight heparin sodium of ultralow free sulphur acidic group |
| CN106986954A (en) * | 2017-04-19 | 2017-07-28 | 烟台东诚药业集团股份有限公司 | A kind of Dalteparin Sodium and Nadroparin Calcium combined preparation process |
| CN106986954B (en) * | 2017-04-19 | 2020-12-25 | 烟台东诚药业集团股份有限公司 | Joint preparation method of dalteparin sodium and nadroparin calcium |
| CN108409890A (en) * | 2018-03-16 | 2018-08-17 | 湖北亿诺瑞生物制药有限公司 | A method of producing calciparine from heparin sodium crude |
| CN109575156A (en) * | 2018-11-05 | 2019-04-05 | 上海宝维医药技术有限公司 | A kind of purification process of low molecular weight heparin |
| CN109575156B (en) * | 2018-11-05 | 2021-02-23 | 上海宝维医药技术有限公司 | Purification method of low-molecular heparin |
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