DK156402B - Process for the preparation of low-molecular heparin - Google Patents
Process for the preparation of low-molecular heparin Download PDFInfo
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- DK156402B DK156402B DK217187A DK217187A DK156402B DK 156402 B DK156402 B DK 156402B DK 217187 A DK217187 A DK 217187A DK 217187 A DK217187 A DK 217187A DK 156402 B DK156402 B DK 156402B
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- heparin
- molecular weight
- depolymerization
- heparinase
- average molecular
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- 238000000034 method Methods 0.000 title claims description 25
- 239000003055 low molecular weight heparin Substances 0.000 title description 3
- 238000002360 preparation method Methods 0.000 title description 2
- 229920000669 heparin Polymers 0.000 claims description 71
- 229960002897 heparin Drugs 0.000 claims description 70
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 claims description 68
- 108010022901 Heparin Lyase Proteins 0.000 claims description 20
- 238000010521 absorption reaction Methods 0.000 claims description 15
- 230000000694 effects Effects 0.000 claims description 14
- 238000012691 depolymerization reaction Methods 0.000 claims description 8
- 239000011541 reaction mixture Substances 0.000 claims description 6
- 239000012736 aqueous medium Substances 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 230000031700 light absorption Effects 0.000 claims description 2
- 238000000354 decomposition reaction Methods 0.000 claims 1
- 239000000047 product Substances 0.000 description 19
- 230000002255 enzymatic effect Effects 0.000 description 11
- 230000000875 corresponding effect Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 6
- 239000000758 substrate Substances 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 238000006116 polymerization reaction Methods 0.000 description 5
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 108010074860 Factor Xa Proteins 0.000 description 3
- 230000002785 anti-thrombosis Effects 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 239000002634 heparin fragment Substances 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 102000004411 Antithrombin III Human genes 0.000 description 2
- 108090000935 Antithrombin III Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 208000007536 Thrombosis Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000004019 antithrombin Substances 0.000 description 2
- 229960005348 antithrombin iii Drugs 0.000 description 2
- 238000004061 bleaching Methods 0.000 description 2
- 150000001720 carbohydrates Chemical group 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 239000007857 degradation product Substances 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 229940127215 low-molecular weight heparin Drugs 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000011146 sterile filtration Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 241000589565 Flavobacterium Species 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 238000010268 HPLC based assay Methods 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- AEMOLEFTQBMNLQ-HNFCZKTMSA-N L-idopyranuronic acid Chemical class OC1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-HNFCZKTMSA-N 0.000 description 1
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102100023038 WD and tetratricopeptide repeats protein 1 Human genes 0.000 description 1
- 230000003024 amidolytic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 238000005422 blasting Methods 0.000 description 1
- 208000034158 bleeding Diseases 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 1
- 239000001639 calcium acetate Substances 0.000 description 1
- 229960005147 calcium acetate Drugs 0.000 description 1
- 235000011092 calcium acetate Nutrition 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229920001577 copolymer Chemical class 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000004836 empirical method Methods 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 description 1
- 239000002565 heparin fraction Substances 0.000 description 1
- -1 heparin oligosaccharides Chemical class 0.000 description 1
- 229960001008 heparin sodium Drugs 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 229910052588 hydroxylapatite Inorganic materials 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 230000001151 other effect Effects 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- XYJRXVWERLGGKC-UHFFFAOYSA-D pentacalcium;hydroxide;triphosphate Chemical compound [OH-].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O XYJRXVWERLGGKC-UHFFFAOYSA-D 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 208000010110 spontaneous platelet aggregation Diseases 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- Polysaccharides And Polysaccharide Derivatives (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
1 DK 156402 B1 DK 156402 B
Den foreliggende opfindelse angâr en fremgangsmâde til fremstilling af lavmolekylær heparin (LMW-heparin) ved enzymatisk depolymerisering af heparin.The present invention relates to a process for the preparation of low molecular weight heparin (LMW-heparin) by enzymatic depolymerization of heparin.
Konventionel heparin er en heterogen blanding af muco-5 polysaccharider, der dækker et molekylvægtomrâde fra 5000 - 50.000 dalton med en antalsmiddelmolekylvægt pâ ca. 10 - 14.000 dalton.Conventional heparin is a heterogeneous mixture of mucopoly polysaccharides covering a molecular weight range of 5000 - 50,000 daltons with a number average molecular weight of approx. 10 - 14,000 daltons.
Heparin indvirker direkte eller indirekte pâ funktionen af adskillige proteiner, især enzymer fra koagulationskaskaden.Heparin directly or indirectly affects the function of several proteins, especially enzymes from the coagulation cascade.
10 Adskillige faktorer, sâsom fordelingen af funktionelle grupper i molekylet og molekylvægten, har indflydelse pâ effekten af heparin. Det er sâledes velkendt, at molekylvægten spiller en vigtig rolle med hensyn til heparinaktivitet, især inaktivering af thrombin og faktor Xa medieret af antithrombin III.10 Several factors, such as the distribution of functional groups in the molecule and molecular weight, influence the effect of heparin. It is thus well known that molecular weight plays an important role in heparin activity, especially inactivation of thrombin and factor Xa mediated by antithrombin III.
15 Antithrombinaktivitet kræver en mindsteheparinmole- kylvægt svarende til ca. 18 monosaccharider, d.v.s. ca. 5400 dalton, hvorimod antifaktor Xa aktivitet kan udtrykkes med hepa-rinmolekyler sâ smâ som 5-6 saccharidenheder, 1500 - 1800 dalton.Antithrombin activity requires a minimum heparin molecular weight equal to approx. 18 monosaccharides, i.e. ca. 5400 daltons, whereas antifactor Xa activity can be expressed with heparin molecules as small as 5-6 saccharide units, 1500 - 1800 daltons.
