CN103540630B - A kind of preparation method of low molecular weight heparin - Google Patents
A kind of preparation method of low molecular weight heparin Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of low molecular weight heparin, be to degrade under certain temperature of reaction heparin with histidine-tagged Heparinase I, namely reaction solution is purified obtains low molecular weight heparin, in described method, certain temperature of reaction is 15-40 DEG C, and described reaction process is the Δ A at reaction solution
232stop when reaching 15-50.The present invention adopts suitable temperature of reaction, ensure that to have high enzyme activity and faster reaction speed; Accurately can judge reaction end simultaneously, the degree controlling the number-average molecular weight DeR of low molecular weight heparin is then realized by the absorbancy detecting 232nm place, thus make the molecular weight of the low molecular weight heparin obtained in target zone, improve the efficiency of degraded heparin.The Histidine Heparinase I utilizing method of the present invention to produce with the good low molecular weight heparin of lower cost preparation quality, can have huge industrial application value.
Description
Technical field
The present invention relates to a kind of preparation method of low molecular weight heparin, belong to biomedicine field.
Background technology
Heparin is the mucopolysaccharide having hexuronic acid (L-iduronic acid, D-Glucose aldehydic acid) and D-Glucosamine Sulphate alternately to be formed with 1 → 4 glycosidic link, its molecular weight is between 3000-40000Da, molecular-weight average is 15000Da, heparin has history for many years as anti-freezing reagent and anti-bolt reagent, and heparin also has the vital function of the aspects such as anti-inflammatory, antianaphylaxis, antiviral, anticancer, Adjust-blood lipid in addition.But because heparin has anticoagulant active, so a large amount of uses can cause bleeding and the side effect such as induced platelet minimizing, osteoporosis, thus limit heparin application clinically greatly.
Low molecular weight heparin (low-molecular-weightheparin, LMWH) (.Linhardt, R.J.Gunay, the component in unfraction heparin with lower molecular weight N.S.Productionandchemicalprocessingoflowmolecularweighth eparins.Sem.Thromb.Hem.25 (3): 5-16), or the small molecule component obtained after depolymerized heparin or fragment, molecular weight is between 3000-8000Da.Compared with heparin, the anticoagulation of LMWH is weaker than heparin, but its anti thrombotic action is obviously better than heparin, and also has other advantages, as molecular weight is little, bioavailability is high, and Half-life in vivo is long, by body weight administration, anticoagulant effect can be predicted, is lessly combined with thrombocyte, and bleeding tendency is little.So LMWH is one antithrombotic reagent safely and effectively, heparin can be replaced clinically.Molecular weight is homogeneous, and the LMWH drug effect clinically of narrow distribution range is obviously better, and thus carrying out in quality control to LMWH, pharmacopoeia of each country all defines its molecular-weight average and range of molecular weight distributions.So, prepare the suitable and low molecular weight heparin of narrow ditribution of molecular weight and have great importance.
At present, the preparation method of LMWH mainly contains chemical cleavage method and enzyme liberating method.Although chemical cleavage method technical process is simpler, but the strong oxidizer added in cracking process can cause the reduction of anticoagulant heparin activity with the sulfate group effect in heparin, in addition this method also may introduce poisonous substance and contaminate environment, and enzyme liberating method due to the reaction conditions of its gentleness and nontoxicity easily realize production serialization and more and more obtain the concern of people.United States Patent (USP) (US5106734,1992) can prepare the low molecular weight heparin product of suitable molecular weight by the absorbancy detecting 235nm place.In enzyme liberating method, main research adopts and prepares Low molecular heparin from the Heparinase I of Flavobacterium heparinum (Flavobacteriumheparinum), because this production of enzyme is low, purification difficult, cause the cost of enzyme very expensive, limit the development that enzyme process prepares Low molecular heparin.(Yu Guangli, Wang Qun, Guan Huashi, the Xu Jiamin such as Yu Guangli, RobertJ.Linhardt, the preparation of ox lung Heparin Oligosaccharides, Qingdao Marine University's journal, 2002,32(2): 231-235) utilize heparinase controlling degraded ox lung heparin, obtain the oligosaccharides sterling that the polymerization degree is 2-20.(the YeFC such as Xing Xinhui, KuangY, ChenS, ZhangC, ChenY, XingXH.Characteristicsoflowmolecularweightheparinproduct ionbyanultrafiltrationmembranebioreactorusingmaltosebind ingproteinfusedheparinaseI.BIOCHEMENGJ.2009, maltose binding protein fusion 46:193-198) is utilized to express Heparinase I, and utilize the enzyme liberating heparin produced, obtain the low molecular weight heparin of a series of different molecular weight.Although these output, way of purification and preparation systems of studying Heparinase I are all improved, but still there is the problem of certain poor stability in the Heparinase I used in enzymolysis heparin, cause the cost of enzyme still expensive, limit the development that enzyme process prepares low molecular weight heparin.Utilizing recombinant bacterial strain to produce heparinase and in enzyme, add stablizer is the effective way reducing enzyme cost.