20 En række andre effekter af heparin, f.eks. antithrombo- tisk effekt (heparinoligosaccharider indeholdende 18 monosaccha-rider eller mindre synes at hâve lav antithrombotisk aktivitet), indflydelse pâ ADP-induceret thrombocytaggregering, biotilgænge-lighed efter subcutan indgift, inhibering med PF^ (blodpladefak-25 tor 4) og HRG (histidinrig glycoprotein) (Thrombosis andA number of other effects of heparin, e.g. antithrombotic effect (heparin oligosaccharides containing 18 monosaccharides or less appear to have low antithrombotic activity), influence on ADP-induced platelet aggregation, bioavailability after subcutaneous administration, inhibition with PF 2 (platelet factor HRG), and platelet factor G 25 glycoprotein) (Thrombosis and
Haemostasis 58 (1987), 190) sâvel som aktiviteten over for koagu-leringsenzymer fra det interne System, som er ansvarlig for dan-nelse af faktor Xa, pâvirkes stærkt af heparins molekylvægt.Haemostasis 58 (1987), 190) as well as the activity against coagulation enzymes of the internal System responsible for the formation of factor Xa is strongly influenced by the molecular weight of heparin.
I de senere âr har interessen samlet sig om heparin-30 fragmenter eller -fraktioner med et hpjt forhold mellem antifaktor Xa aktivitet og antithrombinaktivitet med molekylvægt fra 4000 dalton til op imod 6000, da sâdanne stoffer har vist sig at hâve god antithrombotisk effekt og pâ samme tid ingen eller ringe tendens til at forârsage blodningskomplikationer. De udviser ogsâ 35 foroget biotilgængelighed, især efter subkutan indgift.In recent years, interest has accumulated in heparin fragments or fractions with a high ratio of antifactor Xa activity to antithrombin activity of molecular weight from 4000 daltons up to 6000, as such substances have been shown to have good antithrombotic effects and the same time no or little tendency to cause bleeding complications. They also exhibit increased bioavailability, especially after subcutaneous administration.
2 DK 156402 B2 DK 156402 B
**
Eftersom selektiviteten af heparinvirkning er korrele-ret med molekylvægten, er det sandsynligt, at der er et relativt snævert molekylvægtomrâde, indenfor hvilket heparinaktiviteten er optimal.Since the selectivity of heparin action is correlated with molecular weight, it is likely that there is a relatively narrow molecular weight range within which heparin activity is optimal.
5 En fremgangsmâde til fremstilling af LMW-heparin med en specifik, tilstræbt molekylvægt og en snæver molekylvægtforde-ling, d.v.s. lav polydispersitet ville derfor være fordelagtig.A process for preparing LMW heparin with a specific, targeted molecular weight and a narrow molecular weight distribution, i.e. low polydispersity would therefore be advantageous.
Fremgangsmâden if0lge den foreliggende opfindelse muligg0r opnâelse af et vilkârligt 0nsket molekylvægtomrâde af et 10 depolymeriseringsprodukt af heparin.The process of the present invention enables the obtaining of any desired molecular weight range of a depolymerization product of heparin.
LMW-heparin kan fremstilles i lavt udbytte fra konven-tionel heparin ved fraktionering (DE offentligg0relsesskrift nr. 2.944.792 og nr. 2.945.595). St0rstedelen af LMW-heparin fremstilles imidlertid ved depolymerisering af heparin enten ved 15 kemiske eller enzymatiske fremgangsmâder eventuelt efterfulgt af fraktionering (jfr. A. A. Horner: Heparin, Chemistry and Clinical Usage, V.V. Kakkar og D.P. Thomas (eds.) Academie Press (1976), side 37-48 og Perlin et al., Carbohydrate Research 18^, (1971), 185-194.LMW heparin can be prepared in low yield from conventional heparin by fractionation (DE Publication No. 2,944,792 and 2,945,595). However, the majority of LMW heparin is prepared by depolymerizing heparin either by 15 chemical or enzymatic methods, optionally followed by fractionation (cf. AA Horner: Heparin, Chemistry and Clinical Usage, VV Kakkar and DP Thomas (eds.) Academie Press (1976), pages 37-48 and Perlin et al., Carbohydrate Research 18 ^, (1971), 185-194.
20 Kemisk depolymerisering af heparin er beskrevet i offentliggjorte EP-patentans0gninger nr. 0037.319, 0076.279 og 0014.184, US patentskrift nr. 4.351.938 og GB patentskrift nr. 2.002.406.Chemical depolymerization of heparin is disclosed in published European Patent Application Nos. 0037,319, 0076,279 and 0014,184, U.S. Patent No. 4,351,938 and GB Patent No. 2,002,406.
Enzymatisk depolymerisering er beskrevet i US patent-25 skrift nr. 3.766.167, GB patentskrift nr. 2.002.406, offentlig-gjort EP patentans0gning nr. 0014.184 og US patentskrift nr. 4.396.762.Enzymatic depolymerization is disclosed in US Patent No. 3,766,167, GB Patent No. 2,002,406, EP Patent Application No. 0014,184, and U.S. Patent No. 4,396,762.
Et væsentligt problem forbundet med aile de kendte batchdepolymeriseringsprocesser er at standse depolymeriserings-30 reaktionen ved den korrekte middelmolekylvægt. Ydermere resul-terer depolymeriseringsreaktionen i heparinfragmenter med mindre eller st0rre st0rrelse end den 0nskede molekylvægt, selv i mangel af sidereaktioner.A major problem associated with all of the known batch depolymerization processes is stopping the depolymerization reaction at the correct average molecular weight. Furthermore, the depolymerization reaction results in heparin fragments of less or larger size than the desired molecular weight, even in the absence of side reactions.
I de kendte depolymeriseringsprocesser til depolymeri-35 sering af heparin, som anvender uorganiske depolymeriseringsrea-genser (salpetersyre, hydrogenperoxid etc.) er der ingen præfe-rence med hensyn til st0rrelsen af det angrebne molekyle ellerIn the known depolymerization processes for the depolymerization of heparin using inorganic depolymerization reagents (nitric acid, hydrogen peroxide etc.), there is no preference as to the size of the affected molecule or
3 DK 156402 B3 DK 156402 B
med hensyn til positionen indenfor molekylet af det bând, der skal brydes. Ifolge R.J. Linhardt et al., Biochem.Biophys.Acta 702 (1982) 197-203 skelner sâledes ikke engang enzymet heparin-ase, idet heparinasens virkemâde er endolytisk tilfældig.with respect to the position within the molecule of the band to be broken. According to R.J. Thus, Linhardt et al., Biochem.Biophys.Acta 702 (1982) 197-203 do not even distinguish the enzyme heparinase, since the mode of action of the heparinase is endolytically random.
5 Det betyder, at polydispersiteten af en vilkârlig hepa- rindepolymeriseringsblanding udvikles pâ en statistisk forud-sigelig mâde som en funktion af graden af depolymerisering. Især pâ det tidspunkt, hvor middelmolekylvægten er lige over den 0n-skede værdi, har en stor del af fragmenterne den Onskede molekyl-10 vægt; men pâ grund af depolymeriseringens endolytisk tilfældige natur har de ogsâ en forholdsvis stor chance for at blive yder-ligere depolymeriseret til dannelse af fragmenter af suboptimal storrelse. En batchdepolymerisering bor derfor stoppes pâ dette tidspunkt.This means that the polydispersity of any heparin polymerization mixture is developed in a statistically predictable manner as a function of the degree of depolymerization. Particularly, at the time when the average molecular weight is just above the zero value, a large proportion of the fragments have the desired molecular weight; but due to the endolytically random nature of the depolymerization, they also have a relatively high chance of being further depolymerized to form suboptimal size fragments. A batch depolymerization should therefore be stopped at this time.
15 Der er imidlertid hidtil ikke udviklet tilfredsstillen- de metoder til kontrol af depolymeriseringen af heparin til op-nâelse af ho je udbytter af et forudbes teint LMW-heparinprodukt.However, to date, no satisfactory methods have been developed to control the depolymerization of heparin to obtain high yields of a predetermined LMW heparin product.
Det er et formâl med den foreliggende opfindelse at tilvejebringe en metode til kontrol af en enzymatisk depolymeri-20 sering af heparin med heparinase i vandigt medium.It is an object of the present invention to provide a method for controlling an enzymatic depolymerization of heparin with heparinase in aqueous medium.
Det er yderligere et formâl med den foreliggende opfindelse at tilvejebringe en metode til fremstilling af LMW-heparin med en Onsket middelmolekylvægt.It is a further object of the present invention to provide a method for preparing LMW heparin having a desired average molecular weight.
Den foreliggende opfindelse tilvejebringer en frem- 25 gangsmâde til fremstilling af LMW-heparin, ved hvilken heparin delvist depolymeriseres med heparinase i vandigt medium, og denne fremgangsmâde er ejendommelig ved, at man mâler forogelsen i lysabsorptionen under forlobet af en sâdan depolymerisering, hvilken forogelse er forârsaget af dannelsen af stigende mængder 30 af umættede heparinnedbrydningsprodukter, efterhânden som den enzymatiske depolymerisering skrider frem, og at den enzymatiske depolymerisering standses, nâr stigningen i absorption har nâet en værdi svarende til den onskede antalsmiddelmolekylvægt Mn og den tilsvarende onskede vægtmiddelmolekylvægt M , hvorpâ LMW- w 35 heparinproduktet udvindes fra reaktionsblandingen.The present invention provides a method for preparing LMW heparin in which heparin is partially depolymerized with heparinase in aqueous medium and this process is characterized by measuring the increase in light absorption during the course of such depolymerization, which increase is caused by the formation of increasing amounts of unsaturated heparin degradation products as the enzymatic depolymerization progresses and the enzymatic depolymerization ceases when the increase in absorption has reached a value corresponding to the desired number average molecular weight Mn and the corresponding desired weight average Mn The heparin product is recovered from the reaction mixture.
4 DK 156402 B4 DK 156402 B
Middelmolekylvægten af en heparindepolymeriseringsreak-tion kan bestemmes pâ en række mâder baseret pâ f.eks. GPC-HPLC, viskositetsmâling, lysspredning eller kemisk eller fysisk-kemisk bestemmelse af funktionelle grupper opstaet under depolymerise-5 ringsprocessen.The average molecular weight of a heparin polymerization reaction can be determined in a number of ways based on e.g. GPC-HPLC, viscosity measurement, light scattering, or chemical or physicochemical determination of functional groups formed during the depolymerization process.