Summary of the invention
The object of this invention is to provide and a kind ofly adopt suitable temperature of reaction, there is high enzyme activity, accurately can judge reaction end, control the preparation method of the low molecular weight heparin of the number-average molecular weight of low molecular weight heparin.
The preparation method of low molecular weight heparin provided by the present invention, to degrade under certain temperature of reaction heparin with histidine-tagged Heparinase I, namely reaction solution is purified obtains low molecular weight heparin, it is characterized in that: in described method, certain temperature of reaction is 15-40 DEG C, and described reaction process is the Δ A at reaction solution
232stop when reaching 15-50, described Δ A
232at the end of being the reaction of histidine-tagged Heparinase I degraded heparin, reaction solution is compared to the increased value of the 232nm absorbancy of reaction solution time initial.
In described method, described histidine-tagged Heparinase I be utilize escherichia coli expression with 6 histidine-tagged restructuring Heparinase Is, type of service is the solution resuspended in water of the histidine-tagged Heparinase I lyophilized powder containing dithiothreitol (DTT) and tween 80.
Described heparin prepares with the following method: at 100mL containing 5mMCaCl
2add a certain amount of heparin with in the water of 100mMNaCl, make the concentration of heparin in heparin solution be 1-100mg/mL, be preferably 40mg/mL, pH HCl and be adjusted to 6.0-8.0, be preferably 7.0.In described method, the proportioning (enzyme of Histidine Heparinase I lives mIU: heparin solution mg/mL) of histidine-tagged Heparinase I and heparin is 1:1-4:1, preferably 2:1.
In described method, temperature of reaction is 15-40 DEG C, is preferably 20-35 DEG C.Investigate the temperature of reaction of histidine-tagged Heparinase I, find, between 15-40 DEG C, have certain activities present, and in 20-35 DEG C of interval, enzyme lives performance well, optimal reactive temperature is about 30 DEG C or 32 DEG C.From the stability of enzyme, temperature is higher, and enzyme deactivation speed is faster, and the transformation period is shorter.From reaction efficiency, the temperature high then required reaction times is short.Consider stability and the reaction efficiency of enzyme, the scope of selective reaction temperature be 20-35 DEG C comparatively suitable, make its heparin of can effectively degrading can ensure enough reaction efficiencies again.
In described method, reaction process is at the Δ A of reaction solution
232stopping when reaching 15-50, stopping by regulating pH to 2.0 to realize.The molecular weight of product determines the quality of product, and the accurate control realizing molecular weight then accurately must judge terminal.The control of reaction end realizes by Real-Time Monitoring level of response, and level of response and Δ A232 are proportionate, the value directly can monitoring Δ A232 to control level of response, to guarantee the accurate of reaction end.
In described method, comprise low molecular weight heparin and the Δ A of the number-average molecular weight shown in equation 2
232relational expression, by the Δ A of reaction solution at the end of the reaction process of control group His tag Heparinase I degraded heparin
232, obtain the low molecular weight heparin of number of targets average molecular weight;
1/Mn=1/13000+ Δ A
232/ (7200*c)---equation 2
In described equation 2, Mn is the number-average molecular weight of reaction solution, Δ A
232at the end of being the reaction of histidine-tagged Heparinase I degraded heparin, reaction solution compares the increased value with the 232nm absorbancy of reaction solution time initial.
In described method, purifying low molecular weight heparin in accordance with the following methods: after termination reaction, by reaction solution with the Fibrous membrane filtration of 0.22 micron except Deproteinization, collecting just filtrate molecular weight cut-off is the ultrafiltration membrance filter of 3000-10000Da, centrifugal add the ethanol mixing of filtrate volume 3-4 times in filtrate after, collecting precipitation adds washing with acetone, and precipitation evaporated under reduced pressure can be obtained low molecular weight heparin product.
Method of the present invention adopts histidine-tagged Heparinase I to prepare low molecular weight heparin, be made up of 6 Histidines due to histidine-tagged, molecular weight is only 0.84kDa, steric effect is more weak, substantially can ignore the space conformation of Heparinase I and the impact of zymologic property, remain the enzyme of Heparinase I to heparin largely and cut effect, and Histidine Heparinase I is solubility expression, just can be obtained the proteolytic enzyme of more than 90% by single step purification, thus ensure that the quality of low molecular weight heparin again reduces the production cost of enzyme.In addition adopt suitable temperature of reaction, ensure that there is high enzyme activity and faster reaction speed; Accurately can judge reaction end simultaneously, the degree controlling the number-average molecular weight DeR of low molecular weight heparin is then realized by the absorbancy detecting 232nm place, thus make the molecular weight of the low molecular weight heparin obtained in target zone, improve the efficiency of degraded heparin.The Histidine Heparinase I utilizing method of the present invention to produce with the good low molecular weight heparin of lower cost preparation quality, can have huge industrial application value.
Accompanying drawing explanation
Fig. 1 is that the absorbancy of the 232nm of reaction solution in reaction process is schemed over time
Fig. 2 is reaction solution number-average molecular weight Mn and A
232the graph of a relation of value
Fig. 3 is reaction solution number-average molecular weight Mn and weight-average molecular weight Mw graph of a relation
Embodiment
Following embodiment will illustrate working method of the present invention, but can not as limitation of the invention.
Experimental technique in following embodiment, if no special instructions, is ordinary method.
Percentage composition in following embodiment, if no special instructions, is mass percentage.
(embodiment 1) histidine-tagged Heparinase I degraded heparin prepares reaction solution Δ A in low molecular weight heparin process
232change, number-average molecular weight Mn and Δ A
232the relation of value and the relation of liquid number-average molecular weight Mn and weight-average molecular weight Mw
(1). histidine-tagged Heparinase I degraded heparin prepares reaction solution Δ A in low molecular weight heparin process
232change
With unfraction heparin (purchased from Sangon Biotech (Shanghai) Co., Ltd.) for raw material, 2000mg unfraction heparin is joined 100mL containing 5mMCaCl
2with in the water of 100mMNaCl, pH HCl is adjusted to 7.0, obtains heparin solution.
Appeal 100mL heparin solution is placed in 30 DEG C of constant temperature, the histidine-tagged Heparinase I adding 4000mIU reacts, take out part reaction solution at regular intervals, measure the absorbancy of its 232nm and measure its number-average molecular weight Mn and weight-average molecular weight Mw according to measuring method shown in European Pharmacopoeia 5.0.
The A of the reaction solution of the differential responses time obtained is tested in appeal
232value changing conditions as shown in Figure 1.Fig. 1 shows, the A of reaction solution
232value increases along with the increase of time, and the relation of the two roughly meets first order reaction kinetics.
(2). histidine-tagged Heparinase I degraded heparin prepares reaction solution number-average molecular weight Mn and Δ A in low molecular weight heparin process
232the relation of value
The number-average molecular weight Mn of reaction solution and Δ A
232relation see US Patent No. 005106734:
1/Mn=1/Mn, u+ Δ A
232/ (ε * c) equation 1
In described equation 1, Mn is the number-average molecular weight of reaction solution, and Mn, u are the number-average molecular weight of unfraction heparin, Δ A
232for reaction solution autoreaction initial time 232nm absorbancy increased value, c is the concentration (mg/mL) of initial unfraction heparin, and ε is molar absorptivity.