De fleste nævnte fremgangsmâder sâsom GPC-HPLC er tid-r0vende og vanskeligt anvendelige i produktion i stor mâlestok, og storsteparten af de kendte metoder, der anvendes ved produktion af LMW-heparin er faktisk baseret pâ empiriske metoder, der 10 bygger pâ omhyggelig kontrol af udgangsmaterialet og reaktions-betingelserne for at opnâ den onskede molekylvægt ved afslutnin-gen af depolymeriseringsreaktionen. Imidlertid kan slutproduktets molekylvægt variere fra batch til batch pâ grund af uundgâelige variationer under depolymeriseringsreaktionen, f.eks. variationer 15 i enzymaktivitet, og det kan være vanskeligt at opnâ et ensartet produkt.Most of the mentioned methods such as GPC-HPLC are time consuming and difficult to use in large scale production, and most of the known methods used in the production of LMW heparin are actually based on empirical methods based on careful control of the starting material and reaction conditions to achieve the desired molecular weight at the end of the depolymerization reaction. However, the molecular weight of the final product may vary from batch to batch due to inevitable variations during the depolymerization reaction, e.g. variations in enzyme activity and it can be difficult to obtain a uniform product.
Hvis et produkt med den korrekte gennemsnitsmolekylvægt skal fremstilles fra hver produktionsbatch, skal depolymeriseringsreaktionen standses straks, nâr den onskede middelmolekyl-20 vægt er nâet i depolymeriseringsblandingen. Dette kræver, at ændringen i middelmolekylvægten overvâges ved at anvende metoder til molekylvægtbestemmelse med ringe eller ingen forsinkelse.If a product with the correct average molecular weight is to be prepared from each production batch, the depolymerization reaction must be stopped immediately when the desired average molecular weight is reached in the depolymerization mixture. This requires that the change in the average molecular weight be monitored by using small or no delay molecular weight methods.
Hidtil har der ikke eksisteret en hurtig, praktisk metode til bestemmelse af molekylvægten af en depolymeriserings-25 reaktionsblanding indeholdende LMW-heparin. Ifolge et aspekt af den foreliggende opfindelse tilvejebringes der en sâdan metode.To date, no rapid, practical method for determining the molecular weight of a depolymerization reaction mixture containing LMW heparin has existed. According to one aspect of the present invention, such a method is provided.
Middelmolekylvægten af heparin eller en LMW-heparin kan angives som antalsmiddelmolekylvægten (Mn), d.v.s. vægt/antal molekyler, eller som vægtmiddelmolekylvægt (M ) eller peak mole- w 30 kylvægt (Μ , ). M eller M , anvendes normalt til karakte-The average molecular weight of heparin or an LMW heparin may be indicated as the number average molecular weight (Mn), i.e. weight / number of molecules, or as weight average molecular weight (M) or peak molecular weight (Μ,). M or M, is usually used for character-
pSciJÇ W pSclKpSciJÇ W pSclK
risering af heparin eller LMW-heparinprodukter.heparin or LMW heparin products.
Det er nu blevet bekræftet eksperimentelt, at polydis-persiteten (D), d.v.s. Mw/Mn' af en ÇTiven heparindepolymerise-ringsblanding ændres pâ en regelbunden mâde under den enzymatiske 35 depolymeriseringsreaktion af heparin som vist i fig. 1.It has now been experimentally confirmed that the polydispersity (D), i.e. Mw / Mn 'of a ßive heparin polymerization mixture is changed in a regular manner during the enzymatic depolymerization reaction of heparin as shown in FIG. First
5 DK 156402 BDK 156402 B
Den foreliggende opfindelse bygger pâ det forhold, at der er udviklet en hurtig og pâlidelig metode til bestemmelse af under depolymeriseringen af heparin med heparinase som forkla-ret i det folgende.The present invention is based on the fact that a rapid and reliable method has been developed for determining during the depolymerization of heparin with heparinase as explained below.
5 Da fig. 1 angiver korrelationen mellem M og M , kan w n depolymeriseringen af heparin til LMW-heparin med en given, 0n-sket opnâs ved depolymerisering til den tilsvarende antals-middelmolekylvægt Mn.5 As FIG. 1 indicates the correlation between M and M, w n the depolymerization of heparin to LMW heparin with a given 0n step can be obtained by depolymerization to the corresponding number average molecular weight Mn.
Den enzymatiske depolymeriseringsproces med hepa-10 rinase egner sig til en spektrophotometrisk antalsmiddelmole-kylvægtsbestemmelse (Mn) eftersom den enzymatiske procès er eli-minativ og danner en reducerende endegruppe og en endegruppe bestâende af et /\4,5 umættet-iduronsyrederivat med en distinkt UV-absorption ved 230-235 nm. Den molære absorptionskoefficient 15 for et antal LMW-heparinfragmenter af di-, tetra-, hexa- og oligosaccharider blev offentliggjort af Linker og Hovingh (Biochem. 11 (1972), 563 - 568). Gennemsnitsværdien af den publicerede molære absorptionskoefficient er 5500.The enzymatic depolymerization process with heparinase is suitable for a spectrophotometric number average molecular weight (Mn) since the enzymatic process forms a reducing end group and an end group consisting of a / 4.5 unsaturated iduronic acid derivative with a distinct UV absorption at 230-235 nm. The molar absorption coefficient 15 for a number of LMW heparin fragments of di-, tetra-, hexa- and oligosaccharides was published by Linker and Hovingh (Biochem. 11 (1972), 563-556). The average value of the published molar absorption coefficient is 5500.