The number-average molecular weight of the reaction solution of the differential responses time utilizing example 1 to obtain and the Δ A of reaction solution
232value, meeting the relation shown in equation 1, is 1/Mn=1/13000+ Δ A
232/ (7200*c) equation 2
Then 1/Mn and Δ A
232relation as shown in Figure 2.Δ A can be obtained according to number-average molecular weight
232, thus the degree of reaction is by detecting Δ A
232value realize.
(3). histidine-tagged Heparinase I degraded heparin prepares the relation of reaction solution number-average molecular weight Mn and weight-average molecular weight Mw in low molecular weight heparin process
The number-average molecular weight of the reaction solution of the differential responses time utilizing example 1 to obtain and weight-average molecular weight, take weight-average molecular weight as X-coordinate, number-average molecular weight is ordinate zou mapping, and as shown in Figure 3, then reacting the weight-average molecular weight terminating rear reaction solution can try to achieve according to Fig. 3 result.
(embodiment 2) histidine-tagged Heparinase I controlling degraded heparin prepares low molecular weight heparin 1
Preparation 20mg/mL heparin, 5mMCaCl
2, 100mMNaCl, pH be the reaction soln of 7.0, after being placed on 15 DEG C of thermostat water bath preheating 12min, the histidine-tagged Heparinase I adding 2000mIU reacts, and after reaction for some time, records its Δ A
232value is 34.8, add salt acid for adjusting pH to 2.0 termination reaction, by reaction solution with the Fibrous membrane filtration of 0.22 micron except Deproteinization, collecting just filtrate molecular weight cut-off is the ultrafiltration membrance filter of 3000Da, centrifugal add the ethanol mixing of filtrate volume 3-4 times in filtrate after, collecting precipitation adds washing with acetone, measures its number-average molecular weight Mn and weight-average molecular weight Mw by after precipitation evaporated under reduced pressure.Mn value is 4322Da, Mw value is 6433Da, and distribution coefficient is 1.48, and molecular weight meets the requirement of pharmacopeia to low molecular weight heparin product.
(embodiment 3) histidine-tagged Heparinase I controlling degraded heparin prepares low molecular weight heparin 2
Preparation 20mg/mL heparin, 5mMCaCl
2, 100mMNaCl, pH be the reaction soln of 7.0, after being placed on 20 DEG C of thermostat water bath preheating 10min, the histidine-tagged Heparinase I adding 2000mIU reacts, and after reaction for some time, records its Δ A
232value is 34.8, add salt acid for adjusting pH to 2.0 termination reaction, by reaction solution with the Fibrous membrane filtration of 0.22 micron except Deproteinization, collecting just filtrate molecular weight cut-off is the ultrafiltration membrance filter of 3000Da, centrifugal add the ethanol mixing of filtrate volume 3-4 times in filtrate after, collecting precipitation adds washing with acetone, measures its number-average molecular weight Mn and weight-average molecular weight Mw by after precipitation evaporated under reduced pressure.Mn value is 4322Da, Mw value is 6433Da, and distribution coefficient is 1.48, and molecular weight meets the requirement of pharmacopeia to low molecular weight heparin product.
(embodiment 4) histidine-tagged Heparinase I controlling degraded heparin prepares low molecular weight heparin 3
Preparation 20mg/mL heparin, 5mMCaCl
2, 100mMNaCl, pH be the reaction soln of 7.0, after being placed on 30 DEG C of thermostat water bath preheating 10min, adding enzyme concn is that the histidine-tagged Heparinase I solution of 4000mIU reacts, after reaction for some time, as its Δ A
232when value is 42.7, add salt acid for adjusting pH to 2.0 termination reaction, equally reaction solution is removed Deproteinization with the Fibrous membrane filtration of 0.22 micron, collecting just filtrate molecular weight cut-off is the ultrafiltration membrance filter of 3000Da, centrifugal add the ethanol mixing of filtrate volume 3-4 times in filtrate after, collecting precipitation adds washing with acetone, measures its number-average molecular weight Mn and weight-average molecular weight Mw by after precipitation evaporated under reduced pressure.Mn value is 3590Da, Mw value is 5488Da, and distribution coefficient is 1.52, and molecular weight also meets the requirement of pharmacopeia to low molecular weight heparin product.
(embodiment 5) histidine-tagged Heparinase I controlling degraded heparin prepares low molecular weight heparin 4
Preparation 10mg/mL heparin, 5mMCaCl
2, 100mMNaCl, pH be the reaction soln of 7.0, after being placed on 25 DEG C of thermostat water bath preheating 10min, adding enzyme concn is that the histidine-tagged Heparinase I solution of 4000mIU reacts, after reaction for some time, as its Δ A
232when value is 19.2, add salt acid for adjusting pH to 2.0 termination reaction, equally reaction solution is removed Deproteinization with the Fibrous membrane filtration of 0.22 micron, collecting just filtrate molecular weight cut-off is the ultrafiltration membrance filter of 3000-10000Da, centrifugal add the ethanol mixing of filtrate volume 3-4 times in filtrate after, collecting precipitation adds washing with acetone, measures its number-average molecular weight Mn and weight-average molecular weight Mw by after precipitation evaporated under reduced pressure.Mn value is 4021Da, Mw value is 6150Da, and distribution coefficient is 1.52, and molecular weight also meets the requirement of pharmacopeia to low molecular weight heparin product.
(embodiment 6) histidine-tagged Heparinase I controlling degraded heparin prepares low molecular weight heparin 5
Preparation 30mg/mL heparin, 5mMCaCl
2, 100mMNaCl, pH be the reaction soln of 7.0, after being placed on 28 DEG C of thermostat water bath preheating 10min, adding enzyme concn is that the histidine-tagged Heparinase I solution of 3000mIU reacts, after reaction for some time, as its Δ A
232when value is 23.1, add salt acid for adjusting pH to 2.0 termination reaction, equally reaction solution is removed Deproteinization with the Fibrous membrane filtration of 0.22 micron, collecting just filtrate molecular weight cut-off is the ultrafiltration membrance filter of 3000-10000Da, centrifugal add the ethanol mixing of filtrate volume 3-4 times in filtrate after, collecting precipitation adds washing with acetone, measures its number-average molecular weight Mn and weight-average molecular weight Mw by after precipitation evaporated under reduced pressure.Mn value is 3991Da, Mw value is 5720Da, and distribution coefficient is 1.43, and molecular weight also meets the requirement of pharmacopeia to low molecular weight heparin product.Illustrate that the present invention can prepare the suitable low molecular weight heparin product of molecular weight effectively.
(embodiment 7) histidine-tagged Heparinase I controlling degraded heparin prepares low molecular weight heparin 6
Preparation 30mg/mL heparin, 5mMCaCl
2, 100mMNaCl, pH be the reaction soln of 7.0, after being placed on 32 DEG C of thermostat water bath preheating 10min, adding enzyme concn is that the histidine-tagged Heparinase I solution of 3000mIU reacts, after reaction for some time, as its Δ A
232when value is 23.1, add salt acid for adjusting pH to 2.0 termination reaction, equally reaction solution is removed Deproteinization with the Fibrous membrane filtration of 0.22 micron, collecting just filtrate molecular weight cut-off is the ultrafiltration membrance filter of 3000-10000Da, centrifugal add the ethanol mixing of filtrate volume 3-4 times in filtrate after, collecting precipitation adds washing with acetone, measures its number-average molecular weight Mn and weight-average molecular weight Mw by after precipitation evaporated under reduced pressure.Mn value is 3991Da, Mw value is 5720Da, and distribution coefficient is 1.43, and molecular weight also meets the requirement of pharmacopeia to low molecular weight heparin product.Illustrate that the present invention can prepare the suitable low molecular weight heparin product of molecular weight effectively.