En ligning som 1_ = 1__ + AA235 (1) 20 Μ M c · Ç n n,u t der giver sammenhængen mellem antalsmiddelmolekylvægt (Mn), og stigningen i absorption ved 235 nm kan nemt udledes.An equation such as 1_ = 1__ + AA235 (1) 20 Μ M c · Ç n n, which gives the relationship between number average molecular weight (Mn) and the increase in absorption at 235 nm can be easily deduced.
I formel (1) er Μβ antalsmiddelmolekylvægten af det depolymeriserede produkt, Mn u er antalsmiddelmolekylvægten af 25 heparinsubstratet, c er substratkoncentrationen (g/1), ΔΑ235 er stigningen i absorptionen ved 235 nm og £ er den molære absorptionskoefficient .In formula (1), Μβ is the number average molecular weight of the depolymerized product, Mn u is the number average molecular weight of the heparin substrate, c is the substrate concentration (g / 1), ΔΑ235 is the increase in absorption at 235 nm, and er is the molar absorption coefficient.
Beregning af Mn er mulig, nâr Mn af heparinsubstratet (M ), substratkoncentration (c, g/1) og absorptionskoefficien-n, u 30 ten (£) af de umættede depolymeriseringsprodukter er kendt, og Δα235 mâles.Calculation of Mn is possible when Mn of the heparin substrate (M), substrate concentration (c, g / 1) and the absorption coefficient n, 30 (£) of the unsaturated depolymerization products are known and Δα235 is measured.
I en række fors0g blev heparin depolymeriseret med heparinase delvist renset med hydroxylapatit'kromatografi ifolge Linker og Hovingh (Methods in Enzymology 28^ (1972), 902 - 911).In a number of experiments, heparin was depolymerized with heparinase partially purified by hydroxylapatite chromatography according to Linker and Hovingh (Methods in Enzymology 28 ^ (1972), 902-911).
6 DK 156402 B6 DK 156402 B
Antalsmiddelmolekylvægten Mn blev beregnet ved at an-vende ligningen (1) og anvende den publicerede værdi for £ = 5500 og blev sammenlignet med Mn bestemt ved GPC-HPLC.The number average molecular weight Mn was calculated using Equation (1) and using the published value for £ = 5500 and was compared with Mn determined by GPC-HPLC.
Det blev imidlertid gentagne gange konstateret, at den 5 beregnede værdi af Mn (Mn(^A)) varierede fra den værdi for Mn, der blev fundet med HPLC (Mn(HPLC)), med op til 20%.However, it was repeatedly found that the calculated value of Mn (Mn (^ A)) varied from the value of Mn found by HPLC (Mn (HPLC)) by up to 20%.
Omarbejdning af (1) til ΛΑ235 = c-£ (^_ " ^_) (2) Μ M n n,u 10 muliggor beregning af en absorptionsstigning ΔΑ235 svaren<^e til en onsket antalsmiddelmolekylvægt Mn· Men forsog viste igen, at hvis depolymeriseringen blev standset ved den beregnede værdi af var den faktiske M bestemt ved HPLC betydeligt hojere end den onskede Mn, hvis værdien af c = 5500 fundet af Hovingh og 15 Linker blev anvendt.Recasting (1) to ΛΑ235 = c- £ (^ _ "^ _) (2) Μ M nn, u 10 allows calculation of an absorption increase ΔΑ235 the answer <^ e to a desired number average molecular weight Mn · But experiments again showed that if the depolymerization was stopped at the calculated value of, the actual M determined by HPLC was significantly higher than the desired Mn if the value of c = 5500 found by Hovingh and 15 Linker was used.
Det blev derfor konkluderet, at den dârlige overens-stemmelse mellem Mn(/\A) og Mn(HPLC) skyldtes anvendelsen af værdien 55 00 for£ .It was therefore concluded that the poor correlation between Mn (/ A) and Mn (HPLC) was due to the use of the value 5500 for £.
Omarbejdning af ligning (1) til 20 £ = ΔΑ235 Mn Mn,u (3) c (M - M ) n,u n' viser, at £ kan beregnes ved at anvende kendte værdier for c og ^ og samtidigt mâlte værdier for ΔΑ235 og Mn(HPLC).Recasting Equation (1) to £ 20 = ΔΑ235 Mn Mn, u (3) c (M - M) n, un 'shows that £ can be calculated using known values of c and ^ and simultaneously measured values of ΔΑ235 and Mn (HPLC).
Pâ denne mâde fandtes en værdi for £ = 7600, som gav 25 tæt korrelation mellem beregnet M^(AA) og observeret Mn(HPLC) i en række forsog.In this way, a value of £ = 7600 was found which gave a close correlation between calculated M ^ (AA) and observed Mn (HPLC) in a number of experiments.
Muligheden for at beregne en korrekt værdi af Mn base-ret pâ den letmâlelige ΔΑ235 er grundlaget for udovelsen af den foreliggende opfindelse, ifolge hvilken en batchenzymatiskThe possibility of calculating a correct value of Mn based on the easily measurable ΔΑ235 is the basis for the practice of the present invention, according to which a batch enzymatic
7 DK 156402 B7 DK 156402 B
depolymerisering af heparin fâr lov til at forl0be, indtil den beregnede værdi af ΔΑ235 er nâet, hvorpâ heparindepolymerise-ringsreaktionen standses, og reaktionsblandingen oparbejdes.depolymerization of heparin is allowed to proceed until the calculated value of ΔΑ235 is reached, whereby the heparin polymerization reaction is stopped and the reaction mixture is worked up.