(embodiment 8) histidine-tagged Heparinase I controlling degraded heparin prepares low molecular weight heparin 7
Preparation 10mg/mL heparin, 5mMCaCl
2, 100mMNaCl, pH be the reaction soln of 7.0, after being placed on 25 DEG C of thermostat water bath preheating 10min, adding enzyme concn is that the histidine-tagged Heparinase I solution of 4000mIU reacts, after reaction for some time, as its Δ A
232when value is 19.2, add salt acid for adjusting pH to 2.0 termination reaction, equally reaction solution is removed Deproteinization with the Fibrous membrane filtration of 0.22 micron, collecting just filtrate molecular weight cut-off is the ultrafiltration membrance filter of 3000-10000Da, centrifugal add the ethanol mixing of filtrate volume 3-4 times in filtrate after, collecting precipitation adds washing with acetone, measures its number-average molecular weight Mn and weight-average molecular weight Mw by after precipitation evaporated under reduced pressure.Mn value is 4021Da, Mw value is 6150Da, and distribution coefficient is 1.52, and molecular weight also meets the requirement of pharmacopeia to low molecular weight heparin product.
Claims (6)
1. the preparation method of a low molecular weight heparin, to degrade under certain temperature of reaction heparin with histidine-tagged Heparinase I, namely reaction solution is purified obtains low molecular weight heparin, it is characterized in that: in described method, certain temperature of reaction is 15-40 DEG C, and described reaction process is the Δ A at reaction solution
232stop when reaching 15-50, described Δ A
232at the end of being the reaction of histidine-tagged Heparinase I degraded heparin, reaction solution is compared to the increased value of the 232nm absorbancy of reaction solution time initial; Described histidine-tagged Heparinase I be utilize escherichia coli expression with 6 histidine-tagged restructuring Heparinase Is, type of service is the solution resuspended in water of the histidine-tagged Heparinase I lyophilized powder containing dithiothreitol (DTT) and tween 80; Described dithiothreitol (DTT) and the concentration of tween 80 before the freeze-drying of histidine-tagged Heparinase I solution are respectively 1mM and 0.001%; By the Δ A of reaction solution at the end of the reaction process of control group His tag Heparinase I degraded heparin
232, obtain the low molecular weight heparin of number of targets average molecular weight; The number-average molecular weight of described low molecular weight heparin with Δ A
232relational expression be equation 2, that is:
1/Mn=1/13000+ Δ A
232/ (7200*c) equation 2
In described equation 2, Mn is number-average molecular weight, Δ A
232at the end of being the reaction of histidine-tagged Heparinase I degraded heparin, reaction solution is compared to the increased value of the 232nm absorbancy of reaction solution time initial, and c is the concentration of initial unfraction heparin;
The proportioning of histidine-tagged Heparinase I and heparin and the enzyme of Histidine Heparinase I mIU alive: heparin solution mg/mL is 2:1;
Wherein, histidine-tagged molecular weight is 0.84kDa.
2. the preparation method of low molecular weight heparin according to claim 1, is characterized in that: in described method, and certain temperature of reaction is 20-35 DEG C.
3. the preparation method of low molecular weight heparin according to claim 1, is characterized in that: in described method, and certain temperature of reaction is 30 DEG C or 32 DEG C.
4. according to the preparation method of the arbitrary described low molecular weight heparin of claims 1 to 3, it is characterized in that: reaction terminating realizes by regulating reaction solution pH to 2.0.
5. the preparation method of low molecular weight heparin according to claim 4, is characterized in that: described heparin prepares with the following method: at 100mL containing 5mMCaCl
2add a certain amount of heparin with in the water of 100mMNaCl, make the concentration of heparin in heparin solution be that 1-100mg/mL, pH HCl is adjusted to 6.0-8.0.
6. the preparation method of low molecular weight heparin according to claim 5, is characterized in that: the concentration of heparin in heparin solution is that 40mg/mL, pH HCl is adjusted to 7.0.
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