Heparinase anvendt if0lge den foreliggende opfin-5 delse fremstilles pâ en i og for sig kendt mâde som beskrevet af Hovingh og Linker (Methods in Enzymology 2_8 (1972), 902 - 911 og J.Biol.Chem. 245 (1970), 6170 - 6175) ved dyrkning af Flavobacterium heparium pâ et heparinholdigt substrat, host af celler og sprængning af celler ved sonikering og rensning blandt andet 10 ved kromatografi pâ hydroxyapatit. Nedbrydningen af heparin med heparinase udfpres i vandigt medium som beskrevet af f.eks.Heparinase used according to the present invention is prepared in a manner known per se as described by Hovingh and Linker (Methods in Enzymology 2_8 (1972), 902-911 and J.Biol.Chem. 245 (1970), 6170). 6175) by growing Flavobacterium heparium on a heparin-containing substrate, coughing cells, and blasting cells by sonication and purification, among other things, 10 by hydroxyapatite chromatography. The degradation of heparin with heparinase is expressed in aqueous medium as described by e.g.
Hovingh og Linker (J.Biol.Chem. 240 (1965), 3724-3728). Nâr den pnskede Mn værdi af depolymeriseringsblandingen er nâet, inakti-veres heparinase pâ kendt mâde, f.eks. ved sænkning af pH eller 15 kort varmebehandling.Hovingh and Linker (J. Biol. Chem. 240 (1965), 3724-3728). When the desired Mn value of the depolymerization mixture is reached, heparinase is inactivated in known manner, e.g. by lowering the pH or 15 short heat treatment.
LMW-heparinproduktet udfældes derpâ pâ kendt mâde, f.eks. ved udfældning med alkohol, og oprenses pâ kendt mâde, f.eks. blegning, sterilfiltrering og alkoholudfældning.The LMW heparin product is then precipitated in a known manner, e.g. by precipitation with alcohol, and purified in known manner, e.g. bleaching, sterile filtration and alcohol precipitation.
Beregning af stigningen i absorption ved 20 230 - 235 nm ΔΑ233 svarende til den 0nskede af produktet foretages som folger: a) aflæsning pâ fig. 1 af Mn svarende til den onskede M og w y b) beregning af AA__r svarende til M fra a) ved 23b n 25 hjælp af den ovenfor nævnte formel (2) ved anvendelse af værdien 7600 for £.Calculation of the increase in absorption at 20 230 - 235 nm ΔΑ233 corresponding to the desired of the product is made as follows: a) reading in fig. 1 of Mn corresponding to the desired M and w y b) calculating AA__r corresponding to M from a) at 23b n 25 using the above formula (2) using the value 7600 for £.
ΔΑ235 mâles med et spectrophotometer efter syrning af proven, fortrinsvis til pH mindre end 2,5. Det er indlysende for en fagmand, at stigningen i UV-absorption forârsaget af dannelsen 30 af umættede nedbrydningsprodukter ved heparinasepâvirkning af heparin kan mâles ved andre b0lgelængder end her angivet. Absorp-tionskoefficienten mâles imidlertid fortrinsvis ved 235 nm, da den har sit maksimum ved denne b0lgelængde.ΔΑ235 is measured with a spectrophotometer after acidification of the sample, preferably to pH less than 2.5. It will be appreciated by those skilled in the art that the increase in UV absorption caused by the formation of unsaturated degradation products by the effect of heparinase on heparin can be measured at wavelengths other than those set forth herein. However, the absorption coefficient is preferably measured at 235 nm as it has its maximum at this wavelength.
Depolymeriseringen standses, nâr ΛΑ235 har nâet den 35 beregnede værdi, hvorpâ LMW-heparinproduktet udfældes ved tilsæt-ning af alkohol (fortrinsvis 0,6 - 10 vol/vol)..Depolymerization is stopped when ΛΑ235 has reached the calculated value at which the LMW heparin product is precipitated by the addition of alcohol (preferably 0.6 - 10 vol / vol).
8 DK 156402 B8 DK 156402 B
Heparindepolymeriseringsreaktion udf0res fortrinsvis ved en temperatur pâ 25 - 40°C og ved en pH-værdi pâ 6-8.Heparin polymerization reaction is preferably carried out at a temperature of 25 - 40 ° C and at a pH of 6-8.
Sk0nt den foreliggende opfindelse illustreres ved en procès, hvor heparinase anvendes i fri form, kan immobiliseret 5 heparinase ogsâ anvendes.Although the present invention is illustrated by a process in which heparinase is used in free form, immobilized heparinase can also be used.
Eksempel 1 illustrerer overensstemmelsen mellem malt ved A og mâlinger i forhold til HPLC-mâlinger (M^( /\A) i forhold til Mn(HPLC)) af pr0ver gennem hele forl0bet af en batch-depolymerisering af heparin.Example 1 illustrates the similarity between malt at A and measurements relative to HPLC measurements (M ^ (/ \ A) versus Mn (HPLC)) of samples throughout the course of a batch depolymerization of heparin.
10 Eksempel 1 2,5 g heparinnatrium, USP, M = 17.300, M = 12.400 w n dalton blev opl0st i 25 ml 0,1 M natriumacetat, 0,01 M calcium-acetat pH 7,0.Example 1 2.5 g of heparin sodium, USP, M = 17,300, M = 12,400 w n dalton was dissolved in 25 ml of 0.1 M sodium acetate, 0.01 M calcium acetate pH 7.0.
Heparinase, 0,8 ml, 1500 enheder/ml, specifik aktivitet 15 1050 enheder/mg blev opl0st i 25 ml 0,1 M natriumacetat.Heparinase, 0.8 ml, 1500 units / ml, specific activity 1050 units / mg was dissolved in 25 ml 0.1 M sodium acetate.
En heparinaseenhed defineres if0lge Hovingh og Linker, Methods in Enzymol., 2j3 (19 72), 902 - 911.A heparinase unit is defined according to Hovingh and Linker, Methods in Enzymol., 2j3 (19 72), 902 - 911.
Heparinsubstratet og enzymopl0sningen blev blandet og inkuberet ved let omr0ring i et vandbad holdt pâ 30°C.The heparin substrate and enzyme solution were mixed and incubated by light stirring in a water bath maintained at 30 ° C.
20 En pr0ve udtaget umiddelbart efter blanding blev for- tyndet med 1,7 M perchlorsyre, pH mindre end 2,5, og filtreret, og absorptionen blev mâlt ved 235 nm.A sample taken immediately after mixing was diluted with 1.7 M perchloric acid, pH less than 2.5, and filtered and the absorbance measured at 235 nm.
Absorptionsmâlingen blev gentaget et antal gange som vist i tabellen herunder, og samtidigt udtagne pr0ver blev kort 25 opvarmet over et kogende vandbad for at 0delægge enzymaktivite-ten, afk0let, fortyndet 5 gange med 0,5 M natriumsulfat og filtreret, og molekylvasgten blev analyser et - ved GPC-HPLC.The absorption measurement was repeated a number of times as shown in the table below, and at the same time, samples were briefly heated over a boiling water bath to destroy the enzyme activity, cooled, diluted 5 times with 0.5 M sodium sulfate and filtered, and the molecular weight was analyzed. - by GPC-HPLC.
GPC-HPLC analysen blev udfdrt under anvendelse af Waters 1-125 og 1-60 s0jler i serier med 0,5 M natriumsulfat som 30 eluent, 0,5 ml/min., monitoreret ved brydningsindeksbestemmelse, og molekylvægten blev beregnet ud fra retentionstiden, ved anvendelse af en ikke-lineær standardkurve baseret pâ dextran- og hep ar inf r agment s t an dar der.The GPC-HPLC assay was performed using Waters 1-125 and 1-60 columns in series with 0.5 M sodium sulfate as 30 eluent, 0.5 ml / min, monitored by refractive index determination, and molecular weight was calculated from the retention time. using a non-linear standard curve based on dextran and hep ar infr agment assumptions.
Antalsmiddelmolekylvægten, Mn, blev beregnet if0lge 35 ligning (1) under anvendelse af absorptionsstigningen ved 235 nm, £ = 7600, c = 50 og M = 12.400.The number average molecular weight, Mn, was calculated according to Equation (1) using the absorbance increase at 235 nm, £ = 7600, c = 50 and M = 12,400.
n / iln / il
9 DK 156402 B9 DK 156402 B
Resultaterne er anf0rt i tabellen herunder, og som det fremgâr, opnâedes der tæt overensstemmelse mellem den beregnede og den iagttagne antalsmiddelmolekylvægt.The results are given in the table below and, as can be seen, close agreement was obtained between the calculated and the observed number average molecular weight.
Tabel 5 Tid A235 ΔΑ235 Μη(ΔΑ) Mn(HPLC) (AA)/Mn(HPLC) ( timer ) _(dalton) (dalton)_% __ 0 4,5 4,5 1.0 28,83 24,33 6912 6750 102,4 2.0 47,67 43,17 5148 4661 110,4 10 3,0 63,43 58,93 4242 4261 99,6 3.5 70,03 65,53 3951 3863 102,3 4.0 77,5 73,00 3666 3830 95,7 4.5 83,2 78,7 3475 3565 97,5 5.5 94,4 89,9 3153 3145 100,3 15 22 242,5 238 1415 1405 100,7 101,1 ± 4,4 (S.D.)Table 5 Time A235 ΔΑ235 Μη (ΔΑ) Mn (HPLC) (AA) / Mn (HPLC) (hours) _ (dalton) (dalton) _% __ 0 4.5 4.5 1.0 28.83 24.33 6912 6750 102.4 2.0 47.67 43.17 5148 4661 110.4 10 3.0 63.43 58.93 4242 4261 99.6 3.5 70.03 65.53 3951 3863 102.3 4.0 77.5 73.00 3666 3830 95.7 4.5 83.2 78.7 3475 3565 97.5 5.5 94.4 89.9 3153 3145 100.3 15 22 242.5 238 1415 1405 100.7 101.1 ± 4.4 (SD)
Eksempel 2Example 2
Formâlet med eksperimenterne beskrevet nedenfor var at 20 producere LMW-heparin med en middelmolekylvægt (M ) pâ 6500 ± 500 w dalton.The purpose of the experiments described below was to produce LMW heparin with a mean molecular weight (M) of 6500 ± 500 w daltons.
Fra fig. 1 blev antalsmiddelmolekylvægten (Mn) svarende til M = 6500 dalton fundet til at være ca. 3500 dalton.From FIG. 1, the number average molecular weight (Mn) corresponding to M = 6500 daltons was found to be approx. 3500 daltons.
ww
Fem forskellige hepariner (se tabellen nedenfor) blev 25 valgt til enzymatisk depolymerisering. Ændringen i optisk densi-tet ved 235 nm (ΔΑ233 beregnet) ) svarende til en Mn værdi i reaktionsblandingen pâ 3500 dalton (i et eksperiment 3400 dalton)Five different heparins (see table below) were selected for enzymatic depolymerization. The change in optical density at 235 nm (ΔΑ233 calculated)) corresponding to an Mn value in the reaction mixture of 3500 daltons (in an experiment 3400 daltons)
10 DK 156402 BDK 156402 B
blev beregnet for.hver heparin fra ligning 2 under anvendelse af c = 50 mg/ml, £ = 7600 og M -værdien af de aktuelle heparin-prover (se tabel).was calculated for each heparin from Equation 2 using c = 50 mg / ml, £ = 7600 and the M value of the actual heparin samples (see Table).
Den enzymatiske nedbrydning af heparinerne blev udfort 5 som folger:The enzymatic degradation of the heparins was carried out as follows:
Heparin blev oplost i en koncentrâtion pâ 50 mg/ml i 0,1 M natriumacetatpuffer, pH 7,0 indeholdende spor af calcium (0,0005 - 0,01 M). Oplosningen blev opvarmet til 30°C, og hepa-rinase blev tilsat i en mængde nodvendig for at depolymerisere 10 heparin til den onskede Mn_værdi pâ ca. 48 timer.Heparin was dissolved in a concentration of 50 mg / ml in 0.1 M sodium acetate buffer, pH 7.0 containing traces of calcium (0.0005 - 0.01 M). The solution was heated to 30 ° C and heparinase was added in an amount necessary to depolymerize 10 heparin to the desired Mn value of approx. 48 hours.
Ændringen i optisk densitet ved 235 nm (ΔΑ235^ blev malt lobende i prover af reaktionsblandingerne efter fortynding med 1,7 M perchlorsyre, pH under 2,5. Nâr ΔΑ235 var me<^ ΔΑ235 (beregnet), blev den enzymatiske depolymerisering stand-15 set.The change in optical density at 235 nm (ΔΑ235 ^ was painted continuously in samples of the reaction mixtures after dilution with 1.7 M perchloric acid, pH below 2.5. When ΔΑ235 was with ^ΑΔ352 (calculated), the enzymatic depolymerization was stopped seen.
Nâr ΔΑ235 ^avc^e n^et den beregnede værdi, blev LMW-heparinproduktet udfældet ved tilsætning af alkohol, og det de-polymeriserede produkt blev oprenset pâ kendt mâde, f.eks. ved blegning, sterilfiltrering og alkoholudfældning. Egenskaber af 20 produkterne fra 5 uafhængige forsog er vist i den folgende tabel.When ΔΑ235 ° C reached the calculated value, the LMW heparin product was precipitated by the addition of alcohol and the de-polymerized product was purified in known manner, e.g. by bleaching, sterile filtration and alcohol precipitation. Properties of the 20 products from 5 independent experiments are shown in the following table.
Tabel IITable II
Forsog Batch- M af 0nsket Mængde anvendtAttempt Batch-M of desired quantity used
Π/U JU / U J
nr. storrelse heparin Mr i (bereg- heparinase (g) (dalton) produkt net) (NOVO enheder/g 25 __(dalton) _heparin)_ 1 1000 10100 3500 70,94 53 2 1000 12400 3500 77,93 65 3 1200 13900 3400 84,43 73 4 1000 11600 3500 75,81 60 30 5 1000 11200 3500 74,64 62No. Size Heparin Mr i (Calculate Heparinase (g) (Dalton) Product Grid) (NOVO Units / g 25 __ (Dalton) _ Heparin) _ 1 1000 10100 3500 70.94 53 2 1000 12400 3500 77.93 65 3 1200 13900 3400 84.43 73 4 1000 11600 3500 75.81 60 30 5 1000 11200 3500 74.64 62
11 DK 156402 B11 DK 156402 B
Forseg Slut Udbytte Mi Mi Biologisk II w nr. ΔΑ235 1 af Pro~ produkt produkt aktivitet af reaktions- dukt (dalton) (dalton) produkt _% (w/w)__IU/mg_ 5 1 71,76 87,8 3690 6150 2 77,40 93,9 3840 6510 76 3 83,97 90,6 3710 6630 77 4 75,07 90,5 3660 6180 82 5 74,80 93,0 3610 6610 82 10 Biologisk aktivitet i et amidolytisk antifaktor Xa assay modificeret for udforelse med automatisk analyseapperatur, men ellers som beskrevet af A.N. Teien et al., Thrombosis Research (1976), 413. Der anvendtes renfremstillet antithrombin III (AT III) og faktor Xa og den 1. internationale LMW-heparin-15 standard (National Institute for Biological Standards and Controls, London) som referencestandard.Seal End Yield Mi Mi Biological II w No. ΔΑ235 1 of Pro ~ product Product Activity of Reaction Product (Dalton) (Dalton) Product _% (w / w) __ IU / mg_ 5 1 71.76 87.8 3690 6150 2 77.40 93.9 3840 6510 76 3 83.97 90.6 3710 6630 77 4 75.07 90.5 3660 6180 82 5 74.80 93.0 3610 6610 82 10 Biological activity in an amidolytic antifactor Xa assay modified for execution with automatic analysis apparatus, but otherwise as described by AN Teien et al., Thrombosis Research (1976), 413. Pure antithrombin III (AT III) and factor Xa and the 1st International LMW Heparin Standard (National Institute for Biological Standards and Controls, London) were used as the reference standard.
Det fremgâr af ovennævnte, at aile slut-LMW-heparinpro-dukterne har en vægtmiddelmolekylvægt (M^) inden for det onskede omrâde.It is apparent from the above that all end LMW heparin products have a weight average molecular weight (M 2) within the desired range.
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